Datasets:

xNoper

/

pubmed-orig-test

like 0

[ "wild animals are exposed to environmental contaminants of both natural and anthropogenic origin and are suitable bioindicators of environmental pollution ( obrien et al .", "concentrations of toxic compounds in body tissues are determined by the degree of exposure , the compound s bioavailability and half - life , as well as the type of tissue ( nordberg et al .", "pollutants can be transported over significant distances by air or water , and they are found even in regions situated remotely from industrial centers ( ansara - ross et al .", "therefore , due to the rapid development and wide diversity of human activities , animals are increasingly exposed to harmful effects of compounds of anthropogenic origin ( obrien et al .", "1993 ) . with growing public awareness of environmental pollution , regular monitoring of pollutants in the environment is essential to ensure levels of pollutants are below harmful or recommended , legal values ( nasreddine and parent - massin 2002 ; european commission 2006 ) .", "such monitoring should be carried out not only in industrialized areas but also in natural and agricultural ecosystems away from the source of emissions ( nasreddine and parent - massin 2002 ; szkoda et al . 2012 ) .", "metals include toxic elements such as cadmium ( cd ) and lead ( pb ) , as well as essential elements in which toxicity is determined by the dose , as copper ( cu ) and zinc ( zn ) .", "although these four elements occur naturally in the environment , they are supplied in large quantities through domestic and industrial pollution ( bjerregaard and andersen 2011 ) .", "heavy metals are also persistent environmental contaminants whose physiochemical properties contribute to their mobility in the natural environment , posing a significant threat for living organisms and ecosystems ( bjerregaard and andersen 2011 ) .", "previous studies reported essential and toxic metals in beaver ( family : castoridae ) tissues obtained from industrial areas , generally regarded as polluted ( hillis and parker 1993 ; zalewski et al . 2012 ) , agricultural regions ( fimreite et al .", "2001 ) , and natural ecosystems ( wren 1984 ; zalewski et al . 2012 ; giejewska et al .", "beavers are semi - aquatic , territorial herbivorous rodents feeding on the bark , shoots , and leaves of a wide variety of woody plants including willow ( salix sp . ) and aspen ( populus tremuloides ) , as well as on non - woody terrestrial and aquatic plants ( haarberg and rosell 2006 ; krojerov - prokeov et al .", "beavers catholic diet and sedentary behavior make them suitable bioindicators of local environmental pollution . in poland ,", "the status of eurasian beavers ( castor fiber ) as a partially protected species ( polish minister of the environment 2011 ) contributed to rapid population growth .", "it is currently estimated that 78,000 beavers occur in the country , and beaver populations continue spreading to new areas , including near human settlements , where they are likely to get exposed to anthropogenic pollutants ( flis 2013 ) . in this study ,", "our aim was to use the eurasian beaver as a bioindicator organism ( i ) to assess trace element contents in a region away from major industrial centers and ( ii ) to evaluate the degree of contamination against legal cd and pb levels recommended by the european commission in farm animal edible tissues ( european commission 2006 ) with regard to the lack of an official threshold for wildlife ( giejewska et al .", "2014 ) . specifically , we measured concentrations of cd , pb , cu , and zn in liver and kidney of wild beavers . we predicted ( i ) metal concentrations to differ between liver and kidney according to element - specific metabolism , ( ii ) no sexual difference in metal concentrations in this non - dimorphic species , and ( iii ) higher metal concentrations in adult than in juvenile individuals because of bioaccumulation processes .", "we compared our results with previous research , and we discuss the implications for future use of beavers as bioindicators for the monitoring of metal pollution .", "n ) , warmia and mazury region , northeastern poland , in summer 2012 . warmia and mazury is a traditional agricultural region with extensive forests and numerous lakes .", "the region is an important recreational center for local and international tourists who seek activities in a natural environment .", "the sampling site was away from a major industrial complex , which could putatively be a source of pollutants in their vicinity .", "animals were collected upon approval of the regional nature conservation authority ( opinion no . rdo-28-oop-6631 - 0007 - 638/09/10/pj 2010 ) and the local ethics committee ( opinion no .", "all beavers were collected during daytime with a net and transported to the laboratory in cages specially adapted for this purpose ( zalewski et al .", "2010 ) , and they were classified into two age classes : ( i ) juveniles , 2 months old , and ( ii ) adults , > 24 months old ( table 1 ) .", "sex was determined based on the color of anal gland secretions ( schulte et al .", "beavers were anesthetized with xylazine and ketamine in doses appropriate to body weight , and body measurements taken.table 1biometric data of 10 eurasian beavers from wiajny municipality , northeastern poland , 2012individualsexage classbody weight ( kg)body length ( cm)m1juvenile2.562m2juvenile2.862f3juvenile2.864f4juvenile2.760m5adult18.6116m6adult22.6114f7adult17.3115f8adult21.1117f9adult22.7118f10adult22.0121 biometric data of 10 eurasian beavers from wiajny municipality , northeastern poland , 2012 then , beavers were sacrificed by decapitation under full anesthesia .", "liver and kidney samples of c. 15 g each were collected and stored in polyethylene bags at 25 c until analysis ( zalewski et al .", "each sample was homogenized and two subsamples were prepared from each individual organ and analyzed twice each as replicates .", "all tissue samples were mineralized in the muffle furnace at 450 c for 24 h. ashes were dissolved in 1 m hno3 and deionized water was added to 25 ml ( uczyska et al .", "we performed graphite furnace atomic absorption spectroscopy to determine cd and pb concentrations and flame atomic absorption spectroscopy to determine cu and zn concentrations ( whiteside and milner 1984 ) using an ice 3000 series spectrometer ( thermo scientific , uk ) .", "analysis for each element was performed based on calibration curves plotted from working standards . for calibration , we used commercial stock solutions ( 1000 mg", "working standards were prepared by dissolving stock solutions with 0.1 m hno3 and deionized water .", "calibration ranges were 0.00050.004 g / g for cd , 0.0010.01 g / g for pb , 0.050.8 g / g for cu , and 0.10.8 g / g for zn , respectively .", "limits of detection were 0.002 g / g for cd , 0.001 g / g for pb , and 0.05 g / g for cu and zn .", "quality control of analytical measurements was performed using certified reference material ( bcr-185r bovine liver , irrm , belgium ) .", "recoveries of all four elements were within the range 86109 % and relative standard deviations were below 10 % .", "metal concentrations were expressed in micrograms per gram of wet weight , with precision consistent with limits of quantitation0.001 g / g for cd and pb and 0.1 g / g for cu and zn .", "concentrations in beaver livers and kidneys were compared between age or sex classes using a non - parametric mann - whitney u test .", "pair - wise comparisons between element concentrations in each organ were performed using a spearman rank correlation analysis .", "we report arithmetic means , standard deviations ( sd ) , median , and range . where necessary , data were transformed to meet statistical assumptions .", "significance level was set at p < 0.05 , and all statistical analyses were run using r version 3.0.0 ( r core team 2013 ) .", "we captured six female ( four adults , two juveniles ) and four male ( two adults , two juveniles ) beavers ( table 1 ) . mean concentrations of cd , pb , cu , and zn determined in liver and kidney samples are presented in table 2 and compared with the literature ( table 3 ) .", "higher concentrations in liver than in kidney were found for cu ( t = 4.534 ; df = 9 ; p = 0.001 ) and zn ( t = 19.826 ; df = 9 ; p < 0.001 )", ". additionally , zn levels were significantly correlated to cd levels in kidney ( n = 10 , rs = 0.939 ; p < 0.001).table 2summary statistics of the concentration ( g / g wet weight ) of four metals ( cd , pb , cu , and zn ) in liver and kidney of 10 eurasian beavers , ne poland , 2012metalmean sdmedianrangemaximum legal level \n liver cd0.21 ", " 0.440.060.031.440.5 pb0.08 0.030.060.050.120.5 cu9.2 4.56.95.216.4 \n zn35.7 3.536.231.440.6 \n kidney cd2.81 4.521.370.0214.881.0 pb0.08 0.030.070.050.140.5 cu3.7 1.13.53.03.8 \n zn21.5 2.720.219.027.9 \n\n maximum levels set by the european commission ( 2006 ) for contaminants of toxic elements in food of animal origin \n no regulationtable 3average concentrations ( g / g wet weight ) of cd , pb , zn , and cu in liver and kidney of beavers ( family : castoridae ) given in the literaturespeciesliverkidneysitesreferencecdpbcuzncdpbcuzn \n c. fiber \n 0.210.089.235.72.810.083.721.5unpollutedthis study \n c. fiber \n 0.880.117.930.06unpollutedgiejewska et al .", "( 2001 ) \n c. canadensis \n 2.32.7880.818.83.49.399.315 km from ore smelterhillis and parker ( 1993 ) \n c. canadensis \n 1.42.190 km from ore smelterhillis and parker ( 1993 ) \n c. canadensis \n 0.192.929.61.44325.4175 km from ore smelterwren ( 1984 ) summary statistics of the concentration ( g / g wet weight ) of four metals ( cd , pb , cu , and zn ) in liver and kidney of 10 eurasian beavers , ne poland , 2012 \n maximum levels set by the european commission ( 2006 ) for contaminants of toxic elements in food of animal origin average concentrations ( g / g wet weight ) of cd , pb , zn , and cu in liver and kidney of beavers ( family : castoridae ) given in the literature we found no differences between female ( n = 6 ) and male ( n = 4 ) beavers for any of the four metal concentrations in liver or kidney ( fig . 1 ) .", "contrastingly , we found significant differences in cd and cu concentrations between adult and juvenile beavers ( fig . 2 ) .", "cadmium concentrations were significantly higher in adults ( n = 6 ) than in juveniles ( n = 4 ) in both liver ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and kidney ( u = 0.0 ; z = 2.452 ; p = 0.014 ) . in liver ,", "cu levels were significantly lower in adults than in juveniles ( u = 0.0 ; z = 2.452 ; p = 0.014 ) , and in kidney , cu levels were significantly higher in adults than in juveniles ( u = 2.0 ; z = 2.025 ; p = 0.043).fig .", "1concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six female and four male eurasian beavers , ne poland , 2012 .", "no statistical significant difference in metal concentrations between sex classes was found ( mann - whitney u test , all p > 0.5 ) .", "box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) . for clarity , extreme outliers", "2concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six adult and four juvenile eurasian beavers , ne poland , 2012 .", "significant statistical differences in metal concentrations between age classes were found in liver cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and in kidney cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 2.0 ; z = 2.025 ; p = 0.043 ) . box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) , and statistical outliers ( open circles ) . for clarity ,", "extreme outliers are not shown concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six female and four male eurasian beavers , ne poland , 2012 .", "no statistical significant difference in metal concentrations between sex classes was found ( mann - whitney u test , all p > 0.5 ) . box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) . for clarity ,", "extreme outliers are not shown concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six adult and four juvenile eurasian beavers , ne poland , 2012 .", "significant statistical differences in metal concentrations between age classes were found in liver cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and in kidney cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 2.0 ; z = 2.025 ; p = 0.043 ) .", "box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) , and statistical outliers ( open circles ) . for clarity , extreme outliers", "our study revealed the presence of cd , pb , cu , and zn in liver and kidney of all sampled beavers . no relationship was observed between metal concentrations and the beavers sex .", "although we acknowledge the limitations of the tests due to a reduced number of individuals in each group , these results were expected as the species present no sexual dimorphism and no partitioning in diet ( haarberg and rosell 2006 ; krojerov - prokeov et al .", "contrastingly , some metal concentrations were affected by the animals age . higher liver and kidney cd concentrations were found in adult beavers than in juveniles , indicating that this metal bioaccumulates over time ( parker and hamr 2001 ; bilandi et al .", "cd levels in all six adult beavers exceed the maximum threshold of 1.0 g / g in kidney recommended in farm animal edible tissues . in liver , however , only one adult female ( f7 ) exceeded the recommended 0.5 g / kg cd limit ( european commission 2006 ) .", "such differences in cd levels with age might be due to this metal accumulating over an animal s entire life span , especially in kidney , as previously described in other species ( e.g. , red fox ( vulpes vulpes ) and stone marten ( martes foina ) : bilandi et al .", "2010 ; african grass owl ( tyto capensis ) : ansara - ross et al . 2013 ; penguins ( pygoscelis spp . ) : jerez et al .", "average cd content in kidney was similar to that observed in 2003 in beavers from srokowo forest division , an uncontaminated area in northeastern poland ( zalewski et al .", "2012 ) . however , our results were approximately four- to fivefold lower in liver and three- to fourfold lower in kidney than levels measured in earlier studies on beaver at unpolluted sites in poland ( giejewska et al .", "2014 ) , in norway ( fimreite et al . 2001 ) , and in a putatively contaminated former air base in poland ( zalewski et al .", "contrastingly , cd concentrations in liver and kidney were higher than those reported in canadian beavers ( castor canadensis ) in habitats located 175 km from an ore smelter in ontario , canada ( wren 1984 ) .", "cadmium concentration in wildlife tissue is attributed to its levels in the environment and additionally with animals age .", "previous studies concerned with cd content in unpolluted areas presented values that could be attributed to metal transport over long distances from the pollution source as well as increased mobility due to soil and water acidification ( wren 1984 ) .", "although metal concentrations in food sources typically consumed by beavers were not examined in the present study , it has been reported that beavers have a strong preference for aspen ( p. tremuloides ) and willow ( salix sp . ) , trees that accumulate high levels of cd particularly on acidic soils that increase the mobility of heavy metals ( wren 1984 ) .", "cd is easily absorbed and accumulates even at low levels of chronic exposure ( nordberg et al .", "interspecific differences , intraspecific variations among populations , or physiological determinants of metal absorption and excretion can also contribute to the observed differences ( nordberg et al .", "all values of pb found in beavers were below the 0.5 g / g threshold recommended for both organs in farm animal edible tissues ( european commission 2006 ) .", "lead concentrations in kidney ranged from 0.052 g / g ( f9 ) to 0.142 g / g ( f10 ) .", "lead passes the placenta and young animals have a higher rate of absorption from the intestinal tract ( nordberg et al .", "as this metal is not metabolically regulated in organisms , pb concentrations in animal tissues are related to its presence in the ecosystem ( jerez et al .", "soil intentionally or accidentally consumed by beavers could constitute an additional source of contamination as pb can be bound in soil and bottom sediments and secondarily released ( rajaganapathy et al .", "lead concentrations in our study were low compared to previous research ( table 3 ) .", "this could be attributed to the absence of major industrial sites close to the sample site , seasonality , or individual variability in diet ( brekken and steinnes 2004 ) .", "copper absorption is regulated by homeostatic mechanisms in the liver and is mainly excreted through the bile .", "absorbed zn is bound to plasma albumin and macroglobulins and distributed to the liver where it rapidly accumulates ( parker and hamr 2001 ) .", "although cu has higher affinity than zn to metallothionein ( low molecular weight metal - binding protein ) which binds cu and zn at low levels of cd ( hillis and parker 1993 ; nordberg et al .", "2011a ) , hepatic metallothionein seems to have an important role in accumulation and storage of both cu and zn in the liver ( parker and hamr 2001 ) .", "copper concentrations were higher in juvenile than in adult beavers in liver , indicating age - specific metabolism capabilities of this microelement ( nordberg et al .", "higher cu levels in juveniles liver can be attributed to increased requirements for this element in growing organisms ( parker and hamr 2001 ; jerez et al .", "our cu values in both liver and kidney were higher than those in previous studies ( table 3 ) .", "copper as an essential microelement has effective regulation mechanisms of uptake and excretion , and beaver diet contains high amounts of cu and zn ( brekken and steinnes 2004 ) .", "therefore , we assume that our values were at normal physiological levels ( nordberg et al", "zinc concentrations did not differ between organs and sex or age classes in our study .", "however , excluding beavers collected in the vicinity of the ore smelter in ontario , canada , where zn concentrations reached 80.8 ", "g / g in the liver ( hillis and parker 1993 ) , we found zn levels in liver higher than in previous studies ( table 3 ) . in kidney ,", "zn levels were highly correlated to cd levels regardless of the beaver age or sex .", "zinc and cadmium have similar chemical properties , and they are usually found as a mineral combination in the environment ( nordberg et al .", "metallothionein can bind cd , cu , and zn , and this protein will bind most of the cd and store it in kidney . exposure to low cd levels may cause a redistribution of zn in the organism , increasing zn concentrations in kidney ( nordberg et al .", "higher kidney zn concentrations were reported in beavers from ontario ( hillis and parker 1993 ) .", "the highest zn concentrations ( liver 40.6 g / g ; kidney 27.9 g / g ) we found in adult female f7 could also possibly be attributed to a diet rich in this microelement ( brekken and steinnes 2004 ) .", "general metal concentrations in beaver tissues from northeastern poland were similar to or lower than those found in other countries .", "however , the fact that high cd concentrations and pb were present in a putatively unpolluted area calls for a regular monitoring of environmental pollutants in agricultural and natural areas . future investigation of trace elements in beaver tissues sampled from industrial regions is needed for comparison and to draw further conclusion about levels of contamination .", "significant expansion of beaver populations in poland contributes to growing human - beaver conflict , increasing the amount of compensation paid to farmers and landowners .", "although beaver is currently not considered as a consumptive species in poland , beaver is classified as a game species in six european countries ( belarus , estonia , lithuania , latvia , norway , and sweden ) as well as in canada and russia ( jankowska et al .", "hunting beavers for consumption has been suggested as a tool for population management , and beaver consumption could occur in the near future in poland .", "research to ascertain tissues are safe from a toxicological point of view is therefore timely . in this context ,", "the fact that cd concentrations in adult beavers exceeded limits recommended in farm animal tissues warrants caution .", "also , it would be necessary to investigate levels of persistent organic pollutants ( pops ) in beaver tissues .", "pops are hydrophobic and lipophilic persistent compounds that may accumulate along the food chain and can cause severe metabolic disturbance , such as endocrinal disturbance ( jones and de voogt 1999 ) .", "the presence of pops in remote areas such as the arctic is a major environmental issue ( brault et al .", "consequently , we recommend future research and regular monitoring to identify the source of contaminants in the ecosystem and possible mitigation measures , and to ensure that contamination levels are within a safe range ." ]

[ "<S> heavy metals are persistent environmental contaminants , and wild animals are increasingly exposed to the harmful effects of compounds of anthropogenic origin , even in areas distant from industrial centers . </S>", "<S> we used atomic absorption spectrometry to determine levels of cadmium ( cd ) , lead ( pb ) , copper ( cu ) , and zinc ( zn ) in liver and kidney of wild eurasian beavers ( castor fiber ) in poland . </S>", "<S> cd concentrations in liver ( 0.21 0.44 g / g ) and in kidney ( 2.81 4.52 g / g ) were lower in juvenile than in adult beavers . </S>", "<S> pb concentrations in liver ( 0.08 0.03 g / g ) and kidney ( 0.08 0.03 g / g ) were similar among all individuals , while both cu and zn levels were higher in liver ( cu 9.2 4.5 g / g ; zn 35.7 3.5 g / g ) than in kidney ( cu 3.7 1.1 g / g ; zn 21.5 2.7 g / g ) . </S>", "<S> cu levels also differed between juveniles and adults . </S>", "<S> we reviewed the literature reporting metal concentrations in beavers . </S>", "<S> our results indicate metal contamination in beavers away from important industrial emission sources and suggest the natural environment should be regularly monitored to ensure their levels are below recommended , legal values . </S>" ]

[ "Introduction", "Material and methods", "Results", "Discussion", "Conclusion and outlook" ]

[ [ "wild animals are exposed to environmental contaminants of both natural and anthropogenic origin and are suitable bioindicators of environmental pollution ( obrien et al .", "concentrations of toxic compounds in body tissues are determined by the degree of exposure , the compound s bioavailability and half - life , as well as the type of tissue ( nordberg et al .", "pollutants can be transported over significant distances by air or water , and they are found even in regions situated remotely from industrial centers ( ansara - ross et al .", "therefore , due to the rapid development and wide diversity of human activities , animals are increasingly exposed to harmful effects of compounds of anthropogenic origin ( obrien et al .", "1993 ) . with growing public awareness of environmental pollution , regular monitoring of pollutants in the environment is essential to ensure levels of pollutants are below harmful or recommended , legal values ( nasreddine and parent - massin 2002 ; european commission 2006 ) .", "such monitoring should be carried out not only in industrialized areas but also in natural and agricultural ecosystems away from the source of emissions ( nasreddine and parent - massin 2002 ; szkoda et al . 2012 ) .", "metals include toxic elements such as cadmium ( cd ) and lead ( pb ) , as well as essential elements in which toxicity is determined by the dose , as copper ( cu ) and zinc ( zn ) .", "although these four elements occur naturally in the environment , they are supplied in large quantities through domestic and industrial pollution ( bjerregaard and andersen 2011 ) .", "heavy metals are also persistent environmental contaminants whose physiochemical properties contribute to their mobility in the natural environment , posing a significant threat for living organisms and ecosystems ( bjerregaard and andersen 2011 ) .", "previous studies reported essential and toxic metals in beaver ( family : castoridae ) tissues obtained from industrial areas , generally regarded as polluted ( hillis and parker 1993 ; zalewski et al . 2012 ) , agricultural regions ( fimreite et al .", "2001 ) , and natural ecosystems ( wren 1984 ; zalewski et al . 2012 ; giejewska et al .", "beavers are semi - aquatic , territorial herbivorous rodents feeding on the bark , shoots , and leaves of a wide variety of woody plants including willow ( salix sp . ) and aspen ( populus tremuloides ) , as well as on non - woody terrestrial and aquatic plants ( haarberg and rosell 2006 ; krojerov - prokeov et al .", "beavers catholic diet and sedentary behavior make them suitable bioindicators of local environmental pollution . in poland ,", "the status of eurasian beavers ( castor fiber ) as a partially protected species ( polish minister of the environment 2011 ) contributed to rapid population growth .", "it is currently estimated that 78,000 beavers occur in the country , and beaver populations continue spreading to new areas , including near human settlements , where they are likely to get exposed to anthropogenic pollutants ( flis 2013 ) . in this study ,", "our aim was to use the eurasian beaver as a bioindicator organism ( i ) to assess trace element contents in a region away from major industrial centers and ( ii ) to evaluate the degree of contamination against legal cd and pb levels recommended by the european commission in farm animal edible tissues ( european commission 2006 ) with regard to the lack of an official threshold for wildlife ( giejewska et al .", "2014 ) . specifically , we measured concentrations of cd , pb , cu , and zn in liver and kidney of wild beavers . we predicted ( i ) metal concentrations to differ between liver and kidney according to element - specific metabolism , ( ii ) no sexual difference in metal concentrations in this non - dimorphic species , and ( iii ) higher metal concentrations in adult than in juvenile individuals because of bioaccumulation processes .", "we compared our results with previous research , and we discuss the implications for future use of beavers as bioindicators for the monitoring of metal pollution ." ], [ "n ) , warmia and mazury region , northeastern poland , in summer 2012 . warmia and mazury is a traditional agricultural region with extensive forests and numerous lakes .", "the region is an important recreational center for local and international tourists who seek activities in a natural environment .", "the sampling site was away from a major industrial complex , which could putatively be a source of pollutants in their vicinity .", "animals were collected upon approval of the regional nature conservation authority ( opinion no . rdo-28-oop-6631 - 0007 - 638/09/10/pj 2010 ) and the local ethics committee ( opinion no .", "all beavers were collected during daytime with a net and transported to the laboratory in cages specially adapted for this purpose ( zalewski et al .", "2010 ) , and they were classified into two age classes : ( i ) juveniles , 2 months old , and ( ii ) adults , > 24 months old ( table 1 ) .", "sex was determined based on the color of anal gland secretions ( schulte et al .", "beavers were anesthetized with xylazine and ketamine in doses appropriate to body weight , and body measurements taken.table 1biometric data of 10 eurasian beavers from wiajny municipality , northeastern poland , 2012individualsexage classbody weight ( kg)body length ( cm)m1juvenile2.562m2juvenile2.862f3juvenile2.864f4juvenile2.760m5adult18.6116m6adult22.6114f7adult17.3115f8adult21.1117f9adult22.7118f10adult22.0121 biometric data of 10 eurasian beavers from wiajny municipality , northeastern poland , 2012 then , beavers were sacrificed by decapitation under full anesthesia .", "liver and kidney samples of c. 15 g each were collected and stored in polyethylene bags at 25 c until analysis ( zalewski et al .", "each sample was homogenized and two subsamples were prepared from each individual organ and analyzed twice each as replicates .", "all tissue samples were mineralized in the muffle furnace at 450 c for 24 h. ashes were dissolved in 1 m hno3 and deionized water was added to 25 ml ( uczyska et al .", "we performed graphite furnace atomic absorption spectroscopy to determine cd and pb concentrations and flame atomic absorption spectroscopy to determine cu and zn concentrations ( whiteside and milner 1984 ) using an ice 3000 series spectrometer ( thermo scientific , uk ) .", "analysis for each element was performed based on calibration curves plotted from working standards . for calibration , we used commercial stock solutions ( 1000 mg", "working standards were prepared by dissolving stock solutions with 0.1 m hno3 and deionized water .", "calibration ranges were 0.00050.004 g / g for cd , 0.0010.01 g / g for pb , 0.050.8 g / g for cu , and 0.10.8 g / g for zn , respectively .", "limits of detection were 0.002 g / g for cd , 0.001 g / g for pb , and 0.05 g / g for cu and zn .", "quality control of analytical measurements was performed using certified reference material ( bcr-185r bovine liver , irrm , belgium ) .", "recoveries of all four elements were within the range 86109 % and relative standard deviations were below 10 % .", "metal concentrations were expressed in micrograms per gram of wet weight , with precision consistent with limits of quantitation0.001 g / g for cd and pb and 0.1 g / g for cu and zn .", "concentrations in beaver livers and kidneys were compared between age or sex classes using a non - parametric mann - whitney u test .", "pair - wise comparisons between element concentrations in each organ were performed using a spearman rank correlation analysis .", "we report arithmetic means , standard deviations ( sd ) , median , and range . where necessary , data were transformed to meet statistical assumptions .", "significance level was set at p < 0.05 , and all statistical analyses were run using r version 3.0.0 ( r core team 2013 ) ." ], [ "we captured six female ( four adults , two juveniles ) and four male ( two adults , two juveniles ) beavers ( table 1 ) . mean concentrations of cd , pb , cu , and zn determined in liver and kidney samples are presented in table 2 and compared with the literature ( table 3 ) .", "higher concentrations in liver than in kidney were found for cu ( t = 4.534 ; df = 9 ; p = 0.001 ) and zn ( t = 19.826 ; df = 9 ; p < 0.001 )", ". additionally , zn levels were significantly correlated to cd levels in kidney ( n = 10 , rs = 0.939 ; p < 0.001).table 2summary statistics of the concentration ( g / g wet weight ) of four metals ( cd , pb , cu , and zn ) in liver and kidney of 10 eurasian beavers , ne poland , 2012metalmean sdmedianrangemaximum legal level \n liver cd0.21 ", " 0.440.060.031.440.5 pb0.08 0.030.060.050.120.5 cu9.2 4.56.95.216.4 \n zn35.7 3.536.231.440.6 \n kidney cd2.81 4.521.370.0214.881.0 pb0.08 0.030.070.050.140.5 cu3.7 1.13.53.03.8 \n zn21.5 2.720.219.027.9 \n\n maximum levels set by the european commission ( 2006 ) for contaminants of toxic elements in food of animal origin \n no regulationtable 3average concentrations ( g / g wet weight ) of cd , pb , zn , and cu in liver and kidney of beavers ( family : castoridae ) given in the literaturespeciesliverkidneysitesreferencecdpbcuzncdpbcuzn \n c. fiber \n 0.210.089.235.72.810.083.721.5unpollutedthis study \n c. fiber \n 0.880.117.930.06unpollutedgiejewska et al .", "( 2001 ) \n c. canadensis \n 2.32.7880.818.83.49.399.315 km from ore smelterhillis and parker ( 1993 ) \n c. canadensis \n 1.42.190 km from ore smelterhillis and parker ( 1993 ) \n c. canadensis \n 0.192.929.61.44325.4175 km from ore smelterwren ( 1984 ) summary statistics of the concentration ( g / g wet weight ) of four metals ( cd , pb , cu , and zn ) in liver and kidney of 10 eurasian beavers , ne poland , 2012 \n maximum levels set by the european commission ( 2006 ) for contaminants of toxic elements in food of animal origin average concentrations ( g / g wet weight ) of cd , pb , zn , and cu in liver and kidney of beavers ( family : castoridae ) given in the literature we found no differences between female ( n = 6 ) and male ( n = 4 ) beavers for any of the four metal concentrations in liver or kidney ( fig . 1 ) .", "contrastingly , we found significant differences in cd and cu concentrations between adult and juvenile beavers ( fig . 2 ) .", "cadmium concentrations were significantly higher in adults ( n = 6 ) than in juveniles ( n = 4 ) in both liver ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and kidney ( u = 0.0 ; z = 2.452 ; p = 0.014 ) . in liver ,", "cu levels were significantly lower in adults than in juveniles ( u = 0.0 ; z = 2.452 ; p = 0.014 ) , and in kidney , cu levels were significantly higher in adults than in juveniles ( u = 2.0 ; z = 2.025 ; p = 0.043).fig .", "1concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six female and four male eurasian beavers , ne poland , 2012 .", "no statistical significant difference in metal concentrations between sex classes was found ( mann - whitney u test , all p > 0.5 ) .", "box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) . for clarity , extreme outliers", "2concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six adult and four juvenile eurasian beavers , ne poland , 2012 .", "significant statistical differences in metal concentrations between age classes were found in liver cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and in kidney cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 2.0 ; z = 2.025 ; p = 0.043 ) . box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) , and statistical outliers ( open circles ) . for clarity ,", "extreme outliers are not shown concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six female and four male eurasian beavers , ne poland , 2012 .", "no statistical significant difference in metal concentrations between sex classes was found ( mann - whitney u test , all p > 0.5 ) . box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) . for clarity ,", "extreme outliers are not shown concentrations ( g / g wet weight ) of cd , pb , cu , and zn in a liver and b kidney of six adult and four juvenile eurasian beavers , ne poland , 2012 .", "significant statistical differences in metal concentrations between age classes were found in liver cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and in kidney cd ( u = 0.0 ; z = 2.452 ; p = 0.014 ) and cu ( u = 2.0 ; z = 2.025 ; p = 0.043 ) .", "box and whisker plots show median ( horizontal line within box ) , 25 and 75 % percentiles ( box ) , 1.5 interquartile range ( whiskers ) , and statistical outliers ( open circles ) . for clarity , extreme outliers" ], [ "our study revealed the presence of cd , pb , cu , and zn in liver and kidney of all sampled beavers . no relationship was observed between metal concentrations and the beavers sex .", "although we acknowledge the limitations of the tests due to a reduced number of individuals in each group , these results were expected as the species present no sexual dimorphism and no partitioning in diet ( haarberg and rosell 2006 ; krojerov - prokeov et al .", "contrastingly , some metal concentrations were affected by the animals age . higher liver and kidney cd concentrations were found in adult beavers than in juveniles , indicating that this metal bioaccumulates over time ( parker and hamr 2001 ; bilandi et al .", "cd levels in all six adult beavers exceed the maximum threshold of 1.0 g / g in kidney recommended in farm animal edible tissues . in liver , however , only one adult female ( f7 ) exceeded the recommended 0.5 g / kg cd limit ( european commission 2006 ) .", "such differences in cd levels with age might be due to this metal accumulating over an animal s entire life span , especially in kidney , as previously described in other species ( e.g. , red fox ( vulpes vulpes ) and stone marten ( martes foina ) : bilandi et al .", "2010 ; african grass owl ( tyto capensis ) : ansara - ross et al . 2013 ; penguins ( pygoscelis spp . ) : jerez et al .", "average cd content in kidney was similar to that observed in 2003 in beavers from srokowo forest division , an uncontaminated area in northeastern poland ( zalewski et al .", "2012 ) . however , our results were approximately four- to fivefold lower in liver and three- to fourfold lower in kidney than levels measured in earlier studies on beaver at unpolluted sites in poland ( giejewska et al .", "2014 ) , in norway ( fimreite et al . 2001 ) , and in a putatively contaminated former air base in poland ( zalewski et al .", "contrastingly , cd concentrations in liver and kidney were higher than those reported in canadian beavers ( castor canadensis ) in habitats located 175 km from an ore smelter in ontario , canada ( wren 1984 ) .", "cadmium concentration in wildlife tissue is attributed to its levels in the environment and additionally with animals age .", "previous studies concerned with cd content in unpolluted areas presented values that could be attributed to metal transport over long distances from the pollution source as well as increased mobility due to soil and water acidification ( wren 1984 ) .", "although metal concentrations in food sources typically consumed by beavers were not examined in the present study , it has been reported that beavers have a strong preference for aspen ( p. tremuloides ) and willow ( salix sp . ) , trees that accumulate high levels of cd particularly on acidic soils that increase the mobility of heavy metals ( wren 1984 ) .", "cd is easily absorbed and accumulates even at low levels of chronic exposure ( nordberg et al .", "interspecific differences , intraspecific variations among populations , or physiological determinants of metal absorption and excretion can also contribute to the observed differences ( nordberg et al .", "all values of pb found in beavers were below the 0.5 g / g threshold recommended for both organs in farm animal edible tissues ( european commission 2006 ) .", "lead concentrations in kidney ranged from 0.052 g / g ( f9 ) to 0.142 g / g ( f10 ) .", "lead passes the placenta and young animals have a higher rate of absorption from the intestinal tract ( nordberg et al .", "as this metal is not metabolically regulated in organisms , pb concentrations in animal tissues are related to its presence in the ecosystem ( jerez et al .", "soil intentionally or accidentally consumed by beavers could constitute an additional source of contamination as pb can be bound in soil and bottom sediments and secondarily released ( rajaganapathy et al .", "lead concentrations in our study were low compared to previous research ( table 3 ) .", "this could be attributed to the absence of major industrial sites close to the sample site , seasonality , or individual variability in diet ( brekken and steinnes 2004 ) .", "copper absorption is regulated by homeostatic mechanisms in the liver and is mainly excreted through the bile .", "absorbed zn is bound to plasma albumin and macroglobulins and distributed to the liver where it rapidly accumulates ( parker and hamr 2001 ) .", "although cu has higher affinity than zn to metallothionein ( low molecular weight metal - binding protein ) which binds cu and zn at low levels of cd ( hillis and parker 1993 ; nordberg et al .", "2011a ) , hepatic metallothionein seems to have an important role in accumulation and storage of both cu and zn in the liver ( parker and hamr 2001 ) .", "copper concentrations were higher in juvenile than in adult beavers in liver , indicating age - specific metabolism capabilities of this microelement ( nordberg et al .", "higher cu levels in juveniles liver can be attributed to increased requirements for this element in growing organisms ( parker and hamr 2001 ; jerez et al .", "our cu values in both liver and kidney were higher than those in previous studies ( table 3 ) .", "copper as an essential microelement has effective regulation mechanisms of uptake and excretion , and beaver diet contains high amounts of cu and zn ( brekken and steinnes 2004 ) .", "therefore , we assume that our values were at normal physiological levels ( nordberg et al", "zinc concentrations did not differ between organs and sex or age classes in our study .", "however , excluding beavers collected in the vicinity of the ore smelter in ontario , canada , where zn concentrations reached 80.8 ", "g / g in the liver ( hillis and parker 1993 ) , we found zn levels in liver higher than in previous studies ( table 3 ) . in kidney ,", "zn levels were highly correlated to cd levels regardless of the beaver age or sex .", "zinc and cadmium have similar chemical properties , and they are usually found as a mineral combination in the environment ( nordberg et al .", "metallothionein can bind cd , cu , and zn , and this protein will bind most of the cd and store it in kidney . exposure to low cd levels may cause a redistribution of zn in the organism , increasing zn concentrations in kidney ( nordberg et al .", "higher kidney zn concentrations were reported in beavers from ontario ( hillis and parker 1993 ) .", "the highest zn concentrations ( liver 40.6 g / g ; kidney 27.9 g / g ) we found in adult female f7 could also possibly be attributed to a diet rich in this microelement ( brekken and steinnes 2004 ) ." ], [ "general metal concentrations in beaver tissues from northeastern poland were similar to or lower than those found in other countries .", "however , the fact that high cd concentrations and pb were present in a putatively unpolluted area calls for a regular monitoring of environmental pollutants in agricultural and natural areas . future investigation of trace elements in beaver tissues sampled from industrial regions is needed for comparison and to draw further conclusion about levels of contamination .", "significant expansion of beaver populations in poland contributes to growing human - beaver conflict , increasing the amount of compensation paid to farmers and landowners .", "although beaver is currently not considered as a consumptive species in poland , beaver is classified as a game species in six european countries ( belarus , estonia , lithuania , latvia , norway , and sweden ) as well as in canada and russia ( jankowska et al .", "hunting beavers for consumption has been suggested as a tool for population management , and beaver consumption could occur in the near future in poland .", "research to ascertain tissues are safe from a toxicological point of view is therefore timely . in this context ,", "the fact that cd concentrations in adult beavers exceeded limits recommended in farm animal tissues warrants caution .", "also , it would be necessary to investigate levels of persistent organic pollutants ( pops ) in beaver tissues .", "pops are hydrophobic and lipophilic persistent compounds that may accumulate along the food chain and can cause severe metabolic disturbance , such as endocrinal disturbance ( jones and de voogt 1999 ) .", "the presence of pops in remote areas such as the arctic is a major environmental issue ( brault et al .", "consequently , we recommend future research and regular monitoring to identify the source of contaminants in the ecosystem and possible mitigation measures , and to ensure that contamination levels are within a safe range ." ] ]

[ "parturient with congenital heart block may be asymptomatic but can present with sudden vascular collapse , especially during labour .", "few patients with congenital heart block may have sudden cardiac death ( scd ) for which there are no predictors .", "we present our experience of spinal anaesthesia in a 29-year - old female with congenital complete heart block for lower segment caesarean section ( lscs ) .", "a 29-year - old parturient ( gravida 3 , para 0 , abortion 2 ) was admitted to our hospital with 9 months of amenorrhea .", "she had undergone appendicectomy 8 years back under spinal anaesthesia , and the procedure was uneventful .", "past obstetric history revealed two abortions and an episode of perioperative seizures during intravenous sedation for medical termination of pregnancy .", "the episode was associated with bradycardia non - responsive to atropine , during which she was evaluated as having a congenital complete heart block .", "she had been prescribed orciprenaline by a local practitioner and had received orciprenaline 10 mg bd for 2 months preceding the present pregnancy and till 10 days before presenting to the hospital .", "it was stopped by our cardiologist as it does not help to increase heart rate in complete heart block .", "her general condition was stable ; pulse rate was 46/min and blood pressure ( bp ) was 110/70 mmhg . clinically , cardiorespiratory and central nervous system examinations were normal .", "per - abdomen examination showed a foetus in cephalic presentation , uterine height at 38 weeks , foetal heart rate 136 beats / min and regular with clinical and ultrasonographic evidence of reduced liquor .", "electrocardiography showed a complete heart block with an atrial rate of 80/min , ventricular rate of 46/min and a narrow qrs complex [ figure 1 ] .", "she was accepted for anaesthesia under asa ii and was explained about the anaesthetic technique . patient was kept nil oral for 8 h. tab .", "metoclopramide 10 mg the next morning 2 h before the surgery . on the morning of the surgery", ", the patient was taken for cardiac catheterization in supine position with a wedge under the right buttock and temporary pacemaker insertion ( ventricular , ventricle inhibition ", "the position of the lead in the right ventricle was confirmed by fluoroscopy , with a lead shield cover over the abdomen of the patient .", "a pacemaker can also be inserted using electrocardiographic and echocardiographic guidance to avoid foetal exposure to ionizing radiation , but our cardiologist could not get enough window to do the same . immediately after the procedure ,", "a spinal anaesthesia was given in the l3-l4 interspace with a total of 1.5 ml , which is a combination of a 1.0 ml hyperbaric 0.5% bupivacaine with 0.5 ml fentanyl ( 25 g ) .", "intra - operatively , the first episode of hypotension after the spinal anaesthesia was treated by increasing the pacing rate to 70 beats / min and the second episode was treated by 3 mg of intravenous ( iv ) ephedrine .", "a healthy male baby weighing 2.5 kg was born with an apgar score of 8/10 in the 1 minute and 9/10 in the 5 minute .", "post - operatively , the pacemaker rate was changed to 60 beats / min and the patient was shifted to the post - operative ward for continuous monitoring .", "post - operative pain relief was achieved with iv tramadol 50 mg and diclofenac 75 mg iv alternatively 8 hourly .", "the temporary pacemaker was removed after 24 h and the patient was haemodynamically stable after the removal .", "her post - natal period was uneventful and she was discharged on the 5 day with an advice for the placement of permanent pacemaker as early as possible .", "heart block may be congenital or acquired . congenital heart block may occur alone or in association with other cardiac abnormalities .", "conversely , in isolated complete heart block , 8590% of all births live beyond the neonatal period , even up to late adulthood .", "if congenital complete heart block occurs alone , then it is relatively benign , as the block to conduction is at the level of the av node .", "the ventricular pacemaker is proximal to the bifurcation of the bundle of his , and therefore the qrs complexes are narrow , and the ventricular conduction system intact .", "the rate is relatively high and can vary from 40 to 80 beats / min , and may increase with exercise , atropine or sympathomimetics .", "acquired heart block in children or early adulthood is mostly secondary to cardiac surgery involving closure of perimembranous or infundibular ventricular septal defect ( vsd ) or muscle bundle resection near the conduction tissues , but can occur as an isolated condition also . in the chronic type ,", "the atrio ventricular ( av ) junction or bundle branches are usually involved , the qrs complexes are wide and the heart rate is lower and is not increased by exercise or atropine .", "prophylactic placement of a pacemaker is not indicated in an asymptomatic pregnant patient with complete heart block as it does not cause unusual problems.[35 ] if the patient is symptomatic during her first and second trimesters , then the placement of a permanent pacemaker is indicated .", "we used temporary pacing in this patient because the patient 's heart rate was resistant to exercise and atropine , and the same can not adapt to her changing bp .", "increase in the heart rate during labour is essential to increase in the cardiac output and to maintain the haemodynamics , which is not possible in the patient .", "hence , for a safe delivery , temporary pacemaker insertion was essential , which ideally we did before operative delivery .", "there are quite a few anaesthetic problems in patients with complete heart block undergoing incidental surgeries .", "the anaesthetic technique that least alters the cardiac stability should be wisely planned and executed for the procedure .", "general anaesthesia carries a potential risk to these patients because both the inhalational and the intravenous agents alter the haemodynamics to such an extent to put them in peril.[68 ] if general anaesthesia is planned , drugs with minimal effects in depressing the heart rate have to be preferred , such as ketamine for induction , pancuronium for relaxation and isoflurane for maintenance .", "combined spinal epidural is another option , but we did not opt for it due to cost constraints and brevity of surgery .", "modi et al . successfully managed such a case with the epidural anaesthetic technique . in our patient ,", "we opted for intrathecal opioids , especially fentanyl , which gives adequate anaesthesia with minimal effects on the cardiovascular system .", "we added 25 g of fentanyl with almost half the usual dose of hyperbaric 0.5% bupivacaine .", "we did use 3 mg of vasopressor ephedrine , but such a single low dose to maintain adequate haemodynamic stability was found satisfactory to us .", "we did insert a temporary pacemaker to compensate for any possible haemodynamic eventuality that can occur during anaesthesia , and the immediate post - partum period .", "the pacemaker rate was set at 50/min in order to preserve her native rhythm till her blood pressure is maintained in the intra - operative period .", "long - term pacing suppresses the native rhythm , and our intention was to avoid it .", "also , the fluoroscopy time for positioning the lead was minimized to less than 10 s. the patient was discharged with an advice for permanent pacemaker implantation ( ppi ) . as per the recent guidelines , ppi is indicated for all congenital complete heart blocks ( class 2a / b ) as trials have shown that there is a subgroup of patients who may have scd for which there are no predictors available .", "to conclude , we successfully managed a case of congenital complete heart block for operative delivery with temporary pacemaker in situ with intrathecal low - dose bupivacaine " ]

[ "<S> congenital complete heart block could be absolutely asymptomatic . increased awareness of suspecting an atrioventricular heart block in patients with slow heart rate and electrocardiograph examination </S>", "<S> will ensure recognition of this problem . </S>", "<S> the possibility of sudden cardiac death in these patients should not be forgotten . </S>", "<S> the goal in the peri - operative anaesthetic management is to preserve the heart rate and maintain haemodynamic stability . </S>", "<S> herein , we present a case of congenital complete heart block posted for elective caesarean section for an obstetric indication . </S>", "<S> we would like to highlight the advantage of bupivacaine fentanyl combination in maintaining haemodynamic stability and peri - operative heart rate control with temporary pacemaker . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "parturient with congenital heart block may be asymptomatic but can present with sudden vascular collapse , especially during labour .", "few patients with congenital heart block may have sudden cardiac death ( scd ) for which there are no predictors .", "we present our experience of spinal anaesthesia in a 29-year - old female with congenital complete heart block for lower segment caesarean section ( lscs ) ." ], [ "a 29-year - old parturient ( gravida 3 , para 0 , abortion 2 ) was admitted to our hospital with 9 months of amenorrhea .", "she had undergone appendicectomy 8 years back under spinal anaesthesia , and the procedure was uneventful .", "past obstetric history revealed two abortions and an episode of perioperative seizures during intravenous sedation for medical termination of pregnancy .", "the episode was associated with bradycardia non - responsive to atropine , during which she was evaluated as having a congenital complete heart block .", "she had been prescribed orciprenaline by a local practitioner and had received orciprenaline 10 mg bd for 2 months preceding the present pregnancy and till 10 days before presenting to the hospital .", "it was stopped by our cardiologist as it does not help to increase heart rate in complete heart block .", "her general condition was stable ; pulse rate was 46/min and blood pressure ( bp ) was 110/70 mmhg . clinically , cardiorespiratory and central nervous system examinations were normal .", "per - abdomen examination showed a foetus in cephalic presentation , uterine height at 38 weeks , foetal heart rate 136 beats / min and regular with clinical and ultrasonographic evidence of reduced liquor .", "electrocardiography showed a complete heart block with an atrial rate of 80/min , ventricular rate of 46/min and a narrow qrs complex [ figure 1 ] .", "she was accepted for anaesthesia under asa ii and was explained about the anaesthetic technique . patient was kept nil oral for 8 h. tab .", "metoclopramide 10 mg the next morning 2 h before the surgery . on the morning of the surgery", ", the patient was taken for cardiac catheterization in supine position with a wedge under the right buttock and temporary pacemaker insertion ( ventricular , ventricle inhibition ", "the position of the lead in the right ventricle was confirmed by fluoroscopy , with a lead shield cover over the abdomen of the patient .", "a pacemaker can also be inserted using electrocardiographic and echocardiographic guidance to avoid foetal exposure to ionizing radiation , but our cardiologist could not get enough window to do the same . immediately after the procedure ,", "a spinal anaesthesia was given in the l3-l4 interspace with a total of 1.5 ml , which is a combination of a 1.0 ml hyperbaric 0.5% bupivacaine with 0.5 ml fentanyl ( 25 g ) .", "intra - operatively , the first episode of hypotension after the spinal anaesthesia was treated by increasing the pacing rate to 70 beats / min and the second episode was treated by 3 mg of intravenous ( iv ) ephedrine .", "a healthy male baby weighing 2.5 kg was born with an apgar score of 8/10 in the 1 minute and 9/10 in the 5 minute .", "post - operatively , the pacemaker rate was changed to 60 beats / min and the patient was shifted to the post - operative ward for continuous monitoring .", "post - operative pain relief was achieved with iv tramadol 50 mg and diclofenac 75 mg iv alternatively 8 hourly .", "the temporary pacemaker was removed after 24 h and the patient was haemodynamically stable after the removal .", "her post - natal period was uneventful and she was discharged on the 5 day with an advice for the placement of permanent pacemaker as early as possible ." ], [ "heart block may be congenital or acquired . congenital heart block may occur alone or in association with other cardiac abnormalities .", "conversely , in isolated complete heart block , 8590% of all births live beyond the neonatal period , even up to late adulthood .", "if congenital complete heart block occurs alone , then it is relatively benign , as the block to conduction is at the level of the av node .", "the ventricular pacemaker is proximal to the bifurcation of the bundle of his , and therefore the qrs complexes are narrow , and the ventricular conduction system intact .", "the rate is relatively high and can vary from 40 to 80 beats / min , and may increase with exercise , atropine or sympathomimetics .", "acquired heart block in children or early adulthood is mostly secondary to cardiac surgery involving closure of perimembranous or infundibular ventricular septal defect ( vsd ) or muscle bundle resection near the conduction tissues , but can occur as an isolated condition also . in the chronic type ,", "the atrio ventricular ( av ) junction or bundle branches are usually involved , the qrs complexes are wide and the heart rate is lower and is not increased by exercise or atropine .", "prophylactic placement of a pacemaker is not indicated in an asymptomatic pregnant patient with complete heart block as it does not cause unusual problems.[35 ] if the patient is symptomatic during her first and second trimesters , then the placement of a permanent pacemaker is indicated .", "we used temporary pacing in this patient because the patient 's heart rate was resistant to exercise and atropine , and the same can not adapt to her changing bp .", "increase in the heart rate during labour is essential to increase in the cardiac output and to maintain the haemodynamics , which is not possible in the patient .", "hence , for a safe delivery , temporary pacemaker insertion was essential , which ideally we did before operative delivery .", "there are quite a few anaesthetic problems in patients with complete heart block undergoing incidental surgeries .", "the anaesthetic technique that least alters the cardiac stability should be wisely planned and executed for the procedure .", "general anaesthesia carries a potential risk to these patients because both the inhalational and the intravenous agents alter the haemodynamics to such an extent to put them in peril.[68 ] if general anaesthesia is planned , drugs with minimal effects in depressing the heart rate have to be preferred , such as ketamine for induction , pancuronium for relaxation and isoflurane for maintenance .", "combined spinal epidural is another option , but we did not opt for it due to cost constraints and brevity of surgery .", "modi et al . successfully managed such a case with the epidural anaesthetic technique . in our patient ,", "we opted for intrathecal opioids , especially fentanyl , which gives adequate anaesthesia with minimal effects on the cardiovascular system .", "we added 25 g of fentanyl with almost half the usual dose of hyperbaric 0.5% bupivacaine .", "we did use 3 mg of vasopressor ephedrine , but such a single low dose to maintain adequate haemodynamic stability was found satisfactory to us .", "we did insert a temporary pacemaker to compensate for any possible haemodynamic eventuality that can occur during anaesthesia , and the immediate post - partum period .", "the pacemaker rate was set at 50/min in order to preserve her native rhythm till her blood pressure is maintained in the intra - operative period .", "long - term pacing suppresses the native rhythm , and our intention was to avoid it .", "also , the fluoroscopy time for positioning the lead was minimized to less than 10 s. the patient was discharged with an advice for permanent pacemaker implantation ( ppi ) . as per the recent guidelines , ppi is indicated for all congenital complete heart blocks ( class 2a / b ) as trials have shown that there is a subgroup of patients who may have scd for which there are no predictors available .", "to conclude , we successfully managed a case of congenital complete heart block for operative delivery with temporary pacemaker in situ with intrathecal low - dose bupivacaine " ] ]

[ "ocular surface is protected by tears , and tear abnormalities or ocular surface disorders could result in dry eye syndrome ( des ) .", "des is a common and complex condition with reduced ocular comfort and impaired visual performance .", "nowadays , des is regarded as one of the most common ocular disorders throughout the world .", "the treatment of des is based on local application of eye drops , including artificial tears and anti - inflammatory agents .", "although most des patients treated with eye drops will feel more comfortable , it is hard to cure des .", "in addition , for some individuals , there are no satisfactory therapeutics that can provide relief from des ocular discomfort symptoms .", "thus , better understandings of the risk factors and pathogenesis of des would help in primary prevention of des and effective therapy development .", "the generally recognized risk factors for des included elder age , reproductive factors , tobacco smoking , contact lenses use , ocular surgery , systemic diseases , and dry environment contact .", "better understandings of risk factors and related lifestyle changes of des patients can be helpful in the des related discomforts management .", "vitamin d is a fat soluble vitamin that is involved in a number of physiological and pathological processes .", "vitamin d formulations have been widely used in different diseases and have been reported to be effective in the treatment or prevention of osteoporosis , cancers , and cardiovascular disorders [ 1012 ] .", "in advanced in - vivo and in - vitro studies , vitamin d has been reported to enhance immunity , relieve inflammatory reaction and regulate cell cycles [ 1315 ] .", "the application of vitamin d in treatment of ocular surface disorders has been reported . in a previous study", ", it was suggested that there is a possible role for vitamin d in modulating corneal wound healing , thus have important implications for therapeutic use of vitamin d at the ocular surface . in another study based on mouse , rabbit , and human samples ,", "it was determined that 25-hydroxyvitamin d[(3)(25(oh)d(3 ) ] and/or its active metabolite , 1,25-dihydroxyvitamin d[(3)(1,25(oh)(2)d(3 ) ] can enhance corneal epithelial barrier function .", "several epidemiological studies were conducted to evaluate the association between vitamin d or vitamin d deficiency and incidence rate of des [ 1821 ] , however , no conclusions were reached . a cross - sectional study that included 17,542 adults from korea found that lower serum 25(oh)d levels were associated with incidence rates of des . however , in another cross - sectional study containing male patients , it was reported that vitamin d levels were not significantly associated with the presence or severity of des . the purpose of our case - control study was to determine the effect of serum 25(oh)d on des incidence .", "the study was also designed to determine whether serum 25(oh)d levels were associated with ocular parameters of des patients .", "ethical approval of this current study was obtained from the human research ethics committee of changshu no 2 people s hospital . in this study ,", "a total of 70 des patients and 70 matched controls were included in this study .", "all the des patients were diagnosed from may 2015 to january 2016 at the out - patient clinic of the department of ophthalmology , changshu no 2 people s hospital , changshu , people s republic of china .", "the inclusion criteria were : ( 1 ) matched to the diagnosis criteria of des , ( 2 ) aged over 18 years , and ( 3 ) signed the informed consents before inclusion in the study .", "the exclusion criteria contained : ( 1 ) unable to co - operate in the study handling , ( 2 ) sjogren syndrome , rheumatoid arthritis , lupus erythematosus , or any other immune diseases ; ( 3 ) mental disorder or serious systemic disease , such cancer , hematological system disease , or hyperthyroidism ; ( 4 ) pregnancy or breast - feeding in women ; ( 5 ) participation in other ophthalmic clinical trials ; ( 6 ) other ocular disease , such as ocular surgery history within the recent six months , use of any ophthalmic eye drops or contact lenses within recent one month , eyelid or eyelash abnormalities , nasolacrimal apparatus abnormalities glaucoma , uveitis , retinal hemorrhage , or optic neuritis", ". patients with des were diagnosed using experts consensus about clinical diagnosis and treatment of dry eye ( 2013 ) .", "the diagnostic criteria includes : ( 1 ) subjective symptoms of dryness , foreign body sensation , burning sensation , fatigue , discomfort , visual acuity fluctuation , and tear film breakup time ( tbut ) test 5 seconds or schirmer i test ( without surface anesthesia ) 5 mm/5 minutes ; ( 2 ) the subjective symptoms of ocular discomfort and tbut test 510 seconds or schirmer i test ( without surface anesthesia ) 510 mm/5 minutes combined with corneal fluorescein staining positive . clinical data , including body mass index ( bmi , kg / m ) , smoking history , diabetes status , and blood pressure were obtained through questioning of the participants or reviewing their medical records . the ocular surface disease index ( osdi ) quantitation was conducted by trained investigators .", "the osdi scale questionnaire included 12 questions , containing three aspects of eye symptoms , visual function , and environmental stimulation .", "detailed information was collected from each participant regarding ocular dryness , foreign body , visual fatigue , and other subjective discomfort symptoms .", "the response grades were divided into no symptoms ( 0 point ) , occasional presence ( 0.5 points ) , intermittent ( 1 point ) , and persistent discomfort ( 2 point ) .", "the examinations were conducted in the following order : tbut , keratoepitheliopathy examination , and schirmer i test .", "all the clinical data was collected and recorded in electronic tables . the longer half - life of 25(oh)d ,", "allows it to remains stable in serum and as such it can be used to reflect the level of vitamin d. in this study , 25(oh)d was chosen as a main study parameter .", "peripheral blood samples from each participant were collected and serum samples were frozen at 70c until 25(oh)d detection was conducted .", "serum 25(oh)d levels were measured by a reverse phase liquid chromatography method ( agilent technologies , ca , usa ) . the accuracy quality control of low , medium , and high quality was to be 85~115% with the accuracy value within 15% .", "the statistical analysis was performed using package for spss version 17.0 software ( spss inc . ,", "the data of each index is shown as the mean standard deviation ( sd ) .", "comparison of discontinuous variable parameters was done using the chi - square test or fisher s exact test .", "the differences in the continuous variables were detected using non - paired t - test .", "pearson correlation analysis was used in the detection of the associations between different continuous variables .", "a total of 70 des patients and 70 matched controls were included in this study .", "all the des patients were diagnosed from may 2015 to january 2016 at the out - patient clinic of the department of ophthalmology , changshu no 2 people s hospital , changshu , people s republic of china .", "the inclusion criteria were : ( 1 ) matched to the diagnosis criteria of des , ( 2 ) aged over 18 years , and ( 3 ) signed the informed consents before inclusion in the study .", "the exclusion criteria contained : ( 1 ) unable to co - operate in the study handling , ( 2 ) sjogren syndrome , rheumatoid arthritis , lupus erythematosus , or any other immune diseases ; ( 3 ) mental disorder or serious systemic disease , such cancer , hematological system disease , or hyperthyroidism ; ( 4 ) pregnancy or breast - feeding in women ; ( 5 ) participation in other ophthalmic clinical trials ; ( 6 ) other ocular disease , such as ocular surgery history within the recent six months , use of any ophthalmic eye drops or contact lenses within recent one month , eyelid or eyelash abnormalities , nasolacrimal apparatus abnormalities glaucoma , uveitis , retinal hemorrhage , or optic neuritis .", "patients with des were diagnosed using experts consensus about clinical diagnosis and treatment of dry eye ( 2013 ) .", "the diagnostic criteria includes : ( 1 ) subjective symptoms of dryness , foreign body sensation , burning sensation , fatigue , discomfort , visual acuity fluctuation , and tear film breakup time ( tbut ) test 5 seconds or schirmer i test ( without surface anesthesia ) 5 mm/5 minutes ; ( 2 ) the subjective symptoms of ocular discomfort and tbut test 510 seconds or schirmer i test ( without surface anesthesia ) 510 mm/5 minutes combined with corneal fluorescein staining positive .", "clinical data , including body mass index ( bmi , kg / m ) , smoking history , diabetes status , and blood pressure were obtained through questioning of the participants or reviewing their medical records .", "the osdi scale questionnaire included 12 questions , containing three aspects of eye symptoms , visual function , and environmental stimulation .", "detailed information was collected from each participant regarding ocular dryness , foreign body , visual fatigue , and other subjective discomfort symptoms .", "the response grades were divided into no symptoms ( 0 point ) , occasional presence ( 0.5 points ) , intermittent ( 1 point ) , and persistent discomfort ( 2 point ) .", "the examinations were conducted in the following order : tbut , keratoepitheliopathy examination , and schirmer i test .", "the longer half - life of 25(oh)d , allows it to remains stable in serum and as such it can be used to reflect the level of vitamin d. in this study , 25(oh)d was chosen as a main study parameter .", "peripheral blood samples from each participant were collected and serum samples were frozen at 70c until 25(oh)d detection was conducted .", "serum 25(oh)d levels were measured by a reverse phase liquid chromatography method ( agilent technologies , ca , usa ) . the accuracy quality control of low , medium , and high quality was to be 85~115% with the accuracy value within 15% .", "the statistical analysis was performed using package for spss version 17.0 software ( spss inc . ,", "the data of each index is shown as the mean standard deviation ( sd ) .", "comparison of discontinuous variable parameters was done using the chi - square test or fisher s exact test .", "the differences in the continuous variables were detected using non - paired t - test .", "pearson correlation analysis was used in the detection of the associations between different continuous variables .", "basic clinical characteristics of patients with des and healthy controls are presented in table 1 . in this study , 70 des cases ( 43 males and 27 females ) and 70 matched controls ( 36 males and 34 females ) were included .", "the mean bmi in the des group and the control group was 23.24.4 kg / m and 23.65.1 kg / m , respectively .", "there were no differences in smoking status , diabetes prevalence , and blood pressure between the des group and the control group .", "when the schirmer i test was considered , the des group ( 9.43.9 mm/5 minutes ) demonstrated a significantly lower value compared with the control group ( 13.95.3 mm/5 minutes , p<0.001 ) .", "in addition , a significantly reduced tbut was detected in the des cases ( des group , 6.12.4 second ; control group , 13.43.8 seconds , p<0.001 ) .", "compared with the control group , the mean osdi value was significantly higher in the des group ( p<0.001 ) .", "the serum 25(oh)d levels in patients with des and the control patients are shown in table 2 .", "serum 25(oh)d levels were significantly lower in the des group than in the healthy control group ( des group , 19.35.8 ; control group , 31.67.3 , p<0.001 ) .", "furthermore , serum 25(oh)d levels in both male and female patients were lower compared with the healthy controls ( p<0.05 ) . when the participants were divided into different vitamin d status ( 25(oh)d , < 10 ng / ml , 10~20 ng / ml , 20~30 ng / ml , and 30 ng / ml ) , deficiency of vitamin d was more common in the des group ( p<0.001 ) . considering", "that serum 25(oh)d level was significantly lower in the des group , we conducted additional analyses on the association between serum 25(oh ) d levels and des parameters . through pearson correlation analysis", ", it was found that serum 25(oh)d level was associated with increased schimer test i ( r=0.8248 , p<0.001 ) .", "in addition , there was an inverse correlation between serum 25(oh)d and odsi scores ( r=0.3348 , p=0.005 ) and tbut ( r=0.6806 , p<0.001 ) .", "basic clinical characteristics of patients with des and healthy controls are presented in table 1 . in this study , 70 des cases ( 43 males and 27 females ) and 70 matched controls ( 36 males and 34 females ) were included .", "the mean bmi in the des group and the control group was 23.24.4 kg / m and 23.65.1 kg / m , respectively .", "there were no differences in smoking status , diabetes prevalence , and blood pressure between the des group and the control group .", "when the schirmer i test was considered , the des group ( 9.43.9 mm/5 minutes ) demonstrated a significantly lower value compared with the control group ( 13.95.3 mm/5 minutes , p<0.001 ) .", "in addition , a significantly reduced tbut was detected in the des cases ( des group , 6.12.4 second ; control group , 13.43.8 seconds , p<0.001 ) .", "compared with the control group , the mean osdi value was significantly higher in the des group ( p<0.001 ) .", "the serum 25(oh)d levels in patients with des and the control patients are shown in table 2 .", "serum 25(oh)d levels were significantly lower in the des group than in the healthy control group ( des group , 19.35.8 ; control group , 31.67.3 , p<0.001 ) .", "furthermore , serum 25(oh)d levels in both male and female patients were lower compared with the healthy controls ( p<0.05 ) . when the participants were divided into different vitamin d status ( 25(oh)d , < 10 ng / ml , 10~20 ng / ml , 20~30 ng / ml , and 30 ng / ml ) , deficiency of vitamin d was more common in the des group ( p<0.001 ) .", "considering that serum 25(oh)d level was significantly lower in the des group , we conducted additional analyses on the association between serum 25(oh ) d levels and des parameters . through pearson correlation analysis", ", it was found that serum 25(oh)d level was associated with increased schimer test i ( r=0.8248 , p<0.001 ) . in addition", ", there was an inverse correlation between serum 25(oh)d and odsi scores ( r=0.3348 , p=0.005 ) and tbut ( r=0.6806 , p<0.001 ) .", "in this study , it was shown that 25(oh)d levels were lower in patients with des than in healthy controls . when the 25(oh)d levels was stratified , vitamin d deficiency was more common in the des cases .", "in further analysis , it was found that there were statistical significant associations between serum 25(oh ) d levels and the schimer test , tbut value and osdi scale .", "des is a multifactorial disorder and various risk factors were associated with the incidence of des .", "nowadays , there is more interest in the association between vitamin d and des cases .", "the major conclusions of this case - control study were consistent with previous cross - sectional study conclusions , in that low serum 25(oh)d levels were associated with des . in previous cross - sectional data analysis , it was found that low serum 25(oh)d levels were associated with des in korean adults .", "these results provide clues that vitamin d supplementation might be used in des treatment . in another case - control study with 34 patients with serum vitamin d deficiency and 21 control patients with normal vitamin d levels", ", it was found that vitamin d deficiency decreases the tbut and schirmer test values . in our study ,", "significant lower vitamin d level was detected in des patients and it was found that significant associations were detected in both male and female groups .", "in addition , the advanced correlation analysis results showed more interesting and meaningful association between serum 25(oh ) d and severity and ocular discomfort in des cases .", "rather than considering vitamin d deficiency as a risk factor for des as in a previous study , more attention was focused on the association between vitamin d and ocular physical examination and indisposed symptoms in this current study .", "positive correlation between serum vitamin d level and des severity suggests there is the potential for application of vitamin d for treatment or prevention of des . ass shown in the first study on the effect of vitamin d on the des in chinese population , this study provides additional knowledge on the effect of vitamin d on the des development .", "however , even though the association of vitamin d and des was detected in different studies , the study of the detailed mechanism was lacking .", "there were several possible explains for the effect of vitamin d on the des development .", "it has been recognized that systemic autoimmune diseases or immune abnormalities could result in the increased risk of des .", "considering the remarkable effect of vitamin d on the immune system , it could be conjectured that the immunoregulatory effect of vitamin d might influence the development of des .", "for instance , in a case - control study with 107 sjogren syndrome ( ss ) patients and 74 healthy controls , plasma vitamin d levels in ss patient group were significantly lower than in the control group .", "as dry eye was one of the most frequent symptoms of ss , and vitamin d deficiency was frequent found in patients with ss , vitamin d might be associated with the ss - related des incidence .", "besides , there were several previous studies that reported on the associations between des and systemic autoimmune disorders , including rheumatism and lupus erythematosus .", "abnormal autoimmune status might result in a higher incidence of des and the protective immunomodulatory effect of vitamin d in the regulation of immune cells might help in the prevention of des incidence .", "there was a previous study that reported that vitamin d deficiency decreased the tbut and schirmer test values and may be associated with dry - eye symptoms in non - sjogren syndrome , thus suggesting a potential mechanism beyond the autoimmune effect of vitamin d may exist .", "chronic inflammation induced by autoimmune disorders or environmental stimulating factors in ocular surface cells has been considered to be involved in des .", "for instance , vitamin d could decrease the inflammatory response of cancer and inhibited the proliferation of cancer cells in - vitro . considering that inflammation was one of the key pathogenesis and phenotype of des ,", "higher level vitamin d would decreased the inflammatory response in the ocular surface and then improve the severity of des .", "the production of vitamin d in the metabolic system is from two main sources . in general ,", "vitamin d is mainly produced by cutaneous synthesis upon skin exposure to ultraviolet light and partial vitamin d is gained from food sources .", "because of the short half - life of 1,25(oh)d ( 57 days ) and the long half - life of 25(oh)d ( 20~30 days ) , the serum 5(oh)d level is commonly used as the indicator of vitamin d status and it was used in the present study .", "vitamin d receptor ( vdr ) is a member of the nuclear hormone receptor superfamily and plays an important role in vitamin d function . in a case - control study of 64 des cases and 51 controls , the single nucleotide polymorphisms ( snps ) in the vdr genes also varied between des cases and controls .", "thus , the influence of snps in the vitamin d related gene might explain the association between serum vitamin d and des incidence .", "a major strength of our study was that the method used to assess serum vitamin d levels was sensitive and therefore might produce more robust conclusions .", "first , the sample size of this study was relatively small and it has been demonstrated that larger sample sizes are requirements for advanced study analysis .", "considering that this was a hospital - based study , it was hard to include a large sample size in the participant selection process . additionally , population - based studies are required for the detection of the association between vitamin d and des status .", "second , even though 5(oh)d is the most commonly used index for vitamin d detection , there are other indices reflecting the intracellular effect of vitamin d that were not reported in this study", ". the association of these related indices might provide additional knowledge to aid in our understanding of the mechanism of vitamin d on des .", "future prospective studies were needed to assess other serum markers of vitamin d in addition to 25(oh)d in the des cases . in advanced studies ,", "the effect of dietary vitamin d intake , vitamin d supplementation , and ultraviolet radiation exposure on the development of des should be examined .", "a third limitation of our study was that that using a case - control study design , selection bias could influence the conclusions .", "a significant association between serum 25(oh)d level and des incidence was detected in this study .", "in addition , serum 25(oh)d was associated with the des parameters and ocular discomfort status .", "it could be conjectured that vitamin d might be a potentially favorable adjunctive option for patients with des .", "considering the relatively small sample size of this study , larger studies with longer follow - up duration are needed ." ]

[ "<S> backgroundto determine the association between serum 25(oh)d and dry eye syndrome ( des ) incidence . </S>", "<S> this study was also designed to determine whether serum 25(oh)d levels were associated with ocular parameter of des patients.material/methodsthis is a case - control study with 70 des cases and 70 healthy controls . </S>", "<S> clinical data included body mass index ( bmi , kg / m2 ) , smoking history , diabetes , and blood pressure . </S>", "<S> serum 25(oh)d was chosen as the main parameter and reflected the level of vitamin d. the des parameters included ocular surface disease index ( osdi ) scales , tear film breakup time ( tbut ) and schirmer test i. the differences in each parameter between case and control groups were detected and the association of serum 25(oh)d and des parameter were detected.resultsit was shown that 25(oh)d levels were lower in patients with des than in healthy controls . when the 25(oh)d levels was stratified , vitamin d deficiency was more common in the des cases . </S>", "<S> in advanced studies , it was found that there were statistically significant associations between serum 25(oh ) d levels and the schimer test , tbut , and osdi scales.conclusionsa significant association between serum 25(oh)d level and des incidence was detected in this study . </S>", "<S> considering the relatively small sample size of this study , larger studies are needed in the future . </S>" ]

[ "Background", "Material and Methods", "Patients", "DES diagnosis", "Clinical data collection", "Vitamin D detection", "Statistical analysis", "Results", "Clinical characteristics", "25(OH)D levels in patients with DES", "Association between serum 25(OH) D levels and DES parameters", "Discussion", "Conclusions" ]

[ [ "ocular surface is protected by tears , and tear abnormalities or ocular surface disorders could result in dry eye syndrome ( des ) .", "des is a common and complex condition with reduced ocular comfort and impaired visual performance .", "nowadays , des is regarded as one of the most common ocular disorders throughout the world .", "the treatment of des is based on local application of eye drops , including artificial tears and anti - inflammatory agents .", "although most des patients treated with eye drops will feel more comfortable , it is hard to cure des .", "in addition , for some individuals , there are no satisfactory therapeutics that can provide relief from des ocular discomfort symptoms .", "thus , better understandings of the risk factors and pathogenesis of des would help in primary prevention of des and effective therapy development .", "the generally recognized risk factors for des included elder age , reproductive factors , tobacco smoking , contact lenses use , ocular surgery , systemic diseases , and dry environment contact .", "better understandings of risk factors and related lifestyle changes of des patients can be helpful in the des related discomforts management .", "vitamin d is a fat soluble vitamin that is involved in a number of physiological and pathological processes .", "vitamin d formulations have been widely used in different diseases and have been reported to be effective in the treatment or prevention of osteoporosis , cancers , and cardiovascular disorders [ 1012 ] .", "in advanced in - vivo and in - vitro studies , vitamin d has been reported to enhance immunity , relieve inflammatory reaction and regulate cell cycles [ 1315 ] .", "the application of vitamin d in treatment of ocular surface disorders has been reported . in a previous study", ", it was suggested that there is a possible role for vitamin d in modulating corneal wound healing , thus have important implications for therapeutic use of vitamin d at the ocular surface . in another study based on mouse , rabbit , and human samples ,", "it was determined that 25-hydroxyvitamin d[(3)(25(oh)d(3 ) ] and/or its active metabolite , 1,25-dihydroxyvitamin d[(3)(1,25(oh)(2)d(3 ) ] can enhance corneal epithelial barrier function .", "several epidemiological studies were conducted to evaluate the association between vitamin d or vitamin d deficiency and incidence rate of des [ 1821 ] , however , no conclusions were reached . a cross - sectional study that included 17,542 adults from korea found that lower serum 25(oh)d levels were associated with incidence rates of des . however , in another cross - sectional study containing male patients , it was reported that vitamin d levels were not significantly associated with the presence or severity of des . the purpose of our case - control study was to determine the effect of serum 25(oh)d on des incidence .", "the study was also designed to determine whether serum 25(oh)d levels were associated with ocular parameters of des patients ." ], [ "ethical approval of this current study was obtained from the human research ethics committee of changshu no 2 people s hospital . in this study ,", "a total of 70 des patients and 70 matched controls were included in this study .", "all the des patients were diagnosed from may 2015 to january 2016 at the out - patient clinic of the department of ophthalmology , changshu no 2 people s hospital , changshu , people s republic of china .", "the inclusion criteria were : ( 1 ) matched to the diagnosis criteria of des , ( 2 ) aged over 18 years , and ( 3 ) signed the informed consents before inclusion in the study .", "the exclusion criteria contained : ( 1 ) unable to co - operate in the study handling , ( 2 ) sjogren syndrome , rheumatoid arthritis , lupus erythematosus , or any other immune diseases ; ( 3 ) mental disorder or serious systemic disease , such cancer , hematological system disease , or hyperthyroidism ; ( 4 ) pregnancy or breast - feeding in women ; ( 5 ) participation in other ophthalmic clinical trials ; ( 6 ) other ocular disease , such as ocular surgery history within the recent six months , use of any ophthalmic eye drops or contact lenses within recent one month , eyelid or eyelash abnormalities , nasolacrimal apparatus abnormalities glaucoma , uveitis , retinal hemorrhage , or optic neuritis", ". patients with des were diagnosed using experts consensus about clinical diagnosis and treatment of dry eye ( 2013 ) .", "the diagnostic criteria includes : ( 1 ) subjective symptoms of dryness , foreign body sensation , burning sensation , fatigue , discomfort , visual acuity fluctuation , and tear film breakup time ( tbut ) test 5 seconds or schirmer i test ( without surface anesthesia ) 5 mm/5 minutes ; ( 2 ) the subjective symptoms of ocular discomfort and tbut test 510 seconds or schirmer i test ( without surface anesthesia ) 510 mm/5 minutes combined with corneal fluorescein staining positive . clinical data , including body mass index ( bmi , kg / m ) , smoking history , diabetes status , and blood pressure were obtained through questioning of the participants or reviewing their medical records . the ocular surface disease index ( osdi ) quantitation was conducted by trained investigators .", "the osdi scale questionnaire included 12 questions , containing three aspects of eye symptoms , visual function , and environmental stimulation .", "detailed information was collected from each participant regarding ocular dryness , foreign body , visual fatigue , and other subjective discomfort symptoms .", "the response grades were divided into no symptoms ( 0 point ) , occasional presence ( 0.5 points ) , intermittent ( 1 point ) , and persistent discomfort ( 2 point ) .", "the examinations were conducted in the following order : tbut , keratoepitheliopathy examination , and schirmer i test .", "all the clinical data was collected and recorded in electronic tables . the longer half - life of 25(oh)d ,", "allows it to remains stable in serum and as such it can be used to reflect the level of vitamin d. in this study , 25(oh)d was chosen as a main study parameter .", "peripheral blood samples from each participant were collected and serum samples were frozen at 70c until 25(oh)d detection was conducted .", "serum 25(oh)d levels were measured by a reverse phase liquid chromatography method ( agilent technologies , ca , usa ) . the accuracy quality control of low , medium , and high quality was to be 85~115% with the accuracy value within 15% .", "the statistical analysis was performed using package for spss version 17.0 software ( spss inc . ,", "the data of each index is shown as the mean standard deviation ( sd ) .", "comparison of discontinuous variable parameters was done using the chi - square test or fisher s exact test .", "the differences in the continuous variables were detected using non - paired t - test .", "pearson correlation analysis was used in the detection of the associations between different continuous variables ." ], [ "a total of 70 des patients and 70 matched controls were included in this study .", "all the des patients were diagnosed from may 2015 to january 2016 at the out - patient clinic of the department of ophthalmology , changshu no 2 people s hospital , changshu , people s republic of china .", "the inclusion criteria were : ( 1 ) matched to the diagnosis criteria of des , ( 2 ) aged over 18 years , and ( 3 ) signed the informed consents before inclusion in the study .", "the exclusion criteria contained : ( 1 ) unable to co - operate in the study handling , ( 2 ) sjogren syndrome , rheumatoid arthritis , lupus erythematosus , or any other immune diseases ; ( 3 ) mental disorder or serious systemic disease , such cancer , hematological system disease , or hyperthyroidism ; ( 4 ) pregnancy or breast - feeding in women ; ( 5 ) participation in other ophthalmic clinical trials ; ( 6 ) other ocular disease , such as ocular surgery history within the recent six months , use of any ophthalmic eye drops or contact lenses within recent one month , eyelid or eyelash abnormalities , nasolacrimal apparatus abnormalities glaucoma , uveitis , retinal hemorrhage , or optic neuritis ." ], [ "patients with des were diagnosed using experts consensus about clinical diagnosis and treatment of dry eye ( 2013 ) .", "the diagnostic criteria includes : ( 1 ) subjective symptoms of dryness , foreign body sensation , burning sensation , fatigue , discomfort , visual acuity fluctuation , and tear film breakup time ( tbut ) test 5 seconds or schirmer i test ( without surface anesthesia ) 5 mm/5 minutes ; ( 2 ) the subjective symptoms of ocular discomfort and tbut test 510 seconds or schirmer i test ( without surface anesthesia ) 510 mm/5 minutes combined with corneal fluorescein staining positive ." ], [ "clinical data , including body mass index ( bmi , kg / m ) , smoking history , diabetes status , and blood pressure were obtained through questioning of the participants or reviewing their medical records .", "the osdi scale questionnaire included 12 questions , containing three aspects of eye symptoms , visual function , and environmental stimulation .", "detailed information was collected from each participant regarding ocular dryness , foreign body , visual fatigue , and other subjective discomfort symptoms .", "the response grades were divided into no symptoms ( 0 point ) , occasional presence ( 0.5 points ) , intermittent ( 1 point ) , and persistent discomfort ( 2 point ) .", "the examinations were conducted in the following order : tbut , keratoepitheliopathy examination , and schirmer i test ." ], [ "the longer half - life of 25(oh)d , allows it to remains stable in serum and as such it can be used to reflect the level of vitamin d. in this study , 25(oh)d was chosen as a main study parameter .", "peripheral blood samples from each participant were collected and serum samples were frozen at 70c until 25(oh)d detection was conducted .", "serum 25(oh)d levels were measured by a reverse phase liquid chromatography method ( agilent technologies , ca , usa ) . the accuracy quality control of low , medium , and high quality was to be 85~115% with the accuracy value within 15% ." ], [ "the statistical analysis was performed using package for spss version 17.0 software ( spss inc . ,", "the data of each index is shown as the mean standard deviation ( sd ) .", "comparison of discontinuous variable parameters was done using the chi - square test or fisher s exact test .", "the differences in the continuous variables were detected using non - paired t - test .", "pearson correlation analysis was used in the detection of the associations between different continuous variables ." ], [ "basic clinical characteristics of patients with des and healthy controls are presented in table 1 . in this study , 70 des cases ( 43 males and 27 females ) and 70 matched controls ( 36 males and 34 females ) were included .", "the mean bmi in the des group and the control group was 23.24.4 kg / m and 23.65.1 kg / m , respectively .", "there were no differences in smoking status , diabetes prevalence , and blood pressure between the des group and the control group .", "when the schirmer i test was considered , the des group ( 9.43.9 mm/5 minutes ) demonstrated a significantly lower value compared with the control group ( 13.95.3 mm/5 minutes , p<0.001 ) .", "in addition , a significantly reduced tbut was detected in the des cases ( des group , 6.12.4 second ; control group , 13.43.8 seconds , p<0.001 ) .", "compared with the control group , the mean osdi value was significantly higher in the des group ( p<0.001 ) .", "the serum 25(oh)d levels in patients with des and the control patients are shown in table 2 .", "serum 25(oh)d levels were significantly lower in the des group than in the healthy control group ( des group , 19.35.8 ; control group , 31.67.3 , p<0.001 ) .", "furthermore , serum 25(oh)d levels in both male and female patients were lower compared with the healthy controls ( p<0.05 ) . when the participants were divided into different vitamin d status ( 25(oh)d , < 10 ng / ml , 10~20 ng / ml , 20~30 ng / ml , and 30 ng / ml ) , deficiency of vitamin d was more common in the des group ( p<0.001 ) . considering", "that serum 25(oh)d level was significantly lower in the des group , we conducted additional analyses on the association between serum 25(oh ) d levels and des parameters . through pearson correlation analysis", ", it was found that serum 25(oh)d level was associated with increased schimer test i ( r=0.8248 , p<0.001 ) .", "in addition , there was an inverse correlation between serum 25(oh)d and odsi scores ( r=0.3348 , p=0.005 ) and tbut ( r=0.6806 , p<0.001 ) ." ], [ "basic clinical characteristics of patients with des and healthy controls are presented in table 1 . in this study , 70 des cases ( 43 males and 27 females ) and 70 matched controls ( 36 males and 34 females ) were included .", "the mean bmi in the des group and the control group was 23.24.4 kg / m and 23.65.1 kg / m , respectively .", "there were no differences in smoking status , diabetes prevalence , and blood pressure between the des group and the control group .", "when the schirmer i test was considered , the des group ( 9.43.9 mm/5 minutes ) demonstrated a significantly lower value compared with the control group ( 13.95.3 mm/5 minutes , p<0.001 ) .", "in addition , a significantly reduced tbut was detected in the des cases ( des group , 6.12.4 second ; control group , 13.43.8 seconds , p<0.001 ) .", "compared with the control group , the mean osdi value was significantly higher in the des group ( p<0.001 ) ." ], [ "the serum 25(oh)d levels in patients with des and the control patients are shown in table 2 .", "serum 25(oh)d levels were significantly lower in the des group than in the healthy control group ( des group , 19.35.8 ; control group , 31.67.3 , p<0.001 ) .", "furthermore , serum 25(oh)d levels in both male and female patients were lower compared with the healthy controls ( p<0.05 ) . when the participants were divided into different vitamin d status ( 25(oh)d , < 10 ng / ml , 10~20 ng / ml , 20~30 ng / ml , and 30 ng / ml ) , deficiency of vitamin d was more common in the des group ( p<0.001 ) ." ], [ "considering that serum 25(oh)d level was significantly lower in the des group , we conducted additional analyses on the association between serum 25(oh ) d levels and des parameters . through pearson correlation analysis", ", it was found that serum 25(oh)d level was associated with increased schimer test i ( r=0.8248 , p<0.001 ) . in addition", ", there was an inverse correlation between serum 25(oh)d and odsi scores ( r=0.3348 , p=0.005 ) and tbut ( r=0.6806 , p<0.001 ) ." ], [ "in this study , it was shown that 25(oh)d levels were lower in patients with des than in healthy controls . when the 25(oh)d levels was stratified , vitamin d deficiency was more common in the des cases .", "in further analysis , it was found that there were statistical significant associations between serum 25(oh ) d levels and the schimer test , tbut value and osdi scale .", "des is a multifactorial disorder and various risk factors were associated with the incidence of des .", "nowadays , there is more interest in the association between vitamin d and des cases .", "the major conclusions of this case - control study were consistent with previous cross - sectional study conclusions , in that low serum 25(oh)d levels were associated with des . in previous cross - sectional data analysis , it was found that low serum 25(oh)d levels were associated with des in korean adults .", "these results provide clues that vitamin d supplementation might be used in des treatment . in another case - control study with 34 patients with serum vitamin d deficiency and 21 control patients with normal vitamin d levels", ", it was found that vitamin d deficiency decreases the tbut and schirmer test values . in our study ,", "significant lower vitamin d level was detected in des patients and it was found that significant associations were detected in both male and female groups .", "in addition , the advanced correlation analysis results showed more interesting and meaningful association between serum 25(oh ) d and severity and ocular discomfort in des cases .", "rather than considering vitamin d deficiency as a risk factor for des as in a previous study , more attention was focused on the association between vitamin d and ocular physical examination and indisposed symptoms in this current study .", "positive correlation between serum vitamin d level and des severity suggests there is the potential for application of vitamin d for treatment or prevention of des . ass shown in the first study on the effect of vitamin d on the des in chinese population , this study provides additional knowledge on the effect of vitamin d on the des development .", "however , even though the association of vitamin d and des was detected in different studies , the study of the detailed mechanism was lacking .", "there were several possible explains for the effect of vitamin d on the des development .", "it has been recognized that systemic autoimmune diseases or immune abnormalities could result in the increased risk of des .", "considering the remarkable effect of vitamin d on the immune system , it could be conjectured that the immunoregulatory effect of vitamin d might influence the development of des .", "for instance , in a case - control study with 107 sjogren syndrome ( ss ) patients and 74 healthy controls , plasma vitamin d levels in ss patient group were significantly lower than in the control group .", "as dry eye was one of the most frequent symptoms of ss , and vitamin d deficiency was frequent found in patients with ss , vitamin d might be associated with the ss - related des incidence .", "besides , there were several previous studies that reported on the associations between des and systemic autoimmune disorders , including rheumatism and lupus erythematosus .", "abnormal autoimmune status might result in a higher incidence of des and the protective immunomodulatory effect of vitamin d in the regulation of immune cells might help in the prevention of des incidence .", "there was a previous study that reported that vitamin d deficiency decreased the tbut and schirmer test values and may be associated with dry - eye symptoms in non - sjogren syndrome , thus suggesting a potential mechanism beyond the autoimmune effect of vitamin d may exist .", "chronic inflammation induced by autoimmune disorders or environmental stimulating factors in ocular surface cells has been considered to be involved in des .", "for instance , vitamin d could decrease the inflammatory response of cancer and inhibited the proliferation of cancer cells in - vitro . considering that inflammation was one of the key pathogenesis and phenotype of des ,", "higher level vitamin d would decreased the inflammatory response in the ocular surface and then improve the severity of des .", "the production of vitamin d in the metabolic system is from two main sources . in general ,", "vitamin d is mainly produced by cutaneous synthesis upon skin exposure to ultraviolet light and partial vitamin d is gained from food sources .", "because of the short half - life of 1,25(oh)d ( 57 days ) and the long half - life of 25(oh)d ( 20~30 days ) , the serum 5(oh)d level is commonly used as the indicator of vitamin d status and it was used in the present study .", "vitamin d receptor ( vdr ) is a member of the nuclear hormone receptor superfamily and plays an important role in vitamin d function . in a case - control study of 64 des cases and 51 controls , the single nucleotide polymorphisms ( snps ) in the vdr genes also varied between des cases and controls .", "thus , the influence of snps in the vitamin d related gene might explain the association between serum vitamin d and des incidence .", "a major strength of our study was that the method used to assess serum vitamin d levels was sensitive and therefore might produce more robust conclusions .", "first , the sample size of this study was relatively small and it has been demonstrated that larger sample sizes are requirements for advanced study analysis .", "considering that this was a hospital - based study , it was hard to include a large sample size in the participant selection process . additionally , population - based studies are required for the detection of the association between vitamin d and des status .", "second , even though 5(oh)d is the most commonly used index for vitamin d detection , there are other indices reflecting the intracellular effect of vitamin d that were not reported in this study", ". the association of these related indices might provide additional knowledge to aid in our understanding of the mechanism of vitamin d on des .", "future prospective studies were needed to assess other serum markers of vitamin d in addition to 25(oh)d in the des cases . in advanced studies ,", "the effect of dietary vitamin d intake , vitamin d supplementation , and ultraviolet radiation exposure on the development of des should be examined .", "a third limitation of our study was that that using a case - control study design , selection bias could influence the conclusions ." ], [ "a significant association between serum 25(oh)d level and des incidence was detected in this study .", "in addition , serum 25(oh)d was associated with the des parameters and ocular discomfort status .", "it could be conjectured that vitamin d might be a potentially favorable adjunctive option for patients with des .", "considering the relatively small sample size of this study , larger studies with longer follow - up duration are needed ." ] ]

[ "congenital scrotal anomalies are unusual and include penoscrotal transposition , bifid scrotum , ectopic scrotum , and accessory scrotum ( as ) . among these , as is the least frequent , with only 42 cases reported in the english literature . as is characterized by additional scrotal tissue lacking a testis , besides a normally developed scrotum .", "various associated anomalies have been reported . in particular , contiguous subcutaneous tumor is the most frequently associated abnormality and is reported to be related to the etiology of as . although prenatal screening techniques have advanced , most reported cases of congenital perineal mass have been identified after birth .", "a 28-year - old woman was referred to our hospital for the evaluation of a fetal perineal mass at a gestational age of 31 weeks .", "prenatal ultrasonography and magnetic resonance imaging ( mri ) showed a mass of 1.0 1.2 cm located posterior to the scrotum in a male fetus .", "1 ) the likely diagnosis was lipoma and the mass maintained a stable appearance until delivery .", "the male newborn was delivered vaginally at 38 weeks of gestation and his body weight was 2208 g. there were no specific symptoms after birth .", "arrowheads indicate a 1.0 1.2 cm mass located posterior to the scrotum . on physical examination , the soft peduncular mass , measuring 2.0 cm in diameter , was attached to a midperineal skin tag .", "there was also a rugged pigmented swelling on the mass , measuring 0.7 cm in diameter , which resembled the scrotum .", "the perineal mass showed high signal intensity on t1- and t2- weighted images and the signal intensity was suppressed on fat - suppressed t1-weighted images .", "mri revealed no associated abnormalities of the intraabdominal organs , musculoskeletal system , or genitourinary system .", "a. a soft peduncular mass with a rugged and pigmented swelling is located posterior to the normally developed scrotum . c. the mass is attached at the midperineum with a skin tag .", "the preoperative diagnosis was as with perineal lipoma , and we completely excised the mass under general anesthesia at one month of age .", "the postoperative course was uneventful , and the patient was discharged on the day after the operation .", "there was no recurrence or functional sequelae within a follow - up period of six months .", "a histological examination revealed that the peduncular mass consisted of mature adipose tissue . in this case", ", it was difficult to distinguish between lipoma and normal adipose tissue pathologically . however , our clinical diagnosis was lipoma because the peduncular mass was separated from normal perineal region by the skin tag .", "the rugged swelling on the peduncular mass showed smooth muscle fibers in the subcutaneous layer , which represented the tunica dartos .", "3 ) the swelling was definitively diagnosed as as . histological examination . b. the peduncular mass", "smooth muscle fibers in the subcutaneous layer of the rugged swelling represent the tunica dartos .", "these swellings appear at four weeks of gestation and migrate to the caudal portion after 12 weeks of gestation .", "abnormal migration or early division of the labioscrotal swellings is possibly related to the etiology of congenital scrotal anomalies .", "the least frequent congenital scrotal anomaly is as , characterized by additional scrotal tissue without a testis , besides a normally developed scrotum .", "( table ) characteristics of accessory scrotum various anomalies associated with as have been reported . in particular , contiguous subcutaneous tumor has a high incidence ( 72.5% ) of association with as .", "histologically , one case of subcutaneous tumor was lipoblastoma , three cases were hamartoma , and the others were consistent with lipoma .", "( table ) it is assumed that the contiguous subcutaneous tumor is related to the etiology of as .", "sule hypothesized that as develops when intervening mesenchymal tissue ( i.e. , the developing subcutaneous tumor ) disrupts the continuity of the developing caudal labioscrotal swelling .", "however , the complete etiology of as is not explained by this hypothesis because as can occur with no contiguous tumor .", "takayasu hypothesized that as develops from the early division and teratoid growth of pluripotential labioscrotal tissue elements .", "however , two cases associated with skeletal abnormalities were located in the pubic area , and one case was located on the distal penile shaft .", "our case was detected at a gestational age of 31 weeks , and the other two reported cases were detected at 24 weeks and 32 weeks of gestation .", "a congenital perineal mass is unusual in itself , and most reported cases have been diagnosed after birth . however , detection is possible with careful prenatal screening .", "the differential diagnosis of a fetal perineal mass includes lipoma , lipoblastoma , infantile hemangioma , hamartoma , and choristoma . if a fetal perineal mass is detected during the antenatal period , it is important to look for any associated congenital anomalies .", "the prognoses of surgically treated patients are good , and only one has died , from an associated anomaly before surgery .", "the reported ages at surgery range from four days to 46 years ( median , nine months ) , and three adult cases are recorded in the literature .", "our patient was operated upon in the neonatal period because the mass was considered to be excisable without complications and the associated subcutaneous tumor had a low probability of malignancy . \n", "although a fetal perineal mass is difficult to diagnose , it can be detected with careful prenatal screening .", "many ass are associated with contiguous subcutaneous tumors , which are assumed to be related to the etiology of as ." ]

[ "<S> abstractwe report a case of accessory scrotum ( as ) in the perineal region with peduncular lipoma , diagnosed prenatally . </S>", "<S> a male fetus of 31 weeks gestation was referred to our department with a perineal mass . </S>", "<S> prenatal ultrasonography and magnetic resonance imaging showed a mass of 1.0 1.2 cm located posterior to the scrotum . </S>", "<S> no other abnormalities were noted during pregnancy . </S>", "<S> the patient was delivered vaginally at 38 weeks of gestation . on physical examination , a soft peduncular mass with a rugged and pigmented swelling was located between the normally developed scrotum and the anus . </S>", "<S> there were no specific symptoms or any other associated congenital anomalies . </S>", "<S> we completely excised the mass at one month of age . </S>", "<S> a histological examination revealed lipoma , with tissue suggestive of scrotum , so a definite diagnosis of as was made . as is a rare congenital anomaly of the scrotum . </S>", "<S> we review the literature . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION", "CONFLICT OF INTEREST" ]

[ [ "congenital scrotal anomalies are unusual and include penoscrotal transposition , bifid scrotum , ectopic scrotum , and accessory scrotum ( as ) . among these , as is the least frequent , with only 42 cases reported in the english literature . as is characterized by additional scrotal tissue lacking a testis , besides a normally developed scrotum .", "various associated anomalies have been reported . in particular , contiguous subcutaneous tumor is the most frequently associated abnormality and is reported to be related to the etiology of as . although prenatal screening techniques have advanced , most reported cases of congenital perineal mass have been identified after birth ." ], [ "a 28-year - old woman was referred to our hospital for the evaluation of a fetal perineal mass at a gestational age of 31 weeks .", "prenatal ultrasonography and magnetic resonance imaging ( mri ) showed a mass of 1.0 1.2 cm located posterior to the scrotum in a male fetus .", "1 ) the likely diagnosis was lipoma and the mass maintained a stable appearance until delivery .", "the male newborn was delivered vaginally at 38 weeks of gestation and his body weight was 2208 g. there were no specific symptoms after birth .", "arrowheads indicate a 1.0 1.2 cm mass located posterior to the scrotum . on physical examination , the soft peduncular mass , measuring 2.0 cm in diameter , was attached to a midperineal skin tag .", "there was also a rugged pigmented swelling on the mass , measuring 0.7 cm in diameter , which resembled the scrotum .", "the perineal mass showed high signal intensity on t1- and t2- weighted images and the signal intensity was suppressed on fat - suppressed t1-weighted images .", "mri revealed no associated abnormalities of the intraabdominal organs , musculoskeletal system , or genitourinary system .", "a. a soft peduncular mass with a rugged and pigmented swelling is located posterior to the normally developed scrotum . c. the mass is attached at the midperineum with a skin tag .", "the preoperative diagnosis was as with perineal lipoma , and we completely excised the mass under general anesthesia at one month of age .", "the postoperative course was uneventful , and the patient was discharged on the day after the operation .", "there was no recurrence or functional sequelae within a follow - up period of six months .", "a histological examination revealed that the peduncular mass consisted of mature adipose tissue . in this case", ", it was difficult to distinguish between lipoma and normal adipose tissue pathologically . however , our clinical diagnosis was lipoma because the peduncular mass was separated from normal perineal region by the skin tag .", "the rugged swelling on the peduncular mass showed smooth muscle fibers in the subcutaneous layer , which represented the tunica dartos .", "3 ) the swelling was definitively diagnosed as as . histological examination . b. the peduncular mass", "smooth muscle fibers in the subcutaneous layer of the rugged swelling represent the tunica dartos ." ], [ "these swellings appear at four weeks of gestation and migrate to the caudal portion after 12 weeks of gestation .", "abnormal migration or early division of the labioscrotal swellings is possibly related to the etiology of congenital scrotal anomalies .", "the least frequent congenital scrotal anomaly is as , characterized by additional scrotal tissue without a testis , besides a normally developed scrotum .", "( table ) characteristics of accessory scrotum various anomalies associated with as have been reported . in particular , contiguous subcutaneous tumor has a high incidence ( 72.5% ) of association with as .", "histologically , one case of subcutaneous tumor was lipoblastoma , three cases were hamartoma , and the others were consistent with lipoma .", "( table ) it is assumed that the contiguous subcutaneous tumor is related to the etiology of as .", "sule hypothesized that as develops when intervening mesenchymal tissue ( i.e. , the developing subcutaneous tumor ) disrupts the continuity of the developing caudal labioscrotal swelling .", "however , the complete etiology of as is not explained by this hypothesis because as can occur with no contiguous tumor .", "takayasu hypothesized that as develops from the early division and teratoid growth of pluripotential labioscrotal tissue elements .", "however , two cases associated with skeletal abnormalities were located in the pubic area , and one case was located on the distal penile shaft .", "our case was detected at a gestational age of 31 weeks , and the other two reported cases were detected at 24 weeks and 32 weeks of gestation .", "a congenital perineal mass is unusual in itself , and most reported cases have been diagnosed after birth . however , detection is possible with careful prenatal screening .", "the differential diagnosis of a fetal perineal mass includes lipoma , lipoblastoma , infantile hemangioma , hamartoma , and choristoma . if a fetal perineal mass is detected during the antenatal period , it is important to look for any associated congenital anomalies .", "the prognoses of surgically treated patients are good , and only one has died , from an associated anomaly before surgery .", "the reported ages at surgery range from four days to 46 years ( median , nine months ) , and three adult cases are recorded in the literature .", "our patient was operated upon in the neonatal period because the mass was considered to be excisable without complications and the associated subcutaneous tumor had a low probability of malignancy . \n" ], [ "although a fetal perineal mass is difficult to diagnose , it can be detected with careful prenatal screening .", "many ass are associated with contiguous subcutaneous tumors , which are assumed to be related to the etiology of as ." ], [ "" ] ]

[ "sanitation , immunization and antibiotics , and improved nutrition have increased life expectancy and dramatically changed the patterns of disease in many countries . along with these benefits , however , development , including economic development , population growth , industrial development , urbanization , and increased use of motorized transportation , has been accompanied by global environmental deterioration , which poses a threat to current and future human health , especially in developing and underdeveloped countries . economic disadvantage ( areas with low socioeconomic status )", "augments the deleterious effects of air pollution on human health , a situation designated as environmental inequity ( 1 ) .", "environmental quality has been considered to be dependent on regional characteristics , among which social and economic factors play a pivotal role ( 2,3 ) .", "environmental disparities may increase as the result of the expansion of the interest of the automotive industry , predominantly diesel engine manufacturers , in the growing markets in latin america , africa , and asia , where environmental and health policies are not strongly consolidated ( 4 ) . due to their high toxicity , diesel emissions have been subjected to increasingly restrictive regulations over the last several decades at a cost of higher investments in fuel ( low sulfur content ) and engine technologies in developed countries ( 5 ) .", "sulfur is a naturally occurring component of crude oil and is found in both gasoline and diesel .", "when these fuels are burned , sulfur is emitted as sulfur dioxide ( so2 ) or sulfate particulate matter ( 6 ) . since 1993", ", the european union ( eu ) has established standards to regulate the quality of automotive fuels and vehicle emissions to combat the atmospheric pollution caused by emissions from motor vehicles .", "a series of directives resulted in the progressive introduction of increasingly stringent standards ( euro standards ) that define the acceptable limits for exhaust emissions of vehicles sold in eu member states ( 7 ) .", "unfortunately , the benefits of cleaner technologies are lagging in less developed regions . in the usa ,", "trucks sold after 2007 emit 0.01 g / bhp - hr pm , and in germany , trucks can emit only 0.02 g / kwh ( defined in the euro v standard ) .", "in contrast , in brazil , india , and china , the allowed level of emissions is 0.1 g / kwh pm ( euro iii standard ) ( 8) .", "these differences in emissions standards mean that heavy - duty diesel engines produced by the same manufacturer emit markedly different levels of pollutants depending on the regional market in which the engines are sold .", "economic issues have been evoked to support the aforementioned regulatory differences in environmental emission standards .", "the trade - offs associated with these regulatory differences are relevant to the situation in brazil because the implementation of a policy requiring cleaner diesel technologies ( cdt ; euro iv standards for vehicles produced in 2009 and low sulphur diesel ; diesel with 50 ppm of sulphur ) was postponed until 2012 without a comprehensive analysis of the health consequences ( 9 ) .", "to evaluate the health impact of the decision to delay implementation of the cdt policy , we estimated the monetary health costs of the non - abatement of ambient pm2.5 emissions due to this postponement in brazil .", "pm2.5 ) as the estimator of exposure to air pollution . diesel emissions contribute significantly to ambient pm2.5 concentrations in urban areas , and this class of pollutant exhibits a robust association with adverse health effects , especially lung cancer and cardio - respiratory diseases ( 10,11 ) . during 2007 - 2008 , we performed daily measurements of the ambient levels of pm2.5 in six brazilian metropolitan regions : curitiba , so paulo , belo horizonte , rio de janeiro , recife , and porto alegre .", "these cities have a combined population of 46,811,100 inhabitants ( 25% of the brazilian population ) and generate 37.3% of the country 's gross national product ( 12,13 ) .", "the daily concentration of particulate matter ( pm2.5 ) was obtained by a gravimetric method using a harvard sampler with a pm2.5 impactor ( air diagnostics and engineering inc . , harrison , me , usa ) at a flow rate of 10 l.min using a polycarbonate membrane .", "each membrane was weighed before and after sampling using an ultra - microbalance ( mettler toledo umx2 , readability 1 g , zurich , switzerland ) to determine the daily mass trapped by each membrane , allowing the calculation of the daily mean concentration of pm2.5 ( 13 ) .", "the black carbon concentrations were measured using the reflectance method ( 14 ) using the same membranes , and 1/3 of the membranes were submitted to x - ray fluorescence spectrometry ( 15 ) and ion chromatography ( 16 ) analysis to determine the concentrations of the predominant chemical elements ( na , al , si , p , s , k , ca , ti , v , fe , ni , cu , zn , and pb ) , nitrates and sulfates .", "the daily mean concentrations of pm2.5 were used to calculate the annual daily mean of pm2.5 as described by the who ( 30 ) .", "all of the samplers were installed in central areas of the metropolitan regions at least 200 m from major traffic routes .", "sources and their relative contributions to the ambient pm2.5 concentrations were identified using receptor modeling techniques based on the chemical characteristics of the particles collected at the receptor sites ( in this case , the different metropolitan areas ) .", "in addition , pmf ( positive matrix factorization ) , a factor analysis method , was applied to identify sources and determine the contribution of each identified source to the ambient concentration of pm 2.5 ( 17 ) .", "the data used in this study were obtained from a recent research project conducted by our group ( 18 ) .", "the non - abatement of ambient pm2.5 concentrations due to the delay in the adoption of more stringent emissions standards was estimated by considering the total emissions inventory agreed upon by the brazilian ministry of environment and truck manufacturers and oil companies operating in brazil ( 19 ) . the projected reduction in the pm2.5 concentration that would have been achieved if the cdt policy had been implemented and those expected considering the delay are presented in figure 1 ( 20 ) .", "the differences between the emissions curves presented in figure 1 are assumed to be proportional to the changes in the proportion of pm2.5 generated by diesel engines for each metropolitan area .", "the delay in the decline in the level of diesel - generated particles was computed until 2040 based on the present ambient concentrations of pm2.5 and the relative contribution of diesel to pm2.5 determined for each study site .", "the impact on morbidity of the additional pm2.5 due to the delay in the implementation of the cdt policy was estimated in terms of hospitalizations due to respiratory and cardiovascular events .", "the impact was determined separately for different age groups , employing coefficients from time series studies ( table 1 ) , favoring studies that were conducted locally ( 21 - 26 ) .", "functions associating increases in air pollution with adverse health effects may be derived based on short - term or long - term exposures . when there is no clear evidence of a chronic effect , the short - term dose response is preferable and more conservative . for pm2.5 ,", "therefore , we used the annual mean pm2.5 concentration , as recommended by the who ( 30 ) . for mortality studies , we decided to consider the coefficients that associate chronic exposure to pm2.5 with all causes of mortality in adults to avoid possible bias in establishing the cause of death based on death certificates ( who ) .", "the numbers of respiratory and cardiovascular hospital admissions in the public health system for each metropolitan area for 2007 were obtained from the brazilian health ministry database ( 27 ) and are presented in table 2 .", "we estimated the number of hospital admissions in the private system by extrapolation based on the health insurance coverage rate ( 28 ) of the private system for each metropolitan area ( as presented in table 6- supplemental data ) .", "the number of hospital admissions in the private health system was computed based on the relationship described in the following equation ( 1 ) : nps is the number of hospital admissions in the private system , npublic is the number of hospital admissions in the public system , and cr is the proportion of the population with access to private health insurance . after determining the baseline number of hospital admissions for the relevant health outcomes and the age groups of interest and after establishing the coefficients that relate changes in the pm2.5 concentration to hospital admissions ,", "the projected morbidity effects of the delay in implementing the cdt policy were computed using the following equation ( 2 ) : hadm ( concyear ) = hospital admissions due to the delay in reducing the ambient concentration of pm2.5 in a given year ;", " = coefficient relating pm2.5 to hospital admissions ( table 1 ) ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; and totadm = total hospital admissions for a given year .", "equation ( 2 ) was used to estimate the effects of the additional pm2.5 on the different health outcomes listed in table 2 .", "the value of hadm ( concyear ) was integrated over 40 years , as shown in figure 1 .", "the effects of the delay in implementing the cdt policy were also expressed in terms of mortality .", "data on mortality due to natural causes in individuals over 40 years of age were obtained from datasus ( 29 ) .", "the additional mortality burden due to the delay in implementing the cdt policy was estimated as suggested by the who ( 30 ) using the following equation : m ( concyear ) = number of deaths in a given year due to the delay in reducing the concentration of pm2.5 ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; 0.006 = relative risk of adult mortality for each 1.0 g / m of pm2.5 ; and totalm = the baseline mortality counts for each metropolitan area , as presented in table 3 .", "morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs ( productivity loss ) .", "the mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus ( 31 ) .", "the cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo .", "the cost associated with lost productivity considers the number of days of absence due to hospitalization ( data were obtained from datasus ( 31 ) ) and the mean gross income for each age group ( data obtained from the ibge ( the brazilian statistical and geographic agency ) , which was responsible for the public economic census conducted in 2000 ( 32 ) .", "the economic valuation of mortality was performed based on the disability - adjusted life years ( daly ) method ( 33 ) , which combines an ambient factor , in this case the pm2.5 concentration , with a health indicator ( mortality ) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy .", "the number of ( daly ) ( 33 ) was calculated using an equation that estimates years of life lost ( yll ) , the daly component that refers to time lost due to premature mortality .", "the yll was estimated based on the age at death and the life expectancy in southeastern brazilian .", "the values of the other parameters ( the discount rate and the age - weighted modulation factor ) were adopted as recommended by murray and lopez ( 33 ) .", "the total years of life lost were converted into monetary values as described previously ( 34 ) , and the values for the years of life lost ( yll ) were provided by externe ( 35 ) .", "the life expectancy values were obtained from the ibge ( the brazilian statistical and geographic agency ) ( 12 ) for each metropolitan region .", "pm2.5 ) as the estimator of exposure to air pollution . diesel emissions contribute significantly to ambient pm2.5 concentrations in urban areas , and this class of pollutant exhibits a robust association with adverse health effects , especially lung cancer and cardio - respiratory diseases ( 10,11 ) . during 2007 - 2008 , we performed daily measurements of the ambient levels of pm2.5 in six brazilian metropolitan regions : curitiba , so paulo , belo horizonte , rio de janeiro , recife , and porto alegre .", "these cities have a combined population of 46,811,100 inhabitants ( 25% of the brazilian population ) and generate 37.3% of the country 's gross national product ( 12,13 ) .", "the daily concentration of particulate matter ( pm2.5 ) was obtained by a gravimetric method using a harvard sampler with a pm2.5 impactor ( air diagnostics and engineering inc . , harrison , me , usa ) at a flow rate of 10 l.min using a polycarbonate membrane .", "each membrane was weighed before and after sampling using an ultra - microbalance ( mettler toledo umx2 , readability 1 g , zurich , switzerland ) to determine the daily mass trapped by each membrane , allowing the calculation of the daily mean concentration of pm2.5 ( 13 ) .", "the black carbon concentrations were measured using the reflectance method ( 14 ) using the same membranes , and 1/3 of the membranes were submitted to x - ray fluorescence spectrometry ( 15 ) and ion chromatography ( 16 ) analysis to determine the concentrations of the predominant chemical elements ( na , al , si , p , s , k , ca , ti , v , fe , ni , cu , zn , and pb ) , nitrates and sulfates .", "the daily mean concentrations of pm2.5 were used to calculate the annual daily mean of pm2.5 as described by the who ( 30 ) .", "all of the samplers were installed in central areas of the metropolitan regions at least 200 m from major traffic routes .", "sources and their relative contributions to the ambient pm2.5 concentrations were identified using receptor modeling techniques based on the chemical characteristics of the particles collected at the receptor sites ( in this case , the different metropolitan areas ) .", "in addition , pmf ( positive matrix factorization ) , a factor analysis method , was applied to identify sources and determine the contribution of each identified source to the ambient concentration of pm 2.5 ( 17 ) .", "the data used in this study were obtained from a recent research project conducted by our group ( 18 ) .", "the non - abatement of ambient pm2.5 concentrations due to the delay in the adoption of more stringent emissions standards was estimated by considering the total emissions inventory agreed upon by the brazilian ministry of environment and truck manufacturers and oil companies operating in brazil ( 19 ) . the projected reduction in the pm2.5 concentration that would have been achieved if the cdt policy had been implemented and those expected considering the delay are presented in figure 1 ( 20 ) .", "the differences between the emissions curves presented in figure 1 are assumed to be proportional to the changes in the proportion of pm2.5 generated by diesel engines for each metropolitan area .", "the delay in the decline in the level of diesel - generated particles was computed until 2040 based on the present ambient concentrations of pm2.5 and the relative contribution of diesel to pm2.5 determined for each study site .", "the impact on morbidity of the additional pm2.5 due to the delay in the implementation of the cdt policy was estimated in terms of hospitalizations due to respiratory and cardiovascular events .", "the impact was determined separately for different age groups , employing coefficients from time series studies ( table 1 ) , favoring studies that were conducted locally ( 21 - 26 ) .", "functions associating increases in air pollution with adverse health effects may be derived based on short - term or long - term exposures . when there is no clear evidence of a chronic effect , the short - term dose response is preferable and more conservative . for pm2.5 ,", "therefore , we used the annual mean pm2.5 concentration , as recommended by the who ( 30 ) . for mortality studies", ", we decided to consider the coefficients that associate chronic exposure to pm2.5 with all causes of mortality in adults to avoid possible bias in establishing the cause of death based on death certificates ( who ) .", "the numbers of respiratory and cardiovascular hospital admissions in the public health system for each metropolitan area for 2007 were obtained from the brazilian health ministry database ( 27 ) and are presented in table 2 .", "we estimated the number of hospital admissions in the private system by extrapolation based on the health insurance coverage rate ( 28 ) of the private system for each metropolitan area ( as presented in table 6- supplemental data ) .", "the number of hospital admissions in the private health system was computed based on the relationship described in the following equation ( 1 ) : nps is the number of hospital admissions in the private system , npublic is the number of hospital admissions in the public system , and cr is the proportion of the population with access to private health insurance . after determining the baseline number of hospital admissions for the relevant health outcomes and the age groups of interest and after establishing the coefficients that relate changes in the pm2.5 concentration to hospital admissions ,", "the projected morbidity effects of the delay in implementing the cdt policy were computed using the following equation ( 2 ) : hadm ( concyear ) = hospital admissions due to the delay in reducing the ambient concentration of pm2.5 in a given year ;", " = coefficient relating pm2.5 to hospital admissions ( table 1 ) ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; and totadm = total hospital admissions for a given year .", "equation ( 2 ) was used to estimate the effects of the additional pm2.5 on the different health outcomes listed in table 2 .", "the value of hadm ( concyear ) was integrated over 40 years , as shown in figure 1 .", "the effects of the delay in implementing the cdt policy were also expressed in terms of mortality .", "data on mortality due to natural causes in individuals over 40 years of age were obtained from datasus ( 29 ) .", "the additional mortality burden due to the delay in implementing the cdt policy was estimated as suggested by the who ( 30 ) using the following equation : m ( concyear ) = number of deaths in a given year due to the delay in reducing the concentration of pm2.5 ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; 0.006 = relative risk of adult mortality for each 1.0 g / m of pm2.5 ; and totalm = the baseline mortality counts for each metropolitan area , as presented in table 3 .", "morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs ( productivity loss ) .", "the mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus ( 31 ) .", "the cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo .", "the cost associated with lost productivity considers the number of days of absence due to hospitalization ( data were obtained from datasus ( 31 ) ) and the mean gross income for each age group ( data obtained from the ibge ( the brazilian statistical and geographic agency ) , which was responsible for the public economic census conducted in 2000 ( 32 ) .", "the economic valuation of mortality was performed based on the disability - adjusted life years ( daly ) method ( 33 ) , which combines an ambient factor , in this case the pm2.5 concentration , with a health indicator ( mortality ) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy .", "the number of ( daly ) ( 33 ) was calculated using an equation that estimates years of life lost ( yll ) , the daly component that refers to time lost due to premature mortality .", "the yll was estimated based on the age at death and the life expectancy in southeastern brazilian .", "the values of the other parameters ( the discount rate and the age - weighted modulation factor ) were adopted as recommended by murray and lopez ( 33 ) .", "the total years of life lost were converted into monetary values as described previously ( 34 ) , and the values for the years of life lost ( yll ) were provided by externe ( 35 ) .", "the life expectancy values were obtained from the ibge ( the brazilian statistical and geographic agency ) ( 12 ) for each metropolitan region .", "morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs ( productivity loss ) .", "the mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus ( 31 ) .", "the cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo .", "the cost associated with lost productivity considers the number of days of absence due to hospitalization ( data were obtained from datasus ( 31 ) ) and the mean gross income for each age group ( data obtained from the ibge ( the brazilian statistical and geographic agency ) , which was responsible for the public economic census conducted in 2000 ( 32 ) .", "the economic valuation of mortality was performed based on the disability - adjusted life years ( daly ) method ( 33 ) , which combines an ambient factor , in this case the pm2.5 concentration , with a health indicator ( mortality ) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy .", "the number of ( daly ) ( 33 ) was calculated using an equation that estimates years of life lost ( yll ) , the daly component that refers to time lost due to premature mortality .", "the yll was estimated based on the age at death and the life expectancy in southeastern brazilian .", "the values of the other parameters ( the discount rate and the age - weighted modulation factor ) were adopted as recommended by murray and lopez ( 33 ) .", "the total years of life lost were converted into monetary values as described previously ( 34 ) , and the values for the years of life lost ( yll ) were provided by externe ( 35 ) .", "the life expectancy values were obtained from the ibge ( the brazilian statistical and geographic agency ) ( 12 ) for each metropolitan region .", "our approach indicated that diesel sources contributed approximately 40% of the total mass of pm2.5 , considering both primary and secondary aerosol formation .", "this estimate was consistent with that of the so paulo state sanitation agency ( cetesb ) and previous studies focused on the city of so paulo ( 37 - 38 ) .", "table 4 presents the estimated excess hospital admissions due to respiratory and cardiovascular diseases in the public and private health systems and the estimated excess mortality for the six metropolitan areas for the years 2009 to 2040 resulting from the delay in the implementation of the cdt policy .", "table 5 presents the costs associated with the excess hospital admissions due to respiratory and cardiovascular diseases attributable to pm2.5 .", "mortality costs due to respiratory and cardiovascular diseases attributable to pm2.5 were estimated based on the total years of life lost ( ylls ) .", "for the period 2009 - 2040 , the non - abatement of pm2.5 accounted for 162,878 ylls , which represents a total cost of us$ 11.4 billion .", "our approach indicated that diesel sources contributed approximately 40% of the total mass of pm2.5 , considering both primary and secondary aerosol formation .", "this estimate was consistent with that of the so paulo state sanitation agency ( cetesb ) and previous studies focused on the city of so paulo ( 37 - 38 ) .", "table 4 presents the estimated excess hospital admissions due to respiratory and cardiovascular diseases in the public and private health systems and the estimated excess mortality for the six metropolitan areas for the years 2009 to 2040 resulting from the delay in the implementation of the cdt policy .", "table 5 presents the costs associated with the excess hospital admissions due to respiratory and cardiovascular diseases attributable to pm2.5 .", "mortality costs due to respiratory and cardiovascular diseases attributable to pm2.5 were estimated based on the total years of life lost ( ylls ) .", "for the period 2009 - 2040 , the non - abatement of pm2.5 accounted for 162,878 ylls , which represents a total cost of us$ 11.4 billion .", "our results indicate that considerable health and economic burdens resulted from the delay in implementing the cdt policy .", "air pollution in urban areas has negative health effects , including difficulty breathing , wheezing , coughing , eye irritation , aggravation of pre - existing respiratory and cardiac diseases , and even premature death .", "increases in hospital admissions and medication use in areas with moderate or high levels of air pollution are commonly reported in different parts of the world ( 40 ) . the use of motorized transportation in brazil increases every year . according to denatran ( national traffic department ) ,", "the brazilian fleet has doubled in the last ten years ; there are currently almost 65,000,000 registered vehicles in brazil , including cars , trucks , buses and motorcycles ( 41 ) .", "the states of so paulo , minas gerais , and paran have the largest fleets , with nearly 20 , 7 , and 5 million cars , respectively . although this growth in motorized transportation is a result of and an indicator of the growing economy and a higher standard of living , it is accompanied by environmental and social challenges , including air pollution and traffic congestion , which pose threats to the health and well - being of the residents .", "to address these problems , different countries have conducted economic valuations of public health endpoints to substantiate environmental and health politics ( 42 ) .", "the translation of the social and health benefits of cleaner fuel and vehicle regulations into monetary amounts is necessary to direct the decision - making process regarding actions that have the potential to affect air quality in urban areas ( 43 ) .", "unfortunately , in brazil , the decision to postpone the adoption of the cdt policy was not based on any cost - benefit analysis .", "the displacement of the curve for the reductions in diesel emissions ( figure 1 ) agreed upon by industries and the brazilian authorities was considered not to be significant . however , the more detailed analysis of the health consequences of this delay performed in our study indicated that because of the large number of exposed people , the delay in implementing the cdt policy will be responsible for a considerable number of hospitalizations and a presumable excess of approximately 14,000 premature deaths .", "most likely , the authorities that approved the postponed implementation of the cdt policy would have made a different decision if the health consequences had been fully considered in the decision - making process . because cost - benefit analyses may be highly influenced by regional characteristics , in the present investigation , we collected primary data to minimize uncertainties in the estimates .", "for example , the concentration response curves for morbidity preferentially used data from local studies .", "a comprehensive sampling and chemical analysis of the particle composition and source apportionment techniques were applied to determine the contribution of the present diesel technology to ambient particle levels in the areas of study .", "vehicles , fuels , air pollution , and health should be treated and analyzed as a system .", "changes in fuel composition can immediately impact air pollution and consequently influence air pollution control ( 44 - 45 ) .", "there are many examples of how cleaner diesel fuel contributes to reductions in air pollution . in hong kong , a one - weekend intervention study that required all power plants and road vehicles to use fuel oil with a low sulfur content led to an immediate decrease in the level of ambient sulfur dioxide ( so2 ) and provided direct evidence that this type of change has immediate and long - term health benefits , thus reducing mortality rates ( 46 ) .", "donald mccubbin and mark delucchi evaluated the health costs of a kilogram of various air pollutants , including co , nox ( nitric oxides ) , pm2.5 , sox ( sulfur oxides ) , and vocs ( volatile organic compounds ) .", "they estimated health costs based on such factors as hospitalization , chronic illness , asthma attacks , and lost work days for us urban areas .", "these researchers found that the ranges in health costs per kilogram were from us$0.01 to us$0.10 for co , us$1.59 to us$23.34 for nox , us$14.81 to us$225.36 for pm2.5 , us$9.62 to us$90.94 for sox , and us$ 0.13 to us$ 1.45 for vocs ( 47 ) . in the indian context", ", the health cost of urban air pollution was estimated to be us$ 1.4 billion ( 48 ) .", "our results are consistent with the results of previous studies and indicated that the sulfur content of diesel represents a considerable source of ambient fine particles and that considerable health benefits would result from implementing the cdt policy .", "indeed , a previous study performed in so paulo indicated that the use of improved diesel technology exhibited the best cost - benefit ratio among all other policies considered to reduce the health effects of air pollution ( 49 ) .", "interestingly , although this report was fully available when the decision to delay the implementation of the cdt policy was made , the relevant authorities did not consider the recommendations presented in that report .", "this failure to consider published data - based recommendations indicates that the health consequences of industrial and fuel policies are not usually considered in situations in which economic pressures are high . in conclusion ,", "our results indicate that it is necessary to increase the level of awareness about the health consequences of policies that regulate transportation and fuel technology and that human health must play a role in the environmental agenda in urban areas .", "we thank the following researchers and institutions for monitoring the pm2.5 concentration and for the sampling infrastructure : prof dr elisabeth neves from the department of anatomy , center for biological sciences ( ufpe , federal university of pernambuco ) ; prof dr walter zin from the respiratory physiology laboratory , carlos chagas filho institute of biophysics ( ufrj , federal university of rio de janeiro ) ; prof dr geraldo brasileiro filho from the pathology department , school of medicine ( ufmg , federal university of minas gerais ) ; prof dr orliney maciel guimares from the chemistry institute ( ufpr , federal university of paran ) ; and prof dr cludia ramos rhoden from the department of physiological sciences ( ufcspa , porto alegre federal foundation for medical sciences ) ." ]

[ "<S> objective : due to their toxicity , diesel emissions have been submitted to progressively more restrictive regulations in developed countries . however , in brazil , the implementation of the cleaner diesel technologies policy ( euro iv standards for vehicles produced in 2009 and low - sulfur diesel with 50 ppm of sulfur ) was postponed until 2012 without a comprehensive analysis of the effect of this delay on public health parameters . </S>", "<S> we aimed to evaluate the impact of the delay in implementing the cleaner diesel technologies policy on health indicators and monetary health costs in brazil.methods:the primary estimator of exposure to air pollution was the concentration of ambient fine particulate matter ( particles with aerodynamic diameters < 2.5 m , [ pm2.5 ] ) . </S>", "<S> this parameter was measured daily in six brazilian metropolitan areas during 2007 - 2008 . </S>", "<S> we calculated 1 ) the projected reduction in the pm2.5 that would have been achieved if the euro iv standards had been implemented in 2009 and 2 ) the expected reduction after implementation in 2012 . </S>", "<S> the difference between these two time curves was transformed into health outcomes using previous dose - response curves . </S>", "<S> the economic valuation was performed based on the daly ( disability - adjusted life years ) method.results:the delay in implementing the cleaner diesel technologies policy will result in an estimated excess of 13,984 deaths up to 2040 . </S>", "<S> health expenditures are projected to be increased by nearly us$ 11.5 billion for the same period.conclusions:the present results indicate that a significant health burden will occur because of the postponement in implementing the cleaner diesel technologies policy . </S>", "<S> these results also reinforce the concept that health effects must be considered when revising fuel and emission policies . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Air pollution analysis and emission source determination", "Delay in the adoption of the CDT policy and estimate of the ambient PM2.5 concentrations", "Estimation of the health consequences of the implementation of the CDT policy", "Respiratory and cardiovascular hospital admissions", "Mortality", "Economic valuation of the health effects expected to occur as a consequence of the delay in the implementation of the CDT policy", "RESULTS", "Source", "Hospital Admissions and Mortality", "Health and mortality costs", "DISCUSSION", "ACKNOWLEDGMENTS" ]

[ [ "sanitation , immunization and antibiotics , and improved nutrition have increased life expectancy and dramatically changed the patterns of disease in many countries . along with these benefits , however , development , including economic development , population growth , industrial development , urbanization , and increased use of motorized transportation , has been accompanied by global environmental deterioration , which poses a threat to current and future human health , especially in developing and underdeveloped countries . economic disadvantage ( areas with low socioeconomic status )", "augments the deleterious effects of air pollution on human health , a situation designated as environmental inequity ( 1 ) .", "environmental quality has been considered to be dependent on regional characteristics , among which social and economic factors play a pivotal role ( 2,3 ) .", "environmental disparities may increase as the result of the expansion of the interest of the automotive industry , predominantly diesel engine manufacturers , in the growing markets in latin america , africa , and asia , where environmental and health policies are not strongly consolidated ( 4 ) . due to their high toxicity , diesel emissions have been subjected to increasingly restrictive regulations over the last several decades at a cost of higher investments in fuel ( low sulfur content ) and engine technologies in developed countries ( 5 ) .", "sulfur is a naturally occurring component of crude oil and is found in both gasoline and diesel .", "when these fuels are burned , sulfur is emitted as sulfur dioxide ( so2 ) or sulfate particulate matter ( 6 ) . since 1993", ", the european union ( eu ) has established standards to regulate the quality of automotive fuels and vehicle emissions to combat the atmospheric pollution caused by emissions from motor vehicles .", "a series of directives resulted in the progressive introduction of increasingly stringent standards ( euro standards ) that define the acceptable limits for exhaust emissions of vehicles sold in eu member states ( 7 ) .", "unfortunately , the benefits of cleaner technologies are lagging in less developed regions . in the usa ,", "trucks sold after 2007 emit 0.01 g / bhp - hr pm , and in germany , trucks can emit only 0.02 g / kwh ( defined in the euro v standard ) .", "in contrast , in brazil , india , and china , the allowed level of emissions is 0.1 g / kwh pm ( euro iii standard ) ( 8) .", "these differences in emissions standards mean that heavy - duty diesel engines produced by the same manufacturer emit markedly different levels of pollutants depending on the regional market in which the engines are sold .", "economic issues have been evoked to support the aforementioned regulatory differences in environmental emission standards .", "the trade - offs associated with these regulatory differences are relevant to the situation in brazil because the implementation of a policy requiring cleaner diesel technologies ( cdt ; euro iv standards for vehicles produced in 2009 and low sulphur diesel ; diesel with 50 ppm of sulphur ) was postponed until 2012 without a comprehensive analysis of the health consequences ( 9 ) .", "to evaluate the health impact of the decision to delay implementation of the cdt policy , we estimated the monetary health costs of the non - abatement of ambient pm2.5 emissions due to this postponement in brazil ." ], [ "pm2.5 ) as the estimator of exposure to air pollution . diesel emissions contribute significantly to ambient pm2.5 concentrations in urban areas , and this class of pollutant exhibits a robust association with adverse health effects , especially lung cancer and cardio - respiratory diseases ( 10,11 ) . during 2007 - 2008 , we performed daily measurements of the ambient levels of pm2.5 in six brazilian metropolitan regions : curitiba , so paulo , belo horizonte , rio de janeiro , recife , and porto alegre .", "these cities have a combined population of 46,811,100 inhabitants ( 25% of the brazilian population ) and generate 37.3% of the country 's gross national product ( 12,13 ) .", "the daily concentration of particulate matter ( pm2.5 ) was obtained by a gravimetric method using a harvard sampler with a pm2.5 impactor ( air diagnostics and engineering inc . , harrison , me , usa ) at a flow rate of 10 l.min using a polycarbonate membrane .", "each membrane was weighed before and after sampling using an ultra - microbalance ( mettler toledo umx2 , readability 1 g , zurich , switzerland ) to determine the daily mass trapped by each membrane , allowing the calculation of the daily mean concentration of pm2.5 ( 13 ) .", "the black carbon concentrations were measured using the reflectance method ( 14 ) using the same membranes , and 1/3 of the membranes were submitted to x - ray fluorescence spectrometry ( 15 ) and ion chromatography ( 16 ) analysis to determine the concentrations of the predominant chemical elements ( na , al , si , p , s , k , ca , ti , v , fe , ni , cu , zn , and pb ) , nitrates and sulfates .", "the daily mean concentrations of pm2.5 were used to calculate the annual daily mean of pm2.5 as described by the who ( 30 ) .", "all of the samplers were installed in central areas of the metropolitan regions at least 200 m from major traffic routes .", "sources and their relative contributions to the ambient pm2.5 concentrations were identified using receptor modeling techniques based on the chemical characteristics of the particles collected at the receptor sites ( in this case , the different metropolitan areas ) .", "in addition , pmf ( positive matrix factorization ) , a factor analysis method , was applied to identify sources and determine the contribution of each identified source to the ambient concentration of pm 2.5 ( 17 ) .", "the data used in this study were obtained from a recent research project conducted by our group ( 18 ) .", "the non - abatement of ambient pm2.5 concentrations due to the delay in the adoption of more stringent emissions standards was estimated by considering the total emissions inventory agreed upon by the brazilian ministry of environment and truck manufacturers and oil companies operating in brazil ( 19 ) . the projected reduction in the pm2.5 concentration that would have been achieved if the cdt policy had been implemented and those expected considering the delay are presented in figure 1 ( 20 ) .", "the differences between the emissions curves presented in figure 1 are assumed to be proportional to the changes in the proportion of pm2.5 generated by diesel engines for each metropolitan area .", "the delay in the decline in the level of diesel - generated particles was computed until 2040 based on the present ambient concentrations of pm2.5 and the relative contribution of diesel to pm2.5 determined for each study site .", "the impact on morbidity of the additional pm2.5 due to the delay in the implementation of the cdt policy was estimated in terms of hospitalizations due to respiratory and cardiovascular events .", "the impact was determined separately for different age groups , employing coefficients from time series studies ( table 1 ) , favoring studies that were conducted locally ( 21 - 26 ) .", "functions associating increases in air pollution with adverse health effects may be derived based on short - term or long - term exposures . when there is no clear evidence of a chronic effect , the short - term dose response is preferable and more conservative . for pm2.5 ,", "therefore , we used the annual mean pm2.5 concentration , as recommended by the who ( 30 ) . for mortality studies , we decided to consider the coefficients that associate chronic exposure to pm2.5 with all causes of mortality in adults to avoid possible bias in establishing the cause of death based on death certificates ( who ) .", "the numbers of respiratory and cardiovascular hospital admissions in the public health system for each metropolitan area for 2007 were obtained from the brazilian health ministry database ( 27 ) and are presented in table 2 .", "we estimated the number of hospital admissions in the private system by extrapolation based on the health insurance coverage rate ( 28 ) of the private system for each metropolitan area ( as presented in table 6- supplemental data ) .", "the number of hospital admissions in the private health system was computed based on the relationship described in the following equation ( 1 ) : nps is the number of hospital admissions in the private system , npublic is the number of hospital admissions in the public system , and cr is the proportion of the population with access to private health insurance . after determining the baseline number of hospital admissions for the relevant health outcomes and the age groups of interest and after establishing the coefficients that relate changes in the pm2.5 concentration to hospital admissions ,", "the projected morbidity effects of the delay in implementing the cdt policy were computed using the following equation ( 2 ) : hadm ( concyear ) = hospital admissions due to the delay in reducing the ambient concentration of pm2.5 in a given year ;", " = coefficient relating pm2.5 to hospital admissions ( table 1 ) ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; and totadm = total hospital admissions for a given year .", "equation ( 2 ) was used to estimate the effects of the additional pm2.5 on the different health outcomes listed in table 2 .", "the value of hadm ( concyear ) was integrated over 40 years , as shown in figure 1 .", "the effects of the delay in implementing the cdt policy were also expressed in terms of mortality .", "data on mortality due to natural causes in individuals over 40 years of age were obtained from datasus ( 29 ) .", "the additional mortality burden due to the delay in implementing the cdt policy was estimated as suggested by the who ( 30 ) using the following equation : m ( concyear ) = number of deaths in a given year due to the delay in reducing the concentration of pm2.5 ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; 0.006 = relative risk of adult mortality for each 1.0 g / m of pm2.5 ; and totalm = the baseline mortality counts for each metropolitan area , as presented in table 3 .", "morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs ( productivity loss ) .", "the mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus ( 31 ) .", "the cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo .", "the cost associated with lost productivity considers the number of days of absence due to hospitalization ( data were obtained from datasus ( 31 ) ) and the mean gross income for each age group ( data obtained from the ibge ( the brazilian statistical and geographic agency ) , which was responsible for the public economic census conducted in 2000 ( 32 ) .", "the economic valuation of mortality was performed based on the disability - adjusted life years ( daly ) method ( 33 ) , which combines an ambient factor , in this case the pm2.5 concentration , with a health indicator ( mortality ) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy .", "the number of ( daly ) ( 33 ) was calculated using an equation that estimates years of life lost ( yll ) , the daly component that refers to time lost due to premature mortality .", "the yll was estimated based on the age at death and the life expectancy in southeastern brazilian .", "the values of the other parameters ( the discount rate and the age - weighted modulation factor ) were adopted as recommended by murray and lopez ( 33 ) .", "the total years of life lost were converted into monetary values as described previously ( 34 ) , and the values for the years of life lost ( yll ) were provided by externe ( 35 ) .", "the life expectancy values were obtained from the ibge ( the brazilian statistical and geographic agency ) ( 12 ) for each metropolitan region ." ], [ "pm2.5 ) as the estimator of exposure to air pollution . diesel emissions contribute significantly to ambient pm2.5 concentrations in urban areas , and this class of pollutant exhibits a robust association with adverse health effects , especially lung cancer and cardio - respiratory diseases ( 10,11 ) . during 2007 - 2008 , we performed daily measurements of the ambient levels of pm2.5 in six brazilian metropolitan regions : curitiba , so paulo , belo horizonte , rio de janeiro , recife , and porto alegre .", "these cities have a combined population of 46,811,100 inhabitants ( 25% of the brazilian population ) and generate 37.3% of the country 's gross national product ( 12,13 ) .", "the daily concentration of particulate matter ( pm2.5 ) was obtained by a gravimetric method using a harvard sampler with a pm2.5 impactor ( air diagnostics and engineering inc . , harrison , me , usa ) at a flow rate of 10 l.min using a polycarbonate membrane .", "each membrane was weighed before and after sampling using an ultra - microbalance ( mettler toledo umx2 , readability 1 g , zurich , switzerland ) to determine the daily mass trapped by each membrane , allowing the calculation of the daily mean concentration of pm2.5 ( 13 ) .", "the black carbon concentrations were measured using the reflectance method ( 14 ) using the same membranes , and 1/3 of the membranes were submitted to x - ray fluorescence spectrometry ( 15 ) and ion chromatography ( 16 ) analysis to determine the concentrations of the predominant chemical elements ( na , al , si , p , s , k , ca , ti , v , fe , ni , cu , zn , and pb ) , nitrates and sulfates .", "the daily mean concentrations of pm2.5 were used to calculate the annual daily mean of pm2.5 as described by the who ( 30 ) .", "all of the samplers were installed in central areas of the metropolitan regions at least 200 m from major traffic routes .", "sources and their relative contributions to the ambient pm2.5 concentrations were identified using receptor modeling techniques based on the chemical characteristics of the particles collected at the receptor sites ( in this case , the different metropolitan areas ) .", "in addition , pmf ( positive matrix factorization ) , a factor analysis method , was applied to identify sources and determine the contribution of each identified source to the ambient concentration of pm 2.5 ( 17 ) .", "the data used in this study were obtained from a recent research project conducted by our group ( 18 ) ." ], [ "the non - abatement of ambient pm2.5 concentrations due to the delay in the adoption of more stringent emissions standards was estimated by considering the total emissions inventory agreed upon by the brazilian ministry of environment and truck manufacturers and oil companies operating in brazil ( 19 ) . the projected reduction in the pm2.5 concentration that would have been achieved if the cdt policy had been implemented and those expected considering the delay are presented in figure 1 ( 20 ) ." ], [ "the differences between the emissions curves presented in figure 1 are assumed to be proportional to the changes in the proportion of pm2.5 generated by diesel engines for each metropolitan area .", "the delay in the decline in the level of diesel - generated particles was computed until 2040 based on the present ambient concentrations of pm2.5 and the relative contribution of diesel to pm2.5 determined for each study site .", "the impact on morbidity of the additional pm2.5 due to the delay in the implementation of the cdt policy was estimated in terms of hospitalizations due to respiratory and cardiovascular events .", "the impact was determined separately for different age groups , employing coefficients from time series studies ( table 1 ) , favoring studies that were conducted locally ( 21 - 26 ) .", "functions associating increases in air pollution with adverse health effects may be derived based on short - term or long - term exposures . when there is no clear evidence of a chronic effect , the short - term dose response is preferable and more conservative . for pm2.5 ,", "therefore , we used the annual mean pm2.5 concentration , as recommended by the who ( 30 ) . for mortality studies", ", we decided to consider the coefficients that associate chronic exposure to pm2.5 with all causes of mortality in adults to avoid possible bias in establishing the cause of death based on death certificates ( who ) ." ], [ "the numbers of respiratory and cardiovascular hospital admissions in the public health system for each metropolitan area for 2007 were obtained from the brazilian health ministry database ( 27 ) and are presented in table 2 .", "we estimated the number of hospital admissions in the private system by extrapolation based on the health insurance coverage rate ( 28 ) of the private system for each metropolitan area ( as presented in table 6- supplemental data ) .", "the number of hospital admissions in the private health system was computed based on the relationship described in the following equation ( 1 ) : nps is the number of hospital admissions in the private system , npublic is the number of hospital admissions in the public system , and cr is the proportion of the population with access to private health insurance . after determining the baseline number of hospital admissions for the relevant health outcomes and the age groups of interest and after establishing the coefficients that relate changes in the pm2.5 concentration to hospital admissions ,", "the projected morbidity effects of the delay in implementing the cdt policy were computed using the following equation ( 2 ) : hadm ( concyear ) = hospital admissions due to the delay in reducing the ambient concentration of pm2.5 in a given year ;", " = coefficient relating pm2.5 to hospital admissions ( table 1 ) ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; and totadm = total hospital admissions for a given year .", "equation ( 2 ) was used to estimate the effects of the additional pm2.5 on the different health outcomes listed in table 2 .", "the value of hadm ( concyear ) was integrated over 40 years , as shown in figure 1 ." ], [ "the effects of the delay in implementing the cdt policy were also expressed in terms of mortality .", "data on mortality due to natural causes in individuals over 40 years of age were obtained from datasus ( 29 ) .", "the additional mortality burden due to the delay in implementing the cdt policy was estimated as suggested by the who ( 30 ) using the following equation : m ( concyear ) = number of deaths in a given year due to the delay in reducing the concentration of pm2.5 ; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay ; 0.006 = relative risk of adult mortality for each 1.0 g / m of pm2.5 ; and totalm = the baseline mortality counts for each metropolitan area , as presented in table 3 .", "morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs ( productivity loss ) .", "the mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus ( 31 ) .", "the cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo .", "the cost associated with lost productivity considers the number of days of absence due to hospitalization ( data were obtained from datasus ( 31 ) ) and the mean gross income for each age group ( data obtained from the ibge ( the brazilian statistical and geographic agency ) , which was responsible for the public economic census conducted in 2000 ( 32 ) .", "the economic valuation of mortality was performed based on the disability - adjusted life years ( daly ) method ( 33 ) , which combines an ambient factor , in this case the pm2.5 concentration , with a health indicator ( mortality ) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy .", "the number of ( daly ) ( 33 ) was calculated using an equation that estimates years of life lost ( yll ) , the daly component that refers to time lost due to premature mortality .", "the yll was estimated based on the age at death and the life expectancy in southeastern brazilian .", "the values of the other parameters ( the discount rate and the age - weighted modulation factor ) were adopted as recommended by murray and lopez ( 33 ) .", "the total years of life lost were converted into monetary values as described previously ( 34 ) , and the values for the years of life lost ( yll ) were provided by externe ( 35 ) .", "the life expectancy values were obtained from the ibge ( the brazilian statistical and geographic agency ) ( 12 ) for each metropolitan region ." ], [ "morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs ( productivity loss ) .", "the mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus ( 31 ) .", "the cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo .", "the cost associated with lost productivity considers the number of days of absence due to hospitalization ( data were obtained from datasus ( 31 ) ) and the mean gross income for each age group ( data obtained from the ibge ( the brazilian statistical and geographic agency ) , which was responsible for the public economic census conducted in 2000 ( 32 ) .", "the economic valuation of mortality was performed based on the disability - adjusted life years ( daly ) method ( 33 ) , which combines an ambient factor , in this case the pm2.5 concentration , with a health indicator ( mortality ) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy .", "the number of ( daly ) ( 33 ) was calculated using an equation that estimates years of life lost ( yll ) , the daly component that refers to time lost due to premature mortality .", "the yll was estimated based on the age at death and the life expectancy in southeastern brazilian .", "the values of the other parameters ( the discount rate and the age - weighted modulation factor ) were adopted as recommended by murray and lopez ( 33 ) .", "the total years of life lost were converted into monetary values as described previously ( 34 ) , and the values for the years of life lost ( yll ) were provided by externe ( 35 ) .", "the life expectancy values were obtained from the ibge ( the brazilian statistical and geographic agency ) ( 12 ) for each metropolitan region ." ], [ "our approach indicated that diesel sources contributed approximately 40% of the total mass of pm2.5 , considering both primary and secondary aerosol formation .", "this estimate was consistent with that of the so paulo state sanitation agency ( cetesb ) and previous studies focused on the city of so paulo ( 37 - 38 ) .", "table 4 presents the estimated excess hospital admissions due to respiratory and cardiovascular diseases in the public and private health systems and the estimated excess mortality for the six metropolitan areas for the years 2009 to 2040 resulting from the delay in the implementation of the cdt policy .", "table 5 presents the costs associated with the excess hospital admissions due to respiratory and cardiovascular diseases attributable to pm2.5 .", "mortality costs due to respiratory and cardiovascular diseases attributable to pm2.5 were estimated based on the total years of life lost ( ylls ) .", "for the period 2009 - 2040 , the non - abatement of pm2.5 accounted for 162,878 ylls , which represents a total cost of us$ 11.4 billion ." ], [ "our approach indicated that diesel sources contributed approximately 40% of the total mass of pm2.5 , considering both primary and secondary aerosol formation .", "this estimate was consistent with that of the so paulo state sanitation agency ( cetesb ) and previous studies focused on the city of so paulo ( 37 - 38 ) ." ], [ "table 4 presents the estimated excess hospital admissions due to respiratory and cardiovascular diseases in the public and private health systems and the estimated excess mortality for the six metropolitan areas for the years 2009 to 2040 resulting from the delay in the implementation of the cdt policy ." ], [ "table 5 presents the costs associated with the excess hospital admissions due to respiratory and cardiovascular diseases attributable to pm2.5 .", "mortality costs due to respiratory and cardiovascular diseases attributable to pm2.5 were estimated based on the total years of life lost ( ylls ) .", "for the period 2009 - 2040 , the non - abatement of pm2.5 accounted for 162,878 ylls , which represents a total cost of us$ 11.4 billion ." ], [ "our results indicate that considerable health and economic burdens resulted from the delay in implementing the cdt policy .", "air pollution in urban areas has negative health effects , including difficulty breathing , wheezing , coughing , eye irritation , aggravation of pre - existing respiratory and cardiac diseases , and even premature death .", "increases in hospital admissions and medication use in areas with moderate or high levels of air pollution are commonly reported in different parts of the world ( 40 ) . the use of motorized transportation in brazil increases every year . according to denatran ( national traffic department ) ,", "the brazilian fleet has doubled in the last ten years ; there are currently almost 65,000,000 registered vehicles in brazil , including cars , trucks , buses and motorcycles ( 41 ) .", "the states of so paulo , minas gerais , and paran have the largest fleets , with nearly 20 , 7 , and 5 million cars , respectively . although this growth in motorized transportation is a result of and an indicator of the growing economy and a higher standard of living , it is accompanied by environmental and social challenges , including air pollution and traffic congestion , which pose threats to the health and well - being of the residents .", "to address these problems , different countries have conducted economic valuations of public health endpoints to substantiate environmental and health politics ( 42 ) .", "the translation of the social and health benefits of cleaner fuel and vehicle regulations into monetary amounts is necessary to direct the decision - making process regarding actions that have the potential to affect air quality in urban areas ( 43 ) .", "unfortunately , in brazil , the decision to postpone the adoption of the cdt policy was not based on any cost - benefit analysis .", "the displacement of the curve for the reductions in diesel emissions ( figure 1 ) agreed upon by industries and the brazilian authorities was considered not to be significant . however , the more detailed analysis of the health consequences of this delay performed in our study indicated that because of the large number of exposed people , the delay in implementing the cdt policy will be responsible for a considerable number of hospitalizations and a presumable excess of approximately 14,000 premature deaths .", "most likely , the authorities that approved the postponed implementation of the cdt policy would have made a different decision if the health consequences had been fully considered in the decision - making process . because cost - benefit analyses may be highly influenced by regional characteristics , in the present investigation , we collected primary data to minimize uncertainties in the estimates .", "for example , the concentration response curves for morbidity preferentially used data from local studies .", "a comprehensive sampling and chemical analysis of the particle composition and source apportionment techniques were applied to determine the contribution of the present diesel technology to ambient particle levels in the areas of study .", "vehicles , fuels , air pollution , and health should be treated and analyzed as a system .", "changes in fuel composition can immediately impact air pollution and consequently influence air pollution control ( 44 - 45 ) .", "there are many examples of how cleaner diesel fuel contributes to reductions in air pollution . in hong kong , a one - weekend intervention study that required all power plants and road vehicles to use fuel oil with a low sulfur content led to an immediate decrease in the level of ambient sulfur dioxide ( so2 ) and provided direct evidence that this type of change has immediate and long - term health benefits , thus reducing mortality rates ( 46 ) .", "donald mccubbin and mark delucchi evaluated the health costs of a kilogram of various air pollutants , including co , nox ( nitric oxides ) , pm2.5 , sox ( sulfur oxides ) , and vocs ( volatile organic compounds ) .", "they estimated health costs based on such factors as hospitalization , chronic illness , asthma attacks , and lost work days for us urban areas .", "these researchers found that the ranges in health costs per kilogram were from us$0.01 to us$0.10 for co , us$1.59 to us$23.34 for nox , us$14.81 to us$225.36 for pm2.5 , us$9.62 to us$90.94 for sox , and us$ 0.13 to us$ 1.45 for vocs ( 47 ) . in the indian context", ", the health cost of urban air pollution was estimated to be us$ 1.4 billion ( 48 ) .", "our results are consistent with the results of previous studies and indicated that the sulfur content of diesel represents a considerable source of ambient fine particles and that considerable health benefits would result from implementing the cdt policy .", "indeed , a previous study performed in so paulo indicated that the use of improved diesel technology exhibited the best cost - benefit ratio among all other policies considered to reduce the health effects of air pollution ( 49 ) .", "interestingly , although this report was fully available when the decision to delay the implementation of the cdt policy was made , the relevant authorities did not consider the recommendations presented in that report .", "this failure to consider published data - based recommendations indicates that the health consequences of industrial and fuel policies are not usually considered in situations in which economic pressures are high . in conclusion ,", "our results indicate that it is necessary to increase the level of awareness about the health consequences of policies that regulate transportation and fuel technology and that human health must play a role in the environmental agenda in urban areas ." ], [ "we thank the following researchers and institutions for monitoring the pm2.5 concentration and for the sampling infrastructure : prof dr elisabeth neves from the department of anatomy , center for biological sciences ( ufpe , federal university of pernambuco ) ; prof dr walter zin from the respiratory physiology laboratory , carlos chagas filho institute of biophysics ( ufrj , federal university of rio de janeiro ) ; prof dr geraldo brasileiro filho from the pathology department , school of medicine ( ufmg , federal university of minas gerais ) ; prof dr orliney maciel guimares from the chemistry institute ( ufpr , federal university of paran ) ; and prof dr cludia ramos rhoden from the department of physiological sciences ( ufcspa , porto alegre federal foundation for medical sciences ) ." ] ]

[ "cancer vaccine concept have been encompasses a broad spectrum of platforms intended to trigger or increase an adaptive immune response against a malignant tumor .", "thus cancer vaccines are aimed as active immunotherapeutic interventions mainly in an established disease state in which the malignancy is expressing all its functional capabilities or hallmarks .", "understanding of relationship between structure and function in cancer vaccines is essential to interpret their opportunities to impact into prolonging survival and increasing quality of life in cancer patients .", "the structure of cancer vaccines is referred to a tumor associated ( or specific ) antigen shaped as peptides , recombinant vectors , whole tumor cells or antigen presenting cells linked or supported by an adequate immunopotentiating platform , which should be inserted in a context of therapeutic maneuvers .", "the expected function of cancer vaccines is basically to circumvent the immunologic tolerance in the tumor microenvironment inducing a lowest rate of tumor progression and increasing the host survival .", "so , cancer vaccines function is expected to be relevant at all levels of biological organization of the malignancy host , from the tumor microenvironment up to the whole organism .", "then , to success with active immunotherapy strategy two items need to be considered : the role of target antigens in tumor biology ( cancer hallmark related ) and the immunogenic capacity of vaccine composition .", "once selected the right antigen and the most adequate platform for immune potentiating , the battle field for cancer vaccines is the immunosuppressive microenvironment induced by the malignant machinery ; the corner between structure and function in cancer vaccines is accurately the interaction between the tumors hallmarks and the host immune response .", "prostate cancer has been proposed as a prototype for vaccine therapy scenario . besides the increasing spectrum of therapeutic tools for the disease control at the advanced disease stage , meaning new androgen deprivation modalities such as abiraterone , advanced chemotherapeutic agents such as cabazitaxel and small tyrosine kinase inhibitors such as cabozantinib , a cancer vaccine appear in the prostate carcinoma treatments alternative .", "evidences supporting that sipuleucel - t , a vaccine based on dendritic cells pulsed with prostatic acid phosphatase linked to granulocyte ", "macrophage colony - stimulating factor , prolonged survival in minimal or non - symptomatic mcrpc patients and its fda approval ( april , 2010 ) , constitute the proof of concept of cancer vaccines application in the scenario of a solid tumor with long term disease course , characterized by well described tumor associated antigens , a defined biomarker ( serum psa ) and a good physicians tool to follow up the patients therapeutic response ( halabi nomogram ) .", "non- small cell lung cancer ( nsclc ) is the first cause of cancer associated death worldwide , the 60- 80% of patients are diagnosed at the advanced stages ( iiib / iv ) in which their life expectancy is not too far from 12 months median survival even second and emerging third line of onco - specific treatments . in this particular case the active immune intervention using cancer vaccines is strongly challenged by the tumor induced immunologic tolerance .", "there are several evidences of a marked infiltration of different types of immune cells in the nsclc microenvironment , and the distribution , tissue localization , and cell types are significantly associated with progression and patient s survival .", "the clinical relevance of the microenvironmental immunological milieu in this malignacy is highlighted by the facts that higher foxp3+/cd8 + ratio in tumor sites is an independent factor for poor response to platinum - based neoadjuvant chemotherapy in a setting of advanced stages patients .", "this review is aimed to focus on nsclc as an emerging and promising model for active immunotherapy and the challenges for its inclusion in the current clinical scenario , considering the increasing body of evidences of the interaction between inflammatory cells and tumor cells , facilitating the lung carcinogenesis and tumor hallmarks expression ( angiogenesis and tumor cell migration among others ) , in which antigen - specific antitumor responses are overtly present . in this context", ", the clinical relevance of cancer vaccines should be predicted pointing out the emerging determinants of its structure and function relationships in the particular situation of nsclc malignancy hallmarks .", "the kinetics of the active immunizations encompasses a sequential cascade of clinical endpoints : starting by the activation of the immune system , followed by the antitumor response and finalizing with the consequential impact on patients overall survival .", "today this cascade of clinical endpoints is the backbone for active immunization assessment and moreover the concept of cancer vaccines , applied in the nsclc setting , is just evolving as a complex therapeutic strategy , in which the opportunities for cancer vaccines start from the selection of the target cancer hallmark , followed by the vaccine formulation and its platforms for immune potentiating , also encompass the successful insertion in the standard of care , the chronic administration beyond progression disease , the personalization based on predictors of response and the potential combination with other targeted therapies .", "as an inherently condition of all malignant processes , nsclc carcinogenesis is resulting from the interaction between the epithelial mucosa genome and the lung microenvironment . in this context , the intrinsic cell capacity for dna repair , the nutrients accessibility , the angiogenesis state and the hypoxia levels , seems to be the common context of selective pressures for the cascade of mutations during tumor progression and coming out of the malignant cells phenotypes .", "particularly , the continuing exposure of dna to metabolically activated carcinogens , throughout the exposition to air contaminants or cigarette- smoke , resulting in the formation of dna adducts and consequential genetic instability .", "the rate of dna injure , taking place day after day over many years , is fully consistent with multiple genetic changes in lung cancer and is associated with increasingly severe histopathological phenotypes .", "the importance of the target antigens to tumor biology is considered relevant for the success of active immunotherapy .", "indeed , the selection of vaccine antigens must be focused mainly on those molecules significantly associated with the hallmarks of cancer , also called oncoantigens ; those specific or tumor associated antigens that are highly correlated with the phenotypic consolidation of the malignancy and tumor progression [ 20 , 21 ] .", "the egfr network induce a broad range of biological effects by controlling the cellular outcome , through a multiple ligands activation - dependent regulatory loops , which fall into time dependants temporal domains ; early loops comprise protein modifications mechanisms , while delayed loops involve transcription regulation mechanisms ; both became aberrant and without feedback control in malignant cells . during development and progression of lung neoplasms", "the egfr is overexpressed in the following proportions : 62% of all nsclc tumors , 89% of squamous cell tumors , 41% of adenocarcinomas and 80% of bronchoalveolar tumors . in the case of adenocarcinoma in the peripheral compartment of the lung ( terminal bronquiole and alveoli ) in never smokers ,", "the egfr overexpression is associated with a lower methylation index of the p53 cancer suppressor gene through high proportion of transition mutations ( ie , purine for purine or pyrimidine for pyrimidine ) .", "the degree of egfr expression has been reported as a predictive factor of response to biological therapy in nsclc patients .", "there are two classes of anti - egfr agents that have shown clinical activity in nsclc .", "these are either monoclonal antibodies directed at the extracellular domain of the egfr and inhibiting the binding of natural ligands to the receptor , or low molecular weight tyrosine kinase inhibitors ( tkis ) that inhibit the tyrosine kinase activity of egfr , generally by competing reversibly with atp for the atp binding site .", "targeting the egfr with a cancer vaccine is a newly strategy currently in clinical scrutiny using the active immune deprivation of egf ligand as important tumor growth factor ( cimavax egf ) [ 27 , 28 ] .", "muc1 is over - expressed in almost all nsclc patients , being associated with bad prognosis .", "the role in tumor biology of the nglycosylated muc1c ( muc1 c - terminal transmembrane subunit ) seems to be associated to a functionally important extracellular bridge mediated by galectin 3 between this recognized mucin antigen and tyrosine kinases receptors , such as the egfr .", "muc1 looks to be related not only with promoting cell growth and survival but also with t cell proliferation inhibition and accordingly , immunosuppression . in this context", ", other molecules became aberrant expressed in the cascade of events associated with the sustained proliferation and its negative regulation , apoptosis evasion and the limitless replicative potential .", "tumor suppressor gene p53 receives stress signals from excessive dna damage and suboptimal nutrients triggering dna repairing mechanism or apoptosis .", "loss of p53 function by mutations are frequent events in human oncogenesis , which leads to persistent aberrant expression in about 60% of nsclc , correlated with poor cht response and lowest overall sv of the patients .", "this conduced to exploration of a mutant p53 peptide pulsed dendritic cell vaccine in a phase ii clinical trial with stage iii nsclc patients .", "the specialized dna polymerase , named telomerase , which adds telomere repeat segments to the end of telomeric dna is nearly absent in nonimmortalized cells but expressed at functionally significant levels in 90% of human tumors .", "telomerase- based vaccine gv1001 is starting the clinical scrutiny , supported also by the facts that its presence in sputum samples has been identified as a potential specific marker for early detection of lung cancer .", "the activation of cdk1-cyclin b1 triggers the mitosis progression and its translocation from the cytoplasm to the nucleus .", "positive feedback loops regulate its biological activity and spatial localization , ensuring a rapid , complete , robust , and irreversible transition from interphase to mitosis .", "there is reported the aberrant expression of these regulators of the g2 m cell cycle checkpoint in many neoplasms , including nsclc and significant association between increased cyclin b1 expression and reduced patients survival in stage i/ ii nsclc has been described . furthermore , cyclin b1 peptide - pulsed autologous dendritic cell vaccine is early of clinical evaluation in resectable nsclc patients .", "the ras genes , including h - ras , k - ras and n - ras , encode a family of proteins regulating cell growth , differentiation and apoptosis .", "mutations in the k - ras gene , mainly in codons 12 and 13 , have been found in 20 - 30% of nsclc tumor samples and occur most commonly , but not exclusively , in adenocarcinoma histology and in heavy smokers .", "active immune strategies aimed at interfering with the ras - raf - mek pathway in nsclc are also incipient exploring the use of a ras peptide based vaccine .", "the continuous cascade of molecular events starting from at least a cell clone and characterized by unregulated cellular proliferation and clonal heterogeneity , is resulting in a characteristic nsclc malignant phenotype .", "based only on histology nsclc comprises i.e. for adenocarcinoma , squamous - cell carcinoma and large - cell carcinoma subsets , while , based on the molecular subsets expressed in the resultant malignant phenotypes , nsclc drive a broad spectrum of diseases subsets : egfr , her2 , kras , alk , braf , pik3ca , akt1 , ros1 , nras , map2k1 and the recently identified kif5b - ret fusion subset , each one with dissimilar pattern of incidence in a population .", "the personalization of cancer vaccines therapy is a pending chapter in this background , and the selection of the right vaccine for the right patient is becoming as challenge .", "the design of cancer vaccines attempt to harness the specificity and resistance potentials of the human immune system . with the aim to stimulate the immune system to recognize , attack and destroy tumor cells", "the personalization treatment is intrinsic for mhc context dependent antigens vaccines ( peptides based vaccines ) and for cell based cancer vaccines . in the context of the adaptive immune response", "one foremost challenge for clinical researchers is to classify the patient s population according the pre - existing immune response in potential responders or non- responders to the target antigen . ", "it is widely accepted that the failure of the cancer vaccines in the nsclc scenario is related with its introduction in the advanced disease stages and poor performance status of the patients due to the combination of the tumor induced immunosuppression with the immune senescence .", "the facts that nsclc occur in subjects who have never smoked and have no evident cause for chronic inflammatory changes in the lung parenchyma , suggest that the interactions between the broncho - alveolar compartment and the in situ immune regulators are involved in facilitating tumor occurrence , growth , and spread .", "it is progressively ostensible that crosstalk between cancer cells and cells of tumor stroma is involved in the acquired capability for invasive growth and metastases .", "tumor stroma has been described as pivotal in the established hostile immune environment through the secretion of immunosuppressive factors and the recruitment of suppressive immune cells of myeloid and lymphoid origin , while the tumor cells directly contributing to immune resistance through the secretion of immunosuppressive mediators , such as transforming growth factor ( tgf ) or natural killer ( nk ) cell receptor decoys , and through the expression of ligands for immune checkpoint ( or co - inhibitory ) receptors , such as programmed cell death ligand 1 ( pdl1 ) .", "the distribution , tissue localization , and cell types infiltrating lung tumor microenvironment are significantly associated with the disease progression and patient s survival .", "for example dendritic cell defects increases immature forms that may play an immunosuppressive role and facilitate cancer migration , invasion , and epithelial - to - mesenchymal transition ( schneider et al . 2011 ) .", "also , genetic variations in the transforming growth factor - beta pathway are considered as predictors of survival in advanced non - small cell lung cancer and higher foxp3+/cd8 + ratio in tumor sites is described as an independent factor for poor response to platinum - based neoadjuvant chemotherapy in a setting of advanced stages patients . summarizing ,", "a variety of cellular immune abnormalities , antigen processing and presentation machinery defects , cytokine alterations and also individual conditions , increase the challenge for nsclc therapeutic vaccines .", "vaccines candidates should activate the immune system and elicit a protective antitumor response even when chronic inflammation , the initiator of malignancy , is prevalent in tissues , modulate the tumor microenvironment and circumvent the dominant tolerance to tumor antigens to induce a tumor rejection .", "overcoming the immunosuppressive microenvironment with cancer vaccines in nsclc , became an active immunotherapeutic intervention in an established malignant disease , which structurally imply the precise antigen selection , supported by at least two immune potentiating platforms ; the correct immune system activation , and the accurate therapeutic maneuvers , aimed for induce the lowest rate of tumor progression and increase the host survival with ethical acceptable quality of life . ", "hopeful , certain immunomodulating agents , including a monoclonal antibody directed against ctla-4 ( ipilimumab ) , pd-1 and pd - l1 blocking antibodies and talactoferrin , a dendritic cell activator , are just in scrutiny and have shown clinical activity in nsclc patients , highlighting the space of the immunotherapy as valid choice in this malignant condition [ 47 , 48 ] .", "the well - known tumor associated antigens mage , muc1 , ngc gm3ganglioside , cea and ny - eso-1 are overtly in the resultant malignant phenotype of nsclc , supporting an initial generation of tumor antigen - specific cancer vaccines presently under pressure for achieve highest level of clinical evidences .", "vaccines as mage - a3 , muc1 ( emepepimut - s , tg4010 ) and racotumumab ( anti - idiotipic mab ngc gm3 specific ) are currently ongoing phase iii proof of efficacy clinical trials , whilst cea based cancer vaccines are just in ongoing phase i or ii studies . other vaccines as ny - eso- 1 plasmid dna cancer vaccine have been explored in a phase i clinical trial .", "cell - based cancer vaccines ( cdx-1401 , l - vax , dribble , mrc-5 , tergenpumatucel - l , and belagenpumatucel - l ) became as a second generation of active immunization approaches that becoming the scrutiny for clinically applicability in nsclc and belagenpumatucel - l or lucanix ( allogeneic cells tgf - b2 antisense gene modification ) seem to be the more advanced competitors with ongoing phase iii clinical trials .", "as shown , the strategies improved for overcome the tumor immunosuppressive machinery in lung cancer vaccines are supported on the primary platform comprising peptides ( ras peptides , epemimut - s , tg4010 from muc-1 peptides and mage-12 peptides ) , proteins ( cimavax egf and ny - eso-1 ) , neo - antigen anti - idiotypes ( racotumumab ) , recombinant vectors ( mrc-5 ) , whole tumor cells ( dribble , l - vax , hyperacute , tergenpumatucel - l and belagenpumatucel - l ) or antigen presenting cells ( p53 , cyclin b1 , cdx-1401 ) [ 39 , 52 , 53 ] .", "the above primary structure of the immunization strategy is commonly linked or supported by an adequate secondary platform for the correct apc presentation and immune potentiation .", "it is the case of the autologous egf antigen coupled with non - self - molecule from neisseria meningitides , emulsified with an oil adjuvant ( cimavax egf ) , or the case of a fusion protein between the ny - eso-1 tumor antigen and a fully human monoclonal antibody specific for the dendritic cell receptor dec-205 ( cdx-1401 vaccine ) .", "other examples are the emepepimut - s vaccine that targets the exposed core peptide of muc1 by a liposomal formulation and racotumumab anti - idiotipic vaccine , which act as a surrogate of neugm3 generating specific autologous antibodies [ 54 , 55 ] . once selected the right antigen and the most adequate platform for immune potentiating , those primary and secondary platforms , are not in condition to be winners in the battle field of the immunosuppressive microenvironment induced by the malignant machinery , and should be additionally inserted in a context of therapeutic maneuvers such as the priming and boosting strategies , the pre - treatment with a single low dose of cyclophosphamide ( cimavax egf , epemimut - s ) , the chemotherapy induced lymphopenia and combinations with gm - csf , il-2 , resiquimod and bcg , among others , for improve a tertiary platform for immune potentiation ( table 1 ) [ 56 , 57 ] .", "nsclc vaccines should be able to elicit both potent cd4 + and cd8 + t - cells to achieve an antitumor response .", "the most useful reaction of the immune system against tumors is to kill the abnormal cells using ctls .", "but a broad spectrum of mechanism of action , from cellular to humoral effector , is present in more advances vaccines candidates , mainly depending on targeted antigen .", "success of cimavax egf vaccine , which is oriented to castrate the autologous egf from the serum to subtract this growth factor from the tumor microenvironment , is obtained by inducing high titers of sequestering antibodies . in this case", "the cellular response seems to be not relevant for the vaccine functionality . using cimavax egf", "higher anti - egf antibodies titers correlate with lowest serum egf and longer patient s survival .", "mage - a3-vaccine , based on a fusion protein containing the mage - a3 antigen and protein d , use a second - generation adjuvant system as02b , a saponin based adjuvant containing monophosphoryl lipid a. this vaccine induces both humoral and cellular immune response , but appears to be effective in patients expressing hla - a1 allele , only present in the 20% of patients , by the induction of specific ctl response .", "the use of the adjuvant have been proved to be a prerequisite for cellular response to l - blp25 vaccine ( stimuvax ) , based on muc1 lipopeptide ( 25 aminoacids ) , use a liposomal delivery system and monophosphoryl lipid a , added to enhance the immune response .", "vaccination is assumed to provoke internalization mediated by liposomal structure inducing cd4 + and cd8 + t cells , and also b cell response .", "induced antibodies mediate antibody dependent cellular cytotoxicity ( adcc ) against tumor . up to date", "the clinical insertions of cancer vaccines as therapeutic interventions in nsclc start in the more advanced disease settings in which the malignancy is already established and acquired all its functional capabilities , while the scenario in which the tumor is surgically removed is fewer explored .", "it is important to point out that in this setting the immunosupresive machinery of the malignancy is extremely developed .", "additionally cancer condition is most common in elderly persons in which the immune response could be modified by the natural immunosenescence mechanisms .", "this situation is very controversial and should be carefully considered to design the immunotherapeutic intervention .", "cancer vaccines for nsclc have been focused as a therapeutic option based on the identification of a tumor hallmark and the active immunization with the related molecules that triggers cellular and/or humoral responses that consequently destroy or delay the rate of malignant progression .", "this therapeutic intervention in an established disease state has been aimed to impact into prolonging patients survival with ethically accepted quality of life .", "currently mage - a3 , stimuvax , lucanix , tg4010 , racotumumab and cimavax egf are just in advanced proof of efficacy phase iii clinical trials in nsclc . while mage - a3 improve a trend to benefit the disease free interval in the post - resection of early disease stages pib to ii ( hr 0.73 ; p= 0.109 ) , the advanced disease setting remain the most clinically explored . in this disease setting tg4010 showed a trend to benefit pfs at 6 months with rates of 44% for vaccinated versus 35% for controls ( p= 0.13 ) and in terms of trends for benefit patient s overall survival stimuvax show a hr 0.74 with p = 0.112 , lucanix achieve a clinical response of 15% and better survival in the high - dose group , racotumumab achieve numerical differences of 9.93 months for vaccinated group vs. 4.53 months for control group and cimavax egf achieve 3.5 month of benefit for the subset of 60 years old and younger patients ( table 2 ) . despite first , second and emerging third line of onco - specific treatments", "the life expectancy for nsclc patients diagnosed at advanced stages is surrounding the 12 months of median survival and in facts the today real circumstances are extremely demanding for the success inclusion of cancer vaccines as therapeutic choice in the clinical scenario . as corollary of the described development , an increasing interest in utilizing the multiple new agents that show activity in nsclc and have a tolerable side - effect profile , to maintain response to initial therapy after treatment with platinum - based doublets is rising .", "however considerable controversy , maintenance therapy has been considered a suitable treatment alternative and becoming a plausible therapeutic space for cancer vaccines , in the two proposed modalities ; continuous maintenance or switch maintenance . in both cases the goal is to delay the second line therapy without treatment holidays for the malignancy .", "the incorporation of the active vaccination in the current standard of care based generally in chemotherapeutic agents is yet poorly exploit in the clinical setting .", "there is accumulating evidence that conventional therapies may profit the antitumor effects from the participation of the immune system by several mechanisms such as the immunogenic tumour - cell death . in a phase", "i / ii clinical trial , 20 patients diagnosed with advanced nsclc , received a schedule of two vaccinations of cimavax egf before the platinum based first line chemotherapy and subsequent monthly vaccinations were improved once concluded the chemotherapy cycles .", "this small study evidenced that anti - egf antibody titers were not affected and correlate with a reduction of serum egf concentration , while the patients overall survival also correlate with the highest anti - egf antibody titers without modification of the safety profile .", "the therapeutic vaccine tg4010 was added to nsclc first - line chemotherapy and used until documentation of progression disease in a phase iib , open - label , controlled trial , recruiting 148 chemotherapy - naive patients with diseases stages iiib or iv .", "this study show that 43.2% of patients who receiving the combination therapy were progression free , compared with 35.1% of the chemotherapy - alone group at 6 months of follow up .", "these results assessed the addition of a therapeutic cancer vaccine to first - line chemotherapy in advanced nsclc as another choice for insertion in the current standard of care .", "the emergency during the last five years of a methodological framework to enhance the clinical success of cancer immunotherapy is strong - minded the therapeutic insertion of cancer vaccines in nsclc . from this perspective , the kinetics of active immunizations is understood as a sequential cascade of clinical endpoints .", "starting by the initiation of vaccinations that leads to activation of the immune system which results in a cellular immune response developed in days to weeks ; followed by the antitumor response that becomes evident weeks to months after first immunization and finalizing with the consequential impact on patients overall survival after several months of treatment .", "the immunotherapists and oncologists communities are recognizing this cascade of clinical endpoints as a backbone for active immunization assessment and each one of these three clinical ends points implies their own specific challenges and opportunities .", "connected with the circumstances of the currently clinical scenario , the exactly position of cancer vaccines in the advanced nsclc setting is foremost challenging the current criteria for clinical evaluation of tumor response , because the possibility of pseudo- progression induced by immunotherapy , due to the elicited antitumor cd4 + and cd8 + t - cells infiltration , justifying the forthcoming immune related criteria of tumor progression and therefore the immunization beyond disease progression .", "cimavax egf have been explored after first line therapy and administered beyond diseases progression in a referred phase ii , open , controlled trial in 80 patients after first line platinum based treatment with promising results .", "the metanalysis of 58 patients who were vaccinated monthly for more than one or two years evidenced that long term vaccination was feasible and safe , without evidences of cumulative toxicity or immune response exhaustion .", "the results of the planned interim analysis with the first 226 patients recruited in the ongoing randomized phase iii trial , vaccinating the patients who respond to first line chemotherapy with at least stable disease , in the modality of switch maintenance , beyond progression disease and until change their performance status as per physician criteria , confirm previous results in benefit the patient s overall survival by intent to treat analysis ( manuscript in preparation ) .", "so , chronic vaccination improved beyond progression disease in advanced stages ; seems to be one of the transitions points for positioning cancer vaccines in the current and imminent nsclc therapeutic scenario . regarding", "the insertion of cancer vaccines in the standard of care for advanced nsclc is important to highlight the appearance of an algorithm proposed for choose a first line chemotherapy alternative . in this algorithm", "the presence of positive egfr mutations or alk fusions , the clinical conditions of the patients ( performance status ) and the tumor histological type ( non squamous or squamous ) , are driving the selection of erlotinib , crizotinib , bevacizumab combined or not with platinum / pemetrexed , platinum / gemcitabine doublets or single- agent chemotherapy .", "besides , another transition points for the inclusion of cancer vaccines in the current nsclc therapeutic context is the opportunity to personalize this immunotherapy , based on biomolecules that will be predictive of the potential efficacy for a predetermined subset of patients . up to now during active immunization some patients become not responders due to their specific pattern of immune response .", "for instance , the case of cimavax egf in which those patients who reach the super responder condition , based on their anti- egf antibodies titers , achieve significant benefit in overall survival , but so far there is not predictable this subset of patients before start immunizations . on the other hand , due to the intrinsic relation with the vaccine mechanism of action , intended to induce a humoral immunity that sequestering the circulating egf and its possible effect on patients outcome , today the correlation between the basal serum egf concentrations and the patients survival after vaccine treatment , is been exhaustive evaluated for", "today is widely accepted that the blockade of a single hallmark is not enough to achieve clinical noteworthy benefit on patients survival . because the complexity and redundancy of the molecular pathways that promote tumour growth and maintenance , the generation of potent anti - tumour immune responses requires the blockade of multiple steps .", "an additional transition points for the successful inclusion of cancer vaccines in the current nsclc clinical scenario is the combination with other targeted therapies or the called combinatorial immunotherapy .", "this concept supports the criteria that optimizing immunotherapy requires treatments that affect multiple aspects of the immune response . a small trial combining autologous granulocyte ", "macrophage colony - stimulating factor ( gm - csf)-secreting tumour cell vaccines with ctla4 blockade found increased inflammatory infiltrates and tumour regression , suggesting that vaccine - induced anti - tumour t cells were present within the tumour but anergized owing to ctla4 co - inhibition . the combinatorial approach was explored in prostate cancer preclinical models using anti - ctla-4 mab in combination with rv - cea - tricom cancer vaccine to augment antigen - specific t - cell responses and an independent prognostic effect of ctla-4 overexpression in radically resected nsclc has been recently described pointing out the potentials of this checkpoint modulation in this malignancy [ 70 , 71 ] . the clinical development of combinatorial approaches has been moved forward after the fda approval of antictla4 therapy , quickly followed by reports of encouraging preliminary clinical data for antipd1 therapies [ 47 , 48 ] .", "immune checkpoints are a tiny fraction of the receptors and ligands that have been defined by genetic and biological analyses to inhibit specific types of immune responses at various levels and its immunomodulatory manipulation may results in enhancing efficacy of therapeutic vaccinations .", "thus , the blockade of immune checkpoints promises broad and diverse opportunities to enhance antitumour immunity with the potential durable clinical responses", ". moreover combinatorial immunotherapies such as bispecific t cell engagers ( bites ) and immunotoxins fc - fusion proteins are entering clinical testing in acute lymphoblastic leukemia and breast cancer patients respectively ; opening a new era for cancer active immunotherapy [ 73 , 74 ] . targeted therapies and immunotherapy offer a number of possible synergies in treatment when used together ; however , these combinations should be more intensive studied and dose optimization , timing and sequence will required rationally design in future clinical trials with the aim of maximize anti - tumour efficacy whereas minimizing every possible immunosuppressive adverse reactions .", "the critical obstacles for cancer immunotherapy have been defined , so , for the medical positioning of the cancer vaccines in the current nsclc clinical scenario is important to understand that this concept is evolving into a complex therapeutic strategy .", "support the use of therapeutic vaccines in nsclc is essential to consider the following : insertion in the standard of care . during past two decades the onco - specific treatments advanced from the unique option of platinum doublets to the routine use of first - line , second - line , and even third - line treatment .", "second ; immunotherapies may induce tumor pseudo- progression , challenging current criteria for clinical evaluation of tumor response and allowing the forthcoming immune related criteria of tumor progression .", "personalization based on predictors of response . targeted drugs such as erlotinib and crizotinib are inserted in the therapeutic context of nsclc using specific biomolecules indicatives of therapeutic efficacy for selected subset of patients .", "the blockade of a single hallmark is not enough to achieve clinical noteworthy benefit on patients survival .", "combinations may achieve a synergism , strength of antitumor effects and also avoid or overcome the tumor mechanism of resistance .", " = antigen presenting cells = eastern cooperative oncology group = epidermal growth factor = epidermal growth factor receptor = non- small cell lung cancer = metastatic castration resistant prostate cancer = p64k carrier protein from neisseria meningitides = performance status = randomised controlled trial = tirosine kinases inhibitors" ]

[ "<S> this review is aimed to focus on nsclc as an emerging and promising model for active immunotherapy and the challenges for its inclusion in the current clinical scenario . </S>", "<S> cancer vaccines for nsclc have been focused as a therapeutic option based on the identification of a tumor hallmark and the active immunization with the related molecules that triggers cellular and/or humoral responses that consequently destroy or delay the rate of malignant progression . </S>", "<S> this therapeutic intervention in an established disease state has been aimed to impact into prolonging patients survival with ethically accepted quality of life . </S>", "<S> understanding of relationship between structure and function in cancer vaccines is essential to interpret their opportunities to impact into prolonging survival and increasing quality of life in cancer patients . </S>", "<S> it is widely accepted that the failure of the cancer vaccines in the nsclc scenario is related with its introduction in the advanced disease stages and poor performance status of the patients due to the combination of the tumor induced immunosuppression with the immune senescence . despite first </S>", "<S> , second and emerging third line of onco - specific treatments the life expectancy for nsclc patients diagnosed at advanced stages is surrounding the 12 months of median survival and in facts the today real circumstances are extremely demanding for the success inclusion of cancer vaccines as therapeutic choice in the clinical scenario . </S>", "<S> the kinetics of the active immunizations encompasses a sequential cascade of clinical endpoints : starting by the activation of the immune system , followed by the antitumor response and finalizing with the consequential impact on patients overall survival . today </S>", "<S> this cascade of clinical endpoints is the backbone for active immunization assessment and moreover the concept of cancer vaccines , applied in the nsclc setting , is just evolving as a complex therapeutic strategy , in which the opportunities for cancer vaccines start from the selection of the target cancer hallmark , followed by the vaccine formulation and its platforms for immune potentiating , also cover the successful insertion in the standard of care , the chronic administration beyond progression disease , the personalization based on predictors of response and the potential combination with other targeted therapies . </S>" ]

[ "INTRODUCTION", "NSCLC BIOLOGY AND TARGET ANTIGENS FOR CANCER VACCINES", "CANCER VACCINES DESIGNED TO FACE UP THE IMMUNOSUPPRESSIVE NSCLC MICROENVIRONMENT", "CANCER VACCINES MEDICAL POSITIONING IN THE CURRENT NSCLC CLINICAL SCENARIO", "CONFLICT OF INTEREST", "ABBREVIATIONS" ]

[ [ "cancer vaccine concept have been encompasses a broad spectrum of platforms intended to trigger or increase an adaptive immune response against a malignant tumor .", "thus cancer vaccines are aimed as active immunotherapeutic interventions mainly in an established disease state in which the malignancy is expressing all its functional capabilities or hallmarks .", "understanding of relationship between structure and function in cancer vaccines is essential to interpret their opportunities to impact into prolonging survival and increasing quality of life in cancer patients .", "the structure of cancer vaccines is referred to a tumor associated ( or specific ) antigen shaped as peptides , recombinant vectors , whole tumor cells or antigen presenting cells linked or supported by an adequate immunopotentiating platform , which should be inserted in a context of therapeutic maneuvers .", "the expected function of cancer vaccines is basically to circumvent the immunologic tolerance in the tumor microenvironment inducing a lowest rate of tumor progression and increasing the host survival .", "so , cancer vaccines function is expected to be relevant at all levels of biological organization of the malignancy host , from the tumor microenvironment up to the whole organism .", "then , to success with active immunotherapy strategy two items need to be considered : the role of target antigens in tumor biology ( cancer hallmark related ) and the immunogenic capacity of vaccine composition .", "once selected the right antigen and the most adequate platform for immune potentiating , the battle field for cancer vaccines is the immunosuppressive microenvironment induced by the malignant machinery ; the corner between structure and function in cancer vaccines is accurately the interaction between the tumors hallmarks and the host immune response .", "prostate cancer has been proposed as a prototype for vaccine therapy scenario . besides the increasing spectrum of therapeutic tools for the disease control at the advanced disease stage , meaning new androgen deprivation modalities such as abiraterone , advanced chemotherapeutic agents such as cabazitaxel and small tyrosine kinase inhibitors such as cabozantinib , a cancer vaccine appear in the prostate carcinoma treatments alternative .", "evidences supporting that sipuleucel - t , a vaccine based on dendritic cells pulsed with prostatic acid phosphatase linked to granulocyte ", "macrophage colony - stimulating factor , prolonged survival in minimal or non - symptomatic mcrpc patients and its fda approval ( april , 2010 ) , constitute the proof of concept of cancer vaccines application in the scenario of a solid tumor with long term disease course , characterized by well described tumor associated antigens , a defined biomarker ( serum psa ) and a good physicians tool to follow up the patients therapeutic response ( halabi nomogram ) .", "non- small cell lung cancer ( nsclc ) is the first cause of cancer associated death worldwide , the 60- 80% of patients are diagnosed at the advanced stages ( iiib / iv ) in which their life expectancy is not too far from 12 months median survival even second and emerging third line of onco - specific treatments . in this particular case the active immune intervention using cancer vaccines is strongly challenged by the tumor induced immunologic tolerance .", "there are several evidences of a marked infiltration of different types of immune cells in the nsclc microenvironment , and the distribution , tissue localization , and cell types are significantly associated with progression and patient s survival .", "the clinical relevance of the microenvironmental immunological milieu in this malignacy is highlighted by the facts that higher foxp3+/cd8 + ratio in tumor sites is an independent factor for poor response to platinum - based neoadjuvant chemotherapy in a setting of advanced stages patients .", "this review is aimed to focus on nsclc as an emerging and promising model for active immunotherapy and the challenges for its inclusion in the current clinical scenario , considering the increasing body of evidences of the interaction between inflammatory cells and tumor cells , facilitating the lung carcinogenesis and tumor hallmarks expression ( angiogenesis and tumor cell migration among others ) , in which antigen - specific antitumor responses are overtly present . in this context", ", the clinical relevance of cancer vaccines should be predicted pointing out the emerging determinants of its structure and function relationships in the particular situation of nsclc malignancy hallmarks .", "the kinetics of the active immunizations encompasses a sequential cascade of clinical endpoints : starting by the activation of the immune system , followed by the antitumor response and finalizing with the consequential impact on patients overall survival .", "today this cascade of clinical endpoints is the backbone for active immunization assessment and moreover the concept of cancer vaccines , applied in the nsclc setting , is just evolving as a complex therapeutic strategy , in which the opportunities for cancer vaccines start from the selection of the target cancer hallmark , followed by the vaccine formulation and its platforms for immune potentiating , also encompass the successful insertion in the standard of care , the chronic administration beyond progression disease , the personalization based on predictors of response and the potential combination with other targeted therapies ." ], [ "as an inherently condition of all malignant processes , nsclc carcinogenesis is resulting from the interaction between the epithelial mucosa genome and the lung microenvironment . in this context , the intrinsic cell capacity for dna repair , the nutrients accessibility , the angiogenesis state and the hypoxia levels , seems to be the common context of selective pressures for the cascade of mutations during tumor progression and coming out of the malignant cells phenotypes .", "particularly , the continuing exposure of dna to metabolically activated carcinogens , throughout the exposition to air contaminants or cigarette- smoke , resulting in the formation of dna adducts and consequential genetic instability .", "the rate of dna injure , taking place day after day over many years , is fully consistent with multiple genetic changes in lung cancer and is associated with increasingly severe histopathological phenotypes .", "the importance of the target antigens to tumor biology is considered relevant for the success of active immunotherapy .", "indeed , the selection of vaccine antigens must be focused mainly on those molecules significantly associated with the hallmarks of cancer , also called oncoantigens ; those specific or tumor associated antigens that are highly correlated with the phenotypic consolidation of the malignancy and tumor progression [ 20 , 21 ] .", "the egfr network induce a broad range of biological effects by controlling the cellular outcome , through a multiple ligands activation - dependent regulatory loops , which fall into time dependants temporal domains ; early loops comprise protein modifications mechanisms , while delayed loops involve transcription regulation mechanisms ; both became aberrant and without feedback control in malignant cells . during development and progression of lung neoplasms", "the egfr is overexpressed in the following proportions : 62% of all nsclc tumors , 89% of squamous cell tumors , 41% of adenocarcinomas and 80% of bronchoalveolar tumors . in the case of adenocarcinoma in the peripheral compartment of the lung ( terminal bronquiole and alveoli ) in never smokers ,", "the egfr overexpression is associated with a lower methylation index of the p53 cancer suppressor gene through high proportion of transition mutations ( ie , purine for purine or pyrimidine for pyrimidine ) .", "the degree of egfr expression has been reported as a predictive factor of response to biological therapy in nsclc patients .", "there are two classes of anti - egfr agents that have shown clinical activity in nsclc .", "these are either monoclonal antibodies directed at the extracellular domain of the egfr and inhibiting the binding of natural ligands to the receptor , or low molecular weight tyrosine kinase inhibitors ( tkis ) that inhibit the tyrosine kinase activity of egfr , generally by competing reversibly with atp for the atp binding site .", "targeting the egfr with a cancer vaccine is a newly strategy currently in clinical scrutiny using the active immune deprivation of egf ligand as important tumor growth factor ( cimavax egf ) [ 27 , 28 ] .", "muc1 is over - expressed in almost all nsclc patients , being associated with bad prognosis .", "the role in tumor biology of the nglycosylated muc1c ( muc1 c - terminal transmembrane subunit ) seems to be associated to a functionally important extracellular bridge mediated by galectin 3 between this recognized mucin antigen and tyrosine kinases receptors , such as the egfr .", "muc1 looks to be related not only with promoting cell growth and survival but also with t cell proliferation inhibition and accordingly , immunosuppression . in this context", ", other molecules became aberrant expressed in the cascade of events associated with the sustained proliferation and its negative regulation , apoptosis evasion and the limitless replicative potential .", "tumor suppressor gene p53 receives stress signals from excessive dna damage and suboptimal nutrients triggering dna repairing mechanism or apoptosis .", "loss of p53 function by mutations are frequent events in human oncogenesis , which leads to persistent aberrant expression in about 60% of nsclc , correlated with poor cht response and lowest overall sv of the patients .", "this conduced to exploration of a mutant p53 peptide pulsed dendritic cell vaccine in a phase ii clinical trial with stage iii nsclc patients .", "the specialized dna polymerase , named telomerase , which adds telomere repeat segments to the end of telomeric dna is nearly absent in nonimmortalized cells but expressed at functionally significant levels in 90% of human tumors .", "telomerase- based vaccine gv1001 is starting the clinical scrutiny , supported also by the facts that its presence in sputum samples has been identified as a potential specific marker for early detection of lung cancer .", "the activation of cdk1-cyclin b1 triggers the mitosis progression and its translocation from the cytoplasm to the nucleus .", "positive feedback loops regulate its biological activity and spatial localization , ensuring a rapid , complete , robust , and irreversible transition from interphase to mitosis .", "there is reported the aberrant expression of these regulators of the g2 m cell cycle checkpoint in many neoplasms , including nsclc and significant association between increased cyclin b1 expression and reduced patients survival in stage i/ ii nsclc has been described . furthermore , cyclin b1 peptide - pulsed autologous dendritic cell vaccine is early of clinical evaluation in resectable nsclc patients .", "the ras genes , including h - ras , k - ras and n - ras , encode a family of proteins regulating cell growth , differentiation and apoptosis .", "mutations in the k - ras gene , mainly in codons 12 and 13 , have been found in 20 - 30% of nsclc tumor samples and occur most commonly , but not exclusively , in adenocarcinoma histology and in heavy smokers .", "active immune strategies aimed at interfering with the ras - raf - mek pathway in nsclc are also incipient exploring the use of a ras peptide based vaccine .", "the continuous cascade of molecular events starting from at least a cell clone and characterized by unregulated cellular proliferation and clonal heterogeneity , is resulting in a characteristic nsclc malignant phenotype .", "based only on histology nsclc comprises i.e. for adenocarcinoma , squamous - cell carcinoma and large - cell carcinoma subsets , while , based on the molecular subsets expressed in the resultant malignant phenotypes , nsclc drive a broad spectrum of diseases subsets : egfr , her2 , kras , alk , braf , pik3ca , akt1 , ros1 , nras , map2k1 and the recently identified kif5b - ret fusion subset , each one with dissimilar pattern of incidence in a population .", "the personalization of cancer vaccines therapy is a pending chapter in this background , and the selection of the right vaccine for the right patient is becoming as challenge .", "the design of cancer vaccines attempt to harness the specificity and resistance potentials of the human immune system . with the aim to stimulate the immune system to recognize , attack and destroy tumor cells", "the personalization treatment is intrinsic for mhc context dependent antigens vaccines ( peptides based vaccines ) and for cell based cancer vaccines . in the context of the adaptive immune response", "one foremost challenge for clinical researchers is to classify the patient s population according the pre - existing immune response in potential responders or non- responders to the target antigen . ", "it is widely accepted that the failure of the cancer vaccines in the nsclc scenario is related with its introduction in the advanced disease stages and poor performance status of the patients due to the combination of the tumor induced immunosuppression with the immune senescence .", "the facts that nsclc occur in subjects who have never smoked and have no evident cause for chronic inflammatory changes in the lung parenchyma , suggest that the interactions between the broncho - alveolar compartment and the in situ immune regulators are involved in facilitating tumor occurrence , growth , and spread .", "it is progressively ostensible that crosstalk between cancer cells and cells of tumor stroma is involved in the acquired capability for invasive growth and metastases .", "tumor stroma has been described as pivotal in the established hostile immune environment through the secretion of immunosuppressive factors and the recruitment of suppressive immune cells of myeloid and lymphoid origin , while the tumor cells directly contributing to immune resistance through the secretion of immunosuppressive mediators , such as transforming growth factor ( tgf ) or natural killer ( nk ) cell receptor decoys , and through the expression of ligands for immune checkpoint ( or co - inhibitory ) receptors , such as programmed cell death ligand 1 ( pdl1 ) .", "the distribution , tissue localization , and cell types infiltrating lung tumor microenvironment are significantly associated with the disease progression and patient s survival .", "for example dendritic cell defects increases immature forms that may play an immunosuppressive role and facilitate cancer migration , invasion , and epithelial - to - mesenchymal transition ( schneider et al . 2011 ) .", "also , genetic variations in the transforming growth factor - beta pathway are considered as predictors of survival in advanced non - small cell lung cancer and higher foxp3+/cd8 + ratio in tumor sites is described as an independent factor for poor response to platinum - based neoadjuvant chemotherapy in a setting of advanced stages patients . summarizing ,", "a variety of cellular immune abnormalities , antigen processing and presentation machinery defects , cytokine alterations and also individual conditions , increase the challenge for nsclc therapeutic vaccines .", "vaccines candidates should activate the immune system and elicit a protective antitumor response even when chronic inflammation , the initiator of malignancy , is prevalent in tissues , modulate the tumor microenvironment and circumvent the dominant tolerance to tumor antigens to induce a tumor rejection ." ], [ "overcoming the immunosuppressive microenvironment with cancer vaccines in nsclc , became an active immunotherapeutic intervention in an established malignant disease , which structurally imply the precise antigen selection , supported by at least two immune potentiating platforms ; the correct immune system activation , and the accurate therapeutic maneuvers , aimed for induce the lowest rate of tumor progression and increase the host survival with ethical acceptable quality of life . ", "hopeful , certain immunomodulating agents , including a monoclonal antibody directed against ctla-4 ( ipilimumab ) , pd-1 and pd - l1 blocking antibodies and talactoferrin , a dendritic cell activator , are just in scrutiny and have shown clinical activity in nsclc patients , highlighting the space of the immunotherapy as valid choice in this malignant condition [ 47 , 48 ] .", "the well - known tumor associated antigens mage , muc1 , ngc gm3ganglioside , cea and ny - eso-1 are overtly in the resultant malignant phenotype of nsclc , supporting an initial generation of tumor antigen - specific cancer vaccines presently under pressure for achieve highest level of clinical evidences .", "vaccines as mage - a3 , muc1 ( emepepimut - s , tg4010 ) and racotumumab ( anti - idiotipic mab ngc gm3 specific ) are currently ongoing phase iii proof of efficacy clinical trials , whilst cea based cancer vaccines are just in ongoing phase i or ii studies . other vaccines as ny - eso- 1 plasmid dna cancer vaccine have been explored in a phase i clinical trial .", "cell - based cancer vaccines ( cdx-1401 , l - vax , dribble , mrc-5 , tergenpumatucel - l , and belagenpumatucel - l ) became as a second generation of active immunization approaches that becoming the scrutiny for clinically applicability in nsclc and belagenpumatucel - l or lucanix ( allogeneic cells tgf - b2 antisense gene modification ) seem to be the more advanced competitors with ongoing phase iii clinical trials .", "as shown , the strategies improved for overcome the tumor immunosuppressive machinery in lung cancer vaccines are supported on the primary platform comprising peptides ( ras peptides , epemimut - s , tg4010 from muc-1 peptides and mage-12 peptides ) , proteins ( cimavax egf and ny - eso-1 ) , neo - antigen anti - idiotypes ( racotumumab ) , recombinant vectors ( mrc-5 ) , whole tumor cells ( dribble , l - vax , hyperacute , tergenpumatucel - l and belagenpumatucel - l ) or antigen presenting cells ( p53 , cyclin b1 , cdx-1401 ) [ 39 , 52 , 53 ] .", "the above primary structure of the immunization strategy is commonly linked or supported by an adequate secondary platform for the correct apc presentation and immune potentiation .", "it is the case of the autologous egf antigen coupled with non - self - molecule from neisseria meningitides , emulsified with an oil adjuvant ( cimavax egf ) , or the case of a fusion protein between the ny - eso-1 tumor antigen and a fully human monoclonal antibody specific for the dendritic cell receptor dec-205 ( cdx-1401 vaccine ) .", "other examples are the emepepimut - s vaccine that targets the exposed core peptide of muc1 by a liposomal formulation and racotumumab anti - idiotipic vaccine , which act as a surrogate of neugm3 generating specific autologous antibodies [ 54 , 55 ] . once selected the right antigen and the most adequate platform for immune potentiating , those primary and secondary platforms , are not in condition to be winners in the battle field of the immunosuppressive microenvironment induced by the malignant machinery , and should be additionally inserted in a context of therapeutic maneuvers such as the priming and boosting strategies , the pre - treatment with a single low dose of cyclophosphamide ( cimavax egf , epemimut - s ) , the chemotherapy induced lymphopenia and combinations with gm - csf , il-2 , resiquimod and bcg , among others , for improve a tertiary platform for immune potentiation ( table 1 ) [ 56 , 57 ] .", "nsclc vaccines should be able to elicit both potent cd4 + and cd8 + t - cells to achieve an antitumor response .", "the most useful reaction of the immune system against tumors is to kill the abnormal cells using ctls .", "but a broad spectrum of mechanism of action , from cellular to humoral effector , is present in more advances vaccines candidates , mainly depending on targeted antigen .", "success of cimavax egf vaccine , which is oriented to castrate the autologous egf from the serum to subtract this growth factor from the tumor microenvironment , is obtained by inducing high titers of sequestering antibodies . in this case", "the cellular response seems to be not relevant for the vaccine functionality . using cimavax egf", "higher anti - egf antibodies titers correlate with lowest serum egf and longer patient s survival .", "mage - a3-vaccine , based on a fusion protein containing the mage - a3 antigen and protein d , use a second - generation adjuvant system as02b , a saponin based adjuvant containing monophosphoryl lipid a. this vaccine induces both humoral and cellular immune response , but appears to be effective in patients expressing hla - a1 allele , only present in the 20% of patients , by the induction of specific ctl response .", "the use of the adjuvant have been proved to be a prerequisite for cellular response to l - blp25 vaccine ( stimuvax ) , based on muc1 lipopeptide ( 25 aminoacids ) , use a liposomal delivery system and monophosphoryl lipid a , added to enhance the immune response .", "vaccination is assumed to provoke internalization mediated by liposomal structure inducing cd4 + and cd8 + t cells , and also b cell response .", "induced antibodies mediate antibody dependent cellular cytotoxicity ( adcc ) against tumor . up to date", "the clinical insertions of cancer vaccines as therapeutic interventions in nsclc start in the more advanced disease settings in which the malignancy is already established and acquired all its functional capabilities , while the scenario in which the tumor is surgically removed is fewer explored .", "it is important to point out that in this setting the immunosupresive machinery of the malignancy is extremely developed .", "additionally cancer condition is most common in elderly persons in which the immune response could be modified by the natural immunosenescence mechanisms .", "this situation is very controversial and should be carefully considered to design the immunotherapeutic intervention ." ], [ "cancer vaccines for nsclc have been focused as a therapeutic option based on the identification of a tumor hallmark and the active immunization with the related molecules that triggers cellular and/or humoral responses that consequently destroy or delay the rate of malignant progression .", "this therapeutic intervention in an established disease state has been aimed to impact into prolonging patients survival with ethically accepted quality of life .", "currently mage - a3 , stimuvax , lucanix , tg4010 , racotumumab and cimavax egf are just in advanced proof of efficacy phase iii clinical trials in nsclc . while mage - a3 improve a trend to benefit the disease free interval in the post - resection of early disease stages pib to ii ( hr 0.73 ; p= 0.109 ) , the advanced disease setting remain the most clinically explored . in this disease setting tg4010 showed a trend to benefit pfs at 6 months with rates of 44% for vaccinated versus 35% for controls ( p= 0.13 ) and in terms of trends for benefit patient s overall survival stimuvax show a hr 0.74 with p = 0.112 , lucanix achieve a clinical response of 15% and better survival in the high - dose group , racotumumab achieve numerical differences of 9.93 months for vaccinated group vs. 4.53 months for control group and cimavax egf achieve 3.5 month of benefit for the subset of 60 years old and younger patients ( table 2 ) . despite first , second and emerging third line of onco - specific treatments", "the life expectancy for nsclc patients diagnosed at advanced stages is surrounding the 12 months of median survival and in facts the today real circumstances are extremely demanding for the success inclusion of cancer vaccines as therapeutic choice in the clinical scenario . as corollary of the described development , an increasing interest in utilizing the multiple new agents that show activity in nsclc and have a tolerable side - effect profile , to maintain response to initial therapy after treatment with platinum - based doublets is rising .", "however considerable controversy , maintenance therapy has been considered a suitable treatment alternative and becoming a plausible therapeutic space for cancer vaccines , in the two proposed modalities ; continuous maintenance or switch maintenance . in both cases the goal is to delay the second line therapy without treatment holidays for the malignancy .", "the incorporation of the active vaccination in the current standard of care based generally in chemotherapeutic agents is yet poorly exploit in the clinical setting .", "there is accumulating evidence that conventional therapies may profit the antitumor effects from the participation of the immune system by several mechanisms such as the immunogenic tumour - cell death . in a phase", "i / ii clinical trial , 20 patients diagnosed with advanced nsclc , received a schedule of two vaccinations of cimavax egf before the platinum based first line chemotherapy and subsequent monthly vaccinations were improved once concluded the chemotherapy cycles .", "this small study evidenced that anti - egf antibody titers were not affected and correlate with a reduction of serum egf concentration , while the patients overall survival also correlate with the highest anti - egf antibody titers without modification of the safety profile .", "the therapeutic vaccine tg4010 was added to nsclc first - line chemotherapy and used until documentation of progression disease in a phase iib , open - label , controlled trial , recruiting 148 chemotherapy - naive patients with diseases stages iiib or iv .", "this study show that 43.2% of patients who receiving the combination therapy were progression free , compared with 35.1% of the chemotherapy - alone group at 6 months of follow up .", "these results assessed the addition of a therapeutic cancer vaccine to first - line chemotherapy in advanced nsclc as another choice for insertion in the current standard of care .", "the emergency during the last five years of a methodological framework to enhance the clinical success of cancer immunotherapy is strong - minded the therapeutic insertion of cancer vaccines in nsclc . from this perspective , the kinetics of active immunizations is understood as a sequential cascade of clinical endpoints .", "starting by the initiation of vaccinations that leads to activation of the immune system which results in a cellular immune response developed in days to weeks ; followed by the antitumor response that becomes evident weeks to months after first immunization and finalizing with the consequential impact on patients overall survival after several months of treatment .", "the immunotherapists and oncologists communities are recognizing this cascade of clinical endpoints as a backbone for active immunization assessment and each one of these three clinical ends points implies their own specific challenges and opportunities .", "connected with the circumstances of the currently clinical scenario , the exactly position of cancer vaccines in the advanced nsclc setting is foremost challenging the current criteria for clinical evaluation of tumor response , because the possibility of pseudo- progression induced by immunotherapy , due to the elicited antitumor cd4 + and cd8 + t - cells infiltration , justifying the forthcoming immune related criteria of tumor progression and therefore the immunization beyond disease progression .", "cimavax egf have been explored after first line therapy and administered beyond diseases progression in a referred phase ii , open , controlled trial in 80 patients after first line platinum based treatment with promising results .", "the metanalysis of 58 patients who were vaccinated monthly for more than one or two years evidenced that long term vaccination was feasible and safe , without evidences of cumulative toxicity or immune response exhaustion .", "the results of the planned interim analysis with the first 226 patients recruited in the ongoing randomized phase iii trial , vaccinating the patients who respond to first line chemotherapy with at least stable disease , in the modality of switch maintenance , beyond progression disease and until change their performance status as per physician criteria , confirm previous results in benefit the patient s overall survival by intent to treat analysis ( manuscript in preparation ) .", "so , chronic vaccination improved beyond progression disease in advanced stages ; seems to be one of the transitions points for positioning cancer vaccines in the current and imminent nsclc therapeutic scenario . regarding", "the insertion of cancer vaccines in the standard of care for advanced nsclc is important to highlight the appearance of an algorithm proposed for choose a first line chemotherapy alternative . in this algorithm", "the presence of positive egfr mutations or alk fusions , the clinical conditions of the patients ( performance status ) and the tumor histological type ( non squamous or squamous ) , are driving the selection of erlotinib , crizotinib , bevacizumab combined or not with platinum / pemetrexed , platinum / gemcitabine doublets or single- agent chemotherapy .", "besides , another transition points for the inclusion of cancer vaccines in the current nsclc therapeutic context is the opportunity to personalize this immunotherapy , based on biomolecules that will be predictive of the potential efficacy for a predetermined subset of patients . up to now during active immunization some patients become not responders due to their specific pattern of immune response .", "for instance , the case of cimavax egf in which those patients who reach the super responder condition , based on their anti- egf antibodies titers , achieve significant benefit in overall survival , but so far there is not predictable this subset of patients before start immunizations . on the other hand , due to the intrinsic relation with the vaccine mechanism of action , intended to induce a humoral immunity that sequestering the circulating egf and its possible effect on patients outcome , today the correlation between the basal serum egf concentrations and the patients survival after vaccine treatment , is been exhaustive evaluated for", "today is widely accepted that the blockade of a single hallmark is not enough to achieve clinical noteworthy benefit on patients survival . because the complexity and redundancy of the molecular pathways that promote tumour growth and maintenance , the generation of potent anti - tumour immune responses requires the blockade of multiple steps .", "an additional transition points for the successful inclusion of cancer vaccines in the current nsclc clinical scenario is the combination with other targeted therapies or the called combinatorial immunotherapy .", "this concept supports the criteria that optimizing immunotherapy requires treatments that affect multiple aspects of the immune response . a small trial combining autologous granulocyte ", "macrophage colony - stimulating factor ( gm - csf)-secreting tumour cell vaccines with ctla4 blockade found increased inflammatory infiltrates and tumour regression , suggesting that vaccine - induced anti - tumour t cells were present within the tumour but anergized owing to ctla4 co - inhibition . the combinatorial approach was explored in prostate cancer preclinical models using anti - ctla-4 mab in combination with rv - cea - tricom cancer vaccine to augment antigen - specific t - cell responses and an independent prognostic effect of ctla-4 overexpression in radically resected nsclc has been recently described pointing out the potentials of this checkpoint modulation in this malignancy [ 70 , 71 ] . the clinical development of combinatorial approaches has been moved forward after the fda approval of antictla4 therapy , quickly followed by reports of encouraging preliminary clinical data for antipd1 therapies [ 47 , 48 ] .", "immune checkpoints are a tiny fraction of the receptors and ligands that have been defined by genetic and biological analyses to inhibit specific types of immune responses at various levels and its immunomodulatory manipulation may results in enhancing efficacy of therapeutic vaccinations .", "thus , the blockade of immune checkpoints promises broad and diverse opportunities to enhance antitumour immunity with the potential durable clinical responses", ". moreover combinatorial immunotherapies such as bispecific t cell engagers ( bites ) and immunotoxins fc - fusion proteins are entering clinical testing in acute lymphoblastic leukemia and breast cancer patients respectively ; opening a new era for cancer active immunotherapy [ 73 , 74 ] . targeted therapies and immunotherapy offer a number of possible synergies in treatment when used together ; however , these combinations should be more intensive studied and dose optimization , timing and sequence will required rationally design in future clinical trials with the aim of maximize anti - tumour efficacy whereas minimizing every possible immunosuppressive adverse reactions .", "the critical obstacles for cancer immunotherapy have been defined , so , for the medical positioning of the cancer vaccines in the current nsclc clinical scenario is important to understand that this concept is evolving into a complex therapeutic strategy .", "support the use of therapeutic vaccines in nsclc is essential to consider the following : insertion in the standard of care . during past two decades the onco - specific treatments advanced from the unique option of platinum doublets to the routine use of first - line , second - line , and even third - line treatment .", "second ; immunotherapies may induce tumor pseudo- progression , challenging current criteria for clinical evaluation of tumor response and allowing the forthcoming immune related criteria of tumor progression .", "personalization based on predictors of response . targeted drugs such as erlotinib and crizotinib are inserted in the therapeutic context of nsclc using specific biomolecules indicatives of therapeutic efficacy for selected subset of patients .", "the blockade of a single hallmark is not enough to achieve clinical noteworthy benefit on patients survival .", "combinations may achieve a synergism , strength of antitumor effects and also avoid or overcome the tumor mechanism of resistance ." ], [ "" ], [ " = antigen presenting cells = eastern cooperative oncology group = epidermal growth factor = epidermal growth factor receptor = non- small cell lung cancer = metastatic castration resistant prostate cancer = p64k carrier protein from neisseria meningitides = performance status = randomised controlled trial = tirosine kinases inhibitors" ] ]

[ "the recognition that elevated intracranial pressure ( icp ) is transmitted through the optic nerve and its sheath has been known for many years . this physiological process is the basis for the physical exam finding of papilledema on fundoscopic examination .", "recently , interest has turned to measurement of the optic nerve sheath diameter ( onsd ) through non - invasive imaging technologies to provide surrogate markers for early elevated icp . in this issue of critical care , geeraerts and colleagues ", "present their research correlating magnetic resonance imaging ( mri ) measurements of onsd with icp . in a retrospective review of 38 patients with traumatic brain injury requiring both invasive icp monitoring and mri", ", they found a significant positive relationship between onsd measured by mri and icp ( r = 0.71 ) .", "the best cut - off value to detect an icp > 20 cm h2o based on a receiver operating characteristic curve was found to be onsd = 5.82 mm with a sensitivity of 90% and a specificity of 92% .", "the optic nerve is surrounded by cerebrospinal fluid ( csf ) , which is contiguous with intracranial csf .", "increased icp is transmitted through this subarachnoid space causing distention of the dural optic nerve sheath , especially the retrobulbar segment .", "the optic nerve and its surrounding sheath can be imaged and measured on mri using a fat - suppressed t2-weighted sequence .", "mri has been used to demonstrate increased onsd in idiopathic intracranial hypertension , and interestingly , decreased onsd in csf hypotension .", "the onsd has also been shown on mri to decrease after drainage of subdural hematomas .", "the research presented by geeraerts and colleagues is unique in its comparison of onsd with simultaneous direct measurements of icp through invasive monitoring .", "their findings generally correlate with a growing body of research using bedside ultrasound measurements of onsd to detected elevated icp .", "original research with lumbar intrathecal infusions performed by hansen and helmke demonstrated rapid changes in the onsd with alteration of csf pressures . in emergency department patients with traumatic brain injury , the onsd correlates with signs of elevated icp on computed tomography scans .", "more recently , researches have compared bedside ultrasound measurements of onsd to invasive icp [ 11 - 13 ] .", "while there is some variation in the optimal cut - off value , the correlation between onsd and icp remains consistent . in their current article ,", "geeraets and colleagues provide further evidence of this physiological relationship and an intriguing possibility for non - invasive assessment of icp using mri . the obvious drawbacks to mri include its expense , long acquisition times , need for patient transport , and limited availability .", "however , some research has shown that mri may provide more precise measurements then ultrasound . geeraerts and colleagues used a conventional t2 sequence with relatively large slice thickness and interslice spacing , resulting in an overall feasibility of measuring the onsd in 95% of patients .", "greater accuracy and reliability would be expected in coronal t2 slices with thinner slices . as mri becomes more accessible and faster , non - invasive mri measurements", "may prove to be useful in certain clinical settings and as a potential reference standard for further research . continued research with larger studies is required to confirm the precision and accuracy of mri measurements of onsd , as well as the optimal measurement technique .", "additionally , the time course of onsd distention and reduction needs to be further delineated . currently , non - invasive assessments of icp do not obviate the need for invasive icp monitoring .", "invasive monitoring detects minute to minute variations in icp and , in the case of intraventricular drains , can also be therapeutic .", "however , non - invasive screening tests may be useful in select populations who would not otherwise require invasive monitoring and could undergo mri scans , such as patients with liver failure , meningitis , stroke , and moderate traumatic brain injury . in summary ,", "the study by geeraerts and colleagues adds to a growing body of research demonstrating a correlation between increased onsd and elevated icp . by demonstrating the correlation of mri measurements of the onsd with invasive icp monitoring", ", they illustrate the potential of yet another non - invasive method to screen for elevated icp .", "while this technique will not replace invasive icp monitoring , it may be useful in select patient populations that would not otherwise have invasive monitoring but are at high risk for elevated icp .", "further research is required before we can use measurements of the onsd to predict exact values of icp , but it may be useful as a screening test to estimate the probability of elevated icp .", "csf : cerebrospinal fluid ; icp : intracranial pressure ; mri : magnetic resonance imaging ; onsd : optic nerve sheath diameter ." ]

[ "<S> the current gold standard for the diagnosis of elevated intracranial pressure ( icp ) remains invasive monitoring . </S>", "<S> given that invasive monitoring is not always available or clinically feasible , there is growing interest in non - invasive methods of assessing icp using diagnostic modalities such as ultrasound or magnetic resonance imaging ( mri ) . </S>", "<S> increased icp is transmitted through the cerebrospinal fluid surrounding the optic nerve , causing distention of the optic nerve sheath diameter ( onsd ) . in this issue of critical care , geeraerts and colleagues </S>", "<S> describe a non - invasive method of diagnosing elevated icp using mri to measure the onsd . </S>", "<S> they report a positive correlation between measurements of the onsd on mri and invasive icp measurements . </S>", "<S> if the findings of this study can be replicated in larger populations , this technique may be a useful non - invasive screening test for elevated icp in select populations . </S>" ]

[ "None", "Abbreviations", "Competing interests" ]

[ [ "the recognition that elevated intracranial pressure ( icp ) is transmitted through the optic nerve and its sheath has been known for many years . this physiological process is the basis for the physical exam finding of papilledema on fundoscopic examination .", "recently , interest has turned to measurement of the optic nerve sheath diameter ( onsd ) through non - invasive imaging technologies to provide surrogate markers for early elevated icp . in this issue of critical care , geeraerts and colleagues ", "present their research correlating magnetic resonance imaging ( mri ) measurements of onsd with icp . in a retrospective review of 38 patients with traumatic brain injury requiring both invasive icp monitoring and mri", ", they found a significant positive relationship between onsd measured by mri and icp ( r = 0.71 ) .", "the best cut - off value to detect an icp > 20 cm h2o based on a receiver operating characteristic curve was found to be onsd = 5.82 mm with a sensitivity of 90% and a specificity of 92% .", "the optic nerve is surrounded by cerebrospinal fluid ( csf ) , which is contiguous with intracranial csf .", "increased icp is transmitted through this subarachnoid space causing distention of the dural optic nerve sheath , especially the retrobulbar segment .", "the optic nerve and its surrounding sheath can be imaged and measured on mri using a fat - suppressed t2-weighted sequence .", "mri has been used to demonstrate increased onsd in idiopathic intracranial hypertension , and interestingly , decreased onsd in csf hypotension .", "the onsd has also been shown on mri to decrease after drainage of subdural hematomas .", "the research presented by geeraerts and colleagues is unique in its comparison of onsd with simultaneous direct measurements of icp through invasive monitoring .", "their findings generally correlate with a growing body of research using bedside ultrasound measurements of onsd to detected elevated icp .", "original research with lumbar intrathecal infusions performed by hansen and helmke demonstrated rapid changes in the onsd with alteration of csf pressures . in emergency department patients with traumatic brain injury , the onsd correlates with signs of elevated icp on computed tomography scans .", "more recently , researches have compared bedside ultrasound measurements of onsd to invasive icp [ 11 - 13 ] .", "while there is some variation in the optimal cut - off value , the correlation between onsd and icp remains consistent . in their current article ,", "geeraets and colleagues provide further evidence of this physiological relationship and an intriguing possibility for non - invasive assessment of icp using mri . the obvious drawbacks to mri include its expense , long acquisition times , need for patient transport , and limited availability .", "however , some research has shown that mri may provide more precise measurements then ultrasound . geeraerts and colleagues used a conventional t2 sequence with relatively large slice thickness and interslice spacing , resulting in an overall feasibility of measuring the onsd in 95% of patients .", "greater accuracy and reliability would be expected in coronal t2 slices with thinner slices . as mri becomes more accessible and faster , non - invasive mri measurements", "may prove to be useful in certain clinical settings and as a potential reference standard for further research . continued research with larger studies is required to confirm the precision and accuracy of mri measurements of onsd , as well as the optimal measurement technique .", "additionally , the time course of onsd distention and reduction needs to be further delineated . currently , non - invasive assessments of icp do not obviate the need for invasive icp monitoring .", "invasive monitoring detects minute to minute variations in icp and , in the case of intraventricular drains , can also be therapeutic .", "however , non - invasive screening tests may be useful in select populations who would not otherwise require invasive monitoring and could undergo mri scans , such as patients with liver failure , meningitis , stroke , and moderate traumatic brain injury . in summary ,", "the study by geeraerts and colleagues adds to a growing body of research demonstrating a correlation between increased onsd and elevated icp . by demonstrating the correlation of mri measurements of the onsd with invasive icp monitoring", ", they illustrate the potential of yet another non - invasive method to screen for elevated icp .", "while this technique will not replace invasive icp monitoring , it may be useful in select patient populations that would not otherwise have invasive monitoring but are at high risk for elevated icp .", "further research is required before we can use measurements of the onsd to predict exact values of icp , but it may be useful as a screening test to estimate the probability of elevated icp ." ], [ "csf : cerebrospinal fluid ; icp : intracranial pressure ; mri : magnetic resonance imaging ; onsd : optic nerve sheath diameter ." ], [ "" ] ]

[ "there is no doubt that dog is the most favorite pet animal throughout the world .", "further , due to its physiological similarity with human being , it has turned out to be an ideal model for studying human diseases like diabetes and cancer .", "therefore , a better understanding about the normal physiological events of dogs might have paramount significance for clinicians , researchers , and pet owners .", "however , in many cases it is very difficult to have an accurate age estimation of a dog .", "this is because : ( i ) dog owners fail to keep the birth record of their pets , and it is not possible in stray dogs , ( ii ) dogs like any other companion animal is sold and resold more than once during their lifetime ; hence , change of ownership may cause loss of data regarding age , ( iii ) normal available techniques are not very accurate and/or affordable , and ( iv ) very less information is available about age - related biomarkers in dogs . in a dog , denture wear , loss of elasticity of skin , rough hair coat , and so on are the common external appearances in old age . during this period ,", "hematological alterations like anemia , lowered pcv ( packed cell volume ) , and so on and biochemical changes like decreased serum protein , increased cholesterol , and bun ( blood urea nitrogen ) level , and so on are evident which can be correlated with dysfunction of different organs like liver , kidney , heart , spleen , nervous , and musculoskeletal system , and so on .", "many disease conditions and/or diseases are also common in geriatric dogs like glaucoma , arthritis , skin disease , arteriosclerosis , prostrate hyperplasia , brown pigmentation of internal organs , and so on .", "however , all these age - related alterations are not very specific for age determination .", "importantly , most of these only indicate about the presence or absence of old age .", "they do not give an idea about the intermediate stages of age progression from young to old age .", "therefore , efforts should be made to identify novel age - related biomarkers . in many organs ,", "somatic cells undergo a state of permanent growth arrest and altered function after a finite number of divisions , and this is known as replicative senescence .", "the process of replicative senescence was being established by using the human cells , which showed limited number of doublings before arrest of cell cycle .", "senescent cells are resistant to apoptosis for a long period of time but are metabolically active .", "there is also evidence that senescent cells can accumulate in renewable tissues with age and at sites showing age - related pathologies like osteoarthritis and atherosclerosis . by only looking at cell morphology", ", senescent cells can not be distinguished from quiescent or terminally differentiated cells in tissues .", "various studies have confirmed that senescent - associated gal ( sa-gal ) is same as normal lysosomal gal . however ; in senescence , overproduction of this protein increases the overall activity of this enzyme .", "the normal lysosomal gal is active at ph 4 , but sa-gal activity can be easily detected at ph 6", ". this might be due to presence of high concentration of sa-gal enzymes in senescent cells .", "on the basis of this observation that at a higher ph , sa-gal activity can be detected , various protocols have been adapted to detect sa-gal activity in tissues and cell cultures .", "particularly , the chromogenic substrate 5-bromo-4-chloro-3-indolyl -d - galactosidase ( x - gal ) has been extensively used to detect the sa-gal activity in vivo .", "gal cleaves x - gal and produce an insoluble blue compound , which is detectable in situ .", "the x - gal staining is a very useful assay to design a reliable protocol for the identification of senescent cells quickly with simple sectioning and staining procedure .", "importantly , an increase in sa-gal activity detected in human skin tissues correlated with the old age .", "we were able to detect sa-gal activity successfully in skin samples obtained through rapid necropsy of dead dogs .", "gluteraldehyde , 50% solution , reagent grade and magnesium chloride ; anhydrous ; high purity grade was obtained from amresco ( solon , ohio , usa ) .", "potassium ferrocyanide ( extrapure analytical reagent ) , potassium ferricyanide , and egta ( ethylene glycol tetraacetic acid ) were obtained from sisco research lab pvt ltd ( mumbai , india ) .", "the fixative solution was prepared with gluteraldehyde ( 0.50% ) , egta ( 1.25 mm ; ph 6 or 7.3 ) , mgcl2 ( 2.00 mm ) and 1 phosphate buffered saline ( pbs ) ( ph 6 or 7.3 ) .", "the wash buffer was made up with mgcl2 ( 2 mm ) , np-40 ( 0.02% ) and 1 pbs . and the x - gal stain was prepared with x - gal ( 0.6 mg / ml ) , k ferrocyanide ( 4 mm ) , k ferricyanide ( 4 mm ) , and wash buffer .", "all the tissue samples used in this study were collected during necropsy of dogs that have died just 10 - 15 min before the necropsy procedure .", "particularly , we collected sample from those dogs whose exact birth record was available with the owner .", "we also restricted us in collecting and using samples from dogs that have died not due to any chronic disease .", "most of the samples were collected from dogs that encountered accident and presented at the college of veterinary science and animal husbandry , orissa for treatment , but later on succumbed to death .", "these cases were referred to the department of veterinary pathology for postmortem / necropsy . in this way", ", we were able to collect samples from three dogs ( two old and one young ) .", "further tissue storage and processing was done at the institute of life sciences , bhubaneswar . as samples were collected from dead animals and used in a nonprofitable research study", "however , before collecting the samples , we took the prior consent of the corresponding owners .", "previous studies have shown that storage of snap frozen tissues might reduce the activity of sa-gal activity .", "therefore , the snap frozen tissues were processed on the same day for the x - gal staining . out of curiosity", ", we also kept one fragment of the snap frozen tissue in -80c and 7 days later processed for further sectioning and staining as described below .", "tissues were taken out of liquid nitrogen or -80c and immediately embedded in tissue freezing medium ( leica microsystems nussloch gmbh ) .", "tissue sections of 7 - 15 micron thickness were made by using cryomicrotome ( leica cm1850 ) .", "after sectioning , tissues were fixed over the charged slides . until beginning of the staining procedure ,", "for the x - gal staining procedure all the working solutions were made fresh from corresponding stocks .", "this was followed by incubation of slides with x - gal staining solution for more than 12 h in 37c incubator .", "two beakers containing water were kept inside the incubator for maintaining the humidity and protecting from drying of staining solution ( x - gal ) .", "last , slides were mounted with aqueous mounting solution ( vectamount aq ; vector laboratories , inc . ;", "a consecutive frozen slide was stained using h and e. all slides were observed under leica dm500 light microscope and representative photographs were taken .", "gluteraldehyde , 50% solution , reagent grade and magnesium chloride ; anhydrous ; high purity grade was obtained from amresco ( solon , ohio , usa ) .", "potassium ferrocyanide ( extrapure analytical reagent ) , potassium ferricyanide , and egta ( ethylene glycol tetraacetic acid ) were obtained from sisco research lab pvt ltd ( mumbai , india ) .", "the fixative solution was prepared with gluteraldehyde ( 0.50% ) , egta ( 1.25 mm ; ph 6 or 7.3 ) , mgcl2 ( 2.00 mm ) and 1 phosphate buffered saline ( pbs ) ( ph 6 or 7.3 ) .", "the wash buffer was made up with mgcl2 ( 2 mm ) , np-40 ( 0.02% ) and 1 pbs . and the x - gal stain was prepared with x - gal ( 0.6 mg / ml ) , k ferrocyanide ( 4 mm ) , k ferricyanide ( 4 mm ) , and wash buffer .", "all the tissue samples used in this study were collected during necropsy of dogs that have died just 10 - 15 min before the necropsy procedure . particularly , we collected sample from those dogs whose exact birth record was available with the owner .", "we also restricted us in collecting and using samples from dogs that have died not due to any chronic disease .", "most of the samples were collected from dogs that encountered accident and presented at the college of veterinary science and animal husbandry , orissa for treatment , but later on succumbed to death .", "these cases were referred to the department of veterinary pathology for postmortem / necropsy . in this way", ", we were able to collect samples from three dogs ( two old and one young ) .", "further tissue storage and processing was done at the institute of life sciences , bhubaneswar .", "as samples were collected from dead animals and used in a nonprofitable research study , we did not seek any animal ethical committee approval .", "however , before collecting the samples , we took the prior consent of the corresponding owners .", "previous studies have shown that storage of snap frozen tissues might reduce the activity of sa-gal activity .", "therefore , the snap frozen tissues were processed on the same day for the x - gal staining .", "out of curiosity , we also kept one fragment of the snap frozen tissue in -80c and 7 days later processed for further sectioning and staining as described below .", "tissues were taken out of liquid nitrogen or -80c and immediately embedded in tissue freezing medium ( leica microsystems nussloch gmbh ) .", "tissue sections of 7 - 15 micron thickness were made by using cryomicrotome ( leica cm1850 ) .", "after sectioning , tissues were fixed over the charged slides . until beginning of the staining procedure ,", "slides with the tissue were kept on ice . for the x - gal staining procedure", "initially , slides were placed in fixative for 30 min at 4c . then slides were rinsed with wash buffer for 4 5 min .", "this was followed by incubation of slides with x - gal staining solution for more than 12 h in 37c incubator .", "two beakers containing water were kept inside the incubator for maintaining the humidity and protecting from drying of staining solution ( x - gal ) .", "last , slides were mounted with aqueous mounting solution ( vectamount aq ; vector laboratories , inc . ;", "a consecutive frozen slide was stained using h and e. all slides were observed under leica dm500 light microscope and representative photographs were taken .", "as discussed above , the key to distinguish a lysosomal gal activity from sa-gal activity is staining at different ph - range ( 4 or more than 6 ) .", "in the current study , we noticed x - gal staining both at ph 6 and ph 7.3 .", "as reported earlier , we observed a reduction of sa-gal activity after freezing of tissues in -80c .", "however , even after 5 - 6 days of storage in -80c , we were able to detect x - gal staining ; though of lower intensity .", "this observation is not in accordance with a previously reported statement that even overnight storage of tissue samples at -80c can destroy the enzyme activity . ", "the staining was detectable after minimum of 10 h incubation and staining intensity went on increasing with an increase in incubation time .", "staining was mostly noticed at the inner epithelial layers of hair follicles and sebaceous glands [ figure 1a and b ] .", "further , the x - gal staining intensity was more intense at ph 6.0 than ph 7.3 [ figure 2a and b ] .", "overall , the number of hair follicles positive for x - gal was more at ph 6.0 than ph 7.3 .", "we did not have enough samples to make a comparison of x - gal positive cells ( % ) in young and old age animals ; however , in the tissue of only one young dog available with us , we did not see any staining even after 20 h of incubation ( data not shown ) .", "histology of a dog skin tissue stained with h and e or x - gal ( a ) h and e stained dog skin tissue shows presence of various cellular components of epidermis and dermis .", "arrows ( yellow ) indicate follicular epithelia cells ( compound follicle ) , stars indicate stromal cells , and triangle indicates epithelial cells of a sebaceous gland . for h and e staining , frozen skin sample of a greatdane dog ( ~5 years old ) was cryosectioned and processed by usual h and e staining procedure .", "( b ) x - gal stain of a corresponding tissue shows clear x - gal staining ( red arrow ) in the region of follicular epithelium and sebaceous gland .", "the tissue was from the same dog and processed for x - gal staining at ph 7.3 .", "note : tissue used in this experiment was preserved at -80c for 5 days after its snap freezing in liquid nitrogen x - gal staining at different ph ( a ) gross appearance of canine skin tissues stained with x - gal .", "all the staining regents were adjusted to ph 6 or 7.3 . cryosectioned skin tissues were stained for x - gal at different ph , and after overnight ( 12 h ) staining , photos of those slides were taken by normal digital camera .", "staining at ph 6 produced more intense color ( blue ) than at ph 7.3 .", "( b ) microscopic images of tissues stained at different ph of staining solutions . more intense", "x - gal staining ( blue ) was observed at ph 6 than ph 7.3 knowledge about the age of a dog might have multiple uses in clinic and research .", "currently , available techniques for the detection of a dog 's age are not very reliable .", "the measurement of gal activity has been proposed to be a marker for the estimation of a human age .", "however , not much effort has been made to detect the expression of this protein in other species like dog .", "to best of our knowledge , the current study is the first report regarding possible detection of endogenous gal activity in dog skin tissue .", "detection of gal activity in skin tissues obtained through rapid necropsy showed the feasibility of detecting this molecule 's activity from just died and most potentially from live animal 's skin .", "further , detection of x - gal staining efficiently in the epidermis of skin suggests requirement of a small amount of superficial skin for carrying out this analysis .", "x - gal staining of canine tissues might be instrumental in estimating the proper age of a dog", "it has also potential use in forensic science including wildlife forensic , where age estimation is warranted . for better use of this technique", ", an extensive analysis is required to get the actual idea about sa-gal activity in different age , sex , and breed of dogs .", "we also believe that skin tissues from different regions of the same animal might have different number of senescent cells ; therefore , a more in - depth study is required before the adaptation of x - gal staining as a marker for old age of dogs .", "lac - z , the bacterial gal , has been widely used as a reporter gene in many animal models including dogs . in most of the gene therapy studies in dog ,", "lac - z has a similarity with endogenous gal of other species ; therefore , an idea about expression level of endogenous gal expression / activity in different organs of this animal , might help to carry out x - gal staining in a more careful fashion and proper interpretation of the results . in various literatures ,", "most of the protocols recommend ph 7.3/7.4 for lac - z staining ; however , at this ph we were also able to detect endogenous -gal activity .", "therefore , during lac - z staining of canine tissues , tissue from the control animals should be used to avoid confusion of background staining . furthermore , detection of senescent cells through x - gal staining in canine tissues will help in checking the coexistence and functional significance of these cells in different chronic diseases like cancer . taken together , we believe that our current report has shown the possibility of carrying out x - gal staining with canine tissues and has created an opportunity to exploit this technique for several clinical and research studies .", "on the basis of the current study , following conclusions are made : \n sa-gal activity in dog is detectable through x - gal staining in situ;use of x - gal staining reagents with higher ph ( 7.3 ) might help in detecting more specifically senescent cells than previously suggested ph ( 6);x - gal staining is possible with tissues collected immediately after death;storage of tissues at -80c reduce sa-gal activity ; however , x - gal staining is still feasible in those frozen tissues;x - gal staining of dog skin tissue has the potential to be explored as a marker of senescence or old age . \n ", "sa-gal activity in dog is detectable through x - gal staining in situ ; use of x - gal staining reagents with higher ph ( 7.3 ) might help in detecting more specifically senescent cells than previously suggested ph ( 6 ) ; x - gal staining is possible with tissues collected immediately after death ; storage of tissues at -80c reduce sa-gal activity ; however , x - gal staining is still feasible in those frozen tissues ; x - gal staining of dog skin tissue has the potential to be explored as a marker of senescence or old age ." ]

[ "<S> background : estimation of -galactosidase ( gal ) activity in human cells and tissues indicate its possible use as a marker of senescence.objectives:this study was done to detect senescence - associated gal ( sa-gal ) activity in canine skin tissue by using its substrate 5-bromo-4-chloro-3-indolyl -d - galactosidase ( x - gal).materials and methods : skin samples were collected through rapid necropsy process . </S>", "<S> the x - gal staining was done by altering different factors of the staining procedure like ph of the reagents and incubation time . </S>", "<S> further , effect of tissue preservation procedure was also evaluated.results:typical x - gal staining was detected in old dogs skin samples and it was detectable both at ph 6 and ph 7.3 . </S>", "<S> the cells present in the inner lining of the hair follicles and sebaceous glands are the major cells that have high sa-gal activity . </S>", "<S> the x - gal staining intensity was more prominent in tissues preserved in liquid nitrogen at -196c than in -80c freezer . </S>", "<S> prolonged incubation period increased the intensity of staining.conclusions:this study indicates possibility of x - gal staining in canine tissues and opens an avenue for further in - depth studies that might be useful for different research and clinical studies like determination of dog 's approximate age . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Reagents and solutions", "Procurement and preservation of tissue sample", "Cryosectioning and staining", "RESULTS AND DISCUSSIONS", "CONCLUSION" ]

[ [ "there is no doubt that dog is the most favorite pet animal throughout the world .", "further , due to its physiological similarity with human being , it has turned out to be an ideal model for studying human diseases like diabetes and cancer .", "therefore , a better understanding about the normal physiological events of dogs might have paramount significance for clinicians , researchers , and pet owners .", "however , in many cases it is very difficult to have an accurate age estimation of a dog .", "this is because : ( i ) dog owners fail to keep the birth record of their pets , and it is not possible in stray dogs , ( ii ) dogs like any other companion animal is sold and resold more than once during their lifetime ; hence , change of ownership may cause loss of data regarding age , ( iii ) normal available techniques are not very accurate and/or affordable , and ( iv ) very less information is available about age - related biomarkers in dogs . in a dog , denture wear , loss of elasticity of skin , rough hair coat , and so on are the common external appearances in old age . during this period ,", "hematological alterations like anemia , lowered pcv ( packed cell volume ) , and so on and biochemical changes like decreased serum protein , increased cholesterol , and bun ( blood urea nitrogen ) level , and so on are evident which can be correlated with dysfunction of different organs like liver , kidney , heart , spleen , nervous , and musculoskeletal system , and so on .", "many disease conditions and/or diseases are also common in geriatric dogs like glaucoma , arthritis , skin disease , arteriosclerosis , prostrate hyperplasia , brown pigmentation of internal organs , and so on .", "however , all these age - related alterations are not very specific for age determination .", "importantly , most of these only indicate about the presence or absence of old age .", "they do not give an idea about the intermediate stages of age progression from young to old age .", "therefore , efforts should be made to identify novel age - related biomarkers . in many organs ,", "somatic cells undergo a state of permanent growth arrest and altered function after a finite number of divisions , and this is known as replicative senescence .", "the process of replicative senescence was being established by using the human cells , which showed limited number of doublings before arrest of cell cycle .", "senescent cells are resistant to apoptosis for a long period of time but are metabolically active .", "there is also evidence that senescent cells can accumulate in renewable tissues with age and at sites showing age - related pathologies like osteoarthritis and atherosclerosis . by only looking at cell morphology", ", senescent cells can not be distinguished from quiescent or terminally differentiated cells in tissues .", "various studies have confirmed that senescent - associated gal ( sa-gal ) is same as normal lysosomal gal . however ; in senescence , overproduction of this protein increases the overall activity of this enzyme .", "the normal lysosomal gal is active at ph 4 , but sa-gal activity can be easily detected at ph 6", ". this might be due to presence of high concentration of sa-gal enzymes in senescent cells .", "on the basis of this observation that at a higher ph , sa-gal activity can be detected , various protocols have been adapted to detect sa-gal activity in tissues and cell cultures .", "particularly , the chromogenic substrate 5-bromo-4-chloro-3-indolyl -d - galactosidase ( x - gal ) has been extensively used to detect the sa-gal activity in vivo .", "gal cleaves x - gal and produce an insoluble blue compound , which is detectable in situ .", "the x - gal staining is a very useful assay to design a reliable protocol for the identification of senescent cells quickly with simple sectioning and staining procedure .", "importantly , an increase in sa-gal activity detected in human skin tissues correlated with the old age .", "we were able to detect sa-gal activity successfully in skin samples obtained through rapid necropsy of dead dogs ." ], [ "gluteraldehyde , 50% solution , reagent grade and magnesium chloride ; anhydrous ; high purity grade was obtained from amresco ( solon , ohio , usa ) .", "potassium ferrocyanide ( extrapure analytical reagent ) , potassium ferricyanide , and egta ( ethylene glycol tetraacetic acid ) were obtained from sisco research lab pvt ltd ( mumbai , india ) .", "the fixative solution was prepared with gluteraldehyde ( 0.50% ) , egta ( 1.25 mm ; ph 6 or 7.3 ) , mgcl2 ( 2.00 mm ) and 1 phosphate buffered saline ( pbs ) ( ph 6 or 7.3 ) .", "the wash buffer was made up with mgcl2 ( 2 mm ) , np-40 ( 0.02% ) and 1 pbs . and the x - gal stain was prepared with x - gal ( 0.6 mg / ml ) , k ferrocyanide ( 4 mm ) , k ferricyanide ( 4 mm ) , and wash buffer .", "all the tissue samples used in this study were collected during necropsy of dogs that have died just 10 - 15 min before the necropsy procedure .", "particularly , we collected sample from those dogs whose exact birth record was available with the owner .", "we also restricted us in collecting and using samples from dogs that have died not due to any chronic disease .", "most of the samples were collected from dogs that encountered accident and presented at the college of veterinary science and animal husbandry , orissa for treatment , but later on succumbed to death .", "these cases were referred to the department of veterinary pathology for postmortem / necropsy . in this way", ", we were able to collect samples from three dogs ( two old and one young ) .", "further tissue storage and processing was done at the institute of life sciences , bhubaneswar . as samples were collected from dead animals and used in a nonprofitable research study", "however , before collecting the samples , we took the prior consent of the corresponding owners .", "previous studies have shown that storage of snap frozen tissues might reduce the activity of sa-gal activity .", "therefore , the snap frozen tissues were processed on the same day for the x - gal staining . out of curiosity", ", we also kept one fragment of the snap frozen tissue in -80c and 7 days later processed for further sectioning and staining as described below .", "tissues were taken out of liquid nitrogen or -80c and immediately embedded in tissue freezing medium ( leica microsystems nussloch gmbh ) .", "tissue sections of 7 - 15 micron thickness were made by using cryomicrotome ( leica cm1850 ) .", "after sectioning , tissues were fixed over the charged slides . until beginning of the staining procedure ,", "for the x - gal staining procedure all the working solutions were made fresh from corresponding stocks .", "this was followed by incubation of slides with x - gal staining solution for more than 12 h in 37c incubator .", "two beakers containing water were kept inside the incubator for maintaining the humidity and protecting from drying of staining solution ( x - gal ) .", "last , slides were mounted with aqueous mounting solution ( vectamount aq ; vector laboratories , inc . ;", "a consecutive frozen slide was stained using h and e. all slides were observed under leica dm500 light microscope and representative photographs were taken ." ], [ "gluteraldehyde , 50% solution , reagent grade and magnesium chloride ; anhydrous ; high purity grade was obtained from amresco ( solon , ohio , usa ) .", "potassium ferrocyanide ( extrapure analytical reagent ) , potassium ferricyanide , and egta ( ethylene glycol tetraacetic acid ) were obtained from sisco research lab pvt ltd ( mumbai , india ) .", "the fixative solution was prepared with gluteraldehyde ( 0.50% ) , egta ( 1.25 mm ; ph 6 or 7.3 ) , mgcl2 ( 2.00 mm ) and 1 phosphate buffered saline ( pbs ) ( ph 6 or 7.3 ) .", "the wash buffer was made up with mgcl2 ( 2 mm ) , np-40 ( 0.02% ) and 1 pbs . and the x - gal stain was prepared with x - gal ( 0.6 mg / ml ) , k ferrocyanide ( 4 mm ) , k ferricyanide ( 4 mm ) , and wash buffer ." ], [ "all the tissue samples used in this study were collected during necropsy of dogs that have died just 10 - 15 min before the necropsy procedure . particularly , we collected sample from those dogs whose exact birth record was available with the owner .", "we also restricted us in collecting and using samples from dogs that have died not due to any chronic disease .", "most of the samples were collected from dogs that encountered accident and presented at the college of veterinary science and animal husbandry , orissa for treatment , but later on succumbed to death .", "these cases were referred to the department of veterinary pathology for postmortem / necropsy . in this way", ", we were able to collect samples from three dogs ( two old and one young ) .", "further tissue storage and processing was done at the institute of life sciences , bhubaneswar .", "as samples were collected from dead animals and used in a nonprofitable research study , we did not seek any animal ethical committee approval .", "however , before collecting the samples , we took the prior consent of the corresponding owners .", "previous studies have shown that storage of snap frozen tissues might reduce the activity of sa-gal activity .", "therefore , the snap frozen tissues were processed on the same day for the x - gal staining .", "out of curiosity , we also kept one fragment of the snap frozen tissue in -80c and 7 days later processed for further sectioning and staining as described below ." ], [ "tissues were taken out of liquid nitrogen or -80c and immediately embedded in tissue freezing medium ( leica microsystems nussloch gmbh ) .", "tissue sections of 7 - 15 micron thickness were made by using cryomicrotome ( leica cm1850 ) .", "after sectioning , tissues were fixed over the charged slides . until beginning of the staining procedure ,", "slides with the tissue were kept on ice . for the x - gal staining procedure", "initially , slides were placed in fixative for 30 min at 4c . then slides were rinsed with wash buffer for 4 5 min .", "this was followed by incubation of slides with x - gal staining solution for more than 12 h in 37c incubator .", "two beakers containing water were kept inside the incubator for maintaining the humidity and protecting from drying of staining solution ( x - gal ) .", "last , slides were mounted with aqueous mounting solution ( vectamount aq ; vector laboratories , inc . ;", "a consecutive frozen slide was stained using h and e. all slides were observed under leica dm500 light microscope and representative photographs were taken ." ], [ "as discussed above , the key to distinguish a lysosomal gal activity from sa-gal activity is staining at different ph - range ( 4 or more than 6 ) .", "in the current study , we noticed x - gal staining both at ph 6 and ph 7.3 .", "as reported earlier , we observed a reduction of sa-gal activity after freezing of tissues in -80c .", "however , even after 5 - 6 days of storage in -80c , we were able to detect x - gal staining ; though of lower intensity .", "this observation is not in accordance with a previously reported statement that even overnight storage of tissue samples at -80c can destroy the enzyme activity . ", "the staining was detectable after minimum of 10 h incubation and staining intensity went on increasing with an increase in incubation time .", "staining was mostly noticed at the inner epithelial layers of hair follicles and sebaceous glands [ figure 1a and b ] .", "further , the x - gal staining intensity was more intense at ph 6.0 than ph 7.3 [ figure 2a and b ] .", "overall , the number of hair follicles positive for x - gal was more at ph 6.0 than ph 7.3 .", "we did not have enough samples to make a comparison of x - gal positive cells ( % ) in young and old age animals ; however , in the tissue of only one young dog available with us , we did not see any staining even after 20 h of incubation ( data not shown ) .", "histology of a dog skin tissue stained with h and e or x - gal ( a ) h and e stained dog skin tissue shows presence of various cellular components of epidermis and dermis .", "arrows ( yellow ) indicate follicular epithelia cells ( compound follicle ) , stars indicate stromal cells , and triangle indicates epithelial cells of a sebaceous gland . for h and e staining , frozen skin sample of a greatdane dog ( ~5 years old ) was cryosectioned and processed by usual h and e staining procedure .", "( b ) x - gal stain of a corresponding tissue shows clear x - gal staining ( red arrow ) in the region of follicular epithelium and sebaceous gland .", "the tissue was from the same dog and processed for x - gal staining at ph 7.3 .", "note : tissue used in this experiment was preserved at -80c for 5 days after its snap freezing in liquid nitrogen x - gal staining at different ph ( a ) gross appearance of canine skin tissues stained with x - gal .", "all the staining regents were adjusted to ph 6 or 7.3 . cryosectioned skin tissues were stained for x - gal at different ph , and after overnight ( 12 h ) staining , photos of those slides were taken by normal digital camera .", "staining at ph 6 produced more intense color ( blue ) than at ph 7.3 .", "( b ) microscopic images of tissues stained at different ph of staining solutions . more intense", "x - gal staining ( blue ) was observed at ph 6 than ph 7.3 knowledge about the age of a dog might have multiple uses in clinic and research .", "currently , available techniques for the detection of a dog 's age are not very reliable .", "the measurement of gal activity has been proposed to be a marker for the estimation of a human age .", "however , not much effort has been made to detect the expression of this protein in other species like dog .", "to best of our knowledge , the current study is the first report regarding possible detection of endogenous gal activity in dog skin tissue .", "detection of gal activity in skin tissues obtained through rapid necropsy showed the feasibility of detecting this molecule 's activity from just died and most potentially from live animal 's skin .", "further , detection of x - gal staining efficiently in the epidermis of skin suggests requirement of a small amount of superficial skin for carrying out this analysis .", "x - gal staining of canine tissues might be instrumental in estimating the proper age of a dog", "it has also potential use in forensic science including wildlife forensic , where age estimation is warranted . for better use of this technique", ", an extensive analysis is required to get the actual idea about sa-gal activity in different age , sex , and breed of dogs .", "we also believe that skin tissues from different regions of the same animal might have different number of senescent cells ; therefore , a more in - depth study is required before the adaptation of x - gal staining as a marker for old age of dogs .", "lac - z , the bacterial gal , has been widely used as a reporter gene in many animal models including dogs . in most of the gene therapy studies in dog ,", "lac - z has a similarity with endogenous gal of other species ; therefore , an idea about expression level of endogenous gal expression / activity in different organs of this animal , might help to carry out x - gal staining in a more careful fashion and proper interpretation of the results . in various literatures ,", "most of the protocols recommend ph 7.3/7.4 for lac - z staining ; however , at this ph we were also able to detect endogenous -gal activity .", "therefore , during lac - z staining of canine tissues , tissue from the control animals should be used to avoid confusion of background staining . furthermore , detection of senescent cells through x - gal staining in canine tissues will help in checking the coexistence and functional significance of these cells in different chronic diseases like cancer . taken together , we believe that our current report has shown the possibility of carrying out x - gal staining with canine tissues and has created an opportunity to exploit this technique for several clinical and research studies ." ], [ "on the basis of the current study , following conclusions are made : \n sa-gal activity in dog is detectable through x - gal staining in situ;use of x - gal staining reagents with higher ph ( 7.3 ) might help in detecting more specifically senescent cells than previously suggested ph ( 6);x - gal staining is possible with tissues collected immediately after death;storage of tissues at -80c reduce sa-gal activity ; however , x - gal staining is still feasible in those frozen tissues;x - gal staining of dog skin tissue has the potential to be explored as a marker of senescence or old age . \n ", "sa-gal activity in dog is detectable through x - gal staining in situ ; use of x - gal staining reagents with higher ph ( 7.3 ) might help in detecting more specifically senescent cells than previously suggested ph ( 6 ) ; x - gal staining is possible with tissues collected immediately after death ; storage of tissues at -80c reduce sa-gal activity ; however , x - gal staining is still feasible in those frozen tissues ; x - gal staining of dog skin tissue has the potential to be explored as a marker of senescence or old age ." ] ]

[ "three - dimensional ( 3d ) image analysis of the mandible is possible due to the wide availability of computed tomography ( ct ) imaging technology .", "accordingly , there have been several anatomical analyses of the mandibular foramen ( mnf),1234 most involving distance - only measurement methods .", "moreover , the analytical data for the mandible are limited with respect to their relationship with skeletal malocclusion , the size of the mandible , and the measurement planes and reference points for locating the position of the mnf .", "the position of the mnf has been studied for mandibular treatment ( i.e. ramal surgery , including osteotomy ; inferior alveolar nerve block).56 the present study used 3d images of the mandibles of patients diagnosed with skeletal class iii malocclusion , and analyzed the position of the mnf ( i.e. its proportional position in the mandible ) using measurement planes and anatomical reference points .", "the purpose of this study was to produce a useful guide of mnf positions in patients diagnosed with skeletal class iii malocclusion .", "specifically , the study used ct images of mandibles that were taken during preoperative diagnosis work - ups .", "the ct images were taken with a siemens sensation 64 system ( 1.0 mm slice thickness , siemens sensation 64 ct scanner ; siemens ag , erlangen , germany ) using a pixel size of 0.4375 mm and a field - of - view of 22.40 cm .", "we excluded ct data from facial asymmetry patients with a mandibular shift greater than 5 mm ( considered more than a moderate state of asymmetry).7 the ct data were collected for a total of 85 patients , and both right and left mnf images were analyzed .", "simplant version 14.0 ( materialise dental , leuven , belgium ) was used to reconstruct the ct images into 3d images .", "the anatomical reference points , lines , and planes were then set onto the image of the mandible .", "the 3d position of the mnf was measured based on the established reference points and planes .", "this process was intended to help the surgical team locate the mnf during mandibular ramus osteotomy .", "the anatomic points , lines , and planes that were used , as well as the measurements , are described below .", "the mnf , coronoid process , sigmoid notch , condyle , gonion , antegonial notch , menton , mesiobuccal cusp of the mandibular first molar , and the most superior incisal contact point of the lower central incisor were identified in the images .", "in addition , the points of the anterior and posterior ramus were defined ( table 1 ) .", "the anatomic lines used for defining the reference planes in this study are shown in table 2 .", "three vertical lines were constructed : one connecting the sigmoid notch points and antegonial notch points , termed the sn - agn line ; another connecting a sigmoid notch point and gonion point , termed the sn - gn line ; and the other connecting a condyle point and a gonion point , termed the cond - gn line . in addition", ", five horizontal lines were constructed that connected each of the anterior ramus points and each of the posterior ramus points . the occlusal plane , mandibular plane , and five reference horizontal planes were defined ( table 3 , fig .", "five vertical coronal planes were also defined , as shown in table 3 and figure 1b .", "the vertical heights of the anatomic points were measured according to each of the five horizontal planes ( fig .", "the vertical distances from the coronoid process , sigmoid notch , and condyle to each horizontal plane were added to the vertical distances from the gonion to each horizontal plane , and these distances were set as the vertical heights of the coronoid process , sigmoid notch , and condyle in the mandible for each horizontal plane ( table 4 ) .", "the vertical distances from the gonion to the horizontal planes were set as the heights of the mnf in the mandible , according to each horizontal plane .", "based on each of the five horizontal planes that pass through the mnf , the distance on each plane from the anterior ramus point to the posterior ramus point was designated the anteroposterior horizontal distance of the ramus ( table 4 ) .", "the perpendicular distance from each anterior ramus point to each vertical plane was designated the horizontal distance from the anterior ramus to the mnf ( fig .", "3 ) . to investigate the positional relationship of the mnf in the mandible , regression analysis was used to examine the rational relationships between the vertical heights of the coronoid process , sigmoid notch , and condyle points and the vertical height of the mnf .", "regression analysis was also used to determine the rational relationship between the anteroposterior horizontal distance of the ramus from each of the five horizontal planes passing through the mnf and the horizontal distance from the anterior ramus point to the mnf .", ", armonk , ny , usa ) , and statistical significance was established at p<0.05 . the intra - observer error for anatomic point determination was calculated via repeated measurement ( 10 measurements ) of the mnf .", "simplant version 14.0 ( materialise dental , leuven , belgium ) was used to reconstruct the ct images into 3d images .", "the anatomical reference points , lines , and planes were then set onto the image of the mandible .", "the 3d position of the mnf was measured based on the established reference points and planes .", "this process was intended to help the surgical team locate the mnf during mandibular ramus osteotomy .", "the anatomic points , lines , and planes that were used , as well as the measurements , are described below .", "the mnf , coronoid process , sigmoid notch , condyle , gonion , antegonial notch , menton , mesiobuccal cusp of the mandibular first molar , and the most superior incisal contact point of the lower central incisor were identified in the images .", "in addition , the points of the anterior and posterior ramus were defined ( table 1 ) .", "the anatomic lines used for defining the reference planes in this study are shown in table 2 .", "three vertical lines were constructed : one connecting the sigmoid notch points and antegonial notch points , termed the sn - agn line ; another connecting a sigmoid notch point and gonion point , termed the sn - gn line ; and the other connecting a condyle point and a gonion point , termed the cond - gn line . in addition", ", five horizontal lines were constructed that connected each of the anterior ramus points and each of the posterior ramus points .", "the occlusal plane , mandibular plane , and five reference horizontal planes were defined ( table 3 , fig .", "five vertical coronal planes were also defined , as shown in table 3 and figure 1b .", "the vertical heights of the anatomic points were measured according to each of the five horizontal planes ( fig .", "the vertical distances from the coronoid process , sigmoid notch , and condyle to each horizontal plane were added to the vertical distances from the gonion to each horizontal plane , and these distances were set as the vertical heights of the coronoid process , sigmoid notch , and condyle in the mandible for each horizontal plane ( table 4 ) .", "the vertical distances from the gonion to the horizontal planes were set as the heights of the mnf in the mandible , according to each horizontal plane . based on each of the five horizontal planes that pass through the mnf , the distance on each plane from the anterior ramus point to the posterior ramus point was designated the anteroposterior horizontal distance of the ramus ( table 4 ) .", "the perpendicular distance from each anterior ramus point to each vertical plane was designated the horizontal distance from the anterior ramus to the mnf ( fig .", "to investigate the positional relationship of the mnf in the mandible , regression analysis was used to examine the rational relationships between the vertical heights of the coronoid process , sigmoid notch , and condyle points and the vertical height of the mnf .", "regression analysis was also used to determine the rational relationship between the anteroposterior horizontal distance of the ramus from each of the five horizontal planes passing through the mnf and the horizontal distance from the anterior ramus point to the mnf .", ", armonk , ny , usa ) , and statistical significance was established at p<0.05 .", "the intra - observer error for anatomic point determination was calculated via repeated measurement ( 10 measurements ) of the mnf .", "the sex ratio and age of the 85 patients with skeletal class iii mandibular prognathism were 46 : 39 ( men : women ) and 22.25.5 years .", "the condyle height relative to the mnf - mandibular plane was the smallest ( 53.456.45 mm ) .", "the heights of the coronoid process and the mnf were the greatest when measured relative to the mnf - mandibular plane ( 60.095.96 mm and 21.313.83 mm , respectively ) .", "the height of the sigmoid notch was the greatest when based on the mnf - sn / gn p plane ( 43.895.02 mm ) .", "the height of the condyle was the greatest when based on the mnf - cond / gn p plane ( 63.805.79 mm ) .", "the heights of the mnf , coronoid process , and sigmoid notch were the smallest when based on the mnf - cond / gn p plane ( 18.604.56 mm , 50.086.89 mm , and 40.505.45 mm , respectively ) .", "the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf were the longest when they were based on the mnf - mandibular plane ( 42.985.17 mm and 27.604.22 mm , respectively ; table 5 ) .", "the heights of the coronoid process , sigmoid notch , and condylar head on the five horizontal planes were all significantly associated with the height of the mnf according to the regression analysis .", "the regression relationship was the strongest for the coronoid process , sigmoid notch , and condylar head when the measurements were based on the mnf - mandibular plane ( coefficients of determination ( r ) of 0.424 , 0.597 , and 0.604 respectively ) .", "the heights of the coronoid process and the mnf were significantly associated according to the regression analysis ( table 6 ) .", "the non - standardized regression coefficients were significant for the relationships between the heights of the coronoid process and the mnf based on all the horizontal reference planes .", "the highest coefficients , based on the mnf - mandibular plane , were a standardized regression coefficient of 0.654 and determination coefficient ( r ) of 0.424 .", "the heights of the sigmoid notch and the mnf were significantly associated in the regression analysis ( table 7 ) .", "the non - standardized regression coefficients for the relationships between the heights of the sigmoid notch and the mnf were significant for all the horizontal reference planes .", "the regression constant was highly significant when it was based on the mnf - mandibular plane ( p=0.002 ) .", "the highest coefficients were a standardized regression coefficient of 0.774 and determination coefficient of 0.597 , and these were based on the mnf - mandibular plane .", "the heights of the condylar head and the mnf were significantly related in the regression analysis ( table 8) .", "for all the horizontal reference planes , the non - standardized regression coefficients for the relationships between the heights of the condylar head and the mnf were significant .", "the regression constant was significant when it was based on all of the horizontal planes , except for the mnf - sn / gn p plane .", "the highest coefficients were a standardized regression coefficient of 0.779 and a determination coefficient of 0.604 , and were based on the mnf - mandibular plane . using the five horizontal planes as references , the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf", "for all the horizontal reference planes , the non - standardized regression coefficients were significant for the relationships of the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf .", "the regression constants were significant when the mnf - mandibular plane and mnf - sn / gn p plane were used as references .", "the standardized regression coefficients for all five horizontal planes used as references were greater than 0.73 , and the coefficient was 0.877 using the mnf - mandibular plane and 0.901 using the mnf - sn / gn p plane .", "the determination coefficient was 0.766 using the mnf - mandibular plane and 0.811 using the mnf - sn / gn p plane .", "notably , there were 53 missing values for measurements using the mnf - mandibular plane and 3 missing values for the mnf - sn / gn p plane .", "for the mnf - mandibular plane , which is parallel to the mnf , there were missing values when the anterior part of the plane sloped downward , thus leading directly to the mandibular body instead of the anterior ramus .", "similarly , there were missing values for the mnf - sn / gn p plane , although there were fewer than for the mnf - mandibular plane .", "the heights of the coronoid process , sigmoid notch , and condylar head on the five horizontal planes were all significantly associated with the height of the mnf according to the regression analysis .", "the regression relationship was the strongest for the coronoid process , sigmoid notch , and condylar head when the measurements were based on the mnf - mandibular plane ( coefficients of determination ( r ) of 0.424 , 0.597 , and 0.604 respectively ) .", "the heights of the coronoid process and the mnf were significantly associated according to the regression analysis ( table 6 ) .", "the non - standardized regression coefficients were significant for the relationships between the heights of the coronoid process and the mnf based on all the horizontal reference planes .", "the highest coefficients , based on the mnf - mandibular plane , were a standardized regression coefficient of 0.654 and determination coefficient ( r ) of 0.424 .", "the heights of the sigmoid notch and the mnf were significantly associated in the regression analysis ( table 7 ) .", "the non - standardized regression coefficients for the relationships between the heights of the sigmoid notch and the mnf were significant for all the horizontal reference planes .", "the regression constant was highly significant when it was based on the mnf - mandibular plane ( p=0.002 ) .", "the highest coefficients were a standardized regression coefficient of 0.774 and determination coefficient of 0.597 , and these were based on the mnf - mandibular plane .", "the heights of the condylar head and the mnf were significantly related in the regression analysis ( table 8) .", "for all the horizontal reference planes , the non - standardized regression coefficients for the relationships between the heights of the condylar head and the mnf were significant .", "the regression constant was significant when it was based on all of the horizontal planes , except for the mnf - sn / gn p plane .", "the highest coefficients were a standardized regression coefficient of 0.779 and a determination coefficient of 0.604 , and were based on the mnf - mandibular plane .", "using the five horizontal planes as references , the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf were significantly associated according to the regression analysis ( table 9 ) .", "for all the horizontal reference planes , the non - standardized regression coefficients were significant for the relationships of the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf .", "the regression constants were significant when the mnf - mandibular plane and mnf - sn / gn p plane were used as references .", "the standardized regression coefficients for all five horizontal planes used as references were greater than 0.73 , and the coefficient was 0.877 using the mnf - mandibular plane and 0.901 using the mnf - sn / gn p plane .", "the determination coefficient was 0.766 using the mnf - mandibular plane and 0.811 using the mnf - sn / gn p plane .", "notably , there were 53 missing values for measurements using the mnf - mandibular plane and 3 missing values for the mnf - sn / gn p plane .", "for the mnf - mandibular plane , which is parallel to the mnf , there were missing values when the anterior part of the plane sloped downward , thus leading directly to the mandibular body instead of the anterior ramus .", "similarly , there were missing values for the mnf - sn / gn p plane , although there were fewer than for the mnf - mandibular plane .", "the size of the human mandible varies greatly according to age , ethnicity , and sex.2 accordingly , the present study investigated the relative position of the mnf rather than simply measuring its position within the surrounding anatomical structures . using specific anatomic planes as references , we measured the position of the mnf in patients diagnosed with skeletal class iii malocclusion to determine the 3d position in the mandible .", "we determined the position of the mnf was related vertically to the heights of the mandibular condyle , sigmoid notch , and coronoid process and horizontally to the anteroposterior length of the mandibular ramus .", "further , we found that the mandibular plane was best for determining the relationships of the heights of the mandibular condyle , sigmoid notch , and coronoid process with the height of the mnf .", "this suggests that the mandibular plane can be utilized in clinical cases , not only for analyzing maxillofacial malformations but also for tracing the position of the mnf . during mandibular prognathism surgery , the vertical position of the mnf", "can be estimated using the exposed sigmoid notch area . with reference to the mandibular plane , the height ( in millimeters ) of the mnf", "can be estimated with the following equation : 0.621(the height of the sigmoid notch ) - 5.318 . as an alternative to the mandibular plane", ", one can use the horizontal plane perpendicular to the sn - gn line that connects the sigmoid notch and gonion ; this plane ( the mnf - sn / gn p plane ) is second to the mandibular plane in terms of usefulness for estimating the height of the mnf .", "when using the condyle height , the occlusal plane can be a useful alternative for estimating the height of the mnf .", "vertical ramus osteotomy is a surgical procedure that can be used for patients with mandibular prognathism when the mandibular ramus posterior to the mnf is cut .", "the relationship between the anteroposterior length of the mandibular ramus and the position of the mnf was best shown when the horizontal mnf - sn / gn p and mnf - mandibular planes were used as references . the mandibular ramus area at the mnf level is exposed during mandibular prognathism surgery .", "therefore , the relationship of the horizontal position of the mnf with the anteroposterior length of the ramus was investigated . using the mnf - sn /", "gn p plane as a reference , the horizontal length ( in mm ) from the anterior ramus to the mnf can be estimated with the following equation : 0.728(the anteroposterior distance of the mandibular ramus)- 3.948 . using the mnf - mandibular plane as the reference , the horizontal length ( in millimeters ) from the anterior ramus to the mnf", "can be estimated with the following equation : 0.716(the anteroposterior distance of the ascending ramus)- 3.148 . however , there are cases in which the mandibular plane leans in the anteroinferior direction , and the mnf - mandibular plane can not pass through the anterior ascending ramus area . in the present study", ", there were 53 missing values for the anterior ramus point when using the mnf - mandibular plane as the reference , and there were 3 missing values when using the mnf - sn / gn p plane . missing values may limit the usefulness of these calculations since they may occur in actual clinical cases .", "if this happens , it is recommended that the mnf - sn / gn p plane be used , and the use of the occlusal plane may be more helpful for determining the horizontal position of the mnf when the anterior ramus point can not be defined using the mnf - mandibular plane .", "the vertical height of the mnf from the gonion ranged from 18 mm to 21 mm , and the anteroposterior length of the ramus was about 33 mm to 42 mm .", "the horizontal distance from the anterior ramus point to the mnf was about 20 mm to 27 mm .", "the wide availability of ct scans has resulted in useful 3d images of the mandible . in a study that analyzed the 3d position of the mnf using cone - beam ct ,", "the average distance from the gonion to the mnf was about 24 mm and the average distance from the anterior ramus to the mnf in the axial plane was about 15 mm.2 the difference between the results of that study versus the present study is likely due to the use of the axial and sagittal planes of the ct images in the former . in another study that analyzed the 3d position of the mnf using multiple detector ct ,", "the average height from the gonion to the mnf relative to the occlusal plane was about 18 mm , the average anteroposterior length of the ascending ramus was 37 mm , and the average distance from the anterior ramus to the mnf on a horizontal plane ( parallel to the occlusal plane that passes through the mnf ) was about 22 mm.8 these results are similar to those of the present study , although there are minor differences because the present study only examined mandibles with skeletal iii class malocclusion , and the horizontal length from the anterior ramus to the mnf was measured as the perpendicular distance projected onto each vertical plane that passed through the mnf .", "simply measuring the vertical and horizontal distances may not be that beneficial in actual clinical cases because human mandibles vary in size .", "however , assessing the positional relationships using 3d planes can be useful clinically , as this shows the objective relationship with respect to the reference planes used in the analyses .", "image - assisted surgical techniques are increasingly used for maxillofacial operations ; accordingly , the present study is valuable in that it provides 3d image data that can be utilized in such surgical procedures .", "this study 's research methods and results , with respect to the positional 3d analysis of the mnf , will be useful for computer - assisted surgical methods , such as surgical simulation , navigation , augmented reality models , and robotic surgery .", "previous studies of the mandible were performed on cadavers,910 and often just a few distances were measured in 3d reconstructed images even though ct images were examined.234 importantly , previous studies performed quantitative analyses without considering the different sizes of the mandibles . moreover , even when 3d reconstruction images were used , few studies performed vertical and horizontal assessments using 3d reference planes .", "the present study found that the position of the mnf was related to the vertical heights of the sigmoid notch , coronoid process , and condyle and to the horizontal anteroposterior length of the ascending ramus .", "these findings indicate that the mnf , at least to some extent , maintains its relative position with the growth of the rami .", "in addition , the results indicate that the mnf is posterior to the ascending ramus .", "we expect that future studies will contribute further knowledge about the position of the mnf in the mandible via 3d image analysis of mandibles with skeletal class ii malocclusion for ramus surgery . in conclusion , the 3d mandibular image analysis data reported in this study will be useful for defining and describing the position of the mnf in mandibles of different sizes and shapes and in different ethnic groups .", "the present study showed that the proportional position of the mnf was significantly related to the vertical heights of the sigmoid notch , coronoid process , and condyle and to the horizontal anteroposterior length of the ascending ramus .", "furthermore , the methods used here and the positional 3d analyses of the mnf can be used clinically for computer - assisted surgical methods , especially for ramal surgery in patients with skeletal class iii mandibular prognathism ." ]

[ "<S> purposeto analyze the relative position of the mandibular foramina ( mnfs ) in patients diagnosed with skeletal class iii malocclusion.materials and methodscomputed tomography ( ct ) images were collected from 85 patients . </S>", "<S> the vertical lengths of each anatomic point from the five horizontal planes passing through the mnf were measured at the coronoid process , sigmoid notch , condyle , and the gonion . the distance from the anterior ramus point to the posterior ramus point on the five horizontal planes </S>", "<S> was designated the anteroposterior horizontal distance of the ramus for each plane . </S>", "<S> the perpendicular distance from each anterior ramus point to each vertical plane through the mnf was designated the horizontal distance from the anterior ramus to the mnf . </S>", "<S> the horizontal and vertical positions were examined by regression analysis.resultsregression analysis showed the heights of the coronoid process , sigmoid notch , and condyle for the five horizontal planes were significantly related to the height of the mnf , with the highest significance associated with the mnf - mandibular plane ( coefficients of determination ( r2 ) : 0.424 , 0.597 , and 0.604 , respectively ) . </S>", "<S> the horizontal anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf were significant by regression analysis.conclusionthe relative position of the mnf was significantly related to the vertical heights of the sigmoid notch , coronoid process , and condyle as well as to the horizontal anteroposterior length of the ascending ramus . </S>", "<S> these findings should be clinically useful for patients with skeletal class iii mandibular prognathism . </S>" ]

[ "Introduction", "Materials and Methods", "Points, lines, and planes for measurements", "The vertical heights of anatomic points and horizontal length of the ramus", "Analysis of the measurement values", "Results", "Vertical position of the MnF", "Horizontal position of the MnF", "Discussion" ]

[ [ "three - dimensional ( 3d ) image analysis of the mandible is possible due to the wide availability of computed tomography ( ct ) imaging technology .", "accordingly , there have been several anatomical analyses of the mandibular foramen ( mnf),1234 most involving distance - only measurement methods .", "moreover , the analytical data for the mandible are limited with respect to their relationship with skeletal malocclusion , the size of the mandible , and the measurement planes and reference points for locating the position of the mnf .", "the position of the mnf has been studied for mandibular treatment ( i.e. ramal surgery , including osteotomy ; inferior alveolar nerve block).56 the present study used 3d images of the mandibles of patients diagnosed with skeletal class iii malocclusion , and analyzed the position of the mnf ( i.e. its proportional position in the mandible ) using measurement planes and anatomical reference points .", "the purpose of this study was to produce a useful guide of mnf positions in patients diagnosed with skeletal class iii malocclusion ." ], [ "specifically , the study used ct images of mandibles that were taken during preoperative diagnosis work - ups .", "the ct images were taken with a siemens sensation 64 system ( 1.0 mm slice thickness , siemens sensation 64 ct scanner ; siemens ag , erlangen , germany ) using a pixel size of 0.4375 mm and a field - of - view of 22.40 cm .", "we excluded ct data from facial asymmetry patients with a mandibular shift greater than 5 mm ( considered more than a moderate state of asymmetry).7 the ct data were collected for a total of 85 patients , and both right and left mnf images were analyzed .", "simplant version 14.0 ( materialise dental , leuven , belgium ) was used to reconstruct the ct images into 3d images .", "the anatomical reference points , lines , and planes were then set onto the image of the mandible .", "the 3d position of the mnf was measured based on the established reference points and planes .", "this process was intended to help the surgical team locate the mnf during mandibular ramus osteotomy .", "the anatomic points , lines , and planes that were used , as well as the measurements , are described below .", "the mnf , coronoid process , sigmoid notch , condyle , gonion , antegonial notch , menton , mesiobuccal cusp of the mandibular first molar , and the most superior incisal contact point of the lower central incisor were identified in the images .", "in addition , the points of the anterior and posterior ramus were defined ( table 1 ) .", "the anatomic lines used for defining the reference planes in this study are shown in table 2 .", "three vertical lines were constructed : one connecting the sigmoid notch points and antegonial notch points , termed the sn - agn line ; another connecting a sigmoid notch point and gonion point , termed the sn - gn line ; and the other connecting a condyle point and a gonion point , termed the cond - gn line . in addition", ", five horizontal lines were constructed that connected each of the anterior ramus points and each of the posterior ramus points . the occlusal plane , mandibular plane , and five reference horizontal planes were defined ( table 3 , fig .", "five vertical coronal planes were also defined , as shown in table 3 and figure 1b .", "the vertical heights of the anatomic points were measured according to each of the five horizontal planes ( fig .", "the vertical distances from the coronoid process , sigmoid notch , and condyle to each horizontal plane were added to the vertical distances from the gonion to each horizontal plane , and these distances were set as the vertical heights of the coronoid process , sigmoid notch , and condyle in the mandible for each horizontal plane ( table 4 ) .", "the vertical distances from the gonion to the horizontal planes were set as the heights of the mnf in the mandible , according to each horizontal plane .", "based on each of the five horizontal planes that pass through the mnf , the distance on each plane from the anterior ramus point to the posterior ramus point was designated the anteroposterior horizontal distance of the ramus ( table 4 ) .", "the perpendicular distance from each anterior ramus point to each vertical plane was designated the horizontal distance from the anterior ramus to the mnf ( fig .", "3 ) . to investigate the positional relationship of the mnf in the mandible , regression analysis was used to examine the rational relationships between the vertical heights of the coronoid process , sigmoid notch , and condyle points and the vertical height of the mnf .", "regression analysis was also used to determine the rational relationship between the anteroposterior horizontal distance of the ramus from each of the five horizontal planes passing through the mnf and the horizontal distance from the anterior ramus point to the mnf .", ", armonk , ny , usa ) , and statistical significance was established at p<0.05 . the intra - observer error for anatomic point determination was calculated via repeated measurement ( 10 measurements ) of the mnf ." ], [ "simplant version 14.0 ( materialise dental , leuven , belgium ) was used to reconstruct the ct images into 3d images .", "the anatomical reference points , lines , and planes were then set onto the image of the mandible .", "the 3d position of the mnf was measured based on the established reference points and planes .", "this process was intended to help the surgical team locate the mnf during mandibular ramus osteotomy .", "the anatomic points , lines , and planes that were used , as well as the measurements , are described below .", "the mnf , coronoid process , sigmoid notch , condyle , gonion , antegonial notch , menton , mesiobuccal cusp of the mandibular first molar , and the most superior incisal contact point of the lower central incisor were identified in the images .", "in addition , the points of the anterior and posterior ramus were defined ( table 1 ) .", "the anatomic lines used for defining the reference planes in this study are shown in table 2 .", "three vertical lines were constructed : one connecting the sigmoid notch points and antegonial notch points , termed the sn - agn line ; another connecting a sigmoid notch point and gonion point , termed the sn - gn line ; and the other connecting a condyle point and a gonion point , termed the cond - gn line . in addition", ", five horizontal lines were constructed that connected each of the anterior ramus points and each of the posterior ramus points .", "the occlusal plane , mandibular plane , and five reference horizontal planes were defined ( table 3 , fig .", "five vertical coronal planes were also defined , as shown in table 3 and figure 1b ." ], [ "the vertical heights of the anatomic points were measured according to each of the five horizontal planes ( fig .", "the vertical distances from the coronoid process , sigmoid notch , and condyle to each horizontal plane were added to the vertical distances from the gonion to each horizontal plane , and these distances were set as the vertical heights of the coronoid process , sigmoid notch , and condyle in the mandible for each horizontal plane ( table 4 ) .", "the vertical distances from the gonion to the horizontal planes were set as the heights of the mnf in the mandible , according to each horizontal plane . based on each of the five horizontal planes that pass through the mnf , the distance on each plane from the anterior ramus point to the posterior ramus point was designated the anteroposterior horizontal distance of the ramus ( table 4 ) .", "the perpendicular distance from each anterior ramus point to each vertical plane was designated the horizontal distance from the anterior ramus to the mnf ( fig ." ], [ "to investigate the positional relationship of the mnf in the mandible , regression analysis was used to examine the rational relationships between the vertical heights of the coronoid process , sigmoid notch , and condyle points and the vertical height of the mnf .", "regression analysis was also used to determine the rational relationship between the anteroposterior horizontal distance of the ramus from each of the five horizontal planes passing through the mnf and the horizontal distance from the anterior ramus point to the mnf .", ", armonk , ny , usa ) , and statistical significance was established at p<0.05 .", "the intra - observer error for anatomic point determination was calculated via repeated measurement ( 10 measurements ) of the mnf ." ], [ "the sex ratio and age of the 85 patients with skeletal class iii mandibular prognathism were 46 : 39 ( men : women ) and 22.25.5 years .", "the condyle height relative to the mnf - mandibular plane was the smallest ( 53.456.45 mm ) .", "the heights of the coronoid process and the mnf were the greatest when measured relative to the mnf - mandibular plane ( 60.095.96 mm and 21.313.83 mm , respectively ) .", "the height of the sigmoid notch was the greatest when based on the mnf - sn / gn p plane ( 43.895.02 mm ) .", "the height of the condyle was the greatest when based on the mnf - cond / gn p plane ( 63.805.79 mm ) .", "the heights of the mnf , coronoid process , and sigmoid notch were the smallest when based on the mnf - cond / gn p plane ( 18.604.56 mm , 50.086.89 mm , and 40.505.45 mm , respectively ) .", "the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf were the longest when they were based on the mnf - mandibular plane ( 42.985.17 mm and 27.604.22 mm , respectively ; table 5 ) .", "the heights of the coronoid process , sigmoid notch , and condylar head on the five horizontal planes were all significantly associated with the height of the mnf according to the regression analysis .", "the regression relationship was the strongest for the coronoid process , sigmoid notch , and condylar head when the measurements were based on the mnf - mandibular plane ( coefficients of determination ( r ) of 0.424 , 0.597 , and 0.604 respectively ) .", "the heights of the coronoid process and the mnf were significantly associated according to the regression analysis ( table 6 ) .", "the non - standardized regression coefficients were significant for the relationships between the heights of the coronoid process and the mnf based on all the horizontal reference planes .", "the highest coefficients , based on the mnf - mandibular plane , were a standardized regression coefficient of 0.654 and determination coefficient ( r ) of 0.424 .", "the heights of the sigmoid notch and the mnf were significantly associated in the regression analysis ( table 7 ) .", "the non - standardized regression coefficients for the relationships between the heights of the sigmoid notch and the mnf were significant for all the horizontal reference planes .", "the regression constant was highly significant when it was based on the mnf - mandibular plane ( p=0.002 ) .", "the highest coefficients were a standardized regression coefficient of 0.774 and determination coefficient of 0.597 , and these were based on the mnf - mandibular plane .", "the heights of the condylar head and the mnf were significantly related in the regression analysis ( table 8) .", "for all the horizontal reference planes , the non - standardized regression coefficients for the relationships between the heights of the condylar head and the mnf were significant .", "the regression constant was significant when it was based on all of the horizontal planes , except for the mnf - sn / gn p plane .", "the highest coefficients were a standardized regression coefficient of 0.779 and a determination coefficient of 0.604 , and were based on the mnf - mandibular plane . using the five horizontal planes as references , the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf", "for all the horizontal reference planes , the non - standardized regression coefficients were significant for the relationships of the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf .", "the regression constants were significant when the mnf - mandibular plane and mnf - sn / gn p plane were used as references .", "the standardized regression coefficients for all five horizontal planes used as references were greater than 0.73 , and the coefficient was 0.877 using the mnf - mandibular plane and 0.901 using the mnf - sn / gn p plane .", "the determination coefficient was 0.766 using the mnf - mandibular plane and 0.811 using the mnf - sn / gn p plane .", "notably , there were 53 missing values for measurements using the mnf - mandibular plane and 3 missing values for the mnf - sn / gn p plane .", "for the mnf - mandibular plane , which is parallel to the mnf , there were missing values when the anterior part of the plane sloped downward , thus leading directly to the mandibular body instead of the anterior ramus .", "similarly , there were missing values for the mnf - sn / gn p plane , although there were fewer than for the mnf - mandibular plane ." ], [ "the heights of the coronoid process , sigmoid notch , and condylar head on the five horizontal planes were all significantly associated with the height of the mnf according to the regression analysis .", "the regression relationship was the strongest for the coronoid process , sigmoid notch , and condylar head when the measurements were based on the mnf - mandibular plane ( coefficients of determination ( r ) of 0.424 , 0.597 , and 0.604 respectively ) .", "the heights of the coronoid process and the mnf were significantly associated according to the regression analysis ( table 6 ) .", "the non - standardized regression coefficients were significant for the relationships between the heights of the coronoid process and the mnf based on all the horizontal reference planes .", "the highest coefficients , based on the mnf - mandibular plane , were a standardized regression coefficient of 0.654 and determination coefficient ( r ) of 0.424 .", "the heights of the sigmoid notch and the mnf were significantly associated in the regression analysis ( table 7 ) .", "the non - standardized regression coefficients for the relationships between the heights of the sigmoid notch and the mnf were significant for all the horizontal reference planes .", "the regression constant was highly significant when it was based on the mnf - mandibular plane ( p=0.002 ) .", "the highest coefficients were a standardized regression coefficient of 0.774 and determination coefficient of 0.597 , and these were based on the mnf - mandibular plane .", "the heights of the condylar head and the mnf were significantly related in the regression analysis ( table 8) .", "for all the horizontal reference planes , the non - standardized regression coefficients for the relationships between the heights of the condylar head and the mnf were significant .", "the regression constant was significant when it was based on all of the horizontal planes , except for the mnf - sn / gn p plane .", "the highest coefficients were a standardized regression coefficient of 0.779 and a determination coefficient of 0.604 , and were based on the mnf - mandibular plane ." ], [ "using the five horizontal planes as references , the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf were significantly associated according to the regression analysis ( table 9 ) .", "for all the horizontal reference planes , the non - standardized regression coefficients were significant for the relationships of the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf .", "the regression constants were significant when the mnf - mandibular plane and mnf - sn / gn p plane were used as references .", "the standardized regression coefficients for all five horizontal planes used as references were greater than 0.73 , and the coefficient was 0.877 using the mnf - mandibular plane and 0.901 using the mnf - sn / gn p plane .", "the determination coefficient was 0.766 using the mnf - mandibular plane and 0.811 using the mnf - sn / gn p plane .", "notably , there were 53 missing values for measurements using the mnf - mandibular plane and 3 missing values for the mnf - sn / gn p plane .", "for the mnf - mandibular plane , which is parallel to the mnf , there were missing values when the anterior part of the plane sloped downward , thus leading directly to the mandibular body instead of the anterior ramus .", "similarly , there were missing values for the mnf - sn / gn p plane , although there were fewer than for the mnf - mandibular plane ." ], [ "the size of the human mandible varies greatly according to age , ethnicity , and sex.2 accordingly , the present study investigated the relative position of the mnf rather than simply measuring its position within the surrounding anatomical structures . using specific anatomic planes as references , we measured the position of the mnf in patients diagnosed with skeletal class iii malocclusion to determine the 3d position in the mandible .", "we determined the position of the mnf was related vertically to the heights of the mandibular condyle , sigmoid notch , and coronoid process and horizontally to the anteroposterior length of the mandibular ramus .", "further , we found that the mandibular plane was best for determining the relationships of the heights of the mandibular condyle , sigmoid notch , and coronoid process with the height of the mnf .", "this suggests that the mandibular plane can be utilized in clinical cases , not only for analyzing maxillofacial malformations but also for tracing the position of the mnf . during mandibular prognathism surgery , the vertical position of the mnf", "can be estimated using the exposed sigmoid notch area . with reference to the mandibular plane , the height ( in millimeters ) of the mnf", "can be estimated with the following equation : 0.621(the height of the sigmoid notch ) - 5.318 . as an alternative to the mandibular plane", ", one can use the horizontal plane perpendicular to the sn - gn line that connects the sigmoid notch and gonion ; this plane ( the mnf - sn / gn p plane ) is second to the mandibular plane in terms of usefulness for estimating the height of the mnf .", "when using the condyle height , the occlusal plane can be a useful alternative for estimating the height of the mnf .", "vertical ramus osteotomy is a surgical procedure that can be used for patients with mandibular prognathism when the mandibular ramus posterior to the mnf is cut .", "the relationship between the anteroposterior length of the mandibular ramus and the position of the mnf was best shown when the horizontal mnf - sn / gn p and mnf - mandibular planes were used as references . the mandibular ramus area at the mnf level is exposed during mandibular prognathism surgery .", "therefore , the relationship of the horizontal position of the mnf with the anteroposterior length of the ramus was investigated . using the mnf - sn /", "gn p plane as a reference , the horizontal length ( in mm ) from the anterior ramus to the mnf can be estimated with the following equation : 0.728(the anteroposterior distance of the mandibular ramus)- 3.948 . using the mnf - mandibular plane as the reference , the horizontal length ( in millimeters ) from the anterior ramus to the mnf", "can be estimated with the following equation : 0.716(the anteroposterior distance of the ascending ramus)- 3.148 . however , there are cases in which the mandibular plane leans in the anteroinferior direction , and the mnf - mandibular plane can not pass through the anterior ascending ramus area . in the present study", ", there were 53 missing values for the anterior ramus point when using the mnf - mandibular plane as the reference , and there were 3 missing values when using the mnf - sn / gn p plane . missing values may limit the usefulness of these calculations since they may occur in actual clinical cases .", "if this happens , it is recommended that the mnf - sn / gn p plane be used , and the use of the occlusal plane may be more helpful for determining the horizontal position of the mnf when the anterior ramus point can not be defined using the mnf - mandibular plane .", "the vertical height of the mnf from the gonion ranged from 18 mm to 21 mm , and the anteroposterior length of the ramus was about 33 mm to 42 mm .", "the horizontal distance from the anterior ramus point to the mnf was about 20 mm to 27 mm .", "the wide availability of ct scans has resulted in useful 3d images of the mandible . in a study that analyzed the 3d position of the mnf using cone - beam ct ,", "the average distance from the gonion to the mnf was about 24 mm and the average distance from the anterior ramus to the mnf in the axial plane was about 15 mm.2 the difference between the results of that study versus the present study is likely due to the use of the axial and sagittal planes of the ct images in the former . in another study that analyzed the 3d position of the mnf using multiple detector ct ,", "the average height from the gonion to the mnf relative to the occlusal plane was about 18 mm , the average anteroposterior length of the ascending ramus was 37 mm , and the average distance from the anterior ramus to the mnf on a horizontal plane ( parallel to the occlusal plane that passes through the mnf ) was about 22 mm.8 these results are similar to those of the present study , although there are minor differences because the present study only examined mandibles with skeletal iii class malocclusion , and the horizontal length from the anterior ramus to the mnf was measured as the perpendicular distance projected onto each vertical plane that passed through the mnf .", "simply measuring the vertical and horizontal distances may not be that beneficial in actual clinical cases because human mandibles vary in size .", "however , assessing the positional relationships using 3d planes can be useful clinically , as this shows the objective relationship with respect to the reference planes used in the analyses .", "image - assisted surgical techniques are increasingly used for maxillofacial operations ; accordingly , the present study is valuable in that it provides 3d image data that can be utilized in such surgical procedures .", "this study 's research methods and results , with respect to the positional 3d analysis of the mnf , will be useful for computer - assisted surgical methods , such as surgical simulation , navigation , augmented reality models , and robotic surgery .", "previous studies of the mandible were performed on cadavers,910 and often just a few distances were measured in 3d reconstructed images even though ct images were examined.234 importantly , previous studies performed quantitative analyses without considering the different sizes of the mandibles . moreover , even when 3d reconstruction images were used , few studies performed vertical and horizontal assessments using 3d reference planes .", "the present study found that the position of the mnf was related to the vertical heights of the sigmoid notch , coronoid process , and condyle and to the horizontal anteroposterior length of the ascending ramus .", "these findings indicate that the mnf , at least to some extent , maintains its relative position with the growth of the rami .", "in addition , the results indicate that the mnf is posterior to the ascending ramus .", "we expect that future studies will contribute further knowledge about the position of the mnf in the mandible via 3d image analysis of mandibles with skeletal class ii malocclusion for ramus surgery . in conclusion , the 3d mandibular image analysis data reported in this study will be useful for defining and describing the position of the mnf in mandibles of different sizes and shapes and in different ethnic groups .", "the present study showed that the proportional position of the mnf was significantly related to the vertical heights of the sigmoid notch , coronoid process , and condyle and to the horizontal anteroposterior length of the ascending ramus .", "furthermore , the methods used here and the positional 3d analyses of the mnf can be used clinically for computer - assisted surgical methods , especially for ramal surgery in patients with skeletal class iii mandibular prognathism ." ] ]

[ "dengue has become the most common mosquito - borne viral disease worldwide . according to estimates by the world health organization ( who )", ", the global incidence of dengue infections ranges between 50 million and 200 million every year .", "however , the real disease burden of dengue may be underestimated because of inadequate disease surveillance , misdiagnosis , and low levels of reporting .", "one recent study , based on 1780 country - years of mortality data from 130 countries and 1636 country - years of dengue case reports from 76 countries , estimated an average of 9221 dengue deaths per year between 1990 and 2013 .", "taiwan is no exception , and several dengue infection outbreaks have recently been reported here . in 2015 ,", "a large outbreak of dengue infections caused by dengue serotype 2 developed in tainan , taiwan , and a significant portion of patients with severe dengue infections required intensive care unit ( icu ) admission . because the outcomes of adult patients with dengue infections requiring icu admissions have rarely been investigated , we did this retrospective study to assess the clinical manifestations and the prognostic factors of patients critically ill with severe dengue infections .", "this study was done at chi mei medical center , a tertiary referral hospital with 96 adult icu beds . in this retrospective study ,", "all of the patients with laboratory - confirmed severe dengue infections and admitted to the icu were enrolled between july 31 and november 31 , 2015 , during the large outbreak period .", "the data were routinely collected and the analyses were done retrospectively . therefore , the study was approved by the institutional review board of chi mei medical center ( irb10503 - 005 ) , and informed consent was waived .", "the following information was collected : age , gender , and severity scores the acute physiology and chronic health evaluation ii ( apache ii ) score , therapeutic intervention scoring system ( tiss ) , and glasgow coma scale ( gcs ) score .", "underlying comorbidities included congestive heart failure , chronic lung diseases , end - stage renal disease , liver cirrhosis , diabetes mellitus , and cancer .", "we also measured immunocompromised conditions , associated infections and organisms , the number of multiorgan failures , the use of a mechanical ventilator and continuous renal replacement therapy ( crrt ) , associated laboratory data at or during admission , symptoms presented in the emergency room , and patient outcomes .", "laboratory - confirmed dengue cases included those with at least one of the following positive laboratory results : nonstructural protein 1 antigen test , dengue immunoglobulin - m , dengue immunoglobulin - g , a dengue polymerase chain reaction , or viral isolation .", "thoracic failure was defined as the ratio of the partial pressure of oxygen in the patient 's arterial blood to the fraction of oxygen in the inspired air less than 200 .", "cardiovascular failure was defined as a systolic blood pressure ( sbp ) of 90 mm hg or a mean arterial pressure ( map ) 65 mm hg for at least 1 hour despite adequate fluid resuscitation ; or the need for vasoactive agents ( dopamine 5 ", "mg / kg / min ) to maintain sbp 90 mm hg or map 65 mm hg .", "metabolic acidosis was defined as a ph 7.30 or a base deficit 5.0 meq / l and a plasma lactate level > 3 mmol / l .", "hematologic failure was considered a platelet count < 80,000/mm or a 50% decrease in the platelet count from the highest value recorded over the previous 3 days .", "kidney failure was considered oliguria with an average urine output < 0.5 ml / kg / h for 4 hours despite adequate fluid resuscitation or a creatinine level 2 mg / dl .", "hepatic failure was defined as a markedly increased serum bilirubin level 4 mg / dl . according to the who 2009 criteria", ", patients were classified as having dengue with warning signs if there were reports of abdominal pain or tenderness , vomiting , clinical fluid accumulation , mucosal bleeding , lethargy or restlessness , hepatomegaly , and a rise in hematocrit concurrent with a rapid drop in the platelet count .", "severe dengue ( group c ) criteria included the following symptoms : severe plasma leakage leading to shock or fluid accumulation with respiratory distress , severe bleeding , severe organ involvement , or transaminase levels > 1000 ", "those who were diagnosed with severe dengue by emergency physicians were admitted to the icu and included in this study .", "the focus of bacterial infections was diagnosed on the basis of clinical , laboratory , and radiologic findings .", "patients with bacteremia were divided into 2 subgroups : secondary bacteremia caused by another primary focus , such as a respiratory tract or urinary tract infection , and if no primary focus could be identified , the bacteremia was classified as primary .", "continuous variables are expressed as means standard deviation . the differences between groups , and between survivors and nonsurvivors at hospital discharge ,", "continuous data were compared using student t test or the wilcoxon rank - sum test .", "patients significantly associated with in - hospital mortality in univariate analysis ( p < ", "this study was done at chi mei medical center , a tertiary referral hospital with 96 adult icu beds . in this retrospective study ,", "all of the patients with laboratory - confirmed severe dengue infections and admitted to the icu were enrolled between july 31 and november 31 , 2015 , during the large outbreak period .", "the data were routinely collected and the analyses were done retrospectively . therefore , the study was approved by the institutional review board of chi mei medical center ( irb10503 - 005 ) , and informed consent was waived .", "the following information was collected : age , gender , and severity scores the acute physiology and chronic health evaluation ii ( apache ii ) score , therapeutic intervention scoring system ( tiss ) , and glasgow coma scale ( gcs ) score .", "underlying comorbidities included congestive heart failure , chronic lung diseases , end - stage renal disease , liver cirrhosis , diabetes mellitus , and cancer .", "we also measured immunocompromised conditions , associated infections and organisms , the number of multiorgan failures , the use of a mechanical ventilator and continuous renal replacement therapy ( crrt ) , associated laboratory data at or during admission , symptoms presented in the emergency room , and patient outcomes .", "laboratory - confirmed dengue cases included those with at least one of the following positive laboratory results : nonstructural protein 1 antigen test , dengue immunoglobulin - m , dengue immunoglobulin - g , a dengue polymerase chain reaction , or viral isolation .", "thoracic failure was defined as the ratio of the partial pressure of oxygen in the patient 's arterial blood to the fraction of oxygen in the inspired air less than 200 .", "cardiovascular failure was defined as a systolic blood pressure ( sbp ) of 90 mm hg or a mean arterial pressure ( map ) 65 mm hg for at least 1 hour despite adequate fluid resuscitation ; or the need for vasoactive agents ( dopamine 5 ", "mg / kg / min ) to maintain sbp 90 mm hg or map 65 mm hg .", "metabolic acidosis was defined as a ph 7.30 or a base deficit 5.0 meq / l and a plasma lactate level > 3 mmol / l .", "hematologic failure was considered a platelet count < 80,000/mm or a 50% decrease in the platelet count from the highest value recorded over the previous 3 days .", "kidney failure was considered oliguria with an average urine output < 0.5 ml / kg / h for 4 hours despite adequate fluid resuscitation or a creatinine level 2 mg / dl .", "hepatic failure was defined as a markedly increased serum bilirubin level 4 mg / dl . according to the who 2009 criteria", ", patients were classified as having dengue with warning signs if there were reports of abdominal pain or tenderness , vomiting , clinical fluid accumulation , mucosal bleeding , lethargy or restlessness , hepatomegaly , and a rise in hematocrit concurrent with a rapid drop in the platelet count .", "severe dengue ( group c ) criteria included the following symptoms : severe plasma leakage leading to shock or fluid accumulation with respiratory distress , severe bleeding , severe organ involvement , or transaminase levels > 1000 ", "those who were diagnosed with severe dengue by emergency physicians were admitted to the icu and included in this study .", "the focus of bacterial infections was diagnosed on the basis of clinical , laboratory , and radiologic findings .", "patients with bacteremia were divided into 2 subgroups : secondary bacteremia caused by another primary focus , such as a respiratory tract or urinary tract infection , and if no primary focus could be identified , the bacteremia was classified as primary .", "continuous variables are expressed as means standard deviation . the differences between groups , and between survivors and nonsurvivors at hospital discharge ,", "continuous data were compared using student t test or the wilcoxon rank - sum test .", "patients significantly associated with in - hospital mortality in univariate analysis ( p < ", "during the study period , there were 4787 patients with dengue infections , 143 ( 2.99% ) of whom were critically ill and required icu admission ( mean age : 69.7 years ; age range : 694 years ) ( table 1 ) .", "thirty - three of these patients died ( mortality rate : 23.1% for icu patients and", "the average apache ii , tiss , and gcs scores were 17.9 , 22.8 , and 12.0 , respectively .", "hypertension ( n = 90 ; 62.9% ) and diabetes mellitus ( n = 70 ; 49.0% ) were the 2 most common comorbidities .", "eighty patients had cobacterial infections : the lungs ( pneumonia ) ( n = 40 ) and the urinary tract ( n = 33 ) were the most common sites .", "thirty - three of the 80 patients had cobacteremia : 11 primary bacteremia cases and 22 secondary bacteremia cases ; 59 ( 41.3% ) had multiorgan failure : the hematologic system failed most frequently , followed by thoracic and cardiovascular systems .", "overall , there were 50 ( 35.0% ) patients who required mechanical ventilation and 20 ( 14.0% ) who required crrt .", "the patients who died in hospital had significantly higher apache ii , tiss , and gcs scores , and more hypertension , bacteremia , pneumonia , and organ failure ( cardiovascular , thoracic , metabolic , renal and hepatic failures , and multiorgan failure ) and needed more mechanical ventilation and crrt than did survivors .", ". the levels of procalcitonin and c - reactive protein ( crp ) were 12.3 ( 34.7 ) g / l and 49.0 ( 72.8 ) mg / l , respectively ( table 2 ) .", "the mean levels of the n - terminal prohormone brain natriuretic peptide and of lactate were 4788.5 ( 7492.8 ) and 3.4 ( 4.1 ) , respectively .", "the lowest platelet count was 35,000 ( 43,000)/mm , and it recovered to 151,000 ( 107,000)/mm before the patients were discharged from the icu .", "the highest levels of aspartate aminotransferase ( ast / got ) and alanine aminotransferase ( alt / gpt ) were 1126.4 ( 2488.5 ) ", "the patients who died had significantly higher activated partial thromboplastin time , lactate , ast / got , and alt / gpt levels than did survivors .", "the patients who died , however , had significantly lower albumin , hemoglobin , and hematocrit , and lower platelet counts in the trough and recovery stage before they were discharged from the icu than did survivors .", "fever was the most common presentation symptom ( n = 112 ; 78.3% ) , followed by anorexia ( n = 47 ; 32.9% ) and abdominal pain ( n = 46 ; 32.2% ) ( table 3 ) . the gastrointestinal tract ( n = 45 ; 31.5% ) , the genitourinary tract ( n = 26 ; 18.2% ) , and the gums ( n = 3 ; 2.1% ) were the most common sites , in that order , for bleeding .", "there were no significant differences in the symptoms , the overall icu 7.8 ( 10.3 ) and hospital - stay 14.7 ( 16.1 ) days , or hospital costs between survivors and patients who died .", "mortality was highest for 60- to 69-year - olds ( 8 died [ 36.4% ] ; 14 survived ) , 70- to 79-year - olds ( 16 died [ 24.2% ] ; 50 survived ) , and 80- to 89-year - olds ( 7 died [ 23.3% ] ; 23 survived ) ( fig .", "multiple logistic regression analysis showed that icu mortality was significantly positively associated with the following independent predictors : lower gcss , lower platelet counts , and more organ failures before icu discharge ( table 4 ) .", "this is the first study that focuses on dengue patients in taiwan who require icu admission and is one of few studies that investigates this topic anywhere in the world .", "first , we found that the mortality of this specific group remains as high as 23.1% , which is higher than reported by other studies . in 1 study of 72 critically ill patients with dengue in northern india , 8 patients died ( mortality : 11.1% ) . in another of 198 icu patients with dengue in new delhi , 12 patients died ( mortality : 6.1% ) .", "in contrast , another multicenter study of 42 adults with dengue hemorrhagic fever or dengue shock syndrome admitted to icu in india reported 8 in - icu deaths ( mortality : 19% ) . a study in brazil of 97 adult patients with dengue admitted to the icu reported in - icu and in - hospital mortality rates of 18.6% and 19.6% , respectively .", "the differences between our study and others might be the differences in disease severity . in our study , the mean apache ii score was 17.9 9.6 , higher than the 7.5 and the 11 in 2 of the others .", "in addition , only group c ( severe dengue patients with warning signs and organ failure ) were enrolled in our study , which probably explains why the outcomes of our study were worse .", "in addition , the mean age of our enrolled patients was about 70 years , older than 2 of the other studies ; age itself might also partially contribute to the higher mortality rate .", "second , univariate analysis showed that the patients who died had cases of dengue that were more severe , based on apache ii , tiss , and gcs scores , than did survivors .", "this is similar with other reports that in - hospital mortality was associated with apache ii scores and sequential organ failure assessment scores .", "in contrast , after multivariate analysis , only the association between lower gcs scores and in - hospital mortality remained significant in the present study .", "our finding might indicate that gcs scores , in addition to other commonly used severity score systems , are good predictors of the outcomes of patients with severe dengue .", "third , a multivariate analysis showed that a higher number of organ failures was independently associated with mortality in the present study .", "like another study which reported that some common etiologies of multiple organ dysfunction dengue shock syndrome and multiple organ dysfunction syndrome directly related to the organ failure index present a significant risk of mortality , we found that patients who died had more thoracic ( 72.2% vs 20.0% ) , cardiovascular ( 72.7% vs 17.3% ) , and multiorgan ( 93.9% vs 25.5% ) failures than did survivors ( both p < 0.0001 ) .", "other studies have reported that patients who die were more likely to have cardiovascular ( 100% vs 12% ) or respiratory ( 88% vs 12% ) failures than were survivors ( both p < 0.01 ) , that a dengue viral infection with an acute respiratory failure is a significant mortality risk , and that a lower platelet count before being discharged from the icu was associated with higher in - hospital mortality .", "finally , we found that 80 ( 55.9% ) of our patients developed bacterial infections in the icu .", "moreover , 15 ( 45.5% ) of the 33 patients who died had pneumonia , and 13 ( 39.4% ) had bacteremia . in another study , 45 ( 46.4% )", "patients had had been treated with antibiotics , and 7 ( 36.8% ) of 19 deaths were presumably attributed to bacterial infections associated with dengue . in the present study , the most common pathogen in the 33 cases of confirmed bacteremia was escherichia coli , followed by staphylococcus aureus ( s aureus ) , and streptococcus pp . despite our finding that s aureus was the most", "commonly reported copathogen , different from that of other reports , the present study and other reports might indicate the clinical significance of bacterial infections in patients with severe dengue .", "it should at least emphasize the importance of an early diagnosis of cobacterial infections and prompt antibiotic treatment of patients critically ill with dengue . in the present study ,", "crp levels were nonsignificantly higher in patients who died ( 66.9 ) than in survivors ( 43.4 ) , which does not support the hypothesis that crp is an early predictor of dengue severity in adult patients .", "we enrolled only patients with severe dengue in the present study and found that crp was a limited outcome predictor in patients with severe dengue .", "we also found that albumin levels were significantly lower ( 2.8 0.6 vs 3.1 0.5 ) and lactate significantly higher ( 6.2 6.7 vs 2.4 1.9 ) in patients who died than in survivors , which was consistent with other studies that low serum albumin levels on icu admission were associated with worse outcomes .", "although a multivariate analysis eliminated the significance in our study , it still indicates an association between albumin levels and the outcomes in patients with severe dengue .", "first , because it is a single - center study , the number of cases is limited .", "second , because of the study design of a retrospective investigation , our data collection might have been inadequate .", "third , we did not test the serotypes of dengue virus because our laboratory can not do that type of examination .", "however , several studies have reported the association between dengue virus serotypes and the severity of dengue infection .", "in addition , the predictors of icu mortality by multiple logistic regression analysis should be considered as the association with mortality but not the biological causes of death . finally , despite several reports showed the association between different medications , such as n - acetyl cysteine , factor vii , diuretic , and fluid administration , and the outcome of dengue patients", "the mortality of severe dengue patients admitted to the icu remains high , and the mortality was associated with lower gcs scores , lower platelet count before being discharged from the icu discharge , and more organ failures . in addition , a significant portion of patients with severe dengue who die in the icu have bacterial infections ." ]

[ "<S> abstractoutcomes of adult patients with dengue infections requiring intensive care unit ( icu ) admissions remain unclear . </S>", "<S> we assessed the clinical manifestations and prognostic factors of patients critically ill with severe dengue.this retrospective study was done in a tertiary referral hospital with 96 adult icu beds . </S>", "<S> all of the patients with laboratory - confirmed severe dengue infections and admitted to the icu were enrolled between july 31 and november 31 , 2015 , during the large outbreak period . </S>", "<S> the medical records of all the recruited patients were reviewed for the following information : age , gender , clinical manifestations , disease severity scores , underlying conditions , laboratory examinations , and outcomes . </S>", "<S> the primary endpoint was to find the predictors of icu mortality.during the study period , 4787 patients with dengue infections required icu admission . </S>", "<S> one hundred forty - three ( 2.99% ) were critically ill ( mean age : 69.7 years ) . </S>", "<S> hypertension ( n = 90 , 62.9% ) and diabetes mellitus ( n = 70 , 49.0% ) were the 2 most common underlying diseases . </S>", "<S> eighty critically ill patients ( 55.9% ) had cobacterial infections , and 33 had cobacteremia . </S>", "<S> the hematologic system failed most often , followed by thoracic and cardiovascular systems . </S>", "<S> fever was the most common presentation ( n = 112 ; 78.3% ) , followed by anorexia ( n = 47 ; 32.9% ) and abdominal pain ( n = 46 ; 32.2% ) . </S>", "<S> overall , 33 patients died ( mortality rate : 23.1% ) . </S>", "<S> multivariate analysis showed that icu mortality was significantly associated with lower glasgow coma scale ( gcs ) scores , lower platelet counts before icu discharge , and more organ failures.the number of severe dengue patients who require icu admission remains high . </S>", "<S> the mortality rate was associated with lower gcs scores , lower platelet counts , and more organ failures . in addition </S>", "<S> , more than half of the critically ill dengue patients had comorbid bacterial infections . </S>" ]

[ "Introduction", "Methods", "Patients and hospital setting", "Variables measured", "Definitions", "Statistical analysis", "Results", "Discussion" ]

[ [ "dengue has become the most common mosquito - borne viral disease worldwide . according to estimates by the world health organization ( who )", ", the global incidence of dengue infections ranges between 50 million and 200 million every year .", "however , the real disease burden of dengue may be underestimated because of inadequate disease surveillance , misdiagnosis , and low levels of reporting .", "one recent study , based on 1780 country - years of mortality data from 130 countries and 1636 country - years of dengue case reports from 76 countries , estimated an average of 9221 dengue deaths per year between 1990 and 2013 .", "taiwan is no exception , and several dengue infection outbreaks have recently been reported here . in 2015 ,", "a large outbreak of dengue infections caused by dengue serotype 2 developed in tainan , taiwan , and a significant portion of patients with severe dengue infections required intensive care unit ( icu ) admission . because the outcomes of adult patients with dengue infections requiring icu admissions have rarely been investigated , we did this retrospective study to assess the clinical manifestations and the prognostic factors of patients critically ill with severe dengue infections ." ], [ "this study was done at chi mei medical center , a tertiary referral hospital with 96 adult icu beds . in this retrospective study ,", "all of the patients with laboratory - confirmed severe dengue infections and admitted to the icu were enrolled between july 31 and november 31 , 2015 , during the large outbreak period .", "the data were routinely collected and the analyses were done retrospectively . therefore , the study was approved by the institutional review board of chi mei medical center ( irb10503 - 005 ) , and informed consent was waived .", "the following information was collected : age , gender , and severity scores the acute physiology and chronic health evaluation ii ( apache ii ) score , therapeutic intervention scoring system ( tiss ) , and glasgow coma scale ( gcs ) score .", "underlying comorbidities included congestive heart failure , chronic lung diseases , end - stage renal disease , liver cirrhosis , diabetes mellitus , and cancer .", "we also measured immunocompromised conditions , associated infections and organisms , the number of multiorgan failures , the use of a mechanical ventilator and continuous renal replacement therapy ( crrt ) , associated laboratory data at or during admission , symptoms presented in the emergency room , and patient outcomes .", "laboratory - confirmed dengue cases included those with at least one of the following positive laboratory results : nonstructural protein 1 antigen test , dengue immunoglobulin - m , dengue immunoglobulin - g , a dengue polymerase chain reaction , or viral isolation .", "thoracic failure was defined as the ratio of the partial pressure of oxygen in the patient 's arterial blood to the fraction of oxygen in the inspired air less than 200 .", "cardiovascular failure was defined as a systolic blood pressure ( sbp ) of 90 mm hg or a mean arterial pressure ( map ) 65 mm hg for at least 1 hour despite adequate fluid resuscitation ; or the need for vasoactive agents ( dopamine 5 ", "mg / kg / min ) to maintain sbp 90 mm hg or map 65 mm hg .", "metabolic acidosis was defined as a ph 7.30 or a base deficit 5.0 meq / l and a plasma lactate level > 3 mmol / l .", "hematologic failure was considered a platelet count < 80,000/mm or a 50% decrease in the platelet count from the highest value recorded over the previous 3 days .", "kidney failure was considered oliguria with an average urine output < 0.5 ml / kg / h for 4 hours despite adequate fluid resuscitation or a creatinine level 2 mg / dl .", "hepatic failure was defined as a markedly increased serum bilirubin level 4 mg / dl . according to the who 2009 criteria", ", patients were classified as having dengue with warning signs if there were reports of abdominal pain or tenderness , vomiting , clinical fluid accumulation , mucosal bleeding , lethargy or restlessness , hepatomegaly , and a rise in hematocrit concurrent with a rapid drop in the platelet count .", "severe dengue ( group c ) criteria included the following symptoms : severe plasma leakage leading to shock or fluid accumulation with respiratory distress , severe bleeding , severe organ involvement , or transaminase levels > 1000 ", "those who were diagnosed with severe dengue by emergency physicians were admitted to the icu and included in this study .", "the focus of bacterial infections was diagnosed on the basis of clinical , laboratory , and radiologic findings .", "patients with bacteremia were divided into 2 subgroups : secondary bacteremia caused by another primary focus , such as a respiratory tract or urinary tract infection , and if no primary focus could be identified , the bacteremia was classified as primary .", "continuous variables are expressed as means standard deviation . the differences between groups , and between survivors and nonsurvivors at hospital discharge ,", "continuous data were compared using student t test or the wilcoxon rank - sum test .", "patients significantly associated with in - hospital mortality in univariate analysis ( p < " ], [ "this study was done at chi mei medical center , a tertiary referral hospital with 96 adult icu beds . in this retrospective study ,", "all of the patients with laboratory - confirmed severe dengue infections and admitted to the icu were enrolled between july 31 and november 31 , 2015 , during the large outbreak period .", "the data were routinely collected and the analyses were done retrospectively . therefore , the study was approved by the institutional review board of chi mei medical center ( irb10503 - 005 ) , and informed consent was waived ." ], [ "the following information was collected : age , gender , and severity scores the acute physiology and chronic health evaluation ii ( apache ii ) score , therapeutic intervention scoring system ( tiss ) , and glasgow coma scale ( gcs ) score .", "underlying comorbidities included congestive heart failure , chronic lung diseases , end - stage renal disease , liver cirrhosis , diabetes mellitus , and cancer .", "we also measured immunocompromised conditions , associated infections and organisms , the number of multiorgan failures , the use of a mechanical ventilator and continuous renal replacement therapy ( crrt ) , associated laboratory data at or during admission , symptoms presented in the emergency room , and patient outcomes ." ], [ "laboratory - confirmed dengue cases included those with at least one of the following positive laboratory results : nonstructural protein 1 antigen test , dengue immunoglobulin - m , dengue immunoglobulin - g , a dengue polymerase chain reaction , or viral isolation .", "thoracic failure was defined as the ratio of the partial pressure of oxygen in the patient 's arterial blood to the fraction of oxygen in the inspired air less than 200 .", "cardiovascular failure was defined as a systolic blood pressure ( sbp ) of 90 mm hg or a mean arterial pressure ( map ) 65 mm hg for at least 1 hour despite adequate fluid resuscitation ; or the need for vasoactive agents ( dopamine 5 ", "mg / kg / min ) to maintain sbp 90 mm hg or map 65 mm hg .", "metabolic acidosis was defined as a ph 7.30 or a base deficit 5.0 meq / l and a plasma lactate level > 3 mmol / l .", "hematologic failure was considered a platelet count < 80,000/mm or a 50% decrease in the platelet count from the highest value recorded over the previous 3 days .", "kidney failure was considered oliguria with an average urine output < 0.5 ml / kg / h for 4 hours despite adequate fluid resuscitation or a creatinine level 2 mg / dl .", "hepatic failure was defined as a markedly increased serum bilirubin level 4 mg / dl . according to the who 2009 criteria", ", patients were classified as having dengue with warning signs if there were reports of abdominal pain or tenderness , vomiting , clinical fluid accumulation , mucosal bleeding , lethargy or restlessness , hepatomegaly , and a rise in hematocrit concurrent with a rapid drop in the platelet count .", "severe dengue ( group c ) criteria included the following symptoms : severe plasma leakage leading to shock or fluid accumulation with respiratory distress , severe bleeding , severe organ involvement , or transaminase levels > 1000 ", "those who were diagnosed with severe dengue by emergency physicians were admitted to the icu and included in this study .", "the focus of bacterial infections was diagnosed on the basis of clinical , laboratory , and radiologic findings .", "patients with bacteremia were divided into 2 subgroups : secondary bacteremia caused by another primary focus , such as a respiratory tract or urinary tract infection , and if no primary focus could be identified , the bacteremia was classified as primary ." ], [ "continuous variables are expressed as means standard deviation . the differences between groups , and between survivors and nonsurvivors at hospital discharge ,", "continuous data were compared using student t test or the wilcoxon rank - sum test .", "patients significantly associated with in - hospital mortality in univariate analysis ( p < " ], [ "during the study period , there were 4787 patients with dengue infections , 143 ( 2.99% ) of whom were critically ill and required icu admission ( mean age : 69.7 years ; age range : 694 years ) ( table 1 ) .", "thirty - three of these patients died ( mortality rate : 23.1% for icu patients and", "the average apache ii , tiss , and gcs scores were 17.9 , 22.8 , and 12.0 , respectively .", "hypertension ( n = 90 ; 62.9% ) and diabetes mellitus ( n = 70 ; 49.0% ) were the 2 most common comorbidities .", "eighty patients had cobacterial infections : the lungs ( pneumonia ) ( n = 40 ) and the urinary tract ( n = 33 ) were the most common sites .", "thirty - three of the 80 patients had cobacteremia : 11 primary bacteremia cases and 22 secondary bacteremia cases ; 59 ( 41.3% ) had multiorgan failure : the hematologic system failed most frequently , followed by thoracic and cardiovascular systems .", "overall , there were 50 ( 35.0% ) patients who required mechanical ventilation and 20 ( 14.0% ) who required crrt .", "the patients who died in hospital had significantly higher apache ii , tiss , and gcs scores , and more hypertension , bacteremia , pneumonia , and organ failure ( cardiovascular , thoracic , metabolic , renal and hepatic failures , and multiorgan failure ) and needed more mechanical ventilation and crrt than did survivors .", ". the levels of procalcitonin and c - reactive protein ( crp ) were 12.3 ( 34.7 ) g / l and 49.0 ( 72.8 ) mg / l , respectively ( table 2 ) .", "the mean levels of the n - terminal prohormone brain natriuretic peptide and of lactate were 4788.5 ( 7492.8 ) and 3.4 ( 4.1 ) , respectively .", "the lowest platelet count was 35,000 ( 43,000)/mm , and it recovered to 151,000 ( 107,000)/mm before the patients were discharged from the icu .", "the highest levels of aspartate aminotransferase ( ast / got ) and alanine aminotransferase ( alt / gpt ) were 1126.4 ( 2488.5 ) ", "the patients who died had significantly higher activated partial thromboplastin time , lactate , ast / got , and alt / gpt levels than did survivors .", "the patients who died , however , had significantly lower albumin , hemoglobin , and hematocrit , and lower platelet counts in the trough and recovery stage before they were discharged from the icu than did survivors .", "fever was the most common presentation symptom ( n = 112 ; 78.3% ) , followed by anorexia ( n = 47 ; 32.9% ) and abdominal pain ( n = 46 ; 32.2% ) ( table 3 ) . the gastrointestinal tract ( n = 45 ; 31.5% ) , the genitourinary tract ( n = 26 ; 18.2% ) , and the gums ( n = 3 ; 2.1% ) were the most common sites , in that order , for bleeding .", "there were no significant differences in the symptoms , the overall icu 7.8 ( 10.3 ) and hospital - stay 14.7 ( 16.1 ) days , or hospital costs between survivors and patients who died .", "mortality was highest for 60- to 69-year - olds ( 8 died [ 36.4% ] ; 14 survived ) , 70- to 79-year - olds ( 16 died [ 24.2% ] ; 50 survived ) , and 80- to 89-year - olds ( 7 died [ 23.3% ] ; 23 survived ) ( fig .", "multiple logistic regression analysis showed that icu mortality was significantly positively associated with the following independent predictors : lower gcss , lower platelet counts , and more organ failures before icu discharge ( table 4 ) ." ], [ "this is the first study that focuses on dengue patients in taiwan who require icu admission and is one of few studies that investigates this topic anywhere in the world .", "first , we found that the mortality of this specific group remains as high as 23.1% , which is higher than reported by other studies . in 1 study of 72 critically ill patients with dengue in northern india , 8 patients died ( mortality : 11.1% ) . in another of 198 icu patients with dengue in new delhi , 12 patients died ( mortality : 6.1% ) .", "in contrast , another multicenter study of 42 adults with dengue hemorrhagic fever or dengue shock syndrome admitted to icu in india reported 8 in - icu deaths ( mortality : 19% ) . a study in brazil of 97 adult patients with dengue admitted to the icu reported in - icu and in - hospital mortality rates of 18.6% and 19.6% , respectively .", "the differences between our study and others might be the differences in disease severity . in our study , the mean apache ii score was 17.9 9.6 , higher than the 7.5 and the 11 in 2 of the others .", "in addition , only group c ( severe dengue patients with warning signs and organ failure ) were enrolled in our study , which probably explains why the outcomes of our study were worse .", "in addition , the mean age of our enrolled patients was about 70 years , older than 2 of the other studies ; age itself might also partially contribute to the higher mortality rate .", "second , univariate analysis showed that the patients who died had cases of dengue that were more severe , based on apache ii , tiss , and gcs scores , than did survivors .", "this is similar with other reports that in - hospital mortality was associated with apache ii scores and sequential organ failure assessment scores .", "in contrast , after multivariate analysis , only the association between lower gcs scores and in - hospital mortality remained significant in the present study .", "our finding might indicate that gcs scores , in addition to other commonly used severity score systems , are good predictors of the outcomes of patients with severe dengue .", "third , a multivariate analysis showed that a higher number of organ failures was independently associated with mortality in the present study .", "like another study which reported that some common etiologies of multiple organ dysfunction dengue shock syndrome and multiple organ dysfunction syndrome directly related to the organ failure index present a significant risk of mortality , we found that patients who died had more thoracic ( 72.2% vs 20.0% ) , cardiovascular ( 72.7% vs 17.3% ) , and multiorgan ( 93.9% vs 25.5% ) failures than did survivors ( both p < 0.0001 ) .", "other studies have reported that patients who die were more likely to have cardiovascular ( 100% vs 12% ) or respiratory ( 88% vs 12% ) failures than were survivors ( both p < 0.01 ) , that a dengue viral infection with an acute respiratory failure is a significant mortality risk , and that a lower platelet count before being discharged from the icu was associated with higher in - hospital mortality .", "finally , we found that 80 ( 55.9% ) of our patients developed bacterial infections in the icu .", "moreover , 15 ( 45.5% ) of the 33 patients who died had pneumonia , and 13 ( 39.4% ) had bacteremia . in another study , 45 ( 46.4% )", "patients had had been treated with antibiotics , and 7 ( 36.8% ) of 19 deaths were presumably attributed to bacterial infections associated with dengue . in the present study , the most common pathogen in the 33 cases of confirmed bacteremia was escherichia coli , followed by staphylococcus aureus ( s aureus ) , and streptococcus pp . despite our finding that s aureus was the most", "commonly reported copathogen , different from that of other reports , the present study and other reports might indicate the clinical significance of bacterial infections in patients with severe dengue .", "it should at least emphasize the importance of an early diagnosis of cobacterial infections and prompt antibiotic treatment of patients critically ill with dengue . in the present study ,", "crp levels were nonsignificantly higher in patients who died ( 66.9 ) than in survivors ( 43.4 ) , which does not support the hypothesis that crp is an early predictor of dengue severity in adult patients .", "we enrolled only patients with severe dengue in the present study and found that crp was a limited outcome predictor in patients with severe dengue .", "we also found that albumin levels were significantly lower ( 2.8 0.6 vs 3.1 0.5 ) and lactate significantly higher ( 6.2 6.7 vs 2.4 1.9 ) in patients who died than in survivors , which was consistent with other studies that low serum albumin levels on icu admission were associated with worse outcomes .", "although a multivariate analysis eliminated the significance in our study , it still indicates an association between albumin levels and the outcomes in patients with severe dengue .", "first , because it is a single - center study , the number of cases is limited .", "second , because of the study design of a retrospective investigation , our data collection might have been inadequate .", "third , we did not test the serotypes of dengue virus because our laboratory can not do that type of examination .", "however , several studies have reported the association between dengue virus serotypes and the severity of dengue infection .", "in addition , the predictors of icu mortality by multiple logistic regression analysis should be considered as the association with mortality but not the biological causes of death . finally , despite several reports showed the association between different medications , such as n - acetyl cysteine , factor vii , diuretic , and fluid administration , and the outcome of dengue patients", "the mortality of severe dengue patients admitted to the icu remains high , and the mortality was associated with lower gcs scores , lower platelet count before being discharged from the icu discharge , and more organ failures . in addition , a significant portion of patients with severe dengue who die in the icu have bacterial infections ." ] ]

[ "implant - supported fixed dental prosthesis is a standardized restoration method that is often used when an aesthetically critical anterior single tooth is missing .", "it has similar 5-year survival rates as tooth - supported fixed dental prosthesis.12 although the success rate of an all - ceramic crown was reported to be lower than that of a metal - ceramic crown , all - ceramic crowns displayed similar success rates in both tooth - supported fixed dental prosthesis and implant - supported fixed dental prosthesis.234 titanium is a stable implant material , and titanium abutments have the advantage of supporting gingival health and preventing galvanic reaction between the fixture and abutment.56 however , when a titanium abutment is used in thin peri - implant mucosa in the anterior area , the metal part can be detected through the mucosa .", "for this reason , studies on alumina and zirconia , which are high - strength ceramics , have been conducted , and the study on implant abutments using zirconia has advanced , showing excellent material properties and biocompatibility .", "78 the influence of the type of connection between a zirconia abutment and a titanium fixture were addressed previously .", "the types of implant - abutment connection can be divided into two major groups - internal connection and external connection .", "sailer et al.910 reported that the internal implant - abutment connection type , including zirconia abutment with titanium insert , showed the highest strength , followed by external implant - abutment connection type and 1-piece internal type , when artificial aging was not conducted .", "however , in the above - mentioned experiment , only the implant - abutment fracture load was measured , without involvement of the crowns .", "therefore , the fracture load was measured under non - physiological conditions , and intra - oral temperature changes and dynamic functional load were not applied .", "they showed that the fracture load was dependent on the types of connection , which was the same result as that of the former studies , while the strength was lower after artificial aging .", "however , that study faced the limitation that it can not be applied to actual clinical situations , because the fracture load of the zirconia abutment was measured without crown restoration , and the fracture loads before and after artificial aging were not examined . due to the development of cad / cam systems ( computer - aided design / computer - aided manufacturing system )", ", the prosthesis using zirconia has replaced the conventional ceramic restoration and is now additionally being applied to implant abutments.1213 park et al.14 investigated the fit of a customized zirconia abutment manufactured by the cad / cam system .", "they reported that while the fit was less precise than that of a prefabricated zirconia abutment , it was within the clinically acceptable range .", "the strength of the customized zirconia abutment was significantly higher than that of the prefabricated zirconia abutment . when restoring all - ceramic implant crowns , shoulder or deep chamfer preparation of zirconia abutment", "koutayas et al.15 measured the fracture loads of 1-piece internal full zirconia abutments with three preparation depths ( 0.5 mm , 0.7 mm , and 0.9 mm ) that were restored using lithium disilicate .", "they reported that the fracture loads of all three specimens were higher than physiologic masticatory force , but preparation depths of over 0.7 mm were not recommended .", "subsequent to that report , mitsias et al.16 reported measurement of the fracture load of 1-piece internal full zirconia abutment with artificial aging .", "preparation depths under 0.9 mm were found to have had no influence on the fracture load .", "many literature reports have stated that the optimum preparation depth of zirconia abutment ranged from 0.5 mm to 1.0 mm.171819202122 however , further studies regarding standard indicators are still needed .", "therefore , the purpose of this study was to investigate the change of fracture load of customized zirconia abutments with titanium insert according to the different preparation depths , with aging and chewing simulation .", "the null hypotheses were that the fracture loads of customized zirconia abutment with titanium inserts will not change depending on the various preparation depths , or as a result of aging and chewing simulation .", "a commercial titanium fixture of 4.5 mm in diameter and 10 mm in length ( anyridge , megagen , gyeongsan , korea ) was used .", "the fixture was embedded in acrylic resin ( orthoresin dentsply , weybridge , surrey , uk ) according to the iso - normed protocol [ iso 14801].23 prefabricated titanium inserts that fit into the titanium fixture ( prop abutment , megagen , gyeongsan , korea ) were used , and the zirconia supra - structures to be attached to the insert were fabricated with zirconia blocks ( zenostarzr translucent , wieland , germany ) ( table 1 ) .", "the preparation depths of the zirconia suprastructure were designed to 0.5 mm , 0.7 mm and 0.9 mm using the cad system ( 3shape dental designer premium 2013 , 3shape , denmark ) , and the same - sized zirconia structures were manufactured using the cam system ( zenotec t1 , wieland dental + technik gmbh , pforzheim , germany ) .", "the customized zirconia suprastructures and prefabricated titanium inserts were then bonded using dual cure self - adhesive resin cement ( rely x unicem , 3 m espe , st .", "crowns fabricated to replace a maxillary right central incisor in asian adult ( 8.6 mm in width and 11.9 mm in length ) were made using lithium disilicate ( ips e.max press , ivoclar - vivadent , schaan , liechtenstein ) ( table 1).24 crowns were designed to fit over the customized zirconia abutments using the cad system ( 3shape dental designer premium 2013 , 3shape , denmark ) .", "rapid prototype model of crowns with the same overall size and different preparation depths were fabricated by 3d printer ( digital dental printer , envisiontec , gladbeck , germany ) .", "1 ) . customized zirconia abutments with titanium insert were sandblasted for 30 seconds under the pressure of 0.5 bar using 50 m al2o3 particles ( cobra , renfert , germany ) .", "after the surface processing , ultrasonic cleansing was performed on every specimen for 10 minutes using acetone and alcohol , after which the specimens were dried at room temperature .", "next , zirconia primer ( metal / zirconia primer , ivoclar - vivadent , schaan , liechtenstein ) was applied to the bonding surface . following the manufacturer 's recommendations ,", "the inner surfaces of the lithium disilicate crowns were etched with hydrofluoric acid and silane finished ( monobond - s , ivo c l a r - vivadent , schaan , liechtenstein ) for 1 minute .", "the fixture and abutment were then tightened with a torque of 30 ncm , and the inside of the abutment was filled with cotton and temporary restorative material ( caviton , gc , tokyo , japan ) .", "the abutment and crown were then bonded using dual cure self - adhesive resin cement ( rely x unicem , 3 m espe , st .", "paul , mn , usa ) , and finger pressure was applied for 5 minutes .", "half of the samples ( n=18 ) were put into distilled water at 37 or 24 hours , after which thermocycling was conducted in water baths set to 5 0.5 and 55 0.5 , according to the international standard iso normed protocol ( iso 10477).25 thermocycling were conducted to obtain 5-year artificial aging , based on previous reports ( fig .", "2).2627 after thermocycling , the samples were placed at a 45 degree angle on a chewing simulator ( chewing simulator cs-4 , sd mechatronic gmbh , germany ) using a customized jig . chewing simulation was performed 1,200,000 cycles at 1.67 hz and 49 n , which corresponds to 5-year artificial aging.27 a cobalt - chrome steel indenter with a rounded tip ( 8 mm in diameter ) was used as an antagonist .", "the fracture load of the abutment was measured using the universal testing machine ( rb model 301 unitech m , r and b , korea ) .", "the maximum fracture load was determined by applying load on the palatal 2 mm of the incisal part of the abutment at the cross head speed of 0.5 mm / min . following the a previously published method ,", "0.5 mm of aluminum foil was inserted between the sample and the testing machine for even distribution of the load.1128 depending on the preparation depths and performance of artificial aging , the specimens were classified into 6 groups , including three non - artificial aging groups ( n5 , n7 , n9 ) and three artificial aging groups ( a5 , a7 , a9 ) ( table 2 ) . to evaluate the fracture mode ,", "3 ) . the fracture loads were processed statistically using the spss program ( spss version 21.0 , spss inc . ,", "after confirmation of the equal variance assumption by levene 's test , two - way anova for analysis and tukey hsd post - hoc tests were conducted to determine the interactions between the changes in preparation depth and artificial aging on the fracture load of the customized zirconia abutment with titanium insert ( p>.05 ) . under the equal variance assumption , one - way anova was performed to analyze the fracture load depending on the preparation depths of the samples with artificial aging ( p<.05 ) , while the non - parametric kruskal - wallis test was used in groups without artificial aging because there was no assumption of equal variance ( p<.017 ) .", "the differences in fracture load between the artificial aging group and non - artificial aging group at each preparation depth were statistically analyzed using the independent t - test ( p<.05 ) .", "multiple linear regression analysis was conducted to determine the relationships between variables and the interaction between artificial aging and changes in preparation depths or fracture load ( p<.05 ) .", "the means and standard deviations of the fracture loads of whole specimens are shown in table 3 .", "one sample in group a5 was broken during the chewing simulation , as it could not resist the masticatory force .", "therefore , there were 35 valid samples , for which each fracture load was measured .", "the mean fracture loads in groups n5 , n7 , and n9 ( non - artificial aging groups ) were 539.28 63.11 n , 406.56 28.94 n , and 366.66 30.19 n , respectively .", "the fracture area of this group , without artificial aging , was found to be located in the implant - abutment titanium insert internal connection area , and all specimens showed the typical fracture or deformation .", "3a ) the kruskal - wallis test indicated that the fracture load , depending on preparation depth , was higher in the n5 group than in the n7 and n9 groups ( p<.017 ) , while no significant difference in fracture load was observed between groups n7 and n9 ( p>.017 ) ( fig .", "4 ) . the fracture loads of groups a5 , a7 , and a9 ( with artificial aging ) were 392.616 50.57 n , 317.94 30.05 n , and 292.74 37.15 n , respectively . like the groups without artificial aging ,", "the fracture areas of these groups were also located in the implant - abutment titanium insert internal connection area , and all specimens showed the typical fracture or deformation ( fig .", "3b ) according to one - way anova , there were significant differences in the fracture load depending on the preparation depth ( p<.05 ) .", "tukey hsd post hoc test indicated that the fracture load , depending on preparation depth , was significantly higher in the a5 group than the a7 and a9 groups , while no meaningful difference was found in the fracture load between groups a7 and a9 ( p>.05 ) ( fig . 5 ) .", "the difference in fracture load was evaluated between each paired group ( n5/a5 , n7/a7 , n9/a9 ) , depending on whether artificial aging was performed or not .", "significant differences in the fracture load were observed for every pair of preparation depth through independent t - test ( p<.05 ) ( fig .", "multiple linear regression analysis was conducted to predict the changes in fracture load depending on the three preparation depths and the performance of artificial aging , as well as to examine the influence of fracture load according to each variable .", "the regression model had a power of explanation of 73.4% , and was significant when assumed through analysis of variance ( p<.05 ) .", "coefficient analysis revealed that fracture load decreased by 102.456 n in the case of artificial aging , and by about 68.56 n with increase in the preparation depth of 0.2 mm .", "overall , changes in the preparation depth were found to have more influence on the fracture load than artificial aging ( table 4 ) .", "in the present study , the fracture loads of customized zirconia abutments with titanium inserts were investigated according to preparation depths and the performance of artificial aging ( thermocycling & chewing simulations ) . moreover , for representation of the ' crown - abutment - fixture complex ' , lithium disilicate crowns were fabricated , and the optimum preparation depth of the crown was also assessed .", "the results indicated that the fracture load was significantly influenced by the variance of preparation depth , as well as by artificial aging .", "the fracture load of preparation depth 0.5 mm ( groups n5 and a5 ) was significantly higher than those of the other preparation groups ( groups n7 , n9 , a7 , and a9 ) regardless of artificial aging . after artificial aging and chewing simulations , all groups were significantly weakened .", "the width and length ratio of the maxillary anterior crown is known to be an important factor for anterior esthetic restorations .", "reported that the average size of the central incisor in adult asians was 8.6 mm in width and 11.9 mm in length.24 culp and mclaran reported the use of lithium disilicate crowns with a variety of transparencies for maximization of esthetics.29 many studies in the literature reported that the 6-degree angle of axial preparation offered adequate mechanical retention of the crowns and improved the adaptation of the crown margins .", "30313233 in the present experiments , the crowns and abutments were fabricated based on the reported values .", "for restoration of all - ceramic implant crowns , zirconia abutments require shoulder or deep chamfer margins .", "selected three preparation depths which were thinner than the natural teeth.15 although such specific limitation of the preparation depth resulted in a restricted space for the veneering materials , this disadvantage was outweighed by the favorable color of the underlying zirconia abutment .", "koutayas et al . also stated that when static load was applied to the implant axis at 135 degrees , the thinnest portion of the abutment and fixture connection was fractured due to the levering effect within the internal connection of the 1-piece zirconia abutment.15 they thus named the thinnest point as the \" weakest link \" .", "mitsias et al . also reported that all specimen of 1-piece zirconia abutment presented the typical fracture at the implant - abutment internal connection after dynamic loading.16 substituting the link with metal materials may allow strengthening of the fractured joint.11 however , despite replacement of the link , all specimens in the present study presented fractures in the same area as the previous reports .", "it should be noted that fracture did not occurin all samples , as titanium insert and screw deformation appeared in some of the specimens . the physiological force during mastication and the swallowing of food ranges from 10 - 120 n , with the maximum masticatory force of between 108 - 299 n.3435 herein , except for some specimens in the a7 and a9 groups , most specimens demonstrated tolerance of the physiological and maximum masticatory force .", "exposure of ceramic materials to mechanical stress and moisture results in low - temperature degradation,36 and artificial aging has an adverse impact on the mechanical properties of zirconia material .", "moreover , the phase of zirconia could be transformed from tetragonal to monoclinic.37 mitsias et al .", "reported that the fracture strength and durability of 1-piece full zirconia abutment was increased after dynamic loading in the chewing simulator .", "however , they stated that such results could not be supported by evidence - based scientific data.16 in the present study , the fracture loads of all preparation groups were decreased after artificial aging and chewing simulation . therefore , unlike the results of the abovementioned study , the fracture load of zirconia abutment with titanium insert examined herein was reduced after the 5-year cyclic loading .", "however , it could be expected that 5-year cyclic loading might weaken the physical properties of the zirconia abutment with titanium insert .", "therefore , in order to determine the exact mechanisms involved , more advanced study will be needed . for the zirconia , phase aging and chewing simulation , as well as xrd ( x - ray diffusion ) analyses will be required . in order to evaluate the correlation of the independent variables ( preparation depth and artificial aging ) on the dependent variable ( fracture load ) , multiple linear regression analysis was performed .", "the results revealed that increase of the preparation depth by 0.2 mm tended to decrease the fracture load by 68 n , while the aging and chewing simulations tended to cause decreases of 102 n. changes in the preparation depth of 0.2 mm had a higher effect than the aging and chewing simulations .", "thus , appropriate setting of the preparation depth of zirconia abutment is critical to the survival rate , and hence , the clinical applicability .", "within the limitations of this in vitro study , the following conclusions could be drawn : regardless of chewing simulation , the circumferential preparation depth of 0.5 mm of zirconia abutments had a significantly higher fracture load than other groups .", "artificial aging caused a significant decrease of the fracture load for all groups of different preparation depths .", "the change of preparation depth was more influential than the chewing simulation on the fracture load of the customized zirconia abutment with titanium insert .", "single implants restored with lithium disilicate crowns and zirconia abutments with titanium insert could with - stand maximum masticatory force in the incisive area when the preparation depth was 0.5 mm ." ]

[ "<S> purposethis study evaluated the fracture load of customized zirconia abutments with titanium insert according to preparation depths , with or without 5-year artificial aging.materials and methodsthirty - six identical lithium disilicate crowns ( ips e.max press ) were fabricated to replace a maxillary right central incisor and cemented to the customized zirconia abutment with titanium insert on a 4.510 mm titanium fixture . </S>", "<S> abutments were fabricated with 3 preparation depths ( 0.5 mm , 0.7 mm , and 0.9 mm ) . </S>", "<S> half of the samples were then processed using thermocycling ( temperature : 5 - 55 , dwelling time : 120s ) and chewing simulation ( 1,200,000 cycles , 49 n load ) . </S>", "<S> all specimens were classified into 6 groups depending on the preparation depth and artificial aging ( non - artificial aging groups : n5 , n7 , n9 ; artificial aging groups : a5 , a7 , a9 ) . </S>", "<S> static load was applied at 135 degrees to the implant axis in a universal testing machine . </S>", "<S> statistical analyses of the results were performed using 1-way anova , 2-way anova , independent t - test and multiple linear regression.resultsthe fracture loads were 539.28 63.11 n ( n5 ) , 406.56 28.94 n ( n7 ) , 366.66 30.19 n ( n9 ) , 392.61 50.57 n ( a5 ) , 317.94 30.05 n ( a7 ) , and 292.74 37.15 n ( a9 ) . </S>", "<S> the fracture load of group n5 was significantly higher than those of group n7 and n9 ( p<.017 ) . </S>", "<S> consequently , the fracture load of group a5 was also significantly higher than those of group a7 and a9 ( p<.05 ) . </S>", "<S> after artificial aging , the fracture load was significantly decreased in all groups with various preparation depths ( p<.05).conclusionthe fracture load of a single anterior implant restored with lithium disilicate crown on zirconia abutment with titanium insert differed depending on the preparation depths . </S>", "<S> after 5-year artificial aging , the fracture loads of all preparation groups decreased significantly . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION" ]

[ [ "implant - supported fixed dental prosthesis is a standardized restoration method that is often used when an aesthetically critical anterior single tooth is missing .", "it has similar 5-year survival rates as tooth - supported fixed dental prosthesis.12 although the success rate of an all - ceramic crown was reported to be lower than that of a metal - ceramic crown , all - ceramic crowns displayed similar success rates in both tooth - supported fixed dental prosthesis and implant - supported fixed dental prosthesis.234 titanium is a stable implant material , and titanium abutments have the advantage of supporting gingival health and preventing galvanic reaction between the fixture and abutment.56 however , when a titanium abutment is used in thin peri - implant mucosa in the anterior area , the metal part can be detected through the mucosa .", "for this reason , studies on alumina and zirconia , which are high - strength ceramics , have been conducted , and the study on implant abutments using zirconia has advanced , showing excellent material properties and biocompatibility .", "78 the influence of the type of connection between a zirconia abutment and a titanium fixture were addressed previously .", "the types of implant - abutment connection can be divided into two major groups - internal connection and external connection .", "sailer et al.910 reported that the internal implant - abutment connection type , including zirconia abutment with titanium insert , showed the highest strength , followed by external implant - abutment connection type and 1-piece internal type , when artificial aging was not conducted .", "however , in the above - mentioned experiment , only the implant - abutment fracture load was measured , without involvement of the crowns .", "therefore , the fracture load was measured under non - physiological conditions , and intra - oral temperature changes and dynamic functional load were not applied .", "they showed that the fracture load was dependent on the types of connection , which was the same result as that of the former studies , while the strength was lower after artificial aging .", "however , that study faced the limitation that it can not be applied to actual clinical situations , because the fracture load of the zirconia abutment was measured without crown restoration , and the fracture loads before and after artificial aging were not examined . due to the development of cad / cam systems ( computer - aided design / computer - aided manufacturing system )", ", the prosthesis using zirconia has replaced the conventional ceramic restoration and is now additionally being applied to implant abutments.1213 park et al.14 investigated the fit of a customized zirconia abutment manufactured by the cad / cam system .", "they reported that while the fit was less precise than that of a prefabricated zirconia abutment , it was within the clinically acceptable range .", "the strength of the customized zirconia abutment was significantly higher than that of the prefabricated zirconia abutment . when restoring all - ceramic implant crowns , shoulder or deep chamfer preparation of zirconia abutment", "koutayas et al.15 measured the fracture loads of 1-piece internal full zirconia abutments with three preparation depths ( 0.5 mm , 0.7 mm , and 0.9 mm ) that were restored using lithium disilicate .", "they reported that the fracture loads of all three specimens were higher than physiologic masticatory force , but preparation depths of over 0.7 mm were not recommended .", "subsequent to that report , mitsias et al.16 reported measurement of the fracture load of 1-piece internal full zirconia abutment with artificial aging .", "preparation depths under 0.9 mm were found to have had no influence on the fracture load .", "many literature reports have stated that the optimum preparation depth of zirconia abutment ranged from 0.5 mm to 1.0 mm.171819202122 however , further studies regarding standard indicators are still needed .", "therefore , the purpose of this study was to investigate the change of fracture load of customized zirconia abutments with titanium insert according to the different preparation depths , with aging and chewing simulation .", "the null hypotheses were that the fracture loads of customized zirconia abutment with titanium inserts will not change depending on the various preparation depths , or as a result of aging and chewing simulation ." ], [ "a commercial titanium fixture of 4.5 mm in diameter and 10 mm in length ( anyridge , megagen , gyeongsan , korea ) was used .", "the fixture was embedded in acrylic resin ( orthoresin dentsply , weybridge , surrey , uk ) according to the iso - normed protocol [ iso 14801].23 prefabricated titanium inserts that fit into the titanium fixture ( prop abutment , megagen , gyeongsan , korea ) were used , and the zirconia supra - structures to be attached to the insert were fabricated with zirconia blocks ( zenostarzr translucent , wieland , germany ) ( table 1 ) .", "the preparation depths of the zirconia suprastructure were designed to 0.5 mm , 0.7 mm and 0.9 mm using the cad system ( 3shape dental designer premium 2013 , 3shape , denmark ) , and the same - sized zirconia structures were manufactured using the cam system ( zenotec t1 , wieland dental + technik gmbh , pforzheim , germany ) .", "the customized zirconia suprastructures and prefabricated titanium inserts were then bonded using dual cure self - adhesive resin cement ( rely x unicem , 3 m espe , st .", "crowns fabricated to replace a maxillary right central incisor in asian adult ( 8.6 mm in width and 11.9 mm in length ) were made using lithium disilicate ( ips e.max press , ivoclar - vivadent , schaan , liechtenstein ) ( table 1).24 crowns were designed to fit over the customized zirconia abutments using the cad system ( 3shape dental designer premium 2013 , 3shape , denmark ) .", "rapid prototype model of crowns with the same overall size and different preparation depths were fabricated by 3d printer ( digital dental printer , envisiontec , gladbeck , germany ) .", "1 ) . customized zirconia abutments with titanium insert were sandblasted for 30 seconds under the pressure of 0.5 bar using 50 m al2o3 particles ( cobra , renfert , germany ) .", "after the surface processing , ultrasonic cleansing was performed on every specimen for 10 minutes using acetone and alcohol , after which the specimens were dried at room temperature .", "next , zirconia primer ( metal / zirconia primer , ivoclar - vivadent , schaan , liechtenstein ) was applied to the bonding surface . following the manufacturer 's recommendations ,", "the inner surfaces of the lithium disilicate crowns were etched with hydrofluoric acid and silane finished ( monobond - s , ivo c l a r - vivadent , schaan , liechtenstein ) for 1 minute .", "the fixture and abutment were then tightened with a torque of 30 ncm , and the inside of the abutment was filled with cotton and temporary restorative material ( caviton , gc , tokyo , japan ) .", "the abutment and crown were then bonded using dual cure self - adhesive resin cement ( rely x unicem , 3 m espe , st .", "paul , mn , usa ) , and finger pressure was applied for 5 minutes .", "half of the samples ( n=18 ) were put into distilled water at 37 or 24 hours , after which thermocycling was conducted in water baths set to 5 0.5 and 55 0.5 , according to the international standard iso normed protocol ( iso 10477).25 thermocycling were conducted to obtain 5-year artificial aging , based on previous reports ( fig .", "2).2627 after thermocycling , the samples were placed at a 45 degree angle on a chewing simulator ( chewing simulator cs-4 , sd mechatronic gmbh , germany ) using a customized jig . chewing simulation was performed 1,200,000 cycles at 1.67 hz and 49 n , which corresponds to 5-year artificial aging.27 a cobalt - chrome steel indenter with a rounded tip ( 8 mm in diameter ) was used as an antagonist .", "the fracture load of the abutment was measured using the universal testing machine ( rb model 301 unitech m , r and b , korea ) .", "the maximum fracture load was determined by applying load on the palatal 2 mm of the incisal part of the abutment at the cross head speed of 0.5 mm / min . following the a previously published method ,", "0.5 mm of aluminum foil was inserted between the sample and the testing machine for even distribution of the load.1128 depending on the preparation depths and performance of artificial aging , the specimens were classified into 6 groups , including three non - artificial aging groups ( n5 , n7 , n9 ) and three artificial aging groups ( a5 , a7 , a9 ) ( table 2 ) . to evaluate the fracture mode ,", "3 ) . the fracture loads were processed statistically using the spss program ( spss version 21.0 , spss inc . ,", "after confirmation of the equal variance assumption by levene 's test , two - way anova for analysis and tukey hsd post - hoc tests were conducted to determine the interactions between the changes in preparation depth and artificial aging on the fracture load of the customized zirconia abutment with titanium insert ( p>.05 ) . under the equal variance assumption , one - way anova was performed to analyze the fracture load depending on the preparation depths of the samples with artificial aging ( p<.05 ) , while the non - parametric kruskal - wallis test was used in groups without artificial aging because there was no assumption of equal variance ( p<.017 ) .", "the differences in fracture load between the artificial aging group and non - artificial aging group at each preparation depth were statistically analyzed using the independent t - test ( p<.05 ) .", "multiple linear regression analysis was conducted to determine the relationships between variables and the interaction between artificial aging and changes in preparation depths or fracture load ( p<.05 ) ." ], [ "the means and standard deviations of the fracture loads of whole specimens are shown in table 3 .", "one sample in group a5 was broken during the chewing simulation , as it could not resist the masticatory force .", "therefore , there were 35 valid samples , for which each fracture load was measured .", "the mean fracture loads in groups n5 , n7 , and n9 ( non - artificial aging groups ) were 539.28 63.11 n , 406.56 28.94 n , and 366.66 30.19 n , respectively .", "the fracture area of this group , without artificial aging , was found to be located in the implant - abutment titanium insert internal connection area , and all specimens showed the typical fracture or deformation .", "3a ) the kruskal - wallis test indicated that the fracture load , depending on preparation depth , was higher in the n5 group than in the n7 and n9 groups ( p<.017 ) , while no significant difference in fracture load was observed between groups n7 and n9 ( p>.017 ) ( fig .", "4 ) . the fracture loads of groups a5 , a7 , and a9 ( with artificial aging ) were 392.616 50.57 n , 317.94 30.05 n , and 292.74 37.15 n , respectively . like the groups without artificial aging ,", "the fracture areas of these groups were also located in the implant - abutment titanium insert internal connection area , and all specimens showed the typical fracture or deformation ( fig .", "3b ) according to one - way anova , there were significant differences in the fracture load depending on the preparation depth ( p<.05 ) .", "tukey hsd post hoc test indicated that the fracture load , depending on preparation depth , was significantly higher in the a5 group than the a7 and a9 groups , while no meaningful difference was found in the fracture load between groups a7 and a9 ( p>.05 ) ( fig . 5 ) .", "the difference in fracture load was evaluated between each paired group ( n5/a5 , n7/a7 , n9/a9 ) , depending on whether artificial aging was performed or not .", "significant differences in the fracture load were observed for every pair of preparation depth through independent t - test ( p<.05 ) ( fig .", "multiple linear regression analysis was conducted to predict the changes in fracture load depending on the three preparation depths and the performance of artificial aging , as well as to examine the influence of fracture load according to each variable .", "the regression model had a power of explanation of 73.4% , and was significant when assumed through analysis of variance ( p<.05 ) .", "coefficient analysis revealed that fracture load decreased by 102.456 n in the case of artificial aging , and by about 68.56 n with increase in the preparation depth of 0.2 mm .", "overall , changes in the preparation depth were found to have more influence on the fracture load than artificial aging ( table 4 ) ." ], [ "in the present study , the fracture loads of customized zirconia abutments with titanium inserts were investigated according to preparation depths and the performance of artificial aging ( thermocycling & chewing simulations ) . moreover , for representation of the ' crown - abutment - fixture complex ' , lithium disilicate crowns were fabricated , and the optimum preparation depth of the crown was also assessed .", "the results indicated that the fracture load was significantly influenced by the variance of preparation depth , as well as by artificial aging .", "the fracture load of preparation depth 0.5 mm ( groups n5 and a5 ) was significantly higher than those of the other preparation groups ( groups n7 , n9 , a7 , and a9 ) regardless of artificial aging . after artificial aging and chewing simulations , all groups were significantly weakened .", "the width and length ratio of the maxillary anterior crown is known to be an important factor for anterior esthetic restorations .", "reported that the average size of the central incisor in adult asians was 8.6 mm in width and 11.9 mm in length.24 culp and mclaran reported the use of lithium disilicate crowns with a variety of transparencies for maximization of esthetics.29 many studies in the literature reported that the 6-degree angle of axial preparation offered adequate mechanical retention of the crowns and improved the adaptation of the crown margins .", "30313233 in the present experiments , the crowns and abutments were fabricated based on the reported values .", "for restoration of all - ceramic implant crowns , zirconia abutments require shoulder or deep chamfer margins .", "selected three preparation depths which were thinner than the natural teeth.15 although such specific limitation of the preparation depth resulted in a restricted space for the veneering materials , this disadvantage was outweighed by the favorable color of the underlying zirconia abutment .", "koutayas et al . also stated that when static load was applied to the implant axis at 135 degrees , the thinnest portion of the abutment and fixture connection was fractured due to the levering effect within the internal connection of the 1-piece zirconia abutment.15 they thus named the thinnest point as the \" weakest link \" .", "mitsias et al . also reported that all specimen of 1-piece zirconia abutment presented the typical fracture at the implant - abutment internal connection after dynamic loading.16 substituting the link with metal materials may allow strengthening of the fractured joint.11 however , despite replacement of the link , all specimens in the present study presented fractures in the same area as the previous reports .", "it should be noted that fracture did not occurin all samples , as titanium insert and screw deformation appeared in some of the specimens . the physiological force during mastication and the swallowing of food ranges from 10 - 120 n , with the maximum masticatory force of between 108 - 299 n.3435 herein , except for some specimens in the a7 and a9 groups , most specimens demonstrated tolerance of the physiological and maximum masticatory force .", "exposure of ceramic materials to mechanical stress and moisture results in low - temperature degradation,36 and artificial aging has an adverse impact on the mechanical properties of zirconia material .", "moreover , the phase of zirconia could be transformed from tetragonal to monoclinic.37 mitsias et al .", "reported that the fracture strength and durability of 1-piece full zirconia abutment was increased after dynamic loading in the chewing simulator .", "however , they stated that such results could not be supported by evidence - based scientific data.16 in the present study , the fracture loads of all preparation groups were decreased after artificial aging and chewing simulation . therefore , unlike the results of the abovementioned study , the fracture load of zirconia abutment with titanium insert examined herein was reduced after the 5-year cyclic loading .", "however , it could be expected that 5-year cyclic loading might weaken the physical properties of the zirconia abutment with titanium insert .", "therefore , in order to determine the exact mechanisms involved , more advanced study will be needed . for the zirconia , phase aging and chewing simulation , as well as xrd ( x - ray diffusion ) analyses will be required . in order to evaluate the correlation of the independent variables ( preparation depth and artificial aging ) on the dependent variable ( fracture load ) , multiple linear regression analysis was performed .", "the results revealed that increase of the preparation depth by 0.2 mm tended to decrease the fracture load by 68 n , while the aging and chewing simulations tended to cause decreases of 102 n. changes in the preparation depth of 0.2 mm had a higher effect than the aging and chewing simulations .", "thus , appropriate setting of the preparation depth of zirconia abutment is critical to the survival rate , and hence , the clinical applicability ." ], [ "within the limitations of this in vitro study , the following conclusions could be drawn : regardless of chewing simulation , the circumferential preparation depth of 0.5 mm of zirconia abutments had a significantly higher fracture load than other groups .", "artificial aging caused a significant decrease of the fracture load for all groups of different preparation depths .", "the change of preparation depth was more influential than the chewing simulation on the fracture load of the customized zirconia abutment with titanium insert .", "single implants restored with lithium disilicate crowns and zirconia abutments with titanium insert could with - stand maximum masticatory force in the incisive area when the preparation depth was 0.5 mm ." ] ]

[ "according to the paradigm of antigen processing by the antigen presenting cells ( apc ) , peptides arising from intracellular proteins are presented via class i mhc molecule .", "this occurs after they are degraded via proteasome and transported by transporters associated with antigen processing ( taps ) .", "differently , peptides that originate from extracellular antigens are delivered to the late endosomal / lysosomal compartment , where they are degraded by the lysosomal proteases and then presented in association with class ii mhc . in the lysosomes ,", "the invariant chain , which blocks class ii mhc , is also degraded , rendering the mhc molecules available for peptide loading .", "besides the differences in the sites of origin and processing , peptides presented via class i or ii mhc activate different populations of t lymphocytes , which are cd8 or cd4 , respectively .", "however , exceptions to this model have been described and , for example , dendritic cells ( dcs ) , the most powerful apc , are able to present extracellular antigens also via class i mhc .", "this event , known as cross - presentation , allows dcs to activate both cd4 and cd8 t cells , in response to an extracellular antigen . on the other hand ,", "antigens of intracellular origin can be presented also in class ii mhc , after being delivered to the lysosomes , through double membrane vesicles , called autophagosomes .", "autophagosomes are formed during the induction of autophagy , a self - eating mechanism through which cells recycle their own constituents and survive in stressful conditions .", "some of them , such as atg1 , atg11 , and atg13 , regulate autophagosome formation ; others ( e.g. , atg2 , atg9 , and atg18 ) are required for membrane flow to the expanding phagophore .", "the vesicle nucleation is instead dependent on the class - iii phosphatidylinositol 3 kinase ( ptdins3k ) complex formed by vps34 , vps15 , vps30/atg6 , and atg14 .", "atg6 , also named beclin 1 , represents a protein with a pivotal role in the autophagy induction and the microtubule - associated protein light chain 3 ( lc3 ) , or atg8 , is a marker of the autophagic vacuoles .", "lc3 is expressed as full - length cytosolic protein and upon autophagy induction is cleaved by atg4 to form lc3i , which is then conjugated to phosphatidylethanolamine ( pe ) , generating lc3ii .", "this molecule , whose formation indicates autophagy induction , is associated with the internal and external membrane of autophagosome and , by interacting with adaptor molecules , such as p62/sqstm1 , promotes uptake and degradation of both cargo and adaptors into the lysosomes .", "indeed , autophagosomes fuse with lysosomes transporting intracellular proteins in the last autophagic steps . among them , also viral antigens can end up in the lysosomes , in virally infected cells . here", "peptides derived from their degradation may be complexed and presented in association with class ii mhc molecules", ". another important role of autophagosomes is to transport entire viral particles to the endosomal / lysosomal compartment , to be degraded and eventually eliminated , a process known as xenophagy . after entering the cells ,", "alternatively , phagosomes as well as free viral particles can be engulfed by the double membrane vesicles of autophagosomes , before reaching the lysosomes .", "the latter process is under the control of the atg genes , even if it represents a particular form of autophagy aimed at the antimicrobial defense .", "in addition , autophagy facilitates the pathogen engagement of intracellular tlrs and the consequent release of cytokines , such as type i ifn .", "the binding of tlrs as well as other molecules involved in the immune response , for example , cd46 , by ligands or pathogens that use them as receptors , also triggers autophagy .", "this has been reported for epstein - barr virus ( ebv ) that binds tlrs and for measles virus that uses cd46 as receptor .", "intriguingly , since cd46 represents the cellular receptor also for human herpes - virus 6 ( hhv6 ) , it would be interesting to investigate the impact on autophagy of cd46 engagement by hhv6 . as if the role of autophagy in the immune response was not enough important , it has been recently reported that autophagy is induced by gm - csf in monocytes and that it stimulates monocyte differentiation into functional macrophages and dcs .", "besides that , autophagy also plays an essential role in preventing apoptosis of these cells .", "among the strategies that allow viruses to escape from the immune control , the impairment of monocyte differentiation into functional dcs represents a common one [ 1719 ] . based on the recent finding , suggesting that autophagy is involved in monocyte differentiation in macrophages and dcs , the interference with the autophagic process could represent one of the underlying mechanisms responsible for such impairment .", "this strategy has been recently reported to be exploited by human hepatitis c virus ( hcv ) , whose infection of human monocytes results in a reduction of lysosomal cathepsins , in an autophagic block at the late steps , and , as a consequence , in an impairment of dc differentiation .", "hcv - mediated reduction of lysosomal acidification , as result of cathepsin release , has been reported also in other cell types .", "however , autophagy can contribute to hcv replication since the autophagosomal membranes can be used for viral production .", "given the importance of autophagy in the immune response , it is not surprising that viruses have evolved strategies to interfere with it , in order to avoid their elimination into the lysosomes , impair the production of antiviral cytokines , reduce the presentation of their antigens , and , as described for hcv , alter dc differentiation .", "this is a must for viruses to persist in the infected host , sometimes with pathological consequences .", "all the steps of the autophagic process , from the autophagosome formation to the lysosomal degradation of their content , can be manipulated by viruses [ 24 , 25 ] ( figure 1 ) .", "it has been described that it depends on the virus types , on the phase of their life cycle , and on the host cell that they infect .", "for example , human immunodeficiency virus , a single - stranded rna lentivirus belonging to the retroviridae family , is able to induce the initial phases of autophagy by env , an envelope fusogenic protein . on the other hand ,", "it blocks the late autophagic phases to enhance viral production by expressing nef , an accessory protein that interferes with the autophagosomal maturation [ 24 , 25 ] . during the replicative phase of their life cycle ,", "also herpesviruses such as ebv and kaposi 's sarcoma human herpesvirus ( kshv ) can promote autophagy and exploit the autophagic machinery to enhance their replication [ 2628 ] .", "to do so , they promote the first autophagic steps and block the last ones .", "this strategy allows them to avoid being delivered into the destructive environment of the lysosomes and to usurp the autophagic machinery for viral transportation through the cell cytoplasm [ 22 , 26 , 29 ] .", "pathways involved in the autophagy induction , such as pkr / eif2 alpha and m - tor , can be also targeted by viruses belonging to the herpesvirus family , for example , herpes simplex virus-1 ( hsv-1 ) [ 3032 ] and cytomegalovirus ( hcmv ) .", "also beclin 1 , a key molecule involved in several steps of the autophagic process , can be bound and altered in its function by viral proteins such as vbcl2 of kshv or icp 34.5 of hsv-1 [ 3436 ] .", "all these strategies allow viruses to block autophagy induction during infection of the host cells .", "differently , another herpesvirus , varicella - zoster virus ( vzv ) , has been recently reported to successfully infect target cells without blocking the autophagic process .", "indeed icp 34.5 or us11 , the two proteins responsible for the block of autophagy by hsv1 , are not present in vzv although both viruses belong to the same subfamily .", "herpesviruses are large , double strand dna viruses having a common particle structure . to date eight human herpesviruses", "have been identified and classified into three subfamilies ( alpha , beta , and gamma ) based on their growth characteristics and tissue tropism .", "the -subfamily includes the neurotropic viruses herpes simplex viruses ( hsv ) 1 and 2 and varicella - zoster virus ( vzv ) .", "human cytomegalovirus ( hcmv ) and the human herpesviruses 6 and 7 are members of the -subfamily , while ebv and kshv are members of the -subfamily . a common feature shared by all human herpesviruses", "is the viral persistence into host and the possibility to undergo two alternative life cycle programs , namely , latency and lytic replication . during latency", "the viral genome is retained as a circular episome in the nucleus and no viral progeny is produced .", "furthermore , in the course of latent infection a limited set of genes is expressed in order to reduce immune recognition .", "in contrast to latency , the herpesvirus lytic program is characterized by a regulated cascade of viral gene expression accompanied by viral production and killing of infected cells .", "although most herpesviruses have been reported to interfere with the autophagic process , the next part of this review will focus on the interplay between autophagy and -herpesviruses , given that our laboratory has for long time worked on the virus - host interaction of these viruses associated with several human cancers .", "as for other herpesviruses , ebv infection can be latent or lytic [ 41 , 42 ] . during latency", "the ebv genome is retained as a circular episome in the cell nuclei and upon appropriate stimuli the virus switches into the lytic replication program .", "it is known that ebv infects primarily human b lymphocytes and epithelial cells ; however , its infection can also occur in other cells with a central role in the immune response , such as monocytes and dcs [ 44 , 45 ] .", "the in vitro infection of monocytes induces apoptosis and results in an impairment of dc development [ 45 , 46 ] , although the underlying mechanisms have not been investigated in these studies .", "therefore , the autophagic pathway could be investigated in these cells since , as previously described , a block of autophagy can switch cell differentiation into cell death in monocytes dcs .", "these are the most powerful cells in the priming of the cd8 t cell response , playing a pivotal role in control of the ebv infection . besides cytotoxic t cells , dcs are able to activate a cd4 t cell - mediated immune response against the ebv antigens , such as ebna1 , through the mechanism of cross - presentation .", "autophagy has been shown to be essential for class ii mhc presentation of ebna1 protein .", "the block of autophagy resulted in an accumulation of this protein in the intracellular autophagosomes and , more importantly , in a reduction of ebna1 recognition by ebna1 specific cd4 t cells .", "the involvement of autophagy in ebna 1 presentation was later confirmed by a more recent study . regarding the ebv interaction with plasmacytoid subpopulation of dcs ( pdcs ) , the main type i ifn producing cells", ", it has been reported that autophagy is essential for ifn release in response to the ebv infection .", "autophagy is stimulated by the virus and facilitates its interaction with tlr7 and tlr9 pprs , both located in the endosomal / lysosomal compartment and essential for ebv recognition by these cells .", "however , although ebv stimulates autophagy and ifn release , its infection results in an impairment of pdc maturation . since it has been reported that ebv downregulates tlr 9 in b cells and that this effect is mediated by two ebv proteins , namely , latent membrane protein 1 ( lmp1 ) and bglf5 , it would be interesting to investigate tlr9 expression levels in the pdcs ebv - infected versus uninfected control cells .", "ebv is associated with several different human cancers of b and epithelial cell origin , such as posttransplant lymphoproliferative disorder ( ptld ) , hodgkin and non - hodgkin lymphomas , nasopharyngeal carcinoma ( npc ) , and some forms of gastric carcinoma . besides its strong association with some human cancers , ebv infects and establishes a life - long asymptomatic infection in 95% of adult healthy population , reducing to the minimum or not expressing any protein , in order to escape from the immune recognition . among the viral antigens ,", "only the ebv latent nuclear antigen 1 ( ebna1 ) expression is required for the maintenance of the viral episome in the ebv infected human b cells and this is the only protein always expressed in ebv - associated malignancies .", "this protein has been demonstrated to activate both cd4 t cells , being presented via class ii mhc and cd8 t cells , although for long time it has been considered invisible to the immune system . as previously described , autophagy is essential for ebna1 antigen presentation via class ii mhc while , interestingly , it does not seem to play a role in the class ii mhc presentation of two other ebv latent nuclear proteins , ebna2 and ebna 3c , both expressed only in pathological conditions .", "the most oncogenic ebv latent protein , lmp1 , has been reported to regulate its own expression by inducing or inhibiting autophagy .", "when lmp1 is highly expressed in b cells , autophagy is stimulated and promotes degradation .", "given that lmp1 plays an important role in ebv - induced oncogenesis , the stimulation of autophagy could be used as a strategy to reduce the expression of this protein and consequently to affect the ebv - driven tumorigenesis .", "kshv or human herpesvirus-8 ( hhv-8 ) is the last human -herpesvirus identified to date .", "generally , latency is the kshv default program within 4872 h postinfection depending on target cells . in vivo", ", kshv mainly infects endothelial and b cells and establishes a lifelong latency in b lymphocytes of infected individuals , escaping from the host immune response [ 65 , 66 ] .", "several kshv latent and lytic proteins are involved in immune evasion , in preventing apoptosis and blocking autophagy as well as in transformation .", "kshv is the etiologic agent of kaposi 's sarcoma ( ks ) , a multifocal angioproliferative disorder arising from kshv - infected endothelial cell , and of lymphoproliferative disorders , such as primary effusion lymphomas ( pels ) , a non - hodgkin b cell lymphoma localized in body cavities , and the plasma cell variant of multicentric castleman disease ( mcd ) .", "it is important to underline that , although kshv is detectable in all ks lesions , regardless of disease stage or clinical variant , the virus is necessary but not sufficient for the development of this tumor [ 67 , 68 ] .", "the majority of spindle cells are latently infected by kshv and only a small percentage of these cells express viral lytic antigens [ 68 , 70 ] .", "however , it is currently believed that lytic replication is necessary to support ks lesion formation and maintenance [ 68 , 7174 ] .", "in addition , viral replication allows the secretion of proinflammatory and/or proangiogenic factors that create the inflammatory microenvironment essential in ks pathogenesis [ 68 , 75 ] .", "monocytes are among the inflammatory cells found in ks lesions and a study , based on immunohistochemical staining and in situ hybridization , reported that these cells are kshv infected .", "the virus has also been detected in peripheral t - cells and macrophages [ 77 , 78 ] .", "as other herpesviruses , kshv has to cope with innate and adaptive immunity in order to establish a persistent infection in immunocompetent host .", "latency is one of the strategies used by kshv since viral gene expression is reduced .", "conversely , during its lytic cycle , a high number of immunogenic proteins are produced [ 71 , 79 ] .", "the relevance of the equilibrium between kshv lytic cycle and host immunity is indicated by the increased viral replication and the development of kshv - related malignancies in immunosuppressed patients [ 8082 ] . about 25% of kshv proteins", "are involved in kshv immune evasion mechanisms and , although most of these belong to lytic cycle , both latent and lytic proteins are able to hijack innate and adaptive immune response .", "they can indeed inhibit complement - mediated lysis of infected cells and the ifn i signaling , deregulate the inflammatory cytokine / chemokine networks , and interfere with antigen presentation [ 67 , 79 , 8385 ] .", "pdcs , monocyte - derived dendritic cells , and monocytes are among the cell types infected by kshv [ 19 , 86 , 87 ] . as consequence of the infection , a reduction of costimulatory molecules as well as a deregulation of the cytokine release and an impairment of allostimulatory capacity can occur [ 19 , 76 , 8790 ] . in vivo , dc functional impairment has been reported in patients with classical ks and a reduction of pdcs was observed in aids - ks in comparison to ks negative hiv-1 infected individuals .", "moreover , the number of langerhans cells is also decreased in ks lesions compared to normal skin .", "furthermore , it has been reported that the signal transducer and activator of transcription 3 ( stat3 ) activation in noninfected monocytes / macrophages leads to a block of autophagy and consequent dysfunction , due to the cytokines released during hiv infection .", "we showed that khsv is able to activate stat3 pathway in dcs by binding to its receptor on these cells , namely , the type ii c - type lectin , dendritic cell - specific icam-3 grabbing nonintegrin ( dc - sign ; cd209 ) [ 87 , 96 ] . besides , we showed that stat3 activation led to a block of the autophagic flux , as demonstrated by the reduced expression of lc3ii and the increased level of p62 .", "in addition , looking for a possible mechanism responsible for the block of autophagy mediated by stat3 activation in dcs exposed to kshv , we found an upregulation of mcl-1 [ 96 , 97 ] .", "this is one of the proteins able to bind and sequester beclin 1 , hampering its essential role in autophagosome formation .", "remarkably , stat3 inhibition by ag490 was able to prevent the effects on p62 , lc3ii , and mcl-1 . in agreement", ", a previous paper has shown that the inhibition of stat3 by sorafenib resulted in a downregulation of mcl-1 , disruption of beclin 1-mcl-1 complex , and reversion of the autophagic block in hepatocarcinoma cells .", "of note , the autophagic block occurred also in the presence of uv - inactivated kshv , indicating that neither vbcl2 nor vflip expression was required [ 34 , 96 , 99 , 100 ] .", "concomitantly to the autophagic block induced by kshv - mediated stat3 activation , we also observed a reduction of il12p70 release in response to lps stimulation and a higher production of il-10 , il-6 , and il-23 .", "this cytokine pattern skews the th1/th2 profile towards th2 and/or th17 , promoting immunosuppression and inflammation .", "therefore , stat3 activation could be one of the molecular mechanisms underlying kshv - mediated immunosuppression in dcs .", "indeed , stat3 activation correlates with an immunosuppressive phenotype and dc dysfunction in the ks microenvironment and in the peripheral blood of tumor bearing patients [ 103 , 104 ] .", "several papers have investigated the interplay between kshv and the autophagic pathway . during the latent phase of its life - cycle", ", kshv expresses the fadd - like interleukin-1 beta - converting enzyme inhibitory protein ( v - flip ) , a truncated homolog of the cellular flip , which besides having an antiapoptotic activity [ 105 , 106 ] has been recently shown to block the autophagic flux .", "it competes with lc3 for binding to atg3 , thereby preventing lc3 binding and processing during autophagosome biogenesis .", "furthermore , the inhibition of vflip binding to atg3 reduced the size of kshv positive tumor in mice .", "it has also been demonstrated that v - flip suppression of autophagy counteracts v - cyclin - induced cellular senescence [ 107 , 108 ] .", "v - cyclin is a kshv latent protein that deregulates cell cycle , causes aberrant host dna replication , and triggers the dna damage responses ( ddrs ) . besides vflip , two kshv proteins expressed during the lytic cycle , vbcl2 and k7 , can interfere with autophagy .", "vbcl2 is a homolog of cellular bcl-2 that inhibits both apoptosis [ 99 , 110 ] and autophagy [ 34 , 35 ] . as its cellular counterpart", ", vbcl2 negatively regulates autophagy by binding beclin 1 . always within the context of the lytic cycle", ", recent evidence suggests that kshv k7 protein prevents autophagosome maturation , by interacting with rubicon autophagic protein .", "k7 is involved in apoptosis suppression [ 112 , 113 ] while rubicon is a subunit of the beclin 1/uvrag / vps34 autophagy complex , which regulates the autophagosome maturation and the endocytic trafficking [ 114116 ] .", "as shown by authors , k7 transfection in epithelial cell lines promoted a greater interaction between rubicon and beclin 1/uvrag / vps34 complex resulting in a block of vps34 enzymatic activity and , consequently , of autophagosome maturation .", "interestingly , during the lytic cycle kshv exploits autophagy to enhance its reactivation mediated by rta immediate lytic protein .", "altogether , the data here reviewed suggest that kshv has evolved several strategies to circumvent autophagy - mediated immune responses , to persist in infected hosts .", "moreover , the autophagic block during latency and the autophagy induction during the lytic cycle may contribute to the pathogenesis of kshv associated malignancies .", "a better understanding of how autophagy manipulation could influence antiviral immune response and control virus - induced tumorigenesis might help to discover strategies improving the outcome of the treatments of virus - associated malignancies .", "moreover , since autophagy is activated and is involved in ebv and kshv replication [ 20 , 27 ] , its manipulation could also affect the viral particle release that plays a role in -herpesvirus - associated cancers ." ]

[ "<S> we summarized the most recent findings on the role of autophagy in antiviral immune response . </S>", "<S> we described how viruses have developed strategies to subvert the autophagic process . </S>", "<S> a particular attention has been given to epstein - barr and kaposi 's sarcoma associated herpesvirus , viruses studied for many years in our laboratory . </S>", "<S> these two viruses belong to -herpesvirus subfamily and are associated with several human cancers . besides the effects on the immune response , </S>", "<S> we have described how autophagy subversion by viruses may also concur to the enhancement of their replication and to viral tumorigenesis . </S>" ]

[ "1. Autophagy and the Immune Response", "2. Autophagy Manipulation by Viruses", "3. Herpesviruses", "4. Epstein-Barr Virus (EBV)", "5. Kaposi's Sarcoma-Associated Herpesvirus (KSHV)", "6. Concluding Remarks" ]

[ [ "according to the paradigm of antigen processing by the antigen presenting cells ( apc ) , peptides arising from intracellular proteins are presented via class i mhc molecule .", "this occurs after they are degraded via proteasome and transported by transporters associated with antigen processing ( taps ) .", "differently , peptides that originate from extracellular antigens are delivered to the late endosomal / lysosomal compartment , where they are degraded by the lysosomal proteases and then presented in association with class ii mhc . in the lysosomes ,", "the invariant chain , which blocks class ii mhc , is also degraded , rendering the mhc molecules available for peptide loading .", "besides the differences in the sites of origin and processing , peptides presented via class i or ii mhc activate different populations of t lymphocytes , which are cd8 or cd4 , respectively .", "however , exceptions to this model have been described and , for example , dendritic cells ( dcs ) , the most powerful apc , are able to present extracellular antigens also via class i mhc .", "this event , known as cross - presentation , allows dcs to activate both cd4 and cd8 t cells , in response to an extracellular antigen . on the other hand ,", "antigens of intracellular origin can be presented also in class ii mhc , after being delivered to the lysosomes , through double membrane vesicles , called autophagosomes .", "autophagosomes are formed during the induction of autophagy , a self - eating mechanism through which cells recycle their own constituents and survive in stressful conditions .", "some of them , such as atg1 , atg11 , and atg13 , regulate autophagosome formation ; others ( e.g. , atg2 , atg9 , and atg18 ) are required for membrane flow to the expanding phagophore .", "the vesicle nucleation is instead dependent on the class - iii phosphatidylinositol 3 kinase ( ptdins3k ) complex formed by vps34 , vps15 , vps30/atg6 , and atg14 .", "atg6 , also named beclin 1 , represents a protein with a pivotal role in the autophagy induction and the microtubule - associated protein light chain 3 ( lc3 ) , or atg8 , is a marker of the autophagic vacuoles .", "lc3 is expressed as full - length cytosolic protein and upon autophagy induction is cleaved by atg4 to form lc3i , which is then conjugated to phosphatidylethanolamine ( pe ) , generating lc3ii .", "this molecule , whose formation indicates autophagy induction , is associated with the internal and external membrane of autophagosome and , by interacting with adaptor molecules , such as p62/sqstm1 , promotes uptake and degradation of both cargo and adaptors into the lysosomes .", "indeed , autophagosomes fuse with lysosomes transporting intracellular proteins in the last autophagic steps . among them , also viral antigens can end up in the lysosomes , in virally infected cells . here", "peptides derived from their degradation may be complexed and presented in association with class ii mhc molecules", ". another important role of autophagosomes is to transport entire viral particles to the endosomal / lysosomal compartment , to be degraded and eventually eliminated , a process known as xenophagy . after entering the cells ,", "alternatively , phagosomes as well as free viral particles can be engulfed by the double membrane vesicles of autophagosomes , before reaching the lysosomes .", "the latter process is under the control of the atg genes , even if it represents a particular form of autophagy aimed at the antimicrobial defense .", "in addition , autophagy facilitates the pathogen engagement of intracellular tlrs and the consequent release of cytokines , such as type i ifn .", "the binding of tlrs as well as other molecules involved in the immune response , for example , cd46 , by ligands or pathogens that use them as receptors , also triggers autophagy .", "this has been reported for epstein - barr virus ( ebv ) that binds tlrs and for measles virus that uses cd46 as receptor .", "intriguingly , since cd46 represents the cellular receptor also for human herpes - virus 6 ( hhv6 ) , it would be interesting to investigate the impact on autophagy of cd46 engagement by hhv6 . as if the role of autophagy in the immune response was not enough important , it has been recently reported that autophagy is induced by gm - csf in monocytes and that it stimulates monocyte differentiation into functional macrophages and dcs .", "besides that , autophagy also plays an essential role in preventing apoptosis of these cells ." ], [ "among the strategies that allow viruses to escape from the immune control , the impairment of monocyte differentiation into functional dcs represents a common one [ 1719 ] . based on the recent finding , suggesting that autophagy is involved in monocyte differentiation in macrophages and dcs , the interference with the autophagic process could represent one of the underlying mechanisms responsible for such impairment .", "this strategy has been recently reported to be exploited by human hepatitis c virus ( hcv ) , whose infection of human monocytes results in a reduction of lysosomal cathepsins , in an autophagic block at the late steps , and , as a consequence , in an impairment of dc differentiation .", "hcv - mediated reduction of lysosomal acidification , as result of cathepsin release , has been reported also in other cell types .", "however , autophagy can contribute to hcv replication since the autophagosomal membranes can be used for viral production .", "given the importance of autophagy in the immune response , it is not surprising that viruses have evolved strategies to interfere with it , in order to avoid their elimination into the lysosomes , impair the production of antiviral cytokines , reduce the presentation of their antigens , and , as described for hcv , alter dc differentiation .", "this is a must for viruses to persist in the infected host , sometimes with pathological consequences .", "all the steps of the autophagic process , from the autophagosome formation to the lysosomal degradation of their content , can be manipulated by viruses [ 24 , 25 ] ( figure 1 ) .", "it has been described that it depends on the virus types , on the phase of their life cycle , and on the host cell that they infect .", "for example , human immunodeficiency virus , a single - stranded rna lentivirus belonging to the retroviridae family , is able to induce the initial phases of autophagy by env , an envelope fusogenic protein . on the other hand ,", "it blocks the late autophagic phases to enhance viral production by expressing nef , an accessory protein that interferes with the autophagosomal maturation [ 24 , 25 ] . during the replicative phase of their life cycle ,", "also herpesviruses such as ebv and kaposi 's sarcoma human herpesvirus ( kshv ) can promote autophagy and exploit the autophagic machinery to enhance their replication [ 2628 ] .", "to do so , they promote the first autophagic steps and block the last ones .", "this strategy allows them to avoid being delivered into the destructive environment of the lysosomes and to usurp the autophagic machinery for viral transportation through the cell cytoplasm [ 22 , 26 , 29 ] .", "pathways involved in the autophagy induction , such as pkr / eif2 alpha and m - tor , can be also targeted by viruses belonging to the herpesvirus family , for example , herpes simplex virus-1 ( hsv-1 ) [ 3032 ] and cytomegalovirus ( hcmv ) .", "also beclin 1 , a key molecule involved in several steps of the autophagic process , can be bound and altered in its function by viral proteins such as vbcl2 of kshv or icp 34.5 of hsv-1 [ 3436 ] .", "all these strategies allow viruses to block autophagy induction during infection of the host cells .", "differently , another herpesvirus , varicella - zoster virus ( vzv ) , has been recently reported to successfully infect target cells without blocking the autophagic process .", "indeed icp 34.5 or us11 , the two proteins responsible for the block of autophagy by hsv1 , are not present in vzv although both viruses belong to the same subfamily ." ], [ "herpesviruses are large , double strand dna viruses having a common particle structure . to date eight human herpesviruses", "have been identified and classified into three subfamilies ( alpha , beta , and gamma ) based on their growth characteristics and tissue tropism .", "the -subfamily includes the neurotropic viruses herpes simplex viruses ( hsv ) 1 and 2 and varicella - zoster virus ( vzv ) .", "human cytomegalovirus ( hcmv ) and the human herpesviruses 6 and 7 are members of the -subfamily , while ebv and kshv are members of the -subfamily . a common feature shared by all human herpesviruses", "is the viral persistence into host and the possibility to undergo two alternative life cycle programs , namely , latency and lytic replication . during latency", "the viral genome is retained as a circular episome in the nucleus and no viral progeny is produced .", "furthermore , in the course of latent infection a limited set of genes is expressed in order to reduce immune recognition .", "in contrast to latency , the herpesvirus lytic program is characterized by a regulated cascade of viral gene expression accompanied by viral production and killing of infected cells .", "although most herpesviruses have been reported to interfere with the autophagic process , the next part of this review will focus on the interplay between autophagy and -herpesviruses , given that our laboratory has for long time worked on the virus - host interaction of these viruses associated with several human cancers ." ], [ "as for other herpesviruses , ebv infection can be latent or lytic [ 41 , 42 ] . during latency", "the ebv genome is retained as a circular episome in the cell nuclei and upon appropriate stimuli the virus switches into the lytic replication program .", "it is known that ebv infects primarily human b lymphocytes and epithelial cells ; however , its infection can also occur in other cells with a central role in the immune response , such as monocytes and dcs [ 44 , 45 ] .", "the in vitro infection of monocytes induces apoptosis and results in an impairment of dc development [ 45 , 46 ] , although the underlying mechanisms have not been investigated in these studies .", "therefore , the autophagic pathway could be investigated in these cells since , as previously described , a block of autophagy can switch cell differentiation into cell death in monocytes dcs .", "these are the most powerful cells in the priming of the cd8 t cell response , playing a pivotal role in control of the ebv infection . besides cytotoxic t cells , dcs are able to activate a cd4 t cell - mediated immune response against the ebv antigens , such as ebna1 , through the mechanism of cross - presentation .", "autophagy has been shown to be essential for class ii mhc presentation of ebna1 protein .", "the block of autophagy resulted in an accumulation of this protein in the intracellular autophagosomes and , more importantly , in a reduction of ebna1 recognition by ebna1 specific cd4 t cells .", "the involvement of autophagy in ebna 1 presentation was later confirmed by a more recent study . regarding the ebv interaction with plasmacytoid subpopulation of dcs ( pdcs ) , the main type i ifn producing cells", ", it has been reported that autophagy is essential for ifn release in response to the ebv infection .", "autophagy is stimulated by the virus and facilitates its interaction with tlr7 and tlr9 pprs , both located in the endosomal / lysosomal compartment and essential for ebv recognition by these cells .", "however , although ebv stimulates autophagy and ifn release , its infection results in an impairment of pdc maturation . since it has been reported that ebv downregulates tlr 9 in b cells and that this effect is mediated by two ebv proteins , namely , latent membrane protein 1 ( lmp1 ) and bglf5 , it would be interesting to investigate tlr9 expression levels in the pdcs ebv - infected versus uninfected control cells .", "ebv is associated with several different human cancers of b and epithelial cell origin , such as posttransplant lymphoproliferative disorder ( ptld ) , hodgkin and non - hodgkin lymphomas , nasopharyngeal carcinoma ( npc ) , and some forms of gastric carcinoma . besides its strong association with some human cancers , ebv infects and establishes a life - long asymptomatic infection in 95% of adult healthy population , reducing to the minimum or not expressing any protein , in order to escape from the immune recognition . among the viral antigens ,", "only the ebv latent nuclear antigen 1 ( ebna1 ) expression is required for the maintenance of the viral episome in the ebv infected human b cells and this is the only protein always expressed in ebv - associated malignancies .", "this protein has been demonstrated to activate both cd4 t cells , being presented via class ii mhc and cd8 t cells , although for long time it has been considered invisible to the immune system . as previously described , autophagy is essential for ebna1 antigen presentation via class ii mhc while , interestingly , it does not seem to play a role in the class ii mhc presentation of two other ebv latent nuclear proteins , ebna2 and ebna 3c , both expressed only in pathological conditions .", "the most oncogenic ebv latent protein , lmp1 , has been reported to regulate its own expression by inducing or inhibiting autophagy .", "when lmp1 is highly expressed in b cells , autophagy is stimulated and promotes degradation .", "given that lmp1 plays an important role in ebv - induced oncogenesis , the stimulation of autophagy could be used as a strategy to reduce the expression of this protein and consequently to affect the ebv - driven tumorigenesis ." ], [ "kshv or human herpesvirus-8 ( hhv-8 ) is the last human -herpesvirus identified to date .", "generally , latency is the kshv default program within 4872 h postinfection depending on target cells . in vivo", ", kshv mainly infects endothelial and b cells and establishes a lifelong latency in b lymphocytes of infected individuals , escaping from the host immune response [ 65 , 66 ] .", "several kshv latent and lytic proteins are involved in immune evasion , in preventing apoptosis and blocking autophagy as well as in transformation .", "kshv is the etiologic agent of kaposi 's sarcoma ( ks ) , a multifocal angioproliferative disorder arising from kshv - infected endothelial cell , and of lymphoproliferative disorders , such as primary effusion lymphomas ( pels ) , a non - hodgkin b cell lymphoma localized in body cavities , and the plasma cell variant of multicentric castleman disease ( mcd ) .", "it is important to underline that , although kshv is detectable in all ks lesions , regardless of disease stage or clinical variant , the virus is necessary but not sufficient for the development of this tumor [ 67 , 68 ] .", "the majority of spindle cells are latently infected by kshv and only a small percentage of these cells express viral lytic antigens [ 68 , 70 ] .", "however , it is currently believed that lytic replication is necessary to support ks lesion formation and maintenance [ 68 , 7174 ] .", "in addition , viral replication allows the secretion of proinflammatory and/or proangiogenic factors that create the inflammatory microenvironment essential in ks pathogenesis [ 68 , 75 ] .", "monocytes are among the inflammatory cells found in ks lesions and a study , based on immunohistochemical staining and in situ hybridization , reported that these cells are kshv infected .", "the virus has also been detected in peripheral t - cells and macrophages [ 77 , 78 ] .", "as other herpesviruses , kshv has to cope with innate and adaptive immunity in order to establish a persistent infection in immunocompetent host .", "latency is one of the strategies used by kshv since viral gene expression is reduced .", "conversely , during its lytic cycle , a high number of immunogenic proteins are produced [ 71 , 79 ] .", "the relevance of the equilibrium between kshv lytic cycle and host immunity is indicated by the increased viral replication and the development of kshv - related malignancies in immunosuppressed patients [ 8082 ] . about 25% of kshv proteins", "are involved in kshv immune evasion mechanisms and , although most of these belong to lytic cycle , both latent and lytic proteins are able to hijack innate and adaptive immune response .", "they can indeed inhibit complement - mediated lysis of infected cells and the ifn i signaling , deregulate the inflammatory cytokine / chemokine networks , and interfere with antigen presentation [ 67 , 79 , 8385 ] .", "pdcs , monocyte - derived dendritic cells , and monocytes are among the cell types infected by kshv [ 19 , 86 , 87 ] . as consequence of the infection , a reduction of costimulatory molecules as well as a deregulation of the cytokine release and an impairment of allostimulatory capacity can occur [ 19 , 76 , 8790 ] . in vivo , dc functional impairment has been reported in patients with classical ks and a reduction of pdcs was observed in aids - ks in comparison to ks negative hiv-1 infected individuals .", "moreover , the number of langerhans cells is also decreased in ks lesions compared to normal skin .", "furthermore , it has been reported that the signal transducer and activator of transcription 3 ( stat3 ) activation in noninfected monocytes / macrophages leads to a block of autophagy and consequent dysfunction , due to the cytokines released during hiv infection .", "we showed that khsv is able to activate stat3 pathway in dcs by binding to its receptor on these cells , namely , the type ii c - type lectin , dendritic cell - specific icam-3 grabbing nonintegrin ( dc - sign ; cd209 ) [ 87 , 96 ] . besides , we showed that stat3 activation led to a block of the autophagic flux , as demonstrated by the reduced expression of lc3ii and the increased level of p62 .", "in addition , looking for a possible mechanism responsible for the block of autophagy mediated by stat3 activation in dcs exposed to kshv , we found an upregulation of mcl-1 [ 96 , 97 ] .", "this is one of the proteins able to bind and sequester beclin 1 , hampering its essential role in autophagosome formation .", "remarkably , stat3 inhibition by ag490 was able to prevent the effects on p62 , lc3ii , and mcl-1 . in agreement", ", a previous paper has shown that the inhibition of stat3 by sorafenib resulted in a downregulation of mcl-1 , disruption of beclin 1-mcl-1 complex , and reversion of the autophagic block in hepatocarcinoma cells .", "of note , the autophagic block occurred also in the presence of uv - inactivated kshv , indicating that neither vbcl2 nor vflip expression was required [ 34 , 96 , 99 , 100 ] .", "concomitantly to the autophagic block induced by kshv - mediated stat3 activation , we also observed a reduction of il12p70 release in response to lps stimulation and a higher production of il-10 , il-6 , and il-23 .", "this cytokine pattern skews the th1/th2 profile towards th2 and/or th17 , promoting immunosuppression and inflammation .", "therefore , stat3 activation could be one of the molecular mechanisms underlying kshv - mediated immunosuppression in dcs .", "indeed , stat3 activation correlates with an immunosuppressive phenotype and dc dysfunction in the ks microenvironment and in the peripheral blood of tumor bearing patients [ 103 , 104 ] .", "several papers have investigated the interplay between kshv and the autophagic pathway . during the latent phase of its life - cycle", ", kshv expresses the fadd - like interleukin-1 beta - converting enzyme inhibitory protein ( v - flip ) , a truncated homolog of the cellular flip , which besides having an antiapoptotic activity [ 105 , 106 ] has been recently shown to block the autophagic flux .", "it competes with lc3 for binding to atg3 , thereby preventing lc3 binding and processing during autophagosome biogenesis .", "furthermore , the inhibition of vflip binding to atg3 reduced the size of kshv positive tumor in mice .", "it has also been demonstrated that v - flip suppression of autophagy counteracts v - cyclin - induced cellular senescence [ 107 , 108 ] .", "v - cyclin is a kshv latent protein that deregulates cell cycle , causes aberrant host dna replication , and triggers the dna damage responses ( ddrs ) . besides vflip , two kshv proteins expressed during the lytic cycle , vbcl2 and k7 , can interfere with autophagy .", "vbcl2 is a homolog of cellular bcl-2 that inhibits both apoptosis [ 99 , 110 ] and autophagy [ 34 , 35 ] . as its cellular counterpart", ", vbcl2 negatively regulates autophagy by binding beclin 1 . always within the context of the lytic cycle", ", recent evidence suggests that kshv k7 protein prevents autophagosome maturation , by interacting with rubicon autophagic protein .", "k7 is involved in apoptosis suppression [ 112 , 113 ] while rubicon is a subunit of the beclin 1/uvrag / vps34 autophagy complex , which regulates the autophagosome maturation and the endocytic trafficking [ 114116 ] .", "as shown by authors , k7 transfection in epithelial cell lines promoted a greater interaction between rubicon and beclin 1/uvrag / vps34 complex resulting in a block of vps34 enzymatic activity and , consequently , of autophagosome maturation .", "interestingly , during the lytic cycle kshv exploits autophagy to enhance its reactivation mediated by rta immediate lytic protein .", "altogether , the data here reviewed suggest that kshv has evolved several strategies to circumvent autophagy - mediated immune responses , to persist in infected hosts .", "moreover , the autophagic block during latency and the autophagy induction during the lytic cycle may contribute to the pathogenesis of kshv associated malignancies ." ], [ "a better understanding of how autophagy manipulation could influence antiviral immune response and control virus - induced tumorigenesis might help to discover strategies improving the outcome of the treatments of virus - associated malignancies .", "moreover , since autophagy is activated and is involved in ebv and kshv replication [ 20 , 27 ] , its manipulation could also affect the viral particle release that plays a role in -herpesvirus - associated cancers ." ] ]

[ "seven able - bodied male subjects ( age 30.7 4.2 yrs ; height 178.9 10.2 cm ; mass 73.9 12.2 kg , mean sd ) participated in this study", "the study was approved by the local ethics committee ( ethics committee of the swiss canton of bern , kek bern ) .", "the knee dynamometer is a custom made measurement device , which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension .", "the lower leg is fixed with a brace to an aluminium load cell ( lcb130 , me - mesysteme gmbh , germany ) , which moves , via a lever arm , a chain drive system connected to a magnetostrictive torque sensor ( s-2220 - 75 , ncte ag germany ) . the torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . a brushless motor ( ec45 , 250 w , maxon motor ag , switzerland )", "is used with a planetary gear head ( gear ratio : 156:1 , gp42 , maxon motor ag , switzerland ) .", "the actuator can generate a maximum continuous torque of 90 nm . a position sensor ( vert - x 28 , contelec gmbh , switzerland ) is used for angle measurement with a resolution of 0.648 deg to control the motor torque .", "the measurements were performed at a constant angular velocity of ~110 /s , which is equivalent to a cycling cadence of 50 rpm , and the range of motion was set from 45 to 130 knee extension ( where 180 is full extension ) .", "the electrical pulses were generated with an eight channel stimulator ( rehastim , hasomed gmbh , germany ) .", "co. , ltd , usa ) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point ( fig .", "1 ) . the skin was cleaned and the body hair shaved at the position of the electrodes .", "muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen ( motor point pen , compex , switzerland ) .", "subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma .", "current was applied using an sdss electrode setup , which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode ( 9 5 cm ) with the same total area .", "each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90 , which corresponds to an overall stimulation frequency of 35 hz .", "two different sdss electrode placements were investigated : distal versus proximal sdss electrodes . in both cases the active part", "the mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup .", "each subject participated in two sessions with only one electrode placement tested for each leg within each session .", "between the two measurements in each session , subjects had a break of 15 minutes . stimulation setup order and the leg were chosen randomly . before each measurement", "subjects were placed on the dynamometer and individual adjustments to body proportions were made . then a two - minute phase was started in which the measured leg was moved by the device without stimulation ( non - stimulation [ ns ] phase ) . then the pulse width was manually increased after every third extension , starting at 0 s . pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed .", "80 % of this maximal pulse width ( pwmax ) was then used for the test measurements . after a rest period of 10 minutes ,", "the measurement started with an ns - phase of two minutes followed by a stimulation phase ( st - phase ) of 6 minutes .", "each session was conducted on a different day with at least one day of rest in between .", "electrode positions were marked to ensure identical placement each day only the extension phase of joint motion was evaluated , as the setup was to simulate cycling motion .", "the measured torque ( ) , together with the angular speed was used to calculate the instantaneous output power ( pm ) .", "the power used to move the leg during the ns - phase was denoted as pns .", "the net effective power output of one stimulation cycle is thus pstim = pns pm . for every knee extension", "the following outcomes were calculated : ( a ) mean power output during one extension ( pmean ) , ( b ) peak power output ( ppeak ) and ( c ) the time from onset of the stimulation to 80% of ppeak ( tpeak80 ) . to compare the different stimulation setups ,", "pstim was scaled using a reference pulse width of 100 s ( pstim , s ) , e.g. subject a had a pulse width of 80 s , so the scaled mean power output pmean , s of that subject is pmean \n ( 100/80 ) and the scaled peak power output ppeak , s is ppeak \n ( 100/80 ) .", "all outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions .", "a fatigue index ( fi ) describes the percentage reduction in pmean from the initial phase ( pinit ) to the final phase ( pfinal ) : fi=1-(pinit - pfinal)/pinit .", "the higher the value , the higher the fatigue resistance ; fi=1 means no fatigue .", "the data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means .", "seven able - bodied male subjects ( age 30.7 4.2 yrs ; height 178.9 10.2 cm ; mass 73.9 12.2 kg , mean sd ) participated in this study", "the study was approved by the local ethics committee ( ethics committee of the swiss canton of bern , kek bern ) .", "the knee dynamometer is a custom made measurement device , which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension .", "the lower leg is fixed with a brace to an aluminium load cell ( lcb130 , me - mesysteme gmbh , germany ) , which moves , via a lever arm , a chain drive system connected to a magnetostrictive torque sensor ( s-2220 - 75 , ncte ag germany ) .", "the torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . a brushless motor ( ec45 , 250 w , maxon motor ag , switzerland )", "is used with a planetary gear head ( gear ratio : 156:1 , gp42 , maxon motor ag , switzerland ) .", "a position sensor ( vert - x 28 , contelec gmbh , switzerland ) is used for angle measurement with a resolution of 0.648 deg to control the motor torque .", "the measurements were performed at a constant angular velocity of ~110 /s , which is equivalent to a cycling cadence of 50 rpm , and the range of motion was set from 45 to 130 knee extension ( where 180 is full extension ) .", "the electrical pulses were generated with an eight channel stimulator ( rehastim , hasomed gmbh , germany ) .", "co. , ltd , usa ) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point ( fig .", "1 ) . the skin was cleaned and the body hair shaved at the position of the electrodes .", "muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen ( motor point pen , compex , switzerland ) .", "subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma .", "current was applied using an sdss electrode setup , which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode ( 9 5 cm ) with the same total area .", "each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90 , which corresponds to an overall stimulation frequency of 35 hz .", "two different sdss electrode placements were investigated : distal versus proximal sdss electrodes . in both cases the active part was placed on the previously detected motor point .", "the mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup .", "each subject participated in two sessions with only one electrode placement tested for each leg within each session . between the two measurements in each session , subjects had a break of 15 minutes . stimulation setup order and the leg", "subjects were placed on the dynamometer and individual adjustments to body proportions were made . then a two - minute phase was started in which the measured leg was moved by the device without stimulation ( non - stimulation [ ns ] phase ) . then the pulse width was manually increased after every third extension , starting at 0 s . pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed .", "80 % of this maximal pulse width ( pwmax ) was then used for the test measurements . after a rest period of 10 minutes ,", "the measurement started with an ns - phase of two minutes followed by a stimulation phase ( st - phase ) of 6 minutes .", "each session was conducted on a different day with at least one day of rest in between .", "only the extension phase of joint motion was evaluated , as the setup was to simulate cycling motion .", "the measured torque ( ) , together with the angular speed was used to calculate the instantaneous output power ( pm ) .", "the power used to move the leg during the ns - phase was denoted as pns .", "the net effective power output of one stimulation cycle is thus pstim = pns pm . for every knee extension", "the following outcomes were calculated : ( a ) mean power output during one extension ( pmean ) , ( b ) peak power output ( ppeak ) and ( c ) the time from onset of the stimulation to 80% of ppeak ( tpeak80 ) . to compare the different stimulation setups ,", "pstim was scaled using a reference pulse width of 100 s ( pstim , s ) , e.g. subject a had a pulse width of 80 s , so the scaled mean power output pmean , s of that subject is pmean \n ( 100/80 ) and the scaled peak power output ppeak , s is ppeak \n ( 100/80 ) .", "all outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions . a fatigue index ( fi )", "describes the percentage reduction in pmean from the initial phase ( pinit ) to the final phase ( pfinal ) : fi=1-(pinit - pfinal)/pinit .", "the higher the value , the higher the fatigue resistance ; fi=1 means no fatigue .", "the data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means .", "figure 2 shows the development of pmean , s , ppeak , s and tpeak80 over the 6-minute stimulation phase .", "the corresponding outcome measures for the initial and final stimulation phases are summarised in tab .", "1 . no significant differences between distal and proximal electrode placement were found for any outcome measures during the initial stimulation phase . in the final stimulation phase , ppeak and ppeak ,", "s showed significantly higher values for the distal sdss setup : 25.4 8.1 w vs. 28.2 6.2 w , p=0.0062 and 34.8 9.5 w vs. 38.9 6.7 w , p=0.021 , respectively . in the final phase", "3 ) and pmean , s with the distal sdss placement ( 11.8 3.8 w vs. 12.7 3.3 w , p=0.071 and 16.2 4.5 w vs. 17.4 3.4 w , p=0.14 ) , and of longer tpeak80 for distal sdss ( 347.6 29.2 ms vs. 359.4 38.2 ms , p=0.096 ) . the modestly higher mean power output in the final stage with distal sdss , and a lower dispersion of power values , can be conveniently visualised ( fig .", "fatigue resistance was not different between the two stimulation setups ( fi 0.61 0.14 vs. 0.64 0.9 , p=0.38 ) .", "the aim of this study was to compare the power output , fatigue and activation properties of proximally versus distally placed sdss electrodes in an isokinetic knee extension task simulating knee movement during recumbent cycling .", "overall , especially in the final phase of stimulation , the distal sdss setup showed higher power outputs ( the only exception was pmean , s in the initial phase , which was minimally lower for distal sdss ) .", "this might be regarded as surprising , since the active electrode was placed exactly on the motor point in the proximal sdss setup .", "splitting one large electrode into four small ones of the same overall size , and using a sequential stimulation strategy , was previously shown to give increased power output and better fatigue resistance compared to a standard electrode setup .", "the temporal and spatial shift had a significant impact on muscle activation in a dynamic knee extension task .", "using a standard electrode setup ( ses ) , it should not make a difference which electrode ( active , dispersive ) is positioned at the motor point : the electrical field is the same and the current direction should not activate the muscle fibres differently or change the number of recruited motor units . on the other hand , the electrical field changes with the size of the electrodes , and the distance between the active and dispersive electrodes has a substantial influence on the torque . to control for these factors in the two setups investigated in the present study , the distance was not changed and the active electrode was placed distally .", "that no substantial or significant difference was observed for any power output parameter in the initial phase of the task may indicate that both setups recruited and activated a similar number of motor units . the similar development over time of pmean and ppeak , as well as the different development of tpeak80 ( fig .", "2a ) between setups , shows that the muscle fibre recruitment and the power curve of a single extension is not the same over time . although fi was not significantly different , the examination of power development over time ( fig .", "2b , c ) shows that distal placement seems to have a slower power decrease and a higher power output in the final stimulation phase .", "smaller electrodes increase the current density compared to larger electrodes using the same amplitude and pulse width .", "thus , the non - equal size of the electrodes ( active , dispersive ) leads to an asymmetric electrical field , which seems to influence muscle activation .", "in contrast to the distal placement , where four small active electrodes are placed around the motor point , in the proximal setup one large active electrode is placed exactly on the motor point . in consequence , the change of the electrical field at this sensitive position is lower in this setup .", "less change over time in the pattern of activation has been shown to favourably affect fatigue and power output development ( 13 , 14 ) .", "activation time , i.e. the time from stimulation onset to 80% of peak power , plays a crucial role when electrical stimulation is used to produce a functional movement .", "usually , more than just one muscle group is involved , so that both the coordination of the force and the activation of the different muscles are of importance . in fes - cycling ,", "often only three major muscle groups are involved ( m. quadriceps , m. hamstrings and m. gluteus ) and the coordination of these muscles is one factor for achievement of high power output . during repetitive activation , muscles not only fatigue , but", "tpeak80 for the proximal placement starts to flatten out after about 30 extensions , whereas tpeak80 for the distal placement is still increasing at this time and only begins to flatten out after about 65 extensions ( fig .", "the mean difference for tpeak80 of 11.8 ms in the final phase corresponds to a phase shift of 6 when cycling 50 rpm , which might have an influence on the overall cycling performance . by positioning the electrodes more precisely in relation to the motor points ( proximal setup ) ,", "the activation becomes more efficient and the muscle activation time is less affected by the duration of the task . in conclusion ,", "the sdss approach to muscle stimulation seems to provide substantial performance benefits , but the placement of the electrodes is still a crucial factor .", "distal placement of the sdss electrodes showed higher power output values in the final stimulation phase but also a slightly increased activation time .", "the development of new array electrodes , specifically for sdss , where the initial pulse is applied directly on the motor point and the following pulses are randomly distributed , may combine the positive effects of the proximal and distal electrode placements .", "based on the evidence presented here , for practical fes applications , distal placement of the sdss electrodes appears to be preferable ." ]

[ "<S> spatially distributed sequential stimulation ( sdss ) has demonstrated substantial power output and fatigue benefits compared to single electrode stimulation ( ses ) in the application of functional electrical stimulation ( fes ) . </S>", "<S> this asymmetric electrode setup brings new possibilities but also new questions since precise placement of the electrodes is one critical factor for good muscle activation . </S>", "<S> the aim of this study was to compare the power output , fatigue and activation properties of proximally versus distally placed sdss electrodes in an isokinetic knee extension task simulating knee movement during recumbent cycling . m. vastus lateralis and medialis of seven able - bodied subjects were stimulated with rectangular bi - phasic pulses of constant amplitude of 40 ma and at an sdss frequency of 35 hz for 6 min on both legs with both setups ( i.e. n=14 ) . </S>", "<S> torque was measured during knee - extension movement by a dynamometer at an angular velocity of 110 deg / s . </S>", "<S> mean power , peak power and activation time were calculated and compared for the initial and final stimulation phases , together with an overall fatigue index . </S>", "<S> power output values ( pmean , ppeak ) were scaled to a standardised reference input pulse width of 100 s ( pmean , s , ppeak , s ) . </S>", "<S> the initial evaluation phase showed no significant differences between the two setups for all outcome measures . </S>", "<S> ppeak and ppeak , s were both significantly higher in the final phase for the distal setup ( 25.4 8.1 w vs. 28.2 6.2 w , p=0.0062 and 34.8 9.5 w vs. 38.9 6.7 w , p=0.021 , respectively ) . with distal sdss </S>", "<S> , there was modest evidence of higher pmean and pmean , s ( p=0.071 , p=0.14 , respectively ) but of longer activation time ( p=0.096 ) . </S>", "<S> the rate of fatigue was similar for both setups . for practical fes applications , distal placement of the sdss electrodes </S>", "<S> is preferable . </S>" ]

[ "Materials and Methods", "Subjects", "Device", "Stimulation", "Procedure", "Outcomes and statistical analysis", "Results", "Discussion" ]

[ [ "seven able - bodied male subjects ( age 30.7 4.2 yrs ; height 178.9 10.2 cm ; mass 73.9 12.2 kg , mean sd ) participated in this study", "the study was approved by the local ethics committee ( ethics committee of the swiss canton of bern , kek bern ) .", "the knee dynamometer is a custom made measurement device , which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension .", "the lower leg is fixed with a brace to an aluminium load cell ( lcb130 , me - mesysteme gmbh , germany ) , which moves , via a lever arm , a chain drive system connected to a magnetostrictive torque sensor ( s-2220 - 75 , ncte ag germany ) . the torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . a brushless motor ( ec45 , 250 w , maxon motor ag , switzerland )", "is used with a planetary gear head ( gear ratio : 156:1 , gp42 , maxon motor ag , switzerland ) .", "the actuator can generate a maximum continuous torque of 90 nm . a position sensor ( vert - x 28 , contelec gmbh , switzerland ) is used for angle measurement with a resolution of 0.648 deg to control the motor torque .", "the measurements were performed at a constant angular velocity of ~110 /s , which is equivalent to a cycling cadence of 50 rpm , and the range of motion was set from 45 to 130 knee extension ( where 180 is full extension ) .", "the electrical pulses were generated with an eight channel stimulator ( rehastim , hasomed gmbh , germany ) .", "co. , ltd , usa ) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point ( fig .", "1 ) . the skin was cleaned and the body hair shaved at the position of the electrodes .", "muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen ( motor point pen , compex , switzerland ) .", "subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma .", "current was applied using an sdss electrode setup , which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode ( 9 5 cm ) with the same total area .", "each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90 , which corresponds to an overall stimulation frequency of 35 hz .", "two different sdss electrode placements were investigated : distal versus proximal sdss electrodes . in both cases the active part", "the mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup .", "each subject participated in two sessions with only one electrode placement tested for each leg within each session .", "between the two measurements in each session , subjects had a break of 15 minutes . stimulation setup order and the leg were chosen randomly . before each measurement", "subjects were placed on the dynamometer and individual adjustments to body proportions were made . then a two - minute phase was started in which the measured leg was moved by the device without stimulation ( non - stimulation [ ns ] phase ) . then the pulse width was manually increased after every third extension , starting at 0 s . pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed .", "80 % of this maximal pulse width ( pwmax ) was then used for the test measurements . after a rest period of 10 minutes ,", "the measurement started with an ns - phase of two minutes followed by a stimulation phase ( st - phase ) of 6 minutes .", "each session was conducted on a different day with at least one day of rest in between .", "electrode positions were marked to ensure identical placement each day only the extension phase of joint motion was evaluated , as the setup was to simulate cycling motion .", "the measured torque ( ) , together with the angular speed was used to calculate the instantaneous output power ( pm ) .", "the power used to move the leg during the ns - phase was denoted as pns .", "the net effective power output of one stimulation cycle is thus pstim = pns pm . for every knee extension", "the following outcomes were calculated : ( a ) mean power output during one extension ( pmean ) , ( b ) peak power output ( ppeak ) and ( c ) the time from onset of the stimulation to 80% of ppeak ( tpeak80 ) . to compare the different stimulation setups ,", "pstim was scaled using a reference pulse width of 100 s ( pstim , s ) , e.g. subject a had a pulse width of 80 s , so the scaled mean power output pmean , s of that subject is pmean \n ( 100/80 ) and the scaled peak power output ppeak , s is ppeak \n ( 100/80 ) .", "all outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions .", "a fatigue index ( fi ) describes the percentage reduction in pmean from the initial phase ( pinit ) to the final phase ( pfinal ) : fi=1-(pinit - pfinal)/pinit .", "the higher the value , the higher the fatigue resistance ; fi=1 means no fatigue .", "the data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means ." ], [ "seven able - bodied male subjects ( age 30.7 4.2 yrs ; height 178.9 10.2 cm ; mass 73.9 12.2 kg , mean sd ) participated in this study", "the study was approved by the local ethics committee ( ethics committee of the swiss canton of bern , kek bern ) ." ], [ "the knee dynamometer is a custom made measurement device , which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension .", "the lower leg is fixed with a brace to an aluminium load cell ( lcb130 , me - mesysteme gmbh , germany ) , which moves , via a lever arm , a chain drive system connected to a magnetostrictive torque sensor ( s-2220 - 75 , ncte ag germany ) .", "the torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . a brushless motor ( ec45 , 250 w , maxon motor ag , switzerland )", "is used with a planetary gear head ( gear ratio : 156:1 , gp42 , maxon motor ag , switzerland ) .", "a position sensor ( vert - x 28 , contelec gmbh , switzerland ) is used for angle measurement with a resolution of 0.648 deg to control the motor torque .", "the measurements were performed at a constant angular velocity of ~110 /s , which is equivalent to a cycling cadence of 50 rpm , and the range of motion was set from 45 to 130 knee extension ( where 180 is full extension ) ." ], [ "the electrical pulses were generated with an eight channel stimulator ( rehastim , hasomed gmbh , germany ) .", "co. , ltd , usa ) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point ( fig .", "1 ) . the skin was cleaned and the body hair shaved at the position of the electrodes .", "muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen ( motor point pen , compex , switzerland ) .", "subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma .", "current was applied using an sdss electrode setup , which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode ( 9 5 cm ) with the same total area .", "each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90 , which corresponds to an overall stimulation frequency of 35 hz .", "two different sdss electrode placements were investigated : distal versus proximal sdss electrodes . in both cases the active part was placed on the previously detected motor point .", "the mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup ." ], [ "each subject participated in two sessions with only one electrode placement tested for each leg within each session . between the two measurements in each session , subjects had a break of 15 minutes . stimulation setup order and the leg", "subjects were placed on the dynamometer and individual adjustments to body proportions were made . then a two - minute phase was started in which the measured leg was moved by the device without stimulation ( non - stimulation [ ns ] phase ) . then the pulse width was manually increased after every third extension , starting at 0 s . pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed .", "80 % of this maximal pulse width ( pwmax ) was then used for the test measurements . after a rest period of 10 minutes ,", "the measurement started with an ns - phase of two minutes followed by a stimulation phase ( st - phase ) of 6 minutes .", "each session was conducted on a different day with at least one day of rest in between ." ], [ "only the extension phase of joint motion was evaluated , as the setup was to simulate cycling motion .", "the measured torque ( ) , together with the angular speed was used to calculate the instantaneous output power ( pm ) .", "the power used to move the leg during the ns - phase was denoted as pns .", "the net effective power output of one stimulation cycle is thus pstim = pns pm . for every knee extension", "the following outcomes were calculated : ( a ) mean power output during one extension ( pmean ) , ( b ) peak power output ( ppeak ) and ( c ) the time from onset of the stimulation to 80% of ppeak ( tpeak80 ) . to compare the different stimulation setups ,", "pstim was scaled using a reference pulse width of 100 s ( pstim , s ) , e.g. subject a had a pulse width of 80 s , so the scaled mean power output pmean , s of that subject is pmean \n ( 100/80 ) and the scaled peak power output ppeak , s is ppeak \n ( 100/80 ) .", "all outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions . a fatigue index ( fi )", "describes the percentage reduction in pmean from the initial phase ( pinit ) to the final phase ( pfinal ) : fi=1-(pinit - pfinal)/pinit .", "the higher the value , the higher the fatigue resistance ; fi=1 means no fatigue .", "the data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means ." ], [ "figure 2 shows the development of pmean , s , ppeak , s and tpeak80 over the 6-minute stimulation phase .", "the corresponding outcome measures for the initial and final stimulation phases are summarised in tab .", "1 . no significant differences between distal and proximal electrode placement were found for any outcome measures during the initial stimulation phase . in the final stimulation phase , ppeak and ppeak ,", "s showed significantly higher values for the distal sdss setup : 25.4 8.1 w vs. 28.2 6.2 w , p=0.0062 and 34.8 9.5 w vs. 38.9 6.7 w , p=0.021 , respectively . in the final phase", "3 ) and pmean , s with the distal sdss placement ( 11.8 3.8 w vs. 12.7 3.3 w , p=0.071 and 16.2 4.5 w vs. 17.4 3.4 w , p=0.14 ) , and of longer tpeak80 for distal sdss ( 347.6 29.2 ms vs. 359.4 38.2 ms , p=0.096 ) . the modestly higher mean power output in the final stage with distal sdss , and a lower dispersion of power values , can be conveniently visualised ( fig .", "fatigue resistance was not different between the two stimulation setups ( fi 0.61 0.14 vs. 0.64 0.9 , p=0.38 ) ." ], [ "the aim of this study was to compare the power output , fatigue and activation properties of proximally versus distally placed sdss electrodes in an isokinetic knee extension task simulating knee movement during recumbent cycling .", "overall , especially in the final phase of stimulation , the distal sdss setup showed higher power outputs ( the only exception was pmean , s in the initial phase , which was minimally lower for distal sdss ) .", "this might be regarded as surprising , since the active electrode was placed exactly on the motor point in the proximal sdss setup .", "splitting one large electrode into four small ones of the same overall size , and using a sequential stimulation strategy , was previously shown to give increased power output and better fatigue resistance compared to a standard electrode setup .", "the temporal and spatial shift had a significant impact on muscle activation in a dynamic knee extension task .", "using a standard electrode setup ( ses ) , it should not make a difference which electrode ( active , dispersive ) is positioned at the motor point : the electrical field is the same and the current direction should not activate the muscle fibres differently or change the number of recruited motor units . on the other hand , the electrical field changes with the size of the electrodes , and the distance between the active and dispersive electrodes has a substantial influence on the torque . to control for these factors in the two setups investigated in the present study , the distance was not changed and the active electrode was placed distally .", "that no substantial or significant difference was observed for any power output parameter in the initial phase of the task may indicate that both setups recruited and activated a similar number of motor units . the similar development over time of pmean and ppeak , as well as the different development of tpeak80 ( fig .", "2a ) between setups , shows that the muscle fibre recruitment and the power curve of a single extension is not the same over time . although fi was not significantly different , the examination of power development over time ( fig .", "2b , c ) shows that distal placement seems to have a slower power decrease and a higher power output in the final stimulation phase .", "smaller electrodes increase the current density compared to larger electrodes using the same amplitude and pulse width .", "thus , the non - equal size of the electrodes ( active , dispersive ) leads to an asymmetric electrical field , which seems to influence muscle activation .", "in contrast to the distal placement , where four small active electrodes are placed around the motor point , in the proximal setup one large active electrode is placed exactly on the motor point . in consequence , the change of the electrical field at this sensitive position is lower in this setup .", "less change over time in the pattern of activation has been shown to favourably affect fatigue and power output development ( 13 , 14 ) .", "activation time , i.e. the time from stimulation onset to 80% of peak power , plays a crucial role when electrical stimulation is used to produce a functional movement .", "usually , more than just one muscle group is involved , so that both the coordination of the force and the activation of the different muscles are of importance . in fes - cycling ,", "often only three major muscle groups are involved ( m. quadriceps , m. hamstrings and m. gluteus ) and the coordination of these muscles is one factor for achievement of high power output . during repetitive activation , muscles not only fatigue , but", "tpeak80 for the proximal placement starts to flatten out after about 30 extensions , whereas tpeak80 for the distal placement is still increasing at this time and only begins to flatten out after about 65 extensions ( fig .", "the mean difference for tpeak80 of 11.8 ms in the final phase corresponds to a phase shift of 6 when cycling 50 rpm , which might have an influence on the overall cycling performance . by positioning the electrodes more precisely in relation to the motor points ( proximal setup ) ,", "the activation becomes more efficient and the muscle activation time is less affected by the duration of the task . in conclusion ,", "the sdss approach to muscle stimulation seems to provide substantial performance benefits , but the placement of the electrodes is still a crucial factor .", "distal placement of the sdss electrodes showed higher power output values in the final stimulation phase but also a slightly increased activation time .", "the development of new array electrodes , specifically for sdss , where the initial pulse is applied directly on the motor point and the following pulses are randomly distributed , may combine the positive effects of the proximal and distal electrode placements .", "based on the evidence presented here , for practical fes applications , distal placement of the sdss electrodes appears to be preferable ." ] ]

[ "bipolar disorder is a psychiatric disease state manifesting as two different mood extremes , mania and depression .", "manic symptoms often include hyperactivity , hyper - talkativeness , decreased need for sleep , grandiosity , increased risk taking , and being easily distracted ( apa 2000 ) .", "depression symptoms typically consist of a depressed mood , decreased energy , feelings of guilt , hopelessness , helplessness , crying , and even suicidal ideations ( apa 2000 ) .", "symptoms of bipolar disorder typically present during late adolescence between the ages of 15 and 19 years , however it is not uncommon for proper diagnosis to be delayed for 510 years ( bowden et al 2002 ) .", "bipolar i disorder is estimated to be prevalent in 1% of the us population , however recent findings from a large - scale survey suggest an adjusted prevalence rate near 3.7% ( bowden et al 2002 ; hirschfeld , calabrese et al 2003 ) . additionally , it has been found through survey that there is often a gap of up to 10 years between the first visit and subsequent proper diagnosis , with the majority either not being diagnosed at all or inappropriately diagnosed with unipolar depression ( hirschfeld , calabrese et al 2003 ; hirschfeld , lewis et al 2003 ) .", "identification of which type of bipolar disorder is present is complicated by the fact that both bipolar i and bipolar ii patients spend more time symptomatic in the depressed phase than the manic or hypomanic states ( judd et al 2002 ) .", "this is significant when considering the potential risk of switching a bipolar patient from a depressed state to manic state with antidepressant therapy .", "contrary to this is that failure to address a depressive episode of bipolar disorder presents a dramatic increase in suicide risk . additional things to consider when treating a patient with bipolar disorder are the high rates of concurrent substance abuse and anxiety disorders ( bowden et al 2002 ; mcelroy 2004 ) .", "several hypotheses have been proposed concerning the mechanism of action in epilepsy and bipolar disorder . the most well studied and understood", "mechanism of valproate is its ability to potentiate or mimic the effects of the inhibitory neurotransmitter , gamma - aminobutyric acid ( gaba ) ( loscher 2002 ; casey et al 2003 ; salloum et al 2005 ) .", "indirectly , this potentiation of gaba has been hypothesized to produce inhibitory effects on central dopamine ( casey et al 2002 ; loscher 2002 ) . some of the other and less well understood mechanisms involve the inhibition of neuronal excitability and a resultant anti - kindling effect ( loscher 2002 ) .", "one specific area of study has focused on the inhibition of protein kinase c epsilon ( pkc - epsilon ) ( brunello 2004 ; toth 2005 ) .", "pkc - epsilon has been linked to the stimulation of intracellular calcium release and an increase in cortical excitation and instability .", "valproate has also exhibited effects producing the blockade of voltage - dependent sodium channels ( loscher 2002 ; owens and nemeroff 2003 ) .", "another proposed mechanism , though controversial , is one likening valproate to lithium as a potential inhibitor of inositol synthesis through inhibition of myo - inositol-1phosphate ( mip ) ( harwood and agam 2003 ; shaltiel et al 2004 ; harwood 2005 ) .", "it is not well understood if valproate inhibits mip directly , but it has been shown to deplete inositol ( harwood and agam 2003 ; shaltiel et al 2004 ; harwood 2005 ) .", "delayed - release divalproex ( dr ) is an enteric - coated compound consisting of sodium valproate and valproic acid in a 1:1 molar ratio ( abbott laboratories 2006 ) .", "divalproex is metabolized in the liver , primarily via glucuronidation to active metabolites , with the major active metabolite being trans-2-en - valproate ( loscher 2002 ) . the estimated half - life of divalproex ranges from 9 to 16 hours ( abbott laboratories 2006 ) .", "the usual dosing regimen for dr is on a two or three times daily schedule , often with the larger dose given at bedtime . the therapeutic range for divalproex sodium in acute mania according to primary literature suggests improvement is greatest at concentrations above 50 g / ml and that adverse effects increase significantly at concentrations above 125 g / ml ( bowden et al 2002 ) .", "the more common side effects of dr are transient nausea ( 31% ) , asthenia ( 20% ) , somnolence ( 17% ) , dyspepsia ( 13% ) , dizziness ( 12% ) , diarrhea ( 12% ) , vomiting ( 11% ) , and tremor ( 9% ) ( abbott laboratories 2006 ) . some long - term side effects that have been associated with dr use are alopecia and weight gain .", "therapeutic drug monitoring for valproic acid or any formulation of divalproex requires periodic monitoring of liver enzymes as serious liver toxicity has been reported , especially with use in children under the age of two and in patients receiving multiple antiepileptic medications .", "valproic acid / divalproex have also been shown to produce a dose - related thrombocytopenia , thus periodic monitoring of platelets is also suggested .", "the primary advantage of the dr formulation over immediate release valproic acid ( ir ) is the enteric coating which helps reduce the incidence of gastrointestinal complaints .", "prevalence of hepatic enzyme elevation , tremor , ataxia , increased appetite , weight gain , and alopecia have not been shown to be substantially different in clinical trials between dr and ir preparations .", "the newest formulation of divalproex is in the form of an extended - release tablet , depakote er ( er ) , which provides a once - daily administration option for the treatment of acute manic or mixed episodes of bipolar disorder , independent of the presence of psychotic features ( abbott laboratories 2006 ) .", "the er formulation uses a hydrophilic polymer matrix controlled - release tablet system to provide controlled continued release of medication .", "after oral administration and entry into the stomach , the outer coating of the tablet dissolves and exposes the polymer matrix .", "the outer layer of the matrix becomes hydrated and forms a gel layer from which drug is released .", "in addition to bipolar disorder , er is also approved for the prophylactic management of migraine headaches in adults , as well as monotherapy and adjunctive therapy in adults and children 10 years of age and older with complex partial seizures , adults and children 10 years of age or older with simple and complex absence seizures , and adults and children 10 years of age and older with multiple seizure types including absence seizures ( abbott laboratories 2006 ) .", "the two primary kinetic differences are that the er preparation results in an average bioavailability of 81%89% relative to dr and er has produces a 10%20% lower fluctuation in peak serum concentration as compared to dr ( abbott laboratories 2006 ) .", "these findings suggest that when converting patients from dr to er that the er dose may need to be increased between 8% and 20% to produce an equivalent serum concentration .", "further evidence for pharmacokinetic differences were supported in a study using a healthy adult population and comparing the bioavailability of unequal doses of dr and er ( dutta et al 2002 ) .", "the results from this study determined that daily doses of er dosed 14% and 20% higher than dr ( 1000 mg / day er vs 875 mg / day dr and 1500 mg / day er vs 1250 mg / day dr ) produced equivalent serum concentrations as determined by area - under - the - curve ( auc ) ( dutta and et al 2002 ) .", "peak - to - trough a higher cmin fluctuations were 42%48% lower for the er preparation ( dutta et al 2002 ) .", "this study also showed that the increase in dose required with the er dosage form was well tolerated ( dutta et al 2002 ) .", "while evidence is increasing , there continues to be a relative paucity of information available describing the use of er in treating mood related symptoms within the psychiatric population .", "delayed - release divalproex ( dr ) is an enteric - coated compound consisting of sodium valproate and valproic acid in a 1:1 molar ratio ( abbott laboratories 2006 ) .", "divalproex is metabolized in the liver , primarily via glucuronidation to active metabolites , with the major active metabolite being trans-2-en - valproate ( loscher 2002 ) . the estimated half - life of divalproex ranges from 9 to 16 hours ( abbott laboratories 2006 ) .", "the usual dosing regimen for dr is on a two or three times daily schedule , often with the larger dose given at bedtime . the therapeutic range for divalproex sodium in acute mania according to primary literature suggests improvement is greatest at concentrations above 50 g / ml and that adverse effects increase significantly at concentrations above 125 g / ml ( bowden et al 2002 ) .", "the more common side effects of dr are transient nausea ( 31% ) , asthenia ( 20% ) , somnolence ( 17% ) , dyspepsia ( 13% ) , dizziness ( 12% ) , diarrhea ( 12% ) , vomiting ( 11% ) , and tremor ( 9% ) ( abbott laboratories 2006 ) . some long - term side effects that have been associated with dr use are alopecia and weight gain .", "therapeutic drug monitoring for valproic acid or any formulation of divalproex requires periodic monitoring of liver enzymes as serious liver toxicity has been reported , especially with use in children under the age of two and in patients receiving multiple antiepileptic medications .", "valproic acid / divalproex have also been shown to produce a dose - related thrombocytopenia , thus periodic monitoring of platelets is also suggested .", "the primary advantage of the dr formulation over immediate release valproic acid ( ir ) is the enteric coating which helps reduce the incidence of gastrointestinal complaints .", "prevalence of hepatic enzyme elevation , tremor , ataxia , increased appetite , weight gain , and alopecia have not been shown to be substantially different in clinical trials between dr and ir preparations .", "the newest formulation of divalproex is in the form of an extended - release tablet , depakote er ( er ) , which provides a once - daily administration option for the treatment of acute manic or mixed episodes of bipolar disorder , independent of the presence of psychotic features ( abbott laboratories 2006 ) .", "the er formulation uses a hydrophilic polymer matrix controlled - release tablet system to provide controlled continued release of medication .", "after oral administration and entry into the stomach , the outer coating of the tablet dissolves and exposes the polymer matrix .", "the outer layer of the matrix becomes hydrated and forms a gel layer from which drug is released .", "in addition to bipolar disorder , er is also approved for the prophylactic management of migraine headaches in adults , as well as monotherapy and adjunctive therapy in adults and children 10 years of age and older with complex partial seizures , adults and children 10 years of age or older with simple and complex absence seizures , and adults and children 10 years of age and older with multiple seizure types including absence seizures ( abbott laboratories 2006 ) .", "the two primary kinetic differences are that the er preparation results in an average bioavailability of 81%89% relative to dr and er has produces a 10%20% lower fluctuation in peak serum concentration as compared to dr ( abbott laboratories 2006 ) .", "these findings suggest that when converting patients from dr to er that the er dose may need to be increased between 8% and 20% to produce an equivalent serum concentration .", "further evidence for pharmacokinetic differences were supported in a study using a healthy adult population and comparing the bioavailability of unequal doses of dr and er ( dutta et al 2002 ) .", "the results from this study determined that daily doses of er dosed 14% and 20% higher than dr ( 1000 mg / day er vs 875 mg / day dr and 1500 mg / day er vs 1250 mg / day dr ) produced equivalent serum concentrations as determined by area - under - the - curve ( auc ) ( dutta and et al 2002 ) .", "peak - to - trough a higher cmin fluctuations were 42%48% lower for the er preparation ( dutta et al 2002 ) .", "this study also showed that the increase in dose required with the er dosage form was well tolerated ( dutta et al 2002 ) .", "while evidence is increasing , there continues to be a relative paucity of information available describing the use of er in treating mood related symptoms within the psychiatric population .", "there have been a number of recent additions to the list of approved medications for the treatment of bipolar disorder ( see table 1 ) . despite these new advancements in drug delivery and efficacy for the treatment of bipolar disorder", ", dr continues to be a highly utilized mood stabilizer for the treatment of acute mania ( abbott laboratories 2006 ) . in many instances ,", "bipolar disorder treatment guidelines recommend valproate as a preferred agent , with dr usually being the suggested formulation of valproate / valproic acid ( divalproex ) due to the presumed improved tolerability ( zarate et al 1999 ; bowden et al 2002 ; keck et al 2004 ; suppes et al 2005 ) .", "the disease states where evidence based guidelines support the use of valproate as a first - line choice include : acute manic exacerbations , euphoric mania , dysphoric or mixed mania , and in patients being treated for rapid cycling bipolar disorder ( see table 2 ) ( bowden et al 2002 ; keck et al 2004 ; suppes et al 2005 ) .", "valproate is also considered an appropriate agent for maintenance therapy , though it lacks the fda maintenance indication ( taylor and goodwin 2006 ) .", "other fda approved indications for dr include the treatment of complex partial , simple absence , and complex absence seizures in addition to prophylaxis of migraine headaches ( abbott laboratories 2006 ) .", "dr is not fda approved for aggression , though it is routinely used as monotherapy and is considered appropriate adjunct therapy to reduce hostility associated with schizophrenia ( citrome , casey et al 2004 ; lehman et al 2004 ) .", "in order to identify published , primary literature studying the er formulation in bipolar disorder we conducted an ovid medline search .", "our search terms included bipolar disorder , mania , depression , extended - release divalproex , delayed - release divalproex , divalproex , and schizophrenia .", "we did not include any information that was not published in primary literature form so any data on file with abbott laboratories that is not published is not included in this review .", "our search yielded a small number of clinical trials reporting on the use of extended - release divalproex in bipolar disorder .", "three of the trials are open - label with a study enrollment ranging from 10 to 55 patients .", "one additional study was included that examined the use of extended - release divalproex for mood stabilization and antipsychotic augmentation in schizophrenia .", "the first trial discussed is an open - label , seven day study evaluating the efficacy and safety of converting psychiatric patients from dr to er ( horne and cunanan 2003 ) .", "the majority of participants carried a diagnosis of bipolar disorder or major depression , 36% and 27% respectively .", "a total of 55 patients were included in the conversion , 75% outpatients and 25% inpatients hospitalized for acute symptoms .", "participants had been treated with dr from 2 days to > 4 years at doses of 500 to 5,000 mg / day .", "concomitant medications were described and included agents such as antipsychotics , antidepressants , and anxiolytics . following baseline measurements of plasma valproic acid concentrations ,", "study participants were switched to er at a dose equal to their total daily dose of dr .", "subsequent therapeutic drug monitoring included assessment of plasma valproic acid concentrations which were obtained 10 to 12 hours after the last dose on study days 3 , 5 , and 7 .", "over half the patients in the study ( 58% ) experienced an increase in valproic acid plasma concentration when switched from dr to er dosage forms . in all but three cases", ", plasma concentrations remained within the therapeutic range of 50125 g / ml . in two of the cases the plasma levels increased following the initiation of the er dosage form with no signs of toxicity and returned to values within therapeutic range with dosage reduction . in the third case , the patient s serum valproic acid level decreased below the lower limit of normal but increased following dosage titration .", "efficacy was assessed with the positive and negative syndrome scale ( panss ) at baseline and endpoint . upon analysis of the total patient population", ", a statistically significant improvement was observed in mean panss total score , positive subscale and general psychopathology subscale from baseline to endpoint .", "mean total panss scores at baseline were 71.5 21.4 with a mean change of 4.3 11.1 at endpoint . while a statistical change was appreciated , clinical impact of improvement was most likely small .", "adverse events were assessed with the udvalg for kliniske undersogelser ( uku ) side effect rating scale .", "patients reported a decrease in the number and severity of adverse effects at endpoint . following the cessation of the study ,", "this design characteristic becomes less of an issue for outcome measures in which statistical improvement is observed .", "overall , conversion from dr to er in this inpatient and outpatient psychiatric population was not associated with deterioration in mental status . in addition , a reduction in both the incidence and severity of adverse effects was appreciated with the er dosing formulation , most likely thought to be the result of lower peak concentrations .", "a second published , open - label study highlighted the conversion of dr to er in ten subjects over a four - week time period ( stoner et al 2004 ) .", "subjects were deemed eligible if they had been on dr at least 8 weeks and were considered ", "subjects were also required to be experiencing two mild adverse events or one moderate adverse event that was considered a potential side effect of dr .", "all subjects were using dr for mood or behavioral related symptoms , with most diagnosed with schizophrenia , bipolar disorder , or schizoaffective disorder , bipolar type .", "seven of the ten subjects were converted on an equal milligram - per - milligram basis , while the other three received a 250 mg dose increase to 500 mg as at the time of the study only 500 mg er tablets were available .", "the mean dr dose at baseline was 2,475 1,010 mg / day with a slightly higher mean dose of er observed at study endpoint , 2,550 985 mg / day .", "the study group included six males and four females with an average age of 39.4 years and an average length of mental illness of 21.4 years .", "subjects were diagnosed primarily with schizophrenia ( n = 4 ) , bipolar disorder ( n = 2 ) , and schizoaffective disorder ( n = 2 ) .", "the primary outcome measure in this study was the 18-item brief psychiatric rating scale ( bprs ) , selected to identify any changes in psychiatric , behavioral , or mood related symptoms .", "the bprs was completed at baseline and then at days 7 , 14 , 21 , and 28 .", "of particular interest , no significant changes were appreciated in weekly bprs scores , although numerically the mean scores improved from baseline ( 29.10 6.28 ) to endpoint ( 26.5 7.14 , p = 0.208 ) .", "no individual bprs items showed a statistically significant change , however a trend suggested a decrease in somatic complaints ( p = 0.057 ) .", "eleven - hour post - dose valproic acid serum concentrations were collected at days 14 and 28 .", "no statistically significant difference was found in dr baseline serum valproic acid concentrations ( 90.5 29.11 g / ml ) and day 28 11-hour post - dose er valproic acid serum concentrations ( 95.50 13.68 ; p = 0.493 ) .", "additional monitoring parameters included assessment of weight changes , and collection hematologic , renal , hepatic , electrolyte , lipid , and glucose labs at baseline and study endpoint .", "serum chemistry monitoring showed statistically significant decreases in ldl cholesterol and potassium , although the decline in potassium was not clinically significant . there were no significant changes in platelet counts observed during the course of the study .", "tolerability and adverse event assessment was as measured by the systematic assessment for treatment emergent effects ( saftee ) at baseline and then days 7 , 14 , 21 , and 28 .", "saftee results showed statistically significant reductions in complaints of sedation , stomach and abdominal discomfort , and tremor from baseline to study endpoint .", "this study was limited by a small sample size and the inclusion of only stable patients , thus not allowing the clinical application the er findings to the acute phase of treatment .", "additionally , the study subjects showed a broad variation of axis i diagnosis , not limited to bipolar disorder . despite these limitations ,", "this study does provide some level of evidence that er can be used in place of dr to help maintain psychiatric stability .", "a third open - label , six day study was designed to compare the conversion of stable bipolar i or ii or schizoaffective subjects ( n = 12 ) from the dr to the er formulation ( centorrino et al 2003 ) . upon entry into the study ,", "subjects were required to have baseline serum valproic acid levels within the therapeutic range of 50120 g / ml and had to have been receiving stable does of medications for at least four weeks prior to study initiation .", "participants were switched to er with a goal of maintaining stable valproic acid serum concentrations .", "as the er formulation was only available in the 500 mg tablet dosage form at the time of the study , doses were rounded to the nearest 500 mg / day .", "serum valproic acid levels were collected at baseline , day 7 , week 6 , and one week following a medication adjustment . in this cohort of patients", ", it was observed that er doses needed to be 20.7% higher than the previous dr doses to maintain serum valproic acid levels , a finding consistent with package labeling for er .", "numerous efficacy measurements were evaluated at baseline and weekly thereafter and included the young mania rating scale ( ymrs ) , the 17-item hamilton depression rating scale ( ham - d 17 ) , clinical global impression of severity ( cgi - s ) and improvement ( cgi - i ) , global assessment of functioning scale ( gaf ) , and the 17-item brief psychiatric rating scale ( bprs ) .", "the mean baseline ymrs score was 3.00 3.86 and at endpoint it increased to 3.42 2.53 .", "the baseline mean ham - d 17 score was 11.2 9.3 and at endpoint the mean improved to 7.67 6.97 .", "the mean cgi of severity score at baseline was 2.58 0.79 and at endpoint was 2.75 0.65 .", "the mean baseline gaf score was 68.3 6.2 and at endpoint improved marginally with a value of 69.2 6.0 .", "the baseline mean bprs score was 39.8 10.2 and at endpoint was 37.8 7.82 .", "none of the observed changes were deemed to be significant in regards to psychiatric stability .", "the most commonly reported adverse effects at both baseline and endpoint were impaired concentration , fatigue , depression , and decreased salivation .", "the only statistically significant adverse event that was seen more frequently with the er dosage form as compared to the dr was an increase in polyuria - polydipsia .", "all participants elected to continue treatment with the er dosage formulation at the conclusion of the study .", "this study was also not without limitations , most notably a small sample size and the inclusion of only stable patients .", "three small studies which have been published as letter to the editors are useful in reporting conversions from dr to er dosage formulations in psychiatric outpatients ( longo 2005 ; minirth and veal 2005 ; jackson et al 2006 ) . in the first of these studies , a small , 12-week , open - label , pilot trial examined outpatients diagnosed with bipolar i or ii disorder or schizoaffective disorder ( longo 2005 ) .", "the patients described in this study were being treated with the dr formulation , but were reporting associated adverse events .", "the dose conversion was carried out per package labeling which recommends up to a 20% dose increase in converting from dr to er .", "the primary outcome measures were the clinical global impression scale ( cgi ) and the global assessment of functioning scales ( gaf ) . according to these outcome measures ,", "9 of 10 patients were considered to exhibit no change or slight improvement in their symptoms , while 5 of 10 reported improvements in adverse events .", "baseline psychometric assessment scoring , dosing information , length of prior treatment , and therapeutic drug monitoring parameters were collected , however none of the values were reported .", "this study possesses several limitations , however it does provide some information regarding practical experience with converting patients .", "publications described a retrospective chart review that focused on the evaluation of efficacy , tolerability , and impact on adherence when switching patients from dr to er ( minirth and veal 2005 ) .", "psychiatric patients , including those diagnosed with bipolar disorder types i and ii , were included .", "participants eligible for enrollment had to have been taking dr for at least three months prior to the switch .", "patients were evaluated using the cgi - s scale on the day of the switch from dr to er and again during the follow - up visit .", "additional secondary assessments which were evaluated at baseline and endpoint included self - rating report of symptoms and review of clinician s notes regarding patient symptomatology .", "the study was conducted at a single study site and included the records of 32 patients .", "doses of divalproex sodium dr ranged from 125 to 1000 mg / day and following the milligram - for - milligram switch patients were maintained on 5002000 mg / day of er . while this report suggests clinical improvement ,", "the ability to critically evaluate the study is limited by a lack of data showing baseline and endpoint scores for the outcome measures .", "therapeutic drug monitoring , particularly serum valproic acid levels , were not mentioned in the design of the study and were not reported .", "adherence , one of the secondary outcome measures , was patient rated and evaluated by a telephone interview conducted by the raters .", " briefly described the conversion of 52 patients stabilized on dr who were converted on an equal milligram - per - milligram basis to the er formulation for up to 24 weeks ( jackson et al 2006 ) .", "psychometric assessment measures included the ham - d 21 ( 21-item scale ) and ymrs . utilizing the statistical method of repeated measures analysis ,", "statistically significant improvement was noted from the time of conversion to study endpoint with both the ham - d 21 and ymrs .", "no significant changes were identified in therapeutic drug monitoring lab values and overall patients reported improved tolerability of the er formulation .", "the use of er in the management of psychiatric symptoms has not been limited to bipolar disorder alone .", "one 4-week , open - label dr to er conversion study included thirty patients diagnosed with schizophrenia ( citrome , tremeau et al 2004 ) . to be included in the study , patients had to be on a stable dose ( 1,000 to 3,000 mg / day ) of dr for at least 4 weeks .", "patients were converted at a 1:1 mg ratio ( n = 12 ) for dr to er if the baseline serum concentration of valproate was 85 g / ml and 1:1.2 mg ratio ( n = 18 ) for dr to er if the baseline serum concentration of valproate was < 85 g / ml .", "dosing required the use of 500 mg increments due to the lack of availability of the 250 mg tablet at the time of the study .", "the bprs was the primary outcome measure with side effects assessed with the uku side effect rating scale .", "the baseline mean bprs total score was 37.9 9.2 ( n = 30 ) and the mean endpoint bprs score was 35.7 11.2 ( n = 29 ) , producing a significant mean reduction of 2.3 5.4 points ( p = 0.0322 ) .", "significant improvement was noted for the 1:1 mg conversion group ( p = 0.0561 ) , but not for the 1:1.2 mg group ( p = 0.2223 ) .", "mean uku scores also showed significant improvement , dropping from a mean of 8.8 6.7 ( n = 29 ) at baseline to 7.5 5.8 ( n = 28 ) at endpoint , though for patients with evaluable baseline and endpoint scores the mean change was a reduction of 2.2 4.1 ( n = 27 , p = 0.0111 ) .", "the mean dr dose at study entry was 1,592 mg 498 mg / day which produced a mean baseline trough ( 12-hours post - dose ) valproate concentration of 80.1 20.4 g / ml . the er dosing at study endpoint was 1,950 mg 592 mg / day which produced a mean trough concentration ( 24-hours post - dose ) of 73.1 24.2 g / ml . the 1:1 mg conversion group produced trough levels that were significantly lower at endpoint compared to baseline ( p = 0.0006 ) , though the difference in baseline and endpoint levels for the 1:1.2 conversion group were not ( p = 0.7102 ) . the conversion of dr to er was not associated with any reports of psychiatric decompensation . while the bprs was noted to improve across the group , the small reduction should not be interpreted as being more effective as this study is limited by being open - label , small sample size , short duration , and the actual improvement was only a 6% reduction in total bprs score .", "the improvement of uku scores are consistent with other reports of improved tolerability associated with the er formulation .", "current literature evaluating the use of er in psychiatric patients with bipolar disorder suggests the er formulation has advantages over the dr formulation . in contrast to dr ,", "the er formulation provides a dosage form of divalproex that is pharmacokinetically supported for once - daily dosing .", "while the studies included in this review were not specifically designed to examine differences in medication compliance rates , a correlation between once daily dosing and medication adherence has been established . of additional benefit , er", "had improved tolerability over dr , likely the result of lower peak plasma concentrations without apparent risk of lost efficacy .", "the studies included in this review article are greatly limited when utilizing a strict evidence based medicine review procedure .", "the studies included are not blinded or controlled , they are limited by small sample sizes , they are of short - duration , and are not powered . there is broad variation in the samples making cross - comparisons unreasonable . from a statistical standpoint ,", "there is potential for a beta error , a declaration of no difference when a difference indeed exists , could result in a study not adequately designed to evaluate for mathematical significance . despite these severe limitations , there is value in the information that has been reported in these study findings .", "these studies provide practical practice information on converting from dr to er in clinical settings and what can be expected in terms of efficacy , tolerability , and therapeutic drug monitoring . while there is no data indicating that the er formulation provides any enhanced efficacy over dr there is also no compelling data suggesting there is a risk to using the er over the dr . formulation .", "the current treatment guidelines included in this review do not specifically recommend the er formulation and from an evidence perspective one can not say there is strong enough evidence to make it a first - line choice over other treatment options .", "however , given that the molecule is the same ( divalproex ) and that the primary differences are rate of release and a reduced peak concentration , the er formulation should be considered a safe alternative to dr , particularly when there is a history or concern of dose related ( peak concentration ) adverse events or when once a day dosing is preferred in an effort to enhance compliance ." ]

[ "<S> bipolar disorder can be a devastating disease state for individuals with the disease and also for family members . </S>", "<S> proper recognition and treatment is vital to the successful management of this disease state . through increased community and practitioner awareness , along with efforts to increase awareness for proper assessment , the rate of diagnosed bipolar disorder is increasing . </S>", "<S> recent years have brought about the introduction of several new medications with approved indications for the treatment of bipolar disorder . </S>", "<S> in addition to new agents , traditional mood stabilizing medications have also been released in different formulations to better enhance tolerability without jeopardizing efficacy . </S>", "<S> one particular product is extended - release divalproex sodium . in the following article </S>", "<S> , we review the clinical presentation of bipolar disorder , its epidemiology , and the pharmacokinetics and mechanism of action for divalproex . </S>", "<S> in addition , we specifically review the role of extended - release divalproex in bipolar disorder through a critical analysis of the currently available published primary literature . </S>" ]

[ "Bipolar disorder", "Valproate mechanism", "Divalproex pharmacology", "Delayed-release divalproex", "Extended-release divalproex", "Role of divalproex in bipolar disorder", "Clinical trials efficacy", "Conclusion" ]

[ [ "bipolar disorder is a psychiatric disease state manifesting as two different mood extremes , mania and depression .", "manic symptoms often include hyperactivity , hyper - talkativeness , decreased need for sleep , grandiosity , increased risk taking , and being easily distracted ( apa 2000 ) .", "depression symptoms typically consist of a depressed mood , decreased energy , feelings of guilt , hopelessness , helplessness , crying , and even suicidal ideations ( apa 2000 ) .", "symptoms of bipolar disorder typically present during late adolescence between the ages of 15 and 19 years , however it is not uncommon for proper diagnosis to be delayed for 510 years ( bowden et al 2002 ) .", "bipolar i disorder is estimated to be prevalent in 1% of the us population , however recent findings from a large - scale survey suggest an adjusted prevalence rate near 3.7% ( bowden et al 2002 ; hirschfeld , calabrese et al 2003 ) . additionally , it has been found through survey that there is often a gap of up to 10 years between the first visit and subsequent proper diagnosis , with the majority either not being diagnosed at all or inappropriately diagnosed with unipolar depression ( hirschfeld , calabrese et al 2003 ; hirschfeld , lewis et al 2003 ) .", "identification of which type of bipolar disorder is present is complicated by the fact that both bipolar i and bipolar ii patients spend more time symptomatic in the depressed phase than the manic or hypomanic states ( judd et al 2002 ) .", "this is significant when considering the potential risk of switching a bipolar patient from a depressed state to manic state with antidepressant therapy .", "contrary to this is that failure to address a depressive episode of bipolar disorder presents a dramatic increase in suicide risk . additional things to consider when treating a patient with bipolar disorder are the high rates of concurrent substance abuse and anxiety disorders ( bowden et al 2002 ; mcelroy 2004 ) ." ], [ "several hypotheses have been proposed concerning the mechanism of action in epilepsy and bipolar disorder . the most well studied and understood", "mechanism of valproate is its ability to potentiate or mimic the effects of the inhibitory neurotransmitter , gamma - aminobutyric acid ( gaba ) ( loscher 2002 ; casey et al 2003 ; salloum et al 2005 ) .", "indirectly , this potentiation of gaba has been hypothesized to produce inhibitory effects on central dopamine ( casey et al 2002 ; loscher 2002 ) . some of the other and less well understood mechanisms involve the inhibition of neuronal excitability and a resultant anti - kindling effect ( loscher 2002 ) .", "one specific area of study has focused on the inhibition of protein kinase c epsilon ( pkc - epsilon ) ( brunello 2004 ; toth 2005 ) .", "pkc - epsilon has been linked to the stimulation of intracellular calcium release and an increase in cortical excitation and instability .", "valproate has also exhibited effects producing the blockade of voltage - dependent sodium channels ( loscher 2002 ; owens and nemeroff 2003 ) .", "another proposed mechanism , though controversial , is one likening valproate to lithium as a potential inhibitor of inositol synthesis through inhibition of myo - inositol-1phosphate ( mip ) ( harwood and agam 2003 ; shaltiel et al 2004 ; harwood 2005 ) .", "it is not well understood if valproate inhibits mip directly , but it has been shown to deplete inositol ( harwood and agam 2003 ; shaltiel et al 2004 ; harwood 2005 ) ." ], [ "delayed - release divalproex ( dr ) is an enteric - coated compound consisting of sodium valproate and valproic acid in a 1:1 molar ratio ( abbott laboratories 2006 ) .", "divalproex is metabolized in the liver , primarily via glucuronidation to active metabolites , with the major active metabolite being trans-2-en - valproate ( loscher 2002 ) . the estimated half - life of divalproex ranges from 9 to 16 hours ( abbott laboratories 2006 ) .", "the usual dosing regimen for dr is on a two or three times daily schedule , often with the larger dose given at bedtime . the therapeutic range for divalproex sodium in acute mania according to primary literature suggests improvement is greatest at concentrations above 50 g / ml and that adverse effects increase significantly at concentrations above 125 g / ml ( bowden et al 2002 ) .", "the more common side effects of dr are transient nausea ( 31% ) , asthenia ( 20% ) , somnolence ( 17% ) , dyspepsia ( 13% ) , dizziness ( 12% ) , diarrhea ( 12% ) , vomiting ( 11% ) , and tremor ( 9% ) ( abbott laboratories 2006 ) . some long - term side effects that have been associated with dr use are alopecia and weight gain .", "therapeutic drug monitoring for valproic acid or any formulation of divalproex requires periodic monitoring of liver enzymes as serious liver toxicity has been reported , especially with use in children under the age of two and in patients receiving multiple antiepileptic medications .", "valproic acid / divalproex have also been shown to produce a dose - related thrombocytopenia , thus periodic monitoring of platelets is also suggested .", "the primary advantage of the dr formulation over immediate release valproic acid ( ir ) is the enteric coating which helps reduce the incidence of gastrointestinal complaints .", "prevalence of hepatic enzyme elevation , tremor , ataxia , increased appetite , weight gain , and alopecia have not been shown to be substantially different in clinical trials between dr and ir preparations .", "the newest formulation of divalproex is in the form of an extended - release tablet , depakote er ( er ) , which provides a once - daily administration option for the treatment of acute manic or mixed episodes of bipolar disorder , independent of the presence of psychotic features ( abbott laboratories 2006 ) .", "the er formulation uses a hydrophilic polymer matrix controlled - release tablet system to provide controlled continued release of medication .", "after oral administration and entry into the stomach , the outer coating of the tablet dissolves and exposes the polymer matrix .", "the outer layer of the matrix becomes hydrated and forms a gel layer from which drug is released .", "in addition to bipolar disorder , er is also approved for the prophylactic management of migraine headaches in adults , as well as monotherapy and adjunctive therapy in adults and children 10 years of age and older with complex partial seizures , adults and children 10 years of age or older with simple and complex absence seizures , and adults and children 10 years of age and older with multiple seizure types including absence seizures ( abbott laboratories 2006 ) .", "the two primary kinetic differences are that the er preparation results in an average bioavailability of 81%89% relative to dr and er has produces a 10%20% lower fluctuation in peak serum concentration as compared to dr ( abbott laboratories 2006 ) .", "these findings suggest that when converting patients from dr to er that the er dose may need to be increased between 8% and 20% to produce an equivalent serum concentration .", "further evidence for pharmacokinetic differences were supported in a study using a healthy adult population and comparing the bioavailability of unequal doses of dr and er ( dutta et al 2002 ) .", "the results from this study determined that daily doses of er dosed 14% and 20% higher than dr ( 1000 mg / day er vs 875 mg / day dr and 1500 mg / day er vs 1250 mg / day dr ) produced equivalent serum concentrations as determined by area - under - the - curve ( auc ) ( dutta and et al 2002 ) .", "peak - to - trough a higher cmin fluctuations were 42%48% lower for the er preparation ( dutta et al 2002 ) .", "this study also showed that the increase in dose required with the er dosage form was well tolerated ( dutta et al 2002 ) .", "while evidence is increasing , there continues to be a relative paucity of information available describing the use of er in treating mood related symptoms within the psychiatric population ." ], [ "delayed - release divalproex ( dr ) is an enteric - coated compound consisting of sodium valproate and valproic acid in a 1:1 molar ratio ( abbott laboratories 2006 ) .", "divalproex is metabolized in the liver , primarily via glucuronidation to active metabolites , with the major active metabolite being trans-2-en - valproate ( loscher 2002 ) . the estimated half - life of divalproex ranges from 9 to 16 hours ( abbott laboratories 2006 ) .", "the usual dosing regimen for dr is on a two or three times daily schedule , often with the larger dose given at bedtime . the therapeutic range for divalproex sodium in acute mania according to primary literature suggests improvement is greatest at concentrations above 50 g / ml and that adverse effects increase significantly at concentrations above 125 g / ml ( bowden et al 2002 ) .", "the more common side effects of dr are transient nausea ( 31% ) , asthenia ( 20% ) , somnolence ( 17% ) , dyspepsia ( 13% ) , dizziness ( 12% ) , diarrhea ( 12% ) , vomiting ( 11% ) , and tremor ( 9% ) ( abbott laboratories 2006 ) . some long - term side effects that have been associated with dr use are alopecia and weight gain .", "therapeutic drug monitoring for valproic acid or any formulation of divalproex requires periodic monitoring of liver enzymes as serious liver toxicity has been reported , especially with use in children under the age of two and in patients receiving multiple antiepileptic medications .", "valproic acid / divalproex have also been shown to produce a dose - related thrombocytopenia , thus periodic monitoring of platelets is also suggested .", "the primary advantage of the dr formulation over immediate release valproic acid ( ir ) is the enteric coating which helps reduce the incidence of gastrointestinal complaints .", "prevalence of hepatic enzyme elevation , tremor , ataxia , increased appetite , weight gain , and alopecia have not been shown to be substantially different in clinical trials between dr and ir preparations ." ], [ "the newest formulation of divalproex is in the form of an extended - release tablet , depakote er ( er ) , which provides a once - daily administration option for the treatment of acute manic or mixed episodes of bipolar disorder , independent of the presence of psychotic features ( abbott laboratories 2006 ) .", "the er formulation uses a hydrophilic polymer matrix controlled - release tablet system to provide controlled continued release of medication .", "after oral administration and entry into the stomach , the outer coating of the tablet dissolves and exposes the polymer matrix .", "the outer layer of the matrix becomes hydrated and forms a gel layer from which drug is released .", "in addition to bipolar disorder , er is also approved for the prophylactic management of migraine headaches in adults , as well as monotherapy and adjunctive therapy in adults and children 10 years of age and older with complex partial seizures , adults and children 10 years of age or older with simple and complex absence seizures , and adults and children 10 years of age and older with multiple seizure types including absence seizures ( abbott laboratories 2006 ) .", "the two primary kinetic differences are that the er preparation results in an average bioavailability of 81%89% relative to dr and er has produces a 10%20% lower fluctuation in peak serum concentration as compared to dr ( abbott laboratories 2006 ) .", "these findings suggest that when converting patients from dr to er that the er dose may need to be increased between 8% and 20% to produce an equivalent serum concentration .", "further evidence for pharmacokinetic differences were supported in a study using a healthy adult population and comparing the bioavailability of unequal doses of dr and er ( dutta et al 2002 ) .", "the results from this study determined that daily doses of er dosed 14% and 20% higher than dr ( 1000 mg / day er vs 875 mg / day dr and 1500 mg / day er vs 1250 mg / day dr ) produced equivalent serum concentrations as determined by area - under - the - curve ( auc ) ( dutta and et al 2002 ) .", "peak - to - trough a higher cmin fluctuations were 42%48% lower for the er preparation ( dutta et al 2002 ) .", "this study also showed that the increase in dose required with the er dosage form was well tolerated ( dutta et al 2002 ) .", "while evidence is increasing , there continues to be a relative paucity of information available describing the use of er in treating mood related symptoms within the psychiatric population ." ], [ "there have been a number of recent additions to the list of approved medications for the treatment of bipolar disorder ( see table 1 ) . despite these new advancements in drug delivery and efficacy for the treatment of bipolar disorder", ", dr continues to be a highly utilized mood stabilizer for the treatment of acute mania ( abbott laboratories 2006 ) . in many instances ,", "bipolar disorder treatment guidelines recommend valproate as a preferred agent , with dr usually being the suggested formulation of valproate / valproic acid ( divalproex ) due to the presumed improved tolerability ( zarate et al 1999 ; bowden et al 2002 ; keck et al 2004 ; suppes et al 2005 ) .", "the disease states where evidence based guidelines support the use of valproate as a first - line choice include : acute manic exacerbations , euphoric mania , dysphoric or mixed mania , and in patients being treated for rapid cycling bipolar disorder ( see table 2 ) ( bowden et al 2002 ; keck et al 2004 ; suppes et al 2005 ) .", "valproate is also considered an appropriate agent for maintenance therapy , though it lacks the fda maintenance indication ( taylor and goodwin 2006 ) .", "other fda approved indications for dr include the treatment of complex partial , simple absence , and complex absence seizures in addition to prophylaxis of migraine headaches ( abbott laboratories 2006 ) .", "dr is not fda approved for aggression , though it is routinely used as monotherapy and is considered appropriate adjunct therapy to reduce hostility associated with schizophrenia ( citrome , casey et al 2004 ; lehman et al 2004 ) ." ], [ "in order to identify published , primary literature studying the er formulation in bipolar disorder we conducted an ovid medline search .", "our search terms included bipolar disorder , mania , depression , extended - release divalproex , delayed - release divalproex , divalproex , and schizophrenia .", "we did not include any information that was not published in primary literature form so any data on file with abbott laboratories that is not published is not included in this review .", "our search yielded a small number of clinical trials reporting on the use of extended - release divalproex in bipolar disorder .", "three of the trials are open - label with a study enrollment ranging from 10 to 55 patients .", "one additional study was included that examined the use of extended - release divalproex for mood stabilization and antipsychotic augmentation in schizophrenia .", "the first trial discussed is an open - label , seven day study evaluating the efficacy and safety of converting psychiatric patients from dr to er ( horne and cunanan 2003 ) .", "the majority of participants carried a diagnosis of bipolar disorder or major depression , 36% and 27% respectively .", "a total of 55 patients were included in the conversion , 75% outpatients and 25% inpatients hospitalized for acute symptoms .", "participants had been treated with dr from 2 days to > 4 years at doses of 500 to 5,000 mg / day .", "concomitant medications were described and included agents such as antipsychotics , antidepressants , and anxiolytics . following baseline measurements of plasma valproic acid concentrations ,", "study participants were switched to er at a dose equal to their total daily dose of dr .", "subsequent therapeutic drug monitoring included assessment of plasma valproic acid concentrations which were obtained 10 to 12 hours after the last dose on study days 3 , 5 , and 7 .", "over half the patients in the study ( 58% ) experienced an increase in valproic acid plasma concentration when switched from dr to er dosage forms . in all but three cases", ", plasma concentrations remained within the therapeutic range of 50125 g / ml . in two of the cases the plasma levels increased following the initiation of the er dosage form with no signs of toxicity and returned to values within therapeutic range with dosage reduction . in the third case , the patient s serum valproic acid level decreased below the lower limit of normal but increased following dosage titration .", "efficacy was assessed with the positive and negative syndrome scale ( panss ) at baseline and endpoint . upon analysis of the total patient population", ", a statistically significant improvement was observed in mean panss total score , positive subscale and general psychopathology subscale from baseline to endpoint .", "mean total panss scores at baseline were 71.5 21.4 with a mean change of 4.3 11.1 at endpoint . while a statistical change was appreciated , clinical impact of improvement was most likely small .", "adverse events were assessed with the udvalg for kliniske undersogelser ( uku ) side effect rating scale .", "patients reported a decrease in the number and severity of adverse effects at endpoint . following the cessation of the study ,", "this design characteristic becomes less of an issue for outcome measures in which statistical improvement is observed .", "overall , conversion from dr to er in this inpatient and outpatient psychiatric population was not associated with deterioration in mental status . in addition , a reduction in both the incidence and severity of adverse effects was appreciated with the er dosing formulation , most likely thought to be the result of lower peak concentrations .", "a second published , open - label study highlighted the conversion of dr to er in ten subjects over a four - week time period ( stoner et al 2004 ) .", "subjects were deemed eligible if they had been on dr at least 8 weeks and were considered ", "subjects were also required to be experiencing two mild adverse events or one moderate adverse event that was considered a potential side effect of dr .", "all subjects were using dr for mood or behavioral related symptoms , with most diagnosed with schizophrenia , bipolar disorder , or schizoaffective disorder , bipolar type .", "seven of the ten subjects were converted on an equal milligram - per - milligram basis , while the other three received a 250 mg dose increase to 500 mg as at the time of the study only 500 mg er tablets were available .", "the mean dr dose at baseline was 2,475 1,010 mg / day with a slightly higher mean dose of er observed at study endpoint , 2,550 985 mg / day .", "the study group included six males and four females with an average age of 39.4 years and an average length of mental illness of 21.4 years .", "subjects were diagnosed primarily with schizophrenia ( n = 4 ) , bipolar disorder ( n = 2 ) , and schizoaffective disorder ( n = 2 ) .", "the primary outcome measure in this study was the 18-item brief psychiatric rating scale ( bprs ) , selected to identify any changes in psychiatric , behavioral , or mood related symptoms .", "the bprs was completed at baseline and then at days 7 , 14 , 21 , and 28 .", "of particular interest , no significant changes were appreciated in weekly bprs scores , although numerically the mean scores improved from baseline ( 29.10 6.28 ) to endpoint ( 26.5 7.14 , p = 0.208 ) .", "no individual bprs items showed a statistically significant change , however a trend suggested a decrease in somatic complaints ( p = 0.057 ) .", "eleven - hour post - dose valproic acid serum concentrations were collected at days 14 and 28 .", "no statistically significant difference was found in dr baseline serum valproic acid concentrations ( 90.5 29.11 g / ml ) and day 28 11-hour post - dose er valproic acid serum concentrations ( 95.50 13.68 ; p = 0.493 ) .", "additional monitoring parameters included assessment of weight changes , and collection hematologic , renal , hepatic , electrolyte , lipid , and glucose labs at baseline and study endpoint .", "serum chemistry monitoring showed statistically significant decreases in ldl cholesterol and potassium , although the decline in potassium was not clinically significant . there were no significant changes in platelet counts observed during the course of the study .", "tolerability and adverse event assessment was as measured by the systematic assessment for treatment emergent effects ( saftee ) at baseline and then days 7 , 14 , 21 , and 28 .", "saftee results showed statistically significant reductions in complaints of sedation , stomach and abdominal discomfort , and tremor from baseline to study endpoint .", "this study was limited by a small sample size and the inclusion of only stable patients , thus not allowing the clinical application the er findings to the acute phase of treatment .", "additionally , the study subjects showed a broad variation of axis i diagnosis , not limited to bipolar disorder . despite these limitations ,", "this study does provide some level of evidence that er can be used in place of dr to help maintain psychiatric stability .", "a third open - label , six day study was designed to compare the conversion of stable bipolar i or ii or schizoaffective subjects ( n = 12 ) from the dr to the er formulation ( centorrino et al 2003 ) . upon entry into the study ,", "subjects were required to have baseline serum valproic acid levels within the therapeutic range of 50120 g / ml and had to have been receiving stable does of medications for at least four weeks prior to study initiation .", "participants were switched to er with a goal of maintaining stable valproic acid serum concentrations .", "as the er formulation was only available in the 500 mg tablet dosage form at the time of the study , doses were rounded to the nearest 500 mg / day .", "serum valproic acid levels were collected at baseline , day 7 , week 6 , and one week following a medication adjustment . in this cohort of patients", ", it was observed that er doses needed to be 20.7% higher than the previous dr doses to maintain serum valproic acid levels , a finding consistent with package labeling for er .", "numerous efficacy measurements were evaluated at baseline and weekly thereafter and included the young mania rating scale ( ymrs ) , the 17-item hamilton depression rating scale ( ham - d 17 ) , clinical global impression of severity ( cgi - s ) and improvement ( cgi - i ) , global assessment of functioning scale ( gaf ) , and the 17-item brief psychiatric rating scale ( bprs ) .", "the mean baseline ymrs score was 3.00 3.86 and at endpoint it increased to 3.42 2.53 .", "the baseline mean ham - d 17 score was 11.2 9.3 and at endpoint the mean improved to 7.67 6.97 .", "the mean cgi of severity score at baseline was 2.58 0.79 and at endpoint was 2.75 0.65 .", "the mean baseline gaf score was 68.3 6.2 and at endpoint improved marginally with a value of 69.2 6.0 .", "the baseline mean bprs score was 39.8 10.2 and at endpoint was 37.8 7.82 .", "none of the observed changes were deemed to be significant in regards to psychiatric stability .", "the most commonly reported adverse effects at both baseline and endpoint were impaired concentration , fatigue , depression , and decreased salivation .", "the only statistically significant adverse event that was seen more frequently with the er dosage form as compared to the dr was an increase in polyuria - polydipsia .", "all participants elected to continue treatment with the er dosage formulation at the conclusion of the study .", "this study was also not without limitations , most notably a small sample size and the inclusion of only stable patients .", "three small studies which have been published as letter to the editors are useful in reporting conversions from dr to er dosage formulations in psychiatric outpatients ( longo 2005 ; minirth and veal 2005 ; jackson et al 2006 ) . in the first of these studies , a small , 12-week , open - label , pilot trial examined outpatients diagnosed with bipolar i or ii disorder or schizoaffective disorder ( longo 2005 ) .", "the patients described in this study were being treated with the dr formulation , but were reporting associated adverse events .", "the dose conversion was carried out per package labeling which recommends up to a 20% dose increase in converting from dr to er .", "the primary outcome measures were the clinical global impression scale ( cgi ) and the global assessment of functioning scales ( gaf ) . according to these outcome measures ,", "9 of 10 patients were considered to exhibit no change or slight improvement in their symptoms , while 5 of 10 reported improvements in adverse events .", "baseline psychometric assessment scoring , dosing information , length of prior treatment , and therapeutic drug monitoring parameters were collected , however none of the values were reported .", "this study possesses several limitations , however it does provide some information regarding practical experience with converting patients .", "publications described a retrospective chart review that focused on the evaluation of efficacy , tolerability , and impact on adherence when switching patients from dr to er ( minirth and veal 2005 ) .", "psychiatric patients , including those diagnosed with bipolar disorder types i and ii , were included .", "participants eligible for enrollment had to have been taking dr for at least three months prior to the switch .", "patients were evaluated using the cgi - s scale on the day of the switch from dr to er and again during the follow - up visit .", "additional secondary assessments which were evaluated at baseline and endpoint included self - rating report of symptoms and review of clinician s notes regarding patient symptomatology .", "the study was conducted at a single study site and included the records of 32 patients .", "doses of divalproex sodium dr ranged from 125 to 1000 mg / day and following the milligram - for - milligram switch patients were maintained on 5002000 mg / day of er . while this report suggests clinical improvement ,", "the ability to critically evaluate the study is limited by a lack of data showing baseline and endpoint scores for the outcome measures .", "therapeutic drug monitoring , particularly serum valproic acid levels , were not mentioned in the design of the study and were not reported .", "adherence , one of the secondary outcome measures , was patient rated and evaluated by a telephone interview conducted by the raters .", " briefly described the conversion of 52 patients stabilized on dr who were converted on an equal milligram - per - milligram basis to the er formulation for up to 24 weeks ( jackson et al 2006 ) .", "psychometric assessment measures included the ham - d 21 ( 21-item scale ) and ymrs . utilizing the statistical method of repeated measures analysis ,", "statistically significant improvement was noted from the time of conversion to study endpoint with both the ham - d 21 and ymrs .", "no significant changes were identified in therapeutic drug monitoring lab values and overall patients reported improved tolerability of the er formulation .", "the use of er in the management of psychiatric symptoms has not been limited to bipolar disorder alone .", "one 4-week , open - label dr to er conversion study included thirty patients diagnosed with schizophrenia ( citrome , tremeau et al 2004 ) . to be included in the study , patients had to be on a stable dose ( 1,000 to 3,000 mg / day ) of dr for at least 4 weeks .", "patients were converted at a 1:1 mg ratio ( n = 12 ) for dr to er if the baseline serum concentration of valproate was 85 g / ml and 1:1.2 mg ratio ( n = 18 ) for dr to er if the baseline serum concentration of valproate was < 85 g / ml .", "dosing required the use of 500 mg increments due to the lack of availability of the 250 mg tablet at the time of the study .", "the bprs was the primary outcome measure with side effects assessed with the uku side effect rating scale .", "the baseline mean bprs total score was 37.9 9.2 ( n = 30 ) and the mean endpoint bprs score was 35.7 11.2 ( n = 29 ) , producing a significant mean reduction of 2.3 5.4 points ( p = 0.0322 ) .", "significant improvement was noted for the 1:1 mg conversion group ( p = 0.0561 ) , but not for the 1:1.2 mg group ( p = 0.2223 ) .", "mean uku scores also showed significant improvement , dropping from a mean of 8.8 6.7 ( n = 29 ) at baseline to 7.5 5.8 ( n = 28 ) at endpoint , though for patients with evaluable baseline and endpoint scores the mean change was a reduction of 2.2 4.1 ( n = 27 , p = 0.0111 ) .", "the mean dr dose at study entry was 1,592 mg 498 mg / day which produced a mean baseline trough ( 12-hours post - dose ) valproate concentration of 80.1 20.4 g / ml . the er dosing at study endpoint was 1,950 mg 592 mg / day which produced a mean trough concentration ( 24-hours post - dose ) of 73.1 24.2 g / ml . the 1:1 mg conversion group produced trough levels that were significantly lower at endpoint compared to baseline ( p = 0.0006 ) , though the difference in baseline and endpoint levels for the 1:1.2 conversion group were not ( p = 0.7102 ) . the conversion of dr to er was not associated with any reports of psychiatric decompensation . while the bprs was noted to improve across the group , the small reduction should not be interpreted as being more effective as this study is limited by being open - label , small sample size , short duration , and the actual improvement was only a 6% reduction in total bprs score .", "the improvement of uku scores are consistent with other reports of improved tolerability associated with the er formulation ." ], [ "current literature evaluating the use of er in psychiatric patients with bipolar disorder suggests the er formulation has advantages over the dr formulation . in contrast to dr ,", "the er formulation provides a dosage form of divalproex that is pharmacokinetically supported for once - daily dosing .", "while the studies included in this review were not specifically designed to examine differences in medication compliance rates , a correlation between once daily dosing and medication adherence has been established . of additional benefit , er", "had improved tolerability over dr , likely the result of lower peak plasma concentrations without apparent risk of lost efficacy .", "the studies included in this review article are greatly limited when utilizing a strict evidence based medicine review procedure .", "the studies included are not blinded or controlled , they are limited by small sample sizes , they are of short - duration , and are not powered . there is broad variation in the samples making cross - comparisons unreasonable . from a statistical standpoint ,", "there is potential for a beta error , a declaration of no difference when a difference indeed exists , could result in a study not adequately designed to evaluate for mathematical significance . despite these severe limitations , there is value in the information that has been reported in these study findings .", "these studies provide practical practice information on converting from dr to er in clinical settings and what can be expected in terms of efficacy , tolerability , and therapeutic drug monitoring . while there is no data indicating that the er formulation provides any enhanced efficacy over dr there is also no compelling data suggesting there is a risk to using the er over the dr . formulation .", "the current treatment guidelines included in this review do not specifically recommend the er formulation and from an evidence perspective one can not say there is strong enough evidence to make it a first - line choice over other treatment options .", "however , given that the molecule is the same ( divalproex ) and that the primary differences are rate of release and a reduced peak concentration , the er formulation should be considered a safe alternative to dr , particularly when there is a history or concern of dose related ( peak concentration ) adverse events or when once a day dosing is preferred in an effort to enhance compliance ." ] ]

[ "children spend from 31 to 60% of their school day either \n writing or performing other fine motor tasks2 .", "the preparatory skills \n for writing are coordination of multiple joints , visual perception , vision - motor \n integration , and proprioception3 .", "together with stereognosis , \n proprioception allows smooth joint movement when vision is impaired or absent5 .", "children with poor proprioception have \n problems with handwriting legibility because their grip on a pen is too strong or too \n weak4 . generally , proprioception can be \n measured with joint position sense ( jps ) and kinesthetic sense ( ks)6 . while there are many studies investigating children s \n handwriting , quantitative studies on the association between handwriting and proprioception \n involving jps and ks are lacking7 .", "the aim of this study was to investigate the association between proprioception , involving \n jps and ks , and handwriting legibility in children .", "nineteen healthy children ( 15 boys and 4 girls ) with an average age of 9.7 0.36 years \n participated in this study .", "prior to the study , the children and their parents were informed \n about the purpose of the study and the general procedures to be undertaken . all children and \n", "the study was approved by the kaya university of \n human health science studies committee .", "the investigator moved the child s right arm passively \n through 80 of flexion at the elbow ( from 30 to 110 ) .", "the children were then directed to \n repeat this motion 10 times following a metronome set at 1 s intervals .", "their movements were \n recorded by a compact measuring system ( cms ) 10 for 3d motion analysis ( using the winarm \n software zebris medical gmbh , germany ) .", "cms markers were placed at the greater tubercle and \n lateral epicondyle of the right humerus and at the right wrist .", "angles of deviation from the \n targeted range of flexion were analyzed using matlab version 2014a ( the match works inc . , \n 2014 ) .", "ks was measured by the kinesthesia item in the sensory integration and praxis tests ( sipt , \n wps , torrance , ca , usa ) .", "the investigator moved a finger of the subject passively along a line from \n beginning to end , allowing the child to learn the line direction and distance .", "then , \n blindfolded and after one trial , the children were directed to trace five lines for each \n hand . only the results of the right hand were analyzed in this study .", "the investigator then \n measured the distance between the test endpoints and the real endpoints of the lines .", "legibility was evaluated using \n form , alignment , space , size , and slope8 .", "the legibility score was calculated as the ratio of \n the number of clearly written words to the total number of words ( score for legibility ( % ) = \n number of letters that received 5 points/30 100 ) .", "all data were analyzed using ibm spss statistics 20.0 ( ibm corp . , armonk , ny , usa ) . \n", "spearman s rank correlation was used to determine the relationships between legibility of \n handwriting and jps and ks , with significance defined as p<0.05 .", "values obtained for jps and ks in relation to handwriting legibility are shown in table 1table 1.handwriting legibility and proprioception test results ( n=19)mean sdjoint position sense 110 ( degree)22.530 10.063joint position sense 30 ( degree)14.256 10.408kinesthetic sense ( cm)2.044 0.703handwriting legibility ( % ) 33.521 15.852*standard deviation .", "there was no correlation between writing legibility and jps at either 30 or \n 110 of elbow flexion ( p>0.05 ) .", "a high ks correlated significantly with legible \n handwriting ( p<0.05 , table 2table 2.correlation between handwriting legibility and proprioception ( n=19)handwriting legibility ( % ) joint position sense 110 ( degree)0.016joint position sense 30 ( degree)0.009kinesthetic sense ( cm)0.370*p<0.05 ) .", "this study investigated the association between the legibility of handwriting and jps and \n ks in young children .", "children aged between 7 and 8 years are expected to be proficient in \n building up their speed of handwriting , ensuring consistency in size and proportions of \n letters as well as the spacing between letters and words9 .", "the average age of the participants in this study was 9.7 years \n old .", "our results showed that a highly accurate ks was \n associated with higher legibility scores .", "ks provides ongoing error information and memory storage to be recalled when \n writing is repeated .", "a high ks leads to programmed error correction , and the upgraded \n program generates better writing legibility .", "the results of this study support the \n hypothesis that ks reinforces the linkage between visual and motor control required for \n clear handwriting15 .", "we \n studies jps at the elbow joint because proprioception at the elbow joint is necessary for \n performing fine manipulative tasks including handwriting16 .", "studies have reported that proprioception at the wrist joint17 and finger joints18 influences handwriting legibility .", "we looked at children within a limited age range between 9 and 10 years of age . \n", "further , we did not control for other factors that affect writing legibility , such as fine \n motor control and visual - motor integration19,20,21 . \n", "finally , although handwriting quality is measured in terms of legibility and speed , this \n study investigated only writing legibility ." ]

[ "<S> [ purpose ] this study investigated the association between proprioception , including joint \n position sense and kinetic sense , and handwriting legibility in healthy children . \n [ subjects and methods ] assessment of joint position sense , kinetic sense , and handwriting \n legibility was conducted for 19 healthy children . </S>", "<S> joint position sense was assessed by \n asking the children to flex their right elbow between 30 to 110 while blindfolded . </S>", "<S> the \n range of elbow movement was analyzed with compact measuring system 10 for 3d motion \n analysis . </S>", "<S> kinetic sense was assessed using the sensory integration and praxis test . </S>", "<S> the \n children were directed to write 30 words from the korean alphabet , and the legibility of \n their handwriting was scored for form , alignment , space , size , and shape . to analyze the \n data , descriptive statistics and spearman correlation analysis </S>", "<S> were conducted using ibm \n spss statistics 20.0 . </S>", "<S> [ results ] there was significant negative correlation between \n handwriting legibility and kinetic sense . a significant correlation between handwriting \n legibility and joint position sense </S>", "<S> was not found . </S>", "<S> [ conclusion ] this study showed that a \n higher kinetic sense was associated with better legibility of handwriting . </S>", "<S> further work is \n needed to determine the association of handwriting legibility and speed with joint \n position sense of the elbow , wrist , and fingers . </S>" ]

[ "INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "children spend from 31 to 60% of their school day either \n writing or performing other fine motor tasks2 .", "the preparatory skills \n for writing are coordination of multiple joints , visual perception , vision - motor \n integration , and proprioception3 .", "together with stereognosis , \n proprioception allows smooth joint movement when vision is impaired or absent5 .", "children with poor proprioception have \n problems with handwriting legibility because their grip on a pen is too strong or too \n weak4 . generally , proprioception can be \n measured with joint position sense ( jps ) and kinesthetic sense ( ks)6 . while there are many studies investigating children s \n handwriting , quantitative studies on the association between handwriting and proprioception \n involving jps and ks are lacking7 .", "the aim of this study was to investigate the association between proprioception , involving \n jps and ks , and handwriting legibility in children ." ], [ "nineteen healthy children ( 15 boys and 4 girls ) with an average age of 9.7 0.36 years \n participated in this study .", "prior to the study , the children and their parents were informed \n about the purpose of the study and the general procedures to be undertaken . all children and \n", "the study was approved by the kaya university of \n human health science studies committee .", "the investigator moved the child s right arm passively \n through 80 of flexion at the elbow ( from 30 to 110 ) .", "the children were then directed to \n repeat this motion 10 times following a metronome set at 1 s intervals .", "their movements were \n recorded by a compact measuring system ( cms ) 10 for 3d motion analysis ( using the winarm \n software zebris medical gmbh , germany ) .", "cms markers were placed at the greater tubercle and \n lateral epicondyle of the right humerus and at the right wrist .", "angles of deviation from the \n targeted range of flexion were analyzed using matlab version 2014a ( the match works inc . , \n 2014 ) .", "ks was measured by the kinesthesia item in the sensory integration and praxis tests ( sipt , \n wps , torrance , ca , usa ) .", "the investigator moved a finger of the subject passively along a line from \n beginning to end , allowing the child to learn the line direction and distance .", "then , \n blindfolded and after one trial , the children were directed to trace five lines for each \n hand . only the results of the right hand were analyzed in this study .", "the investigator then \n measured the distance between the test endpoints and the real endpoints of the lines .", "legibility was evaluated using \n form , alignment , space , size , and slope8 .", "the legibility score was calculated as the ratio of \n the number of clearly written words to the total number of words ( score for legibility ( % ) = \n number of letters that received 5 points/30 100 ) .", "all data were analyzed using ibm spss statistics 20.0 ( ibm corp . , armonk , ny , usa ) . \n", "spearman s rank correlation was used to determine the relationships between legibility of \n handwriting and jps and ks , with significance defined as p<0.05 ." ], [ "values obtained for jps and ks in relation to handwriting legibility are shown in table 1table 1.handwriting legibility and proprioception test results ( n=19)mean sdjoint position sense 110 ( degree)22.530 10.063joint position sense 30 ( degree)14.256 10.408kinesthetic sense ( cm)2.044 0.703handwriting legibility ( % ) 33.521 15.852*standard deviation .", "there was no correlation between writing legibility and jps at either 30 or \n 110 of elbow flexion ( p>0.05 ) .", "a high ks correlated significantly with legible \n handwriting ( p<0.05 , table 2table 2.correlation between handwriting legibility and proprioception ( n=19)handwriting legibility ( % ) joint position sense 110 ( degree)0.016joint position sense 30 ( degree)0.009kinesthetic sense ( cm)0.370*p<0.05 ) ." ], [ "this study investigated the association between the legibility of handwriting and jps and \n ks in young children .", "children aged between 7 and 8 years are expected to be proficient in \n building up their speed of handwriting , ensuring consistency in size and proportions of \n letters as well as the spacing between letters and words9 .", "the average age of the participants in this study was 9.7 years \n old .", "our results showed that a highly accurate ks was \n associated with higher legibility scores .", "ks provides ongoing error information and memory storage to be recalled when \n writing is repeated .", "a high ks leads to programmed error correction , and the upgraded \n program generates better writing legibility .", "the results of this study support the \n hypothesis that ks reinforces the linkage between visual and motor control required for \n clear handwriting15 .", "we \n studies jps at the elbow joint because proprioception at the elbow joint is necessary for \n performing fine manipulative tasks including handwriting16 .", "studies have reported that proprioception at the wrist joint17 and finger joints18 influences handwriting legibility .", "we looked at children within a limited age range between 9 and 10 years of age . \n", "further , we did not control for other factors that affect writing legibility , such as fine \n motor control and visual - motor integration19,20,21 . \n", "finally , although handwriting quality is measured in terms of legibility and speed , this \n study investigated only writing legibility ." ] ]

[ "it includes hidradenitis suppurativa , acne conglobata , dissecting cellulitis of the scalp and pilonidal sinus .", "though each of these conditions are commonly encountered on their own , as a symptom complex follicular occlusion tetrad has rarely been reported in the literature . here", "we present a case of hidradenitis suppurativa in a 36-year - old male patient who also had the above mentioned associations .", "a 36-yeasr - old male patient presented to us with a history of recurrent boils since18 years .", "they initially started in the groin and buttocks with boils and gradually broke down to ooze pus .", "the boils healed with scarring after weeks to months . the process progressively started to involve the axillae and then the chest .", "the patient was treated with multiple antibiotics for over a decade that gave only symptomatic relief .", "the patient was diagnosed with pilonidal sinus at the age of 28 and he underwent surgery for the same .", "there was no family history of similar complaints , though his father suffered from psoriasis .", "on general examination , there were multiple areas of cicatricial alopecia on the scalp [ figure 1 ] and nodular scars on lower half of the face [ figure 2 ] .", "on query , the patient gave history of severe acne with nodules and pustules healing with scars on the face .", "he also had patchy swellings over the scalp region associated with pain in the past , which was diagnosed as folliculitis by a physician .", "patch of cicatricial alopecia on the occiput region of the scalp pustules , cysts and nodule - reminiscent of acne conglobata - on the lower half of the face on local examination , there were skin colored - to - erythematous nodules , grouped comedones and band like scars in the groin , buttocks , axillae and chest [ figure 3 and 4 ] .", "the patient had difficulty in lifting arms due to pain and scars in the axillae .", "multiple nodules , cysts and band like scars in the axillary region multiple nodules and pustules over the buttocks with surgical scar ( done for pilonidal sinus ) seen in the upper part of gluteal cleft a thorough general examination was followed by lab work - up .", "liver function tests and renal function tests were within normal limits and so was fbs , ppbs and fasting lipid profile .", "as the patient was obese , he was advised to follow a strict diet chart to lose weight .", "we also started him on oral isotretinoin 0.5 mg / kg body weight once daily .", "the patient came back after six weeks for prescription refill but was thereafter lost for follow - up .", "follicular occlusion tetrad is a condition that includes hidradenitis suppurativa ( hs ) , acne conglobata , dissecting cellulitis of the scalp and pilonidal sinus .", "hidradenitis suppurativa was first described in 1839 by velpeau ; verneuil gave it its name in 1854 and associated it with the sweat glands .", "later , hs was classified as a member of the follicular occlusion triad , along with acne conglobata and dissecting cellulitis of the scalp . in 1975", ", pilonidal cyst was added as a member to this triad , forming the follicular occlusion tetrad . in 1989 , plewig and steger introduced the term acne inversa to substitute the term hs .", "hidradenitis suppurativa or acne inversa is a chronic , inflammatory , recurrent , debilitating skin disease that usually presents after puberty with painful , deep - seated , inflamed lesions in the apocrine gland - bearing areas of the body , most commonly the axillae , inguinal and anogenital regions .", "the exact pathogenesis of this group of diseases is unknown but evidence suggests that they share the same pathological process initiated by follicular occlusion .", "the pathogenesis and aetiology are unknown , but is thought to originate from poral occlusion of the pilosebaceous units .", "diagnostic criteria of hidradenitis suppurativa ( adopted by the 2 international conference on hidradenitis suppurativa , march 5,2009 , san francisco , ca us ) . \n typical lesions , i.e. deep - seated painful nodules : ", "blind boils in early lesions ; abscesses , draining sinus , bridged scars and tombstonedouble - ended pseudo - comedones in secondary lesionstypical topography , i.e. axillae , groins , perineal and perianal region , buttocks , infra and inter mammary foldschronicity and recurrences . \n ", "blind boils in early lesions ; abscesses , draining sinus , bridged scars and tombstonedouble - ended pseudo - comedones in secondary lesions typical topography , i.e. axillae , groins , perineal and perianal region , buttocks , infra and inter mammary folds chronicity and recurrences .", "dissecting cellulitis(also called perifolliculitis capitis abscedens et suffodiens ) manifests with perifollicular pustules , nodules , abscesses and sinuses that evolve into scarring alopecia .", "it predominantly occurs in african american men between 20 - 40 years of age , but can occasionally affect other races and women too", "the tendency of dissecting cellulitis to cause severe alopecia , fluctuant nodules , and sinus tracts helps to distinguish it from acne keloidalis nuchae .", "culture of dissecting cellulitis does not produce a positive fungal culture and nuchal palpation does not reveal palpable lymph nodes , though reports have noted an inflammatory tinea capitis ( kerion ) that mimicked dissecting cellulitis in adolescents .", "its follicular papules and pustules spread peripherally , leaving central scarred patches of alopecia without nodules or sinuses . tufted folliculitis resolves with patches of scarring alopecia within which multiple hair tufts emerge from dilated follicular orifices .", "acne conglobata is an uncommon nodulocystictype of acne vulgaris that is often resistant to therapy .", "this disorder typically begins in adulthood and presents as numerous comedones , papules , pustules , nodules , abscesses , and draining sinus tracts involving the chest , back and buttocks .", "these lesions frequently become secondarily infected with gram - positive bacteria and often heal with scarring .", "pathology usually reveals inflammatory infiltrate around follicles , which can often disrupt the normal dermal architecture .", "acne conglobata is particularly disfiguring and socially detrimental to patients because of its chronicity , severity , and treatment challenge .", "acne conglobata ( ac ) resembles acne fulminans because both cause numerous inflammatory nodules on the trunk .", "unlike acne conglobata , large nodules of acne fulminans tend to become painful ulcers with overhanging borders surrounding exudative necrotic plaques that become confluent .", "the therapy of choice for ac includes isotretinoin 0.5 - 1 mg / kg for 4 - 6 months .", "simultaneous use of systemic steroids such as prednisone 1 mg / kg / d for 2 - 4 weeks may also prove beneficial , particularly if systemic symptoms are evident .", "oral tetracycline antibiotics should not be combined with oral isotretinoin because of an increased risk of pseudotumor cerebri . for treatment - resistant cases ,", "dapsone 50 - 150 mg / d is recommended ; this treatment should be carefully monitored .", "pilonidal sinus was described as far back as 1833 when mayo described a hair - containing cyst located just below the coccyx .", "no specific laboratory studies or tests are needed to diagnose pilonidal disease and its sequelae or differentiate it from other disease entities ; it is a clinical diagnosis best elicited by history and physical examination findings .", "treatment of infection or abscess with oral antibiotics followed by surgical excision of the sinus is practiced ." ]

[ "<S> follicular occlusion tetrad is a symptom complex consisting of four conditions having a similar pathophysiology . </S>", "<S> it includes hidradenitis suppurativa , acne conglobata , dissecting cellulitis of the scalp and pilonidal sinus . </S>", "<S> the exact pathogenesis of this group of disease is unknown but evidence suggests that they share the same pathological process initiated by follicular occlusion in apocrine gland bearing areas . </S>", "<S> though each of these conditions is commonly encountered singly , follicular occlusion tetrad as a symptom complex has been rarely reported in the literature . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "it includes hidradenitis suppurativa , acne conglobata , dissecting cellulitis of the scalp and pilonidal sinus .", "though each of these conditions are commonly encountered on their own , as a symptom complex follicular occlusion tetrad has rarely been reported in the literature . here", "we present a case of hidradenitis suppurativa in a 36-year - old male patient who also had the above mentioned associations ." ], [ "a 36-yeasr - old male patient presented to us with a history of recurrent boils since18 years .", "they initially started in the groin and buttocks with boils and gradually broke down to ooze pus .", "the boils healed with scarring after weeks to months . the process progressively started to involve the axillae and then the chest .", "the patient was treated with multiple antibiotics for over a decade that gave only symptomatic relief .", "the patient was diagnosed with pilonidal sinus at the age of 28 and he underwent surgery for the same .", "there was no family history of similar complaints , though his father suffered from psoriasis .", "on general examination , there were multiple areas of cicatricial alopecia on the scalp [ figure 1 ] and nodular scars on lower half of the face [ figure 2 ] .", "on query , the patient gave history of severe acne with nodules and pustules healing with scars on the face .", "he also had patchy swellings over the scalp region associated with pain in the past , which was diagnosed as folliculitis by a physician .", "patch of cicatricial alopecia on the occiput region of the scalp pustules , cysts and nodule - reminiscent of acne conglobata - on the lower half of the face on local examination , there were skin colored - to - erythematous nodules , grouped comedones and band like scars in the groin , buttocks , axillae and chest [ figure 3 and 4 ] .", "the patient had difficulty in lifting arms due to pain and scars in the axillae .", "multiple nodules , cysts and band like scars in the axillary region multiple nodules and pustules over the buttocks with surgical scar ( done for pilonidal sinus ) seen in the upper part of gluteal cleft a thorough general examination was followed by lab work - up .", "liver function tests and renal function tests were within normal limits and so was fbs , ppbs and fasting lipid profile .", "as the patient was obese , he was advised to follow a strict diet chart to lose weight .", "we also started him on oral isotretinoin 0.5 mg / kg body weight once daily .", "the patient came back after six weeks for prescription refill but was thereafter lost for follow - up ." ], [ "follicular occlusion tetrad is a condition that includes hidradenitis suppurativa ( hs ) , acne conglobata , dissecting cellulitis of the scalp and pilonidal sinus .", "hidradenitis suppurativa was first described in 1839 by velpeau ; verneuil gave it its name in 1854 and associated it with the sweat glands .", "later , hs was classified as a member of the follicular occlusion triad , along with acne conglobata and dissecting cellulitis of the scalp . in 1975", ", pilonidal cyst was added as a member to this triad , forming the follicular occlusion tetrad . in 1989 , plewig and steger introduced the term acne inversa to substitute the term hs .", "hidradenitis suppurativa or acne inversa is a chronic , inflammatory , recurrent , debilitating skin disease that usually presents after puberty with painful , deep - seated , inflamed lesions in the apocrine gland - bearing areas of the body , most commonly the axillae , inguinal and anogenital regions .", "the exact pathogenesis of this group of diseases is unknown but evidence suggests that they share the same pathological process initiated by follicular occlusion .", "the pathogenesis and aetiology are unknown , but is thought to originate from poral occlusion of the pilosebaceous units .", "diagnostic criteria of hidradenitis suppurativa ( adopted by the 2 international conference on hidradenitis suppurativa , march 5,2009 , san francisco , ca us ) . \n typical lesions , i.e. deep - seated painful nodules : ", "blind boils in early lesions ; abscesses , draining sinus , bridged scars and tombstonedouble - ended pseudo - comedones in secondary lesionstypical topography , i.e. axillae , groins , perineal and perianal region , buttocks , infra and inter mammary foldschronicity and recurrences . \n ", "blind boils in early lesions ; abscesses , draining sinus , bridged scars and tombstonedouble - ended pseudo - comedones in secondary lesions typical topography , i.e. axillae , groins , perineal and perianal region , buttocks , infra and inter mammary folds chronicity and recurrences .", "dissecting cellulitis(also called perifolliculitis capitis abscedens et suffodiens ) manifests with perifollicular pustules , nodules , abscesses and sinuses that evolve into scarring alopecia .", "it predominantly occurs in african american men between 20 - 40 years of age , but can occasionally affect other races and women too", "the tendency of dissecting cellulitis to cause severe alopecia , fluctuant nodules , and sinus tracts helps to distinguish it from acne keloidalis nuchae .", "culture of dissecting cellulitis does not produce a positive fungal culture and nuchal palpation does not reveal palpable lymph nodes , though reports have noted an inflammatory tinea capitis ( kerion ) that mimicked dissecting cellulitis in adolescents .", "its follicular papules and pustules spread peripherally , leaving central scarred patches of alopecia without nodules or sinuses . tufted folliculitis resolves with patches of scarring alopecia within which multiple hair tufts emerge from dilated follicular orifices .", "acne conglobata is an uncommon nodulocystictype of acne vulgaris that is often resistant to therapy .", "this disorder typically begins in adulthood and presents as numerous comedones , papules , pustules , nodules , abscesses , and draining sinus tracts involving the chest , back and buttocks .", "these lesions frequently become secondarily infected with gram - positive bacteria and often heal with scarring .", "pathology usually reveals inflammatory infiltrate around follicles , which can often disrupt the normal dermal architecture .", "acne conglobata is particularly disfiguring and socially detrimental to patients because of its chronicity , severity , and treatment challenge .", "acne conglobata ( ac ) resembles acne fulminans because both cause numerous inflammatory nodules on the trunk .", "unlike acne conglobata , large nodules of acne fulminans tend to become painful ulcers with overhanging borders surrounding exudative necrotic plaques that become confluent .", "the therapy of choice for ac includes isotretinoin 0.5 - 1 mg / kg for 4 - 6 months .", "simultaneous use of systemic steroids such as prednisone 1 mg / kg / d for 2 - 4 weeks may also prove beneficial , particularly if systemic symptoms are evident .", "oral tetracycline antibiotics should not be combined with oral isotretinoin because of an increased risk of pseudotumor cerebri . for treatment - resistant cases ,", "dapsone 50 - 150 mg / d is recommended ; this treatment should be carefully monitored .", "pilonidal sinus was described as far back as 1833 when mayo described a hair - containing cyst located just below the coccyx .", "no specific laboratory studies or tests are needed to diagnose pilonidal disease and its sequelae or differentiate it from other disease entities ; it is a clinical diagnosis best elicited by history and physical examination findings .", "treatment of infection or abscess with oral antibiotics followed by surgical excision of the sinus is practiced ." ] ]

[ "a consecutive series of 569 patients with shoulder pain who underwent ultrasonography between december 2008 and january 2010 were included retrospectively . a single orthopaedic surgeon performed ultrasonographic examination in cases where the authors suspected specific shoulder diseases on plain radiograph and physical examinations , such as rotator cuff tear , frozen shoulder , calcific tendinitis , or biceps tendinitis .", "the exclusion criteria were as follows : patients under 18 years of age , a history of fracture and shoulder surgery , a history of intra - articular injection before ultrasonographic examination , and biceps deformation including partially or completely ruptured blht .", "the patients ' average age was 57.22 years ( range , 26 to 88 years ) .", "men comprised 37.95% ( n = 115 ) of the patients studied , whereas women comprised 62.05% ( n = 188 ) .", "for the diagnosis of rotator cuff tear , the authors first used ultrasonography and then confirmed the final diagnosis by magnetic resonance imaging ( mri ) .", "on ultrasonographic examination , a hyper - echogenic signal change or focal decrease in the thickness of the tendon was considered to be a partial - thickness rotator cuff tear , and a hyper - echogenic gap in the tendon substance with retraction was considered to be a full - thickness rotator cuff tear .", "for the diagnosis of calcific tendinitis , the authors made the diagnosis by plain radiograph .", "on standard anteroposterior radiographs , radio - opaque lesions visible in the rotator cuff area were considered to be calcific tendinitis .", "furthermore , the authors checked the rotator cuff integrity on ultrasonography to rule out rotator cuff tearing .", "the authors considered the diagnosis of biceps tendinitis in patients with point tenderness in the bicipital groove and positive findings on speed 's test .", "furthermore , the authors performed mri in these patients to confirm biceps tendinitis and to rule out other pathologic conditions , including rotator cuff tear . on mri", ", biceps tendinitis demonstrated a signal change within the tendon or an irregular margin and a change in the diameter of the tendon .", "adhesive capsulitis is well known to be characterized by a functional restriction of both active and passive shoulder motions , and the radiographs of the glenohumeral joint in this condition are essentially unremarkable.13 ) however , there is no confirmed definition of the degree of shoulder motion restriction . in the present study , the authors defined shoulder stiffness as forward flexion that was less than 120 on passive motion , or external rotation with the arm at the side of less than 30 on passive motion , or internal rotation at the back that was lower than the third lumbar vertebral level passively.14 ) the authors performed ultrasonography and mri on all patients with adhesive capsulitis to rule out combined shoulder lesions .", "if shoulder stiffness was combined with other lesions , it was categorized as part of the main shoulder disease , and not as adhesive capsulitis ; for example , a rotator cuff tear with stiffness was considered part of the rotator cuff tear group , and not as part of the adhesive capsulitis group .", "ultrasonographic examination was performed by a single orthopaedic surgeon experienced in ultrasonography and blinded to the clinical test . a philips hd11 xe ultrasound machine ( amsterdam , the netherlands )", "was used with a high - frequency 12 to 5 mhz linear - array transducer .", "the patients were seated in the upright position with the arm in the neutral position , the elbow flexed to 90 and the palm face up .", "the ultrasonographic transducer was placed in the short axis of the blht at about 1 cm inferior to the coracoid process .", "if low - echogenic fluid was detected around the blht in the short axis view , the authors considered that there would be an appreciably sized glenohumeral joint effusion present .", "we measured the amount of effusion within the blht sheath as the length of effusion in the short axis view , that is , the longest distance from the sheath to the tendon margin was evaluated ( fig .", "zubler et al.12 ) reported that in their ultrasonographic study , the fluid collection in the blht sheath was 0.6 mm and that it increased to more than 0.9 mm after an injection of 8 to 12 ml fluid into the glenohumeral joint .", "therefore , we considered shoulders showing an effusion of more than 0.9 mm to indicate effusion within the blht sheath .", "the clinical data and functional scores were checked at the same time of the ultrasonographic examination by another single orthopaedic surgeon .", "the clinical data were assessed by the visual analogue scale ( vas ) for pain and the passive range of motion of the affected shoulder : forward flexion with fixed scapular motion , external rotation in 90 abduction , external rotation at the side , and internal rotation at the back .", "the vas for pain ranged from 0 to 10 , with 10 being the worst pain .", "the passive range of motion was measured with a full - circle manual goniometer for forward flexion and external rotation .", "the internal rotation level was checked by an indirect method , where the hand was passively placed behind the back and the vertebral level that was reached by the tip of the extended thumb was recorded . for easy statistical analysis", ", we converted the internal rotation levels into contiguously numbered groups : 0 for sacral level , 1 to 5 for l5 to l1 , and 6 to 12 for t12 to t6 .", "the functional scores , including the american shoulder and elbow surgery ( ases ) score , simple shoulder test ( sst ) score and the korean shoulder score ( kss ) were also evaluated at the time of the ultrasonographic examination .", "mary 's hospital , college of medicine , the catholic university of korea ( study no .", "22.0 ( ibm co. , armonk , ny , usa ) , and p - value < 0.05 was considered statistically significant .", "the one - way analysis of variance and student t - test were performed to determine the difference in the amount of effusion within the blht sheath between the diseases .", "pearson correlation coefficients ( pcc ) were calculated to determine the correlation between the amount of the effusion within the blht sheath and the functional outcome .", "a consecutive series of 569 patients with shoulder pain who underwent ultrasonography between december 2008 and january 2010 were included retrospectively . a single orthopaedic surgeon performed ultrasonographic examination in cases where the authors suspected specific shoulder diseases on plain radiograph and physical examinations , such as rotator cuff tear , frozen shoulder , calcific tendinitis , or biceps tendinitis .", "the exclusion criteria were as follows : patients under 18 years of age , a history of fracture and shoulder surgery , a history of intra - articular injection before ultrasonographic examination , and biceps deformation including partially or completely ruptured blht .", "the patients ' average age was 57.22 years ( range , 26 to 88 years ) .", "men comprised 37.95% ( n = 115 ) of the patients studied , whereas women comprised 62.05% ( n = 188 ) .", "for the diagnosis of rotator cuff tear , the authors first used ultrasonography and then confirmed the final diagnosis by magnetic resonance imaging ( mri ) .", "on ultrasonographic examination , a hyper - echogenic signal change or focal decrease in the thickness of the tendon was considered to be a partial - thickness rotator cuff tear , and a hyper - echogenic gap in the tendon substance with retraction was considered to be a full - thickness rotator cuff tear .", "for the diagnosis of calcific tendinitis , the authors made the diagnosis by plain radiograph .", "on standard anteroposterior radiographs , radio - opaque lesions visible in the rotator cuff area were considered to be calcific tendinitis .", "furthermore , the authors checked the rotator cuff integrity on ultrasonography to rule out rotator cuff tearing .", "the authors considered the diagnosis of biceps tendinitis in patients with point tenderness in the bicipital groove and positive findings on speed 's test .", "furthermore , the authors performed mri in these patients to confirm biceps tendinitis and to rule out other pathologic conditions , including rotator cuff tear . on mri", ", biceps tendinitis demonstrated a signal change within the tendon or an irregular margin and a change in the diameter of the tendon .", "adhesive capsulitis is well known to be characterized by a functional restriction of both active and passive shoulder motions , and the radiographs of the glenohumeral joint in this condition are essentially unremarkable.13 ) however , there is no confirmed definition of the degree of shoulder motion restriction . in the present study , the authors defined shoulder stiffness as forward flexion that was less than 120 on passive motion , or external rotation with the arm at the side of less than 30 on passive motion , or internal rotation at the back that was lower than the third lumbar vertebral level passively.14 ) the authors performed ultrasonography and mri on all patients with adhesive capsulitis to rule out combined shoulder lesions .", "if shoulder stiffness was combined with other lesions , it was categorized as part of the main shoulder disease , and not as adhesive capsulitis ; for example , a rotator cuff tear with stiffness was considered part of the rotator cuff tear group , and not as part of the adhesive capsulitis group .", "ultrasonographic examination was performed by a single orthopaedic surgeon experienced in ultrasonography and blinded to the clinical test . a philips hd11 xe ultrasound machine ( amsterdam , the netherlands )", "was used with a high - frequency 12 to 5 mhz linear - array transducer .", "the patients were seated in the upright position with the arm in the neutral position , the elbow flexed to 90 and the palm face up .", "the ultrasonographic transducer was placed in the short axis of the blht at about 1 cm inferior to the coracoid process .", "if low - echogenic fluid was detected around the blht in the short axis view , the authors considered that there would be an appreciably sized glenohumeral joint effusion present .", "we measured the amount of effusion within the blht sheath as the length of effusion in the short axis view , that is , the longest distance from the sheath to the tendon margin was evaluated ( fig .", "zubler et al.12 ) reported that in their ultrasonographic study , the fluid collection in the blht sheath was 0.6 mm and that it increased to more than 0.9 mm after an injection of 8 to 12 ml fluid into the glenohumeral joint .", "therefore , we considered shoulders showing an effusion of more than 0.9 mm to indicate effusion within the blht sheath .", "the clinical data and functional scores were checked at the same time of the ultrasonographic examination by another single orthopaedic surgeon .", "the clinical data were assessed by the visual analogue scale ( vas ) for pain and the passive range of motion of the affected shoulder : forward flexion with fixed scapular motion , external rotation in 90 abduction , external rotation at the side , and internal rotation at the back .", "the vas for pain ranged from 0 to 10 , with 10 being the worst pain .", "the passive range of motion was measured with a full - circle manual goniometer for forward flexion and external rotation .", "the internal rotation level was checked by an indirect method , where the hand was passively placed behind the back and the vertebral level that was reached by the tip of the extended thumb was recorded . for easy statistical analysis", ", we converted the internal rotation levels into contiguously numbered groups : 0 for sacral level , 1 to 5 for l5 to l1 , and 6 to 12 for t12 to t6 .", "the functional scores , including the american shoulder and elbow surgery ( ases ) score , simple shoulder test ( sst ) score and the korean shoulder score ( kss ) were also evaluated at the time of the ultrasonographic examination .", "mary 's hospital , college of medicine , the catholic university of korea ( study no .", "22.0 ( ibm co. , armonk , ny , usa ) , and p - value < 0.05 was considered statistically significant .", "the one - way analysis of variance and student t - test were performed to determine the difference in the amount of effusion within the blht sheath between the diseases .", "pearson correlation coefficients ( pcc ) were calculated to determine the correlation between the amount of the effusion within the blht sheath and the functional outcome .", "there were 157 shoulders with rotator cuff tear , 104 shoulders with adhesive capsulitis , 39 shoulders with calcific tendinitis , and three shoulders with biceps tendinitis .", "all patients with calcific tendinitis showed no combined rotator cuff tear , and rotator cuff tear combined with small calcification less than 2 mm were categorized to rotator cuff tear group .", "all shoulders with biceps tendinitis showed signal changes with the fraying of the biceps tendon on mri .", "the average of the vas for pain was found to be 4.8 2.7 in the rotator cuff tear group , 5.3 2.9 in the adhesive capsulitis group , 4.7 2.8 in the calcific tendinitis group , and 4.5 in the biceps tendinitis group .", "the overall results for the ases score were 58.5 points in the rotator cuff tear group , 58.8 points in the adhesive capsulitis group , 58.1 points in the calcific tendinitis group , and 53.3 points in the biceps tendinitis group .", "for the sst score , the results were as follows : 58.0 points in the rotator cuff tear group , 45.6 points in the adhesive capsulitis group , 51.9 points in the calcific tendinitis group , and 58.3 points in the biceps tendinitis group .", "for the kss score , the results were as follows : 42.4 points in the rotator cuff tear group , 40.8 points in the adhesive capsulitis group , 41.8 points in the calcific tendinitis group , and 41.0 points in the biceps tendinitis group .", "effusion within the blht sheath was detected in 58.42% ( n = 178 ) of the patients in this study : 69.23% ( n = 72 ) of patients with adhesive capsulitis , 56.69% ( n = 89 ) of patients with rotator cuff tear , 41.03% ( n = 16 ) of patients with calcific tendinitis , and 33.33% ( n = 1 ) of patients with biceps tendinitis ( fig .", "the average amount of the effusion within the blht sheath was 1.7 1.6 mm : 2.0 1.6 mm in patients with adhesive capsulitis , 1.6 1.6 mm in patients with rotator cuff tear , 1.2 1.6 mm in patients with calcific tendinitis , and 1.1 1.8 mm in patients with biceps tendinitis .", "the amount of the effusion within the blht sheath in patients with adhesive capsulitis was significantly larger than that in patients with calcific tendinitis ( p = 0.03 ) .", "however , there were no statistically significant differences between the other shoulder diseases ( fig .", "the amount of the effusion within the blht sheath overall had a negative correlation with the range of motion in all patients : forward flexion ( pcc = -0.491 ; p < 0.05 ) , external rotation in 90 abduction ( pcc = -0.381 ; p < 0.05 ) , external rotation at the side ( pcc = -0.356 ; p < 0.05 ) , and internal rotation ( pcc = -0.486 ; p < 0.05 ) . in patients with adhesive capsulitis ,", "pcc was -0.349 with forward flexion ( p < 0.05 ) , -0.310 with external rotation in 90 abduction ( p < 0.05 ) , -0.280 with external rotation at the side ( p < 0.05 ) , and -0.385 with internal rotation ( p < 0.05 ) . in patients with rotator cuff tear ,", "the pcc was -0.509 with forward flexion ( p < 0.05 ) , -0.379 with external rotation at 90 abduction ( p < 0.05 ) , -0.364 with external rotation at the side ( p < 0.05 ) , and -0.506 with internal rotation ( p < 0.05 ) . in patients with calcific tendinitis ,", "pcc was -0.769 with forward flexion ( p < 0.05 ) , -0.456 with external rotation in 90 abduction ( p < 0.05 ) , -0.432 with external rotation at the side ( p < 0.05 ) , and -0.537 with internal rotation ( p < 0.05 ) ( table 1 ) .", "the amount of the effusion within the blht sheath had a negative correlation with the functional score in each disease group .", "however , the correlation coefficients were relatively low : in patients with adhesive capsulitis , ases pcc = -0.311 ( p < 0.05 ) , sst pcc = -0.268 ( p < 0.05 ) , and kss pcc = -0.342 ( p < 0.05 ) ; in patients with rotator cuff tear , ases pcc = -0.449 ( p < 0.05 ) , sst pcc = -0.402 ( p < 0.05 ) , and kss pcc = -0.380 ( p < 0.05 ) ; and in patients with calcific tendinitis , ases pcc = -0.400 ( p < 0.05 ) , sst pcc = -0.275 ( p < 0.05 ) , and kss pcc = -0.343 ( p < 0.05 ) . furthermore", ", the vas for pain showed a weak positive correlation with the amount of the effusion within the blht sheath in each shoulder disease : adhesive capsulitis pcc = 0.109 ( p < 0.05 ) , rotator cuff tear pcc = 0.183 ( p < 0.05 ) , and calcific tendinitis pcc = 0.188 ( p < 0.05 ) ( table 2 ) .", "there were 157 shoulders with rotator cuff tear , 104 shoulders with adhesive capsulitis , 39 shoulders with calcific tendinitis , and three shoulders with biceps tendinitis .", "all patients with calcific tendinitis showed no combined rotator cuff tear , and rotator cuff tear combined with small calcification less than 2 mm were categorized to rotator cuff tear group .", "all shoulders with biceps tendinitis showed signal changes with the fraying of the biceps tendon on mri .", "the average of the vas for pain was found to be 4.8 2.7 in the rotator cuff tear group , 5.3 2.9 in the adhesive capsulitis group , 4.7 2.8 in the calcific tendinitis group , and 4.5 in the biceps tendinitis group .", "the overall results for the ases score were 58.5 points in the rotator cuff tear group , 58.8 points in the adhesive capsulitis group , 58.1 points in the calcific tendinitis group , and 53.3 points in the biceps tendinitis group .", "for the sst score , the results were as follows : 58.0 points in the rotator cuff tear group , 45.6 points in the adhesive capsulitis group , 51.9 points in the calcific tendinitis group , and 58.3 points in the biceps tendinitis group .", "for the kss score , the results were as follows : 42.4 points in the rotator cuff tear group , 40.8 points in the adhesive capsulitis group , 41.8 points in the calcific tendinitis group , and 41.0 points in the biceps tendinitis group .", "effusion within the blht sheath was detected in 58.42% ( n = 178 ) of the patients in this study : 69.23% ( n = 72 ) of patients with adhesive capsulitis , 56.69% ( n = 89 ) of patients with rotator cuff tear , 41.03% ( n = 16 ) of patients with calcific tendinitis , and 33.33% ( n = 1 ) of patients with biceps tendinitis ( fig .", "the average amount of the effusion within the blht sheath was 1.7 1.6 mm : 2.0 1.6 mm in patients with adhesive capsulitis , 1.6 1.6 mm in patients with rotator cuff tear , 1.2 1.6 mm in patients with calcific tendinitis , and 1.1 1.8 mm in patients with biceps tendinitis .", "the amount of the effusion within the blht sheath in patients with adhesive capsulitis was significantly larger than that in patients with calcific tendinitis ( p = 0.03 ) .", "however , there were no statistically significant differences between the other shoulder diseases ( fig .", "the amount of the effusion within the blht sheath overall had a negative correlation with the range of motion in all patients : forward flexion ( pcc = -0.491 ; p < 0.05 ) , external rotation in 90 abduction ( pcc = -0.381 ; p < 0.05 ) , external rotation at the side ( pcc = -0.356 ; p < 0.05 ) , and internal rotation ( pcc = -0.486 ; p < 0.05 ) . in patients with adhesive capsulitis ,", "pcc was -0.349 with forward flexion ( p < 0.05 ) , -0.310 with external rotation in 90 abduction ( p < 0.05 ) , -0.280 with external rotation at the side ( p < 0.05 ) , and -0.385 with internal rotation ( p < 0.05 ) . in patients with rotator cuff tear ,", "the pcc was -0.509 with forward flexion ( p < 0.05 ) , -0.379 with external rotation at 90 abduction ( p < 0.05 ) , -0.364 with external rotation at the side ( p < 0.05 ) , and -0.506 with internal rotation ( p < 0.05 ) . in patients with calcific tendinitis ,", "pcc was -0.769 with forward flexion ( p < 0.05 ) , -0.456 with external rotation in 90 abduction ( p < 0.05 ) , -0.432 with external rotation at the side ( p < 0.05 ) , and -0.537 with internal rotation ( p < 0.05 ) ( table 1 ) .", "the amount of the effusion within the blht sheath had a negative correlation with the functional score in each disease group . however , the correlation coefficients were relatively low : in patients with adhesive capsulitis , ases pcc = -0.311 ( p < 0.05 ) , sst pcc = -0.268 ( p < 0.05 ) , and kss pcc = -0.342 ( p < 0.05 ) ; in patients with rotator cuff tear , ases pcc = -0.449 ( p < 0.05 ) , sst pcc = -0.402 ( p < 0.05 ) , and kss pcc = -0.380 ( p < 0.05 ) ; and in patients with calcific tendinitis , ases pcc = -0.400 ( p < 0.05 ) , sst pcc = -0.275 ( p < 0.05 ) , and kss pcc = -0.343 ( p < 0.05 )", ". furthermore , the vas for pain showed a weak positive correlation with the amount of the effusion within the blht sheath in each shoulder disease : adhesive capsulitis pcc = 0.109 ( p < 0.05 ) , rotator cuff tear pcc = 0.183 ( p < 0.05 ) , and calcific tendinitis pcc = 0.188 ( p < 0.05 ) ( table 2 ) .", "the effusion within the blht sheath was detected in 58.42% of all patients and the average amount was 1.7 1.6 mm in all patients of the present study . among them , patients with adhesive capsulitis showed effusion within the blht sheath most frequently , and the average amount of that group was larger than that of the other shoulder diseases . because the effusion within the blht sheath looks like a fried egg ,", "the amount of effusion within the blht sheath showed a negative correlation with the range of motion and functional scores in each group .", "in particular , the forward flexion and internal rotation levels were most closely correlated with the amount of effusion within the blht sheath in each group .", "the glenohumeral joint cavity communicates with the blht sheath ; thus , profuse effusion of the glenohumeral joint may lead to an increase in the effusion within the tendon sheath .", "zubler et al.12 ) reported that in their ultrasonographic study , the width of the anechoic fluid collection in the blht sheath was increased after 8 to 12 ml of fluid was injected into the glenohumeral joint in most cases .", "these findings suggest that the amount of hypoechogenic fluid in the blht sheath may represent the ratio of the amount of glenohumeral joint effusion to the volume of the glenohumeral joint cavity . in the other words ,", "however , zubler et al.12 ) did not evaluate the correlation between the effusion within the blht sheath and clinical outcomes , they suggested that the effusion within the blht sheath might represent glenohumeral joint effusion . in the present study ,", "the effusion within the blht sheath was more frequently observed in adhesive capsulitis and the amount of effusion within blht sheath showed a negative correlation with the range of motion .", "the axillary fold is contracted , and that reduces the joint volume to below 15 ml.15 ) furthermore , joint shrinkage might push the joint fluid to another space , the blht sheath .", "therefore , the more the joint shrinks , the more joint fluid is collected in the blht sheath .", "the amount of effusion within the blht sheath showed a higher correlation with the range of motion than with the variable functional score or shoulder pain .", "the effusion within the blht sheath was thought to be related to the status of synovitis or capsulitis .", "several studies with synovial or capsular biopsy specimens from patients with adhesive capsulitis have demonstrated that variable proinflammatory cytokines and neovascularizations are involved in synovial hyperplasia and capsular fibrosis.1617 ) adhesive capsulitis , initially described by neviaser,18 ) is thought to be a combination of synovial inflammation and capsular fibrosis.15 ) thus , the limitation of the range of motion is thought to be closely correlated with joint synovitis , and in the present study , the range of motion was also closely correlated with the amount of effusion within the blht sheath .", "therefore , in adhesive capsulitis , combined synovitis and capsulitis can lead to increased joint effusion and joint cavity shrinkage , and these conditions might produce the appearance of the effusion within the blht sheath in shoulders with adhesive capsulitis .", "functional scores and shoulder pain are mainly affected by the severity of each disease ; however , in this study , we aimed to evaluate the clinical meaning of the effusion within the blht sheath , something we frequently encountered during ultrasonographic examinations .", "effusion within the blht sheath was detected in 56.69% of patients with rotator cuff tear .", "several studies showed that low- to mild - grade synovial inflammation is present in shoulders with rotator cuff tears.31920 ) the expression levels of interleukin 1-beta and several degradative enzymes were highly upregulated in the synovium of patients with rotator cuff lesions , and these findings suggest that chronic synovitis is associated with rotator cuff tears.19 ) therefore , chronic synovitis combined with rotator cuff tears might lead to an increase in joint effusion , and these findings would be detected as effusion within the blht sheath . in calcific tendinitis , there was a stronger correlation between the amount of effusion within the blht sheath and range of motion than in rotator cuff tears .", "calcific tendinitis can be divided into three distinct stages : precalcific , calcific , and postcalcific.21 ) among these stages , the postcalcific stage usually shows milky or creamy calcific deposits which frequently induce acute sharp pain , combined with acute synovitis or bursitis .", "thus , acute synovitis in calcific tendinitis might induce increased joint effusion and severely painful limited range of motion .", "this may be the reason for the stronger correlation between the amount of effusion within the blht sheath and the range of motion in calcific tendinitis .", "several studies have suggested that a fluid collection in the biceps tendon sheath may be induced by biceps tendinitis.2223 ) in the present study , only three patients had biceps tendinitis , and among them , only one patient showed effusion within the blht sheath .", "we had only three cases of biceps tendinitis , because our categorization , exclusion criteria , and low incidence of ultrasonographic examination in isolated biceps lesions .", "because of low sample size of biceps lesions in the present study , the authors could not evaluate this lesion to a statistical degree . however , it is believed that the fluid collection in the biceps tendon sheath may be influenced by joint effusion , rather than by the condition of the local bicep .", "although , the final diagnosis was confirmed at the last follow - up , the authors did not trace the patients ' symptoms and functional outcomes after the ultrasonographic examination .", "furthermore , because patients underwent different treatments according to their individual diagnosis , the correlation between the effusion within the blht sheath and disease prognosis could not be evaluated .", "second , in the present study , if shoulder stiffness was combined with another shoulder disease , it was categorized as the main shoulder disease , and not as adhesive capsulitis .", "we simply categorized the rotator cuff tear patients with stiffness into the rotator cuff tear group , for example .", "however , it is well known that there are many differences between rotator cuff tear with stiffness and rotator cuff tear without stiffness , including symptoms and treatment .", "we did not evaluate normal shoulders as control group . because , there would be asymptomatic effusion within blht sheath", ", the percentage of ' fried egg sign ' in each disease and conclusion of the present study might be different if the results were compared with control group .", "lastly , we evaluated the effusion within the blht sheath using ultrasonography , so a bias might occur according to direction and location of ultrasonographic transducer ( the intraobserver reliability was 0.80 ) . the location and thickness of the biceps tendon within the blht sheath also could provide a bias . to reduce the variance , the examiner tried to locate the transducer at the same location in the same shoulder position and shoulders with biceps deformation , especially with biceps tendinitis or biceps tear , were excluded in the present study . in conclusion , the effusion within the blht sheath is closely related to the range of motion and clinical scores in patients with painful shoulders .", "the ultrasonographic detection of the effusion within the blht sheath might be useful to evaluate shoulder functions .", "however , further studies are required to understand the correlation between the effusion within the blht sheath and disease prognosis ." ]

[ "<S> backgroundmany shoulder diseases are related to glenohumeral joint synovitis and effusion . </S>", "<S> the purpose of the present study is to detect effusion within the biceps long head tendon sheath as the sign of glenohumeral joint synovitis using ultrasonography , and to evaluate the clinical meaning of effusion within the biceps long head tendon sheath.methodsa consecutive series of 569 patients who underwent ultrasonography for shoulder pain were reviewed retrospectively and ultimately , 303 patients were included . </S>", "<S> the authors evaluated the incidence and amount of the effusion within the biceps long head tendon sheath on the ultrasonographic short axis view . </S>", "<S> furthermore , the authors evaluated the correlation between the amount of effusion within the biceps long head tendon sheath and the range of motion and the functional score.resultsthe effusion within the biceps long head tendon sheath was detected in 58.42% of the patients studied : 69.23% in adhesive capsulitis , 56.69% in rotator cuff tear , 41.03% in calcific tendinitis , and 33.33% in biceps tendinitis . </S>", "<S> the average amount of the effusion within the biceps long head tendon sheath was 1.7 1.6 mm , and it was measured to be the largest in adhesive capsulitis . </S>", "<S> the amount of effusion within biceps long head tendon sheath showed a moderate to high degree of correlation with the range of motion , and a low degree of correlation with the functional score and visual analogue scale for pain in each type of shoulder disease.conclusionsthe effusion within the biceps long head tendon sheath is closely related to the range of motion and clinical scores in patients with painful shoulders . </S>", "<S> ultrasonographic detection of the effusion within the biceps long head tendon sheath might be a simple and easy method to evaluate shoulder function . </S>" ]

[ "METHODS", "Patient Population", "Ultrasonography Assessment", "Clinical Evaluation", "Statistical Analysis", "RESULTS", "Clinical Data", "Ultrasonographic Data", "Correlation between the Amount of Effusion within the Biceps Long Head Tendon Sheath and Range of Motion and Functional Outcome", "DISCUSSION" ]

[ [ "a consecutive series of 569 patients with shoulder pain who underwent ultrasonography between december 2008 and january 2010 were included retrospectively . a single orthopaedic surgeon performed ultrasonographic examination in cases where the authors suspected specific shoulder diseases on plain radiograph and physical examinations , such as rotator cuff tear , frozen shoulder , calcific tendinitis , or biceps tendinitis .", "the exclusion criteria were as follows : patients under 18 years of age , a history of fracture and shoulder surgery , a history of intra - articular injection before ultrasonographic examination , and biceps deformation including partially or completely ruptured blht .", "the patients ' average age was 57.22 years ( range , 26 to 88 years ) .", "men comprised 37.95% ( n = 115 ) of the patients studied , whereas women comprised 62.05% ( n = 188 ) .", "for the diagnosis of rotator cuff tear , the authors first used ultrasonography and then confirmed the final diagnosis by magnetic resonance imaging ( mri ) .", "on ultrasonographic examination , a hyper - echogenic signal change or focal decrease in the thickness of the tendon was considered to be a partial - thickness rotator cuff tear , and a hyper - echogenic gap in the tendon substance with retraction was considered to be a full - thickness rotator cuff tear .", "for the diagnosis of calcific tendinitis , the authors made the diagnosis by plain radiograph .", "on standard anteroposterior radiographs , radio - opaque lesions visible in the rotator cuff area were considered to be calcific tendinitis .", "furthermore , the authors checked the rotator cuff integrity on ultrasonography to rule out rotator cuff tearing .", "the authors considered the diagnosis of biceps tendinitis in patients with point tenderness in the bicipital groove and positive findings on speed 's test .", "furthermore , the authors performed mri in these patients to confirm biceps tendinitis and to rule out other pathologic conditions , including rotator cuff tear . on mri", ", biceps tendinitis demonstrated a signal change within the tendon or an irregular margin and a change in the diameter of the tendon .", "adhesive capsulitis is well known to be characterized by a functional restriction of both active and passive shoulder motions , and the radiographs of the glenohumeral joint in this condition are essentially unremarkable.13 ) however , there is no confirmed definition of the degree of shoulder motion restriction . in the present study , the authors defined shoulder stiffness as forward flexion that was less than 120 on passive motion , or external rotation with the arm at the side of less than 30 on passive motion , or internal rotation at the back that was lower than the third lumbar vertebral level passively.14 ) the authors performed ultrasonography and mri on all patients with adhesive capsulitis to rule out combined shoulder lesions .", "if shoulder stiffness was combined with other lesions , it was categorized as part of the main shoulder disease , and not as adhesive capsulitis ; for example , a rotator cuff tear with stiffness was considered part of the rotator cuff tear group , and not as part of the adhesive capsulitis group .", "ultrasonographic examination was performed by a single orthopaedic surgeon experienced in ultrasonography and blinded to the clinical test . a philips hd11 xe ultrasound machine ( amsterdam , the netherlands )", "was used with a high - frequency 12 to 5 mhz linear - array transducer .", "the patients were seated in the upright position with the arm in the neutral position , the elbow flexed to 90 and the palm face up .", "the ultrasonographic transducer was placed in the short axis of the blht at about 1 cm inferior to the coracoid process .", "if low - echogenic fluid was detected around the blht in the short axis view , the authors considered that there would be an appreciably sized glenohumeral joint effusion present .", "we measured the amount of effusion within the blht sheath as the length of effusion in the short axis view , that is , the longest distance from the sheath to the tendon margin was evaluated ( fig .", "zubler et al.12 ) reported that in their ultrasonographic study , the fluid collection in the blht sheath was 0.6 mm and that it increased to more than 0.9 mm after an injection of 8 to 12 ml fluid into the glenohumeral joint .", "therefore , we considered shoulders showing an effusion of more than 0.9 mm to indicate effusion within the blht sheath .", "the clinical data and functional scores were checked at the same time of the ultrasonographic examination by another single orthopaedic surgeon .", "the clinical data were assessed by the visual analogue scale ( vas ) for pain and the passive range of motion of the affected shoulder : forward flexion with fixed scapular motion , external rotation in 90 abduction , external rotation at the side , and internal rotation at the back .", "the vas for pain ranged from 0 to 10 , with 10 being the worst pain .", "the passive range of motion was measured with a full - circle manual goniometer for forward flexion and external rotation .", "the internal rotation level was checked by an indirect method , where the hand was passively placed behind the back and the vertebral level that was reached by the tip of the extended thumb was recorded . for easy statistical analysis", ", we converted the internal rotation levels into contiguously numbered groups : 0 for sacral level , 1 to 5 for l5 to l1 , and 6 to 12 for t12 to t6 .", "the functional scores , including the american shoulder and elbow surgery ( ases ) score , simple shoulder test ( sst ) score and the korean shoulder score ( kss ) were also evaluated at the time of the ultrasonographic examination .", "mary 's hospital , college of medicine , the catholic university of korea ( study no .", "22.0 ( ibm co. , armonk , ny , usa ) , and p - value < 0.05 was considered statistically significant .", "the one - way analysis of variance and student t - test were performed to determine the difference in the amount of effusion within the blht sheath between the diseases .", "pearson correlation coefficients ( pcc ) were calculated to determine the correlation between the amount of the effusion within the blht sheath and the functional outcome ." ], [ "a consecutive series of 569 patients with shoulder pain who underwent ultrasonography between december 2008 and january 2010 were included retrospectively . a single orthopaedic surgeon performed ultrasonographic examination in cases where the authors suspected specific shoulder diseases on plain radiograph and physical examinations , such as rotator cuff tear , frozen shoulder , calcific tendinitis , or biceps tendinitis .", "the exclusion criteria were as follows : patients under 18 years of age , a history of fracture and shoulder surgery , a history of intra - articular injection before ultrasonographic examination , and biceps deformation including partially or completely ruptured blht .", "the patients ' average age was 57.22 years ( range , 26 to 88 years ) .", "men comprised 37.95% ( n = 115 ) of the patients studied , whereas women comprised 62.05% ( n = 188 ) .", "for the diagnosis of rotator cuff tear , the authors first used ultrasonography and then confirmed the final diagnosis by magnetic resonance imaging ( mri ) .", "on ultrasonographic examination , a hyper - echogenic signal change or focal decrease in the thickness of the tendon was considered to be a partial - thickness rotator cuff tear , and a hyper - echogenic gap in the tendon substance with retraction was considered to be a full - thickness rotator cuff tear .", "for the diagnosis of calcific tendinitis , the authors made the diagnosis by plain radiograph .", "on standard anteroposterior radiographs , radio - opaque lesions visible in the rotator cuff area were considered to be calcific tendinitis .", "furthermore , the authors checked the rotator cuff integrity on ultrasonography to rule out rotator cuff tearing .", "the authors considered the diagnosis of biceps tendinitis in patients with point tenderness in the bicipital groove and positive findings on speed 's test .", "furthermore , the authors performed mri in these patients to confirm biceps tendinitis and to rule out other pathologic conditions , including rotator cuff tear . on mri", ", biceps tendinitis demonstrated a signal change within the tendon or an irregular margin and a change in the diameter of the tendon .", "adhesive capsulitis is well known to be characterized by a functional restriction of both active and passive shoulder motions , and the radiographs of the glenohumeral joint in this condition are essentially unremarkable.13 ) however , there is no confirmed definition of the degree of shoulder motion restriction . in the present study , the authors defined shoulder stiffness as forward flexion that was less than 120 on passive motion , or external rotation with the arm at the side of less than 30 on passive motion , or internal rotation at the back that was lower than the third lumbar vertebral level passively.14 ) the authors performed ultrasonography and mri on all patients with adhesive capsulitis to rule out combined shoulder lesions .", "if shoulder stiffness was combined with other lesions , it was categorized as part of the main shoulder disease , and not as adhesive capsulitis ; for example , a rotator cuff tear with stiffness was considered part of the rotator cuff tear group , and not as part of the adhesive capsulitis group ." ], [ "ultrasonographic examination was performed by a single orthopaedic surgeon experienced in ultrasonography and blinded to the clinical test . a philips hd11 xe ultrasound machine ( amsterdam , the netherlands )", "was used with a high - frequency 12 to 5 mhz linear - array transducer .", "the patients were seated in the upright position with the arm in the neutral position , the elbow flexed to 90 and the palm face up .", "the ultrasonographic transducer was placed in the short axis of the blht at about 1 cm inferior to the coracoid process .", "if low - echogenic fluid was detected around the blht in the short axis view , the authors considered that there would be an appreciably sized glenohumeral joint effusion present .", "we measured the amount of effusion within the blht sheath as the length of effusion in the short axis view , that is , the longest distance from the sheath to the tendon margin was evaluated ( fig .", "zubler et al.12 ) reported that in their ultrasonographic study , the fluid collection in the blht sheath was 0.6 mm and that it increased to more than 0.9 mm after an injection of 8 to 12 ml fluid into the glenohumeral joint .", "therefore , we considered shoulders showing an effusion of more than 0.9 mm to indicate effusion within the blht sheath ." ], [ "the clinical data and functional scores were checked at the same time of the ultrasonographic examination by another single orthopaedic surgeon .", "the clinical data were assessed by the visual analogue scale ( vas ) for pain and the passive range of motion of the affected shoulder : forward flexion with fixed scapular motion , external rotation in 90 abduction , external rotation at the side , and internal rotation at the back .", "the vas for pain ranged from 0 to 10 , with 10 being the worst pain .", "the passive range of motion was measured with a full - circle manual goniometer for forward flexion and external rotation .", "the internal rotation level was checked by an indirect method , where the hand was passively placed behind the back and the vertebral level that was reached by the tip of the extended thumb was recorded . for easy statistical analysis", ", we converted the internal rotation levels into contiguously numbered groups : 0 for sacral level , 1 to 5 for l5 to l1 , and 6 to 12 for t12 to t6 .", "the functional scores , including the american shoulder and elbow surgery ( ases ) score , simple shoulder test ( sst ) score and the korean shoulder score ( kss ) were also evaluated at the time of the ultrasonographic examination .", "mary 's hospital , college of medicine , the catholic university of korea ( study no ." ], [ "22.0 ( ibm co. , armonk , ny , usa ) , and p - value < 0.05 was considered statistically significant .", "the one - way analysis of variance and student t - test were performed to determine the difference in the amount of effusion within the blht sheath between the diseases .", "pearson correlation coefficients ( pcc ) were calculated to determine the correlation between the amount of the effusion within the blht sheath and the functional outcome ." ], [ "there were 157 shoulders with rotator cuff tear , 104 shoulders with adhesive capsulitis , 39 shoulders with calcific tendinitis , and three shoulders with biceps tendinitis .", "all patients with calcific tendinitis showed no combined rotator cuff tear , and rotator cuff tear combined with small calcification less than 2 mm were categorized to rotator cuff tear group .", "all shoulders with biceps tendinitis showed signal changes with the fraying of the biceps tendon on mri .", "the average of the vas for pain was found to be 4.8 2.7 in the rotator cuff tear group , 5.3 2.9 in the adhesive capsulitis group , 4.7 2.8 in the calcific tendinitis group , and 4.5 in the biceps tendinitis group .", "the overall results for the ases score were 58.5 points in the rotator cuff tear group , 58.8 points in the adhesive capsulitis group , 58.1 points in the calcific tendinitis group , and 53.3 points in the biceps tendinitis group .", "for the sst score , the results were as follows : 58.0 points in the rotator cuff tear group , 45.6 points in the adhesive capsulitis group , 51.9 points in the calcific tendinitis group , and 58.3 points in the biceps tendinitis group .", "for the kss score , the results were as follows : 42.4 points in the rotator cuff tear group , 40.8 points in the adhesive capsulitis group , 41.8 points in the calcific tendinitis group , and 41.0 points in the biceps tendinitis group .", "effusion within the blht sheath was detected in 58.42% ( n = 178 ) of the patients in this study : 69.23% ( n = 72 ) of patients with adhesive capsulitis , 56.69% ( n = 89 ) of patients with rotator cuff tear , 41.03% ( n = 16 ) of patients with calcific tendinitis , and 33.33% ( n = 1 ) of patients with biceps tendinitis ( fig .", "the average amount of the effusion within the blht sheath was 1.7 1.6 mm : 2.0 1.6 mm in patients with adhesive capsulitis , 1.6 1.6 mm in patients with rotator cuff tear , 1.2 1.6 mm in patients with calcific tendinitis , and 1.1 1.8 mm in patients with biceps tendinitis .", "the amount of the effusion within the blht sheath in patients with adhesive capsulitis was significantly larger than that in patients with calcific tendinitis ( p = 0.03 ) .", "however , there were no statistically significant differences between the other shoulder diseases ( fig .", "the amount of the effusion within the blht sheath overall had a negative correlation with the range of motion in all patients : forward flexion ( pcc = -0.491 ; p < 0.05 ) , external rotation in 90 abduction ( pcc = -0.381 ; p < 0.05 ) , external rotation at the side ( pcc = -0.356 ; p < 0.05 ) , and internal rotation ( pcc = -0.486 ; p < 0.05 ) . in patients with adhesive capsulitis ,", "pcc was -0.349 with forward flexion ( p < 0.05 ) , -0.310 with external rotation in 90 abduction ( p < 0.05 ) , -0.280 with external rotation at the side ( p < 0.05 ) , and -0.385 with internal rotation ( p < 0.05 ) . in patients with rotator cuff tear ,", "the pcc was -0.509 with forward flexion ( p < 0.05 ) , -0.379 with external rotation at 90 abduction ( p < 0.05 ) , -0.364 with external rotation at the side ( p < 0.05 ) , and -0.506 with internal rotation ( p < 0.05 ) . in patients with calcific tendinitis ,", "pcc was -0.769 with forward flexion ( p < 0.05 ) , -0.456 with external rotation in 90 abduction ( p < 0.05 ) , -0.432 with external rotation at the side ( p < 0.05 ) , and -0.537 with internal rotation ( p < 0.05 ) ( table 1 ) .", "the amount of the effusion within the blht sheath had a negative correlation with the functional score in each disease group .", "however , the correlation coefficients were relatively low : in patients with adhesive capsulitis , ases pcc = -0.311 ( p < 0.05 ) , sst pcc = -0.268 ( p < 0.05 ) , and kss pcc = -0.342 ( p < 0.05 ) ; in patients with rotator cuff tear , ases pcc = -0.449 ( p < 0.05 ) , sst pcc = -0.402 ( p < 0.05 ) , and kss pcc = -0.380 ( p < 0.05 ) ; and in patients with calcific tendinitis , ases pcc = -0.400 ( p < 0.05 ) , sst pcc = -0.275 ( p < 0.05 ) , and kss pcc = -0.343 ( p < 0.05 ) . furthermore", ", the vas for pain showed a weak positive correlation with the amount of the effusion within the blht sheath in each shoulder disease : adhesive capsulitis pcc = 0.109 ( p < 0.05 ) , rotator cuff tear pcc = 0.183 ( p < 0.05 ) , and calcific tendinitis pcc = 0.188 ( p < 0.05 ) ( table 2 ) ." ], [ "there were 157 shoulders with rotator cuff tear , 104 shoulders with adhesive capsulitis , 39 shoulders with calcific tendinitis , and three shoulders with biceps tendinitis .", "all patients with calcific tendinitis showed no combined rotator cuff tear , and rotator cuff tear combined with small calcification less than 2 mm were categorized to rotator cuff tear group .", "all shoulders with biceps tendinitis showed signal changes with the fraying of the biceps tendon on mri .", "the average of the vas for pain was found to be 4.8 2.7 in the rotator cuff tear group , 5.3 2.9 in the adhesive capsulitis group , 4.7 2.8 in the calcific tendinitis group , and 4.5 in the biceps tendinitis group .", "the overall results for the ases score were 58.5 points in the rotator cuff tear group , 58.8 points in the adhesive capsulitis group , 58.1 points in the calcific tendinitis group , and 53.3 points in the biceps tendinitis group .", "for the sst score , the results were as follows : 58.0 points in the rotator cuff tear group , 45.6 points in the adhesive capsulitis group , 51.9 points in the calcific tendinitis group , and 58.3 points in the biceps tendinitis group .", "for the kss score , the results were as follows : 42.4 points in the rotator cuff tear group , 40.8 points in the adhesive capsulitis group , 41.8 points in the calcific tendinitis group , and 41.0 points in the biceps tendinitis group ." ], [ "effusion within the blht sheath was detected in 58.42% ( n = 178 ) of the patients in this study : 69.23% ( n = 72 ) of patients with adhesive capsulitis , 56.69% ( n = 89 ) of patients with rotator cuff tear , 41.03% ( n = 16 ) of patients with calcific tendinitis , and 33.33% ( n = 1 ) of patients with biceps tendinitis ( fig .", "the average amount of the effusion within the blht sheath was 1.7 1.6 mm : 2.0 1.6 mm in patients with adhesive capsulitis , 1.6 1.6 mm in patients with rotator cuff tear , 1.2 1.6 mm in patients with calcific tendinitis , and 1.1 1.8 mm in patients with biceps tendinitis .", "the amount of the effusion within the blht sheath in patients with adhesive capsulitis was significantly larger than that in patients with calcific tendinitis ( p = 0.03 ) .", "however , there were no statistically significant differences between the other shoulder diseases ( fig ." ], [ "the amount of the effusion within the blht sheath overall had a negative correlation with the range of motion in all patients : forward flexion ( pcc = -0.491 ; p < 0.05 ) , external rotation in 90 abduction ( pcc = -0.381 ; p < 0.05 ) , external rotation at the side ( pcc = -0.356 ; p < 0.05 ) , and internal rotation ( pcc = -0.486 ; p < 0.05 ) . in patients with adhesive capsulitis ,", "pcc was -0.349 with forward flexion ( p < 0.05 ) , -0.310 with external rotation in 90 abduction ( p < 0.05 ) , -0.280 with external rotation at the side ( p < 0.05 ) , and -0.385 with internal rotation ( p < 0.05 ) . in patients with rotator cuff tear ,", "the pcc was -0.509 with forward flexion ( p < 0.05 ) , -0.379 with external rotation at 90 abduction ( p < 0.05 ) , -0.364 with external rotation at the side ( p < 0.05 ) , and -0.506 with internal rotation ( p < 0.05 ) . in patients with calcific tendinitis ,", "pcc was -0.769 with forward flexion ( p < 0.05 ) , -0.456 with external rotation in 90 abduction ( p < 0.05 ) , -0.432 with external rotation at the side ( p < 0.05 ) , and -0.537 with internal rotation ( p < 0.05 ) ( table 1 ) .", "the amount of the effusion within the blht sheath had a negative correlation with the functional score in each disease group . however , the correlation coefficients were relatively low : in patients with adhesive capsulitis , ases pcc = -0.311 ( p < 0.05 ) , sst pcc = -0.268 ( p < 0.05 ) , and kss pcc = -0.342 ( p < 0.05 ) ; in patients with rotator cuff tear , ases pcc = -0.449 ( p < 0.05 ) , sst pcc = -0.402 ( p < 0.05 ) , and kss pcc = -0.380 ( p < 0.05 ) ; and in patients with calcific tendinitis , ases pcc = -0.400 ( p < 0.05 ) , sst pcc = -0.275 ( p < 0.05 ) , and kss pcc = -0.343 ( p < 0.05 )", ". furthermore , the vas for pain showed a weak positive correlation with the amount of the effusion within the blht sheath in each shoulder disease : adhesive capsulitis pcc = 0.109 ( p < 0.05 ) , rotator cuff tear pcc = 0.183 ( p < 0.05 ) , and calcific tendinitis pcc = 0.188 ( p < 0.05 ) ( table 2 ) ." ], [ "the effusion within the blht sheath was detected in 58.42% of all patients and the average amount was 1.7 1.6 mm in all patients of the present study . among them , patients with adhesive capsulitis showed effusion within the blht sheath most frequently , and the average amount of that group was larger than that of the other shoulder diseases . because the effusion within the blht sheath looks like a fried egg ,", "the amount of effusion within the blht sheath showed a negative correlation with the range of motion and functional scores in each group .", "in particular , the forward flexion and internal rotation levels were most closely correlated with the amount of effusion within the blht sheath in each group .", "the glenohumeral joint cavity communicates with the blht sheath ; thus , profuse effusion of the glenohumeral joint may lead to an increase in the effusion within the tendon sheath .", "zubler et al.12 ) reported that in their ultrasonographic study , the width of the anechoic fluid collection in the blht sheath was increased after 8 to 12 ml of fluid was injected into the glenohumeral joint in most cases .", "these findings suggest that the amount of hypoechogenic fluid in the blht sheath may represent the ratio of the amount of glenohumeral joint effusion to the volume of the glenohumeral joint cavity . in the other words ,", "however , zubler et al.12 ) did not evaluate the correlation between the effusion within the blht sheath and clinical outcomes , they suggested that the effusion within the blht sheath might represent glenohumeral joint effusion . in the present study ,", "the effusion within the blht sheath was more frequently observed in adhesive capsulitis and the amount of effusion within blht sheath showed a negative correlation with the range of motion .", "the axillary fold is contracted , and that reduces the joint volume to below 15 ml.15 ) furthermore , joint shrinkage might push the joint fluid to another space , the blht sheath .", "therefore , the more the joint shrinks , the more joint fluid is collected in the blht sheath .", "the amount of effusion within the blht sheath showed a higher correlation with the range of motion than with the variable functional score or shoulder pain .", "the effusion within the blht sheath was thought to be related to the status of synovitis or capsulitis .", "several studies with synovial or capsular biopsy specimens from patients with adhesive capsulitis have demonstrated that variable proinflammatory cytokines and neovascularizations are involved in synovial hyperplasia and capsular fibrosis.1617 ) adhesive capsulitis , initially described by neviaser,18 ) is thought to be a combination of synovial inflammation and capsular fibrosis.15 ) thus , the limitation of the range of motion is thought to be closely correlated with joint synovitis , and in the present study , the range of motion was also closely correlated with the amount of effusion within the blht sheath .", "therefore , in adhesive capsulitis , combined synovitis and capsulitis can lead to increased joint effusion and joint cavity shrinkage , and these conditions might produce the appearance of the effusion within the blht sheath in shoulders with adhesive capsulitis .", "functional scores and shoulder pain are mainly affected by the severity of each disease ; however , in this study , we aimed to evaluate the clinical meaning of the effusion within the blht sheath , something we frequently encountered during ultrasonographic examinations .", "effusion within the blht sheath was detected in 56.69% of patients with rotator cuff tear .", "several studies showed that low- to mild - grade synovial inflammation is present in shoulders with rotator cuff tears.31920 ) the expression levels of interleukin 1-beta and several degradative enzymes were highly upregulated in the synovium of patients with rotator cuff lesions , and these findings suggest that chronic synovitis is associated with rotator cuff tears.19 ) therefore , chronic synovitis combined with rotator cuff tears might lead to an increase in joint effusion , and these findings would be detected as effusion within the blht sheath . in calcific tendinitis , there was a stronger correlation between the amount of effusion within the blht sheath and range of motion than in rotator cuff tears .", "calcific tendinitis can be divided into three distinct stages : precalcific , calcific , and postcalcific.21 ) among these stages , the postcalcific stage usually shows milky or creamy calcific deposits which frequently induce acute sharp pain , combined with acute synovitis or bursitis .", "thus , acute synovitis in calcific tendinitis might induce increased joint effusion and severely painful limited range of motion .", "this may be the reason for the stronger correlation between the amount of effusion within the blht sheath and the range of motion in calcific tendinitis .", "several studies have suggested that a fluid collection in the biceps tendon sheath may be induced by biceps tendinitis.2223 ) in the present study , only three patients had biceps tendinitis , and among them , only one patient showed effusion within the blht sheath .", "we had only three cases of biceps tendinitis , because our categorization , exclusion criteria , and low incidence of ultrasonographic examination in isolated biceps lesions .", "because of low sample size of biceps lesions in the present study , the authors could not evaluate this lesion to a statistical degree . however , it is believed that the fluid collection in the biceps tendon sheath may be influenced by joint effusion , rather than by the condition of the local bicep .", "although , the final diagnosis was confirmed at the last follow - up , the authors did not trace the patients ' symptoms and functional outcomes after the ultrasonographic examination .", "furthermore , because patients underwent different treatments according to their individual diagnosis , the correlation between the effusion within the blht sheath and disease prognosis could not be evaluated .", "second , in the present study , if shoulder stiffness was combined with another shoulder disease , it was categorized as the main shoulder disease , and not as adhesive capsulitis .", "we simply categorized the rotator cuff tear patients with stiffness into the rotator cuff tear group , for example .", "however , it is well known that there are many differences between rotator cuff tear with stiffness and rotator cuff tear without stiffness , including symptoms and treatment .", "we did not evaluate normal shoulders as control group . because , there would be asymptomatic effusion within blht sheath", ", the percentage of ' fried egg sign ' in each disease and conclusion of the present study might be different if the results were compared with control group .", "lastly , we evaluated the effusion within the blht sheath using ultrasonography , so a bias might occur according to direction and location of ultrasonographic transducer ( the intraobserver reliability was 0.80 ) . the location and thickness of the biceps tendon within the blht sheath also could provide a bias . to reduce the variance , the examiner tried to locate the transducer at the same location in the same shoulder position and shoulders with biceps deformation , especially with biceps tendinitis or biceps tear , were excluded in the present study . in conclusion , the effusion within the blht sheath is closely related to the range of motion and clinical scores in patients with painful shoulders .", "the ultrasonographic detection of the effusion within the blht sheath might be useful to evaluate shoulder functions .", "however , further studies are required to understand the correlation between the effusion within the blht sheath and disease prognosis ." ] ]

[ "this was a retrospective cohort study approved by our institutional review board as a quality improvement research .", "the records of catheter days 1 year before the introduction of midlines ( group a : august 1 , 2011 , to july 31 , 2012 ) and catheter days of 1 year after the regular use of midlines ( group b : november 1 , 2012 , to october 31 , 2013 ) in the ventilator unit were collected ( table 1 ) . during the period from august 2012 to november 2012", ", internal medicine residents and nursing staff underwent training on the use and insertion of midline catheters and their care .", "this dedicated team was responsible for replacing cvcs by midlines as per our new practices .", "the midlines that we used were powerwand ( access scientific , san diego , calif ) . before midline catheter ,", "patient days : number of days the patient was in the ventilator unit ; catheter days : number of days the patient had a central venous catheter and after midline catheter , patient days : number of days the patient was in the ventilator unit ; catheter days : number of days the patient had a midline catheter we chose this unit because it has a constant denominator of the number and type of patients .", "most of the patients in this unit are ventilator dependent and being treated for problems such as health care associated pneumonia , urinary tract infections , wound and pressure ulcer infections , or other long - term conditions requiring acute care that can not be administered at a nursing home facility .", "the patients here have longer length of stay compared with other units and generally have difficult venous access .", "the most common reason for cvc use in this unit is for difficult and long - term intravenous access .", "indications of cvcs in these patients included antibiotic therapy , infusion therapy , diagnostic procedures , transfusions , and blood draws .", "the department of infection control of the hospital reports all the clabsi per 1000 catheter days in the hospital , categorized by location and unit .", "the number of clabsi during this period along with the type of bacteria and the date of culture was obtained from the microbiology department .", "we compared the clabsi rates between group a and group b to see whether it decreased through the use of midlines in comparison with central lines .", "we trained a team of residents for a period of 3 months in the insertion of ultrasound - guided midline catheters , which would replace cvcs by midline catheters whenever possible .", "the same antiseptic precautions used in cvc catheter placements under ultrasound guidance were applied to the placement of midlines .", "the policy for replacing the central lines included the following guidelines : any patient with a cvc in a femoral vein had their line removed and replaced by a midline catheter .", "all patients who were not on an ionotropic agent or total parenteral nutrition had their cvc replaced by midlines .", "a midline catheter replaced any cvc in place longer than a week . a patient on antibiotic therapy being sent to nursing home for further management received a midline catheter .", "the total number of catheter days was compared with the rate of clabsi in the 2 groups .", "catheter days were calculated as the number of central line catheters on the unit every day .", "adding the total number of catheters on the unit per day and adding this daily number for the length of time of the study help calculate catheter days .", "central line associated bsi was reported as rate per 1000 catheter days and can be calculated as follows : ( total number of clabsi / total number of catheter days ) 1000 .", "we used a test to compare the number of catheter days per patient days in the 2 groups as well as to compare infections based on the number of catheter days in each group ( table 2 ) . comparing the number of catheter days per patient days in the 2 groups , as well as comparing infections based on the number of catheter days in each group", "there was a significant decrease in the total number of catheter days on the ventilator unit in group a from 2408 catheter days in the 1 year ( august 1 , 2011 , to july 31 , 2012 ) before the introduction of midline catheters to 1521 catheter days in group b in the following year ( november 1 , 2012 , to october 31 , 2013 ; p < 0.05 in both groups ) .", "the total number of clabsi infections in these periods was also significantly decreased from 8 to 0 ( table 3 ) .", "this calculates to 3.32 clabsi per 1000 catheter days and 0 clabsi per 1000 catheter days , respectively ( add ) .", "the number of inpatient days during these periods was 3058 and 2948 days ( table 4 and 5 ) .", "continuation of the test test : observed frequency test : expected ( theoretical ) frequency , asserted by the null hypothesis there were no bsis associated with midlines in this study .", "the centers for disease control and prevention has put in place guidelines to reduce the incidence of clabsi .", "these guidelines brought about by numerous studies for years have helped bring down the rate of clabsi .", "these measures include the following : reducing the number of dwell days by removing catheters as early as possible ; reducing the use of central lines by using other means of venous access ; proper care and technique for cvcs ; maximum sterile barrier precautions during insertion ; use of chlorhexidine for skin disinfection before catheter insertion ; avoidance of the femoral insertion site ; and use of recommended insertion site dressing care practices .", "prolonged dwell time has been shown to increase clabsi rates rapidly after 9 catheter days .", "replacing central lines with midlines decreases the dwell time and also the total number of catheter days .", "it may be argued that the decrease in the use of central lines and thereby reducing the number may be responsible in decreasing clabsi ; however , our denominator is constant for comparison .", "the use of midlines reduces the use of and the dwell days for central lines , and with this study , we show that it can be a factor in reducing clabsi as well .", "another study showed that clabsi rates were higher in patients who had central lines for longer than 7 days . in our study , by replacing central lines with midlines in patients , we essentially decreased risk factors such as dressing changes , catheter care , and duration of central line use .", "a more obvious outcome is the decrease in use of central lines itself , causing a drop in the infection rates .", "the fall in infection rate may be attributed to the fewer number of catheters in place for longer than 7 days . before the introduction of midlines , difficult intravenous access in these chronically ill patients mandated the use of central lines .", "there are other complications of central lines , including inadvertent arterial puncture ( 3% ) , hemothorax , or pneumothorax ( 1%-2% ) .", "central lines and picc lines require x - ray confirmation of tip placement , exposing the patient to radiation .", "midlines have complications as well ; our most common problem was the loss or malfunction of the midline including extravasations in 2 patients .", "the bsi rate of midlines in various studies has been reported to be between 0% and 0.9% .", "the combination of better available products and the increasing use of ultrasound guidance for intravenous catheter placement has renewed interest in midline catheters", ". some limitations of midline catheters are inability to use for vasopressors , total parenteral nutrition or when large peripheral veins are not available such as in amputates and patients with arteriovenous fistulas .", "we conclude that the use of midline catheters to replace central lines for difficult intravenous access decreases the rate of clabsi in a ventilator unit in a community hospital ." ]

[ "<S> hypothesisour objective was to evaluate whether the use of midline venous catheters in place of central line venous catheters , when appropriate , decreased the overall incidence of central line associated bacteremia in a ventilator unit.methodsthe time interval between february 2012 and february 2013 was divided into 2 periods . </S>", "<S> group a was the first half of the year , before the introduction of midline catheters , and group b was the second half of the year , 6 months after their introduction . </S>", "<S> central line associated bloodstream infection ( clabsi ) was calculated using the equation : ( total number of clabsi / total number of catheter days ) 1000 . </S>", "<S> the z test was used for proportions between independent groups to compare the significance in the difference in clabsi between groups a and b.resultsthere was a significant decrease in the total number of catheter days on the ventilator unit in group a from 2408 catheter days in 1 year ( august 1 , 2011 , to july 31 , 2012 ) before the introduction of midline catheters to 1521 catheter days in group b in the following year ( november 1 , 2012 , to october 31 , 2013 ; p < 0.05 for both groups).conclusionsmidline catheters in place of central lines decrease the rate of clabsi in a ventilator unit . </S>", "<S> in addition , no bloodstream infections were associated with midline catheters . </S>" ]

[ "MATERIALS AND METHODS", "STATISTICS", "RESULTS", "DISCUSSION", "CONCLUSIONS" ]

[ [ "this was a retrospective cohort study approved by our institutional review board as a quality improvement research .", "the records of catheter days 1 year before the introduction of midlines ( group a : august 1 , 2011 , to july 31 , 2012 ) and catheter days of 1 year after the regular use of midlines ( group b : november 1 , 2012 , to october 31 , 2013 ) in the ventilator unit were collected ( table 1 ) . during the period from august 2012 to november 2012", ", internal medicine residents and nursing staff underwent training on the use and insertion of midline catheters and their care .", "this dedicated team was responsible for replacing cvcs by midlines as per our new practices .", "the midlines that we used were powerwand ( access scientific , san diego , calif ) . before midline catheter ,", "patient days : number of days the patient was in the ventilator unit ; catheter days : number of days the patient had a central venous catheter and after midline catheter , patient days : number of days the patient was in the ventilator unit ; catheter days : number of days the patient had a midline catheter we chose this unit because it has a constant denominator of the number and type of patients .", "most of the patients in this unit are ventilator dependent and being treated for problems such as health care associated pneumonia , urinary tract infections , wound and pressure ulcer infections , or other long - term conditions requiring acute care that can not be administered at a nursing home facility .", "the patients here have longer length of stay compared with other units and generally have difficult venous access .", "the most common reason for cvc use in this unit is for difficult and long - term intravenous access .", "indications of cvcs in these patients included antibiotic therapy , infusion therapy , diagnostic procedures , transfusions , and blood draws .", "the department of infection control of the hospital reports all the clabsi per 1000 catheter days in the hospital , categorized by location and unit .", "the number of clabsi during this period along with the type of bacteria and the date of culture was obtained from the microbiology department .", "we compared the clabsi rates between group a and group b to see whether it decreased through the use of midlines in comparison with central lines .", "we trained a team of residents for a period of 3 months in the insertion of ultrasound - guided midline catheters , which would replace cvcs by midline catheters whenever possible .", "the same antiseptic precautions used in cvc catheter placements under ultrasound guidance were applied to the placement of midlines .", "the policy for replacing the central lines included the following guidelines : any patient with a cvc in a femoral vein had their line removed and replaced by a midline catheter .", "all patients who were not on an ionotropic agent or total parenteral nutrition had their cvc replaced by midlines .", "a midline catheter replaced any cvc in place longer than a week . a patient on antibiotic therapy being sent to nursing home for further management received a midline catheter ." ], [ "the total number of catheter days was compared with the rate of clabsi in the 2 groups .", "catheter days were calculated as the number of central line catheters on the unit every day .", "adding the total number of catheters on the unit per day and adding this daily number for the length of time of the study help calculate catheter days .", "central line associated bsi was reported as rate per 1000 catheter days and can be calculated as follows : ( total number of clabsi / total number of catheter days ) 1000 .", "we used a test to compare the number of catheter days per patient days in the 2 groups as well as to compare infections based on the number of catheter days in each group ( table 2 ) . comparing the number of catheter days per patient days in the 2 groups , as well as comparing infections based on the number of catheter days in each group" ], [ "there was a significant decrease in the total number of catheter days on the ventilator unit in group a from 2408 catheter days in the 1 year ( august 1 , 2011 , to july 31 , 2012 ) before the introduction of midline catheters to 1521 catheter days in group b in the following year ( november 1 , 2012 , to october 31 , 2013 ; p < 0.05 in both groups ) .", "the total number of clabsi infections in these periods was also significantly decreased from 8 to 0 ( table 3 ) .", "this calculates to 3.32 clabsi per 1000 catheter days and 0 clabsi per 1000 catheter days , respectively ( add ) .", "the number of inpatient days during these periods was 3058 and 2948 days ( table 4 and 5 ) .", "continuation of the test test : observed frequency test : expected ( theoretical ) frequency , asserted by the null hypothesis there were no bsis associated with midlines in this study ." ], [ "the centers for disease control and prevention has put in place guidelines to reduce the incidence of clabsi .", "these guidelines brought about by numerous studies for years have helped bring down the rate of clabsi .", "these measures include the following : reducing the number of dwell days by removing catheters as early as possible ; reducing the use of central lines by using other means of venous access ; proper care and technique for cvcs ; maximum sterile barrier precautions during insertion ; use of chlorhexidine for skin disinfection before catheter insertion ; avoidance of the femoral insertion site ; and use of recommended insertion site dressing care practices .", "prolonged dwell time has been shown to increase clabsi rates rapidly after 9 catheter days .", "replacing central lines with midlines decreases the dwell time and also the total number of catheter days .", "it may be argued that the decrease in the use of central lines and thereby reducing the number may be responsible in decreasing clabsi ; however , our denominator is constant for comparison .", "the use of midlines reduces the use of and the dwell days for central lines , and with this study , we show that it can be a factor in reducing clabsi as well .", "another study showed that clabsi rates were higher in patients who had central lines for longer than 7 days . in our study , by replacing central lines with midlines in patients , we essentially decreased risk factors such as dressing changes , catheter care , and duration of central line use .", "a more obvious outcome is the decrease in use of central lines itself , causing a drop in the infection rates .", "the fall in infection rate may be attributed to the fewer number of catheters in place for longer than 7 days . before the introduction of midlines , difficult intravenous access in these chronically ill patients mandated the use of central lines .", "there are other complications of central lines , including inadvertent arterial puncture ( 3% ) , hemothorax , or pneumothorax ( 1%-2% ) .", "central lines and picc lines require x - ray confirmation of tip placement , exposing the patient to radiation .", "midlines have complications as well ; our most common problem was the loss or malfunction of the midline including extravasations in 2 patients .", "the bsi rate of midlines in various studies has been reported to be between 0% and 0.9% .", "the combination of better available products and the increasing use of ultrasound guidance for intravenous catheter placement has renewed interest in midline catheters", ". some limitations of midline catheters are inability to use for vasopressors , total parenteral nutrition or when large peripheral veins are not available such as in amputates and patients with arteriovenous fistulas ." ], [ "we conclude that the use of midline catheters to replace central lines for difficult intravenous access decreases the rate of clabsi in a ventilator unit in a community hospital ." ] ]

[ "additional information about study design , ethical considerations , and laboratory methods can be found in supplementary materials .", "we studied 72 children ( n = 21 ) and adults with type 1 diabetes without known renal impairment ( estimated glomerular filtration rate < 60 ml / min/1.73 m ) ( supplementary tables 1 and 2 ) . patients underwent a standard mmtt ( 1 ) .", "additional samples were taken at 30 , 60 , and 120 min in pediatric patients ( n = 18 ) , allowing area under the curve ( auc ) to be calculated .", "urine was collected as a fasting second morning void immediately before the start of the mmtt ( 0 min ) and after 120 min .", "nmol / l , in accordance with the diabetes control and complications trial ( 8) .", "urine was collected in boric acid 120 min after the evening meal following a premeal void .", "adult patients collected further home urine samples 120 min after a standard 60-g carbohydrate breakfast and following the patients own lunch .", "urine samples were brought to the research center within 24 h , measured in aliquots , and frozen at 80c .", "we assessed the association between 90-min scp ( 1 ) and both the mmtt 120-min ucpcr and after the home evening meal ( spearman rank correlation coefficient ) . in the pediatric cohort , correlations were also determined between auc scp and 120-min ucpcr .", "ucpcr ( 120 min ) following a home evening meal was compared with that after a mmtt ( wilcoxon test for paired samples ) .", "additional samples were taken at 30 , 60 , and 120 min in pediatric patients ( n = 18 ) , allowing area under the curve ( auc ) to be calculated .", "urine was collected as a fasting second morning void immediately before the start of the mmtt ( 0 min ) and after 120 min .", "nmol / l , in accordance with the diabetes control and complications trial ( 8) .", "urine was collected in boric acid 120 min after the evening meal following a premeal void .", "adult patients collected further home urine samples 120 min after a standard 60-g carbohydrate breakfast and following the patients own lunch .", "urine samples were brought to the research center within 24 h , measured in aliquots , and frozen at 80c .", "we assessed the association between 90-min scp ( 1 ) and both the mmtt 120-min ucpcr and after the home evening meal ( spearman rank correlation coefficient ) . in the pediatric cohort , correlations were also determined between auc scp and 120-min ucpcr .", "ucpcr ( 120 min ) following a home evening meal was compared with that after a mmtt ( wilcoxon test for paired samples ) .", "mmtt 120-min ucpcr was highly correlated with the 90-min scp ( r = 0.97 ; p < 0.0001 ) .", "nmol / l ) was 0.53 nmol / mmol , with 94% sensitivity and 100% specificity ( fig .", "a strong correlation was also seen between auc for scp and 120-min ucpcr during the mmtt ( r = 0.96 ; p < 0.0001 ) .", "scatter diagram showing the relationship between 90-min scp and 120-min ucpcr in the mmtt ( a ) and following the patients own evening meal at home ( b ) .", "a : 120-min ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.97 ; p < 0.0001 ) .", "b : 120-min postprandial ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.91 ) .", "home postprandial evening meal ucpcr ( 120 min ) was well correlated with 90-min scp ( r = 0.91 ; p < 0.0001 ) ( fig .", "the equivalent ucpcr cutoff was 0.37 nmol / mmol , with 84% sensitivity and 97% specificity ( fig .", "1b ) . using the ucpcr cutoff 0.53 nmol / mmol in the home evening meal samples yielded lower levels of sensitivity ( 71% ) and specificity ( 97% ) for significant endogenous insulin secretion .", "this is probably explained by a lower stimulus , as shown by the lower 120-min ucpcr in the home postprandial samples than in those in the mmtt ( 0.16 nmol / mmol [ interquartile range 0.010.76 ] vs. 0.35 nmol / mmol [ 0.041.41 ] ; p < 0.0001 ) .", "the correlations were similar in adults and children when analyzed separately ( supplementary tables 4 and 5 ) .", "result tables for combined ( supplementary table 3 ) and separate analysis of adults ( supplementary table 4 ) and children ( supplementary table 5 ) are given in the supplementary materials .", "mmtt 120-min ucpcr was highly correlated with the 90-min scp ( r = 0.97 ; p < 0.0001 ) .", "nmol / l ) was 0.53 nmol / mmol , with 94% sensitivity and 100% specificity ( fig .", "a strong correlation was also seen between auc for scp and 120-min ucpcr during the mmtt ( r = 0.96 ; p < 0.0001 ) .", "scatter diagram showing the relationship between 90-min scp and 120-min ucpcr in the mmtt ( a ) and following the patients own evening meal at home ( b ) .", "a : 120-min ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.97 ; p < 0.0001 ) .", "b : 120-min postprandial ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.91 ) .", "home postprandial evening meal ucpcr ( 120 min ) was well correlated with 90-min scp ( r = 0.91 ; p < 0.0001 ) ( fig .", "the equivalent ucpcr cutoff was 0.37 nmol / mmol , with 84% sensitivity and 97% specificity ( fig .", "1b ) . using the ucpcr cutoff 0.53 nmol / mmol in the home evening meal samples yielded lower levels of sensitivity ( 71% ) and specificity ( 97% ) for significant endogenous insulin secretion .", "this is probably explained by a lower stimulus , as shown by the lower 120-min ucpcr in the home postprandial samples than in those in the mmtt ( 0.16 nmol / mmol [ interquartile range 0.010.76 ] vs. 0.35 nmol / mmol [ 0.041.41 ] ; p < 0.0001 ) .", "the correlations were similar in adults and children when analyzed separately ( supplementary tables 4 and 5 ) .", "result tables for combined ( supplementary table 3 ) and separate analysis of adults ( supplementary table 4 ) and children ( supplementary table 5 ) are given in the supplementary materials .", "ucpcr measured during an mmtt or after a home meal is highly correlated with mmtt scp .", "our results showed strong correlations between stimulated ucpcr and serum c - peptide ( r = 0.910.97 ) during an mmtt .", "because ucpcr is stable at room temperature for 3 days in boric acid preservative ( 7 ) , home samples could be collected following a liquid mixed meal or the patients own home meal and a spot urine sample collected and posted for analysis directly .", "this would allow assessment to be done at home and to be noninvasive a particular advantage for children .", "as would be predicted , ucpcr values were lower after a meal compared with the standard mmtt , and so a lower concentration of ucpcr was required to suggest clinically significant insulin deficiency .", "the slight loss of precision compared with the standard mmtt needs to be balanced by the practicality of this approach because it would remove the need for inpatient testing and allow widespread screening .", "the strong correlation of ucpcr with serum c - peptide in the mmtt is supported by previous studies that have shown that timed measures of urinary c - peptide are a useful marker of endogenous insulin secretion ( 7,913 ) .", "this allowed spot samples to be taken rather than sampling over 24 h , in which case complete collection is difficult .", "this is similar to the practical reason why spot albumin creatinine ratio is used as opposed to 24 h urine collections in the assessment of renal protein excretion .", "a strong correlation between auc c - peptide and 120-min ucpcr was demonstrated ( r = 0.96 ) ; however , numbers were small ( n = 18 ) and further work is needed to explore this .", "the test can be difficult in young children , especially those who are still in nappies .", "the ease of use means that , if used in conjunction with formal mmtt , ucpcr may be useful for screening patients for initial inclusion and also follow - up during intervention trials . in conclusion , our study demonstrates that in children and adults with type 1 diabetes , ucpcr may be a practical noninvasive alternative to the mmtt for use in routine clinical practice .", "our results showed strong correlations between stimulated ucpcr and serum c - peptide ( r = 0.910.97 ) during an mmtt .", "because ucpcr is stable at room temperature for 3 days in boric acid preservative ( 7 ) , home samples could be collected following a liquid mixed meal or the patients own home meal and a spot urine sample collected and posted for analysis directly .", "this would allow assessment to be done at home and to be noninvasive a particular advantage for children .", "as would be predicted , ucpcr values were lower after a meal compared with the standard mmtt , and so a lower concentration of ucpcr was required to suggest clinically significant insulin deficiency .", "the slight loss of precision compared with the standard mmtt needs to be balanced by the practicality of this approach because it would remove the need for inpatient testing and allow widespread screening .", "the strong correlation of ucpcr with serum c - peptide in the mmtt is supported by previous studies that have shown that timed measures of urinary c - peptide are a useful marker of endogenous insulin secretion ( 7,913 ) .", "this allowed spot samples to be taken rather than sampling over 24 h , in which case complete collection is difficult .", "this is similar to the practical reason why spot albumin creatinine ratio is used as opposed to 24 h urine collections in the assessment of renal protein excretion .", "a strong correlation between auc c - peptide and 120-min ucpcr was demonstrated ( r = 0.96 ) ; however , numbers were small ( n = 18 ) and further work is needed to explore this .", "the test can be difficult in young children , especially those who are still in nappies .", "the ease of use means that , if used in conjunction with formal mmtt , ucpcr may be useful for screening patients for initial inclusion and also follow - up during intervention trials . in conclusion , our study demonstrates that in children and adults with type 1 diabetes , ucpcr may be a practical noninvasive alternative to the mmtt for use in routine clinical practice ." ]

[ "<S> objectivestimulated serum c - peptide ( scp ) during a mixed - meal tolerance test ( mmtt ) is the gold standard measure of endogenous insulin secretion , but practical issues limit its use . </S>", "<S> we assessed urine c - peptide creatinine ratio ( ucpcr ) as an alternative.research design and methodsseventy - two type 1 diabetic patients ( age of diagnosis median 14 years [ interquartile range 1022 ] ; diabetes duration 6.5 [ 2.332.7 ] ) had an mmtt . </S>", "<S> scp was collected at 90 min . </S>", "<S> urine for ucpcr was collected at 120 min and following a home evening meal.resultsmmtt 120-min ucpcr was highly correlated to 90-min scp ( r = 0.97 ; p < 0.0001 ) . </S>", "<S> ucpcr 0.53 nmol / mmol had 94% sensitivity/100% specificity for significant endogenous insulin secretion ( 90-min scp 0.2 </S>", "<S> nmol / l ) . </S>", "<S> the 120-min postprandial evening meal ucpcr was highly correlated to 90-min scp ( r = 0.91 ; p < 0.0001 ) . </S>", "<S> ucpcr 0.37 nmol / mmol had 84% sensitivity/97% specificity for scp 0.2 nmol / l.conclusionsucpcr testing is a sensitive and specific method for detecting insulin secretion . </S>", "<S> ucpcr may be a practical alternative to serum c - peptide testing , avoiding the need for inpatient investigation . </S>" ]

[ "RESEARCH DESIGN AND METHODS", "MMTT", "Home urine collections", "Statistical analysis", "RESULTS", "UCPCR correlations with serum C-peptide", "CONCLUSIONS", "UCPCR as a practical alternative to serum C-peptide measurement", "Other measures of urinary C-peptide", "Study limitations", "Implications", "Supplementary Material" ]

[ [ "additional information about study design , ethical considerations , and laboratory methods can be found in supplementary materials .", "we studied 72 children ( n = 21 ) and adults with type 1 diabetes without known renal impairment ( estimated glomerular filtration rate < 60 ml / min/1.73 m ) ( supplementary tables 1 and 2 ) . patients underwent a standard mmtt ( 1 ) .", "additional samples were taken at 30 , 60 , and 120 min in pediatric patients ( n = 18 ) , allowing area under the curve ( auc ) to be calculated .", "urine was collected as a fasting second morning void immediately before the start of the mmtt ( 0 min ) and after 120 min .", "nmol / l , in accordance with the diabetes control and complications trial ( 8) .", "urine was collected in boric acid 120 min after the evening meal following a premeal void .", "adult patients collected further home urine samples 120 min after a standard 60-g carbohydrate breakfast and following the patients own lunch .", "urine samples were brought to the research center within 24 h , measured in aliquots , and frozen at 80c .", "we assessed the association between 90-min scp ( 1 ) and both the mmtt 120-min ucpcr and after the home evening meal ( spearman rank correlation coefficient ) . in the pediatric cohort , correlations were also determined between auc scp and 120-min ucpcr .", "ucpcr ( 120 min ) following a home evening meal was compared with that after a mmtt ( wilcoxon test for paired samples ) ." ], [ "additional samples were taken at 30 , 60 , and 120 min in pediatric patients ( n = 18 ) , allowing area under the curve ( auc ) to be calculated .", "urine was collected as a fasting second morning void immediately before the start of the mmtt ( 0 min ) and after 120 min .", "nmol / l , in accordance with the diabetes control and complications trial ( 8) ." ], [ "urine was collected in boric acid 120 min after the evening meal following a premeal void .", "adult patients collected further home urine samples 120 min after a standard 60-g carbohydrate breakfast and following the patients own lunch .", "urine samples were brought to the research center within 24 h , measured in aliquots , and frozen at 80c ." ], [ "we assessed the association between 90-min scp ( 1 ) and both the mmtt 120-min ucpcr and after the home evening meal ( spearman rank correlation coefficient ) . in the pediatric cohort , correlations were also determined between auc scp and 120-min ucpcr .", "ucpcr ( 120 min ) following a home evening meal was compared with that after a mmtt ( wilcoxon test for paired samples ) ." ], [ "mmtt 120-min ucpcr was highly correlated with the 90-min scp ( r = 0.97 ; p < 0.0001 ) .", "nmol / l ) was 0.53 nmol / mmol , with 94% sensitivity and 100% specificity ( fig .", "a strong correlation was also seen between auc for scp and 120-min ucpcr during the mmtt ( r = 0.96 ; p < 0.0001 ) .", "scatter diagram showing the relationship between 90-min scp and 120-min ucpcr in the mmtt ( a ) and following the patients own evening meal at home ( b ) .", "a : 120-min ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.97 ; p < 0.0001 ) .", "b : 120-min postprandial ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.91 ) .", "home postprandial evening meal ucpcr ( 120 min ) was well correlated with 90-min scp ( r = 0.91 ; p < 0.0001 ) ( fig .", "the equivalent ucpcr cutoff was 0.37 nmol / mmol , with 84% sensitivity and 97% specificity ( fig .", "1b ) . using the ucpcr cutoff 0.53 nmol / mmol in the home evening meal samples yielded lower levels of sensitivity ( 71% ) and specificity ( 97% ) for significant endogenous insulin secretion .", "this is probably explained by a lower stimulus , as shown by the lower 120-min ucpcr in the home postprandial samples than in those in the mmtt ( 0.16 nmol / mmol [ interquartile range 0.010.76 ] vs. 0.35 nmol / mmol [ 0.041.41 ] ; p < 0.0001 ) .", "the correlations were similar in adults and children when analyzed separately ( supplementary tables 4 and 5 ) .", "result tables for combined ( supplementary table 3 ) and separate analysis of adults ( supplementary table 4 ) and children ( supplementary table 5 ) are given in the supplementary materials ." ], [ "mmtt 120-min ucpcr was highly correlated with the 90-min scp ( r = 0.97 ; p < 0.0001 ) .", "nmol / l ) was 0.53 nmol / mmol , with 94% sensitivity and 100% specificity ( fig .", "a strong correlation was also seen between auc for scp and 120-min ucpcr during the mmtt ( r = 0.96 ; p < 0.0001 ) .", "scatter diagram showing the relationship between 90-min scp and 120-min ucpcr in the mmtt ( a ) and following the patients own evening meal at home ( b ) .", "a : 120-min ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.97 ; p < 0.0001 ) .", "b : 120-min postprandial ucpcr is well correlated with 90-min scp in the mmtt ( r = 0.91 ) .", "home postprandial evening meal ucpcr ( 120 min ) was well correlated with 90-min scp ( r = 0.91 ; p < 0.0001 ) ( fig .", "the equivalent ucpcr cutoff was 0.37 nmol / mmol , with 84% sensitivity and 97% specificity ( fig .", "1b ) . using the ucpcr cutoff 0.53 nmol / mmol in the home evening meal samples yielded lower levels of sensitivity ( 71% ) and specificity ( 97% ) for significant endogenous insulin secretion .", "this is probably explained by a lower stimulus , as shown by the lower 120-min ucpcr in the home postprandial samples than in those in the mmtt ( 0.16 nmol / mmol [ interquartile range 0.010.76 ] vs. 0.35 nmol / mmol [ 0.041.41 ] ; p < 0.0001 ) .", "the correlations were similar in adults and children when analyzed separately ( supplementary tables 4 and 5 ) .", "result tables for combined ( supplementary table 3 ) and separate analysis of adults ( supplementary table 4 ) and children ( supplementary table 5 ) are given in the supplementary materials ." ], [ "ucpcr measured during an mmtt or after a home meal is highly correlated with mmtt scp .", "our results showed strong correlations between stimulated ucpcr and serum c - peptide ( r = 0.910.97 ) during an mmtt .", "because ucpcr is stable at room temperature for 3 days in boric acid preservative ( 7 ) , home samples could be collected following a liquid mixed meal or the patients own home meal and a spot urine sample collected and posted for analysis directly .", "this would allow assessment to be done at home and to be noninvasive a particular advantage for children .", "as would be predicted , ucpcr values were lower after a meal compared with the standard mmtt , and so a lower concentration of ucpcr was required to suggest clinically significant insulin deficiency .", "the slight loss of precision compared with the standard mmtt needs to be balanced by the practicality of this approach because it would remove the need for inpatient testing and allow widespread screening .", "the strong correlation of ucpcr with serum c - peptide in the mmtt is supported by previous studies that have shown that timed measures of urinary c - peptide are a useful marker of endogenous insulin secretion ( 7,913 ) .", "this allowed spot samples to be taken rather than sampling over 24 h , in which case complete collection is difficult .", "this is similar to the practical reason why spot albumin creatinine ratio is used as opposed to 24 h urine collections in the assessment of renal protein excretion .", "a strong correlation between auc c - peptide and 120-min ucpcr was demonstrated ( r = 0.96 ) ; however , numbers were small ( n = 18 ) and further work is needed to explore this .", "the test can be difficult in young children , especially those who are still in nappies .", "the ease of use means that , if used in conjunction with formal mmtt , ucpcr may be useful for screening patients for initial inclusion and also follow - up during intervention trials . in conclusion , our study demonstrates that in children and adults with type 1 diabetes , ucpcr may be a practical noninvasive alternative to the mmtt for use in routine clinical practice ." ], [ "our results showed strong correlations between stimulated ucpcr and serum c - peptide ( r = 0.910.97 ) during an mmtt .", "because ucpcr is stable at room temperature for 3 days in boric acid preservative ( 7 ) , home samples could be collected following a liquid mixed meal or the patients own home meal and a spot urine sample collected and posted for analysis directly .", "this would allow assessment to be done at home and to be noninvasive a particular advantage for children .", "as would be predicted , ucpcr values were lower after a meal compared with the standard mmtt , and so a lower concentration of ucpcr was required to suggest clinically significant insulin deficiency .", "the slight loss of precision compared with the standard mmtt needs to be balanced by the practicality of this approach because it would remove the need for inpatient testing and allow widespread screening ." ], [ "the strong correlation of ucpcr with serum c - peptide in the mmtt is supported by previous studies that have shown that timed measures of urinary c - peptide are a useful marker of endogenous insulin secretion ( 7,913 ) .", "this allowed spot samples to be taken rather than sampling over 24 h , in which case complete collection is difficult .", "this is similar to the practical reason why spot albumin creatinine ratio is used as opposed to 24 h urine collections in the assessment of renal protein excretion ." ], [ "a strong correlation between auc c - peptide and 120-min ucpcr was demonstrated ( r = 0.96 ) ; however , numbers were small ( n = 18 ) and further work is needed to explore this .", "the test can be difficult in young children , especially those who are still in nappies ." ], [ "the ease of use means that , if used in conjunction with formal mmtt , ucpcr may be useful for screening patients for initial inclusion and also follow - up during intervention trials . in conclusion , our study demonstrates that in children and adults with type 1 diabetes , ucpcr may be a practical noninvasive alternative to the mmtt for use in routine clinical practice ." ], [ "" ] ]

[ "metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus ( tcr c-17317 and tcr v-rp24 - 74e19 ) , the igh locus ( igh v-224m14 ) , igh c ( from c1- 3of c ) and a telomere - repeat specific peptide nucleic acid ( pna ) probe ( applied biosystems ) .", "three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described .", "confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat ( n.a .", "1.4 ) objective lens with 0.07 m x - y pixel sampling , optical slice thickness of 0.8 m , and z - step size of 0.2 m .", "volumes of interest ( vois ) were drawn around fish spots to be measured . within the individual vois ,", "the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software ( mipav , cit / nih ) .", "tetraspeck fluorescent microspheres ( molecular probes / invitrogen ) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope , fluorescence channel alignment , and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object .", "the minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . for distance measurements", "the following tcr probe sets were used : rp24 - 334b8 and rp23 - 255n13 ( proximal ) , rp24 - 74e19 and 17317 ( middle ) , rp23 - 304l21 and rp23 - 10k20 ( distal ) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d .", "thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4 , cd8 , tcr ( h57 - 597 ) , tcr ( gl3 ) ( bd pharmingen , san diego , ca ) .", "genomic dna was isolated from thymocytes using a qiagen dna isolation kit ( qiagen , valencia , ca ) .", "50ng dna was pcr amplified with a combination of gene - specific primers ( supplementary table 3 ) using platinum taq polymerase ( invitrogen , carlsbad , ca ) for standard , or the power sybr green master mix kit ( applied biosystems , foster city , ca ) for qpcr .", "real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software ( applied biosystems , foster city , ca ) . for each assay , aliquots of dna were analyzed for a control , non - rearranging dna 3 of j2 .", "the cycle threshold numbers for each primer combinations ( ct ) and for the control amplification ( ct ) were used to calculate the absolute amount of pcr signal .", "the relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . for sequence analysis ,", "150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit ( invitrogen ) .", "amplify tcr v - dj coding joint sequences , v primers were used as forward primers with the primer 3j1 as reverse primer ( supplementary table 4 ) .", "junctions from tcr mice were amplified using the indicated primers ( supplementary table 4 ) .", "briefly , 10 g of total thymus genomic dna was digested with stui ( new england biolabs ) .", "the digest products were run out on a 1% agarose tae gel , transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends .", "one g of genomic dna was treated with terminal deoxynucleotidyl transferase ( new england biolabs ) per manufacturer s protocol at a final concentration of 5 m datp .", "two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers .", "conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute , 57c for 45 seconds , and 72c for 45 seconds and then a final 5 minute extension step at 72c .", "two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water .", "one l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii .", "reaction conditions were the same as for the primary reactions , but the total number of cycles was increased to 30 cycles total .", "15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane ( bio - rad ) .", "loading control pcr reactions , aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described .", "10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane ( bio - rad ) .", "the oligonucleotides d1 , d2 , univ - bglii , t17-univ and ds850 are listed in supplementary table 4 .", "thymi were fixed in buffered 10% formalin , and paraffin sections were stained with hematoxylin - eosin .", "metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus ( tcr c-17317 and tcr v-rp24 - 74e19 ) , the igh locus ( igh v-224m14 ) , igh c ( from c1- 3of c ) and a telomere - repeat specific peptide nucleic acid ( pna ) probe ( applied biosystems ) .", "three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described .", "confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat ( n.a .", "1.4 ) objective lens with 0.07 m x - y pixel sampling , optical slice thickness of 0.8 m , and z - step size of 0.2 m .", "volumes of interest ( vois ) were drawn around fish spots to be measured . within the individual vois ,", "the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software ( mipav , cit / nih ) .", "tetraspeck fluorescent microspheres ( molecular probes / invitrogen ) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope , fluorescence channel alignment , and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object .", "the minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . for distance measurements", "the following tcr probe sets were used : rp24 - 334b8 and rp23 - 255n13 ( proximal ) , rp24 - 74e19 and 17317 ( middle ) , rp23 - 304l21 and rp23 - 10k20 ( distal ) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d .", "thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4 , cd8 , tcr ( h57 - 597 ) , tcr ( gl3 ) ( bd pharmingen , san diego , ca ) .", "genomic dna was isolated from thymocytes using a qiagen dna isolation kit ( qiagen , valencia , ca ) .", "50ng dna was pcr amplified with a combination of gene - specific primers ( supplementary table 3 ) using platinum taq polymerase ( invitrogen , carlsbad , ca ) for standard , or the power sybr green master mix kit ( applied biosystems , foster city , ca ) for qpcr .", "real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software ( applied biosystems , foster city , ca ) . for each assay , aliquots of dna were analyzed for a control , non - rearranging dna 3 of j2 .", "the cycle threshold numbers for each primer combinations ( ct ) and for the control amplification ( ct ) were used to calculate the absolute amount of pcr signal .", "the relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . for sequence analysis ,", "150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit ( invitrogen ) .", "amplify tcr v - dj coding joint sequences , v primers were used as forward primers with the primer 3j1 as reverse primer ( supplementary table 4 ) .", "junctions from tcr mice were amplified using the indicated primers ( supplementary table 4 ) .", "briefly , 10 g of total thymus genomic dna was digested with stui ( new england biolabs ) .", "the digest products were run out on a 1% agarose tae gel , transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends .", "one g of genomic dna was treated with terminal deoxynucleotidyl transferase ( new england biolabs ) per manufacturer s protocol at a final concentration of 5 m datp .", "two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers .", "conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute , 57c for 45 seconds , and 72c for 45 seconds and then a final 5 minute extension step at 72c .", "two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water .", "one l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii .", "reaction conditions were the same as for the primary reactions , but the total number of cycles was increased to 30 cycles total .", "15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane ( bio - rad ) .", "the membrane was subsequently hybridized with p - labeled ds850 oligonucleotide . for il-2 loading control pcr reactions , aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described .", "10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane ( bio - rad ) .", "the oligonucleotides d1 , d2 , univ - bglii , t17-univ and ds850 are listed in supplementary table 4 .", "thymi were fixed in buffered 10% formalin , and paraffin sections were stained with hematoxylin - eosin ." ]

[ "<S> v(d)j recombination and class switch recombination employ overlapping but distinct non - homologous end - joining ( nhej ) pathways to repair dna double strand break ( dsb ) intermediates . </S>", "<S> 53bp1 is a dna damage response protein that is rapidly recruited to sites of chromosomal dsbs , where it appears to function in a subset of ataxia - telangiectasia mutated ( atm ) kinase , h2ax- and mdc1- dependent events1,2 . </S>", "<S> a 53bp1 dependent end joining pathway has been described that is dispensable for v(d)j recombination but essential for class - switch recombination csr3 , 4 . here , we report a previously unrecognized defect in the joining phase of v(d)j recombination in 53bp1 deficient lymphocytes distinct from that found in classical nhej- , h2ax- , mdc1- and atm - deficient mice . </S>", "<S> absence of 53bp1 leads to impairment of distal v - dj joining with extensive degradation of un - repaired coding ends and episomal signal joint reintegration at v(d)j junctions . </S>", "<S> this results in apoptosis , loss of t - cell receptor alpha locus integrity and lymphopenia . </S>", "<S> further impairment of the apoptotic checkpoint causes propagation of lymphocytes bearing antigen receptor breaks . </S>", "<S> these data suggest a more general role for 53bp1 in maintaining genomic stability during long range joining of dna breaks . </S>" ]

[ "Online Methods", "Metaphase and interphase FISH", "Flow cytometry", "PCR and junction analysis", "Southern blot analysis", "TdT-mediated polyadenylation and PCR amplification of free J coding ends", "TUNEL staining", "Supplementary Material" ]

[ [ "metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus ( tcr c-17317 and tcr v-rp24 - 74e19 ) , the igh locus ( igh v-224m14 ) , igh c ( from c1- 3of c ) and a telomere - repeat specific peptide nucleic acid ( pna ) probe ( applied biosystems ) .", "three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described .", "confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat ( n.a .", "1.4 ) objective lens with 0.07 m x - y pixel sampling , optical slice thickness of 0.8 m , and z - step size of 0.2 m .", "volumes of interest ( vois ) were drawn around fish spots to be measured . within the individual vois ,", "the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software ( mipav , cit / nih ) .", "tetraspeck fluorescent microspheres ( molecular probes / invitrogen ) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope , fluorescence channel alignment , and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object .", "the minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . for distance measurements", "the following tcr probe sets were used : rp24 - 334b8 and rp23 - 255n13 ( proximal ) , rp24 - 74e19 and 17317 ( middle ) , rp23 - 304l21 and rp23 - 10k20 ( distal ) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d .", "thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4 , cd8 , tcr ( h57 - 597 ) , tcr ( gl3 ) ( bd pharmingen , san diego , ca ) .", "genomic dna was isolated from thymocytes using a qiagen dna isolation kit ( qiagen , valencia , ca ) .", "50ng dna was pcr amplified with a combination of gene - specific primers ( supplementary table 3 ) using platinum taq polymerase ( invitrogen , carlsbad , ca ) for standard , or the power sybr green master mix kit ( applied biosystems , foster city , ca ) for qpcr .", "real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software ( applied biosystems , foster city , ca ) . for each assay , aliquots of dna were analyzed for a control , non - rearranging dna 3 of j2 .", "the cycle threshold numbers for each primer combinations ( ct ) and for the control amplification ( ct ) were used to calculate the absolute amount of pcr signal .", "the relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . for sequence analysis ,", "150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit ( invitrogen ) .", "amplify tcr v - dj coding joint sequences , v primers were used as forward primers with the primer 3j1 as reverse primer ( supplementary table 4 ) .", "junctions from tcr mice were amplified using the indicated primers ( supplementary table 4 ) .", "briefly , 10 g of total thymus genomic dna was digested with stui ( new england biolabs ) .", "the digest products were run out on a 1% agarose tae gel , transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends .", "one g of genomic dna was treated with terminal deoxynucleotidyl transferase ( new england biolabs ) per manufacturer s protocol at a final concentration of 5 m datp .", "two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers .", "conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute , 57c for 45 seconds , and 72c for 45 seconds and then a final 5 minute extension step at 72c .", "two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water .", "one l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii .", "reaction conditions were the same as for the primary reactions , but the total number of cycles was increased to 30 cycles total .", "15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane ( bio - rad ) .", "loading control pcr reactions , aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described .", "10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane ( bio - rad ) .", "the oligonucleotides d1 , d2 , univ - bglii , t17-univ and ds850 are listed in supplementary table 4 .", "thymi were fixed in buffered 10% formalin , and paraffin sections were stained with hematoxylin - eosin ." ], [ "metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus ( tcr c-17317 and tcr v-rp24 - 74e19 ) , the igh locus ( igh v-224m14 ) , igh c ( from c1- 3of c ) and a telomere - repeat specific peptide nucleic acid ( pna ) probe ( applied biosystems ) .", "three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described .", "confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat ( n.a .", "1.4 ) objective lens with 0.07 m x - y pixel sampling , optical slice thickness of 0.8 m , and z - step size of 0.2 m .", "volumes of interest ( vois ) were drawn around fish spots to be measured . within the individual vois ,", "the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software ( mipav , cit / nih ) .", "tetraspeck fluorescent microspheres ( molecular probes / invitrogen ) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope , fluorescence channel alignment , and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object .", "the minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . for distance measurements", "the following tcr probe sets were used : rp24 - 334b8 and rp23 - 255n13 ( proximal ) , rp24 - 74e19 and 17317 ( middle ) , rp23 - 304l21 and rp23 - 10k20 ( distal ) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d ." ], [ "thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4 , cd8 , tcr ( h57 - 597 ) , tcr ( gl3 ) ( bd pharmingen , san diego , ca ) ." ], [ "genomic dna was isolated from thymocytes using a qiagen dna isolation kit ( qiagen , valencia , ca ) .", "50ng dna was pcr amplified with a combination of gene - specific primers ( supplementary table 3 ) using platinum taq polymerase ( invitrogen , carlsbad , ca ) for standard , or the power sybr green master mix kit ( applied biosystems , foster city , ca ) for qpcr .", "real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software ( applied biosystems , foster city , ca ) . for each assay , aliquots of dna were analyzed for a control , non - rearranging dna 3 of j2 .", "the cycle threshold numbers for each primer combinations ( ct ) and for the control amplification ( ct ) were used to calculate the absolute amount of pcr signal .", "the relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . for sequence analysis ,", "150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit ( invitrogen ) .", "amplify tcr v - dj coding joint sequences , v primers were used as forward primers with the primer 3j1 as reverse primer ( supplementary table 4 ) .", "junctions from tcr mice were amplified using the indicated primers ( supplementary table 4 ) ." ], [ "briefly , 10 g of total thymus genomic dna was digested with stui ( new england biolabs ) .", "the digest products were run out on a 1% agarose tae gel , transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends ." ], [ "one g of genomic dna was treated with terminal deoxynucleotidyl transferase ( new england biolabs ) per manufacturer s protocol at a final concentration of 5 m datp .", "two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers .", "conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute , 57c for 45 seconds , and 72c for 45 seconds and then a final 5 minute extension step at 72c .", "two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water .", "one l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii .", "reaction conditions were the same as for the primary reactions , but the total number of cycles was increased to 30 cycles total .", "15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane ( bio - rad ) .", "the membrane was subsequently hybridized with p - labeled ds850 oligonucleotide . for il-2 loading control pcr reactions , aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described .", "10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane ( bio - rad ) .", "the oligonucleotides d1 , d2 , univ - bglii , t17-univ and ds850 are listed in supplementary table 4 ." ], [ "thymi were fixed in buffered 10% formalin , and paraffin sections were stained with hematoxylin - eosin ." ], [ "" ] ]

[ "it integrates signals from cortical areas and subserves important functions like motor control ( tecuapetla et al . , 2014 , kravitz et al . , 2010 ) and reinforcement / punishment coding ( kravitz et al . , 2012 ) .", "striatal neurons comprise gabaergic spiny projection neurons ( spns , 95% ) and interneurons ( 5% ) .", "spns are classified into direct pathway spns ( dspns ) , which project to the substantia nigra reticulata and external and internal segments of the globus pallidus ( gpe and gpi , respectively ) , and indirect pathway spns ( ispns ) , which project to gpe ( bolam et al . , 2000 , wu et al . , 2000", "striatal interneurons include large aspiny cholinergic neurons and different populations of gabaergic interneurons ( kawaguchi et al . , 1995 ) .", "excitatory glutamatergic neurons from virtually all cortical areas send projections to the striatum ( mcgeorge and faull , 1989 ) , and several studies suggest their recruitment during action selection ( xiong et al . , 2015 , znamenskiy and zador , 2013 , koralek et al . , 2012 ) .", "in contrast , cortical gabaergic neurons projecting to the striatum were not considered to be a component of the canonical corticostriatal network because they were identified only in the prefrontal , somatosensory , and retrosplenial cortices ( lee et al . , 2014 ,", "jinno and kosaka , 2004 ) . only recently were direct gabaergic neurons projecting to the striatum also described in the motor and auditory cortices ( rock et al . , 2016 ) .", "the authors identified the long - range projecting neurons as somatostatin - positive ( som ) and , furthermore , reported that inhibition conveyed by these neurons was onto both dspns and ispns .", "we previously showed that long - range gabaergic neurons connecting several brain structures comprise different molecular subtypes .", "for instance , connectivity between the hippocampus and medial entorhinal cortex is supported by parvalbumin - positive ( pv ) and som neurons ( melzer et al . , 2012 ) .", "moreover , projections from the septum to the medial entorhinal cortex are pv and calbindin , and they inhibit specific interneurons differentially ( fuchs et al . , 2016 ) .", "hence , we wondered whether long - range gabaergic projecting neurons from the motor cortex to the striatum are diverse with respect to their molecular identity , target specificity , and function at the behavioral level .", "based on virus - mediated tracing , optogenetics , patch - clamp recordings in vitro , and behavioral essays , we identified two distinct populations of long - range projecting gabaergic neurons in the primary ( m1 ) and secondary ( m2 ) motor cortex targeting the dorsal striatum and established that these two populations exhibit target cell preference in the striatum and affect locomotion differentially .", "som cells are a major source of intracortical and corticofugal long - range gabaergic projections ( tomioka et al . , 2005 , rock et al . , 2016 ) .", "to substantiate and extend these studies focusing on long - range gabaergic neurons connecting the motor cortex and the striatum , we injected adeno - associated virus ( aav ) double - floxed inverse open reading frame ( dio ) chr2-mcherry into the m1 and m2 area of som mice .", "this resulted in labeling of a subpopulation of gabaergic neurons ( figures 1a and 1b ; figure s1a ) and revealed projections in several ipsilateral cortical and subcortical areas and , to a lesser extent , in contralateral cortices ( table s1 ) .", "there was consistent innervation of the ipsilateral dorsal striatum ( figure 1b ; table s1 ) .", "motor cortex som neuron projections traversed the dorsal striatum and branched preferentially ventro - laterally , sparing the most rostral and caudal part of the dorsal striatum ( figure 1b).figure 1motor cortex som gabaergic neurons innervate the striatum(a ) schematic drawing of the injection site and the location of long - range projections in the striatum shown in ( b ) .", "viral constructs encoding chr2-mcherry were injected into the motor cortex of som mice.(b ) bright - field images of dab - stained sections showing the injection site in the motor cortex ( left ) and mcherry - labeled axons in the striatum ( center ) following aav dio chr2-mcherry injection into the motor cortex of som mice . a higher magnification of the boxed area is shown on the right.(c and d ) confocal images showing a retrogradely labeled area ( c ) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex , visualized via fish for sst and gad1/2 ( d).(e h ) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of som mice , and rabies virus was injected into the striatum .", "( e ) shows differential interference contrast ( dic ) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in ( f ) .", "( g ) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in ( h).str , striatum .", "motor cortex som gabaergic neurons innervate the striatum ( a ) schematic drawing of the injection site and the location of long - range projections in the striatum shown in ( b ) .", "( b ) bright - field images of dab - stained sections showing the injection site in the motor cortex ( left ) and mcherry - labeled axons in the striatum ( center ) following aav dio chr2-mcherry injection into the motor cortex of som mice .", "( c and d ) confocal images showing a retrogradely labeled area ( c ) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex , visualized via fish for sst and gad1/2 ( d ) . ( e ", "h ) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of som mice , and rabies virus was injected into the striatum .", "( e ) shows differential interference contrast ( dic ) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in ( f ) .", "( g ) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in ( h ) .", "see also figure s1 and tables s1 and s2 . to further substantiate the presence of gabaergic corticostriatal projections , we performed retrograde labeling .", "we injected cholera toxin b ( ctb ) subunit 647 into the ventro - lateral part of the dorsal striatum and analyzed retrogradely labeled cells in the m1 region ( figures s1b and s1c ) .", "as expected , a dense band of retrogradely labeled cells became visible in cortical l5 ( figure 1c ) ; i.e. , in the layer that is the major source of corticostriatal excitatory projections ( wilson , 1987 , cowan and wilson , 1994 ) . to visualize gabaergic cells among the m1 retrogradely labeled cells , we performed multi - fluorescence in situ hybridization ( fish ) for sst ( encoding som ) and gad1/2 ( encoding gad67/65 ) .", "we found 13 retrogradely labeled cells in m1 that were clearly positive for gad1/2 ( n = 3,582 ctb cells and 5,064 gad1/2 cells , cell counts across all layers in 35 slices from 4 hemispheres in 4 mice ) , 8 of which co - labeled for sst ( figure 1d ) .", "to confirm a direct long - range gabaergic connection between the motor cortex ( m1/m2 ) and the dorsal striatum , we performed retrograde monosynaptic tracing with rabies virus ( wickersham et al . , 2007 ) .", "we injected aavs encoding cre - dependent avian virus receptor ( avian tumor virus receptor a mcherry [ tcb ] ; weissbourd et al . , 2014 ) and", "rabies glycoprotein ( rg ) into the striatum of a2a - cre mice that express cre recombinase specifically in ispns ( gong et al . , 2003 ) .", "subsequent injection of rg - deleted envelope protein from avian aslv type a ( enva)-pseudotyped rabies virus ( sadg - egfp(enva ) ) into the striatum resulted in transsynaptically retrogradely labeled cells in the cortex ( figures s1e and s1f ) .", "fish for rabies virus - specific mrna ( rabv - gp1 ) and gad1/2 revealed double - positive neurons in the motor cortex ( 7 cells in 34 slices from 4 hemispheres in 4 mice ; figure s1", "the average number of labeled cells per slice was lower than after ctb647 injections , suggesting that ispns were not the only striatal target cells of gabaergic projecting neurons and/or reflecting lower efficiency of transsynaptic tracing ( marshel et al . ,", ", we expressed tcb cre - dependently in the motor cortex ( m1/m2 ) of som mice and injected sadg - egfp(enva ) rabies virus into the striatum .", "tcb retrogradely labeled cells in the motor cortex had a classical or burst accommodating firing pattern ( n = 11 cells from 5 hemispheres in 4 mice ; figures 1e and 1f ; figure s1h ) similar to non - retrogradely labeled tcb cells ( table s2 ) .", "reconstructed cells had a martinotti cell - like morphology ( wang et al . , 2004 ) with axonal projections extending over all cortical layers ( three reconstructions from three hemispheres in two mice ; figures 1 g and 1h ; figures s1i ", "we next tested whether som projecting neurons form functional synapses onto striatal neurons and whether the connectivity exhibits target specificity .", "we injected aav dio chr2-mcherry into m1/m2 of som mice and combined optogenetic stimulation of long - range projections with patch - clamp recordings of putative postsynaptic cells in the striatum ( figure 2a ) .", "all injections ( n = 36 hemispheres ) resulted in labeled axons that projected to the dorsal striatum .", "of 305 patched neurons ( in 27 mice ) , 50 responded with short - latency postsynaptic currents ( pscs ) to 5-ms photostimulation of motor cortex som neuron projections ( figure 2b ) .", "as a specificity control , we repeated the experiment in wild - type mice injected with aav dio chr2-mcherry and found neither mcherry fibers in the dorsal striatum nor a response after photostimulation ( n = 58 cells in 2 mice ) .", "responses in som mice could not be blocked with 6-cyano-2,3-dihydroxy-7-nitro - quinoxaline ( cnqx ) and d-2-amino-5-phosphonovaleric acid ( d - ap5 ) ( 165.6 32.7 pa baseline versus 169.5 31.1 pa with drugs [ mean sem ] , paired t test , t(14 ) = 0.49 , p = 1 , n = 15 cells in 11 mice ; figure s2a ) but with gabazine ( 117.8 [ 134.3 ] pa versus 1.6 [ 1.6 ] pa [ median interquartile range ( iqr ) ] , wilcoxon signed - rank test , w = 210 , p = 0.0002 , n = 20 cells in 15 mice ; figures s2a and s2b ) .", "responses reversed around the reversal potential of gabaergic receptors ( n = 14 cells in 9 mice ; figure 2b ) , thus confirming the gabaergic nature of motor cortex som neuron projections.figure 2motor cortex som neuron projections form functional synapses on striatal output and cholinergic neurons(a ) schematic drawing indicating the injection site ( left ) and location of an exemplary patched neuron in the striatum ( right ) .", "aav dio chr2-mcherry was injected into the motor cortex of som mice , and target cells were patched in the striatum ( dic image).(b ) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation ( blue ticks ) of motor cortex som neuron projections .", "responses were blocked with gabazine but not d - ap5/cnqx.(c ) firing pattern ( membrane potential upon 50-pa current injection and at the action potential [ ap ] threshold ) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections.(d ) firing pattern ( spontaneous activity and maximal firing frequency ) and dic image of a representative cholinergic interneuron ( arrow ) that was responsive to photostimulation of motor cortex som neuron projections.(e ) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections ( from top to bottom : maximal firing frequency , ap threshold , and 50-pa current injection).(f ) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections .", "number of mice : spns , 19 ; cholinergic , 11 ; gabaergic interneurons , 19.(g ) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice.(h ) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp ( left ) and som / drd2-egfp ( right ) mice exhibiting differential egfp expression in the globus pallidus ( arrow ) and substantia nigra ( arrowhead).(i ) exemplary traces of dspn and ispn responses to photostimulation ( blue ticks ) of m1 and m2 som neuron projections using cs - based low cl intracellular solution ( from top to bottom : 0 mv , reversal potential , and 95 mv holding potential).(j ) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections .", "number of mice : m1-dspns , 7 ; m1-ispns , 16 ; m2-dspns , 8 ; m2-ispns , 17 ; m1-cholinergic , 19 ; m2-cholinergic , 13.(k ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m2 to dspn ; dark green , m1 to ispn ; light green , m2 to ispn ; purple , m1 to cholinergic interneuron ; red , m2 to cholinergic interneuron.hp , hippocampus ; ach , cholinergic interneuron .", "som neuron projections form functional synapses on striatal output and cholinergic neurons ( a ) schematic drawing indicating the injection site ( left ) and location of an exemplary patched neuron in the striatum ( right ) .", "aav dio chr2-mcherry was injected into the motor cortex of som mice , and target cells were patched in the striatum ( dic image ) .", "( b ) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation ( blue ticks ) of motor cortex som neuron projections .", "( c ) firing pattern ( membrane potential upon 50-pa current injection and at the action potential [ ap ] threshold ) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections .", "( d ) firing pattern ( spontaneous activity and maximal firing frequency ) and dic image of a representative cholinergic interneuron ( arrow ) that was responsive to photostimulation of motor cortex som neuron projections .", "( e ) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections ( from top to bottom : maximal firing frequency , ap threshold , and 50-pa current injection ) .", "( f ) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections .", "number of mice : spns , 19 ; cholinergic , 11 ; gabaergic interneurons , 19 .", "( g ) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice .", "( h ) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp ( left ) and som / drd2-egfp ( right ) mice exhibiting differential egfp expression in the globus pallidus ( arrow ) and substantia nigra ( arrowhead ) .", "( i ) exemplary traces of dspn and ispn responses to photostimulation ( blue ticks ) of m1 and m2 som neuron projections using cs - based low cl intracellular solution ( from top to bottom : 0 mv , reversal potential , and 95 mv holding potential ) .", "( j ) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections .", "number of mice : m1-dspns , 7 ; m1-ispns , 16 ; m2-dspns , 8 ; m2-ispns , 17 ; m1-cholinergic , 19 ; m2-cholinergic , 13 .", "( k ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m2 to dspn ; dark green , m1 to ispn ; light green , m2 to ispn ; purple , m1 to cholinergic interneuron ; red , m2 to cholinergic interneuron .", "hp , hippocampus ; ach , cholinergic interneuron . see also figures s2 and s3 and tables s3 and s4 . to scrutinize target specificity ,", "striatal neurons were sorted into spns and cholinergic and gabaergic interneurons based on their electrophysiological properties and cell soma shape ( planert et al . , 2013 , gertler et al . , 2008 , kawaguchi , 1992 , bennett and wilson , 1999 , kawaguchi et al . , 1995 ; experimental procedures ; figures 2c2e ; table s3 ) .", "we found that 22.6% of spns , 33.3% of cholinergic cells , and only 2% of gabaergic interneurons responded to 5-ms photostimulation of motor cortex som neuron projections ( figure 2f ; figures s2c and s2d ; table s3 ) .", "together , these data indicate that spns and cholinergic cells are the main target of motor cortex som neuron projections . to answer whether dspns and ispns are differentially targeted by motor cortex som projecting neurons , we cross - bred som mice to drd1a - egfp and drd2-egfp mice in which dspns and ispns , respectively , are selectively labeled ( gong et al . , 2003 ;", "we injected aav dio chr2-mcherry into m1 or m2 of som / drd1a - egfp or som / drd2-egfp mice ( figure 2 g ; figure s2e ) and combined photostimulation of m1 or m2 som neuron projections with patch - clamp recordings of striatal neurons ( figure s2f ) .", "we found that m1 som projecting neurons innervated a comparable proportion of dspns ( 29% ) , ispns ( 22% ) , and cholinergic cells ( 40% ) ( figures 2i and 2j ; fisher s exact test , p = 0.19 ) .", "m2 som projecting neurons targeted dspns ( 25% ) and ispns ( 16% ) to a similar extent , whereas cholinergic cells tended to be innervated less frequently ( 4% ) ( figure 2j ; fisher s exact test , p = 0.08 ) .", "inhibition of cholinergic cells by m2 was significantly less frequent than by m1 ( figure 2j ; fisher s exact test , m1 dspns versus m2 dspns : p = 1 ; m1 ispns versus m2 ispns : p = 1 ; m1 cholinergic versus m2 cholinergic : p = 0.006 ) .", "responses had a latency of 2.5 ( 1.3 ) ms ( median [ iqr ] ; n = 47 responding cells ; see figure s3a and table s4 for more details ) and a reversal potential of 58.0 1.4 mv ( mean sem ; n = 22 responding cells ; see figure s3b and table s4 for more details ) .", "the strength of m1 and m2 som neuron connections to spns was similar ( table s4 ) .", "however , when comparing inputs with dspns and ispns both from m1 and m2 , the response amplitudes were significantly larger for ispns ( amplitudes at 0 mv with cesium [ cs ] internal solution : 15.7 pa versus 44.0 [ 36.5 ] pa [ median ( iqr ) ] in dspns and ispns respectively ; mann - whitney u test , u = 102 , p = 0.02 , n = 14 and 26 responding cells , respectively ; figure s3c ) .", "detected target cells were located preferentially in the ventro - lateral part of the dorsal striatum ( figure 2k ; figures s3d ", "targeted dspns and ispns were intermingled and localized 2.4 ( 0.7 ) mm lateral , 0.1 ( 0.7 ) mm posterior to the bregma and 4 ( 0.7 ) mm deep ( median [ iqr ] ) .", "spns targeted by m1 projections were more lateral than spns targeted by m2 projections ( mann - whitney u test , u = 142 , p = 0.04 ; table s4 ) .", "the retrograde labeling experiments suggested that som neurons are not the only m1 gabaergic population projecting to the striatum .", "pv neurons appeared to be attractive candidates because we identified them before as a major class of neurons providing long - range inhibition in the entorhinal cortex - hippocampal formation ( melzer et al . , 2012 ) .", "testing for the presence of gabaergic long - range projecting pv cells based on virus tracing in pv mice may be complicated by the fact that a fraction of cortical pv cells are glutamatergic ( jinno and kosaka , 2004 ) .", "hence , we first tested the gabaergic nature of pv cells in m1/m2 by counting the number of double - positive cells in injected pv / gad67 mice .", "99.5% of pv cells were gad67 ( 759 of 763 pv cells from 9 slices in 3 mice ) .", "this result is in agreement with previous evidence showing the absence of pv glutamatergic neurons in the motor cortex ( jinno and kosaka , 2004 ) . in the mouse neocortex ,", "pv and som neurons constitute two non - overlapping neuronal entities ( xu et al . , 2010 , tasic et al . , 2016 ) .", "thus , we injected aav dio chr2-mcherry into m1 of pv mice , and , indeed , we detected labeled axons in the dorsal striatum that branched preferentially ventro - laterally ( in 12 of 17 injected hemispheres ) .", "in contrast , injections into m2 of pv mice were less likely to result in labeled striatal projections ( 5 of 12 injections ) , and projections when present were sparser than the ones detected after m1 injections .", "thus , for a more detailed analysis of innervation patterns , we focused on projections from m1 .", "we injected aav dio chr2-mcherry into m1 of pv / drd1a - egfp or pv / drd2-egfp mice ( figure 3a ) and found that 31% of dspns , 6% of ispns , and 6% of cholinergic and 0% of gabaergic interneurons responded to photostimulation of m1 pv neuron projections ( figures 3b and 3c ) .", "thus , pv projecting neurons preferentially innervated dspns ( fisher s exact test ; dspns versus ispns : p = 0.009 , dspns versus cholinergic : p = 0.04 , ispns versus cholinergic : p = 1 ; figure 3b ) .", "spns targeted by m1 pv projecting neurons had a response amplitude comparable with that following stimulation of m1 som projections ( figure s3c ; table s4 ) .", "although responses could not be blocked by cnqx / d - ap5 ( although one of five cells showed a clear decrease in amplitude ) , subsequent application of gabazine led to a significant amplitude decrease ( 68.9 [ 147.3 ] pa baseline versus 70.3 [ 86.1 ] pa with cnqx / d - ap5 versus 0.5 [ 3.2 ] pa with gabazine , friedman test , p = 0.02 , post hoc conover s test with bonferroni correction , baseline versus cnqx / d - ap5 : p = 1 , baseline versus gabazine : p = 0.004 , cnqx / d - ap5 versus gabazine : p = 0.009 , n = 5 cells in 4 mice ; figure 3d ; figures s3 g and s3h ) .", "the reversal potential of the responses was 60.3 2.5 mv ( mean sem , n = 4 cells in 4 mice ; figure s3b ; table s4 ) , reconfirming the gabaergic nature .", "spns targeted by m1 pv projecting neurons were located 0.6 ( 0.7 ) mm posterior to the bregma , 3.0 ( 0.6 ) mm lateral , and 3.7 ( 0.8 ) deep ( median [ iqr ] ) ( figure 3e ; figures s3d ", "s3f ; table s4).figure 3m1 pv neuron projections preferentially target dspns in the striatum(a ) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice.(b ) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections .", "number of mice : dspns , 8 ; ispns , 7 ; cholinergic , 13 ; gabaergic interneurons , 9.(c ) firing pattern ( upon 50 pa current injection and at the ap threshold ) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections.(d ) responses of the dspn shown in ( c ) at 70 mv holding potential using high cl intracellular solution .", "responses were blocked with gabazine but not d - ap5/cnqx.(e ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m1 to ispns ; gray , unidentified responding neurons.(f and g ) epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex ( arrow ) ( f ) with the corresponding firing pattern ( g ) identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of pv mice , and rabies virus was injected into the striatum.(h ) dot plot of the action potential half - width for retrogradely labeled som ( n = 11 cells from 5 hemispheres in 4 mice ) and pv neurons ( n = 3 cells from 3 hemispheres in 2 mice).see also figure s3 and tables s2 and s4 .", "m1 pv neuron projections preferentially target dspns in the striatum ( a ) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice .", "( b ) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections .", "number of mice : dspns , 8 ; ispns , 7 ; cholinergic , 13 ; gabaergic interneurons , 9 .", "( c ) firing pattern ( upon 50 pa current injection and at the ap threshold ) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections .", "( d ) responses of the dspn shown in ( c ) at 70 mv holding potential using high cl intracellular solution .", "( e ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m1 to ispns ; gray , unidentified responding neurons .", "( f and g ) epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex ( arrow ) ( f ) with the corresponding firing pattern ( g ) identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of pv mice , and rabies virus was injected into the striatum .", "( h ) dot plot of the action potential half - width for retrogradely labeled som ( n = 11 cells from 5 hemispheres in 4 mice ) and pv neurons ( n = 3 cells from 3 hemispheres in 2 mice ) .", "the different innervation patterns of som and pv projecting neurons suggested that the two cell types represent distinct subpopulations in the motor cortex .", "we hence analyzed whether the firing pattern of pv projecting neurons differed from that of som projecting neurons and resembled that of classical pv interneurons .", "the detection of projecting neurons was assisted by retrograde tracing with sadg - egfp(enva ) rabies virus injections into the striatum of pv mice expressing cre - dependent tcb in motor cortex neurons .", "indeed , all tcb / rbv - egfp non - pyramidal neurons in the motor cortex exhibited a fast spiking firing pattern with faster action potentials than som projecting neurons ( figures 3f3h ; figure s3i ; table s2 ) . to investigate the behavioral effect of activating the newly discovered motor cortex som and pv projecting neurons ( striatal targeting is summarized in figure 4a ) , we implanted optic fibers bilaterally into the dorsal striatum of pv and som mice that were injected with aav dio chr2-mcherry into the motor cortex ( figures 4b4d ) .", "we compared the performance of four groups of animals : pv mice injected in m1 ( pv - m1 ) , som mice injected in m1 ( som - m1 ) , som mice injected in m2 ( som - m2 ) , and control mice ( pv and som mice injected in m1 or m2 with aav dio eyfp and wild - type mice injected in m1 or m2 with aav tomato ) .", "we first asked whether activation of motor cortex pv and som neuron projections in the striatum modulated spontaneous locomotion .", "mice were allowed to explore a circular arena , and locomotion was measured before and during light stimulation of corticostriatal projections ( 5-ms pulses at 20 hz ; power , 3 mw ) .", "we next calculated the difference between motion ( defined as the distance moved in 5 s ) during and before light stimulation on four epochs of different durations ; i.e. , 10 , 30 , 60 , and 120 s , starting at light stimulation onset .", "the performance of control mice was similar before and during light stimulation ( figures 4e4 g ) ( the slight increase observed during the 10-s epoch was not significantly different from zero ; one - sample t test ( null hypothesis [ h0 ] = 0 ) , t(12 ) = 2.1 , p = 0.06 ) .", "conversely , the performance of som - m1 , pv - m1 , and som - m2 changed upon photostimulation ( figures 4e and 4f ) . during the 10- , 30- , and 60-s epochs", ", som - m1 mice showed a significant motion increase , whereas pv - m1 and som - m2 mice showed a significant motion decrease with respect to control mice ( figures 4e and 4f ; one - way anova followed by post hoc comparisons , 10 s : f(3 , 31 ) = 7.71 , p = 0.0005 , control versus pv - m1 : p = 0.03 , control versus som - m1 : p = 0.02 , control versus som - m2 : p = 0.04 ; 30 s : f(3 , 31 ) = 10.48 , p = 0.0001 , control versus pv - m1 : p = 0.04 , control versus som - m1 : p = 0.002 , control versus som - m2 : p = 0.05 ; 60 s : f(3 , 31 ) = 12.52 , p = 0.0001 , control versus pv - m1 : p = 0.002 , control versus som - m1 : p = 0.01 , control versus som - m2 : p = 0.008 ) .", "for the 120-s epoch , only pv - m1 mice still showed a motion decrease with respect to control mice , whereas som - m1 and som - m2 performance could not be distinguished from that of control mice ( figures 4e and 4f ; one - way anova followed by post hoc comparisons , f(3 , 31 ) = 3.96 , p = 0.01 , control versus pv - m1 : p = 0.01 , control versus som - m1 : p = 0.42 , control versus som - m2 : p = 0.13).figure 4motor cortex som and pv projecting neurons mediate locomotion change(a ) schematic drawing summarizing the newly identified corticostriatal gabaergic projections .", "m1 som neurons innervate dspns , ispns , and cholinergic interneurons ( blue ) ; m2 som neurons preferentially innervate dspns and ispns ( green ) ; m1 pv neurons preferentially innervate dspns ( pink ) ; m2 pv neuron projections are scarce ( purple ) .", "d1 , dspns ; d2 , ispns ; in , gabaergic interneuron.(b ) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice ( top ) and of optic fiber implantation in the striatum ( bottom).(c and d ) bright - field images of dab - stained sections showing m1 and m2 injection sites ( c ) , optic fiber position , and mcherry - labeled axons in the striatum ( d).(e ) exemplary locomotion traces ( in cm/40 ms ) of control , som - m1 , pv - m1 , and som - m2 mice before ( black ) and during light stimulation ( blue ) ( 5-ms pulses delivered at 20 hz ; light power , 3 mw).(f ) mean sem difference between motion levels ( in cm/5 s ) during and before light stimulation for 10- , 30- , 60- , and 120-s epochs , starting at light stimulation onset.(g ", "j ) mean sem cumulative relative frequency of running speed ( top ) , and mean sem number , speed ( in cm / s ) , and duration ( in seconds ) of mobility and immobility bouts 60 s before ( black lines and white bars ) and during 60-s light stimulation ( blue lines and bars ) for control mice ( g ) and som - m1 ( h ) , pv - m1 ( i ) , and som - m2 ( j ) mice .", "control , n = 13 mice ; som - m1 , n = 7 mice ; pv - m1 , n = 9 mice ; som - m2 , n = 6 mice .", "motor cortex som and pv projecting neurons mediate locomotion change ( a ) schematic drawing summarizing the newly identified corticostriatal gabaergic projections .", "m1 som neurons innervate dspns , ispns , and cholinergic interneurons ( blue ) ; m2 som neurons preferentially innervate dspns and ispns ( green ) ; m1 pv neurons preferentially innervate dspns ( pink ) ; m2 pv neuron projections are scarce ( purple ) .", "( b ) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice ( top ) and of optic fiber implantation in the striatum ( bottom ) .", "( c and d ) bright - field images of dab - stained sections showing m1 and m2 injection sites ( c ) , optic fiber position , and mcherry - labeled axons in the striatum ( d ) .", "( e ) exemplary locomotion traces ( in cm/40 ms ) of control , som - m1 , pv - m1 , and som - m2 mice before ( black ) and during light stimulation ( blue ) ( 5-ms pulses delivered at 20 hz ; light power , 3 mw ) .", "( f ) mean sem difference between motion levels ( in cm/5 s ) during and before light stimulation for 10- , 30- , 60- , and 120-s epochs , starting at light stimulation onset .", "( g j ) mean sem cumulative relative frequency of running speed ( top ) , and mean sem number , speed ( in cm / s ) , and duration ( in seconds ) of mobility and immobility bouts 60 s before ( black lines and white bars ) and during 60-s light stimulation ( blue lines and bars ) for control mice ( g ) and som - m1 ( h ) , pv - m1 ( i ) , and som - m2 ( j ) mice .", "control , n = 13 mice ; som - m1 , n = 7 mice ; pv - m1 , n = 9 mice ; som - m2 , n = 6 mice .", "we characterized the photostimulation - induced motion changes occurring during the 60-s epoch in more detail .", "we first analyzed the cumulative frequency distribution of running speed 60 s before and for 60 s during photostimulation ( figures 4g4j ) .", "as expected , in control mice , no difference was found between the cumulative distribution curves before and during photostimulation ( figure 4 g , top ; wilcoxon matched - pairs signed - rank test , w = 193 , p = 0.15 ) . in som - m1 mice , the curve during photostimulation was shifted to the right , and , accordingly , the median running speed increased significantly upon photostimulation ( figure 4h , top ; wilcoxon matched - pairs signed - rank test , w = 215 , p = 0.01 ) . in pv - m1 and som - m2 mice , the cumulative distribution curve during photostimulation was shifted to the left , and the median running speed was significantly lower during than before photostimulation ( figures 4i and 4j , top ; wilcoxon matched - pairs signed - rank test , pv - m1 : w = 113 , p = 0.005 ; som - m2 : w = 90 , p = 0.02 ) .", "finally , based on running speed , we defined mobility and immobility bouts ( experimental procedures ) and measured their number , speed , and duration before and during photostimulation . in control mice , the properties of mobility and immobility bouts did not change upon photostimulation ( figure 4 g ; paired t test , number of bouts ( nr ) mobility : t(12 ) = 0.76 , p = 0.46 ; speed mobility : t(12 ) = 1.33 , p = 0.21 ; nr immobility : t(12 ) = 0.51 , p = 0.62 ; speed immobility : t(12 ) = 0.64 , p = 0.53 ; wilcoxon matched - pairs signed - rank test : duration mobility : w = 19 , p = 0.54 ; duration immobility : w = 6 , p = 0.83 ) . in som - m1 mice , photostimulation elicited significant changes in mobility bouts : the number decreased , whereas the speed and duration increased ( figure 4h ; paired t test : nr", ": t(6 ) = 3.17 , p = 0.02 ; speed : t(6 ) = 2.97 , p = 0.02 ; wilcoxon matched - pairs signed - rank test , duration : w = 21 , p = 0.03 ) .", "immobility bouts remained unaffected ( figure 4h ; paired t test : nr : t(6 ) = 1.46 , p = 0.19 ; speed : t(6 ) = 0.97 , p = 0.37 ; wilcoxon matched - pairs signed - rank test , duration : w = 9 , p = 0.44 ) . in pv - m1 mice ,", "the duration of mobility bouts decreased significantly , and the duration of immobility bouts increased significantly upon photostimulation ( figure 4i ; wilcoxon matched - pairs signed - rank test , duration mobility : w = 35 , p = 0.04 , duration immobility : w = 39 , p = 0.02 ) , whereas their number and speed remained unchanged ( figure 4i ; paired t test : nr mobility : t(8 ) = 0.97 , p = 0.36 ; speed mobility : t(8 ) = 1.57 , p = 0.14 ; nr immobility : t(8 ) = 1.62 , p = 0.19 ; speed immobility : t(6 ) = 0.78 , p = 0.46 ) . in som - m2 mice , the speed of mobility bouts was significantly reduced upon photostimulation ( figure 4j ; paired t test : t(5 ) = 3.48 , p = 0.02 ) , whereas all other variables remained unchanged ( paired t test : nr mobility : t(5 ) = 0.14 , p = 0.89 ; nr immobility : t(5 ) = 0.03 , p = 0.97 ; speed immobility : t(5 ) = 0.26 , p = 0.8 ; wilcoxon matched - pairs signed - rank test , duration mobility : w = 1 , p = 0.99 ; duration immobility : w = 11 , p = 0.31 ) . in sum , we conclude that stimulation of striatal long - range projections of m1 som neurons increased locomotion by increasing the duration and speed of mobility bouts , that stimulation of striatal long - range projections of m1 pv neurons reduced locomotion by increasing the duration of immobility bouts , and that stimulation of striatal long - range projections of m2 som neurons reduced locomotion by decreasing the speed of mobility bouts . it has been proposed that movement control and reinforcement coding are mediated by common corticostriatal circuits ( kravitz and kreitzer , 2012 ) .", "thus , we next asked whether stimulation of motor cortex pv and som neuron projections in the striatum also affect reinforcement / punishment coding .", "we tested the mice in a place preference task ( figure s4 ) .", "the task lasted 3 days , during which we recorded the time mice spent in each compartment . during the first and second days ( habituation and baseline , respectively ) , place preference was measured without photostimulation . during the third day ( test ) ,", "one of the compartments ( stimulation side ) was paired with photostimulation ( 5-ms pulses at 20 hz ; power , 3 mw ) , and place preference was measured .", "we calculated a difference score as the percentage of time spent on the stimulation side during baseline minus the percentage of time spent on the same side during the test .", "we found that the difference score obtained for pv - m1 , som - m1 , and som - m2 mice was similar to that of control mice ( figure s4b ; one - way anova : f(3,21 ) = 0.67 , p = 0.58 ) .", "hence , stimulation of striatal long - range projections of motor cortex pv and som neurons did not elicit place preference by employing this paradigm .", "here we show that distinct long - range gabaergic neurons connect m1 and m2 with the dorsal striatum .", "the newly identified long - range gabaergic neurons express either som or pv and differ with respect to target cell preference and the modulatory effect on motor activity .", "our results indicate that both m1 and m2 harbor long - range gabaergic neurons that target the dorsal striatum and thus extend recent findings by rock et al .", "furthermore , we report the following new findings . first , m1 and m2 contribute differentially to gabaergic corticostriatal projections .", "second , we identified and characterized an additional projection formed by pv neurons that differs significantly from that formed by som neurons .", "multiple reasons can explain why these connections have not been noticed until recently ( rock et al . , 2016 ) .", "first , the scarcity of long - range gabaergic neurons constitutes a challenge as to their detection by anterograde or retrograde labeling , considering the high number of excitatory neurons that are also labeled in the same area with their axons extending along a similar path .", "second , most studies focused on more dorso - anterior areas of the striatum . hence , retrograde tracer injections are unlikely to reveal gabaergic projecting neurons in motor cortices because their axons target preferentially more lateral , posterior , and ventral parts of the dorsal striatum", ". however , jinno and kosaka ( 2004 ) did not detect motor cortical long - range gabaergic neurons even though injections included target areas that were innervated in our study .", "a possible reason may be low uptake efficiency and transport of the tracer fluorogold in gabaergic neurons .", "first , there was robust axon labeling in the striatum even with regionally restricted minimal anterograde injections .", "second , long - range gabaergic neurons were retrogradely labeled with ctb from the striatum .", "third , long - range gabaergic neurons were transsynaptically retrogradely labeled with rabies virus that infected only striatal starter cells .", "finally , the electrophysiological and pharmacological results provide strong evidence for the gabaergic nature of pv and som projecting neurons .", "although we have no indication for any glutamatergic inputs deriving from som cells , based on our immunocytochemistry , pharmacology , and rabies virus tracing , we can not exclude that glutamatergic transmission has a minor contribution to the behavioral effects seen upon stimulation of striatal long - range projections from m1 pv neurons .", "it is important to note that the number of long - range gabaergic neurons presented in this study remains an underestimation because quantitative evaluations are currently hampered by a number of technical constraints .", "first , experiments entail conservative / limited virus injection to prevent viral spread beyond the target area .", "second , labeling by retrograde tracing is strongly dependent on the tracer and cell type ; e.g. , pv neurons could be detected following transsynaptic virus - mediated tracing but not by ctb labeling . for quantitative studies", ", it would be highly desirable to identify markers / promoters for long - range gabaergic neurons .", "characterization of motor cortex gabaergic projecting neurons revealed that m1 and m2 som and pv cells differentially innervate striatal neurons .", "moreover , bilateral stimulation of corticostriatal long - range gabaergic projections modulates motor activity in spite of the scarcity of gabaergic corticostriatal neurons and the relatively small amplitude responses of targeted striatal neurons .", "thus , stimulation of m1 som neuron projections , targeting dspns , ispns , and cholinergic cells , leads to an increase in locomotion .", "in contrast , stimulation of m2 som neuron projections , targeting preferentially dspns and ispns , and of m1 pv neuron projections , targeting preferentially dspns , leads to a decrease in locomotion .", "decreased locomotion upon preferential inhibition of dspns ( pv - m1 ) is in line with previous studies showing either bradykinesia upon deletion of dspns ( drago et al . , 1998 ) or increased locomotion upon stimulation of dspns ( kravitz et al .", "notably , there was a similar effect on locomotion upon preferential inhibition of dspns ( pv - m1 ) or of both dspns and ispns ( som - m2 ) .", "comparable effects were also reported when optogenetically silencing either dspn or both dspns and ispns ( tecuapetla et al . , 2014 ) .", "increased locomotion upon stimulation of m1 som neuron projections most likely reflects the participation of a larger fraction of cholinergic cells .", "these striatal interneurons , constituting 1%3% of all striatal neurons , are tonically active and provide powerful feedforward inhibition to spns ( english et al . , 2011 , nelson et al . ,", "2009 ) , and enhance dopamine release ( threlfell et al . , 2012 ) . direct activation or inhibition of cholinergic striatal interneurons in the dorsal anterior striatum", "had no effect on locomotor activity ( maurice et al . , 2015 ) .", "however , based on our results , it is tempting to speculate that cholinergic cells in more ventral and posterior striatal areas receiving input from m1 som neurons are involved in motor control . at present", ", we can not resolve whether the observed change in locomotor activity results from long - range axon activation in the striatum only or whether activation of putative collaterals via back - propagating action potentials also plays a role . in either case ,", "our results show that the activity of som and pv projecting neurons in the motor cortex differentially modulates locomotor activity .", "this study is also relevant when interpreting data regarding silencing of cortical areas by manipulating gabaergic neurons because the effects may also involve long - distance targets .", "it has been proposed that motor control and reward coding are mediated by common corticostriatal circuits ( kravitz and kreitzer , 2012 ) .", "our data indicate that , although activation of motor cortex pv and som neuron projections in the dorsal striatum affected locomotor activity , it did not affect place preference , although we can not exclude their implication in reward coding more generally . on the other hand , stimulation of gabaergic projections from the prefrontal cortex to the ventral striatum induces avoidance behavior , suggesting that they are involved in the coding of punishment ( lee et al . , 2014 ) .", "further experiments will be required to elucidate whether , and , if so , which , corticostriatal gabaergic projections mediate both locomotion and reward coding .", "this study adds to the increasing evidence that long - range gabaergic neurons are more frequent than previously thought .", "the heterogeneity of long - range gabaergic neurons described here is in line with previous studies indicating neurochemical diversity of long - range gabaergic neurons in the cortex and hippocampus ( jinno et al .", ", 2007 , higo et al . , 2007 , lee et al . , 2014 ,", "notably , we demonstrate that distinct long - range gabaergic neurons exhibit specific functional properties and differential connectivity . finally , it will be of interest to study long - range gabaergic neurons in the context of movement disorders that are thought to be caused by an imbalance of dspn and ispn activity . thus , parkinsonian - like movements can be reproduced by increased ispn activity ( kravitz et al .", ", 2010 ) and can be reduced by selective inhibition of striatal cholinergic interneurons ( maurice et al . ,", "huntington s disease is marked by an early degeneration of ispns ( vonsattel et al .", ", 1985 , mitchell et al . , 1999 ) and an imbalance of excitation and inhibition of dspns and ispns ( andr et al . , 2011 ) . in light of our findings , it is tempting to speculate that motor cortex gabaergic projections to the striatum might be a potential target for restoring the balance of striatal output .", "all experiments were performed in 8- to 20-week - old male mice and were approved by the regierungsprsidium karlsruhe in compliance with the european guidelines for the care and use of laboratory animals ( licenses g74/13 and g248/14 ) . for anterograde tracing experiments , in vitro patch - clamp recordings , and behavioral experiments , aav dio chr2-mcherry was injected into the primary and/or secondary motor cortex of som ( melzer et al . , 2012 ) , pv ( hippenmeyer et al . , 2005 ) , pv / gad67-egfp ( tamamaki et al . , 2003 ) , som / drd1a - egfp , pv / drd1a - egfp , som / drd2-egfp ( gong et al . , 2003 ) , and pv / drd2-egfp mice with a c57bl/6 background . for retrograde tracing experiments , ctb", "647 was injected into the dorsal striatum of wild - type mice . for retrograde transsynaptic rabies virus tracing , aav - cag - flex - tcb , aav - cag - flex - rg , and sadg - egfp(enva )", "were injected into the dorsal striatum of a2a - cre mice . for monosynaptic retrograde rabies virus tracing , aav - cag - flex - tcb", "was injected into the motor cortex of som ( sst , jackson laboratory ) and pv mice , followed by sadg - egfp(enva ) injection into the dorsal striatum . in all cases ,", "anesthesia was induced and maintained with isoflurane ( 1%2.5% ) , and the virus was delivered through a small craniotomy at the appropriate coordinates by a glass micropipette . for behavioral experiments ,", "immunofluorescence and dab staining were performed on sagittal and coronal brain sections using standard protocols .", "fresh - frozen 20-m sections were stained with fish using the rnascope fluorescent multiplex kit ( advanced cell diagnostics ) .", "mice were deeply anesthetized with isoflurane , transcardially perfused with 30 ml ice - cold sucrose solution , and 300-m - thick brain sections were cut .", "chr2-expressing long - range axonal fibers were stimulated with 5-ms photostimulation ( 473 nm , 120 mw / mm laser intensity ) .", "pscs were measured at 0-mv holding potential ( using cs - based intracellular solution ) or at 70-mv holding potential ( using k - based , high cl intracellular solution ) . for firing pattern analysis , incrementally increasing currents of 1-s duration were injected in current clamp mode starting at 50 or 200 pa . series resistances of 37 megohm were accepted for analysis of pscs . stimulus delivery and data acquisition were performed using pulse software .", "mice were video - tracked at 25 frames / s , and their movements were subsequently analyzed using a position tracking system ( ethovision xt9 , noldus ) .", "the implanted optic fiber cannulas were connected to two optic fibers attached to a rotary joint ( doric lenses ) .", "a patch cord connected the optic fibers to a diode - pumped , solid - state , 473-nm laser ( crystalaser ) .", "we used a pulse generator ( master 8) and a transistor - transistor logic ( ttl ) control box ( universal serial bus input / output [ usb - io ] box , noldus ) to automatically control the photostimulation ( 5-ms pulses delivered at 20 hz ; laser power , 3 mw ) .", "evaluation of locomotor activity was performed in a circular arena ( 40 40 cm ) placed in a dimly lit room where mice were allowed to run freely for 21 min .", "it lasted 2 min and was repeated three times with an inter - stimulation period of 4 min .", "brown - forsythe and f tests were used to test the homogeneity of variances . for non - pairwise comparisons , unpaired t tests ( either for equal or unequal variance ) or mann - whitney u tests were used . for pairwise comparisons , paired t tests or wilcoxon matched - pairs signed - rank tests were used .", "one - way anova followed by tukey s multiple comparisons tests was used to compare motion differences .", "performed in vitro electrophysiology , analysis , immunohistochemistry , reconstructions , and retrograde tracing studies ." ]

[ "<S> summarythe motor cortico - basal ganglion loop is critical for motor planning , execution , and learning . </S>", "<S> balanced excitation and inhibition in this loop is crucial for proper motor output . </S>", "<S> excitatory neurons have been thought to be the only source of motor cortical input to the striatum . here , we identify long - range projecting gabaergic neurons in the primary ( m1 ) and secondary ( m2 ) motor cortex that target the dorsal striatum . </S>", "<S> this population of projecting gabaergic neurons comprises both somatostatin - positive ( som+ ) and parvalbumin - positive ( pv+ ) neurons that target direct and indirect pathway striatal output neurons as well as cholinergic interneurons differentially . </S>", "<S> notably , optogenetic stimulation of m1 pv+ and m2 som+ projecting neurons reduced locomotion , whereas stimulation of m1 som+ projecting neurons enhanced locomotion . </S>", "<S> thus , corticostriatal gabaergic projections modulate striatal output and motor activity . </S>" ]

[ "Introduction", "Results", "Discussion", "Experimental Procedures", "Author Contributions" ]

[ [ "it integrates signals from cortical areas and subserves important functions like motor control ( tecuapetla et al . , 2014 , kravitz et al . , 2010 ) and reinforcement / punishment coding ( kravitz et al . , 2012 ) .", "striatal neurons comprise gabaergic spiny projection neurons ( spns , 95% ) and interneurons ( 5% ) .", "spns are classified into direct pathway spns ( dspns ) , which project to the substantia nigra reticulata and external and internal segments of the globus pallidus ( gpe and gpi , respectively ) , and indirect pathway spns ( ispns ) , which project to gpe ( bolam et al . , 2000 , wu et al . , 2000", "striatal interneurons include large aspiny cholinergic neurons and different populations of gabaergic interneurons ( kawaguchi et al . , 1995 ) .", "excitatory glutamatergic neurons from virtually all cortical areas send projections to the striatum ( mcgeorge and faull , 1989 ) , and several studies suggest their recruitment during action selection ( xiong et al . , 2015 , znamenskiy and zador , 2013 , koralek et al . , 2012 ) .", "in contrast , cortical gabaergic neurons projecting to the striatum were not considered to be a component of the canonical corticostriatal network because they were identified only in the prefrontal , somatosensory , and retrosplenial cortices ( lee et al . , 2014 ,", "jinno and kosaka , 2004 ) . only recently were direct gabaergic neurons projecting to the striatum also described in the motor and auditory cortices ( rock et al . , 2016 ) .", "the authors identified the long - range projecting neurons as somatostatin - positive ( som ) and , furthermore , reported that inhibition conveyed by these neurons was onto both dspns and ispns .", "we previously showed that long - range gabaergic neurons connecting several brain structures comprise different molecular subtypes .", "for instance , connectivity between the hippocampus and medial entorhinal cortex is supported by parvalbumin - positive ( pv ) and som neurons ( melzer et al . , 2012 ) .", "moreover , projections from the septum to the medial entorhinal cortex are pv and calbindin , and they inhibit specific interneurons differentially ( fuchs et al . , 2016 ) .", "hence , we wondered whether long - range gabaergic projecting neurons from the motor cortex to the striatum are diverse with respect to their molecular identity , target specificity , and function at the behavioral level .", "based on virus - mediated tracing , optogenetics , patch - clamp recordings in vitro , and behavioral essays , we identified two distinct populations of long - range projecting gabaergic neurons in the primary ( m1 ) and secondary ( m2 ) motor cortex targeting the dorsal striatum and established that these two populations exhibit target cell preference in the striatum and affect locomotion differentially ." ], [ "som cells are a major source of intracortical and corticofugal long - range gabaergic projections ( tomioka et al . , 2005 , rock et al . , 2016 ) .", "to substantiate and extend these studies focusing on long - range gabaergic neurons connecting the motor cortex and the striatum , we injected adeno - associated virus ( aav ) double - floxed inverse open reading frame ( dio ) chr2-mcherry into the m1 and m2 area of som mice .", "this resulted in labeling of a subpopulation of gabaergic neurons ( figures 1a and 1b ; figure s1a ) and revealed projections in several ipsilateral cortical and subcortical areas and , to a lesser extent , in contralateral cortices ( table s1 ) .", "there was consistent innervation of the ipsilateral dorsal striatum ( figure 1b ; table s1 ) .", "motor cortex som neuron projections traversed the dorsal striatum and branched preferentially ventro - laterally , sparing the most rostral and caudal part of the dorsal striatum ( figure 1b).figure 1motor cortex som gabaergic neurons innervate the striatum(a ) schematic drawing of the injection site and the location of long - range projections in the striatum shown in ( b ) .", "viral constructs encoding chr2-mcherry were injected into the motor cortex of som mice.(b ) bright - field images of dab - stained sections showing the injection site in the motor cortex ( left ) and mcherry - labeled axons in the striatum ( center ) following aav dio chr2-mcherry injection into the motor cortex of som mice . a higher magnification of the boxed area is shown on the right.(c and d ) confocal images showing a retrogradely labeled area ( c ) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex , visualized via fish for sst and gad1/2 ( d).(e h ) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of som mice , and rabies virus was injected into the striatum .", "( e ) shows differential interference contrast ( dic ) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in ( f ) .", "( g ) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in ( h).str , striatum .", "motor cortex som gabaergic neurons innervate the striatum ( a ) schematic drawing of the injection site and the location of long - range projections in the striatum shown in ( b ) .", "( b ) bright - field images of dab - stained sections showing the injection site in the motor cortex ( left ) and mcherry - labeled axons in the striatum ( center ) following aav dio chr2-mcherry injection into the motor cortex of som mice .", "( c and d ) confocal images showing a retrogradely labeled area ( c ) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex , visualized via fish for sst and gad1/2 ( d ) . ( e ", "h ) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of som mice , and rabies virus was injected into the striatum .", "( e ) shows differential interference contrast ( dic ) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in ( f ) .", "( g ) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in ( h ) .", "see also figure s1 and tables s1 and s2 . to further substantiate the presence of gabaergic corticostriatal projections , we performed retrograde labeling .", "we injected cholera toxin b ( ctb ) subunit 647 into the ventro - lateral part of the dorsal striatum and analyzed retrogradely labeled cells in the m1 region ( figures s1b and s1c ) .", "as expected , a dense band of retrogradely labeled cells became visible in cortical l5 ( figure 1c ) ; i.e. , in the layer that is the major source of corticostriatal excitatory projections ( wilson , 1987 , cowan and wilson , 1994 ) . to visualize gabaergic cells among the m1 retrogradely labeled cells , we performed multi - fluorescence in situ hybridization ( fish ) for sst ( encoding som ) and gad1/2 ( encoding gad67/65 ) .", "we found 13 retrogradely labeled cells in m1 that were clearly positive for gad1/2 ( n = 3,582 ctb cells and 5,064 gad1/2 cells , cell counts across all layers in 35 slices from 4 hemispheres in 4 mice ) , 8 of which co - labeled for sst ( figure 1d ) .", "to confirm a direct long - range gabaergic connection between the motor cortex ( m1/m2 ) and the dorsal striatum , we performed retrograde monosynaptic tracing with rabies virus ( wickersham et al . , 2007 ) .", "we injected aavs encoding cre - dependent avian virus receptor ( avian tumor virus receptor a mcherry [ tcb ] ; weissbourd et al . , 2014 ) and", "rabies glycoprotein ( rg ) into the striatum of a2a - cre mice that express cre recombinase specifically in ispns ( gong et al . , 2003 ) .", "subsequent injection of rg - deleted envelope protein from avian aslv type a ( enva)-pseudotyped rabies virus ( sadg - egfp(enva ) ) into the striatum resulted in transsynaptically retrogradely labeled cells in the cortex ( figures s1e and s1f ) .", "fish for rabies virus - specific mrna ( rabv - gp1 ) and gad1/2 revealed double - positive neurons in the motor cortex ( 7 cells in 34 slices from 4 hemispheres in 4 mice ; figure s1", "the average number of labeled cells per slice was lower than after ctb647 injections , suggesting that ispns were not the only striatal target cells of gabaergic projecting neurons and/or reflecting lower efficiency of transsynaptic tracing ( marshel et al . ,", ", we expressed tcb cre - dependently in the motor cortex ( m1/m2 ) of som mice and injected sadg - egfp(enva ) rabies virus into the striatum .", "tcb retrogradely labeled cells in the motor cortex had a classical or burst accommodating firing pattern ( n = 11 cells from 5 hemispheres in 4 mice ; figures 1e and 1f ; figure s1h ) similar to non - retrogradely labeled tcb cells ( table s2 ) .", "reconstructed cells had a martinotti cell - like morphology ( wang et al . , 2004 ) with axonal projections extending over all cortical layers ( three reconstructions from three hemispheres in two mice ; figures 1 g and 1h ; figures s1i ", "we next tested whether som projecting neurons form functional synapses onto striatal neurons and whether the connectivity exhibits target specificity .", "we injected aav dio chr2-mcherry into m1/m2 of som mice and combined optogenetic stimulation of long - range projections with patch - clamp recordings of putative postsynaptic cells in the striatum ( figure 2a ) .", "all injections ( n = 36 hemispheres ) resulted in labeled axons that projected to the dorsal striatum .", "of 305 patched neurons ( in 27 mice ) , 50 responded with short - latency postsynaptic currents ( pscs ) to 5-ms photostimulation of motor cortex som neuron projections ( figure 2b ) .", "as a specificity control , we repeated the experiment in wild - type mice injected with aav dio chr2-mcherry and found neither mcherry fibers in the dorsal striatum nor a response after photostimulation ( n = 58 cells in 2 mice ) .", "responses in som mice could not be blocked with 6-cyano-2,3-dihydroxy-7-nitro - quinoxaline ( cnqx ) and d-2-amino-5-phosphonovaleric acid ( d - ap5 ) ( 165.6 32.7 pa baseline versus 169.5 31.1 pa with drugs [ mean sem ] , paired t test , t(14 ) = 0.49 , p = 1 , n = 15 cells in 11 mice ; figure s2a ) but with gabazine ( 117.8 [ 134.3 ] pa versus 1.6 [ 1.6 ] pa [ median interquartile range ( iqr ) ] , wilcoxon signed - rank test , w = 210 , p = 0.0002 , n = 20 cells in 15 mice ; figures s2a and s2b ) .", "responses reversed around the reversal potential of gabaergic receptors ( n = 14 cells in 9 mice ; figure 2b ) , thus confirming the gabaergic nature of motor cortex som neuron projections.figure 2motor cortex som neuron projections form functional synapses on striatal output and cholinergic neurons(a ) schematic drawing indicating the injection site ( left ) and location of an exemplary patched neuron in the striatum ( right ) .", "aav dio chr2-mcherry was injected into the motor cortex of som mice , and target cells were patched in the striatum ( dic image).(b ) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation ( blue ticks ) of motor cortex som neuron projections .", "responses were blocked with gabazine but not d - ap5/cnqx.(c ) firing pattern ( membrane potential upon 50-pa current injection and at the action potential [ ap ] threshold ) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections.(d ) firing pattern ( spontaneous activity and maximal firing frequency ) and dic image of a representative cholinergic interneuron ( arrow ) that was responsive to photostimulation of motor cortex som neuron projections.(e ) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections ( from top to bottom : maximal firing frequency , ap threshold , and 50-pa current injection).(f ) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections .", "number of mice : spns , 19 ; cholinergic , 11 ; gabaergic interneurons , 19.(g ) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice.(h ) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp ( left ) and som / drd2-egfp ( right ) mice exhibiting differential egfp expression in the globus pallidus ( arrow ) and substantia nigra ( arrowhead).(i ) exemplary traces of dspn and ispn responses to photostimulation ( blue ticks ) of m1 and m2 som neuron projections using cs - based low cl intracellular solution ( from top to bottom : 0 mv , reversal potential , and 95 mv holding potential).(j ) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections .", "number of mice : m1-dspns , 7 ; m1-ispns , 16 ; m2-dspns , 8 ; m2-ispns , 17 ; m1-cholinergic , 19 ; m2-cholinergic , 13.(k ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m2 to dspn ; dark green , m1 to ispn ; light green , m2 to ispn ; purple , m1 to cholinergic interneuron ; red , m2 to cholinergic interneuron.hp , hippocampus ; ach , cholinergic interneuron .", "som neuron projections form functional synapses on striatal output and cholinergic neurons ( a ) schematic drawing indicating the injection site ( left ) and location of an exemplary patched neuron in the striatum ( right ) .", "aav dio chr2-mcherry was injected into the motor cortex of som mice , and target cells were patched in the striatum ( dic image ) .", "( b ) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation ( blue ticks ) of motor cortex som neuron projections .", "( c ) firing pattern ( membrane potential upon 50-pa current injection and at the action potential [ ap ] threshold ) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections .", "( d ) firing pattern ( spontaneous activity and maximal firing frequency ) and dic image of a representative cholinergic interneuron ( arrow ) that was responsive to photostimulation of motor cortex som neuron projections .", "( e ) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections ( from top to bottom : maximal firing frequency , ap threshold , and 50-pa current injection ) .", "( f ) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections .", "number of mice : spns , 19 ; cholinergic , 11 ; gabaergic interneurons , 19 .", "( g ) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice .", "( h ) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp ( left ) and som / drd2-egfp ( right ) mice exhibiting differential egfp expression in the globus pallidus ( arrow ) and substantia nigra ( arrowhead ) .", "( i ) exemplary traces of dspn and ispn responses to photostimulation ( blue ticks ) of m1 and m2 som neuron projections using cs - based low cl intracellular solution ( from top to bottom : 0 mv , reversal potential , and 95 mv holding potential ) .", "( j ) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections .", "number of mice : m1-dspns , 7 ; m1-ispns , 16 ; m2-dspns , 8 ; m2-ispns , 17 ; m1-cholinergic , 19 ; m2-cholinergic , 13 .", "( k ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m2 to dspn ; dark green , m1 to ispn ; light green , m2 to ispn ; purple , m1 to cholinergic interneuron ; red , m2 to cholinergic interneuron .", "hp , hippocampus ; ach , cholinergic interneuron . see also figures s2 and s3 and tables s3 and s4 . to scrutinize target specificity ,", "striatal neurons were sorted into spns and cholinergic and gabaergic interneurons based on their electrophysiological properties and cell soma shape ( planert et al . , 2013 , gertler et al . , 2008 , kawaguchi , 1992 , bennett and wilson , 1999 , kawaguchi et al . , 1995 ; experimental procedures ; figures 2c2e ; table s3 ) .", "we found that 22.6% of spns , 33.3% of cholinergic cells , and only 2% of gabaergic interneurons responded to 5-ms photostimulation of motor cortex som neuron projections ( figure 2f ; figures s2c and s2d ; table s3 ) .", "together , these data indicate that spns and cholinergic cells are the main target of motor cortex som neuron projections . to answer whether dspns and ispns are differentially targeted by motor cortex som projecting neurons , we cross - bred som mice to drd1a - egfp and drd2-egfp mice in which dspns and ispns , respectively , are selectively labeled ( gong et al . , 2003 ;", "we injected aav dio chr2-mcherry into m1 or m2 of som / drd1a - egfp or som / drd2-egfp mice ( figure 2 g ; figure s2e ) and combined photostimulation of m1 or m2 som neuron projections with patch - clamp recordings of striatal neurons ( figure s2f ) .", "we found that m1 som projecting neurons innervated a comparable proportion of dspns ( 29% ) , ispns ( 22% ) , and cholinergic cells ( 40% ) ( figures 2i and 2j ; fisher s exact test , p = 0.19 ) .", "m2 som projecting neurons targeted dspns ( 25% ) and ispns ( 16% ) to a similar extent , whereas cholinergic cells tended to be innervated less frequently ( 4% ) ( figure 2j ; fisher s exact test , p = 0.08 ) .", "inhibition of cholinergic cells by m2 was significantly less frequent than by m1 ( figure 2j ; fisher s exact test , m1 dspns versus m2 dspns : p = 1 ; m1 ispns versus m2 ispns : p = 1 ; m1 cholinergic versus m2 cholinergic : p = 0.006 ) .", "responses had a latency of 2.5 ( 1.3 ) ms ( median [ iqr ] ; n = 47 responding cells ; see figure s3a and table s4 for more details ) and a reversal potential of 58.0 1.4 mv ( mean sem ; n = 22 responding cells ; see figure s3b and table s4 for more details ) .", "the strength of m1 and m2 som neuron connections to spns was similar ( table s4 ) .", "however , when comparing inputs with dspns and ispns both from m1 and m2 , the response amplitudes were significantly larger for ispns ( amplitudes at 0 mv with cesium [ cs ] internal solution : 15.7 pa versus 44.0 [ 36.5 ] pa [ median ( iqr ) ] in dspns and ispns respectively ; mann - whitney u test , u = 102 , p = 0.02 , n = 14 and 26 responding cells , respectively ; figure s3c ) .", "detected target cells were located preferentially in the ventro - lateral part of the dorsal striatum ( figure 2k ; figures s3d ", "targeted dspns and ispns were intermingled and localized 2.4 ( 0.7 ) mm lateral , 0.1 ( 0.7 ) mm posterior to the bregma and 4 ( 0.7 ) mm deep ( median [ iqr ] ) .", "spns targeted by m1 projections were more lateral than spns targeted by m2 projections ( mann - whitney u test , u = 142 , p = 0.04 ; table s4 ) .", "the retrograde labeling experiments suggested that som neurons are not the only m1 gabaergic population projecting to the striatum .", "pv neurons appeared to be attractive candidates because we identified them before as a major class of neurons providing long - range inhibition in the entorhinal cortex - hippocampal formation ( melzer et al . , 2012 ) .", "testing for the presence of gabaergic long - range projecting pv cells based on virus tracing in pv mice may be complicated by the fact that a fraction of cortical pv cells are glutamatergic ( jinno and kosaka , 2004 ) .", "hence , we first tested the gabaergic nature of pv cells in m1/m2 by counting the number of double - positive cells in injected pv / gad67 mice .", "99.5% of pv cells were gad67 ( 759 of 763 pv cells from 9 slices in 3 mice ) .", "this result is in agreement with previous evidence showing the absence of pv glutamatergic neurons in the motor cortex ( jinno and kosaka , 2004 ) . in the mouse neocortex ,", "pv and som neurons constitute two non - overlapping neuronal entities ( xu et al . , 2010 , tasic et al . , 2016 ) .", "thus , we injected aav dio chr2-mcherry into m1 of pv mice , and , indeed , we detected labeled axons in the dorsal striatum that branched preferentially ventro - laterally ( in 12 of 17 injected hemispheres ) .", "in contrast , injections into m2 of pv mice were less likely to result in labeled striatal projections ( 5 of 12 injections ) , and projections when present were sparser than the ones detected after m1 injections .", "thus , for a more detailed analysis of innervation patterns , we focused on projections from m1 .", "we injected aav dio chr2-mcherry into m1 of pv / drd1a - egfp or pv / drd2-egfp mice ( figure 3a ) and found that 31% of dspns , 6% of ispns , and 6% of cholinergic and 0% of gabaergic interneurons responded to photostimulation of m1 pv neuron projections ( figures 3b and 3c ) .", "thus , pv projecting neurons preferentially innervated dspns ( fisher s exact test ; dspns versus ispns : p = 0.009 , dspns versus cholinergic : p = 0.04 , ispns versus cholinergic : p = 1 ; figure 3b ) .", "spns targeted by m1 pv projecting neurons had a response amplitude comparable with that following stimulation of m1 som projections ( figure s3c ; table s4 ) .", "although responses could not be blocked by cnqx / d - ap5 ( although one of five cells showed a clear decrease in amplitude ) , subsequent application of gabazine led to a significant amplitude decrease ( 68.9 [ 147.3 ] pa baseline versus 70.3 [ 86.1 ] pa with cnqx / d - ap5 versus 0.5 [ 3.2 ] pa with gabazine , friedman test , p = 0.02 , post hoc conover s test with bonferroni correction , baseline versus cnqx / d - ap5 : p = 1 , baseline versus gabazine : p = 0.004 , cnqx / d - ap5 versus gabazine : p = 0.009 , n = 5 cells in 4 mice ; figure 3d ; figures s3 g and s3h ) .", "the reversal potential of the responses was 60.3 2.5 mv ( mean sem , n = 4 cells in 4 mice ; figure s3b ; table s4 ) , reconfirming the gabaergic nature .", "spns targeted by m1 pv projecting neurons were located 0.6 ( 0.7 ) mm posterior to the bregma , 3.0 ( 0.6 ) mm lateral , and 3.7 ( 0.8 ) deep ( median [ iqr ] ) ( figure 3e ; figures s3d ", "s3f ; table s4).figure 3m1 pv neuron projections preferentially target dspns in the striatum(a ) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice.(b ) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections .", "number of mice : dspns , 8 ; ispns , 7 ; cholinergic , 13 ; gabaergic interneurons , 9.(c ) firing pattern ( upon 50 pa current injection and at the ap threshold ) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections.(d ) responses of the dspn shown in ( c ) at 70 mv holding potential using high cl intracellular solution .", "responses were blocked with gabazine but not d - ap5/cnqx.(e ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m1 to ispns ; gray , unidentified responding neurons.(f and g ) epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex ( arrow ) ( f ) with the corresponding firing pattern ( g ) identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of pv mice , and rabies virus was injected into the striatum.(h ) dot plot of the action potential half - width for retrogradely labeled som ( n = 11 cells from 5 hemispheres in 4 mice ) and pv neurons ( n = 3 cells from 3 hemispheres in 2 mice).see also figure s3 and tables s2 and s4 .", "m1 pv neuron projections preferentially target dspns in the striatum ( a ) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice .", "( b ) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections .", "number of mice : dspns , 8 ; ispns , 7 ; cholinergic , 13 ; gabaergic interneurons , 9 .", "( c ) firing pattern ( upon 50 pa current injection and at the ap threshold ) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections .", "( d ) responses of the dspn shown in ( c ) at 70 mv holding potential using high cl intracellular solution .", "( e ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section .", "color code : orange , m1 to dspn ; yellow , m1 to ispns ; gray , unidentified responding neurons .", "( f and g ) epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex ( arrow ) ( f ) with the corresponding firing pattern ( g ) identified by retrograde tracing with sadg - egfp(enva ) rabies virus .", "tcb was expressed cre - dependently in the motor cortex of pv mice , and rabies virus was injected into the striatum .", "( h ) dot plot of the action potential half - width for retrogradely labeled som ( n = 11 cells from 5 hemispheres in 4 mice ) and pv neurons ( n = 3 cells from 3 hemispheres in 2 mice ) .", "the different innervation patterns of som and pv projecting neurons suggested that the two cell types represent distinct subpopulations in the motor cortex .", "we hence analyzed whether the firing pattern of pv projecting neurons differed from that of som projecting neurons and resembled that of classical pv interneurons .", "the detection of projecting neurons was assisted by retrograde tracing with sadg - egfp(enva ) rabies virus injections into the striatum of pv mice expressing cre - dependent tcb in motor cortex neurons .", "indeed , all tcb / rbv - egfp non - pyramidal neurons in the motor cortex exhibited a fast spiking firing pattern with faster action potentials than som projecting neurons ( figures 3f3h ; figure s3i ; table s2 ) . to investigate the behavioral effect of activating the newly discovered motor cortex som and pv projecting neurons ( striatal targeting is summarized in figure 4a ) , we implanted optic fibers bilaterally into the dorsal striatum of pv and som mice that were injected with aav dio chr2-mcherry into the motor cortex ( figures 4b4d ) .", "we compared the performance of four groups of animals : pv mice injected in m1 ( pv - m1 ) , som mice injected in m1 ( som - m1 ) , som mice injected in m2 ( som - m2 ) , and control mice ( pv and som mice injected in m1 or m2 with aav dio eyfp and wild - type mice injected in m1 or m2 with aav tomato ) .", "we first asked whether activation of motor cortex pv and som neuron projections in the striatum modulated spontaneous locomotion .", "mice were allowed to explore a circular arena , and locomotion was measured before and during light stimulation of corticostriatal projections ( 5-ms pulses at 20 hz ; power , 3 mw ) .", "we next calculated the difference between motion ( defined as the distance moved in 5 s ) during and before light stimulation on four epochs of different durations ; i.e. , 10 , 30 , 60 , and 120 s , starting at light stimulation onset .", "the performance of control mice was similar before and during light stimulation ( figures 4e4 g ) ( the slight increase observed during the 10-s epoch was not significantly different from zero ; one - sample t test ( null hypothesis [ h0 ] = 0 ) , t(12 ) = 2.1 , p = 0.06 ) .", "conversely , the performance of som - m1 , pv - m1 , and som - m2 changed upon photostimulation ( figures 4e and 4f ) . during the 10- , 30- , and 60-s epochs", ", som - m1 mice showed a significant motion increase , whereas pv - m1 and som - m2 mice showed a significant motion decrease with respect to control mice ( figures 4e and 4f ; one - way anova followed by post hoc comparisons , 10 s : f(3 , 31 ) = 7.71 , p = 0.0005 , control versus pv - m1 : p = 0.03 , control versus som - m1 : p = 0.02 , control versus som - m2 : p = 0.04 ; 30 s : f(3 , 31 ) = 10.48 , p = 0.0001 , control versus pv - m1 : p = 0.04 , control versus som - m1 : p = 0.002 , control versus som - m2 : p = 0.05 ; 60 s : f(3 , 31 ) = 12.52 , p = 0.0001 , control versus pv - m1 : p = 0.002 , control versus som - m1 : p = 0.01 , control versus som - m2 : p = 0.008 ) .", "for the 120-s epoch , only pv - m1 mice still showed a motion decrease with respect to control mice , whereas som - m1 and som - m2 performance could not be distinguished from that of control mice ( figures 4e and 4f ; one - way anova followed by post hoc comparisons , f(3 , 31 ) = 3.96 , p = 0.01 , control versus pv - m1 : p = 0.01 , control versus som - m1 : p = 0.42 , control versus som - m2 : p = 0.13).figure 4motor cortex som and pv projecting neurons mediate locomotion change(a ) schematic drawing summarizing the newly identified corticostriatal gabaergic projections .", "m1 som neurons innervate dspns , ispns , and cholinergic interneurons ( blue ) ; m2 som neurons preferentially innervate dspns and ispns ( green ) ; m1 pv neurons preferentially innervate dspns ( pink ) ; m2 pv neuron projections are scarce ( purple ) .", "d1 , dspns ; d2 , ispns ; in , gabaergic interneuron.(b ) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice ( top ) and of optic fiber implantation in the striatum ( bottom).(c and d ) bright - field images of dab - stained sections showing m1 and m2 injection sites ( c ) , optic fiber position , and mcherry - labeled axons in the striatum ( d).(e ) exemplary locomotion traces ( in cm/40 ms ) of control , som - m1 , pv - m1 , and som - m2 mice before ( black ) and during light stimulation ( blue ) ( 5-ms pulses delivered at 20 hz ; light power , 3 mw).(f ) mean sem difference between motion levels ( in cm/5 s ) during and before light stimulation for 10- , 30- , 60- , and 120-s epochs , starting at light stimulation onset.(g ", "j ) mean sem cumulative relative frequency of running speed ( top ) , and mean sem number , speed ( in cm / s ) , and duration ( in seconds ) of mobility and immobility bouts 60 s before ( black lines and white bars ) and during 60-s light stimulation ( blue lines and bars ) for control mice ( g ) and som - m1 ( h ) , pv - m1 ( i ) , and som - m2 ( j ) mice .", "control , n = 13 mice ; som - m1 , n = 7 mice ; pv - m1 , n = 9 mice ; som - m2 , n = 6 mice .", "motor cortex som and pv projecting neurons mediate locomotion change ( a ) schematic drawing summarizing the newly identified corticostriatal gabaergic projections .", "m1 som neurons innervate dspns , ispns , and cholinergic interneurons ( blue ) ; m2 som neurons preferentially innervate dspns and ispns ( green ) ; m1 pv neurons preferentially innervate dspns ( pink ) ; m2 pv neuron projections are scarce ( purple ) .", "( b ) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice ( top ) and of optic fiber implantation in the striatum ( bottom ) .", "( c and d ) bright - field images of dab - stained sections showing m1 and m2 injection sites ( c ) , optic fiber position , and mcherry - labeled axons in the striatum ( d ) .", "( e ) exemplary locomotion traces ( in cm/40 ms ) of control , som - m1 , pv - m1 , and som - m2 mice before ( black ) and during light stimulation ( blue ) ( 5-ms pulses delivered at 20 hz ; light power , 3 mw ) .", "( f ) mean sem difference between motion levels ( in cm/5 s ) during and before light stimulation for 10- , 30- , 60- , and 120-s epochs , starting at light stimulation onset .", "( g j ) mean sem cumulative relative frequency of running speed ( top ) , and mean sem number , speed ( in cm / s ) , and duration ( in seconds ) of mobility and immobility bouts 60 s before ( black lines and white bars ) and during 60-s light stimulation ( blue lines and bars ) for control mice ( g ) and som - m1 ( h ) , pv - m1 ( i ) , and som - m2 ( j ) mice .", "control , n = 13 mice ; som - m1 , n = 7 mice ; pv - m1 , n = 9 mice ; som - m2 , n = 6 mice .", "we characterized the photostimulation - induced motion changes occurring during the 60-s epoch in more detail .", "we first analyzed the cumulative frequency distribution of running speed 60 s before and for 60 s during photostimulation ( figures 4g4j ) .", "as expected , in control mice , no difference was found between the cumulative distribution curves before and during photostimulation ( figure 4 g , top ; wilcoxon matched - pairs signed - rank test , w = 193 , p = 0.15 ) . in som - m1 mice , the curve during photostimulation was shifted to the right , and , accordingly , the median running speed increased significantly upon photostimulation ( figure 4h , top ; wilcoxon matched - pairs signed - rank test , w = 215 , p = 0.01 ) . in pv - m1 and som - m2 mice , the cumulative distribution curve during photostimulation was shifted to the left , and the median running speed was significantly lower during than before photostimulation ( figures 4i and 4j , top ; wilcoxon matched - pairs signed - rank test , pv - m1 : w = 113 , p = 0.005 ; som - m2 : w = 90 , p = 0.02 ) .", "finally , based on running speed , we defined mobility and immobility bouts ( experimental procedures ) and measured their number , speed , and duration before and during photostimulation . in control mice , the properties of mobility and immobility bouts did not change upon photostimulation ( figure 4 g ; paired t test , number of bouts ( nr ) mobility : t(12 ) = 0.76 , p = 0.46 ; speed mobility : t(12 ) = 1.33 , p = 0.21 ; nr immobility : t(12 ) = 0.51 , p = 0.62 ; speed immobility : t(12 ) = 0.64 , p = 0.53 ; wilcoxon matched - pairs signed - rank test : duration mobility : w = 19 , p = 0.54 ; duration immobility : w = 6 , p = 0.83 ) . in som - m1 mice , photostimulation elicited significant changes in mobility bouts : the number decreased , whereas the speed and duration increased ( figure 4h ; paired t test : nr", ": t(6 ) = 3.17 , p = 0.02 ; speed : t(6 ) = 2.97 , p = 0.02 ; wilcoxon matched - pairs signed - rank test , duration : w = 21 , p = 0.03 ) .", "immobility bouts remained unaffected ( figure 4h ; paired t test : nr : t(6 ) = 1.46 , p = 0.19 ; speed : t(6 ) = 0.97 , p = 0.37 ; wilcoxon matched - pairs signed - rank test , duration : w = 9 , p = 0.44 ) . in pv - m1 mice ,", "the duration of mobility bouts decreased significantly , and the duration of immobility bouts increased significantly upon photostimulation ( figure 4i ; wilcoxon matched - pairs signed - rank test , duration mobility : w = 35 , p = 0.04 , duration immobility : w = 39 , p = 0.02 ) , whereas their number and speed remained unchanged ( figure 4i ; paired t test : nr mobility : t(8 ) = 0.97 , p = 0.36 ; speed mobility : t(8 ) = 1.57 , p = 0.14 ; nr immobility : t(8 ) = 1.62 , p = 0.19 ; speed immobility : t(6 ) = 0.78 , p = 0.46 ) . in som - m2 mice , the speed of mobility bouts was significantly reduced upon photostimulation ( figure 4j ; paired t test : t(5 ) = 3.48 , p = 0.02 ) , whereas all other variables remained unchanged ( paired t test : nr mobility : t(5 ) = 0.14 , p = 0.89 ; nr immobility : t(5 ) = 0.03 , p = 0.97 ; speed immobility : t(5 ) = 0.26 , p = 0.8 ; wilcoxon matched - pairs signed - rank test , duration mobility : w = 1 , p = 0.99 ; duration immobility : w = 11 , p = 0.31 ) . in sum , we conclude that stimulation of striatal long - range projections of m1 som neurons increased locomotion by increasing the duration and speed of mobility bouts , that stimulation of striatal long - range projections of m1 pv neurons reduced locomotion by increasing the duration of immobility bouts , and that stimulation of striatal long - range projections of m2 som neurons reduced locomotion by decreasing the speed of mobility bouts . it has been proposed that movement control and reinforcement coding are mediated by common corticostriatal circuits ( kravitz and kreitzer , 2012 ) .", "thus , we next asked whether stimulation of motor cortex pv and som neuron projections in the striatum also affect reinforcement / punishment coding .", "we tested the mice in a place preference task ( figure s4 ) .", "the task lasted 3 days , during which we recorded the time mice spent in each compartment . during the first and second days ( habituation and baseline , respectively ) , place preference was measured without photostimulation . during the third day ( test ) ,", "one of the compartments ( stimulation side ) was paired with photostimulation ( 5-ms pulses at 20 hz ; power , 3 mw ) , and place preference was measured .", "we calculated a difference score as the percentage of time spent on the stimulation side during baseline minus the percentage of time spent on the same side during the test .", "we found that the difference score obtained for pv - m1 , som - m1 , and som - m2 mice was similar to that of control mice ( figure s4b ; one - way anova : f(3,21 ) = 0.67 , p = 0.58 ) .", "hence , stimulation of striatal long - range projections of motor cortex pv and som neurons did not elicit place preference by employing this paradigm ." ], [ "here we show that distinct long - range gabaergic neurons connect m1 and m2 with the dorsal striatum .", "the newly identified long - range gabaergic neurons express either som or pv and differ with respect to target cell preference and the modulatory effect on motor activity .", "our results indicate that both m1 and m2 harbor long - range gabaergic neurons that target the dorsal striatum and thus extend recent findings by rock et al .", "furthermore , we report the following new findings . first , m1 and m2 contribute differentially to gabaergic corticostriatal projections .", "second , we identified and characterized an additional projection formed by pv neurons that differs significantly from that formed by som neurons .", "multiple reasons can explain why these connections have not been noticed until recently ( rock et al . , 2016 ) .", "first , the scarcity of long - range gabaergic neurons constitutes a challenge as to their detection by anterograde or retrograde labeling , considering the high number of excitatory neurons that are also labeled in the same area with their axons extending along a similar path .", "second , most studies focused on more dorso - anterior areas of the striatum . hence , retrograde tracer injections are unlikely to reveal gabaergic projecting neurons in motor cortices because their axons target preferentially more lateral , posterior , and ventral parts of the dorsal striatum", ". however , jinno and kosaka ( 2004 ) did not detect motor cortical long - range gabaergic neurons even though injections included target areas that were innervated in our study .", "a possible reason may be low uptake efficiency and transport of the tracer fluorogold in gabaergic neurons .", "first , there was robust axon labeling in the striatum even with regionally restricted minimal anterograde injections .", "second , long - range gabaergic neurons were retrogradely labeled with ctb from the striatum .", "third , long - range gabaergic neurons were transsynaptically retrogradely labeled with rabies virus that infected only striatal starter cells .", "finally , the electrophysiological and pharmacological results provide strong evidence for the gabaergic nature of pv and som projecting neurons .", "although we have no indication for any glutamatergic inputs deriving from som cells , based on our immunocytochemistry , pharmacology , and rabies virus tracing , we can not exclude that glutamatergic transmission has a minor contribution to the behavioral effects seen upon stimulation of striatal long - range projections from m1 pv neurons .", "it is important to note that the number of long - range gabaergic neurons presented in this study remains an underestimation because quantitative evaluations are currently hampered by a number of technical constraints .", "first , experiments entail conservative / limited virus injection to prevent viral spread beyond the target area .", "second , labeling by retrograde tracing is strongly dependent on the tracer and cell type ; e.g. , pv neurons could be detected following transsynaptic virus - mediated tracing but not by ctb labeling . for quantitative studies", ", it would be highly desirable to identify markers / promoters for long - range gabaergic neurons .", "characterization of motor cortex gabaergic projecting neurons revealed that m1 and m2 som and pv cells differentially innervate striatal neurons .", "moreover , bilateral stimulation of corticostriatal long - range gabaergic projections modulates motor activity in spite of the scarcity of gabaergic corticostriatal neurons and the relatively small amplitude responses of targeted striatal neurons .", "thus , stimulation of m1 som neuron projections , targeting dspns , ispns , and cholinergic cells , leads to an increase in locomotion .", "in contrast , stimulation of m2 som neuron projections , targeting preferentially dspns and ispns , and of m1 pv neuron projections , targeting preferentially dspns , leads to a decrease in locomotion .", "decreased locomotion upon preferential inhibition of dspns ( pv - m1 ) is in line with previous studies showing either bradykinesia upon deletion of dspns ( drago et al . , 1998 ) or increased locomotion upon stimulation of dspns ( kravitz et al .", "notably , there was a similar effect on locomotion upon preferential inhibition of dspns ( pv - m1 ) or of both dspns and ispns ( som - m2 ) .", "comparable effects were also reported when optogenetically silencing either dspn or both dspns and ispns ( tecuapetla et al . , 2014 ) .", "increased locomotion upon stimulation of m1 som neuron projections most likely reflects the participation of a larger fraction of cholinergic cells .", "these striatal interneurons , constituting 1%3% of all striatal neurons , are tonically active and provide powerful feedforward inhibition to spns ( english et al . , 2011 , nelson et al . ,", "2009 ) , and enhance dopamine release ( threlfell et al . , 2012 ) . direct activation or inhibition of cholinergic striatal interneurons in the dorsal anterior striatum", "had no effect on locomotor activity ( maurice et al . , 2015 ) .", "however , based on our results , it is tempting to speculate that cholinergic cells in more ventral and posterior striatal areas receiving input from m1 som neurons are involved in motor control . at present", ", we can not resolve whether the observed change in locomotor activity results from long - range axon activation in the striatum only or whether activation of putative collaterals via back - propagating action potentials also plays a role . in either case ,", "our results show that the activity of som and pv projecting neurons in the motor cortex differentially modulates locomotor activity .", "this study is also relevant when interpreting data regarding silencing of cortical areas by manipulating gabaergic neurons because the effects may also involve long - distance targets .", "it has been proposed that motor control and reward coding are mediated by common corticostriatal circuits ( kravitz and kreitzer , 2012 ) .", "our data indicate that , although activation of motor cortex pv and som neuron projections in the dorsal striatum affected locomotor activity , it did not affect place preference , although we can not exclude their implication in reward coding more generally . on the other hand , stimulation of gabaergic projections from the prefrontal cortex to the ventral striatum induces avoidance behavior , suggesting that they are involved in the coding of punishment ( lee et al . , 2014 ) .", "further experiments will be required to elucidate whether , and , if so , which , corticostriatal gabaergic projections mediate both locomotion and reward coding .", "this study adds to the increasing evidence that long - range gabaergic neurons are more frequent than previously thought .", "the heterogeneity of long - range gabaergic neurons described here is in line with previous studies indicating neurochemical diversity of long - range gabaergic neurons in the cortex and hippocampus ( jinno et al .", ", 2007 , higo et al . , 2007 , lee et al . , 2014 ,", "notably , we demonstrate that distinct long - range gabaergic neurons exhibit specific functional properties and differential connectivity . finally , it will be of interest to study long - range gabaergic neurons in the context of movement disorders that are thought to be caused by an imbalance of dspn and ispn activity . thus , parkinsonian - like movements can be reproduced by increased ispn activity ( kravitz et al .", ", 2010 ) and can be reduced by selective inhibition of striatal cholinergic interneurons ( maurice et al . ,", "huntington s disease is marked by an early degeneration of ispns ( vonsattel et al .", ", 1985 , mitchell et al . , 1999 ) and an imbalance of excitation and inhibition of dspns and ispns ( andr et al . , 2011 ) . in light of our findings , it is tempting to speculate that motor cortex gabaergic projections to the striatum might be a potential target for restoring the balance of striatal output ." ], [ "all experiments were performed in 8- to 20-week - old male mice and were approved by the regierungsprsidium karlsruhe in compliance with the european guidelines for the care and use of laboratory animals ( licenses g74/13 and g248/14 ) . for anterograde tracing experiments , in vitro patch - clamp recordings , and behavioral experiments , aav dio chr2-mcherry was injected into the primary and/or secondary motor cortex of som ( melzer et al . , 2012 ) , pv ( hippenmeyer et al . , 2005 ) , pv / gad67-egfp ( tamamaki et al . , 2003 ) , som / drd1a - egfp , pv / drd1a - egfp , som / drd2-egfp ( gong et al . , 2003 ) , and pv / drd2-egfp mice with a c57bl/6 background . for retrograde tracing experiments , ctb", "647 was injected into the dorsal striatum of wild - type mice . for retrograde transsynaptic rabies virus tracing , aav - cag - flex - tcb , aav - cag - flex - rg , and sadg - egfp(enva )", "were injected into the dorsal striatum of a2a - cre mice . for monosynaptic retrograde rabies virus tracing , aav - cag - flex - tcb", "was injected into the motor cortex of som ( sst , jackson laboratory ) and pv mice , followed by sadg - egfp(enva ) injection into the dorsal striatum . in all cases ,", "anesthesia was induced and maintained with isoflurane ( 1%2.5% ) , and the virus was delivered through a small craniotomy at the appropriate coordinates by a glass micropipette . for behavioral experiments ,", "immunofluorescence and dab staining were performed on sagittal and coronal brain sections using standard protocols .", "fresh - frozen 20-m sections were stained with fish using the rnascope fluorescent multiplex kit ( advanced cell diagnostics ) .", "mice were deeply anesthetized with isoflurane , transcardially perfused with 30 ml ice - cold sucrose solution , and 300-m - thick brain sections were cut .", "chr2-expressing long - range axonal fibers were stimulated with 5-ms photostimulation ( 473 nm , 120 mw / mm laser intensity ) .", "pscs were measured at 0-mv holding potential ( using cs - based intracellular solution ) or at 70-mv holding potential ( using k - based , high cl intracellular solution ) . for firing pattern analysis , incrementally increasing currents of 1-s duration were injected in current clamp mode starting at 50 or 200 pa . series resistances of 37 megohm were accepted for analysis of pscs . stimulus delivery and data acquisition were performed using pulse software .", "mice were video - tracked at 25 frames / s , and their movements were subsequently analyzed using a position tracking system ( ethovision xt9 , noldus ) .", "the implanted optic fiber cannulas were connected to two optic fibers attached to a rotary joint ( doric lenses ) .", "a patch cord connected the optic fibers to a diode - pumped , solid - state , 473-nm laser ( crystalaser ) .", "we used a pulse generator ( master 8) and a transistor - transistor logic ( ttl ) control box ( universal serial bus input / output [ usb - io ] box , noldus ) to automatically control the photostimulation ( 5-ms pulses delivered at 20 hz ; laser power , 3 mw ) .", "evaluation of locomotor activity was performed in a circular arena ( 40 40 cm ) placed in a dimly lit room where mice were allowed to run freely for 21 min .", "it lasted 2 min and was repeated three times with an inter - stimulation period of 4 min .", "brown - forsythe and f tests were used to test the homogeneity of variances . for non - pairwise comparisons , unpaired t tests ( either for equal or unequal variance ) or mann - whitney u tests were used . for pairwise comparisons , paired t tests or wilcoxon matched - pairs signed - rank tests were used .", "one - way anova followed by tukey s multiple comparisons tests was used to compare motion differences ." ], [ "performed in vitro electrophysiology , analysis , immunohistochemistry , reconstructions , and retrograde tracing studies ." ] ]

[ "pectus excavatum ( pe ) , characterized by posterior displacement of the sternum and cartilaginous - rib attachments , is one of the most common congenital chest wall deformities.1)2 ) the physiological impact of pe varies .", "symptomatic patients frequently complain of dyspnea with exertion , progressive loss of endurance , tachycardia , palpitations and chest discomfort.3 - 5 ) in a critical location , even small depressions of the chest can create significant cardiac dysfunction when the right heart and pulmonary outflow tract are compressed to varying degrees by the depressed sternum.5 - 7 ) an index of severity can be calculated by measuring the inner width of the chest ( at the lowest level of the pectus defect ) and dividing it by the distance between the posterior surface of the sternum ( at the lowest part of the defect ) and the anterior surface of the spine ( with normal being approximately 2.52).2)3 ) in general , an index of 3.2 or greater is considered severe however symptoms do not necessarily correlate with severity of index.2 - 4 ) echocardiography plays a significant role in the evaluation of patients with pe .", "the degree of right heart compression however is often difficult to assess by transthoracic echo ( tte ) especially with severe deformities that prevent the operator from obtaining the normal transthoracic views.7 - 9 )", "a 32-year - old woman presented to out - patient clinic for further evaluation of a 1-year history of progressive chest pain , fatigue , dizziness , paroxysmal tachycardia and dyspnea with exertion . with even mild exertional effort she experienced sharp , \" stabbing \" chest pain along the left lower and mid - sternum .", "a 12-lead electrocardiogram demonstrated a right - bundle branch block with left posterior fascicular block .", "she had a history of severe pe ( index of > 4 ) for which she had undergone operative repair 18 months prior by an open resection of cartilage attachments and sternal \" flip \" as described by hawkins & colleagues.10 ) prior to her first correction , she had some dyspnea with exertion , however , cardiac work - up , including echocardiogram had been reported to not show any abnormalities .", "she noted onset of her current symptoms approximately 6 months after her pe repair . a subsequent operation with superficial anterior remodeling of the chest wall cartilage on the left side of the sternum had failed to relieve her progression of pain and symptoms .", "she exhibited post - operative abnormal remodeling of the chest wall with residual as well as some recurrent pe .", "the sternum protruded anterior with bilateral depression of the costal - sternal attachments creating a \" wave - like \" appearance ( fig .", "close attention to the anteroposterior planes ( best seen from the apical four - chamber views ) clearly demonstrated extrinsic compression and deformation of the lower mid - right ventricle ( rv ) by the chest wall which is more obvious during diastole ( fig .", "3b ) . biplane and live 3-d images of the preoperative transesophageal echocardiogram ( tee ) improved the visualization and localization of extrinsic compression of the right ventricle ( fig .", "tricuspid valve prolapse is seen likely due to partial compression of the rv resulting in distortion of the tricuspid annulus ( fig .", "open revision of her chest wall deformity was performed with placement of two stainless steel support bars ( lorentz surgical , jacksonville , fl , usa ) and a trabecular metal implant ( zimmer , inc . ,", "minneapolis , mn , usa ) which elevated the sternum and depressed regions 3 - 4 centimeters anterior to the rv with good cosmetic results ( fig .", "symptomatic pe patients often present for cardiovascular evaluation with tte being the most commonly utilized diagnostic modality .", "documenting the effects of a depressed chest wall on the right heart and outflow tract as well as any associated interference with diastolic filling is critical for decision making in patient treatment and the need for potential surgical intervention .", "modifying standard views such as biplane transthoracic and transesophageal views may be necessary in some patients due to limitations from the abnormal anatomy of the deformed anterior chest wall . in some cases , rv compression is often difficult to assess by tte especially with severe deformities that prevent the operator from obtaining the normal transthoracic window views .", "technical tips for assessment of the rv include 1 ) narrowing the 2-d sector width to optimize only rv structures ; 2 ) use of harmonic imaging and adjustment of gain and compression for good contrast and endocrinal edge detection ; 3 ) making all measurements at end - diastole or the frame demonstrating the largest chamber dimension ; and 4 ) for 2-d measurements , obtain all acquisitions during quiet respiration or full - expiration .", "apical four - chamber views when seen clearly can usually visualize any extrinsic compression to the rv of the heart .", "use of all three possible traditional acoustic windows ( parasternal short - axis , apical and subcostal ) may be necessary . in an effort to minimize foreshortening of the rv , the transducer can be positioned down an intercostal space and laterally until the rv apex is clearly seen . in some patients", "a transesophageal and transgastric view may be necessary to better evaluate the right heart chambers and rv outflow obstruction .", "many patients with pe have associated alterations in rv morphology and function . assessment for localized sacculation of the rv wall , global dilation of the ventricle , prominent trabeculae , and/or hypertrophy of the moderator band is important . identifying abnormalities , including mitral valve prolapse and aortic root measurements are especially critical in suspected or confirmed cases of marfan syndrome .", "resolution of mitral valve prolapse with release of the chest wall entrapment is seen in more than half of patients after pe correction.7)11)12 ) more importantly , many patients improve their symptoms such as exertional dyspnea and chest pain which may be related to extrinsic compression of the rv and reduced preload.4)5)7 ) further studies are needed to better understand the pathophyiology of symptoms in relation to cardiac and chest wall function in patients with pe . in conclusion ,", "echocardiographic evaluation is critical in patients with symptomatic pe to assess the degree of rv compression .", "we presented a case of a pe in a patient with symptoms including severe chest pain with exertion that was diagnosed with the expertise of echocardiography and subsequently surgically corrected .", "subtle abnormalities in cardiac structure and chest wall compression may result in debilitating symptoms in a small population of patients.2 - 4 ) when astutely performed , echocardiography can accurately provide clinically relevant information about cardiac size and any hemodynamic compromise in patients with pe ." ]

[ "<S> pectus excavatum exists as varying anatomic deformities and compression of the right heart by the chest wall can lead to patient symptoms including dyspnea and chest pain with exertion . </S>", "<S> echocardiography can be difficult but is critical to the evaluation and diagnosis of this patient population . </S>", "<S> modifying standard views such as biplane transthoracic and 3-d transesophageal views may be necessary in some patients due to limitations from the abnormal anatomy of the deformed anterior chest wall . </S>", "<S> apical four - chamber views when seen clearly can usually visualize any extrinsic compression to the right ventricle of the heart . </S>" ]

[ "Introduction", "Case", "Discussion" ]

[ [ "pectus excavatum ( pe ) , characterized by posterior displacement of the sternum and cartilaginous - rib attachments , is one of the most common congenital chest wall deformities.1)2 ) the physiological impact of pe varies .", "symptomatic patients frequently complain of dyspnea with exertion , progressive loss of endurance , tachycardia , palpitations and chest discomfort.3 - 5 ) in a critical location , even small depressions of the chest can create significant cardiac dysfunction when the right heart and pulmonary outflow tract are compressed to varying degrees by the depressed sternum.5 - 7 ) an index of severity can be calculated by measuring the inner width of the chest ( at the lowest level of the pectus defect ) and dividing it by the distance between the posterior surface of the sternum ( at the lowest part of the defect ) and the anterior surface of the spine ( with normal being approximately 2.52).2)3 ) in general , an index of 3.2 or greater is considered severe however symptoms do not necessarily correlate with severity of index.2 - 4 ) echocardiography plays a significant role in the evaluation of patients with pe .", "the degree of right heart compression however is often difficult to assess by transthoracic echo ( tte ) especially with severe deformities that prevent the operator from obtaining the normal transthoracic views.7 - 9 )" ], [ "a 32-year - old woman presented to out - patient clinic for further evaluation of a 1-year history of progressive chest pain , fatigue , dizziness , paroxysmal tachycardia and dyspnea with exertion . with even mild exertional effort she experienced sharp , \" stabbing \" chest pain along the left lower and mid - sternum .", "a 12-lead electrocardiogram demonstrated a right - bundle branch block with left posterior fascicular block .", "she had a history of severe pe ( index of > 4 ) for which she had undergone operative repair 18 months prior by an open resection of cartilage attachments and sternal \" flip \" as described by hawkins & colleagues.10 ) prior to her first correction , she had some dyspnea with exertion , however , cardiac work - up , including echocardiogram had been reported to not show any abnormalities .", "she noted onset of her current symptoms approximately 6 months after her pe repair . a subsequent operation with superficial anterior remodeling of the chest wall cartilage on the left side of the sternum had failed to relieve her progression of pain and symptoms .", "she exhibited post - operative abnormal remodeling of the chest wall with residual as well as some recurrent pe .", "the sternum protruded anterior with bilateral depression of the costal - sternal attachments creating a \" wave - like \" appearance ( fig .", "close attention to the anteroposterior planes ( best seen from the apical four - chamber views ) clearly demonstrated extrinsic compression and deformation of the lower mid - right ventricle ( rv ) by the chest wall which is more obvious during diastole ( fig .", "3b ) . biplane and live 3-d images of the preoperative transesophageal echocardiogram ( tee ) improved the visualization and localization of extrinsic compression of the right ventricle ( fig .", "tricuspid valve prolapse is seen likely due to partial compression of the rv resulting in distortion of the tricuspid annulus ( fig .", "open revision of her chest wall deformity was performed with placement of two stainless steel support bars ( lorentz surgical , jacksonville , fl , usa ) and a trabecular metal implant ( zimmer , inc . ,", "minneapolis , mn , usa ) which elevated the sternum and depressed regions 3 - 4 centimeters anterior to the rv with good cosmetic results ( fig ." ], [ "symptomatic pe patients often present for cardiovascular evaluation with tte being the most commonly utilized diagnostic modality .", "documenting the effects of a depressed chest wall on the right heart and outflow tract as well as any associated interference with diastolic filling is critical for decision making in patient treatment and the need for potential surgical intervention .", "modifying standard views such as biplane transthoracic and transesophageal views may be necessary in some patients due to limitations from the abnormal anatomy of the deformed anterior chest wall . in some cases , rv compression is often difficult to assess by tte especially with severe deformities that prevent the operator from obtaining the normal transthoracic window views .", "technical tips for assessment of the rv include 1 ) narrowing the 2-d sector width to optimize only rv structures ; 2 ) use of harmonic imaging and adjustment of gain and compression for good contrast and endocrinal edge detection ; 3 ) making all measurements at end - diastole or the frame demonstrating the largest chamber dimension ; and 4 ) for 2-d measurements , obtain all acquisitions during quiet respiration or full - expiration .", "apical four - chamber views when seen clearly can usually visualize any extrinsic compression to the rv of the heart .", "use of all three possible traditional acoustic windows ( parasternal short - axis , apical and subcostal ) may be necessary . in an effort to minimize foreshortening of the rv , the transducer can be positioned down an intercostal space and laterally until the rv apex is clearly seen . in some patients", "a transesophageal and transgastric view may be necessary to better evaluate the right heart chambers and rv outflow obstruction .", "many patients with pe have associated alterations in rv morphology and function . assessment for localized sacculation of the rv wall , global dilation of the ventricle , prominent trabeculae , and/or hypertrophy of the moderator band is important . identifying abnormalities , including mitral valve prolapse and aortic root measurements are especially critical in suspected or confirmed cases of marfan syndrome .", "resolution of mitral valve prolapse with release of the chest wall entrapment is seen in more than half of patients after pe correction.7)11)12 ) more importantly , many patients improve their symptoms such as exertional dyspnea and chest pain which may be related to extrinsic compression of the rv and reduced preload.4)5)7 ) further studies are needed to better understand the pathophyiology of symptoms in relation to cardiac and chest wall function in patients with pe . in conclusion ,", "echocardiographic evaluation is critical in patients with symptomatic pe to assess the degree of rv compression .", "we presented a case of a pe in a patient with symptoms including severe chest pain with exertion that was diagnosed with the expertise of echocardiography and subsequently surgically corrected .", "subtle abnormalities in cardiac structure and chest wall compression may result in debilitating symptoms in a small population of patients.2 - 4 ) when astutely performed , echocardiography can accurately provide clinically relevant information about cardiac size and any hemodynamic compromise in patients with pe ." ] ]

[ "the adenoid cystic carcinoma ( acc ) is a relatively rare epithelial tumor of the salivary glands .", "it accounts for about 5 - 10% of all salivary gland neoplasms , representing 2 - 4% of malignant occurrences of the head and neck area .", "approximately , 31% of lesions affect minor salivary glands , particularly the palate , although they can also be observed in the sub - mandibular and parotid glands .", "the frequency reported in the tongue is 19.8% , with 85% observed at the base of the tongue .", "we report one such rare case of tongue neoplasm which turned out to be acc in a middle aged lady .", "a 45-year - old - female patient presented with an asymptomatic growth of the tongue , which was perceived just 2 weeks before consultation .", "the intra - oral examination at that time revealed a mass in the dorsum of the tongue with light pain to pressure , without any evidence of cervical lymphadenopathy .", "the mass was firm , same color as that of the surrounding mucosa and asymptomatic otherwise [ figure 1 ] .", "as a pre - operative assessment of the lesion , a fine needle aspiration was done and the smear revealed a salivary neoplasm consisting of well delineated , tightly cohesive clusters of basaloid cells surrounding mucoid , hyaline globules , or clear spaces also forming honeycomb ( cribriform ) pattern [ figure 2 ] . at places", "dense aggregates of monomorphic small cells with uniform round to oval hyperchromatic nuclei and scanty cytoplasm were seen .", "smears also showed individual tumor cells with high n : c ratio and nuclear moulding .", "macroscopically , the mass had firm consistency with an irregular form and surface , brown color and measured 2.5 1.5 1.0 cm .", "the histopathologic study revealed multiple pseudocystic spaces of variable sizes surrounded by cuboidal cells with scarce cytoplasm and oval nuclei , filled with eosinophilic material and hence was consistent with the diagnosis of acc [ figure 3 ] .", "however , there was no evidence of perineural infiltration on serial sections . clinical photograph showing a swelling on the dorsum of the tongue cytological smears show well - delineated clusters of basaloid cells surrounding hyaline globules with uniform round to oval hyperchromatic nuclei and scanty cytoplasm ( papanicolaou stain , 400 ) histopathological section showing multiple pseudocystic cavities of variable size composed of cuboidal cells with scarce cytoplasm and oval nuclei ( h and e , 400 )", "minor salivary gland neoplasms occur less commonly than the major salivary gland tumors and tongue is a relatively uncommon site for salivary gland neoplasms", "acc is a malignant neoplasm that originates in both the minor and major salivary glands , characterized by slow growth , diffuse invasion , and potential to produce distant metastases , mainly to the lungs and bones .", "it is an infrequent lesion , as it represents approximately 1 - 2% of all malignant neoplasms of the head and neck , and up to 10 - 15% of all malignant salivary gland neoplasms .", "the most common intra - oral site for minor salivary gland tumors is the hard palate , followed by the base of the tongue where up to 96% of all tumors are malignant , and acc represents 30% of them . on the other hand ,", "one of the least frequent sites of presentation for acc is the mobile tongue , as several authors have reported an incidence of only approximately 3% of the cases .", ", analyzed 178 cases of salivary gland tumors , out of which only six cases were located on the tongue .", "cytologically , cribriform variety of acc can be diagnosed by hypercellular smears composed of clusters of small , relatively monomorphic epithelial cells with hyperchromatic nuclei .", "these appear bright magenta in may - grunwald giemsa mgg stains and pale blue with papanicolaou stain .", "finger - like process of similar material can also be found in between the groups of cells in tubular variety .", "the solid variant of acc also exhibits the same material and the cells resemble that of small cells of anaplastic carcinoma .", "the globules of amorphous material surrounded by the monomorphic hyperchromatic cells was a clue to the diagnosis of acc in our case , but since the hyaline globules are also found in other tumors like basal cell adenoma , pleomorphic adenoma , polymorphous low grade adenocarcinoma , epithelial myoepithelial carcinoma etc .", "it is important to distinguish the adenomas from acc because of the conservative mode of management in case of adenomas .", "we ruled out the adenomas because of the nature of the globules and the cytological morphology . unlike the adenomas , the hyaline globules were dense and stained intensely with mgg and the cells were relatively monomorphic , hyperchromatic with coarse chromatin and irregular nuclear membrane like that of acc .", "of three histologic variants - tubular , cribriform and solid ; in our case cribriform pattern was the dominant one without any evidence of perineural infiltration .", "the main factors associated with patient survival were tumor location , clinical stage , and the observed histologic variable .", "conversely , spiro et al . , have not found histologic classification to be of any benefit , and deny a correlation between microscopic appearance and prognosis . due to the slow growth pattern of the tumor", "however , due to local recurrence and late metastasis , surgery remains the mainstay of management with or without radiotherapy ." ]

[ "<S> adenoid cystic carcinoma is a relatively rare epithelial tumor of the salivary glands accounting for about 5 - 10% of all salivary gland neoplasms . </S>", "<S> approximately , 31% of salivary gland neoplasms affect minor salivary glands particularly the palate . </S>", "<S> it involves tongue in only 19.8% of cases and even rarely the dorsum of the tongue . </S>", "<S> we report such a rare case that affected dorsum of the tongue in a 45-year - old - female patient . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "the adenoid cystic carcinoma ( acc ) is a relatively rare epithelial tumor of the salivary glands .", "it accounts for about 5 - 10% of all salivary gland neoplasms , representing 2 - 4% of malignant occurrences of the head and neck area .", "approximately , 31% of lesions affect minor salivary glands , particularly the palate , although they can also be observed in the sub - mandibular and parotid glands .", "the frequency reported in the tongue is 19.8% , with 85% observed at the base of the tongue .", "we report one such rare case of tongue neoplasm which turned out to be acc in a middle aged lady ." ], [ "a 45-year - old - female patient presented with an asymptomatic growth of the tongue , which was perceived just 2 weeks before consultation .", "the intra - oral examination at that time revealed a mass in the dorsum of the tongue with light pain to pressure , without any evidence of cervical lymphadenopathy .", "the mass was firm , same color as that of the surrounding mucosa and asymptomatic otherwise [ figure 1 ] .", "as a pre - operative assessment of the lesion , a fine needle aspiration was done and the smear revealed a salivary neoplasm consisting of well delineated , tightly cohesive clusters of basaloid cells surrounding mucoid , hyaline globules , or clear spaces also forming honeycomb ( cribriform ) pattern [ figure 2 ] . at places", "dense aggregates of monomorphic small cells with uniform round to oval hyperchromatic nuclei and scanty cytoplasm were seen .", "smears also showed individual tumor cells with high n : c ratio and nuclear moulding .", "macroscopically , the mass had firm consistency with an irregular form and surface , brown color and measured 2.5 1.5 1.0 cm .", "the histopathologic study revealed multiple pseudocystic spaces of variable sizes surrounded by cuboidal cells with scarce cytoplasm and oval nuclei , filled with eosinophilic material and hence was consistent with the diagnosis of acc [ figure 3 ] .", "however , there was no evidence of perineural infiltration on serial sections . clinical photograph showing a swelling on the dorsum of the tongue cytological smears show well - delineated clusters of basaloid cells surrounding hyaline globules with uniform round to oval hyperchromatic nuclei and scanty cytoplasm ( papanicolaou stain , 400 ) histopathological section showing multiple pseudocystic cavities of variable size composed of cuboidal cells with scarce cytoplasm and oval nuclei ( h and e , 400 )" ], [ "minor salivary gland neoplasms occur less commonly than the major salivary gland tumors and tongue is a relatively uncommon site for salivary gland neoplasms", "acc is a malignant neoplasm that originates in both the minor and major salivary glands , characterized by slow growth , diffuse invasion , and potential to produce distant metastases , mainly to the lungs and bones .", "it is an infrequent lesion , as it represents approximately 1 - 2% of all malignant neoplasms of the head and neck , and up to 10 - 15% of all malignant salivary gland neoplasms .", "the most common intra - oral site for minor salivary gland tumors is the hard palate , followed by the base of the tongue where up to 96% of all tumors are malignant , and acc represents 30% of them . on the other hand ,", "one of the least frequent sites of presentation for acc is the mobile tongue , as several authors have reported an incidence of only approximately 3% of the cases .", ", analyzed 178 cases of salivary gland tumors , out of which only six cases were located on the tongue .", "cytologically , cribriform variety of acc can be diagnosed by hypercellular smears composed of clusters of small , relatively monomorphic epithelial cells with hyperchromatic nuclei .", "these appear bright magenta in may - grunwald giemsa mgg stains and pale blue with papanicolaou stain .", "finger - like process of similar material can also be found in between the groups of cells in tubular variety .", "the solid variant of acc also exhibits the same material and the cells resemble that of small cells of anaplastic carcinoma .", "the globules of amorphous material surrounded by the monomorphic hyperchromatic cells was a clue to the diagnosis of acc in our case , but since the hyaline globules are also found in other tumors like basal cell adenoma , pleomorphic adenoma , polymorphous low grade adenocarcinoma , epithelial myoepithelial carcinoma etc .", "it is important to distinguish the adenomas from acc because of the conservative mode of management in case of adenomas .", "we ruled out the adenomas because of the nature of the globules and the cytological morphology . unlike the adenomas , the hyaline globules were dense and stained intensely with mgg and the cells were relatively monomorphic , hyperchromatic with coarse chromatin and irregular nuclear membrane like that of acc .", "of three histologic variants - tubular , cribriform and solid ; in our case cribriform pattern was the dominant one without any evidence of perineural infiltration .", "the main factors associated with patient survival were tumor location , clinical stage , and the observed histologic variable .", "conversely , spiro et al . , have not found histologic classification to be of any benefit , and deny a correlation between microscopic appearance and prognosis . due to the slow growth pattern of the tumor", "however , due to local recurrence and late metastasis , surgery remains the mainstay of management with or without radiotherapy ." ] ]

[ "it has been proven to be very effective and became a standard of care in the treatment of most cases with severe prolapsus .", "sacropexy is a surgical repair technique that restores pelvic anatomy by attaching synthetic graft material into the vagina and sacrum .", "erosion of vaginal wall or bowel can be seen as a long term complication . in this paper", ", we present a patient suffering from mesh migration into the rectum after abdominal sacral colpopexy .", "a 69-year - old woman was admitted to the hospital with a complaint of sensation of fullness and a feeling of a foreign material protruding during defecation .", "she had been diagnosed with uterine prolapsus and stress incontinence in 2008 and underwent total abdominal hysterectomy , bilateral salpingo - oophorectomy and sacral colpopexy with prolene mesh .", "the patient was admitted to our clinic five years after the procedure with complaints mentioned above . there was a foreign material palpated in rectum with digital examination .", "prolene mesh was detected in sacral region but resection of the mesh could not be conducted because of high levels of adhesions in that region .", "genital prolapse or genital hernia is described as the protrusion of pelvic organs along the vagina .", "it is one of the common gynecological conditions that affect the quality of life in women .", "it may be seen in up to 50% of multipara women , and its incidence increases with age .", "high rates of recurrence with traditional techniques led to the development of new surgical techniques .", "the use of synthetic mesh has become more popular surgical approach in cystocele and rectocele repair .", "mesh migration is a well - known clinical pathology and have been reported in literature .", "yolen and grossman suggested that intra - abdominal foreign bodies ( like mesh ) transmigrate into the small or large bowel by triggering an inflammatory reaction .", "persistent inflammatory reaction causes an opening into a hollow organ assisted by the peristaltic movement of the bowel .", "insufficient fixation of a mesh is another factor for migration of synthetic materials in some patients .", "larger pores greater than 75 nm permit the migration of macrophage and leukocyte migration and reduce the infection rate .", "large pores also improves flexibility of the mesh and cause tissue ingrowths and healthy collagen deposition .", "complications reported after sacropexy include ileus , intraoperative vessel injury , ureter injuries , recurrent descensus and mesh tearing . the use of a mesh as a graft material results in higher success rates but also causes a higher number of complications , such as mesh erosions or chronic infections .", "taoka reported a case of rectal migration of mesh in a 64-year - old woman who presented with a recto - cutaneous fistula 11 months after a tension - free vaginal ( tvm ) repair ; the patient was treated by removal of the infected mesh and closure of the rectal wall defect under cover of a temporary colostomy . by contrast with", "the troublesome symptoms reported in such patients in the literature , the only presenting complaint of our patient was protrusion of foreign material from the rectum .", "in conclusion , mesh migration is a serious complication after sacral colpopexy . sometimes surgical resection of migrated mesh with laparotomy can be difficult due to dense adhesions .", "scu was responsible for writing , conception and design of the study ; ob contributed toward analysis and interpretation of data ; nas , oa , aa- performed acquisition of data ; bk drafted the manuscript ." ]

[ "<S> introductionpelvic organ prolapse ( pop ) is a common gynecological problem . </S>", "<S> repair with synthetic materials such as prolene mesh has become a popular approach in prolapsus surgery . </S>", "<S> migration of synthetic materials can cause serious complications.presentation of casea 69-year - old woman was admitted to the hospital with a complaint of sensation of fullness and a feeling of a foreign material protruding during defecation . </S>", "<S> the patient underwent exploratory laparotomy . </S>", "<S> prolene mesh was detected in sacral region but resection of the mesh could not be conducted because of dense adhesions causing frozen pelvis . </S>", "<S> the migrated prolene mesh was resected transanally.discussiongenital prolapse or genital hernia is described as the protrusion of pelvic organs along the vagina . </S>", "<S> it is one of the common gynecological conditions that affect the quality of life in women . </S>", "<S> mesh migration is a well - known clinical pathology.conclusionmesh migration is a serious complication after sacral colpopexy . </S>", "<S> surgical resection of migrated mesh can be difficult due to dense adhesions . </S>" ]

[ "Introduction", "Presentation of case", "Discussion", "Conclusion", "Conflict of interest", "Funding", "Ethical approval", "Author contributions" ]

[ [ "the use of synthetic material such as prolene mesh in prolapsus surgery has become a popular approach .", "it has been proven to be very effective and became a standard of care in the treatment of most cases with severe prolapsus .", "sacropexy is a surgical repair technique that restores pelvic anatomy by attaching synthetic graft material into the vagina and sacrum .", "erosion of vaginal wall or bowel can be seen as a long term complication . in this paper", ", we present a patient suffering from mesh migration into the rectum after abdominal sacral colpopexy ." ], [ "a 69-year - old woman was admitted to the hospital with a complaint of sensation of fullness and a feeling of a foreign material protruding during defecation .", "she had been diagnosed with uterine prolapsus and stress incontinence in 2008 and underwent total abdominal hysterectomy , bilateral salpingo - oophorectomy and sacral colpopexy with prolene mesh .", "the patient was admitted to our clinic five years after the procedure with complaints mentioned above . there was a foreign material palpated in rectum with digital examination .", "prolene mesh was detected in sacral region but resection of the mesh could not be conducted because of high levels of adhesions in that region ." ], [ "genital prolapse or genital hernia is described as the protrusion of pelvic organs along the vagina .", "it is one of the common gynecological conditions that affect the quality of life in women .", "it may be seen in up to 50% of multipara women , and its incidence increases with age .", "high rates of recurrence with traditional techniques led to the development of new surgical techniques .", "the use of synthetic mesh has become more popular surgical approach in cystocele and rectocele repair .", "mesh migration is a well - known clinical pathology and have been reported in literature .", "yolen and grossman suggested that intra - abdominal foreign bodies ( like mesh ) transmigrate into the small or large bowel by triggering an inflammatory reaction .", "persistent inflammatory reaction causes an opening into a hollow organ assisted by the peristaltic movement of the bowel .", "insufficient fixation of a mesh is another factor for migration of synthetic materials in some patients .", "larger pores greater than 75 nm permit the migration of macrophage and leukocyte migration and reduce the infection rate .", "large pores also improves flexibility of the mesh and cause tissue ingrowths and healthy collagen deposition .", "complications reported after sacropexy include ileus , intraoperative vessel injury , ureter injuries , recurrent descensus and mesh tearing . the use of a mesh as a graft material results in higher success rates but also causes a higher number of complications , such as mesh erosions or chronic infections .", "taoka reported a case of rectal migration of mesh in a 64-year - old woman who presented with a recto - cutaneous fistula 11 months after a tension - free vaginal ( tvm ) repair ; the patient was treated by removal of the infected mesh and closure of the rectal wall defect under cover of a temporary colostomy . by contrast with", "the troublesome symptoms reported in such patients in the literature , the only presenting complaint of our patient was protrusion of foreign material from the rectum ." ], [ "in conclusion , mesh migration is a serious complication after sacral colpopexy . sometimes surgical resection of migrated mesh with laparotomy can be difficult due to dense adhesions ." ], [ "" ], [ "" ], [ "" ], [ "scu was responsible for writing , conception and design of the study ; ob contributed toward analysis and interpretation of data ; nas , oa , aa- performed acquisition of data ; bk drafted the manuscript ." ] ]

[ "congenital seminal vesicle cysts associated with abnormalities of the upper urinary tract are uncommon.1 they can be asymptomatic and discovered incidentally or can be associated with dysuria , urinary tract infections , and infertility .", "diagnosis is more frequently made in adult life during the period of the greatest sexual or reproductive activity.1 \n 2 we present a case of a congenital seminal vesicle cyst associated with ipsilateral renal agenesis incidentally discovered on ultrasound at the age of 4 years and its natural evolution until adolescence .", "the association of congenital seminal vesicle cyst with ipsilateral renal malformations is rare and was first described by zinner in 1914 .", "it is reported in literature as zinner syndrome.3 this condition is considered as the male equivalent of mayer - rokitansky - kustner - hauser ( mrkh ) syndrome described in females.4 \n", "a 4-year - old boy with known right renal agenesis discovered on antenatal ultrasound , presented in our radiology department for his annual control .", "the cyst was anechoic , measured 8 mm , and was not present on previous ultrasounds .", "it was initially considered as an ureterocele or bladder diverticula , however , neither of these diagnosis was confirmed on intravenous urography performed at that time .", "the cyst remained unchanged on size and aspect during 11 years of follow - up .", "however , at the age of 15 years its aspect on the ultrasound had changed : its content was hyperechoic , it had increased in size ( measuring 1.9 cm ) , and protruded in the bladder .", "it was associated to magma of some echogenic , round retrovesical masses , initially considered as lymph nodes ( fig .", "1b , c ) . magnetic resonance imaging ( mri ) of the urinary tract and pelvis was further performed ( coronal , axial , and sagittal t2 spin - echo and axial t1 with fat saturation before and after gadolinium injection images ) .", "it revealed a dilatation of the seminal vesicles , ipsilateral to renal agenesis ( corresponding to the magma of retrovesical round masses seen on ultrasound ) , ending to a relatively small seminal vesicle cyst ( diameter of 2 cm ) that protruded in the bladder ( fig .", "2 a c ) . \n ultrasound ( a ) at the age of 4 years detects a right retrovesical anechoic cyst ( + ) ( b ) at the age of 15 years the cyst has increased in size and it has become hyperechoic ( white arrow ) .", "( c ) it is associated to magma of round retrovesical masses ( black arrow ) . \n", "( b ) t2 spin - echo axial and ( c ) sagittal images of the pelvis show dilatation of the right seminal vesicles ( white arrow ) ending to a seminal vesicle cyst ( * ) that protrudes in the bladder .", "so far , the patient was asymptomatic and had never presented with dysuria , signs of bladder obstruction or urinary tract infection .", "he had neither any perineal discomfort nor pain , he described normal ejaculation without pain , no hematospermia , and had never experimented any urinary infection or prostatitis and also he was not sexually active . in this particular case , in the absence of symptoms and because of the relatively small size of the cyst a conservative treatment was decided , with an annual clinical and ultrasonographic control , at least until the first symptoms appear .", "the association of congenital seminal vesicle cyst with ipsilateral renal malformations is rare and there are approximately 200 cases reported in the literature.4 \n this association is explained by the common embryologic origin of both the organs ( kidneys seminal vesicles ) from the mesonephric duct1 and is due to an insult in embryogenesis between the 4th and 13th week of gestation.4 the ureteral bud arises from the dorsal part of the distal mesonephric duct and extends dorsocranially to meet and induce differentiation of the metanephric blastema , from which the kidney will develop .", "the mesonephric duct will differentiate to epididymis , ejaculatory duct , vas deferens , seminal vesicle , and hemitrigone .", "complete failure of the mesonephric duct results to absence of ipsilateral kidney , ureter , hemitrigone , and seminal vesicle .", "anomalous development of the distal mesonephric duct results to atresia of the ejaculatory ducts and abnormal ureteral budding ; the former leads to obstruction and cystic dilatation of the seminal vesicles with development of seminal cysts , the latter leads to renal agenesis or dysplasia.2 \n 5 zinner syndrome could be considered as the male equivalent of mrkh syndrome described in females.4 \n the cysts are present since birth but enlarge and become symptomatic on late adolescence or adult life , usually in the third to fifth decade , at the period of greatest reproductive or sexual activity.1 \n 2 at this moment the accumulation of secretions in the seminal vesicle due to atresia of the ejaculatory ducts and consequently insufficient drainage leads to formation of cysts in the seminal vesicle.2 \n cysts smaller than 5 cm are usually asymptomatic and are discovered incidentally .", "larger cysts can irritate the bladder and be related to pain , dysuria , frequency , hematuria , urinary tract infection , epididymitis or prostatitis , infertility , hemospermia , bladder outlet , or colonic obstruction.1 \n 2 \n 6 malignant degeneration of the cysts has been also reported.2 \n 4 \n this progressive dilatation of seminal vesicle remains often asymptomatic until the dilatation increases to 4 to 5 cm,4 \n 5 unless the obstruction triggers vas deferens dilatation at younger age,5 which was not the case for our adolescent .", "this dilatation develops slowly , by accumulation of secretions in the seminal vesicle , for many years after puberty , due to congenital atresia of the ejaculatory ducts and consequently insufficient drainage2 : literature reports a majority of cases becoming symptomatic around the third or fourth decade of life .", "diagnosis can also be made earlier in front of infertility linked to contralateral distal ejaculatory pathway compression by this cystic seminal vesicle.3 \n in our case , the cyst was first seen at the age of 4 years , mimicking an ureterocele on ultrasound . in adolescence", "it increased in size , but remained small and , for this reason , asymptomatic .", "diagnosis was made on mri , based on its anatomic connection and to the similar signal intensity with the seminal vesicles .", "differential diagnosis includes other pelvic cystic masses such as utricular or mullerian cyst , ureterocele , dilated ureter , abscess or lymph nodes,2 \n 4 and acquired seminal vesicle cysts .", "acquired cysts are usually bilateral and concern older patients with chronic prostatitis or postprostate surgery .", "ultrasound raises the suspicion of this malformation but the final diagnosis is usually made by mri ( or computed tomography , with this last method being irradiating ) . in adults", "more invasive methods as cystoscopy and vasovesiculography are also used for diagnosis.2 \n there is a medical consensus to propose conservative follow - up for asymptomatic or minimally symptomatic cysts.1 \n 4 \n 7 treatment is considered only for symptomatic patients and is surgical .", "different surgical options exist , from the less invasive transrectal or transperineal aspiration of the cyst which gives a transitory relief of the symptoms,1 to more aggressive technics .", "transurethral unroofing of the cyst by transurethral resection of the ejaculatory duct provides improvement of the quality of semen and improves the paternity rate,3 but this procedure can create injury to rectum , bladder neck , and external sphincter , and induce consequent retrograde ejaculation , and epididymitis . radical treatment , vesiculectomy with resection of ureteral or renal remnants if present , offers the better outcome .", "it was traditionally performed by open exploration but is now well described laparoscopically , with low morbidity and good results in term of symptoms", "relief and semen parameters.1 \n 4 \n 7 \n 8 \n 9 \n 10 \n 11 few children with surgical indication benefited from this approach.12 \n 13 \n concerning fertility , for patients presenting with difficulty to procreate , with low volume of semen and poor quality of spermogram , treatment shows semen quality improvement , and paternity obtained without other medical help.3 \n 4 assisted reproductive procedures should be kept for patients with infertility persisting after successful surgical procedure.4 \n our case is original as it shows the natural history and evolution of this malformation , from childhood to adolescence .", "cases reported in literature concern , in their high majority the adult population ( or late adolescence ) and these cysts are rarely detected at a younger age .", "congenital seminal vesicle cysts in patients with ipsilateral renal agenesis are rare but this association is known and should be considered in the differential diagnosis of cystic pelvic masses in males with renal agenesis or dysplasia .", "ultrasound is useful for diagnosis but mri provides a more detailed analysis and accurate diagnosis .", "congenital seminal vesicle cysts in patients with ipsilateral renal agenesis are rare but this association is well known .", "pediatric surgeons should be aware and consider this entity in the differential diagnosis of cystic pelvic masses in males with renal agenesis or dysplasia .", "ultrasound is useful for diagnosis but magnetic resonance imaging provides a more detailed analysis and accurate diagnosis ." ]

[ "<S> zinner syndrome , the association of congenital seminal vesicle cyst and ipsilateral renal agenesis , is more often reported in adults or older adolescents . </S>", "<S> we present a case of a boy , followed up in our hospital since birth for right renal agenesis who at the age of 4 years presented a right paravesical cyst on ultrasound . </S>", "<S> the cyst was initially considered as an ureterocele . </S>", "<S> the diagnosis of zinner syndrome was made later , at the age of 15 years by ultrasound and magnetic resonance imaging ; at that moment the cyst had increased in size and had changed in aspect . </S>", "<S> this malformation should be considered in the differential diagnosis of a pelvic cyst in male patients with renal agenesis . </S>" ]

[ "Introduction", "Case Report", "Discussion", "Conclusion", "New Insights and the Importance for the Pediatric Surgeon" ]

[ [ "congenital seminal vesicle cysts associated with abnormalities of the upper urinary tract are uncommon.1 they can be asymptomatic and discovered incidentally or can be associated with dysuria , urinary tract infections , and infertility .", "diagnosis is more frequently made in adult life during the period of the greatest sexual or reproductive activity.1 \n 2 we present a case of a congenital seminal vesicle cyst associated with ipsilateral renal agenesis incidentally discovered on ultrasound at the age of 4 years and its natural evolution until adolescence .", "the association of congenital seminal vesicle cyst with ipsilateral renal malformations is rare and was first described by zinner in 1914 .", "it is reported in literature as zinner syndrome.3 this condition is considered as the male equivalent of mayer - rokitansky - kustner - hauser ( mrkh ) syndrome described in females.4 \n" ], [ "a 4-year - old boy with known right renal agenesis discovered on antenatal ultrasound , presented in our radiology department for his annual control .", "the cyst was anechoic , measured 8 mm , and was not present on previous ultrasounds .", "it was initially considered as an ureterocele or bladder diverticula , however , neither of these diagnosis was confirmed on intravenous urography performed at that time .", "the cyst remained unchanged on size and aspect during 11 years of follow - up .", "however , at the age of 15 years its aspect on the ultrasound had changed : its content was hyperechoic , it had increased in size ( measuring 1.9 cm ) , and protruded in the bladder .", "it was associated to magma of some echogenic , round retrovesical masses , initially considered as lymph nodes ( fig .", "1b , c ) . magnetic resonance imaging ( mri ) of the urinary tract and pelvis was further performed ( coronal , axial , and sagittal t2 spin - echo and axial t1 with fat saturation before and after gadolinium injection images ) .", "it revealed a dilatation of the seminal vesicles , ipsilateral to renal agenesis ( corresponding to the magma of retrovesical round masses seen on ultrasound ) , ending to a relatively small seminal vesicle cyst ( diameter of 2 cm ) that protruded in the bladder ( fig .", "2 a c ) . \n ultrasound ( a ) at the age of 4 years detects a right retrovesical anechoic cyst ( + ) ( b ) at the age of 15 years the cyst has increased in size and it has become hyperechoic ( white arrow ) .", "( c ) it is associated to magma of round retrovesical masses ( black arrow ) . \n", "( b ) t2 spin - echo axial and ( c ) sagittal images of the pelvis show dilatation of the right seminal vesicles ( white arrow ) ending to a seminal vesicle cyst ( * ) that protrudes in the bladder .", "so far , the patient was asymptomatic and had never presented with dysuria , signs of bladder obstruction or urinary tract infection .", "he had neither any perineal discomfort nor pain , he described normal ejaculation without pain , no hematospermia , and had never experimented any urinary infection or prostatitis and also he was not sexually active . in this particular case , in the absence of symptoms and because of the relatively small size of the cyst a conservative treatment was decided , with an annual clinical and ultrasonographic control , at least until the first symptoms appear ." ], [ "the association of congenital seminal vesicle cyst with ipsilateral renal malformations is rare and there are approximately 200 cases reported in the literature.4 \n this association is explained by the common embryologic origin of both the organs ( kidneys seminal vesicles ) from the mesonephric duct1 and is due to an insult in embryogenesis between the 4th and 13th week of gestation.4 the ureteral bud arises from the dorsal part of the distal mesonephric duct and extends dorsocranially to meet and induce differentiation of the metanephric blastema , from which the kidney will develop .", "the mesonephric duct will differentiate to epididymis , ejaculatory duct , vas deferens , seminal vesicle , and hemitrigone .", "complete failure of the mesonephric duct results to absence of ipsilateral kidney , ureter , hemitrigone , and seminal vesicle .", "anomalous development of the distal mesonephric duct results to atresia of the ejaculatory ducts and abnormal ureteral budding ; the former leads to obstruction and cystic dilatation of the seminal vesicles with development of seminal cysts , the latter leads to renal agenesis or dysplasia.2 \n 5 zinner syndrome could be considered as the male equivalent of mrkh syndrome described in females.4 \n the cysts are present since birth but enlarge and become symptomatic on late adolescence or adult life , usually in the third to fifth decade , at the period of greatest reproductive or sexual activity.1 \n 2 at this moment the accumulation of secretions in the seminal vesicle due to atresia of the ejaculatory ducts and consequently insufficient drainage leads to formation of cysts in the seminal vesicle.2 \n cysts smaller than 5 cm are usually asymptomatic and are discovered incidentally .", "larger cysts can irritate the bladder and be related to pain , dysuria , frequency , hematuria , urinary tract infection , epididymitis or prostatitis , infertility , hemospermia , bladder outlet , or colonic obstruction.1 \n 2 \n 6 malignant degeneration of the cysts has been also reported.2 \n 4 \n this progressive dilatation of seminal vesicle remains often asymptomatic until the dilatation increases to 4 to 5 cm,4 \n 5 unless the obstruction triggers vas deferens dilatation at younger age,5 which was not the case for our adolescent .", "this dilatation develops slowly , by accumulation of secretions in the seminal vesicle , for many years after puberty , due to congenital atresia of the ejaculatory ducts and consequently insufficient drainage2 : literature reports a majority of cases becoming symptomatic around the third or fourth decade of life .", "diagnosis can also be made earlier in front of infertility linked to contralateral distal ejaculatory pathway compression by this cystic seminal vesicle.3 \n in our case , the cyst was first seen at the age of 4 years , mimicking an ureterocele on ultrasound . in adolescence", "it increased in size , but remained small and , for this reason , asymptomatic .", "diagnosis was made on mri , based on its anatomic connection and to the similar signal intensity with the seminal vesicles .", "differential diagnosis includes other pelvic cystic masses such as utricular or mullerian cyst , ureterocele , dilated ureter , abscess or lymph nodes,2 \n 4 and acquired seminal vesicle cysts .", "acquired cysts are usually bilateral and concern older patients with chronic prostatitis or postprostate surgery .", "ultrasound raises the suspicion of this malformation but the final diagnosis is usually made by mri ( or computed tomography , with this last method being irradiating ) . in adults", "more invasive methods as cystoscopy and vasovesiculography are also used for diagnosis.2 \n there is a medical consensus to propose conservative follow - up for asymptomatic or minimally symptomatic cysts.1 \n 4 \n 7 treatment is considered only for symptomatic patients and is surgical .", "different surgical options exist , from the less invasive transrectal or transperineal aspiration of the cyst which gives a transitory relief of the symptoms,1 to more aggressive technics .", "transurethral unroofing of the cyst by transurethral resection of the ejaculatory duct provides improvement of the quality of semen and improves the paternity rate,3 but this procedure can create injury to rectum , bladder neck , and external sphincter , and induce consequent retrograde ejaculation , and epididymitis . radical treatment , vesiculectomy with resection of ureteral or renal remnants if present , offers the better outcome .", "it was traditionally performed by open exploration but is now well described laparoscopically , with low morbidity and good results in term of symptoms", "relief and semen parameters.1 \n 4 \n 7 \n 8 \n 9 \n 10 \n 11 few children with surgical indication benefited from this approach.12 \n 13 \n concerning fertility , for patients presenting with difficulty to procreate , with low volume of semen and poor quality of spermogram , treatment shows semen quality improvement , and paternity obtained without other medical help.3 \n 4 assisted reproductive procedures should be kept for patients with infertility persisting after successful surgical procedure.4 \n our case is original as it shows the natural history and evolution of this malformation , from childhood to adolescence .", "cases reported in literature concern , in their high majority the adult population ( or late adolescence ) and these cysts are rarely detected at a younger age ." ], [ "congenital seminal vesicle cysts in patients with ipsilateral renal agenesis are rare but this association is known and should be considered in the differential diagnosis of cystic pelvic masses in males with renal agenesis or dysplasia .", "ultrasound is useful for diagnosis but mri provides a more detailed analysis and accurate diagnosis ." ], [ "congenital seminal vesicle cysts in patients with ipsilateral renal agenesis are rare but this association is well known .", "pediatric surgeons should be aware and consider this entity in the differential diagnosis of cystic pelvic masses in males with renal agenesis or dysplasia .", "ultrasound is useful for diagnosis but magnetic resonance imaging provides a more detailed analysis and accurate diagnosis ." ] ]

[ "tori and exostoses are nodular protuberances of calcified bone and are designated according to their anatomic location.1 torus palatinus ( tp ) and torus mandibularis ( tm ) are the two most common types of intraoral osseous overgrowths.2,3 tp is a sessile , nodular bony mass commonly seen on the midline of the hard palate .", "tm is bony protuberance found on the lingual aspect of the mandible , in the canine and premolar region .", "buccal and palatal exostoses are multiple bony nodular masses found less frequently than tori.1,2,4 buccal exostoses occur as bilateral , smooth bony growth along the facial aspect of the maxillary and/or mandibular alveolus . commonly found to appear in the premolar - molar region . on palpation ,", "ulcerations may be seen as a result of trauma or any injury to the mucosa .", "their size may increase to several centimeters thus contributing to periodontal disease of adjoining teeth by retaining food during chewing instead of flushing away .", "usually no treatment is required , but for those possibly affecting the periodontal condition , or when the protruberances cause pain or discomfort to the patient , or when these bony enlargements cause pseudo swelling over the lip , then conservative surgical excision can be performed .", "some of the suggested causes include genetic factors , environmental factors , masticatory hyperfunction and continued jaw bone growth.5,6 the histologic features of tori and exostoses are identical.2 a very small exostosis and tori consist entirely of compact bone but when large and nodular , it consists of cancellous bone surrounded by cortical bone .", "the diagnosis of a buccal exostosis is based on the clinical examination along with radiographic interpretations .", "clinically , the torus may appear as numerous rounded protruberanes or calcified multiple lobules , whereas the exostosis is a single , smooth broad - based mass , may have a sharp , pointed bony projection producing tenderness just beneath the mucosa.8 lesions may slowly enlarge up to 3 - 4 cm in greatest diameter , but it does not have malignant transformation potential .", "buccal exostoses are usually found only on the facial surface of the maxillary alveolar bone , especially in the posterior segment .", "radiographically , exostosis appears as well - defined round or oval calcified structure superimposing the roots of teeth .", "the patients are having multiple bony growths or lesions which are not in the classic torus or buccal exostosis locations should be evaluated for gardner syndrome .", "this autosomal dominant syndrome shows other features such as intestinal polyposis and cutaneous cysts or fibromas.2,5 no bony exostosis or tori requires treatment unless it becomes large enough to interfere with periodontal health , denture placement , or cause recurrent traumatic ulcerations . when treatment is elected , the lesions should be cut - off or removed from the cortex using bone cutting bur or hand instruments .", "the diagnosis of a buccal exostosis is based on the clinical examination along with radiographic interpretations . clinically , the torus may appear as numerous rounded protruberanes or calcified multiple lobules , whereas the exostosis is a single , smooth broad - based mass , may have a sharp , pointed bony projection producing tenderness just beneath the mucosa.8 lesions may slowly enlarge up to 3 - 4 cm in greatest diameter , but it does not have malignant transformation potential .", "buccal exostoses are usually found only on the facial surface of the maxillary alveolar bone , especially in the posterior segment .", "radiographically , exostosis appears as well - defined round or oval calcified structure superimposing the roots of teeth .", "the patients are having multiple bony growths or lesions which are not in the classic torus or buccal exostosis locations should be evaluated for gardner syndrome .", "this autosomal dominant syndrome shows other features such as intestinal polyposis and cutaneous cysts or fibromas.2,5 no bony exostosis or tori requires treatment unless it becomes large enough to interfere with periodontal health , denture placement , or cause recurrent traumatic ulcerations . when treatment is elected , the lesions should be cut - off or removed from the cortex using bone cutting bur or hand instruments .", "a 20-year - old female patient reported to the department of periodontology , maharana pratap college of dentistry and research centre , gwalior ( madhya pradesh ) with bilateral masses just above the premolar and molar region in maxilla interfering in her smile and aesthetics ( figure 1 ) .", "she had noticed slow , but steady enlargement of the masses over the past 5 years .", "physical examination of the oral cavity revealed large , bilateral overgrowths located on the buccal aspect of the maxilla in the premolar and molar areas ( figure 2 ) .", "the overlying mucosa was thin and blanched , and generalized moderate gingivitis with minimal bone loss was present .", "the exostoses were oblong in shape , measuring approximately 1.7 cm 1 cm on the right side and 2 cm 1 cm on the left side .", "radiographic examination showed a well - defined radiopaque area covering the roots of the premolar and molar teeth .", "these bony protruberance caused by the thickening or enlargement of the cortical plate of the facial surface of the maxilla without any systemic abnormality helped to reach to diagnosis that it was multiple buccal exostoses . bilateral enlargement seen in premolar and molar region .", "generally , no treatment for buccal exostoses is required but as a patient was not happy with the bony masses seen during speech and smile .", "the full thickness flap was raised to expose the exostoses adequately ( figures 3 and 4 ) .", "the bony growth was cut with bone cutting carbide bur , no 702 ss white bur under continuous saline irrigation ( figure 5 ) .", "smoothening of the rough surface was carried out with bone file and granulation tissue were curetted .", "the surgical site was washed thoroughly with a solution of povidine iodine and saline in 1:1 proportions and flap was closed .", "a follow - up appointment was scheduled after 10 days of surgery to check the site and for suture removal .", "the tissue appeared healed , and the patient was totally asymptomatic after 10 days ( figure 6 ) .", "flap reflection revealing the bony mass of 2 cm 1 cm on left side flap reflection revealing bony mass of 1.7 cm 1 cm on right side .", "the multiple masses in the maxilla are consistent with multiple buccal exostoses , which are bony protuberances that arise from the cortical plates in the maxilla and mandible .", "they usually occur in the late teens and early adult years , and many continue to enlarge slowly over time .", "the etiology of the multiple exostoses remains unknown , although it has been suggested to be the outcome of a mild , chronic periosteal inflammation .", "furthermore , the patients with multiple bony growths , not in the classic buccal exostoses locations should be evaluated for gardner s syndrome .", "intestinal polyposis and cutaneous cysts or fibromas are other common features of the autosomal dominant gardner s syndrome.2,5 neither the torus nor the bony exostosis require treatment unless it becomes large enough to interfere with function , denture placement , cause recurring traumatic surface ulceration ( usually from sharp food such as potato chips or fish bones ) or as used to get autograft as it is a potent donor site.7 when treatment is elected , the bony mass may be removed using bone cutting bur or chiseled off through the base of the lesion .", "the case report presented above illustrates a unique and rare presentation of exostoses on the buccal side of the maxillary premolar - molar region , bilaterally .", "exostosis is rarely found on the facial surface of maxilla thus should not be ignored and should be carefully differentially diagnosed ." ]

[ "<S> buccal exostoses are broad - based , non - malignant surface growth occurring on the outer or facial surface of the maxilla and/or mandible , found usually in the premolar and molar region . </S>", "<S> etiology is still not established , but it has been suggested that the bony overgrowth can be because of abnormally increased masticatory forces to the teeth . they tend to appear in early adolescence and may very slowly increase in size with time . </S>", "<S> they are painless , self - limiting and may increase patient concern about poor esthetics , inability to perform oral hygiene procedures , and compromised periodontal health by causing food lodgment . </S>", "<S> the following article presents a very rare case of bilateral buccal - sided maxillary exostoses and its management with surgical exploration . </S>" ]

[ "Introduction", "Diagnosis, treatment and prognosis", "Case Report", "Discussion", "Conclusion" ]

[ [ "tori and exostoses are nodular protuberances of calcified bone and are designated according to their anatomic location.1 torus palatinus ( tp ) and torus mandibularis ( tm ) are the two most common types of intraoral osseous overgrowths.2,3 tp is a sessile , nodular bony mass commonly seen on the midline of the hard palate .", "tm is bony protuberance found on the lingual aspect of the mandible , in the canine and premolar region .", "buccal and palatal exostoses are multiple bony nodular masses found less frequently than tori.1,2,4 buccal exostoses occur as bilateral , smooth bony growth along the facial aspect of the maxillary and/or mandibular alveolus . commonly found to appear in the premolar - molar region . on palpation ,", "ulcerations may be seen as a result of trauma or any injury to the mucosa .", "their size may increase to several centimeters thus contributing to periodontal disease of adjoining teeth by retaining food during chewing instead of flushing away .", "usually no treatment is required , but for those possibly affecting the periodontal condition , or when the protruberances cause pain or discomfort to the patient , or when these bony enlargements cause pseudo swelling over the lip , then conservative surgical excision can be performed .", "some of the suggested causes include genetic factors , environmental factors , masticatory hyperfunction and continued jaw bone growth.5,6 the histologic features of tori and exostoses are identical.2 a very small exostosis and tori consist entirely of compact bone but when large and nodular , it consists of cancellous bone surrounded by cortical bone .", "the diagnosis of a buccal exostosis is based on the clinical examination along with radiographic interpretations .", "clinically , the torus may appear as numerous rounded protruberanes or calcified multiple lobules , whereas the exostosis is a single , smooth broad - based mass , may have a sharp , pointed bony projection producing tenderness just beneath the mucosa.8 lesions may slowly enlarge up to 3 - 4 cm in greatest diameter , but it does not have malignant transformation potential .", "buccal exostoses are usually found only on the facial surface of the maxillary alveolar bone , especially in the posterior segment .", "radiographically , exostosis appears as well - defined round or oval calcified structure superimposing the roots of teeth .", "the patients are having multiple bony growths or lesions which are not in the classic torus or buccal exostosis locations should be evaluated for gardner syndrome .", "this autosomal dominant syndrome shows other features such as intestinal polyposis and cutaneous cysts or fibromas.2,5 no bony exostosis or tori requires treatment unless it becomes large enough to interfere with periodontal health , denture placement , or cause recurrent traumatic ulcerations . when treatment is elected , the lesions should be cut - off or removed from the cortex using bone cutting bur or hand instruments ." ], [ "the diagnosis of a buccal exostosis is based on the clinical examination along with radiographic interpretations . clinically , the torus may appear as numerous rounded protruberanes or calcified multiple lobules , whereas the exostosis is a single , smooth broad - based mass , may have a sharp , pointed bony projection producing tenderness just beneath the mucosa.8 lesions may slowly enlarge up to 3 - 4 cm in greatest diameter , but it does not have malignant transformation potential .", "buccal exostoses are usually found only on the facial surface of the maxillary alveolar bone , especially in the posterior segment .", "radiographically , exostosis appears as well - defined round or oval calcified structure superimposing the roots of teeth .", "the patients are having multiple bony growths or lesions which are not in the classic torus or buccal exostosis locations should be evaluated for gardner syndrome .", "this autosomal dominant syndrome shows other features such as intestinal polyposis and cutaneous cysts or fibromas.2,5 no bony exostosis or tori requires treatment unless it becomes large enough to interfere with periodontal health , denture placement , or cause recurrent traumatic ulcerations . when treatment is elected , the lesions should be cut - off or removed from the cortex using bone cutting bur or hand instruments ." ], [ "a 20-year - old female patient reported to the department of periodontology , maharana pratap college of dentistry and research centre , gwalior ( madhya pradesh ) with bilateral masses just above the premolar and molar region in maxilla interfering in her smile and aesthetics ( figure 1 ) .", "she had noticed slow , but steady enlargement of the masses over the past 5 years .", "physical examination of the oral cavity revealed large , bilateral overgrowths located on the buccal aspect of the maxilla in the premolar and molar areas ( figure 2 ) .", "the overlying mucosa was thin and blanched , and generalized moderate gingivitis with minimal bone loss was present .", "the exostoses were oblong in shape , measuring approximately 1.7 cm 1 cm on the right side and 2 cm 1 cm on the left side .", "radiographic examination showed a well - defined radiopaque area covering the roots of the premolar and molar teeth .", "these bony protruberance caused by the thickening or enlargement of the cortical plate of the facial surface of the maxilla without any systemic abnormality helped to reach to diagnosis that it was multiple buccal exostoses . bilateral enlargement seen in premolar and molar region .", "generally , no treatment for buccal exostoses is required but as a patient was not happy with the bony masses seen during speech and smile .", "the full thickness flap was raised to expose the exostoses adequately ( figures 3 and 4 ) .", "the bony growth was cut with bone cutting carbide bur , no 702 ss white bur under continuous saline irrigation ( figure 5 ) .", "smoothening of the rough surface was carried out with bone file and granulation tissue were curetted .", "the surgical site was washed thoroughly with a solution of povidine iodine and saline in 1:1 proportions and flap was closed .", "a follow - up appointment was scheduled after 10 days of surgery to check the site and for suture removal .", "the tissue appeared healed , and the patient was totally asymptomatic after 10 days ( figure 6 ) .", "flap reflection revealing the bony mass of 2 cm 1 cm on left side flap reflection revealing bony mass of 1.7 cm 1 cm on right side ." ], [ "the multiple masses in the maxilla are consistent with multiple buccal exostoses , which are bony protuberances that arise from the cortical plates in the maxilla and mandible .", "they usually occur in the late teens and early adult years , and many continue to enlarge slowly over time .", "the etiology of the multiple exostoses remains unknown , although it has been suggested to be the outcome of a mild , chronic periosteal inflammation .", "furthermore , the patients with multiple bony growths , not in the classic buccal exostoses locations should be evaluated for gardner s syndrome .", "intestinal polyposis and cutaneous cysts or fibromas are other common features of the autosomal dominant gardner s syndrome.2,5 neither the torus nor the bony exostosis require treatment unless it becomes large enough to interfere with function , denture placement , cause recurring traumatic surface ulceration ( usually from sharp food such as potato chips or fish bones ) or as used to get autograft as it is a potent donor site.7 when treatment is elected , the bony mass may be removed using bone cutting bur or chiseled off through the base of the lesion ." ], [ "the case report presented above illustrates a unique and rare presentation of exostoses on the buccal side of the maxillary premolar - molar region , bilaterally .", "exostosis is rarely found on the facial surface of maxilla thus should not be ignored and should be carefully differentially diagnosed ." ] ]

[ "sleep can be disturbed by several factors such as illness , stress , and noise . over time", "these include poor memory , slower reaction time , emotional disturbances , and changes in the immune response ( 1 , 2 ) .", "however , administration of these drugs is accompanied by side effects including psychomotor impairment , drug dependence , tolerance , amnesia , rebound insomnia , and the potentiating effects of other central depressant drugs ( 3 ) . moreover , some patients with sleep disorders do not respond effectively to current therapeutics .", "therefore , studies to find new hypnotic agents with fewer side effects and more efficacy have continued .", "herbal agents have long been a valuable source for developing new therapeutics for disease treatment .", "ocimum basilicum ( o. basilicum ) is an annual herb belonging to the lamiaceae family and grows mostly in tropical regions such as india , africa , and south asia ( 4 ) . in traditional medicine ,", "o. basilicum is used to treat many disorders such as anxiety , diabetes , cardiovascular diseases , headaches , neurological pain , and seizures ( 5 , 6 ) .", "it has been demonstrated that the ethyl acetate fraction ( eaf ) of o. basilicum decreases ischemia - induced oxidative stress in the brain and improves short - term memory and motor coordination ( 5 ) .", "it was reported in some traditional medical texts that o. basilicum leads to sleep and a sedative state .", "also , some biological activities of o. basilicum were screened by ismail , and it was found that the essential oil of o. basilicum induces anticonvulsant and hypnotic activities ( 7 ) . in folk medicine , maceration is the most widely used form of o. basilicum preparation", ". however , there is no pharmacological evidence for the sedative - hypnotic effect of o. basilicum macerated extract .", "the present study was designed to evaluate the sleep - prolonging effects of hydro - alcoholic extract ( hae ) of o. basilicum ) and its fractions .", "dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st .", "dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) .", "the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade .", "a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) .", "hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours .", "the extract was then filtered through a 0.106 mm filter and dried in a water bath .", "the yield of the dried extract as related to the weight of the dried leaves was 21% . for the preparation of different fractions from hae ,", "10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction ,", "the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) .", "all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used .", "then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso .", "male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22 1c ) with a 12 hours light / dark cycle and free access to water and food .", "the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) .", "first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment , to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) .", "the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice .", "after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) .", "all materials were injected with a volume of 10 mg / kg ( 11 ) .", "the onset of sleep is the time that the mice stayed immobile and lost their righting reflex .", "the time interval between administration of pentobarbital and onset of sleep was considered sleep latency .", "nine groups , each containing two mice were used for determination of ld50 of hae .", "groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle .", "the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded .", "the mean of these two doses was considered the median lethal dose ( 14 , 15 ) .", "the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) .", "pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts .", "also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials .", "the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) .", "then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) .", "the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 .", "then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) .", "briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours .", "statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) .", "dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st .", "dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) .", "the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade . a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) .", "hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours .", "the extract was then filtered through a 0.106 mm filter and dried in a water bath .", "the yield of the dried extract as related to the weight of the dried leaves was 21% .", "for the preparation of different fractions from hae , 10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction ,", "the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) .", "all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used .", "then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso .", "male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22 1c ) with a 12 hours light / dark cycle and free access to water and food .", "the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) .", "first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment ,", "to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) .", "the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice .", "after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) .", "all materials were injected with a volume of 10 mg / kg ( 11 ) .", "the onset of sleep is the time that the mice stayed immobile and lost their righting reflex .", "the time interval between administration of pentobarbital and onset of sleep was considered sleep latency .", "nine groups , each containing two mice were used for determination of ld50 of hae .", "groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle .", "the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded .", "the mean of these two doses was considered the median lethal dose ( 14 , 15 ) .", "the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) .", "pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts .", "also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials .", "the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) .", "then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) .", "the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 .", "then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) .", "briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours .", "statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) .", "sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes ( figure 1 ) .", "as expected , the reference drug diazepam was able to increase the duration of sleep ( 42 4.6 minutes , p < 0.001 vs. control ) .", "the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51 3 minutes ( p < 0.001 ) , 79 3 minutes ( p < 0.001 ) , and 46 5 minutes ( p < 0.001 ) , respectively .", "the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) .", "the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100", "mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) .", "similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79 3 minutes and 30 2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) .", "the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ; p", "values are presented as mean sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity .", "duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40 4.5 minutes ( p < 0.001 vs. control ) , 30 2 minutes ( p < 0.001 vs. vehicle ) and 42 2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep .", "the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p", "< 0.05 vs. eaf group ; values are presented as mean sem ( n = 8) .", "figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7 0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5 0.4 minutes ( p < 0.001 ) .", "the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2 0.2 minutes ( p < 0.001 ) , 4.5 0.5 minutes ( p < 0.01 ) , and 4.4 0.4 minutes ( p < 0.01 ) , respectively . the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae .", "the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf .", "values are presented as mean sem ( n = 8) . among the different fractions of hae ,", "however , nbf significantly decreased the latency time to 4.7 0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7 0.4 minutes ) to 5.2 0.2 minutes , this effect was not statistically significant . the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively .", "the mean of these two doses ( 2.4 g / kg ) was considered as ld50 .", "the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) .", "it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract , cell viability was 100", " 3 , 102 2 , 103 2 , 110 3 , and 113 3% , respectively , as compared to the vehicle ( 100 2.5% ) .", "regarding l929 cells , the viability was 106 1.5 , 108 2 , 112 2 , 113 1 , and 123 2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively .", "again , there was no significant difference in the cell viability when compared to the vehicle ( 100 2% ) .", "the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum .", "sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes ( figure 1 ) .", "as expected , the reference drug diazepam was able to increase the duration of sleep ( 42 4.6 minutes , p < 0.001 vs. control ) .", "the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51 3 minutes ( p < 0.001 ) , 79 3 minutes ( p < 0.001 ) , and 46 5 minutes ( p < 0.001 ) , respectively .", "the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) .", "the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100", "mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) .", "similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79 3 minutes and 30 2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) .", "the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ; p", "values are presented as mean sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity .", "duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40 4.5 minutes ( p < 0.001 vs. control ) , 30 2 minutes ( p < 0.001 vs. vehicle ) and 42 2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep .", "the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p", "< 0.05 vs. eaf group ; values are presented as mean sem ( n = 8) .", "figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7 0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5 0.4 minutes ( p < 0.001 ) .", "the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2 0.2 minutes ( p < 0.001 ) , 4.5 0.5 minutes ( p < 0.01 ) , and 4.4 0.4 minutes ( p < 0.01 ) , respectively .", "the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae .", "the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf .", "values are presented as mean sem ( n = 8) . among the different fractions of hae ,", "however , nbf significantly decreased the latency time to 4.7 0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7 0.4 minutes ) to 5.2 0.2 minutes , this effect was not statistically significant .", "the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively .", "the mean of these two doses ( 2.4 g / kg ) was considered as ld50 .", "the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) .", "it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract ,", "cell viability was 100 3 , 102 2 , 103 2 , 110 3 , and 113 3% , respectively , as compared to the vehicle ( 100 2.5% ) .", "regarding l929 cells , the viability was 106 1.5 , 108 2 , 112 2 , 113 1 , and 123 2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively .", "again , there was no significant difference in the cell viability when compared to the vehicle ( 100 2% ) .", "the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum .", "the present study showed that o. basilicum further enhances sleep behavior , confirming that this plant has a hypnotic action as claimed in traditional medicine . to our knowledge , this is the first pharmacological study showing the effects of the macerated extract of this plant on sleep duration and sleep latency . also , this is the first work to determine the ld50 value for hae of o. basilicum and to assess possible cytotoxicity of this extract on neuronal cells .", "yet , results of this study are preliminary and need to be confirmed by further clinical trials .", "the hypnotic assessment method was based on prolongation of sleep induced by pentobarbital , which is the most commonly used method for screening sedative - hypnotic agents ( 11 - 13 ) . in agreement with the previously published works and as expected , diazepam significantly increased pentobarbital - induced sleeping time , indicating that our study method was optimized ( 17 , 18 ) .", "the effect of hae of o. basilicum on sleep latency was lower than that of diazepam .", "however , the sleep - prolonging effect of hae was comparable to that of diazepam , and , even at a dose of 50 mg / kg , hae had a greater effect . also , the hypnotic effect of hae of o. basilicum is greater when compared with the effect of essential oil of o. basilicum reported by ismail ( 4-fold and 2-fold increase in sleep duration , respectively ) ( 7 ) . according to our results , the hypnotic effect of hae was not dose - dependent in the range of given doses , and the maximum effect occurred with a dose of 50 mg / kg .", "therefore , we used this dose to investigate the effects of different fractions of hae . the fractions of hae were prepared to obtain better insight into the nature of compounds responsible for the hypnotic effect of o. basilicum : 1 , wf which extracts water - soluble constituents ( e.g. , glycosides , quaternary alkaloids , tannins ) ; 2 , eaf which extracts compounds of intermediate polarity ( e.g. , some flavonoids ) ; and 3 , nbf which extracts low polar agents ( e.g. , sterols , alkanes , and some terpenoids ) ( 19 , 20 ) .", "although wf , eaf , and nbf at a dose of 50 mg / kg were all able to increase sleep duration , not one of them could enhance sleep duration to the level induced by 50 mg / kg of hae .", "because hae contains the active constituents of all the above - mentioned fractions , it can be concluded that an additive effect was caused by the interaction between these constituents when hae was administrated .", "therefore , both polar and non - polar constituents in o. basilicum extract are involved in the sleep - prolonging effect of this plant . among wf ,", "eaf , and nbf fractions , nbf not only induced the maximum prolongation of sleep duration , but also was the only fraction to induce a significant decrease in sleep latency . on the other hand", "therefore , nbf contains a higher concentration of constituents responsible for the hypnotic effect of o. basilicum .", "these include terpenoids ( e.g. , linalool , eugenol ) , flavonoids ( e.g. , quercetrin , luteolin ) , alkaloids ( e.g. , rosmarinic acid ) , steroids ( e.g. , beta - sitisterol ) , and saponins ( 21 , 22 ) .", "it has been shown that o. basilicum contains high amounts of linalool , eugenol , and rosmarinic acid ( 7 ) .", "linck and coworkers demonstrated that linalool increases barbiturate - induced sleeping time in mice ( 23 ) .", "similarly , the sleep - prolonging effect of eugenol was reported by sharma et al .", "rosmarinic acid is found in methanolic extract of o. basilicum and can be isolated from the nbf of plant extracts ( 24 , 25 ) .", "it plays a major role in the sedative - hypnotic actions of some medicinal plants ( 26 ) .", "neurons located in the anterior hypothalamus release gamma - aminobutyric acid ( gaba ) to inhibit wake - promoting areas in the hypothalamus and brainstem ( 27 , 28 ) .", "barbiturates such as pentobarbital act on the gaba receptor s ionophore complex and favor the binding of gaba .", "benzodiazepines such as diazepam increase the affinity of gaba for its receptor and thereby enhance pentobarbital - induced sleeping time ( 29 ) .", "consistent with this , we observed that pretreatment of mice with flumazenil decreases the sleep - prolonging effect of diazepam .", "in agreement with our finding , awad et al . reported that rosmarinic acid , which is found in the extract of o. basilicum , can act as a gaba transaminase inhibitor and therefore increases the brain level of gaba ( 30 ) . therefore , it is rational to suggest that the sleep - prolonging effect of o. basilicum is mediated , at least in part , through the potentiating of the gabaergic system .", "it is now accepted that some natural compounds interact with the gabaergic system to increase sleep behavior ( 31 , 32 ) .", "the toxicity assay showed that the ld50 value for hae of o. basilicum is 2.4 g / kg .", "this dose is far from its hypnotic doses ( 25 - 100 mg / kg ) .", "also , hae , even at high concentrations , did not decrease the viability of neuronal and fibroblast cells .", "therefore , it seems that the hypnotic effect of o. basilicum is not accompanied by neurotoxicity . in conclusion", ", present data demonstrated that the macerated extract of o. basilicum potentiates sleeping behaviors without any cytotoxicity ." ]

[ "<S> backgroundsleep disorders are accompanied by several complications , and currently used soporific drugs can induce unwanted effects such as psychomotor impairment , tolerance , amnesia , and rebound insomnia.objectivesthe present study was carried out to investigate if ocimum basilicum has a sleep - prolonging effect.materials and methodsthis work was an experimental study on 72 mice which were randomly divided into 9 groups : saline ( control ) ; diazepam ( 3 mg / kg , positive control ) ; hydro - alcoholic extract ( hae ) of ocimum basilicum ( 25 , 50 , or 100 mg / kg ) ; ethyl acetate fraction ( eaf , 50 mg / kg ) ; n - butanol fraction ( nbf , 50 mg / kg ) ; water fraction ( wf , 50 mg / kg ) ; and saline containing 10% dmso ( vehicle for eaf and nbf ) . </S>", "<S> all the test compounds were injected intraperitoneally ( ip ) 30 minutes before pentobarbital administration ( 30 mg / kg ) . duration and latency of pentobarbital - induced sleep were recorded . </S>", "<S> also , ld50 of hae was determined and the cytotoxicity of hae was tested on neural and fibroblast cells using the mtt assay.resultshae increased the duration of pentobarbital - induced sleep at doses of 25 , 50 , and 100 mg / kg ( p < 0.001 ) . the hypnotic effect of hae was comparable to that induced by diazepam . similarly , wf , eaf , and nbf at 50 mg / kg could increase sleep duration . </S>", "<S> the sleep latency was decreased by hae ( p < 0.01 - p < 0.001 ) and nbf ( p </S>", "<S> < 0.001 ) , but not by wf and eaf . the ld50 value for hae was found to be 2.4 g / kg . </S>", "<S> hae had no effect on the viability of neuronal pc12 cells and l929 fibroblast cells.conclusionsthe present data demonstrated that ocimum basilicum potentiates sleeping behaviors without any cytotoxicity . </S>", "<S> the main component ( s ) responsible for the hypnotic effects of this plant is most likely a non - polar agent ( s ) which is found in nbf . </S>", "<S> isolation of the active constituents may yield a novel sedative drug . </S>" ]

[ "1. Background", "2. Objectives", "3. Materials and Methods", "3.1. Drugs and Chemicals", "3.2. Plant Collection and Extraction", "3.3. Animals", "3.4. Evaluation of Pentobarbital-Induced Sleep", "3.5. LD50 Determination", "3.6. Neurotoxicity Assessment", "3.7. Statistics", "4. Results", "4.1. Effects of O. Basilicum on Duration of Sleep", "4.2. Effects of O. Basilicum on Sleep Latency", "4.3. Toxicity Assessments", "5. Discussion" ]

[ [ "sleep can be disturbed by several factors such as illness , stress , and noise . over time", "these include poor memory , slower reaction time , emotional disturbances , and changes in the immune response ( 1 , 2 ) .", "however , administration of these drugs is accompanied by side effects including psychomotor impairment , drug dependence , tolerance , amnesia , rebound insomnia , and the potentiating effects of other central depressant drugs ( 3 ) . moreover , some patients with sleep disorders do not respond effectively to current therapeutics .", "therefore , studies to find new hypnotic agents with fewer side effects and more efficacy have continued .", "herbal agents have long been a valuable source for developing new therapeutics for disease treatment .", "ocimum basilicum ( o. basilicum ) is an annual herb belonging to the lamiaceae family and grows mostly in tropical regions such as india , africa , and south asia ( 4 ) . in traditional medicine ,", "o. basilicum is used to treat many disorders such as anxiety , diabetes , cardiovascular diseases , headaches , neurological pain , and seizures ( 5 , 6 ) .", "it has been demonstrated that the ethyl acetate fraction ( eaf ) of o. basilicum decreases ischemia - induced oxidative stress in the brain and improves short - term memory and motor coordination ( 5 ) .", "it was reported in some traditional medical texts that o. basilicum leads to sleep and a sedative state .", "also , some biological activities of o. basilicum were screened by ismail , and it was found that the essential oil of o. basilicum induces anticonvulsant and hypnotic activities ( 7 ) . in folk medicine , maceration is the most widely used form of o. basilicum preparation", ". however , there is no pharmacological evidence for the sedative - hypnotic effect of o. basilicum macerated extract ." ], [ "the present study was designed to evaluate the sleep - prolonging effects of hydro - alcoholic extract ( hae ) of o. basilicum ) and its fractions ." ], [ "dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st .", "dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) .", "the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade .", "a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) .", "hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours .", "the extract was then filtered through a 0.106 mm filter and dried in a water bath .", "the yield of the dried extract as related to the weight of the dried leaves was 21% . for the preparation of different fractions from hae ,", "10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction ,", "the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) .", "all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used .", "then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso .", "male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22 1c ) with a 12 hours light / dark cycle and free access to water and food .", "the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) .", "first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment , to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) .", "the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice .", "after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) .", "all materials were injected with a volume of 10 mg / kg ( 11 ) .", "the onset of sleep is the time that the mice stayed immobile and lost their righting reflex .", "the time interval between administration of pentobarbital and onset of sleep was considered sleep latency .", "nine groups , each containing two mice were used for determination of ld50 of hae .", "groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle .", "the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded .", "the mean of these two doses was considered the median lethal dose ( 14 , 15 ) .", "the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) .", "pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts .", "also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials .", "the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) .", "then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) .", "the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 .", "then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) .", "briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours .", "statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) ." ], [ "dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st .", "dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) ." ], [ "the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade . a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) .", "hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours .", "the extract was then filtered through a 0.106 mm filter and dried in a water bath .", "the yield of the dried extract as related to the weight of the dried leaves was 21% .", "for the preparation of different fractions from hae , 10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction ,", "the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) .", "all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used .", "then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso ." ], [ "male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22 1c ) with a 12 hours light / dark cycle and free access to water and food .", "the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) .", "first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment ,", "to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) ." ], [ "the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice .", "after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) .", "all materials were injected with a volume of 10 mg / kg ( 11 ) .", "the onset of sleep is the time that the mice stayed immobile and lost their righting reflex .", "the time interval between administration of pentobarbital and onset of sleep was considered sleep latency ." ], [ "nine groups , each containing two mice were used for determination of ld50 of hae .", "groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle .", "the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded .", "the mean of these two doses was considered the median lethal dose ( 14 , 15 ) ." ], [ "the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) .", "pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts .", "also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials .", "the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) .", "then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) .", "the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 .", "then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) .", "briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours ." ], [ "statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) ." ], [ "sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes ( figure 1 ) .", "as expected , the reference drug diazepam was able to increase the duration of sleep ( 42 4.6 minutes , p < 0.001 vs. control ) .", "the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51 3 minutes ( p < 0.001 ) , 79 3 minutes ( p < 0.001 ) , and 46 5 minutes ( p < 0.001 ) , respectively .", "the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) .", "the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100", "mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) .", "similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79 3 minutes and 30 2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) .", "the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ; p", "values are presented as mean sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity .", "duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40 4.5 minutes ( p < 0.001 vs. control ) , 30 2 minutes ( p < 0.001 vs. vehicle ) and 42 2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep .", "the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p", "< 0.05 vs. eaf group ; values are presented as mean sem ( n = 8) .", "figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7 0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5 0.4 minutes ( p < 0.001 ) .", "the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2 0.2 minutes ( p < 0.001 ) , 4.5 0.5 minutes ( p < 0.01 ) , and 4.4 0.4 minutes ( p < 0.01 ) , respectively . the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae .", "the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf .", "values are presented as mean sem ( n = 8) . among the different fractions of hae ,", "however , nbf significantly decreased the latency time to 4.7 0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7 0.4 minutes ) to 5.2 0.2 minutes , this effect was not statistically significant . the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively .", "the mean of these two doses ( 2.4 g / kg ) was considered as ld50 .", "the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) .", "it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract , cell viability was 100", " 3 , 102 2 , 103 2 , 110 3 , and 113 3% , respectively , as compared to the vehicle ( 100 2.5% ) .", "regarding l929 cells , the viability was 106 1.5 , 108 2 , 112 2 , 113 1 , and 123 2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively .", "again , there was no significant difference in the cell viability when compared to the vehicle ( 100 2% ) .", "the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum ." ], [ "sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes ( figure 1 ) .", "as expected , the reference drug diazepam was able to increase the duration of sleep ( 42 4.6 minutes , p < 0.001 vs. control ) .", "the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51 3 minutes ( p < 0.001 ) , 79 3 minutes ( p < 0.001 ) , and 46 5 minutes ( p < 0.001 ) , respectively .", "the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) .", "the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100", "mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) .", "similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79 3 minutes and 30 2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) .", "the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) .", "flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ; p", "values are presented as mean sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity .", "duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40 4.5 minutes ( p < 0.001 vs. control ) , 30 2 minutes ( p < 0.001 vs. vehicle ) and 42 2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep .", "the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p", "< 0.05 vs. eaf group ; values are presented as mean sem ( n = 8) ." ], [ "figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7 0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5 0.4 minutes ( p < 0.001 ) .", "the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2 0.2 minutes ( p < 0.001 ) , 4.5 0.5 minutes ( p < 0.01 ) , and 4.4 0.4 minutes ( p < 0.01 ) , respectively .", "the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae .", "the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf .", "values are presented as mean sem ( n = 8) . among the different fractions of hae ,", "however , nbf significantly decreased the latency time to 4.7 0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7 0.4 minutes ) to 5.2 0.2 minutes , this effect was not statistically significant ." ], [ "the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively .", "the mean of these two doses ( 2.4 g / kg ) was considered as ld50 .", "the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) .", "it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract ,", "cell viability was 100 3 , 102 2 , 103 2 , 110 3 , and 113 3% , respectively , as compared to the vehicle ( 100 2.5% ) .", "regarding l929 cells , the viability was 106 1.5 , 108 2 , 112 2 , 113 1 , and 123 2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively .", "again , there was no significant difference in the cell viability when compared to the vehicle ( 100 2% ) .", "the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum ." ], [ "the present study showed that o. basilicum further enhances sleep behavior , confirming that this plant has a hypnotic action as claimed in traditional medicine . to our knowledge , this is the first pharmacological study showing the effects of the macerated extract of this plant on sleep duration and sleep latency . also , this is the first work to determine the ld50 value for hae of o. basilicum and to assess possible cytotoxicity of this extract on neuronal cells .", "yet , results of this study are preliminary and need to be confirmed by further clinical trials .", "the hypnotic assessment method was based on prolongation of sleep induced by pentobarbital , which is the most commonly used method for screening sedative - hypnotic agents ( 11 - 13 ) . in agreement with the previously published works and as expected , diazepam significantly increased pentobarbital - induced sleeping time , indicating that our study method was optimized ( 17 , 18 ) .", "the effect of hae of o. basilicum on sleep latency was lower than that of diazepam .", "however , the sleep - prolonging effect of hae was comparable to that of diazepam , and , even at a dose of 50 mg / kg , hae had a greater effect . also , the hypnotic effect of hae of o. basilicum is greater when compared with the effect of essential oil of o. basilicum reported by ismail ( 4-fold and 2-fold increase in sleep duration , respectively ) ( 7 ) . according to our results , the hypnotic effect of hae was not dose - dependent in the range of given doses , and the maximum effect occurred with a dose of 50 mg / kg .", "therefore , we used this dose to investigate the effects of different fractions of hae . the fractions of hae were prepared to obtain better insight into the nature of compounds responsible for the hypnotic effect of o. basilicum : 1 , wf which extracts water - soluble constituents ( e.g. , glycosides , quaternary alkaloids , tannins ) ; 2 , eaf which extracts compounds of intermediate polarity ( e.g. , some flavonoids ) ; and 3 , nbf which extracts low polar agents ( e.g. , sterols , alkanes , and some terpenoids ) ( 19 , 20 ) .", "although wf , eaf , and nbf at a dose of 50 mg / kg were all able to increase sleep duration , not one of them could enhance sleep duration to the level induced by 50 mg / kg of hae .", "because hae contains the active constituents of all the above - mentioned fractions , it can be concluded that an additive effect was caused by the interaction between these constituents when hae was administrated .", "therefore , both polar and non - polar constituents in o. basilicum extract are involved in the sleep - prolonging effect of this plant . among wf ,", "eaf , and nbf fractions , nbf not only induced the maximum prolongation of sleep duration , but also was the only fraction to induce a significant decrease in sleep latency . on the other hand", "therefore , nbf contains a higher concentration of constituents responsible for the hypnotic effect of o. basilicum .", "these include terpenoids ( e.g. , linalool , eugenol ) , flavonoids ( e.g. , quercetrin , luteolin ) , alkaloids ( e.g. , rosmarinic acid ) , steroids ( e.g. , beta - sitisterol ) , and saponins ( 21 , 22 ) .", "it has been shown that o. basilicum contains high amounts of linalool , eugenol , and rosmarinic acid ( 7 ) .", "linck and coworkers demonstrated that linalool increases barbiturate - induced sleeping time in mice ( 23 ) .", "similarly , the sleep - prolonging effect of eugenol was reported by sharma et al .", "rosmarinic acid is found in methanolic extract of o. basilicum and can be isolated from the nbf of plant extracts ( 24 , 25 ) .", "it plays a major role in the sedative - hypnotic actions of some medicinal plants ( 26 ) .", "neurons located in the anterior hypothalamus release gamma - aminobutyric acid ( gaba ) to inhibit wake - promoting areas in the hypothalamus and brainstem ( 27 , 28 ) .", "barbiturates such as pentobarbital act on the gaba receptor s ionophore complex and favor the binding of gaba .", "benzodiazepines such as diazepam increase the affinity of gaba for its receptor and thereby enhance pentobarbital - induced sleeping time ( 29 ) .", "consistent with this , we observed that pretreatment of mice with flumazenil decreases the sleep - prolonging effect of diazepam .", "in agreement with our finding , awad et al . reported that rosmarinic acid , which is found in the extract of o. basilicum , can act as a gaba transaminase inhibitor and therefore increases the brain level of gaba ( 30 ) . therefore , it is rational to suggest that the sleep - prolonging effect of o. basilicum is mediated , at least in part , through the potentiating of the gabaergic system .", "it is now accepted that some natural compounds interact with the gabaergic system to increase sleep behavior ( 31 , 32 ) .", "the toxicity assay showed that the ld50 value for hae of o. basilicum is 2.4 g / kg .", "this dose is far from its hypnotic doses ( 25 - 100 mg / kg ) .", "also , hae , even at high concentrations , did not decrease the viability of neuronal and fibroblast cells .", "therefore , it seems that the hypnotic effect of o. basilicum is not accompanied by neurotoxicity . in conclusion", ", present data demonstrated that the macerated extract of o. basilicum potentiates sleeping behaviors without any cytotoxicity ." ] ]

[ "timber harvesting and logging is one of the vital activities carried out in the forestry sector in malawi .", "this is because it creates a room for establishment of new stand in the process of removing the old age .", "this also reduces the impact which may follow if the stands are left unharvested past their harvesting age .", "timber harvesting activities cause some degree of soil disturbance on soil physical , chemical , and biological properties ; this also reduces site productivity .", "ground - based logging systems can cause serious disturbance to the physical properties of forest soil due to soil compaction .", "bulk density , soil porosity , and temperature are amongst the seven physical properties of soil ; hence harvesting and logging has an impact on them .", "the bulk density of the soil is defined as the mass per unit volume of the soil and represents the ratio of the mass of solids to the total or bulk volume of the soil .", "soil porosity is that part of the bulk volume not occupied by either mineral or organic matter but it is an open space occupied by air or water while soil temperature is the thermal diffusivity or conductivity of soil at different weather conditions .", "for instance , soil bulk density reflects the soil 's ability to function for structural support , water and solute movement , and soil aeration .", "in addition , soil temperature plays an important role in many processes , which take place in the soil such as chemical reactions and biological interactions .", "the metabolic activity of soil microorganisms , seed fermentation , and plant roots is directly influenced by soil temperature in terms of water movement and soil freezing . \n", "pinus kesiya royle ex gordon occurs naturally in himalaya region ( asian ) : burma , china , india , laos , philippines , thailand , tibet , and vietnam .", "this species particularly grows well at altitudes from 600 to 1800 m above sea level .", "the trees can reach heights of 3035 or 45 m with straight , cylindrical trunk .", "pinus kesiya is a major exotic plantation species in malawi and other southern african countries .", "its success as an exotic is due to its fast growth rate and wide adaptability .", "current studies have shown a large degree of site specificity both in soil and tree growth responses to soil compaction [ 12 , 13 ] .", "these research results have encouraged new intuitions on the effects of soil compaction on forest productivity in different soil types and proved the importance of having site - specific soil quality assessments in forests .", "moreover , there is still limited information on the relationships between soil compaction / disturbances and tree growth .", "generalization about negative effects of harvest - related soil disturbance on tree growth may be in error because these impacts depend on their type and severity and on soil properties and climatic conditions [ 12 , 14 ] .", "in addition many studies have assessed tree growth and soil response in logged sites using retrospective approach , which may not allow ascertaining the original type , degree , and extent of disturbance .", "therefore , the present study was undertaken to determine the impacts of soil disturbance and compaction on soil physical properties and tree growth and the effectiveness of tillage in maintaining or enhancing site productivity for intensively managed pinus kesiya sites in the study area using prospective approach .", "the study was conducted in malawi located in southern africa in the tropical savannah region at chongoni plantation ( 14 19 s , 34 16 e , and 1650 m above sea level ) .", "the rain season starts from around mid - november to late april and the rainfall pattern is bimodal with peak periods in january and february .", "chongoni plantation receives 1200 mm to 1800 mm rainfall per annum , with annual temperature ranging from 7c to 25c .", "the dry season runs from may to october . it is situated about 85 km southeast of lilongwe the capital . in addition , the experimental site has homogenous soil and topographic conditions .", "the site is on gentle slope ( < 10% ) ; the soils are deep , well - drained , mostly stone - free , and high in ferralsols , acrisols , and nitosols , with site index of 20 m at 25 years for pinus kesiya .", "the old - growth stand previously occupying the site was planted in 1980 with pinus kesiya and was harvested from may to august 2010 .", "treatments plots are 70 m by 70 m ( 0.49 ha ) .", "the treatments were as follows : t1:stem - only removal with soil compaction;t2:stem - only removal with soil compaction plus tillage;t3:stem - only removal with no soil compaction ( control ) .", "stem - only removal with soil compaction ; stem - only removal with soil compaction plus tillage ; stem - only removal with no soil compaction ( control ) .", "the stem - only removal harvest followed merchantability standards of 5.5 m log length and 8 to 13 cm small end top diameter ( i.e. , cut - to - length ( ctl ) operation ) .", "the harvested trees were directionally hand - felled between june and august 2010 so that all tree tops remained within the plot . in t1 and", "t2 logs were pulled by a tractor from the stump area to the road side before being transported to the sawmill . in t2 further thorough soil tillage to the depth of 60 cm was accomplished by using a plough . in t3 sulkies were used to remove the logs from the stump area to the road side .", "ten equally spaced traffic lanes were identified in each plot to be trafficked to make soil disturbance comparable across blocks .", "every other lane was trafficked twice to more closely mimic traffic patterns that operationally occur when a tractor or a sulky is used during harvesting . in december 2010 , the plots were planted with pinus kesiya seedlings on a 2.75 m by 2.75 m spacing ( 1320 trees ha ) using hole planting .", "all treatments including spot weeding in the three growing seasons after planting were carried out in order to eliminate the confounding effects that soil disturbance and compaction can have on vegetation communities and competition pressure .", "twenty ( 20 ) soil samples from each treatment were collected to a depth of 010 cm and 1020 cm in september 2010 using a 31.2 mm diameter punch tube volumetric sampler . according to walworth ,", "soil samples of 15 to 25 collected from randomly selected locations in a field are a good representative of the entire field .", "samples with significant amounts of sound or decayed wood material were discarded and replaced with new samples . in compacted and compacted and tilled plots , sampling was restricted to traffic lanes .", "soil samples collected were then oven - dried at 105c for determination of bulk density and soil gravimetric and volumetric water content .", "the measurements were taken at the point where each tree was planted and the rut depth from the original soil level was recorded .", "furthermore , around every other measurement tree , the percentage of each disturbance class in a tree centred 2.75 by 2.75 m square area was recorded .", "then the percentage of the ground area and the tree frequency in each soil disturbance class was calculated .", "soil strength was measured in september 2012 in twenty locations per plot using a cone penetrometer at 5 cm intervals to the 60 cm depth on each treatment .", "high outlier values created when roots or buried wood was hit by the penetrometer were noted on the cards in the field and those measurements were discarded from the dataset .", "soil cores were randomly collected in all the treatments using 75 cm cylinder rings centred at 5 cm and 15 cm depths to characterize the 010 cm and 1020 cm depths , respectively .", "soil water retention curves and particle density were determined at science laboratory of malawi college of forestry and wildlife .", "total porosity was determined by both the gravimetric method with water saturation and the particle density method .", "volumetric water contents were determined after equilibrating the soil with water at tensions of 10 , 20 , 200 , 400 , and 1500 kpa .", "pore size distribution and water retention were then determined by the procedure as outlined by ares et al . .", "measurements of soil temperature were taken monthly between 08.00 hours and 10.00 hours from january 2011 to december 2013 at 20 cm depth in all the treatment plots .", "trees in all treatments were measured immediately after planting and yearly in the three consecutive growing seasons .", "the parameters measured were total height ( h ) using a telescopic pole , stem basal diameter ( bd ) measured at a permanently marked location 15 cm above the ground level in growing seasons one through three , and stem diameter ( dbh ) at 1.3 m above ground in the growing seasons two and three .", "stem diameters were measured using a diameter tape . a stem volume index ( svi )", "data obtained were subjected to kolmogorov - smirnov d and normal probability plot tests using statistical analysis of systems software version 9.1.3 .", ". then , harvesting and logging effects on soil strength and bulk density were analysed as a mixed model of repeated measures data with soil depth and soil disturbance as fixed effects and block as a random effect .", "the model was fitted using the mixed procedure in sas , which estimates variance components using restricted maximum likelihood methods .", "differences between treatment means were separated using fischer 's least significant difference ( lsd ) at the 0.05 level . a repeated measures analysis was appropriate because measurements of bulk density and soil strength were done at different depths at the same sampling point , and , therefore , sampling errors were not independent .", "measurements taken at adjacent depths are expected to be more correlated than measurements taken some distance apart .", "the covariance structures associated with the within subject factor were selected by choosing those with lowest value for the bayesian criterion and akaike 's information criterion .", "a mixed model approach was also used to test for soil disturbance effects on tree growth in years 0 to 3 .", "the result for ground area affected by the soil disturbance is presented in figure 1 .", "the results indicate that about fifty - two percent of the area of compacted plots was affected by the vehicular traffic .", "seventy percent of the trees within the measurement plots of the compacted treatment were located on some degree of soil disturbance .", "rut depths were shallow to moderate with averages of 2.8 cm for t2 and 13.1 cm for t3 , respectively .", "soil bulk density as related to soil depth and soil disturbance in the study site is presented in table 1 .", "the results show that there was a significant ( p < 0.001 ) difference for soil bulk density between soil depths .", "the depth of 1020 cm had higher soil bulk density than the 010 cm depth .", "similarly , there was a significant difference for soil bulk density among treatments , with t3 having a higher soil bulk density than t1 and t2 .", "relative to t1 , soil bulk density at the depth of 010 cm increased 28.9% for t2 and 48.9% for t3 . at the depth of 1020 ", "cm , the increase in soil bulk density was 22.2% and 38.9% for t2 and t3 , respectively , in relation to t1 .", "soil bulk density significantly ( p < 0.001 ) increased with both soil disturbance and depth .", "the results indicate that soil strength significantly ( p < 0.001 ) increased with soil disturbance and depth , with t1 producing a lower soil strength than class t2 and t3 .", "however , there was no significant ( p > 0.05 ) difference for soil strength between t2 and t3 with increase in depth .", "total soil porosity for all the treatments related to soil depth in the study sites is given in table 2 .", "the results indicate that there was no significant ( p < 0.05 ) difference for soil porosity between soil depth , even though the depth of 010 cm produced higher soil porosity than 1020 cm depth . there was a significant", "( p < 0.001 ) difference for soil porosity among the different treatments with noncompacted soil producing higher soil porosity than the compacted and compacted plus tillage treatments in both soil depths of 010 cm and 1020 cm , respectively .", "however , there was no significant ( p < 0.05 ) difference between noncompacted and compacted plus tillage treatments in soil porosity in both soil depths of 010 cm and 1020 cm , respectively . on compacted soil ,", "total porosity at 010 cm depth decreased by 13.8% compared with noncompacted soil and by 16.1% at the 1020 cm depth .", "the results indicate that there was a significant ( p < 0.001 ) difference in water retention by different treatments with compacted soil consistently retaining more water at 20 to 400 kpa water potential than noncompacted or compacted plus tillage soil at both 010 cm and 1020 cm depth .", "soil temperature , measured once a month at 020 cm depth , was not significantly ( p > 0.05 ) different between compacted and noncompacted areas ( data not shown ) .", "tree mortality and growth for pinus kesiya throughout the three growing seasons are presented in table 3 .", "the results indicate that there were no significant ( p > 0.05 ) differences in mean height , stem basal diameter , diameter at breast height , stem volume index , and mortality for trees growing on compacted , compacted plus tillage , and noncompacted treatments at years one to three .", "this study illustrates that the amount of soil disturbance in cut - to - length ( ctl ) operation by a tractor is about forty - eight percent .", "tepp reported a 38% soil disturbance using a ctl harvester and forwarder , while geist and cochran found that tractor harvesting using ctl produced 36% soil disturbance .", "however , the present study results are higher than those reported by [ 25 , 26 ] .", "armlovich reported that the amount of soil disturbance in a cut - to - length ( ctl ) operation had an average disturbance of 23.2% while in a study by gingras average disturbance was 25% .", "this difference may arise because in the present study , equipment traffic was applied when soil water contents were at somewhat drier than field water capacity , which is considered to correspond to a given soil water potential [ 12 , 27 ] . according to heninger et al . and", "mcnabb and boersma , the degree of soil disturbance depends on the intensity of site treatment , type of equipment used , time of year when harvesting occurs , frequency of entry , amount of slash or litter on site , soil moisture , and soil type .", "soil bulk density significantly ( p < 0.001 ) increased with both soil disturbance and depth .", "the results are in agreement with those reported by [ 12 , 30 ] . according to froese , in the upper soil , biological activity ( roots , animals , etc . )", "can act to reduce resistance and soil bulk density while at lower depths soil texture , gravel content , and structure may increase soil resistance and soil bulk density ; hence soil bulky density increases with depth .", "soil strength in this study also increased after vehicle trafficking to about 60 cm depth but it was not detrimental for tree root growth of 2000 to 3000 kpa depending on tree species and soil conditions [ 12 , 31 , 33 ] .", "the mean soil strength by depth increment in the study site never exceeded 1200 kpa .", "the compacted soils decreased the total soil porosity at the study site by 13.8% and 16.1% in the depths of 010 cm and 1020 cm , respectively , compared to the noncompacted soils .", "these results are within the range of those reported by [ 12 , 34 ] .", "according to kim , the decrease of soil porosity in t2 as compared to t1 is because of soil compaction . in t2 soil", "is highly compacted due to soil texture and structure by determining the size , number , and interconnection of pores .", "coarse - textured soils have many large ( macro)pores because of the loose arrangement of larger particles with one another .", "because fine - textured soils have both macro- and micropores , they generally have a greater total porosity , or sum of all pores , than coarse - textured soils .", "this is in agreement with the results reported by [ 12 , 38 ] . according to ares et al . ", "compacted soil retained more water than noncompacted soil because compression converted larger pores to smaller ones , especially in the upper part of the soil profile .", "the study reveals that mean height , stem basal diameter , diameter at breast height , stem volume index , and mortality for pinus kesiya trees species were not reduced by soil disturbance or compaction despite the fact that the soil physical properties were affected .", "values for these growth indicators were even greater for trees on disturbed soil than those on nondisturbed soil in years 2 and 3 .", "reported that douglas - fir growth was not reduced by soil disturbance and compaction even though the soil physical properties were affected from ground - based harvest . according to ares et al . ,", "since the growth was not reduced by the soil disturbance or compaction , this is an indication that bulky density , soil strength , and macroporosity did not reach levels in compacted areas that can reduce tree growth . the observed bulk density in the compacted area in this study ranged from 0.45 to 0.66 mg m , which is within the 0.50 to 1.11 mg m range for pinus kesiya seedlings growth [ 40 , 41 ] .", "furthermore , the soil strength observed in this study did not exceed 1200 kpa ; that is , it remained well below the critical threshold for tree root growth considered to be around 2000 to 3000 kpa depending on tree species and soil conditions .", "soil physical properties particularly soil bulk density , soil strength , and total soil porosity were greatly affected by compaction which was brought by harvesting and logging . however , the extent of disturbance was not detrimental for early growth of planted pinus kesiya .", "the high organic matter content , low bulk density , and low compressibility of the soil contributed to buffering the harvest impacts and made this soil conducive for intensive forest management and ground based harvesting systems .", "soil compaction could also be considered to be beneficial to early tree growth at three years .", "increased water available in the 20 to 400 kpa range on compacted traffic lanes may explain this increased growth ." ]

[ "<S> a study was conducted to determine the impacts of soil disturbance and compaction on soil physical properties and tree growth and the effectiveness of tillage in maintaining or enhancing site productivity for intensively managed pinus kesiya royle ex gordon sites in dedza , malawi . </S>", "<S> the results indicate that about fifty - two percent of the area of compacted plots was affected by the vehicular traffic . </S>", "<S> seventy percent of the trees were planted on microsites with some degree of soil disturbance . </S>", "<S> soil bulk density at 020 cm depth increased from 0.45 to 0.66 mg m3 in the most compacted portions of traffic lanes . </S>", "<S> soil strength in traffic lanes increased at all 60 cm depth but never exceeded 1200 kpa . </S>", "<S> volumetric soil water content in compacted traffic lanes was greater than that in noncompacted soil . </S>", "<S> total soil porosity decreased 13.8% to 16.1% with compaction , while available water holding capacity increased . </S>", "<S> the study revealed no detrimental effects on tree height and diameter from soil disturbance or compaction throughout the three growing season . at the ages of two and three , </S>", "<S> a tree volume index was actually greater for trees planted on traffic lanes than those on nondisturbed soil . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion" ]

[ [ "timber harvesting and logging is one of the vital activities carried out in the forestry sector in malawi .", "this is because it creates a room for establishment of new stand in the process of removing the old age .", "this also reduces the impact which may follow if the stands are left unharvested past their harvesting age .", "timber harvesting activities cause some degree of soil disturbance on soil physical , chemical , and biological properties ; this also reduces site productivity .", "ground - based logging systems can cause serious disturbance to the physical properties of forest soil due to soil compaction .", "bulk density , soil porosity , and temperature are amongst the seven physical properties of soil ; hence harvesting and logging has an impact on them .", "the bulk density of the soil is defined as the mass per unit volume of the soil and represents the ratio of the mass of solids to the total or bulk volume of the soil .", "soil porosity is that part of the bulk volume not occupied by either mineral or organic matter but it is an open space occupied by air or water while soil temperature is the thermal diffusivity or conductivity of soil at different weather conditions .", "for instance , soil bulk density reflects the soil 's ability to function for structural support , water and solute movement , and soil aeration .", "in addition , soil temperature plays an important role in many processes , which take place in the soil such as chemical reactions and biological interactions .", "the metabolic activity of soil microorganisms , seed fermentation , and plant roots is directly influenced by soil temperature in terms of water movement and soil freezing . \n", "pinus kesiya royle ex gordon occurs naturally in himalaya region ( asian ) : burma , china , india , laos , philippines , thailand , tibet , and vietnam .", "this species particularly grows well at altitudes from 600 to 1800 m above sea level .", "the trees can reach heights of 3035 or 45 m with straight , cylindrical trunk .", "pinus kesiya is a major exotic plantation species in malawi and other southern african countries .", "its success as an exotic is due to its fast growth rate and wide adaptability .", "current studies have shown a large degree of site specificity both in soil and tree growth responses to soil compaction [ 12 , 13 ] .", "these research results have encouraged new intuitions on the effects of soil compaction on forest productivity in different soil types and proved the importance of having site - specific soil quality assessments in forests .", "moreover , there is still limited information on the relationships between soil compaction / disturbances and tree growth .", "generalization about negative effects of harvest - related soil disturbance on tree growth may be in error because these impacts depend on their type and severity and on soil properties and climatic conditions [ 12 , 14 ] .", "in addition many studies have assessed tree growth and soil response in logged sites using retrospective approach , which may not allow ascertaining the original type , degree , and extent of disturbance .", "therefore , the present study was undertaken to determine the impacts of soil disturbance and compaction on soil physical properties and tree growth and the effectiveness of tillage in maintaining or enhancing site productivity for intensively managed pinus kesiya sites in the study area using prospective approach ." ], [ "the study was conducted in malawi located in southern africa in the tropical savannah region at chongoni plantation ( 14 19 s , 34 16 e , and 1650 m above sea level ) .", "the rain season starts from around mid - november to late april and the rainfall pattern is bimodal with peak periods in january and february .", "chongoni plantation receives 1200 mm to 1800 mm rainfall per annum , with annual temperature ranging from 7c to 25c .", "the dry season runs from may to october . it is situated about 85 km southeast of lilongwe the capital . in addition , the experimental site has homogenous soil and topographic conditions .", "the site is on gentle slope ( < 10% ) ; the soils are deep , well - drained , mostly stone - free , and high in ferralsols , acrisols , and nitosols , with site index of 20 m at 25 years for pinus kesiya .", "the old - growth stand previously occupying the site was planted in 1980 with pinus kesiya and was harvested from may to august 2010 .", "treatments plots are 70 m by 70 m ( 0.49 ha ) .", "the treatments were as follows : t1:stem - only removal with soil compaction;t2:stem - only removal with soil compaction plus tillage;t3:stem - only removal with no soil compaction ( control ) .", "stem - only removal with soil compaction ; stem - only removal with soil compaction plus tillage ; stem - only removal with no soil compaction ( control ) .", "the stem - only removal harvest followed merchantability standards of 5.5 m log length and 8 to 13 cm small end top diameter ( i.e. , cut - to - length ( ctl ) operation ) .", "the harvested trees were directionally hand - felled between june and august 2010 so that all tree tops remained within the plot . in t1 and", "t2 logs were pulled by a tractor from the stump area to the road side before being transported to the sawmill . in t2 further thorough soil tillage to the depth of 60 cm was accomplished by using a plough . in t3 sulkies were used to remove the logs from the stump area to the road side .", "ten equally spaced traffic lanes were identified in each plot to be trafficked to make soil disturbance comparable across blocks .", "every other lane was trafficked twice to more closely mimic traffic patterns that operationally occur when a tractor or a sulky is used during harvesting . in december 2010 , the plots were planted with pinus kesiya seedlings on a 2.75 m by 2.75 m spacing ( 1320 trees ha ) using hole planting .", "all treatments including spot weeding in the three growing seasons after planting were carried out in order to eliminate the confounding effects that soil disturbance and compaction can have on vegetation communities and competition pressure .", "twenty ( 20 ) soil samples from each treatment were collected to a depth of 010 cm and 1020 cm in september 2010 using a 31.2 mm diameter punch tube volumetric sampler . according to walworth ,", "soil samples of 15 to 25 collected from randomly selected locations in a field are a good representative of the entire field .", "samples with significant amounts of sound or decayed wood material were discarded and replaced with new samples . in compacted and compacted and tilled plots , sampling was restricted to traffic lanes .", "soil samples collected were then oven - dried at 105c for determination of bulk density and soil gravimetric and volumetric water content .", "the measurements were taken at the point where each tree was planted and the rut depth from the original soil level was recorded .", "furthermore , around every other measurement tree , the percentage of each disturbance class in a tree centred 2.75 by 2.75 m square area was recorded .", "then the percentage of the ground area and the tree frequency in each soil disturbance class was calculated .", "soil strength was measured in september 2012 in twenty locations per plot using a cone penetrometer at 5 cm intervals to the 60 cm depth on each treatment .", "high outlier values created when roots or buried wood was hit by the penetrometer were noted on the cards in the field and those measurements were discarded from the dataset .", "soil cores were randomly collected in all the treatments using 75 cm cylinder rings centred at 5 cm and 15 cm depths to characterize the 010 cm and 1020 cm depths , respectively .", "soil water retention curves and particle density were determined at science laboratory of malawi college of forestry and wildlife .", "total porosity was determined by both the gravimetric method with water saturation and the particle density method .", "volumetric water contents were determined after equilibrating the soil with water at tensions of 10 , 20 , 200 , 400 , and 1500 kpa .", "pore size distribution and water retention were then determined by the procedure as outlined by ares et al . .", "measurements of soil temperature were taken monthly between 08.00 hours and 10.00 hours from january 2011 to december 2013 at 20 cm depth in all the treatment plots .", "trees in all treatments were measured immediately after planting and yearly in the three consecutive growing seasons .", "the parameters measured were total height ( h ) using a telescopic pole , stem basal diameter ( bd ) measured at a permanently marked location 15 cm above the ground level in growing seasons one through three , and stem diameter ( dbh ) at 1.3 m above ground in the growing seasons two and three .", "stem diameters were measured using a diameter tape . a stem volume index ( svi )", "data obtained were subjected to kolmogorov - smirnov d and normal probability plot tests using statistical analysis of systems software version 9.1.3 .", ". then , harvesting and logging effects on soil strength and bulk density were analysed as a mixed model of repeated measures data with soil depth and soil disturbance as fixed effects and block as a random effect .", "the model was fitted using the mixed procedure in sas , which estimates variance components using restricted maximum likelihood methods .", "differences between treatment means were separated using fischer 's least significant difference ( lsd ) at the 0.05 level . a repeated measures analysis was appropriate because measurements of bulk density and soil strength were done at different depths at the same sampling point , and , therefore , sampling errors were not independent .", "measurements taken at adjacent depths are expected to be more correlated than measurements taken some distance apart .", "the covariance structures associated with the within subject factor were selected by choosing those with lowest value for the bayesian criterion and akaike 's information criterion .", "a mixed model approach was also used to test for soil disturbance effects on tree growth in years 0 to 3 ." ], [ "the result for ground area affected by the soil disturbance is presented in figure 1 .", "the results indicate that about fifty - two percent of the area of compacted plots was affected by the vehicular traffic .", "seventy percent of the trees within the measurement plots of the compacted treatment were located on some degree of soil disturbance .", "rut depths were shallow to moderate with averages of 2.8 cm for t2 and 13.1 cm for t3 , respectively .", "soil bulk density as related to soil depth and soil disturbance in the study site is presented in table 1 .", "the results show that there was a significant ( p < 0.001 ) difference for soil bulk density between soil depths .", "the depth of 1020 cm had higher soil bulk density than the 010 cm depth .", "similarly , there was a significant difference for soil bulk density among treatments , with t3 having a higher soil bulk density than t1 and t2 .", "relative to t1 , soil bulk density at the depth of 010 cm increased 28.9% for t2 and 48.9% for t3 . at the depth of 1020 ", "cm , the increase in soil bulk density was 22.2% and 38.9% for t2 and t3 , respectively , in relation to t1 .", "soil bulk density significantly ( p < 0.001 ) increased with both soil disturbance and depth .", "the results indicate that soil strength significantly ( p < 0.001 ) increased with soil disturbance and depth , with t1 producing a lower soil strength than class t2 and t3 .", "however , there was no significant ( p > 0.05 ) difference for soil strength between t2 and t3 with increase in depth .", "total soil porosity for all the treatments related to soil depth in the study sites is given in table 2 .", "the results indicate that there was no significant ( p < 0.05 ) difference for soil porosity between soil depth , even though the depth of 010 cm produced higher soil porosity than 1020 cm depth . there was a significant", "( p < 0.001 ) difference for soil porosity among the different treatments with noncompacted soil producing higher soil porosity than the compacted and compacted plus tillage treatments in both soil depths of 010 cm and 1020 cm , respectively .", "however , there was no significant ( p < 0.05 ) difference between noncompacted and compacted plus tillage treatments in soil porosity in both soil depths of 010 cm and 1020 cm , respectively . on compacted soil ,", "total porosity at 010 cm depth decreased by 13.8% compared with noncompacted soil and by 16.1% at the 1020 cm depth .", "the results indicate that there was a significant ( p < 0.001 ) difference in water retention by different treatments with compacted soil consistently retaining more water at 20 to 400 kpa water potential than noncompacted or compacted plus tillage soil at both 010 cm and 1020 cm depth .", "soil temperature , measured once a month at 020 cm depth , was not significantly ( p > 0.05 ) different between compacted and noncompacted areas ( data not shown ) .", "tree mortality and growth for pinus kesiya throughout the three growing seasons are presented in table 3 .", "the results indicate that there were no significant ( p > 0.05 ) differences in mean height , stem basal diameter , diameter at breast height , stem volume index , and mortality for trees growing on compacted , compacted plus tillage , and noncompacted treatments at years one to three ." ], [ "this study illustrates that the amount of soil disturbance in cut - to - length ( ctl ) operation by a tractor is about forty - eight percent .", "tepp reported a 38% soil disturbance using a ctl harvester and forwarder , while geist and cochran found that tractor harvesting using ctl produced 36% soil disturbance .", "however , the present study results are higher than those reported by [ 25 , 26 ] .", "armlovich reported that the amount of soil disturbance in a cut - to - length ( ctl ) operation had an average disturbance of 23.2% while in a study by gingras average disturbance was 25% .", "this difference may arise because in the present study , equipment traffic was applied when soil water contents were at somewhat drier than field water capacity , which is considered to correspond to a given soil water potential [ 12 , 27 ] . according to heninger et al . and", "mcnabb and boersma , the degree of soil disturbance depends on the intensity of site treatment , type of equipment used , time of year when harvesting occurs , frequency of entry , amount of slash or litter on site , soil moisture , and soil type .", "soil bulk density significantly ( p < 0.001 ) increased with both soil disturbance and depth .", "the results are in agreement with those reported by [ 12 , 30 ] . according to froese , in the upper soil , biological activity ( roots , animals , etc . )", "can act to reduce resistance and soil bulk density while at lower depths soil texture , gravel content , and structure may increase soil resistance and soil bulk density ; hence soil bulky density increases with depth .", "soil strength in this study also increased after vehicle trafficking to about 60 cm depth but it was not detrimental for tree root growth of 2000 to 3000 kpa depending on tree species and soil conditions [ 12 , 31 , 33 ] .", "the mean soil strength by depth increment in the study site never exceeded 1200 kpa .", "the compacted soils decreased the total soil porosity at the study site by 13.8% and 16.1% in the depths of 010 cm and 1020 cm , respectively , compared to the noncompacted soils .", "these results are within the range of those reported by [ 12 , 34 ] .", "according to kim , the decrease of soil porosity in t2 as compared to t1 is because of soil compaction . in t2 soil", "is highly compacted due to soil texture and structure by determining the size , number , and interconnection of pores .", "coarse - textured soils have many large ( macro)pores because of the loose arrangement of larger particles with one another .", "because fine - textured soils have both macro- and micropores , they generally have a greater total porosity , or sum of all pores , than coarse - textured soils .", "this is in agreement with the results reported by [ 12 , 38 ] . according to ares et al . ", "compacted soil retained more water than noncompacted soil because compression converted larger pores to smaller ones , especially in the upper part of the soil profile .", "the study reveals that mean height , stem basal diameter , diameter at breast height , stem volume index , and mortality for pinus kesiya trees species were not reduced by soil disturbance or compaction despite the fact that the soil physical properties were affected .", "values for these growth indicators were even greater for trees on disturbed soil than those on nondisturbed soil in years 2 and 3 .", "reported that douglas - fir growth was not reduced by soil disturbance and compaction even though the soil physical properties were affected from ground - based harvest . according to ares et al . ,", "since the growth was not reduced by the soil disturbance or compaction , this is an indication that bulky density , soil strength , and macroporosity did not reach levels in compacted areas that can reduce tree growth . the observed bulk density in the compacted area in this study ranged from 0.45 to 0.66 mg m , which is within the 0.50 to 1.11 mg m range for pinus kesiya seedlings growth [ 40 , 41 ] .", "furthermore , the soil strength observed in this study did not exceed 1200 kpa ; that is , it remained well below the critical threshold for tree root growth considered to be around 2000 to 3000 kpa depending on tree species and soil conditions ." ], [ "soil physical properties particularly soil bulk density , soil strength , and total soil porosity were greatly affected by compaction which was brought by harvesting and logging . however , the extent of disturbance was not detrimental for early growth of planted pinus kesiya .", "the high organic matter content , low bulk density , and low compressibility of the soil contributed to buffering the harvest impacts and made this soil conducive for intensive forest management and ground based harvesting systems .", "soil compaction could also be considered to be beneficial to early tree growth at three years .", "increased water available in the 20 to 400 kpa range on compacted traffic lanes may explain this increased growth ." ] ]

[ "physical activity during preschool age has significant effects on physical , social and psychological health of children .", "fundamental movement skills ( fms ) including locomotor ( e.g. run , gallop , hop , leap , horizontal jump and slid ) , object control ( e.g. catch , kick , overhand throw and dribble ) and body management ( e.g. balance , climb and forward roll ) provide a base for more advanced physical skills . fms develop during early childhood and are essential for complex activities at adulthood .", "some studies have shown that the level of locomotor skills of children is positively correlated with the levels of participation in physical activities at adulthood[34 ] .", "d'hondt et al reported that the levels of gross motor skills in obese children are lower than the levels of these skills in normal - weight children .", "therefore it seems likely that more proficient children in fms are more physically active , have more self confidence , are less obese and would be healthier in their adulthood .", "fms would not be developed naturally as a result of growth and maturation in all children .", "it has been suggested that the optimal ages for fms learning are between 2 to 7 years[2 , 6 ] .", "brian w et al have recommended that physical activity promotional programs for preschool children should be on base of children 's natural activities such as being spontaneous and intermittent . also preschool children activities should be enjoyable and contain gross motor plays and locomotor activities .", "therefore , it seems that an effective physical activity program for preschool children needs to be developmentally appropriate for children in the listed age range .", "furthermore , physical , emotional and psychosocial needs of children should be considered in development of appropriate physical activity program for these children . ideally physical activity programs in nursery schools", "also a well developed training course for these instructors is essential in efficient teaching of movement skills to children in nursery schools[1 , 9 ] .", "in addition to the role of fms in long - term health , fms play a cardinal role in physical development of preschool children . to our knowledge", "there is no national curriculum for preschool children physical activity education in iranian nursery schools .", "the objectives of this study were a ) to develop a physical activity program for nursery schools in iran , and b ) to evaluate the effects of this program on improvement of fundamental movement skills of preschool children in selected nursery schools in iran .", "this quasi - experimental study evaluated the effect of a 10 weeks physical activity program on fundamental movement skills levels of nursery children in iran .", "the participants were 147 children with age range between 4 to 6 years that were selected by convenience sampling .", "children selected form five available nursery schools in five different cities in iran . a manual for nursery physical activity instructors was developed by a panel of 20 experts in different related fields in iran including sports medicine specialist , pediatrician , expert nursery teachers , psychologist , nutritionist , physical education specialist , phd in sports physiology , and phd in movement and behavior .", "this manual contained two parts ; a ) instructions for nursery physical activity instructors about goals and structure of the program , and materials on children 's growth and development , children 's nutrition , physical education , psychology of children , and health and safety , b ) twenty four sections which cover different lessens containing physical activity and games aimed at developing locomotor and object control skills of children aged 4 to 6 years .", "some sections of this package were developed based on the iranian traditional games and plays that were appropriate for preschool ages .", "five nursery schools were selected in five different cities ( tehran , isfahan , shahrood , neishaboor and gorgan ) and volunteer nursery teachers of these nursery schools were enrolled in an educational course .", "this one week course was designed to train nursery physical activity instructors to be able to physically train children in nursery based on our designed physical activity program .", "the aim of this short - term educational program was to ensure that all physical activity instructors are alert to their duties about implementing of the developed physical activity manual .", "also physical activity instructors were educated to test children using the test of gross motor development - edition 2 ( tgmd-2 ) before and after intervention .", "persian version of this test has been employed by akbari h , et al . and bakhtiari s , et al . in iran[9 ,", "the levels of gross motor development of all subjects were measured before intervention and after 10 weeks physical activity program employing tgmd-2 .", "tgmd-2 is a valid and reliable ( test - retest reliability = 0.88 - 0.96 ) criterion - referenced instrument designed to assess gross motor development among children .", "each subtest consists of 6 locomotor items ( run , hop , gallop , leap , horizontal jump , and slide ) , and 6 object control items ( throw , catch , kick , strike , dribble , and roll ) .", "subtest raw score was defined by sum of 6 items of each subtests of locomotor or object control ( scored between 0 - 48 ) .", "standard scores of each subtest ( scored between 0 - 20 ) and also gross motor quotient were calculated in accordance with tgmd-2 manual .", "( gmq ) for each subject was reported as very superior ( gmq > 130 ) , superior ( gmq = 121130 ) , above average ( gmq = 111120 ) , average ( gmq = 90110 ) , below average ( gmq = 8089 ) , poor ( gmq = 7079 ) , and very poor ( gmq < 70 ) according to the suggestions of the tgmd-2 manual . according to developed physical activity manual", ", physical activity program was conducted 5 days a week for 10 weeks by trained nursery physical activity instructors .", "paired - samples t test was used to determine whether there were significant differences between the levels of locomotor and object control of participants before and after intervention .", "independent samples t - test was used to compare differences between boys and girls on variables .", "in this quasi - experimental study 147 children with mean ( sd ) age of 4.95 ( 0.8 ) ( range 3 - 6 ) year from five nursery schools in 5 cities in iran were included .", "of all subjects 49% ( 72 ) were girls and 51% ( 75 ) were boys .", "the base line and post intervention tgmd-2 scores of subjects are presented in tables 1 and 2 . in the baseline", ", there were no statistically significant differences between the locomotor and object control raw scores of boys and girls ( p=0.49 and p=0.9 ) respectively . \n ", "base line and post intervention tgmd-2 scores of all subjects sd : standard deviation means ( sd ) of base line and after intervention tgmd-2 raw scores of all subjects by age and gender sd : standard deviation after intervention , differences between the locomotor raw scores of boys and girls were not statistically significant ( p=0.5 ) .", "however , there was a statistically significant difference between boys and girls in the object control raw scores ( p=0.048 ) .", "the gmq of all subjects which is the most reliable score of tgmd-2 was statistically significantly increased after 10 weeks of intervention .", "both subtests of tgmd-2 including locomotor raw score and object control raw score in both genders were statistically significantly increased after 10 weeks of intervention ( table 1 ) .", "descriptive rating of gmq for subjects before and after intervention is shown in table 3 . before intervention only 11.5% of all subjects were rated superior / very superior in gmq scores ( i.e. gmq > 120 ) , however this rate increased to 49.7% of all subjects after 10 weeks of intervention . before the intervention ,", "26.6% of all subjects were rated as poor / very poor , this rate decreased to 2% of subjects after 10 weeks intervention .", "age equivalents of subjects on the locomotor and object control subtests before and after intervention are shown in table 4", ". age equivalents or developmental age defined as developmental level or age that corresponds to a raw score made by an individual . \n ", "descriptive rating of the gross motor quotient of all subjects before and after intervention age equivalents for locomotor and object control raw scores according to age groups of all subjects before and after intervention", "the main finding of this study was that the developed physical activity intervention program that was focused on gross motor skills development had a significant positive effect on proficiency in fundamental movement skills in preschool children from selected nursery schools in five cities in iran .", "differences in tgmd-2 results before and after the intervention were significant in locomotor , object control , sum of standard cores and gross motor quotient in all subjects . to our knowledge", "there has been no published data on the levels of physical activity of iranian preschool children .", "however , base line tgmd-2 subtests scores of our subjects in this study were in the range of reported data from the united states ( table 5 ) . \n ", "comparison of tgmd-2 standard scores of subjects in this study with reported data from the united states , mean ( sd ) sd : standard deviation our data suggested that a supervised physical activity program could increase these scores to be better than normative reported data .", "there was no significant difference in the base line mean of the locomotor and object control raw scores between girls and boys ( 30 vs 29.5 ) and ( 24.8 vs 26 ) , respectively .", "however after the intervention the mean of both object control raw scores ( p=0.048 ) and object control standard scores ( p=0.02 ) in girls were higher than these scores in boys .", "cliff et al have reported that locomotor raw score in preschool children was higher in girls compared with boys but there was no significant difference between girls and boys in the object control raw score . in this study ,", "base line means of standard scores of the locomotor ( 9.3 vs 8.9 ) , object control ( 9.1 vs 8.1 ) and gmq ( 95.5 vs 91.2 ) were not significantly different between girls and boys .", "in contrast cliff et al have reported significant differences between girls and boys in the mean of locomotor standard scores ( 9.9 vs 7.9 ) , object control standard scores ( 10.1 vs 8.6 ) and gmq ( 99.7 vs 88.2 ) .", "one study has reported that locomotor skills proficiency is higher in girls and in contrast boys are more proficient in object control skills .", "some studies have suggested that the levels of moderate and vigorous physical activities in children with better motor performance are significantly higher than the levels of these activities in children with less developed skills[4 , 19 ] .", "therefore , conduction of a physical activity program such as the program used in this study may help children to improve their fms which may help to have a higher physical activity in their future .", "first , same person conducted both training sessions and outcomes measurement in each nursery school .", "furthermore , to our knowledge there was no normative data of tgmd-2 for iranian children to be used for comparison with our data .", "further studies are needed to evaluate the long - term effects of physical activity intervention on fms in iran .", "it seems that our developed physical activity program conducted by trained nursery physical activity instructors could be an effective and practical way of improving gross motor skills of preschool children in short term in iran .", "conduction of this program in nursery schools could indirectly help with increasing health levels and levels of physical activities in the society .", "we recommend using of this kind of physical activity programs in all nursery schools in iran and similar counties ." ]

[ "<S> objectivethe objectives of this study were a ) to develop a physical activity program for nursery schools , and b ) to evaluate the effects of this program on fundamental movement skills of preschool age children in iran.methodsin this quasi - experimental study 147 children from five nursery schools in five different cities in iran were enrolled . </S>", "<S> a physical activity program was developed for nursery children . </S>", "<S> trained nursery physical activity instructors conducted the program for 10 weeks for all subjects . </S>", "<S> the levels of gross motor development of all subjects were measured before intervention and after 10 weeks physical activity program employing the test of gross motor development - edition 2 ( tgmd-2).findingsthe participants in this study had a mean ( sd ) age of 4.95 ( 0.83 ) years . at the end of the study , scores of subjects at all components of tgmd-2 ( including locomotor , object control , sum of standard scores and gross motor quotient ) </S>", "<S> were significantly improved compared to the baseline scores ( p<0.001 ) . </S>", "<S> based on descriptive rating of the \" gross motor quotient \" in the base line , 11.5% of subjects were superior / very superior ( gmq > 120 ) and after 10 weeks intervention this rate was increased to 49.7% of all subjects.conclusionit seems that the developed physical activity program conducted by trained nursery physical activity instructors could be an effective and practical way of increasing levels of fundamental movement skills of preschool children in iran . </S>" ]

[ "Introduction", "Subjects and Methods", "Findings", "Discussion", "Conclusion", "Conflict of Interest" ]

[ [ "physical activity during preschool age has significant effects on physical , social and psychological health of children .", "fundamental movement skills ( fms ) including locomotor ( e.g. run , gallop , hop , leap , horizontal jump and slid ) , object control ( e.g. catch , kick , overhand throw and dribble ) and body management ( e.g. balance , climb and forward roll ) provide a base for more advanced physical skills . fms develop during early childhood and are essential for complex activities at adulthood .", "some studies have shown that the level of locomotor skills of children is positively correlated with the levels of participation in physical activities at adulthood[34 ] .", "d'hondt et al reported that the levels of gross motor skills in obese children are lower than the levels of these skills in normal - weight children .", "therefore it seems likely that more proficient children in fms are more physically active , have more self confidence , are less obese and would be healthier in their adulthood .", "fms would not be developed naturally as a result of growth and maturation in all children .", "it has been suggested that the optimal ages for fms learning are between 2 to 7 years[2 , 6 ] .", "brian w et al have recommended that physical activity promotional programs for preschool children should be on base of children 's natural activities such as being spontaneous and intermittent . also preschool children activities should be enjoyable and contain gross motor plays and locomotor activities .", "therefore , it seems that an effective physical activity program for preschool children needs to be developmentally appropriate for children in the listed age range .", "furthermore , physical , emotional and psychosocial needs of children should be considered in development of appropriate physical activity program for these children . ideally physical activity programs in nursery schools", "also a well developed training course for these instructors is essential in efficient teaching of movement skills to children in nursery schools[1 , 9 ] .", "in addition to the role of fms in long - term health , fms play a cardinal role in physical development of preschool children . to our knowledge", "there is no national curriculum for preschool children physical activity education in iranian nursery schools .", "the objectives of this study were a ) to develop a physical activity program for nursery schools in iran , and b ) to evaluate the effects of this program on improvement of fundamental movement skills of preschool children in selected nursery schools in iran ." ], [ "this quasi - experimental study evaluated the effect of a 10 weeks physical activity program on fundamental movement skills levels of nursery children in iran .", "the participants were 147 children with age range between 4 to 6 years that were selected by convenience sampling .", "children selected form five available nursery schools in five different cities in iran . a manual for nursery physical activity instructors was developed by a panel of 20 experts in different related fields in iran including sports medicine specialist , pediatrician , expert nursery teachers , psychologist , nutritionist , physical education specialist , phd in sports physiology , and phd in movement and behavior .", "this manual contained two parts ; a ) instructions for nursery physical activity instructors about goals and structure of the program , and materials on children 's growth and development , children 's nutrition , physical education , psychology of children , and health and safety , b ) twenty four sections which cover different lessens containing physical activity and games aimed at developing locomotor and object control skills of children aged 4 to 6 years .", "some sections of this package were developed based on the iranian traditional games and plays that were appropriate for preschool ages .", "five nursery schools were selected in five different cities ( tehran , isfahan , shahrood , neishaboor and gorgan ) and volunteer nursery teachers of these nursery schools were enrolled in an educational course .", "this one week course was designed to train nursery physical activity instructors to be able to physically train children in nursery based on our designed physical activity program .", "the aim of this short - term educational program was to ensure that all physical activity instructors are alert to their duties about implementing of the developed physical activity manual .", "also physical activity instructors were educated to test children using the test of gross motor development - edition 2 ( tgmd-2 ) before and after intervention .", "persian version of this test has been employed by akbari h , et al . and bakhtiari s , et al . in iran[9 ,", "the levels of gross motor development of all subjects were measured before intervention and after 10 weeks physical activity program employing tgmd-2 .", "tgmd-2 is a valid and reliable ( test - retest reliability = 0.88 - 0.96 ) criterion - referenced instrument designed to assess gross motor development among children .", "each subtest consists of 6 locomotor items ( run , hop , gallop , leap , horizontal jump , and slide ) , and 6 object control items ( throw , catch , kick , strike , dribble , and roll ) .", "subtest raw score was defined by sum of 6 items of each subtests of locomotor or object control ( scored between 0 - 48 ) .", "standard scores of each subtest ( scored between 0 - 20 ) and also gross motor quotient were calculated in accordance with tgmd-2 manual .", "( gmq ) for each subject was reported as very superior ( gmq > 130 ) , superior ( gmq = 121130 ) , above average ( gmq = 111120 ) , average ( gmq = 90110 ) , below average ( gmq = 8089 ) , poor ( gmq = 7079 ) , and very poor ( gmq < 70 ) according to the suggestions of the tgmd-2 manual . according to developed physical activity manual", ", physical activity program was conducted 5 days a week for 10 weeks by trained nursery physical activity instructors .", "paired - samples t test was used to determine whether there were significant differences between the levels of locomotor and object control of participants before and after intervention .", "independent samples t - test was used to compare differences between boys and girls on variables ." ], [ "in this quasi - experimental study 147 children with mean ( sd ) age of 4.95 ( 0.8 ) ( range 3 - 6 ) year from five nursery schools in 5 cities in iran were included .", "of all subjects 49% ( 72 ) were girls and 51% ( 75 ) were boys .", "the base line and post intervention tgmd-2 scores of subjects are presented in tables 1 and 2 . in the baseline", ", there were no statistically significant differences between the locomotor and object control raw scores of boys and girls ( p=0.49 and p=0.9 ) respectively . \n ", "base line and post intervention tgmd-2 scores of all subjects sd : standard deviation means ( sd ) of base line and after intervention tgmd-2 raw scores of all subjects by age and gender sd : standard deviation after intervention , differences between the locomotor raw scores of boys and girls were not statistically significant ( p=0.5 ) .", "however , there was a statistically significant difference between boys and girls in the object control raw scores ( p=0.048 ) .", "the gmq of all subjects which is the most reliable score of tgmd-2 was statistically significantly increased after 10 weeks of intervention .", "both subtests of tgmd-2 including locomotor raw score and object control raw score in both genders were statistically significantly increased after 10 weeks of intervention ( table 1 ) .", "descriptive rating of gmq for subjects before and after intervention is shown in table 3 . before intervention only 11.5% of all subjects were rated superior / very superior in gmq scores ( i.e. gmq > 120 ) , however this rate increased to 49.7% of all subjects after 10 weeks of intervention . before the intervention ,", "26.6% of all subjects were rated as poor / very poor , this rate decreased to 2% of subjects after 10 weeks intervention .", "age equivalents of subjects on the locomotor and object control subtests before and after intervention are shown in table 4", ". age equivalents or developmental age defined as developmental level or age that corresponds to a raw score made by an individual . \n ", "descriptive rating of the gross motor quotient of all subjects before and after intervention age equivalents for locomotor and object control raw scores according to age groups of all subjects before and after intervention" ], [ "the main finding of this study was that the developed physical activity intervention program that was focused on gross motor skills development had a significant positive effect on proficiency in fundamental movement skills in preschool children from selected nursery schools in five cities in iran .", "differences in tgmd-2 results before and after the intervention were significant in locomotor , object control , sum of standard cores and gross motor quotient in all subjects . to our knowledge", "there has been no published data on the levels of physical activity of iranian preschool children .", "however , base line tgmd-2 subtests scores of our subjects in this study were in the range of reported data from the united states ( table 5 ) . \n ", "comparison of tgmd-2 standard scores of subjects in this study with reported data from the united states , mean ( sd ) sd : standard deviation our data suggested that a supervised physical activity program could increase these scores to be better than normative reported data .", "there was no significant difference in the base line mean of the locomotor and object control raw scores between girls and boys ( 30 vs 29.5 ) and ( 24.8 vs 26 ) , respectively .", "however after the intervention the mean of both object control raw scores ( p=0.048 ) and object control standard scores ( p=0.02 ) in girls were higher than these scores in boys .", "cliff et al have reported that locomotor raw score in preschool children was higher in girls compared with boys but there was no significant difference between girls and boys in the object control raw score . in this study ,", "base line means of standard scores of the locomotor ( 9.3 vs 8.9 ) , object control ( 9.1 vs 8.1 ) and gmq ( 95.5 vs 91.2 ) were not significantly different between girls and boys .", "in contrast cliff et al have reported significant differences between girls and boys in the mean of locomotor standard scores ( 9.9 vs 7.9 ) , object control standard scores ( 10.1 vs 8.6 ) and gmq ( 99.7 vs 88.2 ) .", "one study has reported that locomotor skills proficiency is higher in girls and in contrast boys are more proficient in object control skills .", "some studies have suggested that the levels of moderate and vigorous physical activities in children with better motor performance are significantly higher than the levels of these activities in children with less developed skills[4 , 19 ] .", "therefore , conduction of a physical activity program such as the program used in this study may help children to improve their fms which may help to have a higher physical activity in their future .", "first , same person conducted both training sessions and outcomes measurement in each nursery school .", "furthermore , to our knowledge there was no normative data of tgmd-2 for iranian children to be used for comparison with our data .", "further studies are needed to evaluate the long - term effects of physical activity intervention on fms in iran ." ], [ "it seems that our developed physical activity program conducted by trained nursery physical activity instructors could be an effective and practical way of improving gross motor skills of preschool children in short term in iran .", "conduction of this program in nursery schools could indirectly help with increasing health levels and levels of physical activities in the society .", "we recommend using of this kind of physical activity programs in all nursery schools in iran and similar counties ." ], [ "" ] ]

[ "glomerular visceral epithelial cells ( gecs ; podocytes ) play a key role in the maintenance of glomerular permselectivity [ 14 ] . under normal conditions , podocytes are in contact with extracellular matrix , and there appears to be little turnover of podocytes , as these cells are highly differentiated ( terminally differentiated ) .", "gecs form a tight network of interdigitating foot processes , which are bridged by filtration - slit diaphragms , and permselectivity of the glomerular capillary wall is dependent on the maintenance of appropriate structure of podocytes and of their foot processes .", "the complex cellular architecture of the podocyte is maintained by an organization of actin filaments in the cytoplasm .", "proteins including nephrin , neph1 , fat , podocin , cd2ap and zo-1 are found within or near the slit diaphragm , and nephrin , neph family proteins , and cadherins form complexes with scaffolding proteins , cd2ap and zo-1 [ 2 , 4 ] .", "these connect the slit - diaphragm protein complex with actin filaments , which are joined by -actinin-4 proteins . acquired gec injury", "is frequently associated with effacement of the foot processes , disruption of the filtration - slit diaphragms , and proteinuria [ 1 , 2 ] . moreover , heritable mutations in several gec structural proteins alter podocyte ultrastructure and cause heritable proteinuria , implying that impairment of the slit diaphragm or cytoskeletal structure underlies proteinuria [ 4 , 5 ] . focal segmental glomerulosclerosis ( fsgs ) is an important cause of proteinuria and nephrotic syndrome in humans . in recent years", ", evidence has accumulated to support the view that the early pathogenesis of fsgs is associated with gec injury , leading to apoptosis , detachment of gecs from extracellular matrix , and podocytopenia , which is followed by glomerulosclerosis [ 13 , 68 ] .", "acquired gec injury may be triggered by immunological factors ( e.g. , t cell or other factors affecting permeability ) , oxidants , human immunodeficiency virus , toxins , and other substances .", "in addition , fsgs may be associated with heritable mutations in several distinct proteins that play key roles in maintaining gec ultrastructure , including podocin , -actinin-4 , transient receptor potential cation channel , subfamily c , member 6 ( trpc6 ) , inverted formin 2 and others . in this paper , we focus on mutations in -actinin-4 , which are associated with an autosomal dominant late - onset fsgs in humans .", "we discuss the structure and function of -actinin-4 , and we review mechanisms by which mutations in -actinin-4 may lead to podocyte injury and fsgs .", "there are four -actinin genes ( actn1 - 4 ) that encode highly homologous proteins ( -actinin-1 , 2 , 3 and 4 ) , which form ~100 kda head - to - tail homodimers . -actinins-2 and -3 are ca - insensitive muscle -actinins expressed in z - discs of striated muscle cells , while -actinins-1 and -4 are widely distributed , nonmuscle isoforms .", "all family members are actin crosslinking proteins that share a number of structural features and regulatory regions .", "these include a central rod with four spectrin - like repeats , two n - terminal calponin - homology domains ( ch1 and ch2 ) , which contain three actin - binding sites ( abs1 - 3 ) , a c - terminal calmodulin - like domain , containing two putative ef hands , and a phosphoinositide - binding plextrin homology domain .", "the spectrin - like repeats facilitate homodimerization and may confer a degree of flexibility to the -actinin molecule , allowing it to resist mechanical strain . for its interaction with actin filaments ,", "the ch1 and ch2 domains adopt a closed conformation , where abs 2 and 3 are exposed , while abs1 remains buried and inaccessible to f - actin .", "this association with actin is highly dynamic and can be controlled by the regulatory regions along the -actinin sequence . ", "all -actinin isoforms contain two putative ef - hand motifs at the c - terminus of each monomer , but only isoforms 1 and 4 demonstrate sensitivity to ca [ 15 , 16 ] . for these nonmuscle isoforms , binding of ca", "is believed to reduce the affinity of -actinin for actin , providing a mechanism for -actinin - dependent cytoskeletal remodeling .", "additionally , extensive biochemical studies on the effect of phosphoinositide binding to -actinin-1 have been performed .", "binding of phosphatidylinositol ( 3,4,5)-trisphosphate ( pip3 ) , and to a lesser extent phosphatidylinositol ( 4,5)-bisphosphate ( pip2 ) , decreases the association between -actinin-1 and f - actin , suggesting that phosphoinositides mediate cytoskeletal remodeling [ 17 , 18 ] . for -actinin-4 ,", "in contrast to findings for -actinin-1 , our studies showed that phosphoinositides increase the interaction of -actinin-4 with f - actin .", "tyrosine phosphorylation at position 12 was found to be dependent on focal adhesion kinase ( fak ) , supporting a role for -actinin in cell adhesion .", "phosphorylation of -actinin-1 reduced its association with f - actin in vitro , suggesting that -actinin mediates fak - dependent cytoskeletal remodeling .", "the amino acid sequence of -actinin-4 reveals an analogous tyrosine residue at position 32 , suggesting that phosphorylation may also regulate the binding of -actinin-4 to f - actin .", "however , we were unable to demonstrate fak - dependent tyrosine phosphorylation of -actinin-4 ( unpublished observations ) .", "expression of these isoforms at the cytoplasmic face of several types of cellular interaction sites , including focal adhesions , adherens junctions , and hemidesmosomes suggests a degree of versatility .", "the first -actinin binding partners identified were the subunits of integrins and intercellular adhesion molecule-1 .", "since -actinins also bind to various regulatory molecules , -actinin may also serve as a scaffolding protein to integrate signaling molecules at various adhesion sites .", "-actinin-1 may link membrane proteins such as talin , vinculin , and -integrins with the cortical actin cytoskeleton [ 10 , 2225 ] .", "similarly , the tight junction protein , magi-1 , interacts with the c - terminus of -actinin-4 , providing a physical link from the cell periphery to the cytoskeleton .", "the list of interacting proteins is likely to grow and will provide a better understanding of the diverse functions of -actinins in nonmuscle tissues .", "however , the best defined function of -actinin-4 is to crosslink / bundle actin filaments , and it may enhance cell motility and elicit tumor - suppressor activity . intriguingly , -actinin-4 is reported to shuttle between the cytoplasm and the nucleus .", "cytoplasmic localization was associated with an infiltrative phenotype and correlated significantly with a poorer prognosis in cases of breast cancer . in this context", ", the relevance of nuclear -actinin-4 remains unknown , although it has been suggested that nuclear translocation may attenuate metastatic potential .", "the nuclear localization of -actinin-4 suggests a role for -actinin-4 in gene expression . -actinins-1 and", "-4 were identified as histone deacetylase 7 ( hdac7)-interacting proteins , and the interaction domain was mapped to the c - terminus of -actinin 4 .", "hdac7 can participate in multiple cellular processes , including the regulation of myocyte enhancer factor-2- ( mef2- ) mediated transcription .", "a point mutation in hdac7 disrupted its association with -actinin-4 and mef2 , implying that -actinin-4 and mef2 binding sites overlap .", "overexpression of -actinin-4 also potentiated estrogen receptor -mediated transcription by counteracting a negative regulatory effect of hdac7 , while knockdown of -actinin-4 decreased expression of estrogen receptor target genes and affected proliferation of cultured breast cancer cells .", "another study demonstrated that -actinin-4 colocalized along actin stress fibers and in membrane lamellae with nuclear factor-b ( nf-b ) .", "incubation of cells with epidermal growth factor or tumor necrosis factor- induced translocation of -actinin-4 and the p65 subunit of nf-b into the nucleus .", "as nf-b regulates transcription of a large number of genes in response to diverse stimuli , the study further supports a regulatory role for -actinin-4 in transcription .", "human gecs express only -actinin-4 ( although mouse gecs also express -actinin-1 ) [ 10 , 30 ] .", "immunoelectron microscopy studies showed that -actinin localizes to contractile microfilaments within podocyte foot processes [ 31 , 32 ] .", "in cultured mouse gecs , -actinin-4 was found along actin stress fibers , focal adhesions , and within the cortical actin network at the cell periphery ( figure 1 ) .", "this specific subcellular localization of -actinin-4 facilitates its regulation of adhesion and cytoskeletal dynamics [ 19 , 33 , 36 ] .", "interestingly , -actinin-4 was also present in the nucleus of some rat gecs [ 34 , 35 ] , in keeping with earlier reports in other cell lines .", "normally , -actinin-4 may be required for integrin - dependent adhesion of gecs . indeed ,", "knockout of -actinin-4 in mice resulted in reduced glomerular podocyte number , accompanied by the appearance of urinary wilm 's tumor-1 ( wt-1 ) , a podocyte biomarker .", "such podocyte loss is consistent with defective glomerular basement membrane adhesion , as tunel assays failed to detect any apoptotic cells in -actinin-4 null glomeruli .", "the severity of the resulting phenotype seen in -actinin-4 null mice ( i.e. , glomerular disease , renal failure , and early death ) indicates that -actinin-4-mediated podocyte adhesion is critical for filtration barrier function [ 30 , 37 , 38 ] . on the other hand , transgenic podocyte overexpression of wild - type -actinin-4 in mice", "these findings suggest that podocytes tolerate a wide range of -actinin-4 expression , but that a minimum threshold level is essential for normal gec adhesion to the glomerular basement membrane through its interaction with integrins . ", "deficiency of -actinin-4 leading to human disease is not described , but several point mutations or single amino acid deletions in -actinin-4 are found in heritable forms of human fsgs .", "most mutations in actn4-associated fsgs families congregate at the ch1-ch2 interface [ 9 , 40 ] .", "an example is the autosomal dominant k255e mutation ( k256e is the mouse mutant corresponding to human k255e ) .", "such mutants show increased binding to actin filaments , compared with the wild - type protein [ 30 , 41 , 42 ] .", "interestingly , -actinin-4 k256e is insensitive to regulation by ca and phosphoinositides ( pip2 and pip3 ) , suggesting that its gain of affinity for f - actin blunts its responsiveness to regulatory factors .", "expression of an -actinin-4 k256e transgene in mouse podocytes in vivo ( under the control of the nephrin promoter ) resulted in development of proteinuric fsgs .", "in addition , homozygous ( but not heterozygous ) knock - in of the mutant -actinin-4 allele into the actn4 locus in mice induced proteinuria , confirming the pathogenic potential of -actinin-4 mutations . ", "the underlying biochemical mechanism , whereby fsgs - associated actn4 mutations enhance affinity for actin , was uncovered by pollak 's group .", "they showed that disease - causing mutations disable an important intramolecular hinge that normally holds ch1 and ch2 in a closed conformation .", "the mutant protein adopts an open conformation forcing an interaction of all three actin - binding sites with the actin filament , thereby increasing the binding affinity by lowering its rate of dissociation from actin . ", "although -actinin-4 is widely expressed , human podocytes appear to be selectively vulnerable to mutations in -actinin-4 ( including k256e and other analogous mutants ) , perhaps because podocytes are terminally differentiated cells with limited regenerative capacity .", "the downstream cellular mechanism(s ) by which mutant -actinin-4 leads to podocyte injury and fsgs is poorly understood .", "one possibility is that injury is secondary to alterations in the mechanical properties of the podocyte via increased affinity of mutant -actinin-4 for f - actin ( figure 2 ) .", "gecs that express -actinin-4 k256e show defective spreading and motility ( figure 1 ) . moreover , exposure of -actinin-4 k256e expressing gecs to cyclical equibiaxial stretch , an in vitro mimic of the mechanical forces due to glomerular capillary pressure , significantly reduced cell surface area and caused retraction of cellular processes .", "lastly , the enhanced association with f - actin alters the subcellular localization of -actinin-4 , restricting its presence at the cell periphery ( figure 1 ) , and potentially altering its capacity to interact with various binding partners [ 19 , 33 ] .", "such abnormalities could , at least in part , be attributable to cytoskeletal disruption , as well as loss of integrin engagement , effectively yielding a phenotype similar to that of the -actinin-4 null mouse [ 36 , 43 ] . since the actin cytoskeleton is a key component contributing to the unique morphological characteristics of the podocyte foot processes , as well as being connected to both slit diaphragm and adhesion complexes , altered -actinin-4 biology must have important implications for podocyte health . ", "an alternate and/or parallel mechanism , which we also consider in this paper , is that mutant -actinin-4 induces proteotoxicity in podocytes ( i.e. , an impairment of podocyte function caused by misfolding of a protein ) , ultimately leading to apoptosis ( figure 2 ) .", "consistent with this notion , -actinin-4 k256e forms actin - associated aggregates in cultured gecs and in podocytes of both homozygous k256e knock - in mice and humans with actn4-associated fsgs [ 30 , 33 , 35 ] . such misfolded / aggregated protein may be associated with the activation of stress pathways in podocytes ( see below ) .", "prior to discussing the proteotoxic potential of mutant -actinin-4 , this section provides a brief overview of the ubiquitin - proteasome system and er stress .", "the ubiquitin - proteasome system plays a key role in regulating the half - life of short - lived cellular proteins , and in selective degradation of damaged or abnormal proteins [ 45 , 46 ] .", "the proteasome degrades aberrant cytoplasmic or cytoskeletal proteins , and misfolded er proteins , which are retrotranslocated selectively to the cytoplasm .", "the latter process is known as er - associated degradation ( erad ) [ 47 , 48 ] .", "ubiquitin - proteasome pathway - mediated protein degradation involves tagging of the substrate by covalent attachment of ubiquitin molecules via a three - step reaction , and degradation of the tagged protein by the 26s proteasome complex .", "ubiquitinated proteins typically undergo efficient degradation by the proteasome , but sometimes , large amounts of misfolded proteins are not degraded effectively , and may form covalently - linked aggregates . such misfolded proteins and/or aggregates may impair the function of the proteasome and lead to the activation of stress pathways and cytotoxicity .", "secretory , luminal , and membrane proteins normally attain their correctly folded conformation in the er via er - resident chaperones . to rescue misfolded proteins , the er has in place quality control machinery , including the unfolded protein response ( upr ) [ 4953 ] , and erad [ 47 , 48 ] . in the upr ,", "accumulation of misfolded proteins in the er results in the activation of three er sensors . activating transcription factor-6 moves from the er to the golgi , where it is cleaved by proteases .", "the cleaved cytosolic fragment translocates to the nucleus to activate transcription of er chaperones , for example , the glucose - regulated proteins ( grp ) , grp94 and bip ( grp78 ) , and others . in parallel ,", "inositol requiring-1 activates its endoribonuclease activity , cleaving x - box binding protein-1 mrna and changing the reading frame to yield a potent transcriptional activator .", "normally , er chaperones assist in posttranslational processing of proteins and in their exit from the er , and may complex with defective proteins to target them for degradation . during stress ,", "induction of er chaperones may enhance the protein folding capacity , and limit accumulation of abnormal proteins . ", "misfolded proteins in the er also activate perk ( pkr - like er kinase ) , which then phosphorylates the eukaryotic translation initiation factor-2 subunit ( eif2 ) .", "this process reduces initiation aug codon recognition , and the general rate of translation is blunted ( which decreases the protein load on a damaged er ) .", "the upr allows cells to recover from stress , and may be protective to additional insults , but substantial / prolonged er stress may lead to apoptosis .", "for example , certain mrnas may be translated preferentially after eif2 is phosphorylated . among these is activating transcription factor-4 , which induces expression of several genes , including chop ( c / ebp homologous protein-10 ; also known as gadd153 ) , a gene closely associated with apoptosis and/or growth arrest [ 49 , 51 ] .", "apoptosis may also result from activation of caspase-12 or protein kinases [ 49 , 51 ] .", "impairment of the ubiquitin - proteasome system can be associated with exacerbation of er stress [ 49 , 54 ] , perhaps by interference with erad .", "the potential for mutant -actinin-4 to impair podocyte function is suggested by the characteristics of this mutant protein to form microaggregates , undergo ubiquitination , impair the ubiquitin - proteasome system , enhance er stress , and enhance apoptosis ( figure 2 ) .", "density - gradient centrifugation of the k255e mutant -actinin-4 showed abnormal sedimentation , suggesting that the mutant protein forms high molecular mass aggregates . in keeping with this result , expression of -actinin-4 k255e ( and other fsgs - inducing mutants ) , but", "not wild - type -actinin-4 in cultured mouse gecs resulted in formation of aggregates , as visualized by fluorescence microscopy [ 30 , 33 , 42 , 43 ] .", "analogous results were obtained in rat gecs stably transfected with -actinin-4 k256e , where in some cells , the mutant ( but not the wild type ) protein formed aggregates . unlike the wild - type protein , the mutant was not present in the nucleus of rat gecs [ 34 , 35 ] .", "in addition , -actinin-4 k255e formed aggregates in podocytes of homozygous k255e knock - in mice and humans with actn4-associated fsgs .", "misfolded / aggregated proteins may result in the activation of stress pathways . in pulse - chase metabolic labeling experiments , mutant -actinin-4", "was degraded more rapidly , compared with the wild - type protein , with the mutant showing a half - life of ~15 h , and the wild type of over 30 h .", "consistent with this result , after stable transfection in rat gecs or transient transfection in cos cells ( a monkey kidney cell line , which allows for high levels of protein expression ) , the level of -actinin-4 k256e protein was considerably lower , compared with the wild - type protein ( figure 3 ) [ 34 , 35 ] .", "together , these results suggested that -actinin-4 k256e may undergo ubiquitination prior to degradation via the ubiquitin - proteasome pathway .", "indeed , in transiently transfected cos cells , mutant -actinin-4 was polyubiquitinated , whereas the wild - type protein was not ( figure 3 ) .", "treatment of gecs that express -actinin-4 k256e with proteasome inhibitors enhanced expression of -actinin-4 k256e , in keeping with ubiquitination and degradation of the mutant protein by the proteasome [ 30 , 35 ] . \n", "-actinin-4 k256e and wild - type proteins were transiently overexpressed in cos cells to study their effects on the ubiquitin - proteasome system .", "the function of the ubiquitin - proteasome system was monitored in viable cells by use of a reporter consisting of a short degron , cl1 , fused to the c - terminus of green fluorescent protein ( gfp ) .", "this gfp proteasome reporter is rapidly degraded when ubiquitin - proteasome function is normal , whereas impairment of the ubiquitin - proteasome system , for example , via enhanced flux of a mutant / aggregated protein will reduce degradation of gfp , resulting in an increased level of gfp expression . in cos cells transfected with wild - type -actinin-4 ,", "expression of gfp declined significantly , in keeping with proteasomal degradation of gfp ( figure 3 ) .", "in contrast , expression of -actinin-4 k256e retarded the degradation of gfp , implying that the ubiquitin - proteasome system was impaired in the presence of mutant -actinin-4 . \n", "-actinin-4 is a cytosolic / cytoskeletal protein that normally would not enter the er to undergo posttranslational modification .", "nonetheless , compared with -actinin-4 wild type , transient transfection of -actinin-4 k256e in cos cells enhanced the upr , as evidenced by increased expression of the er chaperone , grp94 , and the proapoptotic gene , chop ( figure 3 ) . in gecs ,", "expression of the k256e mutant or wild type -actinin-4 ( by stable transfection ) did not increase expression of the er chaperone , bip , or chop , compared with parental ( untransfected ) cells .", "this difference between cos cells and gecs may be due to lower expression of stably transfected proteins in the gecs .", "however , after incubation of gecs with tunicamycin ( a drug that blocks n - linked glycosylation and causes an accumulation of misfolded proteins in the er ) , the gecs stably transfected with -actinin-4 k256e showed increases in bip and chop that significantly exceeded the increases seen in cells expressing the wild - type protein , as well as the increases in parental gecs .", "the effect of -actinin-4 k256e on the induction of the upr by tunicamycin was particularly robust , given that expression of the mutant was substantially lower than the wild - type protein .", "thus , although stable expression of -actinin-4 k256e in gecs did not induce the upr directly , the mutant appeared to adversely affect the integrity of the er , which may render these cells more susceptible to additional stress and induction of proapoptotic genes .", "together , these results are in keeping with the view that exacerbation of er stress may be secondary to impairment of the ubiquitin - proteasome system [ 49 , 54 ] . stable expression of -actinin-4 k256e in gecs led to a reduction in cell number , as well as increased apoptosis and caspase-3 activity , compared with gecs that stably express -actinin-4 wild type , or parental ( untransfected ) gecs .", "these changes in cell number and apoptosis in the presence of -actinin-4 k256e occurred despite the significantly lower expression level of the mutant , compared with the wild - type -actinin-4 , highlighting the potential detrimental consequences of this mutation . in cos cells , -actinin-4 k256e had only a slight effect on apoptosis and cell number , suggesting that this cell type may be more resistant to cytotoxicity .", "nevertheless , -actinin-4 k256e markedly exacerbated apoptosis and reduced cell number in the context of mild proteasome inhibition .", "this result supports the view that apoptosis induced by mutant -actinin-4 may be associated with an impairment in proteasome function .", "the effects of -actinin-4 k256e on the upr ( discussed above ) are based on studies in cultured cell lines , but importantly , analogous changes also occurred in vivo .", "as stated above , transgenic mice that express an -actinin-4 k256e transgene in podocytes develop proteinuria and fsgs .", "expression of the er chaperones , bip and grp94 , eif2 phosphorylation , as well as expression of the proapoptotic protein , chop , were increased in glomeruli of transgenic mice , compared with control . based on these results", ", it is reasonable to propose that in the -actinin-4 k256e model of fsgs , there is pronounced er stress , which may be contributing , at least in part , to gec apoptosis .", "the maintenance of a highly dynamic actin - based cytoskeleton is critically important to podocyte morphology and function .", "microfilaments in the foot processes tether the actin cytoskeleton to the slit diaphragm and adhesion complexes , while forming the architectural infrastructure for the foot processes .", "-actinin-4 provides structural support to these microfilaments via its crosslinking and bundling activities , while linking them to components of the slit diaphragm and sites of adhesion .", "the gain affinity mutations in fsgs - associated -actinin-4 substantially alter the properties of the actin cytoskeleton , rendering it more rigid and less dynamic .", "therefore , the underlying pathogenesis of actn4-associated podocyte injury , glomerular filtration barrier dysfunction and the appearance of fsgs lesions are at least partly attributable to an aberrantly high interaction of -actinin-4 with f - actin and its impact upon the cytoskeleton . ", "in addition , the enhanced actin--actinin-4 interaction generates misfolded protein / aggregates , which could provide a parallel mechanism of podocyte dysfunction . as discussed above", ", misfolded proteins may induce dysfunction of the ubiquitin - proteasome system , that is , the misfolded proteins choke or ", "this process may enhance proapoptotic stress in cellular compartments , including the er . in addition , since ubiquitination regulates many essential cellular processes , including normal protein degradation , cell cycle , transcription , dna repair , and protein trafficking , a disrupted ubiquitin - proteasome system may have broader adverse consequences for cell function .", "thus , the pathogenesis of fsgs associated with -actinin-4 k256e may resemble processes in certain age - related or neurodegenerative diseases , where signs of er stress , ups dysfunction , and protein misfolding are observed [ 30 , 45 , 54 , 5658 ] .", "for example , in huntington 's disease , the expansion of a glutamine stretch within the n - terminal region of huntingtin gene generates a protein with severe neurotoxic properties .", "expression of mutant huntingtin leads to a pronounced defect in erad , and upr activation was noted in postmortem huntington 's disease brains .", "familial amyotrophic lateral sclerosis has been linked to mutations in the gene encoding superoxide dismutase-1 , and these mutations induce misfolding and aggregation of this protein , which is believed to contribute to neuronal dysfunction and death .", "activation of the upr is observed in mutant superoxide dismutase-1 transgenic mice , and increased levels of er stress markers , as well as wild - type superoxide dismutase-1 aggregates have been reported in spinal cord tissue of sporadic amyotrophic lateral sclerosis .", "modulation of er stress pathways protected mice with experimental amyotrophic lateral sclerosis from disease progression .", "other examples of neurodegenerative diseases associated with protein misfolding / aggregation and er stress are alzheimer 's , parkinson 's , and prion diseases .", "protein misfolding and ups dysfunction also appears to play a role in desmin - related cardiomyopathies , which result in congestive heart failure . by analogy to experimental neurodegenerative disease models , one must , however , be cautious in extrapolating the cell culture events , which are based on overexpression and a relatively brief experimental time frame , to a disease process that in humans takes many years to become established .", "finally , the shuttling of the wild type , but not mutant -actinin-4 between the cytoplasm and the nucleus , as well as the potential for disruption of gene regulation in the presence of mutant -actinin-4 , will require additional consideration as a potential mechanism of podocyte injury in fsgs ." ]

[ "<S> focal segmental glomerulosclerosis ( fsgs ) is an important cause of proteinuria and nephrotic syndrome in humans . </S>", "<S> the pathogenesis of fsgs may be associated with glomerular visceral epithelial cell ( gec ; podocyte ) injury , leading to apoptosis , detachment , and </S>", "<S> podocytopenia , followed by glomerulosclerosis . </S>", "<S> mutations in -actinin-4 are associated with fsgs in humans . in cultured gecs , </S>", "<S> -actinin-4 mediates adhesion and cytoskeletal dynamics . </S>", "<S> fsgs - associated -actinin-4 mutants show increased binding to actin filaments , compared with the wild - type protein . </S>", "<S> expression of an -actinin-4 mutant in mouse podocytes in vivo resulted in proteinuric fsgs . </S>", "<S> gecs that express mutant -actinin-4 show defective spreading and motility , and such abnormalities could alter the mechanical properties of the podocyte , contribute to cytoskeletal disruption , and lead to injury . the potential for mutant -actinin-4 to injure podocytes </S>", "<S> is also suggested by the characteristics of this mutant protein to form microaggregates , undergo ubiquitination , impair the ubiquitin - proteasome system , enhance endoplasmic reticulum stress , and exacerbate apoptosis . </S>" ]

[ "1. Introduction", "2. Role of ", "3. ", "4. How Do the Biophysical Effects of Mutant ", "5. The Ubiquitin-Proteasome System and Endoplasmic Reticulum (ER) Stress", "6. Evidence for the Proteotoxicity of ", "7. Conclusion" ]

[ [ "glomerular visceral epithelial cells ( gecs ; podocytes ) play a key role in the maintenance of glomerular permselectivity [ 14 ] . under normal conditions , podocytes are in contact with extracellular matrix , and there appears to be little turnover of podocytes , as these cells are highly differentiated ( terminally differentiated ) .", "gecs form a tight network of interdigitating foot processes , which are bridged by filtration - slit diaphragms , and permselectivity of the glomerular capillary wall is dependent on the maintenance of appropriate structure of podocytes and of their foot processes .", "the complex cellular architecture of the podocyte is maintained by an organization of actin filaments in the cytoplasm .", "proteins including nephrin , neph1 , fat , podocin , cd2ap and zo-1 are found within or near the slit diaphragm , and nephrin , neph family proteins , and cadherins form complexes with scaffolding proteins , cd2ap and zo-1 [ 2 , 4 ] .", "these connect the slit - diaphragm protein complex with actin filaments , which are joined by -actinin-4 proteins . acquired gec injury", "is frequently associated with effacement of the foot processes , disruption of the filtration - slit diaphragms , and proteinuria [ 1 , 2 ] . moreover , heritable mutations in several gec structural proteins alter podocyte ultrastructure and cause heritable proteinuria , implying that impairment of the slit diaphragm or cytoskeletal structure underlies proteinuria [ 4 , 5 ] . focal segmental glomerulosclerosis ( fsgs ) is an important cause of proteinuria and nephrotic syndrome in humans . in recent years", ", evidence has accumulated to support the view that the early pathogenesis of fsgs is associated with gec injury , leading to apoptosis , detachment of gecs from extracellular matrix , and podocytopenia , which is followed by glomerulosclerosis [ 13 , 68 ] .", "acquired gec injury may be triggered by immunological factors ( e.g. , t cell or other factors affecting permeability ) , oxidants , human immunodeficiency virus , toxins , and other substances .", "in addition , fsgs may be associated with heritable mutations in several distinct proteins that play key roles in maintaining gec ultrastructure , including podocin , -actinin-4 , transient receptor potential cation channel , subfamily c , member 6 ( trpc6 ) , inverted formin 2 and others . in this paper , we focus on mutations in -actinin-4 , which are associated with an autosomal dominant late - onset fsgs in humans .", "we discuss the structure and function of -actinin-4 , and we review mechanisms by which mutations in -actinin-4 may lead to podocyte injury and fsgs ." ], [ "there are four -actinin genes ( actn1 - 4 ) that encode highly homologous proteins ( -actinin-1 , 2 , 3 and 4 ) , which form ~100 kda head - to - tail homodimers . -actinins-2 and -3 are ca - insensitive muscle -actinins expressed in z - discs of striated muscle cells , while -actinins-1 and -4 are widely distributed , nonmuscle isoforms .", "all family members are actin crosslinking proteins that share a number of structural features and regulatory regions .", "these include a central rod with four spectrin - like repeats , two n - terminal calponin - homology domains ( ch1 and ch2 ) , which contain three actin - binding sites ( abs1 - 3 ) , a c - terminal calmodulin - like domain , containing two putative ef hands , and a phosphoinositide - binding plextrin homology domain .", "the spectrin - like repeats facilitate homodimerization and may confer a degree of flexibility to the -actinin molecule , allowing it to resist mechanical strain . for its interaction with actin filaments ,", "the ch1 and ch2 domains adopt a closed conformation , where abs 2 and 3 are exposed , while abs1 remains buried and inaccessible to f - actin .", "this association with actin is highly dynamic and can be controlled by the regulatory regions along the -actinin sequence . ", "all -actinin isoforms contain two putative ef - hand motifs at the c - terminus of each monomer , but only isoforms 1 and 4 demonstrate sensitivity to ca [ 15 , 16 ] . for these nonmuscle isoforms , binding of ca", "is believed to reduce the affinity of -actinin for actin , providing a mechanism for -actinin - dependent cytoskeletal remodeling .", "additionally , extensive biochemical studies on the effect of phosphoinositide binding to -actinin-1 have been performed .", "binding of phosphatidylinositol ( 3,4,5)-trisphosphate ( pip3 ) , and to a lesser extent phosphatidylinositol ( 4,5)-bisphosphate ( pip2 ) , decreases the association between -actinin-1 and f - actin , suggesting that phosphoinositides mediate cytoskeletal remodeling [ 17 , 18 ] . for -actinin-4 ,", "in contrast to findings for -actinin-1 , our studies showed that phosphoinositides increase the interaction of -actinin-4 with f - actin .", "tyrosine phosphorylation at position 12 was found to be dependent on focal adhesion kinase ( fak ) , supporting a role for -actinin in cell adhesion .", "phosphorylation of -actinin-1 reduced its association with f - actin in vitro , suggesting that -actinin mediates fak - dependent cytoskeletal remodeling .", "the amino acid sequence of -actinin-4 reveals an analogous tyrosine residue at position 32 , suggesting that phosphorylation may also regulate the binding of -actinin-4 to f - actin .", "however , we were unable to demonstrate fak - dependent tyrosine phosphorylation of -actinin-4 ( unpublished observations ) .", "expression of these isoforms at the cytoplasmic face of several types of cellular interaction sites , including focal adhesions , adherens junctions , and hemidesmosomes suggests a degree of versatility .", "the first -actinin binding partners identified were the subunits of integrins and intercellular adhesion molecule-1 .", "since -actinins also bind to various regulatory molecules , -actinin may also serve as a scaffolding protein to integrate signaling molecules at various adhesion sites .", "-actinin-1 may link membrane proteins such as talin , vinculin , and -integrins with the cortical actin cytoskeleton [ 10 , 2225 ] .", "similarly , the tight junction protein , magi-1 , interacts with the c - terminus of -actinin-4 , providing a physical link from the cell periphery to the cytoskeleton .", "the list of interacting proteins is likely to grow and will provide a better understanding of the diverse functions of -actinins in nonmuscle tissues .", "however , the best defined function of -actinin-4 is to crosslink / bundle actin filaments , and it may enhance cell motility and elicit tumor - suppressor activity . intriguingly , -actinin-4 is reported to shuttle between the cytoplasm and the nucleus .", "cytoplasmic localization was associated with an infiltrative phenotype and correlated significantly with a poorer prognosis in cases of breast cancer . in this context", ", the relevance of nuclear -actinin-4 remains unknown , although it has been suggested that nuclear translocation may attenuate metastatic potential .", "the nuclear localization of -actinin-4 suggests a role for -actinin-4 in gene expression . -actinins-1 and", "-4 were identified as histone deacetylase 7 ( hdac7)-interacting proteins , and the interaction domain was mapped to the c - terminus of -actinin 4 .", "hdac7 can participate in multiple cellular processes , including the regulation of myocyte enhancer factor-2- ( mef2- ) mediated transcription .", "a point mutation in hdac7 disrupted its association with -actinin-4 and mef2 , implying that -actinin-4 and mef2 binding sites overlap .", "overexpression of -actinin-4 also potentiated estrogen receptor -mediated transcription by counteracting a negative regulatory effect of hdac7 , while knockdown of -actinin-4 decreased expression of estrogen receptor target genes and affected proliferation of cultured breast cancer cells .", "another study demonstrated that -actinin-4 colocalized along actin stress fibers and in membrane lamellae with nuclear factor-b ( nf-b ) .", "incubation of cells with epidermal growth factor or tumor necrosis factor- induced translocation of -actinin-4 and the p65 subunit of nf-b into the nucleus .", "as nf-b regulates transcription of a large number of genes in response to diverse stimuli , the study further supports a regulatory role for -actinin-4 in transcription ." ], [ "human gecs express only -actinin-4 ( although mouse gecs also express -actinin-1 ) [ 10 , 30 ] .", "immunoelectron microscopy studies showed that -actinin localizes to contractile microfilaments within podocyte foot processes [ 31 , 32 ] .", "in cultured mouse gecs , -actinin-4 was found along actin stress fibers , focal adhesions , and within the cortical actin network at the cell periphery ( figure 1 ) .", "this specific subcellular localization of -actinin-4 facilitates its regulation of adhesion and cytoskeletal dynamics [ 19 , 33 , 36 ] .", "interestingly , -actinin-4 was also present in the nucleus of some rat gecs [ 34 , 35 ] , in keeping with earlier reports in other cell lines .", "normally , -actinin-4 may be required for integrin - dependent adhesion of gecs . indeed ,", "knockout of -actinin-4 in mice resulted in reduced glomerular podocyte number , accompanied by the appearance of urinary wilm 's tumor-1 ( wt-1 ) , a podocyte biomarker .", "such podocyte loss is consistent with defective glomerular basement membrane adhesion , as tunel assays failed to detect any apoptotic cells in -actinin-4 null glomeruli .", "the severity of the resulting phenotype seen in -actinin-4 null mice ( i.e. , glomerular disease , renal failure , and early death ) indicates that -actinin-4-mediated podocyte adhesion is critical for filtration barrier function [ 30 , 37 , 38 ] . on the other hand , transgenic podocyte overexpression of wild - type -actinin-4 in mice", "these findings suggest that podocytes tolerate a wide range of -actinin-4 expression , but that a minimum threshold level is essential for normal gec adhesion to the glomerular basement membrane through its interaction with integrins . ", "deficiency of -actinin-4 leading to human disease is not described , but several point mutations or single amino acid deletions in -actinin-4 are found in heritable forms of human fsgs .", "most mutations in actn4-associated fsgs families congregate at the ch1-ch2 interface [ 9 , 40 ] .", "an example is the autosomal dominant k255e mutation ( k256e is the mouse mutant corresponding to human k255e ) .", "such mutants show increased binding to actin filaments , compared with the wild - type protein [ 30 , 41 , 42 ] .", "interestingly , -actinin-4 k256e is insensitive to regulation by ca and phosphoinositides ( pip2 and pip3 ) , suggesting that its gain of affinity for f - actin blunts its responsiveness to regulatory factors .", "expression of an -actinin-4 k256e transgene in mouse podocytes in vivo ( under the control of the nephrin promoter ) resulted in development of proteinuric fsgs .", "in addition , homozygous ( but not heterozygous ) knock - in of the mutant -actinin-4 allele into the actn4 locus in mice induced proteinuria , confirming the pathogenic potential of -actinin-4 mutations . ", "the underlying biochemical mechanism , whereby fsgs - associated actn4 mutations enhance affinity for actin , was uncovered by pollak 's group .", "they showed that disease - causing mutations disable an important intramolecular hinge that normally holds ch1 and ch2 in a closed conformation .", "the mutant protein adopts an open conformation forcing an interaction of all three actin - binding sites with the actin filament , thereby increasing the binding affinity by lowering its rate of dissociation from actin . " ], [ "although -actinin-4 is widely expressed , human podocytes appear to be selectively vulnerable to mutations in -actinin-4 ( including k256e and other analogous mutants ) , perhaps because podocytes are terminally differentiated cells with limited regenerative capacity .", "the downstream cellular mechanism(s ) by which mutant -actinin-4 leads to podocyte injury and fsgs is poorly understood .", "one possibility is that injury is secondary to alterations in the mechanical properties of the podocyte via increased affinity of mutant -actinin-4 for f - actin ( figure 2 ) .", "gecs that express -actinin-4 k256e show defective spreading and motility ( figure 1 ) . moreover , exposure of -actinin-4 k256e expressing gecs to cyclical equibiaxial stretch , an in vitro mimic of the mechanical forces due to glomerular capillary pressure , significantly reduced cell surface area and caused retraction of cellular processes .", "lastly , the enhanced association with f - actin alters the subcellular localization of -actinin-4 , restricting its presence at the cell periphery ( figure 1 ) , and potentially altering its capacity to interact with various binding partners [ 19 , 33 ] .", "such abnormalities could , at least in part , be attributable to cytoskeletal disruption , as well as loss of integrin engagement , effectively yielding a phenotype similar to that of the -actinin-4 null mouse [ 36 , 43 ] . since the actin cytoskeleton is a key component contributing to the unique morphological characteristics of the podocyte foot processes , as well as being connected to both slit diaphragm and adhesion complexes , altered -actinin-4 biology must have important implications for podocyte health . ", "an alternate and/or parallel mechanism , which we also consider in this paper , is that mutant -actinin-4 induces proteotoxicity in podocytes ( i.e. , an impairment of podocyte function caused by misfolding of a protein ) , ultimately leading to apoptosis ( figure 2 ) .", "consistent with this notion , -actinin-4 k256e forms actin - associated aggregates in cultured gecs and in podocytes of both homozygous k256e knock - in mice and humans with actn4-associated fsgs [ 30 , 33 , 35 ] . such misfolded / aggregated protein may be associated with the activation of stress pathways in podocytes ( see below ) ." ], [ "prior to discussing the proteotoxic potential of mutant -actinin-4 , this section provides a brief overview of the ubiquitin - proteasome system and er stress .", "the ubiquitin - proteasome system plays a key role in regulating the half - life of short - lived cellular proteins , and in selective degradation of damaged or abnormal proteins [ 45 , 46 ] .", "the proteasome degrades aberrant cytoplasmic or cytoskeletal proteins , and misfolded er proteins , which are retrotranslocated selectively to the cytoplasm .", "the latter process is known as er - associated degradation ( erad ) [ 47 , 48 ] .", "ubiquitin - proteasome pathway - mediated protein degradation involves tagging of the substrate by covalent attachment of ubiquitin molecules via a three - step reaction , and degradation of the tagged protein by the 26s proteasome complex .", "ubiquitinated proteins typically undergo efficient degradation by the proteasome , but sometimes , large amounts of misfolded proteins are not degraded effectively , and may form covalently - linked aggregates . such misfolded proteins and/or aggregates may impair the function of the proteasome and lead to the activation of stress pathways and cytotoxicity .", "secretory , luminal , and membrane proteins normally attain their correctly folded conformation in the er via er - resident chaperones . to rescue misfolded proteins , the er has in place quality control machinery , including the unfolded protein response ( upr ) [ 4953 ] , and erad [ 47 , 48 ] . in the upr ,", "accumulation of misfolded proteins in the er results in the activation of three er sensors . activating transcription factor-6 moves from the er to the golgi , where it is cleaved by proteases .", "the cleaved cytosolic fragment translocates to the nucleus to activate transcription of er chaperones , for example , the glucose - regulated proteins ( grp ) , grp94 and bip ( grp78 ) , and others . in parallel ,", "inositol requiring-1 activates its endoribonuclease activity , cleaving x - box binding protein-1 mrna and changing the reading frame to yield a potent transcriptional activator .", "normally , er chaperones assist in posttranslational processing of proteins and in their exit from the er , and may complex with defective proteins to target them for degradation . during stress ,", "induction of er chaperones may enhance the protein folding capacity , and limit accumulation of abnormal proteins . ", "misfolded proteins in the er also activate perk ( pkr - like er kinase ) , which then phosphorylates the eukaryotic translation initiation factor-2 subunit ( eif2 ) .", "this process reduces initiation aug codon recognition , and the general rate of translation is blunted ( which decreases the protein load on a damaged er ) .", "the upr allows cells to recover from stress , and may be protective to additional insults , but substantial / prolonged er stress may lead to apoptosis .", "for example , certain mrnas may be translated preferentially after eif2 is phosphorylated . among these is activating transcription factor-4 , which induces expression of several genes , including chop ( c / ebp homologous protein-10 ; also known as gadd153 ) , a gene closely associated with apoptosis and/or growth arrest [ 49 , 51 ] .", "apoptosis may also result from activation of caspase-12 or protein kinases [ 49 , 51 ] .", "impairment of the ubiquitin - proteasome system can be associated with exacerbation of er stress [ 49 , 54 ] , perhaps by interference with erad ." ], [ "the potential for mutant -actinin-4 to impair podocyte function is suggested by the characteristics of this mutant protein to form microaggregates , undergo ubiquitination , impair the ubiquitin - proteasome system , enhance er stress , and enhance apoptosis ( figure 2 ) .", "density - gradient centrifugation of the k255e mutant -actinin-4 showed abnormal sedimentation , suggesting that the mutant protein forms high molecular mass aggregates . in keeping with this result , expression of -actinin-4 k255e ( and other fsgs - inducing mutants ) , but", "not wild - type -actinin-4 in cultured mouse gecs resulted in formation of aggregates , as visualized by fluorescence microscopy [ 30 , 33 , 42 , 43 ] .", "analogous results were obtained in rat gecs stably transfected with -actinin-4 k256e , where in some cells , the mutant ( but not the wild type ) protein formed aggregates . unlike the wild - type protein , the mutant was not present in the nucleus of rat gecs [ 34 , 35 ] .", "in addition , -actinin-4 k255e formed aggregates in podocytes of homozygous k255e knock - in mice and humans with actn4-associated fsgs .", "misfolded / aggregated proteins may result in the activation of stress pathways . in pulse - chase metabolic labeling experiments , mutant -actinin-4", "was degraded more rapidly , compared with the wild - type protein , with the mutant showing a half - life of ~15 h , and the wild type of over 30 h .", "consistent with this result , after stable transfection in rat gecs or transient transfection in cos cells ( a monkey kidney cell line , which allows for high levels of protein expression ) , the level of -actinin-4 k256e protein was considerably lower , compared with the wild - type protein ( figure 3 ) [ 34 , 35 ] .", "together , these results suggested that -actinin-4 k256e may undergo ubiquitination prior to degradation via the ubiquitin - proteasome pathway .", "indeed , in transiently transfected cos cells , mutant -actinin-4 was polyubiquitinated , whereas the wild - type protein was not ( figure 3 ) .", "treatment of gecs that express -actinin-4 k256e with proteasome inhibitors enhanced expression of -actinin-4 k256e , in keeping with ubiquitination and degradation of the mutant protein by the proteasome [ 30 , 35 ] . \n", "-actinin-4 k256e and wild - type proteins were transiently overexpressed in cos cells to study their effects on the ubiquitin - proteasome system .", "the function of the ubiquitin - proteasome system was monitored in viable cells by use of a reporter consisting of a short degron , cl1 , fused to the c - terminus of green fluorescent protein ( gfp ) .", "this gfp proteasome reporter is rapidly degraded when ubiquitin - proteasome function is normal , whereas impairment of the ubiquitin - proteasome system , for example , via enhanced flux of a mutant / aggregated protein will reduce degradation of gfp , resulting in an increased level of gfp expression . in cos cells transfected with wild - type -actinin-4 ,", "expression of gfp declined significantly , in keeping with proteasomal degradation of gfp ( figure 3 ) .", "in contrast , expression of -actinin-4 k256e retarded the degradation of gfp , implying that the ubiquitin - proteasome system was impaired in the presence of mutant -actinin-4 . \n", "-actinin-4 is a cytosolic / cytoskeletal protein that normally would not enter the er to undergo posttranslational modification .", "nonetheless , compared with -actinin-4 wild type , transient transfection of -actinin-4 k256e in cos cells enhanced the upr , as evidenced by increased expression of the er chaperone , grp94 , and the proapoptotic gene , chop ( figure 3 ) . in gecs ,", "expression of the k256e mutant or wild type -actinin-4 ( by stable transfection ) did not increase expression of the er chaperone , bip , or chop , compared with parental ( untransfected ) cells .", "this difference between cos cells and gecs may be due to lower expression of stably transfected proteins in the gecs .", "however , after incubation of gecs with tunicamycin ( a drug that blocks n - linked glycosylation and causes an accumulation of misfolded proteins in the er ) , the gecs stably transfected with -actinin-4 k256e showed increases in bip and chop that significantly exceeded the increases seen in cells expressing the wild - type protein , as well as the increases in parental gecs .", "the effect of -actinin-4 k256e on the induction of the upr by tunicamycin was particularly robust , given that expression of the mutant was substantially lower than the wild - type protein .", "thus , although stable expression of -actinin-4 k256e in gecs did not induce the upr directly , the mutant appeared to adversely affect the integrity of the er , which may render these cells more susceptible to additional stress and induction of proapoptotic genes .", "together , these results are in keeping with the view that exacerbation of er stress may be secondary to impairment of the ubiquitin - proteasome system [ 49 , 54 ] . stable expression of -actinin-4 k256e in gecs led to a reduction in cell number , as well as increased apoptosis and caspase-3 activity , compared with gecs that stably express -actinin-4 wild type , or parental ( untransfected ) gecs .", "these changes in cell number and apoptosis in the presence of -actinin-4 k256e occurred despite the significantly lower expression level of the mutant , compared with the wild - type -actinin-4 , highlighting the potential detrimental consequences of this mutation . in cos cells , -actinin-4 k256e had only a slight effect on apoptosis and cell number , suggesting that this cell type may be more resistant to cytotoxicity .", "nevertheless , -actinin-4 k256e markedly exacerbated apoptosis and reduced cell number in the context of mild proteasome inhibition .", "this result supports the view that apoptosis induced by mutant -actinin-4 may be associated with an impairment in proteasome function .", "the effects of -actinin-4 k256e on the upr ( discussed above ) are based on studies in cultured cell lines , but importantly , analogous changes also occurred in vivo .", "as stated above , transgenic mice that express an -actinin-4 k256e transgene in podocytes develop proteinuria and fsgs .", "expression of the er chaperones , bip and grp94 , eif2 phosphorylation , as well as expression of the proapoptotic protein , chop , were increased in glomeruli of transgenic mice , compared with control . based on these results", ", it is reasonable to propose that in the -actinin-4 k256e model of fsgs , there is pronounced er stress , which may be contributing , at least in part , to gec apoptosis ." ], [ "the maintenance of a highly dynamic actin - based cytoskeleton is critically important to podocyte morphology and function .", "microfilaments in the foot processes tether the actin cytoskeleton to the slit diaphragm and adhesion complexes , while forming the architectural infrastructure for the foot processes .", "-actinin-4 provides structural support to these microfilaments via its crosslinking and bundling activities , while linking them to components of the slit diaphragm and sites of adhesion .", "the gain affinity mutations in fsgs - associated -actinin-4 substantially alter the properties of the actin cytoskeleton , rendering it more rigid and less dynamic .", "therefore , the underlying pathogenesis of actn4-associated podocyte injury , glomerular filtration barrier dysfunction and the appearance of fsgs lesions are at least partly attributable to an aberrantly high interaction of -actinin-4 with f - actin and its impact upon the cytoskeleton . ", "in addition , the enhanced actin--actinin-4 interaction generates misfolded protein / aggregates , which could provide a parallel mechanism of podocyte dysfunction . as discussed above", ", misfolded proteins may induce dysfunction of the ubiquitin - proteasome system , that is , the misfolded proteins choke or ", "this process may enhance proapoptotic stress in cellular compartments , including the er . in addition , since ubiquitination regulates many essential cellular processes , including normal protein degradation , cell cycle , transcription , dna repair , and protein trafficking , a disrupted ubiquitin - proteasome system may have broader adverse consequences for cell function .", "thus , the pathogenesis of fsgs associated with -actinin-4 k256e may resemble processes in certain age - related or neurodegenerative diseases , where signs of er stress , ups dysfunction , and protein misfolding are observed [ 30 , 45 , 54 , 5658 ] .", "for example , in huntington 's disease , the expansion of a glutamine stretch within the n - terminal region of huntingtin gene generates a protein with severe neurotoxic properties .", "expression of mutant huntingtin leads to a pronounced defect in erad , and upr activation was noted in postmortem huntington 's disease brains .", "familial amyotrophic lateral sclerosis has been linked to mutations in the gene encoding superoxide dismutase-1 , and these mutations induce misfolding and aggregation of this protein , which is believed to contribute to neuronal dysfunction and death .", "activation of the upr is observed in mutant superoxide dismutase-1 transgenic mice , and increased levels of er stress markers , as well as wild - type superoxide dismutase-1 aggregates have been reported in spinal cord tissue of sporadic amyotrophic lateral sclerosis .", "modulation of er stress pathways protected mice with experimental amyotrophic lateral sclerosis from disease progression .", "other examples of neurodegenerative diseases associated with protein misfolding / aggregation and er stress are alzheimer 's , parkinson 's , and prion diseases .", "protein misfolding and ups dysfunction also appears to play a role in desmin - related cardiomyopathies , which result in congestive heart failure . by analogy to experimental neurodegenerative disease models , one must , however , be cautious in extrapolating the cell culture events , which are based on overexpression and a relatively brief experimental time frame , to a disease process that in humans takes many years to become established .", "finally , the shuttling of the wild type , but not mutant -actinin-4 between the cytoplasm and the nucleus , as well as the potential for disruption of gene regulation in the presence of mutant -actinin-4 , will require additional consideration as a potential mechanism of podocyte injury in fsgs ." ] ]

[ "through the last twenty years , the knowledge on thyroid disease during pregnancy has rapidly expanded .", "it is well documented that women with overt hypothyroidism during pregnancy have an increased risk of pregnancy loss and adverse pregnancy outcome [ 13 ] , but the consequences of subclinical hypothyroidism and the significance of concomitant thyroid peroxidase antibodies ( tpoab ) are debated [ 46 ] .", "subclinical hypothyroidism is a condition in which a slightly raised thyroid stimulating hormone ( tsh ) signal is representing an early , mild thyroid failure .", "the persistency of mild ( subclinical ) hypothyroidism may differ according to ethnicity and age ; however , tsh increase with age as well as the prevalence of tpoab positivity is increasing with age .", "further the presence of tpoab does seem important , as resolution to euthyroidism is reported much higher in tpoab negative subjects compared to tpoab positive .", "women with asymptomatic autoimmune thyroid disorders , who are euthyroid in early pregnancy , carry a significant risk of developing hypothyroidism progressively during gestation . the aim of this study was to estimate the significance of tsh , thyroid peroxidase antibody ( tpoab ) , and mild ( subclinical ) hypothyroidism in women from the danish general suburban population study ( gesus ) on the number of children born , the number of pregnancies , and the number of spontaneous abortions .", "the danish general suburban population study ( gesus ) was initiated in january 2010 and finished in october 2013 .", "gesus is a cross sectional study of the adult danish suburban general population in naestved municipality ( 70 km south of copenhagen ) selected on the basis of the danish central population register code .", "the gesus study was designed to invite only 25% of younger women of 2029 years age influencing the distribution of age .", "the health examination included a comprehensive physical examination ( body mass index ( bmi ) ) , biochemical tests and a self - administrated questionnaire ( prevalent disease ( diabetes mellitus , hypothyroidism , and hyperthyroidism ) , antihypertensive and cholesterol lowering medication , thyroid and antithyroid medication , contraception , smoking , income , education , employment , the number of pregnancies , the number of children born , age of first child , and the number of spontaneous abortions ) .", "gesus had an overall participation rate of women of 45% ( n = 11565 ) . in this study", "we included participants of european origin ( 99% danish ) ( n = 11387 ) and excluded participants with missing values of tsh , ft4 , and tt3 ( n = 50 ) and missing values of the number of pregnancies , the number of children born , and the number of spontaneous abortions ( n = 31 ) .", "prevalent hypothyroidism was defined as history of hypothyroidism or intake of t4/t3 medication , and prevalent hyperthyroidism was defined as history of hyperthyroidism or intake of antithyroid medication .", "thyroid hormones within the reference interval were defined as ft4 = 10.024.0 pmol / l , tt3 = 1.202.90 nmol / l , and tsh = 0.43.7 mu / l .", "mild ( subclinical ) hypothyroidism was defined as tsh > 3.7 mu / l and ft4 and tt3 within the reference interval , no history of thyroid disease , and no intake of t4/t3 or antithyroid medication .", "controls were defined as having 0.4 < tsh 3.7 mu / l and ft4 and tt3 within the reference interval , no history of thyroid disease , and no intake of t4/t3 or antithyroid medication [ 13 , 14 ] .", "thyroid peroxidase antibody ( tpoab ) positivity was defined by the cut - off value of tpoab > 60 u / ml . all biochemical tests were performed at the same laboratory at naestved hospital .", "measurements of tsh and of thyroid hormones - free thyroxin ( ft4 ) and total triiodothyronine ( tt3 ) were performed using an electrochemiluminescent immunoassay ( roche cobas 6000 , basel , switzerland ) .", "l ( cv < 5% ) , and tt3 reference range 1.202.90 nmol / l ( cv < 4% ) .", "tpoab was measured by kryptor antitpon ( brahms , henigsdorf , germany ) , with detection limit of 10 ", "all statistical analyses were performed using stata version 13.0 for windows ( statacorp , college station , tx , usa ) . a value of p < 0.05 was considered statistically significant .", "tsh and tpoab were not normally distributed and therefore log - transformed ( logtsh and logtpoab ) .", "we performed the following statistical models : for the full cohort of women : linear regression analyses of tsh and tpoab of children born and the number of pregnancies , logistic regression for spontaneous abortion;for women with prevalent hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion;for women with subclinical hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion.models designed were either age - adjusted or multifactorially adjusted using age , bmi , diabetes , contraception , education , income , employment , smoking , antihypertensive medication , cholesterol lowering medication , and menopause as listed in table 1 .", "for the full cohort of women : linear regression analyses of tsh and tpoab of children born and the number of pregnancies , logistic regression for spontaneous abortion ; linear regression analyses of tsh and tpoab of children born and the number of pregnancies , logistic regression for spontaneous abortion ; for women with prevalent hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion ; logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion ; for women with subclinical hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion . logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion .", "the study was approved by the regional ethics committee of zealand , denmark ( reg .", "number rvk sj-177 , sj-113 , and sj-114 ) , registered with clinicaltrial.gov ( nct01335802 ) , and reported to the danish data protection agency .", "\n table 1 shows characteristics of women . in total , 758 ( 6.7% ) had mild ( subclinical ) hypothyroidism , 9.4% prevalent hypothyroidism , and 4.2% prevalent hyperthyroidism . in women with mild hypothyroidism tpoab", "logtsh and logtpoab were negatively linearly associated with the number of children born and number of pregnancies in the full cohort in age - adjusted and multiadjusted models ( table 2 ) .", "mild ( subclinical ) hypothyroidism was associated with a risk of not having children and a risk of not getting pregnant in age - adjusted and multiadjusted models .", "prevalent hypothyroidism was not associated with the number of children born , the number of pregnancies , or spontaneous abortions ( table 3 ) .", "in the present study , we showed that with higher tsh levels the less number of children born and the less number of pregnancies .", "coefficients for logtsh were higher than for logtpoab ; thus , the effect of tsh seems higher than for tpoab .", "mild hypothyroidism was also associated with a higher age of first child born and risk of not having children and not getting pregnant .", "tpoab was not a significant confounder in the models , and there was no interaction with age .", "this analysis is cross sectional and in a way retrospective for those women who are now menopausal . also , for women who are premenopausal we may not have the life - time full number of children born , the number of pregnancies , and the number of spontaneous abortions .", "we can not adjust for age of the mother at the birth of her 1st child as this only applies for those who have had children ; thus , those with no children have missing values in that category .", "we have included the information in table 1 , and it appears that there is a significant difference between those with mild hypothyroidism and controls .", "furthermore , were we not able to perform analyses of pregnancy and child birth history restricted to only within few years of the tsh examination .", "this may be caused by the fact that , during fertile life , early miscarriage may have gone unnoticed or been forgotten .", "had recently reported that women with a combination of subclinical hypothyroidism and thyroid autoimmunity were found to have the highest risk of miscarriage before 20 weeks of gestation compared to only tpoab positivity or subclinical hypothyroidism .", "in contrast , previous prospective studies have confirmed an association between mild hypothyroidism during pregnancy and pregnancy loss .", "allan et al . showed that stillbirth was significantly more frequent in women having tsh higher than 6.0 ", "reported that the risk of child loss was increased with higher levels of maternal tsh , whereas concentrations of maternal ft4 and child loss were not associated .", "observed that the evolution of pregnancy did not depend on whether the hypothyroidism was overt or mild but depended on adequate treatment during pregnancy .", "these findings correspond to our results , since the odds ratios for 1 pregnancy and child birth versus none were not significant comparing prevalent hypothyroidism versus controls reflecting a well - treated condition .", "furthermore that mild hypothyroidism is representing nonadequate treatment and mild thyroid failure was associated with a risk of not having children and a risk of not getting pregnant .", "accordingly , in our study we observed that elevated tsh above the upper reference limit in mild hypothyroidism correlated with the number of pregnancies and child births .", "several studies have reported a possible association between thyroid dysfunction during pregnancy and specific adverse pregnancy outcome like preeclampsia [ 19 , 20 ] , placental abruption , or preterm delivery [ 21 , 22 ] .", "it has been suggested that higher tsh and tpoab positivity were independently associated with lower likelihood of reversion to euthyroidism . in a 20-year follow - up study ,", "an increased serum tsh level was predictive of progression to overt hypothyroidism , and the annual rate of progression to overt hypothyroidism was more than 4% in women with both raised serum tsh and antithyroid antibodies .", "showed a trend towards slightly higher serum tsh levels in women with thyroid autoimmunity in the first trimester of pregnancy compared to women without thyroid autoimmunity and that women with asymptomatic autoimmune thyroid disease who were euthyroid in early pregnancy carried a significant risk of developing hypothyroidism progressively during gestation , despite a marked reduction in antibody titers .", "this retrospective cross sectional study showed that among women without any history of thyroid disease or antihyperthyroid / antihypothyroid medication 6.7% had mild ( subclinical ) hypothyroidism .", "the prevalence of mild hypothyroidism was comparable to previous studies like the colorado study in which 8.5% had subclinical hypothyroidism increasing with age explained by the chosen value of tsh cut - off defining mild hypothyroidism .", "the presence of mild hypothyroidism influenced the level of ft4 within the reference range as ft4 was decreased in women with mild hypothyroidism .", "this could be explained by an increased intracellular deiodination of t4 to t3 in women with mild hypothyroidism to avoid decreased levels of active intracellular thyroid hormone concentrations .", "we are not able to determine the status during pregnancy as the present study is retrospective , but it seems unlikely that tpoab would have an effect directly on metabolism and peripheral thyroid hormone regulated cellular function .", "however , we can not exclude the possibility that the women presenting with mild hypothyroidism have had a previous normalized tsh level .", "this population study was performed in naestved municipality eastern denmark representing a mild iodine deficiency . in denmark ,", "the iodine intake was stable at a low level for many years and in 2000 , the mandatory iodine fortification of bread salt and household salt began .", "follow - up studies reported an increase in prevalence of hypothyroidism and tpoab positivity especially among younger women .", "the present study is limited by the fact that all clinical observations are self - reported questionnaire data .", "furthermore , we did not have any information about the numbers of induced abortions which could influence the data of child births although it is rarely recommended to induce an abortion only due to high levels of tsh during first trimester of pregnancy .", "in addition , individuals were classified based on a single blood test ; thus , we can not distinguish between transient and permanent tsh elevation ; however , somwaru et al . showed that subclinical hypothyroidism was persistent in 56% after 4-year follow - up .", "finally , due to financial reasons the gesus study was designed to invite only 25% of younger women aged 2030 years old influencing the distribution of age .", "the strength of our study was the high number of participants with blood samples of tsh , ft4 , tt3 , and tpoab in 11254 women .", "furthermore , for the analyses of mild ( subclinical ) hypothyroidism we excluded women with any self - reported thyroid disease or use of t4/t3 or antithyroid medication and only compared euthyroid and mild hypothyroid women .", "taken together , we observed that with higher tsh levels the less number of children born and the less number of pregnancies .", "mild hypothyroidism was also associated with a higher age of first child born and risk of not having children and not getting pregnant . in conclusion", ", we observed that impaired fertility is associated with tsh , tpoab , and mild ( subclinical ) hypothyroidism in a danish population of women ." ]

[ "<S> \n introduction . </S>", "<S> the aim of this study was to estimate the significance of tsh , thyroid peroxidase antibody ( tpoab ) , and mild ( subclinical ) hypothyroidism in women from the danish general suburban population study ( gesus ) on the number of children born , the number of pregnancies , and the number of spontaneous abortions . </S>", "<S> methods . </S>", "<S> retrospective cross sectional study of 11254 women participating in gesus . </S>", "<S> data included biochemical measurements and a self - administrated questionnaire . results . </S>", "<S> 6.7% had mild ( subclinical ) hypothyroidism and 9.4% prevalent hypothyroidism . in women with mild hypothyroidism tpoab </S>", "<S> was significantly elevated and age at first child was older compared to controls . </S>", "<S> tsh and tpoab were negatively linearly associated with the number of children born and the number of pregnancies in the full cohort in age - adjusted and multiadjusted models . </S>", "<S> tsh or tpoab was not associated with spontaneous abortions . </S>", "<S> mild ( subclinical ) hypothyroidism was associated with a risk of not having children and a risk of not getting pregnant in age - adjusted and multiadjusted models . </S>", "<S> prevalent hypothyroidism was not associated with the number of children born , the number of pregnancies , or spontaneous abortions . conclusion . </S>", "<S> impaired fertility is associated with tsh , tpoab , and mild ( subclinical ) hypothyroidism in a danish population of women . </S>" ]

[ "1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion", "5. Conclusion" ]

[ [ "through the last twenty years , the knowledge on thyroid disease during pregnancy has rapidly expanded .", "it is well documented that women with overt hypothyroidism during pregnancy have an increased risk of pregnancy loss and adverse pregnancy outcome [ 13 ] , but the consequences of subclinical hypothyroidism and the significance of concomitant thyroid peroxidase antibodies ( tpoab ) are debated [ 46 ] .", "subclinical hypothyroidism is a condition in which a slightly raised thyroid stimulating hormone ( tsh ) signal is representing an early , mild thyroid failure .", "the persistency of mild ( subclinical ) hypothyroidism may differ according to ethnicity and age ; however , tsh increase with age as well as the prevalence of tpoab positivity is increasing with age .", "further the presence of tpoab does seem important , as resolution to euthyroidism is reported much higher in tpoab negative subjects compared to tpoab positive .", "women with asymptomatic autoimmune thyroid disorders , who are euthyroid in early pregnancy , carry a significant risk of developing hypothyroidism progressively during gestation . the aim of this study was to estimate the significance of tsh , thyroid peroxidase antibody ( tpoab ) , and mild ( subclinical ) hypothyroidism in women from the danish general suburban population study ( gesus ) on the number of children born , the number of pregnancies , and the number of spontaneous abortions ." ], [ "the danish general suburban population study ( gesus ) was initiated in january 2010 and finished in october 2013 .", "gesus is a cross sectional study of the adult danish suburban general population in naestved municipality ( 70 km south of copenhagen ) selected on the basis of the danish central population register code .", "the gesus study was designed to invite only 25% of younger women of 2029 years age influencing the distribution of age .", "the health examination included a comprehensive physical examination ( body mass index ( bmi ) ) , biochemical tests and a self - administrated questionnaire ( prevalent disease ( diabetes mellitus , hypothyroidism , and hyperthyroidism ) , antihypertensive and cholesterol lowering medication , thyroid and antithyroid medication , contraception , smoking , income , education , employment , the number of pregnancies , the number of children born , age of first child , and the number of spontaneous abortions ) .", "gesus had an overall participation rate of women of 45% ( n = 11565 ) . in this study", "we included participants of european origin ( 99% danish ) ( n = 11387 ) and excluded participants with missing values of tsh , ft4 , and tt3 ( n = 50 ) and missing values of the number of pregnancies , the number of children born , and the number of spontaneous abortions ( n = 31 ) .", "prevalent hypothyroidism was defined as history of hypothyroidism or intake of t4/t3 medication , and prevalent hyperthyroidism was defined as history of hyperthyroidism or intake of antithyroid medication .", "thyroid hormones within the reference interval were defined as ft4 = 10.024.0 pmol / l , tt3 = 1.202.90 nmol / l , and tsh = 0.43.7 mu / l .", "mild ( subclinical ) hypothyroidism was defined as tsh > 3.7 mu / l and ft4 and tt3 within the reference interval , no history of thyroid disease , and no intake of t4/t3 or antithyroid medication .", "controls were defined as having 0.4 < tsh 3.7 mu / l and ft4 and tt3 within the reference interval , no history of thyroid disease , and no intake of t4/t3 or antithyroid medication [ 13 , 14 ] .", "thyroid peroxidase antibody ( tpoab ) positivity was defined by the cut - off value of tpoab > 60 u / ml . all biochemical tests were performed at the same laboratory at naestved hospital .", "measurements of tsh and of thyroid hormones - free thyroxin ( ft4 ) and total triiodothyronine ( tt3 ) were performed using an electrochemiluminescent immunoassay ( roche cobas 6000 , basel , switzerland ) .", "l ( cv < 5% ) , and tt3 reference range 1.202.90 nmol / l ( cv < 4% ) .", "tpoab was measured by kryptor antitpon ( brahms , henigsdorf , germany ) , with detection limit of 10 ", "all statistical analyses were performed using stata version 13.0 for windows ( statacorp , college station , tx , usa ) . a value of p < 0.05 was considered statistically significant .", "tsh and tpoab were not normally distributed and therefore log - transformed ( logtsh and logtpoab ) .", "we performed the following statistical models : for the full cohort of women : linear regression analyses of tsh and tpoab of children born and the number of pregnancies , logistic regression for spontaneous abortion;for women with prevalent hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion;for women with subclinical hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion.models designed were either age - adjusted or multifactorially adjusted using age , bmi , diabetes , contraception , education , income , employment , smoking , antihypertensive medication , cholesterol lowering medication , and menopause as listed in table 1 .", "for the full cohort of women : linear regression analyses of tsh and tpoab of children born and the number of pregnancies , logistic regression for spontaneous abortion ; linear regression analyses of tsh and tpoab of children born and the number of pregnancies , logistic regression for spontaneous abortion ; for women with prevalent hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion ; logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion ; for women with subclinical hypothyroidism versus controls : logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion . logistic regression analyses for 1 children born , 1 pregnancies , and spontaneous abortion .", "the study was approved by the regional ethics committee of zealand , denmark ( reg .", "number rvk sj-177 , sj-113 , and sj-114 ) , registered with clinicaltrial.gov ( nct01335802 ) , and reported to the danish data protection agency ." ], [ "\n table 1 shows characteristics of women . in total , 758 ( 6.7% ) had mild ( subclinical ) hypothyroidism , 9.4% prevalent hypothyroidism , and 4.2% prevalent hyperthyroidism . in women with mild hypothyroidism tpoab", "logtsh and logtpoab were negatively linearly associated with the number of children born and number of pregnancies in the full cohort in age - adjusted and multiadjusted models ( table 2 ) .", "mild ( subclinical ) hypothyroidism was associated with a risk of not having children and a risk of not getting pregnant in age - adjusted and multiadjusted models .", "prevalent hypothyroidism was not associated with the number of children born , the number of pregnancies , or spontaneous abortions ( table 3 ) ." ], [ "in the present study , we showed that with higher tsh levels the less number of children born and the less number of pregnancies .", "coefficients for logtsh were higher than for logtpoab ; thus , the effect of tsh seems higher than for tpoab .", "mild hypothyroidism was also associated with a higher age of first child born and risk of not having children and not getting pregnant .", "tpoab was not a significant confounder in the models , and there was no interaction with age .", "this analysis is cross sectional and in a way retrospective for those women who are now menopausal . also , for women who are premenopausal we may not have the life - time full number of children born , the number of pregnancies , and the number of spontaneous abortions .", "we can not adjust for age of the mother at the birth of her 1st child as this only applies for those who have had children ; thus , those with no children have missing values in that category .", "we have included the information in table 1 , and it appears that there is a significant difference between those with mild hypothyroidism and controls .", "furthermore , were we not able to perform analyses of pregnancy and child birth history restricted to only within few years of the tsh examination .", "this may be caused by the fact that , during fertile life , early miscarriage may have gone unnoticed or been forgotten .", "had recently reported that women with a combination of subclinical hypothyroidism and thyroid autoimmunity were found to have the highest risk of miscarriage before 20 weeks of gestation compared to only tpoab positivity or subclinical hypothyroidism .", "in contrast , previous prospective studies have confirmed an association between mild hypothyroidism during pregnancy and pregnancy loss .", "allan et al . showed that stillbirth was significantly more frequent in women having tsh higher than 6.0 ", "reported that the risk of child loss was increased with higher levels of maternal tsh , whereas concentrations of maternal ft4 and child loss were not associated .", "observed that the evolution of pregnancy did not depend on whether the hypothyroidism was overt or mild but depended on adequate treatment during pregnancy .", "these findings correspond to our results , since the odds ratios for 1 pregnancy and child birth versus none were not significant comparing prevalent hypothyroidism versus controls reflecting a well - treated condition .", "furthermore that mild hypothyroidism is representing nonadequate treatment and mild thyroid failure was associated with a risk of not having children and a risk of not getting pregnant .", "accordingly , in our study we observed that elevated tsh above the upper reference limit in mild hypothyroidism correlated with the number of pregnancies and child births .", "several studies have reported a possible association between thyroid dysfunction during pregnancy and specific adverse pregnancy outcome like preeclampsia [ 19 , 20 ] , placental abruption , or preterm delivery [ 21 , 22 ] .", "it has been suggested that higher tsh and tpoab positivity were independently associated with lower likelihood of reversion to euthyroidism . in a 20-year follow - up study ,", "an increased serum tsh level was predictive of progression to overt hypothyroidism , and the annual rate of progression to overt hypothyroidism was more than 4% in women with both raised serum tsh and antithyroid antibodies .", "showed a trend towards slightly higher serum tsh levels in women with thyroid autoimmunity in the first trimester of pregnancy compared to women without thyroid autoimmunity and that women with asymptomatic autoimmune thyroid disease who were euthyroid in early pregnancy carried a significant risk of developing hypothyroidism progressively during gestation , despite a marked reduction in antibody titers .", "this retrospective cross sectional study showed that among women without any history of thyroid disease or antihyperthyroid / antihypothyroid medication 6.7% had mild ( subclinical ) hypothyroidism .", "the prevalence of mild hypothyroidism was comparable to previous studies like the colorado study in which 8.5% had subclinical hypothyroidism increasing with age explained by the chosen value of tsh cut - off defining mild hypothyroidism .", "the presence of mild hypothyroidism influenced the level of ft4 within the reference range as ft4 was decreased in women with mild hypothyroidism .", "this could be explained by an increased intracellular deiodination of t4 to t3 in women with mild hypothyroidism to avoid decreased levels of active intracellular thyroid hormone concentrations .", "we are not able to determine the status during pregnancy as the present study is retrospective , but it seems unlikely that tpoab would have an effect directly on metabolism and peripheral thyroid hormone regulated cellular function .", "however , we can not exclude the possibility that the women presenting with mild hypothyroidism have had a previous normalized tsh level .", "this population study was performed in naestved municipality eastern denmark representing a mild iodine deficiency . in denmark ,", "the iodine intake was stable at a low level for many years and in 2000 , the mandatory iodine fortification of bread salt and household salt began .", "follow - up studies reported an increase in prevalence of hypothyroidism and tpoab positivity especially among younger women .", "the present study is limited by the fact that all clinical observations are self - reported questionnaire data .", "furthermore , we did not have any information about the numbers of induced abortions which could influence the data of child births although it is rarely recommended to induce an abortion only due to high levels of tsh during first trimester of pregnancy .", "in addition , individuals were classified based on a single blood test ; thus , we can not distinguish between transient and permanent tsh elevation ; however , somwaru et al . showed that subclinical hypothyroidism was persistent in 56% after 4-year follow - up .", "finally , due to financial reasons the gesus study was designed to invite only 25% of younger women aged 2030 years old influencing the distribution of age .", "the strength of our study was the high number of participants with blood samples of tsh , ft4 , tt3 , and tpoab in 11254 women .", "furthermore , for the analyses of mild ( subclinical ) hypothyroidism we excluded women with any self - reported thyroid disease or use of t4/t3 or antithyroid medication and only compared euthyroid and mild hypothyroid women ." ], [ "taken together , we observed that with higher tsh levels the less number of children born and the less number of pregnancies .", "mild hypothyroidism was also associated with a higher age of first child born and risk of not having children and not getting pregnant . in conclusion", ", we observed that impaired fertility is associated with tsh , tpoab , and mild ( subclinical ) hypothyroidism in a danish population of women ." ] ]

[ "in many instances , oral implants provide ideal replacement of missing teeth . although the overall implant outcome is favourable , implant - related mechanical and biological complications are still frequently encountered .", "one of the contributing factors that influence the longevity of implant prosthesis is dental occlusion .", "uncontrolled occlusal forces can lead to implant overloading and detrimental stresses development.1 strains beyond the physiological limit of bone can evoke bone loss around implants and eventual failure of the implant.2 mechanically , excessive strains on implants can cause different failure types involving the implant , its components , or the prosthesis.13 as a result , it is reasonable to propose an occlusion scheme that will minimize the stresses applied on the implant , without compromising the function and the esthetics of the prosthesis.123 in the literature , several occlusion schemes were described for natural teeth.45678 although they are similar in maximal intercuspation ( mi ) , they vary significantly during excursion .", "the most commonly described occlusion schemes are canine - guided ( cg ) occlusion , group function ( gf ) occlusion , and balanced occlusion.4 cg occlusion occurs when the overlap between the maxillary and mandibular canine teeth disengages the posterior teeth during excursive movements.9 it relies on the anatomy and proprioceptive abilities of canine teeth.10 gf occlusion is described as multiple lateral occlusal contacts on the working side.9 the simultaneous contacts are thought to distribute occlusal forces.56 balanced occlusion is distinguished with the presence of simultaneous contacts on working and non - working sides during excursion.9 however , due to the expected risks associated with balanced occlusion , there has been a recommendation to avoid this scheme.11 another occlusion scheme described in the literature and has been observed naturally is the long centric ( lc ) occlusion , where many teeth maintain the contact in the initial stages of excursion , followed by cg occlusion at the later stages of excursion.12 these schemes are relevant as they have been commonly used to restore teeth and exist in the natural dentitions.13 biomechanically , key differences exist between natural teeth and osseointegrated implants .", "the natural tooth root is retained within the alveolar bone by periodontal ligament ( pdl ) , which absorbs and distributes forces applied on the tooth .", "furthermore , the pdl maintains inherent mobility of the natural tooth.1 in addition , the pdl provides proprioception feature , which allows for protection against occlusal overload . on the other hand", ", pdl is missing between alveolar bone and implant , and the implant is directly anchored within the bone with a movement of 20 times less than natural tooth movement.1 due to the lack of pdl , the proprioception around the implant is reduced.1 therefore , due to the inherent lack of implant mobility and reduced proprioception , the implant and its prosthesis are prone to overloading . during normal mastication", ", teeth become compressed in their sockets , leaving the implant prosthesis in hyperocclusion.13 as a consequence of overloading , the implant may suffer from alveolar bone loss14 and mechanical complications , such as ceramic chipping , screw loosening , or components fracture.21516 to overcome the inherent limitations of implant prosthesis , occlusal considerations were discussed in the literature for the purpose of reducing the implant overloading.1718192021 an occlusion scheme specific to implant prosthesis is termed implant - protected ( ip ) occlusion.123 in light occlusion , the prosthesis should not be touching the opposing tooth . however , under heavy biting forces , the prosthesis should be in light contact as the natural teeth are intruded.1 in the excursions , there should be no contact on the implant prosthesis laterally . instead", "however , in order to provide objective recommendation , it is beneficial to validate the impact of different occlusion schemes .", "therefore , the aim of this study is to investigate the effect of four different occlusion schemes and different excursive positions on peri - implant strains of a maxillary canine implant .", "the hypotheses are ( 1 ) there is an effect of occlusion scheme on peri - implant strains , and ( 2 ) there is an effect of the excursive position on peri - implant strains .", "the right canine of the maxillary model was trimmed . the maxillary and mandibular models were replicated by laboratory silicone ( pinkysil addition cured silicone ; barnes , bankstown , nsw ) .", "methylmethacrylate resin ( vertex self - curing resin ; henry schein , waterloo , nsw ) was poured in the silicone moulds and heat flasked according to the manufacturer 's instructions .", "methylmethacrylate resin was selected because it 's young 's modulus is relatively similar to human bone .", "the generated maxillary and mandibular resin models were articulated on a hanau modular articulator ( 014503 - 000 ; whip mix , louisville , kentucky ) using average values .", "the occlusion was rechecked for maximum intercuspation and lateral gf occlusion was achieved on both sides .", "initial gf occlusion was selected as it allowed alteration of the occlusion scheme by modifying the canine palatal anatomy .", "implant drills were used to create an ideal vertical hole in the area of the maxillary right canine , which was slightly larger than the implant diameter .", "the hole was filled with freshly mixed methylmethacrylate resin and an implant ( 5.0 mm diameter , 13 mm long branemark mk iii external hex implant , nobel biocare ) was inserted in the hole simultaneously .", "the model was returned to the heat flask to complete the curing of the resin .", "four different crowns were fabricated for the purpose of achieving four different lateral occlusion schemes on the right side .", "a wide platform prefabricated titanium abutment ( branemark snappy abutment , nobel biocare , macquarie park , nsw , australia ) was attached on the implant and torqued to a recommended value of 35 ncm .", "a cross - pin design was used to avoid screw access in the area of loading and to allow ease of changing the canine crowns while maintaining the same loading set - up ( fig .", "the abutment was scanned by 3shape scanner ( 3shape d-640 ; wieland - imes , pforzheim , germany ) and each crown was digitally designed to represent a defined lateral occlusion scheme .", "the applied lateral occlusion schemes were : ( 1 ) cg occlusion , where the overlap between the maxillary and mandibular canines evokes immediate disclusion of the rest of the dentition upon excursion ; ( 2 ) gf occlusion , where all the teeth , including the canines , are in contact during excursion ; ( 3 ) ip occlusion , where the contact between the maxillary and mandibular canines exists only at maximal interscuspation , but not in excursion ; and ( 4 ) lc occlusion , where the occlusion is similar to gf occlusion for the first 1 mm of excursion , after which it changes to cg occlusion .", "the digital fabrication method was followed to ensure the differences between the crowns were restricted on the palatal guiding surface .", "a resin pattern for each crown was 3d printed , fitted on the abutment , and verified on the articulator .", "the most apical part of the crowns was broadened to allow incorporation of the cross - pin .", "the first point was on the cingulum and corresponded to the maximal intercuspation ( mi ) position .", "the other points were 1 mm and 2 mm buccal from the mi , which corresponded to the loading after 1 mm and 2 mm excursion , respectively .", "the three points were selected to allow simulation of occlusal contacts that occurred during functional and parafunctional range of movement.2223 one rectangular stacked 45-degree rosette strain gauge ( vishay precision group , raleigh , nc , usa ) was attached with cyanoacrylate resin ( m bond 200 adhesive vishay micro - measurements , raleigh , nc , usa ) to the mid - buccal crestal area overlaying the implant body .", "the gauge was composed of three strain foils : horizontal , vertical , and diagonal foils .", "the model was not moved until the completion of the loading . a linear variable differential transformer ( ldvt ) horizontal arm ( solartron , qc systems pty ltd . , vic , australia )", "was placed against the most convex area on the buccal surface of the crown to measure lateral movement under loading .", "the purpose of the ldvt was to detect any displacement of the crowns during loading .", "the ldvt was attached to a signal conditioning units ( solartron metrology pty , vic , australia ) . eventually , the data recorded from each measuring tool were monitored by computer software ( lab view 7 express , national instruments corp . , austin ,", "tx , usa ) . for each crown , a computer - controlled precision universal testing machine ( mts 810 materials test system ; mts systems corp .", ", eden prairie , mn , usa ) was used to apply repeated vertical static load of 140 n at the three marked points ( fig .", "a 140 n load was chosen as it fits within the physiological load range during mastication.1424 each crown was cycled 10 times to maximum load at each loading site .", "therefore , a total of 30 readings were generated for every lateral occlusion scheme . for each loading condition ,", "the strain generated from each gauge foil ( vertical , horizontal , and diagonal ) was recorded .", "subsequently , maximum and minimum principal strain values were calculated for each loading according to the following equations : maximum principal strain=1+32 + 12(1-2)2+(2-3)2 \n minimum principal strain=1+32 - 12(1-2)2+(2-3)2 where 1 is the horizontal gauge strain , 2 is the vertical gauge strain , and 3 is the diagonal gauge strain .", "the shear strain was subsequently measured by calculating the difference between the maximum and minimum principal strain values : shear strain = maximum principal strain - minimum principal strain the average shear strain and standard deviation were calculated for the 10 readings of each crown and loading site .", "the effects of crown morphology ( cg , gf , lc , ip ) and loading site ( mi , 1 mm , 2 mm ) were evaluated and the interactions between them were investigated by the two - way analysis of variance test followed by tukey post hoc test at a significance level of 5% .", "the statistical analysis was performed via spss software package ( ibm spss statistics , version 22 , spss inc . , chicago , il , usa ) .", "the shear strain values for each crown were plotted in a box and whisker diagram .", "for all the crowns and excursive positions , the lateral displacement did not exceed 70 m . there was no significant influence of the different occlusion schemes and excursive positions on the lateral displacement .", "therefore , major crown displacement that could influence the strains measurements was not observed in any loading condition .", "the shear strain data indicate that the occlusion scheme and the excursive position had effects on localized peri - implant strain magnitude .", "however , greater impact was detected from altering the excursive position than from altering the occlusion scheme .", "for all the occlusion schemes , it is clear that as the excursive position moves buccally , the peri - implant strains increases ( fig .", ", there was a significant difference among the occlusion schemes ( p < .005 ) .", "gf was associated with the least strains , followed by ip , cg , and lc . at 1 mm ,", "this was followed by ip , cg , and lc ( p < .005 ) . however , the difference was not significant between gf and ip ( p = .65 ) and between cg and lc ( p = .32 ) . at 2 mm , the difference was significant between them ( p < .005 ) .", "a significant difference existed within the different schemes , except between the ip and lc occlusions ( p = .16 ) .", "overall , the outcome reflected that at the earlier stages of excursion , gf and ip were associated with the least strain values .", "however , as the excursion increased , the strains generated from ip became considerably high .", "regardless of the occlusion scheme , as the excursion increased , the peri - implant strains increased linearly .", "for all the schemes , the difference in peri - implant strains within the different positions was significant ( p < .005 ) .", "the 2-way analysis of variance test revealed that there was a significant interaction between the occlusion scheme and the site of loading .", "the schemes that had less strain values at the mi position tended to have less strain values for the rest of the positions .", "this study confirms that variation in occlusion scheme and excursive position influences the peri - implant strains .", "thus , the hypotheses that the peri - implant strain magnitudes would be influenced by the occlusion scheme and the excursive location were accepted .", "axial forces are preferred as they place the peri - implant bone , implant components , and prosthesis under compression .", "in addition , the forces are evenly distributed within the peri - implant bone.131516 on the contrary , the non - axial forces are associated with localized stress concentration 1516 and can eventually evoke greater peri - implant strains.14 much of the peri - implant bone strains will be in the crestal region , which is more susceptible to resorption.1516 in this investigation , the occlusion scheme was altered by modifying the canine palatal contour . furthermore , varying the site of load application simulated implant crown loading in maximal intercuspation and excursive positions .", "it is very clear from this study that the palatal morphology and the excursive position can influence the peri - implant strains , which are most likely related to altering the proportion of axial and non - axial forces on the implant . applying forces on inclined morphology was proven to increase the non - axial forces and the bending moment applied on the implant and the crown,219 as opposed to applying forces on flat surface . as the inclination increases , the lever arm between the point of load application and the fulcrum point at centre of implant increases , which will eventually accentuate the torque .", "in addition , the non - axial forces will increase by lateral loading of the implant crown .", "as the occlusal forces move laterally from the implant centre , the implant and the relevant components will be subjected to additional flexion and torqueing.20 tooth morphology plays a considerable role in occlusion .", "this is particularly evident for the canines due to their location in the corner of the arch , which allows them to control the eccentric movements and the pattern of lateral contacts.410 as an effect of altering the canine morphology , the lateral occlusion scheme can range from cg occlusion to gf or ip occlusions .", "cg and lc occlusions are associated with greater overlap between the maxillary and mandibular canines to allow for posterior teeth disclusion.17 this has been established in this study by increasing the incline of the canine palatal contour . as a result , these occlusion schemes were associated with greater peri - implant strains at mi and the early stage of excursion . on the contrary , shallow canine palatal morphology , such as in gf or ip occlusions , tends to have less strains at mi and the early stage of excursion ( 1 mm ) .", ", who found that premolar implant crowns with steeper cusp inclination had greater peri - implant strains than crowns with shallower cusp inclination.19 however , as the excursion extends to 2 mm buccally , a different pattern was observed , and the ip occlusion had experienced a considerable localized increase of peri - implant strains .", "although ip occlusion is thought to evoke less peri - implant strains , this was not consistently observed through the whole excursion path .", "this reflects the complexity associated with dental occlusion and palatal contour that can cause variation in the steepness along the excursion path .", "the crowns were constructed to test the effect of bucco - lingual morphology , but the different crowns exhibited different mesio - distal morphology , which might have had effects on the results .", "differences in the mesio - distal morphology can cause the load pointer to shift down the ridge mesially or distally . while it may appear minimal", ", it will lead to strain fluctuation in experiments due to additional torsional strains being superimposed .", "in addition , although the differences between the occlusion schemes are statistically significant , it is very difficult to speculate the clinical impact of such difference . the high statistical difference can be due to repeating the load application on single crowns . as a result , a low standard deviation was observed .", "additional experimentation should evaluate multiple crowns in each scheme . in order to simulate the natural pattern of occlusal contacts , each crown was loaded in mi and two excursive positions .", "these were adopted because it was thought to be clinically relevant and reflect the functional loading on the crown , as several reports pointed that functional occlusal contacts occurred within a range of few millimetres.2223 functional movements , such as chewing and physiological grinding , occur naturally within 0.5 mm from the mi position.78 the excursive range from 0.5 mm to edge - to - edge position is used during parafunctional activities .", "therefore , the experiment covers the possible occlusal force occurrence during functional and parafunctional range of activities .", "it is very clear that regardless of occlusion scheme , as the excursion increases , the peri - implant strains increase as well . at mi", ", the force is directed parallel to the implant which will render the force of compressive nature . however , when the excursion occurs , the force direction will shift buccally , producing a coupling force to the centre of the implant and resulting in a larger bending moment1521 .", "the increase of peri - implant strains in the excursive positions illustrates the negative consequences of localized stresses during parafucntional activities .", "it is likely that the implemented occlusion scheme is a less important for implant overloading than the presence of parafunctional activities .", "this is in accordance with a clinical study that reported a direct relationship between bruxism and increased implant prostheses complications.18 contrary to the occlusion scheme , the excursive position has a greater impact on peri - implant strains .", "this indicates that the location of the load is much more significant in strain development than the palatal contour of the crowns .", "therefore , altering the occlusion scheme for the purpose of changing the lateral contact pattern is more important than modifying the crown morphology and cuspal inclination .", "while this study had loaded all the crowns in all the excursive locations , the load might be different in clinical situations . from the clinical perspective", ", the canines in cg occlusion and lc occlusions are be the only teeth subjected to lateral forces in excursive positions.9 on the other hand , in gf occlusion , the lateral forces are distributed between the canine and the rest of the posterior teeth on the working side.9 an earlier study had reported that implant prostheses with gf was more comfortable than cg over a period of few months,17 which may be attributed to less restricted mandibular movement.5 while it is attractive to assume that gf is a safer occlusion , other investigators have reported increased chance to grind patients ' teeth in eccentric positions .", "this has been observed by elevated emg readings of masticatory muscles and increased condylar displacement with gf occlusion.56 cg occlusion exhibits a protective mechanism from the proprioceptive feature of canine teeth . however , whether the proprioceptive mechanism will offset the negative effect of palatal steepness is yet to be determined .", "nevertheless , ip occlusion will be safer for the implant due to the absence of lateral occlusal contacts . for implant prostheses ,", "several authors suggested using the natural teeth for occlusion guidance and alleviation of occlusal contacts on the implant prosthesis during excursive movement.120 this is envisioned to minimize the nonaxial forces on implant components and reduce the risk of mechanical complications through micromovement or flexure.20 although ip is likely to be beneficial in eliminating lateral contacts on implant restoration , utilizing this scheme for anterior implant restoration may result in shallow morphology and shortened clinical crown that can hinder the dental esthetics .", "a recent systematic review did not confirm the superiority of any occlusion scheme for teeth or implant prostheses.11 in a retrospective study , kinsel and lin evaluated the effect of lateral occlusion on implant - supported prosthesis complications .", "patients with gf occlusion had significantly greater chance of ceramic chipping than those with cg .", "however , the investigators did not relate the ceramic chipping solely to the occlusion scheme .", "in addition , they have reported more critical factors , such as bruxism and opposing implant prosthesis . on the other hand ,", "several authors have reported there is no specific occlusion scheme that should be implemented for implant restorations . instead ,", "the lateral occlusion scheme for implant prostheses can mimic the the occlusion scheme on tooth supported prostheses.113 as a result , no specific occlusion scheme will be selected for all implant prostheses . while this study has the advantage of simulating a clinical scenario , it has some limitations .", "for example , a true representation of natural cortical bone and trabecular bone is impossible in a laboratory study .", "this experiment assumes isotropic mechanical behaviour of bone . however , bone is inhomogeneous due to the variations in cortical bone thickness and trabecular bone porosity . as a result ,", "variation in bone quality may react differently to occlusal forces.14 in addition , laboratory experimentations assume complete implant integration , which does not represent actual implant osseointegration .", "within the limitations of this preliminary study , it can be concluded that the occlusion scheme and the excursive position influence the peri - implant strains . according to the present experimental set - up", ", it appears that the impact of eccentric position is greater than that of the occlusion scheme . implementing an occlusion concept that reduces the occurrence of occlusal contacts in excursive positions is preferable to reduce the strains within the peri - implant region ." ]

[ "<S> purposethis study aims to investigate the effects of four different lateral occlusion schemes and different excursions on peri - implant strains of a maxillary canine implant.materials and methodsfour metal crowns with different occlusion schemes were attached to an implant in the maxillary canine region of a resin model . </S>", "<S> the included schemes were canine - guided ( cg ) occlusion , group function ( gf ) occlusion , long centric ( lc ) occlusion , and implant - protected ( ip ) occlusion . </S>", "<S> each crown was loaded in three sites that correspond to maximal intercuspation ( mi ) , 1 mm excursion , and 2 mm excursion . </S>", "<S> a load of 140 n was applied on each site and was repeated 10 times . </S>", "<S> the peri - implant strain was recorded by a rosette strain gauge that was attached on the resin model buccal to the implant . for each loading condition , the maximum shear strain value was calculated.resultsthe different schemes and excursive positions had impact on the peri - implant strains . at mi and 1 mm positions , </S>", "<S> the gf had the least strains , followed by ip , cg , and lc . at 2 mm , </S>", "<S> the least strains were associated with gf , followed by cg , lc , and ip . </S>", "<S> however , regardless of the occlusion scheme , as the excursion increases , a linear increase of peri - implant strains was detected.conclusionthe peri - implant strain is susceptible to occlusal factors . </S>", "<S> the eccentric location appears to be more influential on peri - implant strains than the occlusion scheme . </S>", "<S> therefore , adopting an occlusion scheme that can reduce the occurrence of occlusal contacts laterally may be beneficial in reducing peri - implant strains . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION" ]

[ [ "in many instances , oral implants provide ideal replacement of missing teeth . although the overall implant outcome is favourable , implant - related mechanical and biological complications are still frequently encountered .", "one of the contributing factors that influence the longevity of implant prosthesis is dental occlusion .", "uncontrolled occlusal forces can lead to implant overloading and detrimental stresses development.1 strains beyond the physiological limit of bone can evoke bone loss around implants and eventual failure of the implant.2 mechanically , excessive strains on implants can cause different failure types involving the implant , its components , or the prosthesis.13 as a result , it is reasonable to propose an occlusion scheme that will minimize the stresses applied on the implant , without compromising the function and the esthetics of the prosthesis.123 in the literature , several occlusion schemes were described for natural teeth.45678 although they are similar in maximal intercuspation ( mi ) , they vary significantly during excursion .", "the most commonly described occlusion schemes are canine - guided ( cg ) occlusion , group function ( gf ) occlusion , and balanced occlusion.4 cg occlusion occurs when the overlap between the maxillary and mandibular canine teeth disengages the posterior teeth during excursive movements.9 it relies on the anatomy and proprioceptive abilities of canine teeth.10 gf occlusion is described as multiple lateral occlusal contacts on the working side.9 the simultaneous contacts are thought to distribute occlusal forces.56 balanced occlusion is distinguished with the presence of simultaneous contacts on working and non - working sides during excursion.9 however , due to the expected risks associated with balanced occlusion , there has been a recommendation to avoid this scheme.11 another occlusion scheme described in the literature and has been observed naturally is the long centric ( lc ) occlusion , where many teeth maintain the contact in the initial stages of excursion , followed by cg occlusion at the later stages of excursion.12 these schemes are relevant as they have been commonly used to restore teeth and exist in the natural dentitions.13 biomechanically , key differences exist between natural teeth and osseointegrated implants .", "the natural tooth root is retained within the alveolar bone by periodontal ligament ( pdl ) , which absorbs and distributes forces applied on the tooth .", "furthermore , the pdl maintains inherent mobility of the natural tooth.1 in addition , the pdl provides proprioception feature , which allows for protection against occlusal overload . on the other hand", ", pdl is missing between alveolar bone and implant , and the implant is directly anchored within the bone with a movement of 20 times less than natural tooth movement.1 due to the lack of pdl , the proprioception around the implant is reduced.1 therefore , due to the inherent lack of implant mobility and reduced proprioception , the implant and its prosthesis are prone to overloading . during normal mastication", ", teeth become compressed in their sockets , leaving the implant prosthesis in hyperocclusion.13 as a consequence of overloading , the implant may suffer from alveolar bone loss14 and mechanical complications , such as ceramic chipping , screw loosening , or components fracture.21516 to overcome the inherent limitations of implant prosthesis , occlusal considerations were discussed in the literature for the purpose of reducing the implant overloading.1718192021 an occlusion scheme specific to implant prosthesis is termed implant - protected ( ip ) occlusion.123 in light occlusion , the prosthesis should not be touching the opposing tooth . however , under heavy biting forces , the prosthesis should be in light contact as the natural teeth are intruded.1 in the excursions , there should be no contact on the implant prosthesis laterally . instead", "however , in order to provide objective recommendation , it is beneficial to validate the impact of different occlusion schemes .", "therefore , the aim of this study is to investigate the effect of four different occlusion schemes and different excursive positions on peri - implant strains of a maxillary canine implant .", "the hypotheses are ( 1 ) there is an effect of occlusion scheme on peri - implant strains , and ( 2 ) there is an effect of the excursive position on peri - implant strains ." ], [ "the right canine of the maxillary model was trimmed . the maxillary and mandibular models were replicated by laboratory silicone ( pinkysil addition cured silicone ; barnes , bankstown , nsw ) .", "methylmethacrylate resin ( vertex self - curing resin ; henry schein , waterloo , nsw ) was poured in the silicone moulds and heat flasked according to the manufacturer 's instructions .", "methylmethacrylate resin was selected because it 's young 's modulus is relatively similar to human bone .", "the generated maxillary and mandibular resin models were articulated on a hanau modular articulator ( 014503 - 000 ; whip mix , louisville , kentucky ) using average values .", "the occlusion was rechecked for maximum intercuspation and lateral gf occlusion was achieved on both sides .", "initial gf occlusion was selected as it allowed alteration of the occlusion scheme by modifying the canine palatal anatomy .", "implant drills were used to create an ideal vertical hole in the area of the maxillary right canine , which was slightly larger than the implant diameter .", "the hole was filled with freshly mixed methylmethacrylate resin and an implant ( 5.0 mm diameter , 13 mm long branemark mk iii external hex implant , nobel biocare ) was inserted in the hole simultaneously .", "the model was returned to the heat flask to complete the curing of the resin .", "four different crowns were fabricated for the purpose of achieving four different lateral occlusion schemes on the right side .", "a wide platform prefabricated titanium abutment ( branemark snappy abutment , nobel biocare , macquarie park , nsw , australia ) was attached on the implant and torqued to a recommended value of 35 ncm .", "a cross - pin design was used to avoid screw access in the area of loading and to allow ease of changing the canine crowns while maintaining the same loading set - up ( fig .", "the abutment was scanned by 3shape scanner ( 3shape d-640 ; wieland - imes , pforzheim , germany ) and each crown was digitally designed to represent a defined lateral occlusion scheme .", "the applied lateral occlusion schemes were : ( 1 ) cg occlusion , where the overlap between the maxillary and mandibular canines evokes immediate disclusion of the rest of the dentition upon excursion ; ( 2 ) gf occlusion , where all the teeth , including the canines , are in contact during excursion ; ( 3 ) ip occlusion , where the contact between the maxillary and mandibular canines exists only at maximal interscuspation , but not in excursion ; and ( 4 ) lc occlusion , where the occlusion is similar to gf occlusion for the first 1 mm of excursion , after which it changes to cg occlusion .", "the digital fabrication method was followed to ensure the differences between the crowns were restricted on the palatal guiding surface .", "a resin pattern for each crown was 3d printed , fitted on the abutment , and verified on the articulator .", "the most apical part of the crowns was broadened to allow incorporation of the cross - pin .", "the first point was on the cingulum and corresponded to the maximal intercuspation ( mi ) position .", "the other points were 1 mm and 2 mm buccal from the mi , which corresponded to the loading after 1 mm and 2 mm excursion , respectively .", "the three points were selected to allow simulation of occlusal contacts that occurred during functional and parafunctional range of movement.2223 one rectangular stacked 45-degree rosette strain gauge ( vishay precision group , raleigh , nc , usa ) was attached with cyanoacrylate resin ( m bond 200 adhesive vishay micro - measurements , raleigh , nc , usa ) to the mid - buccal crestal area overlaying the implant body .", "the gauge was composed of three strain foils : horizontal , vertical , and diagonal foils .", "the model was not moved until the completion of the loading . a linear variable differential transformer ( ldvt ) horizontal arm ( solartron , qc systems pty ltd . , vic , australia )", "was placed against the most convex area on the buccal surface of the crown to measure lateral movement under loading .", "the purpose of the ldvt was to detect any displacement of the crowns during loading .", "the ldvt was attached to a signal conditioning units ( solartron metrology pty , vic , australia ) . eventually , the data recorded from each measuring tool were monitored by computer software ( lab view 7 express , national instruments corp . , austin ,", "tx , usa ) . for each crown , a computer - controlled precision universal testing machine ( mts 810 materials test system ; mts systems corp .", ", eden prairie , mn , usa ) was used to apply repeated vertical static load of 140 n at the three marked points ( fig .", "a 140 n load was chosen as it fits within the physiological load range during mastication.1424 each crown was cycled 10 times to maximum load at each loading site .", "therefore , a total of 30 readings were generated for every lateral occlusion scheme . for each loading condition ,", "the strain generated from each gauge foil ( vertical , horizontal , and diagonal ) was recorded .", "subsequently , maximum and minimum principal strain values were calculated for each loading according to the following equations : maximum principal strain=1+32 + 12(1-2)2+(2-3)2 \n minimum principal strain=1+32 - 12(1-2)2+(2-3)2 where 1 is the horizontal gauge strain , 2 is the vertical gauge strain , and 3 is the diagonal gauge strain .", "the shear strain was subsequently measured by calculating the difference between the maximum and minimum principal strain values : shear strain = maximum principal strain - minimum principal strain the average shear strain and standard deviation were calculated for the 10 readings of each crown and loading site .", "the effects of crown morphology ( cg , gf , lc , ip ) and loading site ( mi , 1 mm , 2 mm ) were evaluated and the interactions between them were investigated by the two - way analysis of variance test followed by tukey post hoc test at a significance level of 5% .", "the statistical analysis was performed via spss software package ( ibm spss statistics , version 22 , spss inc . , chicago , il , usa ) .", "the shear strain values for each crown were plotted in a box and whisker diagram ." ], [ "for all the crowns and excursive positions , the lateral displacement did not exceed 70 m . there was no significant influence of the different occlusion schemes and excursive positions on the lateral displacement .", "therefore , major crown displacement that could influence the strains measurements was not observed in any loading condition .", "the shear strain data indicate that the occlusion scheme and the excursive position had effects on localized peri - implant strain magnitude .", "however , greater impact was detected from altering the excursive position than from altering the occlusion scheme .", "for all the occlusion schemes , it is clear that as the excursive position moves buccally , the peri - implant strains increases ( fig .", ", there was a significant difference among the occlusion schemes ( p < .005 ) .", "gf was associated with the least strains , followed by ip , cg , and lc . at 1 mm ,", "this was followed by ip , cg , and lc ( p < .005 ) . however , the difference was not significant between gf and ip ( p = .65 ) and between cg and lc ( p = .32 ) . at 2 mm , the difference was significant between them ( p < .005 ) .", "a significant difference existed within the different schemes , except between the ip and lc occlusions ( p = .16 ) .", "overall , the outcome reflected that at the earlier stages of excursion , gf and ip were associated with the least strain values .", "however , as the excursion increased , the strains generated from ip became considerably high .", "regardless of the occlusion scheme , as the excursion increased , the peri - implant strains increased linearly .", "for all the schemes , the difference in peri - implant strains within the different positions was significant ( p < .005 ) .", "the 2-way analysis of variance test revealed that there was a significant interaction between the occlusion scheme and the site of loading .", "the schemes that had less strain values at the mi position tended to have less strain values for the rest of the positions ." ], [ "this study confirms that variation in occlusion scheme and excursive position influences the peri - implant strains .", "thus , the hypotheses that the peri - implant strain magnitudes would be influenced by the occlusion scheme and the excursive location were accepted .", "axial forces are preferred as they place the peri - implant bone , implant components , and prosthesis under compression .", "in addition , the forces are evenly distributed within the peri - implant bone.131516 on the contrary , the non - axial forces are associated with localized stress concentration 1516 and can eventually evoke greater peri - implant strains.14 much of the peri - implant bone strains will be in the crestal region , which is more susceptible to resorption.1516 in this investigation , the occlusion scheme was altered by modifying the canine palatal contour . furthermore , varying the site of load application simulated implant crown loading in maximal intercuspation and excursive positions .", "it is very clear from this study that the palatal morphology and the excursive position can influence the peri - implant strains , which are most likely related to altering the proportion of axial and non - axial forces on the implant . applying forces on inclined morphology was proven to increase the non - axial forces and the bending moment applied on the implant and the crown,219 as opposed to applying forces on flat surface . as the inclination increases , the lever arm between the point of load application and the fulcrum point at centre of implant increases , which will eventually accentuate the torque .", "in addition , the non - axial forces will increase by lateral loading of the implant crown .", "as the occlusal forces move laterally from the implant centre , the implant and the relevant components will be subjected to additional flexion and torqueing.20 tooth morphology plays a considerable role in occlusion .", "this is particularly evident for the canines due to their location in the corner of the arch , which allows them to control the eccentric movements and the pattern of lateral contacts.410 as an effect of altering the canine morphology , the lateral occlusion scheme can range from cg occlusion to gf or ip occlusions .", "cg and lc occlusions are associated with greater overlap between the maxillary and mandibular canines to allow for posterior teeth disclusion.17 this has been established in this study by increasing the incline of the canine palatal contour . as a result , these occlusion schemes were associated with greater peri - implant strains at mi and the early stage of excursion . on the contrary , shallow canine palatal morphology , such as in gf or ip occlusions , tends to have less strains at mi and the early stage of excursion ( 1 mm ) .", ", who found that premolar implant crowns with steeper cusp inclination had greater peri - implant strains than crowns with shallower cusp inclination.19 however , as the excursion extends to 2 mm buccally , a different pattern was observed , and the ip occlusion had experienced a considerable localized increase of peri - implant strains .", "although ip occlusion is thought to evoke less peri - implant strains , this was not consistently observed through the whole excursion path .", "this reflects the complexity associated with dental occlusion and palatal contour that can cause variation in the steepness along the excursion path .", "the crowns were constructed to test the effect of bucco - lingual morphology , but the different crowns exhibited different mesio - distal morphology , which might have had effects on the results .", "differences in the mesio - distal morphology can cause the load pointer to shift down the ridge mesially or distally . while it may appear minimal", ", it will lead to strain fluctuation in experiments due to additional torsional strains being superimposed .", "in addition , although the differences between the occlusion schemes are statistically significant , it is very difficult to speculate the clinical impact of such difference . the high statistical difference can be due to repeating the load application on single crowns . as a result , a low standard deviation was observed .", "additional experimentation should evaluate multiple crowns in each scheme . in order to simulate the natural pattern of occlusal contacts , each crown was loaded in mi and two excursive positions .", "these were adopted because it was thought to be clinically relevant and reflect the functional loading on the crown , as several reports pointed that functional occlusal contacts occurred within a range of few millimetres.2223 functional movements , such as chewing and physiological grinding , occur naturally within 0.5 mm from the mi position.78 the excursive range from 0.5 mm to edge - to - edge position is used during parafunctional activities .", "therefore , the experiment covers the possible occlusal force occurrence during functional and parafunctional range of activities .", "it is very clear that regardless of occlusion scheme , as the excursion increases , the peri - implant strains increase as well . at mi", ", the force is directed parallel to the implant which will render the force of compressive nature . however , when the excursion occurs , the force direction will shift buccally , producing a coupling force to the centre of the implant and resulting in a larger bending moment1521 .", "the increase of peri - implant strains in the excursive positions illustrates the negative consequences of localized stresses during parafucntional activities .", "it is likely that the implemented occlusion scheme is a less important for implant overloading than the presence of parafunctional activities .", "this is in accordance with a clinical study that reported a direct relationship between bruxism and increased implant prostheses complications.18 contrary to the occlusion scheme , the excursive position has a greater impact on peri - implant strains .", "this indicates that the location of the load is much more significant in strain development than the palatal contour of the crowns .", "therefore , altering the occlusion scheme for the purpose of changing the lateral contact pattern is more important than modifying the crown morphology and cuspal inclination .", "while this study had loaded all the crowns in all the excursive locations , the load might be different in clinical situations . from the clinical perspective", ", the canines in cg occlusion and lc occlusions are be the only teeth subjected to lateral forces in excursive positions.9 on the other hand , in gf occlusion , the lateral forces are distributed between the canine and the rest of the posterior teeth on the working side.9 an earlier study had reported that implant prostheses with gf was more comfortable than cg over a period of few months,17 which may be attributed to less restricted mandibular movement.5 while it is attractive to assume that gf is a safer occlusion , other investigators have reported increased chance to grind patients ' teeth in eccentric positions .", "this has been observed by elevated emg readings of masticatory muscles and increased condylar displacement with gf occlusion.56 cg occlusion exhibits a protective mechanism from the proprioceptive feature of canine teeth . however , whether the proprioceptive mechanism will offset the negative effect of palatal steepness is yet to be determined .", "nevertheless , ip occlusion will be safer for the implant due to the absence of lateral occlusal contacts . for implant prostheses ,", "several authors suggested using the natural teeth for occlusion guidance and alleviation of occlusal contacts on the implant prosthesis during excursive movement.120 this is envisioned to minimize the nonaxial forces on implant components and reduce the risk of mechanical complications through micromovement or flexure.20 although ip is likely to be beneficial in eliminating lateral contacts on implant restoration , utilizing this scheme for anterior implant restoration may result in shallow morphology and shortened clinical crown that can hinder the dental esthetics .", "a recent systematic review did not confirm the superiority of any occlusion scheme for teeth or implant prostheses.11 in a retrospective study , kinsel and lin evaluated the effect of lateral occlusion on implant - supported prosthesis complications .", "patients with gf occlusion had significantly greater chance of ceramic chipping than those with cg .", "however , the investigators did not relate the ceramic chipping solely to the occlusion scheme .", "in addition , they have reported more critical factors , such as bruxism and opposing implant prosthesis . on the other hand ,", "several authors have reported there is no specific occlusion scheme that should be implemented for implant restorations . instead ,", "the lateral occlusion scheme for implant prostheses can mimic the the occlusion scheme on tooth supported prostheses.113 as a result , no specific occlusion scheme will be selected for all implant prostheses . while this study has the advantage of simulating a clinical scenario , it has some limitations .", "for example , a true representation of natural cortical bone and trabecular bone is impossible in a laboratory study .", "this experiment assumes isotropic mechanical behaviour of bone . however , bone is inhomogeneous due to the variations in cortical bone thickness and trabecular bone porosity . as a result ,", "variation in bone quality may react differently to occlusal forces.14 in addition , laboratory experimentations assume complete implant integration , which does not represent actual implant osseointegration ." ], [ "within the limitations of this preliminary study , it can be concluded that the occlusion scheme and the excursive position influence the peri - implant strains . according to the present experimental set - up", ", it appears that the impact of eccentric position is greater than that of the occlusion scheme . implementing an occlusion concept that reduces the occurrence of occlusal contacts in excursive positions is preferable to reduce the strains within the peri - implant region ." ] ]

[ "hemangiomas are categorized as racemose , capillary , cavernous , and venous , according to the size of their vascular spaces .", "cavernous and venous hemangiomas have low blood flow , because they lack arterial or capillary components .", "histopathologically , venous hemangiomas contain dilated vessels with thick , fibrous walls , whereas cavernous hemangiomas have capillary - sized vessels lined by flat endothelial cells .", "we report the first case of a venous hemangioma of the pps and discuss its typical radiographic findings .", "a 49-year - old female patient was referred for evaluation of swelling in the right submandibular region .", "she had swelling for the first time 3 weeks earlier , and it had not improved after medical treatment .", "clinical examination revealed a soft mass in the right submandibular region and a bulge at the right lateral pharyngeal wall . a contrast - enhanced computed tomography ( ct ) scan revealed a cystic lesion , 4.63.0 cm , with contrast non - enhancement in the parapharyngeal space ( pps ) .", "mri showed a well - circumscribed non - enhancing mass with high signal intensity on t2-weighted images ( fig .", "the cystic lesion of the pps was surgically removed using a transcervical approach . during surgery", "the lesion bled profusely , but the mass was bluntly dissected from the surrounding structures .", "the surgical specimen , measuring 4.34.52 cm , showed a pale - to - dark brown soft cut surface with a blood - filled spongy vascular lesion .", "microscopic examination revealed blood - filled sinusoidal spaces with large irregular lumens and thick walls lined by endothelial cells ( fig .", "postoperative recovery was uneventful , and there has been no evidence of cranial nerve palsy or tumor recurrence after one year .", "pps has a complex anatomy and close proximity to vital anatomical structures , with which it may become involved by various pathological processes . presenting symptoms of pps tumors may be attributed to the size of the mass and compression of neighboring structures ( 1 ) .", "we had difficulty with the preoperative diagnosis , due to the absence of typical symptoms or signs of a vascular lesion in the pps , such as pulsation or bruit .", "fine - needle aspiration cytology ( fnac ) was also not helpful in reaching a diagnosis .", "although an exact tissue characterization of hemangioma in the pps could not be made on fnac , contrast - enhanced ct and magnetic resonance imaging ( mri ) should be included in differentiating these lesions from other tumors of the pps . in general ,", "mr imaging is superior to ct imaging in its ability to ascertain the soft tissue characteristics of pps tumors ( 2 ) .", "changes in the blood flow dynamics within a hemangioma result in thrombus formation and phleboliths ( 3 ) .", "phleboliths are calcified nodules that can be regarded as a characteristic property of venous or cavernous hemangiomas .", "ct with contrast is an excellent imaging technique for revealing phleboliths . although mri is not sensitive for the detection of small amounts of calcification , large amounts of calcification show discrete low signal intensity on all pulse sequences", "mri can produce high signal intensities , representing the blood , as well as focal heterogeneities , representing areas of thrombosis , fibrosis , or calcification ( 4 ) .", "although mri is very useful for the detection of vascular lesions , the detectability of phleboliths on ct images is superior to that by mri .", "some reports have shown that plain x - ray films may also reveal calcified lesions .", "when imaging shows calcification in the pps , the differential diagnosis may include pleomorphic adenoma of the deep lobe of the parotid gland and metastatic thyroid carcinoma of the pps .", "pleomorphic adenoma with foci of chondroid or osteoid stroma can demonstrate opacities on imaging , but usually shows minute , scattered flecks .", "additionally , in cases of metastatic thyroid carcinoma of the pps , large flocculent calcification is seen on ct images ( 5 ) .", "venous hemangioma can not be clinically or radiologically differentiated from cavernous hemangioma , because of their similar characteristics .", "however , perfusion and blood pool scintigraphy has been demonstrated to have high sensitivity for detecting head and neck hemangioma , and can also differentiate between cavernous and venous hemangiomas ( 6 ) .", "they tend to be larger and less well circumscribed , and show no tendency to regress ( 7 ) .", "attention needs to be given to the tumor location , extent , growth rate , and accessibility as well as the patient 's age and esthetic concerns .", "the excision of large tumors in the pps can be challenging , given the risk for severe hemorrhage and nerve injury .", "a surgical approach should be chosen according to the tumor size and location , its relationship to the great vessels , and any suspicion of malignancy .", "generally , hemangiomas in the pps , including venous hemangioma , can be resected by using a transcervical approach .", "profuse bleeding often occurs during surgery , but after all of the pathology has been removed , the bleeding will cease . when surgery is impossible , the use of corticosteroids , cryotherapy ,", "feeding vessel ligation , embolization , and fibrosing agents can be considered as alternatives ( 8) . in summary , we present the first reported case of venous hemangioma occurring in the parapharyngeal space . considering that a presumptive diagnosis of most pps tumors can be made based on imaging studies , preoperative imaging findings are very important in approaching these lesions .", "it may be that multiple , spotty , calcific nodules , 13 mm in size , within a cystic lesion on ct images indicate the pathognomonic finding of hemangioma in the pps ." ]

[ "<S> a hemangioma of the parapharyngeal space ( pps ) is an extremely rare tumor and is responsible for 0.5 - 1% of all tumors occurring in the pps . </S>", "<S> we report a case of pps venous hemangioma in a 49-year - old woman presenting with diffuse swelling in the submandibular region . a preoperative computed tomography ( ct ) scan showed a cystic mass with multiple calcifications in the pps . </S>", "<S> the calcific nodules were round and about 2 mm in diameter . </S>", "<S> the hemangioma was completely resected via a transcervical approach . during surgery </S>", "<S> , we found several calcific nodules , which represented phleoboliths or areas of thrombosis with dystrophic calcification . despite its rarity , </S>", "<S> a venous hemangioma of the pps should be considered in a differential diagnosis when a cystic mass with calcification is found by ct scan . to our knowledge , this is the first reported case of a pps venous hemangioma ; we describe its pathognomonic findings on imaging . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "hemangiomas are categorized as racemose , capillary , cavernous , and venous , according to the size of their vascular spaces .", "cavernous and venous hemangiomas have low blood flow , because they lack arterial or capillary components .", "histopathologically , venous hemangiomas contain dilated vessels with thick , fibrous walls , whereas cavernous hemangiomas have capillary - sized vessels lined by flat endothelial cells .", "we report the first case of a venous hemangioma of the pps and discuss its typical radiographic findings ." ], [ "a 49-year - old female patient was referred for evaluation of swelling in the right submandibular region .", "she had swelling for the first time 3 weeks earlier , and it had not improved after medical treatment .", "clinical examination revealed a soft mass in the right submandibular region and a bulge at the right lateral pharyngeal wall . a contrast - enhanced computed tomography ( ct ) scan revealed a cystic lesion , 4.63.0 cm , with contrast non - enhancement in the parapharyngeal space ( pps ) .", "mri showed a well - circumscribed non - enhancing mass with high signal intensity on t2-weighted images ( fig .", "the cystic lesion of the pps was surgically removed using a transcervical approach . during surgery", "the lesion bled profusely , but the mass was bluntly dissected from the surrounding structures .", "the surgical specimen , measuring 4.34.52 cm , showed a pale - to - dark brown soft cut surface with a blood - filled spongy vascular lesion .", "microscopic examination revealed blood - filled sinusoidal spaces with large irregular lumens and thick walls lined by endothelial cells ( fig .", "postoperative recovery was uneventful , and there has been no evidence of cranial nerve palsy or tumor recurrence after one year ." ], [ "pps has a complex anatomy and close proximity to vital anatomical structures , with which it may become involved by various pathological processes . presenting symptoms of pps tumors may be attributed to the size of the mass and compression of neighboring structures ( 1 ) .", "we had difficulty with the preoperative diagnosis , due to the absence of typical symptoms or signs of a vascular lesion in the pps , such as pulsation or bruit .", "fine - needle aspiration cytology ( fnac ) was also not helpful in reaching a diagnosis .", "although an exact tissue characterization of hemangioma in the pps could not be made on fnac , contrast - enhanced ct and magnetic resonance imaging ( mri ) should be included in differentiating these lesions from other tumors of the pps . in general ,", "mr imaging is superior to ct imaging in its ability to ascertain the soft tissue characteristics of pps tumors ( 2 ) .", "changes in the blood flow dynamics within a hemangioma result in thrombus formation and phleboliths ( 3 ) .", "phleboliths are calcified nodules that can be regarded as a characteristic property of venous or cavernous hemangiomas .", "ct with contrast is an excellent imaging technique for revealing phleboliths . although mri is not sensitive for the detection of small amounts of calcification , large amounts of calcification show discrete low signal intensity on all pulse sequences", "mri can produce high signal intensities , representing the blood , as well as focal heterogeneities , representing areas of thrombosis , fibrosis , or calcification ( 4 ) .", "although mri is very useful for the detection of vascular lesions , the detectability of phleboliths on ct images is superior to that by mri .", "some reports have shown that plain x - ray films may also reveal calcified lesions .", "when imaging shows calcification in the pps , the differential diagnosis may include pleomorphic adenoma of the deep lobe of the parotid gland and metastatic thyroid carcinoma of the pps .", "pleomorphic adenoma with foci of chondroid or osteoid stroma can demonstrate opacities on imaging , but usually shows minute , scattered flecks .", "additionally , in cases of metastatic thyroid carcinoma of the pps , large flocculent calcification is seen on ct images ( 5 ) .", "venous hemangioma can not be clinically or radiologically differentiated from cavernous hemangioma , because of their similar characteristics .", "however , perfusion and blood pool scintigraphy has been demonstrated to have high sensitivity for detecting head and neck hemangioma , and can also differentiate between cavernous and venous hemangiomas ( 6 ) .", "they tend to be larger and less well circumscribed , and show no tendency to regress ( 7 ) .", "attention needs to be given to the tumor location , extent , growth rate , and accessibility as well as the patient 's age and esthetic concerns .", "the excision of large tumors in the pps can be challenging , given the risk for severe hemorrhage and nerve injury .", "a surgical approach should be chosen according to the tumor size and location , its relationship to the great vessels , and any suspicion of malignancy .", "generally , hemangiomas in the pps , including venous hemangioma , can be resected by using a transcervical approach .", "profuse bleeding often occurs during surgery , but after all of the pathology has been removed , the bleeding will cease . when surgery is impossible , the use of corticosteroids , cryotherapy ,", "feeding vessel ligation , embolization , and fibrosing agents can be considered as alternatives ( 8) . in summary , we present the first reported case of venous hemangioma occurring in the parapharyngeal space . considering that a presumptive diagnosis of most pps tumors can be made based on imaging studies , preoperative imaging findings are very important in approaching these lesions .", "it may be that multiple , spotty , calcific nodules , 13 mm in size , within a cystic lesion on ct images indicate the pathognomonic finding of hemangioma in the pps ." ] ]

[ "the tetracycline class of antibacterial agents has seen widespread clinical use for over 50 years due to its broad spectrum antibacterial activity.(1 ) tetracyclines inhibit bacterial growth by preventing protein biosynthesis through binding to the 30s ribosome , thereby blocking the binding of aminoacyl - trna to the acceptor site.(2 ) this class includes a number of naturally occurring compounds , such as chlorotetracycline ( 1)(3 ) and tetracycline ( 2),(4 ) as well as several semisynthetic analogues , such as doxycycline ( 3)(5 ) and minocycline ( 4 ) ( figure 1).(6 ) in addition to their use as antibiotics for serious infections , tetracyclines have also been prescribed for use in acne and rosacea.(7 ) as with many classes of antibiotics , widespread use of tetracyclines has led to resistance and has limited their use in more complicated bacterial infections .", "examples of naturally occurring ( 1 and 2 ) and semisynthetic ( 36 ) tetracyclines .", "two major bacterial resistance mechanisms have been identified for tetracyclines : ( 1 ) active transport from bacterial cells via efflux pumps(10 ) and ( 2 ) ribosomal protection.(11 ) the efflux pumps are comprised of inner membrane tetracycline efflux proteins that were first identified in gram - negative organisms ( tet(ae ) ) and were later found in gram - positive bacteria ( tet(kl)).(10c ) the ribosomal protection mechanism ( tet(mo ) ) involves the expression of proteins that bind to the ribosome and allow protein synthesis to proceed even in the presence of tetracyclines .", "since these early discoveries , the number of efflux and ribosomal protection mechanisms identified has increased.(12 ) the presence of the strongly activating 10-hydroxy group readily enables chemical modification of the tetracycline d - ring at the 7- and 9-positions .", "this strategy led to the introduction of the 7-dimethylamino group in compounds 46 , all of which have demonstrated improved activity in bacterial strains bearing efflux pump resistance mechanisms .", "more recently , the 9-substituted glycylcyclines , such as tigecycline ( 5),(13 ) and the aminomethylcyclines , such as amadacycline ( 6),(14 ) have shown improved activity for resistant organisms bearing the ribosomal protection mechanism . in 2005 , myers and co - workers published a fully synthetic route to the tetracyclines , the key step of which involves the michaeldieckmann condensation of the ab precursor 8 with structurally diverse d - ring precursors .", "this route provides potential access to a broad range of tetracyclines that are inaccessible via traditional semisynthesis.(17 ) one example of this is the introduction of a nitrogen atom into the d - ring , resulting in azatetracyclines . ", "one 7-azatetracycline and one 9-azatetracycline were disclosed in myers original work , resulting in compounds with only modest antibacterial activity .", "herein we demonstrate this powerful approach using pyridyl d - ring precursors 7 to generate novel 8-azatetracyclines , 10 ( scheme 1 ) .", "to explore 7- and 9-substituted-8-azatetracyclines , we required intermediates that could be readily modified either prior to or after construction of the protected compound 9 . thus , the halopyridines 15 and 16 were prepared according to schemes 2 and 3 .", "3,5-dichloroisonicotinic acid was treated with sodium benzylate to give compound 12 , which was esterified to the phenyl ester 13 by conversion to the acid chloride , followed by treatment with phenol and 4-(dimethylamino)pyridine ( dmap ) .", "suzuki coupling(18 ) using methylboronic acid and pdcl2cy2 catalyst(19 ) gave the parent d - ring precursor 14 in good yield .", "n - oxide formation with hydrogen peroxide in acetic acid followed by treatment with either phosphorousoxychloride or phosphorousoxybromide gave 15a and 15b , respectively . for 15a ,", "the reaction gave a 4.5:1.0 ratio of the desired regioisomer , while a 25:1 ratio was observed for 15b .", "the regiochemistry was assigned by conversion to the dimethylamino compound 15c through a palladium - mediated amination.(20)h nmr studies indicated an noe between the methyl groups of the amine and the adjacent ring methyl group .", "the methoxy derivative 15d was prepared from 15b by treatment with sodium methoxide in 2-methylpyrrolidinone ( nmp ) at 120 c .", "this gave the acid directly , which was re - esterified to the phenyl ester .", "reagents and conditions : ( i ) nah ( 2 equiv ) , bnoh , nmp , 80 c ; ( ii ) ( cocl)2 , cat .", "dmf , ch2cl2 ; ( iii ) phoh , et3n , dmap , ch2cl2 ; ( iv ) ch3b(oh)2 , pdcl2cy2 , k3po4 , toluene , water , 100 c ; ( v ) h2o2 , acoh , 80 c ; ( vi ) pocl3 , toluene , 100 c ; ( vii ) pobr3 , toluene , 100 c ; ( viii ) dimethylamine in thf , pd2dba3 , xantphos , k3po4 , 1,4-dioxane , 100 c ( sealed ) ;", "reagents and conditions : ( i ) for 15a , mcpba , ch2cl2 ; for 15b , h2o2 , acoh , 80 c ; ( ii ) pobr3 , toluene , 100 c ; ( iii ) bocnh2 , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 80 c ; ( iv ) hcl , 1,4-dioxane ; ( v ) hf - pyridine , nano2 , pyridine ; ( vi ) dimethylamine in thf , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 60 c ( sealed ) .", "the dihalo compounds 16a and 16b were prepared by n - oxidation of 15a and 15b followed by treatment with pobr3 ( scheme 3 ) .", "yields were typically moderate ( 4550% ) , with reduction of the n - oxide to give the starting pyridines 15 observed as major side reactions in both cases .", "palladium - mediated coupling of 16b with t - butylcarbamate gave a 2.5:1 mixture of regioisomers , which were readily separated after conversion to the free amine 16c upon treatment with hcl .", "treatment of 16c with sodium nitrite in the presence of hf - pyridine gave the fluoro derivative 16d in good yield.(21 ) similarly , a 2.4:1.0 ratio of regioisomers was observed in the palladium - mediated reaction of 16b with dimethylamine to give 16e and 16f .", "interestingly , the regioselectivity could be reversed by heating 16b in the presence of dimethylamine without catalyst to give 16f as the major isomer in a 3:1 ratio .", "the 9-amino d - ring precursors 18a and 18b were prepared according to scheme 4 .", "once again , palladium - mediated couplings using t - butylcarbamate proved to be highly effective for introduction of the amino group . further protection of the mono - boc intermediates 17 with boc2o and dmap in dmf gave the di - boc precursors 18 .", "reagents and conditions : ( i ) bocnh2 , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 80 c ; ( ii ) boc2o , dmap , dmf .", "most of the d - ring precursors were lithiated with lithium diisopropylamide ( lda ) at 78 c , followed by treatment with the enone 8 .", "warming to 20 c yielded the fully protected tetracycline precursors 19 . for d - ring precursors 16a , 16d , and 18b , a one - pot reaction was performed in which lithium bis(trimethylsilyl)amide ( lhmds ) was added to a 78 c solution of the precursor and the enone 8 followed by warming to 20 c .", "yields varied from 20 to 84% depending on substrate , with larger reaction scales generally providing better results .", "the lhmds procedure also typically gave higher yields but was only possible on the more highly electron deficient d - ring precursors .", "the 7-bromo intermediate 19c was further derivatized at this stage via suzuki couplings with methylboronic acid and phenylboronic acid to give 19d and 19e , respectively .", "similarly , palladium - mediated amination with 19h and t - butylcarbamate provided the 7-chloro-9-amino compound 19j .", "deprotection of the intermediates 19 with hf followed by hydrogenation in the presence of palladium on carbon gave the final 8-azatetracyclines 20ak .", "reagents and conditions : ( i ) for 14 , 15bd , 16e , and 18a , lda , tmeda , 78 c , thf then 8 , 78 c to 20 c ; ( ii ) for 16a , 16d , and 18b , lhmds , 8 , thf , 78", "c to 20 c ; ( iii ) ch3b(oh)2 or phb(oh)2 , pdcl2dppf2-ch2cl2 , k3po4 , toluene , 1,4-dioxane , water , 100 c ; ( iv ) bocnh2 , pd2dba3 , xantphos , k3po4 , 1,4-dioxane , 100 c ; ( v ) aqueous hf , ch3cn or 1,4-dioxane ; ( vi ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane . to compare the 8-azatetracyclines directly to tigecycline , the 9-glycylamido-8-azatetracyclines 21ac were prepared ( scheme 6 ) .", "treatment with bromoacetylchloride in thf followed by excess t - butylamine gave the acylated intermediates , which were deprotected under standard conditions to give the final analogues 21ac .", "reagents and conditions : ( i ) hcl , 1,4-dioxane ; ( ii ) bromoacetylchloride , thf then t - bunh2 , 50 c ; ( iii ) aqueous hf , ch3cn or 1,4-dioxane ; ( iv ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane .", "the bromopyridine intermediates proved to be useful for the introduction of alkylamines via palladium - mediated aminations and alkyl groups via suzuki couplings , ultimately providing the 9-alkylamino-8-azatetracyclines 24 and 9-alkyl-8-azatetracyclines 26 ( scheme 7 ) .", "thus , 19h was treated with various amines in the presence of pd2dba3 and 9,9-dimethyl-4,5-bis-(diphenylphosphino)xanthenes ( xantphos ) in 1,4-dioxane at 100 c .", "potassium phosphate was found to be the most effective base for this transformation , with cesium carbonate and sodium t - butoxide leading to rapid decomposition .", "low yields were generally observed due to decomposition of both the starting material and products upon heating .", "reaction times of 24 h were eventually settled on as a balance between product formation and decomposition .", "copper - catalyzed aminations were also explored , but rapid decomposition of the starting material was observed .", "hindered amines generally gave low yields as did reactions with the 7-dimethylamino compound 19 m .", "alternatively , the alkylamines could be introduced prior to the michaeldieckmann reaction using either palladium or copper - mediated aminations(22 ) with 16a , 16d , and 16e .", "protection of the resulting aminopyridines upon treatment with lhmds followed by boc2o or cbzcl gave the d - ring precursors 22 .", "suzuki reactions with 19h or 19 m and various boronic acids were also carried out in moderate yields , providing the 9-alkyl intermediates 25 .", "reagents and conditions : ( i ) r2r3nh2 , pd2dba3 , xantphos , k3po4 or cs2co3 , 1,4-dioxane , 100 c or n - propylamine , cui , 2-acetylcyclohexanone , k3po4 , dmf , 100 c ; ( ii ) lhmds , thf then boc2o or cbzcl ; ( iii ) lda , tmeda , 78 c , thf then 8 , 78 c to 20 c or lhmds , 8 , thf , 78", "c to 20 c ; ( iv ) aqueous hf , ch3cn , or 1,4-dioxane ; ( v ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane ; ( vi ) r2b(oh)2 , ( ph3p)4pd , k3po4 , toluene , 1,4-dioxane , h2o , 90 c . the in vitro antibacterial activities of all analogues were determined for a panel of gram - positive ( e.g. , staphylococcus aureus and streptococcus pneumoniae ) and gram - negative ( e.g. , escherichia coli and klebsiella pneumoniae ) bacteria .", "the activities of the 7-substituted-8-azatetracycline as well as several control tetracyclines for a representative set of these organisms are found in table 1 .", "the parent 8-azatetracycline 20a showed comparable activity to tetracycline , with reasonable minimum inhibitory concentrations ( mics ) of 0.251 g / ml for the tetracycline - susceptible strains . lacking a substituent at either c-7 or c-9", ", 20a demonstrated poor activity for the strains bearing tet(m ) , tet(k ) , and tet(a ) .", "overall , the 7-position appears to be very tolerant of substitution , allowing for bulky ( 20e ) , electron withdrawing ( 20b and 20i ) , and electron donating ( 20f and 20 g ) groups while maintaining activity against the tetracycline - susceptible strains . as with minocycline , introduction of the 7-dimethylamino group in 20f led to improved activities for most strains tested .", "modest ( 12 dilution ) improvements in mics were observed for the tet(m ) strains , while a more significant improvement was seen for the strain bearing the tet(k ) resistance mechanism . with the exception of the 7-chloro analogue , all of the 7-substituted-8-aza compounds exhibited improved mics for the tet(k ) strain , correlating well with literature reports on 7-substituted tetracyclines .", "unfortunately , all of the 8-azatetracyclines appear to be substrates for the tet(a ) efflux pump , exhibiting no antibacterial activity at the highest concentration tested ( 32 g / ml ) .", "in general , substitution at the 7-position appears to be inefficient at overcoming the ribosomal protection resistance mechanism of the tet(m ) strains .", "the only compound with two dilution improvements for both tet(m ) strains was compound 20i , the 7-fluoro analogue .", "obtained from marilyn roberts lab at the university of washington . to make a direct comparison to tigecycline , the 9-glycylamido-8-azatetracyclines were prepared ( table 2 ) .", "whereas tigecycline is a very potent antibacterial agent against all of the organisms in our panel , including the four strains bearing tetracycline resistance mechanisms , the corresponding 8-azatetracycline analogues 21ac were found to have poor overall activity .", "the aniline precursors 20j and 20k , however , showed significant improvements in mics for both tet(m ) strains relative to the corresponding 9-unsubstituted compounds 20b and 20i .", "for the 7-cl analogue 20j , antibacterial activity also improved for the tet(k ) and wild type strains .", "these compounds were also assessed for their ability to inhibit protein synthesis in a cell - free transcription / translation assay ( table 3 ) . in this assay ,", "compounds 20j and 20k were about 2-fold more potent than tetracycline , in agreement with their relative mics for e. coli .", "compounds 21ac , on the other hand , inhibited protein synthesis with ic50s between 0.42 and 0.90 g / ml , similar to tigecycline , despite having only modest antibacterial activity in the whole - cell assays .", "these data suggest that the compounds are active at the target , but that compounds 21ac are not capable of getting to the target in the whole - cell assay , possibly due to poor cell permeability .", "atcc 25922 is a tetracycline - susceptible strain . on the basis of the results of compounds 20j and 20k ,", "compounds bearing a small , unbranched alkylamino group at the 9-position ( 24ac ) proved to be very potent antibacterial agents against the two tetracycline - susceptible gram - positive strains , with mics between 0.016 and 0.25 g / ml . a 4-fold improvement in activity against the tet(m ) s. aureus strain", "was observed for all of these compounds relative to 20j , while mics for the s. pneumoniae tet(m ) strain were largely unchanged . for the tet(k )", "s. aureus strain , antibacterial activity improved with increasing size of the alkyl group . among the tetracycline - susceptible gram - negative strains ,", "however , there was a clear loss in potency as the size of the alkyl group increased , a trend that continued for the larger branched alkyl compounds 24df .", "this is not surprising , as it is known that gram - negative antibacterial agents tend to be smaller and more polar.(23 ) these larger side chains also adversely affected the antibacterial activity for s. pneumoniae while having less effect on the mics for the s. aureus strains .", "the 7-fluoro-9-n - propylamino ( 24n ) and 7-dimethylamino-9-n - propylamino ( 24o ) analogues were prepared as comparisons to 24c . while the 7-fluoro compound had mics that tracked fairly closely to the 7-chloro analogue , the 7-dimethylamino compound exhibited no antibacterial activity for any of the strains at the highest concentration tested ( 32 g / ml ) with the exception of the tetracycline - susceptible s. pneumoniae strain .", "all three of the compounds had similar , albeit weak , activity in the transcription / translation assay , indicating the differences in mics might be due to poor permeability of the 7-dimethylamino analogue .", "introduction of a more polar heteroatom into the side chain ( 24gi , k ) resulted in improved antibacterial activity against the s. pneumoniae and gram - negative strains but had minimal effect on activity toward either resistant s. aureus organisms .", "interestingly , the oxygen bearing side chains ( 24 g and 24h ) yielded significantly improved antibacterial activity toward tetracycline - susceptible gram - positive strains relative to the all carbon side chains ( 24e and 24f ) , while the nitrogen analogues ( 24k and 24i ) were less potent against s. aureus and showed more modest improvements against s. pneumoniae .", "the combination of a branched side chain with a distal amino group gave the most balanced antibacterial compounds in this series ( 24l , 24 m , and 24j ) .", "these compounds combine good antibacterial activity toward both gram - positive and gram - negative organisms , with modest activity against tet(a ) e. coli . both compounds 24l and 24 m are less potent than tigecycline but compare favorably to amadacycline .", "the antibacterial activity of the 9-alkyl and phenyl substituted compounds were generally modest ( table 5 ) . here again , a larger substituent at c-9 yielded improved activity for tet(m ) and tet(k ) strains but resulted in loss of activity for wild type s. pneumoniae and gram - negative strains .", "introduction of a 7-cl or 7-dimethylamino group also gave improved activity for most of the strains in the panel .", "when comparing the 7-cl-9-alkyl compounds ( 26b ) to the 7-cl-9-aminoalkyl compounds ( 24c ) , it is clear that the nitrogen confers significant improvements in antibacterial activity . in the 7-dimethylamino case , however , the 7-dimethylamino-9-alkyl compound 26a is a balanced gram - positive antibacterial agent while the 9-aminoalkyl compound 24o has no significant activity .", "were selected for further profiling . both compounds inhibited protein synthesis in the transcription / translation assay with ic50s comparable to tigecycline .", "susceptibility testing was performed , and mic50 and mic90 values were calculated for recent clinical isolates of s. aureus , s. pneumoniae , haemophilus influenzae , and e. coli ( table 6 ) .", "the panels contained both tetracycline - susceptible and resistant strains , including 12 methicillin - resistant s. aureus ( mrsa ) strains . as expected from the initial mic data , compound 20f had very potent antibacterial activity for the tetracycline - susceptible strains but performed poorly against the resistant organisms .", "the compound did have mic90s that were significantly lower than tetracycline . despite having higher mics in the primary screen relative to tigecycline , compound 24l performed similarly in the s. aureus , s. pneumoniae , and h. influenzae panels , exhibiting clinically relevant mic90s in each case .", "these organisms are particularly relevant , as they are responsible for infections in a large portion of community - acquired bacterial pneumonia cases .", "the pharmacokinetic profiles of the two 8-azatetracyclines were determined in spraguedawley rats ( table 7 ) .", "compound 20f had very similar auc , clearance , and volume of distribution profiles to both tetracycline and tigecycline , although the half - life was somewhat shorter .", "low oral bioavailability ( % f ) of 12.7% was observed but was comparable to tetracycline ( 12.1% ) . in humans , tetracycline is generally reported to have oral bioavailability of 5070%.(24 ) we and others have found the oral bioavailability of several tetracycline derivatives to be significantly lower in rat than reported data in humans .", "compound 24l had a 4-fold higher auc and 5-fold lower clearance than the other three compounds as well as a significantly lower volume of distribution .", "compounds were dosed at 1 mg / kg iv and 10 mg / kg po with sterile water as vehicle .", "the compounds were also investigated in mouse s. aureus and e. coli septicemia models ( table 8) . both 8-azatetracyclines performed well in the s. aureus model following iv administration , with pd50 values comparable to the two marketed control tetracyclines .", "20f , the most potent of the four compounds , also provided the best protection , with 100% survival at the lowest dose at which it was administered ( 0.30 mg / kg ) . despite having an", "8-fold higher mic , compound 24l provided equivalent protection relative to tigecycline , likely due to its higher auc and lower clearance.(25 ) the e. coli model proved to be more challenging , requiring higher doses for protection .", "compound 20f had a pd50 of 4.3 mg / kg compared to 2.1 mg / kg for tigecycline .", "compound 24l was 4-fold less potent than 20f and was comparable to tetracycline in this model .", "s. aureus atcc 13709 ( smith ) or e. coli atcc 25922 was mixed with 5% mucin and inoculated by intraperitoneal injection at 2.1 10 cfu / mouse ( s. aureus ) or 2.0 10 cfu / mouse ( e. coli ) .", "one hour postchallenge , mice received iv treatment ( vehicle = sterile water ) with the test article at concentrations ranging from 0.0510 mg / kg ( s. aureus ) or 0.3030 mg / kg ( e. coli ) .", "the pd50 in mg / kg was calculated as survival after 48 h. lowest dose was 0.30 mg / kg with 100% survival at all doses .", "a novel , fully synthetic series of 8-azatetracycline analogues was prepared and optimized . through modification of the 7- and 9-positions , compounds were found that overcome both tet(k ) efflux and tet(m)-mediated ribosomal protection .", "the 7-chloro-9-alkylamino-8-azatetracycline 24l exhibits clinically relevant mic90 profiles against both gram - positive and gram - negative organisms that include these resistance mechanisms .", "this compound also demonstrated in vivo efficacy in murine models of infection that is equivalent to currently marketed tetracycline antibacterial agents .", "the 7-dimethylamino-8-azatetracycline 20f was efficacious when administered intravenously and had oral bioavailability in rat equivalent to tetracycline .", "the current work demonstrates the ability of this chemistry platform to generate antibacterial compounds that are inaccessible through traditional semisynthesis , providing the medicinal chemist a broader array of tools through which essential antibacterial properties can be modulated .", "importantly , this includes the ability to overcome ever evolving resistance mechanisms and to identify structureactivity relationships to deliver compounds with desired pharmacokinetic properties .", "air and moisture sensitive liquids and reagents were transferred via syringe or cannula and were introduced into flame - dried glassware under a positive pressure of dry nitrogen through rubber septa .", "analytical thin - layer chromatography was performed on em science precoated glass - backed silica gel 60 f-254 250 m plates .", "visualization of the plates was effected by ultraviolet illumination and/or immersion of the plate in a basic solution of potassium permanganate in water followed by heating .", "column chromatography was performed on a flashmaster personal system using isolute flash si ii silica gel prepacked cartridges ( available from biotage ) .", "preparative reversed - phase hplc chromatography ( hplc ) was accomplished using a waters autopurification system with mass - directed fraction collection .", "all intermediate compounds were purified with a waters sunfire prep c18 obd column ( 5 m , 19 mm 50 mm ; flow rate = 20 ml / min ) using a mobile phase mixture of h2o ( a ) and ch3cn ( b ) containing 0.1% hco2h .", "the final 8-azatetracycline compounds were purified using a phenomenex polymerx 10 rp 100a column ( 10 m , 30 mm 21.2 mm ; flow rate = 20 ml / min ) using a mobile phase mixture of 0.05n hcl in h2o ( a ) and ch3cn ( b ) .", "all final 8-azatetracycline compounds were isolated as mono- , di- , or trihydrochloride salts following freeze - drying .", "h nmr spectra were recorded on a jeol ecx-400 ( 400 mhz ) spectrometer and are reported in ppm using residual solvent as the internal standard ( cdcl3 at 7.24 ppm , dmso - d6 at 2.50 ppm , or cd3od at 3.31 ppm ) .", "high performance liquid chromatographyelectrospray mass spectra ( lc - ms ) were obtained using a waters alliance hplc system equipped with a binary pump , a diode array detector , a waters sunfire c18 ( 5 m , 4.6 mm i.d .", " 50 mm ) column , and a waters 3100 series mass spectrometer with electrospray ionization .", "the eluent was a mixture of h2o ( a ) and ch3cn ( b ) containing 0.1% hco2h at a flow rate of 1 ml / min .", "purity of the final compounds was assessed by the following method : ( a ) time = 0 , 100% a ; ( b ) time = 0.5 min , 100% a ; ( c ) time = 3.5 min , 100% b ; ( d ) time = 5 min , 100% b ( e ) time = 6 min , 100% a ; ( f ) time = 7 min , 100% a. all final products were 95% purity as assessed by this method .", "sodium hydride ( 60% dispersion in mineral oil , 4.37 g , 109 mmol ) was added portionwise to a solution of 3,5-dichloroisonicotinic acid ( 10.2 g , 53.3 mmol ) in nmp ( 100 ml ) .", "after gas evolution ceased , benzyl alcohol ( 5.52 ml , 53.3 mmol ) was added dropwise .", "after 1 h , the reaction was complete and was allowed to cool to room temperature and stand overnight .", "the reaction mixture was diluted with water ( 300 ml ) and was washed with et2o ( 2 100 ml , discarded ) .", "the aqueous layer was brought to ph 2 with conc hcl , causing a precipitate to form .", "the mixture was diluted with brine ( 100 ml ) and was allowed to stand for 30 min .", "the solid was collected by filtration , was washed with water ( 3 ) , and was dried in a 45 c vacuum oven overnight .", "this gave 9.36 g ( 67% ) of the title compound as a white solid .", "h nmr ( 400 mhz , dmso - d6 ) 14.3014.10 ( bs , 1 h ) , 8.52 ( s , 1h ) , 8.34 ( s , 1 h ) , 7.507.30 ( m , 5 h ) , 5.33 ( s , 2 h ) .", "oxalyl chloride ( 4.9 ml , 56 mmol ) was added to a suspension of 12 ( 3.71 g , 14.1 mmol ) in ch2cl2 ( 50 ml ) over 2 min .", "dmf was added dropwise in 20 l portions every 5 min until complete solution was achieved . after stirring for an additional 30 min ,", "the resulting solid was dissolved in ch2cl2 ( 50 ml ) , and phenol ( 2.65 g , 28.1 mmol ) , dmap ( 0.172 g , 1.41 mmol ) , and et3n ( 9.76 ml , 70.4 mmol ) were added .", "after 1 h , the reaction mixture was diluted with ch2cl2 ( 50 ml ) and was washed with water ( 2 100 ml ) and nahco3 ( saturated , aqueous , 100 ml ) .", "the material was purified by column chromatography ( biotage 50 g column , 025% etoac in hexanes gradient ) , yielding 4.20 g ( 88% ) of the product as an off - white solid .", "h nmr ( 400 mhz , dmso - d6 ) 8.70 ( s , 1h ) , 8.48 ( s , 1 h ) , 7.527.30 ( m , 8 h ) , 7.157.08 ( m , 2 h ) , 5.44 ( s , 2 h ) .", "compound 13 ( 2.01 g , 5.92 mmol ) , methyl boronic acid ( 1.06 g , 17.7 mmol ) , dichlorobis(tricyclohexylphosphine)palladium(ii ) ( 102 mg , 0.296 mmol ) , and k3po4 ( 3.76 g , 17.7 mmol ) were heated to 100 c in toluene ( 20 ml ) and water ( 2 ml ) . after 16 h", ", the reaction mixture was allowed to cool to room temperature and was diluted with etoac ( 20 ml ) .", "this was washed with water ( 20 ml ) and brine ( 20 ml ) , dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 040% etoac in hexanes gradient ) , yielding 1.66 g ( 88% ) of the product as a white solid .", "h nmr ( 400 mhz , dmso - d6 ) 8.50 ( s , 1h ) , 8.26 ( s , 1 h ) , 7.527.30 ( m , 8 h ) , 7.207.10 ( m , 2 h ) , 5.37 ( s , 2 h ) , 2.38 ( s , 3 h ) . ms ( esi ) m / z 320.27 ( m + h ) .", "compound 14 ( 9.66 g , 30.3 mmol ) was heated to 80 c in acetic acid ( 50 ml ) and hydrogen peroxide ( 30% aqueous solution , 17 ml ) .", "after 6 h , lc / ms indicated that the starting material was consumed and the n - oxide present .", "most of the acetic acid and water were removed under reduced pressure . the material was then diluted with etoac ( 200 ml ) , and the mixture was washed with nahco3 ( saturated aqueous solution , 3 50 ml ) .", "the organics were dried over na2so4 , filtered , and concentrated under reduced pressure , yielding 9.37 g ( 92% ) of the crude n - oxide .", "the material was dissolved in toluene ( 60 ml ) , phosphorousoxychloride ( 7.69 g , 50.1 mmol ) was added , and the reaction mixture was heated to 100 c .", "after 2 h , lc / ms indicated that the reaction was complete . upon cooling to room temperature", ", the reaction mixture was poured into a solution of k2co3 ( 40 g ) in water ( 200 ml ) .", "this mixture was extracted with etoac ( 3 150 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was redissolved in etoac ( 30 ml ) with heating and was then allowed to cool to room temperature and stand overnight .", "another 1.92 g ( 19% ) of the white crystalline solid was obtained from the mother liquor after standing in a refrigerator overnight .", "h nmr ( 400 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) ,", "7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) . compound 14 ( 25.9 g , 81.1 mmol ) was heated to 80 c in acetic acid ( 160 ml ) and hydrogen peroxide ( 30% aqueous solution , 40 ml ) .", "after stirring overnight , lc / ms indicated that the starting material was consumed and the n - oxide present .", "the reaction mixture was concentrated under reduced pressure , was diluted with etoac , and was washed with nahco3 ( saturated aqueous solution , 3 ) .", "the organics were dried over na2so4 , filtered , and concentrated under reduced pressure , yielding 25.4 g ( 93% crude ) of the n - oxide as a tan solid .", "the material was suspended in toluene ( 200 ml ) and was heated to 80 c , at which point most of the solid had dissolved .", "upon cooling to room temperature , the reaction mixture was poured into a solution of k2co3 ( 83 g ) in water ( 300 ml ) .", "the combined extracts were dried over mgso4 , filtered through celite , and concentrated under reduced pressure .", "the material was recrystallized from etoac / hexanes ( 1:1 ) , yielding 20.6 g ( 64% ) of the product as an off - white solid .", "h nmr ( 400 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) . compound 15b ( 1.06 g , 2.66 mmol ) , dimethylamine ( 2.0 m solution in thf , 4.0 ml , 8.0 mmol ) , k3po4 ( 1.7 g , 8.0 mmol ) , tris(dibenzylideneacetone)dipalladium ( 120 mg , 0.13 mmol ) , and xantphos ( 220 mg , 0.40 mmol ) were heated to 100 c in 1,4-dioxane ( 10 ml ) in a sealed pressure vessel .", "after heating overnight , the reaction mixture was cooled to room temperature , was diluted with etoac ( 100 ml ) , and was washed with water ( 3 50 ml ) and brine ( 50 ml ) .", "the material was purified by column chromatography ( biotage 50 g column , 015% etoac in hexanes gradient ) , yielding 388 mg ( 40% ) of the title compound as a white solid .", "h nmr ( 400 mhz , cdcl3 ) 7.96 ( s , 1h ) , 7.457.20 ( m , 8 h ) , 7.08 ( d , j = 8.2 hz , 2 h ) , 5.16 ( s , 2 h ) , 2.76 ( s , 6 h ) , 2.37 ( s , 3 h ) . ms ( esi ) m / z 363.45 ( m + h ) . sodium methoxide ( 38 mg , 0.70 mmol ) was added to a solution of compound 15b ( 118 mg , 0.351 mmol ) in nmp ( 2 ml ) , and the reaction mixture was heated to 100 c . after heating overnight", ", additional sodium methoxide ( 57 mg , 1.1 mmol ) was added and heating was continued at 100 c .", "after an additional 1 h , the reaction mixture was cooled to room temperature , water ( 20 ml ) was added , and the ph was adjusted to 2 with 6 n aqueous hcl .", "this was extracted with etoac ( 3 20 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the crude acid intermediate was dissolved in ch2cl2 ( 10 ml ) , and oxalyl chloride ( 0.123 ml , 1.40 mmol ) was added .", "the material was dissolved in ch2cl2 ( 10 ml ) , and phenol ( 66 mg , 0.70 mmol ) , dmap ( 4 mg , 0.04 mmol ) , and et3n ( 0.245 ml , 1.76 mmol ) were added .", "after 1 h , the reaction mixture was diluted with etoac ( 50 ml ) and was washed with water ( 2 30 ml ) and brine ( 30 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 20.3 mg ( 17% ) of clean product .", "h nmr ( 400 mhz , cdcl3 ) 7.75 ( s , 1h ) , 7.457.24 ( m , 9 h ) , 7.10 ( d , j = 7.3 hz , 2 h ) , 5.13 ( s , 2 h ) , 3.91 ( s , 3 h ) , 2.67 ( s , 3 h ) . ms ( esi ) m / z 336.1 , 338.1 ( m + h ) .", "compound 15a ( 4.99 g , 14.1 mmol ) was dissolved in ch2cl2 ( 30 ml ) , and 3-chloroperbenzoic acid ( 77% , 6.33 g , 28.2 mmol ) was added in one portion . after 6 h , an additional portion of 3-chloroperbenzoic acid ( 3.15 g , 14.1 mmol ) was added , and the reaction was stirred at room temperature overnight .", "the reaction mixture was diluted with ch2cl2 ( 200 ml ) and was washed with na2co3 ( saturated aqueous solution , 100 ml ) and brine .", "the material was purified by column chromatography ( biotage 70 g column , 550% etoac in hexanes gradient ) , yielding 3.90 g ( 75% ) of the pure n - oxide as an off - white solid ( rf = 0.33 in 66% etoac / hexanes ) .", "the n - oxide was dissolved in toluene ( 30 ml ) , phosphorousoxybromide ( 6.36 g , 22.1 mmol ) was added , and the reaction mixture was heated to 80 c .", "after 1 h , the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 ( 10 g ) in water ( 50 ml ) .", "this mixture was extracted with etoac ( 3 100 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 010% etoac in hexanes gradient ) , yielding 2.06 g ( 45% ) of the desired product 16a as an oil which slowly solidified on standing overnight .", "h nmr ( 400 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) .", "compound 15b ( 8.54 g , 21.4 mmol ) was heated to 80 c in acetic acid ( 120 ml ) and hydrogen peroxide ( 30% aqueous solution , 30 ml ) . after stirring overnight ,", "lc / ms indicated that the starting material was mostly consumed and the n - oxide present .", "the reaction mixture was cooled to room temperature and was diluted with brine ( 150 ml ) .", "the resulting precipitate was collected by filtration and was washed with water / brine ( 1:1 , 2 ) .", "the solid was dissolved in ch2cl2 and was dried over na2so4 , filtered , and concentrated under reduced pressure .", "this gave 7.83 g ( 88% crude ) of the n - oxide as a tan solid .", "the material was suspended in toluene ( 200 ml ) and was heated to 100 c , at which point most of the solid had dissolved .", "a solution of phosphorousoxybromide ( 10.8 g , 37.8 mmol ) in toluene ( 10 ml ) was added rapidly .", "after 45 min , the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 ( 40 g ) in water ( 150 ml ) . after stirring for 30 min at room temperature ,", "the combined extracts were dried over mgso4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 08% etoac in hexanes gradient to isolate the product then 15% etoac in hexanes to recover 15b ) , yielding 5.02 g ( 49% ) of the product as an off - white solid .", "h nmr ( 400 mhz , dmso - d6 ) 7.497.32 ( m , 8 h ) , 7.10 ( d , j = 7.3 hz , 2 h ) , 5.15 ( s , 2 h ) , 2.41 ( s , 3 h ) . ms ( esi ) m / z 476.18 , 478.16 , 480.16 ( m + h ) .", "compound 16b ( 2.15 g , 4.50 mmol ) , t - butylcarbamate ( 633 mg , 5.40 mmol ) , cs2co3 ( 2.93 g , 9.00 mmol ) , tris(dibenzylideneacetone)dipalladium ( 206 mg , 0.225 mmol ) , and xantphos ( 380 mg , 0.673 mmol ) were weighed into a flask .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 15 ml ) was added .", "the reaction mixture was cooled to room temperature , was diluted with etoac ( 100 ml ) , and was washed with water ( 2 50 ml ) .", "the material was purified by column chromatography ( biotage 50 g column , 012% etoac in hexanes gradient ) , yielding 1.43 g ( 62% ) of a 4:1 mixture of the two regioisomeric compounds .", "h nmr ( 400 mhz , cdcl3 ) 7.517.46 ( m , 2 h ) , 7.427.32 ( m , 7 h ) , 7.307.24 ( m , 1 h ) , 7.12 ( d , j = 8.2 hz , 0.5 h ) , 7.03 ( d , j = 8.2 hz , 2 h ) , 6.88 ( s , 0.25 h ) , 6.71 ( s , 1 h ) , 5.12 ( s , 2 h ) , 5.02 ( s , 0.5 h ) , 2.43 ( s , 0.75 h ) , 2.34 ( s , 3 h ) , 1.50 ( s , 9 h ) , 1.47 ( s , 2.25 h ) . ms ( esi ) m / z 535.10 , 537.10 ( m + na ) . the above regioisomeric mixture ( 1.43 g , 2.78 mmol ) was stirred in 4 m hcl in 1,4-dioxane ( 20 ml ) and 1,4-dioxane ( 5 ml ) overnight .", "the reaction mixture was diluted with etoac ( 100 ml ) and was washed with nahco3 ( saturated aqueous solution , 2 100 ml ) .", "the material was purified by column chromatography ( biotage 20 g column , 030% etoac in hexanes gradient ) , yielding 805 mg ( 70% ) of the major regioisomer 16c and 270 mg ( 24% ) of the minor regioisomer .", "h nmr ( 400 mhz , cdcl3 ) 7.507.42 ( m , 2 h ) , 7.407.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.04 ( d , j = 8.2 hz , 2 h ) , 5.06 ( s , 2 h ) , 4.56 ( s , 2 h ) , 2.16 ( s , 3 h ) . ms ( esi ) m / z 413.11 , 415.01 ( m + h ) .", "nmr ( 400 mhz , cdcl3 ) 7.427.34 ( m , 8 h ) , 7.137.10 ( m , 2 h ) , 5.01 ( s , 2 h ) , 4.64 ( s , 2 h ) , 2.35 ( s , 3 h ) ; ms ( esi ) m / z 413.09 , 415.09 ( m + h ) .", "hf - pyridine solution ( 70% hf , 2 ml ) was added to a 0 c solution of compound 16c ( 884 mg , 2.14 mmol ) in pyridine ( 1 ml ) .", "sodium nitrite ( 177 mg , 2.57 mmol ) was added ( bubbling observed ) , and the reaction mixture was allowed to slowly warm to room temperature . after 3 days , the reaction mixture was poured into na2co3 ( saturated aqueous solution , 30 ml ) and was extracted with etoac ( 3 30 ml ) .", "the combined extracts were washed with hcl ( 1 n aqueous solution , 2 50 ml ) and were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 20 g column , 06% etoac in hexanes gradient ) , yielding 687 mg ( 77% ) of the product as a colorless oil that slowly solidified on standing .", "h nmr ( 400 mhz , cdcl3 ) 7.507.42 ( m , 2 h ) , 7.407.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.14 ( s , 2 h ) , 2.33 ( s , 3 h ) . ms ( esi ) m / z 416.17 , 418.15 ( m + h ) .", "compound 16b ( 1.51 g , 3.16 mmol ) , cs2co3 ( 3.1 g , 9.5 mmol ) , tris(dibenzylideneacetone)dipalladium ( 145 mg , 0.158 mmol ) , and xantphos ( 267 mg , 0.474 mmol ) were weighed into a vial equipped with a septum .", "this was evacuated and flushed with nitrogen ( 3 ) , and 1,4-dioxane ( 7 ml ) and dimethylamine ( 2.0 m solution in thf , 4.75 ml , 9.50 mmol ) were added .", "the reaction mixture was heated to 60 c and was stirred for 4 h. the reaction mixture was allowed to cool to room temperature , was filtered through celite , and was concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 010% etoac in hexanes gradient ) , yielding 868 mg ( 58% ) of the major regioisomer 16e and 354 mg ( 24% ) of the minor regioisomer 16f .", "data for 16e : h nmr ( 400 mhz , cdcl3 ) 7.507.46 ( m , 2 h ) , 7.417.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.08 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.32 ( s , 3 h ) ; ms ( esi ) m / z 441.19 , 443.18", "( m + h ) ; rf = 0.29 in 10% etoac / hexanes . the regiochemistry was confirmed by an noe between the methyl protons and the n , n - dimethylamino protons ( 0.61% ) .", "data for 16f : h nmr ( 400 mhz , cdcl3 ) 7.417.31 ( m , 7 h ) , 7.307.20 ( m , 1 h ) , 7.01 ( d , j = 7.3 hz , 2 h ) , 4.94 ( s , 2 h ) , 3.04 ( s , 6 h ) , 2.33 ( s , 3 h ) ; ms ( esi ) m / z 441.19 , 443.18", "( m + h ) ; rf = 0.40 in 10% etoac / hexanes . the regiochemistry was confirmed by noe between the benzylic protons of the o - benzyl group and the n , n - dimethylamino protons ( 0.44% ) .", "compound 16d ( 538 mg , 1.29 mmol ) , t - butylcarbamate ( 302 mg , 2.58 mmol ) , cs2co3 ( 840 mg , 2.58 mmol ) , tris(dibenzylideneacetone)dipalladium ( 59 mg , 0.065 mmol ) , and xantphos ( 109 mg , 0.190 mmol ) were weighed into a flask .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 3 ml ) was added .", "after 4 h , the reaction mixture was cooled to room temperature , was diluted with etoac ( 50 ml ) , and was washed with water ( 2 25 ml ) .", "the material was purified by column chromatography ( biotage 20 g column , 012% etoac in hexanes gradient ) , yielding 451 mg ( 77% ) of the product .", "rf = 0.35 in 20% etoac / hexanes . h nmr ( 400 mhz , cdcl3 ) 7.427.24 ( m , 8 h ) , 7.046.99 ( m , 2 h ) , 4.99 ( s , 2 h ) , 2.38 ( s , 3 h ) , 1.40 ( s , 18 h ) .", "compound 17a ( 451 mg , 1.00 mmol ) was treated with di - t - butyldicarbonate ( 1.09 g , 5.00 mmol ) and dmap ( 24 mg , 0.20 mmol ) in dmf ( 15 ml ) .", "after 30 min , the reaction mixture was diluted with etoac ( 100 ml ) and was washed with water ( 3 50 ml ) and brine ( 50 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 08% etoac in hexanes gradient ) , yielding 481 mg ( 87% ) of the title compound .", "h nmr ( 400 mhz , cdcl3 ) 7.427.24 ( m , 8 h ) , 7.046.99 ( m , 2 h ) , 4.99 ( s , 2 h ) , 2.38 ( s , 3 h ) , 1.40 ( s , 18 h ) .", "compound 16e ( 467 mg , 1.06 mmol ) , t - butylcarbamate ( 372 mg , 3.17 mmol ) , cs2co3 ( 1.04 g , 3.17 mmol ) , tris(dibenzylideneacetone)dipalladium ( 48 mg , 0.053 mmol ) , and xantphos ( 89.3 mg , 0.159 mmol ) were weighed into a vial .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 3 ml ) was added .", "after 4 h , the reaction mixture was cooled to room temperature and was filtered through celite .", "the filtrate was concentrated under reduced pressure , and the material was purified by column chromatography ( biotage 20 g column , 016% etoac in hexanes gradient ) , yielding 493 mg ( 98% ) of the product containing 20% of an unidentified impurity .", "h nmr ( 400 mhz , cdcl3 ) 7.437.32 ( m , 8 h ) , 7.307.24 ( m , 1 h ) , 7.157.10 ( m , 2 h ) , 6.80 ( s , 1 h ) , 4.94 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.31 ( s , 3 h ) , 1.48 ( s , 9 h ) . ms ( esi ) m / z 478.27 ( m + h ) .", "compound 17b ( 490 mg , 1.03 mmol ) was treated with di - t - butyldicarbonate ( 672 mg , 3.08 mmol ) and dmap ( 12.5 mg , 0.103 mmol ) in dmf ( 5 ml ) .", "after stirring overnight , the reaction mixture was diluted with etoac ( 25 ml ) and was washed with water ( 3 20 ml ) and brine ( 20 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 010% etoac in hexanes gradient ) , yielding 424 mg ( 72% ) of the product .", "h nmr ( 400 mhz , cdcl3 ) 7.427.24 ( m , 8 h ) , 7.067.00 ( m , 2 h ) , 4.92 ( s , 2 h ) , 2.79 ( s , 6 h ) , 2.36 ( s , 3 h ) , 1.39 ( s , 18 h ) . ms ( esi ) m / z 578.33 ( m + h ) .", "lda was prepared at 78 c from n - butyllithium ( 1.6 m solution in hexane , 0.495 ml , 0.792 mmol ) and diisopropylamine ( 0.112 ml , 0.792 mmol ) in thf ( 5 ml ) .", "tmeda ( 0.318 ml , 2.11 mmol ) was added , followed by dropwise addition of a solution of compound 14 ( 169 mg , 0.529 mmol ) in thf ( 2 ml ) .", "after 5 min , a solution of 8 ( 128 mg , 0.264 mmol ) in thf ( 2 ml ) was added .", "after complete addition , the reaction mixture was allowed to warm to 20 c over 1 h. the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution , 20 ml ) and was extracted with etoac ( 2 20 ml ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 65.5 mg ( 35% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.77 ( s , 1 h ) , 8.36 ( s , 1 h ) , 8.16 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.405.25 ( m , 4 h ) , 3.93 ( d , j = 11.0 hz , 1 h ) , 3.163.04 ( m , 1 h ) , 2.982.90 ( m , 1 h ) , 2.762.64 ( m , 1 h ) , 2.602.40 ( m , 8 h ) , 2.12 ( d , j = 14 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 708.72 ( m + h ) .", "the following compounds were synthesized by methods similar to 19a . prepared from 15a in 38% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.6 ( s , 1 h ) , 8.12 ( s , 1 h ) , 7.567.49 ( m , 4 h ) , 7.427.32 ( m , 6 h ) , 5.37 ( s , 2 h ) , 5.07 ( s , 2 h ) , 3.91 ( d , j = 11.0 hz , 1 h ) , 3.133.02 ( m , 1 h ) , 2.97 ( dd , j = 15.6 4.9 hz , 1 h ) , 2.652.45 ( m , 3 h ) , 2.50 ( s , 6 h ) , 2.14 ( d , j = 15.6 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.27 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 742.56 ( m + h ) . prepared from 15b in 71% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.55 ( s , 1 h ) , 8.12 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.405.22 ( m , 4 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.25 ( dd , j = 16.5 hz , j = 4.88 hz , 1 h ) , 3.123.02 ( m , 1 h ) , 2.622.42 ( m , 9 h ) , 2.14 ( d , j = 14.0 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 786.63 , 788.63 ( m + h ) . prepared from 15c in 50% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.65 ( s , 1 h ) , 8.01 ( s , 1 h ) , 7.527.22 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 11.0 hz , 2 h ) , 4.02 ( d , j = 11.0 hz , 1 h ) , 3.05 ( dd , j = 17.2 hz , j = 11.6 hz , 1 h ) , 2.982.86 ( m , 1 h ) , 2.73 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.12 ( d , j = 13.4 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 751.79 ( m + h ) . prepared from 15d in 27% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.70 ( s , 1 h ) , 7.83 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 13.4 hz , 2 h ) , 3.98 ( d , j = 11.0 hz , 1 h ) , 3.72 ( s , 3 h ) , 3.18 ( d , j = 16.5 hz , 1 h ) , 3.04 2.96 ( m , 1 h ) , 2.602.30 ( m , 9 h ) , 2.15 ( d , j = 14 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 738.66 ( m + h ) .", "h nmr ( 400 mhz , cdcl3 ) 15.59 ( s , 1 h ) , 7.567.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.92 ( q , j = 67.8 hz , j = 9.16 hz , 2 h ) , 3.91 ( d , j = 11.0 hz , 1 h ) , 3.203.02 ( m , 2 h ) , 2.622.45 ( m , 9 h ) , 2.192.12 ( m , 1 h ) , 1.37 , ( s , 18 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 941.59 ( m + h ) . prepared from 16e in 54% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.65 ( s , 1 h ) , 7.527.22 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 11.0 hz , 2 h ) , 4.01 ( d , j = 11.0 hz , 1 h ) , 3.05 ( dd , j = 17.2 hz , j = 11.6 hz , 1 h ) , 2.982.86 ( m , 1 h ) , 2.73 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.12 ( d , j = 13.4 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 829.49 , 831.48 ( m + h ) . compound 16a ( 793 mg , 1.84 mmol ) and compound 8 ( 885 mg , 1.84 mmol ) were dissolved in thf ( 16 ml ) , and the solution was cooled to 78 c .", "lhmds ( 1.0 m solution in thf , 5.5 ml , 5.5 mmol ) was added slowly via syringe .", "after 10 min , the reaction mixture was allowed to slowly warm to 0 c over 1 h. a phosphate buffer solution ( ph 7.0 , 20 ml ) was added , followed by the addition of ammonium chloride ( saturated , aqueous solution , 50 ml ) .", "the resulting mixture was extracted with ch2cl2 ( 3 50 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the resulting brown solid was washed with cold methanol ( 3 5 ml ) to afford 1.11 g ( 74% ) of the title compound as a yellowbrown powder .", "the organics were concentrated under reduced pressure and purified by column chromatography ( biotage 20 g column , 520% etoac in hexanes gradient ) , yielding an additional 150 mg ( 10% ) of the product .", "h nmr ( 400 mhz , cdcl3 ) 15.45 ( br , 1 h ) , 7.547.48 ( m , 4 h ) , 7.407.30 ( m , 6 h ) , 5.36 ( s , 2 h ) , 5.03 ( abq , j = 10.4 hz , 2 h ) , 3.87 ( d , j = 11.0 hz , 1 h ) , 3.273.23 ( m , 1 h ) , 3.103.00 ( m , 1 h ) , 2.652.57 ( m , 1 h ) , 2.572.43 ( m , 8 h ) , 2.16 ( d , j = 11.0 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 820.37 , 822.37 ( m + h ) .", "the following compounds were prepared by methods similar to 19h . prepared from 16d in 45% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.55 ( s , 1 h ) , 7.567.26 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.02 ( s , 2 h ) , 3.88 ( d , j = 10.4 hz , 1 h ) , 3.183.04 ( m , 2 h ) , 2.622.58 ( m , 1 h ) , 2.582.40 ( m , 8 h ) , 2.17 ( d , j = 14.6 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 804.34 , 806.34 ( m + h ) . prepared from 18b in 53% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.50 ( s , 1 h ) , 7.527.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.85 ( q , j = 86.1 hz , j = 9.76 hz , 2 h ) , 4.02 ( d , j = 10.4 hz , 1 h ) , 3.083.00 ( m , 1 h ) , 3.002.82 ( m , 1 h ) , 2.77 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.202.12 ( m , 1 h ) , 1.35 , ( br s , 18 h ) , 0.79 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 966.59 ( m + h ) .", "a solution of compound 19c ( 52 mg , 0.067 mmol ) , methylboronic acid ( 40 mg , 0.67 mmol ) , dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii ) dichloromethane adduct ( 3 mg , 0.003 mmol ) , and k3po4 ( 142 mg , 0.667 mmol ) in toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.2 ml ) was heated to 70 c . after 2 h , the reaction mixture was heated to 100 c .", "after an additional 2 h , the reaction mixture was diluted with etoac ( 20 ml ) and was washed with water ( 15 ml ) and brine ( 15 ml ) .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 18.3 mg ( 38% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.71 ( s , 1 h ) , 8.24 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.305.20 ( m , 2 h ) , 3.95 ( d , j = 10.4 hz , 1 h ) , 3.102.92 ( m , 2 h ) , 2.622.42 ( m , 12 h ) , 2.12 ( d , j = 14.0 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.14 ( s , 3 h ) . ms ( esi ) m / z 722.72 ( m + h ) . a solution of compound 19c ( 31 mg , 0.040 mmol ) , phenylboronic acid ( 24.5 mg , 0.201 mmol ) , dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii ) dichloromethane adduct ( 1.6 mg , 0.002 mmol ) , and sodium carbonate ( 21.3 mg , 0.201 mmol ) in toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.2 ml ) was heated to 100 c via microwave reactor for 10 min .", "the reaction mixture was diluted with etoac ( 10 ml ) and was washed with water ( 5 ml ) and brine ( 5 ml ) .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 16.9 mg ( 54% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.66 ( s , 1 h ) , 8.48 ( s , 1 h ) , 7.527.24 ( m , 15 h ) , 5.425.30 ( m , 4 h ) , 3.97 ( d , j = 10.4 hz , 1 h ) , 3.002.86 ( m , 2 h ) , 2.782.62 ( m , 1 h ) , 2.582.30 ( m , 8 h ) , 2.00 ( d , j = 14.0 hz , 1 h ) , 0.80 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.14 ( s , 3 h ) . ms ( esi ) m / z 784.75 ( m + h ) . compound 19h ( 100 mg , 0.122 mmol ) , t - butylcarbamate ( 42.9 mg , 0.366 mmol ) , k3po4 ( 77.7 mg , 0.366 mmol ) , tris(dibenzylideneacetone)dipalladium ( 5.6 mg , 0.006 mmol ) , and xantphos ( 10.3 mg , 0.018 mmol ) were weighed into a vial equipped with a septum .", "the vial was evacuated and flushed with nitrogen ( 3 ) , and 1,4-dioxane ( 0.5 ml ) was added .", "the reaction mixture was heated to 100 c and stirred for 2 h. the reaction mixture was allowed to cool to room temperature , was filtered through celite , and was concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 41.7 mg ( 40% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.6515.45 ( br s , 1 h ) , 7.567.50 ( m , 2 h ) , 7.437.34 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 5.38 ( s , 2 h ) , 4.94 ( dd , j = 29.3 hz , j = 11.0 hz , 2 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.27 ( dd , j = 16.4 hz , j = 11.6 hz , 1 h ) , 3.123.03 ( m , 1 h ) , 2.682.60 ( m , 1 h ) , 2.602.45 ( m , 8 h ) , 2.18 ( d , j = 14.6 hz , 1 h ) , 1.47 ( s , 9 h ) , 0.85 ( s , 9 h ) , 0.29 ( s , 3 h ) , 0.15 ( s , 3 h ) . ms ( esi ) m / z 857.67 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 19a ( 65.5 mg , 0.093 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water ( 20 ml ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 2 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 18.4 mg ( 41% , 2 steps ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.55 ( s , 1 h ) , 8.36 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.262.98 ( m , 9 h ) , 2.71 ( t , j = 14.2 hz , 1 h ) , 2.392.32 ( m , 1 h ) , 1.751.63 ( m , 1 h ) .", "the following compounds were synthesized by similar methods to 20a . prepared from 19b in 24% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 8.08 ( d , j = 6.0 hz , 1 h ) , 4.09 ( s , 1 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.70 1.58 ( m , 1 h ) . ms ( esi ) m / z 450.18 ( m + h ) . prepared from 19d in 34% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.35 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.252.95 ( m , 9 h ) , 2.802.25 ( m , 5 h ) , 1.801.63 ( m , 1 h ) .", "ms ( esi ) m / z 430.46 ( m + h ) . prepared from 19e in 24% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.53 ( s , 1 h ) , 7.62 ( s , 5 h ) , 4.18 ( s , 1 h ) , 3.142.82 ( m , 9 h ) , 2.812.66 ( m , 1 h ) , 2.242.14 ( m , 1 h ) , 1.661.52 ( m , 1 h ) .", "ms ( esi ) m / z 492.48 ( m + h ) . prepared from 19f in 96% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.22 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.383.30 ( m , 2 h ) , 3.24 ( s , 6 h ) , 3.122.95 ( m , 7 h ) , 2.60 ( t , j = 14.2 hz , 1 h ) , 2.422.34 ( m , 1 h ) , 1.751.63 ( m , 1 h ) .", "ms ( esi ) m / z 459.50 ( m + h ) . prepared from 19 g in 51% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 7.80 ( s , 1 h ) , 4.15 ( s , 1 h ) , 3.97 ( s , 3 h ) , 3.402.98 ( m , 9 h ) , 2.322.20 ( m , 2 h ) , 1.721.56 ( m , 1 h ) .", "ms ( esi ) m / z 446.39 ( m + h ) . prepared from 19i in 68% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 7.81 ( s , 1 h ) , 4.17 ( s , 1 h ) , 3.262.96 ( m , 9 h ) , 2.422.25 ( m , 2 h ) , 1.701.58 ( m , 1 h ) .", "ms ( esi ) m / z 434.27 ( m + h ) . prepared from 19j in 68% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.19 ( s , 1 h ) , 3.202.98 ( m , 8 h ) , 2.352.22 ( m , 2 h ) , 1.681.56 ( m , 1 h ) .", "ms ( esi ) m / z 465.32 ( m + h ) . prepared from 19k in 49% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.20 ( s , 1 h ) , 3.402.90 ( m , 9 h ) , 2.422.22 ( m , 2 h ) , 1.701.54 ( m , 1 h ) .", "compound 19j ( 68.3 mg , 0.080 mmol ) was stirred in 4 m hcl in 1,4-dioxane for 2 h. the reaction mixture was concentrated under reduced pressure . the material was dissolved in thf ( 2 ml ) , and bromoacetylchloride ( 12.5 mg , 0.080 mmol ) was added . after stirring overnight , an additional portion of bromoacetylchloride ( 0.020 ml ) was added .", "t - butylamine ( 0.063 ml , 0.60 mmol ) was added to a solution of the intermediate ( 35 mg , 0.040 mmol ) in thf ( 1 ml ) , and the reaction mixture was heated to 50 c . after stirring overnight ,", "the reaction mixture was concentrated under reduced pressure and was purified by preparative hplc ( sunfire prep c18 column , 50100% b gradient ) .", "this gave 16.8 mg ( 49% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 8.20 ( br s , 1 h ) , 7.527.46 ( m , 2 h ) , 7.437.30 ( m , 8 h ) , 5.36 ( s , 2 h ) , 4.95 ( dd , j = 40.6 hz , j = 11.0 hz , 2 h ) , 3.88 ( d , j = 11.0 hz , 1 h ) , 3.323.24 ( m , 1 h ) , 3.123.03 ( m , 1 h ) , 2.662.59 ( m , 1 h ) , 2.582.42 ( m , 8 h ) , 2.18 ( d , j = 14.6 hz , 1 h ) , 1.301.10 ( br s , 9 h ) , 0.82 ( s , 9 h ) , 0.27 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 870.60 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of the preceding intermediate ( 16.8 mg , 0.019 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 7.8 g ) in water ( 15 ml ) .", "sodium chloride ( 10 g ) was added to the aqueous layer , and this was extracted with etoac ( 2 ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx 10 rp 100a column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 1.2 mg ( 10% , 2 steps ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.304.20 ( s , 2 h ) , 4.19 ( s , 1 h ) , 3.252.96 ( m , 9 h ) , 2.482.28 ( m , 2 h ) , 1.721.58 ( m , 1 h ) , 1.43 ( s , 9 h ) .", "the following compounds were prepared according to methods similar to 21a : prepared from 19k in 68% yield for the acylation / amine displacement and 58% yield for the deprotections , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.244.65 ( m , 3 h ) , 3.262.97 ( m , 9 h ) , 2.382.28 ( m , 2 h ) , 1.691.56 ( m , 1 h ) , 1.44 ( s , 9 h ) .", "ms ( esi ) m / z 562.37 ( m + h ) . prepared from 19l in 43% yield for the acylation / amine displacement and 55% yield for the deprotections , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.304.40 ( m , 3 h ) , 3.403.25 ( m , 8 h ) , 3.123.04 ( m , 4 h ) , 2.99 ( s , 3 h ) , 2.622.52 ( m , 1 h ) , 2.412.35 ( m , 1 h ) , 1.731.60 ( m , 1 h ) , 1.45 ( s , 9 h ) .", "compound 19h ( 50 mg , 0.061 mmol ) , k3po4 ( 39 mg , 0.18 mmol ) , tris(dibenzylideneacetone)dipalladium ( 2.8 mg , 0.003 mmol ) , and xantphos ( 5.1 mg , 0.009 mmol ) were added to a small vial equipped with a septum . after the flask was evacuated and flushed with nitrogen ( 3 ) , 1,4-dioxane ( 0.5 ml ) and ethylamine ( 2.0 m solution in thf , 0.091 ml , 0.18 mmol ) were added .", "the reaction mixture was heated to 100 c and stirred for 3 h. the reaction mixture was allowed to cool to room temperature and was filtered through celite .", "the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 11 mg ( 23% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.6 ( br , 1 h ) , 7.567.49 ( m , 4 h ) , 7.427.30 ( m , 6 h ) , 5.37 ( s , 2 h ) , 5.03 ( s , 2 h ) , 3.89 ( d , j = 11.0 hz , 1 h ) , 3.243.04 ( m , 3 h ) , 2.97 ( dd , j = 15.6 , 4.9 hz , 1 h ) , 2.642.43 ( m , 3 h ) , 2.49 ( s , 6 h ) , 2.14 ( d , j = 15.6 hz , 1 h ) , 1.25 ( t , j = 6.8 hz , 3 h ) , 0.81 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.11 ( s , 3 h ) . ms ( esi ) m / z 785.57 ( m + h ) .", "aqueous hf ( 48% , 0.3 ml ) was added to a solution of the preceding intermediate ( 11 mg , 0.014 mmol ) in ch3cn ( 7 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 2 g ) in water ( 10 ml ) .", "the material was dissolved in meoh ( 2 ml ) and 1,4-dioxane ( 2 ml ) , and palladium on carbon ( degussa , 10 wt % , 5.6 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx 10 rp 100a column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 3.2 mg ( 46% , 2 steps ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.08 ( s , 1 h ) , 3.43 ( q , j = 7.4 hz , 2 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.671.55 ( m , 1 h ) , 1.22 ( t , j = 7.4 hz , 3h ) . ms ( esi ) m / z 493.24 ( m + h ) .", "the following compounds were prepared by similar methods to 24a : prepared from 19h and methylamine ( 1.0 m solution in thf ) in 21% yield for the amination step , 19% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.08 ( s , 1 h ) , 3.41 ( s , 3 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.671.55 ( m , 1 h ) .", "ms ( esi ) m / z 479.22 ( m + h ) . prepared from 19h and n - propylamine in 24% yield for the amination step , 40% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.16 ( s , 1h ) , 3.47 ( t , j = 7.3 hz , 2 h ) , 3.152.96 ( m , 9 h ) , 2.322.20 ( m , 2 h ) , 1.781.55 ( m , 3 h ) , 1.01 ( t , j = 7.4 hz , 3 h ) .", "ms ( esi ) m / z 507.29 ( m + h ) . prepared from 19h and 2-propylamine in 11% yield for the amination step , 16% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.214.13 ( m , 1 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.312.15 ( m , 2 h ) , 1.671.56 ( m , 1 h ) , 1.23 ( dd , j = 6.4 , 1.8 hz , 6 h ) .", "ms ( esi ) m / z 507.24 ( m + h ) . prepared from 19h and 2-methylpropan-1-amine in 15% yield for the amination step , 57% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.18 ( s , 1h ) , 3.38 ( d , j = 7.3 hz , 2 h ) , 3.202.96 ( m , 9 h ) , 2.342.20 ( m , 2 h ) , 2.101.98 ( m , 1h ) , 1.681.54 ( m , 1 h ) , 1.00 ( d , j = 6.4 hz , 6 h ) . ms ( esi ) m / z 521.40 ( m + h ) . prepared from 19h and 3-methyl - butan-1-amine in 25% yield for the amination step , 66% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.453.40 ( m , 2 h ) , 3.20 ( s , 1 h ) , 3.082.93 ( m , 9 h ) , 2.302.16 ( m , 2 h ) , 1.721.58 ( m , 2 h ) , 1.551.48 ( m , 2 h ) , 0.96 ( d , j = 6.4 hz , 6 h ) . ms ( esi ) m / z 535.30 ( m + h ) . prepared from 19h and 2-methoxyethylamine in 19% yield for the amination step , 44% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.13 ( s , 1h ) , 3.723.60 ( m , 4h ) , 3.40 ( s , 3 h ) , 3.122.95 ( m , 9 h ) , 2.322.21 ( m , 2 h ) , 1.691.56 ( m , 1 h ) .", "ms ( esi ) m / z 523.32 ( m + h ) . prepared from 19h and 2-(propan-2-yloxy)ethan-1-amine in 9% yield for the amination step , 30% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.19 ( s , 1h ) , 3.823.69 ( m , 5h ) , 3.202.98 ( m , 9 h ) , 2.352.22 ( m , 2 h ) , 1.681.56 ( m , 1 h ) , 1.18 ( d , j = 6.4 hz , 6h ) . ms ( esi ) m / z 551.41 ( m + h ) . prepared from 19h and ( 3-aminopropyl)dimethylamine in 9% yield for the amination step , 21% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.703.46 ( m , 4 h ) , 3.102.90 ( m , 15 h ) , 2.312.17 ( m , 2 h ) , 2.102.00 ( m , 2 h ) , 1.681.56 ( m , 1 h ) .", "compound 16d ( 100 mg , 0.24 mmol ) , cs2co3 ( 235 mg , 0.720 mmol ) , tris(dibenzylideneacetone)dipalladium ( 11 mg , 0.012 mmol ) , and xantphos ( 20 mg , 0.036 mmol ) were weighed into a flask .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 0.5 ml ) and n , n - dimethylneopentanediamine ( 0.057 ml , 0.36 mmol ) were added .", "after 1.5 h , the reaction mixture was cooled to room temperature and was filtered through celite .", "the filtrate was concentrated under reduced pressure and was purified by column chromatography ( biotage 10 g column , 03% meoh in ch2cl2 gradient ) , yielding 76.9 mg ( 69% ) of the intermediate .", "lhmds ( 1.0 m in thf , 0.22 ml , 0.22 mmol ) was added dropwise to a 78 c solution of the above intermediate ( 93 mg , 0.20 mmol ) in thf ( 5 ml ) . after 5 min , benzyl chloroformate ( 0.084 ml , 0.60 mmol )", "after 15 min , the reaction mixture was quenched by the addition of nh4cl ( saturated , aqueous solution , 15 ml ) and was extracted with etoac .", "the material was purified by column chromatography ( biotage 10 g column , 07% meoh in ch2cl2 gradient ) , yielding 111 mg ( 93% ) of the product .", "nmr ( 400 mhz , cdcl3 ) 7.457.20 ( m , 13 h ) , 7.066.98 ( m , 2 h ) , 5.16 ( s , 2 h ) , 4.87 ( s , 2 h ) , 3.78 ( br s , 2 h ) , 2.38 ( s , 3 h ) , 2.18 ( br s , 6 h ) , 1.62 ( br s , 2 h ) , 0.90 ( br s , 6 h ) . ms ( esi ) m / z 600.28 ( m + h ) .", "a solution of compound 22a ( 54 mg , 0.090 mmol ) in thf ( 0.5 ml ) was added dropwise to a 78 c solution of lda ( 2.0 m solution in thf , 0.112 ml , 0.224 mmol ) and tmeda ( 0.081 ml , 0.54 mmol ) in thf ( 2 ml ) , resulting in an orange - colored solution .", "after 10 min , a solution of 8 ( 43 mg , 0.090 mmol ) in thf ( 0.5 ml ) was added dropwise over 30 s. after complete addition , the reaction mixture was allowed to warm to 10 c over 1 h. the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) , was diluted with water , and was extracted with etoac ( 2 ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 20100% b gradient ) , yielding 22.9 mg ( 26% ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.55 ( br s , 1 h ) , 7.527.20 ( m , 15 h ) , 5.36 ( s , 2 h ) , 5.205.00 ( m , 2 h ) , 4.80 ( s , 2 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.193.05 ( m , 2 h ) , 2.622.57 ( m , 1 h ) , 2.562.12 ( m , 19 h ) , 0.940.74 ( m , 15 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 988.59 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 23a ( 22.9 mg , 0.0232 mmol ) in ch3cn ( 0.8 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water .", "the mixture was extracted with etoac , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) , 0.5 m hcl in meoh ( 0.2 ml ) , and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 2 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 3 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 9.9 mg ( 67% , 2 steps ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.16 ( s , 1 h ) , 3.43 ( s , 2 h ) , 3.152.90 ( m , 17 h ) , 2.302.22 ( m , 1 h ) , 2.222.12 ( m , 1 h ) , 1.651.54 ( m , 1 h ) , 1.17 , ( s , 6 h ) .", "the following compounds were prepared according to methods similar to 24j : prepared from 16a and n , n - dimethylethylenediamine .", "h nmr ( 400 mhz , cd3od ) 4.13 ( s , 1 h ) , 3.843.77 ( m , 2 h ) , 3.443.38 ( m , 2 h ) , 3.102.92 ( m , 15 h ) , 2.322.20 ( m , 2 h ) , 1.681.55 ( m , 1 h ) .", "ms ( esi ) m / z 536.25 ( m + h ) . prepared from 16a and n , n - dimethylneopentanediamine .", "h nmr ( 400 mhz , cd3od ) 4.10 ( s , 1 h ) , 3.513.36 ( m , 4 h ) , 3.102.92 ( m , 15 h ) , 2.332.18 ( m , 2 h ) , 1.681.57 ( m , 1 h ) , 1.174 ( s , 3 h ) , 1.169 ( s , 3 h ) .", "ms ( esi ) m / z 578.35 ( m + h ) . prepared from 16a and 2,2-dimethyl-3-(pyrrolidin-1-yl)propan-1-amine .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.883.78 ( m , 2 h ) , 3.503.39 ( m , 2 h ) , 3.18 2.92 ( m , 9 h ) , 2.322.08 ( m , 6 h ) , 1.671.56 ( m , 1 h ) , 1.15 ( s , 6 h ) . ms ( esi ) m / z 604.29 ( m + h ) . prepared from 16d and n - propylamine .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.33 ( t , j = 6.9 hz , 2 h ) 3.423.38 ( m , 2 h ) , 3.20 ( s , 1 h ) , 3.072.94 ( m , 8 h ) , 2.91 ( dd , j = 14.6 , 4.6 hz , 1 h ) , 2.232.12 ( m , 2 h ) , 1.681.55 ( m , 3 h ) , 0.96 ( t , j = 7.3 hz , 3 h ) .", "copper(i ) iodide ( 4.7 mg , 0.025 mmol ) and k3po4 ( 209 mg , 0.984 mmol ) were added to a vial equipped with a septum . after the vial was evacuated and flushed with nitrogen ( 3 ) , a solution of compound 16e ( 217 mg , 0.492 mmol ) in dmf ( 2 ml ) , 1-propylamine ( 0.162 ml , 1.97 mmol ) , and 2-acetylcyclohexanone ( 0.013 ml , 0.098 mmol ) were added .", "the reaction mixture was heated to 100 c for 3 h. the reaction mixture was allowed to cool to room temperature , was diluted with etoac ( 50 ml ) , and was washed with water ( 3 40 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 74.6 mg ( 36% ) of the title compound as a thick oil .", "h nmr ( 400 mhz , cdcl3 ) 7.507.46 ( m , 2 h ) , 7.417.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.08 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.32 ( s , 3 h ) . ms ( esi ) m / z 420.34 ( m + h ) .", "lhmds ( 1.0 m solution in thf , 0.167 ml , 0.167 mmol ) was added to a solution of phenyl 5-benzyloxy-2-(dimethylamino)-3-methyl-6-(propylamino)isonicotinate ( 63.5 mg , 0.152 mmol ) in thf ( 2 ml ) , resulting in a bright - yellow solution .", "a solution of di - t - butyldicarbonate ( 99.5 mg , 0.456 mmol ) in thf ( 1 ml ) was added .", "after 10 min , the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) and was extracted with etoac ( 2 ) .", "the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 13.6 mg ( 17% ) of the product .", "h nmr ( 400 mhz , cdcl3 ) 7.407.28 ( m , 7 h ) , 7.287.22 ( m , 1 h ) , 7.057.00 ( m , 2 h ) , 4.90 ( s , 2 h ) , 2.79 ( s , 6 h ) , 2.34 ( s , 3 h ) , 1.761.66 ( m , 2 h ) , 1.561.50 ( m , 2 h ) , 0.91 ( t , j = 7.56 hz , 3 h ) . ms ( esi ) m / z 520.38 ( m + h ) .", "a solution of 22b ( 24 mg , 0.047 mmol ) in thf ( 0.5 ml ) was added dropwise to a 78 c solution of lda ( 0.5 m solution in thf , 0.102 ml , 0.051 mmol ) and tmeda ( 0.041 ml , 0.27 mmol ) in thf ( 2 ml ) , resulting in a yellowishorange colored solution .", "after 10 min , a solution of 8 ( 16.4 mg , 0.034 mmol ) in thf ( 0.5 ml ) was added dropwise .", "after complete addition , the reaction mixture was allowed to warm to 20 c over 45 min .", "the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) and was extracted with etoac ( 2 ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient ) , yielding 6.1 mg ( 20% ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.56 ( s , 1 h ) , 7.527.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.994.64 ( m , 2 h ) , 4.02 ( d , j = 11.0 hz , 1 h ) , 3.102.81 ( m , 2 h ) , 2.78 ( s , 6 h ) , 2.622.40 ( m , 10 h ) , 2.202.14 ( m , 1 h ) , 1.801.45 ( m , 2 h ) , 1.441.15 ( m , 11 h ) , 1.000.70 ( m , 12 h ) , 0.25 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 908.65 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 20o ( 6.1 mg , 0.007 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water ( 15 ml ) .", "the combined etoac extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 2 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 4 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", ". fractions with the desired mw were collected and freeze - dried to yield 2.8 mg ( 70% , 2 steps ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.19 ( s , 1 h ) , 3.44 ( t , j = 7.6 hz , 2 h ) , 3.243.12 ( m , 7 h ) , 3.122.97 ( m , 8 h ) , 2.392.26 ( m , 2 h ) , 1.731.56 ( m , 3 h ) , 0.98 ( t , j = 8.4 hz , 3 h ) ; ms ( esi ) m / z 516.42 ( m + h ) . compound 19 m ( 105 mg , 0.126 mmol ) , tetrakis(triphenylphosphine)palladium(0 ) ( 15 mg , 0.013 mmol ) , and k3po4 ( 80 mg , 0.38 mmol ) were weighed into an 8 ml vial .", "this was sealed with a septum and was evacuated and backflushed with nitrogen ( 3 ) .", "toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.5 ml ) were added , followed by allylboronic acid pinacol ester ( 63.7 mg , 0.379 mmol ) .", "after 2.5 h , the reaction mixture was cooled to room temperature and was concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient ) , yielding 28.6 mg ( 29% ) of the title compound as a yellow solid .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 25a ( 28.6 mg , 0.036 mmol ) in 1,4-dioxane ( 1 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water .", "the mixture was extracted with etoac , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) , 0.5 m hcl in meoh ( 0.5 ml ) , and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx column , 070% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 14.3 mg ( 69% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.20 ( s , 1 h ) , 3.383.15 ( m , 7 h ) , 3.142.94 ( m , 8 h ) , 2.912.82 ( m , 2 h ) , 2.56 ( t , j = 13.8 hz , 1 h ) , 2.402.32 ( m , 1 h ) , 1.861.76 ( m , 2 h ) , 1.741.62 ( m , 1 h ) , 1.01 ( t , j = 7.34 hz , 3 h ) .", "the following compounds were prepared according to methods similar to 26a : prepared from 19h and allylboronic acid pinacol ester , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.10 ( s , 1 h ) , 3.222.92 ( m , 9 h ) , 2.812.74 ( m , 2 h ) , 2.492.38 ( m , 1 h ) , 2.262.19 ( m , 1 h ) , 1.77 1.60 ( m , 3 h ) , 0.97 ( t , j = 7.3 hz , 3 h ) .", "ms ( esi ) m / z 492.27 ( m + h ) . prepared from 19h and methylboronic acid , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.222.92 ( m , 9 h ) , 2.85 ( s , 3 h ) , 2.492.38 ( m , 1 h ) , 2.262.19 ( m , 1 h ) , 1.77 1.60 ( m , 1h ) .", "ms ( esi ) m / z 464.30 ( m + h ) . prepared from 19h as an over - reduced side product in the synthesis of 26c .", "h nmr ( 400 mhz , cd3od ) 7.78 ( s , 1 h ) , 4.12 ( s , 1 h ) , 3.222.91 ( m , 9 h ) , 2.78 ( s , 3 h ) , 2.492.21 ( m , 1 h ) , 1.771.62 ( m , 1h ) .", "ms ( esi ) m / z 430.46 ( m + h ) . prepared from 19h and phenylboronic acid .", "h nmr ( 400 mhz , cd3od ) 8.047.98 ( m , 2 h ) , 7.477.39 ( m , 3 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.572.47 ( m , 1 h ) , 2.282.07 ( m , 1 h ) , 1.731.65 ( m , 1h ) .", "ms ( esi ) m / z 526.26 ( m + h ) . prepared from 19h as an over - reduced side product in the synthesis of 26e .", "h nmr ( 400 mhz , cd3od ) 8.047.98 ( m , 2 h ) , 7.477.39 ( m , 3 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.572.47 ( m , 1 h ) , 2.282.07 ( m , 1 h ) , 1.731.65 ( m , 1h ) .", "tetracycline - susceptible isolates sa100 ( s. aureus atcc 13709 , smith ) , sa101 ( s. aureus atcc 29213 ) , sp106 ( s. pneumoniae atcc 49619 ) , ef103 ( e. faecalis atcc 29212 ) , ec107 ( e. coli atcc 25922 ) , kp109 ( k. pneumoniae atcc 13883 ) , ab110 ( acinetobacter baumannii atcc 19606 ) , ec108 ( enterobacter cloacae atcc 13047 ) , and pa111 ( pseudomonas aeruginosa atcc 27853 ) were obtained from the american type culture collection ( atcc , manassas , va ) .", "tetracycline - resistant isolates s. aureus sa158 ( tet(k ) ) , s. pneumoniae sp160 ( tet(m ) ) , e. faecalis ef159 ( tet(m ) ) , e. coli ec155 ( tet(a ) ) , k. pneumoniae kp153 ( tet(a ) ) were obtained from marilyn roberts lab at the university of washington .", "s. aureus sa161 ( tet(m ) ) was obtained from micromyx ( kalamazoo , mi ) .", "minimal inhibitory concentration ( mic ) determinations were performed in liquid medium in 96-well microtiter plates according to the methods described by the clinical and laboratory standards institute ( clsi).(26 ) cation - adjusted mueller hinton broth was obtained from bbl ( cat .", "212322 , becton dickinson , sparks , md ) , prepared fresh and kept at 4 c prior to testing .", "a0432 , pml microbiologicals , wilsonville , or ) was used to supplement medium , as appropriate .", "all test methods met acceptable standards based on recommended quality control ranges for all comparator antibiotics and the appropriate atcc quality control strains .", "compound stocks prepared and diluted in sterile deionized water were assayed for inhibition of coupled in vitro transcription / translation using an e. coli s30 extract system with a firefly luciferase readout from promega ( cat .", "briefly , compounds were diluted into water and added to reaction mix aliquoted to black - walled 96-well microtiter plates ( cat .", "an appropriate three - point titration was used for each compound , and reactions were run in duplicate .", "plates were incubated at 37 c for one hour and then placed on ice for 5 min to arrest transcription / translation .", "e1500 ) was added to each well and luminescence was detected on a bmg labtech lumistar - optima instrument .", "positive assay control values , from reactions without inhibitor , were averaged per plate to determine percent inhibition of luciferase production .", "results were plotted using microsoft excel and 50% inhibition values ( ic50 ) were determined ." ]

[ "<S> a novel series of fully synthetic 8-azatetracyclines was prepared and evaluated for antibacterial activity . </S>", "<S> compounds were identified that overcome both efflux ( tet(k ) ) and ribosomal protection ( tet(m ) ) tetracycline resistance mechanisms and are active against gram - positive and gram - negative organisms . </S>", "<S> two compounds were identified that exhibit comparable efficacy to marketed tetracyclines in in vivo models of bacterial infection . </S>" ]

[ "Introduction", "Results and Discussion", "Conclusions", "Experimental Procedures" ]

[ [ "the tetracycline class of antibacterial agents has seen widespread clinical use for over 50 years due to its broad spectrum antibacterial activity.(1 ) tetracyclines inhibit bacterial growth by preventing protein biosynthesis through binding to the 30s ribosome , thereby blocking the binding of aminoacyl - trna to the acceptor site.(2 ) this class includes a number of naturally occurring compounds , such as chlorotetracycline ( 1)(3 ) and tetracycline ( 2),(4 ) as well as several semisynthetic analogues , such as doxycycline ( 3)(5 ) and minocycline ( 4 ) ( figure 1).(6 ) in addition to their use as antibiotics for serious infections , tetracyclines have also been prescribed for use in acne and rosacea.(7 ) as with many classes of antibiotics , widespread use of tetracyclines has led to resistance and has limited their use in more complicated bacterial infections .", "examples of naturally occurring ( 1 and 2 ) and semisynthetic ( 36 ) tetracyclines .", "two major bacterial resistance mechanisms have been identified for tetracyclines : ( 1 ) active transport from bacterial cells via efflux pumps(10 ) and ( 2 ) ribosomal protection.(11 ) the efflux pumps are comprised of inner membrane tetracycline efflux proteins that were first identified in gram - negative organisms ( tet(ae ) ) and were later found in gram - positive bacteria ( tet(kl)).(10c ) the ribosomal protection mechanism ( tet(mo ) ) involves the expression of proteins that bind to the ribosome and allow protein synthesis to proceed even in the presence of tetracyclines .", "since these early discoveries , the number of efflux and ribosomal protection mechanisms identified has increased.(12 ) the presence of the strongly activating 10-hydroxy group readily enables chemical modification of the tetracycline d - ring at the 7- and 9-positions .", "this strategy led to the introduction of the 7-dimethylamino group in compounds 46 , all of which have demonstrated improved activity in bacterial strains bearing efflux pump resistance mechanisms .", "more recently , the 9-substituted glycylcyclines , such as tigecycline ( 5),(13 ) and the aminomethylcyclines , such as amadacycline ( 6),(14 ) have shown improved activity for resistant organisms bearing the ribosomal protection mechanism . in 2005 , myers and co - workers published a fully synthetic route to the tetracyclines , the key step of which involves the michaeldieckmann condensation of the ab precursor 8 with structurally diverse d - ring precursors .", "this route provides potential access to a broad range of tetracyclines that are inaccessible via traditional semisynthesis.(17 ) one example of this is the introduction of a nitrogen atom into the d - ring , resulting in azatetracyclines . ", "one 7-azatetracycline and one 9-azatetracycline were disclosed in myers original work , resulting in compounds with only modest antibacterial activity .", "herein we demonstrate this powerful approach using pyridyl d - ring precursors 7 to generate novel 8-azatetracyclines , 10 ( scheme 1 ) ." ], [ "to explore 7- and 9-substituted-8-azatetracyclines , we required intermediates that could be readily modified either prior to or after construction of the protected compound 9 . thus , the halopyridines 15 and 16 were prepared according to schemes 2 and 3 .", "3,5-dichloroisonicotinic acid was treated with sodium benzylate to give compound 12 , which was esterified to the phenyl ester 13 by conversion to the acid chloride , followed by treatment with phenol and 4-(dimethylamino)pyridine ( dmap ) .", "suzuki coupling(18 ) using methylboronic acid and pdcl2cy2 catalyst(19 ) gave the parent d - ring precursor 14 in good yield .", "n - oxide formation with hydrogen peroxide in acetic acid followed by treatment with either phosphorousoxychloride or phosphorousoxybromide gave 15a and 15b , respectively . for 15a ,", "the reaction gave a 4.5:1.0 ratio of the desired regioisomer , while a 25:1 ratio was observed for 15b .", "the regiochemistry was assigned by conversion to the dimethylamino compound 15c through a palladium - mediated amination.(20)h nmr studies indicated an noe between the methyl groups of the amine and the adjacent ring methyl group .", "the methoxy derivative 15d was prepared from 15b by treatment with sodium methoxide in 2-methylpyrrolidinone ( nmp ) at 120 c .", "this gave the acid directly , which was re - esterified to the phenyl ester .", "reagents and conditions : ( i ) nah ( 2 equiv ) , bnoh , nmp , 80 c ; ( ii ) ( cocl)2 , cat .", "dmf , ch2cl2 ; ( iii ) phoh , et3n , dmap , ch2cl2 ; ( iv ) ch3b(oh)2 , pdcl2cy2 , k3po4 , toluene , water , 100 c ; ( v ) h2o2 , acoh , 80 c ; ( vi ) pocl3 , toluene , 100 c ; ( vii ) pobr3 , toluene , 100 c ; ( viii ) dimethylamine in thf , pd2dba3 , xantphos , k3po4 , 1,4-dioxane , 100 c ( sealed ) ;", "reagents and conditions : ( i ) for 15a , mcpba , ch2cl2 ; for 15b , h2o2 , acoh , 80 c ; ( ii ) pobr3 , toluene , 100 c ; ( iii ) bocnh2 , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 80 c ; ( iv ) hcl , 1,4-dioxane ; ( v ) hf - pyridine , nano2 , pyridine ; ( vi ) dimethylamine in thf , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 60 c ( sealed ) .", "the dihalo compounds 16a and 16b were prepared by n - oxidation of 15a and 15b followed by treatment with pobr3 ( scheme 3 ) .", "yields were typically moderate ( 4550% ) , with reduction of the n - oxide to give the starting pyridines 15 observed as major side reactions in both cases .", "palladium - mediated coupling of 16b with t - butylcarbamate gave a 2.5:1 mixture of regioisomers , which were readily separated after conversion to the free amine 16c upon treatment with hcl .", "treatment of 16c with sodium nitrite in the presence of hf - pyridine gave the fluoro derivative 16d in good yield.(21 ) similarly , a 2.4:1.0 ratio of regioisomers was observed in the palladium - mediated reaction of 16b with dimethylamine to give 16e and 16f .", "interestingly , the regioselectivity could be reversed by heating 16b in the presence of dimethylamine without catalyst to give 16f as the major isomer in a 3:1 ratio .", "the 9-amino d - ring precursors 18a and 18b were prepared according to scheme 4 .", "once again , palladium - mediated couplings using t - butylcarbamate proved to be highly effective for introduction of the amino group . further protection of the mono - boc intermediates 17 with boc2o and dmap in dmf gave the di - boc precursors 18 .", "reagents and conditions : ( i ) bocnh2 , pd2dba3 , xantphos , cs2co3 , 1,4-dioxane , 80 c ; ( ii ) boc2o , dmap , dmf .", "most of the d - ring precursors were lithiated with lithium diisopropylamide ( lda ) at 78 c , followed by treatment with the enone 8 .", "warming to 20 c yielded the fully protected tetracycline precursors 19 . for d - ring precursors 16a , 16d , and 18b , a one - pot reaction was performed in which lithium bis(trimethylsilyl)amide ( lhmds ) was added to a 78 c solution of the precursor and the enone 8 followed by warming to 20 c .", "yields varied from 20 to 84% depending on substrate , with larger reaction scales generally providing better results .", "the lhmds procedure also typically gave higher yields but was only possible on the more highly electron deficient d - ring precursors .", "the 7-bromo intermediate 19c was further derivatized at this stage via suzuki couplings with methylboronic acid and phenylboronic acid to give 19d and 19e , respectively .", "similarly , palladium - mediated amination with 19h and t - butylcarbamate provided the 7-chloro-9-amino compound 19j .", "deprotection of the intermediates 19 with hf followed by hydrogenation in the presence of palladium on carbon gave the final 8-azatetracyclines 20ak .", "reagents and conditions : ( i ) for 14 , 15bd , 16e , and 18a , lda , tmeda , 78 c , thf then 8 , 78 c to 20 c ; ( ii ) for 16a , 16d , and 18b , lhmds , 8 , thf , 78", "c to 20 c ; ( iii ) ch3b(oh)2 or phb(oh)2 , pdcl2dppf2-ch2cl2 , k3po4 , toluene , 1,4-dioxane , water , 100 c ; ( iv ) bocnh2 , pd2dba3 , xantphos , k3po4 , 1,4-dioxane , 100 c ; ( v ) aqueous hf , ch3cn or 1,4-dioxane ; ( vi ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane . to compare the 8-azatetracyclines directly to tigecycline , the 9-glycylamido-8-azatetracyclines 21ac were prepared ( scheme 6 ) .", "treatment with bromoacetylchloride in thf followed by excess t - butylamine gave the acylated intermediates , which were deprotected under standard conditions to give the final analogues 21ac .", "reagents and conditions : ( i ) hcl , 1,4-dioxane ; ( ii ) bromoacetylchloride , thf then t - bunh2 , 50 c ; ( iii ) aqueous hf , ch3cn or 1,4-dioxane ; ( iv ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane .", "the bromopyridine intermediates proved to be useful for the introduction of alkylamines via palladium - mediated aminations and alkyl groups via suzuki couplings , ultimately providing the 9-alkylamino-8-azatetracyclines 24 and 9-alkyl-8-azatetracyclines 26 ( scheme 7 ) .", "thus , 19h was treated with various amines in the presence of pd2dba3 and 9,9-dimethyl-4,5-bis-(diphenylphosphino)xanthenes ( xantphos ) in 1,4-dioxane at 100 c .", "potassium phosphate was found to be the most effective base for this transformation , with cesium carbonate and sodium t - butoxide leading to rapid decomposition .", "low yields were generally observed due to decomposition of both the starting material and products upon heating .", "reaction times of 24 h were eventually settled on as a balance between product formation and decomposition .", "copper - catalyzed aminations were also explored , but rapid decomposition of the starting material was observed .", "hindered amines generally gave low yields as did reactions with the 7-dimethylamino compound 19 m .", "alternatively , the alkylamines could be introduced prior to the michaeldieckmann reaction using either palladium or copper - mediated aminations(22 ) with 16a , 16d , and 16e .", "protection of the resulting aminopyridines upon treatment with lhmds followed by boc2o or cbzcl gave the d - ring precursors 22 .", "suzuki reactions with 19h or 19 m and various boronic acids were also carried out in moderate yields , providing the 9-alkyl intermediates 25 .", "reagents and conditions : ( i ) r2r3nh2 , pd2dba3 , xantphos , k3po4 or cs2co3 , 1,4-dioxane , 100 c or n - propylamine , cui , 2-acetylcyclohexanone , k3po4 , dmf , 100 c ; ( ii ) lhmds , thf then boc2o or cbzcl ; ( iii ) lda , tmeda , 78 c , thf then 8 , 78 c to 20 c or lhmds , 8 , thf , 78", "c to 20 c ; ( iv ) aqueous hf , ch3cn , or 1,4-dioxane ; ( v ) 10% pd / c , h2 , hcl , meoh , 1,4-dioxane ; ( vi ) r2b(oh)2 , ( ph3p)4pd , k3po4 , toluene , 1,4-dioxane , h2o , 90 c . the in vitro antibacterial activities of all analogues were determined for a panel of gram - positive ( e.g. , staphylococcus aureus and streptococcus pneumoniae ) and gram - negative ( e.g. , escherichia coli and klebsiella pneumoniae ) bacteria .", "the activities of the 7-substituted-8-azatetracycline as well as several control tetracyclines for a representative set of these organisms are found in table 1 .", "the parent 8-azatetracycline 20a showed comparable activity to tetracycline , with reasonable minimum inhibitory concentrations ( mics ) of 0.251 g / ml for the tetracycline - susceptible strains . lacking a substituent at either c-7 or c-9", ", 20a demonstrated poor activity for the strains bearing tet(m ) , tet(k ) , and tet(a ) .", "overall , the 7-position appears to be very tolerant of substitution , allowing for bulky ( 20e ) , electron withdrawing ( 20b and 20i ) , and electron donating ( 20f and 20 g ) groups while maintaining activity against the tetracycline - susceptible strains . as with minocycline , introduction of the 7-dimethylamino group in 20f led to improved activities for most strains tested .", "modest ( 12 dilution ) improvements in mics were observed for the tet(m ) strains , while a more significant improvement was seen for the strain bearing the tet(k ) resistance mechanism . with the exception of the 7-chloro analogue , all of the 7-substituted-8-aza compounds exhibited improved mics for the tet(k ) strain , correlating well with literature reports on 7-substituted tetracyclines .", "unfortunately , all of the 8-azatetracyclines appear to be substrates for the tet(a ) efflux pump , exhibiting no antibacterial activity at the highest concentration tested ( 32 g / ml ) .", "in general , substitution at the 7-position appears to be inefficient at overcoming the ribosomal protection resistance mechanism of the tet(m ) strains .", "the only compound with two dilution improvements for both tet(m ) strains was compound 20i , the 7-fluoro analogue .", "obtained from marilyn roberts lab at the university of washington . to make a direct comparison to tigecycline , the 9-glycylamido-8-azatetracyclines were prepared ( table 2 ) .", "whereas tigecycline is a very potent antibacterial agent against all of the organisms in our panel , including the four strains bearing tetracycline resistance mechanisms , the corresponding 8-azatetracycline analogues 21ac were found to have poor overall activity .", "the aniline precursors 20j and 20k , however , showed significant improvements in mics for both tet(m ) strains relative to the corresponding 9-unsubstituted compounds 20b and 20i .", "for the 7-cl analogue 20j , antibacterial activity also improved for the tet(k ) and wild type strains .", "these compounds were also assessed for their ability to inhibit protein synthesis in a cell - free transcription / translation assay ( table 3 ) . in this assay ,", "compounds 20j and 20k were about 2-fold more potent than tetracycline , in agreement with their relative mics for e. coli .", "compounds 21ac , on the other hand , inhibited protein synthesis with ic50s between 0.42 and 0.90 g / ml , similar to tigecycline , despite having only modest antibacterial activity in the whole - cell assays .", "these data suggest that the compounds are active at the target , but that compounds 21ac are not capable of getting to the target in the whole - cell assay , possibly due to poor cell permeability .", "atcc 25922 is a tetracycline - susceptible strain . on the basis of the results of compounds 20j and 20k ,", "compounds bearing a small , unbranched alkylamino group at the 9-position ( 24ac ) proved to be very potent antibacterial agents against the two tetracycline - susceptible gram - positive strains , with mics between 0.016 and 0.25 g / ml . a 4-fold improvement in activity against the tet(m ) s. aureus strain", "was observed for all of these compounds relative to 20j , while mics for the s. pneumoniae tet(m ) strain were largely unchanged . for the tet(k )", "s. aureus strain , antibacterial activity improved with increasing size of the alkyl group . among the tetracycline - susceptible gram - negative strains ,", "however , there was a clear loss in potency as the size of the alkyl group increased , a trend that continued for the larger branched alkyl compounds 24df .", "this is not surprising , as it is known that gram - negative antibacterial agents tend to be smaller and more polar.(23 ) these larger side chains also adversely affected the antibacterial activity for s. pneumoniae while having less effect on the mics for the s. aureus strains .", "the 7-fluoro-9-n - propylamino ( 24n ) and 7-dimethylamino-9-n - propylamino ( 24o ) analogues were prepared as comparisons to 24c . while the 7-fluoro compound had mics that tracked fairly closely to the 7-chloro analogue , the 7-dimethylamino compound exhibited no antibacterial activity for any of the strains at the highest concentration tested ( 32 g / ml ) with the exception of the tetracycline - susceptible s. pneumoniae strain .", "all three of the compounds had similar , albeit weak , activity in the transcription / translation assay , indicating the differences in mics might be due to poor permeability of the 7-dimethylamino analogue .", "introduction of a more polar heteroatom into the side chain ( 24gi , k ) resulted in improved antibacterial activity against the s. pneumoniae and gram - negative strains but had minimal effect on activity toward either resistant s. aureus organisms .", "interestingly , the oxygen bearing side chains ( 24 g and 24h ) yielded significantly improved antibacterial activity toward tetracycline - susceptible gram - positive strains relative to the all carbon side chains ( 24e and 24f ) , while the nitrogen analogues ( 24k and 24i ) were less potent against s. aureus and showed more modest improvements against s. pneumoniae .", "the combination of a branched side chain with a distal amino group gave the most balanced antibacterial compounds in this series ( 24l , 24 m , and 24j ) .", "these compounds combine good antibacterial activity toward both gram - positive and gram - negative organisms , with modest activity against tet(a ) e. coli . both compounds 24l and 24 m are less potent than tigecycline but compare favorably to amadacycline .", "the antibacterial activity of the 9-alkyl and phenyl substituted compounds were generally modest ( table 5 ) . here again , a larger substituent at c-9 yielded improved activity for tet(m ) and tet(k ) strains but resulted in loss of activity for wild type s. pneumoniae and gram - negative strains .", "introduction of a 7-cl or 7-dimethylamino group also gave improved activity for most of the strains in the panel .", "when comparing the 7-cl-9-alkyl compounds ( 26b ) to the 7-cl-9-aminoalkyl compounds ( 24c ) , it is clear that the nitrogen confers significant improvements in antibacterial activity . in the 7-dimethylamino case , however , the 7-dimethylamino-9-alkyl compound 26a is a balanced gram - positive antibacterial agent while the 9-aminoalkyl compound 24o has no significant activity .", "were selected for further profiling . both compounds inhibited protein synthesis in the transcription / translation assay with ic50s comparable to tigecycline .", "susceptibility testing was performed , and mic50 and mic90 values were calculated for recent clinical isolates of s. aureus , s. pneumoniae , haemophilus influenzae , and e. coli ( table 6 ) .", "the panels contained both tetracycline - susceptible and resistant strains , including 12 methicillin - resistant s. aureus ( mrsa ) strains . as expected from the initial mic data , compound 20f had very potent antibacterial activity for the tetracycline - susceptible strains but performed poorly against the resistant organisms .", "the compound did have mic90s that were significantly lower than tetracycline . despite having higher mics in the primary screen relative to tigecycline , compound 24l performed similarly in the s. aureus , s. pneumoniae , and h. influenzae panels , exhibiting clinically relevant mic90s in each case .", "these organisms are particularly relevant , as they are responsible for infections in a large portion of community - acquired bacterial pneumonia cases .", "the pharmacokinetic profiles of the two 8-azatetracyclines were determined in spraguedawley rats ( table 7 ) .", "compound 20f had very similar auc , clearance , and volume of distribution profiles to both tetracycline and tigecycline , although the half - life was somewhat shorter .", "low oral bioavailability ( % f ) of 12.7% was observed but was comparable to tetracycline ( 12.1% ) . in humans , tetracycline is generally reported to have oral bioavailability of 5070%.(24 ) we and others have found the oral bioavailability of several tetracycline derivatives to be significantly lower in rat than reported data in humans .", "compound 24l had a 4-fold higher auc and 5-fold lower clearance than the other three compounds as well as a significantly lower volume of distribution .", "compounds were dosed at 1 mg / kg iv and 10 mg / kg po with sterile water as vehicle .", "the compounds were also investigated in mouse s. aureus and e. coli septicemia models ( table 8) . both 8-azatetracyclines performed well in the s. aureus model following iv administration , with pd50 values comparable to the two marketed control tetracyclines .", "20f , the most potent of the four compounds , also provided the best protection , with 100% survival at the lowest dose at which it was administered ( 0.30 mg / kg ) . despite having an", "8-fold higher mic , compound 24l provided equivalent protection relative to tigecycline , likely due to its higher auc and lower clearance.(25 ) the e. coli model proved to be more challenging , requiring higher doses for protection .", "compound 20f had a pd50 of 4.3 mg / kg compared to 2.1 mg / kg for tigecycline .", "compound 24l was 4-fold less potent than 20f and was comparable to tetracycline in this model .", "s. aureus atcc 13709 ( smith ) or e. coli atcc 25922 was mixed with 5% mucin and inoculated by intraperitoneal injection at 2.1 10 cfu / mouse ( s. aureus ) or 2.0 10 cfu / mouse ( e. coli ) .", "one hour postchallenge , mice received iv treatment ( vehicle = sterile water ) with the test article at concentrations ranging from 0.0510 mg / kg ( s. aureus ) or 0.3030 mg / kg ( e. coli ) .", "the pd50 in mg / kg was calculated as survival after 48 h. lowest dose was 0.30 mg / kg with 100% survival at all doses ." ], [ "a novel , fully synthetic series of 8-azatetracycline analogues was prepared and optimized . through modification of the 7- and 9-positions , compounds were found that overcome both tet(k ) efflux and tet(m)-mediated ribosomal protection .", "the 7-chloro-9-alkylamino-8-azatetracycline 24l exhibits clinically relevant mic90 profiles against both gram - positive and gram - negative organisms that include these resistance mechanisms .", "this compound also demonstrated in vivo efficacy in murine models of infection that is equivalent to currently marketed tetracycline antibacterial agents .", "the 7-dimethylamino-8-azatetracycline 20f was efficacious when administered intravenously and had oral bioavailability in rat equivalent to tetracycline .", "the current work demonstrates the ability of this chemistry platform to generate antibacterial compounds that are inaccessible through traditional semisynthesis , providing the medicinal chemist a broader array of tools through which essential antibacterial properties can be modulated .", "importantly , this includes the ability to overcome ever evolving resistance mechanisms and to identify structureactivity relationships to deliver compounds with desired pharmacokinetic properties ." ], [ "air and moisture sensitive liquids and reagents were transferred via syringe or cannula and were introduced into flame - dried glassware under a positive pressure of dry nitrogen through rubber septa .", "analytical thin - layer chromatography was performed on em science precoated glass - backed silica gel 60 f-254 250 m plates .", "visualization of the plates was effected by ultraviolet illumination and/or immersion of the plate in a basic solution of potassium permanganate in water followed by heating .", "column chromatography was performed on a flashmaster personal system using isolute flash si ii silica gel prepacked cartridges ( available from biotage ) .", "preparative reversed - phase hplc chromatography ( hplc ) was accomplished using a waters autopurification system with mass - directed fraction collection .", "all intermediate compounds were purified with a waters sunfire prep c18 obd column ( 5 m , 19 mm 50 mm ; flow rate = 20 ml / min ) using a mobile phase mixture of h2o ( a ) and ch3cn ( b ) containing 0.1% hco2h .", "the final 8-azatetracycline compounds were purified using a phenomenex polymerx 10 rp 100a column ( 10 m , 30 mm 21.2 mm ; flow rate = 20 ml / min ) using a mobile phase mixture of 0.05n hcl in h2o ( a ) and ch3cn ( b ) .", "all final 8-azatetracycline compounds were isolated as mono- , di- , or trihydrochloride salts following freeze - drying .", "h nmr spectra were recorded on a jeol ecx-400 ( 400 mhz ) spectrometer and are reported in ppm using residual solvent as the internal standard ( cdcl3 at 7.24 ppm , dmso - d6 at 2.50 ppm , or cd3od at 3.31 ppm ) .", "high performance liquid chromatographyelectrospray mass spectra ( lc - ms ) were obtained using a waters alliance hplc system equipped with a binary pump , a diode array detector , a waters sunfire c18 ( 5 m , 4.6 mm i.d .", " 50 mm ) column , and a waters 3100 series mass spectrometer with electrospray ionization .", "the eluent was a mixture of h2o ( a ) and ch3cn ( b ) containing 0.1% hco2h at a flow rate of 1 ml / min .", "purity of the final compounds was assessed by the following method : ( a ) time = 0 , 100% a ; ( b ) time = 0.5 min , 100% a ; ( c ) time = 3.5 min , 100% b ; ( d ) time = 5 min , 100% b ( e ) time = 6 min , 100% a ; ( f ) time = 7 min , 100% a. all final products were 95% purity as assessed by this method .", "sodium hydride ( 60% dispersion in mineral oil , 4.37 g , 109 mmol ) was added portionwise to a solution of 3,5-dichloroisonicotinic acid ( 10.2 g , 53.3 mmol ) in nmp ( 100 ml ) .", "after gas evolution ceased , benzyl alcohol ( 5.52 ml , 53.3 mmol ) was added dropwise .", "after 1 h , the reaction was complete and was allowed to cool to room temperature and stand overnight .", "the reaction mixture was diluted with water ( 300 ml ) and was washed with et2o ( 2 100 ml , discarded ) .", "the aqueous layer was brought to ph 2 with conc hcl , causing a precipitate to form .", "the mixture was diluted with brine ( 100 ml ) and was allowed to stand for 30 min .", "the solid was collected by filtration , was washed with water ( 3 ) , and was dried in a 45 c vacuum oven overnight .", "this gave 9.36 g ( 67% ) of the title compound as a white solid .", "h nmr ( 400 mhz , dmso - d6 ) 14.3014.10 ( bs , 1 h ) , 8.52 ( s , 1h ) , 8.34 ( s , 1 h ) , 7.507.30 ( m , 5 h ) , 5.33 ( s , 2 h ) .", "oxalyl chloride ( 4.9 ml , 56 mmol ) was added to a suspension of 12 ( 3.71 g , 14.1 mmol ) in ch2cl2 ( 50 ml ) over 2 min .", "dmf was added dropwise in 20 l portions every 5 min until complete solution was achieved . after stirring for an additional 30 min ,", "the resulting solid was dissolved in ch2cl2 ( 50 ml ) , and phenol ( 2.65 g , 28.1 mmol ) , dmap ( 0.172 g , 1.41 mmol ) , and et3n ( 9.76 ml , 70.4 mmol ) were added .", "after 1 h , the reaction mixture was diluted with ch2cl2 ( 50 ml ) and was washed with water ( 2 100 ml ) and nahco3 ( saturated , aqueous , 100 ml ) .", "the material was purified by column chromatography ( biotage 50 g column , 025% etoac in hexanes gradient ) , yielding 4.20 g ( 88% ) of the product as an off - white solid .", "h nmr ( 400 mhz , dmso - d6 ) 8.70 ( s , 1h ) , 8.48 ( s , 1 h ) , 7.527.30 ( m , 8 h ) , 7.157.08 ( m , 2 h ) , 5.44 ( s , 2 h ) .", "compound 13 ( 2.01 g , 5.92 mmol ) , methyl boronic acid ( 1.06 g , 17.7 mmol ) , dichlorobis(tricyclohexylphosphine)palladium(ii ) ( 102 mg , 0.296 mmol ) , and k3po4 ( 3.76 g , 17.7 mmol ) were heated to 100 c in toluene ( 20 ml ) and water ( 2 ml ) . after 16 h", ", the reaction mixture was allowed to cool to room temperature and was diluted with etoac ( 20 ml ) .", "this was washed with water ( 20 ml ) and brine ( 20 ml ) , dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 040% etoac in hexanes gradient ) , yielding 1.66 g ( 88% ) of the product as a white solid .", "h nmr ( 400 mhz , dmso - d6 ) 8.50 ( s , 1h ) , 8.26 ( s , 1 h ) , 7.527.30 ( m , 8 h ) , 7.207.10 ( m , 2 h ) , 5.37 ( s , 2 h ) , 2.38 ( s , 3 h ) . ms ( esi ) m / z 320.27 ( m + h ) .", "compound 14 ( 9.66 g , 30.3 mmol ) was heated to 80 c in acetic acid ( 50 ml ) and hydrogen peroxide ( 30% aqueous solution , 17 ml ) .", "after 6 h , lc / ms indicated that the starting material was consumed and the n - oxide present .", "most of the acetic acid and water were removed under reduced pressure . the material was then diluted with etoac ( 200 ml ) , and the mixture was washed with nahco3 ( saturated aqueous solution , 3 50 ml ) .", "the organics were dried over na2so4 , filtered , and concentrated under reduced pressure , yielding 9.37 g ( 92% ) of the crude n - oxide .", "the material was dissolved in toluene ( 60 ml ) , phosphorousoxychloride ( 7.69 g , 50.1 mmol ) was added , and the reaction mixture was heated to 100 c .", "after 2 h , lc / ms indicated that the reaction was complete . upon cooling to room temperature", ", the reaction mixture was poured into a solution of k2co3 ( 40 g ) in water ( 200 ml ) .", "this mixture was extracted with etoac ( 3 150 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was redissolved in etoac ( 30 ml ) with heating and was then allowed to cool to room temperature and stand overnight .", "another 1.92 g ( 19% ) of the white crystalline solid was obtained from the mother liquor after standing in a refrigerator overnight .", "h nmr ( 400 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) ,", "7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) . compound 14 ( 25.9 g , 81.1 mmol ) was heated to 80 c in acetic acid ( 160 ml ) and hydrogen peroxide ( 30% aqueous solution , 40 ml ) .", "after stirring overnight , lc / ms indicated that the starting material was consumed and the n - oxide present .", "the reaction mixture was concentrated under reduced pressure , was diluted with etoac , and was washed with nahco3 ( saturated aqueous solution , 3 ) .", "the organics were dried over na2so4 , filtered , and concentrated under reduced pressure , yielding 25.4 g ( 93% crude ) of the n - oxide as a tan solid .", "the material was suspended in toluene ( 200 ml ) and was heated to 80 c , at which point most of the solid had dissolved .", "upon cooling to room temperature , the reaction mixture was poured into a solution of k2co3 ( 83 g ) in water ( 300 ml ) .", "the combined extracts were dried over mgso4 , filtered through celite , and concentrated under reduced pressure .", "the material was recrystallized from etoac / hexanes ( 1:1 ) , yielding 20.6 g ( 64% ) of the product as an off - white solid .", "h nmr ( 400 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) . compound 15b ( 1.06 g , 2.66 mmol ) , dimethylamine ( 2.0 m solution in thf , 4.0 ml , 8.0 mmol ) , k3po4 ( 1.7 g , 8.0 mmol ) , tris(dibenzylideneacetone)dipalladium ( 120 mg , 0.13 mmol ) , and xantphos ( 220 mg , 0.40 mmol ) were heated to 100 c in 1,4-dioxane ( 10 ml ) in a sealed pressure vessel .", "after heating overnight , the reaction mixture was cooled to room temperature , was diluted with etoac ( 100 ml ) , and was washed with water ( 3 50 ml ) and brine ( 50 ml ) .", "the material was purified by column chromatography ( biotage 50 g column , 015% etoac in hexanes gradient ) , yielding 388 mg ( 40% ) of the title compound as a white solid .", "h nmr ( 400 mhz , cdcl3 ) 7.96 ( s , 1h ) , 7.457.20 ( m , 8 h ) , 7.08 ( d , j = 8.2 hz , 2 h ) , 5.16 ( s , 2 h ) , 2.76 ( s , 6 h ) , 2.37 ( s , 3 h ) . ms ( esi ) m / z 363.45 ( m + h ) . sodium methoxide ( 38 mg , 0.70 mmol ) was added to a solution of compound 15b ( 118 mg , 0.351 mmol ) in nmp ( 2 ml ) , and the reaction mixture was heated to 100 c . after heating overnight", ", additional sodium methoxide ( 57 mg , 1.1 mmol ) was added and heating was continued at 100 c .", "after an additional 1 h , the reaction mixture was cooled to room temperature , water ( 20 ml ) was added , and the ph was adjusted to 2 with 6 n aqueous hcl .", "this was extracted with etoac ( 3 20 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the crude acid intermediate was dissolved in ch2cl2 ( 10 ml ) , and oxalyl chloride ( 0.123 ml , 1.40 mmol ) was added .", "the material was dissolved in ch2cl2 ( 10 ml ) , and phenol ( 66 mg , 0.70 mmol ) , dmap ( 4 mg , 0.04 mmol ) , and et3n ( 0.245 ml , 1.76 mmol ) were added .", "after 1 h , the reaction mixture was diluted with etoac ( 50 ml ) and was washed with water ( 2 30 ml ) and brine ( 30 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 20.3 mg ( 17% ) of clean product .", "h nmr ( 400 mhz , cdcl3 ) 7.75 ( s , 1h ) , 7.457.24 ( m , 9 h ) , 7.10 ( d , j = 7.3 hz , 2 h ) , 5.13 ( s , 2 h ) , 3.91 ( s , 3 h ) , 2.67 ( s , 3 h ) . ms ( esi ) m / z 336.1 , 338.1 ( m + h ) .", "compound 15a ( 4.99 g , 14.1 mmol ) was dissolved in ch2cl2 ( 30 ml ) , and 3-chloroperbenzoic acid ( 77% , 6.33 g , 28.2 mmol ) was added in one portion . after 6 h , an additional portion of 3-chloroperbenzoic acid ( 3.15 g , 14.1 mmol ) was added , and the reaction was stirred at room temperature overnight .", "the reaction mixture was diluted with ch2cl2 ( 200 ml ) and was washed with na2co3 ( saturated aqueous solution , 100 ml ) and brine .", "the material was purified by column chromatography ( biotage 70 g column , 550% etoac in hexanes gradient ) , yielding 3.90 g ( 75% ) of the pure n - oxide as an off - white solid ( rf = 0.33 in 66% etoac / hexanes ) .", "the n - oxide was dissolved in toluene ( 30 ml ) , phosphorousoxybromide ( 6.36 g , 22.1 mmol ) was added , and the reaction mixture was heated to 80 c .", "after 1 h , the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 ( 10 g ) in water ( 50 ml ) .", "this mixture was extracted with etoac ( 3 100 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 010% etoac in hexanes gradient ) , yielding 2.06 g ( 45% ) of the desired product 16a as an oil which slowly solidified on standing overnight .", "h nmr ( 400 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.437.30 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 7.07 ( d , j = 8.2 hz , 2 h ) , 5.21 ( s , 2 h ) , 2.46 ( s , 3 h ) . ms ( esi ) m / z 398.22 , 400.22 ( m + h ) .", "compound 15b ( 8.54 g , 21.4 mmol ) was heated to 80 c in acetic acid ( 120 ml ) and hydrogen peroxide ( 30% aqueous solution , 30 ml ) . after stirring overnight ,", "lc / ms indicated that the starting material was mostly consumed and the n - oxide present .", "the reaction mixture was cooled to room temperature and was diluted with brine ( 150 ml ) .", "the resulting precipitate was collected by filtration and was washed with water / brine ( 1:1 , 2 ) .", "the solid was dissolved in ch2cl2 and was dried over na2so4 , filtered , and concentrated under reduced pressure .", "this gave 7.83 g ( 88% crude ) of the n - oxide as a tan solid .", "the material was suspended in toluene ( 200 ml ) and was heated to 100 c , at which point most of the solid had dissolved .", "a solution of phosphorousoxybromide ( 10.8 g , 37.8 mmol ) in toluene ( 10 ml ) was added rapidly .", "after 45 min , the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 ( 40 g ) in water ( 150 ml ) . after stirring for 30 min at room temperature ,", "the combined extracts were dried over mgso4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 08% etoac in hexanes gradient to isolate the product then 15% etoac in hexanes to recover 15b ) , yielding 5.02 g ( 49% ) of the product as an off - white solid .", "h nmr ( 400 mhz , dmso - d6 ) 7.497.32 ( m , 8 h ) , 7.10 ( d , j = 7.3 hz , 2 h ) , 5.15 ( s , 2 h ) , 2.41 ( s , 3 h ) . ms ( esi ) m / z 476.18 , 478.16 , 480.16 ( m + h ) .", "compound 16b ( 2.15 g , 4.50 mmol ) , t - butylcarbamate ( 633 mg , 5.40 mmol ) , cs2co3 ( 2.93 g , 9.00 mmol ) , tris(dibenzylideneacetone)dipalladium ( 206 mg , 0.225 mmol ) , and xantphos ( 380 mg , 0.673 mmol ) were weighed into a flask .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 15 ml ) was added .", "the reaction mixture was cooled to room temperature , was diluted with etoac ( 100 ml ) , and was washed with water ( 2 50 ml ) .", "the material was purified by column chromatography ( biotage 50 g column , 012% etoac in hexanes gradient ) , yielding 1.43 g ( 62% ) of a 4:1 mixture of the two regioisomeric compounds .", "h nmr ( 400 mhz , cdcl3 ) 7.517.46 ( m , 2 h ) , 7.427.32 ( m , 7 h ) , 7.307.24 ( m , 1 h ) , 7.12 ( d , j = 8.2 hz , 0.5 h ) , 7.03 ( d , j = 8.2 hz , 2 h ) , 6.88 ( s , 0.25 h ) , 6.71 ( s , 1 h ) , 5.12 ( s , 2 h ) , 5.02 ( s , 0.5 h ) , 2.43 ( s , 0.75 h ) , 2.34 ( s , 3 h ) , 1.50 ( s , 9 h ) , 1.47 ( s , 2.25 h ) . ms ( esi ) m / z 535.10 , 537.10 ( m + na ) . the above regioisomeric mixture ( 1.43 g , 2.78 mmol ) was stirred in 4 m hcl in 1,4-dioxane ( 20 ml ) and 1,4-dioxane ( 5 ml ) overnight .", "the reaction mixture was diluted with etoac ( 100 ml ) and was washed with nahco3 ( saturated aqueous solution , 2 100 ml ) .", "the material was purified by column chromatography ( biotage 20 g column , 030% etoac in hexanes gradient ) , yielding 805 mg ( 70% ) of the major regioisomer 16c and 270 mg ( 24% ) of the minor regioisomer .", "h nmr ( 400 mhz , cdcl3 ) 7.507.42 ( m , 2 h ) , 7.407.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.04 ( d , j = 8.2 hz , 2 h ) , 5.06 ( s , 2 h ) , 4.56 ( s , 2 h ) , 2.16 ( s , 3 h ) . ms ( esi ) m / z 413.11 , 415.01 ( m + h ) .", "nmr ( 400 mhz , cdcl3 ) 7.427.34 ( m , 8 h ) , 7.137.10 ( m , 2 h ) , 5.01 ( s , 2 h ) , 4.64 ( s , 2 h ) , 2.35 ( s , 3 h ) ; ms ( esi ) m / z 413.09 , 415.09 ( m + h ) .", "hf - pyridine solution ( 70% hf , 2 ml ) was added to a 0 c solution of compound 16c ( 884 mg , 2.14 mmol ) in pyridine ( 1 ml ) .", "sodium nitrite ( 177 mg , 2.57 mmol ) was added ( bubbling observed ) , and the reaction mixture was allowed to slowly warm to room temperature . after 3 days , the reaction mixture was poured into na2co3 ( saturated aqueous solution , 30 ml ) and was extracted with etoac ( 3 30 ml ) .", "the combined extracts were washed with hcl ( 1 n aqueous solution , 2 50 ml ) and were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 20 g column , 06% etoac in hexanes gradient ) , yielding 687 mg ( 77% ) of the product as a colorless oil that slowly solidified on standing .", "h nmr ( 400 mhz , cdcl3 ) 7.507.42 ( m , 2 h ) , 7.407.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.14 ( s , 2 h ) , 2.33 ( s , 3 h ) . ms ( esi ) m / z 416.17 , 418.15 ( m + h ) .", "compound 16b ( 1.51 g , 3.16 mmol ) , cs2co3 ( 3.1 g , 9.5 mmol ) , tris(dibenzylideneacetone)dipalladium ( 145 mg , 0.158 mmol ) , and xantphos ( 267 mg , 0.474 mmol ) were weighed into a vial equipped with a septum .", "this was evacuated and flushed with nitrogen ( 3 ) , and 1,4-dioxane ( 7 ml ) and dimethylamine ( 2.0 m solution in thf , 4.75 ml , 9.50 mmol ) were added .", "the reaction mixture was heated to 60 c and was stirred for 4 h. the reaction mixture was allowed to cool to room temperature , was filtered through celite , and was concentrated under reduced pressure .", "the material was purified by column chromatography ( biotage 50 g column , 010% etoac in hexanes gradient ) , yielding 868 mg ( 58% ) of the major regioisomer 16e and 354 mg ( 24% ) of the minor regioisomer 16f .", "data for 16e : h nmr ( 400 mhz , cdcl3 ) 7.507.46 ( m , 2 h ) , 7.417.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.08 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.32 ( s , 3 h ) ; ms ( esi ) m / z 441.19 , 443.18", "( m + h ) ; rf = 0.29 in 10% etoac / hexanes . the regiochemistry was confirmed by an noe between the methyl protons and the n , n - dimethylamino protons ( 0.61% ) .", "data for 16f : h nmr ( 400 mhz , cdcl3 ) 7.417.31 ( m , 7 h ) , 7.307.20 ( m , 1 h ) , 7.01 ( d , j = 7.3 hz , 2 h ) , 4.94 ( s , 2 h ) , 3.04 ( s , 6 h ) , 2.33 ( s , 3 h ) ; ms ( esi ) m / z 441.19 , 443.18", "( m + h ) ; rf = 0.40 in 10% etoac / hexanes . the regiochemistry was confirmed by noe between the benzylic protons of the o - benzyl group and the n , n - dimethylamino protons ( 0.44% ) .", "compound 16d ( 538 mg , 1.29 mmol ) , t - butylcarbamate ( 302 mg , 2.58 mmol ) , cs2co3 ( 840 mg , 2.58 mmol ) , tris(dibenzylideneacetone)dipalladium ( 59 mg , 0.065 mmol ) , and xantphos ( 109 mg , 0.190 mmol ) were weighed into a flask .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 3 ml ) was added .", "after 4 h , the reaction mixture was cooled to room temperature , was diluted with etoac ( 50 ml ) , and was washed with water ( 2 25 ml ) .", "the material was purified by column chromatography ( biotage 20 g column , 012% etoac in hexanes gradient ) , yielding 451 mg ( 77% ) of the product .", "rf = 0.35 in 20% etoac / hexanes . h nmr ( 400 mhz , cdcl3 ) 7.427.24 ( m , 8 h ) , 7.046.99 ( m , 2 h ) , 4.99 ( s , 2 h ) , 2.38 ( s , 3 h ) , 1.40 ( s , 18 h ) .", "compound 17a ( 451 mg , 1.00 mmol ) was treated with di - t - butyldicarbonate ( 1.09 g , 5.00 mmol ) and dmap ( 24 mg , 0.20 mmol ) in dmf ( 15 ml ) .", "after 30 min , the reaction mixture was diluted with etoac ( 100 ml ) and was washed with water ( 3 50 ml ) and brine ( 50 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 08% etoac in hexanes gradient ) , yielding 481 mg ( 87% ) of the title compound .", "h nmr ( 400 mhz , cdcl3 ) 7.427.24 ( m , 8 h ) , 7.046.99 ( m , 2 h ) , 4.99 ( s , 2 h ) , 2.38 ( s , 3 h ) , 1.40 ( s , 18 h ) .", "compound 16e ( 467 mg , 1.06 mmol ) , t - butylcarbamate ( 372 mg , 3.17 mmol ) , cs2co3 ( 1.04 g , 3.17 mmol ) , tris(dibenzylideneacetone)dipalladium ( 48 mg , 0.053 mmol ) , and xantphos ( 89.3 mg , 0.159 mmol ) were weighed into a vial .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 3 ml ) was added .", "after 4 h , the reaction mixture was cooled to room temperature and was filtered through celite .", "the filtrate was concentrated under reduced pressure , and the material was purified by column chromatography ( biotage 20 g column , 016% etoac in hexanes gradient ) , yielding 493 mg ( 98% ) of the product containing 20% of an unidentified impurity .", "h nmr ( 400 mhz , cdcl3 ) 7.437.32 ( m , 8 h ) , 7.307.24 ( m , 1 h ) , 7.157.10 ( m , 2 h ) , 6.80 ( s , 1 h ) , 4.94 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.31 ( s , 3 h ) , 1.48 ( s , 9 h ) . ms ( esi ) m / z 478.27 ( m + h ) .", "compound 17b ( 490 mg , 1.03 mmol ) was treated with di - t - butyldicarbonate ( 672 mg , 3.08 mmol ) and dmap ( 12.5 mg , 0.103 mmol ) in dmf ( 5 ml ) .", "after stirring overnight , the reaction mixture was diluted with etoac ( 25 ml ) and was washed with water ( 3 20 ml ) and brine ( 20 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 010% etoac in hexanes gradient ) , yielding 424 mg ( 72% ) of the product .", "h nmr ( 400 mhz , cdcl3 ) 7.427.24 ( m , 8 h ) , 7.067.00 ( m , 2 h ) , 4.92 ( s , 2 h ) , 2.79 ( s , 6 h ) , 2.36 ( s , 3 h ) , 1.39 ( s , 18 h ) . ms ( esi ) m / z 578.33 ( m + h ) .", "lda was prepared at 78 c from n - butyllithium ( 1.6 m solution in hexane , 0.495 ml , 0.792 mmol ) and diisopropylamine ( 0.112 ml , 0.792 mmol ) in thf ( 5 ml ) .", "tmeda ( 0.318 ml , 2.11 mmol ) was added , followed by dropwise addition of a solution of compound 14 ( 169 mg , 0.529 mmol ) in thf ( 2 ml ) .", "after 5 min , a solution of 8 ( 128 mg , 0.264 mmol ) in thf ( 2 ml ) was added .", "after complete addition , the reaction mixture was allowed to warm to 20 c over 1 h. the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution , 20 ml ) and was extracted with etoac ( 2 20 ml ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 65.5 mg ( 35% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.77 ( s , 1 h ) , 8.36 ( s , 1 h ) , 8.16 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.405.25 ( m , 4 h ) , 3.93 ( d , j = 11.0 hz , 1 h ) , 3.163.04 ( m , 1 h ) , 2.982.90 ( m , 1 h ) , 2.762.64 ( m , 1 h ) , 2.602.40 ( m , 8 h ) , 2.12 ( d , j = 14 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 708.72 ( m + h ) .", "the following compounds were synthesized by methods similar to 19a . prepared from 15a in 38% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.6 ( s , 1 h ) , 8.12 ( s , 1 h ) , 7.567.49 ( m , 4 h ) , 7.427.32 ( m , 6 h ) , 5.37 ( s , 2 h ) , 5.07 ( s , 2 h ) , 3.91 ( d , j = 11.0 hz , 1 h ) , 3.133.02 ( m , 1 h ) , 2.97 ( dd , j = 15.6 4.9 hz , 1 h ) , 2.652.45 ( m , 3 h ) , 2.50 ( s , 6 h ) , 2.14 ( d , j = 15.6 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.27 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 742.56 ( m + h ) . prepared from 15b in 71% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.55 ( s , 1 h ) , 8.12 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.405.22 ( m , 4 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.25 ( dd , j = 16.5 hz , j = 4.88 hz , 1 h ) , 3.123.02 ( m , 1 h ) , 2.622.42 ( m , 9 h ) , 2.14 ( d , j = 14.0 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 786.63 , 788.63 ( m + h ) . prepared from 15c in 50% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.65 ( s , 1 h ) , 8.01 ( s , 1 h ) , 7.527.22 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 11.0 hz , 2 h ) , 4.02 ( d , j = 11.0 hz , 1 h ) , 3.05 ( dd , j = 17.2 hz , j = 11.6 hz , 1 h ) , 2.982.86 ( m , 1 h ) , 2.73 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.12 ( d , j = 13.4 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 751.79 ( m + h ) . prepared from 15d in 27% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.70 ( s , 1 h ) , 7.83 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 13.4 hz , 2 h ) , 3.98 ( d , j = 11.0 hz , 1 h ) , 3.72 ( s , 3 h ) , 3.18 ( d , j = 16.5 hz , 1 h ) , 3.04 2.96 ( m , 1 h ) , 2.602.30 ( m , 9 h ) , 2.15 ( d , j = 14 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 738.66 ( m + h ) .", "h nmr ( 400 mhz , cdcl3 ) 15.59 ( s , 1 h ) , 7.567.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.92 ( q , j = 67.8 hz , j = 9.16 hz , 2 h ) , 3.91 ( d , j = 11.0 hz , 1 h ) , 3.203.02 ( m , 2 h ) , 2.622.45 ( m , 9 h ) , 2.192.12 ( m , 1 h ) , 1.37 , ( s , 18 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 941.59 ( m + h ) . prepared from 16e in 54% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.65 ( s , 1 h ) , 7.527.22 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.17 ( q , j = 11.0 hz , 2 h ) , 4.01 ( d , j = 11.0 hz , 1 h ) , 3.05 ( dd , j = 17.2 hz , j = 11.6 hz , 1 h ) , 2.982.86 ( m , 1 h ) , 2.73 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.12 ( d , j = 13.4 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.13 ( s , 3 h ) . ms ( esi ) m / z 829.49 , 831.48 ( m + h ) . compound 16a ( 793 mg , 1.84 mmol ) and compound 8 ( 885 mg , 1.84 mmol ) were dissolved in thf ( 16 ml ) , and the solution was cooled to 78 c .", "lhmds ( 1.0 m solution in thf , 5.5 ml , 5.5 mmol ) was added slowly via syringe .", "after 10 min , the reaction mixture was allowed to slowly warm to 0 c over 1 h. a phosphate buffer solution ( ph 7.0 , 20 ml ) was added , followed by the addition of ammonium chloride ( saturated , aqueous solution , 50 ml ) .", "the resulting mixture was extracted with ch2cl2 ( 3 50 ml ) , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the resulting brown solid was washed with cold methanol ( 3 5 ml ) to afford 1.11 g ( 74% ) of the title compound as a yellowbrown powder .", "the organics were concentrated under reduced pressure and purified by column chromatography ( biotage 20 g column , 520% etoac in hexanes gradient ) , yielding an additional 150 mg ( 10% ) of the product .", "h nmr ( 400 mhz , cdcl3 ) 15.45 ( br , 1 h ) , 7.547.48 ( m , 4 h ) , 7.407.30 ( m , 6 h ) , 5.36 ( s , 2 h ) , 5.03 ( abq , j = 10.4 hz , 2 h ) , 3.87 ( d , j = 11.0 hz , 1 h ) , 3.273.23 ( m , 1 h ) , 3.103.00 ( m , 1 h ) , 2.652.57 ( m , 1 h ) , 2.572.43 ( m , 8 h ) , 2.16 ( d , j = 11.0 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 820.37 , 822.37 ( m + h ) .", "the following compounds were prepared by methods similar to 19h . prepared from 16d in 45% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.55 ( s , 1 h ) , 7.567.26 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.02 ( s , 2 h ) , 3.88 ( d , j = 10.4 hz , 1 h ) , 3.183.04 ( m , 2 h ) , 2.622.58 ( m , 1 h ) , 2.582.40 ( m , 8 h ) , 2.17 ( d , j = 14.6 hz , 1 h ) , 0.81 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 804.34 , 806.34 ( m + h ) . prepared from 18b in 53% yield , yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.50 ( s , 1 h ) , 7.527.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.85 ( q , j = 86.1 hz , j = 9.76 hz , 2 h ) , 4.02 ( d , j = 10.4 hz , 1 h ) , 3.083.00 ( m , 1 h ) , 3.002.82 ( m , 1 h ) , 2.77 ( s , 6 h ) , 2.622.38 ( m , 9 h ) , 2.202.12 ( m , 1 h ) , 1.35 , ( br s , 18 h ) , 0.79 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 966.59 ( m + h ) .", "a solution of compound 19c ( 52 mg , 0.067 mmol ) , methylboronic acid ( 40 mg , 0.67 mmol ) , dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii ) dichloromethane adduct ( 3 mg , 0.003 mmol ) , and k3po4 ( 142 mg , 0.667 mmol ) in toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.2 ml ) was heated to 70 c . after 2 h , the reaction mixture was heated to 100 c .", "after an additional 2 h , the reaction mixture was diluted with etoac ( 20 ml ) and was washed with water ( 15 ml ) and brine ( 15 ml ) .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 18.3 mg ( 38% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.71 ( s , 1 h ) , 8.24 ( s , 1 h ) , 7.557.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 5.305.20 ( m , 2 h ) , 3.95 ( d , j = 10.4 hz , 1 h ) , 3.102.92 ( m , 2 h ) , 2.622.42 ( m , 12 h ) , 2.12 ( d , j = 14.0 hz , 1 h ) , 0.82 ( s , 9 h ) , 0.26 ( s , 3 h ) , 0.14 ( s , 3 h ) . ms ( esi ) m / z 722.72 ( m + h ) . a solution of compound 19c ( 31 mg , 0.040 mmol ) , phenylboronic acid ( 24.5 mg , 0.201 mmol ) , dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii ) dichloromethane adduct ( 1.6 mg , 0.002 mmol ) , and sodium carbonate ( 21.3 mg , 0.201 mmol ) in toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.2 ml ) was heated to 100 c via microwave reactor for 10 min .", "the reaction mixture was diluted with etoac ( 10 ml ) and was washed with water ( 5 ml ) and brine ( 5 ml ) .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 16.9 mg ( 54% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.66 ( s , 1 h ) , 8.48 ( s , 1 h ) , 7.527.24 ( m , 15 h ) , 5.425.30 ( m , 4 h ) , 3.97 ( d , j = 10.4 hz , 1 h ) , 3.002.86 ( m , 2 h ) , 2.782.62 ( m , 1 h ) , 2.582.30 ( m , 8 h ) , 2.00 ( d , j = 14.0 hz , 1 h ) , 0.80 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.14 ( s , 3 h ) . ms ( esi ) m / z 784.75 ( m + h ) . compound 19h ( 100 mg , 0.122 mmol ) , t - butylcarbamate ( 42.9 mg , 0.366 mmol ) , k3po4 ( 77.7 mg , 0.366 mmol ) , tris(dibenzylideneacetone)dipalladium ( 5.6 mg , 0.006 mmol ) , and xantphos ( 10.3 mg , 0.018 mmol ) were weighed into a vial equipped with a septum .", "the vial was evacuated and flushed with nitrogen ( 3 ) , and 1,4-dioxane ( 0.5 ml ) was added .", "the reaction mixture was heated to 100 c and stirred for 2 h. the reaction mixture was allowed to cool to room temperature , was filtered through celite , and was concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 80100% b gradient ) , yielding 41.7 mg ( 40% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.6515.45 ( br s , 1 h ) , 7.567.50 ( m , 2 h ) , 7.437.34 ( m , 7 h ) , 7.297.20 ( m , 1 h ) , 5.38 ( s , 2 h ) , 4.94 ( dd , j = 29.3 hz , j = 11.0 hz , 2 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.27 ( dd , j = 16.4 hz , j = 11.6 hz , 1 h ) , 3.123.03 ( m , 1 h ) , 2.682.60 ( m , 1 h ) , 2.602.45 ( m , 8 h ) , 2.18 ( d , j = 14.6 hz , 1 h ) , 1.47 ( s , 9 h ) , 0.85 ( s , 9 h ) , 0.29 ( s , 3 h ) , 0.15 ( s , 3 h ) . ms ( esi ) m / z 857.67 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 19a ( 65.5 mg , 0.093 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water ( 20 ml ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 2 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 18.4 mg ( 41% , 2 steps ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.55 ( s , 1 h ) , 8.36 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.262.98 ( m , 9 h ) , 2.71 ( t , j = 14.2 hz , 1 h ) , 2.392.32 ( m , 1 h ) , 1.751.63 ( m , 1 h ) .", "the following compounds were synthesized by similar methods to 20a . prepared from 19b in 24% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 8.08 ( d , j = 6.0 hz , 1 h ) , 4.09 ( s , 1 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.70 1.58 ( m , 1 h ) . ms ( esi ) m / z 450.18 ( m + h ) . prepared from 19d in 34% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.35 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.252.95 ( m , 9 h ) , 2.802.25 ( m , 5 h ) , 1.801.63 ( m , 1 h ) .", "ms ( esi ) m / z 430.46 ( m + h ) . prepared from 19e in 24% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.53 ( s , 1 h ) , 7.62 ( s , 5 h ) , 4.18 ( s , 1 h ) , 3.142.82 ( m , 9 h ) , 2.812.66 ( m , 1 h ) , 2.242.14 ( m , 1 h ) , 1.661.52 ( m , 1 h ) .", "ms ( esi ) m / z 492.48 ( m + h ) . prepared from 19f in 96% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 8.22 ( s , 1 h ) , 4.19 ( s , 1 h ) , 3.383.30 ( m , 2 h ) , 3.24 ( s , 6 h ) , 3.122.95 ( m , 7 h ) , 2.60 ( t , j = 14.2 hz , 1 h ) , 2.422.34 ( m , 1 h ) , 1.751.63 ( m , 1 h ) .", "ms ( esi ) m / z 459.50 ( m + h ) . prepared from 19 g in 51% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 7.80 ( s , 1 h ) , 4.15 ( s , 1 h ) , 3.97 ( s , 3 h ) , 3.402.98 ( m , 9 h ) , 2.322.20 ( m , 2 h ) , 1.721.56 ( m , 1 h ) .", "ms ( esi ) m / z 446.39 ( m + h ) . prepared from 19i in 68% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 7.81 ( s , 1 h ) , 4.17 ( s , 1 h ) , 3.262.96 ( m , 9 h ) , 2.422.25 ( m , 2 h ) , 1.701.58 ( m , 1 h ) .", "ms ( esi ) m / z 434.27 ( m + h ) . prepared from 19j in 68% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.19 ( s , 1 h ) , 3.202.98 ( m , 8 h ) , 2.352.22 ( m , 2 h ) , 1.681.56 ( m , 1 h ) .", "ms ( esi ) m / z 465.32 ( m + h ) . prepared from 19k in 49% yield , 2 steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.20 ( s , 1 h ) , 3.402.90 ( m , 9 h ) , 2.422.22 ( m , 2 h ) , 1.701.54 ( m , 1 h ) .", "compound 19j ( 68.3 mg , 0.080 mmol ) was stirred in 4 m hcl in 1,4-dioxane for 2 h. the reaction mixture was concentrated under reduced pressure . the material was dissolved in thf ( 2 ml ) , and bromoacetylchloride ( 12.5 mg , 0.080 mmol ) was added . after stirring overnight , an additional portion of bromoacetylchloride ( 0.020 ml ) was added .", "t - butylamine ( 0.063 ml , 0.60 mmol ) was added to a solution of the intermediate ( 35 mg , 0.040 mmol ) in thf ( 1 ml ) , and the reaction mixture was heated to 50 c . after stirring overnight ,", "the reaction mixture was concentrated under reduced pressure and was purified by preparative hplc ( sunfire prep c18 column , 50100% b gradient ) .", "this gave 16.8 mg ( 49% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 8.20 ( br s , 1 h ) , 7.527.46 ( m , 2 h ) , 7.437.30 ( m , 8 h ) , 5.36 ( s , 2 h ) , 4.95 ( dd , j = 40.6 hz , j = 11.0 hz , 2 h ) , 3.88 ( d , j = 11.0 hz , 1 h ) , 3.323.24 ( m , 1 h ) , 3.123.03 ( m , 1 h ) , 2.662.59 ( m , 1 h ) , 2.582.42 ( m , 8 h ) , 2.18 ( d , j = 14.6 hz , 1 h ) , 1.301.10 ( br s , 9 h ) , 0.82 ( s , 9 h ) , 0.27 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 870.60 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of the preceding intermediate ( 16.8 mg , 0.019 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 7.8 g ) in water ( 15 ml ) .", "sodium chloride ( 10 g ) was added to the aqueous layer , and this was extracted with etoac ( 2 ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx 10 rp 100a column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 1.2 mg ( 10% , 2 steps ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.304.20 ( s , 2 h ) , 4.19 ( s , 1 h ) , 3.252.96 ( m , 9 h ) , 2.482.28 ( m , 2 h ) , 1.721.58 ( m , 1 h ) , 1.43 ( s , 9 h ) .", "the following compounds were prepared according to methods similar to 21a : prepared from 19k in 68% yield for the acylation / amine displacement and 58% yield for the deprotections , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.244.65 ( m , 3 h ) , 3.262.97 ( m , 9 h ) , 2.382.28 ( m , 2 h ) , 1.691.56 ( m , 1 h ) , 1.44 ( s , 9 h ) .", "ms ( esi ) m / z 562.37 ( m + h ) . prepared from 19l in 43% yield for the acylation / amine displacement and 55% yield for the deprotections , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.304.40 ( m , 3 h ) , 3.403.25 ( m , 8 h ) , 3.123.04 ( m , 4 h ) , 2.99 ( s , 3 h ) , 2.622.52 ( m , 1 h ) , 2.412.35 ( m , 1 h ) , 1.731.60 ( m , 1 h ) , 1.45 ( s , 9 h ) .", "compound 19h ( 50 mg , 0.061 mmol ) , k3po4 ( 39 mg , 0.18 mmol ) , tris(dibenzylideneacetone)dipalladium ( 2.8 mg , 0.003 mmol ) , and xantphos ( 5.1 mg , 0.009 mmol ) were added to a small vial equipped with a septum . after the flask was evacuated and flushed with nitrogen ( 3 ) , 1,4-dioxane ( 0.5 ml ) and ethylamine ( 2.0 m solution in thf , 0.091 ml , 0.18 mmol ) were added .", "the reaction mixture was heated to 100 c and stirred for 3 h. the reaction mixture was allowed to cool to room temperature and was filtered through celite .", "the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 11 mg ( 23% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.6 ( br , 1 h ) , 7.567.49 ( m , 4 h ) , 7.427.30 ( m , 6 h ) , 5.37 ( s , 2 h ) , 5.03 ( s , 2 h ) , 3.89 ( d , j = 11.0 hz , 1 h ) , 3.243.04 ( m , 3 h ) , 2.97 ( dd , j = 15.6 , 4.9 hz , 1 h ) , 2.642.43 ( m , 3 h ) , 2.49 ( s , 6 h ) , 2.14 ( d , j = 15.6 hz , 1 h ) , 1.25 ( t , j = 6.8 hz , 3 h ) , 0.81 ( s , 9 h ) , 0.25 ( s , 3 h ) , 0.11 ( s , 3 h ) . ms ( esi ) m / z 785.57 ( m + h ) .", "aqueous hf ( 48% , 0.3 ml ) was added to a solution of the preceding intermediate ( 11 mg , 0.014 mmol ) in ch3cn ( 7 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 2 g ) in water ( 10 ml ) .", "the material was dissolved in meoh ( 2 ml ) and 1,4-dioxane ( 2 ml ) , and palladium on carbon ( degussa , 10 wt % , 5.6 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx 10 rp 100a column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 3.2 mg ( 46% , 2 steps ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.08 ( s , 1 h ) , 3.43 ( q , j = 7.4 hz , 2 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.671.55 ( m , 1 h ) , 1.22 ( t , j = 7.4 hz , 3h ) . ms ( esi ) m / z 493.24 ( m + h ) .", "the following compounds were prepared by similar methods to 24a : prepared from 19h and methylamine ( 1.0 m solution in thf ) in 21% yield for the amination step , 19% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.08 ( s , 1 h ) , 3.41 ( s , 3 h ) , 3.082.92 ( m , 9 h ) , 2.302.15 ( m , 2 h ) , 1.671.55 ( m , 1 h ) .", "ms ( esi ) m / z 479.22 ( m + h ) . prepared from 19h and n - propylamine in 24% yield for the amination step , 40% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.16 ( s , 1h ) , 3.47 ( t , j = 7.3 hz , 2 h ) , 3.152.96 ( m , 9 h ) , 2.322.20 ( m , 2 h ) , 1.781.55 ( m , 3 h ) , 1.01 ( t , j = 7.4 hz , 3 h ) .", "ms ( esi ) m / z 507.29 ( m + h ) . prepared from 19h and 2-propylamine in 11% yield for the amination step , 16% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.214.13 ( m , 1 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.312.15 ( m , 2 h ) , 1.671.56 ( m , 1 h ) , 1.23 ( dd , j = 6.4 , 1.8 hz , 6 h ) .", "ms ( esi ) m / z 507.24 ( m + h ) . prepared from 19h and 2-methylpropan-1-amine in 15% yield for the amination step , 57% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.18 ( s , 1h ) , 3.38 ( d , j = 7.3 hz , 2 h ) , 3.202.96 ( m , 9 h ) , 2.342.20 ( m , 2 h ) , 2.101.98 ( m , 1h ) , 1.681.54 ( m , 1 h ) , 1.00 ( d , j = 6.4 hz , 6 h ) . ms ( esi ) m / z 521.40 ( m + h ) . prepared from 19h and 3-methyl - butan-1-amine in 25% yield for the amination step , 66% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.453.40 ( m , 2 h ) , 3.20 ( s , 1 h ) , 3.082.93 ( m , 9 h ) , 2.302.16 ( m , 2 h ) , 1.721.58 ( m , 2 h ) , 1.551.48 ( m , 2 h ) , 0.96 ( d , j = 6.4 hz , 6 h ) . ms ( esi ) m / z 535.30 ( m + h ) . prepared from 19h and 2-methoxyethylamine in 19% yield for the amination step , 44% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.13 ( s , 1h ) , 3.723.60 ( m , 4h ) , 3.40 ( s , 3 h ) , 3.122.95 ( m , 9 h ) , 2.322.21 ( m , 2 h ) , 1.691.56 ( m , 1 h ) .", "ms ( esi ) m / z 523.32 ( m + h ) . prepared from 19h and 2-(propan-2-yloxy)ethan-1-amine in 9% yield for the amination step , 30% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.19 ( s , 1h ) , 3.823.69 ( m , 5h ) , 3.202.98 ( m , 9 h ) , 2.352.22 ( m , 2 h ) , 1.681.56 ( m , 1 h ) , 1.18 ( d , j = 6.4 hz , 6h ) . ms ( esi ) m / z 551.41 ( m + h ) . prepared from 19h and ( 3-aminopropyl)dimethylamine in 9% yield for the amination step , 21% for the 2 deprotection steps , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.703.46 ( m , 4 h ) , 3.102.90 ( m , 15 h ) , 2.312.17 ( m , 2 h ) , 2.102.00 ( m , 2 h ) , 1.681.56 ( m , 1 h ) .", "compound 16d ( 100 mg , 0.24 mmol ) , cs2co3 ( 235 mg , 0.720 mmol ) , tris(dibenzylideneacetone)dipalladium ( 11 mg , 0.012 mmol ) , and xantphos ( 20 mg , 0.036 mmol ) were weighed into a flask .", "this was evacuated and backflushed with nitrogen ( 3 ) , and 1,4-dioxane ( 0.5 ml ) and n , n - dimethylneopentanediamine ( 0.057 ml , 0.36 mmol ) were added .", "after 1.5 h , the reaction mixture was cooled to room temperature and was filtered through celite .", "the filtrate was concentrated under reduced pressure and was purified by column chromatography ( biotage 10 g column , 03% meoh in ch2cl2 gradient ) , yielding 76.9 mg ( 69% ) of the intermediate .", "lhmds ( 1.0 m in thf , 0.22 ml , 0.22 mmol ) was added dropwise to a 78 c solution of the above intermediate ( 93 mg , 0.20 mmol ) in thf ( 5 ml ) . after 5 min , benzyl chloroformate ( 0.084 ml , 0.60 mmol )", "after 15 min , the reaction mixture was quenched by the addition of nh4cl ( saturated , aqueous solution , 15 ml ) and was extracted with etoac .", "the material was purified by column chromatography ( biotage 10 g column , 07% meoh in ch2cl2 gradient ) , yielding 111 mg ( 93% ) of the product .", "nmr ( 400 mhz , cdcl3 ) 7.457.20 ( m , 13 h ) , 7.066.98 ( m , 2 h ) , 5.16 ( s , 2 h ) , 4.87 ( s , 2 h ) , 3.78 ( br s , 2 h ) , 2.38 ( s , 3 h ) , 2.18 ( br s , 6 h ) , 1.62 ( br s , 2 h ) , 0.90 ( br s , 6 h ) . ms ( esi ) m / z 600.28 ( m + h ) .", "a solution of compound 22a ( 54 mg , 0.090 mmol ) in thf ( 0.5 ml ) was added dropwise to a 78 c solution of lda ( 2.0 m solution in thf , 0.112 ml , 0.224 mmol ) and tmeda ( 0.081 ml , 0.54 mmol ) in thf ( 2 ml ) , resulting in an orange - colored solution .", "after 10 min , a solution of 8 ( 43 mg , 0.090 mmol ) in thf ( 0.5 ml ) was added dropwise over 30 s. after complete addition , the reaction mixture was allowed to warm to 10 c over 1 h. the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) , was diluted with water , and was extracted with etoac ( 2 ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 20100% b gradient ) , yielding 22.9 mg ( 26% ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.55 ( br s , 1 h ) , 7.527.20 ( m , 15 h ) , 5.36 ( s , 2 h ) , 5.205.00 ( m , 2 h ) , 4.80 ( s , 2 h ) , 3.90 ( d , j = 11.0 hz , 1 h ) , 3.193.05 ( m , 2 h ) , 2.622.57 ( m , 1 h ) , 2.562.12 ( m , 19 h ) , 0.940.74 ( m , 15 h ) , 0.26 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 988.59 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 23a ( 22.9 mg , 0.0232 mmol ) in ch3cn ( 0.8 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water .", "the mixture was extracted with etoac , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) , 0.5 m hcl in meoh ( 0.2 ml ) , and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 2 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 3 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx column , 0100% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 9.9 mg ( 67% , 2 steps ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.16 ( s , 1 h ) , 3.43 ( s , 2 h ) , 3.152.90 ( m , 17 h ) , 2.302.22 ( m , 1 h ) , 2.222.12 ( m , 1 h ) , 1.651.54 ( m , 1 h ) , 1.17 , ( s , 6 h ) .", "the following compounds were prepared according to methods similar to 24j : prepared from 16a and n , n - dimethylethylenediamine .", "h nmr ( 400 mhz , cd3od ) 4.13 ( s , 1 h ) , 3.843.77 ( m , 2 h ) , 3.443.38 ( m , 2 h ) , 3.102.92 ( m , 15 h ) , 2.322.20 ( m , 2 h ) , 1.681.55 ( m , 1 h ) .", "ms ( esi ) m / z 536.25 ( m + h ) . prepared from 16a and n , n - dimethylneopentanediamine .", "h nmr ( 400 mhz , cd3od ) 4.10 ( s , 1 h ) , 3.513.36 ( m , 4 h ) , 3.102.92 ( m , 15 h ) , 2.332.18 ( m , 2 h ) , 1.681.57 ( m , 1 h ) , 1.174 ( s , 3 h ) , 1.169 ( s , 3 h ) .", "ms ( esi ) m / z 578.35 ( m + h ) . prepared from 16a and 2,2-dimethyl-3-(pyrrolidin-1-yl)propan-1-amine .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.883.78 ( m , 2 h ) , 3.503.39 ( m , 2 h ) , 3.18 2.92 ( m , 9 h ) , 2.322.08 ( m , 6 h ) , 1.671.56 ( m , 1 h ) , 1.15 ( s , 6 h ) . ms ( esi ) m / z 604.29 ( m + h ) . prepared from 16d and n - propylamine .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.33 ( t , j = 6.9 hz , 2 h ) 3.423.38 ( m , 2 h ) , 3.20 ( s , 1 h ) , 3.072.94 ( m , 8 h ) , 2.91 ( dd , j = 14.6 , 4.6 hz , 1 h ) , 2.232.12 ( m , 2 h ) , 1.681.55 ( m , 3 h ) , 0.96 ( t , j = 7.3 hz , 3 h ) .", "copper(i ) iodide ( 4.7 mg , 0.025 mmol ) and k3po4 ( 209 mg , 0.984 mmol ) were added to a vial equipped with a septum . after the vial was evacuated and flushed with nitrogen ( 3 ) , a solution of compound 16e ( 217 mg , 0.492 mmol ) in dmf ( 2 ml ) , 1-propylamine ( 0.162 ml , 1.97 mmol ) , and 2-acetylcyclohexanone ( 0.013 ml , 0.098 mmol ) were added .", "the reaction mixture was heated to 100 c for 3 h. the reaction mixture was allowed to cool to room temperature , was diluted with etoac ( 50 ml ) , and was washed with water ( 3 40 ml ) .", "the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 74.6 mg ( 36% ) of the title compound as a thick oil .", "h nmr ( 400 mhz , cdcl3 ) 7.507.46 ( m , 2 h ) , 7.417.30 ( m , 5 h ) , 7.307.24 ( m , 1 h ) , 7.087.02 ( m , 2 h ) , 5.08 ( s , 2 h ) , 2.85 ( s , 6 h ) , 2.32 ( s , 3 h ) . ms ( esi ) m / z 420.34 ( m + h ) .", "lhmds ( 1.0 m solution in thf , 0.167 ml , 0.167 mmol ) was added to a solution of phenyl 5-benzyloxy-2-(dimethylamino)-3-methyl-6-(propylamino)isonicotinate ( 63.5 mg , 0.152 mmol ) in thf ( 2 ml ) , resulting in a bright - yellow solution .", "a solution of di - t - butyldicarbonate ( 99.5 mg , 0.456 mmol ) in thf ( 1 ml ) was added .", "after 10 min , the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) and was extracted with etoac ( 2 ) .", "the material was purified by column chromatography ( biotage 10 g column , 012% etoac in hexanes gradient ) , yielding 13.6 mg ( 17% ) of the product .", "h nmr ( 400 mhz , cdcl3 ) 7.407.28 ( m , 7 h ) , 7.287.22 ( m , 1 h ) , 7.057.00 ( m , 2 h ) , 4.90 ( s , 2 h ) , 2.79 ( s , 6 h ) , 2.34 ( s , 3 h ) , 1.761.66 ( m , 2 h ) , 1.561.50 ( m , 2 h ) , 0.91 ( t , j = 7.56 hz , 3 h ) . ms ( esi ) m / z 520.38 ( m + h ) .", "a solution of 22b ( 24 mg , 0.047 mmol ) in thf ( 0.5 ml ) was added dropwise to a 78 c solution of lda ( 0.5 m solution in thf , 0.102 ml , 0.051 mmol ) and tmeda ( 0.041 ml , 0.27 mmol ) in thf ( 2 ml ) , resulting in a yellowishorange colored solution .", "after 10 min , a solution of 8 ( 16.4 mg , 0.034 mmol ) in thf ( 0.5 ml ) was added dropwise .", "after complete addition , the reaction mixture was allowed to warm to 20 c over 45 min .", "the reaction was quenched by the addition of ammonium chloride ( saturated , aqueous solution ) and was extracted with etoac ( 2 ) .", "the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient ) , yielding 6.1 mg ( 20% ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cdcl3 ) 15.56 ( s , 1 h ) , 7.527.24 ( m , 10 h ) , 5.36 ( s , 2 h ) , 4.994.64 ( m , 2 h ) , 4.02 ( d , j = 11.0 hz , 1 h ) , 3.102.81 ( m , 2 h ) , 2.78 ( s , 6 h ) , 2.622.40 ( m , 10 h ) , 2.202.14 ( m , 1 h ) , 1.801.45 ( m , 2 h ) , 1.441.15 ( m , 11 h ) , 1.000.70 ( m , 12 h ) , 0.25 ( s , 3 h ) , 0.12 ( s , 3 h ) . ms ( esi ) m / z 908.65 ( m + h ) .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 20o ( 6.1 mg , 0.007 mmol ) in ch3cn ( 0.6 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water ( 15 ml ) .", "the combined etoac extracts were dried over na2so4 , filtered , and concentrated under reduced pressure .", "the material was dissolved in meoh ( 1 ml ) and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 2 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 4 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", ". fractions with the desired mw were collected and freeze - dried to yield 2.8 mg ( 70% , 2 steps ) of the desired product as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.19 ( s , 1 h ) , 3.44 ( t , j = 7.6 hz , 2 h ) , 3.243.12 ( m , 7 h ) , 3.122.97 ( m , 8 h ) , 2.392.26 ( m , 2 h ) , 1.731.56 ( m , 3 h ) , 0.98 ( t , j = 8.4 hz , 3 h ) ; ms ( esi ) m / z 516.42 ( m + h ) . compound 19 m ( 105 mg , 0.126 mmol ) , tetrakis(triphenylphosphine)palladium(0 ) ( 15 mg , 0.013 mmol ) , and k3po4 ( 80 mg , 0.38 mmol ) were weighed into an 8 ml vial .", "this was sealed with a septum and was evacuated and backflushed with nitrogen ( 3 ) .", "toluene ( 1 ml ) , 1,4-dioxane ( 1 ml ) , and water ( 0.5 ml ) were added , followed by allylboronic acid pinacol ester ( 63.7 mg , 0.379 mmol ) .", "after 2.5 h , the reaction mixture was cooled to room temperature and was concentrated under reduced pressure .", "the material was purified by preparative hplc ( sunfire prep c18 column , 90100% b gradient ) , yielding 28.6 mg ( 29% ) of the title compound as a yellow solid .", "aqueous hf ( 48% , 0.4 ml ) was added to a solution of 25a ( 28.6 mg , 0.036 mmol ) in 1,4-dioxane ( 1 ml ) in a plastic vial . after 18 h , the reaction mixture was poured into a solution of k2hpo4 ( 4.8 g ) in water .", "the mixture was extracted with etoac , and the combined extracts were dried over na2so4 , filtered , and concentrated under reduced pressure . the material was dissolved in meoh ( 1 ml ) , 0.5 m hcl in meoh ( 0.5 ml ) , and 1,4-dioxane ( 1 ml ) , and palladium on carbon ( degussa , 10 wt % , 5 mg ) was added .", "an atmosphere of hydrogen was introduced , and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite , and the filtrate was concentrated under reduced pressure .", "the material was purified by preparative hplc ( phenomenex polymerx column , 070% b gradient )", ". fractions with the desired mw were collected and freeze - dried to yield 14.3 mg ( 69% ) of the title compound as a yellow solid .", "h nmr ( 400 mhz , cd3od with 1 drop dcl ) 4.20 ( s , 1 h ) , 3.383.15 ( m , 7 h ) , 3.142.94 ( m , 8 h ) , 2.912.82 ( m , 2 h ) , 2.56 ( t , j = 13.8 hz , 1 h ) , 2.402.32 ( m , 1 h ) , 1.861.76 ( m , 2 h ) , 1.741.62 ( m , 1 h ) , 1.01 ( t , j = 7.34 hz , 3 h ) .", "the following compounds were prepared according to methods similar to 26a : prepared from 19h and allylboronic acid pinacol ester , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.10 ( s , 1 h ) , 3.222.92 ( m , 9 h ) , 2.812.74 ( m , 2 h ) , 2.492.38 ( m , 1 h ) , 2.262.19 ( m , 1 h ) , 1.77 1.60 ( m , 3 h ) , 0.97 ( t , j = 7.3 hz , 3 h ) .", "ms ( esi ) m / z 492.27 ( m + h ) . prepared from 19h and methylboronic acid , yellow solid .", "h nmr ( 400 mhz , cd3od ) 4.09 ( s , 1 h ) , 3.222.92 ( m , 9 h ) , 2.85 ( s , 3 h ) , 2.492.38 ( m , 1 h ) , 2.262.19 ( m , 1 h ) , 1.77 1.60 ( m , 1h ) .", "ms ( esi ) m / z 464.30 ( m + h ) . prepared from 19h as an over - reduced side product in the synthesis of 26c .", "h nmr ( 400 mhz , cd3od ) 7.78 ( s , 1 h ) , 4.12 ( s , 1 h ) , 3.222.91 ( m , 9 h ) , 2.78 ( s , 3 h ) , 2.492.21 ( m , 1 h ) , 1.771.62 ( m , 1h ) .", "ms ( esi ) m / z 430.46 ( m + h ) . prepared from 19h and phenylboronic acid .", "h nmr ( 400 mhz , cd3od ) 8.047.98 ( m , 2 h ) , 7.477.39 ( m , 3 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.572.47 ( m , 1 h ) , 2.282.07 ( m , 1 h ) , 1.731.65 ( m , 1h ) .", "ms ( esi ) m / z 526.26 ( m + h ) . prepared from 19h as an over - reduced side product in the synthesis of 26e .", "h nmr ( 400 mhz , cd3od ) 8.047.98 ( m , 2 h ) , 7.477.39 ( m , 3 h ) , 4.08 ( s , 1 h ) , 3.142.92 ( m , 9 h ) , 2.572.47 ( m , 1 h ) , 2.282.07 ( m , 1 h ) , 1.731.65 ( m , 1h ) .", "tetracycline - susceptible isolates sa100 ( s. aureus atcc 13709 , smith ) , sa101 ( s. aureus atcc 29213 ) , sp106 ( s. pneumoniae atcc 49619 ) , ef103 ( e. faecalis atcc 29212 ) , ec107 ( e. coli atcc 25922 ) , kp109 ( k. pneumoniae atcc 13883 ) , ab110 ( acinetobacter baumannii atcc 19606 ) , ec108 ( enterobacter cloacae atcc 13047 ) , and pa111 ( pseudomonas aeruginosa atcc 27853 ) were obtained from the american type culture collection ( atcc , manassas , va ) .", "tetracycline - resistant isolates s. aureus sa158 ( tet(k ) ) , s. pneumoniae sp160 ( tet(m ) ) , e. faecalis ef159 ( tet(m ) ) , e. coli ec155 ( tet(a ) ) , k. pneumoniae kp153 ( tet(a ) ) were obtained from marilyn roberts lab at the university of washington .", "s. aureus sa161 ( tet(m ) ) was obtained from micromyx ( kalamazoo , mi ) .", "minimal inhibitory concentration ( mic ) determinations were performed in liquid medium in 96-well microtiter plates according to the methods described by the clinical and laboratory standards institute ( clsi).(26 ) cation - adjusted mueller hinton broth was obtained from bbl ( cat .", "212322 , becton dickinson , sparks , md ) , prepared fresh and kept at 4 c prior to testing .", "a0432 , pml microbiologicals , wilsonville , or ) was used to supplement medium , as appropriate .", "all test methods met acceptable standards based on recommended quality control ranges for all comparator antibiotics and the appropriate atcc quality control strains .", "compound stocks prepared and diluted in sterile deionized water were assayed for inhibition of coupled in vitro transcription / translation using an e. coli s30 extract system with a firefly luciferase readout from promega ( cat .", "briefly , compounds were diluted into water and added to reaction mix aliquoted to black - walled 96-well microtiter plates ( cat .", "an appropriate three - point titration was used for each compound , and reactions were run in duplicate .", "plates were incubated at 37 c for one hour and then placed on ice for 5 min to arrest transcription / translation .", "e1500 ) was added to each well and luminescence was detected on a bmg labtech lumistar - optima instrument .", "positive assay control values , from reactions without inhibitor , were averaged per plate to determine percent inhibition of luciferase production .", "results were plotted using microsoft excel and 50% inhibition values ( ic50 ) were determined ." ] ]

[ "humans are infected naturally by 3 species of hookworms ; necator americanus , ancylostoma duodenale , and ancylostoma ceylanicum .", "individual people can be infected with 1 species , 2 species , or all 3 species of hookworms [ 1 - 5 ] . although a. ceylanicum is often considered to be a zoonotic pathogen , since it is also found in dogs and cats , its importance as a significant soil - transmitted helminth of humans is increasingly being recognized .", "two haplotypes of a. ceylanicum have been identified ; one largely found in humans and another that infects humans , dogs , and cats .", "a. ceylanicum is considered to be of emerging importance as a helminth of humans in the asia pacific region .", "a fourth hookworm also reported from humans , ancylostoma caninum represents a canine species with zoonotic potential .", "this species has mainly been recorded as causing eosinophilic enteritis in people in tropical australia , but never establishes a patent infection and hence humans are regarded as accidental hosts .", "human hookworm is common in the solomon islands , with an estimated 192,000 people ( 36% of the population ) infected , but the different species involved are yet to be determined [ 9 - 11 ] .", "the report to the rockefeller hookworm campaign in 1928 described only n. americanus as being present on these islands .", "the only previous report of a. ceylanicum from the solomon islands was a 5-year - old boy who imported the species in 1938 from the shortland island group of the solomon islands to australia .", "the solomon islands is a small pacific nation to the north east of australia , with a predominantly melanesian population .", "the regional assistance mission to solomon islands ( ramsi ) occurred between 2003 and 2013 , at the request of the solomon island s government , to assist in stabilizing internal troubles occurring within the nation .", "ramsi consisted of 2,200 police and soldiers from many pacific island nations and was led by australia .", "this case report describes the finding of a. ceylanicum in an australian soldier who developed symptomatic disease after returning from a deployment in solomon islands with ramsi .", "the patient was a 26-year - old male living in townsville , north queensland , australia who presented in march 2004 with central poorly defined abdominal pain of 4 weeks duration and increased frequency of defecation .", "a peripheral eosinophilia had been noted on blood tests in february prior to symptom development , and in march , when eosinophils were 6.2010/l whilst red blood cell indices were normal ( hemoglobin 146 g / l , mcv 93 fl , hematocrit 0.44 , red cell count 4.710/l ) .", "the patient had served with ramsi in the solomon islands from late july to early december 2003 .", "his duties involved travel to various remote areas and provided multiple opportunities to become infected with hookworms .", "routine treatment with albendazole had been given by the australian defense force on return to townsville in december , 2 months prior to development of symptoms .", "initially , in march 2004 no parasite eggs were detected in feces by formalin - ethyl acetate concentration , but infective larvae of hookworms were found on harada - mori culture .", "infective larvae were sheathed , had inconspicuous buccal spears , the intestine was narrower than the esophageal bulb , and inconspicuous transverse striations were noted on the sheath in the tail region .", "the infective larvae , measured after preservation in hot formalin , had the following dimensions , meansd ( range ) : length 657.19.6 m ( 633 - 669 m ) , width 21.40.9 m ( 20.2 - 22.2 m ) , esophagus 156.73.5 m ( 151 - 162 m ) , tail 77.93.8 m ( 69 - 81 m ) , length of sheath 763.78.6 m ( 779 - 746 m ) ; esophagus / length 23.80.5% , and tail / length 11.90.6% . due to the failure of single dose post - deployment albendazole therapy to eradicate this infection , the patient was treated with 100 mg of mebendazole twice daily for 3 days .", "twelve adult hookworms ( 6 females and 6 males ) were recovered by dissection of stools passed during the 48 hr immediately following treatment . the en face view revealed a robust ventral cutting plate bilaterally with a prominent point on the dorsal end and less obvious point on the ventral end ( fig .", "the width of the bursa in lateral view was greater than its length , and the mediolateral and posteriolateral bursal rays were parallel ( fig .", "the morphology of the adult hookworms was consistent with a. ceylanicum with key features being : the large ventral cutting plates terminating at the dorsal end in a large single tooth ; and in the male the parallel mediolateral and posteriolateral bursal rays .", "although the morphology and dimensions of the infective larvae are more consistent with a. ceylanicum than n. americanus , differentiating them from infective larvae of a. duodenale is unreliable .", "although molecular taxonomy is valuable in identifying hookworm species , it was not available in this case and , as the specimen was subsequently accidentally disposed of , molecular taxonomic studies can not be performed now . however , the morphological features were sufficient to identify this species as a. ceylanicum .", "this represents only the second report of human a. ceylanicum infection having been acquired in this nation , with the first being made almost 80 years ago .", "this finding not only expands the known range of human a. ceylanicum infection but also raises the question of whether a. ceylanicum is in fact a common cause of human hookworm infection in some regions of the solomon islands , as it has recently been described as being in many parts of asia . the only previous reported description of hookworm species in the country was almost 80 years ago and was anecdotal , not providing descriptions of number of people tested or how the species identifications were arrived at .", "a. ceylanicum infection has recently been reported from two people in western australia . prior to this , a. ceylanicum had been reported from dogs and cats in northern australia , including those from townsville .", "hence , the infection could have been acquired in north queensland or while deployed with ramsi .", "the likelihood of infection with ancylostoma from humans in the solomon islands is high , but negligible in north queensland since human feces are disposed of safely .", "however , since a. ceylanicum is present in dogs and cats in north queensland , there is a possibility of a zoonotic australian source even though this infection has not been seen in humans in this region .", "many ramsi personnel were infected with hookworms during their mission to the solomon islands , and an isolated case of a. ceylanicum infection had been reported from this area early in the 20th century .", "this adds to the likelihood that the case presented here was acquired in the solomon islands and not elsewhere .", "the clinical symptoms displayed by our patient were consistent with the phase of hookworm infection during which the host attempts to eliminate the parasite using an allergic response , manifesting morphologically as eosinophilic enteritis . for n. americanus", "this response begins about 10 days after infection and persists for up to a month .", "a. caninum and a. duodenale have a more complicated life cycle in which some infective larvae can enter a state of hypobiosis ( arrested development ) and can orchestrate entry to the intestine to coincide with the wet season and physiological events [ 23 - 26 ] . whether a. ceylanicum has", "the failure of a routine post - deployment albendazole treatment to eliminate the infection in our patient supports a hypothesis that the current infection was due to hypobiotic larvae activated after the anthelmintic was given .", "acute enteritis has been described previously in soldiers with a. ceylanicum acquired in west papua ( formerly dutch new guinea ) and in a traveller returning from myanmar to france .", "treatment with albendazole in the latter patient cleared the gut infection but symptoms and parasites recurred after 3 months adding evidence for hypobiosis in a. ceylanicum .", "our patient was treated after diagnosis with mebendazole due to its far greater tissue penetration and therefore greater capacity to kill hypobiotic larvae in the tissue .", "the key points from this case report are : a. ceylanicum should be considered as a cause of acute eosinophilic enteritis in returned travellers , workers , or defense force personnel who have lived or visited tropical and subtropical countries ; laboratory confirmation may require persistence to make the diagnosis of hookworms and will require special tests ( morphology of adults after treatment ; molecular taxonomy on stools or infective larvae ) to identify the species ; and human infection with a. ceylanicum is probably endemic in the solomon islands and further study of the prevalence of different hookworm species present in this country is warranted ." ]

[ "<S> a 26-year - old male member of the australian defense force presented with a history of central abdominal pain of 4 weeks duration and peripheral eosinophilia consistent with eosinophilic enteritis . </S>", "<S> acute hookworm disease was diagnosed as the cause . </S>", "<S> adult worms recovered from feces after therapy with albendazole were morphologically consistent with ancylostoma ceylanicum . </S>", "<S> as the patient had been deployed with the regional assistance mission to solomon islands for 6 months prior to this presentation , it is very likely that the a. ceylanicum was acquired in solomon islands . </S>", "<S> until now , it has been assumed that any ancylostoma spp . recovered from humans in solomon islands </S>", "<S> is a. duodenale . </S>", "<S> however , this case demonstrates that human hookworm infection acquired in the solomon islands could be caused by a. ceylanicum . </S>" ]

[ "INTRODUCTION", "CASE RECORD", "DISCUSSION" ]

[ [ "humans are infected naturally by 3 species of hookworms ; necator americanus , ancylostoma duodenale , and ancylostoma ceylanicum .", "individual people can be infected with 1 species , 2 species , or all 3 species of hookworms [ 1 - 5 ] . although a. ceylanicum is often considered to be a zoonotic pathogen , since it is also found in dogs and cats , its importance as a significant soil - transmitted helminth of humans is increasingly being recognized .", "two haplotypes of a. ceylanicum have been identified ; one largely found in humans and another that infects humans , dogs , and cats .", "a. ceylanicum is considered to be of emerging importance as a helminth of humans in the asia pacific region .", "a fourth hookworm also reported from humans , ancylostoma caninum represents a canine species with zoonotic potential .", "this species has mainly been recorded as causing eosinophilic enteritis in people in tropical australia , but never establishes a patent infection and hence humans are regarded as accidental hosts .", "human hookworm is common in the solomon islands , with an estimated 192,000 people ( 36% of the population ) infected , but the different species involved are yet to be determined [ 9 - 11 ] .", "the report to the rockefeller hookworm campaign in 1928 described only n. americanus as being present on these islands .", "the only previous report of a. ceylanicum from the solomon islands was a 5-year - old boy who imported the species in 1938 from the shortland island group of the solomon islands to australia .", "the solomon islands is a small pacific nation to the north east of australia , with a predominantly melanesian population .", "the regional assistance mission to solomon islands ( ramsi ) occurred between 2003 and 2013 , at the request of the solomon island s government , to assist in stabilizing internal troubles occurring within the nation .", "ramsi consisted of 2,200 police and soldiers from many pacific island nations and was led by australia .", "this case report describes the finding of a. ceylanicum in an australian soldier who developed symptomatic disease after returning from a deployment in solomon islands with ramsi ." ], [ "the patient was a 26-year - old male living in townsville , north queensland , australia who presented in march 2004 with central poorly defined abdominal pain of 4 weeks duration and increased frequency of defecation .", "a peripheral eosinophilia had been noted on blood tests in february prior to symptom development , and in march , when eosinophils were 6.2010/l whilst red blood cell indices were normal ( hemoglobin 146 g / l , mcv 93 fl , hematocrit 0.44 , red cell count 4.710/l ) .", "the patient had served with ramsi in the solomon islands from late july to early december 2003 .", "his duties involved travel to various remote areas and provided multiple opportunities to become infected with hookworms .", "routine treatment with albendazole had been given by the australian defense force on return to townsville in december , 2 months prior to development of symptoms .", "initially , in march 2004 no parasite eggs were detected in feces by formalin - ethyl acetate concentration , but infective larvae of hookworms were found on harada - mori culture .", "infective larvae were sheathed , had inconspicuous buccal spears , the intestine was narrower than the esophageal bulb , and inconspicuous transverse striations were noted on the sheath in the tail region .", "the infective larvae , measured after preservation in hot formalin , had the following dimensions , meansd ( range ) : length 657.19.6 m ( 633 - 669 m ) , width 21.40.9 m ( 20.2 - 22.2 m ) , esophagus 156.73.5 m ( 151 - 162 m ) , tail 77.93.8 m ( 69 - 81 m ) , length of sheath 763.78.6 m ( 779 - 746 m ) ; esophagus / length 23.80.5% , and tail / length 11.90.6% . due to the failure of single dose post - deployment albendazole therapy to eradicate this infection , the patient was treated with 100 mg of mebendazole twice daily for 3 days .", "twelve adult hookworms ( 6 females and 6 males ) were recovered by dissection of stools passed during the 48 hr immediately following treatment . the en face view revealed a robust ventral cutting plate bilaterally with a prominent point on the dorsal end and less obvious point on the ventral end ( fig .", "the width of the bursa in lateral view was greater than its length , and the mediolateral and posteriolateral bursal rays were parallel ( fig ." ], [ "the morphology of the adult hookworms was consistent with a. ceylanicum with key features being : the large ventral cutting plates terminating at the dorsal end in a large single tooth ; and in the male the parallel mediolateral and posteriolateral bursal rays .", "although the morphology and dimensions of the infective larvae are more consistent with a. ceylanicum than n. americanus , differentiating them from infective larvae of a. duodenale is unreliable .", "although molecular taxonomy is valuable in identifying hookworm species , it was not available in this case and , as the specimen was subsequently accidentally disposed of , molecular taxonomic studies can not be performed now . however , the morphological features were sufficient to identify this species as a. ceylanicum .", "this represents only the second report of human a. ceylanicum infection having been acquired in this nation , with the first being made almost 80 years ago .", "this finding not only expands the known range of human a. ceylanicum infection but also raises the question of whether a. ceylanicum is in fact a common cause of human hookworm infection in some regions of the solomon islands , as it has recently been described as being in many parts of asia . the only previous reported description of hookworm species in the country was almost 80 years ago and was anecdotal , not providing descriptions of number of people tested or how the species identifications were arrived at .", "a. ceylanicum infection has recently been reported from two people in western australia . prior to this , a. ceylanicum had been reported from dogs and cats in northern australia , including those from townsville .", "hence , the infection could have been acquired in north queensland or while deployed with ramsi .", "the likelihood of infection with ancylostoma from humans in the solomon islands is high , but negligible in north queensland since human feces are disposed of safely .", "however , since a. ceylanicum is present in dogs and cats in north queensland , there is a possibility of a zoonotic australian source even though this infection has not been seen in humans in this region .", "many ramsi personnel were infected with hookworms during their mission to the solomon islands , and an isolated case of a. ceylanicum infection had been reported from this area early in the 20th century .", "this adds to the likelihood that the case presented here was acquired in the solomon islands and not elsewhere .", "the clinical symptoms displayed by our patient were consistent with the phase of hookworm infection during which the host attempts to eliminate the parasite using an allergic response , manifesting morphologically as eosinophilic enteritis . for n. americanus", "this response begins about 10 days after infection and persists for up to a month .", "a. caninum and a. duodenale have a more complicated life cycle in which some infective larvae can enter a state of hypobiosis ( arrested development ) and can orchestrate entry to the intestine to coincide with the wet season and physiological events [ 23 - 26 ] . whether a. ceylanicum has", "the failure of a routine post - deployment albendazole treatment to eliminate the infection in our patient supports a hypothesis that the current infection was due to hypobiotic larvae activated after the anthelmintic was given .", "acute enteritis has been described previously in soldiers with a. ceylanicum acquired in west papua ( formerly dutch new guinea ) and in a traveller returning from myanmar to france .", "treatment with albendazole in the latter patient cleared the gut infection but symptoms and parasites recurred after 3 months adding evidence for hypobiosis in a. ceylanicum .", "our patient was treated after diagnosis with mebendazole due to its far greater tissue penetration and therefore greater capacity to kill hypobiotic larvae in the tissue .", "the key points from this case report are : a. ceylanicum should be considered as a cause of acute eosinophilic enteritis in returned travellers , workers , or defense force personnel who have lived or visited tropical and subtropical countries ; laboratory confirmation may require persistence to make the diagnosis of hookworms and will require special tests ( morphology of adults after treatment ; molecular taxonomy on stools or infective larvae ) to identify the species ; and human infection with a. ceylanicum is probably endemic in the solomon islands and further study of the prevalence of different hookworm species present in this country is warranted ." ] ]

[ "verruciform xanthoma ( vx ) , which has almost similar histologic features , is also a rare lesion usually found on the oral mucosa or the genital area .", "it is presumably associated with the inflammatory response to mucosal damage.1 however , xanthoma and vx of the esophagus are extremely rare . since the first report by remmele and engelsing2 only 13 cases of esophageal xanthoma have been reported,1,2,3,4,5,6,7,8,9 and since the report by herrera - goepfert et al.,10 only four cases of vx of the esophagus have been reported.10,11,12,13 the etiologies of both lesions are not understood .", "we describe herein a new case , including a review of all reported cases of xanthoma and vx of the esophagus .", "a 70-year - old man with an unremarkable medical history was hospitalized with a complaint of epigastric pain .", "serum total cholesterol , triglyceride , high density lipoprotein cholesterol , and low density lipoprotein cholesterol levels were 151 , 215 , 33 , and 102 mg / dl , respectively .", "multiple shallow gastric ulcers and a duodenal ulcer were detected and suspected to be the cause of the pain .", "besides the ulcers , in the upper esophagus 20 cm from the incisors , a 3-mm yellowish granular elevated mucosal lesion was found and a biopsy was performed ( fig .", "1 ) . microscopically , large round cells were aggregated in the lamina propria immediately beneath the squamous epithelium .", "the etiologies are different , as xanthoma is caused by hyperlipidemia and vx arises presumably as a result of an inflammatory response to continuous mucosal damage.1 however , the etiologies of the two lesions arising in the esophagus are not understood .", "the characteristics of all reported cases of xanthoma and vx of the esophagus are summarized in table 1 .", "fourteen cases of xanthoma and four cases of vx of the esophagus have been reported .", "however , some reports loosely stratified vx into esophageal xanthoma , whereas others have excluded it.6,8 in terms of clinical data , both diseases were found predominantly in men than in women : 9 versus 3 in xanthoma and 3 versus 1 in vx .", "the median age was 59 years ( range , 37 to 74 ) in xanthoma and .", "the predominant location was the lower esophagus for xanthoma ( lower , 7 ; middle , 2 ; upper , 3 ) , whereas vx was not reported in the lower esophagus ( upper , 2 ; middle , 2 ) .", "the median size was not different : 3 mm ( range , 2 to 10 ) for xanthoma and 4 mm ( range , 3 to 20 ) for vx .", "the associated medical conditions were diverse ; however , two patients with malignant tumors were included in each group : hepatocellular carcinoma and ileocecal lymphoma in xanthoma , and gastric cancer and multifocal cancer ( cancer of the glottis , liver , and trachea ) in vx , although there was no definite association .", "vx is characterized by its histologic features , including papillomatosis , acanthosis , and hyperparakeratosis.11 also , the external morphology is verrucoid .", "nevertheless , findings of large round foam cells in the lamina propria under the squamous epithelium are the same as those in xanthoma .", "it is difficult to differentiate between the two lesions on the basis of gross examination when they arise on the esophagus .", "exophytic and verrucoid features seen in vx of the skin were not observed in the esophagus because most of the reported cases were small in size.10,12 considering that xanthoma and vx are nonneoplastic lesions , differentiating between them could be a waste of effort .", "however , these lesions have to be grossly distinguished from ectopic sebaceous glands and small subepithelial tumors such as carcinoid and granular cell tumor because most of the reported esophageal xanthomas are yellowish or white mucosal elevated lesions . in terms of microscopic findings , signet ring cell carcinoma , which contains round cells with abundant cytoplasm ,", "while signet ring cell carcinoma has an eccentrically located nucleus because of the intracellularly abundant mucin , xanthoma has a centrally located and small nucleus .", "positive immunohistochemical staining for cd68 , which indicates a histiocytic origin , is another characteristic finding of xanthoma.5 moreover , esophageal cancer and ectopic sebaceous glands do not commonly stain with lugol 's solution ; thus , endoscopists need to be aware of these lesions for the differential diagnosis.6,14 with more case reports of esophageal xanthoma and vx of the esophagus , the characteristics of both lesions will be more clearly elucidated ." ]

[ "<S> xanthoma is an uncommon nonneoplastic lesion resulting from the accumulation of histiocytes . </S>", "<S> it predominantly shows cutaneous manifestations associated with dyslipidemia . however , xanthoma of the esophagus is extremely rare . to the best of our knowledge , </S>", "<S> only 14 cases have been reported thus far . </S>", "<S> the clinical significance of this lesion has not been established . </S>", "<S> however , this lesion should be distinguished grossly from ectopic sebaceous glands and small subepithelial tumors such as carcinoid and granular cell tumor . moreover , signet ring cell carcinoma , which contains round cells with abundant cytoplasm and has similar histologic features to xanthoma , should be distinguished microscopically . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "verruciform xanthoma ( vx ) , which has almost similar histologic features , is also a rare lesion usually found on the oral mucosa or the genital area .", "it is presumably associated with the inflammatory response to mucosal damage.1 however , xanthoma and vx of the esophagus are extremely rare . since the first report by remmele and engelsing2 only 13 cases of esophageal xanthoma have been reported,1,2,3,4,5,6,7,8,9 and since the report by herrera - goepfert et al.,10 only four cases of vx of the esophagus have been reported.10,11,12,13 the etiologies of both lesions are not understood .", "we describe herein a new case , including a review of all reported cases of xanthoma and vx of the esophagus ." ], [ "a 70-year - old man with an unremarkable medical history was hospitalized with a complaint of epigastric pain .", "serum total cholesterol , triglyceride , high density lipoprotein cholesterol , and low density lipoprotein cholesterol levels were 151 , 215 , 33 , and 102 mg / dl , respectively .", "multiple shallow gastric ulcers and a duodenal ulcer were detected and suspected to be the cause of the pain .", "besides the ulcers , in the upper esophagus 20 cm from the incisors , a 3-mm yellowish granular elevated mucosal lesion was found and a biopsy was performed ( fig .", "1 ) . microscopically , large round cells were aggregated in the lamina propria immediately beneath the squamous epithelium ." ], [ "the etiologies are different , as xanthoma is caused by hyperlipidemia and vx arises presumably as a result of an inflammatory response to continuous mucosal damage.1 however , the etiologies of the two lesions arising in the esophagus are not understood .", "the characteristics of all reported cases of xanthoma and vx of the esophagus are summarized in table 1 .", "fourteen cases of xanthoma and four cases of vx of the esophagus have been reported .", "however , some reports loosely stratified vx into esophageal xanthoma , whereas others have excluded it.6,8 in terms of clinical data , both diseases were found predominantly in men than in women : 9 versus 3 in xanthoma and 3 versus 1 in vx .", "the median age was 59 years ( range , 37 to 74 ) in xanthoma and .", "the predominant location was the lower esophagus for xanthoma ( lower , 7 ; middle , 2 ; upper , 3 ) , whereas vx was not reported in the lower esophagus ( upper , 2 ; middle , 2 ) .", "the median size was not different : 3 mm ( range , 2 to 10 ) for xanthoma and 4 mm ( range , 3 to 20 ) for vx .", "the associated medical conditions were diverse ; however , two patients with malignant tumors were included in each group : hepatocellular carcinoma and ileocecal lymphoma in xanthoma , and gastric cancer and multifocal cancer ( cancer of the glottis , liver , and trachea ) in vx , although there was no definite association .", "vx is characterized by its histologic features , including papillomatosis , acanthosis , and hyperparakeratosis.11 also , the external morphology is verrucoid .", "nevertheless , findings of large round foam cells in the lamina propria under the squamous epithelium are the same as those in xanthoma .", "it is difficult to differentiate between the two lesions on the basis of gross examination when they arise on the esophagus .", "exophytic and verrucoid features seen in vx of the skin were not observed in the esophagus because most of the reported cases were small in size.10,12 considering that xanthoma and vx are nonneoplastic lesions , differentiating between them could be a waste of effort .", "however , these lesions have to be grossly distinguished from ectopic sebaceous glands and small subepithelial tumors such as carcinoid and granular cell tumor because most of the reported esophageal xanthomas are yellowish or white mucosal elevated lesions . in terms of microscopic findings , signet ring cell carcinoma , which contains round cells with abundant cytoplasm ,", "while signet ring cell carcinoma has an eccentrically located nucleus because of the intracellularly abundant mucin , xanthoma has a centrally located and small nucleus .", "positive immunohistochemical staining for cd68 , which indicates a histiocytic origin , is another characteristic finding of xanthoma.5 moreover , esophageal cancer and ectopic sebaceous glands do not commonly stain with lugol 's solution ; thus , endoscopists need to be aware of these lesions for the differential diagnosis.6,14 with more case reports of esophageal xanthoma and vx of the esophagus , the characteristics of both lesions will be more clearly elucidated ." ] ]

[ "the sizing of pda for device implantation is based on the automatic calculations of dimension by the inbuilt sizing software in the cardiac catheterisation laboratory and many experienced interventionists believe their eyeball measurements more .", "the estimations can be erroneous if the calibrations are not proper , leading to catastrophic embolisation of the device , needing emergency surgical removal as was needed in our patient .", "calibration by comparing with the fluoroscopic images of measured metallic sizing devices placed outside body can be misleading because of magnification errors.1 in order to circumvent these problems we suggest some novel ways which take care of magnification errors and avoid the need for aortic root angiography .", "an eight year child with echocardiographically documented 6 mm patent ductus arteriosus ( pda ) was planned for device closure .", "femoral venous access was obtained using 8f sidearm sheath and the artery cannulated with a 6f sidearm sheath .", "a 6f pigtail catheter was advanced through the femoral arterial sheath into the descending aorta at the mouth of the pda and an angiogram obtained in a lateral projection delineating the ductus ( figure 1 ) .", "the minimum diameter at the aortic end was 6 mm and a 8 - 10 mm pda device was selected for deployment .", "a 7f swan - ganz catheter was advanced into the the pulmonary artery through venous route and a 0.35 \" terumo wire advanced through the pda into the descending aorta .the terumo wire was exchanged with an 0.35 \" extra stiff amplatzer wire and the swan - ganz catheter with the pda delivery system . an 8 - 10 mm size pda device was released across the ductus and after confirming the proper position by repeat angiogram which showed a small residual shunt flow which may be expected .", "angiogram in lateral plane showing a small residual left to right shunt with pda device in place before unscrewing of the stylet after a short while , the device suddenly embolised into the left pulmonary artery ( figure 2 ) .", "the patient had a brief episode of mild hypotension which improved spontaneously and his oxygen saturations remained static .", "an attempt was made to retrieve the device percutaneously by a snare and later by a bioptome but the trial was given up as the device tended to go more distally , in which case even surgical removal would be difficult .", "the patient was discussed with the cardiac surgeon who shifted the patient for emergency surgery .", "after considerable debate surgeon decided for a left thoracotomy hoping to avoid cardiopulmonary bypass and closed the ductus . later the left pulmonary artery was separated from the surrounding tissues and umbilical tape passed around it proximally for control of bleeding . a 1.5 cm longitudinal incision was made distal to the umbilical tape which was kept under traction and a long curved forceps passed toward the pulmonary hilum and the device retrieved .", "cine image in lateral plane showing device embolized into left pulmonary artery soon after its release from stylet . showing the pda device being retrieved from left pulmonary artery via left thoracotomy without cardiopulmonary bypass . in order to prevent such eventualities we tried to devise some alternative ways of sizing a pda .the", "first involves measuring a fully inflated tyshak pulmonary balloon outside the human body with a caliper and then inflating the balloon to the same pressure in the right ventricular outflow tract passed over a 0.35 \" extra - stiff wire , and repeating the measurements , and using the same calibration factor for sizing of pda visualized by aortic arch angiogram .", "previously , some radio opaque sizing devices have been used placing them outside human body which can have magnification errors if not placed exactly at the level of heart.2 the second method involves advancing a tyshak balloon 2 - 4 mm bigger than the echocardiographic dimension across the pda and inflating the balloon with dilute dye under low pressure without dilating the ductus so that it forms an impression of the ductus which is recorded in a cine mode in multiple planes , and the plane delineating the exact anatomy of the ductus is used for measurements after proper calibrations as mentioned above without the need for aortic arch angiogram ( figures 4 and 5 ) . showing measurement of diameter of inflated tyshak balloon by a caliper after inflating it with fixed quantity of contrast .", "angiogram in lateral plane showing an inflated tyshak balloon across the pda making an impression of the ductus .", "technological advances have made nonsurgical closure of the pda a simple and a routine percutaneous intervention .", "the use of the amplatzer device occluder ( ado ) has further simplified the method and improved the results with minimal complications .", "however there are situations where complications are encountered and surgical help is required to ameliorate them .", "faella and colleagues reported 15 procedure related complications in 316 patients which included hemolysis , left pulmonary artery artery stenosis , device protrusion into aorta causing coarctation , device misplacement and one death following device embolisation.2 late embolisation of the device to the left pulmonary artery has been reported with impaired left pulmonary perfusion six months after implantation requiring surgical removal .", "m vavuranakis et al reported severe hemolysis with jaundice , anemia and hemogloginura on the second day following deployment of smaller sized coil due to improper sizing which needed removal and replacement by an ado after repeat sizing using balloon tipped catheter.3 the complications can be reduced by proper expertise and optimal sizing of the ductus .", "sizing of atrial septal defect ( asd ) by inflating a balloon across the asd till a circumferential waist is created and measuring the waist ( stretched balloon diameter ) and also inflating the balloon outside by same amount of dye across sized rings , is a standard method.45 using a similar method for the pda is not routine since angiographic visualization is usually adequate .", "however making an impression of the ductus by inflating a balloon across it can give a detailed anatomy about the length and breadth of the ductus in multiple planes without the need for multiple dye injection and may help in the selection of proper sized device ." ]

[ "<S> nonsurgical closure of patent ductus arteriosus ( pda ) using a duct occluder placed percutaneously is currently the first line of therapy and the success rate is quite high . </S>", "<S> several devices are currently available . </S>", "<S> an eight year child underwent device closure of the ductus . </S>", "<S> however after deployment of the device it , became dislodged into the left pulmonary artery . </S>", "<S> several attempts at catheter retrieval failed . </S>", "<S> the child underwent successful surgical removal of the device without cardiopulmonary bypass . </S>" ]

[ "Introduction", "Technique", "Discussion" ]

[ [ "the sizing of pda for device implantation is based on the automatic calculations of dimension by the inbuilt sizing software in the cardiac catheterisation laboratory and many experienced interventionists believe their eyeball measurements more .", "the estimations can be erroneous if the calibrations are not proper , leading to catastrophic embolisation of the device , needing emergency surgical removal as was needed in our patient .", "calibration by comparing with the fluoroscopic images of measured metallic sizing devices placed outside body can be misleading because of magnification errors.1 in order to circumvent these problems we suggest some novel ways which take care of magnification errors and avoid the need for aortic root angiography ." ], [ "an eight year child with echocardiographically documented 6 mm patent ductus arteriosus ( pda ) was planned for device closure .", "femoral venous access was obtained using 8f sidearm sheath and the artery cannulated with a 6f sidearm sheath .", "a 6f pigtail catheter was advanced through the femoral arterial sheath into the descending aorta at the mouth of the pda and an angiogram obtained in a lateral projection delineating the ductus ( figure 1 ) .", "the minimum diameter at the aortic end was 6 mm and a 8 - 10 mm pda device was selected for deployment .", "a 7f swan - ganz catheter was advanced into the the pulmonary artery through venous route and a 0.35 \" terumo wire advanced through the pda into the descending aorta .the terumo wire was exchanged with an 0.35 \" extra stiff amplatzer wire and the swan - ganz catheter with the pda delivery system . an 8 - 10 mm size pda device was released across the ductus and after confirming the proper position by repeat angiogram which showed a small residual shunt flow which may be expected .", "angiogram in lateral plane showing a small residual left to right shunt with pda device in place before unscrewing of the stylet after a short while , the device suddenly embolised into the left pulmonary artery ( figure 2 ) .", "the patient had a brief episode of mild hypotension which improved spontaneously and his oxygen saturations remained static .", "an attempt was made to retrieve the device percutaneously by a snare and later by a bioptome but the trial was given up as the device tended to go more distally , in which case even surgical removal would be difficult .", "the patient was discussed with the cardiac surgeon who shifted the patient for emergency surgery .", "after considerable debate surgeon decided for a left thoracotomy hoping to avoid cardiopulmonary bypass and closed the ductus . later the left pulmonary artery was separated from the surrounding tissues and umbilical tape passed around it proximally for control of bleeding . a 1.5 cm longitudinal incision was made distal to the umbilical tape which was kept under traction and a long curved forceps passed toward the pulmonary hilum and the device retrieved .", "cine image in lateral plane showing device embolized into left pulmonary artery soon after its release from stylet . showing the pda device being retrieved from left pulmonary artery via left thoracotomy without cardiopulmonary bypass . in order to prevent such eventualities we tried to devise some alternative ways of sizing a pda .the", "first involves measuring a fully inflated tyshak pulmonary balloon outside the human body with a caliper and then inflating the balloon to the same pressure in the right ventricular outflow tract passed over a 0.35 \" extra - stiff wire , and repeating the measurements , and using the same calibration factor for sizing of pda visualized by aortic arch angiogram .", "previously , some radio opaque sizing devices have been used placing them outside human body which can have magnification errors if not placed exactly at the level of heart.2 the second method involves advancing a tyshak balloon 2 - 4 mm bigger than the echocardiographic dimension across the pda and inflating the balloon with dilute dye under low pressure without dilating the ductus so that it forms an impression of the ductus which is recorded in a cine mode in multiple planes , and the plane delineating the exact anatomy of the ductus is used for measurements after proper calibrations as mentioned above without the need for aortic arch angiogram ( figures 4 and 5 ) . showing measurement of diameter of inflated tyshak balloon by a caliper after inflating it with fixed quantity of contrast .", "angiogram in lateral plane showing an inflated tyshak balloon across the pda making an impression of the ductus ." ], [ "technological advances have made nonsurgical closure of the pda a simple and a routine percutaneous intervention .", "the use of the amplatzer device occluder ( ado ) has further simplified the method and improved the results with minimal complications .", "however there are situations where complications are encountered and surgical help is required to ameliorate them .", "faella and colleagues reported 15 procedure related complications in 316 patients which included hemolysis , left pulmonary artery artery stenosis , device protrusion into aorta causing coarctation , device misplacement and one death following device embolisation.2 late embolisation of the device to the left pulmonary artery has been reported with impaired left pulmonary perfusion six months after implantation requiring surgical removal .", "m vavuranakis et al reported severe hemolysis with jaundice , anemia and hemogloginura on the second day following deployment of smaller sized coil due to improper sizing which needed removal and replacement by an ado after repeat sizing using balloon tipped catheter.3 the complications can be reduced by proper expertise and optimal sizing of the ductus .", "sizing of atrial septal defect ( asd ) by inflating a balloon across the asd till a circumferential waist is created and measuring the waist ( stretched balloon diameter ) and also inflating the balloon outside by same amount of dye across sized rings , is a standard method.45 using a similar method for the pda is not routine since angiographic visualization is usually adequate .", "however making an impression of the ductus by inflating a balloon across it can give a detailed anatomy about the length and breadth of the ductus in multiple planes without the need for multiple dye injection and may help in the selection of proper sized device ." ] ]

[ "extrahepatic portal venous obstruction ( ehpvo ) is accompanied by replacement of the extrahepatic portal vein by a cavernoma with or without thrombosis of the intrahepatic portal , splenic , or superior mesenteric veins . in developing countries ,", "ehpvo has been reported to be the most common cause of upper gastrointestinal bleeding ( ugib ) in children ( 70% in some reports ) and is also a common cause of variceal bleeding in adults . in western countries , ehpvo is second only to cirrhosis as a cause of portal hypertension , but its relative incidence is much lower compared with that in the developing countries .", "its aetiology is still not clear but has been attributed to umbilical sepsis after birth with thrombosis extending to the portal system via the patent umbilical vein or portal pyaemia following intra - abdominal sepsis .", "however , notwithstanding a lack of knowledge about its cause , most children and adults with ehpvo are generally from the so - called lower economic strata .", "however , the outcome after a bleed is better compared to bleeding in cirrhotics ( if adequate blood replacement facilities are at hand ) , because patients with ehpvo have normal liver function ( and histology ) which helps them to sustain bleeding episodes without decompensation", ". however mortality rates of between 5 and 30% have been reported for a single bleeding episode because of the large volumes of blood lost in patients who do not have access to sophisticated medical facilities including blood transfusion . till the middle of the 20th century , surgery was the only treatment available for these patients . however , with the advent of endoscopic therapy , this soon became the predominant treatment modality for the control of acute bleeding and also an important method for the prevention of a repeated bleeding episode .", "the main disadvantages of endotherapy are that it requires multiple sessions and a long - term followup with a recurrence rate of up to 40% in some studies .", "because the prevalence of ehpvo is the highest in developing countries and the condition affects mainly the poor [ 2 , 19 ] , most of whom do not have access to blood transfusion facilities and are not treatment compliant , the benefits of using a less invasive procedure like endoscopic therapy must be weighed against surgery which , in the best centres carries an operative mortality of 1% , is a onetime treatment , is not associated with encephalopathy and followed by rebleeding rates of less than 10% .", "moreover , operations like a splenectomy and proximal lienorenal shunt eliminate a large painful spleen and hypersplenism and restore a normal growth pattern in children .", "thus , any new treatment for ehpvo must be compared with the results of shunt surgery which have stood the test of time especially if it has been performed by an experienced surgeon .", "a history of major upper gastrointestinal bleeding in a child who has oesophagogastric varices in endoscopy and normal liver function tests should raise the suspicion of extrahepatic portal venous obstruction .", "although a similar picture can also be present in well compensated child a , cirrhosis ehpvo is much more common in children especially in the developing world .", "investigations to confirm the diagnosis are usually simple and readily available at most centres . ", "massive splenomegaly is present in 90% of patients , and a complete blood count may reveal a low haemoglobin level and decreased total leukocyte and platelet counts due to hypersplenism in 50% .", "liver function tests are nearly always normal , unlike in cirrhotics , but in the long - term the prothrombin time and albumin levels may be deranged due to the prolonged decreased portal blood flow and hence decreased synthetic function . \n", "ultrasonic doppler ( usg doppler ) examination of the upper abdomen should be the first radiological investigation performed to confirm the diagnosis as it has a sensitivity of 7090% and a specificity of 99% in diagnosing ehpvo .", "the characteristic findings are the replacement of portal vein by multiple tortuous vessels , also known as cavernous transformation , with hepatopetal blood flow in the collaterals ( figure 1 ) .", "upper gastrointestinal endoscopy ( ugie ) is done to confirm the presence of varices and to grade their size .", "splenoportovenography by splenic puncture or selective coeliac / superior mesenteric angiography provides excellent imaging of the portal venous system but is invasive and has largely been replaced with computed tomography and magnetic resonance angiography . ", "ct arterial portography is highly accurate , not operator dependent , and useful in circumstances when bowel gas obscures the findings on ultrasound examination . however , its high cost , exposure to radiation , and the systemic toxicity of the contrast agents used are its main disadvantages ( figure 2 ) .", "mr angiography is noninvasive and has a diagnostic accuracy that is similar to ct scans .", "however , it has limited availability and also has a high cost . with newer mr techniques ,", "a clear portal venogram can be obtained and the direction and velocity of blood flow in the portal system can be determined . ", "it should be done only if a patient presents with abnormal lfts or has hepatic dysfunction due possibly to coincident hepatitis following repeated blood transfusions for previous bleeding episodes .", "in spite of many years of familiarity with the disease , the causal factors have not been established .", "umbilical sepsis at birth , portal pyaemia , and thrombosis , and so forth are rare clinical features .", "larroche in his landmark paper in 1970 described portal vein thrombosis occurring after umbilical vein catheterization , but most of these resolved within short period of time and did not progress to ehpvo .", "however , there seems to be a strong association with levels of hygiene and poverty as the disease seems to affect the socially disadvantaged and has all but disappeared in most western countries and japan where it was more common in the late nineteenth century .", "there have been occasional reports of tests for dysmorphic megakaryocytes and endogenous erythroid colony assessment as being sensitive in distinguishing these patients from a normal population , but these are not readily available and therefore not commonly used .", "tests for venous thromboembolism such as the presence of factor v leiden , protein c , s and antithrombin iii levels , and prothrombin gene mutation may also be positive in certain adult patients [ 23 , 24 ] but have not had a high yield in the indian scenario .", "variceal bleeding being the most common and most serious presentation of ehpvo , it remains the most common indication for treatment which should be tailored depending on the patient 's social circumstances and his or her access to medical facilities . before the 1980s ,", "conservative medical management was recommended [ 2629 ] which included bed rest and blood transfusions as it was thought that these children would eventually grow out of their disease possibly by forming more collaterals to decompress the raised portal venous pressure .", "however , when it was realized that a single episode of variceal bleeding could carry a mortality rate of up to 30% in western countries , most physicians now follow a more active approach . in india , basu followed 25 patients with ehpvo for five years who had not had any intervention and reported that all had died .", "the main treatment options available today , as with other forms of variceal bleeding , are pharmacotherapy with beta blockers , endoscopy , and surgery .", "prevention of a first episode of variceal bleeding with drugs has been well studied in adults with cirrhosis . in children with child a cirrhosis and portal hypertension , ozsoylu et al", "one of the most probable reasons may be that these patients usually present with bleeding as their first symptom ( rather than splenomegaly ) , and hence primary prevention is difficult to study .", "further studies are required to assess the role of drugs for primary prevention of variceal bleeding in ehpvo .", "secondary prophylaxis with beta blockers has been shown to be effective in reducing presinusoidal portal hypertension in animals and humans , but their efficacy in preventing variceal bleeding in ehpvo has not been proved .", "excellent results have been achieved using endoscopic therapy in the control of acute bleeding , and this has now become the therapeutic modality of choice in this situation with injection sclerotherapy and elastic band ligation being effective in 90% of cases . in", "those few patients who continue to bleed or are bleeding so massively that endoscopic control is not a possible surgical treatment in the form of portosystemic shunts or oesophagogastric devascularisation , it has been shown to be effective [ 3436 ] but carries mortality rates ranging from 1030% . ", "both cirrhotic and noncirrhotic children with portal hypertension found that 42% of patients with oesophageal varices who had no intervention presented with bleeding as compared to 24% patients in whom sclerotherapy was used for primary prevention .", "however , there was no difference in survival and patients with sclerotherapy had more bleeding episodes from ectopic sites after the procedure .", "but since few ehpvo patients in the developing world present before a bleeding episode , endoscopic therapy for primary prophylaxis has not become an established mode of treatment . ", "complete eradication of varices with endoscopic sclerotherapy ( est ) and variceal band ligation ( evl ) occurs in 8090% of patients with ehpvo . at present", ", endoscopic variceal eradication therapy is mainly indicated if it is used as a primary treatment modality , the vessels are too small for anastomosis , extensive thrombosis of the portal venous system which means there are no veins available for shunting , and in those patients who can not tolerate the surgical procedure due to underlying comorbidities . ", "it is used as a primary treatment modality , the vessels are too small for anastomosis , extensive thrombosis of the portal venous system which means there are no veins available for shunting , and in those patients who can not tolerate the surgical procedure due to underlying comorbidities . \n", "however it must be remembered that endotherapy does not relieve the underlying portal hypertension and may result in an increased incidence of gastric and ectopic varices .", "est is associated with higher rates of ulcer and stricture formation than evl which has the additional advantages of eradicating varices in fewer sessions . a randomized study by zargar et al .", "has shown superiority of evl over est , but others have found that evl alone may be associated with increased risk of recurrence of varices ( 40% in one study ) .", "poddar et al . in 2005 showed that evl + est compared to est alone was a better method in treatment of oesophageal varices in children with ehpvo because of fewer treatment sessions and fewer complications .", "variceal recurrence rates were low in both the groups over a followup of 27 months ( 6.6% in evl + est group as compared to 10% in the est group ) .", "further studies are , however , needed to establish the superior efficacy of evl + est over est or evl alone .", "however , est is still favoured over evl in many places due to its low cost . \n", "zargar et al . have shown 88% success with injection sclerotherapy in a followup of 15 yrs .", "most of their patients had recurrent bleeding in the first 4 yrs after variceal eradication which was also managed by endoscopic therapy , and therefore the authors recommended annual endoscopy for the first 4 yrs after variceal eradication .", "in contrast , a similar study from king 's college hospital in london , uk , had previously shown that after a mean followup of 8.7 yrs , recurrent bleeding occurred in 31% .", "we believe that it should be recommended as the treatment of choice for secondary prophylaxis in developing countries where the disease is more common and the accessibility to health care resources is poor . ", "surgical intervention in variceal bleeding in ehpvo is indicated if there isfailure of endoscopic management in acute variceal bleeding , bleeding not amenable to endoscopic treatment such as portal hypertensive gastropathy and ectopic varices , as a onetime treatment for secondary prophylaxis in those who have difficult access to specialized centresfor associated complications like portal biliopathy , growth retardation , hypersplenism , and massive splenomegaly leading to poor quality of life .", "failure of endoscopic management in acute variceal bleeding , bleeding not amenable to endoscopic treatment such as portal hypertensive gastropathy and ectopic varices , as a onetime treatment for secondary prophylaxis in those who have difficult access to specialized centres for associated complications like portal biliopathy , growth retardation , hypersplenism , and massive splenomegaly leading to poor quality of life . \n", "these procedures divert blood flow from the high pressure portal circulation to low pressure systemic circulation by creation of an anastomosis between a tributary of the portal vein ( splenic , superior mesenteric , and left gastric , left gastroepiploic ) and a systemic vein ( renal , inferior vena cava , and adrenal ) .", "the shunts may be selective ( i.e. , only decompressing the varices ) or nonselective ( decompressing the entire portal venous system ) .", "the main requirement for shunt procedure is the presence of a vessel free of thrombus and of sufficient size . for shunt to be effective and", "remain patent , it should be at least 10 mm in diameter although bismuth et al . and prasad et al . ", "have had patency rates ranging from 84 to 96% after anastomosing veins of down to 4 mm in diameter . \n", "these are the most commonly performed procedures and include proximal splenorenal ( psrs ) , mesocaval , and portacaval shunts . the initial results with nonselective shunts were not promising since shunt thrombosis and rebleed and encephalopathy occurred in a large proportion of patients [ 29 , 45 ] .", "but recent series have shown rebleed rates of 211% , a mortality rate of < 2% , and no postshunt encephalopathy [ 9 , 10 ] .", "orloff et al . in 1994 found similar results with proximal side - side splenorenal shunts with or without splenectomy and end - side cavomesenteric shunts .", "they showed survival rates of > 96% after 10 years and shunt thrombosis rates of < 2% over a 15 yr followup . ", "it is advantageous in that , along with diversion of blood flow to decrease portal pressure and control bleeding , it also relieves the patient from symptomatic enlarged spleen and the effects of hypersplenism .", "psrs has shown good long - term results . in a study by prasad et al .", ", the 15 yr survival was 95% in 160 patients of ehpvo treated with psrs with a rebleeding rate of 11% .", "the long - term outcomes with shunts are shown below ( table 2 ) . ", "in a recent prospective randomized study by wani et al . in which the authors compared endoscopic sclerotherapy and shunt surgery revealed that rebleeding rates were significantly lower in the shunt surgery group ( 3.3% versus 22.6% ) .", "treatment failure rates were also much less in the surgery group ( 6.7% versus 19.4% ) .", "a study by krishna et al . evaluating qol after endoscopic or surgical treatment of ehpvo showed that endoscopic variceal eradication had no significant effect on qol , but the postsurgery group had improvement in physical , psychosocial , and total qol scores .", "the risks of overwhelming postsplenectomy infection have been described , but many studies including those from india and mexico have shown low rates of postsplenectomy sepsis [ 9 , 48 , 49 ] . other disadvantage is rebleeding due to shunt thrombosis .", "rates of shunt thrombosis vary from 416% [ 811 ] , and rebleeding in these patients is usually easily controlled by endoscopic therapy .", "surgical shunts in cirrhotics have been shown to be associated with neurological disturbances . in ehpvo", ", however this complication is rare , but there have been occasional reports of abnormal findings on electroencephalography , late cns side effects , and emotional disorders even after 20 yrs .", "minimum hepatic encephalopathy ( mhe ) rates were higher in surgically shunted patients as compared to nonshunted children ( but the difference was not significant ) .", "shunt procedures , however , are not popular in the emergency setting because they are ( i ) time consuming , ( ii ) need technical expertise and associated with ( iii ) high rates of shunt thrombosis , and encephalopathy . however , the rate of shunt thrombosis depends on the experience of surgeon .", "a study by orloff et al . showed shunt thrombosis rates of 0.5% in emergencies . \n", "selective shunts aim to decompress only the gastrosplenic circulation leaving the blood flow to the liver intact , therefore , theoretically at least decreasing the rebleeding risk from oesophagogastric varices whilst preserving hepatopetal flow . the distal splenorenal shunt ( warren shunt ) is the most commonly performed selective shunt although other shunts also have been described like the left gastroepiploic to left renal vein and left gastric to left renal vein shunts .", "the patency rates were 92% , rebleeding rates of 12% , shunt dysfunction occurred in 25% , and encephalopathy in none of their patients .", "such shunts can not be performed in patients with thrombosis of the splenic vein or those who have a history of splenectomy . \n", "superina et al . in a study of 34 patients showed that mesenterico left portal vein bypass ( mlpvb ) or rex shunt was successful in 91% , and they concluded that it was a more physiological shunt for ehpvo .", "but it requires the presence of a patent superior mesenteric vein , intrahepatic left portal vein , and internal jugular vein .", "these procedures are indicated if ( i ) performed as salvage therapy in variceal bleeding not controlled with endoscopic measures , ( ii ) a suitable size vein is not available for a shunt procedure , ( iii ) surgical expertise for a shunt procedure is not available .", "splenectomy alone is not recommended since it does not decompress the portal circulation and also leads to thrombosis of splenic vein which thereafter can not be used for shunting later .", "mathur et al . evaluated the role of oesophagogastric devascularisation with or without gastro - oesophageal stapling in acute variceal bleeding . in their study , 20 patients had ehpvo and the operative mortality was 5% ; recurrent varices occurred in 5% with rebleeding in 11% .", "none of their patients had encephalopathy . in a retrospective study of 24 patients with ehpvo undergoing salvage surgery for variceal bleeding ( 13 had devascularisation procedures ; 11 had proximal splenorenal shunts ) , they achieved control of bleeding in 96% .", "retrospectively reported a four - year followup of 22 patients with noncirrhotic portal hypertension in whom the bleeding was not controlled with endoscopic therapy . in their study , the rebleeding rate was 10% and overall survival was 95% . \n", "both endoscopy and shunt surgeries have shown good long - term results in secondary prophylaxis .", "however , treatment should take into account the socioeconomic status of the patient and facilities available locally .", "splenectomy and proximal lienorenal shunt being a one - time procedure with , if performed by experienced surgeons , low mortality and occlusion rates and an absence of postprocedure encephalopathy should be considered as the main treatment in patients with difficult access to sophisticated medical facilities .", "the role of shunt surgery has expanded since it is also effective in correcting portal biliopathy , hypersplenism , and growth retardation and may improve the quality of life ." ]

[ "<S> extrahepatic portal venous obstruction , although rare in the western world , is a common cause of major and life threatening upper gastrointestinal bleeding among the poor in developing countries . </S>", "<S> patients have large spleens and stunted growth . the diagnosis is easily confirmed by doppler ultrasonography . </S>", "<S> endoscopy sclerotherapy is the best option for the control of acute variceal bleeding . for secondary prophylaxis of bleeding </S>", "<S> , the choice lies between repeated sclerotherapy and a portosystemic shunt . </S>", "<S> we believe that due consideration should be given to performing a splenectomy and a lienorenal shunt . </S>", "<S> performed by experienced surgeons , it carries a low operative mortality of 1% , a rebleeding rate of about 10% , removes the large spleen , reverses hypersplenism , and is not followed by portosystemic encephalopathy . </S>", "<S> most importantly , it is a onetime procedure particularly suited to those who have little access to blood transfusion and sophisticated medical facilities . </S>" ]

[ "1. Introduction", "2. Diagnosis", "3. Tests to Establish the Aetiopathogenesis of EHPVO ", "4. Treatment " ]

[ [ "extrahepatic portal venous obstruction ( ehpvo ) is accompanied by replacement of the extrahepatic portal vein by a cavernoma with or without thrombosis of the intrahepatic portal , splenic , or superior mesenteric veins . in developing countries ,", "ehpvo has been reported to be the most common cause of upper gastrointestinal bleeding ( ugib ) in children ( 70% in some reports ) and is also a common cause of variceal bleeding in adults . in western countries , ehpvo is second only to cirrhosis as a cause of portal hypertension , but its relative incidence is much lower compared with that in the developing countries .", "its aetiology is still not clear but has been attributed to umbilical sepsis after birth with thrombosis extending to the portal system via the patent umbilical vein or portal pyaemia following intra - abdominal sepsis .", "however , notwithstanding a lack of knowledge about its cause , most children and adults with ehpvo are generally from the so - called lower economic strata .", "however , the outcome after a bleed is better compared to bleeding in cirrhotics ( if adequate blood replacement facilities are at hand ) , because patients with ehpvo have normal liver function ( and histology ) which helps them to sustain bleeding episodes without decompensation", ". however mortality rates of between 5 and 30% have been reported for a single bleeding episode because of the large volumes of blood lost in patients who do not have access to sophisticated medical facilities including blood transfusion . till the middle of the 20th century , surgery was the only treatment available for these patients . however , with the advent of endoscopic therapy , this soon became the predominant treatment modality for the control of acute bleeding and also an important method for the prevention of a repeated bleeding episode .", "the main disadvantages of endotherapy are that it requires multiple sessions and a long - term followup with a recurrence rate of up to 40% in some studies .", "because the prevalence of ehpvo is the highest in developing countries and the condition affects mainly the poor [ 2 , 19 ] , most of whom do not have access to blood transfusion facilities and are not treatment compliant , the benefits of using a less invasive procedure like endoscopic therapy must be weighed against surgery which , in the best centres carries an operative mortality of 1% , is a onetime treatment , is not associated with encephalopathy and followed by rebleeding rates of less than 10% .", "moreover , operations like a splenectomy and proximal lienorenal shunt eliminate a large painful spleen and hypersplenism and restore a normal growth pattern in children .", "thus , any new treatment for ehpvo must be compared with the results of shunt surgery which have stood the test of time especially if it has been performed by an experienced surgeon ." ], [ "a history of major upper gastrointestinal bleeding in a child who has oesophagogastric varices in endoscopy and normal liver function tests should raise the suspicion of extrahepatic portal venous obstruction .", "although a similar picture can also be present in well compensated child a , cirrhosis ehpvo is much more common in children especially in the developing world .", "investigations to confirm the diagnosis are usually simple and readily available at most centres . ", "massive splenomegaly is present in 90% of patients , and a complete blood count may reveal a low haemoglobin level and decreased total leukocyte and platelet counts due to hypersplenism in 50% .", "liver function tests are nearly always normal , unlike in cirrhotics , but in the long - term the prothrombin time and albumin levels may be deranged due to the prolonged decreased portal blood flow and hence decreased synthetic function . \n", "ultrasonic doppler ( usg doppler ) examination of the upper abdomen should be the first radiological investigation performed to confirm the diagnosis as it has a sensitivity of 7090% and a specificity of 99% in diagnosing ehpvo .", "the characteristic findings are the replacement of portal vein by multiple tortuous vessels , also known as cavernous transformation , with hepatopetal blood flow in the collaterals ( figure 1 ) .", "upper gastrointestinal endoscopy ( ugie ) is done to confirm the presence of varices and to grade their size .", "splenoportovenography by splenic puncture or selective coeliac / superior mesenteric angiography provides excellent imaging of the portal venous system but is invasive and has largely been replaced with computed tomography and magnetic resonance angiography . ", "ct arterial portography is highly accurate , not operator dependent , and useful in circumstances when bowel gas obscures the findings on ultrasound examination . however , its high cost , exposure to radiation , and the systemic toxicity of the contrast agents used are its main disadvantages ( figure 2 ) .", "mr angiography is noninvasive and has a diagnostic accuracy that is similar to ct scans .", "however , it has limited availability and also has a high cost . with newer mr techniques ,", "a clear portal venogram can be obtained and the direction and velocity of blood flow in the portal system can be determined . ", "it should be done only if a patient presents with abnormal lfts or has hepatic dysfunction due possibly to coincident hepatitis following repeated blood transfusions for previous bleeding episodes ." ], [ "in spite of many years of familiarity with the disease , the causal factors have not been established .", "umbilical sepsis at birth , portal pyaemia , and thrombosis , and so forth are rare clinical features .", "larroche in his landmark paper in 1970 described portal vein thrombosis occurring after umbilical vein catheterization , but most of these resolved within short period of time and did not progress to ehpvo .", "however , there seems to be a strong association with levels of hygiene and poverty as the disease seems to affect the socially disadvantaged and has all but disappeared in most western countries and japan where it was more common in the late nineteenth century .", "there have been occasional reports of tests for dysmorphic megakaryocytes and endogenous erythroid colony assessment as being sensitive in distinguishing these patients from a normal population , but these are not readily available and therefore not commonly used .", "tests for venous thromboembolism such as the presence of factor v leiden , protein c , s and antithrombin iii levels , and prothrombin gene mutation may also be positive in certain adult patients [ 23 , 24 ] but have not had a high yield in the indian scenario ." ], [ "variceal bleeding being the most common and most serious presentation of ehpvo , it remains the most common indication for treatment which should be tailored depending on the patient 's social circumstances and his or her access to medical facilities . before the 1980s ,", "conservative medical management was recommended [ 2629 ] which included bed rest and blood transfusions as it was thought that these children would eventually grow out of their disease possibly by forming more collaterals to decompress the raised portal venous pressure .", "however , when it was realized that a single episode of variceal bleeding could carry a mortality rate of up to 30% in western countries , most physicians now follow a more active approach . in india , basu followed 25 patients with ehpvo for five years who had not had any intervention and reported that all had died .", "the main treatment options available today , as with other forms of variceal bleeding , are pharmacotherapy with beta blockers , endoscopy , and surgery .", "prevention of a first episode of variceal bleeding with drugs has been well studied in adults with cirrhosis . in children with child a cirrhosis and portal hypertension , ozsoylu et al", "one of the most probable reasons may be that these patients usually present with bleeding as their first symptom ( rather than splenomegaly ) , and hence primary prevention is difficult to study .", "further studies are required to assess the role of drugs for primary prevention of variceal bleeding in ehpvo .", "secondary prophylaxis with beta blockers has been shown to be effective in reducing presinusoidal portal hypertension in animals and humans , but their efficacy in preventing variceal bleeding in ehpvo has not been proved .", "excellent results have been achieved using endoscopic therapy in the control of acute bleeding , and this has now become the therapeutic modality of choice in this situation with injection sclerotherapy and elastic band ligation being effective in 90% of cases . in", "those few patients who continue to bleed or are bleeding so massively that endoscopic control is not a possible surgical treatment in the form of portosystemic shunts or oesophagogastric devascularisation , it has been shown to be effective [ 3436 ] but carries mortality rates ranging from 1030% . ", "both cirrhotic and noncirrhotic children with portal hypertension found that 42% of patients with oesophageal varices who had no intervention presented with bleeding as compared to 24% patients in whom sclerotherapy was used for primary prevention .", "however , there was no difference in survival and patients with sclerotherapy had more bleeding episodes from ectopic sites after the procedure .", "but since few ehpvo patients in the developing world present before a bleeding episode , endoscopic therapy for primary prophylaxis has not become an established mode of treatment . ", "complete eradication of varices with endoscopic sclerotherapy ( est ) and variceal band ligation ( evl ) occurs in 8090% of patients with ehpvo . at present", ", endoscopic variceal eradication therapy is mainly indicated if it is used as a primary treatment modality , the vessels are too small for anastomosis , extensive thrombosis of the portal venous system which means there are no veins available for shunting , and in those patients who can not tolerate the surgical procedure due to underlying comorbidities . ", "it is used as a primary treatment modality , the vessels are too small for anastomosis , extensive thrombosis of the portal venous system which means there are no veins available for shunting , and in those patients who can not tolerate the surgical procedure due to underlying comorbidities . \n", "however it must be remembered that endotherapy does not relieve the underlying portal hypertension and may result in an increased incidence of gastric and ectopic varices .", "est is associated with higher rates of ulcer and stricture formation than evl which has the additional advantages of eradicating varices in fewer sessions . a randomized study by zargar et al .", "has shown superiority of evl over est , but others have found that evl alone may be associated with increased risk of recurrence of varices ( 40% in one study ) .", "poddar et al . in 2005 showed that evl + est compared to est alone was a better method in treatment of oesophageal varices in children with ehpvo because of fewer treatment sessions and fewer complications .", "variceal recurrence rates were low in both the groups over a followup of 27 months ( 6.6% in evl + est group as compared to 10% in the est group ) .", "further studies are , however , needed to establish the superior efficacy of evl + est over est or evl alone .", "however , est is still favoured over evl in many places due to its low cost . \n", "zargar et al . have shown 88% success with injection sclerotherapy in a followup of 15 yrs .", "most of their patients had recurrent bleeding in the first 4 yrs after variceal eradication which was also managed by endoscopic therapy , and therefore the authors recommended annual endoscopy for the first 4 yrs after variceal eradication .", "in contrast , a similar study from king 's college hospital in london , uk , had previously shown that after a mean followup of 8.7 yrs , recurrent bleeding occurred in 31% .", "we believe that it should be recommended as the treatment of choice for secondary prophylaxis in developing countries where the disease is more common and the accessibility to health care resources is poor . ", "surgical intervention in variceal bleeding in ehpvo is indicated if there isfailure of endoscopic management in acute variceal bleeding , bleeding not amenable to endoscopic treatment such as portal hypertensive gastropathy and ectopic varices , as a onetime treatment for secondary prophylaxis in those who have difficult access to specialized centresfor associated complications like portal biliopathy , growth retardation , hypersplenism , and massive splenomegaly leading to poor quality of life .", "failure of endoscopic management in acute variceal bleeding , bleeding not amenable to endoscopic treatment such as portal hypertensive gastropathy and ectopic varices , as a onetime treatment for secondary prophylaxis in those who have difficult access to specialized centres for associated complications like portal biliopathy , growth retardation , hypersplenism , and massive splenomegaly leading to poor quality of life . \n", "these procedures divert blood flow from the high pressure portal circulation to low pressure systemic circulation by creation of an anastomosis between a tributary of the portal vein ( splenic , superior mesenteric , and left gastric , left gastroepiploic ) and a systemic vein ( renal , inferior vena cava , and adrenal ) .", "the shunts may be selective ( i.e. , only decompressing the varices ) or nonselective ( decompressing the entire portal venous system ) .", "the main requirement for shunt procedure is the presence of a vessel free of thrombus and of sufficient size . for shunt to be effective and", "remain patent , it should be at least 10 mm in diameter although bismuth et al . and prasad et al . ", "have had patency rates ranging from 84 to 96% after anastomosing veins of down to 4 mm in diameter . \n", "these are the most commonly performed procedures and include proximal splenorenal ( psrs ) , mesocaval , and portacaval shunts . the initial results with nonselective shunts were not promising since shunt thrombosis and rebleed and encephalopathy occurred in a large proportion of patients [ 29 , 45 ] .", "but recent series have shown rebleed rates of 211% , a mortality rate of < 2% , and no postshunt encephalopathy [ 9 , 10 ] .", "orloff et al . in 1994 found similar results with proximal side - side splenorenal shunts with or without splenectomy and end - side cavomesenteric shunts .", "they showed survival rates of > 96% after 10 years and shunt thrombosis rates of < 2% over a 15 yr followup . ", "it is advantageous in that , along with diversion of blood flow to decrease portal pressure and control bleeding , it also relieves the patient from symptomatic enlarged spleen and the effects of hypersplenism .", "psrs has shown good long - term results . in a study by prasad et al .", ", the 15 yr survival was 95% in 160 patients of ehpvo treated with psrs with a rebleeding rate of 11% .", "the long - term outcomes with shunts are shown below ( table 2 ) . ", "in a recent prospective randomized study by wani et al . in which the authors compared endoscopic sclerotherapy and shunt surgery revealed that rebleeding rates were significantly lower in the shunt surgery group ( 3.3% versus 22.6% ) .", "treatment failure rates were also much less in the surgery group ( 6.7% versus 19.4% ) .", "a study by krishna et al . evaluating qol after endoscopic or surgical treatment of ehpvo showed that endoscopic variceal eradication had no significant effect on qol , but the postsurgery group had improvement in physical , psychosocial , and total qol scores .", "the risks of overwhelming postsplenectomy infection have been described , but many studies including those from india and mexico have shown low rates of postsplenectomy sepsis [ 9 , 48 , 49 ] . other disadvantage is rebleeding due to shunt thrombosis .", "rates of shunt thrombosis vary from 416% [ 811 ] , and rebleeding in these patients is usually easily controlled by endoscopic therapy .", "surgical shunts in cirrhotics have been shown to be associated with neurological disturbances . in ehpvo", ", however this complication is rare , but there have been occasional reports of abnormal findings on electroencephalography , late cns side effects , and emotional disorders even after 20 yrs .", "minimum hepatic encephalopathy ( mhe ) rates were higher in surgically shunted patients as compared to nonshunted children ( but the difference was not significant ) .", "shunt procedures , however , are not popular in the emergency setting because they are ( i ) time consuming , ( ii ) need technical expertise and associated with ( iii ) high rates of shunt thrombosis , and encephalopathy . however , the rate of shunt thrombosis depends on the experience of surgeon .", "a study by orloff et al . showed shunt thrombosis rates of 0.5% in emergencies . \n", "selective shunts aim to decompress only the gastrosplenic circulation leaving the blood flow to the liver intact , therefore , theoretically at least decreasing the rebleeding risk from oesophagogastric varices whilst preserving hepatopetal flow . the distal splenorenal shunt ( warren shunt ) is the most commonly performed selective shunt although other shunts also have been described like the left gastroepiploic to left renal vein and left gastric to left renal vein shunts .", "the patency rates were 92% , rebleeding rates of 12% , shunt dysfunction occurred in 25% , and encephalopathy in none of their patients .", "such shunts can not be performed in patients with thrombosis of the splenic vein or those who have a history of splenectomy . \n", "superina et al . in a study of 34 patients showed that mesenterico left portal vein bypass ( mlpvb ) or rex shunt was successful in 91% , and they concluded that it was a more physiological shunt for ehpvo .", "but it requires the presence of a patent superior mesenteric vein , intrahepatic left portal vein , and internal jugular vein .", "these procedures are indicated if ( i ) performed as salvage therapy in variceal bleeding not controlled with endoscopic measures , ( ii ) a suitable size vein is not available for a shunt procedure , ( iii ) surgical expertise for a shunt procedure is not available .", "splenectomy alone is not recommended since it does not decompress the portal circulation and also leads to thrombosis of splenic vein which thereafter can not be used for shunting later .", "mathur et al . evaluated the role of oesophagogastric devascularisation with or without gastro - oesophageal stapling in acute variceal bleeding . in their study , 20 patients had ehpvo and the operative mortality was 5% ; recurrent varices occurred in 5% with rebleeding in 11% .", "none of their patients had encephalopathy . in a retrospective study of 24 patients with ehpvo undergoing salvage surgery for variceal bleeding ( 13 had devascularisation procedures ; 11 had proximal splenorenal shunts ) , they achieved control of bleeding in 96% .", "retrospectively reported a four - year followup of 22 patients with noncirrhotic portal hypertension in whom the bleeding was not controlled with endoscopic therapy . in their study , the rebleeding rate was 10% and overall survival was 95% . \n", "both endoscopy and shunt surgeries have shown good long - term results in secondary prophylaxis .", "however , treatment should take into account the socioeconomic status of the patient and facilities available locally .", "splenectomy and proximal lienorenal shunt being a one - time procedure with , if performed by experienced surgeons , low mortality and occlusion rates and an absence of postprocedure encephalopathy should be considered as the main treatment in patients with difficult access to sophisticated medical facilities .", "the role of shunt surgery has expanded since it is also effective in correcting portal biliopathy , hypersplenism , and growth retardation and may improve the quality of life ." ] ]

[ "cancer genomics has gone through a dramatic period of progress due to the availability of genome - wide technologies .", "( tcga , https://cancergenome.nih.gov/ ) and the international cancer genome consortium ( icgc , http://icgc.org/ ) , involve thousands of patients and have generated petabytes of data .", "one of the major assets of such projects is the public availability of the data allowing their integration with data from other initiatives . in that way", ", data from these initiatives can push a more focused and deeper analysis either in a specific gene set or in a specific cohort of patients / samples .", "the human surfaceome , the collection of cell surface proteins in human cells , has been defined and studied by us previously . by using bioinformatics pipeline and an experimental approach based either on real - time pcr or on other gene expression technologies , we were able to identify potential new biomarkers for few tumor types and have characterized new cell surface putative cancer - testis ( ct ) antigens [ 1 , 2 ] .", "relevant roles of surface proteins include nutrient and ion transport , adhesion to substrates , signaling , and intercellular interaction .", "due to these roles and their subcellular localization , easily accessible to therapeutic agents , surface proteins are important targets for cancer intervention . since our original publication few reports have further explored the human surfaceome [ 26 ] , mostly in the context of a mass - spectrometry - based characterization of the cell surface of tumor cells .", "data from tcga / icgc allow the development of new metrics that evaluate the frequency of gene alterations in different cancer types .", "recently , we developed a new scoring system for the identification and prioritization of cancer genes .", "the s - score method integrates information derived from different omics technologies to generate a gene - specific score that indicates whether that specific gene is a tumor suppressor ( negative s - score ) or an oncogene ( positive s - score ) .", "the numerical value indicates the frequency in which that gene is altered in the cohort of samples used in the calculation .", "we have used the s - score metric to identify cancer genes in a set of human homologs of yeast genes characterized as suppressors of genome instability in yeast .", "the availability of the s - score system provides a quantitative way to identify and prioritize cancer genes in a particular set of samples . here", ", we capitalize on the availability of data from the tcga project to further and deeper investigate the status of the human surfaceome in 15 tumor types , including gbm and colorectal and breast tumors , all analyzed in our previous publications [ 1 , 2 ] .", "this generated a pan - cancer landscape of the human surfaceome with the identification of shared and tumor - specific markers .", "furthermore , the use of the s - score system allowed us to identify gene signatures associated with overall survival in breast cancer patients .", "these signatures can be ultimately used in the development of new and more efficient diagnostic and therapeutic protocols .", "the protein - coding sequences of all human genes were obtained from the ncbi refseq ( release 64 ) .", "the illumina human bodymap 2.0 dataset was obtained from embl - ebi expression atlas ( https://www.ebi.ac.uk/gxa/experiments/e-mtab-513 ) .", "tcga data was retrieved from both , the tcga web site ( https://cancergenome.nih.gov/ ) and the cbio cancer genomics portal ( http://www.cbioportal.org/ ) . to predict plasma membrane subcellular localization ,", "the ncbi reference sequence dataset was submitted to tmhmm version 2.0 ( http://www.cbs.dtu.dk/services/tmhmm/ ) .", "all sequences containing at least one tm domain were selected . to avoid false positives , sequences containing only one tm domain in the first 50 residues , which could be a signal peptide , were excluded and classified as secreted protein .", "furthermore , sequences were also filtered based on the identification of signal peptide cleavage sites by signalp , release 4.1 ( http://www.cbs.dtu.dk/services/signalp/ ) . since tm domains are not exclusive to cell surface proteins , the sequences were grouped according to subcellular localization as defined by gene ontology .", "we excluded sequences that were exclusively located at the following cellular compartments : lysosome , endoplasmic reticulum , mitochondria , cytoskeleton , endosome , liposome , nucleolus , nucleus , and ribosome .", "this step was conducted using in - house perl scripts . to validate the obtained list of surfaceome genes", ", we classified these genes as belonging to the following classes : g - protein - coupled receptors ( gpcrs ) , solute carrier ( slc ) proteins , and cluster of differentiation ( cd ) antigens .", "the gpcr genes were obtained from gpcrdb ( http://gpcrdb.org ) , while cd and slc genes were collected from hgnc ( http://www.genenames.org/genefamilies/a-z#r ) . \n", "s - scores were calculated for the human surfaceome and for the 15 tumor types as previously defined .", "the distribution of s - scores was used to calculate z - scores for all genes using r statistical package .", "the go enrichment analyses of the surfaceome gene cluster were conducted using clusterprofiler , implemented in r , with p values < 0.01 as a cutoff .", "genes with extreme s - score ( s - score < 2 and > 2 for breast tumors ) were selected to test any putative association with overall survival in breast cancer samples derived from tcga ( without subtype distinction ) .", "the altered set comprised samples within which the respective genes were differentially expressed ( z - score > 2 or z - score < 2 , as reported by tcga ) , amplified or deleted , or presenting deleterious mutations ( nonsense , frameshift , and splice - site ) .", "after that , for each gene , the survival analysis was performed using the kaplan - meier method and the difference in survival curves was tested for statistical significance using the log rank test p value .", "we then selected a nonredundant set of 20 genes with the lowest p value ( cutoff of 0.05 ) and tested all possible groups of three genes .", "first of all , we decided to reannotate the set of human genes coding for putative cell surface proteins .", "this was required due to ( i ) an improvement in the annotation of gene and protein databases regarding a protein 's subcellular localization and ( ii ) the inclusion of new human genes in the reference sequence collection . the same approach used by da cunha et al .", "( 2009 ) generated now a set of 3,758 human genes , composing the human surfaceome ( figure 1 ) .", "the complete list of human genes coding for cell surface proteins is provided as supplementary table s1 , in supplementary material available online at http://dx.doi.org/10.1155/2016/8346198 . as expected , the great majority ( 85% ) of surfaceome genes present in our dataset in 2009 remained classified as such in 2016 .", "new genes were added ( 585 ) , mostly due to their inclusion in the reference sequence collection and some other genes ( 529 ) were excluded due mainly to new functional annotation that classified their protein products as belonging to other subcellular compartments . to assess the robustness of our approach", ", we performed the same analysis reported by us in our original 2009 paper checking the representation of three known families of cell surface proteins ( g - protein - coupled receptors ( gpcrs ) , solute carrier ( slc ) proteins and cluster of differentiation ( cd ) antigens ) .", "since these are large and well - studied families of cell surface proteins , we envisaged that they would be appropriate for a benchmark analysis .", "for gpcrs , 98% of their known members were represented in our dataset . for slc proteins and cd antigens we found 77% and 88% represented in the surfaceome set , respectively .", "overall , 90% of members of these three families were represented in our present surfaceome set , compared to 83% in our previous analysis .", "this improvement is expected due to a better annotation of the sequences in public databases .", "capitalizing on the availability of surfaceome sets derived from mass - spectrometry analysis , we decided to compare our dataset to the dataset from bausch - fluck et al . .", "although this type of comparison is problematic for different reasons , including ( i ) the nonexhaustive nature of the wet - based approach ( due to the method itself and the samples screened ) and ( ii ) the different premises of both methods ( the requirement of at least one tm domain per protein in our pipeline and the lack of such requirement in which allowed the authors to characterize gpi - anchored proteins , e.g. ) , the analysis may be illuminating in the sense that it can highlight important differences in both methodologies .", "we found that 66.6% ( 664 out of 996 ) of the proteins classified by bausch - fluck et al . ", "were present in our dataset while only 17.6% ( 664 out of 3758 ) of our proteins were present in their dataset .", "this was expected due to the issues raised above . to illustrate the complex nature of this comparison , 23.8% of all cell surface proteins found in have no tm domain , as identified by tmhmm .", "next , the s - score method was used to identify cancer genes within the surfaceome set .", "s - score threshold was defined for each tumor type as the s - score representing the average s - score plus / minus three standard deviations ( z - score 3 or 3 ) . the list of all cancer genes coding for cell surface proteins in all 15 tumors types is shown in supplementary table s1 . using the above z - score threshold", ", we found 248 surfaceome genes classified as a cancer gene in at least one tumor type . in the heatmap representation of the surfaceome cancer genes ( figure 2(a ) ) we can clearly identify three distinct clusters based on the s - score values for all 15 tumor types .", "although all three groups have a variety of oncogenes and suppressors , some features deserve further comments .", "for example , the first group is mainly composed of suppressors , especially in melanoma and colorectal and lung adenocarcinoma and uterine corpus endometrial carcinoma .", "genes in this group include several members of the cadherin superfamily ( pcdhgb3 , pcdha2 , pcdha7 , pcdh15 , pcdhgb5 , pcdh11x , pcdhac1 , fat1 , fat2 , and fat4 ) .", "there is a set of oncogenes in group 2 shared by almost all tumors and involving 30 genes , including ephb1 and ephb3 .", "there is no clear pattern in group 3 and oncogenes and suppressors seem to be distributed evenly across all tumors . to better understand the pattern presented in figure 2(a ) , we performed a gene ontology ( go ) enrichment analysis ( using the biological process ", "ontology ) for the three different clusters . as expected , all three groups shared go categories associated with the cell surface such as transmembrane transport and cell surface receptor signaling pathway .", "more interestingly , however , is the fact that specific go categories were enriched in individual groups ( figure 2(b ) ) .", "go categories exclusively found in group 1 were clearly associated with nervous system including neuromuscular process ; memory ; and neuronal action potential . ", "the same pattern was observed for group 2 although the go categories represented different aspects of nervous system including the following : sensory perception of pain and other categories related to axonogenesis .", "regarding group 3 , go analysis lent further support for the current concept of ion transport associated with cancer , including manganese ion transport .", " additionally , this group presented genes related to antigen processing and presentation , highlighting that the interplay between immune and tumor cells is complex .", "several of the identified surfaceome cancer genes are known for their involvement in different aspects of cancer biology , especially the ones classified in group 2 .", "abcc5 , a cell surface transporter , was involved in resistance to anticancer drugs and overexpression of atp11b has been linked to drug resistance in ovarian cancer .", "both genes were regarded as oncogenic by our work especially in lung squamous cell carcinoma and ovarian cancer .", "on the other hand , ephb3 has already been suggested as a candidate target gene for both lung small cell carcinoma and colorectal cancer .", "finally , a transferrin receptor ( tfrc ) has shown an increased expression in many malignant tumors and was also found to be highly oncogenic in this work . as previously discussed by us ,", "the s - score method allows the prioritization of cancer genes based on clinical parameters .", "for example , we have identified genes associated with both short- and long - term survival in ovarian cancer . to test whether we could identify genes in the surfaceome set associated with clinical parameters , we decided to look at overall survival in breast tumors , since this type of tumor is the one with the largest cohort in tcga .", "for this specific analysis a more relaxed threshold ( z - score < 2 or > 2 ) was used to classify a gene as a cancer gene in breast tumor to increase the number of genes under test without compromising the quality of the classification ( a heatmap , similar to figure 2(a ) and generated using the dataset with a more relaxed threshold , is presented in supplementary figure 1 ) . for each surfaceome gene classified as oncogene or suppressor in breast tumor", ", we split the breast cancer samples into two groups : altered ( genes with differential expression , genes amplified / deleted , or genes mutated ) and unaltered . for each gene , the two groups", "were then compared by a kaplan - meier analysis to evaluate whether they had significantly different overall survival .", "twenty - three genes , seven oncogenes and 16 suppressors , were significantly ( p - value < 0.05 ) associated with differences in overall survival in breast cancer patients ( supplementary table 2 ) .", "these genes are involved in cell adhesion and ion transport , two of the main categories enriched in our gene ontology analysis .", "next , all possible combinations of these genes were similarly tested for differences in overall survival .", "although we found several combinations with statistically significant differences in overall survival , we have focused on wnt5a , cnga2 , and igsf9b due to statistical significance ( it is the most significant three - gene set in supplementary table 2 ) and novelty .", "patients in which one of the three genes was altered had a significantly shorter survival ( p value = 1.82e)compared to patients where these three genes were unaltered ( figure 3 ) .", "the wnt5a , cnga2 , and igsf9b genes have negative s - scores in breast cancer ( 2.59 , 3.39 , and 2.56 , resp . ) , demonstrating a tumor suppressor profile .", ", the loss of wnt5a has been associated with poor prognosis , in agreement with the suppressor status defined by the respective s - score . on the other hand ,", "wnt5a was recently reported to promote cancer cell lines invasion and proliferation , a feature typical of oncogenes .", "wnt5a is present in pathways where wnt signaling is involved through interaction with frizzleds ( fzd10 , e.g. ) and dishevelled .", "cnga2 , a homotetrameric channel in olfactory sensory neurons , has not been reported in association with cancer .", "however , cnga2 represents the alpha subunit of a cyclic nucleotide - gated olfactory channel possessing a role in calcium signaling pathway acting through calmodulin - like 6 and calcium / calmodulin - dependent protein kinase iv directly involved with protein kinase a ( pka ) , a biological target in cancer therapy .", "igsf9b was only recently identified as an inhibitory synaptic adhesion molecule and no link with cancer was found in the literature .", "we have updated the set of human genes coding for cell surface proteins , the human surfaceome .", "using tcga data for 15 tumor types and a new method of cancer genes classification that integrates information from different omics technologies and allows a ranking based on clinical parameters ( s - score ) , we identified several potential therapeutic targets within the surfaceome set .", "furthermore , we present evidence that a three - gene set wnt5a , cnga2 and igsf9b was associated with shorter survival in breast cancer patients .", "our results clearly show the importance of large - scale genomics datasets from cancer patient cohorts , like the one provided by tcga .", "we envisage that the data we provide here will be extremely useful to researchers who aim to characterize cell surface targets for a variety of tumor types ." ]

[ "<S> it is estimated that 10 to 20% of all genes in the human genome encode cell surface proteins and due to their subcellular localization these proteins represent excellent targets for cancer diagnosis and therapeutics . </S>", "<S> therefore , a precise characterization of the surfaceome set in different types of tumor is needed . </S>", "<S> using tcga data from 15 different tumor types and a new method to identify cancer genes , the s - score , we identified several potential therapeutic targets within the surfaceome set . </S>", "<S> this allowed us to expand a previous analysis from us and provided a clear characterization of the human surfaceome in the tumor landscape . </S>", "<S> moreover , we present evidence that a three - gene set wnt5a , cnga2 , and igsf9b can be used as a signature associated with shorter survival in breast cancer patients . </S>", "<S> the data made available here will help the community to develop more efficient diagnostic and therapeutic tools for a variety of tumor types . </S>" ]

[ "1. Introduction", "2. Material and Methods", "3. Results and Discussion", "4. Conclusion" ]

[ [ "cancer genomics has gone through a dramatic period of progress due to the availability of genome - wide technologies .", "( tcga , https://cancergenome.nih.gov/ ) and the international cancer genome consortium ( icgc , http://icgc.org/ ) , involve thousands of patients and have generated petabytes of data .", "one of the major assets of such projects is the public availability of the data allowing their integration with data from other initiatives . in that way", ", data from these initiatives can push a more focused and deeper analysis either in a specific gene set or in a specific cohort of patients / samples .", "the human surfaceome , the collection of cell surface proteins in human cells , has been defined and studied by us previously . by using bioinformatics pipeline and an experimental approach based either on real - time pcr or on other gene expression technologies , we were able to identify potential new biomarkers for few tumor types and have characterized new cell surface putative cancer - testis ( ct ) antigens [ 1 , 2 ] .", "relevant roles of surface proteins include nutrient and ion transport , adhesion to substrates , signaling , and intercellular interaction .", "due to these roles and their subcellular localization , easily accessible to therapeutic agents , surface proteins are important targets for cancer intervention . since our original publication few reports have further explored the human surfaceome [ 26 ] , mostly in the context of a mass - spectrometry - based characterization of the cell surface of tumor cells .", "data from tcga / icgc allow the development of new metrics that evaluate the frequency of gene alterations in different cancer types .", "recently , we developed a new scoring system for the identification and prioritization of cancer genes .", "the s - score method integrates information derived from different omics technologies to generate a gene - specific score that indicates whether that specific gene is a tumor suppressor ( negative s - score ) or an oncogene ( positive s - score ) .", "the numerical value indicates the frequency in which that gene is altered in the cohort of samples used in the calculation .", "we have used the s - score metric to identify cancer genes in a set of human homologs of yeast genes characterized as suppressors of genome instability in yeast .", "the availability of the s - score system provides a quantitative way to identify and prioritize cancer genes in a particular set of samples . here", ", we capitalize on the availability of data from the tcga project to further and deeper investigate the status of the human surfaceome in 15 tumor types , including gbm and colorectal and breast tumors , all analyzed in our previous publications [ 1 , 2 ] .", "this generated a pan - cancer landscape of the human surfaceome with the identification of shared and tumor - specific markers .", "furthermore , the use of the s - score system allowed us to identify gene signatures associated with overall survival in breast cancer patients .", "these signatures can be ultimately used in the development of new and more efficient diagnostic and therapeutic protocols ." ], [ "the protein - coding sequences of all human genes were obtained from the ncbi refseq ( release 64 ) .", "the illumina human bodymap 2.0 dataset was obtained from embl - ebi expression atlas ( https://www.ebi.ac.uk/gxa/experiments/e-mtab-513 ) .", "tcga data was retrieved from both , the tcga web site ( https://cancergenome.nih.gov/ ) and the cbio cancer genomics portal ( http://www.cbioportal.org/ ) . to predict plasma membrane subcellular localization ,", "the ncbi reference sequence dataset was submitted to tmhmm version 2.0 ( http://www.cbs.dtu.dk/services/tmhmm/ ) .", "all sequences containing at least one tm domain were selected . to avoid false positives , sequences containing only one tm domain in the first 50 residues , which could be a signal peptide , were excluded and classified as secreted protein .", "furthermore , sequences were also filtered based on the identification of signal peptide cleavage sites by signalp , release 4.1 ( http://www.cbs.dtu.dk/services/signalp/ ) . since tm domains are not exclusive to cell surface proteins , the sequences were grouped according to subcellular localization as defined by gene ontology .", "we excluded sequences that were exclusively located at the following cellular compartments : lysosome , endoplasmic reticulum , mitochondria , cytoskeleton , endosome , liposome , nucleolus , nucleus , and ribosome .", "this step was conducted using in - house perl scripts . to validate the obtained list of surfaceome genes", ", we classified these genes as belonging to the following classes : g - protein - coupled receptors ( gpcrs ) , solute carrier ( slc ) proteins , and cluster of differentiation ( cd ) antigens .", "the gpcr genes were obtained from gpcrdb ( http://gpcrdb.org ) , while cd and slc genes were collected from hgnc ( http://www.genenames.org/genefamilies/a-z#r ) . \n", "s - scores were calculated for the human surfaceome and for the 15 tumor types as previously defined .", "the distribution of s - scores was used to calculate z - scores for all genes using r statistical package .", "the go enrichment analyses of the surfaceome gene cluster were conducted using clusterprofiler , implemented in r , with p values < 0.01 as a cutoff .", "genes with extreme s - score ( s - score < 2 and > 2 for breast tumors ) were selected to test any putative association with overall survival in breast cancer samples derived from tcga ( without subtype distinction ) .", "the altered set comprised samples within which the respective genes were differentially expressed ( z - score > 2 or z - score < 2 , as reported by tcga ) , amplified or deleted , or presenting deleterious mutations ( nonsense , frameshift , and splice - site ) .", "after that , for each gene , the survival analysis was performed using the kaplan - meier method and the difference in survival curves was tested for statistical significance using the log rank test p value .", "we then selected a nonredundant set of 20 genes with the lowest p value ( cutoff of 0.05 ) and tested all possible groups of three genes ." ], [ "first of all , we decided to reannotate the set of human genes coding for putative cell surface proteins .", "this was required due to ( i ) an improvement in the annotation of gene and protein databases regarding a protein 's subcellular localization and ( ii ) the inclusion of new human genes in the reference sequence collection . the same approach used by da cunha et al .", "( 2009 ) generated now a set of 3,758 human genes , composing the human surfaceome ( figure 1 ) .", "the complete list of human genes coding for cell surface proteins is provided as supplementary table s1 , in supplementary material available online at http://dx.doi.org/10.1155/2016/8346198 . as expected , the great majority ( 85% ) of surfaceome genes present in our dataset in 2009 remained classified as such in 2016 .", "new genes were added ( 585 ) , mostly due to their inclusion in the reference sequence collection and some other genes ( 529 ) were excluded due mainly to new functional annotation that classified their protein products as belonging to other subcellular compartments . to assess the robustness of our approach", ", we performed the same analysis reported by us in our original 2009 paper checking the representation of three known families of cell surface proteins ( g - protein - coupled receptors ( gpcrs ) , solute carrier ( slc ) proteins and cluster of differentiation ( cd ) antigens ) .", "since these are large and well - studied families of cell surface proteins , we envisaged that they would be appropriate for a benchmark analysis .", "for gpcrs , 98% of their known members were represented in our dataset . for slc proteins and cd antigens we found 77% and 88% represented in the surfaceome set , respectively .", "overall , 90% of members of these three families were represented in our present surfaceome set , compared to 83% in our previous analysis .", "this improvement is expected due to a better annotation of the sequences in public databases .", "capitalizing on the availability of surfaceome sets derived from mass - spectrometry analysis , we decided to compare our dataset to the dataset from bausch - fluck et al . .", "although this type of comparison is problematic for different reasons , including ( i ) the nonexhaustive nature of the wet - based approach ( due to the method itself and the samples screened ) and ( ii ) the different premises of both methods ( the requirement of at least one tm domain per protein in our pipeline and the lack of such requirement in which allowed the authors to characterize gpi - anchored proteins , e.g. ) , the analysis may be illuminating in the sense that it can highlight important differences in both methodologies .", "we found that 66.6% ( 664 out of 996 ) of the proteins classified by bausch - fluck et al . ", "were present in our dataset while only 17.6% ( 664 out of 3758 ) of our proteins were present in their dataset .", "this was expected due to the issues raised above . to illustrate the complex nature of this comparison , 23.8% of all cell surface proteins found in have no tm domain , as identified by tmhmm .", "next , the s - score method was used to identify cancer genes within the surfaceome set .", "s - score threshold was defined for each tumor type as the s - score representing the average s - score plus / minus three standard deviations ( z - score 3 or 3 ) . the list of all cancer genes coding for cell surface proteins in all 15 tumors types is shown in supplementary table s1 . using the above z - score threshold", ", we found 248 surfaceome genes classified as a cancer gene in at least one tumor type . in the heatmap representation of the surfaceome cancer genes ( figure 2(a ) ) we can clearly identify three distinct clusters based on the s - score values for all 15 tumor types .", "although all three groups have a variety of oncogenes and suppressors , some features deserve further comments .", "for example , the first group is mainly composed of suppressors , especially in melanoma and colorectal and lung adenocarcinoma and uterine corpus endometrial carcinoma .", "genes in this group include several members of the cadherin superfamily ( pcdhgb3 , pcdha2 , pcdha7 , pcdh15 , pcdhgb5 , pcdh11x , pcdhac1 , fat1 , fat2 , and fat4 ) .", "there is a set of oncogenes in group 2 shared by almost all tumors and involving 30 genes , including ephb1 and ephb3 .", "there is no clear pattern in group 3 and oncogenes and suppressors seem to be distributed evenly across all tumors . to better understand the pattern presented in figure 2(a ) , we performed a gene ontology ( go ) enrichment analysis ( using the biological process ", "ontology ) for the three different clusters . as expected , all three groups shared go categories associated with the cell surface such as transmembrane transport and cell surface receptor signaling pathway .", "more interestingly , however , is the fact that specific go categories were enriched in individual groups ( figure 2(b ) ) .", "go categories exclusively found in group 1 were clearly associated with nervous system including neuromuscular process ; memory ; and neuronal action potential . ", "the same pattern was observed for group 2 although the go categories represented different aspects of nervous system including the following : sensory perception of pain and other categories related to axonogenesis .", "regarding group 3 , go analysis lent further support for the current concept of ion transport associated with cancer , including manganese ion transport .", " additionally , this group presented genes related to antigen processing and presentation , highlighting that the interplay between immune and tumor cells is complex .", "several of the identified surfaceome cancer genes are known for their involvement in different aspects of cancer biology , especially the ones classified in group 2 .", "abcc5 , a cell surface transporter , was involved in resistance to anticancer drugs and overexpression of atp11b has been linked to drug resistance in ovarian cancer .", "both genes were regarded as oncogenic by our work especially in lung squamous cell carcinoma and ovarian cancer .", "on the other hand , ephb3 has already been suggested as a candidate target gene for both lung small cell carcinoma and colorectal cancer .", "finally , a transferrin receptor ( tfrc ) has shown an increased expression in many malignant tumors and was also found to be highly oncogenic in this work . as previously discussed by us ,", "the s - score method allows the prioritization of cancer genes based on clinical parameters .", "for example , we have identified genes associated with both short- and long - term survival in ovarian cancer . to test whether we could identify genes in the surfaceome set associated with clinical parameters , we decided to look at overall survival in breast tumors , since this type of tumor is the one with the largest cohort in tcga .", "for this specific analysis a more relaxed threshold ( z - score < 2 or > 2 ) was used to classify a gene as a cancer gene in breast tumor to increase the number of genes under test without compromising the quality of the classification ( a heatmap , similar to figure 2(a ) and generated using the dataset with a more relaxed threshold , is presented in supplementary figure 1 ) . for each surfaceome gene classified as oncogene or suppressor in breast tumor", ", we split the breast cancer samples into two groups : altered ( genes with differential expression , genes amplified / deleted , or genes mutated ) and unaltered . for each gene , the two groups", "were then compared by a kaplan - meier analysis to evaluate whether they had significantly different overall survival .", "twenty - three genes , seven oncogenes and 16 suppressors , were significantly ( p - value < 0.05 ) associated with differences in overall survival in breast cancer patients ( supplementary table 2 ) .", "these genes are involved in cell adhesion and ion transport , two of the main categories enriched in our gene ontology analysis .", "next , all possible combinations of these genes were similarly tested for differences in overall survival .", "although we found several combinations with statistically significant differences in overall survival , we have focused on wnt5a , cnga2 , and igsf9b due to statistical significance ( it is the most significant three - gene set in supplementary table 2 ) and novelty .", "patients in which one of the three genes was altered had a significantly shorter survival ( p value = 1.82e)compared to patients where these three genes were unaltered ( figure 3 ) .", "the wnt5a , cnga2 , and igsf9b genes have negative s - scores in breast cancer ( 2.59 , 3.39 , and 2.56 , resp . ) , demonstrating a tumor suppressor profile .", ", the loss of wnt5a has been associated with poor prognosis , in agreement with the suppressor status defined by the respective s - score . on the other hand ,", "wnt5a was recently reported to promote cancer cell lines invasion and proliferation , a feature typical of oncogenes .", "wnt5a is present in pathways where wnt signaling is involved through interaction with frizzleds ( fzd10 , e.g. ) and dishevelled .", "cnga2 , a homotetrameric channel in olfactory sensory neurons , has not been reported in association with cancer .", "however , cnga2 represents the alpha subunit of a cyclic nucleotide - gated olfactory channel possessing a role in calcium signaling pathway acting through calmodulin - like 6 and calcium / calmodulin - dependent protein kinase iv directly involved with protein kinase a ( pka ) , a biological target in cancer therapy .", "igsf9b was only recently identified as an inhibitory synaptic adhesion molecule and no link with cancer was found in the literature ." ], [ "we have updated the set of human genes coding for cell surface proteins , the human surfaceome .", "using tcga data for 15 tumor types and a new method of cancer genes classification that integrates information from different omics technologies and allows a ranking based on clinical parameters ( s - score ) , we identified several potential therapeutic targets within the surfaceome set .", "furthermore , we present evidence that a three - gene set wnt5a , cnga2 and igsf9b was associated with shorter survival in breast cancer patients .", "our results clearly show the importance of large - scale genomics datasets from cancer patient cohorts , like the one provided by tcga .", "we envisage that the data we provide here will be extremely useful to researchers who aim to characterize cell surface targets for a variety of tumor types ." ] ]

[ "a recent national cross - sectional survey showed that the overall prevalence of diabetes was estimated to be 11.6% in the chinese adult population .", "diabetes is a progressive disease , due in part to the loss of -cell function , with the reduction in function probably commencing 10 to 12 years prior to diagnosis and aggravated by increasing fasting plasma glucose ( fpg ) levels .", "furthermore , east asian patients with type 2 diabetes have a higher risk of developing renal complications than europeans , and , with regard to cardiovascular complications , a predisposition for developing strokes .", "these results denote that more studies are needed to explain these interethnic differences , and , most importantly , effective strategies are required to facilitate earlier identification and prevention to combat these growing disease burdens , particularly in china .", "methylenetetrahydrofolate reductase ( mthfr ) is the main regulatory enzyme for folate / homocysteine metabolism .", "mthfr converts 5,10-methylene - tetrahydrofolate ( thf ) into 5-methyl - thf , the dominant circulating form of folate .", "the 5-methyl - thf product donates a methyl group to homocysteine in the generation of s - adenosylmethionine , a major source of methyl groups used for dna methylation .", "polymorphism of mthfr 677ct leads to a reduction in enzyme activity , resulting in increased concentrations of plasma homocysteine and lower levels of serum folate , and thereby confers a higher risk for stroke , particularly in those with low folate intake .", "a recent meta - analysis of case control studies found that the homocysteine concentration in individuals with type 2 diabetes was significantly higher than that in control subjects ( 0.94 mol / l , 95% confidence interval [ ci ] 0.401.48 ) , and the odds ratio ( or ) associated with type 2 diabetes for mthfr 677tt relative to 677cc was 1.38 ( 95% ci 1.181.62 ) .", "our recent study also showed that participants with mthfr 677tt genotype had a higher prevalence of diabetes than those with cc genotype .", "furthermore , plasma homocysteine levels were significantly inversely associated with estimated glomerular filtration rate ( egfr ) , and mthfr 677tt genotype was associated with a higher risk for decreased kidney function independent of homocysteine levels . however ,", "no prospective studies investigating the association between mthfr 677ct polymorphism , homocysteine , renal function , and new - onset diabetes ( nod ) have been conducted .", "furthermore , the frequency of the mthfr 677 tt genotype , which showed marked ethnic variations , was more common in china than in most of the european countries .", "the chinese also had higher homocysteine levels , lower folate concentrations , and did not have a policy of mandatory folic acid fortification in food .", "these characteristics make the chinese population particularly suited for testing the possible genotype ( mthfr 677ct polymorphism ) and phenotype ( homocysteine and egfr levels)-disease association using the same analytical framework .", "therefore , in the current study , we aimed to evaluate the effect of mthfr 677ct polymorphism , homocysteine , and egfr levels on the risk of nod in a rural chinese cohort , and to identify the possible effect modifiers .", "our findings may possibly provide insights into the ethnic differences in diabetic complications seen between east asian patients and europeans .", "this report followed the strobe ( strengthening the reporting of observational studies in epidemiology ) statement for cohort studies .", "study participants were from an epidemiological study of metabolic syndrome conducted during 2003 to 2005 in rural communities ( dongzhi and wangjiang ) in anqing , anhui province of china .", "detailed protocol and the details regarding study population and data collection have been previously described . briefly , 6301 of the study subjects from dongzhi community who received baseline screening examination were invited for a follow - up visit in 2011 , and 2901 ( 46% ) of them responded .", "the nonresponders did not differ from the responders substantially with respect to baseline characteristics ( data not shown ) .", "this study was approved by the institutional review boards from the nanfang hospital in guangzhou and the institute of biomedicine in the anhui medical university . written informed consent was obtained from each study participant .", "fpg , total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein - cholesterol ( hdl - c ) , and homocysteine were measured on the hitachi 7020 automatic analyzer ( hitachi , tokyo , japan ) .", "serum creatinine concentrations were determined using an enzymatic method ( sarcosine oxidase - peroxidase anti - peroxidase ) .", "plasma insulin was measured using an enhanced chemiluminescence method on an elecsys 2010 system ( roche , basel , switzerland ) .", "mthfr 677ct genotype was determined by taqman assay designed and manufactured by applied biosystems ( foster city , ca ) .", "the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated from the fasting concentrations of insulin and glucose using the following formula : fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 .", "we excluded participants with self - reported physician diagnosis of diabetes or fpg 7.0 mmol / l at baseline in the final analysis .", "nod was defined as fpg 7.0 mmol / l or self - reported physician diagnosis of diabetes at follow - up year .", "current alcohol drinking was defined as drinking alcohol at least once per week in the last year .", "the question about insomnia was phrased as follows : do you frequently suffer from insomnia ? , and a choice of 3 responses ( frequent : almost every week , medium : 13 times per month , and seldom ) was provided .", "baseline characteristics are presented as mean or percentage , except for fasting insulin and homocysteine , which are presented as median ( q1q3 ) because of the skewed distribution .", "between - group differences in baseline characteristics were tested using the student t test , the signed rank test , or the test , accordingly .", "the effects of mthfr 677ct polymorphism ( cc , ct , and tt ) , homocysteine ( < 10 , 1016 , and 16 ", "mol / l ) , and egfr ( 120 , 90120 , < 90 ml / min/1.73 m ) on the risks of nod in males and females were estimated using logistic regression models with adjustment for baseline covariates including age ( year ) , baseline fpg ( 5.6 vs < 5.6 mmol / l ) , body mass index ( bmi , 23 vs < 23 kg / m ) , blood pressure ( < 130/85 , 130/85140/90 , 140/90 mm hg ) , tg ( 1.7 vs", "mmol / l ) , hdl - c ( < 1.3 [ females]/1.0 [ males ] vs 1.3/1.0 ", "mmol / l ) , current cigarette smoking , current alcohol drinking , and insomnia ( frequent , medium , and seldom ) .", "r software , version 2.15.1 ( http://www.r-project.org ) was used to perform all statistical analyses .", "overall , 2901 subjects were revisited . in this report , study participants with self - reported physician diagnosis of cardiovascular disease ( cvd , n = 28 ) , diabetes ( n = 10 ) , hypertension ( n = 124 ) , or with any missing data ( n = 186 ) regarding baseline values for age , cigarette smoking status , alcohol drinking status , fasting glucose , homocysteine , mthfr 677ct polymorphism or insomnia , or with any missing data ( n = 61 ) for fpg or self - reported physician diagnosis of diabetes at follow up , or who had an fpg value of 7.0 mmol / l at baseline(n = 70 ) were excluded .", "the prevalence of mthfr 677 cc , 677 ct , and 677 tt genotypes was 36.8% , 47.4% , and 15.8% , respectively .", "this population had no significant deviations in genotype distributions from expected hardy weinberg equilibrium .", "those with mthfr 677 tt genotype had significantly higher homocysteine levels ( median , 12.0 in males and 10.1 ", "mol / l in females ) than those with ct ( 10.4 and 9.1 mol / l ) or cc ( 10.4 and 8.6 ", "mol / l ) genotype ( p < 0.05 for either of these genotypes in males or females ) .", "furthermore , there was an inverse association between homocysteine and egfr levels in males ( r = 0.43 , p < 0.001 ) and females ( r = 0.63 , p < 0.001 ) .", "the follow - up time ranged from 5.81 to 7.57 years , with a mean of 7.02 ( sd 0.31 ) years .", "there were no significant differences in the follow - up time between subjects with and without nod in males ( 6.97 [ 0.31 ] vs 7.02 [ 0.31 ] , p = 0.137 ) and females ( 7.05 [ 0.34 ] vs 7.03 [ 0.32 ] , p = 0.506 )", ". the baseline characteristics of the study subjects stratified by nod status and sex are summarized in table 1 .", "nod patients had significantly higher tg , bmi , insulin levels , and homa - ir compared with those without in both males and females . however , nod patients had significantly higher mthfr 677 tt genotype rates in females only ( table 1 ) .", "baseline characteristics according to nod status by sex the incidence rates of nod in males and females were 8.0% and 7.0% , respectively . compared with subjects with mthfr 677 cc genotype , those with tt genotype had a higher risk of nod in females ( or 2.78 , 95% ci 1.395.56 ) but not in males ( 0.80 , 0.401.61 , p for interaction = 0.008 ) .", "< 10 mol / l ) was not associated with nod in males ( 0.88 , 0.421.85 ) or females ( 1.52 , 0.653.57 ) . however", ", mildly decreased egfr ( < 90 vs 90120 ml / min/1.73 m ) was associated with nod mainly in males ( 1.96 , 1.013.78 ; females , 0.74 , 0.321.72 , p for interaction = 0.134 ) ( table 2 ) .", "relationship of mthfr 677ct polymorphism , baseline homocysteine levels , and egfr with the risk of nod in the full models , we observed that higher tg , bmi , fpg levels , and insomnia , in addition to mthfr c677 t polymorphism in females , and higher fpg levels and insomnia in addition to lower egfr levels in males remained independent risk factors for the risk of nod ( data not shown ) . in the stratified analyses ,", "the mthfr 677ct polymorphism ( cc , ct , and tt genotypes ) was more strongly associated with the risk of nod among females with higher bmi ( 23 vs < 23 kg / m , p for interaction = 0.009 ) or lower hdl - c levels ( < 1.3 vs 1.3 mmol / l , p for interaction = 0.015 ) .", "however , the association of mthfr 677ct polymorphism with increased risks of nod in females appeared to be similar among subgroups classified according to baseline homocysteine ( 10 vs < 10 mol / l , p for interaction = 0.061 ) , egfr ( < 110 vs 110 ml / min/1.73 m , p for interaction = 0.229 ) , fpg ( 5.6 vs", "< 5.6 mmol / l , p for interaction = 0.635 ) , tg levels ( 1.7 vs < 1.7 mmol / l , p for interaction = 0.189 ) , or insomnia ( frequent vs medium or seldom , p for interaction = 0.831 ) . furthermore", ", similar trends for egfr category and increased risk of nod in males were observed among subgroups stratified by insomnia ( p for interaction = 0.946 ) , baseline fpg ( p for interaction = 0.977 ) , homocysteine levels ( p for interaction = 0.573 ) , or mthfr 677ct polymorphism ( p for interaction = 0.880 ) ( table 3 ) .", "stratified analysis of multivariate ors for nod among 1172 women according to mthfr 677ct polymorphism further adjustment for homa - ir ( n = 2144 ) did not substantially change the relationship of mthfr 677ct polymorphism ( tt vs cc , 2.20 ; 1.024.73 ) in females or egfr levels ( < 90 vs 90120 ml / min/1.73 m , 1.85 ; 0.923.71 ) in males with the risk of nod .", "previous studies have shown conflicting results regarding the homocysteine levels in patients with diabetes . a recent meta - analysis of 14 case control studies found that the mean homocysteine concentration was greater in patients with type 2 diabetes than in control subjects . however , the 3 studies that contributed most to the overall estimate included patients with type 2 diabetes accompanied by varying degrees of kidney disorders .", "these results suggest that it may be renal dysfunction but not diabetes that mostly explains the difference in homocysteine levels between type 2 diabetes patients and control subjects in this meta - analysis .", "in fact , in our present prospective study , we did not find significant associations between homocysteine levels and nod in males or females .", "however , consistent with this meta - analysis , female subjects with tt genotype in the current study had a higher risk of nod ( tt vs cc genotype , 2.78 ; 1.395.56 ) . to explain the significant relationship of mthfr gene 677ct polymorphism ( but not homocysteine levels ) with the risk of nod , we speculate that , first , the homocysteine levels ( median 10.7 ", "mol / l in females ) in the present study was possibly not high enough to cause obvious organ damage .", "second , plasma homocysteine may just serve as a reliable functional marker of folate status , and folate may have other actions , including antioxidant actions , effects on cofactor availability , or direct interactions with the enzyme endothelial no synthase , in addition to homocysteine lowering that influence health status .", "a previous report showed that folic acid given to patients with coronary heart disease resulted in improvement in endothelial function without any change in plasma homocysteine concentration .", "unfortunately , we were not able to directly examine the association between mthfr 677ct polymorphism , folate , and the risk of nod in the current study due to the lack of baseline folate data .", "additional studies are required to further address this topic and to evaluate the role of folic acid supplementation in reducing the risk of diabetes , particularly in populations without folic acid fortification .", "meanwhile , we did not have enough information to explain the sex - specific effect of mthfr 677ct polymorphism , which may relate to the possible role of hormones in folate / homocysteine metabolism .", "our current study detected a detrimentally interactive effect between the mthfr 677ct polymorphism and higher bmi ( 23 vs < 23 ", "kg / m ) , or lower hdl - c ( < 1.3 vs 1.3 ", "obese subjects without diabetes have been shown to exhibit an enhanced rate of glucose production .", "we hypothesize that a higher bmi state may augment the mthfr 677ct polymorphism - mediated risk of nod .", "furthermore , mthfr 677ct polymorphism appeared to modify the efficacy of the drug pravastatin in reducing risk of cardiovascular events .", "a significantly protective effect against coronary heart disease ( hazard ratio [ hr ] 0.71 , 95% ci 0.580.87 ) was shown in subjects with cc genotype but not in subjects with ct ( hr 1.25 , 95% ci 0.971.61 ) or tt genotype ( hr 0.80 , 95% ci 0.501.28 , p for interaction = 0.004 ) .", "consistently , we also observed an interaction between the mthfr 677ct polymorphism and hdl - c levels on the risk of nod .", "these results suggest that an investigation of the possible modifying effect of mthfr 677ct polymorphism on cvd associated with hdl - c increasing therapy maybe provide some clues regarding the conflicting results gathered from these kinds of trials .", "overall , our results indicate that the mthfr 677tt genotype , along with homocysteine , bmi , and hdl - c levels , may help to identify apparently healthy females at increased risk for diabetes .", "menon et al reported that hyperhomocysteinemia did not appear to be a risk factor for all - cause or cvd mortality , and prior studies demonstrating an association between homocysteine and cvd risk may have inadequately adjusted for the confounding effects of kidney function .", "we also observed that mildly decreased egfr ( < 90 ml / min/1.73 m ) , but not homocysteine , was associated with the risk of new - onset disease .", "nevertheless , more studies are needed to verify if our findings can be generalized to other populations or ethnicities , particularly individuals with lower egfr levels or obvious chronic kidney disease .", "the strengths of our study include the 7-year prospective follow - up of middle - aged rural chinese men and women , and the comprehensive adjustments for the major traditional risk factors for nod , including baseline fpg and ir .", "first , we did not measure glycosylated hemoglobin levels or perform glucose - tolerance tests .", "second , we did not have information regarding family history of diabetes . however , further adjustment for family history of diabetes did not change the significant association between mthfr 677ct polymorphism and prevalence of diabetes in our previous study .", "lastly , previous studies have shown that nonalcoholic fatty liver disease ( nafld ) was strongly associated with ir and diabetes .", "unfortunately , we were not able to examine this effect in the current study due to the lack of data on nafld .", ", we found that individuals with mthfr 677tt genotype had a significantly higher risk of nod among females , particularly in those with higher bmi or lower hdl - c levels .", "the higher risk of nod associated with mildly decreased egfr ( < 90 ml / min/1.73 m ) also warrants more investigation .", "our study findings , if further confirmed , will provide new strategies to identify apparently healthy population at increased risk for diabetes .", "furthermore , considering the higher frequency of mthfr 677tt genotype in china and the higher stroke risk associated with tt genotype , our results also provide some explanations for the ethnic differences in diabetic complications seen between east asian patients and europeans ." ]

[ "<S> abstracteast asian patients with diabetes have a higher risk for renal complications and strokes than europeans . </S>", "<S> we aimed to evaluate the effect of methylenetetrahydrofolate reductase ( mthfr ) gene 677ct polymorphism , which was associated with a higher stroke risk and was common in the chinese population , as well as homocysteine and estimated glomerular filtration rate ( egfr ) levels on the risk of new - onset diabetes ( nod).a total of 2422 subjects without diabetes were followed - up for 7 years . </S>", "<S> nod was defined as fasting plasma glucose 7.0 mmol / l or self - reported physician diagnosis of diabetes.compared with subjects with mthfr 677cc genotype , those with tt genotype had a higher risk of nod in females ( odds ratio 2.78 , 95% confidence interval 1.395.56 ) but not in males ( 0.80 , 0.401.61 , p for interaction = 0.008 ) . </S>", "<S> furthermore , mthfr 677ct polymorphism was more strongly associated with the risk of nod among females with higher body mass index ( bmi , 23 vs < 23 kg / m2 , p for interaction = 0.009 ) or lower high - density lipoprotein - cholesterol ( hdl - c , < 1.3 vs 1.3 mmol / l , p for interaction = 0.015 ) levels . hyperhomocysteinemia ( 16 vs < 10 </S>", "<S> mol / l ) was not significantly associated with nod in males ( 0.88 , 0.421.85 ) or females ( 1.52 , 0.653.57 ) . </S>", "<S> however , mildly decreased egfr ( < 90 vs 90120 ml / min/1.73 m2 ) was associated with nod mainly in males ( 1.96 , 1.013.78 ; females , 0.74 , 0.321.72 , p for interaction = 0.134).females with mthfr 677tt genotype had a significantly higher risk of nod , particularly those with higher bmi or low hdl - c levels . the higher risk of nod associated with mildly decreased egfr also warrants more investigation . </S>", "<S> our results provide insights into the ethnic differences of diabetic complications between east asian patients and europeans . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "STUDY POPULATION AND DATA COLLECTION", "OUTCOMES", "STATISTICAL ANALYSIS", "RESULTS", "DISCUSSION" ]

[ [ "a recent national cross - sectional survey showed that the overall prevalence of diabetes was estimated to be 11.6% in the chinese adult population .", "diabetes is a progressive disease , due in part to the loss of -cell function , with the reduction in function probably commencing 10 to 12 years prior to diagnosis and aggravated by increasing fasting plasma glucose ( fpg ) levels .", "furthermore , east asian patients with type 2 diabetes have a higher risk of developing renal complications than europeans , and , with regard to cardiovascular complications , a predisposition for developing strokes .", "these results denote that more studies are needed to explain these interethnic differences , and , most importantly , effective strategies are required to facilitate earlier identification and prevention to combat these growing disease burdens , particularly in china .", "methylenetetrahydrofolate reductase ( mthfr ) is the main regulatory enzyme for folate / homocysteine metabolism .", "mthfr converts 5,10-methylene - tetrahydrofolate ( thf ) into 5-methyl - thf , the dominant circulating form of folate .", "the 5-methyl - thf product donates a methyl group to homocysteine in the generation of s - adenosylmethionine , a major source of methyl groups used for dna methylation .", "polymorphism of mthfr 677ct leads to a reduction in enzyme activity , resulting in increased concentrations of plasma homocysteine and lower levels of serum folate , and thereby confers a higher risk for stroke , particularly in those with low folate intake .", "a recent meta - analysis of case control studies found that the homocysteine concentration in individuals with type 2 diabetes was significantly higher than that in control subjects ( 0.94 mol / l , 95% confidence interval [ ci ] 0.401.48 ) , and the odds ratio ( or ) associated with type 2 diabetes for mthfr 677tt relative to 677cc was 1.38 ( 95% ci 1.181.62 ) .", "our recent study also showed that participants with mthfr 677tt genotype had a higher prevalence of diabetes than those with cc genotype .", "furthermore , plasma homocysteine levels were significantly inversely associated with estimated glomerular filtration rate ( egfr ) , and mthfr 677tt genotype was associated with a higher risk for decreased kidney function independent of homocysteine levels . however ,", "no prospective studies investigating the association between mthfr 677ct polymorphism , homocysteine , renal function , and new - onset diabetes ( nod ) have been conducted .", "furthermore , the frequency of the mthfr 677 tt genotype , which showed marked ethnic variations , was more common in china than in most of the european countries .", "the chinese also had higher homocysteine levels , lower folate concentrations , and did not have a policy of mandatory folic acid fortification in food .", "these characteristics make the chinese population particularly suited for testing the possible genotype ( mthfr 677ct polymorphism ) and phenotype ( homocysteine and egfr levels)-disease association using the same analytical framework .", "therefore , in the current study , we aimed to evaluate the effect of mthfr 677ct polymorphism , homocysteine , and egfr levels on the risk of nod in a rural chinese cohort , and to identify the possible effect modifiers .", "our findings may possibly provide insights into the ethnic differences in diabetic complications seen between east asian patients and europeans ." ], [ "this report followed the strobe ( strengthening the reporting of observational studies in epidemiology ) statement for cohort studies ." ], [ "study participants were from an epidemiological study of metabolic syndrome conducted during 2003 to 2005 in rural communities ( dongzhi and wangjiang ) in anqing , anhui province of china .", "detailed protocol and the details regarding study population and data collection have been previously described . briefly , 6301 of the study subjects from dongzhi community who received baseline screening examination were invited for a follow - up visit in 2011 , and 2901 ( 46% ) of them responded .", "the nonresponders did not differ from the responders substantially with respect to baseline characteristics ( data not shown ) .", "this study was approved by the institutional review boards from the nanfang hospital in guangzhou and the institute of biomedicine in the anhui medical university . written informed consent was obtained from each study participant .", "fpg , total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein - cholesterol ( hdl - c ) , and homocysteine were measured on the hitachi 7020 automatic analyzer ( hitachi , tokyo , japan ) .", "serum creatinine concentrations were determined using an enzymatic method ( sarcosine oxidase - peroxidase anti - peroxidase ) .", "plasma insulin was measured using an enhanced chemiluminescence method on an elecsys 2010 system ( roche , basel , switzerland ) .", "mthfr 677ct genotype was determined by taqman assay designed and manufactured by applied biosystems ( foster city , ca ) .", "the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated from the fasting concentrations of insulin and glucose using the following formula : fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 ." ], [ "we excluded participants with self - reported physician diagnosis of diabetes or fpg 7.0 mmol / l at baseline in the final analysis .", "nod was defined as fpg 7.0 mmol / l or self - reported physician diagnosis of diabetes at follow - up year ." ], [ "current alcohol drinking was defined as drinking alcohol at least once per week in the last year .", "the question about insomnia was phrased as follows : do you frequently suffer from insomnia ? , and a choice of 3 responses ( frequent : almost every week , medium : 13 times per month , and seldom ) was provided .", "baseline characteristics are presented as mean or percentage , except for fasting insulin and homocysteine , which are presented as median ( q1q3 ) because of the skewed distribution .", "between - group differences in baseline characteristics were tested using the student t test , the signed rank test , or the test , accordingly .", "the effects of mthfr 677ct polymorphism ( cc , ct , and tt ) , homocysteine ( < 10 , 1016 , and 16 ", "mol / l ) , and egfr ( 120 , 90120 , < 90 ml / min/1.73 m ) on the risks of nod in males and females were estimated using logistic regression models with adjustment for baseline covariates including age ( year ) , baseline fpg ( 5.6 vs < 5.6 mmol / l ) , body mass index ( bmi , 23 vs < 23 kg / m ) , blood pressure ( < 130/85 , 130/85140/90 , 140/90 mm hg ) , tg ( 1.7 vs", "mmol / l ) , hdl - c ( < 1.3 [ females]/1.0 [ males ] vs 1.3/1.0 ", "mmol / l ) , current cigarette smoking , current alcohol drinking , and insomnia ( frequent , medium , and seldom ) .", "r software , version 2.15.1 ( http://www.r-project.org ) was used to perform all statistical analyses ." ], [ "overall , 2901 subjects were revisited . in this report , study participants with self - reported physician diagnosis of cardiovascular disease ( cvd , n = 28 ) , diabetes ( n = 10 ) , hypertension ( n = 124 ) , or with any missing data ( n = 186 ) regarding baseline values for age , cigarette smoking status , alcohol drinking status , fasting glucose , homocysteine , mthfr 677ct polymorphism or insomnia , or with any missing data ( n = 61 ) for fpg or self - reported physician diagnosis of diabetes at follow up , or who had an fpg value of 7.0 mmol / l at baseline(n = 70 ) were excluded .", "the prevalence of mthfr 677 cc , 677 ct , and 677 tt genotypes was 36.8% , 47.4% , and 15.8% , respectively .", "this population had no significant deviations in genotype distributions from expected hardy weinberg equilibrium .", "those with mthfr 677 tt genotype had significantly higher homocysteine levels ( median , 12.0 in males and 10.1 ", "mol / l in females ) than those with ct ( 10.4 and 9.1 mol / l ) or cc ( 10.4 and 8.6 ", "mol / l ) genotype ( p < 0.05 for either of these genotypes in males or females ) .", "furthermore , there was an inverse association between homocysteine and egfr levels in males ( r = 0.43 , p < 0.001 ) and females ( r = 0.63 , p < 0.001 ) .", "the follow - up time ranged from 5.81 to 7.57 years , with a mean of 7.02 ( sd 0.31 ) years .", "there were no significant differences in the follow - up time between subjects with and without nod in males ( 6.97 [ 0.31 ] vs 7.02 [ 0.31 ] , p = 0.137 ) and females ( 7.05 [ 0.34 ] vs 7.03 [ 0.32 ] , p = 0.506 )", ". the baseline characteristics of the study subjects stratified by nod status and sex are summarized in table 1 .", "nod patients had significantly higher tg , bmi , insulin levels , and homa - ir compared with those without in both males and females . however , nod patients had significantly higher mthfr 677 tt genotype rates in females only ( table 1 ) .", "baseline characteristics according to nod status by sex the incidence rates of nod in males and females were 8.0% and 7.0% , respectively . compared with subjects with mthfr 677 cc genotype , those with tt genotype had a higher risk of nod in females ( or 2.78 , 95% ci 1.395.56 ) but not in males ( 0.80 , 0.401.61 , p for interaction = 0.008 ) .", "< 10 mol / l ) was not associated with nod in males ( 0.88 , 0.421.85 ) or females ( 1.52 , 0.653.57 ) . however", ", mildly decreased egfr ( < 90 vs 90120 ml / min/1.73 m ) was associated with nod mainly in males ( 1.96 , 1.013.78 ; females , 0.74 , 0.321.72 , p for interaction = 0.134 ) ( table 2 ) .", "relationship of mthfr 677ct polymorphism , baseline homocysteine levels , and egfr with the risk of nod in the full models , we observed that higher tg , bmi , fpg levels , and insomnia , in addition to mthfr c677 t polymorphism in females , and higher fpg levels and insomnia in addition to lower egfr levels in males remained independent risk factors for the risk of nod ( data not shown ) . in the stratified analyses ,", "the mthfr 677ct polymorphism ( cc , ct , and tt genotypes ) was more strongly associated with the risk of nod among females with higher bmi ( 23 vs < 23 kg / m , p for interaction = 0.009 ) or lower hdl - c levels ( < 1.3 vs 1.3 mmol / l , p for interaction = 0.015 ) .", "however , the association of mthfr 677ct polymorphism with increased risks of nod in females appeared to be similar among subgroups classified according to baseline homocysteine ( 10 vs < 10 mol / l , p for interaction = 0.061 ) , egfr ( < 110 vs 110 ml / min/1.73 m , p for interaction = 0.229 ) , fpg ( 5.6 vs", "< 5.6 mmol / l , p for interaction = 0.635 ) , tg levels ( 1.7 vs < 1.7 mmol / l , p for interaction = 0.189 ) , or insomnia ( frequent vs medium or seldom , p for interaction = 0.831 ) . furthermore", ", similar trends for egfr category and increased risk of nod in males were observed among subgroups stratified by insomnia ( p for interaction = 0.946 ) , baseline fpg ( p for interaction = 0.977 ) , homocysteine levels ( p for interaction = 0.573 ) , or mthfr 677ct polymorphism ( p for interaction = 0.880 ) ( table 3 ) .", "stratified analysis of multivariate ors for nod among 1172 women according to mthfr 677ct polymorphism further adjustment for homa - ir ( n = 2144 ) did not substantially change the relationship of mthfr 677ct polymorphism ( tt vs cc , 2.20 ; 1.024.73 ) in females or egfr levels ( < 90 vs 90120 ml / min/1.73 m , 1.85 ; 0.923.71 ) in males with the risk of nod ." ], [ "previous studies have shown conflicting results regarding the homocysteine levels in patients with diabetes . a recent meta - analysis of 14 case control studies found that the mean homocysteine concentration was greater in patients with type 2 diabetes than in control subjects . however , the 3 studies that contributed most to the overall estimate included patients with type 2 diabetes accompanied by varying degrees of kidney disorders .", "these results suggest that it may be renal dysfunction but not diabetes that mostly explains the difference in homocysteine levels between type 2 diabetes patients and control subjects in this meta - analysis .", "in fact , in our present prospective study , we did not find significant associations between homocysteine levels and nod in males or females .", "however , consistent with this meta - analysis , female subjects with tt genotype in the current study had a higher risk of nod ( tt vs cc genotype , 2.78 ; 1.395.56 ) . to explain the significant relationship of mthfr gene 677ct polymorphism ( but not homocysteine levels ) with the risk of nod , we speculate that , first , the homocysteine levels ( median 10.7 ", "mol / l in females ) in the present study was possibly not high enough to cause obvious organ damage .", "second , plasma homocysteine may just serve as a reliable functional marker of folate status , and folate may have other actions , including antioxidant actions , effects on cofactor availability , or direct interactions with the enzyme endothelial no synthase , in addition to homocysteine lowering that influence health status .", "a previous report showed that folic acid given to patients with coronary heart disease resulted in improvement in endothelial function without any change in plasma homocysteine concentration .", "unfortunately , we were not able to directly examine the association between mthfr 677ct polymorphism , folate , and the risk of nod in the current study due to the lack of baseline folate data .", "additional studies are required to further address this topic and to evaluate the role of folic acid supplementation in reducing the risk of diabetes , particularly in populations without folic acid fortification .", "meanwhile , we did not have enough information to explain the sex - specific effect of mthfr 677ct polymorphism , which may relate to the possible role of hormones in folate / homocysteine metabolism .", "our current study detected a detrimentally interactive effect between the mthfr 677ct polymorphism and higher bmi ( 23 vs < 23 ", "kg / m ) , or lower hdl - c ( < 1.3 vs 1.3 ", "obese subjects without diabetes have been shown to exhibit an enhanced rate of glucose production .", "we hypothesize that a higher bmi state may augment the mthfr 677ct polymorphism - mediated risk of nod .", "furthermore , mthfr 677ct polymorphism appeared to modify the efficacy of the drug pravastatin in reducing risk of cardiovascular events .", "a significantly protective effect against coronary heart disease ( hazard ratio [ hr ] 0.71 , 95% ci 0.580.87 ) was shown in subjects with cc genotype but not in subjects with ct ( hr 1.25 , 95% ci 0.971.61 ) or tt genotype ( hr 0.80 , 95% ci 0.501.28 , p for interaction = 0.004 ) .", "consistently , we also observed an interaction between the mthfr 677ct polymorphism and hdl - c levels on the risk of nod .", "these results suggest that an investigation of the possible modifying effect of mthfr 677ct polymorphism on cvd associated with hdl - c increasing therapy maybe provide some clues regarding the conflicting results gathered from these kinds of trials .", "overall , our results indicate that the mthfr 677tt genotype , along with homocysteine , bmi , and hdl - c levels , may help to identify apparently healthy females at increased risk for diabetes .", "menon et al reported that hyperhomocysteinemia did not appear to be a risk factor for all - cause or cvd mortality , and prior studies demonstrating an association between homocysteine and cvd risk may have inadequately adjusted for the confounding effects of kidney function .", "we also observed that mildly decreased egfr ( < 90 ml / min/1.73 m ) , but not homocysteine , was associated with the risk of new - onset disease .", "nevertheless , more studies are needed to verify if our findings can be generalized to other populations or ethnicities , particularly individuals with lower egfr levels or obvious chronic kidney disease .", "the strengths of our study include the 7-year prospective follow - up of middle - aged rural chinese men and women , and the comprehensive adjustments for the major traditional risk factors for nod , including baseline fpg and ir .", "first , we did not measure glycosylated hemoglobin levels or perform glucose - tolerance tests .", "second , we did not have information regarding family history of diabetes . however , further adjustment for family history of diabetes did not change the significant association between mthfr 677ct polymorphism and prevalence of diabetes in our previous study .", "lastly , previous studies have shown that nonalcoholic fatty liver disease ( nafld ) was strongly associated with ir and diabetes .", "unfortunately , we were not able to examine this effect in the current study due to the lack of data on nafld .", ", we found that individuals with mthfr 677tt genotype had a significantly higher risk of nod among females , particularly in those with higher bmi or lower hdl - c levels .", "the higher risk of nod associated with mildly decreased egfr ( < 90 ml / min/1.73 m ) also warrants more investigation .", "our study findings , if further confirmed , will provide new strategies to identify apparently healthy population at increased risk for diabetes .", "furthermore , considering the higher frequency of mthfr 677tt genotype in china and the higher stroke risk associated with tt genotype , our results also provide some explanations for the ethnic differences in diabetic complications seen between east asian patients and europeans ." ] ]

[ "soft tissue coverage for wounds remains a difficult management problem for patients sustaining traumatic injury and burns .", "there are several methods to achieve wound coverage secondary healing , primary suturing , skin grafting , and flap surgeries as described in reconstruction ladder .", "a skin graft is the most commonly used modality for coverage of wounds in reconstructive plastic surgery . the skin graft needs to undergo various stages of healing for a good take on the recipient bed .", "there are different methods employed to secure the graft to recipient bed for a few days with a basic idea to ensure that the graft is not elevated off the bed by formation of haematoma / seroma under it .", "repeated tie over dressings are required in situations where the dressing needs to be changed more frequently as in cases of infected raw area , bleeding tendency , patients on anticoagulant drugs , and in convex areas of body such as buttocks , breast , and the scalp , where the dressing is difficult to secure .", "in this novel method , a sterile sample container was cut at its upper part [ figures 1 and 2 ] .", "the skin graft was applied on the raw area and fixed with skin staplers and tie over sutures . once paraffin gauze and adequate padding is applied on the raw area , the tie over threads were passed from inside out of the container [ figures 3 and 4 ] and pulled at the appropriate tension to keep the dressing in place .", "the lid of the container was tightened to complete the dressing ensuring that the graft was maintained in close approximation with the wound surface .", "sterile plastic container upper part of the sterile container is cut tie over threads being passed from inside out the lid is tightened over the dressing the dressing can be changed repeatedly with sterile precautions depending on the requirement , as an outpatient procedure by unscrewing the lid [ figures 5 - 8 ] which can then be easily reapplied .", "the procedure can be used on wounds of any size by changing the size of the sterile container chosen .", "post - toilet mastectomy raw area covered with the skin graft the tie over dressing applied in ot tie over dressing repeated in an outpatient department we have used this method in eight cases of the post toilet mastectomy raw areas with very good results [ figures 9 and 10 ] . post - toilet mastectomy raw area", "skin graft once applied has to be covered with petrolatum gauze to avoid its separation from the wound bed at the time of change of dressing .", "an ideal method of graft fixation should be simple , rapid , repeatable , able to be performed in the outpatient department , prevent hematoma or seroma formation , soak the exudates well , and allow the graft bed to be inspected easily .", "there are multiple methods of securing dressings over the skin graft , some of them can be applied only once and some can be repeated .", "the dressings that can be applied only once are like foam , hydro cellular dressing ( highly absorbent and can be easily changed ) , negative pressure therapy dressing ( stabilises the graft , increases the vascularity of bed , takes away toxic chemicals ) , and gas bag ( transparent , can see graft and monitor any haematoma ) .", "these traditional methods can stabilize the graft till the first dressing post operatively . in some contaminated wounds ,", "the dressing needs to be removed earlier , especially if there is drainage or foul smell .", "this approach may also be proper for graft , used to cover defects of some anatomical regions with increases risk of contamination , such as perineal , axillary , and genital or it can be used in areas where base of wound is difficult to immobilize like breast / pectoral region .", "repeated tie over dressings can be done by keeping interrupted sutures long to be used as tie over dressings .", "these ties over dressing can be made of sutures or rubber bands . when taking a tie over stitch , both", "the threads can be left long and only one thread is tied at a time , the other thread is left long for next time .", "these techniques are difficult for small dressings especially the bra hooks ; the silk loops method is very cumbersome and takes long time to do .", "the novel method being discussed has a very small learning curve and is very fast .", "it hardly takes 5 min in the hands of a plastic surgeon to complete the dressing .", "this dressing technique maintains the advantage of conventional tie over dressing with rapidity and repeativity .", "good graft take can be expected whether split - thickness or full - thickness with appropriate methods of stabilisation .", "we recommend a novel , low cost , simple , rapid method of graft fixation that can be used repeatedly and can be applied to a wound of any size ." ]

[ "<S> tie overdressing is commonly used to secure the graft against the raw surface and prevent loss due to of hematoma or seroma . a conventional tie over dressing with silk sutures , </S>", "<S> is a useful method of securing the graft to raw area . </S>", "<S> refixation is difficult when repeated tie over dressings are needed . </S>", "<S> we assessed a low cost repeated tie over dressing method using sterile sample collection containers and silk suture threads in eight patients . </S>", "<S> after the graft is applied on the bed , tie - over stitches are taken , and paraffin gauze is applied over with adequate padding ; the tie over sutures are passed through the container and the lid is tightened over it to complete the dressing . </S>", "<S> the lid can be unscrewed easily at any time to inspect the graft and can be easily reapplied in the outpatient department . </S>", "<S> the skin graft take in all the patients was complete without any seroma or hematoma . </S>", "<S> a novel and low - cost tie over dressing that enables simple fixation of the dressing , to maintain proper position of grafts that require repeated fixation is reported here . </S>" ]

[ "INTRODUCTION", "PROCEDURE", "DISCUSSION", "CONCLUSION" ]

[ [ "soft tissue coverage for wounds remains a difficult management problem for patients sustaining traumatic injury and burns .", "there are several methods to achieve wound coverage secondary healing , primary suturing , skin grafting , and flap surgeries as described in reconstruction ladder .", "a skin graft is the most commonly used modality for coverage of wounds in reconstructive plastic surgery . the skin graft needs to undergo various stages of healing for a good take on the recipient bed .", "there are different methods employed to secure the graft to recipient bed for a few days with a basic idea to ensure that the graft is not elevated off the bed by formation of haematoma / seroma under it .", "repeated tie over dressings are required in situations where the dressing needs to be changed more frequently as in cases of infected raw area , bleeding tendency , patients on anticoagulant drugs , and in convex areas of body such as buttocks , breast , and the scalp , where the dressing is difficult to secure ." ], [ "in this novel method , a sterile sample container was cut at its upper part [ figures 1 and 2 ] .", "the skin graft was applied on the raw area and fixed with skin staplers and tie over sutures . once paraffin gauze and adequate padding is applied on the raw area , the tie over threads were passed from inside out of the container [ figures 3 and 4 ] and pulled at the appropriate tension to keep the dressing in place .", "the lid of the container was tightened to complete the dressing ensuring that the graft was maintained in close approximation with the wound surface .", "sterile plastic container upper part of the sterile container is cut tie over threads being passed from inside out the lid is tightened over the dressing the dressing can be changed repeatedly with sterile precautions depending on the requirement , as an outpatient procedure by unscrewing the lid [ figures 5 - 8 ] which can then be easily reapplied .", "the procedure can be used on wounds of any size by changing the size of the sterile container chosen .", "post - toilet mastectomy raw area covered with the skin graft the tie over dressing applied in ot tie over dressing repeated in an outpatient department we have used this method in eight cases of the post toilet mastectomy raw areas with very good results [ figures 9 and 10 ] . post - toilet mastectomy raw area" ], [ "skin graft once applied has to be covered with petrolatum gauze to avoid its separation from the wound bed at the time of change of dressing .", "an ideal method of graft fixation should be simple , rapid , repeatable , able to be performed in the outpatient department , prevent hematoma or seroma formation , soak the exudates well , and allow the graft bed to be inspected easily .", "there are multiple methods of securing dressings over the skin graft , some of them can be applied only once and some can be repeated .", "the dressings that can be applied only once are like foam , hydro cellular dressing ( highly absorbent and can be easily changed ) , negative pressure therapy dressing ( stabilises the graft , increases the vascularity of bed , takes away toxic chemicals ) , and gas bag ( transparent , can see graft and monitor any haematoma ) .", "these traditional methods can stabilize the graft till the first dressing post operatively . in some contaminated wounds ,", "the dressing needs to be removed earlier , especially if there is drainage or foul smell .", "this approach may also be proper for graft , used to cover defects of some anatomical regions with increases risk of contamination , such as perineal , axillary , and genital or it can be used in areas where base of wound is difficult to immobilize like breast / pectoral region .", "repeated tie over dressings can be done by keeping interrupted sutures long to be used as tie over dressings .", "these ties over dressing can be made of sutures or rubber bands . when taking a tie over stitch , both", "the threads can be left long and only one thread is tied at a time , the other thread is left long for next time .", "these techniques are difficult for small dressings especially the bra hooks ; the silk loops method is very cumbersome and takes long time to do .", "the novel method being discussed has a very small learning curve and is very fast .", "it hardly takes 5 min in the hands of a plastic surgeon to complete the dressing .", "this dressing technique maintains the advantage of conventional tie over dressing with rapidity and repeativity ." ], [ "good graft take can be expected whether split - thickness or full - thickness with appropriate methods of stabilisation .", "we recommend a novel , low cost , simple , rapid method of graft fixation that can be used repeatedly and can be applied to a wound of any size ." ] ]

[ "the stomach is a sensitive digestive organ that is susceptible to exogenous pathogens to which it is exposed by the diet . in response to such pathogens", ", the stomach induces oxidative stress , which might be related to the development of both gastric organic disorders such as gastritis , gastric ulcers , and gastric cancer and functional disorders such as functional dyspepsia [ 2 , 3 ] .", "one of the most important possible causes of gastric ulcers is oxidative stress , which is involved in the pathogenesis of gastric inflammation and ulcerogenesis , but also in lifestyle related diseases including atherosclerosis , hypertension , diabetes mellitus , ischemic heart diseases , and malignancies [ 4 , 5 ] .", "nowadays , researchers and companies are inclined to investigate herbal medicines , more and many plants with anti - ulcerogenic properties have been found by different groups .", "herbs , medicinal plants , spices , vegetables , and crude drug substances are considered to be potential candidates for use in the treatment of gastric ulcer . among them , ganoderma lucidum ( ) has been found to have anti - tumor , anti- cancer , immunomodulatory and immunotherapeutic effects , to inhibit platelet aggregation , and to lower blood pressure , cholesterol and blood sugar .", "recentiy , pharmacopuncture treatment has been widely used and different types of herbs have been found to be effective for treating various diseases . this study was accomplished to investigate the effect of ganoderma lucidum pharmacopuncture ( glp ) in treating chronic gastric ulcer induced in rats by using ethanol ( etoh ) .", "ganoderma lucidum caps , 500 g grown in south korea were washed thoroughly with distilled water , cut into pieces , and submerged in 4 l of 25% alcohol for 10 hours at room temperature to be extracted .", "the alcohol extract was condensed to 500 ml , by using a rotary evaporator , and impurities were removed with a 0.22 m filter , the extracts was then , sterilized and stored at a temperature of 20. the extract was dissolved in ethanol before administrations , and was diluted with 5% dw ( dextrose water , jw pharmacoceutical ) to keep the final concentration of ganoderma lucidum at 10% .", "adult male , sprague - dawley ( sd ) rats ( weighing 220 240 g , 15 weeks old and housed five rats per cage ) were purchased from sam - taco co. they were provided with standard food and water ad libitum and were maintained in an animal house at a controlled temperature ( 22 2 ) with a 12 hours light / dark cycle .", "the rats were divided randomly into 4 groups of 8 rats each : the normal , the control , the normal saline ( np ) and the glp groups .", "the study was approved by the ethics committee for animal experimentation , dong - eui university . in this study ,", "the np and the glp groups were treated with injection of saline and glp respectively .", "two local acupoints cv12 ( ) and st36 ( ) were used . a pharmacopuncture needle ( 29 gauge :", "1 ml disposal insulin injection syringe from hwajin co. busan , korea ) was used .", "all laboratory rats were administered treatment for 15 days . on last day , the rats were sacrificed and their stomachs were immediately excised .", "the reactivities , activities and deaths of rats in each group were observed during the experiment .", "the animals were killed by cervical dislocation after administration of an overdose of ether after the two week experiment .", "the whole stomach was cut and removed 1.5 cm away from the cardia and the pylorus . for general specimen observation", "then , the specimen was placed in formaldehyde and glutaraldehyde , and was stored in a liquid nitrogen solution for later observation .", "the length and the width of the injured gastric mucosa region were measured with a vernier caliper . the gastric mucosa ulcer index ( ui )", "was determined according to the guth standard : spot erosions were recorded as 1 point , erosion lengths < 1 mm were recorded as 2 points , erosion lengths from 1 to 2 mm were recorded as 3 points , those from 2 to 3 mm were recorded as 4 points , and those > 3 mm were recorded as 5 points ; the score was doubled when the erosion width was > 1 mm .", "ulcers of the gastric mucosa appear as elongated bands of hemorrhagic lesions parallel to the long axis of the stomach .", "the inhibition percentage ( % ) was calculated by using the following formula : to investigate whether glp had affeced the activity of radical scavenging enzymes , we measured the activity of superoxidase dismutase ( sod ) in the gastric mucosa according to the method of mccord and fridovich . the standard assay", "was performed in 3 ml of 50 mm potassium phosphate buffer at ph 7.8 containing 0.1 mm ethylene diamine triacetic acid ( edta ) in a cuvette thermostated at 25. the reaction mixture contained 0.1 mm ferricytochrome c , 0.1 mm xanthine , and sufficient xanthine oxidase to produce a reduction rate of ferricytochrome c at 550 nm of 0.025 absorbance unit per min .", "a kinetic spectrophotometric analysis was started , with the addition of xanthine oxidase at 550 nm . under these conditions , the amount of sod required to inhibit the reduction rate of ferricytochrome c by 50% was defined as 1 unit of activity .", "after the opened stomachs had been cut into small pieces , they were preserved in 10% buffered formalin overnight .", "next , the biopsies were embedded in paraffin wax , sectioned into 5 m thicknesses by using a microtome and then stained with haematoxylin and eosin ( h&e ) .", "the tissue sections were assessed for histopathological changes such as congestion , edema , hemorrhage and necrosis by using a light microscope .", "tissue section slides were heated at 60 for approximately 25 minutes in a hot oven .", "immunohistochemical staining was conducted according to the manufacturer s protocol ( dakocytomation , usa ) .", "briefly , endogenous peroxidase was blocked by using a peroxidase block ( 0.03% hydrogen peroxide containing sodium azide ) for 5 minutes .", "tissue sections were washed gently with wash buffer and then incubated with bcl-2-associated x protein ( bax ) ( 1 : 200 ) , b - cell lymphoma 2 ( bcl-2 ) ( 1 : 200 ) , and transforming growth factor - beta 1 ( tgf-1 ) ( 1 : 100 ) biotinylated primary antibodies for 15 minutes . then", ", the tissue sections were rinsed gently in wash buffer and placed in a buffer bath . following washing and counterstaining with hematoxylin for 5 seconds , we added diaminobenzidine substrate chromagen to the tissue sections and incubated them further for 5 minutes .", "the total numbers of immune positive cells per field ( 10 m ) were counted in at least 15 randomly chosen fields at 100 magnification .", "the statistical significances of the differences among groups were assessed with a one way analysis of variance ( anova , post hoc analysis ) . a value of p < 0.05 was considered significant .", "scale bar = 1 cm ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "p < 0.05 vs. control ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture . scale bar = 200 m and magnification = 40 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "p < 0.05 vs. control ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "scale bar = 100 m and magnification = 100 ; bax , bcl-2-associated x ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "scale bar = 100 m and magnification = 100 ; bcl-2 , b - cell lymphoma 2 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "bax , bcl-2-associated x ; bcl-2 , b - cell lymphoma 2 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture . scale bar = 100 m and magnification = 100 ; tgf-1 , transforming growth factor - beta 1 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "p < 0.05 vs. the control ; tgf-1 , transforming growth factor - beta 1 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "the areas of gastric ulcer formation were reduced in the np and the glp groups compared with the control group ( fig .", "the formation of the ulcers was significantly suppressed in the glp group compared to the control group ( p < 0.05 ) .", "however in the control group , scattered mucosal damage and local congestion were observed . in the np and the glp groups ,", "the gastric mucosal injuries were obviously slightly lwss severe than they were in the control group ( fig .", "the treatment with glp caused a significant increase ( p < 0.05 ) in the enzyme activity of sod ( fig .", "an increased level of bax protein was observed in the control group , whereas , the expressions of bax protein in the np group was decreased compared with those in the control group and those in the glp group were more significantly decreased compared with those in the control group ( p < 0.01 ) ( figs .", "bcl-2 protein expression was hardly observed in the control group and the expressions of bcl-2 protein in the np group were increased significantly compared with those in the control group ( p < 0.05 ) ( figs .", "bcl-2 protein expressions in the glp group were more significantly decreased compared those in the control group ( p < 0.01 ) ( figs .", "the increase in the glp group was more significant ( p < 0.01 ) than it was in the control group ( figs .", "the gastric mucosa is one of the most important tissue in an organism , because of its function , structure , and the pathological processes that can take place in this tissue .", "the integrity of the gastric mucosa depends on the protection provided by the gastric mucosal barrier , and the gastric mucosa can be damaged by a variety of internal or external factors with the production of a number of inflammatory mediators and cytokines , resulting in secondary mucosal damage .", "ethanol is an ulcerogenic agent that is known to produce erosions , ulcerative lesions , and petechial bleeding in the mucosa of the stomach .", "ethanol rapidly penetrates the gastric mucosa , causing membrane damage , exfoliation of cells , erosion , and ulcer formation . in the present study , using the chronic injury model , gastric mucosal lesions in rats were induced by using etoh .", "administration of an ethanol solution for 15 days induced hemorrhagic lesions in the gastric mucosa in the control group . in the control group ,", "the np and the glp groups had reduced areas of gastric ulcer formation when compared with the control group ( fig .", "1 ) . in the glp , compared with the np group , the formation of ulcers was significantly suppressed ( fig .", "2 ) . gastric ulcers were induced on the anterior walls of the stomachs in rats by using an etoh solution .", "the rats of the control group showed loss of mucosa , but compared with control group , the mucosal losses were less in the np and the glp groups ( fig .", "ethanol administration reduced sod activity in the control group , compared with the normal group ( fig .", "this might have resulted from the utilization for the decomposition of superoxide anion generated by lipid peroxidation . however , the activities in the glp group were significantly compared to the control group ( fig .", "4 ) . apoptosis , programmed death of cells through dna fragmentation , cell shrinkage , and dilation of endoplasmic reticulum is normally followed by cell degeneration and the formation of membrane vesicles , called apoptosis bodies .", "ethanol by itself was revealed to be able to induce apoptosis in the gastric epithelium .", "the up regulation of the pro apoptotic factor , bax protein , and the down regulation of antiapoptotic bcl-2 are two main indicators for apoptosis .", "the level of bax protein was increased in the control group , but in the np and the glp groups , the level of bax protein were decreased compared with the control group . especially in the glp group ,", "on the other hand , bcl-2 immunoreactivites in the np group were up regulated compared with the control group .", "glp not only up regulated the levels of bcl-2 but also down regulated the levels of bax compared with the control group ( fig .", "therefore , we assume that glp suppressed apoptosis by regulating the mitochondrial damage mediated endogenous pathway , which could be one of the important mechanisms for preventing gastric ulcer disease . tgf-1 is well known to be a multi functional cytokine that regulates many biological processes such as cell proliferation , cell differentiation , adhesion , inter cell signaling , and the also production and the degradation of extracellular matrix proteins , thus playing an essential role during wound healing and tissue repair .", "this indicates that tgf-1 expression is part of the normal healing response of gastric tissue . in this study , the level of tgf-1 was significantly increased in the glp group ( figs .", "the present study indicated the protective efficacy of glp against etoh induced gastric ulcer in sd rats .", "this conclusion was based on gross appearance , histology , and immunohistochemistry staining for bax , bcl-2 and tgf-1 ." ]

[ "<S> objectives : the stomach is a sensitive digestive organ that is susceptible to exogenous pathogens from the diet . in response to such pathogens , the stomach induces oxidative stress , which might be related to the development of both gastric organic disorders such as gastritis , gastric ulcers , and gastric cancer , and functional disorders such as functional dyspepsia . this study was accomplished to investigate the effect of ganoderma lucidum pharmacopuncture ( glp ) on chronic gastric ulcers in rats.methods:the rats were divided into 4 groups of 8 animals each : the normal , the control , the normal saline ( np ) and the glp groups . in this study , </S>", "<S> the modified ethanol gastritis model was used . </S>", "<S> the rats were administrated 56% ethanol orally every other day . </S>", "<S> the dose of ethanol was 8 g / kg body weight . </S>", "<S> the normal group received the same amount of normal saline instead of ethanol . </S>", "<S> the np and the glp groups were treated with injection of saline and glp respectively . </S>", "<S> the control group received no treatment . </S>", "<S> two local acupoints cv12 ( ) and st36 ( ) were used . </S>", "<S> all laboratory rats underwent treatment for 15 days . on last day </S>", "<S> , the rats were sacrificed and their stomachs were immediately excised.results:ulcers of the gastric mucosa appeared as elongated bands of hemorrhagic lesions parallel to the long axis of the stomach . in the np and glp groups , the injuries to the gastric mucosal injuries were not as severe as they were in the control group . </S>", "<S> wound healings of the chronic gastric ulcers was promoted by using glp and significant alterations of the indices in the gastric mucosa were observed . </S>", "<S> such protection was demonstrated by gross appearance , histology and immunehistochemistry staining for bcl-2-associated x ( bax ) , b - cell lymphoma 2 ( bcl-2 ) and transforming growth factor - beta 1 ( tgf-1).conclusion : these results suggest that glp at cv12 and st36 can provide significant protection to the gastric mucosa against an ethanol induced chronic gastric ulcer . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion" ]

[ [ "the stomach is a sensitive digestive organ that is susceptible to exogenous pathogens to which it is exposed by the diet . in response to such pathogens", ", the stomach induces oxidative stress , which might be related to the development of both gastric organic disorders such as gastritis , gastric ulcers , and gastric cancer and functional disorders such as functional dyspepsia [ 2 , 3 ] .", "one of the most important possible causes of gastric ulcers is oxidative stress , which is involved in the pathogenesis of gastric inflammation and ulcerogenesis , but also in lifestyle related diseases including atherosclerosis , hypertension , diabetes mellitus , ischemic heart diseases , and malignancies [ 4 , 5 ] .", "nowadays , researchers and companies are inclined to investigate herbal medicines , more and many plants with anti - ulcerogenic properties have been found by different groups .", "herbs , medicinal plants , spices , vegetables , and crude drug substances are considered to be potential candidates for use in the treatment of gastric ulcer . among them , ganoderma lucidum ( ) has been found to have anti - tumor , anti- cancer , immunomodulatory and immunotherapeutic effects , to inhibit platelet aggregation , and to lower blood pressure , cholesterol and blood sugar .", "recentiy , pharmacopuncture treatment has been widely used and different types of herbs have been found to be effective for treating various diseases . this study was accomplished to investigate the effect of ganoderma lucidum pharmacopuncture ( glp ) in treating chronic gastric ulcer induced in rats by using ethanol ( etoh ) ." ], [ "ganoderma lucidum caps , 500 g grown in south korea were washed thoroughly with distilled water , cut into pieces , and submerged in 4 l of 25% alcohol for 10 hours at room temperature to be extracted .", "the alcohol extract was condensed to 500 ml , by using a rotary evaporator , and impurities were removed with a 0.22 m filter , the extracts was then , sterilized and stored at a temperature of 20. the extract was dissolved in ethanol before administrations , and was diluted with 5% dw ( dextrose water , jw pharmacoceutical ) to keep the final concentration of ganoderma lucidum at 10% .", "adult male , sprague - dawley ( sd ) rats ( weighing 220 240 g , 15 weeks old and housed five rats per cage ) were purchased from sam - taco co. they were provided with standard food and water ad libitum and were maintained in an animal house at a controlled temperature ( 22 2 ) with a 12 hours light / dark cycle .", "the rats were divided randomly into 4 groups of 8 rats each : the normal , the control , the normal saline ( np ) and the glp groups .", "the study was approved by the ethics committee for animal experimentation , dong - eui university . in this study ,", "the np and the glp groups were treated with injection of saline and glp respectively .", "two local acupoints cv12 ( ) and st36 ( ) were used . a pharmacopuncture needle ( 29 gauge :", "1 ml disposal insulin injection syringe from hwajin co. busan , korea ) was used .", "all laboratory rats were administered treatment for 15 days . on last day , the rats were sacrificed and their stomachs were immediately excised .", "the reactivities , activities and deaths of rats in each group were observed during the experiment .", "the animals were killed by cervical dislocation after administration of an overdose of ether after the two week experiment .", "the whole stomach was cut and removed 1.5 cm away from the cardia and the pylorus . for general specimen observation", "then , the specimen was placed in formaldehyde and glutaraldehyde , and was stored in a liquid nitrogen solution for later observation .", "the length and the width of the injured gastric mucosa region were measured with a vernier caliper . the gastric mucosa ulcer index ( ui )", "was determined according to the guth standard : spot erosions were recorded as 1 point , erosion lengths < 1 mm were recorded as 2 points , erosion lengths from 1 to 2 mm were recorded as 3 points , those from 2 to 3 mm were recorded as 4 points , and those > 3 mm were recorded as 5 points ; the score was doubled when the erosion width was > 1 mm .", "ulcers of the gastric mucosa appear as elongated bands of hemorrhagic lesions parallel to the long axis of the stomach .", "the inhibition percentage ( % ) was calculated by using the following formula : to investigate whether glp had affeced the activity of radical scavenging enzymes , we measured the activity of superoxidase dismutase ( sod ) in the gastric mucosa according to the method of mccord and fridovich . the standard assay", "was performed in 3 ml of 50 mm potassium phosphate buffer at ph 7.8 containing 0.1 mm ethylene diamine triacetic acid ( edta ) in a cuvette thermostated at 25. the reaction mixture contained 0.1 mm ferricytochrome c , 0.1 mm xanthine , and sufficient xanthine oxidase to produce a reduction rate of ferricytochrome c at 550 nm of 0.025 absorbance unit per min .", "a kinetic spectrophotometric analysis was started , with the addition of xanthine oxidase at 550 nm . under these conditions , the amount of sod required to inhibit the reduction rate of ferricytochrome c by 50% was defined as 1 unit of activity .", "after the opened stomachs had been cut into small pieces , they were preserved in 10% buffered formalin overnight .", "next , the biopsies were embedded in paraffin wax , sectioned into 5 m thicknesses by using a microtome and then stained with haematoxylin and eosin ( h&e ) .", "the tissue sections were assessed for histopathological changes such as congestion , edema , hemorrhage and necrosis by using a light microscope .", "tissue section slides were heated at 60 for approximately 25 minutes in a hot oven .", "immunohistochemical staining was conducted according to the manufacturer s protocol ( dakocytomation , usa ) .", "briefly , endogenous peroxidase was blocked by using a peroxidase block ( 0.03% hydrogen peroxide containing sodium azide ) for 5 minutes .", "tissue sections were washed gently with wash buffer and then incubated with bcl-2-associated x protein ( bax ) ( 1 : 200 ) , b - cell lymphoma 2 ( bcl-2 ) ( 1 : 200 ) , and transforming growth factor - beta 1 ( tgf-1 ) ( 1 : 100 ) biotinylated primary antibodies for 15 minutes . then", ", the tissue sections were rinsed gently in wash buffer and placed in a buffer bath . following washing and counterstaining with hematoxylin for 5 seconds , we added diaminobenzidine substrate chromagen to the tissue sections and incubated them further for 5 minutes .", "the total numbers of immune positive cells per field ( 10 m ) were counted in at least 15 randomly chosen fields at 100 magnification .", "the statistical significances of the differences among groups were assessed with a one way analysis of variance ( anova , post hoc analysis ) . a value of p < 0.05 was considered significant .", "scale bar = 1 cm ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "p < 0.05 vs. control ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture . scale bar = 200 m and magnification = 40 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "p < 0.05 vs. control ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "scale bar = 100 m and magnification = 100 ; bax , bcl-2-associated x ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "scale bar = 100 m and magnification = 100 ; bcl-2 , b - cell lymphoma 2 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "bax , bcl-2-associated x ; bcl-2 , b - cell lymphoma 2 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture . scale bar = 100 m and magnification = 100 ; tgf-1 , transforming growth factor - beta 1 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture .", "p < 0.05 vs. the control ; tgf-1 , transforming growth factor - beta 1 ; np , injection of saline ; glp , injection of ganoderma lucidum pharmacopuncture ." ], [ "the areas of gastric ulcer formation were reduced in the np and the glp groups compared with the control group ( fig .", "the formation of the ulcers was significantly suppressed in the glp group compared to the control group ( p < 0.05 ) .", "however in the control group , scattered mucosal damage and local congestion were observed . in the np and the glp groups ,", "the gastric mucosal injuries were obviously slightly lwss severe than they were in the control group ( fig .", "the treatment with glp caused a significant increase ( p < 0.05 ) in the enzyme activity of sod ( fig .", "an increased level of bax protein was observed in the control group , whereas , the expressions of bax protein in the np group was decreased compared with those in the control group and those in the glp group were more significantly decreased compared with those in the control group ( p < 0.01 ) ( figs .", "bcl-2 protein expression was hardly observed in the control group and the expressions of bcl-2 protein in the np group were increased significantly compared with those in the control group ( p < 0.05 ) ( figs .", "bcl-2 protein expressions in the glp group were more significantly decreased compared those in the control group ( p < 0.01 ) ( figs .", "the increase in the glp group was more significant ( p < 0.01 ) than it was in the control group ( figs ." ], [ "the gastric mucosa is one of the most important tissue in an organism , because of its function , structure , and the pathological processes that can take place in this tissue .", "the integrity of the gastric mucosa depends on the protection provided by the gastric mucosal barrier , and the gastric mucosa can be damaged by a variety of internal or external factors with the production of a number of inflammatory mediators and cytokines , resulting in secondary mucosal damage .", "ethanol is an ulcerogenic agent that is known to produce erosions , ulcerative lesions , and petechial bleeding in the mucosa of the stomach .", "ethanol rapidly penetrates the gastric mucosa , causing membrane damage , exfoliation of cells , erosion , and ulcer formation . in the present study , using the chronic injury model , gastric mucosal lesions in rats were induced by using etoh .", "administration of an ethanol solution for 15 days induced hemorrhagic lesions in the gastric mucosa in the control group . in the control group ,", "the np and the glp groups had reduced areas of gastric ulcer formation when compared with the control group ( fig .", "1 ) . in the glp , compared with the np group , the formation of ulcers was significantly suppressed ( fig .", "2 ) . gastric ulcers were induced on the anterior walls of the stomachs in rats by using an etoh solution .", "the rats of the control group showed loss of mucosa , but compared with control group , the mucosal losses were less in the np and the glp groups ( fig .", "ethanol administration reduced sod activity in the control group , compared with the normal group ( fig .", "this might have resulted from the utilization for the decomposition of superoxide anion generated by lipid peroxidation . however , the activities in the glp group were significantly compared to the control group ( fig .", "4 ) . apoptosis , programmed death of cells through dna fragmentation , cell shrinkage , and dilation of endoplasmic reticulum is normally followed by cell degeneration and the formation of membrane vesicles , called apoptosis bodies .", "ethanol by itself was revealed to be able to induce apoptosis in the gastric epithelium .", "the up regulation of the pro apoptotic factor , bax protein , and the down regulation of antiapoptotic bcl-2 are two main indicators for apoptosis .", "the level of bax protein was increased in the control group , but in the np and the glp groups , the level of bax protein were decreased compared with the control group . especially in the glp group ,", "on the other hand , bcl-2 immunoreactivites in the np group were up regulated compared with the control group .", "glp not only up regulated the levels of bcl-2 but also down regulated the levels of bax compared with the control group ( fig .", "therefore , we assume that glp suppressed apoptosis by regulating the mitochondrial damage mediated endogenous pathway , which could be one of the important mechanisms for preventing gastric ulcer disease . tgf-1 is well known to be a multi functional cytokine that regulates many biological processes such as cell proliferation , cell differentiation , adhesion , inter cell signaling , and the also production and the degradation of extracellular matrix proteins , thus playing an essential role during wound healing and tissue repair .", "this indicates that tgf-1 expression is part of the normal healing response of gastric tissue . in this study , the level of tgf-1 was significantly increased in the glp group ( figs ." ], [ "the present study indicated the protective efficacy of glp against etoh induced gastric ulcer in sd rats .", "this conclusion was based on gross appearance , histology , and immunohistochemistry staining for bax , bcl-2 and tgf-1 ." ] ]

[ "\n improper nutritional knowledge is one of the most important causes of nutritional problems , which can affect practice and cause more complications .", "it has been recommended that food choices and dietary behaviors can be impressed by knowledge about diet ( 1 ) . to effectively improve healthy eating ,", "it is necessary to understand the nutritional attitudes and beliefs of the general community ( 2 ) . \n \n", "age , education level , gender and marital status can influence nutritional knowledge , attitude and practice ( kap ) ( 3 ) .", "the association of socioeconomic status ( ses ) with nutrition knowledge and beliefs has been confirmed ( 4 ) .", "many studies have found that nutrient intakes and dietary patterns of people in low ses groups threaten the general health and raise the risk of nutritional disease ( 5,6 ) .", "people in low ses group , due to lack of access to health care , improper living conditions , less knowledge and greater psychological stress , may be at a higher risk of poorer health conditions more than others ( 7 - 10 ) .", "with respect to the association of ses index with health behaviors ( 11 ) and undesirable health consequences , it is of prime importance to evaluate the effect of ses index on health - related behaviors . \n \n in this study ,", "the association between nutritional kap with ses was assessed among the iranians in urban and rural regions of 31 provinces of iran . \n", "\n the study population consisted of 14,136 iranian households who lived in urban and rural regions of 31 provinces , selected by multi - stage cluster sampling .", "non - iranian households were not included in the survey , and the households who were absent for tree times at the time of the interview were also excluded .", "the method of study has been published in a previous survey ( 12 ) . \n \n", "mothers or any over 15-year old member of the households , who were in charge of cooking for the entire family , were considered as statistical units of the study . a structured questionnaire and interview with a qualified person in families", "nutritional knowledge questions inquired about main food groups , causes of consuming food , role of main food groups , sources of protein intake , and role of dietary fiber as well as nutritional attitude toward health - related behaviors and food choices . to assess the practice of the households , the household members were asked about the frequencies of different consumed foods . \n \n", "ses was an index that included household assets ( house ownership , number of rooms in the house , having such equipment as tv , cell phone , car , freezer , washing machine , dish washing machine , phone , microwave , access to internet ) , occupation and education level of the heads of the families and the respondents and number of family members . \n \n", "data were analyzed using the stata version 11.0 ( stata corp , college station , tex . ) ( survey analysis ) .", "\n data were analyzed using the stata version 11.0 ( stata corp , college station , tex . ) ( survey analysis ) .", "the percentage of nutritional knowledge was significantly higher in families with good ses and it linearly increased with family ses .", "most people consumed food to prevent disease and be healthy ( 54.7% , 95% ci : 53.5 , 56.0 ) .", "more than half percent of households were aware of grain , meat and legumes , and dairy group ( 60.5% , 69.7% , and 52.5% , respectively ) .", "the percentage of knowledge about identification of fruit , vegetable and fat groups was less than half percent .", "about 73.1% of the households ( 95% ci : 71.9 , 74.3 ) were familiar with the role of dairy group ( growing and strengthen teeth and bones ) , although less than 10% of them were aware of the role of fruit groups in providing dietary fiber ( 8.6% , 95% ci : 7.1 , 9.2 ) .", "the percent of participants knowledge about legumes and soy , another source of protein , was 45.5% ( 95% ci : 44.2 , 46.8 ) and 40.1% ( 95% ci : 38.8 , 41.5 ) , respectively .", "only 10.1% of the participants were familiar with the concept of dietary fiber ; among them , the most percentage of knowledge belonged to bowel movement . \n \n *", "( % ( 95% ci ) ) , p<0.05 , ses ; socioeconomic status \n \n the percentage of attitude is shown in table 2 .", "more than 97% of the participants had a favorable attitude towards the importance of nutrition and diet in health . respectively , 90.3% , and 70.4% of the participants had a favorable attitude toward importance of nutritional requirements of children rather than adults and the necessity of equal food intake in both genders when there is few food .", "the percentage of a favorable attitude of the family with good , moderate and weak ses about preferring fruit consumption than bread at time of hunger was 39.8% , 50.8% and 58.5% , respectively .", "the percentage of participants who disagreed with consuming steam cooked rice was significantly higher in families with weak ses .", "about 80.5% of the participants had a favorable attitude toward preferring consumption of tuna fish and the lowest percentage was related to weak ses families .", "households with good ses had a more favorable attitude about the necessity of daily consumption of vegetables or salad and the need for milk consumption in any age besides childhood period ( 95.3% , and 93.1% , respectively ) .", "the families with weak ses had the highest favorable attitude about preferring whole meal bread on other kinds of breads .", "the percentage of favorable attitude toward drinking water in the middle of eating food and the necessity of keeping body fitness in girls at puberty was 66.5% and 56.1% , respectively .", "when the participants were asked about the difference between nutrition fact of meat and mushrooms , only 25.4% agreed . \n \n", "* ( % ( 95% ci ) ) , p < 0.05 , ses ; socioeconomic status \n \n table 3 demonstrates the practice of households based on ses .", "most households consumed fruit , vegetable , milk , yoghurt , cheese and sugar daily .", "consumption of foods such as rice , red meat , butter , cream , egg , legumes , dough , chicken and poultry was weekly in most participants .", "the other items such as viscera , tuna , nuts and synthetic juice were rarely or never consumed .", "families with good ses significantly consumed more fruit , vegetable , dairy group , red meat , chicken and poultry , fish and egg , while sugar consumption was significantly higher in families with weak ses . \n \n * ( % ( 95% ci ) ) , p<0.05 , ses ; socioeconomic status \n", "\n the aim of this study was to assess the association between nutritional kap with ses among iranian households .", "the best knowledge in all items was seen in families with good ses index and it linearly increased with family ses . \n \n", "households with weak ses had the best favorable attitude toward the difference between mushroom and meat nutrition fact , preferring whole meal bread on other kinds of breads and preferring fruit consumption than bread at time of hunger .", "the consumption of food items such as red meat , chicken and poultry , fish , egg , dairy group , fruit , vegetable and nuts was significantly higher in households with good ses index while the other items including rice , tuna , legumes and sugar were consumed the most in weak families . \n \n", "these findings are supported by other surveys that have shown that more intake of fruit and vegetable are related to more diet costs , and diet rich in fat and sugar is contributed to lower costs ( 13,14 ) .", "findings from a survey on 4,356 us adults suggested that better ses index independently promotes the possibility of adequate fruits and vegetables intake and overall diet quality .", "they also reported that nutritional knowledge and belief can affect the positive association between ses and diet quality indicators(4 ) . as it is confirmed in other studies ,", "the socio - demographic variation in intake can be associated with nutritional knowledge as a partial mediator in improving diet .", "the result of this study also revealed that healthy eating was significantly associated with knowledge and possibility of meeting current recommendations for fruit , vegetable and fat intake ( 15 ) .", "the association of ses and dietary knowledge or income and diet is supported by other studies ( 16 - 19 ) . another way that ses can influence diet is related to food purchasing differences .", "a study on australians in 2000 showed that food purchasing differences due to household income is related to diet via food - cost concern ( 20 ) .", "food purchase decisions due to a person s attitude toward food price can influence diet quality .", "based on this survey , people who care about food price were more likely to live in low - income , food - insecure households , they had low education , were tenants and did not own homes , and were service workers .", "they were more susceptible to diseases such as overweight , high blood pressure , heart disease and diabetes than the others ( 21 ) . \n \n", "our results showed higher consumption of food items such as sugar , tuna and lower consumption of nuts and protein sources such as meat , fish , egg and dairy product in families with weak ses , which can be associated with an increased rate of some diseases .", "thus , implementing measures to guide people in the line of healthier nutrition is necessary and it can help decrease the rate of diet - related diseases , especially in low ses households . \n", "\n with respect to the increasing nutritional disease and the important role of dietary behaviors , increasing nutritional kap may be a way to change life style and health related behaviors , but it is not enough .", "therefore , targeted policies should be coupled with efforts to promote diet and nutritional kap for those people with unfavorable socio - economic status .", "some cost - effective strategies should be presented for the low - income groups in the society to neutralize the negative effect of income on food purchasing patterns and health related life style . \n" ]

[ "<S> \n background : improper nutritional knowledge is one of the most important causes of nutritional problems , \n which can affect practice and cause more complications . the aim of this study was to assess the association between \n nutritional knowledge , attitude and practice ( kap ) of iranian households with socioeconomic status \n ( ses ) . \n\n </S>", "<S> methods : the study population was 14,136 households ( 57 clusters of 8 individuals in each province ) who \n lived in urban and rural regions of 31 provinces of iran . </S>", "<S> the sample size of the study was selected using multistage \n cluster sampling technique . a structured questionnaire and interview with the qualified person in each \n family </S>", "<S> was used to collect data . </S>", "<S> the questionnaire included demographic , ses and nutritional kap questions . \n </S>", "<S> using principle component analysis , some variables including household assets , occupation and education level \n of the heads of the families and respondents and the number of family members were used to construct family \n ses . </S>", "<S> the ses was categorized as good , moderate and weak . </S>", "<S> pearson s chi - square test was used to analyze categorical \n variables . \n\n </S>", "<S> results : the percentage of knowledge about growing up , acquiring energy and being healthy as reasons for \n eating food was 24.1% , 44.8% and 54.7% , respectively . </S>", "<S> only 69.7% , 60.5% and 52.5% of the participants had \n knowledge of identification of meat and legumes , grain and dairy group , respectively . </S>", "<S> more than 97% of the \n participants had a favorable attitude toward importance of nutrition in health . </S>", "<S> the nutritional knowledge linearly \n increased with increasing ses . </S>", "<S> families with good ses significantly consumed more fruit , vegetable , dairy \n group , red meat , chicken and poultry , fish and egg while sugar consumption was significantly higher in families \n with weak ses ( p<0.05 ) . \n\n </S>", "<S> conclusion : ses can influence the rate of nutritional kap . some policies should be considered to increase nutritional kap especially in lower ses group in the society . \n </S>" ]

[ "Introduction", "Methods", "Statistical Analysis", "Results", "Discussion", "Conclusion", "Acknowledgements", "Conflict of interests" ]

[ [ "\n improper nutritional knowledge is one of the most important causes of nutritional problems , which can affect practice and cause more complications .", "it has been recommended that food choices and dietary behaviors can be impressed by knowledge about diet ( 1 ) . to effectively improve healthy eating ,", "it is necessary to understand the nutritional attitudes and beliefs of the general community ( 2 ) . \n \n", "age , education level , gender and marital status can influence nutritional knowledge , attitude and practice ( kap ) ( 3 ) .", "the association of socioeconomic status ( ses ) with nutrition knowledge and beliefs has been confirmed ( 4 ) .", "many studies have found that nutrient intakes and dietary patterns of people in low ses groups threaten the general health and raise the risk of nutritional disease ( 5,6 ) .", "people in low ses group , due to lack of access to health care , improper living conditions , less knowledge and greater psychological stress , may be at a higher risk of poorer health conditions more than others ( 7 - 10 ) .", "with respect to the association of ses index with health behaviors ( 11 ) and undesirable health consequences , it is of prime importance to evaluate the effect of ses index on health - related behaviors . \n \n in this study ,", "the association between nutritional kap with ses was assessed among the iranians in urban and rural regions of 31 provinces of iran . \n" ], [ "\n the study population consisted of 14,136 iranian households who lived in urban and rural regions of 31 provinces , selected by multi - stage cluster sampling .", "non - iranian households were not included in the survey , and the households who were absent for tree times at the time of the interview were also excluded .", "the method of study has been published in a previous survey ( 12 ) . \n \n", "mothers or any over 15-year old member of the households , who were in charge of cooking for the entire family , were considered as statistical units of the study . a structured questionnaire and interview with a qualified person in families", "nutritional knowledge questions inquired about main food groups , causes of consuming food , role of main food groups , sources of protein intake , and role of dietary fiber as well as nutritional attitude toward health - related behaviors and food choices . to assess the practice of the households , the household members were asked about the frequencies of different consumed foods . \n \n", "ses was an index that included household assets ( house ownership , number of rooms in the house , having such equipment as tv , cell phone , car , freezer , washing machine , dish washing machine , phone , microwave , access to internet ) , occupation and education level of the heads of the families and the respondents and number of family members . \n \n", "data were analyzed using the stata version 11.0 ( stata corp , college station , tex . ) ( survey analysis ) ." ], [ "\n data were analyzed using the stata version 11.0 ( stata corp , college station , tex . ) ( survey analysis ) ." ], [ "the percentage of nutritional knowledge was significantly higher in families with good ses and it linearly increased with family ses .", "most people consumed food to prevent disease and be healthy ( 54.7% , 95% ci : 53.5 , 56.0 ) .", "more than half percent of households were aware of grain , meat and legumes , and dairy group ( 60.5% , 69.7% , and 52.5% , respectively ) .", "the percentage of knowledge about identification of fruit , vegetable and fat groups was less than half percent .", "about 73.1% of the households ( 95% ci : 71.9 , 74.3 ) were familiar with the role of dairy group ( growing and strengthen teeth and bones ) , although less than 10% of them were aware of the role of fruit groups in providing dietary fiber ( 8.6% , 95% ci : 7.1 , 9.2 ) .", "the percent of participants knowledge about legumes and soy , another source of protein , was 45.5% ( 95% ci : 44.2 , 46.8 ) and 40.1% ( 95% ci : 38.8 , 41.5 ) , respectively .", "only 10.1% of the participants were familiar with the concept of dietary fiber ; among them , the most percentage of knowledge belonged to bowel movement . \n \n *", "( % ( 95% ci ) ) , p<0.05 , ses ; socioeconomic status \n \n the percentage of attitude is shown in table 2 .", "more than 97% of the participants had a favorable attitude towards the importance of nutrition and diet in health . respectively , 90.3% , and 70.4% of the participants had a favorable attitude toward importance of nutritional requirements of children rather than adults and the necessity of equal food intake in both genders when there is few food .", "the percentage of a favorable attitude of the family with good , moderate and weak ses about preferring fruit consumption than bread at time of hunger was 39.8% , 50.8% and 58.5% , respectively .", "the percentage of participants who disagreed with consuming steam cooked rice was significantly higher in families with weak ses .", "about 80.5% of the participants had a favorable attitude toward preferring consumption of tuna fish and the lowest percentage was related to weak ses families .", "households with good ses had a more favorable attitude about the necessity of daily consumption of vegetables or salad and the need for milk consumption in any age besides childhood period ( 95.3% , and 93.1% , respectively ) .", "the families with weak ses had the highest favorable attitude about preferring whole meal bread on other kinds of breads .", "the percentage of favorable attitude toward drinking water in the middle of eating food and the necessity of keeping body fitness in girls at puberty was 66.5% and 56.1% , respectively .", "when the participants were asked about the difference between nutrition fact of meat and mushrooms , only 25.4% agreed . \n \n", "* ( % ( 95% ci ) ) , p < 0.05 , ses ; socioeconomic status \n \n table 3 demonstrates the practice of households based on ses .", "most households consumed fruit , vegetable , milk , yoghurt , cheese and sugar daily .", "consumption of foods such as rice , red meat , butter , cream , egg , legumes , dough , chicken and poultry was weekly in most participants .", "the other items such as viscera , tuna , nuts and synthetic juice were rarely or never consumed .", "families with good ses significantly consumed more fruit , vegetable , dairy group , red meat , chicken and poultry , fish and egg , while sugar consumption was significantly higher in families with weak ses . \n \n * ( % ( 95% ci ) ) , p<0.05 , ses ; socioeconomic status \n" ], [ "\n the aim of this study was to assess the association between nutritional kap with ses among iranian households .", "the best knowledge in all items was seen in families with good ses index and it linearly increased with family ses . \n \n", "households with weak ses had the best favorable attitude toward the difference between mushroom and meat nutrition fact , preferring whole meal bread on other kinds of breads and preferring fruit consumption than bread at time of hunger .", "the consumption of food items such as red meat , chicken and poultry , fish , egg , dairy group , fruit , vegetable and nuts was significantly higher in households with good ses index while the other items including rice , tuna , legumes and sugar were consumed the most in weak families . \n \n", "these findings are supported by other surveys that have shown that more intake of fruit and vegetable are related to more diet costs , and diet rich in fat and sugar is contributed to lower costs ( 13,14 ) .", "findings from a survey on 4,356 us adults suggested that better ses index independently promotes the possibility of adequate fruits and vegetables intake and overall diet quality .", "they also reported that nutritional knowledge and belief can affect the positive association between ses and diet quality indicators(4 ) . as it is confirmed in other studies ,", "the socio - demographic variation in intake can be associated with nutritional knowledge as a partial mediator in improving diet .", "the result of this study also revealed that healthy eating was significantly associated with knowledge and possibility of meeting current recommendations for fruit , vegetable and fat intake ( 15 ) .", "the association of ses and dietary knowledge or income and diet is supported by other studies ( 16 - 19 ) . another way that ses can influence diet is related to food purchasing differences .", "a study on australians in 2000 showed that food purchasing differences due to household income is related to diet via food - cost concern ( 20 ) .", "food purchase decisions due to a person s attitude toward food price can influence diet quality .", "based on this survey , people who care about food price were more likely to live in low - income , food - insecure households , they had low education , were tenants and did not own homes , and were service workers .", "they were more susceptible to diseases such as overweight , high blood pressure , heart disease and diabetes than the others ( 21 ) . \n \n", "our results showed higher consumption of food items such as sugar , tuna and lower consumption of nuts and protein sources such as meat , fish , egg and dairy product in families with weak ses , which can be associated with an increased rate of some diseases .", "thus , implementing measures to guide people in the line of healthier nutrition is necessary and it can help decrease the rate of diet - related diseases , especially in low ses households . \n" ], [ "\n with respect to the increasing nutritional disease and the important role of dietary behaviors , increasing nutritional kap may be a way to change life style and health related behaviors , but it is not enough .", "therefore , targeted policies should be coupled with efforts to promote diet and nutritional kap for those people with unfavorable socio - economic status .", "some cost - effective strategies should be presented for the low - income groups in the society to neutralize the negative effect of income on food purchasing patterns and health related life style . \n" ], [ "" ], [ "" ] ]

[ "t - cell activation requires specific recognition of antigen presented as small fragments ( peptides ) bound to major histocompatibility complex ( mhc ) molecules .", "the recognition of a particular peptide - mhc occurs through a highly specific t - cell receptor ( tcr ) , which is selected in the thymus .", "following tcr triggering , costimulation and the presence of polarizing cytokines together determine the t - cell activation pattern and guide ultimate t - cell differentiation .", "classically , cd4t - cells recognize antigens scavenged extracellularly by the antigen presenting cell ( apc ) that are presented in mhc class ii , whereas cd8 t - cells recognize endogenous antigens presented by mhc class i ( mhc - i ) [ 1 , 2 ] . in spite of this widely held view , already decades ago it was shown that also antigens derived from intracellular pathogens such as viruses or intracellular bacteria can be presented in mhc - i .", "more recently , cross - presentation by dendritic cells and autophagy have been elucidated as important mechanisms in this context [ 2 , 4 ] .", "transplantation of hematopoietic cells as well as solid organs and detailed studies of viral infections provided the initial key information leading to the concept of genetic mhc restriction by autologous mhc molecules .", "however , numerous t - cell subsets have been identified that do not fulfil these criteria , including mhc class ib restricted t - cells , cd1 restricted t - cells , mr1 restricted mucosal associated invariant t - cells ( mait ) , nkt - cells , and t - cells , subsets that are collectively called unconventional or donor - unrestricted t - cells ( durt ) .", "unconventional t - cells behave differently in terms of memory , kinetics , and ligands recognized compared to conventional t - cells as recently summarized .", "an intriguing group of durt family cells are the t - cells that are restricted by mhc class ib molecules .", "these cells may share several critical properties with conventional t - cells but most importantly recognize antigens typically in the context of nonpolymorphic mhc - i molecules .", "the human mhc class ib family , also called nonclassical hla class i , is comprised of hla - e , hla - f , and hla - g .", "the major difference with classical class ia molecules is their very low level of allelic variation . whereas hla class ia families", "are composed of several hundred family members for hla - a , hla - b , and hla - c alleles , hla - e , hla - f , and hla - g comprise only 3 , 4 , and 10 family members , respectively , and not all of these are actually expressed as functional proteins .", "immune cells express relatively high levels of hla - e protein , but also tissue cells can express the hla - e protein ( http://www.proteinatlas.org/ ) . although hla - e was originally described to be broadly expressed by almost all cells that also express hla class ia molecules , other studies suggest hla - e expression is restricted to lymphoid and endothelial cells .", "furthermore , pathogens can affect hla - e cell surface expression ; for example , human cytomegalovirus ( cmv ) can upregulate its expression .", "hla - e functions as ligand for cd94-nkg2 receptors and has a peptide - binding groove that is ideally suited for binding peptides derived from the leader sequences of other mhc - i molecules . in this regard ,", "the loss of leader - peptide loaded hla - e expression is a marker for cells having lost expression of hla class ia molecules , which targets these cells for recognition and lysis by natural killer ( nk ) cells .", "in contrast to hla - e , hla - f expression appears to be more restricted and is detected mostly in liver and bladder .", "however , its expression is largely intracellular and in association with other mhc - i molecules , which has led to speculations that hla - f might be involved in the intracellular stabilization of hla class ia molecules .", "the third human mhc class ib family member , hla - g , has an even more narrow tissue distribution ; its expression appears limited to trophoblasts in the placenta , and it has been associated with fetal - maternal tolerance .", "hla - g may function during pregnancy to inhibit nk mediated lysis as trophoblasts lack hla - a and hla - b expression .", "thus , given the intracellular expression of hla - f and the placental restriction of hla - g , limited information is available on t - cells interacting with these molecules , and their relevance to general immunity remains unclear .", "for this reason , the focus of this review will be on hla - e restricted t - cells .", "the role of hla - e in the innate immune response is to present signal sequence - derived peptides of other hla class i molecules to inhibit nk mediated lysis of cells via recognition by cd94/nkg2a .", "however , hla - e can also bind and present other peptide sequences , which can be self or pathogen derived and can be recognized by adaptive t - cells .", "hla - e is thus considered to play a role in both innate and adaptive immunity , via interacting with both nk cells as well as presenting peptides to antigen specific cd8 t - cells ( figure 1 ) .", "eleven alleles have been reported for hla - e , only 3 of which can be translated into proteins , 2 of them being highly dominant , the hla - e ( e01:01 ) and the hla - e ( e01:03 ) variants , which differ only in a single amino acid at position 107 , being arginine ( e01:01 ) or glycine ( e01:03 ) . position 107 is located on the loop between the -strands outside of the 2 domain of the heavy chain , just outside the peptide - binding groove .", "the frequency of the hla - e and hla - e in the population is about equal , suggesting balanced selection in diverse populations [ 14 , 15 ] . whether hla - e and hla - e display functional differences has not been studied in detail [ 14 , 16 ] , but it has been demonstrated that hla - e homozygous cells express higher levels of hla - e and had higher peptide - binding affinity .", "more recently , peptide elution studies revealed a different peptide - binding repertoire eluted from hla - e versus hla - e molecules , indicating that the f - pocket of hla - e bound a smaller variety of peptides and had a stronger preference for a lysine at the p position [ 17 , 18 ] .", "the structural basis of hla - e 's ability to bind signal sequence - derived peptides from hla class ia ( hla - i ) molecules has been studied previously .", "unlike hla class ia that typically contain 2 or 3 anchor residues , hla - e contains 5 anchor residues in the peptide - binding groove that highly constrains the sequence of the bound peptide [ 19 , 20 ] .", "however , hla - e was additionally shown to bind peptides derived from viruses such as influenza m1 protein and ebv bzlf1 . moreover", ", a sequence from cytomegalovirus ( cmv ) glycoprotein ul40 , which is identical to the hla - c03 leader sequence , can bind to hla - e and is capable of preventing nk mediated lysis [ 9 , 22 ] . the same was found for a hcv derived sequence , despite its sequence difference from signal peptides .", "peptide identification and characterization using random peptide approaches also revealed that a leucine on p9 is a critical anchor residue for hla - e binding but did not identify methionine as critical p2 anchor for hla - e binding and folding .", "identification of the motif within the hla class ia leader sequences critical for interaction with the cd94/nkg2 complex suggested anchor residues at positions 2 , 6 , 7 , and 9 , while solvent exposed residues at p5 and p8 were likely important for binding to cd94/nkg2 .", "thus , while p2 and p9 are anchor residues for binding to hla - e and p5 and p8 are involved in the interaction with cd94/nkg2 , p8 is also the critical residue distinguishing self ( signal sequence ) from nonself ( cmv ul40 ) and allowing for tcr recognition of cmv ul40 .", "recent peptide elution studies provided important insights in the types of peptides that are naturally presented by hla - e and identified a larger array of peptides eluted from hla - e than originally discovered [ 17 , 18 , 27 ] .", "eluted peptides were generally short ( 8 - 9 - 10 mers ) , but occasionally also longer peptides were eluted including 1117 mers [ 17 , 18 , 27 ] .", "this is in line with earlier studies , where also peptides greater than 8 amino acids could bind hla - e .", "furthermore , eluted peptides were different from signal sequences , possessing hydrophobic amino acids on p2 and p9 , consistent with a binding motif that was very similar to that of hla - a2 .", "recent studies in rhesus macaques immunized with siv - gag ( simian immunodeficiency virus ) , in specific cmv vectors , revealed a series of peptides that were recognized by cd8 t - cells but only a minority contained the canonical mhc class i antigen e ( mhc - e ) binding motif .", "structural analyses revealed that the peptide - binding cleft of hla - e is rigid but relatively open compared to that of hla class ia family members .", "peptides that lack the canonical residues can adopt a backbone structure that is similar to canonical peptides , allowing them to bind the hla - e molecule .", "these unique binding properties of hla - e may explain the observed epitope diversity and breadth in siv - gag in the rhesus macaque vaccination studies .", "furthermore , the authors suggested that the open structure of hla - e may allow peptide exchange ; this may be in particular relevant for mycobacterium tuberculosis ( mtb ) as hla - e expression is enriched in the mtb phagosome .", "intriguingly , we have identified a large series of mtb epitopes presented in hla - e and recognized by mycobacteria exposed human donors , many of which lack the canonical residues .", "together , these data indicate that hla - e binds signal sequence - derived peptides not only from mhc class ia molecules but also from other self and even pathogen - derived sequences .", "although many peptides contain canonical amino acids for binding hla - e , clear examples exist for peptides which lack canonical residues and can still bind hla - e .", "presentation of nonself sequences , being absent during thymic selection , may elicit adaptive immune responses by cd8 t - cells .", "specific recognition of pathogen - derived sequences presented by the unconventional presentation molecule hla - e by the cd8 tcr could lead to specific activation of adaptive immune responses , independent of classically hla restricted cd4 and cd8 t - cells .", "as viruses require the human host to survive and therefore reside within host cells , their proteins are presented by hla class i molecules , including hla class ib .", "peptides from epstein barr virus ( ebv ) [ 3436 ] , cytomegalovirus ( cmv ) [ 12 , 3741 ] , and hepatitis c virus ( hcv ) can be presented by hla - e and are recognized by virus specific t - cells ( table 1 ) .", "the peptide epitopes studied from cmv ul40 are highly similar to the hla class ia signal sequences , whereas the sequences from ebv bzlf1 and hcv appear more different .", "the functional and phenotypical description of these t - cells is rather limited , but they all express cd8 as expected for hla class i restricted cells . in many studies , hla - e restricted t - cells have been identified and enumerated using hla - e tetramers whereas functional analyses were limited to the demonstration of target cell lysis . likewise , phenotypical characterizations were very limited in scope but when performed showed a cytolytic t - cell phenotype ( perforin , granzyme a / b ) and ifn production in some studies [ 38 , 41 ] .", ". initial studies on possible recognition of hla - e peptide complexes by t - cells in a tcr dependent manner were performed using signal sequences from hla class ia alleles [ 34 , 35 , 42 ] ( table 1 ) .", "it is a priori not clear why healthy human subjects would mount t - cell responses towards signal sequences of conserved class ia molecules . while some of these studies lack information on the hla - typing of the donors , others", "have suggested that these cells are mostly reactivity against nonself target peptides of signal sequences [ 34 , 35 , 42 ] .", "similarly , cytotoxic cd8 t - cells recognizing hla - e binding sequences from tcr v chains were detected in peripheral blood .", "an alternative , nonexclusive , explanation may be that these t - cells are reactive with nonself virally derived antigens that share sequence homology with the mhc class ia derived signal sequences used in these studies .", "nk - ctl due to their ability to lyse a broad range of allogeneic targets , it was subsequently found that these t - cells were specific for the cmv ul40-encoded peptide ( vmaprtlil ) bound to hla - e [ 35 , 42 ] .", "the detected alloreactivity was due to target cells possessing hla - c alleles encoding the same sequence as the ul40 peptide ( e.g. , hla - c03 ) .", "ul40-specific t - cells can reach frequencies in the circulation similar to those restricted by classical hla - i , indicating their potential to play a significant role in cmv immunity .", "the tcr from an ul40-specific t - cell clone , kk50.4 , was cloned , expressed , and analysed for its interaction with hla - e in complex with the ul40-epitope vmaprtlil ( figure 2 ) .", "overall , the structural basis for recognition of hla - e largely overlaps that of tcr recognition of hla class ia .", "however , in order for ul40-specific t - cells to recognize the ul40 antigen or allogeneic hla - i peptides , the ul40-specific tcr need to distinguish between the ul40 epitope ( vmaprtlil ) and nearly identical self peptides which may differ by as little as a single methyl group ( e.g. , vmaprtlvl ) .", "analysis of tcr sequences from ul40 specific t - cell clones suggested there were a limited number of tcrs capable of such discrimination , as all of the clones isolated utilized trbv14 ( v16 ) and there was a characteristic arginine residue present in the cdr3 ( figure 2 ) .", "structural analyses of the kk50.4 clone showed that the convergence of cdr1 , cdr2 , and cdr3 of the kk50.4 chain onto p8 ile determined self / nonself discrimination ( figure 2 ) .", "notably , the highly selected arginine present in the cdr3 made multiple contacts with both hla - e and peptide .", "as the cmv ul40 peptide is highly homologous to hla class ia derived signal sequences , typically these t - cells are only observed in individuals where the ul40 epitope differs from that found in self - hla - c alleles ( e.g. , hla - c07 homozygotes ) .", "however , other pathogen - derived peptides that bind to hla - e are more different from the class ia signal sequences and may therefore depend less on the donors ' hla class ia genotype . ( 2 )", "hiv - nef proteins interact with the intracellular domain of hla - a and hla - b molecules , resulting in downregulation of hla class ia molecules from the cell surface .", "in contrast , hla - c and hla class ib molecules , particularly , hla - e and hla - g , lack these intracellular nef - interaction domains and thus remain expressed normally on the cell surface of hiv infected cells .", "in addition , it has been shown that a peptide from the hiv-1 capsid protein p24 ( aisprtlna ) may further enhance hla - e surface expression .", "however , recently it was shown that this peptide presented in hla - e is not recognized by cd94/nkg2a and that these cells thus were not protected against nk mediated t - cell lysis .", "this is most likely due to lack of homology between the hiv p24 peptide and hla leader peptides , which prevents ligation of cd94-nkg2a to the hla - e peptide complex .", "inhibition of nk mediated lysis of hiv-1 infected t - cells rather appears to be the result of hla - c expression and recognition by nk cells that specifically express the nk cell receptors kir2dl1/2/3 .", "interestingly , it has not yet been investigated whether the hiv-1 p24 peptide , presented in hla - e , may be recognized by the host adaptive immune system and thus may result in specific cd8 t - cells .", "induction of such cd8 t - cell responses would be interesting from a therapeutic as well as vaccination point of view .", "in rhesus macaques , mhc - e ( or mamu - e ) is the homologue of human hla - e , which also showed upregulated expression in hiv / siv infected animals .", "vaccination of rhesus macaques with a cmv - based vector , expressing hiv gag , revealed a very strong cd8 t - cell response ( tcr ) with a large variety of specific interactions , with an estimated induction of 4 distinct epitopes per 100 amino acids in all tested hiv / siv derived antigens . although this massive mhc - e restricted response was due to the specific design of the viral vector , it clearly illustrates the abundance of potential hla - e epitopes in a large array of antigens .", "detailed characterization of the peptide - binding domain revealed a relatively open structure , exposing many side chains for interaction with the tcr .", "interestingly , next to inducing mhc - e restricted t - cells , these cmv recombinant vectors also induced a significant population of other unconventional cd8 t - cells , which were restricted by mhc class ii molecules .", "bacteria like salmonella and mtb are intracellular pathogens that hijack host cells to promote their own survival .", "intriguingly , the expression of hla - e is enriched on mtb phagosomes compared to classical hla class ia family members , thus presumably facilitating hla - e loading by mtb peptides in infected cells . in 1998 , lewinsohn et al . identified mtb specific cd8 t - cell clones that appeared to be restricted to mhc class ib , two of which were hla - e restricted", "unpublished recent data point to a peptide derived from the mtb glycoprotein mpt32 ( david lewinsohn , personal communication , manuscript in preparation ) . in an independent effort", ", we have screened the mtb genome for the presence of peptides that could potentially be presented by hla - e and selected 69 peptides based on 3 different prediction algorithms .", "many of these peptides were recognized by donors that had been previously sensitized by mycobacteria , suggesting in vivo priming and t - cell memory for several hla - e epitopes .", "we have shown that these peptides presented in hla - e elicit cd8 t - cell activation through the tcr , as measured by both zap70 phosphorylation ( the first downstream effect in tcr signalling ) , as well as cd137 expression ( a molecule exclusively expressed following specific antigen recognition on cd8 t - cells ) .", "moreover , hla - e restricted t - cell lines specific for mtb had strongly reduced cytokine production in the presence of blocking antibodies against the tcr or hla - e . altogether , these data support specific recognition of hla - e peptide complexes by mtb specific tcrs .", "hla - e binding peptides from mtb were not capable of preventing nk mediated lysis in a cd94/nkg2a dependent manner , similar to hiv p24 , indicating that only surface expression of peptide containing hla - e may not be sufficient .", "detailed characterization of these t - cells revealed that they are cytolytic or suppressive , and that t - cells reactive against the same peptide can display different functional polarities , indicating that polarity is not determined by the peptide .", "interestingly , t - cell clones with cytolytic activity were also capable of inhibiting intracellular outgrowth of mtb , suggesting that they are potent antimycobacterial effector cells .", "many of the hla - e restricted mtb specific t - cells did not produce typical cytotoxic t - lymphocyte associated cytokines , nor did they produce classical th1 cytokines ( ifn , tnf , and il2 ) , but instead they produced an array of th2 cytokines including il-4 , il-5 , il-10 , and il-13 , as well as the th2 associated transcription factor gata-3 ( table 2 ) [ 47 , 48 ] . in patients with tb disease , hla - e tetramers identified mtb specific hla - e restricted cd8 t - cells , with the highest frequencies at tb diagnosis and waning of the response during successful treatment .", "moreover , in line with the knowledge that hla - e is not susceptible to downregulation by hiv , we were able to detect hla - e specific t - cells in patients that concomitantly were infected with mtb and hiv .", "salmonella peptides presented by hla - e are also recognized by hla - e restricted t - cells .", "volunteers vaccinated with a s. typhi vaccine had a robust hla - e restricted t - cell response , as measured by the cytolytic capacities of these cells , such as granzyme b activity ( table 1 ) .", "kinetic analysis of these responses in a similarly vaccinated cohort revealed that the hla - e restricted t - cells are long - lasting , up to 2 years after vaccination , again suggesting immune memory .", "moreover , following challenge experiments with salmonella in unvaccinated , healthy volunteers , multifunctional hla - e restricted cd8 t - cells were detected and correlated with protection against typhoid disease development .", "interestingly , t - cells reactive with self hsp60sp presented by hla - e have also been identified , both in healthy donors and in patients with type 1 diabetes .", "these cd8 t - cell lines were involved in discriminating self from nonself in the periphery , and defective discrimination between self and nonself was detected in the majority of patients with type 1 diabetes .", "while many viruses are known to interfere with antigen processing and presentation , resulting in peptide presentation in a tap - independent manner , this is also the case in many tumors . as a consequence", ", tumor unique antigens may be presented also in the context of hla - e [ 54 , 55 ] , which may subsequently be recognized by cytotoxic t - cells .", "in contrast to what was expected , in humans the presence of ctls was only beneficial in patients with lung carcinoma if hla - e was not expressed by the tumor , indicating that hla - e restricted ctls may not directly contribute to tumor elimination in these patients .", "hla - e restricted t - cell responses have been studied only to a very limited extent in autoimmune diseases ; however they could potentially play an important role . in patients with multiple sclerosis ( ms ) , increased frequencies of ebv specific , hla - e restricted cd8 t - cells have been found to be associated mostly with the relapsing remitting form of the disease rather than the progressive form .", "moreover , cd8 t - cells induced by glatiramer acetate vaccination appear to be restricted to hla - e and have immunomodulatory capacities , resulting in amelioration of ms [ 5759 ] .", "hla - e restricted cd8 t - cells in patients with ms appeared phenotypically different from healthy controls ; however these t - cells were selected based on the expression of nkg2c and thus may reflect only a small subset of hla - e restricted t - cells . in rheumatoid arthritis ( ra ) , limited information is available , although hla - e polymorphisms may be associated with disease susceptibility and treatment responsiveness . interestingly and similar to the studies in ms ,", "ra like autoimmunity can be strongly inhibited by induction of ( self hsp60 ) peptide specific qa-1 restricted suppressor t - cells in mice .", "furthermore , there is a defect in cd8 t - cell recognition of hla - e / hsp60sp in patients with type i diabetes .", "thus , mhc - e restricted cd8 t - cells with immunoregulatory properties may be critical in amelioration of autoimmune diseases and deserve further detailed characterization .", "surprisingly , little information is available on the phenotype and function of human cd8t - cells recognizing peptides presented by hla - e . in many studies ,", "hla - e restricted t - cells have only been enumerated using tetramer staining , or the presence of hla - e reactivity was demonstrated using cytolytic assays ( table 2 ) .", "analyses of the tcr composition were only performed in a limited number of studies and , as mentioned above , found consistent selection of trav14 ( v16 ) in cmv specific tcrs .", "basic descriptive information of hla - e restricted t - cells , such as memory phenotype , is also largely lacking .", "the limited data that have been published do not suggest specific memory stages to be overrepresented among hla - e restricted cd8t - cell populations , since both cd45ra positive and negative populations were identified , as was also reported for ccr7 ( table 2 ) . generally , the t - cells reported expressed cytolytic molecules , but in some cases these were weakly expressed or even undetectable ( table 2 ) .", "specific target cell lysis was frequently used as read out to demonstrate hla - e restriction of cd8 t - cells .", "recently , we demonstrated for the first time that hla - e restricted cd8 t - cell clones had antibacterial activity against mtb , considered to be an important property in immune control of intracellular pathogens ( table 2 ) .", "other studies have not assessed or reported viral or bacterial inhibition following hla - e restricted cd8 t - cell activation or downstream target cell lysis .", "it will be of interest to identify the mechanism of control of intracellular outgrowth of mtb , as this could be a yet unknown component of the immune system that could be harnessed for preventive or therapeutic interventions .", "cytokine production has been analysed in detail only in the most recent series of papers .", "originally , studies focused on the production of classical th1 cytokines , such as ifn and tnf , and although sometimes detected in hla - e restricted t - cells , not all produced ifn in response to specific peptide stimulation ( table 2 ) .", "as mentioned above , we recently found mtb specific hla - e restricted cd8 t - cells to produce th2 rather than th1 cytokines and demonstrated that these cells utilize il-4 to activate b - cells ( table 2 ) .", "it would be interesting and relevant to investigate th2 cytokine production also in hla - e restricted cd8 t - cells in response to other ligands .", "moreover , an analysis of transcription factor expression in hla - e restricted cd8 t - cells has been limited thus far to our description of mtb specific t - cells expressing gata-3 ( table 2 ) .", "one of the major differences between innate and adaptive immunity is the formation of immunological memory during adaptive immune responses . as hla - e restricted cd8 t - cells", "are activated through their tcr , in an antigen specific manner , it is likely that they also differentiate into memory cells .", "the formation of memory cells following hla - e mediated antigen presentation would be an important prerequisite for successful application of these peptides in future vaccination strategies .", "phenotypically , effector memory and effector memory recently activated [ 38 , 41 ] have been described as indicators of memory .", "moreover , screening for recognition of hla - e restricted mtb peptides by human donors showed recognition only in donors that had been sensitized by mycobacteria ( as measured by ppd recognition ) , suggesting that in vivo priming , and thus memory induction , was critical .", "vaccination of healthy volunteers with a single dose of s. typhi strain ty21a resulted in antigen specific , hla - e restricted cd8 t - cells that were detectable up to 2 years after vaccination , suggesting that hla - e restricted memory t - cells had been induced . also , individuals with latent tb and individuals successfully treated for tb disease still had circulating cd8 t - cells binding to hla - e tetramers ( tm ) loaded with mtb peptides ( notwithstanding that the frequencies detected were highest in patients with active tb disease ) ; the persistence of responses after microbiological cure thus also suggests immune memory .", "the murine homologue of human hla - e is qa-1 , which also presents signal sequences from mhc class i proteins that are called qdm ( qa-1 determinant modifier ) .", "however , also specific recognition of qa-1 peptide complexes by t - cells has been described .", "interestingly , similar to hla - e , qa-1 restricted t - cells have been isolated that are able to detect differences in leader - sequenced derived peptides between mouse strains , although there is no indication they are involved in immunity to mouse cmv . also ,", "similar to hla - e , qa-1 restricted t - cells have been implicated in immunity to pathogens , including listeria monocytogenes and salmonella typhimurium . qa-1", "restricted s. typhimurium specific t - cells are curiously cross - reactive to self hsp-60 derived peptides and thus have been implicated in autoimmune conditions .", "furthermore , there is a large and older body of research focusing on qa-1 restricted suppressor cd8 t - cells .", "these t - cells reportedly recognize qa-1 in a tcr dependent manner and act to suppress autoreactive cd4 t - cells , thereby attenuating the development of autoimmune encephalomyelitis ( a mouse model of multiple sclerosis ) , very similar to the role of hla - e restricted cd8 t - cells evoked by vaccination in human ms [ 6769 ] . in this model ,", "qa-1 deficient mice developed exaggerated secondary cd4 responses , as a result of the lack of a population of regulatory cd8 t - cells , demonstrating the in vivo significance of suppressor qa-1 restricted t - cells .", "their role in infectious diseases and in particular in the elimination of pathogens has not been studied in great detail thus far . moreover ,", "a detailed description of the phenotype and function of these cells is lacking and warrants further investigation .", "hla - e plays a dual role in the innate and adaptive immune system ( figure 1 ) .", "the low polymorphism in hla - e in conjunction with its relative insensitivity to downregulation by , for example , hiv , makes hla - e an interesting target for vaccination strategies against infectious diseases and tumors .", "small sets of peptides should suffice to induce t - cells recognizing foreign antigens and mount effector responses .", "the quality of these t - cell responses needs further investigation , however , given the diversity in functions that have been described thus far , ranging from cytotoxicity and pathogen control to immune suppression .", "various pathogen - derived antigens can bind and stabilize hla - e at the cell surface , for some of which this may be an essential mechanism to prevent nk mediated target cell lysis", ". however , many of these antigens can also be recognized in a tcr dependent manner by cd8 t - cells .", "these t - cells may suppress bystander t - cells or lyse ( infected ) target cells and inhibit intracellular bacteria , indicating an important functional contribution to the immune response .", "nevertheless , the relative frequency of hla - e restricted t - cells and their in vivo relevance in many cases remains unknown and unstudied .", "although these t - cells are donor - unrestricted , they in many aspects display similar functionalities to classical , conventional t - cells .", "pathogen specific hla - e restricted cd8 t - cells are an interesting new player in the field of immunology . future work should address their exact roles and relative contributions in the immune response against infectious diseases ." ]

[ "<S> human hla - e can , in addition to self - antigens , also present pathogen - derived sequences , which elicit specific t - cell responses . </S>", "<S> t - cells recognize their antigen presented by hla - e highly specifically and have unique functional and phenotypical properties . </S>", "<S> pathogen specific hla - e restricted cd8 + t - cells are an interesting new player in the field of immunology . </S>", "<S> future work should address their exact roles and relative contributions in the immune response against infectious diseases . </S>" ]

[ "1. Introduction ", "2. HLA-E", "3. Summary and Implications" ]

[ [ "t - cell activation requires specific recognition of antigen presented as small fragments ( peptides ) bound to major histocompatibility complex ( mhc ) molecules .", "the recognition of a particular peptide - mhc occurs through a highly specific t - cell receptor ( tcr ) , which is selected in the thymus .", "following tcr triggering , costimulation and the presence of polarizing cytokines together determine the t - cell activation pattern and guide ultimate t - cell differentiation .", "classically , cd4t - cells recognize antigens scavenged extracellularly by the antigen presenting cell ( apc ) that are presented in mhc class ii , whereas cd8 t - cells recognize endogenous antigens presented by mhc class i ( mhc - i ) [ 1 , 2 ] . in spite of this widely held view , already decades ago it was shown that also antigens derived from intracellular pathogens such as viruses or intracellular bacteria can be presented in mhc - i .", "more recently , cross - presentation by dendritic cells and autophagy have been elucidated as important mechanisms in this context [ 2 , 4 ] .", "transplantation of hematopoietic cells as well as solid organs and detailed studies of viral infections provided the initial key information leading to the concept of genetic mhc restriction by autologous mhc molecules .", "however , numerous t - cell subsets have been identified that do not fulfil these criteria , including mhc class ib restricted t - cells , cd1 restricted t - cells , mr1 restricted mucosal associated invariant t - cells ( mait ) , nkt - cells , and t - cells , subsets that are collectively called unconventional or donor - unrestricted t - cells ( durt ) .", "unconventional t - cells behave differently in terms of memory , kinetics , and ligands recognized compared to conventional t - cells as recently summarized .", "an intriguing group of durt family cells are the t - cells that are restricted by mhc class ib molecules .", "these cells may share several critical properties with conventional t - cells but most importantly recognize antigens typically in the context of nonpolymorphic mhc - i molecules .", "the human mhc class ib family , also called nonclassical hla class i , is comprised of hla - e , hla - f , and hla - g .", "the major difference with classical class ia molecules is their very low level of allelic variation . whereas hla class ia families", "are composed of several hundred family members for hla - a , hla - b , and hla - c alleles , hla - e , hla - f , and hla - g comprise only 3 , 4 , and 10 family members , respectively , and not all of these are actually expressed as functional proteins .", "immune cells express relatively high levels of hla - e protein , but also tissue cells can express the hla - e protein ( http://www.proteinatlas.org/ ) . although hla - e was originally described to be broadly expressed by almost all cells that also express hla class ia molecules , other studies suggest hla - e expression is restricted to lymphoid and endothelial cells .", "furthermore , pathogens can affect hla - e cell surface expression ; for example , human cytomegalovirus ( cmv ) can upregulate its expression .", "hla - e functions as ligand for cd94-nkg2 receptors and has a peptide - binding groove that is ideally suited for binding peptides derived from the leader sequences of other mhc - i molecules . in this regard ,", "the loss of leader - peptide loaded hla - e expression is a marker for cells having lost expression of hla class ia molecules , which targets these cells for recognition and lysis by natural killer ( nk ) cells .", "in contrast to hla - e , hla - f expression appears to be more restricted and is detected mostly in liver and bladder .", "however , its expression is largely intracellular and in association with other mhc - i molecules , which has led to speculations that hla - f might be involved in the intracellular stabilization of hla class ia molecules .", "the third human mhc class ib family member , hla - g , has an even more narrow tissue distribution ; its expression appears limited to trophoblasts in the placenta , and it has been associated with fetal - maternal tolerance .", "hla - g may function during pregnancy to inhibit nk mediated lysis as trophoblasts lack hla - a and hla - b expression .", "thus , given the intracellular expression of hla - f and the placental restriction of hla - g , limited information is available on t - cells interacting with these molecules , and their relevance to general immunity remains unclear .", "for this reason , the focus of this review will be on hla - e restricted t - cells ." ], [ "the role of hla - e in the innate immune response is to present signal sequence - derived peptides of other hla class i molecules to inhibit nk mediated lysis of cells via recognition by cd94/nkg2a .", "however , hla - e can also bind and present other peptide sequences , which can be self or pathogen derived and can be recognized by adaptive t - cells .", "hla - e is thus considered to play a role in both innate and adaptive immunity , via interacting with both nk cells as well as presenting peptides to antigen specific cd8 t - cells ( figure 1 ) .", "eleven alleles have been reported for hla - e , only 3 of which can be translated into proteins , 2 of them being highly dominant , the hla - e ( e01:01 ) and the hla - e ( e01:03 ) variants , which differ only in a single amino acid at position 107 , being arginine ( e01:01 ) or glycine ( e01:03 ) . position 107 is located on the loop between the -strands outside of the 2 domain of the heavy chain , just outside the peptide - binding groove .", "the frequency of the hla - e and hla - e in the population is about equal , suggesting balanced selection in diverse populations [ 14 , 15 ] . whether hla - e and hla - e display functional differences has not been studied in detail [ 14 , 16 ] , but it has been demonstrated that hla - e homozygous cells express higher levels of hla - e and had higher peptide - binding affinity .", "more recently , peptide elution studies revealed a different peptide - binding repertoire eluted from hla - e versus hla - e molecules , indicating that the f - pocket of hla - e bound a smaller variety of peptides and had a stronger preference for a lysine at the p position [ 17 , 18 ] .", "the structural basis of hla - e 's ability to bind signal sequence - derived peptides from hla class ia ( hla - i ) molecules has been studied previously .", "unlike hla class ia that typically contain 2 or 3 anchor residues , hla - e contains 5 anchor residues in the peptide - binding groove that highly constrains the sequence of the bound peptide [ 19 , 20 ] .", "however , hla - e was additionally shown to bind peptides derived from viruses such as influenza m1 protein and ebv bzlf1 . moreover", ", a sequence from cytomegalovirus ( cmv ) glycoprotein ul40 , which is identical to the hla - c03 leader sequence , can bind to hla - e and is capable of preventing nk mediated lysis [ 9 , 22 ] . the same was found for a hcv derived sequence , despite its sequence difference from signal peptides .", "peptide identification and characterization using random peptide approaches also revealed that a leucine on p9 is a critical anchor residue for hla - e binding but did not identify methionine as critical p2 anchor for hla - e binding and folding .", "identification of the motif within the hla class ia leader sequences critical for interaction with the cd94/nkg2 complex suggested anchor residues at positions 2 , 6 , 7 , and 9 , while solvent exposed residues at p5 and p8 were likely important for binding to cd94/nkg2 .", "thus , while p2 and p9 are anchor residues for binding to hla - e and p5 and p8 are involved in the interaction with cd94/nkg2 , p8 is also the critical residue distinguishing self ( signal sequence ) from nonself ( cmv ul40 ) and allowing for tcr recognition of cmv ul40 .", "recent peptide elution studies provided important insights in the types of peptides that are naturally presented by hla - e and identified a larger array of peptides eluted from hla - e than originally discovered [ 17 , 18 , 27 ] .", "eluted peptides were generally short ( 8 - 9 - 10 mers ) , but occasionally also longer peptides were eluted including 1117 mers [ 17 , 18 , 27 ] .", "this is in line with earlier studies , where also peptides greater than 8 amino acids could bind hla - e .", "furthermore , eluted peptides were different from signal sequences , possessing hydrophobic amino acids on p2 and p9 , consistent with a binding motif that was very similar to that of hla - a2 .", "recent studies in rhesus macaques immunized with siv - gag ( simian immunodeficiency virus ) , in specific cmv vectors , revealed a series of peptides that were recognized by cd8 t - cells but only a minority contained the canonical mhc class i antigen e ( mhc - e ) binding motif .", "structural analyses revealed that the peptide - binding cleft of hla - e is rigid but relatively open compared to that of hla class ia family members .", "peptides that lack the canonical residues can adopt a backbone structure that is similar to canonical peptides , allowing them to bind the hla - e molecule .", "these unique binding properties of hla - e may explain the observed epitope diversity and breadth in siv - gag in the rhesus macaque vaccination studies .", "furthermore , the authors suggested that the open structure of hla - e may allow peptide exchange ; this may be in particular relevant for mycobacterium tuberculosis ( mtb ) as hla - e expression is enriched in the mtb phagosome .", "intriguingly , we have identified a large series of mtb epitopes presented in hla - e and recognized by mycobacteria exposed human donors , many of which lack the canonical residues .", "together , these data indicate that hla - e binds signal sequence - derived peptides not only from mhc class ia molecules but also from other self and even pathogen - derived sequences .", "although many peptides contain canonical amino acids for binding hla - e , clear examples exist for peptides which lack canonical residues and can still bind hla - e .", "presentation of nonself sequences , being absent during thymic selection , may elicit adaptive immune responses by cd8 t - cells .", "specific recognition of pathogen - derived sequences presented by the unconventional presentation molecule hla - e by the cd8 tcr could lead to specific activation of adaptive immune responses , independent of classically hla restricted cd4 and cd8 t - cells .", "as viruses require the human host to survive and therefore reside within host cells , their proteins are presented by hla class i molecules , including hla class ib .", "peptides from epstein barr virus ( ebv ) [ 3436 ] , cytomegalovirus ( cmv ) [ 12 , 3741 ] , and hepatitis c virus ( hcv ) can be presented by hla - e and are recognized by virus specific t - cells ( table 1 ) .", "the peptide epitopes studied from cmv ul40 are highly similar to the hla class ia signal sequences , whereas the sequences from ebv bzlf1 and hcv appear more different .", "the functional and phenotypical description of these t - cells is rather limited , but they all express cd8 as expected for hla class i restricted cells . in many studies , hla - e restricted t - cells have been identified and enumerated using hla - e tetramers whereas functional analyses were limited to the demonstration of target cell lysis . likewise , phenotypical characterizations were very limited in scope but when performed showed a cytolytic t - cell phenotype ( perforin , granzyme a / b ) and ifn production in some studies [ 38 , 41 ] .", ". initial studies on possible recognition of hla - e peptide complexes by t - cells in a tcr dependent manner were performed using signal sequences from hla class ia alleles [ 34 , 35 , 42 ] ( table 1 ) .", "it is a priori not clear why healthy human subjects would mount t - cell responses towards signal sequences of conserved class ia molecules . while some of these studies lack information on the hla - typing of the donors , others", "have suggested that these cells are mostly reactivity against nonself target peptides of signal sequences [ 34 , 35 , 42 ] .", "similarly , cytotoxic cd8 t - cells recognizing hla - e binding sequences from tcr v chains were detected in peripheral blood .", "an alternative , nonexclusive , explanation may be that these t - cells are reactive with nonself virally derived antigens that share sequence homology with the mhc class ia derived signal sequences used in these studies .", "nk - ctl due to their ability to lyse a broad range of allogeneic targets , it was subsequently found that these t - cells were specific for the cmv ul40-encoded peptide ( vmaprtlil ) bound to hla - e [ 35 , 42 ] .", "the detected alloreactivity was due to target cells possessing hla - c alleles encoding the same sequence as the ul40 peptide ( e.g. , hla - c03 ) .", "ul40-specific t - cells can reach frequencies in the circulation similar to those restricted by classical hla - i , indicating their potential to play a significant role in cmv immunity .", "the tcr from an ul40-specific t - cell clone , kk50.4 , was cloned , expressed , and analysed for its interaction with hla - e in complex with the ul40-epitope vmaprtlil ( figure 2 ) .", "overall , the structural basis for recognition of hla - e largely overlaps that of tcr recognition of hla class ia .", "however , in order for ul40-specific t - cells to recognize the ul40 antigen or allogeneic hla - i peptides , the ul40-specific tcr need to distinguish between the ul40 epitope ( vmaprtlil ) and nearly identical self peptides which may differ by as little as a single methyl group ( e.g. , vmaprtlvl ) .", "analysis of tcr sequences from ul40 specific t - cell clones suggested there were a limited number of tcrs capable of such discrimination , as all of the clones isolated utilized trbv14 ( v16 ) and there was a characteristic arginine residue present in the cdr3 ( figure 2 ) .", "structural analyses of the kk50.4 clone showed that the convergence of cdr1 , cdr2 , and cdr3 of the kk50.4 chain onto p8 ile determined self / nonself discrimination ( figure 2 ) .", "notably , the highly selected arginine present in the cdr3 made multiple contacts with both hla - e and peptide .", "as the cmv ul40 peptide is highly homologous to hla class ia derived signal sequences , typically these t - cells are only observed in individuals where the ul40 epitope differs from that found in self - hla - c alleles ( e.g. , hla - c07 homozygotes ) .", "however , other pathogen - derived peptides that bind to hla - e are more different from the class ia signal sequences and may therefore depend less on the donors ' hla class ia genotype . ( 2 )", "hiv - nef proteins interact with the intracellular domain of hla - a and hla - b molecules , resulting in downregulation of hla class ia molecules from the cell surface .", "in contrast , hla - c and hla class ib molecules , particularly , hla - e and hla - g , lack these intracellular nef - interaction domains and thus remain expressed normally on the cell surface of hiv infected cells .", "in addition , it has been shown that a peptide from the hiv-1 capsid protein p24 ( aisprtlna ) may further enhance hla - e surface expression .", "however , recently it was shown that this peptide presented in hla - e is not recognized by cd94/nkg2a and that these cells thus were not protected against nk mediated t - cell lysis .", "this is most likely due to lack of homology between the hiv p24 peptide and hla leader peptides , which prevents ligation of cd94-nkg2a to the hla - e peptide complex .", "inhibition of nk mediated lysis of hiv-1 infected t - cells rather appears to be the result of hla - c expression and recognition by nk cells that specifically express the nk cell receptors kir2dl1/2/3 .", "interestingly , it has not yet been investigated whether the hiv-1 p24 peptide , presented in hla - e , may be recognized by the host adaptive immune system and thus may result in specific cd8 t - cells .", "induction of such cd8 t - cell responses would be interesting from a therapeutic as well as vaccination point of view .", "in rhesus macaques , mhc - e ( or mamu - e ) is the homologue of human hla - e , which also showed upregulated expression in hiv / siv infected animals .", "vaccination of rhesus macaques with a cmv - based vector , expressing hiv gag , revealed a very strong cd8 t - cell response ( tcr ) with a large variety of specific interactions , with an estimated induction of 4 distinct epitopes per 100 amino acids in all tested hiv / siv derived antigens . although this massive mhc - e restricted response was due to the specific design of the viral vector , it clearly illustrates the abundance of potential hla - e epitopes in a large array of antigens .", "detailed characterization of the peptide - binding domain revealed a relatively open structure , exposing many side chains for interaction with the tcr .", "interestingly , next to inducing mhc - e restricted t - cells , these cmv recombinant vectors also induced a significant population of other unconventional cd8 t - cells , which were restricted by mhc class ii molecules .", "bacteria like salmonella and mtb are intracellular pathogens that hijack host cells to promote their own survival .", "intriguingly , the expression of hla - e is enriched on mtb phagosomes compared to classical hla class ia family members , thus presumably facilitating hla - e loading by mtb peptides in infected cells . in 1998 , lewinsohn et al . identified mtb specific cd8 t - cell clones that appeared to be restricted to mhc class ib , two of which were hla - e restricted", "unpublished recent data point to a peptide derived from the mtb glycoprotein mpt32 ( david lewinsohn , personal communication , manuscript in preparation ) . in an independent effort", ", we have screened the mtb genome for the presence of peptides that could potentially be presented by hla - e and selected 69 peptides based on 3 different prediction algorithms .", "many of these peptides were recognized by donors that had been previously sensitized by mycobacteria , suggesting in vivo priming and t - cell memory for several hla - e epitopes .", "we have shown that these peptides presented in hla - e elicit cd8 t - cell activation through the tcr , as measured by both zap70 phosphorylation ( the first downstream effect in tcr signalling ) , as well as cd137 expression ( a molecule exclusively expressed following specific antigen recognition on cd8 t - cells ) .", "moreover , hla - e restricted t - cell lines specific for mtb had strongly reduced cytokine production in the presence of blocking antibodies against the tcr or hla - e . altogether , these data support specific recognition of hla - e peptide complexes by mtb specific tcrs .", "hla - e binding peptides from mtb were not capable of preventing nk mediated lysis in a cd94/nkg2a dependent manner , similar to hiv p24 , indicating that only surface expression of peptide containing hla - e may not be sufficient .", "detailed characterization of these t - cells revealed that they are cytolytic or suppressive , and that t - cells reactive against the same peptide can display different functional polarities , indicating that polarity is not determined by the peptide .", "interestingly , t - cell clones with cytolytic activity were also capable of inhibiting intracellular outgrowth of mtb , suggesting that they are potent antimycobacterial effector cells .", "many of the hla - e restricted mtb specific t - cells did not produce typical cytotoxic t - lymphocyte associated cytokines , nor did they produce classical th1 cytokines ( ifn , tnf , and il2 ) , but instead they produced an array of th2 cytokines including il-4 , il-5 , il-10 , and il-13 , as well as the th2 associated transcription factor gata-3 ( table 2 ) [ 47 , 48 ] . in patients with tb disease , hla - e tetramers identified mtb specific hla - e restricted cd8 t - cells , with the highest frequencies at tb diagnosis and waning of the response during successful treatment .", "moreover , in line with the knowledge that hla - e is not susceptible to downregulation by hiv , we were able to detect hla - e specific t - cells in patients that concomitantly were infected with mtb and hiv .", "salmonella peptides presented by hla - e are also recognized by hla - e restricted t - cells .", "volunteers vaccinated with a s. typhi vaccine had a robust hla - e restricted t - cell response , as measured by the cytolytic capacities of these cells , such as granzyme b activity ( table 1 ) .", "kinetic analysis of these responses in a similarly vaccinated cohort revealed that the hla - e restricted t - cells are long - lasting , up to 2 years after vaccination , again suggesting immune memory .", "moreover , following challenge experiments with salmonella in unvaccinated , healthy volunteers , multifunctional hla - e restricted cd8 t - cells were detected and correlated with protection against typhoid disease development .", "interestingly , t - cells reactive with self hsp60sp presented by hla - e have also been identified , both in healthy donors and in patients with type 1 diabetes .", "these cd8 t - cell lines were involved in discriminating self from nonself in the periphery , and defective discrimination between self and nonself was detected in the majority of patients with type 1 diabetes .", "while many viruses are known to interfere with antigen processing and presentation , resulting in peptide presentation in a tap - independent manner , this is also the case in many tumors . as a consequence", ", tumor unique antigens may be presented also in the context of hla - e [ 54 , 55 ] , which may subsequently be recognized by cytotoxic t - cells .", "in contrast to what was expected , in humans the presence of ctls was only beneficial in patients with lung carcinoma if hla - e was not expressed by the tumor , indicating that hla - e restricted ctls may not directly contribute to tumor elimination in these patients .", "hla - e restricted t - cell responses have been studied only to a very limited extent in autoimmune diseases ; however they could potentially play an important role . in patients with multiple sclerosis ( ms ) , increased frequencies of ebv specific , hla - e restricted cd8 t - cells have been found to be associated mostly with the relapsing remitting form of the disease rather than the progressive form .", "moreover , cd8 t - cells induced by glatiramer acetate vaccination appear to be restricted to hla - e and have immunomodulatory capacities , resulting in amelioration of ms [ 5759 ] .", "hla - e restricted cd8 t - cells in patients with ms appeared phenotypically different from healthy controls ; however these t - cells were selected based on the expression of nkg2c and thus may reflect only a small subset of hla - e restricted t - cells . in rheumatoid arthritis ( ra ) , limited information is available , although hla - e polymorphisms may be associated with disease susceptibility and treatment responsiveness . interestingly and similar to the studies in ms ,", "ra like autoimmunity can be strongly inhibited by induction of ( self hsp60 ) peptide specific qa-1 restricted suppressor t - cells in mice .", "furthermore , there is a defect in cd8 t - cell recognition of hla - e / hsp60sp in patients with type i diabetes .", "thus , mhc - e restricted cd8 t - cells with immunoregulatory properties may be critical in amelioration of autoimmune diseases and deserve further detailed characterization .", "surprisingly , little information is available on the phenotype and function of human cd8t - cells recognizing peptides presented by hla - e . in many studies ,", "hla - e restricted t - cells have only been enumerated using tetramer staining , or the presence of hla - e reactivity was demonstrated using cytolytic assays ( table 2 ) .", "analyses of the tcr composition were only performed in a limited number of studies and , as mentioned above , found consistent selection of trav14 ( v16 ) in cmv specific tcrs .", "basic descriptive information of hla - e restricted t - cells , such as memory phenotype , is also largely lacking .", "the limited data that have been published do not suggest specific memory stages to be overrepresented among hla - e restricted cd8t - cell populations , since both cd45ra positive and negative populations were identified , as was also reported for ccr7 ( table 2 ) . generally , the t - cells reported expressed cytolytic molecules , but in some cases these were weakly expressed or even undetectable ( table 2 ) .", "specific target cell lysis was frequently used as read out to demonstrate hla - e restriction of cd8 t - cells .", "recently , we demonstrated for the first time that hla - e restricted cd8 t - cell clones had antibacterial activity against mtb , considered to be an important property in immune control of intracellular pathogens ( table 2 ) .", "other studies have not assessed or reported viral or bacterial inhibition following hla - e restricted cd8 t - cell activation or downstream target cell lysis .", "it will be of interest to identify the mechanism of control of intracellular outgrowth of mtb , as this could be a yet unknown component of the immune system that could be harnessed for preventive or therapeutic interventions .", "cytokine production has been analysed in detail only in the most recent series of papers .", "originally , studies focused on the production of classical th1 cytokines , such as ifn and tnf , and although sometimes detected in hla - e restricted t - cells , not all produced ifn in response to specific peptide stimulation ( table 2 ) .", "as mentioned above , we recently found mtb specific hla - e restricted cd8 t - cells to produce th2 rather than th1 cytokines and demonstrated that these cells utilize il-4 to activate b - cells ( table 2 ) .", "it would be interesting and relevant to investigate th2 cytokine production also in hla - e restricted cd8 t - cells in response to other ligands .", "moreover , an analysis of transcription factor expression in hla - e restricted cd8 t - cells has been limited thus far to our description of mtb specific t - cells expressing gata-3 ( table 2 ) .", "one of the major differences between innate and adaptive immunity is the formation of immunological memory during adaptive immune responses . as hla - e restricted cd8 t - cells", "are activated through their tcr , in an antigen specific manner , it is likely that they also differentiate into memory cells .", "the formation of memory cells following hla - e mediated antigen presentation would be an important prerequisite for successful application of these peptides in future vaccination strategies .", "phenotypically , effector memory and effector memory recently activated [ 38 , 41 ] have been described as indicators of memory .", "moreover , screening for recognition of hla - e restricted mtb peptides by human donors showed recognition only in donors that had been sensitized by mycobacteria ( as measured by ppd recognition ) , suggesting that in vivo priming , and thus memory induction , was critical .", "vaccination of healthy volunteers with a single dose of s. typhi strain ty21a resulted in antigen specific , hla - e restricted cd8 t - cells that were detectable up to 2 years after vaccination , suggesting that hla - e restricted memory t - cells had been induced . also , individuals with latent tb and individuals successfully treated for tb disease still had circulating cd8 t - cells binding to hla - e tetramers ( tm ) loaded with mtb peptides ( notwithstanding that the frequencies detected were highest in patients with active tb disease ) ; the persistence of responses after microbiological cure thus also suggests immune memory .", "the murine homologue of human hla - e is qa-1 , which also presents signal sequences from mhc class i proteins that are called qdm ( qa-1 determinant modifier ) .", "however , also specific recognition of qa-1 peptide complexes by t - cells has been described .", "interestingly , similar to hla - e , qa-1 restricted t - cells have been isolated that are able to detect differences in leader - sequenced derived peptides between mouse strains , although there is no indication they are involved in immunity to mouse cmv . also ,", "similar to hla - e , qa-1 restricted t - cells have been implicated in immunity to pathogens , including listeria monocytogenes and salmonella typhimurium . qa-1", "restricted s. typhimurium specific t - cells are curiously cross - reactive to self hsp-60 derived peptides and thus have been implicated in autoimmune conditions .", "furthermore , there is a large and older body of research focusing on qa-1 restricted suppressor cd8 t - cells .", "these t - cells reportedly recognize qa-1 in a tcr dependent manner and act to suppress autoreactive cd4 t - cells , thereby attenuating the development of autoimmune encephalomyelitis ( a mouse model of multiple sclerosis ) , very similar to the role of hla - e restricted cd8 t - cells evoked by vaccination in human ms [ 6769 ] . in this model ,", "qa-1 deficient mice developed exaggerated secondary cd4 responses , as a result of the lack of a population of regulatory cd8 t - cells , demonstrating the in vivo significance of suppressor qa-1 restricted t - cells .", "their role in infectious diseases and in particular in the elimination of pathogens has not been studied in great detail thus far . moreover ,", "a detailed description of the phenotype and function of these cells is lacking and warrants further investigation ." ], [ "hla - e plays a dual role in the innate and adaptive immune system ( figure 1 ) .", "the low polymorphism in hla - e in conjunction with its relative insensitivity to downregulation by , for example , hiv , makes hla - e an interesting target for vaccination strategies against infectious diseases and tumors .", "small sets of peptides should suffice to induce t - cells recognizing foreign antigens and mount effector responses .", "the quality of these t - cell responses needs further investigation , however , given the diversity in functions that have been described thus far , ranging from cytotoxicity and pathogen control to immune suppression .", "various pathogen - derived antigens can bind and stabilize hla - e at the cell surface , for some of which this may be an essential mechanism to prevent nk mediated target cell lysis", ". however , many of these antigens can also be recognized in a tcr dependent manner by cd8 t - cells .", "these t - cells may suppress bystander t - cells or lyse ( infected ) target cells and inhibit intracellular bacteria , indicating an important functional contribution to the immune response .", "nevertheless , the relative frequency of hla - e restricted t - cells and their in vivo relevance in many cases remains unknown and unstudied .", "although these t - cells are donor - unrestricted , they in many aspects display similar functionalities to classical , conventional t - cells .", "pathogen specific hla - e restricted cd8 t - cells are an interesting new player in the field of immunology . future work should address their exact roles and relative contributions in the immune response against infectious diseases ." ] ]

[ "clonal proliferation of histiocytes was reported in langerhans cell histiocytosis ( lch ) by willman et al . in 1994 ( 1 ) .", "langerhans cells are a type of nonlymphoid mononuclear cells involved in inflammatory responses and also in neoplastic processes .", "they are immature dendritic cells which express lysosomal enzymes cd1a and s-100 protein and ultrastructurally contain racket shaped organelles of birbeck granules ( 2 ) .", "the etiology is unknown ; however , environmental agents and viruses , especially epstein - barr virus ( ebv ) , or vaccination , have been proposed to play a role in the pathogenesis of lch ( 3 , 4 ) .", "the role of ebv as the etiologic agent of several malignancies is known and herpes viruses are shown to be responsible for persistent infection ( 5 , 6 ) .", "in addition , ebv and cytomegalovirus ( cmv ) are considered to be responsible for hemophagocytic syndromes in human with several inherited immunodeficiencies ( 7 , 8) .", "one study showed 30% positivity for cmv in lch ( 9 ) ; however , others have failed to show any association with adenovirus , cytomegalovirus , epstein - barr virus , herpes simplex virus , human herpes virus type 6 , human immunodeficiency virus , human t - cell leukemia virus types i and ii , and parvovirus using pcr ( 10 - 12 ) .", "in this study , we have investigated the presence of cmv in lch by qualitative pcr method .", "formalin - fixed , paraffin - embedded ( ffpe ) tissue samples of 30 patients with pathologic diagnosis of lch were extracted from archive of pathology department of mofid children s hospital in tehran ( one of the referral centers from all over the country ) for a ten year period ( 2002 to 2012 ) .", "the diagnosis of lch was made by a pediatric pathologist , using the histological criteria mentioned in the pathology textbooks , i.e. granulomas composed of langerhans cells with typical grooved nuclei mixed with eosinophils and other inflammatory cells .", "the diagnoses were confirmed using immunohistochemical technique for cd1a , s-100 protein and cd68 when available .", "thirty tissue samples with non lch diagnoses were also selected ( between the years 2002 and 2012 ) as age and site - matched controls to lch cases .", "paraffin was removed from the ffpe samples after undergoing xylene treatment , followed by two washes with pure ethanol .", "total dna was extracted from the air - dried pellet tissues according to the rtp dna / rna virus mini kit procedure ( stratec molecular , berlin , germany ) .", "the extracted nucleic acid was stored at -20c until it underwent pcr . to assess the quality of the extracted genome , as well as inhibition of pcr test ,", "all extracted and stored nucleic acid underwent beta - globin pcr , using the pco3/pco4 primer set , as described previously with modifications in method ( 13 ) .", "the beta - globin pcr was performed in syber green real - time pcr - melting curve format ( figure 1 ) . to detect cmv ,", "real - time pcr was performed using envelope glycoprotein b ( gpul55 ) gene primer sets amplifying 116 bp gene region of the virus genome ( cmv - r ; 5-aagtacccctatcgcgtgtg-3 ) , cmv - f ; 5-atgatgccctcrtccargtc -3 , with an internal probe , cmv - p ; 5-fam - tggcccagggtacggatcttattcg - bhq1 - 3 ) ( 14 ) .", "amplification of cmv gpul55 genes was performed in reaction volumes of 20 l under the following conditions : first the samples underwent denaturation at 94c for 10 minutes , followed by denaturation at 94c for 10 seconds , followed by annealing and extension at 60c for one minute , 50 cycles ( figure 2 ) .", "real - time pcr system cfx-96 ( bio - rad , usa ) with hs prime taq premix taqman reagent ( genetbio , korea ) was used in real - time pcr assay .", "the limit detection of 5 genome copies of cmv per reaction was determined by the real - time assay , using the serial dilutions of amplirun cytomegalovirus dna control ( vircell , spain ) .", "the graph shows that the melting temperature of beta - globin pcr product is around 82.5c .", "the achieved data were compared by chi - square ( or fisher s exact test if appropriate ) .", "p value of less than 0.05 was considered to indicate statistical significance . in this study", ", no ethical issues were involved ; only the pathology reports were reviewed retrospectively , and the patients were anonymous .", "formalin - fixed , paraffin - embedded ( ffpe ) tissue samples of 30 patients with pathologic diagnosis of lch were extracted from archive of pathology department of mofid children s hospital in tehran ( one of the referral centers from all over the country ) for a ten year period ( 2002 to 2012 ) .", "the diagnosis of lch was made by a pediatric pathologist , using the histological criteria mentioned in the pathology textbooks , i.e. granulomas composed of langerhans cells with typical grooved nuclei mixed with eosinophils and other inflammatory cells .", "the diagnoses were confirmed using immunohistochemical technique for cd1a , s-100 protein and cd68 when available .", "thirty tissue samples with non lch diagnoses were also selected ( between the years 2002 and 2012 ) as age and site - matched controls to lch cases .", "paraffin was removed from the ffpe samples after undergoing xylene treatment , followed by two washes with pure ethanol .", "total dna was extracted from the air - dried pellet tissues according to the rtp dna / rna virus mini kit procedure ( stratec molecular , berlin , germany ) .", "to assess the quality of the extracted genome , as well as inhibition of pcr test , all extracted and stored nucleic acid underwent beta - globin pcr , using the pco3/pco4 primer set , as described previously with modifications in method ( 13 ) .", "the beta - globin pcr was performed in syber green real - time pcr - melting curve format ( figure 1 ) . to detect cmv ,", "real - time pcr was performed using envelope glycoprotein b ( gpul55 ) gene primer sets amplifying 116 bp gene region of the virus genome ( cmv - r ; 5-aagtacccctatcgcgtgtg-3 ) , cmv - f ; 5-atgatgccctcrtccargtc -3 , with an internal probe , cmv - p ; 5-fam - tggcccagggtacggatcttattcg - bhq1 - 3 ) ( 14 ) .", "amplification of cmv gpul55 genes was performed in reaction volumes of 20 l under the following conditions : first the samples underwent denaturation at 94c for 10 minutes , followed by denaturation at 94c for 10 seconds , followed by annealing and extension at 60c for one minute , 50 cycles ( figure 2 ) .", "real - time pcr system cfx-96 ( bio - rad , usa ) with hs prime taq premix taqman reagent ( genetbio , korea ) was used in real - time pcr assay .", "the limit detection of 5 genome copies of cmv per reaction was determined by the real - time assay , using the serial dilutions of amplirun cytomegalovirus dna control ( vircell , spain ) .", "the graph shows that the melting temperature of beta - globin pcr product is around 82.5c .", "the achieved data were compared by chi - square ( or fisher s exact test if appropriate ) .", "p value of less than 0.05 was considered to indicate statistical significance . in this study ,", "no ethical issues were involved ; only the pathology reports were reviewed retrospectively , and the patients were anonymous .", "all patients were iranians ( 16 males and 14 females ) and the age ranged 2 months to 10 years .", "thirty tissue samples with non lch diagnoses were also selected as controls which were age and site - matched to lch cases .", "cmv dna was detected in only 2 ( 6.66% ) lch patients out of 30 cases ( positive results in 6.66% and negative results in 93.34% ) . in control group , we detected cmv virus in 1(3.3% ) sample out of a total of 30 cases ( positive results in 3.3% and negative results in 96.7% of controls ) with a p value of 1.00 ( 1.00 > 0.05 ) and or : 2.07 ; 95% ci of or : 0.18 - 24.15 which shows no significant difference in cmv prevalence results between lch patients and controls .", "the lch patients with positive result for cmv expression were a 2 year old boy with frontal bone lesion and a 1 year old girl with chest skin lesion ; the positive control was in a 6 year - old boy with skin biopsy diagnosed as pilonidal fistula .", "lch is a rare disease involving clonal proliferation of bone marrow derived langerhans cells which mostly affects children , particularly children younger than 5 years with an incidence of about 5 per 1,000,000 ( 2 ) .", "clinically , there are three overlapping clinical syndromes including multifocal multisystemic lch ( letterer - siwe syndrome ) , multifocal unisystemic lch ( hand - schuller - christian syndrome ) and unifocal lch ( solitary eosinophilic granuloma ) ( 2 ) .", "histologically , the findings are the same in various tissues , characterized by granulomas composed of a mixture of langerhans cells , macrophages , eosinophils , multinucleated giant cells and lymphocytes ( 15 , 16 ) .", "most adult patients with lung involvement usually have an indolent course of regress , whereas children need to be treated ( 17 - 20 ) .", "( 9 ) detected cmv in lesional langerhans cells in 30% of 29 patients by immunochemistry , in situ hybridization , and pcr , indicating that release of cytokines stimulates langerhans cell growth and induction of lch .", "( 10 ) , however , did not support the hypothesis of the role of ebv , cmv , or hhv-6 , in the pathogenesis of lch in their study .", "( 11 ) also failed to find evidence of genomes for adenovirus , cmv , ebv , hsv , hhv-6 , human immunodeficiency virus , human t - cell leukemia virus types i and ii , and parvovirus in 56 cases of lch employing in situ hybridization and pcr technique .", "we detected cmv dna in only 2 patients with lch ( positive results in 6.66% and negative results in 93.34% ) .", "only one in our control group had cmv virus ( positive results in 3.3% and negative results in 96.7% of controls ) , which could be attributed to a coincidence .", "the p value of 1.00 ( 1.00 > 0.05 ) and or : 2.07 ; 95% ci of or : 0.18 - 24.15 indicates no significant difference in cmv prevalence results between lch patients and controls .", "this is in keeping with some other results reported in the literature ( 10 , 12 ) . in previous studies ( 9 - 12 ) authors declared no limitations and the methods used included serology , immunochemistry , in situ hybridization , and pcr .", "however , immunochemistry and in situ hybridization for cmv were not available to be employed in our study and the patients were not checked for the evidence of cmv infection serologically ( limitations in our study ) .", "our study is the first study performed in iran on this subject and does not support the hypothesis of a role for cmv in the pathogenesis of lch , in concordance with some other studies in the literature ( 10 - 12 ) .", "our study is the first study performed in iran on this subject and does not support the hypothesis of a role for cmv in the pathogenesis of lch , in concordance with some other studies in the literature ( 10 - 12 ) ." ]

[ "<S> background : langerhans cell histiocytosis is a rare proliferative histiocytic disease of unknown etiology . </S>", "<S> histologically , it is characterized by granuloma - like proliferation of langerhans - type dendritic cells derived from bone marrow . </S>", "<S> many investigators have suggested the possible role of viruses such as epstein - barr virus , human herpesvirus-6 ( hhv-6 ) , herpes simplex virus ( hsv ) types 1 and 2 , and cytomegalovirus in the pathogenesis of langerhans cell histiocytosis.objectives:in this study , we have investigated the presence of cytomegalovirus in langerhans cell histiocytosis in iranian children.patients and methods : in this retrospective study , we have investigated the presence of cytomegalovirus dna expression , using paraffin - embedded tissue samples of 30 patients with langerhans cell histiocytosis and 30 age and site - matched controls by qualitative polymerase chain reaction ( pcr ) method.results:no significant difference in prevalence of cytomegalovirus presence between patients and controls was found . </S>", "<S> cytomegalovirus was found by qualitative pcr in only 2 ( 6.66% ) out of 30 patients and in 1 ( 3.3% ) of 30 control samples with a p value of 1 ( 1.00 > 0.05 ) using chi - square test with or : 2.07 ; 95% ci of or : 0.18 - 24.15.conclusions:our findings do not support the hypothesis of a possible role for cytomegalovirus in the pathogenesis of langerhans cell histiocytosis . </S>" ]

[ "1. Background", "2. Objectives", "3. Patients and Methods", "3.1. Patients and Controls", "3.2. Nucleic Acid Extraction", "3.3. PCR", "3.4. Statistical and Ethical Considerations", "4. Results", "5. Discussion", "5.1. Conclusions" ]

[ [ "clonal proliferation of histiocytes was reported in langerhans cell histiocytosis ( lch ) by willman et al . in 1994 ( 1 ) .", "langerhans cells are a type of nonlymphoid mononuclear cells involved in inflammatory responses and also in neoplastic processes .", "they are immature dendritic cells which express lysosomal enzymes cd1a and s-100 protein and ultrastructurally contain racket shaped organelles of birbeck granules ( 2 ) .", "the etiology is unknown ; however , environmental agents and viruses , especially epstein - barr virus ( ebv ) , or vaccination , have been proposed to play a role in the pathogenesis of lch ( 3 , 4 ) .", "the role of ebv as the etiologic agent of several malignancies is known and herpes viruses are shown to be responsible for persistent infection ( 5 , 6 ) .", "in addition , ebv and cytomegalovirus ( cmv ) are considered to be responsible for hemophagocytic syndromes in human with several inherited immunodeficiencies ( 7 , 8) .", "one study showed 30% positivity for cmv in lch ( 9 ) ; however , others have failed to show any association with adenovirus , cytomegalovirus , epstein - barr virus , herpes simplex virus , human herpes virus type 6 , human immunodeficiency virus , human t - cell leukemia virus types i and ii , and parvovirus using pcr ( 10 - 12 ) ." ], [ "in this study , we have investigated the presence of cmv in lch by qualitative pcr method ." ], [ "formalin - fixed , paraffin - embedded ( ffpe ) tissue samples of 30 patients with pathologic diagnosis of lch were extracted from archive of pathology department of mofid children s hospital in tehran ( one of the referral centers from all over the country ) for a ten year period ( 2002 to 2012 ) .", "the diagnosis of lch was made by a pediatric pathologist , using the histological criteria mentioned in the pathology textbooks , i.e. granulomas composed of langerhans cells with typical grooved nuclei mixed with eosinophils and other inflammatory cells .", "the diagnoses were confirmed using immunohistochemical technique for cd1a , s-100 protein and cd68 when available .", "thirty tissue samples with non lch diagnoses were also selected ( between the years 2002 and 2012 ) as age and site - matched controls to lch cases .", "paraffin was removed from the ffpe samples after undergoing xylene treatment , followed by two washes with pure ethanol .", "total dna was extracted from the air - dried pellet tissues according to the rtp dna / rna virus mini kit procedure ( stratec molecular , berlin , germany ) .", "the extracted nucleic acid was stored at -20c until it underwent pcr . to assess the quality of the extracted genome , as well as inhibition of pcr test ,", "all extracted and stored nucleic acid underwent beta - globin pcr , using the pco3/pco4 primer set , as described previously with modifications in method ( 13 ) .", "the beta - globin pcr was performed in syber green real - time pcr - melting curve format ( figure 1 ) . to detect cmv ,", "real - time pcr was performed using envelope glycoprotein b ( gpul55 ) gene primer sets amplifying 116 bp gene region of the virus genome ( cmv - r ; 5-aagtacccctatcgcgtgtg-3 ) , cmv - f ; 5-atgatgccctcrtccargtc -3 , with an internal probe , cmv - p ; 5-fam - tggcccagggtacggatcttattcg - bhq1 - 3 ) ( 14 ) .", "amplification of cmv gpul55 genes was performed in reaction volumes of 20 l under the following conditions : first the samples underwent denaturation at 94c for 10 minutes , followed by denaturation at 94c for 10 seconds , followed by annealing and extension at 60c for one minute , 50 cycles ( figure 2 ) .", "real - time pcr system cfx-96 ( bio - rad , usa ) with hs prime taq premix taqman reagent ( genetbio , korea ) was used in real - time pcr assay .", "the limit detection of 5 genome copies of cmv per reaction was determined by the real - time assay , using the serial dilutions of amplirun cytomegalovirus dna control ( vircell , spain ) .", "the graph shows that the melting temperature of beta - globin pcr product is around 82.5c .", "the achieved data were compared by chi - square ( or fisher s exact test if appropriate ) .", "p value of less than 0.05 was considered to indicate statistical significance . in this study", ", no ethical issues were involved ; only the pathology reports were reviewed retrospectively , and the patients were anonymous ." ], [ "formalin - fixed , paraffin - embedded ( ffpe ) tissue samples of 30 patients with pathologic diagnosis of lch were extracted from archive of pathology department of mofid children s hospital in tehran ( one of the referral centers from all over the country ) for a ten year period ( 2002 to 2012 ) .", "the diagnosis of lch was made by a pediatric pathologist , using the histological criteria mentioned in the pathology textbooks , i.e. granulomas composed of langerhans cells with typical grooved nuclei mixed with eosinophils and other inflammatory cells .", "the diagnoses were confirmed using immunohistochemical technique for cd1a , s-100 protein and cd68 when available .", "thirty tissue samples with non lch diagnoses were also selected ( between the years 2002 and 2012 ) as age and site - matched controls to lch cases ." ], [ "paraffin was removed from the ffpe samples after undergoing xylene treatment , followed by two washes with pure ethanol .", "total dna was extracted from the air - dried pellet tissues according to the rtp dna / rna virus mini kit procedure ( stratec molecular , berlin , germany ) ." ], [ "to assess the quality of the extracted genome , as well as inhibition of pcr test , all extracted and stored nucleic acid underwent beta - globin pcr , using the pco3/pco4 primer set , as described previously with modifications in method ( 13 ) .", "the beta - globin pcr was performed in syber green real - time pcr - melting curve format ( figure 1 ) . to detect cmv ,", "real - time pcr was performed using envelope glycoprotein b ( gpul55 ) gene primer sets amplifying 116 bp gene region of the virus genome ( cmv - r ; 5-aagtacccctatcgcgtgtg-3 ) , cmv - f ; 5-atgatgccctcrtccargtc -3 , with an internal probe , cmv - p ; 5-fam - tggcccagggtacggatcttattcg - bhq1 - 3 ) ( 14 ) .", "amplification of cmv gpul55 genes was performed in reaction volumes of 20 l under the following conditions : first the samples underwent denaturation at 94c for 10 minutes , followed by denaturation at 94c for 10 seconds , followed by annealing and extension at 60c for one minute , 50 cycles ( figure 2 ) .", "real - time pcr system cfx-96 ( bio - rad , usa ) with hs prime taq premix taqman reagent ( genetbio , korea ) was used in real - time pcr assay .", "the limit detection of 5 genome copies of cmv per reaction was determined by the real - time assay , using the serial dilutions of amplirun cytomegalovirus dna control ( vircell , spain ) .", "the graph shows that the melting temperature of beta - globin pcr product is around 82.5c ." ], [ "the achieved data were compared by chi - square ( or fisher s exact test if appropriate ) .", "p value of less than 0.05 was considered to indicate statistical significance . in this study ,", "no ethical issues were involved ; only the pathology reports were reviewed retrospectively , and the patients were anonymous ." ], [ "all patients were iranians ( 16 males and 14 females ) and the age ranged 2 months to 10 years .", "thirty tissue samples with non lch diagnoses were also selected as controls which were age and site - matched to lch cases .", "cmv dna was detected in only 2 ( 6.66% ) lch patients out of 30 cases ( positive results in 6.66% and negative results in 93.34% ) . in control group , we detected cmv virus in 1(3.3% ) sample out of a total of 30 cases ( positive results in 3.3% and negative results in 96.7% of controls ) with a p value of 1.00 ( 1.00 > 0.05 ) and or : 2.07 ; 95% ci of or : 0.18 - 24.15 which shows no significant difference in cmv prevalence results between lch patients and controls .", "the lch patients with positive result for cmv expression were a 2 year old boy with frontal bone lesion and a 1 year old girl with chest skin lesion ; the positive control was in a 6 year - old boy with skin biopsy diagnosed as pilonidal fistula ." ], [ "lch is a rare disease involving clonal proliferation of bone marrow derived langerhans cells which mostly affects children , particularly children younger than 5 years with an incidence of about 5 per 1,000,000 ( 2 ) .", "clinically , there are three overlapping clinical syndromes including multifocal multisystemic lch ( letterer - siwe syndrome ) , multifocal unisystemic lch ( hand - schuller - christian syndrome ) and unifocal lch ( solitary eosinophilic granuloma ) ( 2 ) .", "histologically , the findings are the same in various tissues , characterized by granulomas composed of a mixture of langerhans cells , macrophages , eosinophils , multinucleated giant cells and lymphocytes ( 15 , 16 ) .", "most adult patients with lung involvement usually have an indolent course of regress , whereas children need to be treated ( 17 - 20 ) .", "( 9 ) detected cmv in lesional langerhans cells in 30% of 29 patients by immunochemistry , in situ hybridization , and pcr , indicating that release of cytokines stimulates langerhans cell growth and induction of lch .", "( 10 ) , however , did not support the hypothesis of the role of ebv , cmv , or hhv-6 , in the pathogenesis of lch in their study .", "( 11 ) also failed to find evidence of genomes for adenovirus , cmv , ebv , hsv , hhv-6 , human immunodeficiency virus , human t - cell leukemia virus types i and ii , and parvovirus in 56 cases of lch employing in situ hybridization and pcr technique .", "we detected cmv dna in only 2 patients with lch ( positive results in 6.66% and negative results in 93.34% ) .", "only one in our control group had cmv virus ( positive results in 3.3% and negative results in 96.7% of controls ) , which could be attributed to a coincidence .", "the p value of 1.00 ( 1.00 > 0.05 ) and or : 2.07 ; 95% ci of or : 0.18 - 24.15 indicates no significant difference in cmv prevalence results between lch patients and controls .", "this is in keeping with some other results reported in the literature ( 10 , 12 ) . in previous studies ( 9 - 12 ) authors declared no limitations and the methods used included serology , immunochemistry , in situ hybridization , and pcr .", "however , immunochemistry and in situ hybridization for cmv were not available to be employed in our study and the patients were not checked for the evidence of cmv infection serologically ( limitations in our study ) .", "our study is the first study performed in iran on this subject and does not support the hypothesis of a role for cmv in the pathogenesis of lch , in concordance with some other studies in the literature ( 10 - 12 ) ." ], [ "our study is the first study performed in iran on this subject and does not support the hypothesis of a role for cmv in the pathogenesis of lch , in concordance with some other studies in the literature ( 10 - 12 ) ." ] ]

[ "glucocorticoids ( gcs ) are among the most effective drugs used in the treatment of hematopoietic malignancies of the lymphoid lineage in virtue of their ability to induce apoptosis of these cancerous cells [ 13 ] .", "the main hematopoietic cancer types that respond well to gc therapy include t acute lymphoblastic leukemia ( t - all ) , chronic b lymphocytic leukemia ( cll ) , multiple myeloma ( mm ) , hodgkin 's lymphoma ( hl ) , and non - hodgkin 's lymphoma ( nhl ) .", "gcs appear , however , to have little value in the treatment of acute or chronic myeloid leukemia ( aml / cml ) .", "a major drawback of gc therapy is the gradual development of resistance to gc during treatment that limits the clinical utility of this drug .", "poor response to a 7-day monotherapy with the gc prednisone is one of the strongest predictors of adverse outcomes in the treatment of pediatric all [ 2 , 4 ] .", "a great challenge today is to develop strategies that can overcome the drug resistant phenotype . for this purpose", "it is important to understand the underlying mechanisms of gc resistance and the signaling pathways regulating apoptosis induced by gcs . besides inducing apoptosis of lymphoid cells , gcs", "gc treatment produces rapid symptomatic improvements , including relief of fever , sweats , lethargy , weakness , and other nonspecific effects of cancer.gcs decrease the severity of chemotherapy - induced emesis .", "gcs are also used in the clinics for other medical conditions such as autoimmune diseases , asthma , ulcerative colitis , chronic obstructive pulmonary disease , kidney diseases , and rheumatologic disorders due to their strong anti - inflammatory and immunosuppressive properties .", "gc therapy is hampered by a variety of metabolic and medical complications , including insulin resistance , diabetes , hypertension , glaucoma , osteoporosis , and osteonecrosis with increased risk of bone fractures [ 510 ] .", "diabetes may develop by direct gc - mediated induction of apoptosis in insulin - producing beta cells of the langerhans islets [ 1113 ] , and osteoporosis may develop due to apoptosis of osteoblasts [ 1416 ] .", "gcs also suppress cell growth and proliferation processes in the brain [ 17 , 18 ] . besides being used as monotherapy at high dosages , gcs", "are frequently combined with other chemotherapeutic drugs to achieve rapid and more efficient therapeutic effects . for the treatment of t - all ,", "gcs such as prednisone , methylprednisolone , and dexamethasone are usually used in combination with other chemotherapeutic drugs such as vincristine , daunorubicine , l - asparaginase , cytosine arabinoside , doxorubicin , and cyclophosphamide .", "this multidrug regimen prolongs remission , minimizes the long - term use of prednisone , and thus reduces the steroid - mediated adverse effects . ", "typical b - cell chronic lymphocytic leukemia ( cll ) in the early stage of progression responds well to combination chemotherapy including an alkylating agent ( such as chlorambucil ) plus or minus prednisolone.advanced stages of the disease often require the addition of an anthracycline and a vinca alkaloid for successful therapy .", "one commonly used combination is cyclophosphamide , doxorubicin , vincristine , and prednisolone , a drug combination termed chop .", "rituximab , a chimeric monoclonal antibody directed against the b - cell specific antigen cd20 , is often added to the therapy , which is here termed r - chop .", "rituximab is also combined with fludarabine and cyclophosphamide in the treatment of cll [ 19 , 20 ] .", "another antibody proved to be efficient against cll in combination with methylprednisolone is alemtuzumab , which targets cd52 .", "the immunomodulatory drug lenalidomide shows also good activity in relapse / refractory or treatment - nave cll [ 23 , 24 ] .", "chop is also used for non - hodgkin 's lymphomas and anaplastic large cell lymphoma ( alcl ) .", ", prednisone has been used in combination with carmustine , vincristine ( oncovin ) , procarbazine ( mopp ) , and rituximab .", "recently , brentuximab vedotin ( adcetris ) , an antibody directed towards cd30 conjugated with the anti - tubulin chemotherapeutic agent monomethyl auristatin e , has been approved for the treatment of hodgkin 's lymphoma and systemic anaplastic large cell lymphoma .", "cd30 expression is restricted to only a relative small population of activated t and b cells , and therefore this treatment is expected to be more selective for cd30-positive tumor cells .", "another monoclonal antibody entered the clinics is epratuzumab , which targets cd22 and is proved to be efficient in the treatment of adult non - hodgkin 's lymphoma as a single agent or in combination with chemotherapy . a phase ii clinical trial showed that combining epratuzumab with rituximab and chop ( er - chop )", "may have a favorable response on diffusing large b - cell non - hodgkin lymphoma ( dlbcl ) . ", "multiple myeloma ( mm ) has frequently been treated with vincristine , doxorubicine ( adriamycin ) , and dexamethasone ( vad ) or prednisone / melphalan .", "bortezomib ( velcade ) , lenalidomide , and to a lesser extend thalidomide have proven efficient in the treatment of mm in combination with dexamethasone .", "lenalidomide is a 4-amino - glutamyl analogue of thalidomide that lacks the neurological side effects of thalidomide and has emerged as a drug with activity against various hematological malignancies [ 27 , 28 ] .", "bortezomib is a selective inhibitor of the 26s proteasome that stabilizes many cell cycle - regulatory proteins .", "the antitumor effects of bortezomib in lymphoid tumors have been attributed to nfb inhibition through stabilization of its inhibitor ib .", "other tumors that have been treated with combination chemotherapy involving a gc include medulloblastoma , primitive neuroectodermal tumors , and ependymomas . one major obstacle in the therapy of lymphoid malignancies is the appearance of gc resistant cells .", "drug resistance may occur at the level of the glucocorticoid receptor ( gr ) or through alterations in downstream regulatory pathways . in", "most gc - resistant all primary biopsy specimens , gr was found to be functional , suggesting that pharmacological intervention may restore drug sensitivity .", "several strategies have been developed that aim to overcome drug resistance through specifically targeting anti - apoptotic pathways .", "gc resistance may occur due to overexpression of anti - apoptotic proteins of the bcl-2 superfamily [ 30 , 31 ] . among these ,", "small molecules that target the anti - apoptotic proteins of the bcl-2 family are attractive drugs that should be able to overcome gc resistance .", "one example is abt-737 , a bh3 mimetic that inhibits the pro - survival function of bcl-2 , bcl - xl , and bcl - w and induces apoptosis in a variety of cancer cell types including leukemias [ 3335 ] .", "treatment of the lymphoma - prone e-myc transgenic mice with abt-737 prevented the development of myc - driven lymphomagenesis , understating the need for these anti - apoptotic proteins .", "combined use of abt-737 and the dual specificity pi3k / mtor inhibitor pi-103 led to loss of c - myc expression and apoptosis of burkitt 's lymphoma cells , whose tumorigenicity is driven by overexpression of the c - myc gene . ", "the pro - apoptotic effect of abt-737 in cll depends on sufficient amount of bcl-2 that tonically sequesters the pro - apoptotic bim protein .", "also , the sensitivity of lymphoma cell lines to bcl-2 antagonism is directly related to the amount of bcl-2 primed with bim .", "the sequestration of bim may explain the marked chemosensitivity of cll and follicular lymphoma ( fl ) that express abundant bcl-2", "abt-737 potentiated the effect of vincristine , dexamethasone , and l - asparaginase ( vxl ) treatment on all cells and could potentiate the effect of the vxl combination in chemoresistant human primary all xenografts .", "this study also shows a synergistic effect between the three components of the vxl regimen .", "an additive effect was observed in primary mm cells when abt-737 was combined with dexamethasone [ 41 , 42 ] . ", "abt-263 ( navitoclax ) is a second generation , orally bioavailable small molecule bcl-2 family protein inhibitor that has entered clinical trials with promising efficacy on cll [ 4346 ] .", "abt-263 has been shown to have synergistic effects with r - chop treatment on mantle cell lymphoma .", "the pro - apoptotic bim that is displaced from bcl-2 by abt-737 , becomes captured by either bfl-1 or mcl-1", ". the resistance could be overcome by decreasing the mcl-1 level with the cyclin - dependent kinase ( cdk ) inhibitors flavopiridol and pha767491 , or by inhibiting mtor complex 1 ( mtorc1 ) or glycolysis [ 49 , 50 ] . another approach to overcome mcl-1-dependent resistance is to use the small molecule obatoclax ( gx15 - 070 ) that has entered clinical trials in the combined treatment of various hematopoietic neoplasms [ 5153 ] .", "obatoclax disrupts the interaction between mcl-1 and its pro - apoptotic counterparts including bak , bax , and noxa [ 54 , 55 ] .", "obatoclax and flavopiridol synergized in overcoming drug resistance in human myeloma cells through a mechanism involving bim and noxa .", "the multikinase inhibitor sorafenib could synergize with obatoclax in inducing apoptosis in acute myeloid leukemia ( aml ) through downregulating mcl-1 .", "obatoclax could overcome gc resistance in all through induction of apoptosis and autophagy , an effect that depends on the pro - apoptotic bak and to a certain extent also on beclin-1 [ 58 , 59 ] , a mammalian orthologue of yeast atg6 that plays a central role in autophagy .", "under certain conditions , cell death induced by obatoclax and gc may be executed in the absence of both bax and bak . under these conditions ,", "necroptosis ensues necroptosis ensues , a process mediated by rip-1 ( receptor - interacting protein-1 ) kinase and the cylindromatosis deubiquitinase cyld .", "it may promote either cell death or cell survival dependent on its ubiquitinated state , which is regulated by cyld and a20 , two nfb target genes .", "altogether , there is a general consensus that obatoclax might be a favorable drug that ought to be combined with dexamethasone / prednisone and/or rapamycin to overcome gc resistance in all cells and other hematological lymphoid malignancies . \n", "1.2.1.3 . overcoming bcl-2-mediated resistance with small molecular inhibitors of xiap ( x - linked inhibitor of apoptosis ) .", "bcl-2-mediated resistance in cll may also be overcome by small molecular inhibitors of the anti - apoptotic xiap ( x - linked inhibitor of apoptosis ) when exposed to trail [ 62 , 63 ] .", "xiap and the cellular ciaps 1 and 2 are expressed at high levels in cll cells [ 62 , 63 ] .", "xiap inhibitors enhanced bcl-2 cleavage and induced a conformational change in bax . similarly , xiap inhibitors sensitized all for cd95-induced apoptosis . in patients with t - all ,", "xiap inhibition using the low - molecular - weight smac mimetic lbw242 resulted in increased prednisone - induced apoptosis in vitro . ", "another anti - apoptotic protein that negatively regulates gc - induced apoptosis is notch1 [ 6668 ] .", "notch1 is indispensable for normal t - cell development [ 6971 ] and is an attractive target in the treatment of hematopoietic malignancies of the t lineage .", "mice transplanted with bone marrow cells transduced with a constitutively active form of notch1 develop t - cell neoplasms , while mice transgenic for constitutively active form of notch3 develop thymic lymphomas .", "acute lymphoblastic t - cell leukemia is frequently associated with increased notch signaling [ 7579 ] , which may be caused by the chromosomal translocation t(7 ; 9)(q34 ; q34.3 ) , gain - of - function mutations of notch1 , and/or mutations in fbw7 ( f - box and wd repeat domain - containing 7 ) , a negative regulator of notch1 . ", "one approach to avoid notch activation is to prevent its cleavage by the -secretase complex using -secretase inhibitors ( gsi ) .", "rather , gsi can enhance the pro - apoptotic effect of gcs and other chemotherapeutic agents including the mtor inhibitor rapamycin [ 84 , 88 ] .", "gsi does not overcome gc resistance in t - all deficient for pten [ 89 , 90 ] , supposedly due to elevated akt activity .", "the constitutive akt activation in the absence of pten leads to increased glucose metabolism and bypasses the requirement of notch signaling to sustain cell growth . in this context", "it should be noted that notch1 by itself may upregulate the p13k / akt pathway via its target gene hes1 .", "as pten is a target of several micrornas that are often expressed abnormally in cancer ( see section 2.4.2.3 ) , resistance to gsi may be far more prevalent .", "nevertheless , gsi compounds , such as pf-03084014 , have entered clinical trials for refractory t - all .", "preclinical data do show a synergistic effect between gsi inhibition and gc in reducing xenografted t - all tumor burden .", "another concern associated with the clinical use of gsis is severe toxicity to various organs at therapeutic doses , which may be explained by the broad action of notch1 as well as -secretase on various biological systems .", "the simultaneous use of gcs may prevent the gsi - induced gastrointestinal toxicity via inhibition of goblet cell metaplasia .", "a more specific inhibition of notch1 can be achieved by the sahm1 peptide that prevents notch - mediated transcription by interfering with the mastermind - notch interaction essential for notch - mediated transcription of target genes .", "the effect of this peptide on gc sensitivity awaits examination as well as its toxicity . since notch signaling", "is intertwined with the pi3k / akt / mtor signaling axis [ 9496 ] , the inhibition of the latter has proven to be more efficient in overcoming gc resistance ( see section 1.2.3 ) and would be a better therapeutic choice . accumulating data show that gc therapy can affect the activity of several protein kinases , and , vice versa , many protein kinases can affect gc - induced apoptosis [ 30 , 31 , 9799 ] .", "the mtor signaling pathway is frequently activated and found to be essential for cell growth and survival in lymphoid malignancies [ 100106 ] .", "gc resistance frequently appears in malignant cells due to aberrant activation of various protein kinases that exert anti - apoptotic effects [ 30 , 31 , 67 , 97 , 107109 ] .", "one strategy to overcome gc resistance would be to prevent the activities of the pi3k / akt / mtor , mek1/erk1/2 , and other activated protein kinase pathways .", "the mtor inhibitor rapamycin especially has proven efficient in sensitizing human gc - resistant t - all , b - all , mm , and npm - alk ( nucleophosmin - anaplastic lymphoma kinase)-dlbcl to gc - induced apoptosis [ 110117 ] .", "the combinatory therapy of rapamycin with dexamethasone was proven to be effective also in pten - negative cells . a lower dose of dexamethasone was sufficient for reducing t - all burden in a xenograft model when used together with rapamycin . one major drawback with rapamycin therapy", "is its immunosuppressive function , which adds to the immunosuppressive function of gcs . the dual pi3k / mtor inhibitor nvp - bez235 synergistically enhanced cytotoxicity of dexamethasone , doxorubicine , and cytosine arabinoside ( arac ) , even in gc - resistant all cells .", "the broad - acting protein kinase staurosporine was especially effective in overcoming gc resistance in mouse lymphomas that overexpressed notch-1 , bcl-2 , and/or bcl - xl .", "this sensitization was achieved through prevention of akt - mediated inhibition of gsk3 and induction of the pro - apoptotic nur77 . however , staurosporine was less effective on human t - all cell lines ( unpublished data ) , which could rather be sensitized to gc by rapamycin . in order to choose the right kinase inhibitor for combinatory therapy ,", "the cyclin - dependent kinase ( cdk ) inhibitors flavopiridol ( alvocidib ) , bms-387032 ( sns-032 ) , sunitinib , and sorafenib are currently under clinical trials for relapsed / refractory cll .", "these include vandetanib ( zd6474 ) , bosutinib ( ski-606 ) , tki258 ( chir-258 ) , pazopanib ( gw786034 ) , and axitinib ( ag013736 ) .", "chir-258 , a potent inhibitor of flt3 ( fms - like tyrosine kinase receptor-3 ) , c - kit tyrosine kinase , and fibroblast growth factor receptor 3 ( fgfr3 ) , prevented cell growth of fgfr3-positive human multiple myeloma cell lines and augmented their sensitivity to gc - induced apoptosis .", "importantly , neither interleukin-6 ( il-6 ) nor stromal cells conferred resistance to chir-258 . other protein kinase inhibitors with more cell - type specific effects have been developed , which are expected to have less adverse effects . the classical example for efficient use of a specific protein kinase inhibitor in the clinics is the bcr - abl kinase inhibitor sti-572 ( imatinib ) used for the treatment of chronic myelogenic leukemia ( cml ) . a similar strong response of a single agent", "was observed in alk - anaplastic large cell lymphoma ( alcl ) patients treated with crizotinib , an inhibitor of the alk tyrosine kinase .", "another promising target is the b - cell receptor ( bcr ) signaling , which is important during b - cell oncogenesis and is a key to the survival of malignant b cells , including cll and dlbcl [ 125 , 126 ] .", "the survival of dlbcl may depend on the nonligand - dependent ( tonic ) signals from the bcr .", "the bcr signaling can be targeted with small molecular inhibitors directed against bruton 's tyrosine kinase ( btk ) , spleen tyrosine kinase ( syk ) , or phosphoinositide 3-kinase ( pi3k ) isoform p110 ( pi3k ) , all being efficient in the treatment of cll . targeting btk with the inhibitor pci-32765 leads to disruption of bcr signaling and was effective in a preclinical model of b cell non - hodgin 's lymphoma [ 127 , 128 ] .", "pci-32765 seems also to be promising for the treatment of cll [ 128131 ] and mm .", "importantly , pci-32765 induced apoptosis in cll cells even in the presence of various exogenous stimuli , including cd40l , baff , il-6 , and il-4 and when cultivated together with stromal cells .", "two other btk inhibitors , ibrutinib and avl-263 , are also under investigation for cll .", "the syk ( spleen tyrosine kinase ) inhibitor fostamatinib had clinical activity in non - hodgkin lymphoma and cll .", "syk is a cytoplasmic tyrosine kinase that is important for immunoreceptor signaling in b cells .", "syk has also been shown to be critical for the survival and maintenance of mature normal and malignant b cells [ 125 , 134 ] and is frequently expressed at high levels in follicular lymphoma .", "the pi3k inhibitor gs-1101 ( cal-101 ) had preclinical and clinical activity against cll , mantle cell lymphoma , and mm [ 121 , 129 , 136138 ] . while the pi3k and isoforms are ubiquitously expressed , pi3k expression is largely restricted to hematopoietic cells , where it plays a role in b - cell homeostasis and function .", "the effect of the btk , syk , and pi3k kinase inhibitors on the sensitivity to gcs warrants investigations . ", "accordi et al . found aberrant activation of protein kinases in poor prognosis pediatric b - cell precursor - all patients .", "the p56 ( lymphocyte cell - specific tyrosine kinase ) activity was enhanced in patients with poor clinical response to prednisone with respect to those with good response .", "p56 is a nonreceptor tyrosine kinase of the src oncogene family mostly expressed in t cells where it plays an essential role in activation and development , and in some b cells .", "its activity is negatively regulated by the membrane - bound tyrosine kinase csk ( c - src tyrosine kinase ) .", "the p56 inhibitor dasatinib ( bms-354825 ) was shown to enhance apoptosis induction by dexamethasone in otherwise gc - resistant cll cells .", "this finding concurs with the observation by sade et al . showing that notch - mediated resistance of a mouse lymphoma cell line", "a synergistic effect was observed between the aurora a kinase inhibitor mnl8237 ( alisertib ) and dexamethasone . ", "ampk ( amp activated protein kinase ) activation has a dual effect on cell death and survival , which contextually depends on signaling alterations with related oncogenic pathways .", "however , in all and cll , activation of ampk by aicar ( 5-aminoimidazole-4-carboxamide riboside or acadesine ) , a cell - permeable nucleotide , induces growth inhibition and apoptosis [ 146148 ] .", "however , aicar prevented glucocorticoid - induced apoptosis and thus can not be combined with steroids in the treatment of lymphoid malignancies . of note , inhibition of either bcl-2 family members , notch1 , or", "the akt / mtor survival pathways was independently sufficient for sensitizing resistant cells to gc , suggesting a tight crosstalk between these pathways , interruption of one of them being sufficient for abrogating the resistant phenotype .", "however , it is likely that using a combination of these three strategies together with gc should lead to a more efficient therapy , which may require lower dosages with reduced adverse effects .", "in order to develop strategies to overcome gc resistance , it is essential to understand the signaling network regulating gc - induced apoptosis .", "main factors affecting the response to gc include the basal and inducible gr expression levels , the induction of and basal expression of genes involved in the intrinsic apoptotic pathway , the ability of gr to translocate to the mitochondria , the activity of gsk3 ( glycogen synthase kinase 3 ) , the general protein kinase activation profile of the cell prior to and following gc therapy , the expression profile of anti - apoptotic proteins , and the activities of pro - survival signaling pathways .", "the main traits will only be briefly described here as these have been extensively reviewed elsewhere [ 30 , 31 , 99 , 150153 ] , and the scope of this paper is to provide updated data with a specific focus on the microrna world that has emerged to comprise important regulators of most biological processes .", "numerous factors have been shown to affect gc responsiveness by regulating glucocorticoid receptor ( gr ) activity and expression level .", "these include gr co - activators and corepressors [ 154 , 155 ] , gr splice variants [ 156159 ] , gr isoforms [ 160 , 161 ] , and regulators of gc nucleocytoplasmic shuttle [ 162164 ] . ", "the transcription of human gr is regulated by at least 11 different promoters ( 1a1 , 1a2 , 1a3 , 1b , 1c , 1d , 1e , 1f , 1h , 1i , and 1j ) [ 155 , 165 ] , seven of them being embedded in a highly enriched cpg island region subjected to methylation and harbor single nucleotide polymorphisms ( snps ) that affect their activity .", "promoter 1a is involved in the upregulation of gr by gc in some kinds of t cells , while downregulated in other cell types [ 167169 ] .", "gc resistance in primary pediatric t- and b - all could not be correlated with either basal or stimulated expression of the 1a- , 1b , or 1c transcripts . ", "the gr expression level prior and following gc therapy affects drug responsiveness . the cellular response", "to gcs depends on sufficient gr expression [ 30 , 171179 ] , and resistance to gc therapy has been associated with downregulation and loss of gr expression in malignant plasma cells [ 180 , 181 ] .", "however , most primary all cells showed upregulation of gr expression upon prednisolone treatment regardless of their phenotype or sensitivity to gc - induced apoptosis , suggesting that other factors are more dominant for conferring a gc - resistant phenotype in these cells [ 29 , 170 , 182184 ] .", "many glucocorticoid - regulated genes ( e.g. , fkbp5 and socs1 ) were upregulated by dexamethasone in all primary all xenografts tested , suggesting for a functional gr in these leukemic cells .", "also , beesley et al . observed that receptor mutation is not a common mechanism of gc resistant in primary all .", "however , the minor c allele of rs10482605 ( 1c ) has been associated with a higher complication rate in childhood all .", "a bcli polymorphism in the nr3c1 gene was associated with increased lymphocyte response to methylprednisolone .", "also , initial good responder cells may develop resistance upon repeated gc dosages , a phenomenon that sometimes occurs due to downregulation of gr [ 156 , 179 , 188 , 189 ] .", "posttranslational modifications of gr are another way of regulating its target gene specificity and involve several cell - signaling cascades [ 30 , 190 , 191 ] .", "gr can be phosphorylated at ser211 by cdks and p38 map kinase , and at ser226 by jnk .", "phosphorylation of gr modulates its transcriptional activity , alters its protein stability and subcellular location [ 192195 ] .", "gr phosphorylation appears to be cell - cycle dependent [ 196 , 197 ] and may affect gc - sensitivity of t - all cells [ 98 , 195 ] .", ", gr is mostly located to the cytosol sequestered to heat - shock protein complexes [ 30 , 162 ] .", "following gc binding to gr , the receptor undergoes phosphorylation , dissociates from the heat - shock complexes , dimerizes , and translocates to the nucleus where it either promotes or represses a whole series of genes .", "transcriptional activation is either directly mediated by binding of gr to glucocorticoid response elements ( gres ) , or through interaction with other transcription factors such as forkhead transcription factors , thereby increasing their transcriptional activity on target genes .", "gr may repress gene expression either through binding to negative gres ( ngres ) or through interaction with and inhibition of the transcription factors activating protein-1 ( ap-1 ) and nfb .", "the o - glcnac transferase ( ogt ) was found to be involved in gc - mediated transrepression .", "hundreds of genes are regulated by gcs [ 199203 ] , and some genes are differentially regulated in gc - sensitive versus gc - resistant cells [ 29 , 199 , 204 ] . ", "of special importance is the induction of the pro - apoptotic bim ( bh3-only b - cell lymphoma 2 ( bcl-2 ) interacting mediator of cell death ; or bcl2l11bcl-2-like apoptosis initiator-11 ) for achieving the propensity to undergo apoptosis in response to gc [ 29 , 30 , 67 , 205208 ] .", "the central role of bim in gc - induced apoptosis is understated by the partial gc response of bim thymocytes , and gc resistance of lymphoma cells after knocking down bim [ 67 , 207 ] .", "bim is often expressed at high basal levels in lymphoid cells [ 30 , 120 , 209 , 210 ] , and in these cells there is no further need for upregulating bim in order to achieve an apoptotic response to gcs [ 30 , 59 ] . however , in several t - all and b - all cells , an upregulation of bim in response to gcs is an absolute must , especially when the basal level is low .", "bim was shown to be upregulated in gc - sensitive primary t - all samples , but not in resistant ones [ 29 , 182 ] . also , a comparison of established t - all cell lines , bim was upregulated in the sensitive ones only . when sufficient bim expression can not be achieved , gc resistance pursued .", "a significantly lower bim expression was detected in high risk childhood all patients who exhibited slow early response to a standard 4-drug induction regimen compared with patients who responded rapidly .", "homozygous deletion of bim has been seen in many mantle cell lymphomas and silencing of bim by promoter methylation and mutation is common in b - cell lymphomas . however , in pediatric all , no correlation between bim cpg methylation and gc resistance was found . rather , gc resistance in primary pediatric all samples correlated with decreased histone h3 acetylation .", "the histone deacetylase inhibitor vorinostat relieved bim repression and exerted synergistic antileukemic efficacy with dexamethasone both in vitro and in vivo using a xenograft model .", "bim has been shown to be a prognostic biomarker for early prednisolone response in pediatric all .", "bim is a potent pro - apoptotic protein belonging to the bcl-2 protein family [ 215 , 216 ] .", "bim binds to the pro - survival proteins bcl-2 , bcl - xl , and mcl-1 , thereby allowing bax and bak to promote apoptosis .", "bim may also directly bind to bax and bak , triggering a conformational change required for their subsequent oligomerization on the mitochondrial outer membrane .", "bim appears in various alternative splice variants , which exhibit different intrinsic toxicities and modes of regulation . in gc - resistant primary cll", ", bim was upregulated by dexamethasone , but failed to activate bax and bak due to exclusive sequestration to bcl-2 .", "bim may cooperate with the pro - apoptotic puma ( p53 upregulated modulator of apoptosis ) in mediating apoptosis induced by dexamethasone . in b - lymphoid cells ,", "other pro - apoptotic members of the bcl-2 family that is not directly upregulated by gcs , but may contribute to the cell death response , include bid , bad , and noxa .", "also the thioredoxin - interacting protein txnip ( vdup1/tbp-2 ) has been shown to be upregulated by gc and could contribute to gc - induced apoptosis in one mouse lymphoma cell line . during gc monotherapy of childhood all , gc was found to repress the expression of the pro - apoptotic pmaip / noxa , which could be one mechanism leading to impaired gc sensitivity .", "bam , is also induced by gcs in all cells , but its importance in gc - induced apoptosis is still not defined .", "bim expression is tightly regulated both at the transcription and posttranscriptional levels [ 215 , 218 ] ( figure 1 ) .", "rather , gc - induced bim expression in lymphoid cells requires p38 activation and is mediated by the forkhead transcription factor foxo3a / fkhr - l1 .", "foxo3a has also been shown to promote bim transcription in various other cellular systems [ 227229 ] and may cooperate with runx1 ( runt - related transcription factor 1 ) .", "differential recruitment of foxo3a to the bim promoter was observed after dexamethasone treatment of gc - sensitive versus gc - resistant childhood all xenografts .", "foxo3 was found to be an immediate early gr target , whose transcription is further enhanced by stimuli that activate the amp - activated protein kinase ampk .", "the activity of foxo transcription factors is tightly regulated , inhibited by akt and erk signaling , while promoted by p38 signaling [ 232236 ] .", "the ribosomal protein s6 kinase ( rsk ) activated downstream of erk1/2 , phosphorylates bimel , providing a binding site for the f - box proteins beta - transducin repeat containing protein ( trcp)1 and trcp2 , which promote the polyubiquitination of bimel .", "the erk1/2-mediated phosphorylation of bimel at ser69 facilitates optimal phosphorylation by rsk at ser93 , ser94 , and ser98 and this motif serves as the binding sites for trcp1/2 . while erk1/2 lowers the affinity of bim for mcl-1 and bcl - xl and targets bim for degradation , phosphorylation of bim by jnk increases the pro - apoptotic activity of bim [ 240 , 241 ] .", "gcs may repress erk1/2 activity through upregulation of mitogen - activated protein kinase phosphatase 1 ( mkp-1 ) .", "several drugs that inhibit the erk1/2 and pkb / akt pathways may facilitate upregulation of bim expression .", "additional signals are required , such as simultaneous inhibition of the pkb / akt pathway or the downstream mammalian target of rapamycin ( mtor ) kinase .", "apoptosis may be induced in a variety of all cells when cotreated with dexamethasone and a mek / erk inhibitor or an akt inhibitor [ 67 , 108 , 243 ] .", "early studies by the thompson research group noticed that c - jun played a role in gc - induced apoptosis .", "an increase in c - jun was observed in gc - sensitive , but not gc - resistant t - all cell lines , while c - fos and jund were unaffected by the steroid .", "chen et al . reconfirmed that c - jun was upregulated by gcs in gc - sensitive , but not gc - resistant all cells .", "they further showed that c - jun is recruited to the ap-1 site of the bim promoter upon gc treatment .", "another study showed that dexamethasone - induced bim expression was decreased in cells harboring a dominant - negative c - jun , suggesting a role for c - jun in the upregulation of bim .", "a p38 inhibitor prevented dexamethasone - induced expression of runx2 , c - jun , and bim , suggesting that p38-mapk activation acts upstream to the induction of these three molecules . another level of bim regulation is through micrornas .", "bim transcription is repressed by the mir-17~92 microrna cluster , which , in turn , is repressed by gcs .", "thus , one mechanism by which gcs upregulate bim is through repression of mir-17~92 . of note , the mir-17~92 cluster is often overexpressed or amplified in human cancers [ 247252 ] , thereby preventing the upregulation of bim required for an apoptotic response .", "another microrna that suppresses bim expression is mir-26a , which is frequently upregulated in t - all patients . in gastric cancer , mir-106a~363 targets bim .", "the mir-106a~363 cluster located at chromosome xq26.2 is the paralogue of mir-17~92 and encodes for mir-363 , mir-106a , and mir-20b . in hepatocellular carcinoma ,", "mir-25 of the mir-106b~25 cluster targets bim . also , the mir-106b~25 cluster , which includes mir-106b , mir-93 and mir-25 , is a paralogue of the mir-17~92 cluster and located on chromosome 7 within the thirteenth intron of the protein - coding gene mcm7 . also , the foxo transcription factors , important for bim upregulation , are regulated by micrornas ( figure 2 ) .", "foxo1 and foxo3 transcripts might be targeted by mir-182 [ 258261 ] , mir-1 , mir-27a , mir-96 , and mir-155 [ 263 , 264 ] .", "mir-155 plays a role in the activation and function of b and t lymphocytes [ 265 , 266 ] ( see section 3.1.6 ) .", "mir-182 expression was higher in gc - resistant cells in comparison to gc sensitive ones .", "increased expression of mir-182 reduced total foxo3a expression in t - all cells with consequent lower bim expression . foxo3a and bim increased upon downregulation of mir-182 , suggesting that mir-182 is involved in conferring gc resistance .", "the expression of the mir-182~96~183 cluster was induced in splenocytes from mouse with experimental systemic lupus erythematosus ( sle ) , suggesting a role of these micrornas in the breakdown of immunological tolerance and the manifestation of chronic autoimmune inflammation .", "this microrna cluster was also upregulated upon t - cell activation by an il-2-dependent manner .", "prevention of the expression of the mir-182~96~183 cluster led to increased foxo1 expression and limited population expansion of activated t - helper cells , due to increased cell death .", "vice versa , foxo3a was found to negatively regulate the oncomir mir-21 , which may be one mechanism by which foxo3a regulates apoptosis .", "as mir-21 targets pten [ 269 , 270 ] , activation of foxo3 by gcs may be one mechanism responsible for the gc - induced reduction in akt activity . besides function as a transcription factor in the nucleus , gr was found to translocate to the mitochondria in gc - sensitive , but not gc - resistant , lymphoma cell lines .", "gr was also found to translocate to the mitochondria in gc - sensitive thymocytes [ 272 , 273 ] .", "although there is one paper describing an interaction between gr and bcl-2 in the mitochondria , gc - induced mitochondrial gr translocation in gc - sensitive thymocytes and lymphoma cells proceeded in the absence of bcl-2 .", "exclusive overexpression of gr in the mitochondria was sufficient for inducing apoptosis , suggesting that mitochondrial gr may contribute to gc - induced apoptosis .", "glucocorticoid treatment inhibited complex i and complex iii of the electron transport chain , and the mitochondria was found to be the primary source of h2o2 production required for gc - induced apoptosis of lymphoma cells [ 275 , 276 ] .", "gcs may interact with the mitochondrial thioredoxin trx2 , a redox regulator , and directly modulate mitochondrial gene transcription .", "several mitochondrial metabolite and protein transporters and two subunits of the atp synthase were downregulated in t - all and precursor b - all cells at the gene expression level by dexamethasone .", "these changes were observed in gc - sensitive , but not gc - resistant , cells .", "corticosterone and other steroids were found to directly act on mitochondria to inhibit mitochondrial atp production by suppressing electron transfer from nadh to the electron transfer chain through complex i .", "the cellular protein kinase network ( kinome ) has critical influence on the gc sensitivity of lymphoid cells [ 30 , 31 , 97 , 281 ] .", "below , i will provide data supporting an involvement of gsk3 ( glycogen synthase kinase 3 ) in gc - induced apoptosis , and the antagonism of its activity by protein kinases such as akt and mtor , which leads to gc resistance . ", "the activity of gsk3 was found to be essential for gc - induced apoptosis [ 67 , 282 ] .", "gsk3 inhibitors prevented gc - induced apoptosis , and gc resistance frequently occurs through inhibition of gsk activity . reactivating gsk3 by using inhibitors of the pi3k - akt or mtor pathways sensitized gc - resistant cells to gc - induced apoptosis [ 67 , 108 , 115 , 116 , 243 , 283 ] .", "gsk3 was found to interact with gr in the absence of ligand and released from gr following exposure to gc .", "gsk3 also regulates gr transcriptional activity of bim , iap1 ( inhibitor of apoptosis 1 ) , and gilz ( glucocorticoid - induced leucine zipper ) [ 282 , 284 ] .", "the pi3k / akt and mtor signaling pathways are frequently hyperactivated in gc - resistant t - all [ 104 , 286 , 287 ] and is associated with poor prognosis and chemotherapeutic resistance in pediatric b - precursor all .", "mtor is a crucial regulator of cell metabolism , growth , and proliferation and mtor is positively regulated by pi3k / akt and notch1 [ 96 , 289 ] , while negatively regulated by the tuberous sclerosis tumor suppressor complex ( tsc1/tsc2 ) .", "activation of mtor contributes to tumor cell survival in alk ( anaplastic lymphoma kinase)-positive alcl ( anaplastic large cell lymphoma ) , mantle cell lymphoma , childhood b - precursor all , t - all , and aml .", "akt and mtor confer drug resistance by phosphorylating a series of targets [ 292 , 293 ] .", "phosphorylation and inactivation of gsk3 is a major cause for gc resistance that can be overcome by reactivating gsk3 , for example , by akt inhibitors or mtor inhibitors .", "as mentioned in section 1.2.3 , the mtor inhibitor rapamycin is efficient in overcoming gc resistance in various lymphoid malignancies .", "gc resistance can also be overcome in akt - active lymphoma cells by inhibiting src members ( e.g. , by pp1 ) , pi3k ( e.g. , wortmannin ) , or an akt inhibitor [ 67 , 68 ] .", "combination of gc with rapamycin or gc with obatoclax led to reduced akt phosphorylation at ser473 , suggesting that mtor may also act upstream to akt .", "gcs could also independent of other cytotoxic agents reduce mtor activity in lymphoid cells . low - dose arsenic trioxide could sensitize gc - resistant all to dex through an akt - dependent pathway .", "inhibition of mtor with rapamycin , which binds to fkbp12 , leads to increased bim expression and overcomes ras - dependent survival signals .", "synergy between mtor inhibitors ( e.g. , rapamycin ( sirolimus ) and cci-779 ( temsirolimus ) ) and other chemotherapeutic agents has been observed in b- and t - lineage all cell lines and preclinical models [ 96 , 298 ] . \n", "the akt activity is negatively regulated by pten ( phosphatase and tensin homolog deleted on chromosome 10 ) , a tumor suppressor gene that is suppressed , mutated , or deleted at high frequency in a large number of cancers .", "pten mutations or deletions are frequent in t - all and pten deletions are associated with less favorable outcome in t - all [ 104 , 300 ] .", "for instance , treatment of t - all with gamma secretase inhibitor ( gsi ) was only efficient if the cells expressed functional pten .", "one mechanism by which notch confers gc resistance is through pten inhibition leading to akt activation .", "pten specifically catalyzes the dephosphorylation of 3-phosphate of the inositol ring in phosphatidylinositol ( 3,4,5)-triphosphate ( pip3 ) resulting in the biphosphate product phosphatidyl ( 4,5)-biphosphate ( pip2 ) .", "pip3 is a second messenger generated by pi3k that binds to the pleckstrin homology ( ph ) domain of akt , which allows its phosphorylation and activation by the 3-phosphoinositide - dependent protein kinase 1 ( pdk1 ) . \n", "regulation of pten stability by phosphorylation and ubiquitination . taken into account the important role of pten in determining drug sensitivity ,", "pten expression has been attributed to epigenetic events such as promoter methylation [ 302 , 303 ] . at the posttranslational level , phosphorylation and ubiquitination decrease pten protein levels , while oxidation and acetylation reduce pten activity .", "rak phosphorylation of pten at tyr336 stabilizes the pten protein , while phosphorylation at thr366 , ser370 , ser380 , thr382 , and ser385 by casein kinase 2 ( ck2 ) and gsk3 reduces its stability [ 305 , 306 ] .", "pten is regulated by the protooncogene ubiquitin ligase nedd4 - 1 ( neural precursor cell expressed , developmentally downregulated 4 ) that promotes pten for proteasomal degradation .", "in multiple human cancer samples where the genetic background of pten was normal , but its protein level was low , nedd4 - 1 was highly expressed . upon tcr / cd28 stimulation of t cells , pten undergoes inactivation by nedd4 - 1 .", "the association between pten and nedd4 could be impeded by the e3 ubiquitin ligase cbl - b ( casitas - b - lineage lymphoma protein - b ) .", "cbl - b t cells show elevated akt activity , which was abrogated by simultaneous deficiency in nedd4 .", "pten is also negatively regulated by the anti - apoptotic xiap ( x - linked inhibitor of apoptosis ) that promotes pten for polyubiquitination and proteosomal degradation .", "induction of apoptosis in b - cll by arsenic trioxide was shown to lead to activation of c - jun - nh2 terminal kinase ( jnk ) , inactivation of akt and nfb , xiap downregulation , and pten upregulation .", "two other e3 ligases downregulating pten include wwp2 ( ww - domain containing protein-2 or aip-2 , atrophin-1-interacting protein 2 ) , and chip ( chaperone - associated e3 ligase c terminus of hsc70-interacting protein ) .", "pten expression can also be repressed by a range of micrornas including the mir-17~92 cluster [ 247 , 248 ] , mir-106b~25 , mir-21 , mir-26a [ 253 , 315 ] , mir-29b , mir-214 [ 317 , 318 ] , mir-216a and mir-217 , mir-212 , mir-221 , and mir-222 ( figure 3 ) .", "bcl-2 and bcl - xl are anti - apoptotic proteins residing in the mitochondrial outer membrane and in the endoplasmic reticulum .", "they prevent apoptosis of various chemotherapeutic drugs including gcs by capturing pro - apoptotic members of the bcl-2 superfamily , including bim , bax , and bak [ 215 , 322 , 323 ] .", "bcl-2 may also regulate gene expression [ 324 , 325 ] , cell cycle [ 326328 ] , activate erk1/2 [ 324 , 329 ] , and modulate the activities of transcription factors such as p53 , e2f , nfb , and notch [ 332 , 333 ] .", "this may be explained by the ability of bcl-2 to modulate p27 expression and promote g0 arrest [ 325 , 327 , 331 , 335 , 336 ] .", "long - term exposure to gcs could overcome resistance caused by either bcl-2 or bcl - xl [ 30 , 120 , 337 ] .", "overexpression of bcl-2 is common in leukemias and lymphomas [ 338341 ] . in follicular lymphoma ( fl ) and", "diffuse large b - cell lymphoma ( dlbcl ) , bcl-2 upregulation is commonly due to the t(14,18)(q32 ; q21 ) translocation , which places the bcl-2 gene under the control of ig heavy chain enhancers [ 342344 ] . \n", "overexpression of bcl-2 is common in cll due to the loss or downregulation of the human chromosome 13q14 locus , which harbors the mir-15a and mir-16 - 1 cluster .", "overexpression of mir-15a and mir-16 - 1 in cll cells led to cleavage of procaspase-9 and parp ( poly - adp - ribose polymerase ) and activation of the intrinsic apoptosis pathway .", "these two micrornas could serve as natural antisense bcl-2 actors that have potential use in the therapy of bcl-2 overexpressing tumors .", "the tumor - suppressor mir-34a , a pivotal member of the p53 network , also downregulates bcl-2 [ 347 , 348 ] , which may be one mechanism by which p53 activation leads to downregulation of bcl-2 .", "it also targets mcl-1 and bcl - w , and indirectly bcl - xl by attenuating il-6/stat-3 ( signal transducer and activator of transcription 3 ) signaling pathway [ 350 , 352 ] .", "mir-125b may function both as tumor suppressor and as an oncogene and has been widely considered as conferring drug resistance , among others by downregulating bak1 ( bcl-2 antagonist killer 1 ) [ 353355 ] and bmf . over - expression of mir-125b could induce leukemia in a mouse model .", "mir-181a / b that shows altered expression in cll could also target bcl-2 , besides acting on mcl-1 and xiap [ 358360 ] .", "bcl - xl can be targeted by the tumor suppressor microrna let-7 and mir-491 .", "a predominant feature of the gene expression signature leading to gc resistance in all was found to be elevated expression of the anti - apoptotic mcl-1 ( myeloid cell leukemia sequence 1 ) [ 364 , 365 ] .", "mcl-1 expression is especially high in mll - rearranged all , which represents an unfavorable type of leukemia that is often highly resistant to gcs .", "mcl-1 is also frequently overexpressed in b - cell and mantle - cell lymphomas , cml , cll , and mm .", "mcl-1 is an anti - apoptotic protein that sequesters the pro - apoptotic proteins tbid , bim , puma , noxa , and bak . besides preventing gc - induced apoptosis", ", mcl-1 confers resistance to trail ( tumor necrosis factor - related apoptosis inducing ligand)-induced cell death . \n", "mcl-1 differs from bcl-2 and bcl - xl in having a short protein turnover regulated by the 26s proteasome and its expression is tightly regulated . unlike bcl-2 ,", "rapamycin , a mtor inhibitor that sensitizes resistant all cells to gc , reduces the expression level of mcl-1 [ 113 , 287 ] .", "the degradation of mcl-1 depends on gsk3-mediated phosphorylation of mcl-1 at ser159 [ 369 , 370 ] .", "e3 ubiquitin ligases implicated in the regulation of mcl-1 include mule ( mcl-1-ubiquitinase ligase e3 ) , scf ( skp1/cul1/f - box protein -transducin repeat - containing protein ) , and fbw7 ( f - box and wd repeat domain - containing 7 ) which is part of the skp1-cullin1-f - box ( scf ) e3 ligase complex . the deubiquitinase usp9x ( ubiquitin specific peptidase 9 x - linked ) is an important regulator of mcl-1 stability . silencing of usp9x resulted in loss of mcl-1 .", "high levels of mcl-1 correlated with elevated usp9x expression in follicular lymphoma , diffuse large b - cell lymphoma , and some other cancer samples . increased expression of usp9x mrna was associated with poor prognosis of multiple myeloma .", "overexpression of noxa triggered an increase in the mule / mcl-1 interaction in parallel to a decrease in mule / usp9x complex formation . in an", "akt - driven , e-myc lymphoma mouse model , translational regulation of mcl-1 by mtor has been implicated in promoting lymphomagenesis .", "as gc may activate gsk3 and gsk3 inhibits mtor through phosphorylation of tsc2 and promotes mcl-1 degradation [ 369 , 370 ] , mcl-1 expressing lymphoid cells may ultimately undergo apoptosis if the exposure time to gc is sufficiently long .", "this may explain why many mcl-1-positive all cells exhibit delayed response to gcs , and not complete resistance [ 67 , 108 ] .", "also , the anti - apoptotic function of mcl-1 appears to require simultaneous expression of other anti - apoptotic bcl-2 family members .", "similarly , overexpression of mcl-1 in bcl-2- and bcl - xl - negative mouse double positive thymic lymphoma cells did not confer gc resistance upon these cells .", "usually , mcl-1 is expressed together with other anti - apoptotic proteins in gc - resistant lymphoid malignancies . \n", "mcl-1 is also regulated by micrornas ( figure 2 ) , including mir-29a , mir-29b [ 381383 ] , mir-101 , mir-125b , mir-181a / b [ 358 , 385 ] , mir-133b , mir-193b , and mir-512 .", "alk - positive anaplastic large cell lymphomas ( alcl ) express low levels of mir-29a , whose downregulation requires an active npm - alk kinase , and may probably also be due to methylation repression . enforced mir-29a expression reduced mcl-1 expression in alcl cells and reduced tumor growth in a xenografted model .", "mir-29b is downregulated in primary mm and aml samples and forced overexpression of mir-29b - induced apoptosis in mm and aml cells [ 381 , 383 ] .", "mir-29b overexpression also downregulated the expression of the dna methyltransferase isoforms dnmt1 , dnmt3a , and 3b .", "the global dna hypomethylation induced by mir-29b led to reexpression of tumor suppressor genes such as the cdk inhibitor p15 .", "altogether , these data propose that targeting mcl-1 with micrornas such as mir-29 represents a potential tool to constrict tumor growth of mcl-1 positive lymphomas .", "gcs release ca from the endoplasmic reticulum into the cytosol , which in turn increases the amount of mitochondrial ca .", "elevated expression of calcium - binding proteins s100a8 and s100a9 and of the anti - apoptotic mcl-1 ( myeloid cell leukemia-1 ) inhibits the free cytosolic ca and mitochondrial ca signals , respectively , thereby imposing gc resistance [ 287 , 365 , 389 , 390 ] .", "downregulation of s100a8 and s100a9 by the src kinase inhibitor pp2 sensitized mll - arranged all cells otherwise resistant to prednisolone - induced cell death .", "bcl-2 inhibits apoptosis in part by decreasing the size of ca stores in the endoplasmic reticulum resulting in reduced ca transfer to the mitochondria [ 391393 ] .", "one mechanism is through interaction of bcl-2 with ip3r ( inositol 1,4,5-triphosphate ( insp3 ) receptor ) , which is the principle er ca release channel in most cell types .", "also , bcl - xl and mcl-1 act in part by inhibiting ip3r [ 393 , 395 , 396 ] .", "an increase in hydrogen peroxide ( h2o2 ) is a necessary signal for gc - induced apoptosis .", "the mitochondria is the source of this signal , gcs inhibit complex i and complex iii of the electron transport chain .", "expression of anti - oxidant defense proteins such as manganese superoxide dismutase , thioredoxin , and catalase prevents gc - induced apoptosis [ 276 , 398400 ] .", "the anti - apoptotic bcl-2 may regulate the mitochondrial redox state in cancer cells [ 323 , 401 ] .", "notch is frequently activated in t - all cells , which may be due to mutations in notch1 ( gain - of - function ) and/or in the e3 ligase fbw7 that targets notch1 for degradation [ 7678 , 80 , 81 , 402405 ] .", "for example lnx1 ( ligand of numb - protein x1 ) is a positive regulator of notch signaling through degradation of numb , a membrane - associated protein that inhibits the function of the notch receptor .", "neuralized ( neur ) and mind bomb ( mib ) promote the monoubiquitination and endocytosis of delta [ 409 , 410 ] .", "itch binds to the n - terminal portion of the notch intracellular domain via its ww domains and promotes ubiquitination of icn - notch1 through its hect ubiquitin ligase domain .", "recent studies showed that notch1 can be activated in leukemic cells through interaction with bone marrow stromal cells that express notch receptors and ligands [ 412 , 413 ] .", "interaction with bone marrow stroma is also a mechanism for notch activation in multiple myeloma .", "cyclin e , which is targeted for degradation by fbw7 [ 415 , 416 ] , is expressed at higher levels in early relapsed pediatric b - cell precursor all patients , who usually show an unfavorable prognosis .", "notch1 prevents gc - induced apoptosis , among others , through activation of p56 , which activates the pi3k - akt axis , and through the transactivation of its target genes deltex and hes1 .", "deltex is a ring - domain ubiquitin ligase that may affect notch activity , and its overexpression prevents gc - induced apoptosis .", "activation of the pro - survival pi3k / akt / mtor pathway by notch has also been observed in other studies [ 95 , 106 , 419 , 420 ] and may be responsible for notch - mediated inhibition of the p53 tumor suppressor gene .", "another mechanism by which notch1 protects t - all cells from gc - induced apoptosis , is through the anti - apoptotic gimap5/ian5 ( gtpase of the immunity - associated protein / immune - associated nucleotide - binding protein 5 ) [ 421 , 422 ] . giamp5/ian5", "interacts with bcl-2 and bcl - xl and inhibits apoptosis during t - cell development and is highly expressed in human b - cell lymphoid malignancies .", "it is localized within the mitochondria and endoplasmic reticulum ( er ) and regulates mitochondrial integrity .", "gimap has been linked to immunological diseases such as t - cell lymphopenia and autoimmune diseases .", "notch also activates nfb signaling [ 74 , 427 ] and induces c - myc expression [ 428430 ] , both contributing to apoptotic resistance .", "this resistance can be overcome by the simultaneous exposure of the cells to src inhibitors , pi3k / akt inhibitors , or mtor inhibitors [ 67 , 68 ] , understating the importance of the protein kinase network in regulating the effects of notch1 on gc - induced apoptosis . ", "a recent report showed that gc sensitivity of t - all is associated with gr - mediated inhibition of notch1 expression .", "the serum- and glucocorticoid - inducible kinase 1 ( sgk1 ) was also shown to control notch1 signaling by downregulating its protein stability through fbw7 ubiquitin ligase .", "sgk1 phosphorylates fbw7 at ser227 , an effect inducing icn - notch1 ubiquitination and degradation . despite gc resistance induced by notch , notch- and fbw7-mutated", "t - all shows in general a favorable response to gc therapy and in some studies , but not all , also exhibits a better prognosis [ 405 , 433436 ] .", "this may be related to the fact that gcs may overcome notch - dependent drug resistance , and in these t - all cases the cell survival depends on notch signaling .", "various micrornas negatively regulate fbw7 expression including mir-27a , mir-182 , mir-363~92 , and mir-223 [ 253 , 438 , 439 ] and may increase the expression of fbw7-regulated target genes including notch1 , mcl-1 , c - jun , c - myc , and cyclin e . mir-451 and mir-709 suppressed oncogenesis in notch1-induced mouse t - all .", "mir-150 , which is upregulated upon thymocyte maturation , targets notch3 and thus regulates t - cell proliferation and survival .", "the p53-induced mir-34a also targets the notch1 receptor as well as its ligand dll1 ( delta like-1 ) [ 443 , 444 ] .", "prevention of notch activation in cutaneous t - cell lymphoma ( ctlc ) by gsi ( -secretase inhibitor ) treatment led to alterations in the microrna profile of the cell . among others ,", "mir-27a , mir92b , mir-181a , mir-18a , mir-19b , mir-222 , and mir-221 were downregulated , while mir-122 and mir-214 upregulated .", "mir-27a targets fbw7/hcdc4 [ 253 , 438 , 439 ] , the substrate recognition component of the scf ( skp1-cullin - f - box ) ubiquitin ligase complex that targets notch1 for degradation . the repressive effect of mir-27a on fbw7 mrna is especially pronounced at the g2/m and early g1 phases .", "other targets of mir-27a includes zbtb10 ( zinc finger and btb domain containing 10 ) , which acts as a repressor of sp ( specificity proteins ) transcription factors and induces g1 arrest , and the myt-1 kinase , which inhibits the transition through g2-m by enhanced phosphorylation and inactivation of cdc2 ( cdk1 , cyclin - dependent kinase 1 ) .", "upregulation of mir-122 by gsi seems to be mediated by p53 and has an antagonistic effect on apoptosis through activation of akt .", "c - myc is , among others , a target of notch [ 428430 ] and has broad effects on tumorigenesis and modulates gc - induced apoptosis [ 99 , 448 ] .", "conditional overexpression of c - myc in hematopoietic cells in mice culminated in the formation of malignant t - cell lymphomas and acute myeloid leukemias .", "these authors demonstrated a role for the pi3k / akt axis in c - myc activation .", "dysregulation of the c - myc gene is a common trait of burkitt 's lymphoma due to chromosomal translocations , the most frequent one being t(8 ; 14)(q24:q32 ) involving c - myc and igh ( immunoglobulin heavy locus ) [ 451453 ] .", "other hematopoietic malignancies characterized with c - myc overexpression include diffuse large b - cell lymphoma ( dlbcl ) , follicular lymphoma , cll , b - cell lymphoma , and aml [ 454459 ] .", "earlier studies have shown that dexamethasone - induced apoptosis of a t - all cell line was associated with c - myc suppression [ 460 , 461 ] .", "the gc - mediated down - regulation of c - myc expression was initially thought to be one mechanism that contributes to apoptosis .", "not all studies have confirmed this finding , which may be explained by the many signaling pathways induced by gcs .", "c - myc uses distinct mechanisms for activating and repressing gene expression . for transcriptional activation , c - myc dimerizes with max and", "transcriptional repression is achieved through protein - protein interactions , where it antagonizes the activity of positive regulators of transcriptions .", "the c - myc - mediated upregulation of mir-17 and mir-20a ( belonging to the mir-17~92 cluster ) negatively regulates e2f1 translation by targeting the 3-utr of e2f1 mrna and may therefore fine tune the direct myc - mediated transcriptional activation of e2f1 , allowing a tightly regulated proliferative signal ( figure 4 ) .", "e2f1 - 3 also binds to the promoter of the mir-17~92 cluster and activates its transcription , thus generating an autoregulatory feedback loop .", "another target of the mir-17~92 cluster is cyclin d1 , which also induces the expression of mir-17 and mir-20a by binding to the promoter regulatory region of the mir-17~92 cluster .", "the gc - induced down - regulation of mir-17~92 should actually stimulate e2f1 expression , which under certain circumstances may exert pro - apoptotic effects .", "e2f1 may promote apoptosis through transcriptional activation of the pro - apoptotic mir-15a~16 cluster and by activating jnk . in a b - cell lymphoma model , c - myc down - regulated a series of micrornas , an action that may contribute to tumorigenesis .", "the c - myc mediated repression of the mir-30 cluster may affect autophagy , as beclin-1 expression is regulated by mir-30a .", "some of the pro - autophagy activity of cancer therapy is mediated through down - regulation of mir-30a .", "also the down - regulation of mir-15a and mir-16 by c - myc is of interest as these micrornas are deleted or downregulated in over two - thirds of individuals with cll , and they target the anti - apoptotic bcl-2 gene [ 345 , 346 ] .", "a third mirna downregulated by c - myc is the tumor suppressor let-7 mirna cluster , which targets , among others , the ras oncogene , hmga2 ( high mobility group a2 ) [ 477 , 478 ] , bcl - xl , cdc25a , cdk6 ( cyclin - dependent kinase 6 ) , and cyclin d2 .", "other mirnas repressed by myc include mir-22 , mir-23a / b , mir-26a / b , mir-29a / b / c , mir-34a , mir-146a , mir-150 , and mir-195 [ 465 , 473 , 480 ] .", "mir-26a levels were found to be reduced in various b - cell lymphomas , especially burkitt lymphoma as well as various solid tumors [ 481 , 482 ] .", "b - cll , which does not have a prominent pathological role of c - myc , showed higher expression of mir-26a than myc - dependent burkitt lymphoma .", "mir-26 restoration in burkitt lymphoma or nasopharyngeal carcinomas reduced proliferation and colony formation through g1 arrest and repression of the histone - lysine n - methyltransferase ezh2 , a global regulator of gene expression [ 465 , 481 , 483 ] . the tumor - suppression function was only seen in myc - transformed cells , but not in v - abl transformed cells [ 465 , 483 ] . however , in t - all , mir-26a was one of five micrornas that independently promoted tumorigenesis through inhibition of pten .", "in the background of activating mutations in notch1 , mir-26a overexpression decreased the latency of t - all .", "forced overexpression of mir-34a , mir-150 , and mir-15a/16 - 1 attenuated in vivo tumor growth of myc - induced b - cell lymphoma .", "mir-34a is a crucial component of the p53 tumor suppressor network with potential anti - proliferative and pro - apoptotic activity [ 484486 ] .", "c - myc transcriptionally induces lin28b , which is an rna - binding protein that suppresses the maturation of let-7 family microrna precursors [ 487 , 488 ] .", "lin28 is involved in stem cell maintenance [ 489491 ] and is a marker of cancer stem cells .", "the effect of autophagy on the cellular response to chemotherapy is dual . under certain conditions ,", "autophagy acts as a pro - survival mechanism to protect cancer cells from chemotherapy , whereas under other circumstances , autophagy mediates the therapeutic effects of the anticancer agents .", "another important regulator of autophagy is the activity of mtor ( mammalian target of rapamycin ) , which is a central element signaling cell growth and enhancing protein translation . when this kinase is inhibited , autophagy is promoted .", "it should be noted that beclin-1 may play a dual role in both regulating autophagy and apoptosis , thus being at the cross - road between these two physiological processes .", "beclin-1 has recently been recognized as a bh3-only protein interacting with bcl-2 , bcl - xl and mcl-1 [ 59 , 60 , 494496 ] .", "one report provides evidence that after initiating apoptosis , beclin-1 is cleaved by caspases and the n - terminal fragment of beclin can inhibit autophagy , while the c - terminal fragment can amplify mitochondrial - mediated apoptosis .", "perturbation of beclin-1 cleavage by knockin mutation phenocopied the autophagy induction observed in apoptosis - defective cancer cells and rendered chemotherapy resistance both in vitro and in vivo . a role for beclin in regulating tumorigenesis has been demonstrated in mice with heterozygous disruption of beclin-1 .", "dlbcl expressing high beclin-1 levels had a favorable clinical outcome with r - chop treatment than those with low beclin-1 expression .", "gcs have been shown to promote autophagy in lymphocyte cell lines and primary t - all cells [ 501 , 502 ] .", "one mechanism for induction of autophagy is through upregulation of the mtor - inhibitory stress protein dig2 ( dexamethasone - induced gene 2 ) , also known as rtp801 and redd1 ( regulated in development and dna damage responses 1 ) .", "mtor inhibition by dexamethasone was demonstrated by reduced phosphorylation of s6k ( 70kd ribosomal protein s6 kinase 1 ) , a member of the rsk family of serine / threonine kinases .", "dig2 releases tsc2 from 14 - 3 - 3 , thereby promoting the assembly of the tsc1/tsc2 complex , which inhibits mtor .", "dig2 knockout thymocytes underwent more extensive dexamethasone - induced cell death , suggesting that autophagy promotes cell survival .", "however , rapamycin , an inhibitor of mtor and inducer of autophagy , strongly sensitizes resistant mm and t - all cells to gc - induced apoptosis [ 59 , 111 , 116 , 117 ] , suggesting that induction of autophagy does not always combat apoptosis .", "it could be that the higher degree of autophagy induced by rapamycin itself may be pro - apoptotic .", "bonapace et al . showed that rapamycin induces an autophagy - dependent necroptosis , which is required for childhood t - all to overcome gc resistance .", "necroptosis is a form of programmed necrosis that occurs when apoptosis is abortive due to caspase inhibition .", "the gc - mediated necroptosis was mediated by rip-1 ( receptor - interacting protein-1 ) and cyld ( cylindromatosis ) .", "mir-19 , which is frequently overexpressed in t - all patients and cell lines , represses cyld expression .", "obatoclax , a putative antagonist of bcl-2 family members , could also sensitize t - all cells to gc - induced apoptosis through induction of autophagy .", "this effect was associated with dissociation of the autophagy inducer beclin-1 from mcl-1 and decreased mtor activity .", "the apoptosis induced by gc in combination with obatoclax or rapamycin could be prevented by the autophagy inhibitors 3-methyladenine and bafilomycin .", "cdkn2/p16 , which acts as a g0/g1 cycle inhibitor , is frequently lost in t - all [ 507 , 508 ] and predicts relapse in children with all [ 508510 ] .", "p16 sensitizes t - all cell lines to gc - induced apoptosis through induction of bbc3/puma and repression of mcl-1 and bcl-2 .", "noxa was repressed in p16 transgenic cells , which could be a result of the simultaneous repression of e2f1 due to retinoblastoma protein and p130 activation .", "diffuse large b - cell lymphoma with cdkn2a deletion had a poor prognosis under r - chop treatment .", "also , myc gene arrangement in diffuse large b - cell lymphoma patients had a poor prognosis with r - chop chemotherapy .", "during the last decade , micrornas have become the focus of having a central role in the pathogenesis of cancer including lymphoid malignancies , besides their role in regulating gene expression during cell division , development , and differentiation [ 514523 ] .", "micrornas are short noncoding rnas that induce posttranscriptional gene silencing through base pairing with the 3 untranslated region ( utr ) of their target mrnas , thereby inhibiting their translation , with subsequent reduced protein levels [ 524 , 525 ]", ". bases 27 or 28 of the microrna are primary contributors to target specificity and are referred to as the microrna seed region .", "the micrornas are usually transcribed by rna polymerase ii , and sometimes by rna polymerase iii , into long primary precursor transcripts referred to as pri - mirnas .", "mirna are encoded by one arm of a stem loop structure embedded in introns or , less frequently , exons of protein - coding or noncoding transcripts . in the nucleus", ", the pri - mirnas stem loop is cleaved by the nuclear rnase iii enzyme drosha together with its cofactor dgcr8 ( digeorge syndrome critical region 8)/pasha ( the microprocessor complex ) to generate ~70 nucleotides long precursors called pre - mirnas . in some cases , an entire intron consists of such a stem loop structure , which is released by the splicing machinery in a drosha - independent manner .", "pre - mirnas are exported by rangtp / exportin-5 to the cytoplasm , where they are further processed by dicer , another rnase iii enzyme , to generate ~22 base pair microrna duplexes that enter effector complexes called mirisc ( mirna - containing rna - induced silencing complex ) . here , they are converted into single - stranded mature mirnas that target mrnas and thereby affect their translation and stability [ 516 , 528 , 529 ] .", "cancer cells frequently display reduced levels of micrornas that act as tumor suppressors , while expressing elevated levels of oncogenic micrornas , called oncomirs that promote tumor development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation and apoptosis .", "there are variations in the microrna expression pattern described between the various scientific reports , which can be explained by the use of different internal standards , different controls for comparison , and the use of sample materials of malignant cells at different developmental stage and at different ontogeny tumor grade .", "virtually every step in hematopoiesis seems to be finely tuned by specific micrornas [ 514 , 530533 ] .", "conditional deletion of dicer expression in the t - cell compartment resulted in impaired t - cell development and diminished regulatory t - cell function [ 534536 ] , and ablation of dicer in the b - cell compartment attenuates b - cell development and alters the antibody repertoire .", "it should be noted that there exists an alternative microrna processing pathway that is independent of dicer , but dependent on argonaute-2 .", "microrna expression is dynamically regulated during thymocyte development , with different enriched micrornas expressed at each developmental stage ( table 1 ) .", "it should be emphasized that the cd4cd8 ( double positive , dp ) thymocytes are the most gc - sensitive thymocyte population [ 540542 ] .", "dicer - deficient dp thymocytes expressed higher levels of cd69 and tcr ( t - cell receptor ) , but lower levels of bcl-2 .", "the dicer - deficient thymocytes were more prone to apoptosis than control cells [ 539 , 543 ] , understating the role of micrornas in regulating cell survival .", "some micrornas , such as mir-146a and mir-182 , play a dominant role in the regulation of the innate and adaptive immune responses , respectively [ 544 , 545 ] . according to neilson et al .", ", the pro - apoptotic mir-15b is almost not expressed at the immature dn1 ( double negative 1 ) thymocyte stage but becomes gradually upregulated in dn3 and dn4 , and further in dp cells .", "the pro - apoptotic mir-16 is also low in dp1 and reaches a maximum in dn4 cells , with a reduction upon transition to dp cells .", "the oncogenic mir-21 is expressed at the highest level in dn1 and becomes reduced upon transition to dn3 and is almost not expressed in dp cells .", "mir-181a / b is expressed at the highest level in dp thymocytes , together with mir-92 and mir-350 .", "it should be noted that in this study the expression of each microrna was determined relative to the general microrna pool of each subpopulation . since the amount of total microrna becomes strongly reduced upon transition from dn4 to dp ( a drop from 32000 to 5200 copies / cell ) ,", "the absolute microrna number in each cell population differs , which can be demonstrated by the mir-181a transcript .", "while mir-181a presents 15.6% of the microrna in dp cells and 6.7% and 5% in dn3 and dn4 , respectively , the numbers of mir-181a copies in these three populations were estimated to be 810 in dp , 1400 in dn3 , and 1600 in dn4 .", ". showed that mir-181a is expressed at dn1 and becomes upregulated during dn2 and dn3 and then downregulated at dn4 .", "mir-181 is still significantly expressed in dp cells , albeit at a slight lesser extent than in dn4 and becomes downregulated upon differentiation to the sp ( single positive ) stage .", "performed an extensive microrna profiling to identify micrornas specifically expressed in b - cell subsets during peripheral b - cell differentiation .", "notably , mir-18a , mir-28 , mir-15a~16 - 1 , and mir-181 are expressed at higher levels in centroblasts ( germinal center b lymphocytes ) compared with memory b cells , whereas mir-101c , mir-150 , mir-29a , b , c , and mir-23a~24 are enriched in memory b cells .", "mir-17~92 , mir-363~106a , and mir25~106b are highly expressed in all b - cell subtypes .", "the high level of mir-15a~16 - 1 in germinal center b - cells corresponds with low bcl-2 expression in these cells .", "mir-223 is highly expressed in nave and memory b cells , but not in centroblasts .", "mir-181a represses the expression of bcl-2 , cd69 , and the t - cell receptor ( tcr ) -chain .", "mir-181a augments the strength of tcr signaling and down - regulates several phosphatases including dusp5 , dusp6 , shp-2 , and ptpn22 that regulate the sensitivity of t cells to antigens .", "the down - regulation of ptpn22 by mir-181a led to elevated phosphorylation of p56 at y394 and the down - regulation of dusp5/6 to increased erk activation .", "the normally high levels of mir-181a maintain t - cell tolerance to self - peptide / mhc molecules , with a reduction in this microrna increasing the number of self - reactive t cells . also , dampening mir-181a expression using antagomir-181a impaired positive selection with about a 70% reduction of mature cd4 sp thymocytes .", "thus , mir-181a plays a role in regulating tcr response during t - cell development .", "recently , mir-181a-1/b-1 , but notmir-181a-2b-2andmir-181-c / d , was found to control the development of normal thymic t cells and leukemia cells .", "conditional deletion of mir-181ab1 allele resulted in 50%75% decrease in cellularity in the thymus and a significant reduction in the percentage of dp cells .", "mir-181a expression decreased during the dn3a to dn3b transition during -selection , and loss ofmir-181ab1resulted in a reduction in the percentage of dn3 and dn4 cells that expressed intracellular tcr- , while pret expression in dn3 thymocytes was normal .", "other alterations occurring upon tcr / cd28 co - stimulation includes the upregulation of the mir-466 family , mir-574 , mir-346 , mir-214 , mir-155 , and mir-709 , and the down - regulation of the mir-29 family , mir-15a , mir-15b , mir-16 , mir-146b , mir-142 , mir-27a , mir-150 , and let-7 family . chen et al", ". showed that mir-181 is expressed in the b - lymphoid cells of the mouse bone marrow , and its ectopic overexpression in hematopoietic stem / progenitor cells significantly increased b - cell production .", "mir-181 also affects the development of nk cells through targeting the nemo - like kinase ( nlk ) , an inhibitor of notch signaling .", "mir-181 targets the rna - binding protein lin28 , thereby disrupting the lin28-let-7 reciprocal regulatory loop , with concomitant upregulation of let-7 and differentiation of megakaryocytes .", "mir-150 is highly expressed in mature and resting lymphocytes , but not in their progenitors [ 547 , 553 ] .", "overexpression of mir-150 led to a block in b - cell formation at the pro - b to pre - b - cell transition by downregulating c - myb , among other targets , suggesting for a role for this microrna in b - cell differentiation . within the lymphoid lineage the choice between t and b cells", "the t - cell population level was unaffected by overexpression of mir-150 in hematopoietic progenitor cells , while the mature b - cell levels were strongly reduced .", "mir-150 drives megakaryocyte - erythrocyte progenitor ( mep ) cells towards megakaryocytes at the expense of erythroid cells .", "mir-150 also regulates the development of nk ( natural killer ) and inkt ( invariant nk ) cells .", "mice with target deletion in mir-150 had a defect in their ability to generate mature nk cells , while overexpression of mir-150 resulted in a substantial reduction in inkt in the thymus and in the peripheral lymphoid organs , supposedly through targeting of c - myb by mir-150 .", "mir-125b affects t - cell differentiation through regulation of ifn ( interferon ) , il-2r , il-10r , and prdm1/blimp1 ( b lymphocyte - induced maturation protein-1 ) .", "ectopic expression of mir-125b in nave lymphocytes inhibited differentiation to effector cells . during normal b - cell development , mir-125b is enriched in germinal center b cells and keeps the transcription factor irf4 and prdm1/blimp1 down , while mir-223 is enriched in memory b cells , where it targets the transcription factor lmo2 , which is specifically expressed in germinal center b cells .", "irf4 and prdm1/blimp1 expression are repressed in centroblasts , but is necessary for differentiation into memory and plasma cells [ 593 , 594 ] .", "overexpression of mir-125b alone in mice causes an aggressive , transplantable myeloid leukemia . before leukemia , these mice did not display elevation of white blood cells in the spleen or bone marrow , rather the hematopoietic compartment showed lineage - skewing , with myeloid cell numbers dramatically increase and b - cell numbers severely diminished .", "mir-125b targets lin28a , an induced pluripotent stem cell gene . knockdown of lin28a led to hematopoietic lineage - skewing similar to ectopic mir-125b overexpression , with increased myeloid and decreased b - cell number .", "mir-155 is also important for lymphopoiesis and for preserving normal immune system responses [ 266 , 597599 ] .", "mir-155 is processed within the second exon of the nonprotein - encoding gene bic ( b - cell integration cluster ) .", "mir-155 is upregulated upon tcr / cd28 costimulation in mouse t cells , and in macrophages by several tlr ( toll - like receptor ) pathways .", "b cells require mir-155 for normal production of isotype - switched , high - affinity antibodies and for a memory response .", "the transcription factor pu.1 , which down - regulates igg1 levels , is a target gene of mir-155 in b cells .", "this may explain the reduced amount of circulating igg1 in mir-155 knockout mice . as with b cells", ", it seems that mir-155 is involved in t - cell differentiation [ 266 , 597 ] .", "nave t cells derived from mir-155 knockout mice showed increased propensity to differentiate into th2 rather than th1 cells , with the concomitant production of th2 cytokines such as il-4 , il-5 , and il-10 [ 266 , 597 ] .", "one explanation for this biased development of th2 cells might be the mir-155 mediated targeting of c - maf ( musculoaponeurotic fibrosarcoma ) , a transcription factor that transactivates the il-4 gene . with regard to the acute immune response , the t cells had an impaired response and showed attenuated il-2 and ifn release in response to antigens [ 266 , 597 ] .", "mir-155 is upregulated by the transcription factor foxp3 and critical for t regulatory cell function .", "mice overexpressing mir-155 in the b - cell lineage results in preleukemic pre - b - cell proliferation in the spleen and bone marrow , followed later in life by b - cell malignancy .", "the mir-17~92 cluster located on chromosome 13 at locus q31.3 is essential for b - cell development .", "the expression of mir-17~92 peaked in pre - b cells , where it inhibited cell death .", "it is expressed at higher levels in normal germinal center b cells compared to nave and memory b cells .", "knockout of mir-17~92 leads to increased bim expression and inhibits b - cell development at the pro - b to pre - b transition , a step also blocked by mir-150 .", "mice overexpressing the mir-17~92 cluster in lymphocytes developed lymphoproliferative disease and autoimmunity and they died prematurely .", "these animals were found to have increased numbers of activated b cells , and a higher ratio of activated cd4 t cells versus cd8 t cells .", "the enhanced proliferation and survival of b and t cells may result from the down - regulation of bim and pten .", "mir-17~92 expression is strongly induced after activation of cd8 t cells , which is critical for the rapid clonal expansion of these cells . however , following the clonal expansion , mir-17~92 is downregulated and further silenced during memory development .", "malignant lymphomas arise from normal b- or t - cell counterparts at different ontogeny stages and commonly continue to express gene signatures inherited from their nontransformed cellular progenitors .", "extensive mirna profiling studies have been performed on various lymphoid malignancies , including t - all , cutaneous t - cell lymphoma , cll [ 557 , 563 ] , pre - b - all [ 557 , 605 , 607 ] , diffuse large b - cell lymphoma ( dlbcl ) [ 564 , 580582 , 608610 ] , anaplastic large cell lymphoma ( alcl ) , multiple myeloma ( mm ) [ 574 , 611 , 612 ] , mantle cell lymphoma ( mcl ) [ 583 , 591 , 613 ] , burkitt lymphoma ( bl ) [ 564 , 614 ] , and follicular lymphoma ( fl ) [ 135 , 582 , 615 ] .", "a comprehensive study aimed to integrate the many mirnas upregulated in t - all into a microrna - transcription factor coregulatory network was performed by ye et al . .", "a short description of some important micrornas in malignant lymphoid diseases is described below and summarized in tables 2 and 3 . in general ,", "t - all is characterized by upregulation of the mir-17~92 cluster , mir-26a , mir-128a / b , mir-146a , mir-181a / b , mir-150 , and mir-155 , while let-7b and mir-223 are downregulated [ 253 , 557561 ] . \n 3.2.1.1 .", "the mir-19 , mir-20a , mir-92a , and mir-17 especially of the mir-17~92 cluster are upregulated in t - all .", "all six mirnas mir-17 , mir-18a , mir-19a , mir-20a , mir-19b , and mir-92a , of the mir-17~92 cluster promoted leukemogenesis in notch1-induced t - all in vivo [ 253 , 616 ] . among them", ", the mir-19 family has been considered the key oncogenic component [ 248 , 506 , 617 ] .", "the mir-17~92 cluster is located within a fragile site that is frequently amplified in a range of hematopoietic malignancies .", "mir-19 represses notch1 , pten , hoxa9 , cyld , runx1 , e2f1 , and bcl2l11 ( bim ) [ 506 , 616 , 620 ] .", "posttranslational inactivation of pten by mir-19 promotes activation of the pi3k / akt pathway , and incontrollable proliferation of t cells [ 104 , 105 ] . increased akt signaling antagonizes gc - induced apoptosis by several mechanisms , including phosphorylation of foxo3a , thus preventing its nuclear translocation and transcriptional activation of bim , and through inactivation of gsk3 , which is essential for gc - induced apoptosis [ 30 , 67 , 97 ] .", "hoxa9 is a leukemogenic homeoprotein in t - all , and a target gene of the oncogenic mll - af4 fusion protein .", "high expression of mir-196b was found in pediatric all with aberrant activation of hoxa genes .", "notch1 plays a vital role in t - cell development and transformation , and about 50% of primary t - all samples show abnormal notch1 expression .", "downstream transcriptional targets of notch1 include hes1 and c - myc , the former affecting the pi3k / akt and nfb signaling pathways [ 624 , 625 ] .", "c - myc is a potent and direct transcriptional activator of mir-17~92 , leading to modulation of e2f1 expression .", "gcs repress the expression of mir-17~92 , which may be one means to overcome the tumorigenicity of t - all cells and to elevate bim expression .", "in contrast to mir-19 , mir-451 , and mir-709 are potent suppressors of oncogenesis in notch1-induced mouse t - all .", "mir-451 represses c - myc , while mir-709 represses ras - grf-1 that acts upstream to ras and prevents akt activation .", "both mir-451 and mir-709 are transcriptional targets of the bhlh e2a tumor suppressor , which is degraded upon notch1 induction in mouse t - all cells [ 626 , 627 ] .", "repression of tumor suppressor mir-451 is essential for notch1-induced oncogenesis in a murine model of t - all .", "human t - alls with activating notch1 mutations have decreased mir-451 and increased c - myc levels compared with t - alls with wild - type notch1 .", "one mechanism of the tumor suppressive action of mir-451 could be through down - regulation of the pi3k / akt survival pathway . \n", "primary t - all cells also express elevated levels of mir-26a that suppresses pten and bim . mir-26a", "mir-26a was found to be repressed by c - myc in a mouse lymphoma model , leading to enhanced expression of the ezh2 oncogene , a component of the polycomb repressive complex 2 .", "mir-146a , mir-181a / c , and mir-221 were associated with overall survival in all patients .", "however , overexpression studies of mir-146a in transplanted bone marrow cells suggest a tumor - suppressive role for this microrna .", "mir-146a knockout mice developed massive myeloproliferation followed by hematopoietic tumors , including myeloid sarcomas and lymphomas [ 635 , 636 ] . the myeloproliferative phenotype correlated with enhanced nfb signaling .", "mir-146a suppresses the nfb activators irak1 ( interleukin 1 receptor - associated kinase 1 ) and traf6 ( tnf receptor - associated factor 6 ) [ 635 , 637 , 638 ] .", "a negative feedback loop exists between nfb and mir-146 . whereas mir-146 represses nfb signaling , nfb signaling upregulates mir-146 . \n", "mir-181a family members are highly expressed in t - all leukemia cells and downregulated during remission .", "deletion ofmir-181a-1/b-1expression inhibits the development ofnotch1 oncogene - induced t - all in a mouse model .", "mir-181a / bcontrols the strength and threshold of notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of notch and pre - t - cell receptor ( tcr ) signaling pathways . \n", "mir-124a has been shown to be downregulated in more than 50% of all cases and associated with higher relapse rate and mortality rate .", "a comparison study of primary cll samples with normal unstimulated or cpg - stimulated b cells showed high similarities between cll and activated b cells , including upregulation of mir-34a , mir-155 , and mir-342 and down - regulation of mir-103 and mir-181a / b .", "activation of normal b cells led to reduced mir-23a , mir-23b , mir-24 , mir-27b , mir-181a / b , and mir-223 and increased mir-155 with all activation agents used .", "mir-150 was reduced during b - cell activation , whereas it was upregulated in most cll cases .", "the latter confirms the study of fulci et al . , but is opposed to wang et al", "ectopic mir-150 expression increased cell death in pro - b cells , while mir-150 deficiency led to b - cell expansion and an enhanced humoral immune response .", "some differences in mir-150 are observed between the mutated versus unmutated igvh ( immunoglobulin heavy chain variable - region genes ) subgroups , where expression is higher at the average in the mutated igvh subgroup .", "cll cases with unmutated igvh or with high expression levels of zap-70 ( 70kd zeta - associated protein ) show an unfavorable course with rapid progression in comparison to patients with a mutated igvh .", "two research groups [ 565 , 566 ] observed decreased levels of mir-29c and mir-223 in cll with zap70 and igvh unmutated status .", "calin et al . observed that the unmutated igvh cll subgroup exhibited high levels of tcl-1 due to low expression of mir-29 and mir-181 that negatively regulate this oncogene . mir-181 and mir-29 might therefore be considered to have tumor - suppressor functions .", "tcl-1 functions as a coactivator of akt , and b - cell forced expression of tcl-1 in transgenic mice resulted in tumors that resembled cll .", "cll with unmutated igvh and high expression of zap-70 showed also relative high expression of mir-15a , mir-16 - 1 , mir-16 - 2 , mir-195 , mir-23b , mir-155 , mir24 - 1 , and mir-146 , while low expression of mir-223 , mir-29a-2 , mir-29b-2 , and mir-29c . in an aggressive subtype of cll with abnormalities in the tp53 gene ,", "cll cases with good prognostic features are typically characterized by down - regulation of mir-15a and mir-16 - 1 [ 643 , 708 ] , located at the 13q14.3 locus .", "these mirnas map to a region between exon 2 and 5 of the leu2 gene .", "deletion of 13q14.3 ( del(13q ) ) is the most common cytogenetic abnormality in cll occurring in more than 50% of the cases and implies for a favorable prognosis .", "this deletion occurs also frequently in mm patients .deletion in mice of the 13q14-minimal deleted region , which encompasses the mir-15a~16 cluster , caused the development of indolent b - cell - autonomous , clonal lymphoproliferative disorders , recapitulating the spectrum of cll - associated phenotypes observed in humans .", "repression of mir-15a and mir-16 - 1 , as well as mir-29b , in cll may also be mediated by histone deacetylases ( hdacs ) .", "hdac inhibition triggered the accumulation of the transcriptionally activating chromatin modification h3k4me2 and restored the expression of mir-15a , mir-16 - 1 , and mir-29b .", "both mir-15a and mir-16 - 1 negatively regulate bcl-2 , and mir-29 targets mcl-1 [ 381 , 382 ] .", "the expression of bcl-2 in cll cases is inversely correlated with the expression of mir-15a and mir-16 - 1 [ 563 , 711 ] .", "other targets of mir-15/16 include chek1 , cyclind1 , cyclind2 , and cdc25a [ 525 , 645 ] .", "overexpression of mir-15a and mir-16 - 1 induced cell cycle arrest at g1/g0 in an rb - dependent manner . a germ - line mutation in the primary precursor of mir-15a/16 - 1 that impairs their processing was observed in familial cll patients .", "targeting deletion of mir-15a~16 in mice led to the development of a spectrum of diseases resembling cll - associated lymphoproliferation in humans , including cll , cd5 monoclonal b - cell lymphocytosis , and cd5 non - hodgkin 's lymphomas .", "the new zealand black ( nzb ) mouse that harbor a point mutation in the 3-flanking region of mir-16 that leads to reduced mir-16 expression and develops symptoms similar to b - cll in humans , further confirming the tumor suppressor function of this locus . \n", "underexpression of mir-181a / b was associated with shorter overall survival in cll , while higher levels of mir-181a were associated with a shorter time from diagnosis to initial therapy . during the course of cll progression ,", "the mir-181a / b levels were decreased , which inversely correlated with increased levels of its target genes mcl-1 and bcl-2 .", "the study of marton et al . showed consistent underexpression of mir-181a , as well as let-7a and mir-30d in all cll cases studied .", "however , increased expression of mir-181a / b was associated with favorable outcome in patients with cytogenetically normal acute myeloid leukemia ( aml ) .", "ectopic overexpression of mir-181a / b into primary cll increased fludarabine - sensitivity in p53 wild - type cells , but not in cll with attenuated p53 response .", "the importance of the mir-181 target mcl-1 in cll survival was demonstrated by rapid apoptosis of cll cells following sirna - mediated down - regulation of mcl-1 , and by the mcl-1 transgenic mice , which developed b - cell lymphoma .", "thus , low mir-181 and mir-29 expression in cll could confer drug resistance through upregulation of mcl-1 expression . \n", "the mir-29 family consisting of mir-29a and mir-29b seems to play a dual role in tumorigenesis . on the one hand , mir-29a and mir-29b", "are downregulated in mantle cell lymphoma , aggressive cll samples ( high zap-70 with unmutated igvh ) [ 659 , 710 , 716 ] , alk - positive anaplastic large cell lymphomas ( alcl ) , mm , and aml . on the other hand , mir-29a and b", "are expressed at higher degree in indolent cll ( low zap-70/mutated igvh ) than in normal cd19 cells [ 563 , 569 , 716 ] . mir-29c", "together with mir-223 down - regulation is associated with higher tumor burden , disease aggressiveness , and poor prognosis in cll . forced overexpression of mir-29b", "the tumor suppressor activity of mir-29 may be achieved through targeting cell cycle regulators and oncogenes such as cdk6 , dna methyltransferase 3a ( dnm3a ) and 3b ( dnmt3b ) , mcl-1 , and tcl1a [ 382 , 569 , 583 , 717 ] .", "another tumor suppressor function of mir-29 is mediated through activation of p53 , which is achieved by targeting p85 ( the regulatory subunit of pi3k kinase ) and cdc42 ( a rho family gtpase ) . however , in another setting mir-29 acts as an oncogene .", "mir29a overexpression in immature and mature b cells promoted cll development , and transplantation of mir-29-transduced hematopoietic stem and progenitor cells into irradiated mice resulted in myeloproliferative disease and aml .", "one mechanism for the oncogenic feature of mir-29 could be through repression of the tumor suppressor cell - adhesion molecule peroxidasin homologue ( pxdn ) .", "thus , depending on the cellular contexts , mir-29 can function as an oncogene or a tumor suppressor . \n", "mir-221 and mir-222 are expressed at higher levels in cll with unmutated igvh and high expression of zap-70 , the most aggressive cll subtype with poor prognosis .", "these micrornas may contribute to oncogenesis by targeting the cdk inhibitor p27 [ 695 , 696 , 718 , 719 ] , foxo3a [ 720 , 721 ] , apaf-1 [ 721 , 722 ] , p57 , bmf , pten , and timp3 ( tissue inhibitor of metalloproteinase 3 ) .", "in other cll cases , the mir-222 was found to be lower than that of normal cd19 cells .", "the p53 target mir-34a is decreased in cll patients with 11q deletions , leading to increased zap-70 expression .", "mir-34a also targets bcl-2 [ 348 , 484 ] , and the e2f1 and b - myb oncogenes in cll .", "members of the mir-17~92 polycistron are upregulated in b - cell lymphoma , as well as mir-155 [ 469 , 568 , 685 ] .", "adoptive transfer of hematopoietic stem cells bearing a truncated portion of the mir-17~92 polycistron in c - myc transgenic mice resulted in a more rapid onset of malignant b - cell lymphomas .", "transgenic overexpression of the entire mir-17~92 in the murine hematopoietic compartment led to the development of lymphoproliferative disease and increased lethality .", "the negative regulation of bim by the mir-17~92 cluster seems to be a major mechanism by which b - cell lymphomas evade apoptosis .", "silencing of mir-17 and mir-20a in mantle cell lymphoma led to upregulation of the cyclin - dependent kinase ( cdk ) inhibitor p21 , suggesting that p21 is an essential target of the mir-17~92 cluster during b - cell lymphomagenesis .", "overexpression of c - myc mrna together with mir-17 - 5p / mir-20a was associated with a more aggressive behavior in mantle cell lymphoma .", "mir-17~92 confers chemoresistance in mantle cell lymphoma through activation of the pi3k / akt pathway .", "mir-21 is commonly upregulated in cll as well as cml and many other cancer cell types .", "forced overexpression of mir-21 under the control of the nestin promoter resulted in severe pre - b - cell lymphoma .", "mir-21 overexpression potentiated lung tumorigenesis of a constitutively activated k - ras proto - oncogene .", "mir-21 is an oncomir that promotes tumorigenesis by targeting a range of genes involved in regulating cell proliferation and/or survival , including pten , sprouty ( spry2 ) , pdcd4 ( programmed cell death 4 ) , tpm1 ( tropomyosin 1 ) , and human dna muts homolog 2 ( hmsh2 ) . in glioblastoma cells , mir-21 also targets a network of p53 pathways , tgf , and mitochondrial tumor suppressor genes .", "pdcd4 inhibits ap-1-mediated transactivation and negatively regulates the pro - survival ral guanine - nucleotide dissociation stimulator ( ralgds ) signaling pathways [ 517 , 729 ] .", "pdcd4 is a tumor suppressor that is upregulated during apoptosis and downregulated in several cancer forms [ 737739 ] .", "spouty , which is downregulated by mir-21 , negatively regulates the c - raf pro - survival signaling pathway . \n", "overexpression of mir-125b in cll - derived cell lines resulted in the repression of many transcripts encoding enzymes implicated in cell metabolism .", "these authors proposed that mir-125b acts as a regulator for the adaptation of cell metabolism to a transformed state . \n", "one microrna consistently downregulated in most b - lymphomas is mir-150 , which is proposed to act as a tumor suppressor [ 523 , 547 , 589 ] .", "mice lacking mir-150 have increased expression of its target transcription factor c - myb , which plays an important role in lymphocyte development and maturation .", "premature expression of mir-150 blocked the transition from pro - b to the pre - b stage .", "overexpression of mir-150 in nk / t lymphomas increased apoptosis and reduced cell proliferation , with concomitant reduction in dkc1 ( dyskeratosis congenita 1 ) and akt2 , reduced akt phosphorylation , and elevated levels of bim and p53 . \n", "mir-155 is overexpressed in many b - cell lymphomas including cll , primary mediastinal b - cell lymphoma ( pmbl ) , aggressive activated b - cell like ( abc ) subtype of dlbcl , hodgkin 's lymphoma , and pediatric burkitt 's lymphoma , but is almost absent in adult burkitt 's lymphoma [ 515 , 566 , 568 , 576 , 583 , 584 , 587 , 602 , 685 , 686 ] .", "c - myb ( v - myb myeloblastosis viral oncogene homolog ) , which is overexpressed in a subset of cll patients , associates with the promoter of mir-155 host genes ( mir155hg , also known as bic , b - cell integration cluster ) and stimulates its transcription .", "forced overexpression of mir-155 in b cells ( e-mir-155 transgenic mice ) led to initial preleukemic pre - b - cell proliferation followed by frank b - cell malignancy .", "the mir-155 orthologue mir - k12 - 11 in kaposi sarcoma - associated herpes virus ( kshv ) has been associated with b - cell tumors .", "mir-155 is essential for immune function and is strongly induced in activated t and b cells .", "mir-155 represses sh2-domain containing inositol-5-phosphatase-1 ( ship-1 ) , which is a critical phosphatase that negatively downmodulates akt pathway and is involved in normal b cell development . thus , sustained overexpression of mir-155 in b cells unblocks akt activity , inducing b - cell development .", "c - maf in lymphocytes , and hgal and smad5 in diffuse large b - cell lymphoma ( dlbcl ) [ 741 , 742 ] .", "hgal , a germinal center ( gc)-specific gene , inhibits lymphocyte and lymphoma cell motility by activating rhoa signaling cascade and by interacting with actin and myosin proteins .", "smad5 is a bone morphogenetic protein ( bmp)-responsive transcription factor and is activated by various cytokines .", "dlbcl expressing high levels of mir-155 concomitant with low hgal expression showed high aggressiveness and cell dissemination .", "thus , down - regulation of smad5 in diffuse large b - cell lymphoma defines a unique mechanism used by the cancerous cells to escape tgf growth inhibitory effects . in breast cancer ,", "as foxo3a is a positive regulator of the pro - apoptotic bim essential for gc - induced apoptosis [ 227229 ] , mir-155 overexpression may prevent bim upregulation . in one study , mir-93 , mir-25 , mir-92 ,", "mir-19a / b , mir-181a / b , and mir-32 were shown to be significantly overexpressed , while let7-b , let7-i , let7-c , mir-29a , and -29b significantly downregulated in mm .", ". found decreased expression of mir-15a~16 and increased expression of mir-222 , mir-221 , mir-382 , and mir-181a / b in their mm samples .", "also , the 13q14.3 locus containing the mir-15a and mir-16 - 1 is sometimes deleted in mm [ 345 , 746748 ] .", "the absence of mir-15a expression and overexpression of mir-181a / b correlated with worse prognosis of mm .", "antagonists especially to mir-19a / b and mir-181a / b ( antagomirs ) suppressed tumor growth of human myeloma cells implanted into nude mice .", "some differential mirna expression was observed between malignant mm and mgus ( monoclonal gammopathy of undetermined significance ) , which is the precancerous state preceding mm .", "mgus show already upregulation of mir-21 , mir-106~25 , mir-181a / b , mir-1 , and mir-133a , while during the progression to malignant multiple myeloma mir-17~92 , mir-32 , mir-193b~365 are upregulated and mir-192~194~215 and mir-15a~16 are downregulated [ 574 , 576 , 577 ] .", "the upregulation of mir-17~92 could be related to the upregulation of c - myc observed during mm progression [ 750 , 751 ] .", "upregulation of mir-1 and mir-133a correlated with t(14 ; 16 ) translocation in mm cases , suggesting that deregulation of microrna expression could be associated with chromosomal abberations .", "higher expression of dicer was associated with improved progression - free survival in symptomatic mm cases . the global increase in microrna expression in high - risk mm patients with poor prognosis", "was associated with increased expression of argonaute ( ago2/elf2c2 ) , a master regulator of mirna maturation and function [ 753 , 754 ] .", "adhesion of multiple myeloma to bone marrow stroma triggers cytokine production and enhances cell proliferation and resistance to chemotherapy through il-6-induced activation of nfb , pi3k / akt , and stat3 pathways .", "it should be noted that these pro - survival pathways antagonize gc - induced apoptosis in mm [ 756760 ] .", "mir-19a and mir-19b that are part of the mir-17~92 cluster downregulate socs-1 ( suppressor of cytokine signaling-1 ) , a gene frequently silenced in mm that plays a critical role as inhibitor of il-6 growth signaling , thus enforcing the il-6-induced survival signals . \n", "the oncogenic mir-21 is upregulated in mm patient samples and cell lines [ 574 , 579 , 652 ] . in il-6-dependent", "ectopic mir-21 expression was sufficient to sustain growth of il-6-dependent cell lines in the absence of il-6 .", "mir-21 is upregulated in a nfb - dependent manner in mm cells upon cell adhesion to bone marrow stromal cells . combining mir-21 inhibition with dexamethasone", "the p300-cbp - associated factor ( pcaf ) was found to be a target of the combined action of the mir106b~25 cluster and mir-32 .", "mir106b~25 , mir-17 , and mir-20a target the cdkn1a1/p21 cell cycle regulator , which prevents cell cycle progression in general and prevents the growth of mm cells [ 764 , 765 ] . \n", "mir-15a~16 is a pro - apoptotic microrna that targets bcl-2 , cyclin d1 , cyclin d2 , and cdc25a [ 346 , 748 , 766768 ] .", "overexpression of mir-15a~16 in mm led to inhibition of akt3 , ribosomal protein s6 , map kinases , and the nfb - activator map3kip3 , ultimately resulting in an antiproliferative effect and apoptosis .", "the anti - mm effect of mir-15a~16 was observed even in the context of the bone marrow microenvironment . mir-15a~16", "reduced vegf secretion from mm cells , thereby reducing mm cell - induced pro - angiogenic activity on endothelial cells .", "vegf represents one of the major pro - angiogenic cytokines responsible for the induction of neoangiogenesis in mm patients [ 769 , 770 ] .", "a distinct microrna profile could distinguish between alk and alk subtypes of alcl , an aggressive form of non - hodgkin 's lymphoma ( nhl ) belonging to the t - cell lineage .", "more than 80% of alk alcl harbor the t(2 ; 5)(p23 ; q35 ) translocation , resulting in the expression of the chimeric nucleophosmin ( npm)-alk .", "the constitutive alk activity leads to the activation of many different growth - promoting and anti - apoptotic pathways including pi3k / akt / mtor , jak / stat , c - jun , junb , and c - myc . the prognosis of alk alcl is worse [ 772 , 773 ] .", "alk alcl has a high cure rate with chop treatment , in contrast to alk cells that are relative resistant .", "five members of the mir-17~92 cluster were expressed higher in alk alcl , whereas mir-155 was expressed more than 10-fold higher in alk alcl .", "the upregulation of mir-17~92 cluster in alk alcl cells is in agreement with the observation that c - myc is expressed in alk alcl and absent from alk samples .", "mir-101 targets mtor , mcl-1 , and the histone methyltransferase ezh2 [ 778 , 779 ] . inhibition of mtor , which is targeted by mir-101 , led to reduced tumor growth in engrafted alcl mouse models .", "mir-29a and mir29b down - regulation in alk alcl confer apoptotic resistance due to mcl-1 upregulation [ 380 , 587 ] .", "another microrna that has been implicated in npm - alk - driven oncogenicity is mir-135b .", "mir-135b inhibition reduced tumor angiogenesis and growth in vivo , suggesting that targeting this microrna has therapeutic potential .", "a 9-mirna signature ( mir-146b-5p , mir-146a , mir-21 , mir-155 , mir-500 , mir-222 , mir-363 , mir-574 - 3p , and mir-574 - 5p ) could differentiate the diffuse large b - cell lymphoma ( dlbcl ) , the most common subtype of non - hodgkin 's lymphoma , into abc ( activated b - cell ) or gcb ( germinal center b - cell ) subtypes , with a general higher expression in the abc subtype .", "another study found that mir-331 , mir-151 , mir-28 , and mir-454 were upregulated in the gcb type , whereas mir-222 , mir-144 , mir-451 , and mir-221 upregulated in the abc type .", "the microrna expression of both gcb - like and abc - like cells was more similar to germinal center lymphocytes than memory b - cells .", "the region encoding the mir-17~92 cluster was more commonly amplified in gcb - like than abc - like dlbcl .", "lawrie et al . identified 3 mirnas , mir-155 , mir-21 , and mir-221 , more highly expressed in abc type than gcb type cells .", "expression of mir-155 and mir-21 was also higher in nonmalignant abc than in gcb cells .", "patients with gcb dlbcl have longer overall survival and event - free survival compared with patients with an abc phenotype when treated with r - chop [ 782 , 783 ] . increased expression of mir-18a in dlbcl was associated with a shorter os ( overall survival ) of patients receiving r - chop regimen .", "increased expression of mir-181a was associated with longer pfs ( progression - free survival ) , while increased expression of mir-222 was associated with shorter pfs . in dlbcl , mir-181a regulates foxp1 ( forkhead box protein p1 ) and mgmt ( o - methylguanine - dna methyltransferase ) expression in dlbcl cells .", "foxp1 is expressed in normal activated b cells , mantle zone b cells , and some germinal center b cells [ 784 , 785 ] .", "foxp1 is recurrently targeted by chromosomal translocations involving the immunoglobulin heavy chain locus in marginal zone lymphomas and dlbcl , suggesting a potential role for foxp1 in lymphomagenesis [ 786 , 787 ] .", "foxp1 has in some studies been shown to be associated with poor prognosis and survival [ 788 , 789 ] .", "the ability of mir-181a to reduce mgmt protein expression may contribute to better cyclophosphamide chemosensitivity .", "mir-222 is part of the mir-221/mir-222 cluster , which is highly expressed in abc - like dlbcl cell lines and abc - like dlbcl tumors .", "mir-222 regulates the expression of the stem cell factor c - kit , and the cyclin - dependent kinase inhibitors p27 and p57 [ 695 , 698 ] .", "high expression of mir-222 was associated with inferior overall survival and progression - free survival .", "fl is characterized by high mir-9 , mir-138 , mir-20a / b , and mir-155 expression [ 135 , 564 , 582 ] . \n", "mir-9 targets also the transcription factor prdm1/blimp1 in lymphoma and may contribute to the phenotype maintenance and pathogenesis of lymphoma cells by interfering with normal b - cell terminal differentiation [ 582 , 608 ] .", "brdm1/blimp1 and bcl6 are critical regulators of germinal center b - cell differentiation [ 594 , 792 , 793 ] .", "brdm1/blimp1 and bcl6 are expressed in a mutual exclusive pattern and evidence suggests that they repress each other in germinal center b cells [ 792 , 794 ] .", "a marked decrease of brdm1/blimp1 and an increase of bcl6 were observed in follicular lymphoma cells , which might be related to the increased mir-9 levels in these cells .", "mutations in brdm1/blimp1 are frequently found in activated b cell ( abc)-like dlbcl [ 790 , 795 ] .", "the malignant hodgkin 's lymphoma cells are usually derived from b cells , but have lost the expression of typical b - cell genes .", "multiple signaling pathways are deregulated , including nfb , jak ( janus kinase)/stat ( signal transducer and activator of transcription ) , pi3k / akt , erk , notch1 , and receptor tyrosine kinases .", "patients with low mir-135a expression had a higher probability of relapse and a shorter disease - free survival .", "mir-135a targets jak2 , a cytoplasmic tyrosine kinase involved in a subset of cytokine receptor signaling pathways", ". transfection of pre - mir-135a into classical hl ( chl ) caused apoptosis and decreased cell growth .", "the mir-135a - mediated jak2 down - regulation led to decreased bcl - xl expression , a downstream effector of jak2 .", "about 40%60% of hodgkin 's lymphomas have ebv ( epstein - barr virus ) associated with the malignant cells .", "since mir-155 is overexpressed in hodgkin 's lymphoma and promotes b - cell lymphoma formation [ 602 , 798 , 799 ] , ebv may be important in the pathogenesis of chl .", "micrornas have been shown to modulate gr expression in neuronal tissue [ 649 , 800 , 801 ] .", "mir-18 and mir-124a especially reduced gr - mediated events in addition to decreasing gr protein levels .", "upregulation of the mir-17~92 has causally been related to small cell lung cancer [ 802 , 803 ] , where reduced gr levels have been associated with gc resistance .", "activation of the gr - responsive glucocorticoid - induced leucine zipper ( gilz ) was impaired by mir-124a and -18 overexpression , while mirs-22 , -328 , and -524 did not have any effect .", "of note , mir-124 regulates hes1 expression in p19 teratocarcinoma cells , a transcription factor that negatively regulate gr expression .", "while mir-130b , -181a , and -636 have putative complimentary binding sites in the 3-utr of gr , only mir-130b was found to down - regulate endogeneous gr protein expression in the multiple myeloma cell line mm.1 .", "the mir-130b , -181a , and -636 were differentially expressed between gc - sensitive and gc - resistant mm.1 cell lines .", "overexpression of mir-130b in mm.1s cells resulted in decreased expression of endogeneous gr , decreased induction of the gr - target gene gilz , and induction of gc resistance .", "expression of mir-130b was therefore suggested to be a potential biomarker for patients who could be refractory to gc therapy . in gastric cancers , mir-130b regulated the tumor suppressor gene runx3 .", "mir-130b may also down - regulate p21 , resulting in inhibition of cellular senescence [ 676 , 806 ] .", "another study showed that elevated mir-142 expression in human t - all cells confers gc resistance by reducing the gr expression level .", "other mechanism for the oncogenic role of mir-142 might be explained by its targeting of adenylyl cyclase 9 mrna leading to reduced production of cyclic adenosine monophosphate ( camp ) production with concomitant inhibition of the protein kinase a ( pka ) signaling pathway .", "the reduction in camp levels and reduced pka activity caused by mir-142 relieve the inhibitory effect of pka on t - leukemic cell proliferation .", "t - all with poor prognosis expressed higher levels of mir-142 than those with good prognosis .", "also , mir-142 was expressed at higher levels in relapsed t - all than newly diagnosed samples .", "transfection of mir-142 inhibitor increased gr expression levels and sensitized t - all cells to gc - induced apoptosis .", "these findings are in accord with previous findings showing a synergistic effect of camp mimetics on gc - induced apoptosis [ 99 , 460 , 807 ] .", "camp signaling can also be negatively regulated by phosphodiesterase 4b ( pde4b ) that is frequently overexpressed in diffuse large b - cell lymphoma ( dlbcl ) .", "pharmacological inhibition of pde4 in a xenograft model of human lymphoma unleashed camp effects , inhibited akt , and restored gc sensitivity .", "pde4 inhibitors may thus improve the clinical outcome of patients with b - cell malignancies .", "triptolide , a drug that overcomes dexamethasone - resistance in human multiple myeloma cells , was found to regulate gr expression in the mm1.s cell line by downregulating the expression of mir-142 and mir-181a . mir-142 and mir-181a mimetics slightly attenuated , whereas mir-142 and mir-181a inhibitors enforced gc - induced apoptosis of mm1.s cells .", "mir-181a / b can also increase gc - induced apoptosis in virtue of their ability to repress the expression of the anti - apoptotic bcl-2 , mcl-1 , and xiap proteins [ 385 , 539 , 810 ] . ", ". showed that broad microrna repression occurs during gc - induced apoptosis of rat thymocytes .", "this repression was associated with reduced expression of both nuclear ( drosha and dgcr8/pasha ) and cytoplasmic ( dicer ) microrna processing enzymes .", "silencing of dicer in two human leukemic cell lines ( cem - c7 and ectopic gr-overexpressed jurkat cells ) led to enhanced sensitivity to gc - induced apoptosis .", "global downregulation of microrna levels , especially the mir-17 family , by gcs was also observed in gc - sensitive all cell lines , with concomitant upregulation of bim .", "later studies showed that gcs selectively upregulate and downmodulate specific mirnas that can not be explained by altered dicer expression .", "one polycistron cluster repressed by gcs is mir-17~92 [ 648 , 657 ] , which regulates bim expression [ 246 , 247 ] .", "this microrna cluster also represses pten , a negative regulator of the pi3k / akt signaling pathway .", "the gc - mediated downregulation of mir-17~92 might be one mechanism responsible for the gc - induced dephosphorylation of akt .", "primary thymocytes derived from mice transgenic for the mir-17~92 polycistron members in the lymphocyte compartment exhibited diminished sensitivity to gc - induced apoptosis in lymphocytes , further supporting a role for gc - induced repression of mir-17~92 in promoting apoptosis .", "observed that gcs reduced mir-17 family expression in 50% of primary gc - sensitive all , but not in any of the gc - resistant ones .", "overexpression of mir-17~92 attenuated gc - induced cell death , while inhibition of mir-17~92 increased the sensitivity to gc .", "they also reported that in a pre - b all cell line , a 10-hour dexamethasone treatment led to a reduction in mir-142 and mir-27a , while mir-9 was induced .", "there is also some evidence that gcs can reduce mir-27a expression in mouse muscle cells .", "rainer et al . reported an induction of the myeloid - specific mir-223 and the apoptosis and cell - cycle arrest inducing mir-15~16 clusters by gc in a subset of b- and t - all cells , together with downregulation of the mir-17~92 complex .", "the mirnas of the mir-15~16 family are encoded in two clusters ( 15a~16 - 1 and 15b~16 - 2 ) embedded in the dleu2 ( deleted in leukemia 2 ) and smc4 loci , respectively [ 766 , 812 ] .", "they have been implicated in cell - cycle arrest and in cell death / survival decisions , the latter supposedly by targeting bcl-2 .", "other micrornas affected by gcs in pediatric all include upregulation of mir-548d-1 and repression of mir-128b along with mir-106b~25~93 , the paralogue of mir-17~92 .", "it is still not known whether the gc - induced upregulation of mir-223 affects gc - induced apoptosis .", "increased expression of mir-223 is involved in the differentiation of myeloid precursors into granulocytes such as neutrophils [ 701 , 814 ] . during granulopoiesis ,", "mir-223 targets e2f1 , which in turn represses mir-223 expression , creating an autoregulatory negative feedback loop .", "a negative feedback loop also exists between mir-223 and the transcription factor nfi - a .", "mir-223 is positively regulated by c / ebp during differentiation to granulocytes and negatively regulated by aml1/eto in leukemia cells .", "moreover , mir-223 targets the myeloid elf-1-like factor ( mef)-2c and igfr ( insulin - like growth factor receptor ) , which may account for some of its negative regulation of granulocyte proliferation . through suppression of igf-1r , the downstream pi3k / akt / mtor / p70s6k pathway is suppressed , with consequent inhibition of cell proliferation . mir-223 attenuates hematopoietic cell proliferation and positively regulates mir-142 through lmo2 isoforms and c / ebp .", "mir-223 knockout mice showed increased numbers of granulocyte progenitors in the bone marrow and hypermature neutrophils in the circulation , suggesting that mir-223 is involved in the negative regulation of maturation rather than differentiation of granulocytes . mir-223", "may also target fbw7 [ 705 , 816 ] , a negative regulator of the anti - apoptotic mcl-1 .", "dexamethasone treatment of thymocytes led to upregulation of mir-150 and mir-342 , while mir-181a and mir-181d were downregulated .", "lif is a member of the il-6 cytokine family expressed in thymic epithelial cells and t lymphocytes , which elevates gc levels following lps exposure and is responsible for thymic atropy induced by stress [ 817819 ] .", "a recent report showed that gcs could prevent lipopolysaccharide ( lps)-mediated inflammatory responses in macrophages by downregulating mir-155 .", "overexpression of mir-155 reversed the suppressive action of gcs , while inhibition of mir-155 exhibited an effect similar to that of gcs on lps - treated macrophages , suggesting that gc - induced repression of mir-155 is one mechanism for the immunosuppressive function of gc .", "mir-155 is transcribed from b - cell integration cluster ( bic ) [ 584 , 691 ] and targets among others socs1 ( suppressor of cytokine signaling 1 ) , which negatively regulates jak / stat signaling .", "gcs also prevented the lps - mediated upregulation of mir-146 , mir-147 , mir-148 , mir-32b , and mir-301 in macrophages . in the brain", ", gcs prevents bdnf ( brain - derived neurotrophic factor)-regulated synaptic function through suppression of mir-132 expression .", "mir-132 is increased by bdnf and is involved in promotion of neuronal outgrowth . in some carcinoma cell lines , dexamethasone was shown to down - regulate mir-27b , mir-148a , and mir-451 . from all we have learned above , any microrna that modulates", "any of the many factors regulating gc - induced apoptosis may affect the apoptotic response to gcs ( figures 16 ) .", "these include micrornas that affect gr expression ( e.g. , mir-18 , mir-124a , mir-130b , mir-142 , and mir-181a ) , those affecting bim expression ( mir-26a , mir-93 , mir-17~92 , mir-106a~363 , and mir-106b~25 ) or its transcription factor foxo3 ( e.g. , mir-1 , mir-21 , mir-27a , mir-96 , mir-135b , mir-155 , and mir-182 ) , those affecting pten expression ( mir-17~92 , mir-106b~25 , mir-21 , mir-26a , mir-29b , mir-214 , mir-216a , mir-217 , mir-221 , and mir-222 ) or mtor ( e.g. , mir-101 ) , and those downregulating directly or indirectly the anti - apoptotic proteins bcl-2 , bcl - xl , mcl-1 , xiap , and cyld ( e.g. , mir-15a~16 , mir-181a / b , mir-34a , mir-125b , mir-29a / b / c , mir-101 , mir-133b , mir-193b , mir-512 , let-7 , and mir-491 ) . the effect of some of these micrornas on gc - sensitivity has already been described above and will not be repeated here .", "rather , i will present here some data from primary samples showing the influence of micrornas on clinical outcome .", "a study searching for differential mirnas expression in all relapse cells versus childhood all with complete remission showed significant associations for mir-708 , mir-223 , and mir-27a with individual relapse - free survival .", "for samples at relapse versus diagnosis , the most differentially expressed micrornas included mir-223 , mir-23a , let-7 g , mir-181 , mir-708 , and mir-130b , while comparison of complete response with diagnostic samples showed differential expression pattern of mir-27a , mir-223 , mir-23a , mir-181 , and mir-128b . among these micrornas , mir-223 , mir-128b , mir-23a , and let-7 g", "were downregulated in the relapse samples compared with complete response samples , while mir-181 family members , mir-708 , and mir-130b were upregulated in the relapse samples .", "it should be remained here that mir-130b targets gr , runx3 , and p21 , and mir-223 is upregulated by gcs and targets igfr and e2f1 .", "e2f1 has a dual role in cell - cycle control , as it affects several cell processes .", "it can either act as a tumor - suppressor or oncogene depending on the cellular context .", "thus , the upregulation of mir-130b together with downregulation of mir-223 may contributes to gc resistance .", "mir-708 was the most upregulated microrna in the relapse samples , whereas mir-223 was significantly downregulated , suggesting that these two micrornas may have important role in pediatric all relapse .", "moreover , upregulation of mir-708 was found to be associated with the in vivo gc therapy response and with disease risk stratification in childhood all .", "standard and middle risk stratification groups had a higher mir-708 expression at diagnosis than the high risk group .", "interestingly , mir-708 was low in high relapse patients at diagnosis , while specimens of relapsed samples showed abundance of mir-708 , suggesting for an upregulation of mir-708 during disease progression .", "foxo3 , that is critical for hematopoietic stem cell self - renewal and mediates the initial apoptotic response [ 825827 ] , contains a conserved mir-708 response element in its 3-utr .", "foxo3 can act as either an oncogene or a tumor suppressor in leukemia [ 828 , 829 ] .", "foxo3 transcriptional activity was found to prevent b - cll and cml proliferation [ 825 , 828 ] .", "foxo3a is also targeted by other micrornas , including mir-27a ( see section 2.2.6 ) .", "moreover , mir-27a directly regulates the drug - resistant factor p - glycoprotein , and overexpression of mir-27a increased sensitivity of leukemia cells to doxorubicin .", "mir-27a is relevant to treatment outcome in vivo and may be involved in relapse of both lymphocytic leukemia and myeloid leukemia .", "low expression of mir-27a might promote all relapse [ 655 , 658 ] . on the contrary ,", "mir-27a exerts oncogenic effects by regulating zbtb10 [ 446 , 830 ] and fbw7 [ 253 , 438 ] .", "mir-128b , which was higher in relapse all and at diagnosis compared to complete response , has been reported to confer drug resistance in many cancers including all [ 672 , 673 ] .", "both mir-27a and mir-128b might target bmi1 , a transcription factor of the polycomb - group gene necessary for hematopoietic stem cell ( hsc ) and leukemia stem - cell self - renewal [ 831 , 832 ] .", "deletion of bmi1 inhibits self - renewal of tumor stem cells and prevents leukemia recurrence .", "a role for mir-128 and mir-221 in regulating gc sensitivity in cells from mll - af4 all patients has been proposed . mir-128b and mir-221 are downregulated in mll - arranged all relative to other types of all . the mll gene is located at 11q23 , a site frequently involved in chromosomal translocations in aggressive human lymphoid and myeloid leukemias . as a result of chromosomal translocations ,", "a portion of mll becomes fused to one among more than 40 different partner proteins .", "mll - af4 all , which results from the translocation between mll and af4 , is associated with gc resistance and has a poor prognosis [ 834 , 835 ] .", "re - expression of mr-128 and mir-221 in cultured mll - af4 all cells sensitized them to gcs .", "mir-128 targets mll , af4 , and the mll - af4 pusion protein resulting in lower expression of hoxa9 , whereas mir-221 downregulates cdkn1b ( cyclin - dependent kinase inhibitor 1b , p27 ) , another gene transcriptionally activated by mll - af4 as well as the wild - type mll protein .", "the targeting of different proteins may explain the cooperative effect of mir-128b and mir-221 on gc sensitization .", "it should be noted that mir-221 in other settings , for example , cll , has anti - apoptotic effects and functions as an oncogene . in light of the multiple effects of various micrornas on cell survival and apoptosis", ", modulating microrna expression in tumor cells is an attractive approach for sensitizing the tumor cells to chemotherapeutic drugs .", "inhibition of specific micrornas is performed by using antisense sequences ( termed antagomirs ) targeting the microrna guide stand that blocks the interaction with the microrna recognition elements within the 3-utr of the target mrna genes . to increase their binding affinity and stability in biological fluids , the antagomirs", "are often modified with 2-o - methyl- , phosphorothioate , or locked nucleic acid substitutions . to overexpress micrornas ,", "one potential use of micrornas is to repress the expression of mll - af4 fusion protein in all that is responsible for gc resistance .", "this fusion protein can be repressed through overexpression of mir-143 , or mir-128 together with mir-221 .", "the latter combination was shown to sensitize the mll - af4-carrying all cells to gcs . ", "another promising approach is to target mir-155 , an oncogenic microrna often correlated with poor prognosis .", "they showed that overexpression of mir-155 in lymphoid tissues resulted in disseminatedlymphomacharacterized by a clonal , transplantable pre - b - cell population of neoplastic lymphocytes .", "systemic delivery of antisense peptide nucleic acids encapsulated in unique polymer nanoparticles inhibited mir-155 and slowed the growth of these pre - b - cell tumors in vivo .", "glucocorticoid - induced apoptosis appears to be a complex process involving several signaling pathways ( figure 6 ) .", "these include ( 1 ) transactivation of pro - apoptotic genes ( importantly bim ) ; ( 2 ) alterations in microrna expression ( upregulation of mir-15~16 that targets the pro - apoptotic bcl-2 ; mir-223 that targets igfr ; mir-150 that targets akt and notch , while suppressing mir-17~92 that prevents bim and pten translation ) ; ( 3 ) direct action of gr on the mitochondria ( including mitochondrial gr translocation and production of reactive oxygen species within the mitochondria ) ; ( 4 ) activation of the protein kinases gsk3 and p38 ; ( 5 ) activation of the foxo3a transcription factor that upregulates bim ; ( 6 ) inhibition of the notch1 , pi3/akt / mtor , and erk1/2 survival pathways .", "altered microrna expression in malignant cells may modulate many of these processes thereby imposing apoptotic resistance ( figures 16 ) .", "gc - resistant lymphoid cells might be divided into two major subgroups according to the underlying mechanism of resistance .", "the first group consists of cancer cells whose drug resistance can be overcome by exposing the cells to gcs in combination with drugs that target protein kinases such as akt , mtor , src , alk , and/or bcr , or drugs antagonizing bcl-2 , bcl - xl , mcl-1 , c - myc , or notch .", "these lymphoid malignancies show in general a more favorable response to combined gc therapy and in many cases may be explained by their growth dependency on these signaling molecules .", "the second group of gc - resistant cells exhibits an intrinsic defect in the gc - mediated apoptotic process and can thus not be turned sensitive to this drug .", "it is important to distinguish between these two subgroups prior to therapy initiation in order to choose the right drug combination .", "a diagnostic test needs to be developed that can distinguish between the different resistance backgrounds .", "recently , burnsides et al . have developed an ex vivo stimulation assay that determines the ability of leukocytes to upregulate anti - inflammatory genes such as gilz and fkbp51 following exposure to dexamethasone .", "it is reasonable that a similar test may be developed to gene profiling lymphoid malignancies prior to and following gc treatment , where upregulation of the pro - apoptotic bim gene would be a favorable predictor .", "also , bim induction may be measured after combining gc with a protein kinase inhibitor .", "simultaneous expression profiling of micrornas , notch1 , and bcl-2 family proteins together with the activated protein kinase status in the malignant cell would provide valuable information for choosing the proper drug combination . a predictor for a good gc response would be to determine the ability of gcs to downregulate mir-17~92 and upregulate mir-15~16 , mir-150 , and mir-223 .", "a tentative therapeutic approach would be to modulate the microrna status of the cell using microrna mimics or antagomirs as described in section 4.4 .", "what we have learned from the studies described in this paper is that it seems that in general it would be favorable to augment the expression of mir-29 , mir-27 , mir-15a~16 , mir-34a , mir-150 , and let-7 , while suppressing mir-155 , mir-181 , mir-182 , mir-21 , and mir-221/222 as well as mir-17~92 .", "obviously , an initial microrna profiling should be performed , and the cancer - type classification should be considered .", "while down - regulation of mir-181 may suppress the growth of t - all and mm , augmented mir-181 expression prevents the growth of unmutated igvh cll cases .", "also , mir-26a has a dual effect . its overexpression prevents growth of c - myc - positive burkitt lymphoma , while it must be downregulated in notch - positive t - all to achieve growth inhibition . mir-451 and mir-709 could prevent growth of notch - positive t - all . a reduction in mir-142 , and maybe also of mir-708 , which is highly expressed in relapsed childhood t - all , is anticipated to improve t - all therapy . for classical hl , mir-135a may cause apoptosis . in conclusion , in certain types of lymphoid malignancies", ", gc resistance may be overcome by relieving the inhibitory effects of protein kinases and bcl-2 family members .", "both the activity of protein kinases and the expression of bcl-2 members are affected by the microrna network .", "modulation of microrna expression might increase gc drug responsiveness and thus improve the therapy of lymphoid malignancies ." ]

[ "<S> the initial response of lymphoid malignancies to glucocorticoids ( gcs ) is a critical parameter predicting successful treatment . </S>", "<S> although being known as a strong inducer of apoptosis in lymphoid cells for almost a century , the signaling pathways regulating the susceptibility of the cells to gcs are only partly revealed . </S>", "<S> there is still a need to develop clinical tests that can predict the outcome of gc therapy . in this paper , </S>", "<S> i discuss important parameters modulating the pro - apoptotic effects of gcs , with a specific emphasis on the microrna world comprised of small players with big impacts . </S>", "<S> the journey through the multifaceted complexity of gc - induced apoptosis brings forth explanations for the differential treatment response and raises potential strategies for overcoming drug resistance . </S>" ]

[ "1. Introduction", "2. Parameters Affecting the Susceptibility of Lymphoid Malignancies to GC-Induced Apoptosis", "3. MicroRNA in Normal and Malignant ", "4. MicroRNA in Regulating GC-Induced ", "5. Summary" ]

[ [ "glucocorticoids ( gcs ) are among the most effective drugs used in the treatment of hematopoietic malignancies of the lymphoid lineage in virtue of their ability to induce apoptosis of these cancerous cells [ 13 ] .", "the main hematopoietic cancer types that respond well to gc therapy include t acute lymphoblastic leukemia ( t - all ) , chronic b lymphocytic leukemia ( cll ) , multiple myeloma ( mm ) , hodgkin 's lymphoma ( hl ) , and non - hodgkin 's lymphoma ( nhl ) .", "gcs appear , however , to have little value in the treatment of acute or chronic myeloid leukemia ( aml / cml ) .", "a major drawback of gc therapy is the gradual development of resistance to gc during treatment that limits the clinical utility of this drug .", "poor response to a 7-day monotherapy with the gc prednisone is one of the strongest predictors of adverse outcomes in the treatment of pediatric all [ 2 , 4 ] .", "a great challenge today is to develop strategies that can overcome the drug resistant phenotype . for this purpose", "it is important to understand the underlying mechanisms of gc resistance and the signaling pathways regulating apoptosis induced by gcs . besides inducing apoptosis of lymphoid cells , gcs", "gc treatment produces rapid symptomatic improvements , including relief of fever , sweats , lethargy , weakness , and other nonspecific effects of cancer.gcs decrease the severity of chemotherapy - induced emesis .", "gcs are also used in the clinics for other medical conditions such as autoimmune diseases , asthma , ulcerative colitis , chronic obstructive pulmonary disease , kidney diseases , and rheumatologic disorders due to their strong anti - inflammatory and immunosuppressive properties .", "gc therapy is hampered by a variety of metabolic and medical complications , including insulin resistance , diabetes , hypertension , glaucoma , osteoporosis , and osteonecrosis with increased risk of bone fractures [ 510 ] .", "diabetes may develop by direct gc - mediated induction of apoptosis in insulin - producing beta cells of the langerhans islets [ 1113 ] , and osteoporosis may develop due to apoptosis of osteoblasts [ 1416 ] .", "gcs also suppress cell growth and proliferation processes in the brain [ 17 , 18 ] . besides being used as monotherapy at high dosages , gcs", "are frequently combined with other chemotherapeutic drugs to achieve rapid and more efficient therapeutic effects . for the treatment of t - all ,", "gcs such as prednisone , methylprednisolone , and dexamethasone are usually used in combination with other chemotherapeutic drugs such as vincristine , daunorubicine , l - asparaginase , cytosine arabinoside , doxorubicin , and cyclophosphamide .", "this multidrug regimen prolongs remission , minimizes the long - term use of prednisone , and thus reduces the steroid - mediated adverse effects . ", "typical b - cell chronic lymphocytic leukemia ( cll ) in the early stage of progression responds well to combination chemotherapy including an alkylating agent ( such as chlorambucil ) plus or minus prednisolone.advanced stages of the disease often require the addition of an anthracycline and a vinca alkaloid for successful therapy .", "one commonly used combination is cyclophosphamide , doxorubicin , vincristine , and prednisolone , a drug combination termed chop .", "rituximab , a chimeric monoclonal antibody directed against the b - cell specific antigen cd20 , is often added to the therapy , which is here termed r - chop .", "rituximab is also combined with fludarabine and cyclophosphamide in the treatment of cll [ 19 , 20 ] .", "another antibody proved to be efficient against cll in combination with methylprednisolone is alemtuzumab , which targets cd52 .", "the immunomodulatory drug lenalidomide shows also good activity in relapse / refractory or treatment - nave cll [ 23 , 24 ] .", "chop is also used for non - hodgkin 's lymphomas and anaplastic large cell lymphoma ( alcl ) .", ", prednisone has been used in combination with carmustine , vincristine ( oncovin ) , procarbazine ( mopp ) , and rituximab .", "recently , brentuximab vedotin ( adcetris ) , an antibody directed towards cd30 conjugated with the anti - tubulin chemotherapeutic agent monomethyl auristatin e , has been approved for the treatment of hodgkin 's lymphoma and systemic anaplastic large cell lymphoma .", "cd30 expression is restricted to only a relative small population of activated t and b cells , and therefore this treatment is expected to be more selective for cd30-positive tumor cells .", "another monoclonal antibody entered the clinics is epratuzumab , which targets cd22 and is proved to be efficient in the treatment of adult non - hodgkin 's lymphoma as a single agent or in combination with chemotherapy . a phase ii clinical trial showed that combining epratuzumab with rituximab and chop ( er - chop )", "may have a favorable response on diffusing large b - cell non - hodgkin lymphoma ( dlbcl ) . ", "multiple myeloma ( mm ) has frequently been treated with vincristine , doxorubicine ( adriamycin ) , and dexamethasone ( vad ) or prednisone / melphalan .", "bortezomib ( velcade ) , lenalidomide , and to a lesser extend thalidomide have proven efficient in the treatment of mm in combination with dexamethasone .", "lenalidomide is a 4-amino - glutamyl analogue of thalidomide that lacks the neurological side effects of thalidomide and has emerged as a drug with activity against various hematological malignancies [ 27 , 28 ] .", "bortezomib is a selective inhibitor of the 26s proteasome that stabilizes many cell cycle - regulatory proteins .", "the antitumor effects of bortezomib in lymphoid tumors have been attributed to nfb inhibition through stabilization of its inhibitor ib .", "other tumors that have been treated with combination chemotherapy involving a gc include medulloblastoma , primitive neuroectodermal tumors , and ependymomas . one major obstacle in the therapy of lymphoid malignancies is the appearance of gc resistant cells .", "drug resistance may occur at the level of the glucocorticoid receptor ( gr ) or through alterations in downstream regulatory pathways . in", "most gc - resistant all primary biopsy specimens , gr was found to be functional , suggesting that pharmacological intervention may restore drug sensitivity .", "several strategies have been developed that aim to overcome drug resistance through specifically targeting anti - apoptotic pathways .", "gc resistance may occur due to overexpression of anti - apoptotic proteins of the bcl-2 superfamily [ 30 , 31 ] . among these ,", "small molecules that target the anti - apoptotic proteins of the bcl-2 family are attractive drugs that should be able to overcome gc resistance .", "one example is abt-737 , a bh3 mimetic that inhibits the pro - survival function of bcl-2 , bcl - xl , and bcl - w and induces apoptosis in a variety of cancer cell types including leukemias [ 3335 ] .", "treatment of the lymphoma - prone e-myc transgenic mice with abt-737 prevented the development of myc - driven lymphomagenesis , understating the need for these anti - apoptotic proteins .", "combined use of abt-737 and the dual specificity pi3k / mtor inhibitor pi-103 led to loss of c - myc expression and apoptosis of burkitt 's lymphoma cells , whose tumorigenicity is driven by overexpression of the c - myc gene . ", "the pro - apoptotic effect of abt-737 in cll depends on sufficient amount of bcl-2 that tonically sequesters the pro - apoptotic bim protein .", "also , the sensitivity of lymphoma cell lines to bcl-2 antagonism is directly related to the amount of bcl-2 primed with bim .", "the sequestration of bim may explain the marked chemosensitivity of cll and follicular lymphoma ( fl ) that express abundant bcl-2", "abt-737 potentiated the effect of vincristine , dexamethasone , and l - asparaginase ( vxl ) treatment on all cells and could potentiate the effect of the vxl combination in chemoresistant human primary all xenografts .", "this study also shows a synergistic effect between the three components of the vxl regimen .", "an additive effect was observed in primary mm cells when abt-737 was combined with dexamethasone [ 41 , 42 ] . ", "abt-263 ( navitoclax ) is a second generation , orally bioavailable small molecule bcl-2 family protein inhibitor that has entered clinical trials with promising efficacy on cll [ 4346 ] .", "abt-263 has been shown to have synergistic effects with r - chop treatment on mantle cell lymphoma .", "the pro - apoptotic bim that is displaced from bcl-2 by abt-737 , becomes captured by either bfl-1 or mcl-1", ". the resistance could be overcome by decreasing the mcl-1 level with the cyclin - dependent kinase ( cdk ) inhibitors flavopiridol and pha767491 , or by inhibiting mtor complex 1 ( mtorc1 ) or glycolysis [ 49 , 50 ] . another approach to overcome mcl-1-dependent resistance is to use the small molecule obatoclax ( gx15 - 070 ) that has entered clinical trials in the combined treatment of various hematopoietic neoplasms [ 5153 ] .", "obatoclax disrupts the interaction between mcl-1 and its pro - apoptotic counterparts including bak , bax , and noxa [ 54 , 55 ] .", "obatoclax and flavopiridol synergized in overcoming drug resistance in human myeloma cells through a mechanism involving bim and noxa .", "the multikinase inhibitor sorafenib could synergize with obatoclax in inducing apoptosis in acute myeloid leukemia ( aml ) through downregulating mcl-1 .", "obatoclax could overcome gc resistance in all through induction of apoptosis and autophagy , an effect that depends on the pro - apoptotic bak and to a certain extent also on beclin-1 [ 58 , 59 ] , a mammalian orthologue of yeast atg6 that plays a central role in autophagy .", "under certain conditions , cell death induced by obatoclax and gc may be executed in the absence of both bax and bak . under these conditions ,", "necroptosis ensues necroptosis ensues , a process mediated by rip-1 ( receptor - interacting protein-1 ) kinase and the cylindromatosis deubiquitinase cyld .", "it may promote either cell death or cell survival dependent on its ubiquitinated state , which is regulated by cyld and a20 , two nfb target genes .", "altogether , there is a general consensus that obatoclax might be a favorable drug that ought to be combined with dexamethasone / prednisone and/or rapamycin to overcome gc resistance in all cells and other hematological lymphoid malignancies . \n", "1.2.1.3 . overcoming bcl-2-mediated resistance with small molecular inhibitors of xiap ( x - linked inhibitor of apoptosis ) .", "bcl-2-mediated resistance in cll may also be overcome by small molecular inhibitors of the anti - apoptotic xiap ( x - linked inhibitor of apoptosis ) when exposed to trail [ 62 , 63 ] .", "xiap and the cellular ciaps 1 and 2 are expressed at high levels in cll cells [ 62 , 63 ] .", "xiap inhibitors enhanced bcl-2 cleavage and induced a conformational change in bax . similarly , xiap inhibitors sensitized all for cd95-induced apoptosis . in patients with t - all ,", "xiap inhibition using the low - molecular - weight smac mimetic lbw242 resulted in increased prednisone - induced apoptosis in vitro . ", "another anti - apoptotic protein that negatively regulates gc - induced apoptosis is notch1 [ 6668 ] .", "notch1 is indispensable for normal t - cell development [ 6971 ] and is an attractive target in the treatment of hematopoietic malignancies of the t lineage .", "mice transplanted with bone marrow cells transduced with a constitutively active form of notch1 develop t - cell neoplasms , while mice transgenic for constitutively active form of notch3 develop thymic lymphomas .", "acute lymphoblastic t - cell leukemia is frequently associated with increased notch signaling [ 7579 ] , which may be caused by the chromosomal translocation t(7 ; 9)(q34 ; q34.3 ) , gain - of - function mutations of notch1 , and/or mutations in fbw7 ( f - box and wd repeat domain - containing 7 ) , a negative regulator of notch1 . ", "one approach to avoid notch activation is to prevent its cleavage by the -secretase complex using -secretase inhibitors ( gsi ) .", "rather , gsi can enhance the pro - apoptotic effect of gcs and other chemotherapeutic agents including the mtor inhibitor rapamycin [ 84 , 88 ] .", "gsi does not overcome gc resistance in t - all deficient for pten [ 89 , 90 ] , supposedly due to elevated akt activity .", "the constitutive akt activation in the absence of pten leads to increased glucose metabolism and bypasses the requirement of notch signaling to sustain cell growth . in this context", "it should be noted that notch1 by itself may upregulate the p13k / akt pathway via its target gene hes1 .", "as pten is a target of several micrornas that are often expressed abnormally in cancer ( see section 2.4.2.3 ) , resistance to gsi may be far more prevalent .", "nevertheless , gsi compounds , such as pf-03084014 , have entered clinical trials for refractory t - all .", "preclinical data do show a synergistic effect between gsi inhibition and gc in reducing xenografted t - all tumor burden .", "another concern associated with the clinical use of gsis is severe toxicity to various organs at therapeutic doses , which may be explained by the broad action of notch1 as well as -secretase on various biological systems .", "the simultaneous use of gcs may prevent the gsi - induced gastrointestinal toxicity via inhibition of goblet cell metaplasia .", "a more specific inhibition of notch1 can be achieved by the sahm1 peptide that prevents notch - mediated transcription by interfering with the mastermind - notch interaction essential for notch - mediated transcription of target genes .", "the effect of this peptide on gc sensitivity awaits examination as well as its toxicity . since notch signaling", "is intertwined with the pi3k / akt / mtor signaling axis [ 9496 ] , the inhibition of the latter has proven to be more efficient in overcoming gc resistance ( see section 1.2.3 ) and would be a better therapeutic choice . accumulating data show that gc therapy can affect the activity of several protein kinases , and , vice versa , many protein kinases can affect gc - induced apoptosis [ 30 , 31 , 9799 ] .", "the mtor signaling pathway is frequently activated and found to be essential for cell growth and survival in lymphoid malignancies [ 100106 ] .", "gc resistance frequently appears in malignant cells due to aberrant activation of various protein kinases that exert anti - apoptotic effects [ 30 , 31 , 67 , 97 , 107109 ] .", "one strategy to overcome gc resistance would be to prevent the activities of the pi3k / akt / mtor , mek1/erk1/2 , and other activated protein kinase pathways .", "the mtor inhibitor rapamycin especially has proven efficient in sensitizing human gc - resistant t - all , b - all , mm , and npm - alk ( nucleophosmin - anaplastic lymphoma kinase)-dlbcl to gc - induced apoptosis [ 110117 ] .", "the combinatory therapy of rapamycin with dexamethasone was proven to be effective also in pten - negative cells . a lower dose of dexamethasone was sufficient for reducing t - all burden in a xenograft model when used together with rapamycin . one major drawback with rapamycin therapy", "is its immunosuppressive function , which adds to the immunosuppressive function of gcs . the dual pi3k / mtor inhibitor nvp - bez235 synergistically enhanced cytotoxicity of dexamethasone , doxorubicine , and cytosine arabinoside ( arac ) , even in gc - resistant all cells .", "the broad - acting protein kinase staurosporine was especially effective in overcoming gc resistance in mouse lymphomas that overexpressed notch-1 , bcl-2 , and/or bcl - xl .", "this sensitization was achieved through prevention of akt - mediated inhibition of gsk3 and induction of the pro - apoptotic nur77 . however , staurosporine was less effective on human t - all cell lines ( unpublished data ) , which could rather be sensitized to gc by rapamycin . in order to choose the right kinase inhibitor for combinatory therapy ,", "the cyclin - dependent kinase ( cdk ) inhibitors flavopiridol ( alvocidib ) , bms-387032 ( sns-032 ) , sunitinib , and sorafenib are currently under clinical trials for relapsed / refractory cll .", "these include vandetanib ( zd6474 ) , bosutinib ( ski-606 ) , tki258 ( chir-258 ) , pazopanib ( gw786034 ) , and axitinib ( ag013736 ) .", "chir-258 , a potent inhibitor of flt3 ( fms - like tyrosine kinase receptor-3 ) , c - kit tyrosine kinase , and fibroblast growth factor receptor 3 ( fgfr3 ) , prevented cell growth of fgfr3-positive human multiple myeloma cell lines and augmented their sensitivity to gc - induced apoptosis .", "importantly , neither interleukin-6 ( il-6 ) nor stromal cells conferred resistance to chir-258 . other protein kinase inhibitors with more cell - type specific effects have been developed , which are expected to have less adverse effects . the classical example for efficient use of a specific protein kinase inhibitor in the clinics is the bcr - abl kinase inhibitor sti-572 ( imatinib ) used for the treatment of chronic myelogenic leukemia ( cml ) . a similar strong response of a single agent", "was observed in alk - anaplastic large cell lymphoma ( alcl ) patients treated with crizotinib , an inhibitor of the alk tyrosine kinase .", "another promising target is the b - cell receptor ( bcr ) signaling , which is important during b - cell oncogenesis and is a key to the survival of malignant b cells , including cll and dlbcl [ 125 , 126 ] .", "the survival of dlbcl may depend on the nonligand - dependent ( tonic ) signals from the bcr .", "the bcr signaling can be targeted with small molecular inhibitors directed against bruton 's tyrosine kinase ( btk ) , spleen tyrosine kinase ( syk ) , or phosphoinositide 3-kinase ( pi3k ) isoform p110 ( pi3k ) , all being efficient in the treatment of cll . targeting btk with the inhibitor pci-32765 leads to disruption of bcr signaling and was effective in a preclinical model of b cell non - hodgin 's lymphoma [ 127 , 128 ] .", "pci-32765 seems also to be promising for the treatment of cll [ 128131 ] and mm .", "importantly , pci-32765 induced apoptosis in cll cells even in the presence of various exogenous stimuli , including cd40l , baff , il-6 , and il-4 and when cultivated together with stromal cells .", "two other btk inhibitors , ibrutinib and avl-263 , are also under investigation for cll .", "the syk ( spleen tyrosine kinase ) inhibitor fostamatinib had clinical activity in non - hodgkin lymphoma and cll .", "syk is a cytoplasmic tyrosine kinase that is important for immunoreceptor signaling in b cells .", "syk has also been shown to be critical for the survival and maintenance of mature normal and malignant b cells [ 125 , 134 ] and is frequently expressed at high levels in follicular lymphoma .", "the pi3k inhibitor gs-1101 ( cal-101 ) had preclinical and clinical activity against cll , mantle cell lymphoma , and mm [ 121 , 129 , 136138 ] . while the pi3k and isoforms are ubiquitously expressed , pi3k expression is largely restricted to hematopoietic cells , where it plays a role in b - cell homeostasis and function .", "the effect of the btk , syk , and pi3k kinase inhibitors on the sensitivity to gcs warrants investigations . ", "accordi et al . found aberrant activation of protein kinases in poor prognosis pediatric b - cell precursor - all patients .", "the p56 ( lymphocyte cell - specific tyrosine kinase ) activity was enhanced in patients with poor clinical response to prednisone with respect to those with good response .", "p56 is a nonreceptor tyrosine kinase of the src oncogene family mostly expressed in t cells where it plays an essential role in activation and development , and in some b cells .", "its activity is negatively regulated by the membrane - bound tyrosine kinase csk ( c - src tyrosine kinase ) .", "the p56 inhibitor dasatinib ( bms-354825 ) was shown to enhance apoptosis induction by dexamethasone in otherwise gc - resistant cll cells .", "this finding concurs with the observation by sade et al . showing that notch - mediated resistance of a mouse lymphoma cell line", "a synergistic effect was observed between the aurora a kinase inhibitor mnl8237 ( alisertib ) and dexamethasone . ", "ampk ( amp activated protein kinase ) activation has a dual effect on cell death and survival , which contextually depends on signaling alterations with related oncogenic pathways .", "however , in all and cll , activation of ampk by aicar ( 5-aminoimidazole-4-carboxamide riboside or acadesine ) , a cell - permeable nucleotide , induces growth inhibition and apoptosis [ 146148 ] .", "however , aicar prevented glucocorticoid - induced apoptosis and thus can not be combined with steroids in the treatment of lymphoid malignancies . of note , inhibition of either bcl-2 family members , notch1 , or", "the akt / mtor survival pathways was independently sufficient for sensitizing resistant cells to gc , suggesting a tight crosstalk between these pathways , interruption of one of them being sufficient for abrogating the resistant phenotype .", "however , it is likely that using a combination of these three strategies together with gc should lead to a more efficient therapy , which may require lower dosages with reduced adverse effects ." ], [ "in order to develop strategies to overcome gc resistance , it is essential to understand the signaling network regulating gc - induced apoptosis .", "main factors affecting the response to gc include the basal and inducible gr expression levels , the induction of and basal expression of genes involved in the intrinsic apoptotic pathway , the ability of gr to translocate to the mitochondria , the activity of gsk3 ( glycogen synthase kinase 3 ) , the general protein kinase activation profile of the cell prior to and following gc therapy , the expression profile of anti - apoptotic proteins , and the activities of pro - survival signaling pathways .", "the main traits will only be briefly described here as these have been extensively reviewed elsewhere [ 30 , 31 , 99 , 150153 ] , and the scope of this paper is to provide updated data with a specific focus on the microrna world that has emerged to comprise important regulators of most biological processes .", "numerous factors have been shown to affect gc responsiveness by regulating glucocorticoid receptor ( gr ) activity and expression level .", "these include gr co - activators and corepressors [ 154 , 155 ] , gr splice variants [ 156159 ] , gr isoforms [ 160 , 161 ] , and regulators of gc nucleocytoplasmic shuttle [ 162164 ] . ", "the transcription of human gr is regulated by at least 11 different promoters ( 1a1 , 1a2 , 1a3 , 1b , 1c , 1d , 1e , 1f , 1h , 1i , and 1j ) [ 155 , 165 ] , seven of them being embedded in a highly enriched cpg island region subjected to methylation and harbor single nucleotide polymorphisms ( snps ) that affect their activity .", "promoter 1a is involved in the upregulation of gr by gc in some kinds of t cells , while downregulated in other cell types [ 167169 ] .", "gc resistance in primary pediatric t- and b - all could not be correlated with either basal or stimulated expression of the 1a- , 1b , or 1c transcripts . ", "the gr expression level prior and following gc therapy affects drug responsiveness . the cellular response", "to gcs depends on sufficient gr expression [ 30 , 171179 ] , and resistance to gc therapy has been associated with downregulation and loss of gr expression in malignant plasma cells [ 180 , 181 ] .", "however , most primary all cells showed upregulation of gr expression upon prednisolone treatment regardless of their phenotype or sensitivity to gc - induced apoptosis , suggesting that other factors are more dominant for conferring a gc - resistant phenotype in these cells [ 29 , 170 , 182184 ] .", "many glucocorticoid - regulated genes ( e.g. , fkbp5 and socs1 ) were upregulated by dexamethasone in all primary all xenografts tested , suggesting for a functional gr in these leukemic cells .", "also , beesley et al . observed that receptor mutation is not a common mechanism of gc resistant in primary all .", "however , the minor c allele of rs10482605 ( 1c ) has been associated with a higher complication rate in childhood all .", "a bcli polymorphism in the nr3c1 gene was associated with increased lymphocyte response to methylprednisolone .", "also , initial good responder cells may develop resistance upon repeated gc dosages , a phenomenon that sometimes occurs due to downregulation of gr [ 156 , 179 , 188 , 189 ] .", "posttranslational modifications of gr are another way of regulating its target gene specificity and involve several cell - signaling cascades [ 30 , 190 , 191 ] .", "gr can be phosphorylated at ser211 by cdks and p38 map kinase , and at ser226 by jnk .", "phosphorylation of gr modulates its transcriptional activity , alters its protein stability and subcellular location [ 192195 ] .", "gr phosphorylation appears to be cell - cycle dependent [ 196 , 197 ] and may affect gc - sensitivity of t - all cells [ 98 , 195 ] .", ", gr is mostly located to the cytosol sequestered to heat - shock protein complexes [ 30 , 162 ] .", "following gc binding to gr , the receptor undergoes phosphorylation , dissociates from the heat - shock complexes , dimerizes , and translocates to the nucleus where it either promotes or represses a whole series of genes .", "transcriptional activation is either directly mediated by binding of gr to glucocorticoid response elements ( gres ) , or through interaction with other transcription factors such as forkhead transcription factors , thereby increasing their transcriptional activity on target genes .", "gr may repress gene expression either through binding to negative gres ( ngres ) or through interaction with and inhibition of the transcription factors activating protein-1 ( ap-1 ) and nfb .", "the o - glcnac transferase ( ogt ) was found to be involved in gc - mediated transrepression .", "hundreds of genes are regulated by gcs [ 199203 ] , and some genes are differentially regulated in gc - sensitive versus gc - resistant cells [ 29 , 199 , 204 ] . ", "of special importance is the induction of the pro - apoptotic bim ( bh3-only b - cell lymphoma 2 ( bcl-2 ) interacting mediator of cell death ; or bcl2l11bcl-2-like apoptosis initiator-11 ) for achieving the propensity to undergo apoptosis in response to gc [ 29 , 30 , 67 , 205208 ] .", "the central role of bim in gc - induced apoptosis is understated by the partial gc response of bim thymocytes , and gc resistance of lymphoma cells after knocking down bim [ 67 , 207 ] .", "bim is often expressed at high basal levels in lymphoid cells [ 30 , 120 , 209 , 210 ] , and in these cells there is no further need for upregulating bim in order to achieve an apoptotic response to gcs [ 30 , 59 ] . however , in several t - all and b - all cells , an upregulation of bim in response to gcs is an absolute must , especially when the basal level is low .", "bim was shown to be upregulated in gc - sensitive primary t - all samples , but not in resistant ones [ 29 , 182 ] . also , a comparison of established t - all cell lines , bim was upregulated in the sensitive ones only . when sufficient bim expression can not be achieved , gc resistance pursued .", "a significantly lower bim expression was detected in high risk childhood all patients who exhibited slow early response to a standard 4-drug induction regimen compared with patients who responded rapidly .", "homozygous deletion of bim has been seen in many mantle cell lymphomas and silencing of bim by promoter methylation and mutation is common in b - cell lymphomas . however , in pediatric all , no correlation between bim cpg methylation and gc resistance was found . rather , gc resistance in primary pediatric all samples correlated with decreased histone h3 acetylation .", "the histone deacetylase inhibitor vorinostat relieved bim repression and exerted synergistic antileukemic efficacy with dexamethasone both in vitro and in vivo using a xenograft model .", "bim has been shown to be a prognostic biomarker for early prednisolone response in pediatric all .", "bim is a potent pro - apoptotic protein belonging to the bcl-2 protein family [ 215 , 216 ] .", "bim binds to the pro - survival proteins bcl-2 , bcl - xl , and mcl-1 , thereby allowing bax and bak to promote apoptosis .", "bim may also directly bind to bax and bak , triggering a conformational change required for their subsequent oligomerization on the mitochondrial outer membrane .", "bim appears in various alternative splice variants , which exhibit different intrinsic toxicities and modes of regulation . in gc - resistant primary cll", ", bim was upregulated by dexamethasone , but failed to activate bax and bak due to exclusive sequestration to bcl-2 .", "bim may cooperate with the pro - apoptotic puma ( p53 upregulated modulator of apoptosis ) in mediating apoptosis induced by dexamethasone . in b - lymphoid cells ,", "other pro - apoptotic members of the bcl-2 family that is not directly upregulated by gcs , but may contribute to the cell death response , include bid , bad , and noxa .", "also the thioredoxin - interacting protein txnip ( vdup1/tbp-2 ) has been shown to be upregulated by gc and could contribute to gc - induced apoptosis in one mouse lymphoma cell line . during gc monotherapy of childhood all , gc was found to repress the expression of the pro - apoptotic pmaip / noxa , which could be one mechanism leading to impaired gc sensitivity .", "bam , is also induced by gcs in all cells , but its importance in gc - induced apoptosis is still not defined .", "bim expression is tightly regulated both at the transcription and posttranscriptional levels [ 215 , 218 ] ( figure 1 ) .", "rather , gc - induced bim expression in lymphoid cells requires p38 activation and is mediated by the forkhead transcription factor foxo3a / fkhr - l1 .", "foxo3a has also been shown to promote bim transcription in various other cellular systems [ 227229 ] and may cooperate with runx1 ( runt - related transcription factor 1 ) .", "differential recruitment of foxo3a to the bim promoter was observed after dexamethasone treatment of gc - sensitive versus gc - resistant childhood all xenografts .", "foxo3 was found to be an immediate early gr target , whose transcription is further enhanced by stimuli that activate the amp - activated protein kinase ampk .", "the activity of foxo transcription factors is tightly regulated , inhibited by akt and erk signaling , while promoted by p38 signaling [ 232236 ] .", "the ribosomal protein s6 kinase ( rsk ) activated downstream of erk1/2 , phosphorylates bimel , providing a binding site for the f - box proteins beta - transducin repeat containing protein ( trcp)1 and trcp2 , which promote the polyubiquitination of bimel .", "the erk1/2-mediated phosphorylation of bimel at ser69 facilitates optimal phosphorylation by rsk at ser93 , ser94 , and ser98 and this motif serves as the binding sites for trcp1/2 . while erk1/2 lowers the affinity of bim for mcl-1 and bcl - xl and targets bim for degradation , phosphorylation of bim by jnk increases the pro - apoptotic activity of bim [ 240 , 241 ] .", "gcs may repress erk1/2 activity through upregulation of mitogen - activated protein kinase phosphatase 1 ( mkp-1 ) .", "several drugs that inhibit the erk1/2 and pkb / akt pathways may facilitate upregulation of bim expression .", "additional signals are required , such as simultaneous inhibition of the pkb / akt pathway or the downstream mammalian target of rapamycin ( mtor ) kinase .", "apoptosis may be induced in a variety of all cells when cotreated with dexamethasone and a mek / erk inhibitor or an akt inhibitor [ 67 , 108 , 243 ] .", "early studies by the thompson research group noticed that c - jun played a role in gc - induced apoptosis .", "an increase in c - jun was observed in gc - sensitive , but not gc - resistant t - all cell lines , while c - fos and jund were unaffected by the steroid .", "chen et al . reconfirmed that c - jun was upregulated by gcs in gc - sensitive , but not gc - resistant all cells .", "they further showed that c - jun is recruited to the ap-1 site of the bim promoter upon gc treatment .", "another study showed that dexamethasone - induced bim expression was decreased in cells harboring a dominant - negative c - jun , suggesting a role for c - jun in the upregulation of bim .", "a p38 inhibitor prevented dexamethasone - induced expression of runx2 , c - jun , and bim , suggesting that p38-mapk activation acts upstream to the induction of these three molecules . another level of bim regulation is through micrornas .", "bim transcription is repressed by the mir-17~92 microrna cluster , which , in turn , is repressed by gcs .", "thus , one mechanism by which gcs upregulate bim is through repression of mir-17~92 . of note , the mir-17~92 cluster is often overexpressed or amplified in human cancers [ 247252 ] , thereby preventing the upregulation of bim required for an apoptotic response .", "another microrna that suppresses bim expression is mir-26a , which is frequently upregulated in t - all patients . in gastric cancer , mir-106a~363 targets bim .", "the mir-106a~363 cluster located at chromosome xq26.2 is the paralogue of mir-17~92 and encodes for mir-363 , mir-106a , and mir-20b . in hepatocellular carcinoma ,", "mir-25 of the mir-106b~25 cluster targets bim . also , the mir-106b~25 cluster , which includes mir-106b , mir-93 and mir-25 , is a paralogue of the mir-17~92 cluster and located on chromosome 7 within the thirteenth intron of the protein - coding gene mcm7 . also , the foxo transcription factors , important for bim upregulation , are regulated by micrornas ( figure 2 ) .", "foxo1 and foxo3 transcripts might be targeted by mir-182 [ 258261 ] , mir-1 , mir-27a , mir-96 , and mir-155 [ 263 , 264 ] .", "mir-155 plays a role in the activation and function of b and t lymphocytes [ 265 , 266 ] ( see section 3.1.6 ) .", "mir-182 expression was higher in gc - resistant cells in comparison to gc sensitive ones .", "increased expression of mir-182 reduced total foxo3a expression in t - all cells with consequent lower bim expression . foxo3a and bim increased upon downregulation of mir-182 , suggesting that mir-182 is involved in conferring gc resistance .", "the expression of the mir-182~96~183 cluster was induced in splenocytes from mouse with experimental systemic lupus erythematosus ( sle ) , suggesting a role of these micrornas in the breakdown of immunological tolerance and the manifestation of chronic autoimmune inflammation .", "this microrna cluster was also upregulated upon t - cell activation by an il-2-dependent manner .", "prevention of the expression of the mir-182~96~183 cluster led to increased foxo1 expression and limited population expansion of activated t - helper cells , due to increased cell death .", "vice versa , foxo3a was found to negatively regulate the oncomir mir-21 , which may be one mechanism by which foxo3a regulates apoptosis .", "as mir-21 targets pten [ 269 , 270 ] , activation of foxo3 by gcs may be one mechanism responsible for the gc - induced reduction in akt activity . besides function as a transcription factor in the nucleus , gr was found to translocate to the mitochondria in gc - sensitive , but not gc - resistant , lymphoma cell lines .", "gr was also found to translocate to the mitochondria in gc - sensitive thymocytes [ 272 , 273 ] .", "although there is one paper describing an interaction between gr and bcl-2 in the mitochondria , gc - induced mitochondrial gr translocation in gc - sensitive thymocytes and lymphoma cells proceeded in the absence of bcl-2 .", "exclusive overexpression of gr in the mitochondria was sufficient for inducing apoptosis , suggesting that mitochondrial gr may contribute to gc - induced apoptosis .", "glucocorticoid treatment inhibited complex i and complex iii of the electron transport chain , and the mitochondria was found to be the primary source of h2o2 production required for gc - induced apoptosis of lymphoma cells [ 275 , 276 ] .", "gcs may interact with the mitochondrial thioredoxin trx2 , a redox regulator , and directly modulate mitochondrial gene transcription .", "several mitochondrial metabolite and protein transporters and two subunits of the atp synthase were downregulated in t - all and precursor b - all cells at the gene expression level by dexamethasone .", "these changes were observed in gc - sensitive , but not gc - resistant , cells .", "corticosterone and other steroids were found to directly act on mitochondria to inhibit mitochondrial atp production by suppressing electron transfer from nadh to the electron transfer chain through complex i .", "the cellular protein kinase network ( kinome ) has critical influence on the gc sensitivity of lymphoid cells [ 30 , 31 , 97 , 281 ] .", "below , i will provide data supporting an involvement of gsk3 ( glycogen synthase kinase 3 ) in gc - induced apoptosis , and the antagonism of its activity by protein kinases such as akt and mtor , which leads to gc resistance . ", "the activity of gsk3 was found to be essential for gc - induced apoptosis [ 67 , 282 ] .", "gsk3 inhibitors prevented gc - induced apoptosis , and gc resistance frequently occurs through inhibition of gsk activity . reactivating gsk3 by using inhibitors of the pi3k - akt or mtor pathways sensitized gc - resistant cells to gc - induced apoptosis [ 67 , 108 , 115 , 116 , 243 , 283 ] .", "gsk3 was found to interact with gr in the absence of ligand and released from gr following exposure to gc .", "gsk3 also regulates gr transcriptional activity of bim , iap1 ( inhibitor of apoptosis 1 ) , and gilz ( glucocorticoid - induced leucine zipper ) [ 282 , 284 ] .", "the pi3k / akt and mtor signaling pathways are frequently hyperactivated in gc - resistant t - all [ 104 , 286 , 287 ] and is associated with poor prognosis and chemotherapeutic resistance in pediatric b - precursor all .", "mtor is a crucial regulator of cell metabolism , growth , and proliferation and mtor is positively regulated by pi3k / akt and notch1 [ 96 , 289 ] , while negatively regulated by the tuberous sclerosis tumor suppressor complex ( tsc1/tsc2 ) .", "activation of mtor contributes to tumor cell survival in alk ( anaplastic lymphoma kinase)-positive alcl ( anaplastic large cell lymphoma ) , mantle cell lymphoma , childhood b - precursor all , t - all , and aml .", "akt and mtor confer drug resistance by phosphorylating a series of targets [ 292 , 293 ] .", "phosphorylation and inactivation of gsk3 is a major cause for gc resistance that can be overcome by reactivating gsk3 , for example , by akt inhibitors or mtor inhibitors .", "as mentioned in section 1.2.3 , the mtor inhibitor rapamycin is efficient in overcoming gc resistance in various lymphoid malignancies .", "gc resistance can also be overcome in akt - active lymphoma cells by inhibiting src members ( e.g. , by pp1 ) , pi3k ( e.g. , wortmannin ) , or an akt inhibitor [ 67 , 68 ] .", "combination of gc with rapamycin or gc with obatoclax led to reduced akt phosphorylation at ser473 , suggesting that mtor may also act upstream to akt .", "gcs could also independent of other cytotoxic agents reduce mtor activity in lymphoid cells . low - dose arsenic trioxide could sensitize gc - resistant all to dex through an akt - dependent pathway .", "inhibition of mtor with rapamycin , which binds to fkbp12 , leads to increased bim expression and overcomes ras - dependent survival signals .", "synergy between mtor inhibitors ( e.g. , rapamycin ( sirolimus ) and cci-779 ( temsirolimus ) ) and other chemotherapeutic agents has been observed in b- and t - lineage all cell lines and preclinical models [ 96 , 298 ] . \n", "the akt activity is negatively regulated by pten ( phosphatase and tensin homolog deleted on chromosome 10 ) , a tumor suppressor gene that is suppressed , mutated , or deleted at high frequency in a large number of cancers .", "pten mutations or deletions are frequent in t - all and pten deletions are associated with less favorable outcome in t - all [ 104 , 300 ] .", "for instance , treatment of t - all with gamma secretase inhibitor ( gsi ) was only efficient if the cells expressed functional pten .", "one mechanism by which notch confers gc resistance is through pten inhibition leading to akt activation .", "pten specifically catalyzes the dephosphorylation of 3-phosphate of the inositol ring in phosphatidylinositol ( 3,4,5)-triphosphate ( pip3 ) resulting in the biphosphate product phosphatidyl ( 4,5)-biphosphate ( pip2 ) .", "pip3 is a second messenger generated by pi3k that binds to the pleckstrin homology ( ph ) domain of akt , which allows its phosphorylation and activation by the 3-phosphoinositide - dependent protein kinase 1 ( pdk1 ) . \n", "regulation of pten stability by phosphorylation and ubiquitination . taken into account the important role of pten in determining drug sensitivity ,", "pten expression has been attributed to epigenetic events such as promoter methylation [ 302 , 303 ] . at the posttranslational level , phosphorylation and ubiquitination decrease pten protein levels , while oxidation and acetylation reduce pten activity .", "rak phosphorylation of pten at tyr336 stabilizes the pten protein , while phosphorylation at thr366 , ser370 , ser380 , thr382 , and ser385 by casein kinase 2 ( ck2 ) and gsk3 reduces its stability [ 305 , 306 ] .", "pten is regulated by the protooncogene ubiquitin ligase nedd4 - 1 ( neural precursor cell expressed , developmentally downregulated 4 ) that promotes pten for proteasomal degradation .", "in multiple human cancer samples where the genetic background of pten was normal , but its protein level was low , nedd4 - 1 was highly expressed . upon tcr / cd28 stimulation of t cells , pten undergoes inactivation by nedd4 - 1 .", "the association between pten and nedd4 could be impeded by the e3 ubiquitin ligase cbl - b ( casitas - b - lineage lymphoma protein - b ) .", "cbl - b t cells show elevated akt activity , which was abrogated by simultaneous deficiency in nedd4 .", "pten is also negatively regulated by the anti - apoptotic xiap ( x - linked inhibitor of apoptosis ) that promotes pten for polyubiquitination and proteosomal degradation .", "induction of apoptosis in b - cll by arsenic trioxide was shown to lead to activation of c - jun - nh2 terminal kinase ( jnk ) , inactivation of akt and nfb , xiap downregulation , and pten upregulation .", "two other e3 ligases downregulating pten include wwp2 ( ww - domain containing protein-2 or aip-2 , atrophin-1-interacting protein 2 ) , and chip ( chaperone - associated e3 ligase c terminus of hsc70-interacting protein ) .", "pten expression can also be repressed by a range of micrornas including the mir-17~92 cluster [ 247 , 248 ] , mir-106b~25 , mir-21 , mir-26a [ 253 , 315 ] , mir-29b , mir-214 [ 317 , 318 ] , mir-216a and mir-217 , mir-212 , mir-221 , and mir-222 ( figure 3 ) .", "bcl-2 and bcl - xl are anti - apoptotic proteins residing in the mitochondrial outer membrane and in the endoplasmic reticulum .", "they prevent apoptosis of various chemotherapeutic drugs including gcs by capturing pro - apoptotic members of the bcl-2 superfamily , including bim , bax , and bak [ 215 , 322 , 323 ] .", "bcl-2 may also regulate gene expression [ 324 , 325 ] , cell cycle [ 326328 ] , activate erk1/2 [ 324 , 329 ] , and modulate the activities of transcription factors such as p53 , e2f , nfb , and notch [ 332 , 333 ] .", "this may be explained by the ability of bcl-2 to modulate p27 expression and promote g0 arrest [ 325 , 327 , 331 , 335 , 336 ] .", "long - term exposure to gcs could overcome resistance caused by either bcl-2 or bcl - xl [ 30 , 120 , 337 ] .", "overexpression of bcl-2 is common in leukemias and lymphomas [ 338341 ] . in follicular lymphoma ( fl ) and", "diffuse large b - cell lymphoma ( dlbcl ) , bcl-2 upregulation is commonly due to the t(14,18)(q32 ; q21 ) translocation , which places the bcl-2 gene under the control of ig heavy chain enhancers [ 342344 ] . \n", "overexpression of bcl-2 is common in cll due to the loss or downregulation of the human chromosome 13q14 locus , which harbors the mir-15a and mir-16 - 1 cluster .", "overexpression of mir-15a and mir-16 - 1 in cll cells led to cleavage of procaspase-9 and parp ( poly - adp - ribose polymerase ) and activation of the intrinsic apoptosis pathway .", "these two micrornas could serve as natural antisense bcl-2 actors that have potential use in the therapy of bcl-2 overexpressing tumors .", "the tumor - suppressor mir-34a , a pivotal member of the p53 network , also downregulates bcl-2 [ 347 , 348 ] , which may be one mechanism by which p53 activation leads to downregulation of bcl-2 .", "it also targets mcl-1 and bcl - w , and indirectly bcl - xl by attenuating il-6/stat-3 ( signal transducer and activator of transcription 3 ) signaling pathway [ 350 , 352 ] .", "mir-125b may function both as tumor suppressor and as an oncogene and has been widely considered as conferring drug resistance , among others by downregulating bak1 ( bcl-2 antagonist killer 1 ) [ 353355 ] and bmf . over - expression of mir-125b could induce leukemia in a mouse model .", "mir-181a / b that shows altered expression in cll could also target bcl-2 , besides acting on mcl-1 and xiap [ 358360 ] .", "bcl - xl can be targeted by the tumor suppressor microrna let-7 and mir-491 .", "a predominant feature of the gene expression signature leading to gc resistance in all was found to be elevated expression of the anti - apoptotic mcl-1 ( myeloid cell leukemia sequence 1 ) [ 364 , 365 ] .", "mcl-1 expression is especially high in mll - rearranged all , which represents an unfavorable type of leukemia that is often highly resistant to gcs .", "mcl-1 is also frequently overexpressed in b - cell and mantle - cell lymphomas , cml , cll , and mm .", "mcl-1 is an anti - apoptotic protein that sequesters the pro - apoptotic proteins tbid , bim , puma , noxa , and bak . besides preventing gc - induced apoptosis", ", mcl-1 confers resistance to trail ( tumor necrosis factor - related apoptosis inducing ligand)-induced cell death . \n", "mcl-1 differs from bcl-2 and bcl - xl in having a short protein turnover regulated by the 26s proteasome and its expression is tightly regulated . unlike bcl-2 ,", "rapamycin , a mtor inhibitor that sensitizes resistant all cells to gc , reduces the expression level of mcl-1 [ 113 , 287 ] .", "the degradation of mcl-1 depends on gsk3-mediated phosphorylation of mcl-1 at ser159 [ 369 , 370 ] .", "e3 ubiquitin ligases implicated in the regulation of mcl-1 include mule ( mcl-1-ubiquitinase ligase e3 ) , scf ( skp1/cul1/f - box protein -transducin repeat - containing protein ) , and fbw7 ( f - box and wd repeat domain - containing 7 ) which is part of the skp1-cullin1-f - box ( scf ) e3 ligase complex . the deubiquitinase usp9x ( ubiquitin specific peptidase 9 x - linked ) is an important regulator of mcl-1 stability . silencing of usp9x resulted in loss of mcl-1 .", "high levels of mcl-1 correlated with elevated usp9x expression in follicular lymphoma , diffuse large b - cell lymphoma , and some other cancer samples . increased expression of usp9x mrna was associated with poor prognosis of multiple myeloma .", "overexpression of noxa triggered an increase in the mule / mcl-1 interaction in parallel to a decrease in mule / usp9x complex formation . in an", "akt - driven , e-myc lymphoma mouse model , translational regulation of mcl-1 by mtor has been implicated in promoting lymphomagenesis .", "as gc may activate gsk3 and gsk3 inhibits mtor through phosphorylation of tsc2 and promotes mcl-1 degradation [ 369 , 370 ] , mcl-1 expressing lymphoid cells may ultimately undergo apoptosis if the exposure time to gc is sufficiently long .", "this may explain why many mcl-1-positive all cells exhibit delayed response to gcs , and not complete resistance [ 67 , 108 ] .", "also , the anti - apoptotic function of mcl-1 appears to require simultaneous expression of other anti - apoptotic bcl-2 family members .", "similarly , overexpression of mcl-1 in bcl-2- and bcl - xl - negative mouse double positive thymic lymphoma cells did not confer gc resistance upon these cells .", "usually , mcl-1 is expressed together with other anti - apoptotic proteins in gc - resistant lymphoid malignancies . \n", "mcl-1 is also regulated by micrornas ( figure 2 ) , including mir-29a , mir-29b [ 381383 ] , mir-101 , mir-125b , mir-181a / b [ 358 , 385 ] , mir-133b , mir-193b , and mir-512 .", "alk - positive anaplastic large cell lymphomas ( alcl ) express low levels of mir-29a , whose downregulation requires an active npm - alk kinase , and may probably also be due to methylation repression . enforced mir-29a expression reduced mcl-1 expression in alcl cells and reduced tumor growth in a xenografted model .", "mir-29b is downregulated in primary mm and aml samples and forced overexpression of mir-29b - induced apoptosis in mm and aml cells [ 381 , 383 ] .", "mir-29b overexpression also downregulated the expression of the dna methyltransferase isoforms dnmt1 , dnmt3a , and 3b .", "the global dna hypomethylation induced by mir-29b led to reexpression of tumor suppressor genes such as the cdk inhibitor p15 .", "altogether , these data propose that targeting mcl-1 with micrornas such as mir-29 represents a potential tool to constrict tumor growth of mcl-1 positive lymphomas .", "gcs release ca from the endoplasmic reticulum into the cytosol , which in turn increases the amount of mitochondrial ca .", "elevated expression of calcium - binding proteins s100a8 and s100a9 and of the anti - apoptotic mcl-1 ( myeloid cell leukemia-1 ) inhibits the free cytosolic ca and mitochondrial ca signals , respectively , thereby imposing gc resistance [ 287 , 365 , 389 , 390 ] .", "downregulation of s100a8 and s100a9 by the src kinase inhibitor pp2 sensitized mll - arranged all cells otherwise resistant to prednisolone - induced cell death .", "bcl-2 inhibits apoptosis in part by decreasing the size of ca stores in the endoplasmic reticulum resulting in reduced ca transfer to the mitochondria [ 391393 ] .", "one mechanism is through interaction of bcl-2 with ip3r ( inositol 1,4,5-triphosphate ( insp3 ) receptor ) , which is the principle er ca release channel in most cell types .", "also , bcl - xl and mcl-1 act in part by inhibiting ip3r [ 393 , 395 , 396 ] .", "an increase in hydrogen peroxide ( h2o2 ) is a necessary signal for gc - induced apoptosis .", "the mitochondria is the source of this signal , gcs inhibit complex i and complex iii of the electron transport chain .", "expression of anti - oxidant defense proteins such as manganese superoxide dismutase , thioredoxin , and catalase prevents gc - induced apoptosis [ 276 , 398400 ] .", "the anti - apoptotic bcl-2 may regulate the mitochondrial redox state in cancer cells [ 323 , 401 ] .", "notch is frequently activated in t - all cells , which may be due to mutations in notch1 ( gain - of - function ) and/or in the e3 ligase fbw7 that targets notch1 for degradation [ 7678 , 80 , 81 , 402405 ] .", "for example lnx1 ( ligand of numb - protein x1 ) is a positive regulator of notch signaling through degradation of numb , a membrane - associated protein that inhibits the function of the notch receptor .", "neuralized ( neur ) and mind bomb ( mib ) promote the monoubiquitination and endocytosis of delta [ 409 , 410 ] .", "itch binds to the n - terminal portion of the notch intracellular domain via its ww domains and promotes ubiquitination of icn - notch1 through its hect ubiquitin ligase domain .", "recent studies showed that notch1 can be activated in leukemic cells through interaction with bone marrow stromal cells that express notch receptors and ligands [ 412 , 413 ] .", "interaction with bone marrow stroma is also a mechanism for notch activation in multiple myeloma .", "cyclin e , which is targeted for degradation by fbw7 [ 415 , 416 ] , is expressed at higher levels in early relapsed pediatric b - cell precursor all patients , who usually show an unfavorable prognosis .", "notch1 prevents gc - induced apoptosis , among others , through activation of p56 , which activates the pi3k - akt axis , and through the transactivation of its target genes deltex and hes1 .", "deltex is a ring - domain ubiquitin ligase that may affect notch activity , and its overexpression prevents gc - induced apoptosis .", "activation of the pro - survival pi3k / akt / mtor pathway by notch has also been observed in other studies [ 95 , 106 , 419 , 420 ] and may be responsible for notch - mediated inhibition of the p53 tumor suppressor gene .", "another mechanism by which notch1 protects t - all cells from gc - induced apoptosis , is through the anti - apoptotic gimap5/ian5 ( gtpase of the immunity - associated protein / immune - associated nucleotide - binding protein 5 ) [ 421 , 422 ] . giamp5/ian5", "interacts with bcl-2 and bcl - xl and inhibits apoptosis during t - cell development and is highly expressed in human b - cell lymphoid malignancies .", "it is localized within the mitochondria and endoplasmic reticulum ( er ) and regulates mitochondrial integrity .", "gimap has been linked to immunological diseases such as t - cell lymphopenia and autoimmune diseases .", "notch also activates nfb signaling [ 74 , 427 ] and induces c - myc expression [ 428430 ] , both contributing to apoptotic resistance .", "this resistance can be overcome by the simultaneous exposure of the cells to src inhibitors , pi3k / akt inhibitors , or mtor inhibitors [ 67 , 68 ] , understating the importance of the protein kinase network in regulating the effects of notch1 on gc - induced apoptosis . ", "a recent report showed that gc sensitivity of t - all is associated with gr - mediated inhibition of notch1 expression .", "the serum- and glucocorticoid - inducible kinase 1 ( sgk1 ) was also shown to control notch1 signaling by downregulating its protein stability through fbw7 ubiquitin ligase .", "sgk1 phosphorylates fbw7 at ser227 , an effect inducing icn - notch1 ubiquitination and degradation . despite gc resistance induced by notch , notch- and fbw7-mutated", "t - all shows in general a favorable response to gc therapy and in some studies , but not all , also exhibits a better prognosis [ 405 , 433436 ] .", "this may be related to the fact that gcs may overcome notch - dependent drug resistance , and in these t - all cases the cell survival depends on notch signaling .", "various micrornas negatively regulate fbw7 expression including mir-27a , mir-182 , mir-363~92 , and mir-223 [ 253 , 438 , 439 ] and may increase the expression of fbw7-regulated target genes including notch1 , mcl-1 , c - jun , c - myc , and cyclin e . mir-451 and mir-709 suppressed oncogenesis in notch1-induced mouse t - all .", "mir-150 , which is upregulated upon thymocyte maturation , targets notch3 and thus regulates t - cell proliferation and survival .", "the p53-induced mir-34a also targets the notch1 receptor as well as its ligand dll1 ( delta like-1 ) [ 443 , 444 ] .", "prevention of notch activation in cutaneous t - cell lymphoma ( ctlc ) by gsi ( -secretase inhibitor ) treatment led to alterations in the microrna profile of the cell . among others ,", "mir-27a , mir92b , mir-181a , mir-18a , mir-19b , mir-222 , and mir-221 were downregulated , while mir-122 and mir-214 upregulated .", "mir-27a targets fbw7/hcdc4 [ 253 , 438 , 439 ] , the substrate recognition component of the scf ( skp1-cullin - f - box ) ubiquitin ligase complex that targets notch1 for degradation . the repressive effect of mir-27a on fbw7 mrna is especially pronounced at the g2/m and early g1 phases .", "other targets of mir-27a includes zbtb10 ( zinc finger and btb domain containing 10 ) , which acts as a repressor of sp ( specificity proteins ) transcription factors and induces g1 arrest , and the myt-1 kinase , which inhibits the transition through g2-m by enhanced phosphorylation and inactivation of cdc2 ( cdk1 , cyclin - dependent kinase 1 ) .", "upregulation of mir-122 by gsi seems to be mediated by p53 and has an antagonistic effect on apoptosis through activation of akt .", "c - myc is , among others , a target of notch [ 428430 ] and has broad effects on tumorigenesis and modulates gc - induced apoptosis [ 99 , 448 ] .", "conditional overexpression of c - myc in hematopoietic cells in mice culminated in the formation of malignant t - cell lymphomas and acute myeloid leukemias .", "these authors demonstrated a role for the pi3k / akt axis in c - myc activation .", "dysregulation of the c - myc gene is a common trait of burkitt 's lymphoma due to chromosomal translocations , the most frequent one being t(8 ; 14)(q24:q32 ) involving c - myc and igh ( immunoglobulin heavy locus ) [ 451453 ] .", "other hematopoietic malignancies characterized with c - myc overexpression include diffuse large b - cell lymphoma ( dlbcl ) , follicular lymphoma , cll , b - cell lymphoma , and aml [ 454459 ] .", "earlier studies have shown that dexamethasone - induced apoptosis of a t - all cell line was associated with c - myc suppression [ 460 , 461 ] .", "the gc - mediated down - regulation of c - myc expression was initially thought to be one mechanism that contributes to apoptosis .", "not all studies have confirmed this finding , which may be explained by the many signaling pathways induced by gcs .", "c - myc uses distinct mechanisms for activating and repressing gene expression . for transcriptional activation , c - myc dimerizes with max and", "transcriptional repression is achieved through protein - protein interactions , where it antagonizes the activity of positive regulators of transcriptions .", "the c - myc - mediated upregulation of mir-17 and mir-20a ( belonging to the mir-17~92 cluster ) negatively regulates e2f1 translation by targeting the 3-utr of e2f1 mrna and may therefore fine tune the direct myc - mediated transcriptional activation of e2f1 , allowing a tightly regulated proliferative signal ( figure 4 ) .", "e2f1 - 3 also binds to the promoter of the mir-17~92 cluster and activates its transcription , thus generating an autoregulatory feedback loop .", "another target of the mir-17~92 cluster is cyclin d1 , which also induces the expression of mir-17 and mir-20a by binding to the promoter regulatory region of the mir-17~92 cluster .", "the gc - induced down - regulation of mir-17~92 should actually stimulate e2f1 expression , which under certain circumstances may exert pro - apoptotic effects .", "e2f1 may promote apoptosis through transcriptional activation of the pro - apoptotic mir-15a~16 cluster and by activating jnk . in a b - cell lymphoma model , c - myc down - regulated a series of micrornas , an action that may contribute to tumorigenesis .", "the c - myc mediated repression of the mir-30 cluster may affect autophagy , as beclin-1 expression is regulated by mir-30a .", "some of the pro - autophagy activity of cancer therapy is mediated through down - regulation of mir-30a .", "also the down - regulation of mir-15a and mir-16 by c - myc is of interest as these micrornas are deleted or downregulated in over two - thirds of individuals with cll , and they target the anti - apoptotic bcl-2 gene [ 345 , 346 ] .", "a third mirna downregulated by c - myc is the tumor suppressor let-7 mirna cluster , which targets , among others , the ras oncogene , hmga2 ( high mobility group a2 ) [ 477 , 478 ] , bcl - xl , cdc25a , cdk6 ( cyclin - dependent kinase 6 ) , and cyclin d2 .", "other mirnas repressed by myc include mir-22 , mir-23a / b , mir-26a / b , mir-29a / b / c , mir-34a , mir-146a , mir-150 , and mir-195 [ 465 , 473 , 480 ] .", "mir-26a levels were found to be reduced in various b - cell lymphomas , especially burkitt lymphoma as well as various solid tumors [ 481 , 482 ] .", "b - cll , which does not have a prominent pathological role of c - myc , showed higher expression of mir-26a than myc - dependent burkitt lymphoma .", "mir-26 restoration in burkitt lymphoma or nasopharyngeal carcinomas reduced proliferation and colony formation through g1 arrest and repression of the histone - lysine n - methyltransferase ezh2 , a global regulator of gene expression [ 465 , 481 , 483 ] . the tumor - suppression function was only seen in myc - transformed cells , but not in v - abl transformed cells [ 465 , 483 ] . however , in t - all , mir-26a was one of five micrornas that independently promoted tumorigenesis through inhibition of pten .", "in the background of activating mutations in notch1 , mir-26a overexpression decreased the latency of t - all .", "forced overexpression of mir-34a , mir-150 , and mir-15a/16 - 1 attenuated in vivo tumor growth of myc - induced b - cell lymphoma .", "mir-34a is a crucial component of the p53 tumor suppressor network with potential anti - proliferative and pro - apoptotic activity [ 484486 ] .", "c - myc transcriptionally induces lin28b , which is an rna - binding protein that suppresses the maturation of let-7 family microrna precursors [ 487 , 488 ] .", "lin28 is involved in stem cell maintenance [ 489491 ] and is a marker of cancer stem cells .", "the effect of autophagy on the cellular response to chemotherapy is dual . under certain conditions ,", "autophagy acts as a pro - survival mechanism to protect cancer cells from chemotherapy , whereas under other circumstances , autophagy mediates the therapeutic effects of the anticancer agents .", "another important regulator of autophagy is the activity of mtor ( mammalian target of rapamycin ) , which is a central element signaling cell growth and enhancing protein translation . when this kinase is inhibited , autophagy is promoted .", "it should be noted that beclin-1 may play a dual role in both regulating autophagy and apoptosis , thus being at the cross - road between these two physiological processes .", "beclin-1 has recently been recognized as a bh3-only protein interacting with bcl-2 , bcl - xl and mcl-1 [ 59 , 60 , 494496 ] .", "one report provides evidence that after initiating apoptosis , beclin-1 is cleaved by caspases and the n - terminal fragment of beclin can inhibit autophagy , while the c - terminal fragment can amplify mitochondrial - mediated apoptosis .", "perturbation of beclin-1 cleavage by knockin mutation phenocopied the autophagy induction observed in apoptosis - defective cancer cells and rendered chemotherapy resistance both in vitro and in vivo . a role for beclin in regulating tumorigenesis has been demonstrated in mice with heterozygous disruption of beclin-1 .", "dlbcl expressing high beclin-1 levels had a favorable clinical outcome with r - chop treatment than those with low beclin-1 expression .", "gcs have been shown to promote autophagy in lymphocyte cell lines and primary t - all cells [ 501 , 502 ] .", "one mechanism for induction of autophagy is through upregulation of the mtor - inhibitory stress protein dig2 ( dexamethasone - induced gene 2 ) , also known as rtp801 and redd1 ( regulated in development and dna damage responses 1 ) .", "mtor inhibition by dexamethasone was demonstrated by reduced phosphorylation of s6k ( 70kd ribosomal protein s6 kinase 1 ) , a member of the rsk family of serine / threonine kinases .", "dig2 releases tsc2 from 14 - 3 - 3 , thereby promoting the assembly of the tsc1/tsc2 complex , which inhibits mtor .", "dig2 knockout thymocytes underwent more extensive dexamethasone - induced cell death , suggesting that autophagy promotes cell survival .", "however , rapamycin , an inhibitor of mtor and inducer of autophagy , strongly sensitizes resistant mm and t - all cells to gc - induced apoptosis [ 59 , 111 , 116 , 117 ] , suggesting that induction of autophagy does not always combat apoptosis .", "it could be that the higher degree of autophagy induced by rapamycin itself may be pro - apoptotic .", "bonapace et al . showed that rapamycin induces an autophagy - dependent necroptosis , which is required for childhood t - all to overcome gc resistance .", "necroptosis is a form of programmed necrosis that occurs when apoptosis is abortive due to caspase inhibition .", "the gc - mediated necroptosis was mediated by rip-1 ( receptor - interacting protein-1 ) and cyld ( cylindromatosis ) .", "mir-19 , which is frequently overexpressed in t - all patients and cell lines , represses cyld expression .", "obatoclax , a putative antagonist of bcl-2 family members , could also sensitize t - all cells to gc - induced apoptosis through induction of autophagy .", "this effect was associated with dissociation of the autophagy inducer beclin-1 from mcl-1 and decreased mtor activity .", "the apoptosis induced by gc in combination with obatoclax or rapamycin could be prevented by the autophagy inhibitors 3-methyladenine and bafilomycin .", "cdkn2/p16 , which acts as a g0/g1 cycle inhibitor , is frequently lost in t - all [ 507 , 508 ] and predicts relapse in children with all [ 508510 ] .", "p16 sensitizes t - all cell lines to gc - induced apoptosis through induction of bbc3/puma and repression of mcl-1 and bcl-2 .", "noxa was repressed in p16 transgenic cells , which could be a result of the simultaneous repression of e2f1 due to retinoblastoma protein and p130 activation .", "diffuse large b - cell lymphoma with cdkn2a deletion had a poor prognosis under r - chop treatment .", "also , myc gene arrangement in diffuse large b - cell lymphoma patients had a poor prognosis with r - chop chemotherapy ." ], [ "during the last decade , micrornas have become the focus of having a central role in the pathogenesis of cancer including lymphoid malignancies , besides their role in regulating gene expression during cell division , development , and differentiation [ 514523 ] .", "micrornas are short noncoding rnas that induce posttranscriptional gene silencing through base pairing with the 3 untranslated region ( utr ) of their target mrnas , thereby inhibiting their translation , with subsequent reduced protein levels [ 524 , 525 ]", ". bases 27 or 28 of the microrna are primary contributors to target specificity and are referred to as the microrna seed region .", "the micrornas are usually transcribed by rna polymerase ii , and sometimes by rna polymerase iii , into long primary precursor transcripts referred to as pri - mirnas .", "mirna are encoded by one arm of a stem loop structure embedded in introns or , less frequently , exons of protein - coding or noncoding transcripts . in the nucleus", ", the pri - mirnas stem loop is cleaved by the nuclear rnase iii enzyme drosha together with its cofactor dgcr8 ( digeorge syndrome critical region 8)/pasha ( the microprocessor complex ) to generate ~70 nucleotides long precursors called pre - mirnas . in some cases , an entire intron consists of such a stem loop structure , which is released by the splicing machinery in a drosha - independent manner .", "pre - mirnas are exported by rangtp / exportin-5 to the cytoplasm , where they are further processed by dicer , another rnase iii enzyme , to generate ~22 base pair microrna duplexes that enter effector complexes called mirisc ( mirna - containing rna - induced silencing complex ) . here , they are converted into single - stranded mature mirnas that target mrnas and thereby affect their translation and stability [ 516 , 528 , 529 ] .", "cancer cells frequently display reduced levels of micrornas that act as tumor suppressors , while expressing elevated levels of oncogenic micrornas , called oncomirs that promote tumor development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation and apoptosis .", "there are variations in the microrna expression pattern described between the various scientific reports , which can be explained by the use of different internal standards , different controls for comparison , and the use of sample materials of malignant cells at different developmental stage and at different ontogeny tumor grade .", "virtually every step in hematopoiesis seems to be finely tuned by specific micrornas [ 514 , 530533 ] .", "conditional deletion of dicer expression in the t - cell compartment resulted in impaired t - cell development and diminished regulatory t - cell function [ 534536 ] , and ablation of dicer in the b - cell compartment attenuates b - cell development and alters the antibody repertoire .", "it should be noted that there exists an alternative microrna processing pathway that is independent of dicer , but dependent on argonaute-2 .", "microrna expression is dynamically regulated during thymocyte development , with different enriched micrornas expressed at each developmental stage ( table 1 ) .", "it should be emphasized that the cd4cd8 ( double positive , dp ) thymocytes are the most gc - sensitive thymocyte population [ 540542 ] .", "dicer - deficient dp thymocytes expressed higher levels of cd69 and tcr ( t - cell receptor ) , but lower levels of bcl-2 .", "the dicer - deficient thymocytes were more prone to apoptosis than control cells [ 539 , 543 ] , understating the role of micrornas in regulating cell survival .", "some micrornas , such as mir-146a and mir-182 , play a dominant role in the regulation of the innate and adaptive immune responses , respectively [ 544 , 545 ] . according to neilson et al .", ", the pro - apoptotic mir-15b is almost not expressed at the immature dn1 ( double negative 1 ) thymocyte stage but becomes gradually upregulated in dn3 and dn4 , and further in dp cells .", "the pro - apoptotic mir-16 is also low in dp1 and reaches a maximum in dn4 cells , with a reduction upon transition to dp cells .", "the oncogenic mir-21 is expressed at the highest level in dn1 and becomes reduced upon transition to dn3 and is almost not expressed in dp cells .", "mir-181a / b is expressed at the highest level in dp thymocytes , together with mir-92 and mir-350 .", "it should be noted that in this study the expression of each microrna was determined relative to the general microrna pool of each subpopulation . since the amount of total microrna becomes strongly reduced upon transition from dn4 to dp ( a drop from 32000 to 5200 copies / cell ) ,", "the absolute microrna number in each cell population differs , which can be demonstrated by the mir-181a transcript .", "while mir-181a presents 15.6% of the microrna in dp cells and 6.7% and 5% in dn3 and dn4 , respectively , the numbers of mir-181a copies in these three populations were estimated to be 810 in dp , 1400 in dn3 , and 1600 in dn4 .", ". showed that mir-181a is expressed at dn1 and becomes upregulated during dn2 and dn3 and then downregulated at dn4 .", "mir-181 is still significantly expressed in dp cells , albeit at a slight lesser extent than in dn4 and becomes downregulated upon differentiation to the sp ( single positive ) stage .", "performed an extensive microrna profiling to identify micrornas specifically expressed in b - cell subsets during peripheral b - cell differentiation .", "notably , mir-18a , mir-28 , mir-15a~16 - 1 , and mir-181 are expressed at higher levels in centroblasts ( germinal center b lymphocytes ) compared with memory b cells , whereas mir-101c , mir-150 , mir-29a , b , c , and mir-23a~24 are enriched in memory b cells .", "mir-17~92 , mir-363~106a , and mir25~106b are highly expressed in all b - cell subtypes .", "the high level of mir-15a~16 - 1 in germinal center b - cells corresponds with low bcl-2 expression in these cells .", "mir-223 is highly expressed in nave and memory b cells , but not in centroblasts .", "mir-181a represses the expression of bcl-2 , cd69 , and the t - cell receptor ( tcr ) -chain .", "mir-181a augments the strength of tcr signaling and down - regulates several phosphatases including dusp5 , dusp6 , shp-2 , and ptpn22 that regulate the sensitivity of t cells to antigens .", "the down - regulation of ptpn22 by mir-181a led to elevated phosphorylation of p56 at y394 and the down - regulation of dusp5/6 to increased erk activation .", "the normally high levels of mir-181a maintain t - cell tolerance to self - peptide / mhc molecules , with a reduction in this microrna increasing the number of self - reactive t cells . also , dampening mir-181a expression using antagomir-181a impaired positive selection with about a 70% reduction of mature cd4 sp thymocytes .", "thus , mir-181a plays a role in regulating tcr response during t - cell development .", "recently , mir-181a-1/b-1 , but notmir-181a-2b-2andmir-181-c / d , was found to control the development of normal thymic t cells and leukemia cells .", "conditional deletion of mir-181ab1 allele resulted in 50%75% decrease in cellularity in the thymus and a significant reduction in the percentage of dp cells .", "mir-181a expression decreased during the dn3a to dn3b transition during -selection , and loss ofmir-181ab1resulted in a reduction in the percentage of dn3 and dn4 cells that expressed intracellular tcr- , while pret expression in dn3 thymocytes was normal .", "other alterations occurring upon tcr / cd28 co - stimulation includes the upregulation of the mir-466 family , mir-574 , mir-346 , mir-214 , mir-155 , and mir-709 , and the down - regulation of the mir-29 family , mir-15a , mir-15b , mir-16 , mir-146b , mir-142 , mir-27a , mir-150 , and let-7 family . chen et al", ". showed that mir-181 is expressed in the b - lymphoid cells of the mouse bone marrow , and its ectopic overexpression in hematopoietic stem / progenitor cells significantly increased b - cell production .", "mir-181 also affects the development of nk cells through targeting the nemo - like kinase ( nlk ) , an inhibitor of notch signaling .", "mir-181 targets the rna - binding protein lin28 , thereby disrupting the lin28-let-7 reciprocal regulatory loop , with concomitant upregulation of let-7 and differentiation of megakaryocytes .", "mir-150 is highly expressed in mature and resting lymphocytes , but not in their progenitors [ 547 , 553 ] .", "overexpression of mir-150 led to a block in b - cell formation at the pro - b to pre - b - cell transition by downregulating c - myb , among other targets , suggesting for a role for this microrna in b - cell differentiation . within the lymphoid lineage the choice between t and b cells", "the t - cell population level was unaffected by overexpression of mir-150 in hematopoietic progenitor cells , while the mature b - cell levels were strongly reduced .", "mir-150 drives megakaryocyte - erythrocyte progenitor ( mep ) cells towards megakaryocytes at the expense of erythroid cells .", "mir-150 also regulates the development of nk ( natural killer ) and inkt ( invariant nk ) cells .", "mice with target deletion in mir-150 had a defect in their ability to generate mature nk cells , while overexpression of mir-150 resulted in a substantial reduction in inkt in the thymus and in the peripheral lymphoid organs , supposedly through targeting of c - myb by mir-150 .", "mir-125b affects t - cell differentiation through regulation of ifn ( interferon ) , il-2r , il-10r , and prdm1/blimp1 ( b lymphocyte - induced maturation protein-1 ) .", "ectopic expression of mir-125b in nave lymphocytes inhibited differentiation to effector cells . during normal b - cell development , mir-125b is enriched in germinal center b cells and keeps the transcription factor irf4 and prdm1/blimp1 down , while mir-223 is enriched in memory b cells , where it targets the transcription factor lmo2 , which is specifically expressed in germinal center b cells .", "irf4 and prdm1/blimp1 expression are repressed in centroblasts , but is necessary for differentiation into memory and plasma cells [ 593 , 594 ] .", "overexpression of mir-125b alone in mice causes an aggressive , transplantable myeloid leukemia . before leukemia , these mice did not display elevation of white blood cells in the spleen or bone marrow , rather the hematopoietic compartment showed lineage - skewing , with myeloid cell numbers dramatically increase and b - cell numbers severely diminished .", "mir-125b targets lin28a , an induced pluripotent stem cell gene . knockdown of lin28a led to hematopoietic lineage - skewing similar to ectopic mir-125b overexpression , with increased myeloid and decreased b - cell number .", "mir-155 is also important for lymphopoiesis and for preserving normal immune system responses [ 266 , 597599 ] .", "mir-155 is processed within the second exon of the nonprotein - encoding gene bic ( b - cell integration cluster ) .", "mir-155 is upregulated upon tcr / cd28 costimulation in mouse t cells , and in macrophages by several tlr ( toll - like receptor ) pathways .", "b cells require mir-155 for normal production of isotype - switched , high - affinity antibodies and for a memory response .", "the transcription factor pu.1 , which down - regulates igg1 levels , is a target gene of mir-155 in b cells .", "this may explain the reduced amount of circulating igg1 in mir-155 knockout mice . as with b cells", ", it seems that mir-155 is involved in t - cell differentiation [ 266 , 597 ] .", "nave t cells derived from mir-155 knockout mice showed increased propensity to differentiate into th2 rather than th1 cells , with the concomitant production of th2 cytokines such as il-4 , il-5 , and il-10 [ 266 , 597 ] .", "one explanation for this biased development of th2 cells might be the mir-155 mediated targeting of c - maf ( musculoaponeurotic fibrosarcoma ) , a transcription factor that transactivates the il-4 gene . with regard to the acute immune response , the t cells had an impaired response and showed attenuated il-2 and ifn release in response to antigens [ 266 , 597 ] .", "mir-155 is upregulated by the transcription factor foxp3 and critical for t regulatory cell function .", "mice overexpressing mir-155 in the b - cell lineage results in preleukemic pre - b - cell proliferation in the spleen and bone marrow , followed later in life by b - cell malignancy .", "the mir-17~92 cluster located on chromosome 13 at locus q31.3 is essential for b - cell development .", "the expression of mir-17~92 peaked in pre - b cells , where it inhibited cell death .", "it is expressed at higher levels in normal germinal center b cells compared to nave and memory b cells .", "knockout of mir-17~92 leads to increased bim expression and inhibits b - cell development at the pro - b to pre - b transition , a step also blocked by mir-150 .", "mice overexpressing the mir-17~92 cluster in lymphocytes developed lymphoproliferative disease and autoimmunity and they died prematurely .", "these animals were found to have increased numbers of activated b cells , and a higher ratio of activated cd4 t cells versus cd8 t cells .", "the enhanced proliferation and survival of b and t cells may result from the down - regulation of bim and pten .", "mir-17~92 expression is strongly induced after activation of cd8 t cells , which is critical for the rapid clonal expansion of these cells . however , following the clonal expansion , mir-17~92 is downregulated and further silenced during memory development .", "malignant lymphomas arise from normal b- or t - cell counterparts at different ontogeny stages and commonly continue to express gene signatures inherited from their nontransformed cellular progenitors .", "extensive mirna profiling studies have been performed on various lymphoid malignancies , including t - all , cutaneous t - cell lymphoma , cll [ 557 , 563 ] , pre - b - all [ 557 , 605 , 607 ] , diffuse large b - cell lymphoma ( dlbcl ) [ 564 , 580582 , 608610 ] , anaplastic large cell lymphoma ( alcl ) , multiple myeloma ( mm ) [ 574 , 611 , 612 ] , mantle cell lymphoma ( mcl ) [ 583 , 591 , 613 ] , burkitt lymphoma ( bl ) [ 564 , 614 ] , and follicular lymphoma ( fl ) [ 135 , 582 , 615 ] .", "a comprehensive study aimed to integrate the many mirnas upregulated in t - all into a microrna - transcription factor coregulatory network was performed by ye et al . .", "a short description of some important micrornas in malignant lymphoid diseases is described below and summarized in tables 2 and 3 . in general ,", "t - all is characterized by upregulation of the mir-17~92 cluster , mir-26a , mir-128a / b , mir-146a , mir-181a / b , mir-150 , and mir-155 , while let-7b and mir-223 are downregulated [ 253 , 557561 ] . \n 3.2.1.1 .", "the mir-19 , mir-20a , mir-92a , and mir-17 especially of the mir-17~92 cluster are upregulated in t - all .", "all six mirnas mir-17 , mir-18a , mir-19a , mir-20a , mir-19b , and mir-92a , of the mir-17~92 cluster promoted leukemogenesis in notch1-induced t - all in vivo [ 253 , 616 ] . among them", ", the mir-19 family has been considered the key oncogenic component [ 248 , 506 , 617 ] .", "the mir-17~92 cluster is located within a fragile site that is frequently amplified in a range of hematopoietic malignancies .", "mir-19 represses notch1 , pten , hoxa9 , cyld , runx1 , e2f1 , and bcl2l11 ( bim ) [ 506 , 616 , 620 ] .", "posttranslational inactivation of pten by mir-19 promotes activation of the pi3k / akt pathway , and incontrollable proliferation of t cells [ 104 , 105 ] . increased akt signaling antagonizes gc - induced apoptosis by several mechanisms , including phosphorylation of foxo3a , thus preventing its nuclear translocation and transcriptional activation of bim , and through inactivation of gsk3 , which is essential for gc - induced apoptosis [ 30 , 67 , 97 ] .", "hoxa9 is a leukemogenic homeoprotein in t - all , and a target gene of the oncogenic mll - af4 fusion protein .", "high expression of mir-196b was found in pediatric all with aberrant activation of hoxa genes .", "notch1 plays a vital role in t - cell development and transformation , and about 50% of primary t - all samples show abnormal notch1 expression .", "downstream transcriptional targets of notch1 include hes1 and c - myc , the former affecting the pi3k / akt and nfb signaling pathways [ 624 , 625 ] .", "c - myc is a potent and direct transcriptional activator of mir-17~92 , leading to modulation of e2f1 expression .", "gcs repress the expression of mir-17~92 , which may be one means to overcome the tumorigenicity of t - all cells and to elevate bim expression .", "in contrast to mir-19 , mir-451 , and mir-709 are potent suppressors of oncogenesis in notch1-induced mouse t - all .", "mir-451 represses c - myc , while mir-709 represses ras - grf-1 that acts upstream to ras and prevents akt activation .", "both mir-451 and mir-709 are transcriptional targets of the bhlh e2a tumor suppressor , which is degraded upon notch1 induction in mouse t - all cells [ 626 , 627 ] .", "repression of tumor suppressor mir-451 is essential for notch1-induced oncogenesis in a murine model of t - all .", "human t - alls with activating notch1 mutations have decreased mir-451 and increased c - myc levels compared with t - alls with wild - type notch1 .", "one mechanism of the tumor suppressive action of mir-451 could be through down - regulation of the pi3k / akt survival pathway . \n", "primary t - all cells also express elevated levels of mir-26a that suppresses pten and bim . mir-26a", "mir-26a was found to be repressed by c - myc in a mouse lymphoma model , leading to enhanced expression of the ezh2 oncogene , a component of the polycomb repressive complex 2 .", "mir-146a , mir-181a / c , and mir-221 were associated with overall survival in all patients .", "however , overexpression studies of mir-146a in transplanted bone marrow cells suggest a tumor - suppressive role for this microrna .", "mir-146a knockout mice developed massive myeloproliferation followed by hematopoietic tumors , including myeloid sarcomas and lymphomas [ 635 , 636 ] . the myeloproliferative phenotype correlated with enhanced nfb signaling .", "mir-146a suppresses the nfb activators irak1 ( interleukin 1 receptor - associated kinase 1 ) and traf6 ( tnf receptor - associated factor 6 ) [ 635 , 637 , 638 ] .", "a negative feedback loop exists between nfb and mir-146 . whereas mir-146 represses nfb signaling , nfb signaling upregulates mir-146 . \n", "mir-181a family members are highly expressed in t - all leukemia cells and downregulated during remission .", "deletion ofmir-181a-1/b-1expression inhibits the development ofnotch1 oncogene - induced t - all in a mouse model .", "mir-181a / bcontrols the strength and threshold of notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of notch and pre - t - cell receptor ( tcr ) signaling pathways . \n", "mir-124a has been shown to be downregulated in more than 50% of all cases and associated with higher relapse rate and mortality rate .", "a comparison study of primary cll samples with normal unstimulated or cpg - stimulated b cells showed high similarities between cll and activated b cells , including upregulation of mir-34a , mir-155 , and mir-342 and down - regulation of mir-103 and mir-181a / b .", "activation of normal b cells led to reduced mir-23a , mir-23b , mir-24 , mir-27b , mir-181a / b , and mir-223 and increased mir-155 with all activation agents used .", "mir-150 was reduced during b - cell activation , whereas it was upregulated in most cll cases .", "the latter confirms the study of fulci et al . , but is opposed to wang et al", "ectopic mir-150 expression increased cell death in pro - b cells , while mir-150 deficiency led to b - cell expansion and an enhanced humoral immune response .", "some differences in mir-150 are observed between the mutated versus unmutated igvh ( immunoglobulin heavy chain variable - region genes ) subgroups , where expression is higher at the average in the mutated igvh subgroup .", "cll cases with unmutated igvh or with high expression levels of zap-70 ( 70kd zeta - associated protein ) show an unfavorable course with rapid progression in comparison to patients with a mutated igvh .", "two research groups [ 565 , 566 ] observed decreased levels of mir-29c and mir-223 in cll with zap70 and igvh unmutated status .", "calin et al . observed that the unmutated igvh cll subgroup exhibited high levels of tcl-1 due to low expression of mir-29 and mir-181 that negatively regulate this oncogene . mir-181 and mir-29 might therefore be considered to have tumor - suppressor functions .", "tcl-1 functions as a coactivator of akt , and b - cell forced expression of tcl-1 in transgenic mice resulted in tumors that resembled cll .", "cll with unmutated igvh and high expression of zap-70 showed also relative high expression of mir-15a , mir-16 - 1 , mir-16 - 2 , mir-195 , mir-23b , mir-155 , mir24 - 1 , and mir-146 , while low expression of mir-223 , mir-29a-2 , mir-29b-2 , and mir-29c . in an aggressive subtype of cll with abnormalities in the tp53 gene ,", "cll cases with good prognostic features are typically characterized by down - regulation of mir-15a and mir-16 - 1 [ 643 , 708 ] , located at the 13q14.3 locus .", "these mirnas map to a region between exon 2 and 5 of the leu2 gene .", "deletion of 13q14.3 ( del(13q ) ) is the most common cytogenetic abnormality in cll occurring in more than 50% of the cases and implies for a favorable prognosis .", "this deletion occurs also frequently in mm patients .deletion in mice of the 13q14-minimal deleted region , which encompasses the mir-15a~16 cluster , caused the development of indolent b - cell - autonomous , clonal lymphoproliferative disorders , recapitulating the spectrum of cll - associated phenotypes observed in humans .", "repression of mir-15a and mir-16 - 1 , as well as mir-29b , in cll may also be mediated by histone deacetylases ( hdacs ) .", "hdac inhibition triggered the accumulation of the transcriptionally activating chromatin modification h3k4me2 and restored the expression of mir-15a , mir-16 - 1 , and mir-29b .", "both mir-15a and mir-16 - 1 negatively regulate bcl-2 , and mir-29 targets mcl-1 [ 381 , 382 ] .", "the expression of bcl-2 in cll cases is inversely correlated with the expression of mir-15a and mir-16 - 1 [ 563 , 711 ] .", "other targets of mir-15/16 include chek1 , cyclind1 , cyclind2 , and cdc25a [ 525 , 645 ] .", "overexpression of mir-15a and mir-16 - 1 induced cell cycle arrest at g1/g0 in an rb - dependent manner . a germ - line mutation in the primary precursor of mir-15a/16 - 1 that impairs their processing was observed in familial cll patients .", "targeting deletion of mir-15a~16 in mice led to the development of a spectrum of diseases resembling cll - associated lymphoproliferation in humans , including cll , cd5 monoclonal b - cell lymphocytosis , and cd5 non - hodgkin 's lymphomas .", "the new zealand black ( nzb ) mouse that harbor a point mutation in the 3-flanking region of mir-16 that leads to reduced mir-16 expression and develops symptoms similar to b - cll in humans , further confirming the tumor suppressor function of this locus . \n", "underexpression of mir-181a / b was associated with shorter overall survival in cll , while higher levels of mir-181a were associated with a shorter time from diagnosis to initial therapy . during the course of cll progression ,", "the mir-181a / b levels were decreased , which inversely correlated with increased levels of its target genes mcl-1 and bcl-2 .", "the study of marton et al . showed consistent underexpression of mir-181a , as well as let-7a and mir-30d in all cll cases studied .", "however , increased expression of mir-181a / b was associated with favorable outcome in patients with cytogenetically normal acute myeloid leukemia ( aml ) .", "ectopic overexpression of mir-181a / b into primary cll increased fludarabine - sensitivity in p53 wild - type cells , but not in cll with attenuated p53 response .", "the importance of the mir-181 target mcl-1 in cll survival was demonstrated by rapid apoptosis of cll cells following sirna - mediated down - regulation of mcl-1 , and by the mcl-1 transgenic mice , which developed b - cell lymphoma .", "thus , low mir-181 and mir-29 expression in cll could confer drug resistance through upregulation of mcl-1 expression . \n", "the mir-29 family consisting of mir-29a and mir-29b seems to play a dual role in tumorigenesis . on the one hand , mir-29a and mir-29b", "are downregulated in mantle cell lymphoma , aggressive cll samples ( high zap-70 with unmutated igvh ) [ 659 , 710 , 716 ] , alk - positive anaplastic large cell lymphomas ( alcl ) , mm , and aml . on the other hand , mir-29a and b", "are expressed at higher degree in indolent cll ( low zap-70/mutated igvh ) than in normal cd19 cells [ 563 , 569 , 716 ] . mir-29c", "together with mir-223 down - regulation is associated with higher tumor burden , disease aggressiveness , and poor prognosis in cll . forced overexpression of mir-29b", "the tumor suppressor activity of mir-29 may be achieved through targeting cell cycle regulators and oncogenes such as cdk6 , dna methyltransferase 3a ( dnm3a ) and 3b ( dnmt3b ) , mcl-1 , and tcl1a [ 382 , 569 , 583 , 717 ] .", "another tumor suppressor function of mir-29 is mediated through activation of p53 , which is achieved by targeting p85 ( the regulatory subunit of pi3k kinase ) and cdc42 ( a rho family gtpase ) . however , in another setting mir-29 acts as an oncogene .", "mir29a overexpression in immature and mature b cells promoted cll development , and transplantation of mir-29-transduced hematopoietic stem and progenitor cells into irradiated mice resulted in myeloproliferative disease and aml .", "one mechanism for the oncogenic feature of mir-29 could be through repression of the tumor suppressor cell - adhesion molecule peroxidasin homologue ( pxdn ) .", "thus , depending on the cellular contexts , mir-29 can function as an oncogene or a tumor suppressor . \n", "mir-221 and mir-222 are expressed at higher levels in cll with unmutated igvh and high expression of zap-70 , the most aggressive cll subtype with poor prognosis .", "these micrornas may contribute to oncogenesis by targeting the cdk inhibitor p27 [ 695 , 696 , 718 , 719 ] , foxo3a [ 720 , 721 ] , apaf-1 [ 721 , 722 ] , p57 , bmf , pten , and timp3 ( tissue inhibitor of metalloproteinase 3 ) .", "in other cll cases , the mir-222 was found to be lower than that of normal cd19 cells .", "the p53 target mir-34a is decreased in cll patients with 11q deletions , leading to increased zap-70 expression .", "mir-34a also targets bcl-2 [ 348 , 484 ] , and the e2f1 and b - myb oncogenes in cll .", "members of the mir-17~92 polycistron are upregulated in b - cell lymphoma , as well as mir-155 [ 469 , 568 , 685 ] .", "adoptive transfer of hematopoietic stem cells bearing a truncated portion of the mir-17~92 polycistron in c - myc transgenic mice resulted in a more rapid onset of malignant b - cell lymphomas .", "transgenic overexpression of the entire mir-17~92 in the murine hematopoietic compartment led to the development of lymphoproliferative disease and increased lethality .", "the negative regulation of bim by the mir-17~92 cluster seems to be a major mechanism by which b - cell lymphomas evade apoptosis .", "silencing of mir-17 and mir-20a in mantle cell lymphoma led to upregulation of the cyclin - dependent kinase ( cdk ) inhibitor p21 , suggesting that p21 is an essential target of the mir-17~92 cluster during b - cell lymphomagenesis .", "overexpression of c - myc mrna together with mir-17 - 5p / mir-20a was associated with a more aggressive behavior in mantle cell lymphoma .", "mir-17~92 confers chemoresistance in mantle cell lymphoma through activation of the pi3k / akt pathway .", "mir-21 is commonly upregulated in cll as well as cml and many other cancer cell types .", "forced overexpression of mir-21 under the control of the nestin promoter resulted in severe pre - b - cell lymphoma .", "mir-21 overexpression potentiated lung tumorigenesis of a constitutively activated k - ras proto - oncogene .", "mir-21 is an oncomir that promotes tumorigenesis by targeting a range of genes involved in regulating cell proliferation and/or survival , including pten , sprouty ( spry2 ) , pdcd4 ( programmed cell death 4 ) , tpm1 ( tropomyosin 1 ) , and human dna muts homolog 2 ( hmsh2 ) . in glioblastoma cells , mir-21 also targets a network of p53 pathways , tgf , and mitochondrial tumor suppressor genes .", "pdcd4 inhibits ap-1-mediated transactivation and negatively regulates the pro - survival ral guanine - nucleotide dissociation stimulator ( ralgds ) signaling pathways [ 517 , 729 ] .", "pdcd4 is a tumor suppressor that is upregulated during apoptosis and downregulated in several cancer forms [ 737739 ] .", "spouty , which is downregulated by mir-21 , negatively regulates the c - raf pro - survival signaling pathway . \n", "overexpression of mir-125b in cll - derived cell lines resulted in the repression of many transcripts encoding enzymes implicated in cell metabolism .", "these authors proposed that mir-125b acts as a regulator for the adaptation of cell metabolism to a transformed state . \n", "one microrna consistently downregulated in most b - lymphomas is mir-150 , which is proposed to act as a tumor suppressor [ 523 , 547 , 589 ] .", "mice lacking mir-150 have increased expression of its target transcription factor c - myb , which plays an important role in lymphocyte development and maturation .", "premature expression of mir-150 blocked the transition from pro - b to the pre - b stage .", "overexpression of mir-150 in nk / t lymphomas increased apoptosis and reduced cell proliferation , with concomitant reduction in dkc1 ( dyskeratosis congenita 1 ) and akt2 , reduced akt phosphorylation , and elevated levels of bim and p53 . \n", "mir-155 is overexpressed in many b - cell lymphomas including cll , primary mediastinal b - cell lymphoma ( pmbl ) , aggressive activated b - cell like ( abc ) subtype of dlbcl , hodgkin 's lymphoma , and pediatric burkitt 's lymphoma , but is almost absent in adult burkitt 's lymphoma [ 515 , 566 , 568 , 576 , 583 , 584 , 587 , 602 , 685 , 686 ] .", "c - myb ( v - myb myeloblastosis viral oncogene homolog ) , which is overexpressed in a subset of cll patients , associates with the promoter of mir-155 host genes ( mir155hg , also known as bic , b - cell integration cluster ) and stimulates its transcription .", "forced overexpression of mir-155 in b cells ( e-mir-155 transgenic mice ) led to initial preleukemic pre - b - cell proliferation followed by frank b - cell malignancy .", "the mir-155 orthologue mir - k12 - 11 in kaposi sarcoma - associated herpes virus ( kshv ) has been associated with b - cell tumors .", "mir-155 is essential for immune function and is strongly induced in activated t and b cells .", "mir-155 represses sh2-domain containing inositol-5-phosphatase-1 ( ship-1 ) , which is a critical phosphatase that negatively downmodulates akt pathway and is involved in normal b cell development . thus , sustained overexpression of mir-155 in b cells unblocks akt activity , inducing b - cell development .", "c - maf in lymphocytes , and hgal and smad5 in diffuse large b - cell lymphoma ( dlbcl ) [ 741 , 742 ] .", "hgal , a germinal center ( gc)-specific gene , inhibits lymphocyte and lymphoma cell motility by activating rhoa signaling cascade and by interacting with actin and myosin proteins .", "smad5 is a bone morphogenetic protein ( bmp)-responsive transcription factor and is activated by various cytokines .", "dlbcl expressing high levels of mir-155 concomitant with low hgal expression showed high aggressiveness and cell dissemination .", "thus , down - regulation of smad5 in diffuse large b - cell lymphoma defines a unique mechanism used by the cancerous cells to escape tgf growth inhibitory effects . in breast cancer ,", "as foxo3a is a positive regulator of the pro - apoptotic bim essential for gc - induced apoptosis [ 227229 ] , mir-155 overexpression may prevent bim upregulation . in one study , mir-93 , mir-25 , mir-92 ,", "mir-19a / b , mir-181a / b , and mir-32 were shown to be significantly overexpressed , while let7-b , let7-i , let7-c , mir-29a , and -29b significantly downregulated in mm .", ". found decreased expression of mir-15a~16 and increased expression of mir-222 , mir-221 , mir-382 , and mir-181a / b in their mm samples .", "also , the 13q14.3 locus containing the mir-15a and mir-16 - 1 is sometimes deleted in mm [ 345 , 746748 ] .", "the absence of mir-15a expression and overexpression of mir-181a / b correlated with worse prognosis of mm .", "antagonists especially to mir-19a / b and mir-181a / b ( antagomirs ) suppressed tumor growth of human myeloma cells implanted into nude mice .", "some differential mirna expression was observed between malignant mm and mgus ( monoclonal gammopathy of undetermined significance ) , which is the precancerous state preceding mm .", "mgus show already upregulation of mir-21 , mir-106~25 , mir-181a / b , mir-1 , and mir-133a , while during the progression to malignant multiple myeloma mir-17~92 , mir-32 , mir-193b~365 are upregulated and mir-192~194~215 and mir-15a~16 are downregulated [ 574 , 576 , 577 ] .", "the upregulation of mir-17~92 could be related to the upregulation of c - myc observed during mm progression [ 750 , 751 ] .", "upregulation of mir-1 and mir-133a correlated with t(14 ; 16 ) translocation in mm cases , suggesting that deregulation of microrna expression could be associated with chromosomal abberations .", "higher expression of dicer was associated with improved progression - free survival in symptomatic mm cases . the global increase in microrna expression in high - risk mm patients with poor prognosis", "was associated with increased expression of argonaute ( ago2/elf2c2 ) , a master regulator of mirna maturation and function [ 753 , 754 ] .", "adhesion of multiple myeloma to bone marrow stroma triggers cytokine production and enhances cell proliferation and resistance to chemotherapy through il-6-induced activation of nfb , pi3k / akt , and stat3 pathways .", "it should be noted that these pro - survival pathways antagonize gc - induced apoptosis in mm [ 756760 ] .", "mir-19a and mir-19b that are part of the mir-17~92 cluster downregulate socs-1 ( suppressor of cytokine signaling-1 ) , a gene frequently silenced in mm that plays a critical role as inhibitor of il-6 growth signaling , thus enforcing the il-6-induced survival signals . \n", "the oncogenic mir-21 is upregulated in mm patient samples and cell lines [ 574 , 579 , 652 ] . in il-6-dependent", "ectopic mir-21 expression was sufficient to sustain growth of il-6-dependent cell lines in the absence of il-6 .", "mir-21 is upregulated in a nfb - dependent manner in mm cells upon cell adhesion to bone marrow stromal cells . combining mir-21 inhibition with dexamethasone", "the p300-cbp - associated factor ( pcaf ) was found to be a target of the combined action of the mir106b~25 cluster and mir-32 .", "mir106b~25 , mir-17 , and mir-20a target the cdkn1a1/p21 cell cycle regulator , which prevents cell cycle progression in general and prevents the growth of mm cells [ 764 , 765 ] . \n", "mir-15a~16 is a pro - apoptotic microrna that targets bcl-2 , cyclin d1 , cyclin d2 , and cdc25a [ 346 , 748 , 766768 ] .", "overexpression of mir-15a~16 in mm led to inhibition of akt3 , ribosomal protein s6 , map kinases , and the nfb - activator map3kip3 , ultimately resulting in an antiproliferative effect and apoptosis .", "the anti - mm effect of mir-15a~16 was observed even in the context of the bone marrow microenvironment . mir-15a~16", "reduced vegf secretion from mm cells , thereby reducing mm cell - induced pro - angiogenic activity on endothelial cells .", "vegf represents one of the major pro - angiogenic cytokines responsible for the induction of neoangiogenesis in mm patients [ 769 , 770 ] .", "a distinct microrna profile could distinguish between alk and alk subtypes of alcl , an aggressive form of non - hodgkin 's lymphoma ( nhl ) belonging to the t - cell lineage .", "more than 80% of alk alcl harbor the t(2 ; 5)(p23 ; q35 ) translocation , resulting in the expression of the chimeric nucleophosmin ( npm)-alk .", "the constitutive alk activity leads to the activation of many different growth - promoting and anti - apoptotic pathways including pi3k / akt / mtor , jak / stat , c - jun , junb , and c - myc . the prognosis of alk alcl is worse [ 772 , 773 ] .", "alk alcl has a high cure rate with chop treatment , in contrast to alk cells that are relative resistant .", "five members of the mir-17~92 cluster were expressed higher in alk alcl , whereas mir-155 was expressed more than 10-fold higher in alk alcl .", "the upregulation of mir-17~92 cluster in alk alcl cells is in agreement with the observation that c - myc is expressed in alk alcl and absent from alk samples .", "mir-101 targets mtor , mcl-1 , and the histone methyltransferase ezh2 [ 778 , 779 ] . inhibition of mtor , which is targeted by mir-101 , led to reduced tumor growth in engrafted alcl mouse models .", "mir-29a and mir29b down - regulation in alk alcl confer apoptotic resistance due to mcl-1 upregulation [ 380 , 587 ] .", "another microrna that has been implicated in npm - alk - driven oncogenicity is mir-135b .", "mir-135b inhibition reduced tumor angiogenesis and growth in vivo , suggesting that targeting this microrna has therapeutic potential .", "a 9-mirna signature ( mir-146b-5p , mir-146a , mir-21 , mir-155 , mir-500 , mir-222 , mir-363 , mir-574 - 3p , and mir-574 - 5p ) could differentiate the diffuse large b - cell lymphoma ( dlbcl ) , the most common subtype of non - hodgkin 's lymphoma , into abc ( activated b - cell ) or gcb ( germinal center b - cell ) subtypes , with a general higher expression in the abc subtype .", "another study found that mir-331 , mir-151 , mir-28 , and mir-454 were upregulated in the gcb type , whereas mir-222 , mir-144 , mir-451 , and mir-221 upregulated in the abc type .", "the microrna expression of both gcb - like and abc - like cells was more similar to germinal center lymphocytes than memory b - cells .", "the region encoding the mir-17~92 cluster was more commonly amplified in gcb - like than abc - like dlbcl .", "lawrie et al . identified 3 mirnas , mir-155 , mir-21 , and mir-221 , more highly expressed in abc type than gcb type cells .", "expression of mir-155 and mir-21 was also higher in nonmalignant abc than in gcb cells .", "patients with gcb dlbcl have longer overall survival and event - free survival compared with patients with an abc phenotype when treated with r - chop [ 782 , 783 ] . increased expression of mir-18a in dlbcl was associated with a shorter os ( overall survival ) of patients receiving r - chop regimen .", "increased expression of mir-181a was associated with longer pfs ( progression - free survival ) , while increased expression of mir-222 was associated with shorter pfs . in dlbcl , mir-181a regulates foxp1 ( forkhead box protein p1 ) and mgmt ( o - methylguanine - dna methyltransferase ) expression in dlbcl cells .", "foxp1 is expressed in normal activated b cells , mantle zone b cells , and some germinal center b cells [ 784 , 785 ] .", "foxp1 is recurrently targeted by chromosomal translocations involving the immunoglobulin heavy chain locus in marginal zone lymphomas and dlbcl , suggesting a potential role for foxp1 in lymphomagenesis [ 786 , 787 ] .", "foxp1 has in some studies been shown to be associated with poor prognosis and survival [ 788 , 789 ] .", "the ability of mir-181a to reduce mgmt protein expression may contribute to better cyclophosphamide chemosensitivity .", "mir-222 is part of the mir-221/mir-222 cluster , which is highly expressed in abc - like dlbcl cell lines and abc - like dlbcl tumors .", "mir-222 regulates the expression of the stem cell factor c - kit , and the cyclin - dependent kinase inhibitors p27 and p57 [ 695 , 698 ] .", "high expression of mir-222 was associated with inferior overall survival and progression - free survival .", "fl is characterized by high mir-9 , mir-138 , mir-20a / b , and mir-155 expression [ 135 , 564 , 582 ] . \n", "mir-9 targets also the transcription factor prdm1/blimp1 in lymphoma and may contribute to the phenotype maintenance and pathogenesis of lymphoma cells by interfering with normal b - cell terminal differentiation [ 582 , 608 ] .", "brdm1/blimp1 and bcl6 are critical regulators of germinal center b - cell differentiation [ 594 , 792 , 793 ] .", "brdm1/blimp1 and bcl6 are expressed in a mutual exclusive pattern and evidence suggests that they repress each other in germinal center b cells [ 792 , 794 ] .", "a marked decrease of brdm1/blimp1 and an increase of bcl6 were observed in follicular lymphoma cells , which might be related to the increased mir-9 levels in these cells .", "mutations in brdm1/blimp1 are frequently found in activated b cell ( abc)-like dlbcl [ 790 , 795 ] .", "the malignant hodgkin 's lymphoma cells are usually derived from b cells , but have lost the expression of typical b - cell genes .", "multiple signaling pathways are deregulated , including nfb , jak ( janus kinase)/stat ( signal transducer and activator of transcription ) , pi3k / akt , erk , notch1 , and receptor tyrosine kinases .", "patients with low mir-135a expression had a higher probability of relapse and a shorter disease - free survival .", "mir-135a targets jak2 , a cytoplasmic tyrosine kinase involved in a subset of cytokine receptor signaling pathways", ". transfection of pre - mir-135a into classical hl ( chl ) caused apoptosis and decreased cell growth .", "the mir-135a - mediated jak2 down - regulation led to decreased bcl - xl expression , a downstream effector of jak2 .", "about 40%60% of hodgkin 's lymphomas have ebv ( epstein - barr virus ) associated with the malignant cells .", "since mir-155 is overexpressed in hodgkin 's lymphoma and promotes b - cell lymphoma formation [ 602 , 798 , 799 ] , ebv may be important in the pathogenesis of chl ." ], [ "micrornas have been shown to modulate gr expression in neuronal tissue [ 649 , 800 , 801 ] .", "mir-18 and mir-124a especially reduced gr - mediated events in addition to decreasing gr protein levels .", "upregulation of the mir-17~92 has causally been related to small cell lung cancer [ 802 , 803 ] , where reduced gr levels have been associated with gc resistance .", "activation of the gr - responsive glucocorticoid - induced leucine zipper ( gilz ) was impaired by mir-124a and -18 overexpression , while mirs-22 , -328 , and -524 did not have any effect .", "of note , mir-124 regulates hes1 expression in p19 teratocarcinoma cells , a transcription factor that negatively regulate gr expression .", "while mir-130b , -181a , and -636 have putative complimentary binding sites in the 3-utr of gr , only mir-130b was found to down - regulate endogeneous gr protein expression in the multiple myeloma cell line mm.1 .", "the mir-130b , -181a , and -636 were differentially expressed between gc - sensitive and gc - resistant mm.1 cell lines .", "overexpression of mir-130b in mm.1s cells resulted in decreased expression of endogeneous gr , decreased induction of the gr - target gene gilz , and induction of gc resistance .", "expression of mir-130b was therefore suggested to be a potential biomarker for patients who could be refractory to gc therapy . in gastric cancers , mir-130b regulated the tumor suppressor gene runx3 .", "mir-130b may also down - regulate p21 , resulting in inhibition of cellular senescence [ 676 , 806 ] .", "another study showed that elevated mir-142 expression in human t - all cells confers gc resistance by reducing the gr expression level .", "other mechanism for the oncogenic role of mir-142 might be explained by its targeting of adenylyl cyclase 9 mrna leading to reduced production of cyclic adenosine monophosphate ( camp ) production with concomitant inhibition of the protein kinase a ( pka ) signaling pathway .", "the reduction in camp levels and reduced pka activity caused by mir-142 relieve the inhibitory effect of pka on t - leukemic cell proliferation .", "t - all with poor prognosis expressed higher levels of mir-142 than those with good prognosis .", "also , mir-142 was expressed at higher levels in relapsed t - all than newly diagnosed samples .", "transfection of mir-142 inhibitor increased gr expression levels and sensitized t - all cells to gc - induced apoptosis .", "these findings are in accord with previous findings showing a synergistic effect of camp mimetics on gc - induced apoptosis [ 99 , 460 , 807 ] .", "camp signaling can also be negatively regulated by phosphodiesterase 4b ( pde4b ) that is frequently overexpressed in diffuse large b - cell lymphoma ( dlbcl ) .", "pharmacological inhibition of pde4 in a xenograft model of human lymphoma unleashed camp effects , inhibited akt , and restored gc sensitivity .", "pde4 inhibitors may thus improve the clinical outcome of patients with b - cell malignancies .", "triptolide , a drug that overcomes dexamethasone - resistance in human multiple myeloma cells , was found to regulate gr expression in the mm1.s cell line by downregulating the expression of mir-142 and mir-181a . mir-142 and mir-181a mimetics slightly attenuated , whereas mir-142 and mir-181a inhibitors enforced gc - induced apoptosis of mm1.s cells .", "mir-181a / b can also increase gc - induced apoptosis in virtue of their ability to repress the expression of the anti - apoptotic bcl-2 , mcl-1 , and xiap proteins [ 385 , 539 , 810 ] . ", ". showed that broad microrna repression occurs during gc - induced apoptosis of rat thymocytes .", "this repression was associated with reduced expression of both nuclear ( drosha and dgcr8/pasha ) and cytoplasmic ( dicer ) microrna processing enzymes .", "silencing of dicer in two human leukemic cell lines ( cem - c7 and ectopic gr-overexpressed jurkat cells ) led to enhanced sensitivity to gc - induced apoptosis .", "global downregulation of microrna levels , especially the mir-17 family , by gcs was also observed in gc - sensitive all cell lines , with concomitant upregulation of bim .", "later studies showed that gcs selectively upregulate and downmodulate specific mirnas that can not be explained by altered dicer expression .", "one polycistron cluster repressed by gcs is mir-17~92 [ 648 , 657 ] , which regulates bim expression [ 246 , 247 ] .", "this microrna cluster also represses pten , a negative regulator of the pi3k / akt signaling pathway .", "the gc - mediated downregulation of mir-17~92 might be one mechanism responsible for the gc - induced dephosphorylation of akt .", "primary thymocytes derived from mice transgenic for the mir-17~92 polycistron members in the lymphocyte compartment exhibited diminished sensitivity to gc - induced apoptosis in lymphocytes , further supporting a role for gc - induced repression of mir-17~92 in promoting apoptosis .", "observed that gcs reduced mir-17 family expression in 50% of primary gc - sensitive all , but not in any of the gc - resistant ones .", "overexpression of mir-17~92 attenuated gc - induced cell death , while inhibition of mir-17~92 increased the sensitivity to gc .", "they also reported that in a pre - b all cell line , a 10-hour dexamethasone treatment led to a reduction in mir-142 and mir-27a , while mir-9 was induced .", "there is also some evidence that gcs can reduce mir-27a expression in mouse muscle cells .", "rainer et al . reported an induction of the myeloid - specific mir-223 and the apoptosis and cell - cycle arrest inducing mir-15~16 clusters by gc in a subset of b- and t - all cells , together with downregulation of the mir-17~92 complex .", "the mirnas of the mir-15~16 family are encoded in two clusters ( 15a~16 - 1 and 15b~16 - 2 ) embedded in the dleu2 ( deleted in leukemia 2 ) and smc4 loci , respectively [ 766 , 812 ] .", "they have been implicated in cell - cycle arrest and in cell death / survival decisions , the latter supposedly by targeting bcl-2 .", "other micrornas affected by gcs in pediatric all include upregulation of mir-548d-1 and repression of mir-128b along with mir-106b~25~93 , the paralogue of mir-17~92 .", "it is still not known whether the gc - induced upregulation of mir-223 affects gc - induced apoptosis .", "increased expression of mir-223 is involved in the differentiation of myeloid precursors into granulocytes such as neutrophils [ 701 , 814 ] . during granulopoiesis ,", "mir-223 targets e2f1 , which in turn represses mir-223 expression , creating an autoregulatory negative feedback loop .", "a negative feedback loop also exists between mir-223 and the transcription factor nfi - a .", "mir-223 is positively regulated by c / ebp during differentiation to granulocytes and negatively regulated by aml1/eto in leukemia cells .", "moreover , mir-223 targets the myeloid elf-1-like factor ( mef)-2c and igfr ( insulin - like growth factor receptor ) , which may account for some of its negative regulation of granulocyte proliferation . through suppression of igf-1r , the downstream pi3k / akt / mtor / p70s6k pathway is suppressed , with consequent inhibition of cell proliferation . mir-223 attenuates hematopoietic cell proliferation and positively regulates mir-142 through lmo2 isoforms and c / ebp .", "mir-223 knockout mice showed increased numbers of granulocyte progenitors in the bone marrow and hypermature neutrophils in the circulation , suggesting that mir-223 is involved in the negative regulation of maturation rather than differentiation of granulocytes . mir-223", "may also target fbw7 [ 705 , 816 ] , a negative regulator of the anti - apoptotic mcl-1 .", "dexamethasone treatment of thymocytes led to upregulation of mir-150 and mir-342 , while mir-181a and mir-181d were downregulated .", "lif is a member of the il-6 cytokine family expressed in thymic epithelial cells and t lymphocytes , which elevates gc levels following lps exposure and is responsible for thymic atropy induced by stress [ 817819 ] .", "a recent report showed that gcs could prevent lipopolysaccharide ( lps)-mediated inflammatory responses in macrophages by downregulating mir-155 .", "overexpression of mir-155 reversed the suppressive action of gcs , while inhibition of mir-155 exhibited an effect similar to that of gcs on lps - treated macrophages , suggesting that gc - induced repression of mir-155 is one mechanism for the immunosuppressive function of gc .", "mir-155 is transcribed from b - cell integration cluster ( bic ) [ 584 , 691 ] and targets among others socs1 ( suppressor of cytokine signaling 1 ) , which negatively regulates jak / stat signaling .", "gcs also prevented the lps - mediated upregulation of mir-146 , mir-147 , mir-148 , mir-32b , and mir-301 in macrophages . in the brain", ", gcs prevents bdnf ( brain - derived neurotrophic factor)-regulated synaptic function through suppression of mir-132 expression .", "mir-132 is increased by bdnf and is involved in promotion of neuronal outgrowth . in some carcinoma cell lines , dexamethasone was shown to down - regulate mir-27b , mir-148a , and mir-451 . from all we have learned above , any microrna that modulates", "any of the many factors regulating gc - induced apoptosis may affect the apoptotic response to gcs ( figures 16 ) .", "these include micrornas that affect gr expression ( e.g. , mir-18 , mir-124a , mir-130b , mir-142 , and mir-181a ) , those affecting bim expression ( mir-26a , mir-93 , mir-17~92 , mir-106a~363 , and mir-106b~25 ) or its transcription factor foxo3 ( e.g. , mir-1 , mir-21 , mir-27a , mir-96 , mir-135b , mir-155 , and mir-182 ) , those affecting pten expression ( mir-17~92 , mir-106b~25 , mir-21 , mir-26a , mir-29b , mir-214 , mir-216a , mir-217 , mir-221 , and mir-222 ) or mtor ( e.g. , mir-101 ) , and those downregulating directly or indirectly the anti - apoptotic proteins bcl-2 , bcl - xl , mcl-1 , xiap , and cyld ( e.g. , mir-15a~16 , mir-181a / b , mir-34a , mir-125b , mir-29a / b / c , mir-101 , mir-133b , mir-193b , mir-512 , let-7 , and mir-491 ) . the effect of some of these micrornas on gc - sensitivity has already been described above and will not be repeated here .", "rather , i will present here some data from primary samples showing the influence of micrornas on clinical outcome .", "a study searching for differential mirnas expression in all relapse cells versus childhood all with complete remission showed significant associations for mir-708 , mir-223 , and mir-27a with individual relapse - free survival .", "for samples at relapse versus diagnosis , the most differentially expressed micrornas included mir-223 , mir-23a , let-7 g , mir-181 , mir-708 , and mir-130b , while comparison of complete response with diagnostic samples showed differential expression pattern of mir-27a , mir-223 , mir-23a , mir-181 , and mir-128b . among these micrornas , mir-223 , mir-128b , mir-23a , and let-7 g", "were downregulated in the relapse samples compared with complete response samples , while mir-181 family members , mir-708 , and mir-130b were upregulated in the relapse samples .", "it should be remained here that mir-130b targets gr , runx3 , and p21 , and mir-223 is upregulated by gcs and targets igfr and e2f1 .", "e2f1 has a dual role in cell - cycle control , as it affects several cell processes .", "it can either act as a tumor - suppressor or oncogene depending on the cellular context .", "thus , the upregulation of mir-130b together with downregulation of mir-223 may contributes to gc resistance .", "mir-708 was the most upregulated microrna in the relapse samples , whereas mir-223 was significantly downregulated , suggesting that these two micrornas may have important role in pediatric all relapse .", "moreover , upregulation of mir-708 was found to be associated with the in vivo gc therapy response and with disease risk stratification in childhood all .", "standard and middle risk stratification groups had a higher mir-708 expression at diagnosis than the high risk group .", "interestingly , mir-708 was low in high relapse patients at diagnosis , while specimens of relapsed samples showed abundance of mir-708 , suggesting for an upregulation of mir-708 during disease progression .", "foxo3 , that is critical for hematopoietic stem cell self - renewal and mediates the initial apoptotic response [ 825827 ] , contains a conserved mir-708 response element in its 3-utr .", "foxo3 can act as either an oncogene or a tumor suppressor in leukemia [ 828 , 829 ] .", "foxo3 transcriptional activity was found to prevent b - cll and cml proliferation [ 825 , 828 ] .", "foxo3a is also targeted by other micrornas , including mir-27a ( see section 2.2.6 ) .", "moreover , mir-27a directly regulates the drug - resistant factor p - glycoprotein , and overexpression of mir-27a increased sensitivity of leukemia cells to doxorubicin .", "mir-27a is relevant to treatment outcome in vivo and may be involved in relapse of both lymphocytic leukemia and myeloid leukemia .", "low expression of mir-27a might promote all relapse [ 655 , 658 ] . on the contrary ,", "mir-27a exerts oncogenic effects by regulating zbtb10 [ 446 , 830 ] and fbw7 [ 253 , 438 ] .", "mir-128b , which was higher in relapse all and at diagnosis compared to complete response , has been reported to confer drug resistance in many cancers including all [ 672 , 673 ] .", "both mir-27a and mir-128b might target bmi1 , a transcription factor of the polycomb - group gene necessary for hematopoietic stem cell ( hsc ) and leukemia stem - cell self - renewal [ 831 , 832 ] .", "deletion of bmi1 inhibits self - renewal of tumor stem cells and prevents leukemia recurrence .", "a role for mir-128 and mir-221 in regulating gc sensitivity in cells from mll - af4 all patients has been proposed . mir-128b and mir-221 are downregulated in mll - arranged all relative to other types of all . the mll gene is located at 11q23 , a site frequently involved in chromosomal translocations in aggressive human lymphoid and myeloid leukemias . as a result of chromosomal translocations ,", "a portion of mll becomes fused to one among more than 40 different partner proteins .", "mll - af4 all , which results from the translocation between mll and af4 , is associated with gc resistance and has a poor prognosis [ 834 , 835 ] .", "re - expression of mr-128 and mir-221 in cultured mll - af4 all cells sensitized them to gcs .", "mir-128 targets mll , af4 , and the mll - af4 pusion protein resulting in lower expression of hoxa9 , whereas mir-221 downregulates cdkn1b ( cyclin - dependent kinase inhibitor 1b , p27 ) , another gene transcriptionally activated by mll - af4 as well as the wild - type mll protein .", "the targeting of different proteins may explain the cooperative effect of mir-128b and mir-221 on gc sensitization .", "it should be noted that mir-221 in other settings , for example , cll , has anti - apoptotic effects and functions as an oncogene . in light of the multiple effects of various micrornas on cell survival and apoptosis", ", modulating microrna expression in tumor cells is an attractive approach for sensitizing the tumor cells to chemotherapeutic drugs .", "inhibition of specific micrornas is performed by using antisense sequences ( termed antagomirs ) targeting the microrna guide stand that blocks the interaction with the microrna recognition elements within the 3-utr of the target mrna genes . to increase their binding affinity and stability in biological fluids , the antagomirs", "are often modified with 2-o - methyl- , phosphorothioate , or locked nucleic acid substitutions . to overexpress micrornas ,", "one potential use of micrornas is to repress the expression of mll - af4 fusion protein in all that is responsible for gc resistance .", "this fusion protein can be repressed through overexpression of mir-143 , or mir-128 together with mir-221 .", "the latter combination was shown to sensitize the mll - af4-carrying all cells to gcs . ", "another promising approach is to target mir-155 , an oncogenic microrna often correlated with poor prognosis .", "they showed that overexpression of mir-155 in lymphoid tissues resulted in disseminatedlymphomacharacterized by a clonal , transplantable pre - b - cell population of neoplastic lymphocytes .", "systemic delivery of antisense peptide nucleic acids encapsulated in unique polymer nanoparticles inhibited mir-155 and slowed the growth of these pre - b - cell tumors in vivo ." ], [ "glucocorticoid - induced apoptosis appears to be a complex process involving several signaling pathways ( figure 6 ) .", "these include ( 1 ) transactivation of pro - apoptotic genes ( importantly bim ) ; ( 2 ) alterations in microrna expression ( upregulation of mir-15~16 that targets the pro - apoptotic bcl-2 ; mir-223 that targets igfr ; mir-150 that targets akt and notch , while suppressing mir-17~92 that prevents bim and pten translation ) ; ( 3 ) direct action of gr on the mitochondria ( including mitochondrial gr translocation and production of reactive oxygen species within the mitochondria ) ; ( 4 ) activation of the protein kinases gsk3 and p38 ; ( 5 ) activation of the foxo3a transcription factor that upregulates bim ; ( 6 ) inhibition of the notch1 , pi3/akt / mtor , and erk1/2 survival pathways .", "altered microrna expression in malignant cells may modulate many of these processes thereby imposing apoptotic resistance ( figures 16 ) .", "gc - resistant lymphoid cells might be divided into two major subgroups according to the underlying mechanism of resistance .", "the first group consists of cancer cells whose drug resistance can be overcome by exposing the cells to gcs in combination with drugs that target protein kinases such as akt , mtor , src , alk , and/or bcr , or drugs antagonizing bcl-2 , bcl - xl , mcl-1 , c - myc , or notch .", "these lymphoid malignancies show in general a more favorable response to combined gc therapy and in many cases may be explained by their growth dependency on these signaling molecules .", "the second group of gc - resistant cells exhibits an intrinsic defect in the gc - mediated apoptotic process and can thus not be turned sensitive to this drug .", "it is important to distinguish between these two subgroups prior to therapy initiation in order to choose the right drug combination .", "a diagnostic test needs to be developed that can distinguish between the different resistance backgrounds .", "recently , burnsides et al . have developed an ex vivo stimulation assay that determines the ability of leukocytes to upregulate anti - inflammatory genes such as gilz and fkbp51 following exposure to dexamethasone .", "it is reasonable that a similar test may be developed to gene profiling lymphoid malignancies prior to and following gc treatment , where upregulation of the pro - apoptotic bim gene would be a favorable predictor .", "also , bim induction may be measured after combining gc with a protein kinase inhibitor .", "simultaneous expression profiling of micrornas , notch1 , and bcl-2 family proteins together with the activated protein kinase status in the malignant cell would provide valuable information for choosing the proper drug combination . a predictor for a good gc response would be to determine the ability of gcs to downregulate mir-17~92 and upregulate mir-15~16 , mir-150 , and mir-223 .", "a tentative therapeutic approach would be to modulate the microrna status of the cell using microrna mimics or antagomirs as described in section 4.4 .", "what we have learned from the studies described in this paper is that it seems that in general it would be favorable to augment the expression of mir-29 , mir-27 , mir-15a~16 , mir-34a , mir-150 , and let-7 , while suppressing mir-155 , mir-181 , mir-182 , mir-21 , and mir-221/222 as well as mir-17~92 .", "obviously , an initial microrna profiling should be performed , and the cancer - type classification should be considered .", "while down - regulation of mir-181 may suppress the growth of t - all and mm , augmented mir-181 expression prevents the growth of unmutated igvh cll cases .", "also , mir-26a has a dual effect . its overexpression prevents growth of c - myc - positive burkitt lymphoma , while it must be downregulated in notch - positive t - all to achieve growth inhibition . mir-451 and mir-709 could prevent growth of notch - positive t - all . a reduction in mir-142 , and maybe also of mir-708 , which is highly expressed in relapsed childhood t - all , is anticipated to improve t - all therapy . for classical hl , mir-135a may cause apoptosis . in conclusion , in certain types of lymphoid malignancies", ", gc resistance may be overcome by relieving the inhibitory effects of protein kinases and bcl-2 family members .", "both the activity of protein kinases and the expression of bcl-2 members are affected by the microrna network .", "modulation of microrna expression might increase gc drug responsiveness and thus improve the therapy of lymphoid malignancies ." ] ]

[ "advances in cancer treatment have resulted in improved relative survival rates . however , many oncology patients are faced with treatment - related symptoms and conditions that lead to increased use of health services , including emergency departments ( eds ) .", "febrile neutropenia ( fn ) is such a condition and remains one of the most common causes of oncological patient presentation to the ed .", "febrile neutropenia is a life - threatening treatment - related condition that requires urgent management in the ed .", "although there have been many advances in the treatment of fn , such patients are known to be at risk of serious infections that are associated with 1242% mortality .", "the management of febrile neutropenic patients should be based on their clinical course and possible infection source .", "early prediction of bacteremia with timely and tailored empiric antimicrobial therapy will improve the prognosis of patients with fn .", "recent progress in the treatment of neutropenic fever has underscored the importance of risk stratification and indicated the need to evaluate predictive factors for outcomes in fn .", "patient - specific risk factors , comorbid conditions , performance status , type of cancer and stage , age , number of previous febrile neutropenic episodes , and the severity and duration of neutropenia have been considered important predictors of outcomes .", "based on the characteristics of fn patients , several predictive models have been developed to classify patients into low- or high - risk groups .", "( 2000 ) developed an internationally validated scoring system called the multinational association of supportive care in cancer ( mascc ) risk index score .", "the mascc score also allows the selection of low - risk patients who can be safely treated with orally administered antibiotics .", "a mascc score 21 indicates that the patient is at low risk of complications and mortality ( table 1 ) . the clinical risk - prediction model proposed by the mascc", "is widely used in clinical practice to define low - risk febrile neutropenia . in the present study", ", we analyzed the clinical factors predictive of poor outcomes in patients with chemotherapy - induced fn at initial patient evaluation in the ed .", "all adult chemotherapy - induced fn patients who presented to the ed of the university of ankara hospital from january 1 , 2011 to december 31 , 2013 were included in this study .", "approval to conduct the study was granted by the ethics committee of the university of ankara .", "overall , 200 chemotherapy - induced fn patients over 18 years of age and with hematological and oncological malignancies were evaluated retrospectively .", "fever was defined as body temperature greater than 38.3c at triage or a temperature of 38c for 1 h or longer during the first 24 h .", "a c - reactive protein ( crp ) cut - off value of 50 mg / dl was used , as proposed in a previous study .", "clinical data were obtained from medical records in the electronic patient record system . upon arrival in the ed ,", "we collected information regarding clinical data ( sex , age , vital signs , underlying systemic diseases ) , laboratory test results ( assessment of complete blood count , blood chemistry with differential , liver and renal function tests , blood glucose , electrolytes , protein , estimated glomerular filtration rate [ egfr ] , blood cultures , crp concentration ) , and radiological examinations ( a chest radiograph of all febrile neutropenic patients ) . egfr was calculated from serum creatinine using the modification of diet in renal disease ( mdrd ) study equation .", "the mascc risk index score was calculated for all patients with chemotherapy - induced neutropenic fever .", "outcomes were categorized as good if the patient could be discharged without any serious complications and poor if there were serious complications during hospitalization or if death occurred .", "serious complications included refractory hypotension ( defined as systolic blood pressure less than 90 mmhg that required treatment with vasopressors agents ) , respiratory failure ( defined as the need for mechanical ventilation ) , need for intensive care unit ( icu ) admission , renal failure , need for treatment with fluids or hemodialysis , severe bleeding and need for transfusion , fungal infection , altered mental status , arrhythmia requiring treatment , chronic heart failure , and allergic reactions to treatment .", "this list of serious medical complications was adapted and modified from klastersky et al . .", "the following data were collected : a ) variables that were available at presentation , such as age , sex , laboratory variables ( hemoglobin , neutrophil count , crp , egfr , protein , platelet count ) , chest x - ray , and comorbidities ( e.g. , diabetes mellitus , heart failure , copd , acute coronary syndromes ) ; b ) other variables that were determined at triage such as vital signs ( respiratory and pulse rates , systolic blood pressure ) ; and c ) variables related to blood cultures and complications during hospitalization .", "differences between the 2 groups were evaluated using the t - test for continuous variables .", "categorical variables were assessed using the chi - square test , and odds ratios were calculated . to define the risk factors of the outcome variables ( mortality and complications ) ,", "the sensitivity , specificity , and positive and negative predicted values were calculated for diagnostic performance of the multiple logistic regression model .", "differences between the 2 groups were evaluated using the t - test for continuous variables .", "categorical variables were assessed using the chi - square test , and odds ratios were calculated . to define the risk factors of the outcome variables ( mortality and complications ) , multiple logistic regression analysis was used , and adjusted odds ratios were calculated .", "the sensitivity , specificity , and positive and negative predicted values were calculated for diagnostic performance of the multiple logistic regression model .", "during the study period , there were 200 episodes of fn in 200 patients with cancer .", "the mean and median age of the study patients were 56.413.46 ( mean standard deviation ) and 57.5 , respectively .", "of the 200 febrile episodes , 72 ( 36% ) had bacteremia and 128 ( 64% ) were categorized as an unexplained fever ( table 2 ) .", "serious medical complications were observed in 105 ( 52.5% ) patients and 64 ( 32% ) patients died during hospitalization ( table 3 ) .", "univariate analyses of the clinical parameters available from the ed and mascc scoring system were performed ( table 4 ) . in the multiple logistic regression analysis , a platelet count < 50 000 cells / mm ( or 3.93 , 95% ci 1.4210.92 ) , serum crp > 50 mg / dl ( or 3.80 , 95% ci 1.688.61 ) , hypoproteinemia ( or 7.81 , 95% ci 3.4317.78 ) , egfr 90 ml / min/1.73 m ( or 3.06 , 95% ci 1.138.26 ) , and mascc risk - index score < 21 ( or 3.45 , 95% ci 1.537.79 ) were determined to be independent risk factors for the prediction of poor clinical outcomes of fn patients admitted to the ed ( table 5 ) .", "these risk factors were predictive of serious complications with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 81% , 78% , 81% , and 79% , respectively .", "as shown in table 6 , the clinical parameters and mascc scores were evaluated with univariate analysis . in the multiple logistic regression analysis , a platelet count < 50 000", "cells / mm ( or 3.90 , 95% ci 1.629.43 ) , pulmonary infiltration ( or 3.45 , 95% ci 1.488.07 ) , hypoproteinemia < 6g / dl ( or 3.30 , 95% ci 1.278.56 ) , respiratory rate > 24/min ( or 8.75 , 95% ci 2.1835.13 ) , and mascc risk - index score < 21 ( or 9.20 , 95% ci 3.9821.26 ) were determined to be independent risk factors for death ( table 7 ) .", "these parameters were predictive of death with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 75% , 89% , 77% , and 88% , respectively .", "during the study period , there were 200 episodes of fn in 200 patients with cancer .", "the mean and median age of the study patients were 56.413.46 ( mean standard deviation ) and 57.5 , respectively .", "of the 200 febrile episodes , 72 ( 36% ) had bacteremia and 128 ( 64% ) were categorized as an unexplained fever ( table 2 ) .", "serious medical complications were observed in 105 ( 52.5% ) patients and 64 ( 32% ) patients died during hospitalization ( table 3 ) .", "univariate analyses of the clinical parameters available from the ed and mascc scoring system were performed ( table 4 ) . in the multiple logistic regression analysis , a platelet count < 50 000 cells / mm ( or 3.93 , 95% ci 1.4210.92 ) , serum crp > 50 mg / dl ( or 3.80 , 95% ci 1.688.61 ) , hypoproteinemia ( or 7.81 , 95% ci 3.4317.78 ) , egfr 90 ml / min/1.73 m ( or 3.06 , 95% ci 1.138.26 ) , and mascc risk - index score < 21 ( or 3.45 , 95% ci 1.537.79 ) were determined to be independent risk factors for the prediction of poor clinical outcomes of fn patients admitted to the ed ( table 5 ) .", "these risk factors were predictive of serious complications with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 81% , 78% , 81% , and 79% , respectively .", "as shown in table 6 , the clinical parameters and mascc scores were evaluated with univariate analysis . in the multiple logistic regression analysis , a platelet count < 50 000 cells / mm ( or 3.90 , 95% ci 1.629.43 ) , pulmonary infiltration ( or 3.45 , 95% ci 1.488.07 ) , hypoproteinemia < 6g / dl ( or 3.30 , 95% ci 1.278.56 ) , respiratory rate > 24/min ( or 8.75 , 95% ci 2.1835.13 ) , and mascc risk - index score < 21 ( or 9.20 , 95% ci 3.9821.26 ) were determined to be independent risk factors for death ( table 7 ) .", "these parameters were predictive of death with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 75% , 89% , 77% , and 88% , respectively .", "the development of multi - drug chemotherapy protocols in the treatment of hematological conditions and solid organ tumors , use of high - dose drugs , and improved supporting treatment options have improved the chances of survival for cancer patients in recent years", ". however , these treatment regimens also result in immunosuppression and neutropenia , conditions that make patients prone to developing severe infections .", "currently , clinicians are aiming to prolong life with these new anti - cancer treatments while also reducing adverse effects such as fn .", "one of the most important subjects of recent research is the ability to predict the prognosis of these patients .", "guidelines have been developed to categorize fn patients into low- or high - risk groups .", "this scoring system , which predicts prognosis , is believed to be more useful in low - risk patients .", "high fever in a neutropenic patient requires prompt treatment with broad - spectrum antibiotics until the culture results are obtained .", "this urgent approach requires emergency physicians to predict independent risk factors for fn apart from those indicated by the massc risk - scoring system .", "however , only a few studies have been conducted in the ed setting to identify independent factors associated with serious complications after the emergent diagnosis of fn . in this study ,", "independent predictive factors for serious complications in patients diagnosed with fn were identified with a sensitivity of 81% and specificity of 78% .", "previous studies that aimed to predict complicated fn in the ed found that laboratory parameters ( e.g. , platelet count , crp , and pulmonary infiltration as revealed by chest x - ray ) identified patients likely to develop complications .", "our results are in agreement with these studies ; platelet counts of 50 000cells / mm and crp , an indicator of inflammation , were associated with serious complications .", "platelets are unique blood cells with specialized molecular repertoires that have evolved to accomplish crucial functions in host integrity , defense , and repair .", "growing evidence shows that platelets play an active role in fighting infection and innate immunity , and thrombocytopenia is frequently observed in systemic infections .", "previous studies have found that the severity of thrombocytopenia is associated with increased mortality rates [ 1315 ] . in our study , the level of serum crp was also recognized as an independent factor . because infections are the major cause of morbidity and mortality in neutropenic patients , considerable attention has focused on the value of acute - phase reactants in identification of high - risk patients .", "however , results from previous studies have been inconsistent , and their cut - off points are variable .", "we used a cut - off value of crp of 50 mg / dl , as proposed in a previous study .", "several studies have reported that crp is a significant predictor of severe sepsis , while other studies have stated that crp was not useful in predicting infectious complications in neutropenic patients . however , in our study , we found crp ( or 3.8 , 95% ci 1.688.61 ) to be a significant predictor of serious complications . in our study , egfr < 90 ml / min/1.73 m is associated with increased risk of serious complications .", "the observed increased risk of serious complications from neutropenic patients with reduced egfr is considerable , and we suggest that decreased kidney function itself is a predictor of poor outcomes .", "first , previous studies have shown that patients with chronic kidney diseases ( ckd ) are less likely to undergo cancer screening , and patients with ckd also have multiple comorbid conditions .", "there is little information regarding the optimal timing and necessary dose adjustment of cytotoxic agents for patients with reduced kidney function . in a previous study , iff et al . stated that an egfr < 60 ml / min/1.73 m is a risk factor for cancer deaths and is also a predictor of poor cancer outcomes in the older population . except for their study , we were unable to find another study that evaluated the egfr threshold associated with an increased risk of cancer death . in another study ,", "g / l ) was found to be associated with severe sepsis in neutropenic patients . in our study , hypoproteinemia was another independent factor that could predict serious complications in febrile neutropenic patients .", "this finding can be explained by the fact that intensive chemotherapy can lead to energy consumption and high protein catabolism .", "these are common features in progressive advanced cancer and are responsible for increased severe hematological toxicity .", "clinical malnutrition , which negatively affects patient response to therapy , increases the incidence of treatment - related adverse effects and can decrease survival .", "numerous studies have shown that patients with a mascc risk index score of less than 21 should be considered at high risk for complications , as we did in our study . in this study ,", "independent predictive factors for death in patients diagnosed with fn have been identified with a sensitivity of 75% and specificity of 89% .", "we found that hypoproteinemia , platelet counts of 50 000 cells / mm , and the mascc risk index score were independent predictive factors .", "additionally , a respiratory rate > 24/min ( or 8.75 , 95% ci 2.1835.13 ) was the only component in vital signs that was predictive of exitus in multivariate analysis .", "in a recent study of patients with hematological malignancies , tachypnea was a predictor of septic shock and of poor prognosis of febrile neutropenic patients . in previous studies , infiltration or abnormality in the chest x - ray", "both aspects could be responsible for the mortality rate of up to 2550% ( 2426 ) . in our study ,", "pulmonary infiltration was significantly predictive for mortality ( or 3.25 , 95% ci 1.478.07 ) .", "this was a retrospective analysis of a small population in a single - center study .", "therefore , our results may not be representative of all other institutions and require validation .", "the results of our study may help emergency medicine physicians to prevent the development of serious complications and death in fn patients by the proper use of simple independent risk factors in addition to the mascc score .", "early stratifications of patients into low- and high - risk groups with timely and tailored empiric antimicrobial therapy can improve the prognosis of patients with fn ." ]

[ "<S> backgroundfebrile neutropenia ( fn ) is a life - threatening condition that requires urgent management in the emergency department ( ed ) . </S>", "<S> recent progress in the treatment of neutropenic fever has underscored the importance of risk stratification . in this study </S>", "<S> , we aimed to determine independent factors for prediction of poor outcomes in patients with fn.material/methodswe retrospectively evaluated 200 chemotherapy - induced febrile neutropenic patients who visited the ed . upon arrival at the ed , clinical data , including sex , age , vital signs , underlying systemic diseases , laboratory test results , estimated gfr , blood cultures , crp , radiologic examinations , and multinational association of supportive care in cancer ( mascc ) score of all febrile neutropenic patients were obtained . </S>", "<S> outcomes were categorized as poor if serious complications during hospitalization , including death , occurred.resultsthe platelet count < 50 000 cells / mm3 ( or 3.90 , 95% ci 1.629.43 ) , pulmonary infiltration ( or 3.45 , 95% ci 1.488.07 ) , hypoproteinemia < 6 g / dl ( or 3.30 , 95% ci 1.278.56 ) , respiratory rate > 24/min ( or 8.75 , 95% ci 2.1835.13 ) , and mascc score < 21 ( or 9.20 , 95% ci 3.9821.26 ) were determined as independent risk factors for the prediction of death . </S>", "<S> the platelet count < 50 000 cells / mm3 ( or 3.93 , 95% ci 1.4210.92 ) , serum crp > 50 mg / dl ( or 3.80 , 95% ci 1.688.61 ) , hypoproteinemia ( or 7.81 , 95% ci 3.4317.78 ) , egfr 90 ml / min/1.73 m2 ( or 3.06 , 95% ci 1.138.26 ) , and mascc score < 21 ( or 3.45 , 95% ci 1.537.79 ) were determined as independent risk factors for the prediction of poor clinical outcomes of fn patients . </S>", "<S> platelet count , protein level , respiratory rate , pulmonary infiltration , crp , mascc score , and egfr were shown to have a significant association with outcome.conclusionsthe results of our study may help emergency medicine physicians to prevent serious complications with proper use of simple independent risk factors besides mascc score . </S>" ]

[ "Background", "Material and Methods", "Statistical analyses", "Results", "Patient characteristics and clinical features", "Risk stratification for serious complications", "Risk stratification for death", "Discussion", "Conclusions" ]

[ [ "advances in cancer treatment have resulted in improved relative survival rates . however , many oncology patients are faced with treatment - related symptoms and conditions that lead to increased use of health services , including emergency departments ( eds ) .", "febrile neutropenia ( fn ) is such a condition and remains one of the most common causes of oncological patient presentation to the ed .", "febrile neutropenia is a life - threatening treatment - related condition that requires urgent management in the ed .", "although there have been many advances in the treatment of fn , such patients are known to be at risk of serious infections that are associated with 1242% mortality .", "the management of febrile neutropenic patients should be based on their clinical course and possible infection source .", "early prediction of bacteremia with timely and tailored empiric antimicrobial therapy will improve the prognosis of patients with fn .", "recent progress in the treatment of neutropenic fever has underscored the importance of risk stratification and indicated the need to evaluate predictive factors for outcomes in fn .", "patient - specific risk factors , comorbid conditions , performance status , type of cancer and stage , age , number of previous febrile neutropenic episodes , and the severity and duration of neutropenia have been considered important predictors of outcomes .", "based on the characteristics of fn patients , several predictive models have been developed to classify patients into low- or high - risk groups .", "( 2000 ) developed an internationally validated scoring system called the multinational association of supportive care in cancer ( mascc ) risk index score .", "the mascc score also allows the selection of low - risk patients who can be safely treated with orally administered antibiotics .", "a mascc score 21 indicates that the patient is at low risk of complications and mortality ( table 1 ) . the clinical risk - prediction model proposed by the mascc", "is widely used in clinical practice to define low - risk febrile neutropenia . in the present study", ", we analyzed the clinical factors predictive of poor outcomes in patients with chemotherapy - induced fn at initial patient evaluation in the ed ." ], [ "all adult chemotherapy - induced fn patients who presented to the ed of the university of ankara hospital from january 1 , 2011 to december 31 , 2013 were included in this study .", "approval to conduct the study was granted by the ethics committee of the university of ankara .", "overall , 200 chemotherapy - induced fn patients over 18 years of age and with hematological and oncological malignancies were evaluated retrospectively .", "fever was defined as body temperature greater than 38.3c at triage or a temperature of 38c for 1 h or longer during the first 24 h .", "a c - reactive protein ( crp ) cut - off value of 50 mg / dl was used , as proposed in a previous study .", "clinical data were obtained from medical records in the electronic patient record system . upon arrival in the ed ,", "we collected information regarding clinical data ( sex , age , vital signs , underlying systemic diseases ) , laboratory test results ( assessment of complete blood count , blood chemistry with differential , liver and renal function tests , blood glucose , electrolytes , protein , estimated glomerular filtration rate [ egfr ] , blood cultures , crp concentration ) , and radiological examinations ( a chest radiograph of all febrile neutropenic patients ) . egfr was calculated from serum creatinine using the modification of diet in renal disease ( mdrd ) study equation .", "the mascc risk index score was calculated for all patients with chemotherapy - induced neutropenic fever .", "outcomes were categorized as good if the patient could be discharged without any serious complications and poor if there were serious complications during hospitalization or if death occurred .", "serious complications included refractory hypotension ( defined as systolic blood pressure less than 90 mmhg that required treatment with vasopressors agents ) , respiratory failure ( defined as the need for mechanical ventilation ) , need for intensive care unit ( icu ) admission , renal failure , need for treatment with fluids or hemodialysis , severe bleeding and need for transfusion , fungal infection , altered mental status , arrhythmia requiring treatment , chronic heart failure , and allergic reactions to treatment .", "this list of serious medical complications was adapted and modified from klastersky et al . .", "the following data were collected : a ) variables that were available at presentation , such as age , sex , laboratory variables ( hemoglobin , neutrophil count , crp , egfr , protein , platelet count ) , chest x - ray , and comorbidities ( e.g. , diabetes mellitus , heart failure , copd , acute coronary syndromes ) ; b ) other variables that were determined at triage such as vital signs ( respiratory and pulse rates , systolic blood pressure ) ; and c ) variables related to blood cultures and complications during hospitalization .", "differences between the 2 groups were evaluated using the t - test for continuous variables .", "categorical variables were assessed using the chi - square test , and odds ratios were calculated . to define the risk factors of the outcome variables ( mortality and complications ) ,", "the sensitivity , specificity , and positive and negative predicted values were calculated for diagnostic performance of the multiple logistic regression model ." ], [ "differences between the 2 groups were evaluated using the t - test for continuous variables .", "categorical variables were assessed using the chi - square test , and odds ratios were calculated . to define the risk factors of the outcome variables ( mortality and complications ) , multiple logistic regression analysis was used , and adjusted odds ratios were calculated .", "the sensitivity , specificity , and positive and negative predicted values were calculated for diagnostic performance of the multiple logistic regression model ." ], [ "during the study period , there were 200 episodes of fn in 200 patients with cancer .", "the mean and median age of the study patients were 56.413.46 ( mean standard deviation ) and 57.5 , respectively .", "of the 200 febrile episodes , 72 ( 36% ) had bacteremia and 128 ( 64% ) were categorized as an unexplained fever ( table 2 ) .", "serious medical complications were observed in 105 ( 52.5% ) patients and 64 ( 32% ) patients died during hospitalization ( table 3 ) .", "univariate analyses of the clinical parameters available from the ed and mascc scoring system were performed ( table 4 ) . in the multiple logistic regression analysis , a platelet count < 50 000 cells / mm ( or 3.93 , 95% ci 1.4210.92 ) , serum crp > 50 mg / dl ( or 3.80 , 95% ci 1.688.61 ) , hypoproteinemia ( or 7.81 , 95% ci 3.4317.78 ) , egfr 90 ml / min/1.73 m ( or 3.06 , 95% ci 1.138.26 ) , and mascc risk - index score < 21 ( or 3.45 , 95% ci 1.537.79 ) were determined to be independent risk factors for the prediction of poor clinical outcomes of fn patients admitted to the ed ( table 5 ) .", "these risk factors were predictive of serious complications with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 81% , 78% , 81% , and 79% , respectively .", "as shown in table 6 , the clinical parameters and mascc scores were evaluated with univariate analysis . in the multiple logistic regression analysis , a platelet count < 50 000", "cells / mm ( or 3.90 , 95% ci 1.629.43 ) , pulmonary infiltration ( or 3.45 , 95% ci 1.488.07 ) , hypoproteinemia < 6g / dl ( or 3.30 , 95% ci 1.278.56 ) , respiratory rate > 24/min ( or 8.75 , 95% ci 2.1835.13 ) , and mascc risk - index score < 21 ( or 9.20 , 95% ci 3.9821.26 ) were determined to be independent risk factors for death ( table 7 ) .", "these parameters were predictive of death with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 75% , 89% , 77% , and 88% , respectively ." ], [ "during the study period , there were 200 episodes of fn in 200 patients with cancer .", "the mean and median age of the study patients were 56.413.46 ( mean standard deviation ) and 57.5 , respectively .", "of the 200 febrile episodes , 72 ( 36% ) had bacteremia and 128 ( 64% ) were categorized as an unexplained fever ( table 2 ) .", "serious medical complications were observed in 105 ( 52.5% ) patients and 64 ( 32% ) patients died during hospitalization ( table 3 ) ." ], [ "univariate analyses of the clinical parameters available from the ed and mascc scoring system were performed ( table 4 ) . in the multiple logistic regression analysis , a platelet count < 50 000 cells / mm ( or 3.93 , 95% ci 1.4210.92 ) , serum crp > 50 mg / dl ( or 3.80 , 95% ci 1.688.61 ) , hypoproteinemia ( or 7.81 , 95% ci 3.4317.78 ) , egfr 90 ml / min/1.73 m ( or 3.06 , 95% ci 1.138.26 ) , and mascc risk - index score < 21 ( or 3.45 , 95% ci 1.537.79 ) were determined to be independent risk factors for the prediction of poor clinical outcomes of fn patients admitted to the ed ( table 5 ) .", "these risk factors were predictive of serious complications with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 81% , 78% , 81% , and 79% , respectively ." ], [ "as shown in table 6 , the clinical parameters and mascc scores were evaluated with univariate analysis . in the multiple logistic regression analysis , a platelet count < 50 000 cells / mm ( or 3.90 , 95% ci 1.629.43 ) , pulmonary infiltration ( or 3.45 , 95% ci 1.488.07 ) , hypoproteinemia < 6g / dl ( or 3.30 , 95% ci 1.278.56 ) , respiratory rate > 24/min ( or 8.75 , 95% ci 2.1835.13 ) , and mascc risk - index score < 21 ( or 9.20 , 95% ci 3.9821.26 ) were determined to be independent risk factors for death ( table 7 ) .", "these parameters were predictive of death with sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 75% , 89% , 77% , and 88% , respectively ." ], [ "the development of multi - drug chemotherapy protocols in the treatment of hematological conditions and solid organ tumors , use of high - dose drugs , and improved supporting treatment options have improved the chances of survival for cancer patients in recent years", ". however , these treatment regimens also result in immunosuppression and neutropenia , conditions that make patients prone to developing severe infections .", "currently , clinicians are aiming to prolong life with these new anti - cancer treatments while also reducing adverse effects such as fn .", "one of the most important subjects of recent research is the ability to predict the prognosis of these patients .", "guidelines have been developed to categorize fn patients into low- or high - risk groups .", "this scoring system , which predicts prognosis , is believed to be more useful in low - risk patients .", "high fever in a neutropenic patient requires prompt treatment with broad - spectrum antibiotics until the culture results are obtained .", "this urgent approach requires emergency physicians to predict independent risk factors for fn apart from those indicated by the massc risk - scoring system .", "however , only a few studies have been conducted in the ed setting to identify independent factors associated with serious complications after the emergent diagnosis of fn . in this study ,", "independent predictive factors for serious complications in patients diagnosed with fn were identified with a sensitivity of 81% and specificity of 78% .", "previous studies that aimed to predict complicated fn in the ed found that laboratory parameters ( e.g. , platelet count , crp , and pulmonary infiltration as revealed by chest x - ray ) identified patients likely to develop complications .", "our results are in agreement with these studies ; platelet counts of 50 000cells / mm and crp , an indicator of inflammation , were associated with serious complications .", "platelets are unique blood cells with specialized molecular repertoires that have evolved to accomplish crucial functions in host integrity , defense , and repair .", "growing evidence shows that platelets play an active role in fighting infection and innate immunity , and thrombocytopenia is frequently observed in systemic infections .", "previous studies have found that the severity of thrombocytopenia is associated with increased mortality rates [ 1315 ] . in our study , the level of serum crp was also recognized as an independent factor . because infections are the major cause of morbidity and mortality in neutropenic patients , considerable attention has focused on the value of acute - phase reactants in identification of high - risk patients .", "however , results from previous studies have been inconsistent , and their cut - off points are variable .", "we used a cut - off value of crp of 50 mg / dl , as proposed in a previous study .", "several studies have reported that crp is a significant predictor of severe sepsis , while other studies have stated that crp was not useful in predicting infectious complications in neutropenic patients . however , in our study , we found crp ( or 3.8 , 95% ci 1.688.61 ) to be a significant predictor of serious complications . in our study , egfr < 90 ml / min/1.73 m is associated with increased risk of serious complications .", "the observed increased risk of serious complications from neutropenic patients with reduced egfr is considerable , and we suggest that decreased kidney function itself is a predictor of poor outcomes .", "first , previous studies have shown that patients with chronic kidney diseases ( ckd ) are less likely to undergo cancer screening , and patients with ckd also have multiple comorbid conditions .", "there is little information regarding the optimal timing and necessary dose adjustment of cytotoxic agents for patients with reduced kidney function . in a previous study , iff et al . stated that an egfr < 60 ml / min/1.73 m is a risk factor for cancer deaths and is also a predictor of poor cancer outcomes in the older population . except for their study , we were unable to find another study that evaluated the egfr threshold associated with an increased risk of cancer death . in another study ,", "g / l ) was found to be associated with severe sepsis in neutropenic patients . in our study , hypoproteinemia was another independent factor that could predict serious complications in febrile neutropenic patients .", "this finding can be explained by the fact that intensive chemotherapy can lead to energy consumption and high protein catabolism .", "these are common features in progressive advanced cancer and are responsible for increased severe hematological toxicity .", "clinical malnutrition , which negatively affects patient response to therapy , increases the incidence of treatment - related adverse effects and can decrease survival .", "numerous studies have shown that patients with a mascc risk index score of less than 21 should be considered at high risk for complications , as we did in our study . in this study ,", "independent predictive factors for death in patients diagnosed with fn have been identified with a sensitivity of 75% and specificity of 89% .", "we found that hypoproteinemia , platelet counts of 50 000 cells / mm , and the mascc risk index score were independent predictive factors .", "additionally , a respiratory rate > 24/min ( or 8.75 , 95% ci 2.1835.13 ) was the only component in vital signs that was predictive of exitus in multivariate analysis .", "in a recent study of patients with hematological malignancies , tachypnea was a predictor of septic shock and of poor prognosis of febrile neutropenic patients . in previous studies , infiltration or abnormality in the chest x - ray", "both aspects could be responsible for the mortality rate of up to 2550% ( 2426 ) . in our study ,", "pulmonary infiltration was significantly predictive for mortality ( or 3.25 , 95% ci 1.478.07 ) .", "this was a retrospective analysis of a small population in a single - center study .", "therefore , our results may not be representative of all other institutions and require validation ." ], [ "the results of our study may help emergency medicine physicians to prevent the development of serious complications and death in fn patients by the proper use of simple independent risk factors in addition to the mascc score .", "early stratifications of patients into low- and high - risk groups with timely and tailored empiric antimicrobial therapy can improve the prognosis of patients with fn ." ] ]

[ "cotton is an economically important plant grown world - wide as a principal source of staple fiber and vegetable oil .", "a great deal of effort has been made to improve cotton cultivation and characteristics by genetic engineering , such as adapting advantageous varieties to new geographical areas , increasing protein and oil contents of seeds , recovering more fertile varieties , and developing disease and insect resistance .", "although widely cultivated , transgenic plants have aroused wide concern among the public [ 46 ] , including the transfer of the introduced genes to wild plants and nontransgenic plants , the indirect effects of the transgenic crops on the environment , modification of the biodiversity of wildlife , unpredicted harmful changes in food products , and so on .", "therefore , there is an increasing demand for monitoring and verifying the presence and the amount of genetically modified organisms ( gmos ) in agricultural crops and in products derived [ 7 , 8 ] . to perform a transgenic analysis ,", "the currently used methods for transgenic product identification include protein - based methods , dna - based methods , microscopy , spectroscopy , and chromatography [ 1 , 11 ] .", "the rationale behind nir transgenic analysis is the spectral absorbance of molecular bonds such as c h , c ", "n , and c o that is related to the phenotypic changes ( expression level ) caused by genotypic changes .", "then , chemometric methods are used to extract detailed information concerning sample genotypes . for transgenic identification ,", "some advantages make nir spectroscopy a useful alternative tool to biological analytical methods : ( 1 ) no or less sample preparation , ( 2 ) reduced analysis time and cost , ( 3 ) simultaneous characterization of multiple components influenced by genotype , and ( 4 ) feasibility of online analysis .", "however , compared with biological analysis methods , nir - transgenic analysis also suffers some disadvantages .", "firstly , due to the baseline , low signal - to - noise ratio ( snr ) and the natural weak absorbance of some components , the sensitivity of nir analysis is much lower .", "to increase the analytical sensitivity , proper data - preprocessing methods , such as smoothing , taking derivatives , and standard normal variate ( snv ) , are required to remove background , improve snr , and enhance spectral resolution .", "another practical problem is the existence of outliers which would degrade or spoil the classification models . considering the multivariate nature and uncertainty of potential spectral outliers", ", it is important to detect and exclude the real outliers before any chemometric models are developed . ", "the aim of this paper is to develop a rapid , accurate , and robust method for genotype analysis of cotton plants ( parent , transgenic , and parent - transgenic hybrid ) by near infrared ( nir ) spectroscopy and robust partial least squares discriminant analysis ( plsda ) methods . to tackle the problem of outliers , robust principal component analysis ( rpca ) ", "the influence of different data - preprocessing methods on model prediction performance was also investigated .", "\n the cotton plants of three different genotypes including parent , transgenic , and parent - transgenic hybrid were collected from two plantations as shown in table 1 .", "the collected leaves were cleaned with water and dried at 60c for 24 hours before grinding . for seed collection ,", "both leaves and seeds were then ground into powders and finally sifted through a 0.45 mm sieve .", "nir spectra were collected using a tensor37 fourier transform nir spectrometer ( bruker , ettlingen , germany ) in the wavelength range of 400012000 cm . for each sample ,", "32 scans were performed with a resolution of 8 cm at 25c using opus6.5 software .", "the average of the 32 scans was used as a raw spectrum for further data analysis .", "firstly , nir spectra are of multivariate nature ; for example , a spectrum can have more than one thousand analytical channels , while the size of training set is usually less than 100 . therefore , in the case of large p , small n problem , sufficient description of the multivariate sample distribution usually requires dimension reduction of the measured data by latent - variable methods , such as pca .", "moreover , when performing outlier detection , the masking effects of multiple outliers need to be considered , so robust class models resistant to outliers are required", ". robust principal component analysis ( rpca ) was based on robust estimators of principal components ( pcs ) and the resulted projection distances and residuals .", "hubert et al . proposed an improved version of rpca algorithm , which was numerically more stable for high - dimensional data and computationally effective . in rpca ,", "score distance ( sd ) is defined as the sample distance from the data center in pc space and orthogonal distance ( od ) as a measure of the pc projection residual .", "an object can be classified into one of the following four groups in terms of od and sd : good pca - leverage points ( with large sd and small od ) , orthogonal outliers ( with small sd and large od ) , bad pca - leverage points ( with large sd and large od ) , and regular objects ( with small sd and small od ) . since pls is a commonly used method in chemometrics , here only a brief introduction to multiclass plsda", "the training nir spectral set can be arranged in an n p matrix x containing the absorbance measurements at p wavelengths for n samples . for multiclass problems ,", "n denotes the total size of all the b ( in this paper , b = 3 ) different classes .", "a response matrix y ( n b ) is constructed containing the category variables of each sample in x , where each row vector in y indicates the class of a sample .", "if a sample belongs to class i(i = 1 : b ) , then the ith element of its response variable is assigned a value of 1 and otherwise 0 .", "then b pls models can be developed to fit each column of y using x. for prediction , an unknown object is classified into class j(j = 1 : b ) when the jth element of its predicted response vector is the nearest to 1 . for plsda ,", "an important problem is to select the number of latent components or determine the model complexity .", "including too many latent variables would lead to an unnecessarily complicated model that tends to overfitting , while selecting too few components would lose useful data information and fail to classify the samples sufficiently .", "therefore , an improved cross - validation algorithm , monte carlo cross - validation ( mccv ) , was used for this purpose . by multiple resampling and leaving out a higher percent of training samples for prediction , mccv has been proved to be a reliable method to estimate model complexity and can reduce the risk of overfitting effectively .", "the rmsemccv ( root mean square errors of mccv ) values with different model complexity were calculated and then tested with a well - established f - test procedure [ 19 , 20 ] . to avoid selecting too many latent variables , this f - test procedure determines model complexity as obtaining an rmsemccv not significantly higher than the lowest rmsemccv with least model complexity . to evaluate the performance of discriminant models , sensitivity and specificity of prediction set for each genotype", "were calculated as follows : \n ( 1)sens.=tptp+fn , spec.=tntn+fp , \n where tp , fn , tn , and fp denote the numbers of true positives , false negatives , true negatives , and false positives , respectively .", "all the data analysis was performed on matlab 7.0.1 ( mathworks , sherborn , ma ) .", "\n some of the measured spectra of cotton seeds and leaves from three different genotypes are shown in figure 1 . the interval between 12000 cm and 10000 cm", "is contaminated with significant noise and was excluded from data analysis . seen from figure 1 , for both seeds and leaves , the spectra of three genotypes assume very similar absorbance bands and the data are characterized by low absorbance and baseline .", "therefore , proper data preprocessing methods were required to reduce various undesirable factors in the raw data .", "figures 2 and 3 show the preprocessed spectra obtained by smoothing and taking second - order derivative and snv transformation for leaves and seeds , respectively .", "smoothed spectra seem to have an improved snr at the cost of losing some detailed information .", "second derivative can effectively improve resolution but has a degraded snr . from figure 3 , it is very obvious the detailed information around 7200 cm in second - order derivative spectra is very useful for classification .", "outlier detection was performed based on rpca of the raw spectral data at a significance level of 0.05 . because each genotype has a different sample distribution , rpca was performed on each of the genotype . to demonstrate the outlier diagnosis ,", "10 components account for 85.77% of the total variances and more components can not decrease the robust cross - validation press ( prediction sum of squares ) value significantly ; therefore , 10 components were selected .", "seen from figure 4(b ) , sample 13 was detected as orthogonal outliers and samples 22 and 35 as good pca - leverage points . to select a representative set covering a wide range of samples , only bad pca - leverage points and orthogonal outliers were excluded and good pca - leverage samples were retained .", "the outlier diagnosis results for three genotypes of leaves and seeds are summarized in table 2 . to select representative training and test sets for model training and validation ,", "k - s algorithm was used to split the samples into a training set and a test set .", "the k - s algorithm selects the set of training samples that covers the overall sample domain based on their distance ( euclidean distance ) from each other .", "because the distributions of different genotypes were different , k - s algorithm was performed on each subclass and then the obtained samples were combined to form a training set and test set", ". table 3 demonstrates the splitting of training and test sets with outliers waded . with different preprocessing methods", "the sampling time of mccv was 100 and the significance level of the f - test was set to be 0.25 as proposed .", "the prediction results of test set are summarized in table 4 . seen from table 4 , second derivative and snv spectra obtained improved prediction accuracy compared with raw and smoothed spectra . for cotton seeds ,", "second - order spectra can correctly classify all the test samples and snv spectra had just one object wrongly predicted . for leave samples ,", "the effects of second - order derivative spectra on classification can be also seen from figure 3(b ) , where the three genotypes can be clearly distinguished from the naked eye by some detailed high - frequency information .", "compared with the raw data , smoothed spectra can not improve classification accuracy , which might be attributed to the loss of high - frequency spectral information .", " rapid and accurate discrimination of three different genotypes of cotton plants were developed by nir analysis of leaves and seeds .", "the best models obtained total classification accuracy of 100% and 97.6% for seeds and leaves , respectively . in order to tackle the problem of spectral outliers , robust pca models were applied to each subclass and were proved to be very effective .", "snv and second - order derivative can significantly improve the classification accuracy by removing background and baseline and enhancing resolution .", "spectral smoothing can not improve prediction performance due to the possible loss of high - frequency information .", "the results also demonstrate the removal of background and baseline plays a more important role than enhancing signal snr for classification ." ]

[ "<S> this paper reports the application of near infrared ( nir ) spectroscopy and pattern recognition methods to rapid and automatic discrimination of the genotypes ( parent , transgenic , and parent - transgenic hybrid ) of cotton plants . diffuse reflectance nir spectra of representative cotton seeds ( n = 120 ) and leaves ( n = 123 ) were measured in the range of 400012000 cm1 . </S>", "<S> a practical problem when developing classification models is the degradation and even breakdown of models caused by outliers . </S>", "<S> considering the high - dimensional nature and uncertainty of potential spectral outliers , robust principal component analysis ( rpca ) was applied to each separate sample group to detect and exclude outliers . </S>", "<S> the influence of different data preprocessing methods on model prediction performance was also investigated . </S>", "<S> the results demonstrate that rpca can effectively detect outliers and maintain the efficiency of discriminant analysis . </S>", "<S> moreover , the classification accuracy can be significantly improved by second - order derivative and standard normal variate ( snv ) . </S>", "<S> the best partial least squares discriminant analysis ( plsda ) models obtained total classification accuracy of 100% and 97.6% for seeds and leaves , respectively . </S>" ]

[ "1. Introduction", "2. Experimental and Methods", "3. Results and Discussion", "4. Conclusions" ]

[ [ "cotton is an economically important plant grown world - wide as a principal source of staple fiber and vegetable oil .", "a great deal of effort has been made to improve cotton cultivation and characteristics by genetic engineering , such as adapting advantageous varieties to new geographical areas , increasing protein and oil contents of seeds , recovering more fertile varieties , and developing disease and insect resistance .", "although widely cultivated , transgenic plants have aroused wide concern among the public [ 46 ] , including the transfer of the introduced genes to wild plants and nontransgenic plants , the indirect effects of the transgenic crops on the environment , modification of the biodiversity of wildlife , unpredicted harmful changes in food products , and so on .", "therefore , there is an increasing demand for monitoring and verifying the presence and the amount of genetically modified organisms ( gmos ) in agricultural crops and in products derived [ 7 , 8 ] . to perform a transgenic analysis ,", "the currently used methods for transgenic product identification include protein - based methods , dna - based methods , microscopy , spectroscopy , and chromatography [ 1 , 11 ] .", "the rationale behind nir transgenic analysis is the spectral absorbance of molecular bonds such as c h , c ", "n , and c o that is related to the phenotypic changes ( expression level ) caused by genotypic changes .", "then , chemometric methods are used to extract detailed information concerning sample genotypes . for transgenic identification ,", "some advantages make nir spectroscopy a useful alternative tool to biological analytical methods : ( 1 ) no or less sample preparation , ( 2 ) reduced analysis time and cost , ( 3 ) simultaneous characterization of multiple components influenced by genotype , and ( 4 ) feasibility of online analysis .", "however , compared with biological analysis methods , nir - transgenic analysis also suffers some disadvantages .", "firstly , due to the baseline , low signal - to - noise ratio ( snr ) and the natural weak absorbance of some components , the sensitivity of nir analysis is much lower .", "to increase the analytical sensitivity , proper data - preprocessing methods , such as smoothing , taking derivatives , and standard normal variate ( snv ) , are required to remove background , improve snr , and enhance spectral resolution .", "another practical problem is the existence of outliers which would degrade or spoil the classification models . considering the multivariate nature and uncertainty of potential spectral outliers", ", it is important to detect and exclude the real outliers before any chemometric models are developed . ", "the aim of this paper is to develop a rapid , accurate , and robust method for genotype analysis of cotton plants ( parent , transgenic , and parent - transgenic hybrid ) by near infrared ( nir ) spectroscopy and robust partial least squares discriminant analysis ( plsda ) methods . to tackle the problem of outliers , robust principal component analysis ( rpca ) ", "the influence of different data - preprocessing methods on model prediction performance was also investigated ." ], [ "\n the cotton plants of three different genotypes including parent , transgenic , and parent - transgenic hybrid were collected from two plantations as shown in table 1 .", "the collected leaves were cleaned with water and dried at 60c for 24 hours before grinding . for seed collection ,", "both leaves and seeds were then ground into powders and finally sifted through a 0.45 mm sieve .", "nir spectra were collected using a tensor37 fourier transform nir spectrometer ( bruker , ettlingen , germany ) in the wavelength range of 400012000 cm . for each sample ,", "32 scans were performed with a resolution of 8 cm at 25c using opus6.5 software .", "the average of the 32 scans was used as a raw spectrum for further data analysis .", "firstly , nir spectra are of multivariate nature ; for example , a spectrum can have more than one thousand analytical channels , while the size of training set is usually less than 100 . therefore , in the case of large p , small n problem , sufficient description of the multivariate sample distribution usually requires dimension reduction of the measured data by latent - variable methods , such as pca .", "moreover , when performing outlier detection , the masking effects of multiple outliers need to be considered , so robust class models resistant to outliers are required", ". robust principal component analysis ( rpca ) was based on robust estimators of principal components ( pcs ) and the resulted projection distances and residuals .", "hubert et al . proposed an improved version of rpca algorithm , which was numerically more stable for high - dimensional data and computationally effective . in rpca ,", "score distance ( sd ) is defined as the sample distance from the data center in pc space and orthogonal distance ( od ) as a measure of the pc projection residual .", "an object can be classified into one of the following four groups in terms of od and sd : good pca - leverage points ( with large sd and small od ) , orthogonal outliers ( with small sd and large od ) , bad pca - leverage points ( with large sd and large od ) , and regular objects ( with small sd and small od ) . since pls is a commonly used method in chemometrics , here only a brief introduction to multiclass plsda", "the training nir spectral set can be arranged in an n p matrix x containing the absorbance measurements at p wavelengths for n samples . for multiclass problems ,", "n denotes the total size of all the b ( in this paper , b = 3 ) different classes .", "a response matrix y ( n b ) is constructed containing the category variables of each sample in x , where each row vector in y indicates the class of a sample .", "if a sample belongs to class i(i = 1 : b ) , then the ith element of its response variable is assigned a value of 1 and otherwise 0 .", "then b pls models can be developed to fit each column of y using x. for prediction , an unknown object is classified into class j(j = 1 : b ) when the jth element of its predicted response vector is the nearest to 1 . for plsda ,", "an important problem is to select the number of latent components or determine the model complexity .", "including too many latent variables would lead to an unnecessarily complicated model that tends to overfitting , while selecting too few components would lose useful data information and fail to classify the samples sufficiently .", "therefore , an improved cross - validation algorithm , monte carlo cross - validation ( mccv ) , was used for this purpose . by multiple resampling and leaving out a higher percent of training samples for prediction , mccv has been proved to be a reliable method to estimate model complexity and can reduce the risk of overfitting effectively .", "the rmsemccv ( root mean square errors of mccv ) values with different model complexity were calculated and then tested with a well - established f - test procedure [ 19 , 20 ] . to avoid selecting too many latent variables , this f - test procedure determines model complexity as obtaining an rmsemccv not significantly higher than the lowest rmsemccv with least model complexity . to evaluate the performance of discriminant models , sensitivity and specificity of prediction set for each genotype", "were calculated as follows : \n ( 1)sens.=tptp+fn , spec.=tntn+fp , \n where tp , fn , tn , and fp denote the numbers of true positives , false negatives , true negatives , and false positives , respectively .", "all the data analysis was performed on matlab 7.0.1 ( mathworks , sherborn , ma ) ." ], [ "\n some of the measured spectra of cotton seeds and leaves from three different genotypes are shown in figure 1 . the interval between 12000 cm and 10000 cm", "is contaminated with significant noise and was excluded from data analysis . seen from figure 1 , for both seeds and leaves , the spectra of three genotypes assume very similar absorbance bands and the data are characterized by low absorbance and baseline .", "therefore , proper data preprocessing methods were required to reduce various undesirable factors in the raw data .", "figures 2 and 3 show the preprocessed spectra obtained by smoothing and taking second - order derivative and snv transformation for leaves and seeds , respectively .", "smoothed spectra seem to have an improved snr at the cost of losing some detailed information .", "second derivative can effectively improve resolution but has a degraded snr . from figure 3 , it is very obvious the detailed information around 7200 cm in second - order derivative spectra is very useful for classification .", "outlier detection was performed based on rpca of the raw spectral data at a significance level of 0.05 . because each genotype has a different sample distribution , rpca was performed on each of the genotype . to demonstrate the outlier diagnosis ,", "10 components account for 85.77% of the total variances and more components can not decrease the robust cross - validation press ( prediction sum of squares ) value significantly ; therefore , 10 components were selected .", "seen from figure 4(b ) , sample 13 was detected as orthogonal outliers and samples 22 and 35 as good pca - leverage points . to select a representative set covering a wide range of samples , only bad pca - leverage points and orthogonal outliers were excluded and good pca - leverage samples were retained .", "the outlier diagnosis results for three genotypes of leaves and seeds are summarized in table 2 . to select representative training and test sets for model training and validation ,", "k - s algorithm was used to split the samples into a training set and a test set .", "the k - s algorithm selects the set of training samples that covers the overall sample domain based on their distance ( euclidean distance ) from each other .", "because the distributions of different genotypes were different , k - s algorithm was performed on each subclass and then the obtained samples were combined to form a training set and test set", ". table 3 demonstrates the splitting of training and test sets with outliers waded . with different preprocessing methods", "the sampling time of mccv was 100 and the significance level of the f - test was set to be 0.25 as proposed .", "the prediction results of test set are summarized in table 4 . seen from table 4 , second derivative and snv spectra obtained improved prediction accuracy compared with raw and smoothed spectra . for cotton seeds ,", "second - order spectra can correctly classify all the test samples and snv spectra had just one object wrongly predicted . for leave samples ,", "the effects of second - order derivative spectra on classification can be also seen from figure 3(b ) , where the three genotypes can be clearly distinguished from the naked eye by some detailed high - frequency information .", "compared with the raw data , smoothed spectra can not improve classification accuracy , which might be attributed to the loss of high - frequency spectral information ." ], [ " rapid and accurate discrimination of three different genotypes of cotton plants were developed by nir analysis of leaves and seeds .", "the best models obtained total classification accuracy of 100% and 97.6% for seeds and leaves , respectively . in order to tackle the problem of spectral outliers , robust pca models were applied to each subclass and were proved to be very effective .", "snv and second - order derivative can significantly improve the classification accuracy by removing background and baseline and enhancing resolution .", "spectral smoothing can not improve prediction performance due to the possible loss of high - frequency information .", "the results also demonstrate the removal of background and baseline plays a more important role than enhancing signal snr for classification ." ] ]

[ "we tested a structured relaxation program on nine patients with a diagnosis of schizophrenia suffering from akathisia .", "all patients were rated on barnes akathisia scale ( bas ) before the relaxation program , immediately after and again one week later .", "the mean bas score was before the relaxation 3.3 which reduced to 1.4 immediately after to finally 1.0 a week later .", "a wilcoxon signed ranks test revealed a significant reduction in bas score from baseline to endpoint ( p = 0.026 ; z = 2.232 ) and a highly significant reduction from baseline to follow - up ( p = 0.008 ; z = 2.636 ) .", "although the study has a number of limitations the relaxation program appears to be a promising alternative to traditional treatment of akathisia .", "the patients appreciated the relaxation session but none of them managed to carry it out on their own without professional encouragement .", "akathisia ( inability to sit still ) is a movement disorder characterized by objective movements and restlessness and/or distress , which is common among patients under - going treatment with psychotropic drugs .", "it is generally agreed that the syndrome of akathisia comprises both an objective and a subjective component.1 although one of the main motivating factors for the development of second generation antipsychotics was to reduce the incidence and severity of treatment - emergent akathisia and other extrapyramidal side effects , these remain problematic and not uncommon:2 furthermore , akathisia can also be associated with antidepressant treatment.3 although often extremely upsetting , the widely held view of an association between akathisia and suicidal behavior has not been proven in larger methodologically sound studies,4 and is mainly based on case reports .", "it is nevertheless a distressing problem that can reduce quality of life and may impede treatment compliance in some patients undergoing pharmacological treatment . in an attempt to make sense of the condition some patients develop a dysfunctional interpretation of the stressful symptoms.5", "there is no general agreement on how to diagnose akathisia , and this has hampered both research and clinical practice.6 if akathisia is recognized , current treatment options include reducing the daily dosage or withdrawal of the implicated medication , or the addition of other medications .", "the most widely used pharmacological interventions include anticholinergic drugs , beta - blockers , and benzodiazepines , but the evidence base to support these interventions is limited and treatment may entail risks such as development of hypotension , tolerance , and dependence.7 in fact the only small , randomized controlled trial on biperiden showed no difference compared to saline water in the treatment of akathisia.8 some of the most important differential diagnoses of akathisia are the presence of agitation and anxiety symptoms , and these can be helped significantly with structured relaxation programs.9 however to our knowledge a relaxation approach has not been examined in patients experiencing akathisia associated with antipsychotic drugs , therefore the aim of this short report is to investigate whether a relaxation approach would reduce akathisia in patients suffering from this syndrome .", "nine patients with a primary diagnosis of schizophrenia and currently experiencing distressing akathisia ( measured on the barnes akathisia scale [ bas ] ) were invited to participate in a structured relaxation program , lasting 12 minutes and consisting of breathing ( four exercises ) and tension - relaxation ( six exercises ) .", "the patients were selected specifically because they suffered from akathisia all participants were receiving , or had recently received , pharmacological interventions for akathisia such as benzodiazepines , beta blockers , or procyclidine ( see table 1 ) leading to an unsatisfactorily response", ". however , the responsible clinicians had been reluctant to lower the dose of antipsychotic medication due to fears of deterioration of psychotic symptoms .", "one patient ( number 9 ) underwent a repeat of the relaxation program with tl during the week before the final follow - up assessment .", "all patients were rated ( by lkh ) using the bas,10 before the intervention ( baseline , bas median , 3 ) , after the relaxation session ( endpoint , bas median 2 ) , and again one week later ( follow - up bas median , 2 ) .", "the short follow - up period was decided on to minimize the risk of changes to the medication regime that could have made the patients unsuitable for the study .", "all patients were given a written version of the program following the sessions , but according to self - reports , none managed to undertake the program on their own in the week before follow - up .", "there were no changes to medication regimes in any patient , during the study period .", "a wilcoxon signed rank test showed a significant reduction in bas score from baseline to endpoint ( p = 0.026 , z = 2.232 ) and a highly significant reduction from baseline to follow - up ( p = 0.008 ; z = 2.636 ) . no significant difference was however found between endpoint and follow - up ( p = 0.257 ; z = 1.134 ) .", "these findings are promising , although it is possible that the benefits were at least partly due to factors such as increased attention and greater contact with other patients and staff .", "other limitations in this report include the small sample size , preliminary pilot nature of the data , use of a potentially biased ( lkh ) rater , and the uneven completion of the relaxation program in some patients .", "although the number of patients was limited , all but one patient appeared to have benefited from the relaxation program .", "the approach was received enthusiastically by the participating patients , but no patient managed to perform the program on their own without professional intervention .", "the structured relaxation program appeared to be well accepted and to be associated with a notable reduction in the severity of symptoms of akathisia in this group of patients with chronic schizophrenia , treated with antipsychotic drugs .", "the pathophysiology underlying akathisia is not fully understood , and current treatment approaches are less than ideal .", "it is important to develop evidence - based alternatives to current interventions that are feasible in routine clinical practice .", "the mechanism underlying the reduction in symptoms of akathisia with the structured relaxation program is uncertain but biofeedback methods may be at least partly responsible for the beneficial effect .", "biofeedback is a process that enables a person to change physiological activity ( eg , heart rate , muscle activity ) .", "it is conceivable that the relaxation program improved the patients ability to minimize the restlessness characteristic of akathisia . the sustained effect at 1-week follow - up is intriguing and warrants further study , possibly comparing structured relaxation with current pharmacological treatment options , such as use of beta - blockers or benzodiazepines in an open label clinical trial ." ]

[ "<S> purposeakathisia remains a common side effect especially from antipsychotic medication . </S>", "<S> if the condition is diagnosed the management options are limited.subjects/methodologywe tested a structured relaxation program on nine patients with a diagnosis of schizophrenia suffering from akathisia . </S>", "<S> all patients were rated on barnes akathisia scale ( bas ) before the relaxation program , immediately after and again one week later.resultsthe mean bas score was before the relaxation 3.3 which reduced to 1.4 immediately after to finally 1.0 a week later . </S>", "<S> a wilcoxon signed ranks test revealed a significant reduction in bas score from baseline to endpoint ( p = 0.026 ; z = 2.232 ) and a highly significant reduction from baseline to follow - up ( p = 0.008 ; z = 2.636).discussionalthough the study has a number of limitations the relaxation program appears to be a promising alternative to traditional treatment of akathisia . </S>", "<S> the patients appreciated the relaxation session but none of them managed to carry it out on their own without professional encouragement . </S>", "<S> the findings in this case series warrant further investigation with larger numbers of patients . </S>" ]

[ "Purpose", "Subjects/methodology", "Results", "Discussion", "Background", "Case series", "Results", "Discussion" ]

[ [ "" ], [ "we tested a structured relaxation program on nine patients with a diagnosis of schizophrenia suffering from akathisia .", "all patients were rated on barnes akathisia scale ( bas ) before the relaxation program , immediately after and again one week later ." ], [ "the mean bas score was before the relaxation 3.3 which reduced to 1.4 immediately after to finally 1.0 a week later .", "a wilcoxon signed ranks test revealed a significant reduction in bas score from baseline to endpoint ( p = 0.026 ; z = 2.232 ) and a highly significant reduction from baseline to follow - up ( p = 0.008 ; z = 2.636 ) ." ], [ "although the study has a number of limitations the relaxation program appears to be a promising alternative to traditional treatment of akathisia .", "the patients appreciated the relaxation session but none of them managed to carry it out on their own without professional encouragement ." ], [ "akathisia ( inability to sit still ) is a movement disorder characterized by objective movements and restlessness and/or distress , which is common among patients under - going treatment with psychotropic drugs .", "it is generally agreed that the syndrome of akathisia comprises both an objective and a subjective component.1 although one of the main motivating factors for the development of second generation antipsychotics was to reduce the incidence and severity of treatment - emergent akathisia and other extrapyramidal side effects , these remain problematic and not uncommon:2 furthermore , akathisia can also be associated with antidepressant treatment.3 although often extremely upsetting , the widely held view of an association between akathisia and suicidal behavior has not been proven in larger methodologically sound studies,4 and is mainly based on case reports .", "it is nevertheless a distressing problem that can reduce quality of life and may impede treatment compliance in some patients undergoing pharmacological treatment . in an attempt to make sense of the condition some patients develop a dysfunctional interpretation of the stressful symptoms.5", "there is no general agreement on how to diagnose akathisia , and this has hampered both research and clinical practice.6 if akathisia is recognized , current treatment options include reducing the daily dosage or withdrawal of the implicated medication , or the addition of other medications .", "the most widely used pharmacological interventions include anticholinergic drugs , beta - blockers , and benzodiazepines , but the evidence base to support these interventions is limited and treatment may entail risks such as development of hypotension , tolerance , and dependence.7 in fact the only small , randomized controlled trial on biperiden showed no difference compared to saline water in the treatment of akathisia.8 some of the most important differential diagnoses of akathisia are the presence of agitation and anxiety symptoms , and these can be helped significantly with structured relaxation programs.9 however to our knowledge a relaxation approach has not been examined in patients experiencing akathisia associated with antipsychotic drugs , therefore the aim of this short report is to investigate whether a relaxation approach would reduce akathisia in patients suffering from this syndrome ." ], [ "nine patients with a primary diagnosis of schizophrenia and currently experiencing distressing akathisia ( measured on the barnes akathisia scale [ bas ] ) were invited to participate in a structured relaxation program , lasting 12 minutes and consisting of breathing ( four exercises ) and tension - relaxation ( six exercises ) .", "the patients were selected specifically because they suffered from akathisia all participants were receiving , or had recently received , pharmacological interventions for akathisia such as benzodiazepines , beta blockers , or procyclidine ( see table 1 ) leading to an unsatisfactorily response", ". however , the responsible clinicians had been reluctant to lower the dose of antipsychotic medication due to fears of deterioration of psychotic symptoms .", "one patient ( number 9 ) underwent a repeat of the relaxation program with tl during the week before the final follow - up assessment ." ], [ "all patients were rated ( by lkh ) using the bas,10 before the intervention ( baseline , bas median , 3 ) , after the relaxation session ( endpoint , bas median 2 ) , and again one week later ( follow - up bas median , 2 ) .", "the short follow - up period was decided on to minimize the risk of changes to the medication regime that could have made the patients unsuitable for the study .", "all patients were given a written version of the program following the sessions , but according to self - reports , none managed to undertake the program on their own in the week before follow - up .", "there were no changes to medication regimes in any patient , during the study period .", "a wilcoxon signed rank test showed a significant reduction in bas score from baseline to endpoint ( p = 0.026 , z = 2.232 ) and a highly significant reduction from baseline to follow - up ( p = 0.008 ; z = 2.636 ) . no significant difference was however found between endpoint and follow - up ( p = 0.257 ; z = 1.134 ) ." ], [ "these findings are promising , although it is possible that the benefits were at least partly due to factors such as increased attention and greater contact with other patients and staff .", "other limitations in this report include the small sample size , preliminary pilot nature of the data , use of a potentially biased ( lkh ) rater , and the uneven completion of the relaxation program in some patients .", "although the number of patients was limited , all but one patient appeared to have benefited from the relaxation program .", "the approach was received enthusiastically by the participating patients , but no patient managed to perform the program on their own without professional intervention .", "the structured relaxation program appeared to be well accepted and to be associated with a notable reduction in the severity of symptoms of akathisia in this group of patients with chronic schizophrenia , treated with antipsychotic drugs .", "the pathophysiology underlying akathisia is not fully understood , and current treatment approaches are less than ideal .", "it is important to develop evidence - based alternatives to current interventions that are feasible in routine clinical practice .", "the mechanism underlying the reduction in symptoms of akathisia with the structured relaxation program is uncertain but biofeedback methods may be at least partly responsible for the beneficial effect .", "biofeedback is a process that enables a person to change physiological activity ( eg , heart rate , muscle activity ) .", "it is conceivable that the relaxation program improved the patients ability to minimize the restlessness characteristic of akathisia . the sustained effect at 1-week follow - up is intriguing and warrants further study , possibly comparing structured relaxation with current pharmacological treatment options , such as use of beta - blockers or benzodiazepines in an open label clinical trial ." ] ]

[ "viruses are divided into two similar - sized groups depending on whether the virus particle contains dna or rna , and , as causes of human fatality , rna viruses are by far the more important ( 1 ) . new viral diseases continue to appear as a result of several changes in human activity : travel , population growth , interaction with wild habitats etc .", "well - known novel , or emergent , rna diseases include severe acute respiratory syndrome ( sars ) ( 2 ) , human immunodeficiency virus 1 ( hiv-1 ) ( 3 ) , and may come to include avian influenza h5n1 virus ( 4 ) .", "these emergent diseases are an important factor behind the increase in the number of genome sequences that ncbi treats as representing new species ( figure 1 ) . in 2005", ", more than 200 new virus species were submitted to genbank ( more recent dates are less reliable because there is typically a delay between submission and public availability ) . as more emergent viruses appear , it is important to have a site that allows their genomes to be compared to those of known viruses . the origin of most major infectious diseases is unknown because of our ignorance of the diversity of pathogens in wild animals .", "this restricts our ability to both predict risks and develop treatments ( 5 ) . \n", "dates are the earliest given in the accession ( either of submission or publication ) .", "submissions after 2006 are excluded because accessions are made public typically only following publication and thus the frequency of submissions is more recent time periods is underestimated .", "dates are the earliest given in the accession ( either of submission or publication ) .", "submissions after 2006 are excluded because accessions are made public typically only following publication and thus the frequency of submissions is more recent time periods is underestimated . despite some advances ( 6,7 ) ,", "the evolutionary history of rna viruses is in general poorly known , especially the deep phylogenetic relationships between virus families ( 8,9 ) .", "we believe that one of the reasons for this is a lack of easily available translated genes and genomes for all species , and the lack of aligned genome sequences representing different isolates of the same species .", "in addition to the need to facilitate greater comparative analysis of rna viruses is the need to link together the existing virus web sites and their underlying databases .", "there are many web sites that provide genomic data , tools for genetic analysis and/or biological information for some viruses ( see links on our site home page ) .", "the rna virus database is intended to complement these other sites by providing basic genomic information and tools for all rna viruses and linking the user to more specialist sites , where they exist , e.g. for hiv-1 and hepatitis c virus ( hcv ) , we provide links to sites such as the los alamos laboratory on the main page for each of these viruses ( find by typing hiv-1 or hcv into the search window in the top toolbar ) . for such viruses ,", "we do not duplicate the work of other groups by attempting to display the available diversity of genomes .", "we intend that the rna virus database should develop further as a hub for other sites and we therefore encourage other workers to contact us with details of their sites that they wish linked to ours . also , we encourage workers to adopt a virus and improve and/or expand the information that we provide for individual species . this can be done by emailing us or getting involved directly in developing the database , which is an open source project available at our googlecode site ( http://code.google.com/p/rnavirusdb ) . some of the data and tools on the rna virus database can be found elsewhere , but not all of them can", ", e.g. ncbi 's genome site provides genomic overviews of virus species and pairwise alignments of other isolates to the reference sequence , but it does not provide multiple alignments or complete translated genomes as we do .", "similarly , its general entrez site provides pair - wise alignments of the query sequence and similar sequences in the database , plus a phylogenetic tree calculated from those distances ; however , no multiple alignment is built .", "we also corrected the ( few ) errors in the genbank entries , and our database records features such as rna editing ( 10 ) that make genome translation problematic .", "we have , therefore , created the rna virus database as a user - friendly site devoted to rna viruses , providing essential genomic data and tools ( discussed in more detail below ) and links to the other virus web sites .", "provide multiple whole - genome alignments , gene and whole - genome translations for all rna virus speciesidentification and taxonomic searching facilityguidance to other web resources .", "provide multiple whole - genome alignments , gene and whole - genome translations for all rna virus species identification and taxonomic searching facility guidance to other web resources .", "we provide multiple alignments of whole genomes ( as nucleotides ) for all species where genbank contains multiple representatives .", "our database , thus , currently has multiple alignments for approximately half the species ( available from the main page of any virus species under ", "these alignments were made by downloading from genbank all complete ( or near - complete ) genomes using the bioperl genbank modules ( 11 ) .", "accidental mismatches were excluded by performing a preliminary alignment using blastalign ( 12 ) , which is designed to cope with non- or poorly homologous sequences and reports such matches .", "the sequences that showed clear homology to the ncbi reference sequences ( we used a cutoff of a maximum of 40% of positions being represented by gaps in the blastalign alignment ) , up to a maximum of 50 , were then aligned using clustalw ( default parameter values ) ( 13 ) . for species for which we have at least three sequences ,", "a neighbor - joining tree was then constructed using paup ( with hky - adjusted genetic distances ) ( 14 ) , and this tree is displayed both as a pdf [ via the treegraph program ( 15 ) ] and using figtree , which is a new java - based tree - drawing application created by one of us ( rambaut , a . , unpublished data ) .", "our site allows virus nucleotide or amino acid sequences submitted by the user to be identified or , if the query is a new species , its closest relative to be found .", "in addition , the genomic location of any matched region of the library sequences is shown .", "for this we use ncbi 's suite of blast programs ( 16 ) ( go to the blast link on the toolbar of the home page ) . once the most closely related reference species has been located , the query sequence can then be placed into a whole - genome multiple alignment for that species ( where such an alignment is present ) in order to show the query 's phylogenetic relationships to the genomes in our database ( go to ", "( i ) a blast of the query to the reference species sequence provides coordinates from the resulting pair - wise alignment .", "these coordinates are then used to select homologous regions from the reference multiple alignment , and a new multiple alignment is then built using clustalw along with a phylogenetic tree using paup as described above .", "( ii ) blastalign ( described above ) is used to generate a new multiple alignment using the query sequence plus the sequences from the reference multiple alignment . \n", "figure 2.screenshots illustrating use of the rna virus database to investigate a submitted virus nucleotide sequence .", "screenshots illustrating use of the rna virus database to investigate a submitted virus nucleotide sequence .", "isolates , includes the biological data of the isolates used in the whole - genome multiple alignments [ except for hiv-1 , hcv and hepatitis b virus ( hbv ) , where we used manually built multiple alignments ; for these three species , accession numbers for the isolates are given in the supplementary data as accessionhivhcvhbv.xls ] .", "we provide amino acid sequences for all virus genes ( or , more strictly , for all open reading frames ) plus complete translated genomes for each virus species ( go to ", "the translated genomes are intended to facilitate phylogenetic analysis of more distantly related viruses ( 9 ) .", "one feature that makes annotation of rna viruses difficult is that most species have some gene overlap ( 17 ) , i.e. where the same nucleotides code for two different genes by being read in two different frames .", "we , therefore , allow the user to select from three possible options for dealing with this feature : ( i ) have overlapped regions excised from the translated genome , ( ii ) have one only of any overlapped amino acid sequences represented or ( iii ) have all the amino acids sequences present , with overlapped sequences placed sequentially ( and thus the nucleotides represented twice ) . using a key word search of the genbank entries , and a standard reference work ( 18 ) where this did not reveal a match ,", "the rna virus database is a php web application on top of a mysql database .", "php is available at http://www.php.net , but should come pre - installed on unix machines .", "all data have been taken from the ncbi 's genome and nucleotide sites at the following two urls . \n", "table names are in large bold font and interlinking column names are in small regular font . relationships of tables in the underlying mysql database .", "table names are in large bold font and interlinking column names are in small regular font .", "species names , nucleotide sequences and accession numbers were downloaded directly from genbank using bioperl modules , while further details of the virus gene coordinates , taxonomic affinities etc.were subsequently extracted from the flatfile of all genbank entries that can be downloaded from the ncbi genome site ( 19 ) .", "our approach was to treat all entries in the ncbi virus genome sites as species and to follow their taxonomic classification , although we only give virus type ( e.g. single - stranded positive - sense , retrotranscribing ) , family and genus at our site .", "we , therefore , follow ncbi 's inclusion of hepadnaviruses , which include hbv , and caulimoviruses among the rna viruses despite their mature virion containing dna rather than rna ( their possession of reverse transcriptase clearly places them biologically and evolutionarily among the reverse - transcribing group of rna viruses ) .", "the php scripts can be accessed using subversion ( http://subversion.tigris.org/ ) from our googlecode site at http://code.google.com/p/rnavirusdb .", "the mysql database ( a gzipped 16 mb dump ) may also be downloaded from the same site for installation on the user 's computer if required .", "if required , the database can subsequently be updated by other users following instructions and perl scripts given in the supplementary material ( perl_scripts.tar ) .", "this updating involves a series of short intervening manual steps ( we find that complete automation of such processes is inefficient ) .", "we intend to update the database on at least a 6-monthly basis in order to include newly discovered viruses , and are currently working to incorporate biological and epidemiological data .", "as discussed in introduction , we are keen to collaborate with other groups over further developments of our database .", "wellcome trust ( to r.b . ) ; eu marie curie fellowship scheme ( to t.d.o . ) ; the royal society ( to a.r . )" ]

[ "<S> the rna virus database is a database and web application describing the genome organization and providing analytical tools for the 938 known species of rna virus . </S>", "<S> it can identify submitted nucleotide sequences , can place them into multiple whole - genome alignments ( in species where more than one isolate has been fully sequenced ) and contains translated genome sequences for all species . </S>", "<S> it has been created for two main purposes : to facilitate the comparative analysis of rna viruses and to become a hub for other , more specialised virus web sites . </S>", "<S> it is available at the following four mirrored sites . \n </S>", "<S> http://virus.zoo.ox.ac.uk/rnavirusdbhttp://hivweb.sanbi.ac.za/rnavirusdbhttp://bioinf.cs.auckland.ac.nz/rnavirusdbhttp://tree.bio.ed.ac.uk/rnavirusdb </S>" ]

[ "INTRODUCTION", "WHOLE-GENOME ALIGNMENTS", "IDENTIFY RELATIONSHIPS OF SUBMITTED SEQUENCES", "TRANSLATED GENES AND GENOMES", "DATABASE STRUCTURE AND DATA SOURCES", "AVAILABILITY, FUTURE EXTENSIONS AND UPDATES", "SUPPLEMENTARY DATA", "FUNDING" ]

[ [ "viruses are divided into two similar - sized groups depending on whether the virus particle contains dna or rna , and , as causes of human fatality , rna viruses are by far the more important ( 1 ) . new viral diseases continue to appear as a result of several changes in human activity : travel , population growth , interaction with wild habitats etc .", "well - known novel , or emergent , rna diseases include severe acute respiratory syndrome ( sars ) ( 2 ) , human immunodeficiency virus 1 ( hiv-1 ) ( 3 ) , and may come to include avian influenza h5n1 virus ( 4 ) .", "these emergent diseases are an important factor behind the increase in the number of genome sequences that ncbi treats as representing new species ( figure 1 ) . in 2005", ", more than 200 new virus species were submitted to genbank ( more recent dates are less reliable because there is typically a delay between submission and public availability ) . as more emergent viruses appear , it is important to have a site that allows their genomes to be compared to those of known viruses . the origin of most major infectious diseases is unknown because of our ignorance of the diversity of pathogens in wild animals .", "this restricts our ability to both predict risks and develop treatments ( 5 ) . \n", "dates are the earliest given in the accession ( either of submission or publication ) .", "submissions after 2006 are excluded because accessions are made public typically only following publication and thus the frequency of submissions is more recent time periods is underestimated .", "dates are the earliest given in the accession ( either of submission or publication ) .", "submissions after 2006 are excluded because accessions are made public typically only following publication and thus the frequency of submissions is more recent time periods is underestimated . despite some advances ( 6,7 ) ,", "the evolutionary history of rna viruses is in general poorly known , especially the deep phylogenetic relationships between virus families ( 8,9 ) .", "we believe that one of the reasons for this is a lack of easily available translated genes and genomes for all species , and the lack of aligned genome sequences representing different isolates of the same species .", "in addition to the need to facilitate greater comparative analysis of rna viruses is the need to link together the existing virus web sites and their underlying databases .", "there are many web sites that provide genomic data , tools for genetic analysis and/or biological information for some viruses ( see links on our site home page ) .", "the rna virus database is intended to complement these other sites by providing basic genomic information and tools for all rna viruses and linking the user to more specialist sites , where they exist , e.g. for hiv-1 and hepatitis c virus ( hcv ) , we provide links to sites such as the los alamos laboratory on the main page for each of these viruses ( find by typing hiv-1 or hcv into the search window in the top toolbar ) . for such viruses ,", "we do not duplicate the work of other groups by attempting to display the available diversity of genomes .", "we intend that the rna virus database should develop further as a hub for other sites and we therefore encourage other workers to contact us with details of their sites that they wish linked to ours . also , we encourage workers to adopt a virus and improve and/or expand the information that we provide for individual species . this can be done by emailing us or getting involved directly in developing the database , which is an open source project available at our googlecode site ( http://code.google.com/p/rnavirusdb ) . some of the data and tools on the rna virus database can be found elsewhere , but not all of them can", ", e.g. ncbi 's genome site provides genomic overviews of virus species and pairwise alignments of other isolates to the reference sequence , but it does not provide multiple alignments or complete translated genomes as we do .", "similarly , its general entrez site provides pair - wise alignments of the query sequence and similar sequences in the database , plus a phylogenetic tree calculated from those distances ; however , no multiple alignment is built .", "we also corrected the ( few ) errors in the genbank entries , and our database records features such as rna editing ( 10 ) that make genome translation problematic .", "we have , therefore , created the rna virus database as a user - friendly site devoted to rna viruses , providing essential genomic data and tools ( discussed in more detail below ) and links to the other virus web sites .", "provide multiple whole - genome alignments , gene and whole - genome translations for all rna virus speciesidentification and taxonomic searching facilityguidance to other web resources .", "provide multiple whole - genome alignments , gene and whole - genome translations for all rna virus species identification and taxonomic searching facility guidance to other web resources ." ], [ "we provide multiple alignments of whole genomes ( as nucleotides ) for all species where genbank contains multiple representatives .", "our database , thus , currently has multiple alignments for approximately half the species ( available from the main page of any virus species under ", "these alignments were made by downloading from genbank all complete ( or near - complete ) genomes using the bioperl genbank modules ( 11 ) .", "accidental mismatches were excluded by performing a preliminary alignment using blastalign ( 12 ) , which is designed to cope with non- or poorly homologous sequences and reports such matches .", "the sequences that showed clear homology to the ncbi reference sequences ( we used a cutoff of a maximum of 40% of positions being represented by gaps in the blastalign alignment ) , up to a maximum of 50 , were then aligned using clustalw ( default parameter values ) ( 13 ) . for species for which we have at least three sequences ,", "a neighbor - joining tree was then constructed using paup ( with hky - adjusted genetic distances ) ( 14 ) , and this tree is displayed both as a pdf [ via the treegraph program ( 15 ) ] and using figtree , which is a new java - based tree - drawing application created by one of us ( rambaut , a . , unpublished data ) ." ], [ "our site allows virus nucleotide or amino acid sequences submitted by the user to be identified or , if the query is a new species , its closest relative to be found .", "in addition , the genomic location of any matched region of the library sequences is shown .", "for this we use ncbi 's suite of blast programs ( 16 ) ( go to the blast link on the toolbar of the home page ) . once the most closely related reference species has been located , the query sequence can then be placed into a whole - genome multiple alignment for that species ( where such an alignment is present ) in order to show the query 's phylogenetic relationships to the genomes in our database ( go to ", "( i ) a blast of the query to the reference species sequence provides coordinates from the resulting pair - wise alignment .", "these coordinates are then used to select homologous regions from the reference multiple alignment , and a new multiple alignment is then built using clustalw along with a phylogenetic tree using paup as described above .", "( ii ) blastalign ( described above ) is used to generate a new multiple alignment using the query sequence plus the sequences from the reference multiple alignment . \n", "figure 2.screenshots illustrating use of the rna virus database to investigate a submitted virus nucleotide sequence .", "screenshots illustrating use of the rna virus database to investigate a submitted virus nucleotide sequence .", "isolates , includes the biological data of the isolates used in the whole - genome multiple alignments [ except for hiv-1 , hcv and hepatitis b virus ( hbv ) , where we used manually built multiple alignments ; for these three species , accession numbers for the isolates are given in the supplementary data as accessionhivhcvhbv.xls ] ." ], [ "we provide amino acid sequences for all virus genes ( or , more strictly , for all open reading frames ) plus complete translated genomes for each virus species ( go to ", "the translated genomes are intended to facilitate phylogenetic analysis of more distantly related viruses ( 9 ) .", "one feature that makes annotation of rna viruses difficult is that most species have some gene overlap ( 17 ) , i.e. where the same nucleotides code for two different genes by being read in two different frames .", "we , therefore , allow the user to select from three possible options for dealing with this feature : ( i ) have overlapped regions excised from the translated genome , ( ii ) have one only of any overlapped amino acid sequences represented or ( iii ) have all the amino acids sequences present , with overlapped sequences placed sequentially ( and thus the nucleotides represented twice ) . using a key word search of the genbank entries , and a standard reference work ( 18 ) where this did not reveal a match ," ], [ "the rna virus database is a php web application on top of a mysql database .", "php is available at http://www.php.net , but should come pre - installed on unix machines .", "all data have been taken from the ncbi 's genome and nucleotide sites at the following two urls . \n", "table names are in large bold font and interlinking column names are in small regular font . relationships of tables in the underlying mysql database .", "table names are in large bold font and interlinking column names are in small regular font .", "species names , nucleotide sequences and accession numbers were downloaded directly from genbank using bioperl modules , while further details of the virus gene coordinates , taxonomic affinities etc.were subsequently extracted from the flatfile of all genbank entries that can be downloaded from the ncbi genome site ( 19 ) .", "our approach was to treat all entries in the ncbi virus genome sites as species and to follow their taxonomic classification , although we only give virus type ( e.g. single - stranded positive - sense , retrotranscribing ) , family and genus at our site .", "we , therefore , follow ncbi 's inclusion of hepadnaviruses , which include hbv , and caulimoviruses among the rna viruses despite their mature virion containing dna rather than rna ( their possession of reverse transcriptase clearly places them biologically and evolutionarily among the reverse - transcribing group of rna viruses ) ." ], [ "the php scripts can be accessed using subversion ( http://subversion.tigris.org/ ) from our googlecode site at http://code.google.com/p/rnavirusdb .", "the mysql database ( a gzipped 16 mb dump ) may also be downloaded from the same site for installation on the user 's computer if required .", "if required , the database can subsequently be updated by other users following instructions and perl scripts given in the supplementary material ( perl_scripts.tar ) .", "this updating involves a series of short intervening manual steps ( we find that complete automation of such processes is inefficient ) .", "we intend to update the database on at least a 6-monthly basis in order to include newly discovered viruses , and are currently working to incorporate biological and epidemiological data .", "as discussed in introduction , we are keen to collaborate with other groups over further developments of our database ." ], [ "" ], [ "wellcome trust ( to r.b . ) ; eu marie curie fellowship scheme ( to t.d.o . ) ; the royal society ( to a.r . )" ] ]

[ "cryptogenic organizing pneumonia ( cop ) , also known as bronchiolitis obliterans organizing pneumonia ( boop ) , is a relatively rare disorder with distinctive clinical , imaging and pathological features .", "the pathological findings are characterized by plugs of granulation tissue lying within small airways , alveolar ducts and alveoli and by chronic inflammatory cell infiltration in the alveolar walls .", "cop is defined as idiopathic and any cause for the development of pneumonia such as infection or underlying tissue disease is excluded .", "patients with cop manifest rapid clinical and imaging improvement with corticosteroid therapy , but suffer from frequent relapses . through histopathological examination and clinical observation , uveitis is usually classified into two categories , granulomatous and nongranulomatous .", "hypopyon is the accumulation of white blood cells in the anterior chamber and is often observed in nongranulomatous uveitis .", "nongranulomatous presentations are mostly idiopathic or due to hla - b27 antigen involvement [ 2 , 3 , 4 ] . it is well known that systemic diseases that can be associated with nongranulomatous uveitis include behet 's disease , ankylosing spondylitis , inflammatory bowel disease and psoriasis . although the cause is not fully disclosed , certain ocular and systemic conditions might be the underlying association with nongranulomatous uveitis . in this report", ", we describe two cases of cop who developed bilateral anterior uveitis with hypopyon unresponsive to topical corticosteroid therapy but responsive to systemic corticosteroid therapy . in addition , the clinical condition of their cop was concomitant to that of their intraocular inflammation .", "a 65-year - old woman was referred for evaluation of bilateral decreased vision and glaucoma .", "she had undergone a series of treatment for breast cancer with surgery , radiation and chemotherapy 14 years earlier .", "concerning her ocular history , she had been diagnosed as having an epiretinal macular membrane on her right eye and received a vitrectomy and phacoemulsification 3 years prior .", "the intraocular inflammation was resistant to topical betamethasone administration but decreased spontaneously in 1 year . despite the uveitis", "being mostly controlled , the intraocular pressure in both eyes had gradually increased and she was referred to our hospital .", "the initial ophthalmic examination disclosed a best - corrected visual acuity ( bcva ) of 20/50 in the right eye and 20/80 in the left eye .", "the intraocular pressure was 25 mm hg in the right and 40 mm hg in the left eye .", "slit - lamp examination demonstrated no sign of uveitis or retinal disease besides pseudophakia in the right eye and a cataract in the left eye .", "the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein .", "serological analyses indicated that there was no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigen type b testing were positive for b-60 and b-61 .", "after admission , the patient underwent phacoemulsification surgery with intraocular lens implantation and a viscocanalostomy in the left eye for her cataract and glaucoma .", "her bcva improved to 20/40 in the right eye and 20/32 in the left eye , and both the intraocular inflammation and intraocular pressure were controlled .", "five months later , the cellular infiltration with hypopyon in the anterior chamber was seen in both eyes ( fig .", "then , the patient received systemic prednisolone ( psl ) 40 mg / day administered orally to treat the aggravated cop , and both lung symptoms and intraocular inflammation diminished .", "when psl dosage was reduced to 5 mg / day during tapering , cellular infiltration in the anterior chamber and hypopyon relapsed and bcva of her eyes , particularly of her left eye , declined despite administration of topical dexamethasone .", "an optical coherence tomography ( oct ) examination showed a macular edema in her left eye ( fig .", "although a subconjunctival triamcinolone injection was given when her left bcva reached 20/200 , there was little improvement .", "then , oral psl was increased to 30 mg / day , and the intraocular inflammation subsequently decreased and her bcva gradually recovered ( fig .", ", yttrium - aluminum - argon ( yag ) laser treatment was performed to treat posterior capsule opacification in her left eye . during the clinical course", ", there was temporal intraocular pressure elevation , which could be maintained by topical antiglaucoma medication . at the middle of the relapse period ,", "cytological examination of infiltrated cells in the anterior chamber showed neutrophils and a few lymphocytes , but no atypical cell .", "the genomic dna in aqueous humor was analyzed to screen infectious pathogens including bacteria , parasites , and viruses by comprehensive polymerase chain reaction .", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms . a 69-year - old woman presented with blurred vision in both eyes .", "her ocular history expressed age - related macular degeneration in both eyes 6 years prior .", "subsequently , she was referred to our hospital . the initial ophthalmic examination disclosed a bcva of 20/25 in the right eye and 20/20 in the left eye .", "slit - lamp examination demonstrated bilateral fine keratic precipitates , hypopyon and intensive cell infiltration in the anterior chamber .", "there was a mild cataract but no sign of posterior uveitis or diabetic change in the retina .", "3 . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor , and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein and hba1c ( 7.1% ) .", "serological analyses showed no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigens type b testing were positive for b-51 and b-61 .", "one month later , she was administered systemic psl 40 mg / day orally to treat cop . the intraocular cell infiltration and hypopyon diminished in 1 month . during psl", "an oct examination showed an epiretinal membrane and macular edema in both eyes ( fig .", "the psl administration was reduced gradually and then halted , with no recurrence of uveitis .", "therefore , the cytological examination of infiltrated cells in the anterior chamber or the genomic dna analysis of infectious pathogens was not performed . during observation", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms .", "a 65-year - old woman was referred for evaluation of bilateral decreased vision and glaucoma .", "she had undergone a series of treatment for breast cancer with surgery , radiation and chemotherapy 14 years earlier .", "concerning her ocular history , she had been diagnosed as having an epiretinal macular membrane on her right eye and received a vitrectomy and phacoemulsification 3 years prior .", "the intraocular inflammation was resistant to topical betamethasone administration but decreased spontaneously in 1 year . despite the uveitis", "being mostly controlled , the intraocular pressure in both eyes had gradually increased and she was referred to our hospital .", "the initial ophthalmic examination disclosed a best - corrected visual acuity ( bcva ) of 20/50 in the right eye and 20/80 in the left eye .", "the intraocular pressure was 25 mm hg in the right and 40 mm hg in the left eye .", "slit - lamp examination demonstrated no sign of uveitis or retinal disease besides pseudophakia in the right eye and a cataract in the left eye .", "the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein .", "serological analyses indicated that there was no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigen type b testing were positive for b-60 and b-61 .", "after admission , the patient underwent phacoemulsification surgery with intraocular lens implantation and a viscocanalostomy in the left eye for her cataract and glaucoma .", "her bcva improved to 20/40 in the right eye and 20/32 in the left eye , and both the intraocular inflammation and intraocular pressure were controlled .", "five months later , the cellular infiltration with hypopyon in the anterior chamber was seen in both eyes ( fig .", "then , the patient received systemic prednisolone ( psl ) 40 mg / day administered orally to treat the aggravated cop , and both lung symptoms and intraocular inflammation diminished .", "when psl dosage was reduced to 5 mg / day during tapering , cellular infiltration in the anterior chamber and hypopyon relapsed and bcva of her eyes , particularly of her left eye , declined despite administration of topical dexamethasone .", "an optical coherence tomography ( oct ) examination showed a macular edema in her left eye ( fig .", "although a subconjunctival triamcinolone injection was given when her left bcva reached 20/200 , there was little improvement .", "then , oral psl was increased to 30 mg / day , and the intraocular inflammation subsequently decreased and her bcva gradually recovered ( fig .", ", yttrium - aluminum - argon ( yag ) laser treatment was performed to treat posterior capsule opacification in her left eye . during the clinical course", ", there was temporal intraocular pressure elevation , which could be maintained by topical antiglaucoma medication . at the middle of the relapse period ,", "cytological examination of infiltrated cells in the anterior chamber showed neutrophils and a few lymphocytes , but no atypical cell .", "the genomic dna in aqueous humor was analyzed to screen infectious pathogens including bacteria , parasites , and viruses by comprehensive polymerase chain reaction .", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms .", "she had undergone treatment for ovarian cancer with surgery 8 years earlier . in addition , she had been regularly examined and treated for cop and nephrolithiasis . her ocular history expressed age - related macular degeneration in both eyes 6 years prior .", "the initial ophthalmic examination disclosed a bcva of 20/25 in the right eye and 20/20 in the left eye .", "slit - lamp examination demonstrated bilateral fine keratic precipitates , hypopyon and intensive cell infiltration in the anterior chamber .", "there was a mild cataract but no sign of posterior uveitis or diabetic change in the retina .", "3 . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor , and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein and hba1c ( 7.1% ) .", "serological analyses showed no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigens type b testing were positive for b-51 and b-61 .", "one month later , she was administered systemic psl 40 mg / day orally to treat cop . the intraocular cell infiltration and hypopyon diminished in 1 month . during psl", "an oct examination showed an epiretinal membrane and macular edema in both eyes ( fig .", "the psl administration was reduced gradually and then halted , with no recurrence of uveitis .", "therefore , the cytological examination of infiltrated cells in the anterior chamber or the genomic dna analysis of infectious pathogens was not performed . during observation", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms .", "in the present study , we described two cases that had common points in their phenotype . first , both cases showed bilateral anterior uveitis with hypopyon .", "their intraocular inflammation was unresponsive to the administration of topical corticosteroids but showed a greater response to systemic corticosteroid administration ( sca ) .", "furthermore , both patients were receiving treatments for cop , and had medical histories of malignant tumors .", "however , neither was identified with any systemic disease that could be associated with uveitis .", "nongranulomatous uveitis is characterized by fine keratic precipitates , and in severe cases , fibrinous clotting or hypopyon in the anterior chamber .", "the most common form of nongranulomatous anterior uveitis is acute anterior uveitis , which is associated with the hla - b27 allele [ 2 , 3 , 5 ] .", "the ocular feature of hla - b27-associated acute anterior uveitis is acute onset of unilateral inflammation , and is common in males between the ages of 2040 years . in this series , both cases were hla - b27 negative , and the uveitis was bilateral .", "other diseases that could be associated with uveitis were screened . by the absence of systemic symptoms such as skin lesion , genital ulcer , oral aphtha and digestive symptoms ,", "the laboratory investigations also eliminated the involvement of ankylosing spondylitis , arthritis , inflammatory bowel disease , sarcoidosis and infection .", "in addition , several drugs including cidofovir and rifabutin are reported to induce uveitis . in the present study", ", neither patient had taken those medicines during the period in which they contracted uveitis .", "it is also known that the malignant neoplasm can introduce hypopyon that resembles intraocular inflammation [ 7 , 8 , 9 ] .", "since they had not shown any recurrence for several years , it was assumed that their malignancies were controlled .", "additionally , the cytological analysis of aqueous humor with cell infiltration in case 1 showed a moderate inflammation pattern without neoplastic cell . through the follow - up period of ovarian cancer without anticancer therapy in case 2", ", there was no sign of recurrent cancer or metastasis . from these facts , it is unlikely that their malignancy is associated with uveitis in their eyes .", "cop is diagnosed by its typical pathological findings of organizing pneumonia and the absence of any identified cause .", "the organizing pneumonia occurs in cases of autoimmune diseases such as rheumatoid arthritis and sjgren 's syndrome .", "several reports have described how cop accompanies uveitis as a part of behet 's disease [ 12 , 13 ] . in those cases ,", "although the uveitis in our report developed concomitant to cop , there was no clinical indication to suggest a diagnosis of behet 's disease . additionally , the anterior uveitis in our patients was resistant to topical corticosteroid drops , which rarely occurs during anterior uveitis care .", "this suggests that anterior uveitis in our cases was associated with cop . at present , there is one report that describes the complications of cop and uveitis without any cause . in this case ,", "bilateral nongranulomatous uveitis was unresponsive to topical corticosteroid treatment , but systemic corticosteroid treatment had a more positive effect .", "the present findings suggest the possibility that there is a novel cause of uveitis that shares the same etiology with cop .", "further investigation with increased numbers of case studies and statistical analysis may result in the discovery of new symptoms for uveitis and cop ." ]

[ "<S> two female patients with histories of cancer who showed cryptogenic organizing pneumonia ( cop ) complications and bilateral anterior uveitis with hypopyon were examined . </S>", "<S> both patients had suffered from cop and received intermitted systemic corticosteroid administration ( sca ) . the first patient , a 65-year - old woman with a history of breast cancer , showed bilateral uveitis with hypopyon . </S>", "<S> the topical corticosteroid treatment was ineffective . </S>", "<S> after sca for the treatment of cop was started , the hypopyon gradually dissipated . upon termination of sca </S>", "<S> , uveitis relapses were controlled by renewed sca . </S>", "<S> the other patient , a 69-year - old woman with a history of ovarian cancer , showed bilateral anterior uveitis with hypopyon . </S>", "<S> her intraocular outcome did not improve by the topical corticosteroid administration , but sca that was applied to treat cop led to remission of uveitis . </S>", "<S> imaging examinations , biochemical analysis , symptoms or hla - b27 antigen screenings in either patient did not explain the development of uveitis . </S>", "<S> bilateral anterior uveitis is commonly related to autoimmune disease or systemic syndrome . </S>", "<S> we report two cases with cop that developed bilateral anterior uveitis with hypopyon resistant to topical administration but responsive to systemic administration of corticosteroid . </S>", "<S> these findings suggest that cop can be associated with the etiology of anterior uveitis . </S>" ]

[ "Introduction", "Case Presentations", "Case 1", "Case 2", "Discussion", "Disclosure Statement" ]

[ [ "cryptogenic organizing pneumonia ( cop ) , also known as bronchiolitis obliterans organizing pneumonia ( boop ) , is a relatively rare disorder with distinctive clinical , imaging and pathological features .", "the pathological findings are characterized by plugs of granulation tissue lying within small airways , alveolar ducts and alveoli and by chronic inflammatory cell infiltration in the alveolar walls .", "cop is defined as idiopathic and any cause for the development of pneumonia such as infection or underlying tissue disease is excluded .", "patients with cop manifest rapid clinical and imaging improvement with corticosteroid therapy , but suffer from frequent relapses . through histopathological examination and clinical observation , uveitis is usually classified into two categories , granulomatous and nongranulomatous .", "hypopyon is the accumulation of white blood cells in the anterior chamber and is often observed in nongranulomatous uveitis .", "nongranulomatous presentations are mostly idiopathic or due to hla - b27 antigen involvement [ 2 , 3 , 4 ] . it is well known that systemic diseases that can be associated with nongranulomatous uveitis include behet 's disease , ankylosing spondylitis , inflammatory bowel disease and psoriasis . although the cause is not fully disclosed , certain ocular and systemic conditions might be the underlying association with nongranulomatous uveitis . in this report", ", we describe two cases of cop who developed bilateral anterior uveitis with hypopyon unresponsive to topical corticosteroid therapy but responsive to systemic corticosteroid therapy . in addition , the clinical condition of their cop was concomitant to that of their intraocular inflammation ." ], [ "a 65-year - old woman was referred for evaluation of bilateral decreased vision and glaucoma .", "she had undergone a series of treatment for breast cancer with surgery , radiation and chemotherapy 14 years earlier .", "concerning her ocular history , she had been diagnosed as having an epiretinal macular membrane on her right eye and received a vitrectomy and phacoemulsification 3 years prior .", "the intraocular inflammation was resistant to topical betamethasone administration but decreased spontaneously in 1 year . despite the uveitis", "being mostly controlled , the intraocular pressure in both eyes had gradually increased and she was referred to our hospital .", "the initial ophthalmic examination disclosed a best - corrected visual acuity ( bcva ) of 20/50 in the right eye and 20/80 in the left eye .", "the intraocular pressure was 25 mm hg in the right and 40 mm hg in the left eye .", "slit - lamp examination demonstrated no sign of uveitis or retinal disease besides pseudophakia in the right eye and a cataract in the left eye .", "the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein .", "serological analyses indicated that there was no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigen type b testing were positive for b-60 and b-61 .", "after admission , the patient underwent phacoemulsification surgery with intraocular lens implantation and a viscocanalostomy in the left eye for her cataract and glaucoma .", "her bcva improved to 20/40 in the right eye and 20/32 in the left eye , and both the intraocular inflammation and intraocular pressure were controlled .", "five months later , the cellular infiltration with hypopyon in the anterior chamber was seen in both eyes ( fig .", "then , the patient received systemic prednisolone ( psl ) 40 mg / day administered orally to treat the aggravated cop , and both lung symptoms and intraocular inflammation diminished .", "when psl dosage was reduced to 5 mg / day during tapering , cellular infiltration in the anterior chamber and hypopyon relapsed and bcva of her eyes , particularly of her left eye , declined despite administration of topical dexamethasone .", "an optical coherence tomography ( oct ) examination showed a macular edema in her left eye ( fig .", "although a subconjunctival triamcinolone injection was given when her left bcva reached 20/200 , there was little improvement .", "then , oral psl was increased to 30 mg / day , and the intraocular inflammation subsequently decreased and her bcva gradually recovered ( fig .", ", yttrium - aluminum - argon ( yag ) laser treatment was performed to treat posterior capsule opacification in her left eye . during the clinical course", ", there was temporal intraocular pressure elevation , which could be maintained by topical antiglaucoma medication . at the middle of the relapse period ,", "cytological examination of infiltrated cells in the anterior chamber showed neutrophils and a few lymphocytes , but no atypical cell .", "the genomic dna in aqueous humor was analyzed to screen infectious pathogens including bacteria , parasites , and viruses by comprehensive polymerase chain reaction .", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms . a 69-year - old woman presented with blurred vision in both eyes .", "her ocular history expressed age - related macular degeneration in both eyes 6 years prior .", "subsequently , she was referred to our hospital . the initial ophthalmic examination disclosed a bcva of 20/25 in the right eye and 20/20 in the left eye .", "slit - lamp examination demonstrated bilateral fine keratic precipitates , hypopyon and intensive cell infiltration in the anterior chamber .", "there was a mild cataract but no sign of posterior uveitis or diabetic change in the retina .", "3 . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor , and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein and hba1c ( 7.1% ) .", "serological analyses showed no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigens type b testing were positive for b-51 and b-61 .", "one month later , she was administered systemic psl 40 mg / day orally to treat cop . the intraocular cell infiltration and hypopyon diminished in 1 month . during psl", "an oct examination showed an epiretinal membrane and macular edema in both eyes ( fig .", "the psl administration was reduced gradually and then halted , with no recurrence of uveitis .", "therefore , the cytological examination of infiltrated cells in the anterior chamber or the genomic dna analysis of infectious pathogens was not performed . during observation", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms ." ], [ "a 65-year - old woman was referred for evaluation of bilateral decreased vision and glaucoma .", "she had undergone a series of treatment for breast cancer with surgery , radiation and chemotherapy 14 years earlier .", "concerning her ocular history , she had been diagnosed as having an epiretinal macular membrane on her right eye and received a vitrectomy and phacoemulsification 3 years prior .", "the intraocular inflammation was resistant to topical betamethasone administration but decreased spontaneously in 1 year . despite the uveitis", "being mostly controlled , the intraocular pressure in both eyes had gradually increased and she was referred to our hospital .", "the initial ophthalmic examination disclosed a best - corrected visual acuity ( bcva ) of 20/50 in the right eye and 20/80 in the left eye .", "the intraocular pressure was 25 mm hg in the right and 40 mm hg in the left eye .", "slit - lamp examination demonstrated no sign of uveitis or retinal disease besides pseudophakia in the right eye and a cataract in the left eye .", "the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein .", "serological analyses indicated that there was no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigen type b testing were positive for b-60 and b-61 .", "after admission , the patient underwent phacoemulsification surgery with intraocular lens implantation and a viscocanalostomy in the left eye for her cataract and glaucoma .", "her bcva improved to 20/40 in the right eye and 20/32 in the left eye , and both the intraocular inflammation and intraocular pressure were controlled .", "five months later , the cellular infiltration with hypopyon in the anterior chamber was seen in both eyes ( fig .", "then , the patient received systemic prednisolone ( psl ) 40 mg / day administered orally to treat the aggravated cop , and both lung symptoms and intraocular inflammation diminished .", "when psl dosage was reduced to 5 mg / day during tapering , cellular infiltration in the anterior chamber and hypopyon relapsed and bcva of her eyes , particularly of her left eye , declined despite administration of topical dexamethasone .", "an optical coherence tomography ( oct ) examination showed a macular edema in her left eye ( fig .", "although a subconjunctival triamcinolone injection was given when her left bcva reached 20/200 , there was little improvement .", "then , oral psl was increased to 30 mg / day , and the intraocular inflammation subsequently decreased and her bcva gradually recovered ( fig .", ", yttrium - aluminum - argon ( yag ) laser treatment was performed to treat posterior capsule opacification in her left eye . during the clinical course", ", there was temporal intraocular pressure elevation , which could be maintained by topical antiglaucoma medication . at the middle of the relapse period ,", "cytological examination of infiltrated cells in the anterior chamber showed neutrophils and a few lymphocytes , but no atypical cell .", "the genomic dna in aqueous humor was analyzed to screen infectious pathogens including bacteria , parasites , and viruses by comprehensive polymerase chain reaction .", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms ." ], [ "she had undergone treatment for ovarian cancer with surgery 8 years earlier . in addition , she had been regularly examined and treated for cop and nephrolithiasis . her ocular history expressed age - related macular degeneration in both eyes 6 years prior .", "the initial ophthalmic examination disclosed a bcva of 20/25 in the right eye and 20/20 in the left eye .", "slit - lamp examination demonstrated bilateral fine keratic precipitates , hypopyon and intensive cell infiltration in the anterior chamber .", "there was a mild cataract but no sign of posterior uveitis or diabetic change in the retina .", "3 . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor , and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein and hba1c ( 7.1% ) .", "serological analyses showed no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus .", "the results of human leukocyte antigens type b testing were positive for b-51 and b-61 .", "one month later , she was administered systemic psl 40 mg / day orally to treat cop . the intraocular cell infiltration and hypopyon diminished in 1 month . during psl", "an oct examination showed an epiretinal membrane and macular edema in both eyes ( fig .", "the psl administration was reduced gradually and then halted , with no recurrence of uveitis .", "therefore , the cytological examination of infiltrated cells in the anterior chamber or the genomic dna analysis of infectious pathogens was not performed . during observation", ", there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms ." ], [ "in the present study , we described two cases that had common points in their phenotype . first , both cases showed bilateral anterior uveitis with hypopyon .", "their intraocular inflammation was unresponsive to the administration of topical corticosteroids but showed a greater response to systemic corticosteroid administration ( sca ) .", "furthermore , both patients were receiving treatments for cop , and had medical histories of malignant tumors .", "however , neither was identified with any systemic disease that could be associated with uveitis .", "nongranulomatous uveitis is characterized by fine keratic precipitates , and in severe cases , fibrinous clotting or hypopyon in the anterior chamber .", "the most common form of nongranulomatous anterior uveitis is acute anterior uveitis , which is associated with the hla - b27 allele [ 2 , 3 , 5 ] .", "the ocular feature of hla - b27-associated acute anterior uveitis is acute onset of unilateral inflammation , and is common in males between the ages of 2040 years . in this series , both cases were hla - b27 negative , and the uveitis was bilateral .", "other diseases that could be associated with uveitis were screened . by the absence of systemic symptoms such as skin lesion , genital ulcer , oral aphtha and digestive symptoms ,", "the laboratory investigations also eliminated the involvement of ankylosing spondylitis , arthritis , inflammatory bowel disease , sarcoidosis and infection .", "in addition , several drugs including cidofovir and rifabutin are reported to induce uveitis . in the present study", ", neither patient had taken those medicines during the period in which they contracted uveitis .", "it is also known that the malignant neoplasm can introduce hypopyon that resembles intraocular inflammation [ 7 , 8 , 9 ] .", "since they had not shown any recurrence for several years , it was assumed that their malignancies were controlled .", "additionally , the cytological analysis of aqueous humor with cell infiltration in case 1 showed a moderate inflammation pattern without neoplastic cell . through the follow - up period of ovarian cancer without anticancer therapy in case 2", ", there was no sign of recurrent cancer or metastasis . from these facts , it is unlikely that their malignancy is associated with uveitis in their eyes .", "cop is diagnosed by its typical pathological findings of organizing pneumonia and the absence of any identified cause .", "the organizing pneumonia occurs in cases of autoimmune diseases such as rheumatoid arthritis and sjgren 's syndrome .", "several reports have described how cop accompanies uveitis as a part of behet 's disease [ 12 , 13 ] . in those cases ,", "although the uveitis in our report developed concomitant to cop , there was no clinical indication to suggest a diagnosis of behet 's disease . additionally , the anterior uveitis in our patients was resistant to topical corticosteroid drops , which rarely occurs during anterior uveitis care .", "this suggests that anterior uveitis in our cases was associated with cop . at present , there is one report that describes the complications of cop and uveitis without any cause . in this case ,", "bilateral nongranulomatous uveitis was unresponsive to topical corticosteroid treatment , but systemic corticosteroid treatment had a more positive effect .", "the present findings suggest the possibility that there is a novel cause of uveitis that shares the same etiology with cop .", "further investigation with increased numbers of case studies and statistical analysis may result in the discovery of new symptoms for uveitis and cop ." ], [ "" ] ]

[ "since 2006 , surveillance physicians have listed and collected blood from those patients encephalitis , defined as fever or history of fever with axillary temperature > 38.5c ( 101.3f ) and altered mental status , new onset of seizures , or new neurologic deficit in patients admitted to 3 nipah surveillance hospitals : rajshahi , rangpur , and faridpur medical college hospitals .", "the institute for epidemiology disease control and research and us centers for disease control and prevention tested serum with an igm - capture enzyme immunoassay to detect niv igm , and we defined laboratory - confirmed niv encephalitis as niv igm in serum . during december 2012march 2013 , surveillance physicians interviewed accompanying caregivers of all hospitalized patients whose illness met the encephalitis case definition on admission in the inpatient ward .", "study physicians asked about patients consumption of raw or fermented date palm sap and contact with other persons with fever and altered mental status in the month before illness onset ; if caregivers were unaware of the patient s exposures , study physicians asked them phone the patient s friends and colleagues about exposures .", "hospital physicians used personal protection equipment and provided it to caregivers of each patient with encephalitis and a history of these exposures . as part of subsequent epidemiologic studies", ", we also conducted detailed case investigations at each niv encephalitis case - patient s household .", "we interviewed surviving patients directly , or appropriate proxies among family , friends and relatives for patients who died , about their exposures to encephalitis patients or to fresh or fermented date palm sap before illness .", "we calculated the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of the screening questions asked on admissions to hospitals by comparing with the niv igm results .", "we repeated the calculations for patients hospitalized during january and february , when the prevalence of niv encephalitis is highest .", "we compared the answers provided by caregivers during patient hospitalization with those provided during interviews in the community as part of our epidemiologic studies .", "icddr , b s ethical review committee reviewed and approved the protocol for niv surveillance and case investigation .", "they collected and tested blood samples from 328 ( 91% ) patients for niv igm .", "seventeen ( 5% ) had niv igm ( table 1 ) , of whom 15 ( 88% ) niv encephalitis case - patients were identified during january and february 2013 . of the 17 confirmed case - patients , family caregivers of 14 reported either a history of drinking raw or fermented date palm sap or contact with other persons with fever and altered mental status in the month before illness onset .", "therefore , the sensitivity of the screening questions was 82% , specificity was 86% , ppv was 24% and npv was 99% ( table 2 ) .", "the sensitivity during january february was 93% , specificity was 82% , ppv was 37% , and npv was 99% . * drinking raw or fermented date palm sap or having contact with encephalitis patients in month before illness onset . at admission , 3 ( 18% )", "niv encephalitis case - patients had no reported history of drinking raw or fermented date palm sap or of contact with persons who had encephalitis ( table 1 ) .", "however , during the epidemiologic investigations in the community , family members of 2 case - patients reported that the patients drank fermented date palm sap in the month before illness onset .", "of the 14 niv encephalitis case - patients who , at admission , had reported 1 of the risk exposures , results were consistent with exposures reported during the epidemiologic investigation .", "screening patients with possible encephalitis at the time they seek hospital care regarding recent exposure to date palm sap and to other patients with encephalitis demonstrated high sensitivity and specificity for detecting niv encephalitis , particularly during peak months of niv encephalitis incidence .", "the high npv of the screening questions suggests that focusing infection control efforts toward patients with these exposures is an efficient use of scarce resources to prevent transmission .", "although three fourths of encephalitis patients had reported histories of exposure , they possibly could have had other infections , including other bat - borne viruses , that were transmitted through similar routes or could have lacked niv igm , despite having niv infection ( 13 ) .", "alternatively , recent consumption of date palm sap by these patients might have been purely coincidental because this practice is common in bangladesh during this season , but nipah infection is rare . for 2 niv", "encephalitis case - patients , caregivers did not report a history of drinking fermented date palm sap during hospital interview , but this behavior was reported in later community investigations . because 90% of bangladeshis are muslim , and consumption of alcohol is prohibited by islam ( 14 ) and illegal in bangladesh", ", patients might be reluctant to report drinking traditional liquor made of fermented date palm sap .", "therefore , caregivers should be asked about socially stigmatized behaviors privately and confidentially to increase the odds that these stigmatized behaviors are reported .", "exposure - based screening can detect patients at high risk for niv encephalitis in low - income , resource - constrained settings , such as bangladesh .", "we deployed screening questions on admission to inpatient wards but screening earlier , at triage in emergency wards , could further reduce risk .", "surveillance for other diseases with well - described exposures that put healthcare workers at risk , such as ebola virus infection , and where laboratory diagnosis is limited or delayed could also deploy this approach ." ]

[ "<S> we measured the performance of exposure screening questions to identify nipah virus encephalitis in hospitalized encephalitis patients during the 201213 nipah virus season in bangladesh . the sensitivity ( 93% ) , </S>", "<S> specificity ( 82% ) , positive predictive value ( 37% ) , and negative predictive value ( 99% ) results suggested that screening questions could more quickly identify persons with nipah virus encephalitis . </S>" ]

[ "The Study", "Conclusions" ]

[ [ "since 2006 , surveillance physicians have listed and collected blood from those patients encephalitis , defined as fever or history of fever with axillary temperature > 38.5c ( 101.3f ) and altered mental status , new onset of seizures , or new neurologic deficit in patients admitted to 3 nipah surveillance hospitals : rajshahi , rangpur , and faridpur medical college hospitals .", "the institute for epidemiology disease control and research and us centers for disease control and prevention tested serum with an igm - capture enzyme immunoassay to detect niv igm , and we defined laboratory - confirmed niv encephalitis as niv igm in serum . during december 2012march 2013 , surveillance physicians interviewed accompanying caregivers of all hospitalized patients whose illness met the encephalitis case definition on admission in the inpatient ward .", "study physicians asked about patients consumption of raw or fermented date palm sap and contact with other persons with fever and altered mental status in the month before illness onset ; if caregivers were unaware of the patient s exposures , study physicians asked them phone the patient s friends and colleagues about exposures .", "hospital physicians used personal protection equipment and provided it to caregivers of each patient with encephalitis and a history of these exposures . as part of subsequent epidemiologic studies", ", we also conducted detailed case investigations at each niv encephalitis case - patient s household .", "we interviewed surviving patients directly , or appropriate proxies among family , friends and relatives for patients who died , about their exposures to encephalitis patients or to fresh or fermented date palm sap before illness .", "we calculated the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of the screening questions asked on admissions to hospitals by comparing with the niv igm results .", "we repeated the calculations for patients hospitalized during january and february , when the prevalence of niv encephalitis is highest .", "we compared the answers provided by caregivers during patient hospitalization with those provided during interviews in the community as part of our epidemiologic studies .", "icddr , b s ethical review committee reviewed and approved the protocol for niv surveillance and case investigation .", "they collected and tested blood samples from 328 ( 91% ) patients for niv igm .", "seventeen ( 5% ) had niv igm ( table 1 ) , of whom 15 ( 88% ) niv encephalitis case - patients were identified during january and february 2013 . of the 17 confirmed case - patients , family caregivers of 14 reported either a history of drinking raw or fermented date palm sap or contact with other persons with fever and altered mental status in the month before illness onset .", "therefore , the sensitivity of the screening questions was 82% , specificity was 86% , ppv was 24% and npv was 99% ( table 2 ) .", "the sensitivity during january february was 93% , specificity was 82% , ppv was 37% , and npv was 99% . * drinking raw or fermented date palm sap or having contact with encephalitis patients in month before illness onset . at admission , 3 ( 18% )", "niv encephalitis case - patients had no reported history of drinking raw or fermented date palm sap or of contact with persons who had encephalitis ( table 1 ) .", "however , during the epidemiologic investigations in the community , family members of 2 case - patients reported that the patients drank fermented date palm sap in the month before illness onset .", "of the 14 niv encephalitis case - patients who , at admission , had reported 1 of the risk exposures , results were consistent with exposures reported during the epidemiologic investigation ." ], [ "screening patients with possible encephalitis at the time they seek hospital care regarding recent exposure to date palm sap and to other patients with encephalitis demonstrated high sensitivity and specificity for detecting niv encephalitis , particularly during peak months of niv encephalitis incidence .", "the high npv of the screening questions suggests that focusing infection control efforts toward patients with these exposures is an efficient use of scarce resources to prevent transmission .", "although three fourths of encephalitis patients had reported histories of exposure , they possibly could have had other infections , including other bat - borne viruses , that were transmitted through similar routes or could have lacked niv igm , despite having niv infection ( 13 ) .", "alternatively , recent consumption of date palm sap by these patients might have been purely coincidental because this practice is common in bangladesh during this season , but nipah infection is rare . for 2 niv", "encephalitis case - patients , caregivers did not report a history of drinking fermented date palm sap during hospital interview , but this behavior was reported in later community investigations . because 90% of bangladeshis are muslim , and consumption of alcohol is prohibited by islam ( 14 ) and illegal in bangladesh", ", patients might be reluctant to report drinking traditional liquor made of fermented date palm sap .", "therefore , caregivers should be asked about socially stigmatized behaviors privately and confidentially to increase the odds that these stigmatized behaviors are reported .", "exposure - based screening can detect patients at high risk for niv encephalitis in low - income , resource - constrained settings , such as bangladesh .", "we deployed screening questions on admission to inpatient wards but screening earlier , at triage in emergency wards , could further reduce risk .", "surveillance for other diseases with well - described exposures that put healthcare workers at risk , such as ebola virus infection , and where laboratory diagnosis is limited or delayed could also deploy this approach ." ] ]

[ "since chitosan is insoluble in water , the use of chitosan in basic environment is limited and hence delivery of drugs to the intestine is not possible .", "a derivative of chitosan , that is cmc , is soluble in water [ 1 , 2 ] .", "amphoteric polyelectrolyte hydrogels possessing both positive and negative charges , and many researchers are using amphoteric polyelectrolyte hydrogels to develop controlled delivery systems such as an insulin pump for diabetics , matrices for molecular recognition or separation , and so forth .", "a lot of research is going on the stimuli - sensitive polymer hydrogels . among stimuli - sensitive systems , ph or temperature - responsive hydrogels", "have been extensively studied in the biomedical field , because these two factors can be easily controlled and are applicable both in vitro and in vivo conditions [ 3 , 4 ] .", "cmc is amphoteric polyelectrolyte and has various applications due to its unique chemical , physical , and biological properties , especially its excellent biocompatibility .", "it is used to prepare wound dressings , artificial bone , and skin , is used as a bacteriostatic agent and blood anticoagulant also .", "it has also demonstrated good ph and ion sensitivity in aqueous solutions due to abundant cooh and nh2 groups .", "recent studies have shown that cmc has been used in preparation of nanoparticles for treatment of cancer [ 6 , 7 ] .", "the use of cmc has also been explored for delivery of antimicrobial agents and proteins . extended release matrix tablets", "they found that hydrogels show minimal swelling in acidic ph . considering this behavior of hydrogels", "carbopol 934 is a polymer which is sensitive to ph and was used in the present study along with chitosan . to combine the advantage of synthetic and natural polymers and at the same time", "maintain the property of natural polymers such as biodegradation and bioactivity , amphoteric polyelectrolyte hydrogels with ph sensitivity were synthesized with cmc and carbopol 934 in this work .", "the release behavior of theophylline was investigated , when it was loaded into the ph - sensitive hydrogels .", "theophylline is a antiasthmatic drug and the dosing of theophylline is complicated because it shows extensive variation in bioavailability among patients .", "about 75% of people with asthma have symptoms that disrupt both the length and depth of their nighttime sleep at least once a week .", "the number of inflammatory cells in the airways is highest in the early morning , with a peak at 4 am . in one study", ", patients with nocturnal asthma were found to have a 20% decrease in lung function overnight compared with 4% in nonasthmatics .", "hencem changing the timing or dosage of the medications may improve symptoms one experiences at night .", "theophylline comes in both short - acting and slow - release formulations , once or twice a day .", "it comes as a pill or in granules which should be swallowed whole , so as not to release too much medication at one time .", "the main drawback of this type of dosage form is that when blood levels are too high , unpleasant side effects may occur , such as nausea , vomiting , abdominal pain , jitteriness , insomnia , rapid or irregular heartbeat .", "theophylline , if delivered to the intestine can be useful in treatment of nocturnal asthma and a single dose of a slow release or extended release theophylline preparation given at night , may provide effective control of nocturnal asthma symptoms . in the present paper", ", an attempt was made to formulate a ph - sensitive hydrogel from cmc containing theophylline which has not been attempted so far and evaluate it in vitro and in vivo .", "the polymer exhibits ph dependent swelling that is , they swell and release the drug depending on ph range , hence sustained or extended drug delivery is possible in the basic environment of gastrointestinal tract .", "cmc since soluble in water , undergoes extensive swelling in basic ph compared to acidic ph and drug release is maximum in intestine , thus it can be highly helpful in controlling symptoms of nocturnal asthma .", "80% deacetylated ) was purchased from sigma aldrich , usa . carbopol 934 was purchased from loba chemie pvt . ltd . ,", "there is no conflict of interests for any financial gain , as the chemicals were purchased from the companies .", "chitosan solution was prepared in acetic acid and methanol , and acetic anhydride was added under stirring at room temperature .", "the prepared gel was agitated with 0.5 m naoh in ethanol at room temperature overnight .", "the product was transferred to 2-propanol and chloroacetic acid was added in portions under stirring . after stirring at room temperature for 2 hr , the reaction mixture was heated to 60c for another 2 hr .", "dialysis was carried out against deionized water for 3 days ; the product obtained was dried in dessicator .", "cmc solution was prepared in distilled water under stirring at 5000 rpm for 30 min .", "theophylline was added to cmc solution and the solution was stirred for 15 min .", "carbopol 934 dissolved in 1.75 m acetic acid was added to cmc solution gradually under stirring .", "the turbid dispersion obtained was immediately poured into petri dish and kept overnight for cross linking at room temp .", "the hydrogel obtained was cut into 1 cm 1 cm pieces and dried for 24 hr under vacuum .", "the prepared hydrogel was subjected to ftir analysis by kbr pellet method using fourier transform infrared ( ftir ) spectrophotometer ( perkin elmer , spectrum-100 , japan ) .", "sem studies were carried out on hydrogel samples after coating with gold palladium on a scanning electron microscope , joel sem analysis instrument , japan .", "differential scanning calorimetry was performed on a pure sample of theophylline and its formulation , using shimadzu dsc-50 apparatus .", "differential scanning calorimetric thermograms of 2 to 3 mg samples were recorded at a heating rate of 5c / min in an open aluminium pan over the range of 25c300c .", "nuclear magnetic resonance studies were carried out on cmc to determine whether the conversion of chitosan has occurred to cmc using c nmr and using an nmr spectrometer ( dsx-300 , bruker , india ) .", "the solid state ( without solvent ) nmr was done at 75 mhz . here", "an amount of hydrogels containing 20 mg of theophylline was placed in 7.4 ph phosphate buffer solution , for 24 hours . in the 7.4 ph phosphate buffer solution", "the hydrogels swell and the drug is released . at the end of 24 hours ,", "amount of theophylline present in 7.4 ph phosphate buffer is determined spectrophotometrically at 272 nm .", "the method obeyed beer 's law in the concentration range 214 g / ml . the ph - dependent swelling property of hydrogel was studied by immersing the dry hydrogels in aqueous solutions of the ph 1.2 hcl buffer for 2 hr and then in ph 7.4 phosphate buffer for another 8 hr .", "after regular intervals of time , hydrogels were removed from the aqueous solution , excess surface water was removed with filter paper , weighed , and returned to the same container until equilibrium was observed [ 13 , 15 ] .", "the degree of swelling ( wt ) was calculated at different times by means of following equation : \n ( 1)s=(weight of swollen hydrogelweight of dry hydrogel)100weight of swollen hydrogel . \n \n in vitro drug release from the hydrogels was carried out in triplicate at 37 0.1c in usp xxii dissolution apparatus type ii ( six basket dissolution tester - usp xxii , tdt-08l , electrolab , mumbai , india ) at a rotation speed of 50 rpm .", "a sample of hydrogel equivalent to 300 mg of theophylline was used in each test .", "drug release from the hydrogel was studied in 900 ml of dissolution medium ( 2 hr in ph 1.2 hcl buffer and 10 hr in ph 7.4 phosphate buffer ) .", "sample of dissolution fluid was withdrawn through a filter ( 0.45 ) m at every hour and was assayed at 272 nm for theophylline content using a shimadzu uv-1700 double beam spectrophotometer .", "the release data obtained were fitted into korsmeyer - peppas equation , log% r = logk + nlogt , where r is the amount of drug released in given time t , k is the release rate constant , and n is the time exponent .", "the intercept on y - axis gave the value of k , the release rate constant and the slope the value of n , the time exponent .", "about 50 hydrogel particles were taken for the study and spread over the sheep 's intestinal mucosa ( 2 2 cm ) taken as a biological substrate for studying mucoadhesive nature of the hydrogels .", "the prepared hydrogel was passed through sieve number 20 ; the particles which were retained on the sieve were coarser and were counted and taken for the study .", "the instrument designed was , disintegration apparatus usp , the 6 tubes were removed and the mocosa was fixed to the base of the apparatus .", "the medium chosen was 7.4 ph phosphate buffer , every 5 min interval hydrogel particles adhering to the mucosa were counted .", "the animals were divided into two groups of 6 rabbits each as a standard and the other as test .", "a written approval was obtained from the institutional ethical committee of jss medical college and hospital and jss college of pharmacy , mysore , india .", "detailed verbal and written information on the study was provided to the veterinary surgeon , central animal facility , jss medical college and hospital and permission was obtained .", "the animals were fasted for 12 hours before the capsules were introduced into the oesophagus and washed using 5 ml of distilled water in order to avoid the possible damage caused by chewing .", "blood samples were collected from ear vein at 1 , 2 , 4 , 8 , 16 , 24 hr after the oral administration .", "the blood samples were centrifuged and plasma was stored at 20c for further analytical determination . to the above samples , isopropyl alcohol was added and vortexed for 30 sec .", "the drug was extracted with 2 ml of chloroform and vortexed at high speed for 1 min .", "after centrifugation at 1000 rpm for 5 min , the organic layer was evaporated and the residue was reconstituted with 100 ml of the mobile phase .", "acetonitrile ( 7.5% ) in 0.2% acetic acid solution was used as the mobile phase .", "the in vivo studies were conducted on prepared optimized hydrogel formulation ( test ) and on marketed sustained release formulation theobid sr tablet from cipla ( standard ) .", "stability studies were conducted on optimized formulation of cmc hydrogels to assess their stability with respect to their physical appearance , drug content , swelling , and drug release characteristics after storing them at 25c/(rh ) 60% , and 30c/(rh ) 65% as per ich q1a ( r2 ) regulations for 6 months .", "when chitosan is changed into o - carboxymethyl chitosan ( o cmc ) by introducing ch2cooh groups onto oh along chitosan molecular chain , an amphoteric polyelectrolyte containing both cationic and anionic fixed charges was prepared . by varying degree of deacetylation and carboxymethyl group substitution of the chitosan", "carboxymethyl substituents were observed on amino and hydroxyl sites on the surface of modified chitosan .", "first , n - acetyl chitosan was prepared using acetic anhydride , then carboxymethylation was done to get cmc . as given in literature at 50% concentration naoh gives better degree of substitution , hence , 50% concentration was used .", "the prepared cmc is white - colored free - flowing powder and shows good solubility in both water and organic solvents , which extends its range of applications .", "this property and water solubility was used in preparing ph - sensitive hydrogels in the present work .", "cmc is amphoteric in nature and contains positively charged groups , they interact with negatively charged carboxylic groups of carbopol and form interpolyelectrolyte complexes ( ipecs ) which were stabilized by cooperative ionic bonds .", "moreover , interpolymer interactions were possible between countercharged groups in the own macromolecule and of course between copolymer chains of different macromolecules . due to its good solubility in wide range of ph values , the cmc solution could be readily blended with polyacrylic acid solution and homogenous hydrogels were obtained .", "different formulations were prepared by varying the concentration of cmc by keeping the concentration of polyacrylic acid constant from f1f5 . for the formulation f1 to f5 the concentration of carboxymethylchitosan", "was increased gradually from 1% , 1.25% , 1.5% , 1.75% , and 2% , respectively .", "ftir studies were carried out for carboxymethylchitosan and chitosan , and the spectra are given in figure 1 .", "spectra showed signals of nonmodified chitosan at 1,653 and 1,560 cm for the c o stretching ( amide ) and n ", "other characteristic peaks of chitosan o h stretch , c h stretch , and c o stretch were present at 3,4003,600 , 2,8002,900 and 1,0201,180 cm , respectively .", "the spectra of carboxymethylchitosan are similar to that of the original chitosan with a new peak appearing at 1,703 cm , which is assigned to the carbonyl groups .", "carboxymethylchitosan showed the disappearance of the nh2 associated band at 1595 cm , which can be ascribed to characteristic vibration deformation of the primary amine n h and the appearance of some new intensive peaks at 29222852 , 1466 , and 721 cm which can be attributed to the methyl groups and the long carbon segment of the quaternary ammonium salt .", "characteristic peaks of the hydroxyl and second hydroxyl groups between 1152 and 1030 cm did not change .", "theophylline ( pure drug ) and hydrogel formulation were subjected for ftir spectroscopic to ascertain whether there is any interaction between the drugs and polymers used .", "the characteristic peaks of the pure drug were compared with the peaks obtained for their formulation .", "it was observed that similar characteristic peaks appear with minor differences , at 1654 cm ( c = o stretching amide ) , 1596 cm ( c = c stretching aromatic ) , 1307 cm ( c o stretching ) for theophylline and for the formulation as shown in figure 2 .", "hence , it can be concluded that the drug is in free state and there is no interaction between drug and polymer used .", "the c nmr of chitosan was studied as given in literature and compared with the spectra of cmc .", "chitosan spectra show peaks at 177.9 ppm and at 25 ppm , which are assigned to the carbonyl carbon of coch3 and the methyl carbon ( ch3 ) , respectively .", "the signal at 101.3 ppm is assigned to the hydrogen bonded to carbon of chitosan and the signals in 59.6 ppm , 73.1 ppm , 81.1 ppm , 78.6 ppm , and 64 ppm are assigned to carbons of glucopyranose .", "spectra show the signal shifted from 101.3 ppm to 105.9 ppm because of the electron - withdrawing effect of the carboxymethyl substituents . since various different units occur in the structure of carboxymethylchitosan , many of the signals in the spectrum of chitosan appear split in the spectrum of carboxymethylchitosan .", "thus , the signals at 60.1 ppm , 73.8 ppm , 73.2 ppm , 82.2 ppm 78.2 ppm , and 63.9 ppm are split and shifted in relation to those detected in the spectrum of the parent chitosan .", "the signal observed at 180.7 ppm is assigned to the carbonyl carbons of carboxymethyl groups while the one detected at 177.9 ppm corresponds to the carbonyl carbon of coch3 of the parent chitosan .", "the methylene groups ( ch2 ) , carbons give rise to the signals at 53 and 57.4 ppm , respectively .", "however , no signal was detected at 53 ppm in the spectrum of carboxymethylchitosan figure 3 and the weak signal at 58.4 ppm can be probably assigned to the methylene ( ch2 ) bonded to the amino group ( nh ) .", "these features are taken as evidence that the carboxymethylation occurred at the hydroxyl as well as in the amino groups of chitosan which is also supported by ftir studies .", "scanning electron microscopy was carried out in order to study surface morphology , texture , and porosity of hydrogels .", "dsc studies of pure drug and f1 were studied to determine the possible interaction between the drug and the hydrogel .", "thermogram of theophylline has shown a sharp endothermic peak at 272.41c , which corresponds to its melting point .", "the evaluation of thermograms obtained from dsc revealed no interaction between the drug and polymers used , as there was no significant change in the melting point of theophylline .", "the test for drug content was carried out to ascertain that the amount of drug in the formulation . from the results obtained", ", it can be inferred that there is proper distribution of theophylline in the hydrogels .", "the drug content analysis showed that the drug is uniformly distributed in the range of 74.588.6% of the total amount of the drug added in different formulations .", "swelling studies were conducted for 12 hrs but swelling did not show significant change after 10 hrs until 12 hrs hence data for 10 hrs is presented .", "the swelling in water mainly depends on the osmotic pressure difference between inside the gel and the surroundings caused by redistribution of mobile ions .", "the swelling was observed to be more at basic ph due to increase in the number of mobile ions inside the gel and large osmotic pressure leads to swelling .", "the results indicate that with an increase in ph from 1.2 to 7.4 , a considerable increase in swelling was observed for all the hydrogel formulations , which may be due to the dissociation of the cooh groups of cmc , thereby increasing the osmotic pressure inside the hydrogels resulting in increased swelling . swelling", "increased when ratio of cmc was changed till 1.5 : 1 with polyacrylic acid , that is , formulation f1f3 but further increase in ratio , that is , 1.75 : 1 and 2.0 : 1 ( f4 and f5 ) swelling decreased .", "this shows that cmc and polyacrylic acid have synergistic effect till certain ratio as a result of which swelling increases but further increase in cmc amount resulted in reduced water uptake which may be attributed to the antagonistic effect resulting in decrease swelling .", "swelling strongly depends on the extent of cross - linking . at lower cross - linking ,", "the network is loose with a greater hydrodynamic free volume , so that the chains can accommodate more of the solvent molecules resulting in higher swelling . in this study", "it was found that the swelling was increased when the ph was changed from acidic to basic , and it conforms that the prepared hydrogel was sensitive to ph .", "the in vitro release studies were carried out for all the formulations in both acidic and basic media .", "the release studies were carried in the ph 1.2 hcl buffer for the first two hours , to mimic the acidic conditions prevailing in the stomach .", "for the next 10 hours , the release studies were carried out in ph 7.4 ph phosphate buffer , to mimic the alkaline conditions of the intestine .", "for the initial 2 hours that is , in the ph 1.2 hcl buffer , the percentage drug release was found to be low in all the cases ; this can be attributed to the fact that the hydrogel swells less in the acidic medium .", "when the dissolution medium was changed to ph 7.4 ph phosphate buffer , the release was found to increase , with time .", "the effect observed is based on swelling , as swelling increases the drug release decreases and again when swelling decreases drug release increases as shown in figure 6 . on the basis of above studies conducted", "f1 was chosen as optimized formulation as it showed desired sustained - release profile along with other drug content and swelling studies results .", "it may be attributed to diffusion of the drug caused by rapid gel swelling and also the release of drug adsorbed towards the surface of the gel matrix .", "the % drug release was found in range of 37.2198.47 at the end of 12 hrs .", "the value of n determined from korsmeyer peppas equation was in the range of 0.50.7 , which indicates the drug release from the hydrogels followed non - fickian or anomalous mechanism ( relaxation controlled ) .", "the mucoadhesive study showed that all the hydrogel particles get detached from the mucosa within 20 min .", "this study shows that carboxymethylchitosan does not have a good mucoadhesion property when compared to well - known mucoadhesive strength of parent chitosan .", "this can be attributed to the better solubility of cmc in water and organic solvents . \n in vivo studies", "were carried out for theobid sr tablet from cipla ( product a ) and theophylline loaded hydrogels of cmc both containing 300 mg of theophylline on albino rabbits .", "blood samples were withdrawn at different time intervals and plasma concentrations of theophylline were estimated , the profile is presented in figure 7 .", "statistical comparison of the mean values of pharmacokinetic parameters derived for both products a and b are given in table 4 .", "from the data obtained , it may be observed that after oral administration , peak plasma concentration cmax of 12.34 2.42 g / ml was observed for product a and 09.69 4.12 ", "g / ml for product b. from the comparison of mean values of plasma concentrations of product a and b , it was observed that product b has lower plasma concentrations .", "it was observed that the plasma concentration of theophylline in all animals after 24 hrs of oral administration was below 20 g / ml for both products .", "it was also observed from the studies that the therapeutic concentration range of theophylline maintained for about 24 hrs following a single oral dose administration for both products . from the data", "obtained , it may be observed that the time taken to reach peak plasma concentration tmax was 5.0 0.81 hrs for product a and 6.0 0.75 hrs for product b. mean elimination rate constant kel was found to be 0.08410 h for product a and 0.07813 h for product b. similarly mean elimination half life t1/2 for product", "a was 8.24 4.7 hrs and for product b 8.87 5.74 hrs .", "the mean auc0 - 24 values for product a was 101.73 16.5 ghr / ml and for product b 123.17 21.5 ", "ghr / ml . the lower cmax , prolonged tmax , of theophylline in rabbits indicated that the drug release from the product b is slow , thereby providing a prolonged and controlled in vivo delivery of the drug .", "these in vivo absorption characteristics are in confirmation with the observed in vitro drug release rate of the drug from the hydrogel .", "stability studies were conducted for different formulations for a period of 6 months . at specific time intervals ,", "the drug content in samples was tested at 2 different conditions along with 95% confidence limits , using sigma plot software 10.0 as shown in figures 8 and 9 .", "stability studies results obtained at various intervals showed that the hydrogel prepared from cmc did not show significant difference in physical appearance at the end of 6 months except in change of colour from light brown to brown . % drug release , % swelling , and mucoadhesion did not change significantly at the end of stability studies . from the data", "from the results obtained , it may be concluded that the prepared hydrogels were ph - sensitive and the degree of swelling of hydrogel depends on the concentration of cross - linking agent as well as on the ph of the environment . as the theophylline is released highly in basic ph the peak theophylline concentration will be achieved at early morning which will be highly beneficial to the patients suffering from nocturnal asthma .", "the in vitro and in vivo studies showed that the drug release is slowed down and prolonged which is better than commercial available formulation and hence better .", "thus , the hydrogel prepared using cmc can be used to deliver theophylline in sustained manner and can be used effectively against nocturnal asthma ; cmc can also be useful for the delivery of drugs which are unstable in acidic ph ." ]

[ "<S> chitosan is a natural polymer which has limited solubility . </S>", "<S> chitosan gets solubilized at acidic ph but is insoluble at basic ph . in the present study , </S>", "<S> carboxymethyl chitosan ( cmc ) was prepared which shows high swelling in basic ph and thus can delay the drug release and can act as matrix for extended release formulation . </S>", "<S> cmc was characterized by ftir and nmr . </S>", "<S> ph - sensitive hydrogels of theophylline were formulated using cmc and carbopol 934 . </S>", "<S> hydrogels were evaluated for swelling , drug content in vitro drug release studies , and in vivo studies on rabbit . </S>", "<S> the swelling studies have shown little swelling in acidic ph 432% at the end of two hours and 1631% in basic ph at the end of 12 hours . </S>", "<S> the release profile of the formulation i containing cmc and carbopol in 1 : 1 ratio showed sustained release . in vivo studies showed that the release of theophylline from the prepared hydrogel formulation ( test ) exhibit better prolonged action when compared to ( standard ) marketed sustained release formulation . </S>", "<S> the studies showed that the ph - sensitive hydrogel of cmc can be used for extended release of theophylline in intestine and can be highly useful in treating symptoms of nocturnal asthma . </S>" ]

[ "1. Introduction", "2. Experimental", "3. Results and Discussion", "4. Conclusion" ]

[ [ "since chitosan is insoluble in water , the use of chitosan in basic environment is limited and hence delivery of drugs to the intestine is not possible .", "a derivative of chitosan , that is cmc , is soluble in water [ 1 , 2 ] .", "amphoteric polyelectrolyte hydrogels possessing both positive and negative charges , and many researchers are using amphoteric polyelectrolyte hydrogels to develop controlled delivery systems such as an insulin pump for diabetics , matrices for molecular recognition or separation , and so forth .", "a lot of research is going on the stimuli - sensitive polymer hydrogels . among stimuli - sensitive systems , ph or temperature - responsive hydrogels", "have been extensively studied in the biomedical field , because these two factors can be easily controlled and are applicable both in vitro and in vivo conditions [ 3 , 4 ] .", "cmc is amphoteric polyelectrolyte and has various applications due to its unique chemical , physical , and biological properties , especially its excellent biocompatibility .", "it is used to prepare wound dressings , artificial bone , and skin , is used as a bacteriostatic agent and blood anticoagulant also .", "it has also demonstrated good ph and ion sensitivity in aqueous solutions due to abundant cooh and nh2 groups .", "recent studies have shown that cmc has been used in preparation of nanoparticles for treatment of cancer [ 6 , 7 ] .", "the use of cmc has also been explored for delivery of antimicrobial agents and proteins . extended release matrix tablets", "they found that hydrogels show minimal swelling in acidic ph . considering this behavior of hydrogels", "carbopol 934 is a polymer which is sensitive to ph and was used in the present study along with chitosan . to combine the advantage of synthetic and natural polymers and at the same time", "maintain the property of natural polymers such as biodegradation and bioactivity , amphoteric polyelectrolyte hydrogels with ph sensitivity were synthesized with cmc and carbopol 934 in this work .", "the release behavior of theophylline was investigated , when it was loaded into the ph - sensitive hydrogels .", "theophylline is a antiasthmatic drug and the dosing of theophylline is complicated because it shows extensive variation in bioavailability among patients .", "about 75% of people with asthma have symptoms that disrupt both the length and depth of their nighttime sleep at least once a week .", "the number of inflammatory cells in the airways is highest in the early morning , with a peak at 4 am . in one study", ", patients with nocturnal asthma were found to have a 20% decrease in lung function overnight compared with 4% in nonasthmatics .", "hencem changing the timing or dosage of the medications may improve symptoms one experiences at night .", "theophylline comes in both short - acting and slow - release formulations , once or twice a day .", "it comes as a pill or in granules which should be swallowed whole , so as not to release too much medication at one time .", "the main drawback of this type of dosage form is that when blood levels are too high , unpleasant side effects may occur , such as nausea , vomiting , abdominal pain , jitteriness , insomnia , rapid or irregular heartbeat .", "theophylline , if delivered to the intestine can be useful in treatment of nocturnal asthma and a single dose of a slow release or extended release theophylline preparation given at night , may provide effective control of nocturnal asthma symptoms . in the present paper", ", an attempt was made to formulate a ph - sensitive hydrogel from cmc containing theophylline which has not been attempted so far and evaluate it in vitro and in vivo .", "the polymer exhibits ph dependent swelling that is , they swell and release the drug depending on ph range , hence sustained or extended drug delivery is possible in the basic environment of gastrointestinal tract .", "cmc since soluble in water , undergoes extensive swelling in basic ph compared to acidic ph and drug release is maximum in intestine , thus it can be highly helpful in controlling symptoms of nocturnal asthma ." ], [ "80% deacetylated ) was purchased from sigma aldrich , usa . carbopol 934 was purchased from loba chemie pvt . ltd . ,", "there is no conflict of interests for any financial gain , as the chemicals were purchased from the companies .", "chitosan solution was prepared in acetic acid and methanol , and acetic anhydride was added under stirring at room temperature .", "the prepared gel was agitated with 0.5 m naoh in ethanol at room temperature overnight .", "the product was transferred to 2-propanol and chloroacetic acid was added in portions under stirring . after stirring at room temperature for 2 hr , the reaction mixture was heated to 60c for another 2 hr .", "dialysis was carried out against deionized water for 3 days ; the product obtained was dried in dessicator .", "cmc solution was prepared in distilled water under stirring at 5000 rpm for 30 min .", "theophylline was added to cmc solution and the solution was stirred for 15 min .", "carbopol 934 dissolved in 1.75 m acetic acid was added to cmc solution gradually under stirring .", "the turbid dispersion obtained was immediately poured into petri dish and kept overnight for cross linking at room temp .", "the hydrogel obtained was cut into 1 cm 1 cm pieces and dried for 24 hr under vacuum .", "the prepared hydrogel was subjected to ftir analysis by kbr pellet method using fourier transform infrared ( ftir ) spectrophotometer ( perkin elmer , spectrum-100 , japan ) .", "sem studies were carried out on hydrogel samples after coating with gold palladium on a scanning electron microscope , joel sem analysis instrument , japan .", "differential scanning calorimetry was performed on a pure sample of theophylline and its formulation , using shimadzu dsc-50 apparatus .", "differential scanning calorimetric thermograms of 2 to 3 mg samples were recorded at a heating rate of 5c / min in an open aluminium pan over the range of 25c300c .", "nuclear magnetic resonance studies were carried out on cmc to determine whether the conversion of chitosan has occurred to cmc using c nmr and using an nmr spectrometer ( dsx-300 , bruker , india ) .", "the solid state ( without solvent ) nmr was done at 75 mhz . here", "an amount of hydrogels containing 20 mg of theophylline was placed in 7.4 ph phosphate buffer solution , for 24 hours . in the 7.4 ph phosphate buffer solution", "the hydrogels swell and the drug is released . at the end of 24 hours ,", "amount of theophylline present in 7.4 ph phosphate buffer is determined spectrophotometrically at 272 nm .", "the method obeyed beer 's law in the concentration range 214 g / ml . the ph - dependent swelling property of hydrogel was studied by immersing the dry hydrogels in aqueous solutions of the ph 1.2 hcl buffer for 2 hr and then in ph 7.4 phosphate buffer for another 8 hr .", "after regular intervals of time , hydrogels were removed from the aqueous solution , excess surface water was removed with filter paper , weighed , and returned to the same container until equilibrium was observed [ 13 , 15 ] .", "the degree of swelling ( wt ) was calculated at different times by means of following equation : \n ( 1)s=(weight of swollen hydrogelweight of dry hydrogel)100weight of swollen hydrogel . \n \n in vitro drug release from the hydrogels was carried out in triplicate at 37 0.1c in usp xxii dissolution apparatus type ii ( six basket dissolution tester - usp xxii , tdt-08l , electrolab , mumbai , india ) at a rotation speed of 50 rpm .", "a sample of hydrogel equivalent to 300 mg of theophylline was used in each test .", "drug release from the hydrogel was studied in 900 ml of dissolution medium ( 2 hr in ph 1.2 hcl buffer and 10 hr in ph 7.4 phosphate buffer ) .", "sample of dissolution fluid was withdrawn through a filter ( 0.45 ) m at every hour and was assayed at 272 nm for theophylline content using a shimadzu uv-1700 double beam spectrophotometer .", "the release data obtained were fitted into korsmeyer - peppas equation , log% r = logk + nlogt , where r is the amount of drug released in given time t , k is the release rate constant , and n is the time exponent .", "the intercept on y - axis gave the value of k , the release rate constant and the slope the value of n , the time exponent .", "about 50 hydrogel particles were taken for the study and spread over the sheep 's intestinal mucosa ( 2 2 cm ) taken as a biological substrate for studying mucoadhesive nature of the hydrogels .", "the prepared hydrogel was passed through sieve number 20 ; the particles which were retained on the sieve were coarser and were counted and taken for the study .", "the instrument designed was , disintegration apparatus usp , the 6 tubes were removed and the mocosa was fixed to the base of the apparatus .", "the medium chosen was 7.4 ph phosphate buffer , every 5 min interval hydrogel particles adhering to the mucosa were counted .", "the animals were divided into two groups of 6 rabbits each as a standard and the other as test .", "a written approval was obtained from the institutional ethical committee of jss medical college and hospital and jss college of pharmacy , mysore , india .", "detailed verbal and written information on the study was provided to the veterinary surgeon , central animal facility , jss medical college and hospital and permission was obtained .", "the animals were fasted for 12 hours before the capsules were introduced into the oesophagus and washed using 5 ml of distilled water in order to avoid the possible damage caused by chewing .", "blood samples were collected from ear vein at 1 , 2 , 4 , 8 , 16 , 24 hr after the oral administration .", "the blood samples were centrifuged and plasma was stored at 20c for further analytical determination . to the above samples , isopropyl alcohol was added and vortexed for 30 sec .", "the drug was extracted with 2 ml of chloroform and vortexed at high speed for 1 min .", "after centrifugation at 1000 rpm for 5 min , the organic layer was evaporated and the residue was reconstituted with 100 ml of the mobile phase .", "acetonitrile ( 7.5% ) in 0.2% acetic acid solution was used as the mobile phase .", "the in vivo studies were conducted on prepared optimized hydrogel formulation ( test ) and on marketed sustained release formulation theobid sr tablet from cipla ( standard ) .", "stability studies were conducted on optimized formulation of cmc hydrogels to assess their stability with respect to their physical appearance , drug content , swelling , and drug release characteristics after storing them at 25c/(rh ) 60% , and 30c/(rh ) 65% as per ich q1a ( r2 ) regulations for 6 months ." ], [ "when chitosan is changed into o - carboxymethyl chitosan ( o cmc ) by introducing ch2cooh groups onto oh along chitosan molecular chain , an amphoteric polyelectrolyte containing both cationic and anionic fixed charges was prepared . by varying degree of deacetylation and carboxymethyl group substitution of the chitosan", "carboxymethyl substituents were observed on amino and hydroxyl sites on the surface of modified chitosan .", "first , n - acetyl chitosan was prepared using acetic anhydride , then carboxymethylation was done to get cmc . as given in literature at 50% concentration naoh gives better degree of substitution , hence , 50% concentration was used .", "the prepared cmc is white - colored free - flowing powder and shows good solubility in both water and organic solvents , which extends its range of applications .", "this property and water solubility was used in preparing ph - sensitive hydrogels in the present work .", "cmc is amphoteric in nature and contains positively charged groups , they interact with negatively charged carboxylic groups of carbopol and form interpolyelectrolyte complexes ( ipecs ) which were stabilized by cooperative ionic bonds .", "moreover , interpolymer interactions were possible between countercharged groups in the own macromolecule and of course between copolymer chains of different macromolecules . due to its good solubility in wide range of ph values , the cmc solution could be readily blended with polyacrylic acid solution and homogenous hydrogels were obtained .", "different formulations were prepared by varying the concentration of cmc by keeping the concentration of polyacrylic acid constant from f1f5 . for the formulation f1 to f5 the concentration of carboxymethylchitosan", "was increased gradually from 1% , 1.25% , 1.5% , 1.75% , and 2% , respectively .", "ftir studies were carried out for carboxymethylchitosan and chitosan , and the spectra are given in figure 1 .", "spectra showed signals of nonmodified chitosan at 1,653 and 1,560 cm for the c o stretching ( amide ) and n ", "other characteristic peaks of chitosan o h stretch , c h stretch , and c o stretch were present at 3,4003,600 , 2,8002,900 and 1,0201,180 cm , respectively .", "the spectra of carboxymethylchitosan are similar to that of the original chitosan with a new peak appearing at 1,703 cm , which is assigned to the carbonyl groups .", "carboxymethylchitosan showed the disappearance of the nh2 associated band at 1595 cm , which can be ascribed to characteristic vibration deformation of the primary amine n h and the appearance of some new intensive peaks at 29222852 , 1466 , and 721 cm which can be attributed to the methyl groups and the long carbon segment of the quaternary ammonium salt .", "characteristic peaks of the hydroxyl and second hydroxyl groups between 1152 and 1030 cm did not change .", "theophylline ( pure drug ) and hydrogel formulation were subjected for ftir spectroscopic to ascertain whether there is any interaction between the drugs and polymers used .", "the characteristic peaks of the pure drug were compared with the peaks obtained for their formulation .", "it was observed that similar characteristic peaks appear with minor differences , at 1654 cm ( c = o stretching amide ) , 1596 cm ( c = c stretching aromatic ) , 1307 cm ( c o stretching ) for theophylline and for the formulation as shown in figure 2 .", "hence , it can be concluded that the drug is in free state and there is no interaction between drug and polymer used .", "the c nmr of chitosan was studied as given in literature and compared with the spectra of cmc .", "chitosan spectra show peaks at 177.9 ppm and at 25 ppm , which are assigned to the carbonyl carbon of coch3 and the methyl carbon ( ch3 ) , respectively .", "the signal at 101.3 ppm is assigned to the hydrogen bonded to carbon of chitosan and the signals in 59.6 ppm , 73.1 ppm , 81.1 ppm , 78.6 ppm , and 64 ppm are assigned to carbons of glucopyranose .", "spectra show the signal shifted from 101.3 ppm to 105.9 ppm because of the electron - withdrawing effect of the carboxymethyl substituents . since various different units occur in the structure of carboxymethylchitosan , many of the signals in the spectrum of chitosan appear split in the spectrum of carboxymethylchitosan .", "thus , the signals at 60.1 ppm , 73.8 ppm , 73.2 ppm , 82.2 ppm 78.2 ppm , and 63.9 ppm are split and shifted in relation to those detected in the spectrum of the parent chitosan .", "the signal observed at 180.7 ppm is assigned to the carbonyl carbons of carboxymethyl groups while the one detected at 177.9 ppm corresponds to the carbonyl carbon of coch3 of the parent chitosan .", "the methylene groups ( ch2 ) , carbons give rise to the signals at 53 and 57.4 ppm , respectively .", "however , no signal was detected at 53 ppm in the spectrum of carboxymethylchitosan figure 3 and the weak signal at 58.4 ppm can be probably assigned to the methylene ( ch2 ) bonded to the amino group ( nh ) .", "these features are taken as evidence that the carboxymethylation occurred at the hydroxyl as well as in the amino groups of chitosan which is also supported by ftir studies .", "scanning electron microscopy was carried out in order to study surface morphology , texture , and porosity of hydrogels .", "dsc studies of pure drug and f1 were studied to determine the possible interaction between the drug and the hydrogel .", "thermogram of theophylline has shown a sharp endothermic peak at 272.41c , which corresponds to its melting point .", "the evaluation of thermograms obtained from dsc revealed no interaction between the drug and polymers used , as there was no significant change in the melting point of theophylline .", "the test for drug content was carried out to ascertain that the amount of drug in the formulation . from the results obtained", ", it can be inferred that there is proper distribution of theophylline in the hydrogels .", "the drug content analysis showed that the drug is uniformly distributed in the range of 74.588.6% of the total amount of the drug added in different formulations .", "swelling studies were conducted for 12 hrs but swelling did not show significant change after 10 hrs until 12 hrs hence data for 10 hrs is presented .", "the swelling in water mainly depends on the osmotic pressure difference between inside the gel and the surroundings caused by redistribution of mobile ions .", "the swelling was observed to be more at basic ph due to increase in the number of mobile ions inside the gel and large osmotic pressure leads to swelling .", "the results indicate that with an increase in ph from 1.2 to 7.4 , a considerable increase in swelling was observed for all the hydrogel formulations , which may be due to the dissociation of the cooh groups of cmc , thereby increasing the osmotic pressure inside the hydrogels resulting in increased swelling . swelling", "increased when ratio of cmc was changed till 1.5 : 1 with polyacrylic acid , that is , formulation f1f3 but further increase in ratio , that is , 1.75 : 1 and 2.0 : 1 ( f4 and f5 ) swelling decreased .", "this shows that cmc and polyacrylic acid have synergistic effect till certain ratio as a result of which swelling increases but further increase in cmc amount resulted in reduced water uptake which may be attributed to the antagonistic effect resulting in decrease swelling .", "swelling strongly depends on the extent of cross - linking . at lower cross - linking ,", "the network is loose with a greater hydrodynamic free volume , so that the chains can accommodate more of the solvent molecules resulting in higher swelling . in this study", "it was found that the swelling was increased when the ph was changed from acidic to basic , and it conforms that the prepared hydrogel was sensitive to ph .", "the in vitro release studies were carried out for all the formulations in both acidic and basic media .", "the release studies were carried in the ph 1.2 hcl buffer for the first two hours , to mimic the acidic conditions prevailing in the stomach .", "for the next 10 hours , the release studies were carried out in ph 7.4 ph phosphate buffer , to mimic the alkaline conditions of the intestine .", "for the initial 2 hours that is , in the ph 1.2 hcl buffer , the percentage drug release was found to be low in all the cases ; this can be attributed to the fact that the hydrogel swells less in the acidic medium .", "when the dissolution medium was changed to ph 7.4 ph phosphate buffer , the release was found to increase , with time .", "the effect observed is based on swelling , as swelling increases the drug release decreases and again when swelling decreases drug release increases as shown in figure 6 . on the basis of above studies conducted", "f1 was chosen as optimized formulation as it showed desired sustained - release profile along with other drug content and swelling studies results .", "it may be attributed to diffusion of the drug caused by rapid gel swelling and also the release of drug adsorbed towards the surface of the gel matrix .", "the % drug release was found in range of 37.2198.47 at the end of 12 hrs .", "the value of n determined from korsmeyer peppas equation was in the range of 0.50.7 , which indicates the drug release from the hydrogels followed non - fickian or anomalous mechanism ( relaxation controlled ) .", "the mucoadhesive study showed that all the hydrogel particles get detached from the mucosa within 20 min .", "this study shows that carboxymethylchitosan does not have a good mucoadhesion property when compared to well - known mucoadhesive strength of parent chitosan .", "this can be attributed to the better solubility of cmc in water and organic solvents . \n in vivo studies", "were carried out for theobid sr tablet from cipla ( product a ) and theophylline loaded hydrogels of cmc both containing 300 mg of theophylline on albino rabbits .", "blood samples were withdrawn at different time intervals and plasma concentrations of theophylline were estimated , the profile is presented in figure 7 .", "statistical comparison of the mean values of pharmacokinetic parameters derived for both products a and b are given in table 4 .", "from the data obtained , it may be observed that after oral administration , peak plasma concentration cmax of 12.34 2.42 g / ml was observed for product a and 09.69 4.12 ", "g / ml for product b. from the comparison of mean values of plasma concentrations of product a and b , it was observed that product b has lower plasma concentrations .", "it was observed that the plasma concentration of theophylline in all animals after 24 hrs of oral administration was below 20 g / ml for both products .", "it was also observed from the studies that the therapeutic concentration range of theophylline maintained for about 24 hrs following a single oral dose administration for both products . from the data", "obtained , it may be observed that the time taken to reach peak plasma concentration tmax was 5.0 0.81 hrs for product a and 6.0 0.75 hrs for product b. mean elimination rate constant kel was found to be 0.08410 h for product a and 0.07813 h for product b. similarly mean elimination half life t1/2 for product", "a was 8.24 4.7 hrs and for product b 8.87 5.74 hrs .", "the mean auc0 - 24 values for product a was 101.73 16.5 ghr / ml and for product b 123.17 21.5 ", "ghr / ml . the lower cmax , prolonged tmax , of theophylline in rabbits indicated that the drug release from the product b is slow , thereby providing a prolonged and controlled in vivo delivery of the drug .", "these in vivo absorption characteristics are in confirmation with the observed in vitro drug release rate of the drug from the hydrogel .", "stability studies were conducted for different formulations for a period of 6 months . at specific time intervals ,", "the drug content in samples was tested at 2 different conditions along with 95% confidence limits , using sigma plot software 10.0 as shown in figures 8 and 9 .", "stability studies results obtained at various intervals showed that the hydrogel prepared from cmc did not show significant difference in physical appearance at the end of 6 months except in change of colour from light brown to brown . % drug release , % swelling , and mucoadhesion did not change significantly at the end of stability studies . from the data" ], [ "from the results obtained , it may be concluded that the prepared hydrogels were ph - sensitive and the degree of swelling of hydrogel depends on the concentration of cross - linking agent as well as on the ph of the environment . as the theophylline is released highly in basic ph the peak theophylline concentration will be achieved at early morning which will be highly beneficial to the patients suffering from nocturnal asthma .", "the in vitro and in vivo studies showed that the drug release is slowed down and prolonged which is better than commercial available formulation and hence better .", "thus , the hydrogel prepared using cmc can be used to deliver theophylline in sustained manner and can be used effectively against nocturnal asthma ; cmc can also be useful for the delivery of drugs which are unstable in acidic ph ." ] ]

[ "dental caries in primary teeth has been widely studied in many countries worldwide since it is known to be one of the most common oral diseases of childhood .", "however , figures to quantify the prevalence of such disease in palestine are almost inexistent .", "it is extremely important to study the prevalence of dental caries in preschool children in palestine to be able to provide a clear picture about the situation of oral and dental health in such population so as to help the local authorities to make future plans to reduce as much as possible the incidence of dental caries .", "many countries have developed different strategies to reduce or even eradicate dental caries from preschool children following dental caries surveys .", "some have already obtained excellent results using water and salt fluoridation , dental health education , school oral health program , and so forth . in palestine ,", "dental caries surveys are rare and are unable to draw the attention of the local health authorities to take any reaction in fighting such common disease .", "the aim of this study is to give an idea about the actual situation of dental caries among preschool children in palestine .", "the results found here would probably be helpful in making future plans concerning the best methods to lower the level of such oral disease and make necessary improvements in the palestinian health system especially in the field of oral and dental health problems .", "the prevalence of dental caries in primary teeth is commonly evaluated using the dmft index . the number of decayed , missing , and filled primary teeth is calculated in each child to obtain a sum that is known to be the mean dmft score in such child .", "children of both sexes aged between 4 years and 5 years ( n = 1376 ) who were accompanied by their parents to the dental centre of the arab american university of jenin during the first 10 months of 2013 were examined clinically by one of the three calibrated examiners in the clinics of pediatric dentistry department after gaining the permission of the parents . as the families come from different geographic areas of northern palestine , the examined population was subdivided as shown in table 1 .", "medical and dental histories were taken for each child by the help of one of the parents before starting the clinical examination .", "the parent was asked about the mother 's level of education ( elementary school , secondary school , college graduated , or postgraduate studies ) .", "children who had previous history of receiving any specific organized preventive treatment were excluded from the study .", "clinical examination intraorally was achieved using sterile dental mirror and sterile dental explorer under dental unit 's light .", "the presence of cavitation has been considered to be indicative of carious lesion in accordance with the criteria recommended by the who in 1997 [ 24 ] .", "teeth which were missing due to trauma or congenitally absent teeth were excluded from the data processing ; therefore , they did not contribute to the final score .", "the number of dental restorations has also been registered for each child which contributed to the f component in the dmft score .", "the dmft scores of the clinically examined children were registered as shown in table 2 .", "it was found that only 330 children ( 24% ) were caries - free ( dmft = 0 ) at the age of 4 - 5 years while the other 1046 children have already experienced dental caries at this age . as for the ( d ) component", ", it was found that 932 children have had at least dental decay or caries in one or more primary teeth at the cavitation stage by the age of 4 - 5 years as shown in figure 1 .", "however , the number of decayed teeth in the whole population studied here was 2895 .", "the mean dt would be 2.10 while the number of children who had at least one missing primary tooth due to dental caries was only 98 representing 7.1% of the overall sample .", "the figure below shows the prevalence of missing teeth ( mt ) in the studied population ( figure 2 ) . the number of missing teeth in the whole studied population was 163 .", "when looking to the number of filled teeth representing the ( f ) component here , it was found that the number of children who had at least one restored primary tooth ( filled tooth ) was 154 representing 11.2% of the whole sample as shown in figure 3 , while the total number of restored teeth was 325 .", "when asked about their level of education , mothers of examined children were divided into 4 groups as shown in table 3 . as mentioned above , the number of children who had at least one restored ( filled ) tooth was 154 .", "mothers of such children have been found to have a high level of education in comparison to mothers of children who have decayed ( cavitated ) teeth or missing teeth ( table 4 ) .", "correlation between mother 's level of education and the presence of fillings in her child 's mouth has been found to be statistically significant ( p value < 0.05 ) .", "the overall caries prevalence in the study population was 76% with an overall mean dmft score of 2.46 of which decayed component is 2.10 , missing component 0.12 , and filled component 0.24 .", "difference between males and females in caries prevalence at the age of 4 - 5 years has not been found to be significant as demonstrated by table 5 . as seen above , caries prevalence in primary teeth of 4- to 5-year - old girls", "is 74.6% which is very close to that of boys at the same age ( 77.2% ) .", "the table below shows the mean dmft scores for both sexes ( table 6 ) .", "the prevalence of dental caries in primary dentition in 4- to 5-year - old children in northern palestine would be about 76% which means that almost 3 children out of 4 have already experienced dental caries by the age of 5 years in northern palestine .", "figures found here seem to be far from the who / fdi goals for 2000 ; that is , 50% of 5- to 6-year - old children should be caries - free . when compared to other developing countries , recent studies in pakistan and india revealed that caries prevalence in preschool children in different regions of both countries is about 5060% which is considered much better than what we found in our study here [ 69 ] .", "on the other hand , caries prevalence in preschool children in some arab countries like saudi arabia has been found to be high ( approaching the 75% ) [ 1012 ] . in the united arab emirates ,", "a high prevalence of caries among preschool children has been registered as well ( 7080% ) [ 13 , 14 ] .", "kuwaiti kindergarten schoolchildren who are caries - free at the age of 4 - 5 years do not represent more than 2432% of such population according to a national epidemiologic survey done in kuwait in 2010 .", "these findings are almost similar to what we found here in northern palestine , although they are still far from the figures published by many developed countries as we could find in the united kingdom ( 4060% caries prevalence in 5-year - old children ) or in sweden ( 69% of 3-year - old preschool children are caries - free in 2003 ) as well as in brisbane , australia ( 66% of 4- to 6-year - old children are caries - free in 2002 ) [ 1618 ] . a probable explanation for such discrepancy can be the following : inequality in economic conditions and resources , effective fluoridation policy , efficiency of healthcare system , availability and consumption of refined sugars , standard of oral health awareness among public , dietary and oral hygiene lifestyles , and motivation status of parents and children .", "as expected , highly educated mothers tend to take their children to the dentist early in their lives so as to make regular check - ups and treat dental caries as soon as it appears , which best explains the high f component ( ft ) in case of children of college - graduated mothers .", "the mean dmft scores as well as caries prevalence in primary teeth of 4- to 5-year - old palestinian children reflect a considerable defect in the oral health care at home and at school , showing a real need to establish school oral health programs in the different regions of northern palestine [ 1 , 7 ] .", "such programs are possible to implement in cooperation with the ministry of health so as to be able to finance them properly .", "it is always possible to prevent dental caries in primary dentition and lower the caries prevalence in young children in northern palestine starting with good dental health education of the parents and teaching them how to take care of their children 's teeth as soon as they start to erupt .", "emphasis on babies feeding habits as well as the use of kids toothpastes is also important [ 1 , 20 ] .", "parents should be encouraged to take their children to the public dental clinics directed by the ministry of health or to their private dentist before the age of 1 year .", "they would be able to have an idea about dental health education programs and topical fluoride application campaigns .", "preventive measures campaigns including topical fluoride application , fissure sealants , and healthy diet promotion would be a lot of help to improve the situation of the oral and dental health in young children who go to nurseries and kindergartens . it is always possible to find volunteer dentists locally or to employ freshly graduated dentists to provide preventive measures to children in nurseries and kindergartens in different northern palestinian districts including fluoride varnish application to all primary teeth as early as possible , educating the teachers as well as the parents about the best hygiene methods and toothpastes to be used by preschool children , making regular dental check - ups to the children , and giving them advices about healthy nutrition ." ]

[ "<S> \n aim . to determine the prevalence of dental caries among a representative sample of preschool children ( 4 - 5 years old ) who were accompanied by their parents to the dental centre of the arab american university in jenin whether they come seeking dental treatment or as visitors with adult patients . </S>", "<S> materials and methods . </S>", "<S> 1376 children of both sexes were investigated by three calibrated and trained examiners for dental caries using the dmft index according to the who method . results . </S>", "<S> 76% of the studied children have already experienced dental caries at the age of 4 - 5 years ( 1046 children ) . </S>", "<S> the mean dmft score was found to be 2.46 while the other 24% of children were caries - free . </S>", "<S> there was no significant difference in caries prevalence between boys and girls ( 77.2% versus 74.6% ) . </S>", "<S> children of highly educated and college graduated mothers were found to have more fillings ( restored teeth ) in comparison to those who belong to mothers who did not finish their secondary ( high school ) education . conclusion . </S>", "<S> the number of caries - free children in northern palestine is still far from numbers found in developed countries . </S>", "<S> there is a real need to make improvements at the level of parents dental health education , application of preventive measures , and dietary habits among preschool children . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion" ]

[ [ "dental caries in primary teeth has been widely studied in many countries worldwide since it is known to be one of the most common oral diseases of childhood .", "however , figures to quantify the prevalence of such disease in palestine are almost inexistent .", "it is extremely important to study the prevalence of dental caries in preschool children in palestine to be able to provide a clear picture about the situation of oral and dental health in such population so as to help the local authorities to make future plans to reduce as much as possible the incidence of dental caries .", "many countries have developed different strategies to reduce or even eradicate dental caries from preschool children following dental caries surveys .", "some have already obtained excellent results using water and salt fluoridation , dental health education , school oral health program , and so forth . in palestine ,", "dental caries surveys are rare and are unable to draw the attention of the local health authorities to take any reaction in fighting such common disease .", "the aim of this study is to give an idea about the actual situation of dental caries among preschool children in palestine .", "the results found here would probably be helpful in making future plans concerning the best methods to lower the level of such oral disease and make necessary improvements in the palestinian health system especially in the field of oral and dental health problems .", "the prevalence of dental caries in primary teeth is commonly evaluated using the dmft index . the number of decayed , missing , and filled primary teeth is calculated in each child to obtain a sum that is known to be the mean dmft score in such child ." ], [ "children of both sexes aged between 4 years and 5 years ( n = 1376 ) who were accompanied by their parents to the dental centre of the arab american university of jenin during the first 10 months of 2013 were examined clinically by one of the three calibrated examiners in the clinics of pediatric dentistry department after gaining the permission of the parents . as the families come from different geographic areas of northern palestine , the examined population was subdivided as shown in table 1 .", "medical and dental histories were taken for each child by the help of one of the parents before starting the clinical examination .", "the parent was asked about the mother 's level of education ( elementary school , secondary school , college graduated , or postgraduate studies ) .", "children who had previous history of receiving any specific organized preventive treatment were excluded from the study .", "clinical examination intraorally was achieved using sterile dental mirror and sterile dental explorer under dental unit 's light .", "the presence of cavitation has been considered to be indicative of carious lesion in accordance with the criteria recommended by the who in 1997 [ 24 ] .", "teeth which were missing due to trauma or congenitally absent teeth were excluded from the data processing ; therefore , they did not contribute to the final score .", "the number of dental restorations has also been registered for each child which contributed to the f component in the dmft score ." ], [ "the dmft scores of the clinically examined children were registered as shown in table 2 .", "it was found that only 330 children ( 24% ) were caries - free ( dmft = 0 ) at the age of 4 - 5 years while the other 1046 children have already experienced dental caries at this age . as for the ( d ) component", ", it was found that 932 children have had at least dental decay or caries in one or more primary teeth at the cavitation stage by the age of 4 - 5 years as shown in figure 1 .", "however , the number of decayed teeth in the whole population studied here was 2895 .", "the mean dt would be 2.10 while the number of children who had at least one missing primary tooth due to dental caries was only 98 representing 7.1% of the overall sample .", "the figure below shows the prevalence of missing teeth ( mt ) in the studied population ( figure 2 ) . the number of missing teeth in the whole studied population was 163 .", "when looking to the number of filled teeth representing the ( f ) component here , it was found that the number of children who had at least one restored primary tooth ( filled tooth ) was 154 representing 11.2% of the whole sample as shown in figure 3 , while the total number of restored teeth was 325 .", "when asked about their level of education , mothers of examined children were divided into 4 groups as shown in table 3 . as mentioned above , the number of children who had at least one restored ( filled ) tooth was 154 .", "mothers of such children have been found to have a high level of education in comparison to mothers of children who have decayed ( cavitated ) teeth or missing teeth ( table 4 ) .", "correlation between mother 's level of education and the presence of fillings in her child 's mouth has been found to be statistically significant ( p value < 0.05 ) .", "the overall caries prevalence in the study population was 76% with an overall mean dmft score of 2.46 of which decayed component is 2.10 , missing component 0.12 , and filled component 0.24 .", "difference between males and females in caries prevalence at the age of 4 - 5 years has not been found to be significant as demonstrated by table 5 . as seen above , caries prevalence in primary teeth of 4- to 5-year - old girls", "is 74.6% which is very close to that of boys at the same age ( 77.2% ) .", "the table below shows the mean dmft scores for both sexes ( table 6 ) ." ], [ "the prevalence of dental caries in primary dentition in 4- to 5-year - old children in northern palestine would be about 76% which means that almost 3 children out of 4 have already experienced dental caries by the age of 5 years in northern palestine .", "figures found here seem to be far from the who / fdi goals for 2000 ; that is , 50% of 5- to 6-year - old children should be caries - free . when compared to other developing countries , recent studies in pakistan and india revealed that caries prevalence in preschool children in different regions of both countries is about 5060% which is considered much better than what we found in our study here [ 69 ] .", "on the other hand , caries prevalence in preschool children in some arab countries like saudi arabia has been found to be high ( approaching the 75% ) [ 1012 ] . in the united arab emirates ,", "a high prevalence of caries among preschool children has been registered as well ( 7080% ) [ 13 , 14 ] .", "kuwaiti kindergarten schoolchildren who are caries - free at the age of 4 - 5 years do not represent more than 2432% of such population according to a national epidemiologic survey done in kuwait in 2010 .", "these findings are almost similar to what we found here in northern palestine , although they are still far from the figures published by many developed countries as we could find in the united kingdom ( 4060% caries prevalence in 5-year - old children ) or in sweden ( 69% of 3-year - old preschool children are caries - free in 2003 ) as well as in brisbane , australia ( 66% of 4- to 6-year - old children are caries - free in 2002 ) [ 1618 ] . a probable explanation for such discrepancy can be the following : inequality in economic conditions and resources , effective fluoridation policy , efficiency of healthcare system , availability and consumption of refined sugars , standard of oral health awareness among public , dietary and oral hygiene lifestyles , and motivation status of parents and children .", "as expected , highly educated mothers tend to take their children to the dentist early in their lives so as to make regular check - ups and treat dental caries as soon as it appears , which best explains the high f component ( ft ) in case of children of college - graduated mothers ." ], [ "the mean dmft scores as well as caries prevalence in primary teeth of 4- to 5-year - old palestinian children reflect a considerable defect in the oral health care at home and at school , showing a real need to establish school oral health programs in the different regions of northern palestine [ 1 , 7 ] .", "such programs are possible to implement in cooperation with the ministry of health so as to be able to finance them properly .", "it is always possible to prevent dental caries in primary dentition and lower the caries prevalence in young children in northern palestine starting with good dental health education of the parents and teaching them how to take care of their children 's teeth as soon as they start to erupt .", "emphasis on babies feeding habits as well as the use of kids toothpastes is also important [ 1 , 20 ] .", "parents should be encouraged to take their children to the public dental clinics directed by the ministry of health or to their private dentist before the age of 1 year .", "they would be able to have an idea about dental health education programs and topical fluoride application campaigns .", "preventive measures campaigns including topical fluoride application , fissure sealants , and healthy diet promotion would be a lot of help to improve the situation of the oral and dental health in young children who go to nurseries and kindergartens . it is always possible to find volunteer dentists locally or to employ freshly graduated dentists to provide preventive measures to children in nurseries and kindergartens in different northern palestinian districts including fluoride varnish application to all primary teeth as early as possible , educating the teachers as well as the parents about the best hygiene methods and toothpastes to be used by preschool children , making regular dental check - ups to the children , and giving them advices about healthy nutrition ." ] ]

[ "stroke , after myocardial infarction ( mi ) , is the second leading reason for mortality in iran as with many countries worldwide .", "the epidemiology of stroke has already been investigated in the american , european , african , and asian countries .", "no comprehensive study has yet investigated the epidemiology of stroke , particularly in mi patients , in iran , one of the largest countries in southwest asia .", "stroke and mi share many risk factors , most prevalent of which are smoking , dyslipidemia , type 2 diabetes , and hypertension .", "the risk factors for stroke and mi , especially smoking , hypertension , and dyslipidemia are highly prevalent in iran , as well . according to projections", "urbanism , increased life expectancy , reduction in childbirth , aging and elderly population , epidemiological changes , socioeconomic status , geographical conditions , and lifestyles such as poor diet , stress , and low mobility are the main causes of the burden of noncommunicable diseases , particularly stroke . because the determinants of stroke in different communities are various , we require knowledge about the risk factors and determinants of mortality in a community for effective planning and selection of appropriate strategies for the prevention and management of stroke and heart attack as the most important causes of death . since no comprehensive study has yet been investigated the status and mortality determinants of stroke in mi patients in iran , this study is conducted to determine and compare the determinants of mortality due to stroke in mi patients .", "in this retrospective cohort study , the data obtained from the mi registry of iran 's cardiovascular diseases surveillance system were analyzed . around", "20,750 hospitalized patients with mi with a new presentation ( hospitalized in 540 hospitals ) between april , 2012 and march , 2013 were enrolled .", "the study was approved by the management center of noncommunicable diseases and the department of cardiovascular diseases prevention of iran 's ministry of health and medical education ( approval no .", "the research followed the principles of the declaration of helsinki ; the researchers did not conduct any interventions on the patients , and an institutional review board approved this research .", "the study protocol was approved by an independent scientific review committee ( at shahid beheshti university medical of sciences ) ( no .", "inclusion criteria were determined according to the world health organization ( who ) and world heart federation definition of mi diagnosis per the international classification of diseases-10 codes i21 and i22 .", "patients with mi history or no definite diagnosis by a cardiologist were excluded from the study . the data on age , gender , education , ischemic heart disease symptoms , hospital stay duration , smoking , dyslipidemia , hypertension , type 2 diabetes , heart failure , and family history of cardiovascular diseases were gathered .", "stroke history was defined based on the who s definition rapidly developing signs of focal ( or global ) disturbance of cerebral function lasting > 24 h ( unless interrupted by surgery or death ) , with no apparent nonvascular cause . ", "therefore , patients with transient cerebral ischemia or stroke events were excluded if they had blood disease or brain tumors . moreover , patients with secondary strokes caused by trauma , acute hemorrhagic stroke , or transient ischemic attack were excluded . after a definite diagnosis of mi by a cardiologist ,", "the data on the left bundle branch block , right bundle branch block , atrial fibrillation , ventricular and atrial tachycardia , type and site of mi , and use of therapeutic regimens such as coronary artery bypass grafting , percutaneous coronary intervention , and thrombolytic therapy were recorded .", "the cohort of patients was defined by the date at mi diagnosis , hospital stay , and follow - up till discharge or death ( outcome ) .", "odds ratio ( or ) of mortality for clinical and demographic risk factors were calculated by logistic regression . or of mortality was calculated as the crude and adjusted ratio for patients with and without stroke by simple and multiple logistic regression models .", "first , univariate analysis was conducted . to control confounders , we entered the significant or approximately significant variables into a multiple regression model . for data", "analysis , chi - square , t - test , and regression model in stata software ( stata corp .", "2011 , stata statistical software : release 14 , college station , tx : stata corp lp , usa ) were used and p < 0.05 was considered the level of significance .", "the cohort of patients was defined by the date at mi diagnosis , hospital stay , and follow - up till discharge or death ( outcome ) .", "odds ratio ( or ) of mortality for clinical and demographic risk factors were calculated by logistic regression . or of mortality was calculated as the crude and adjusted ratio for patients with and without stroke by simple and multiple logistic regression models .", "first , univariate analysis was conducted . to control confounders , we entered the significant or approximately significant variables into a multiple regression model . for data", "analysis , chi - square , t - test , and regression model in stata software ( stata corp .", "2011 , stata statistical software : release 14 , college station , tx : stata corp lp , usa ) were used and p < 0.05 was considered the level of significance .", "the prevalence of stroke in the studied population was derived 20.96% ( confidence interval ci 95% : 20.1321.24 ) .", "mean standard deviation age at mi incidence was 64.38 ( 12.9 ) years in patients with stroke history and 60.37 ( 13.3 ) years in patients without , with a significant difference ( p < 0.001 ) .", "about 64.8% of these patients were male , 52.8% of them were illiterate , and only 4.9% had an academic education .", "the prevalence of diabetes , hypertension , hypercholesterolemia , and heart failure was obtained 30% , 56.5% , 27.3% , and 8.1% in patients with stroke history and 20.1% , 30% , 15.4% , and 8% in patients without stroke history , respectively .", "the ratio of cardiovascular surgery , percutaneous coronary intervention , and thrombolytic therapy was obtained 6.4% , 5.8% , and 41.1% in the former group and 1.6% , 7.1% , and 45.3% in the latter , respectively . the demographic characteristics and individual risk factors in patients with and without stroke", "demographic and medical history of myocardial infarction patients with and without stroke within 1 year of study , 18.8% of the stroke patients deceased .", "the mortality ratio in all patients was derived 12.1% . of the 4293 patients with stroke , 2537 ( 59.1% ) had st - elevation mi ( stemi ) .", "mortality ratio was obtained 18.8% in patients stroke history and 10.3% in the without . the clinical characteristics and risk factors in the patients according to the type of hospital diagnosis and therapeutic measures", "the ratio of cardiovascular surgery , percutaneous coronary intervention , and thrombolytic therapy was obtained , respectively , 6.4% , 5.8% , and 41.1% . by logistic regression ,", "the mortality - associated factors were different in the mi patients with and without stroke history .", "the adjusted or of mortality in patients with stroke history was derived 7.02 ( 95% ci : 5.429 ) for chest pain resistant to treatment , 2.39 ( 95% ci : 1.972.9 ) for stemi , 3.02 ( 95% ci : 2.53.64 ) for lack of thrombolytic therapy , 2.2 ( 95% ci : 1.662.91 ) for heart failure , and 2.17 ( 95% ci : 1.62.9 ) for ventricular tachycardia . considering other potential covariates and the confounders of hospital mortality in patients with stroke , we obtained the highest adjusted or of hospital mortality in patients with stroke for chest pain resistant to treatment followed by lack of thrombolytic therapy and stemi .", "prevalence of clinical risk factors in myocardial infarction patients with and without stroke risk factors for hospital mortality in myocardial infarction patients with and without stroke", "in this study , the risk factors in the mi patients with and without stroke history were investigated and compared for the first time in iran . in reliable , electronic databases such as pubmed and scopus , we found no study to model and compare the characteristics of the two groups of patients as with our study .", "our study indicated that the pattern of determinants of hospital mi mortality with and without a stroke is various .", "the mean age at incidence in our study is similar to a study in singapore comparing the risk factors for stroke in chinese , indian , and malaysian ethnic populations .", "the study in singapore reported the mean age of stroke patients to be 64.1 years . in the study of singapore", "the prevalence of hypertension was obtained 83.2% , 85% , and 84.1% in chinese , malaysian , and indian ethnics , respectively , which is twice higher than the prevalence of hypertension in the patients in iran .", "moreover , the prevalence of smoking was obtained 11.8% , 20% , and 15.9% in chinese , malaysian , and indian ethnics in singapore , respectively .", "the prevalence of smoking was obtained approximately 24% in our study , which is higher than that reported by the study of singapore . in the study of singapore ,", "the prevalence of diabetes was reported to be 67.5% in malaysian ethnics and 39.8% in chinese ethnics while it was 27.9% in the patients of our study . within 1 year of study ,", "hospital mortality was obtained 9.1% , 3.4% , and 2.5% in indian , chinese , and malaysian ethnics , respectively .", "this inconsistency can be explained by the difference in lifestyles , life expectancy , and the incidence of stroke risk factors .", "another explanation can be related to access to therapeutic and hospital services to prevent mortality due to stroke .", "however , it is noteworthy that the sample size in the study of singapore seems unsatisfactorily small , which might be another reason for the inconsistency of the findings .", "a review article reported that the age at stroke incidence was 68.6 years in men and 72.9 years in women , and the incidence and prevalence of stroke were higher in men than women .", "although the age of incidence in our study is younger than the age reported in this review article , the gender differences in our study and this review article are consistent .", "a nationwide multicenter cooperative cohort study of japanese patients with mi and stroke reported stroke history ( 14.7% ) to be more frequent than mi history ( 2.6% ) in patients with stroke , whereas stroke history ( 6.6% ) was less frequent than mi history ( 7.6% ) in patients with mi .", "the mean age at mi incidence in our study is lower than a study in australia that reported the mean age of patients with stroke was 78.6 years .", "the difference in lifestyle , life expectancy , and patterns of risk factors for noncommunicable diseases as well as the larger elderly population of australia can be some reasons for the discrepancy in the mean age at the incidence of stroke . in a study in nigeria , the prevalence of hypertension and dyslipidemia", "the difference in the population and environment of the study may be the main reasons for the inconsistency of the findings . in a study in australia and europe ( finland , norway , belgium , france ,", "germany , the netherlands , switzerland , italy , greece , and hungary ) , 3944 patients with stroke were studied , and smoking , dyslipidemia , and hypertension were reported to be the most prevalent ( 48.7% , 45.8% , and 35.9% , respectively ) risk factors for stroke , which are , except hypertension , higher than our study .", "the prevalence of diabetes and heart failure in the study in europe was obtained 6.511.1% and 3.84.2% , respectively , which is lower than the corresponding figures in our study .", "the study in europe has recommended that adoption of a primary strategy to avoid a stroke in young adolescents and the intervention aimed to change and/or decrease the highly prevalent risk factors that can be mitigated is an important principle of the stroke control . in brazil , the risk factors for stroke were studied , and the prevalence of hypertension , diabetes , and stroke history was reported to be 29.9% , 9.1% , and 2.1% , respectively , which are lower than the present study . in a study in japan ,", "the mean age at incidence was 73.3 years in men and 76.8 years in women .", "moreover , the prevalence of hypertension , diabetes , dyslipidemia , smoking , and heart attack was 55% , 19.3% , 17.9% , 25.9% , and 6.9% , respectively . in another study in japan , the fatality rate due to stroke was obtained 14.9% and 15.7% in men and women , respectively . since life expectancy is much higher in japan than iran and the mean age at stroke incidence", "is expected to be younger in iran than japan , the determinants of hospital mortality in our study are similar to a study reporting that mi history , diabetes , gender , and age are the most common determinants of outcome in intracerebral hemorrhage patients .", "interestingly , the prevalence of risk factors in the mi patients without stroke in iran and japan is similar .", "the prevalence of manageable risk factors is being controlled in patients with stroke and mi and was obtained lower compared to other studies . in a review article , the fatality rate of stroke within a month after the onset of the disease was reported to be 17% in japan , 33% in italy , and 22.9% overall .", "the difference may be explained by the access to better therapeutic services in hospitals and health - care facilities in italy compared with iran . in a study in iran , losing the golden time to start thrombolytic therapy , lack of beds in the intensive care units , and failure to afford the facilities and services were some of the obstacles to initiating the treatment in stroke and mi patients . in iran ,", "our study is the first report in iran and indicated that the pattern of determinants of hospital mi mortality with and without a stroke is various .", "therefore , adopting an appropriate strategy for the treatment and prevention of death in the two groups of the patients requires close attention to the characteristics of each group of the patients .", "in addition , our study demonstrated that the risk factors including hypertension , diabetes , smoking , dyslipidemia , and heart failure were more prevalent in mi patients with stroke history than those without .", "therefore , it is recommended to give greater priority to the mi patients with the previous stroke over those without in iran for training , healthcare , and control of the risk factors such as hypertension , diabetes , and smoking to enhance the cost - effectiveness of the interventions for prevention and treatment and to prevent avoidable mortalities . as no similar study was found to make a closer comparison between our and others findings , similar studies in other countries", "hence , the findings can be generalized only to hospitalized patients . in addition , tissue plasminogen activator thrombolytic therapy was not considered in mi patients with the previous stroke .", "because no similar study , to the best of our knowledge , was found to compare our and others findings more closely , similar studies in other countries should be conducted to give more definite explanations .", "hence , the findings can be generalized only to hospitalized patients . in addition , tissue plasminogen activator thrombolytic therapy was not considered in mi patients with the previous stroke .", "we did not gather any treatment information about these patients before mi . because no similar study , to the best of our knowledge , was found to compare our and others findings more closely", "our study demonstrated that the risk factors including hypertension , diabetes , smoking , dyslipidemia , and heart failure were more prevalent in the mi patients with a stroke history than those without .", "in fact , the mi patients without stroke history appear to control and mitigate the risk factors themselves .", "chest pain resistant to treatment gave the highest adjusted or of hospital mortality in patients with stroke followed by no thrombolytic therapy and stemi .", "it is recommended to give greater priority to the mi patients with the previous stroke over those without in iran for training , healthcare , and control of the risk factors as well as to enhance the cost - effectiveness of the interventions for prevention and treatment .", "aa , ak and ke contributed to the design of the research protocol and supervised its implementation .", "aa and ke analysed the data and aa , ak and ke drafted the article .", "all of the authors approved the final version of of the manuscript before publishing and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved .", "all of the authors reviewed drafts of the article and contributed to the conceptual framework ." ]

[ "<S> background : the data and determinants of mortality due to stroke in myocardial infarction ( mi ) patients are unknown . </S>", "<S> this study was conducted to evaluate the differences in risk factors for hospital mortality among mi patients with and without stroke history.materials and methods : this study was a retrospective , cohort study ; 20,750 new patients with mi from april , 2012 to march , 2013 were followed up and their data were analyzed according to having or not having the stroke history . </S>", "<S> stroke and mi were defined based on the world health organization 's definition . </S>", "<S> the data were analyzed by logistic regression in stata software.results:of the 20,750 studied patients , 4293 had stroke history . </S>", "<S> the prevalence of stroke in the studied population was derived 20.96% ( confidence interval [ ci ] 95% : 20.1321.24 ) . of the patients , 2537 ( 59.1% ) had st - elevation mi ( stemi ) . </S>", "<S> mortality ratio in patients with and without stroke was obtained 18.8% and 10.3% , respectively . </S>", "<S> the prevalence of risk factors in mi patients with and without a stroke is various . </S>", "<S> the adjusted odds ratio of mortality in patients with stroke history was derived 7.02 ( 95% ci : 5.429 ) for chest pain resistant to treatment , 2.39 ( 95% ci : 1.972.9 ) for stemi , 3.02 ( 95% ci : 2.53.64 ) for lack of thrombolytic therapy , 2.2 ( 95% ci : 1.662.91 ) for heart failure , and 2.17 ( 95% ci : 1.62.9 ) for ventricular tachycardia.conclusion:with regards to the factors associated with mortality in this study , it is particularly necessary to control the mortality in mi patients with stroke history . </S>", "<S> more emphasis should be placed on the mi patients with the previous stroke over those without in the interventions developed for prevention and treatment , and for the prevention of avoidable mortalities . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Statistical analysis", "RESULTS", "DISCUSSION", "Limitations of the study", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest", "AUTHORS CONTRIBUTORS" ]

[ [ "stroke , after myocardial infarction ( mi ) , is the second leading reason for mortality in iran as with many countries worldwide .", "the epidemiology of stroke has already been investigated in the american , european , african , and asian countries .", "no comprehensive study has yet investigated the epidemiology of stroke , particularly in mi patients , in iran , one of the largest countries in southwest asia .", "stroke and mi share many risk factors , most prevalent of which are smoking , dyslipidemia , type 2 diabetes , and hypertension .", "the risk factors for stroke and mi , especially smoking , hypertension , and dyslipidemia are highly prevalent in iran , as well . according to projections", "urbanism , increased life expectancy , reduction in childbirth , aging and elderly population , epidemiological changes , socioeconomic status , geographical conditions , and lifestyles such as poor diet , stress , and low mobility are the main causes of the burden of noncommunicable diseases , particularly stroke . because the determinants of stroke in different communities are various , we require knowledge about the risk factors and determinants of mortality in a community for effective planning and selection of appropriate strategies for the prevention and management of stroke and heart attack as the most important causes of death . since no comprehensive study has yet been investigated the status and mortality determinants of stroke in mi patients in iran , this study is conducted to determine and compare the determinants of mortality due to stroke in mi patients ." ], [ "in this retrospective cohort study , the data obtained from the mi registry of iran 's cardiovascular diseases surveillance system were analyzed . around", "20,750 hospitalized patients with mi with a new presentation ( hospitalized in 540 hospitals ) between april , 2012 and march , 2013 were enrolled .", "the study was approved by the management center of noncommunicable diseases and the department of cardiovascular diseases prevention of iran 's ministry of health and medical education ( approval no .", "the research followed the principles of the declaration of helsinki ; the researchers did not conduct any interventions on the patients , and an institutional review board approved this research .", "the study protocol was approved by an independent scientific review committee ( at shahid beheshti university medical of sciences ) ( no .", "inclusion criteria were determined according to the world health organization ( who ) and world heart federation definition of mi diagnosis per the international classification of diseases-10 codes i21 and i22 .", "patients with mi history or no definite diagnosis by a cardiologist were excluded from the study . the data on age , gender , education , ischemic heart disease symptoms , hospital stay duration , smoking , dyslipidemia , hypertension , type 2 diabetes , heart failure , and family history of cardiovascular diseases were gathered .", "stroke history was defined based on the who s definition rapidly developing signs of focal ( or global ) disturbance of cerebral function lasting > 24 h ( unless interrupted by surgery or death ) , with no apparent nonvascular cause . ", "therefore , patients with transient cerebral ischemia or stroke events were excluded if they had blood disease or brain tumors . moreover , patients with secondary strokes caused by trauma , acute hemorrhagic stroke , or transient ischemic attack were excluded . after a definite diagnosis of mi by a cardiologist ,", "the data on the left bundle branch block , right bundle branch block , atrial fibrillation , ventricular and atrial tachycardia , type and site of mi , and use of therapeutic regimens such as coronary artery bypass grafting , percutaneous coronary intervention , and thrombolytic therapy were recorded .", "the cohort of patients was defined by the date at mi diagnosis , hospital stay , and follow - up till discharge or death ( outcome ) .", "odds ratio ( or ) of mortality for clinical and demographic risk factors were calculated by logistic regression . or of mortality was calculated as the crude and adjusted ratio for patients with and without stroke by simple and multiple logistic regression models .", "first , univariate analysis was conducted . to control confounders , we entered the significant or approximately significant variables into a multiple regression model . for data", "analysis , chi - square , t - test , and regression model in stata software ( stata corp .", "2011 , stata statistical software : release 14 , college station , tx : stata corp lp , usa ) were used and p < 0.05 was considered the level of significance ." ], [ "the cohort of patients was defined by the date at mi diagnosis , hospital stay , and follow - up till discharge or death ( outcome ) .", "odds ratio ( or ) of mortality for clinical and demographic risk factors were calculated by logistic regression . or of mortality was calculated as the crude and adjusted ratio for patients with and without stroke by simple and multiple logistic regression models .", "first , univariate analysis was conducted . to control confounders , we entered the significant or approximately significant variables into a multiple regression model . for data", "analysis , chi - square , t - test , and regression model in stata software ( stata corp .", "2011 , stata statistical software : release 14 , college station , tx : stata corp lp , usa ) were used and p < 0.05 was considered the level of significance ." ], [ "the prevalence of stroke in the studied population was derived 20.96% ( confidence interval ci 95% : 20.1321.24 ) .", "mean standard deviation age at mi incidence was 64.38 ( 12.9 ) years in patients with stroke history and 60.37 ( 13.3 ) years in patients without , with a significant difference ( p < 0.001 ) .", "about 64.8% of these patients were male , 52.8% of them were illiterate , and only 4.9% had an academic education .", "the prevalence of diabetes , hypertension , hypercholesterolemia , and heart failure was obtained 30% , 56.5% , 27.3% , and 8.1% in patients with stroke history and 20.1% , 30% , 15.4% , and 8% in patients without stroke history , respectively .", "the ratio of cardiovascular surgery , percutaneous coronary intervention , and thrombolytic therapy was obtained 6.4% , 5.8% , and 41.1% in the former group and 1.6% , 7.1% , and 45.3% in the latter , respectively . the demographic characteristics and individual risk factors in patients with and without stroke", "demographic and medical history of myocardial infarction patients with and without stroke within 1 year of study , 18.8% of the stroke patients deceased .", "the mortality ratio in all patients was derived 12.1% . of the 4293 patients with stroke , 2537 ( 59.1% ) had st - elevation mi ( stemi ) .", "mortality ratio was obtained 18.8% in patients stroke history and 10.3% in the without . the clinical characteristics and risk factors in the patients according to the type of hospital diagnosis and therapeutic measures", "the ratio of cardiovascular surgery , percutaneous coronary intervention , and thrombolytic therapy was obtained , respectively , 6.4% , 5.8% , and 41.1% . by logistic regression ,", "the mortality - associated factors were different in the mi patients with and without stroke history .", "the adjusted or of mortality in patients with stroke history was derived 7.02 ( 95% ci : 5.429 ) for chest pain resistant to treatment , 2.39 ( 95% ci : 1.972.9 ) for stemi , 3.02 ( 95% ci : 2.53.64 ) for lack of thrombolytic therapy , 2.2 ( 95% ci : 1.662.91 ) for heart failure , and 2.17 ( 95% ci : 1.62.9 ) for ventricular tachycardia . considering other potential covariates and the confounders of hospital mortality in patients with stroke , we obtained the highest adjusted or of hospital mortality in patients with stroke for chest pain resistant to treatment followed by lack of thrombolytic therapy and stemi .", "prevalence of clinical risk factors in myocardial infarction patients with and without stroke risk factors for hospital mortality in myocardial infarction patients with and without stroke" ], [ "in this study , the risk factors in the mi patients with and without stroke history were investigated and compared for the first time in iran . in reliable , electronic databases such as pubmed and scopus , we found no study to model and compare the characteristics of the two groups of patients as with our study .", "our study indicated that the pattern of determinants of hospital mi mortality with and without a stroke is various .", "the mean age at incidence in our study is similar to a study in singapore comparing the risk factors for stroke in chinese , indian , and malaysian ethnic populations .", "the study in singapore reported the mean age of stroke patients to be 64.1 years . in the study of singapore", "the prevalence of hypertension was obtained 83.2% , 85% , and 84.1% in chinese , malaysian , and indian ethnics , respectively , which is twice higher than the prevalence of hypertension in the patients in iran .", "moreover , the prevalence of smoking was obtained 11.8% , 20% , and 15.9% in chinese , malaysian , and indian ethnics in singapore , respectively .", "the prevalence of smoking was obtained approximately 24% in our study , which is higher than that reported by the study of singapore . in the study of singapore ,", "the prevalence of diabetes was reported to be 67.5% in malaysian ethnics and 39.8% in chinese ethnics while it was 27.9% in the patients of our study . within 1 year of study ,", "hospital mortality was obtained 9.1% , 3.4% , and 2.5% in indian , chinese , and malaysian ethnics , respectively .", "this inconsistency can be explained by the difference in lifestyles , life expectancy , and the incidence of stroke risk factors .", "another explanation can be related to access to therapeutic and hospital services to prevent mortality due to stroke .", "however , it is noteworthy that the sample size in the study of singapore seems unsatisfactorily small , which might be another reason for the inconsistency of the findings .", "a review article reported that the age at stroke incidence was 68.6 years in men and 72.9 years in women , and the incidence and prevalence of stroke were higher in men than women .", "although the age of incidence in our study is younger than the age reported in this review article , the gender differences in our study and this review article are consistent .", "a nationwide multicenter cooperative cohort study of japanese patients with mi and stroke reported stroke history ( 14.7% ) to be more frequent than mi history ( 2.6% ) in patients with stroke , whereas stroke history ( 6.6% ) was less frequent than mi history ( 7.6% ) in patients with mi .", "the mean age at mi incidence in our study is lower than a study in australia that reported the mean age of patients with stroke was 78.6 years .", "the difference in lifestyle , life expectancy , and patterns of risk factors for noncommunicable diseases as well as the larger elderly population of australia can be some reasons for the discrepancy in the mean age at the incidence of stroke . in a study in nigeria , the prevalence of hypertension and dyslipidemia", "the difference in the population and environment of the study may be the main reasons for the inconsistency of the findings . in a study in australia and europe ( finland , norway , belgium , france ,", "germany , the netherlands , switzerland , italy , greece , and hungary ) , 3944 patients with stroke were studied , and smoking , dyslipidemia , and hypertension were reported to be the most prevalent ( 48.7% , 45.8% , and 35.9% , respectively ) risk factors for stroke , which are , except hypertension , higher than our study .", "the prevalence of diabetes and heart failure in the study in europe was obtained 6.511.1% and 3.84.2% , respectively , which is lower than the corresponding figures in our study .", "the study in europe has recommended that adoption of a primary strategy to avoid a stroke in young adolescents and the intervention aimed to change and/or decrease the highly prevalent risk factors that can be mitigated is an important principle of the stroke control . in brazil , the risk factors for stroke were studied , and the prevalence of hypertension , diabetes , and stroke history was reported to be 29.9% , 9.1% , and 2.1% , respectively , which are lower than the present study . in a study in japan ,", "the mean age at incidence was 73.3 years in men and 76.8 years in women .", "moreover , the prevalence of hypertension , diabetes , dyslipidemia , smoking , and heart attack was 55% , 19.3% , 17.9% , 25.9% , and 6.9% , respectively . in another study in japan , the fatality rate due to stroke was obtained 14.9% and 15.7% in men and women , respectively . since life expectancy is much higher in japan than iran and the mean age at stroke incidence", "is expected to be younger in iran than japan , the determinants of hospital mortality in our study are similar to a study reporting that mi history , diabetes , gender , and age are the most common determinants of outcome in intracerebral hemorrhage patients .", "interestingly , the prevalence of risk factors in the mi patients without stroke in iran and japan is similar .", "the prevalence of manageable risk factors is being controlled in patients with stroke and mi and was obtained lower compared to other studies . in a review article , the fatality rate of stroke within a month after the onset of the disease was reported to be 17% in japan , 33% in italy , and 22.9% overall .", "the difference may be explained by the access to better therapeutic services in hospitals and health - care facilities in italy compared with iran . in a study in iran , losing the golden time to start thrombolytic therapy , lack of beds in the intensive care units , and failure to afford the facilities and services were some of the obstacles to initiating the treatment in stroke and mi patients . in iran ,", "our study is the first report in iran and indicated that the pattern of determinants of hospital mi mortality with and without a stroke is various .", "therefore , adopting an appropriate strategy for the treatment and prevention of death in the two groups of the patients requires close attention to the characteristics of each group of the patients .", "in addition , our study demonstrated that the risk factors including hypertension , diabetes , smoking , dyslipidemia , and heart failure were more prevalent in mi patients with stroke history than those without .", "therefore , it is recommended to give greater priority to the mi patients with the previous stroke over those without in iran for training , healthcare , and control of the risk factors such as hypertension , diabetes , and smoking to enhance the cost - effectiveness of the interventions for prevention and treatment and to prevent avoidable mortalities . as no similar study was found to make a closer comparison between our and others findings , similar studies in other countries", "hence , the findings can be generalized only to hospitalized patients . in addition , tissue plasminogen activator thrombolytic therapy was not considered in mi patients with the previous stroke .", "because no similar study , to the best of our knowledge , was found to compare our and others findings more closely , similar studies in other countries should be conducted to give more definite explanations ." ], [ "hence , the findings can be generalized only to hospitalized patients . in addition , tissue plasminogen activator thrombolytic therapy was not considered in mi patients with the previous stroke .", "we did not gather any treatment information about these patients before mi . because no similar study , to the best of our knowledge , was found to compare our and others findings more closely" ], [ "our study demonstrated that the risk factors including hypertension , diabetes , smoking , dyslipidemia , and heart failure were more prevalent in the mi patients with a stroke history than those without .", "in fact , the mi patients without stroke history appear to control and mitigate the risk factors themselves .", "chest pain resistant to treatment gave the highest adjusted or of hospital mortality in patients with stroke followed by no thrombolytic therapy and stemi .", "it is recommended to give greater priority to the mi patients with the previous stroke over those without in iran for training , healthcare , and control of the risk factors as well as to enhance the cost - effectiveness of the interventions for prevention and treatment ." ], [ "" ], [ "" ], [ "aa , ak and ke contributed to the design of the research protocol and supervised its implementation .", "aa and ke analysed the data and aa , ak and ke drafted the article .", "all of the authors approved the final version of of the manuscript before publishing and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved .", "all of the authors reviewed drafts of the article and contributed to the conceptual framework ." ] ]

[ "angiogenesis , the formation of new blood vessels , is essential for tumor growth and metastasis and inhibition of angiogenesis is an important new approach for therapy of many cancers [ 1 , 2 ] . a principal regulator of angiogenesis is vascular endothelial growth factor ( vegf ) .", "the vegf family is comprised of at least five genes ( vegf a through e ) , of which the most potent activators of angiogenesis are vegf - a and vegf - b .", "analysis of the vegf - a ( termed vegf here and elsewhere in the text ) gene promoter region has revealed numerous potential transcriptional activator sites .", "one of the best studied stimuli for vegf synthesis and secretion is hypoxia , which acts by upregulating hypoxia inducible factor-1 ( hif-1 ) .", "inhibition of hif-1 may therefore decrease angiogenesis by reducing vegf levels and potentially other proangiogenic factors , such as angiopoeitin-1 and -2 ( ang-1 and ang-2 ) [ 68 ] , basic fibroblast growth factor ( bfgf ) , and platelet derived growth factor ( pdgf ) , all well known regulators of angiogenesis .", "t - oligos , telomere homolog oligonucleotides , activate p53 and e2f1 , resulting in apoptosis . ", "however , in p53 null cells like malignant melanoma an cells ( mm - an ) , apoptosis is induced by the p53 homolog p73 , presumably acting coordinately with e2f1 .", "moreover , e2f1 has also been reported to inhibit angiogenesis [ 9 , 12 ] . while e2f1 decreases vegf production in fibroblasts through p53 activation , its effect on angiogenesis in cells that lack functional p53 , such as mm - an cells has not been studied [ 11 , 13 ] .", "several lines of evidence suggest that normal cells have an integrated program of genome - protective responses , functionally analogous to the bacterial sos response , that is based in the telomeres and appears to be abrogated in malignancy .", "telomeres , the ends of chromosomes , are maintained in a loop configuration by insertion of the single - stranded 3 overhang into the proximal telomere duplex .", "disruption of this loop structure by removal of the principal binding protein trf2 ( telomere repeat binding factor 2 ) leads to exposure and digestion of the overhang and activation of atm ( ataxia telangiectasia mutated ) and its effector protein p53 , followed by apoptosis or senescence , depending on cell type .", "knockdown of another telomere - associated protein , the protection of telomeres-1 ( pot-1 ) , also expected to expose the ttaggg telomere repeat sequence , activates atr ( ataxia telangiectasia and rad3-related ) , leading to similar downstream effects [ 16 , 17 ] .", "moreover , treatment of malignant cells with rnai to knockdown the expression of ter , the rna subunit of telomerase , rapidly alters the expression of many genes in a pattern predicted to reduce cancer cell proliferation and invasiveness , then leads to apoptosis in a time course far too rapid to be attributable to the expected loss of telomerase activity and consequent telomere shortening , suggesting that other telomere - based effects are responsible .", "our laboratory has described several anticancer properties of oligonucleotides homologous to the telomere repeat sequence ttaggg ( t - oligos ) [ 10 , 11 , 13 , 15 , 1928 ] .", "t - oligos provided to cultured cells rapidly accumulate in the nucleus and mediate dna damage responses without digestion of the telomere overhang or other detectable effects on genomic dna [ 10 , 13 , 15 , 19 , 23 , 25 , 26 , 29 ] .", "t - oligos activate the atm kinase [ 21 , 27 ] , upregulate and activate p53 [ 30 , 31 ] , as well as upregulate and/or activate its homolog p73 , e2f1 , p16 , p33 , p27 , and p95/nbs1 , and phosphorylate the histone variant protein h2ax [ 11 , 13 , 21 , 22 , 24 , 25 ] .", "in addition , t - oligos promote differentiation of melanoma cells and downregulate the inhibitor of apoptosis protein iap / livin in these cells .", "t - oligo effects require wrn , the protein mutated in the progeroid cancer - prone werner syndrome , and are associated with formation of classic dna damage foci at telomeres . in combination , these signaling cascades result in induction of apoptosis , autophagy , and/or senescence selectively in cancer cells [ 13 , 19 , 27 , 28 ] ; while in normal cells they lead to transient cell cycle arrest , increased dna repair capacity and adaptive differentiation [ 19 , 21 , 23 , 2527 , 30 ] . ", "complementary , unrelated , or scrambled oligonucleotides comparably accumulate in the nucleus , but do not cause dna damage - like signaling or affect growth , differentiation or survival of malignant cells [ 11 , 13 , 15 , 19 , 21 , 24 , 27 , 28 ] . because the t - oligo - induced transcription factors p53 , p73 , and e2f1 are known to affect endothelial cell ( ec ) survival , differentiation , and proliferation during tumor angiogenesis ", "[ 12 , 33 , 34 ] and because blocking angiogenesis would be an additional anticancer mechanism of action for t - oligos , we asked whether t - oligo treatment inhibits tumor angiogenesis .", "we now report that t - oligo inhibits angiogenesis in the aggressive human melanoma cell line mm - an , derived from a metastatic melanoma , by decreasing production and secretion of proangiogenic factors in both tumor cells and ecs . as well", ", t - oligo treatment decreases the number of total and functional ( perfused ) vessels in flank tumors of mm - an cells in scid mice after two systemic injections .", "human microvascular endothelial cells ( hmvecs ) and human umbilical vein endothelial cells ( huvecs ) were obtained at passage 2 and used by passage 46 .", "cells were maintained in egm-2 medium with 2% fbs plus growth factors ( bullet kit ) ( cambrex bio sciences , walkersville , md ) . human melanoma mm - an and ep cells were cultured in modified eagle 's medium ( mem ) ( mediatech , inc . ,", "herndon , va ) supplemented with fetal bovine serum ( fbs 2% ) , calf serum ( cs 8% ) , and antibiotic - antimicotic .", "human breast adenocarcinoma ( mcf-7 ) and ovarian adenocarcinoma ( ovcar3 ) cells were cultured and maintained according to atcc recommendations .", "the oligonucleotide was synthesized by midland certified reagent ( midland , tx ) and then diluted from a 2 mm stock in medium to obtain a final concentration of 40 m .", "mm - an , ep , mcf-7 , and ovcar3 cells were grown in their corresponding media and treated once with diluent or 40 m t - oligo . for qrt - pcr", "the sequences used were as follows : e2f1 forward : cggtgtcgtcgacctgaact , e2f1 reverse : aggacgttggtgatgtcatagatg , e2f1 probe : tgccgaggtgctgaaggtgcag ; ang-1 forward : cagaaaacagtgggagaagatataacc , ang-1 reverse : tgccatcgtgttctggaaga , ang-1 probe : caacatgggcaatgtgcctacactttc ; ang-2 forward : ggctgggcaatgagtttgtc , ang-2 reverse : cccagtccttcagctggatct ; ang-2 probe : accggtcagcaccgctacgtgc .", "cells were treated with diluent or 40 m t - oligo and harvested 0 , 1 , 16 , and 32 hours after treatment .", "emsas were carried out as described using a total of 5 g of nuclear protein per lane .", "the assay was performed using consensus sequences of e2f1 and hif-1 transcription factors ( santa cruz biotechnology , ca ) .", "the specificity of the bands was confirmed by using either 25 or 50 excess of cold probe as competitor and mutant oligos as control . as an additional negative control", ", nuclear extracts were incubated with a specific competing e2f1 antibody before adding the radioactive ( p - labelled ) consensus oligonucleotides .", "cells were treated with t - oligos , harvested at various times , snap - frozen , and stored at 70c .", "total cellular protein was isolated and 50 g of total protein was processed for western blot analysis as described .", "antibody reactions were performed with the following antibodies : anti - e2f1 ( neomarkers , inc . ,", "fremont , ca ) , anti - vegf - a ( sc-507 , santa cruz biotechnology , inc , santa cruz , ca ) that recognizes all vegf - a isoforms , and anti - vegf - r2 ( a kind gift from dr .", "nader rahimi , departments of ophthalmology and biochemistry , boston university school of medicine ) . the human vegf immunoassay kit ( quantikine , r&d systems , minneapolis , mn ) was used to compare the release of vegf into the medium by ecs and mm - an cells", "each cell type was cultured in its appropriate medium and treated with either diluent or t - oligo ( 40 m ) .", "the conditioned medium was collected at 24 , 48 , and 72 hours after treatment and frozen at 70c until it processed for elisa .", "the plate was read using the tecan spectra model 96 well microplate reader ( mtx lab systems , vienna , va ) .", "200 l of matrigel basement membrane matrix ( phenol - red free ) ( bd biosciences , bedford , ma ) was added per 4-well chamber slide and allowed to solidify for 1 hour at 37c .", "huvecs ( 50,000 cells ) were then added to each chamber in 500 l of medium as described .", "cells were treated at the time of plating with diluent , 40 m , 80 m , or 120 m of t - oligo . some of the cells treated with 40 m of t - oligo were retreated a second time at 2 hours postplating , at the time tube - formation", "some cells that were treated twice with 40 m of t - oligo received a third treatment 6 hours after plating .", "tube - formation was observed and photographed at numerous time points starting from 2 hours and up to 24 hours after treatment . of note , after tubular structures formed in control wells , they stayed intact up to 72 hours ( data not shown ) .", "image - tool ( the university of texas health science center in san antonio , san antonio , tx ) software was used to quantify the length of tubular structures . the biocoat matrigel invasion chamber ( bd biosciences , bedford ,", "mm - an cells were treated with diluent or 40 m t - oligo and media were collected after 72 hours , based on elisa data ( mm - an conditioned media ) . conditioned mm - an medium was then used as the chemoattractant for the assay .", "ecs and mm - an cells were grown on the inserts ( upper chambers ) and allowed to invade through the matrigel and attach to the membrane as described in the manufacturer 's protocol .", "invasion was assessed after 22 hours by staining with diff - quick ( fisher , atlanta , ga ) bisected membranes from the bottom of the chambers ( containing the invading cells ) as described in the manufacturer 's protocol . of note , we believe that any effect on ecs or mm - an invasion can not be attributed to residual active t - oligos in the conditioned medium , diffusing into the upper chamber and killing mm - an cells , because t - oligos are rapidly degraded in serum - containing medium . with a measured half - life for a 12-base telomere homolog of 46 hours", ", the 16-base t - oligo would thus have been present in the medium after 72 hours of conditioning at an estimated ( 1/2 ) or 10-fold less than the initial therapeutic concentration .", "2 10 mm - an cells were injected subcutaneously into the flank of 6 week old scid mice ( fox - chase cancer center , philadelphia , pa ) .", "mice ( 5 - 6 per treatment group ) were injected via tail vein with t - oligo or diluent alone when tumors were first palpable ( 2 - 3 mm diameter ) .", "tumor sizes were recorded every 2 - 3 days throughout the experiment using electronic callipers and all animals were sacrificed 4 weeks after tumor inoculation .", "the diagnosis of melanoma was confirmed histologically on sections of each nodule cut through the center of the clinical tumor .", "2 10 mm - an cells were injected subcutaneously into the flank of 6 week old scid mice ( fox - chase cancer center , philadelphia , pa )", ". then mice ( 5 - 6 per treatment group ) were injected via the tail vein with t - oligo or diluent alone ( 60 nmoles / injection , 15 mg / kg ) when tumors were first palpable ( 2 - 3 mm diameter ) .", "microvascular density ( mvd ) in bisected tumors were assessed 24 hours after two systemic injections of t - oligos by using two ec - specific markers cd31 ( pecam-1 ) and bandeurea simplicifolia ( bs)-1 lectin conjugated to rhodamine ( vector laboratories , burlingame , ca ) . to measure functional mvd in the tumor tissue ,", "30 minutes before sacrifice a set of mice ( 5 per treatment group ) were anesthetized and were perfused with 0.5 mg ( in 100 l of isotonic solution ) of rhodamine - conjugated bs-1 lectin as described [ 40 , 41 ] . to measure total mvd , 6 m cross - sections of bisected tumor tissue ( of the same mice perfused with bs-1 lectin )", "were stained with cd31 primary antibody followed by fitc - labelled secondary antibody as described . ", "samples were photographed using a multicolour fluorescence microscope ( nikon , nikon instruments inc , melville , ny ) , and analyzed using a digital image analysis system ( nikon ) .", "anova with post hoc analysis by scheffe and bonferroni - dunn and unpaired t - test were performed using statview ( sas institute , inc . ,", "mm - an cells were treated once with t - oligo or diluent alone , provided in fresh medium , and processed for western blot analysis .", "compared to diluent , t - oligo decreased vegf protein level at 24 hours ( p < .01 ) by ~41% and at 48 hours ( p = ns ) by ~32% ( figures 1(a ) and 1(b ) ) .", "in addition , after 48 hours t - oligo treatment reduced ang-1 mrna ( p < .01 ) by ~60% ( figure 1(c ) ) and ang-2 mrna ( p < .04 ) by ~45% ( figure 1(d ) ) .", "similar decreases in vegf ( supplemental figures 1(a)1(c ) and ang-1 ( supplemental figures 2(a)2(c ) ) mrna were seen in a second human melanoma line ep , as well as in other malignant cell types . paired dishes of mm - an and ecs were treated with 40 m t - oligo or diluent alone once .", "medium was collected after 24 , 48 and 72 hours and vegf level in the medium was measured by elisa . in ecs ( hmvec and huvec ) , there was a ~50%75% variable and statistically insignificant decrease in vegf release into the medium over time in both diluent- and t - oligo - treated cells ( data not shown ) . ", "in contrast , total vegf release from mm - an cells in diluent - treated dishes rose progressively over 72 hours to ~600% ( of 24-hours vegf levels ) , while vegf release in t - oligo - treated dishes was decreased ( p < .0008 ) by ~30% over the 72-hours experiment after a single dosing at time 0 ( figure 1(e ) ) . ", "thus , t - oligo not only decreased vegf protein expression in tumor cells but also the release of this potent angiogenic factor into the surrounding culture medium .", "e2f1 mrna expression was increased at 24 hours ( p < .0001 ) by ~41% and at 48 hours ( p < .0001 ) by ~100% in t - oligo - treated mm - an cells compared to control ( figure 2(a ) ) .", "similar increases in e2f1 mrna were seen in a second human melanoma line ep , as well as in other malignant cell types ( supplemental figures 3(a)3(c ) ) .", "t - oligo treatment also increased e2f1 protein level at 24 hours to ~218% and at 48 hours to ~286% ( p < .02 ) of control values ( figures 2(b ) and 2(c ) ) .", "in addition , there was a doubling in e2f1 dna binding activity 32 hours after treatment compared to diluent - treated cells ( figures 2(d ) and 2(e ) , compare lane 3 versus 4 ) .", "mm - an cells were treated once with t - oligo or diluent alone and nuclear proteins were harvested up to 32 hours after treatment to evaluate dna binding activities of the angiogenic transcription factor hif-1. there was ~80%95% decrease in hif-1 dna binding activity at 1 and 16 hours after treatments ( figures 2(f ) and 2(g ) , lanes 3 versus 4 , and 5 versus 6 , respectively ) .", "these data suggest that t - oligo - mediated inhibition in dna binding activity of hif-1 transcription factor contributes to decreased expression of the angiogenic factors , vegf and ang-1 , whose promoters contain binding sites for hif-1 .", "we used mm - an medium collected 72 hours after addition of t - oligo or diluent alone ( mm - an conditioned media : t - oligo cm or diluent cm ) as the chemoattractant for an in vitro invasion assay .", "mm - an and hmvecs were both evaluated for invasion through matrigel to determine if t - oligo treatment reduces the chemoattractant properties of mm - an cells . as recommended by the manufacturer of the matrigel invasion kit , after 22 hours of incubation the invading cells were fixed , stained and counted for each membrane .", "mm - an cells plated above t - oligo cm had a ~96% decrease in invasion ( p < .03 ) through matrigel ( figures 3(a ) and 3(b ) ) .", "hmvecs plated on inserts coated with matrigel and exposed to t - oligo cm had a ~40% reduction in matrigel invasion compared to controls , but this did not reach statistical significance ( p < .08 ) ( figures 3(c ) and 3(d ) ) .", "these findings demonstrate that t - oligos reduce the migration / invasion of mm - an cells towards chemoattractant stimuli and , possibly , to a lesser degree affect hmvecs .", "this is consistent with the observation that mm - an cells elaborate factors that promote migration and invasion of tumor cells , like vegf and ang-1 , and that these factor(s ) are reduced as a result of t - oligo treatment . because vegf signaling through vegfr-2 is principally responsible for ec survival , proliferation , migration , and angiogenesis [ 4447 ] , we examined the effect of t - oligo treatment on these proteins .", "hmvecs and huvecs were grown and treated with 40 m t - oligo or diluent alone .", "compared to diluent - treated control cells there was a decrease ( p < .004 ) of ~86% in vegf expression at 24 hours and an insignificant decrease of ~50% at 48 hours in t - oligo - treated samples ( figures 4(a)4(c ) ) .", "in addition , in t - oligo - treated hmvecs and huvecs there were ~29% ( p = ns ) and ~59% ( p < .04 ) reduction in the protein level of vegfr-2 at 24 and 48 hours , respectively ( figures 4(d)4(f ) ) .", "thus , t - oligo - mediated antiangiogenic effects on ecs may be , in part , due to inhibition of vegf - vegr2 axis .", "hmvecs were plated on matrigel in 4-well chamber slides in serum - containing medium and treated with diluent alone or increasing concentrations of t - oligo .", "tube - like structure formation was maximal in controls at 22 hours and formal comparisons were made after 222 hours , as customary for this assay . by 22 hours , in cells treated once with either 40 m or 80 m t - oligo there was a ~19% reduction in average tube length compared to cells treated with diluent alone ( p = ns ) ( figures 5(a ) and 5(b ) ) .", "cells treated with two separate doses of 40 m t - oligo at plating and 2 hours after plating showed a ~35% reduction in average tube length ( p < .03 ) .", "cells treated once at plating with 120 m t - oligo showed a ~58% reduction in average tube length compared to the diluent - treated cells ( p < .001 ) . a reduction of ~83% ( p", "< .0001 ) in tube length was observed in cells treated with 3 separate doses of 40 m t - oligo ( 40 m 3 ) added to the medium at the time of plating and then at 2 and 6 hours ( figures 5(a ) and 5(b ) ) .", "these results indicate that t - oligo inhibits ec function in a manner dependent on the dose and frequency of administration , with the caveat that the very large amount of oligos administered , rather that specific telomere - based t - oligo initiated signalling may have contributed to the effect observed . in our future experiments", ", we plan to examine the effect of large and/or fractioned t - oligo doses on survival of ecs . in melanoma tumors inoculated into scid mice , we determined the effect of systemic t - oligo injection on melanoma angiogenesis by evaluating functional and total vessel density in tumor tissue ( 5 mice per group ) . when tumors became palpable", "( 2 - 3 mm in diameter , day 5 to 14 after inoculation ) we injected t - oligo ( 60 nmoles / injection , 15 mg / kg ) via the tail vein ( iv ) and injected again after 6 hours , then harvested tumors 24 hours after the second t - oligo injections .", "representative images of tumor cross - sections were immunostained to identify all vessels as well as functional ( patent ) vessels 24 hours after t - oligo injection ( figure 6(a ) ) . compared to control - injected mice", ".002 ) in functional vessels in t - oligo - injected mice ( figure 6(b ) ) . ", "there was also more than ~60% decrease ( p < .004 ) in total vessels in t - oligo - injected mice ( figure 6(c ) ) . ", "these data corroborate our in vitro findings ( figures 4 and 5 ) and demonstrate that two systemic administrations of t - oligo reduce tumor angiogenesis and vessel patency in vivo ( figures 6(a)6(c ) ) . to determine the effect of systemic t - oligo treatment on melanoma growth , in scid xenograft model mm - an cells", "were injected subcutaneously in the flank . when tumors became palpable ( 2 - 3 mm in diameter , day 5 to 14 ) ,", "the mice received daily systemic injections of t - oligo ( 60 nmoles / injection , 15 mg / kg bid ) or vehicle for 5 days only . in control animals , tumors increased in volume from 6.96 2.9 mm on day 6 to 88.04 19.72 mm on day 27 ( figure 6(d ) , blue line ) .", "in contrast , in t - oligo treated animals , whose tumors were the same size as in controls on day 6 ( 6.48 2.57 ) there was very little tumor growth through day 15 ( 6.48 2.57 versus 9.92 3.8 mm ) and thereafter a slower and statistically insignificant increase in volume to 29.65 10.47 mm by day 27 ( figure 6(d ) , pink line ) .", "thereby , in t - oligo - treated mice tumor growth was inhibited ( p < .003 ) by ~53% after ~4 weeks , when the experiment was terminated .", "these data demonstrate that systemic administration of t - oligo for only 5 days has a persistent inhibitory effect on melanoma growth .", "we have also evaluated t - oligos toxicity in several internal organs of scid mice 24 hours after the last iv injections ( 15 mg / kg bid for 5 days ) .", "no systemic toxicity was observed in bone marrow , liver , intestines , brain , lungs and kidneys of t - oligo - injected mice ( figures 7(a)7(f ) ) .", "tumor angiogenesis is essential for tumor growth and metastasis . without active angiogenesis tumor diameter", "angiogenesis is mediated through release of angiogenic factors by tumor cells and cells in the tumor stroma and microenvironment including , but not limited to , endothelial cells . ", "we now report that telomere homolog oligonucleotides ( t - oligos ) decrease the synthesis and release of angiogenic factors by ecs and melanoma cells , inhibit ec tubulogenesis and impede melanoma cells and ecs from invading matrix ( matrigel ) .", "ec proliferation , in vitro tubulogenesis , and survival are all known to be stimulated in large part by vegf . decreased", "vegf levels or inhibition of receptor activation in ecs often correlate with decrease in tumor size and metastatic potential .", "vegf binds to the extracellular domain of the vegfr-1 ( flt-1 ) and vegfr-2 ( flk-1 ) , inducing receptor dimerization and activation of tyrosine kinases by autophosphorylation , leading to angiogenesis , increased vascular permeability , and ec proliferation and survival .", "we found that t - oligo decreases the expression of vegfr-2 by hmvec and huvec ( figures 4(c ) and 4(f ) ) . ", "other investigators reported that receptor tyrosine kinase inhibitors ( tkis ) such as sunitinib and dasatinib reduce signaling through the raf / mek / erk pathway that is activated by ligand binding to angiogenic receptors like vegfr-2 , pdgfr- , fh-3 and c - kit , indirectly inhibiting tumor growth by affecting tumor angiogenesis . by reducing vegf - r2 level ,", "vegf also induces leakage within tumor vessels , allowing tumor cells to infiltrate blood vessels and migrate into the blood stream .", "hence , changes in angiogenic factors even early in tumor formation can affect metastasis and spread and inhibiting vegf production by t - oligos would be expected to reduce the metastatic potential of tumor cells . additionally , increased blood vessel permeability within the tumor may interfere with adequate delivery and retention of chemotherapeutic agents [ 54 , 55 ] . ", "indeed , certain antiangiogenic agents that prevent tumor vessel leakage ( a phenomenon called vessel normalization ) were shown to enhance the delivery of chemotherapeutic agents into tumors . ", "thus , combination treatment with antiangiogenic factors together with conventional chemo - therapeutic agents may be superior to using the latter alone . ", "furthermore , because vegf is likely required for migration and recruitment of ecs , t - oligo - me diated vegf reduction would also likely decrease the number of blood vessels in the tumor [ 1 , 6 ] . ", "our present findings suggest that t - oligos may induce potent antiangiogenic effects , enhancing their attractiveness as a therapeutic option for cancer .", "in addition to vegf and its receptors , angiopoietin 1 and 2 ( ang-1 and ang-2 ) and their tyrosine kinase receptor tie-2 have been identified as major players in the processes of growth and remodelling of tumor vasculature [ 57 , 58 ] .", "although under certain conditions ang-2 may inhibit ang-1 effect , in an in vivo mouse model for melanoma and in glioma cells increased expression of ang-2 is thought to stimulate angiogenesis . another angiogenic factor", ", hif-1 , a transcription factor that is activated by hypoxia , exerts its effect by upregulating vegf levels . ", "we found that t - oligo treatment inhibits hif-1 activity and ang-1 and ang-2 expression in melanoma cell line and decreases vegf synthesis secretion in these cells , strongly suggesting that t - oligo - mediated effects on tumor angiogenesis are transcriptional and ultimately affect several angiogenic molecules .", "our laboratory has previously shown that t - oligo increases p73 level in the p53 null mm - an cells and that blocking p73 expression by rnai decreases t - oligo - induced apoptosis in these cells . like p53 , p73 is known to inhibit angiogenesis , primarily through vegf down - regulation .", "therefore , we assume that in mm - an cells t - oligo decreases vegf production , in addition to its effect on hif-1 , by activating p73 through induction of e2f1 . ", "however , in cells with functional p53 , we assume that t - oligo - induced p53 and p73 would cooperate to inhibit angiogenesis . ", "furthermore , e2f1 is known to induce apoptosis in part by its effect on p73 . ", "thus , induction of e2f1 by t - oligos also contributes to tumor cell apoptosis . ", "we have previously shown that t - oligo treatment activates ( phosphorylates ) atm [ 21 , 27 ] .", "we therefore suggest that t - oligo regulates e2f1 first via atm - mediated phosphorylation of e2f1 and that e2f1 then further transcriptionally upregulates e2f1 by binding to its own promoter . indeed", ", we show that t - oligo increases e2f1 mrna and protein levels as well as its dna binding activity in human mm - an melanoma cells , further confirming our previous finding that e2f1contributes to t - oligo effects . ", "the t - oligos used in the present experiments have physiologic readily hydrolysable phosphodiester linkage , unlike for example antisense dna in which phosphorothioate or other nonhydrolyzable linkage is employed to increase the molecule 's half - life ( t 1/2 ) . ", "hydrolyzable linkage is required for initiation of t - oligo signalling and , despite the known short t 1/2 , approximately 46 hours in serum - containing medium for a 12-base 100% telomere homolog , nevertheless this allows for the cellular responses observed in the present and previous experiments to evolve over 35 days [ 11 , 13 , 19 , 21 , 22 , 2428 ] . ", "this may reflect the fact that at least in vitro t - oligos rapidly enter the nucleus [ 11 , 22 , 24 , 27 ] and that , once in the nucleus , such oligos have a far longer t 1/2 . ", "the efficacy of these presumptively short - lived t - oligos may also reflect the likelihood that , after interaction of the oligos with the werner protein and formation of dna damage - like foci at telomeres , signalling through the dna damage response pathways may continue even if the t - oligos have been hydrolyzed , at least through 48 hours at which time the dna damage foci can still be observed by immunohistochemistry . in earlier studies , we have shown that t - oligos inhibit tumor growth in scid mouse models by inducing cell cycle arrest , differentiation , apoptosis , and senescence [ 13 , 15 , 27 ] . ", "angiogenesis inhibition encompassing both ecs and melanoma cells , in combination with other t - oligo - mediated anti - tumor effects [ 11 , 13 , 22 , 25 , 27 ] , likely combine to significantly decrease melanoma burden in established scid mouse models , as reported in our earlier publications [ 13 , 27 ] . ", "these multiple diverse responses are all mediated through activation and/or upregulation of dna damage response proteins . ", "they are thus reminiscent of the less complex but very well characterized bacterial sos response , a genome - protective mechanism that enhances survival of prokaryotic organisms in the face of dna damage . ", "the telomere - based dna damage - like signalling initiated by t - oligos may be viewed as an evolutionarily perfected parallel mechanism in mammalian cells that addresses the threat to genomic integrity posed by malignant transformation . in summary , the present paper indicates that t - oligos exert multiple antiangiogenic effects .", "these data reinforce prior evidence that t - oligo therapy may offer a powerful new approach to treatment of human primary melanoma and possibly other human malignancies , with several conceptual advantages over the currently lionized targeted therapy approach ." ]

[ "<S> telomere homolog oligonucleotides ( t - oligos ) activate an innate telomere - based program that leads to multiple anticancer effects . </S>", "<S> t - oligos act at telomeres to initiate signaling through the werner protein and atm kinase . </S>", "<S> we wanted to determine if t - oligos have antiangiogenic effects . </S>", "<S> we found that t - oligo - treated human melanoma ( mm - an ) cells had decreased expression of vascular endothelial growth factor ( vegf ) , vegf receptor 2 , angiopoeitin-1 and -2 and decreased vegf secretion . </S>", "<S> t - oligos activated the transcription factor e2f1 and inhibited the activity of the angiogenic transcription factor , hif-1. t - oligos inhibited ec tubulogenesis and total tumor microvascular density matrix invasion by mm - an cells and ecs in vitro . in melanoma scid xenografts , two systemic t - oligo injections decreased by 60% ( p < .004 ) total tumor microvascular density and the functional vessels density by 80% ( p </S>", "<S> < .002 ) . </S>", "<S> these findings suggest that restriction of tumor angiogenesis is among the host 's innate telomere - based anticancer responses and provide further evidence that t - oligos may offer a powerful new approach for melanoma treatment . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion" ]

[ [ "angiogenesis , the formation of new blood vessels , is essential for tumor growth and metastasis and inhibition of angiogenesis is an important new approach for therapy of many cancers [ 1 , 2 ] . a principal regulator of angiogenesis is vascular endothelial growth factor ( vegf ) .", "the vegf family is comprised of at least five genes ( vegf a through e ) , of which the most potent activators of angiogenesis are vegf - a and vegf - b .", "analysis of the vegf - a ( termed vegf here and elsewhere in the text ) gene promoter region has revealed numerous potential transcriptional activator sites .", "one of the best studied stimuli for vegf synthesis and secretion is hypoxia , which acts by upregulating hypoxia inducible factor-1 ( hif-1 ) .", "inhibition of hif-1 may therefore decrease angiogenesis by reducing vegf levels and potentially other proangiogenic factors , such as angiopoeitin-1 and -2 ( ang-1 and ang-2 ) [ 68 ] , basic fibroblast growth factor ( bfgf ) , and platelet derived growth factor ( pdgf ) , all well known regulators of angiogenesis .", "t - oligos , telomere homolog oligonucleotides , activate p53 and e2f1 , resulting in apoptosis . ", "however , in p53 null cells like malignant melanoma an cells ( mm - an ) , apoptosis is induced by the p53 homolog p73 , presumably acting coordinately with e2f1 .", "moreover , e2f1 has also been reported to inhibit angiogenesis [ 9 , 12 ] . while e2f1 decreases vegf production in fibroblasts through p53 activation , its effect on angiogenesis in cells that lack functional p53 , such as mm - an cells has not been studied [ 11 , 13 ] .", "several lines of evidence suggest that normal cells have an integrated program of genome - protective responses , functionally analogous to the bacterial sos response , that is based in the telomeres and appears to be abrogated in malignancy .", "telomeres , the ends of chromosomes , are maintained in a loop configuration by insertion of the single - stranded 3 overhang into the proximal telomere duplex .", "disruption of this loop structure by removal of the principal binding protein trf2 ( telomere repeat binding factor 2 ) leads to exposure and digestion of the overhang and activation of atm ( ataxia telangiectasia mutated ) and its effector protein p53 , followed by apoptosis or senescence , depending on cell type .", "knockdown of another telomere - associated protein , the protection of telomeres-1 ( pot-1 ) , also expected to expose the ttaggg telomere repeat sequence , activates atr ( ataxia telangiectasia and rad3-related ) , leading to similar downstream effects [ 16 , 17 ] .", "moreover , treatment of malignant cells with rnai to knockdown the expression of ter , the rna subunit of telomerase , rapidly alters the expression of many genes in a pattern predicted to reduce cancer cell proliferation and invasiveness , then leads to apoptosis in a time course far too rapid to be attributable to the expected loss of telomerase activity and consequent telomere shortening , suggesting that other telomere - based effects are responsible .", "our laboratory has described several anticancer properties of oligonucleotides homologous to the telomere repeat sequence ttaggg ( t - oligos ) [ 10 , 11 , 13 , 15 , 1928 ] .", "t - oligos provided to cultured cells rapidly accumulate in the nucleus and mediate dna damage responses without digestion of the telomere overhang or other detectable effects on genomic dna [ 10 , 13 , 15 , 19 , 23 , 25 , 26 , 29 ] .", "t - oligos activate the atm kinase [ 21 , 27 ] , upregulate and activate p53 [ 30 , 31 ] , as well as upregulate and/or activate its homolog p73 , e2f1 , p16 , p33 , p27 , and p95/nbs1 , and phosphorylate the histone variant protein h2ax [ 11 , 13 , 21 , 22 , 24 , 25 ] .", "in addition , t - oligos promote differentiation of melanoma cells and downregulate the inhibitor of apoptosis protein iap / livin in these cells .", "t - oligo effects require wrn , the protein mutated in the progeroid cancer - prone werner syndrome , and are associated with formation of classic dna damage foci at telomeres . in combination , these signaling cascades result in induction of apoptosis , autophagy , and/or senescence selectively in cancer cells [ 13 , 19 , 27 , 28 ] ; while in normal cells they lead to transient cell cycle arrest , increased dna repair capacity and adaptive differentiation [ 19 , 21 , 23 , 2527 , 30 ] . ", "complementary , unrelated , or scrambled oligonucleotides comparably accumulate in the nucleus , but do not cause dna damage - like signaling or affect growth , differentiation or survival of malignant cells [ 11 , 13 , 15 , 19 , 21 , 24 , 27 , 28 ] . because the t - oligo - induced transcription factors p53 , p73 , and e2f1 are known to affect endothelial cell ( ec ) survival , differentiation , and proliferation during tumor angiogenesis ", "[ 12 , 33 , 34 ] and because blocking angiogenesis would be an additional anticancer mechanism of action for t - oligos , we asked whether t - oligo treatment inhibits tumor angiogenesis .", "we now report that t - oligo inhibits angiogenesis in the aggressive human melanoma cell line mm - an , derived from a metastatic melanoma , by decreasing production and secretion of proangiogenic factors in both tumor cells and ecs . as well", ", t - oligo treatment decreases the number of total and functional ( perfused ) vessels in flank tumors of mm - an cells in scid mice after two systemic injections ." ], [ "human microvascular endothelial cells ( hmvecs ) and human umbilical vein endothelial cells ( huvecs ) were obtained at passage 2 and used by passage 46 .", "cells were maintained in egm-2 medium with 2% fbs plus growth factors ( bullet kit ) ( cambrex bio sciences , walkersville , md ) . human melanoma mm - an and ep cells were cultured in modified eagle 's medium ( mem ) ( mediatech , inc . ,", "herndon , va ) supplemented with fetal bovine serum ( fbs 2% ) , calf serum ( cs 8% ) , and antibiotic - antimicotic .", "human breast adenocarcinoma ( mcf-7 ) and ovarian adenocarcinoma ( ovcar3 ) cells were cultured and maintained according to atcc recommendations .", "the oligonucleotide was synthesized by midland certified reagent ( midland , tx ) and then diluted from a 2 mm stock in medium to obtain a final concentration of 40 m .", "mm - an , ep , mcf-7 , and ovcar3 cells were grown in their corresponding media and treated once with diluent or 40 m t - oligo . for qrt - pcr", "the sequences used were as follows : e2f1 forward : cggtgtcgtcgacctgaact , e2f1 reverse : aggacgttggtgatgtcatagatg , e2f1 probe : tgccgaggtgctgaaggtgcag ; ang-1 forward : cagaaaacagtgggagaagatataacc , ang-1 reverse : tgccatcgtgttctggaaga , ang-1 probe : caacatgggcaatgtgcctacactttc ; ang-2 forward : ggctgggcaatgagtttgtc , ang-2 reverse : cccagtccttcagctggatct ; ang-2 probe : accggtcagcaccgctacgtgc .", "cells were treated with diluent or 40 m t - oligo and harvested 0 , 1 , 16 , and 32 hours after treatment .", "emsas were carried out as described using a total of 5 g of nuclear protein per lane .", "the assay was performed using consensus sequences of e2f1 and hif-1 transcription factors ( santa cruz biotechnology , ca ) .", "the specificity of the bands was confirmed by using either 25 or 50 excess of cold probe as competitor and mutant oligos as control . as an additional negative control", ", nuclear extracts were incubated with a specific competing e2f1 antibody before adding the radioactive ( p - labelled ) consensus oligonucleotides .", "cells were treated with t - oligos , harvested at various times , snap - frozen , and stored at 70c .", "total cellular protein was isolated and 50 g of total protein was processed for western blot analysis as described .", "antibody reactions were performed with the following antibodies : anti - e2f1 ( neomarkers , inc . ,", "fremont , ca ) , anti - vegf - a ( sc-507 , santa cruz biotechnology , inc , santa cruz , ca ) that recognizes all vegf - a isoforms , and anti - vegf - r2 ( a kind gift from dr .", "nader rahimi , departments of ophthalmology and biochemistry , boston university school of medicine ) . the human vegf immunoassay kit ( quantikine , r&d systems , minneapolis , mn ) was used to compare the release of vegf into the medium by ecs and mm - an cells", "each cell type was cultured in its appropriate medium and treated with either diluent or t - oligo ( 40 m ) .", "the conditioned medium was collected at 24 , 48 , and 72 hours after treatment and frozen at 70c until it processed for elisa .", "the plate was read using the tecan spectra model 96 well microplate reader ( mtx lab systems , vienna , va ) .", "200 l of matrigel basement membrane matrix ( phenol - red free ) ( bd biosciences , bedford , ma ) was added per 4-well chamber slide and allowed to solidify for 1 hour at 37c .", "huvecs ( 50,000 cells ) were then added to each chamber in 500 l of medium as described .", "cells were treated at the time of plating with diluent , 40 m , 80 m , or 120 m of t - oligo . some of the cells treated with 40 m of t - oligo were retreated a second time at 2 hours postplating , at the time tube - formation", "some cells that were treated twice with 40 m of t - oligo received a third treatment 6 hours after plating .", "tube - formation was observed and photographed at numerous time points starting from 2 hours and up to 24 hours after treatment . of note , after tubular structures formed in control wells , they stayed intact up to 72 hours ( data not shown ) .", "image - tool ( the university of texas health science center in san antonio , san antonio , tx ) software was used to quantify the length of tubular structures . the biocoat matrigel invasion chamber ( bd biosciences , bedford ,", "mm - an cells were treated with diluent or 40 m t - oligo and media were collected after 72 hours , based on elisa data ( mm - an conditioned media ) . conditioned mm - an medium was then used as the chemoattractant for the assay .", "ecs and mm - an cells were grown on the inserts ( upper chambers ) and allowed to invade through the matrigel and attach to the membrane as described in the manufacturer 's protocol .", "invasion was assessed after 22 hours by staining with diff - quick ( fisher , atlanta , ga ) bisected membranes from the bottom of the chambers ( containing the invading cells ) as described in the manufacturer 's protocol . of note , we believe that any effect on ecs or mm - an invasion can not be attributed to residual active t - oligos in the conditioned medium , diffusing into the upper chamber and killing mm - an cells , because t - oligos are rapidly degraded in serum - containing medium . with a measured half - life for a 12-base telomere homolog of 46 hours", ", the 16-base t - oligo would thus have been present in the medium after 72 hours of conditioning at an estimated ( 1/2 ) or 10-fold less than the initial therapeutic concentration .", "2 10 mm - an cells were injected subcutaneously into the flank of 6 week old scid mice ( fox - chase cancer center , philadelphia , pa ) .", "mice ( 5 - 6 per treatment group ) were injected via tail vein with t - oligo or diluent alone when tumors were first palpable ( 2 - 3 mm diameter ) .", "tumor sizes were recorded every 2 - 3 days throughout the experiment using electronic callipers and all animals were sacrificed 4 weeks after tumor inoculation .", "the diagnosis of melanoma was confirmed histologically on sections of each nodule cut through the center of the clinical tumor .", "2 10 mm - an cells were injected subcutaneously into the flank of 6 week old scid mice ( fox - chase cancer center , philadelphia , pa )", ". then mice ( 5 - 6 per treatment group ) were injected via the tail vein with t - oligo or diluent alone ( 60 nmoles / injection , 15 mg / kg ) when tumors were first palpable ( 2 - 3 mm diameter ) .", "microvascular density ( mvd ) in bisected tumors were assessed 24 hours after two systemic injections of t - oligos by using two ec - specific markers cd31 ( pecam-1 ) and bandeurea simplicifolia ( bs)-1 lectin conjugated to rhodamine ( vector laboratories , burlingame , ca ) . to measure functional mvd in the tumor tissue ,", "30 minutes before sacrifice a set of mice ( 5 per treatment group ) were anesthetized and were perfused with 0.5 mg ( in 100 l of isotonic solution ) of rhodamine - conjugated bs-1 lectin as described [ 40 , 41 ] . to measure total mvd , 6 m cross - sections of bisected tumor tissue ( of the same mice perfused with bs-1 lectin )", "were stained with cd31 primary antibody followed by fitc - labelled secondary antibody as described . ", "samples were photographed using a multicolour fluorescence microscope ( nikon , nikon instruments inc , melville , ny ) , and analyzed using a digital image analysis system ( nikon ) .", "anova with post hoc analysis by scheffe and bonferroni - dunn and unpaired t - test were performed using statview ( sas institute , inc . ," ], [ "mm - an cells were treated once with t - oligo or diluent alone , provided in fresh medium , and processed for western blot analysis .", "compared to diluent , t - oligo decreased vegf protein level at 24 hours ( p < .01 ) by ~41% and at 48 hours ( p = ns ) by ~32% ( figures 1(a ) and 1(b ) ) .", "in addition , after 48 hours t - oligo treatment reduced ang-1 mrna ( p < .01 ) by ~60% ( figure 1(c ) ) and ang-2 mrna ( p < .04 ) by ~45% ( figure 1(d ) ) .", "similar decreases in vegf ( supplemental figures 1(a)1(c ) and ang-1 ( supplemental figures 2(a)2(c ) ) mrna were seen in a second human melanoma line ep , as well as in other malignant cell types . paired dishes of mm - an and ecs were treated with 40 m t - oligo or diluent alone once .", "medium was collected after 24 , 48 and 72 hours and vegf level in the medium was measured by elisa . in ecs ( hmvec and huvec ) , there was a ~50%75% variable and statistically insignificant decrease in vegf release into the medium over time in both diluent- and t - oligo - treated cells ( data not shown ) . ", "in contrast , total vegf release from mm - an cells in diluent - treated dishes rose progressively over 72 hours to ~600% ( of 24-hours vegf levels ) , while vegf release in t - oligo - treated dishes was decreased ( p < .0008 ) by ~30% over the 72-hours experiment after a single dosing at time 0 ( figure 1(e ) ) . ", "thus , t - oligo not only decreased vegf protein expression in tumor cells but also the release of this potent angiogenic factor into the surrounding culture medium .", "e2f1 mrna expression was increased at 24 hours ( p < .0001 ) by ~41% and at 48 hours ( p < .0001 ) by ~100% in t - oligo - treated mm - an cells compared to control ( figure 2(a ) ) .", "similar increases in e2f1 mrna were seen in a second human melanoma line ep , as well as in other malignant cell types ( supplemental figures 3(a)3(c ) ) .", "t - oligo treatment also increased e2f1 protein level at 24 hours to ~218% and at 48 hours to ~286% ( p < .02 ) of control values ( figures 2(b ) and 2(c ) ) .", "in addition , there was a doubling in e2f1 dna binding activity 32 hours after treatment compared to diluent - treated cells ( figures 2(d ) and 2(e ) , compare lane 3 versus 4 ) .", "mm - an cells were treated once with t - oligo or diluent alone and nuclear proteins were harvested up to 32 hours after treatment to evaluate dna binding activities of the angiogenic transcription factor hif-1. there was ~80%95% decrease in hif-1 dna binding activity at 1 and 16 hours after treatments ( figures 2(f ) and 2(g ) , lanes 3 versus 4 , and 5 versus 6 , respectively ) .", "these data suggest that t - oligo - mediated inhibition in dna binding activity of hif-1 transcription factor contributes to decreased expression of the angiogenic factors , vegf and ang-1 , whose promoters contain binding sites for hif-1 .", "we used mm - an medium collected 72 hours after addition of t - oligo or diluent alone ( mm - an conditioned media : t - oligo cm or diluent cm ) as the chemoattractant for an in vitro invasion assay .", "mm - an and hmvecs were both evaluated for invasion through matrigel to determine if t - oligo treatment reduces the chemoattractant properties of mm - an cells . as recommended by the manufacturer of the matrigel invasion kit , after 22 hours of incubation the invading cells were fixed , stained and counted for each membrane .", "mm - an cells plated above t - oligo cm had a ~96% decrease in invasion ( p < .03 ) through matrigel ( figures 3(a ) and 3(b ) ) .", "hmvecs plated on inserts coated with matrigel and exposed to t - oligo cm had a ~40% reduction in matrigel invasion compared to controls , but this did not reach statistical significance ( p < .08 ) ( figures 3(c ) and 3(d ) ) .", "these findings demonstrate that t - oligos reduce the migration / invasion of mm - an cells towards chemoattractant stimuli and , possibly , to a lesser degree affect hmvecs .", "this is consistent with the observation that mm - an cells elaborate factors that promote migration and invasion of tumor cells , like vegf and ang-1 , and that these factor(s ) are reduced as a result of t - oligo treatment . because vegf signaling through vegfr-2 is principally responsible for ec survival , proliferation , migration , and angiogenesis [ 4447 ] , we examined the effect of t - oligo treatment on these proteins .", "hmvecs and huvecs were grown and treated with 40 m t - oligo or diluent alone .", "compared to diluent - treated control cells there was a decrease ( p < .004 ) of ~86% in vegf expression at 24 hours and an insignificant decrease of ~50% at 48 hours in t - oligo - treated samples ( figures 4(a)4(c ) ) .", "in addition , in t - oligo - treated hmvecs and huvecs there were ~29% ( p = ns ) and ~59% ( p < .04 ) reduction in the protein level of vegfr-2 at 24 and 48 hours , respectively ( figures 4(d)4(f ) ) .", "thus , t - oligo - mediated antiangiogenic effects on ecs may be , in part , due to inhibition of vegf - vegr2 axis .", "hmvecs were plated on matrigel in 4-well chamber slides in serum - containing medium and treated with diluent alone or increasing concentrations of t - oligo .", "tube - like structure formation was maximal in controls at 22 hours and formal comparisons were made after 222 hours , as customary for this assay . by 22 hours , in cells treated once with either 40 m or 80 m t - oligo there was a ~19% reduction in average tube length compared to cells treated with diluent alone ( p = ns ) ( figures 5(a ) and 5(b ) ) .", "cells treated with two separate doses of 40 m t - oligo at plating and 2 hours after plating showed a ~35% reduction in average tube length ( p < .03 ) .", "cells treated once at plating with 120 m t - oligo showed a ~58% reduction in average tube length compared to the diluent - treated cells ( p < .001 ) . a reduction of ~83% ( p", "< .0001 ) in tube length was observed in cells treated with 3 separate doses of 40 m t - oligo ( 40 m 3 ) added to the medium at the time of plating and then at 2 and 6 hours ( figures 5(a ) and 5(b ) ) .", "these results indicate that t - oligo inhibits ec function in a manner dependent on the dose and frequency of administration , with the caveat that the very large amount of oligos administered , rather that specific telomere - based t - oligo initiated signalling may have contributed to the effect observed . in our future experiments", ", we plan to examine the effect of large and/or fractioned t - oligo doses on survival of ecs . in melanoma tumors inoculated into scid mice , we determined the effect of systemic t - oligo injection on melanoma angiogenesis by evaluating functional and total vessel density in tumor tissue ( 5 mice per group ) . when tumors became palpable", "( 2 - 3 mm in diameter , day 5 to 14 after inoculation ) we injected t - oligo ( 60 nmoles / injection , 15 mg / kg ) via the tail vein ( iv ) and injected again after 6 hours , then harvested tumors 24 hours after the second t - oligo injections .", "representative images of tumor cross - sections were immunostained to identify all vessels as well as functional ( patent ) vessels 24 hours after t - oligo injection ( figure 6(a ) ) . compared to control - injected mice", ".002 ) in functional vessels in t - oligo - injected mice ( figure 6(b ) ) . ", "there was also more than ~60% decrease ( p < .004 ) in total vessels in t - oligo - injected mice ( figure 6(c ) ) . ", "these data corroborate our in vitro findings ( figures 4 and 5 ) and demonstrate that two systemic administrations of t - oligo reduce tumor angiogenesis and vessel patency in vivo ( figures 6(a)6(c ) ) . to determine the effect of systemic t - oligo treatment on melanoma growth , in scid xenograft model mm - an cells", "were injected subcutaneously in the flank . when tumors became palpable ( 2 - 3 mm in diameter , day 5 to 14 ) ,", "the mice received daily systemic injections of t - oligo ( 60 nmoles / injection , 15 mg / kg bid ) or vehicle for 5 days only . in control animals , tumors increased in volume from 6.96 2.9 mm on day 6 to 88.04 19.72 mm on day 27 ( figure 6(d ) , blue line ) .", "in contrast , in t - oligo treated animals , whose tumors were the same size as in controls on day 6 ( 6.48 2.57 ) there was very little tumor growth through day 15 ( 6.48 2.57 versus 9.92 3.8 mm ) and thereafter a slower and statistically insignificant increase in volume to 29.65 10.47 mm by day 27 ( figure 6(d ) , pink line ) .", "thereby , in t - oligo - treated mice tumor growth was inhibited ( p < .003 ) by ~53% after ~4 weeks , when the experiment was terminated .", "these data demonstrate that systemic administration of t - oligo for only 5 days has a persistent inhibitory effect on melanoma growth .", "we have also evaluated t - oligos toxicity in several internal organs of scid mice 24 hours after the last iv injections ( 15 mg / kg bid for 5 days ) .", "no systemic toxicity was observed in bone marrow , liver , intestines , brain , lungs and kidneys of t - oligo - injected mice ( figures 7(a)7(f ) ) ." ], [ "tumor angiogenesis is essential for tumor growth and metastasis . without active angiogenesis tumor diameter", "angiogenesis is mediated through release of angiogenic factors by tumor cells and cells in the tumor stroma and microenvironment including , but not limited to , endothelial cells . ", "we now report that telomere homolog oligonucleotides ( t - oligos ) decrease the synthesis and release of angiogenic factors by ecs and melanoma cells , inhibit ec tubulogenesis and impede melanoma cells and ecs from invading matrix ( matrigel ) .", "ec proliferation , in vitro tubulogenesis , and survival are all known to be stimulated in large part by vegf . decreased", "vegf levels or inhibition of receptor activation in ecs often correlate with decrease in tumor size and metastatic potential .", "vegf binds to the extracellular domain of the vegfr-1 ( flt-1 ) and vegfr-2 ( flk-1 ) , inducing receptor dimerization and activation of tyrosine kinases by autophosphorylation , leading to angiogenesis , increased vascular permeability , and ec proliferation and survival .", "we found that t - oligo decreases the expression of vegfr-2 by hmvec and huvec ( figures 4(c ) and 4(f ) ) . ", "other investigators reported that receptor tyrosine kinase inhibitors ( tkis ) such as sunitinib and dasatinib reduce signaling through the raf / mek / erk pathway that is activated by ligand binding to angiogenic receptors like vegfr-2 , pdgfr- , fh-3 and c - kit , indirectly inhibiting tumor growth by affecting tumor angiogenesis . by reducing vegf - r2 level ,", "vegf also induces leakage within tumor vessels , allowing tumor cells to infiltrate blood vessels and migrate into the blood stream .", "hence , changes in angiogenic factors even early in tumor formation can affect metastasis and spread and inhibiting vegf production by t - oligos would be expected to reduce the metastatic potential of tumor cells . additionally , increased blood vessel permeability within the tumor may interfere with adequate delivery and retention of chemotherapeutic agents [ 54 , 55 ] . ", "indeed , certain antiangiogenic agents that prevent tumor vessel leakage ( a phenomenon called vessel normalization ) were shown to enhance the delivery of chemotherapeutic agents into tumors . ", "thus , combination treatment with antiangiogenic factors together with conventional chemo - therapeutic agents may be superior to using the latter alone . ", "furthermore , because vegf is likely required for migration and recruitment of ecs , t - oligo - me diated vegf reduction would also likely decrease the number of blood vessels in the tumor [ 1 , 6 ] . ", "our present findings suggest that t - oligos may induce potent antiangiogenic effects , enhancing their attractiveness as a therapeutic option for cancer .", "in addition to vegf and its receptors , angiopoietin 1 and 2 ( ang-1 and ang-2 ) and their tyrosine kinase receptor tie-2 have been identified as major players in the processes of growth and remodelling of tumor vasculature [ 57 , 58 ] .", "although under certain conditions ang-2 may inhibit ang-1 effect , in an in vivo mouse model for melanoma and in glioma cells increased expression of ang-2 is thought to stimulate angiogenesis . another angiogenic factor", ", hif-1 , a transcription factor that is activated by hypoxia , exerts its effect by upregulating vegf levels . ", "we found that t - oligo treatment inhibits hif-1 activity and ang-1 and ang-2 expression in melanoma cell line and decreases vegf synthesis secretion in these cells , strongly suggesting that t - oligo - mediated effects on tumor angiogenesis are transcriptional and ultimately affect several angiogenic molecules .", "our laboratory has previously shown that t - oligo increases p73 level in the p53 null mm - an cells and that blocking p73 expression by rnai decreases t - oligo - induced apoptosis in these cells . like p53 , p73 is known to inhibit angiogenesis , primarily through vegf down - regulation .", "therefore , we assume that in mm - an cells t - oligo decreases vegf production , in addition to its effect on hif-1 , by activating p73 through induction of e2f1 . ", "however , in cells with functional p53 , we assume that t - oligo - induced p53 and p73 would cooperate to inhibit angiogenesis . ", "furthermore , e2f1 is known to induce apoptosis in part by its effect on p73 . ", "thus , induction of e2f1 by t - oligos also contributes to tumor cell apoptosis . ", "we have previously shown that t - oligo treatment activates ( phosphorylates ) atm [ 21 , 27 ] .", "we therefore suggest that t - oligo regulates e2f1 first via atm - mediated phosphorylation of e2f1 and that e2f1 then further transcriptionally upregulates e2f1 by binding to its own promoter . indeed", ", we show that t - oligo increases e2f1 mrna and protein levels as well as its dna binding activity in human mm - an melanoma cells , further confirming our previous finding that e2f1contributes to t - oligo effects . ", "the t - oligos used in the present experiments have physiologic readily hydrolysable phosphodiester linkage , unlike for example antisense dna in which phosphorothioate or other nonhydrolyzable linkage is employed to increase the molecule 's half - life ( t 1/2 ) . ", "hydrolyzable linkage is required for initiation of t - oligo signalling and , despite the known short t 1/2 , approximately 46 hours in serum - containing medium for a 12-base 100% telomere homolog , nevertheless this allows for the cellular responses observed in the present and previous experiments to evolve over 35 days [ 11 , 13 , 19 , 21 , 22 , 2428 ] . ", "this may reflect the fact that at least in vitro t - oligos rapidly enter the nucleus [ 11 , 22 , 24 , 27 ] and that , once in the nucleus , such oligos have a far longer t 1/2 . ", "the efficacy of these presumptively short - lived t - oligos may also reflect the likelihood that , after interaction of the oligos with the werner protein and formation of dna damage - like foci at telomeres , signalling through the dna damage response pathways may continue even if the t - oligos have been hydrolyzed , at least through 48 hours at which time the dna damage foci can still be observed by immunohistochemistry . in earlier studies , we have shown that t - oligos inhibit tumor growth in scid mouse models by inducing cell cycle arrest , differentiation , apoptosis , and senescence [ 13 , 15 , 27 ] . ", "angiogenesis inhibition encompassing both ecs and melanoma cells , in combination with other t - oligo - mediated anti - tumor effects [ 11 , 13 , 22 , 25 , 27 ] , likely combine to significantly decrease melanoma burden in established scid mouse models , as reported in our earlier publications [ 13 , 27 ] . ", "these multiple diverse responses are all mediated through activation and/or upregulation of dna damage response proteins . ", "they are thus reminiscent of the less complex but very well characterized bacterial sos response , a genome - protective mechanism that enhances survival of prokaryotic organisms in the face of dna damage . ", "the telomere - based dna damage - like signalling initiated by t - oligos may be viewed as an evolutionarily perfected parallel mechanism in mammalian cells that addresses the threat to genomic integrity posed by malignant transformation . in summary , the present paper indicates that t - oligos exert multiple antiangiogenic effects .", "these data reinforce prior evidence that t - oligo therapy may offer a powerful new approach to treatment of human primary melanoma and possibly other human malignancies , with several conceptual advantages over the currently lionized targeted therapy approach ." ] ]

[ "muscle dysfunction of the leg is very common in children with spastic diplegia cerebral \n palsy ( cp)1 .", "spasticity causes muscle \n stiffness and weakness , and decreases daily functional activities including standing and \n walking2 .", "passive muscle stretching is a \n common physical therapy for decreasing the spasticity of children and adults with cp \n spasticity .", "it has been reported that prolonged passive muscle stretching improves the range \n of movements and reduces spasticity3 . \n", "prolonged passive muscle stretching while standing on a tilt - table decreases the resistance \n to passive ankle joint movements in children with cp4 .", "therefore , it has been suggested as a treatment technique for \n children and adults with cp .", "whole body vibration has also been proposed as a treatment for \n children and adults with spastic cp .", "some studies have shown that whole body vibration decreases the \n spasticity of patients with stroke , spinal cord injury and multiple sclerosis5,6,7 .", "a whole body vibration intervention \n decreases spasticity and improves walking speed , muscle strength and gross motor function \n without any side effects in adults with cp7 . because both muscle stretching and vibration have underlying \n treatment efficacy in the alleviation of spasticity , and promote muscle function in adults \n with cp , a therapeutic program combining passive muscle stretching and whole body vibration \n may show better treatment effects in children and adolescents with spastic cp .", "however , no \n study has yet combined passive muscle stretching with whole body vibration for children and \n adolescents with spastic cp .", "therefore , the aim of this study was to investigate the effect \n of combining passive muscle stretching and whole body vibration on the spasticity , \n functional strength and balance of children and adolescents with spastic cp .", "twelve children and adolescents with spastic cp aged 618 years ( age , 10.58 2.35 years ; \n height , 127.25 9.72 cm ; body mass , 26.48 7.95 kg ; mean sd ) were recruited from khon \n kaen special school .", "subjects were classified for gross motor function at levels i , ii and \n iii according to the gross motor function classification system ( gmfcs ) , and were included \n in the study when their modified ashworth scale ( mas ) scores were greater than or equal to \n 1 .", "exclusion criteria for this study : were receiving pms more than 3 times / week ; medical \n treatment for spasticity such as a drugs , botulinum toxin injection , or orthopedic surgery \n during the last six months ; medical problems such as arthritis , congenital abnormality , \n cardiovascular or pulmonary diseases , or neuromuscular diseases ; musculoskeletal disorders ; \n or infectious disease .", "this study was approved by the local ethics committee for human \n research of khon kaen university .", "parents of the children and the children read and signed \n an informed consent form before the start of the study .", "the study was designed as a two - period cross - over trial in which all subjects received \n both , passive muscle stretching alone as a control group ( cg ) , and a combination of passive \n muscle stretching and whole body vibration as an experimental group ( eg ) , as shown in fig .", "cg : control group ; eg : experimental group ; pms : passive muscle stretching ; wbv : whole \n body vibration .", "both treatment groups were evaluated for immediate effect ( one treatment ) and for \n short - term effect ( 6 weeks treatment ) .", "the sample size was calculated based on a primary \n modified ashworth scale ( mas ) outcome which was determined as the predicted value ( the \n post - test value of mas decreased 20% ) .", "sampling \n randomization was stratified according to the gmfcs from levels i to iii , and ages of \n children ( 6 to < 10 years ) , and adolescents ( 1019 years ) .", "after the baseline assessment \n of all subjects , 12 children were randomly assigned to either eg - cg or cg - eg . in eg - cg , the \n number of subjects with gmfcs levels i , ii and iii was 1 , 1 and 4 , respectively , and 3 of \n the 6 subjects were under the age of 10 while in cg - eg , the number of subjects with gmfcs \n levels i , ii and iii was 1 , 2 and 3 , respectively and 2 of the 6 subjects were under the age \n of 10 .", "the gmfcs levels and ages were similar in both groups and each group was crossed over \n at the end of each phase was so that each group received the same treatment . in phase 1 , \n subjects were evaluated for outcome variables before and immediately after a one - time \n treatment and in phase 2 , the procedure was repeated after group crossover .", "all subjects \n rested for 2 days for a washout between phases 1 and 2 .", "after phase 2 had finished , subjects \n rested for the 2-day washout period , and in phase 3 , all individuals continuously received \n treatment for 6 weeks .", "after phase 3 , a washout period of 2 weeks was conducted before group \n crossover for phase 4 . pre - and post - treatment measurements were conducted in phases 3 and \n 4 .", "cg : control group ; eg : experimental group ; pms : passive muscle stretching ; wbv : whole \n body vibration for cg , passive muscle stretching was performed while the subjects stood on a tilt table \n for 40 minutes per session .", "the table was tilted to 7080 degrees relative to the horizontal \n plane , and two straps around the chest and knees helped to support the body . for eg , a \n combination of passive muscle stretching and whole body vibration was conducted .", "whole body \n vibration was applied at 20 hz on the oscillation platform ( aiko vibrator , etf-001cg , \n thailand ) .", "after passive muscle stretching for 30 minutes , a total of 10 minutes of \n one - minute vibration with one minute rest was performed while the subjects stood with equal \n weight - bearing on both feet .", "subjects stood in a standardized foot position on the platform \n as recommended by the manufacturer with center of the platform located between the legs \n placed shoulder - width apart .", "the amplitude of vibration becomes larger when the feet are \n placed further from the center line . for safety reasons , some subjects held a handle - bar \n during the vibration .", "both the cg and eg interventions were conducted for a total of 40 \n minutes treatment / day , 5 days / week for 6 weeks , in addition to the regular physiotherapy \n program at school of 1 session / week . before and after the single treatments and the 6-week \n treatments , subjects were evaluated using the modified ashworth scale ( mas ) , the five times \n sit - to - stand test ( ftsst ) and the pediatric balance scale ( pbs ) .", "mas is known as an essential \n clinical measure of muscle spasticity in people with neurological conditions8 .", "the topographic distribution of impairment was determined \n as the leg with lower or higher spasticity which represented the weaker or stronger side , \n respectively .", "mas scores were assigned numerical values for the evaluated mas scores ( 0 , 1 , \n 2 , 3 , 4 and 5 ) .", "a value of 0 indicates no increase in muscle tone ; 1 ( 1 point ) is a slight \n increase in muscle tone , manifested by a catch and release or by minimal resistance at the \n end of the range of motion when the affected part is moved in flexion or extension ; 1 + ( 2 \n points ) is a slight increase in muscle tone , manifested by a catch , followed by minimal \n resistance throughout the remainder ( less than half ) of the rom ( range of movement ) ; 2 ( 3 \n points ) is a more marked increase in muscle tone through most of the rom , but the affected \n part is easily moved ; 3 ( 4 points ) is a considerable increase in muscle tone passivity , with \n movement difficult ; 4 ( 5 points ) is an affected part rigid in flexion or extension .", "mas was \n assessed by the same evaluator throughout the experiment , and outcomes were tested for the \n intra - tester reliability . the five times sit - to - stand test ( ftsst ) is a reliable tool for evaluating lower limb \n muscle strength and balance ability9 . \n", "subjects sat on a chair with their arms crossed over the chest , and they were asked to stand \n up and sit down again as quickly as possible five times without using arm support .", "the test \n began with the word go and stopped when subjects sat after the fifth performance ; the time \n was recorded .", "the pediatric balance scale ( pbs ) is a modification of the berg balance scale , which was \n developed for the evaluation of the balance ability of school - age children with mild to \n moderate motor impairments .", "pbs has been demonstrated to have good test - retest and \n inter - rater reliabilities10 .", "there are 14 \n items and each item is scored on a five point ordinal scale from zero to four with a maximum \n score of 56 points . in order to demonstrate each task and give instruction , each child was \n allowed to practice each item once .", "if the child was unable to complete the task based on \n their inability to understand the directions , a second practice trial was allowed , and the \n best score was recorded11 .", "pbs took \n approximately 15 minutes to conduct , and if some children needed more time or felt fatigued \n during the test they were allowed to rest for a few minutes .", "the distribution of the data was examined using the shapiro - wilk s test to examine the \n assumption of normality .", "the \n differences of variables within and between the groups were assessed with the wilcoxon \n signed - ranks test and the mann - whitney u - test , respectively .", "tables 1table 1.pre-and post - intervention evaluation items of the control and experimental \n groupsimmediate effectshort - term effectcontrol groupexperimental groupcontrol groupexperimental groupbeforeafterbeforeafterbeforeafterbeforeaftermas scorestrongerhip adductors 2 ( 1:3.75)2 ( 1:2)2 ( 1:3)1 ( 1:2.75)2 ( 1:3)2 ( 1:2.8)2 ( 1:3)1 ( 1:1)quadriceps 2.5 ( 2:3)1 ( 1:2)2.5 ( 2:3)2 ( 1.25:2)3 ( 1.3:3)2 ( 1.3:2.8)3 ( 1.3:3)1 ( 1:2)hamstrings2 ( 1:3)1 ( 1:2)2 ( 1:3)1 ( 1:1)2.5 ( 2:3)2 ( 1.3:2.8)3 ( 2:3)1 ( 1:1.8)soleus2 ( 1.25:4.75)1.5 ( 1:3)2 ( 1.25:4.75)1 ( 1:2)3 ( 1.3:5)2 ( 1:4)3 ( 2:5)1.5 ( 1:2.8)weakerhip adductors 2 ( 1:3.75)1 ( 1:3)2 ( 1:3.75)1 ( 1:3)1.5 ( 1:2.8)1.5 ( 1:2.0)1.5 ( 1:2.8)1 ( 1:1)quadriceps 2 ( 1:2.75)2 ( 1:2)2 ( 1:2.75)1 ( 1:2)2 ( 1:2)2 ( 1:2)2 ( 1:2)1 ( 1:1.8)hamstrings 1.5 ( 1:2.75)1 ( 1:2)1.5 ( 1:2.75)1 ( 1:1)1.5 ( 1:2)2 ( 1:2.8)1.5 ( 1:2)1 ( 1:1)soleus2 ( 2:3.75)1.5 ( 1:3.75)2 ( 2:3.75)1 ( 1:2.5)2.5 ( 1:4)2 ( 1:3)2 ( 1:3.8)1 ( 1:2.8)ftsst ( s)16.59 ( 13.0:23.1)17.28 ( 10.6:22.7)16.93 ( 12.3:16.9)11.96 ( 10.9:19.9)13.34 ( 10.7:20.6)10.14 ( 9.6:12.7)13.55 ( 11.8:21.9)11.36 ( 8.1:13.4)pbs ( score)26.00 ( 7:42)25.00 ( 7.5:42.25)24.00 ( 8.5:42.25)26.50 ( 10:42.25)26.0 ( 7.3:45.8)27.5 ( 8.5:42.0)24.5 ( 8.5:42.0)31.0 ( 10.0:44.3)data are presented as median ( quartile1:quartile3 ) for all outcomes .", "p \n < 0.05 , p < 0.01 and 2table 2.comparison of the median differences between the control and experimental \n groupsimmediate effectshort - term effectcontrol groupexperimental groupcontrol groupexperimental groupmas scorestrongerhip adductors 0 ( 1.0 : 0)0 ( 1.0 : 0 ) 0 ( 1 : 0.75)0.5 ( 1 : 0)quadriceps 1.0 ( 1.75 : 0)1.0 ( 1 : 0 ) 0 ( 1 : 0)1.0 ( 1 : 1)hamstrings 0.5 ( 1.0 : 0)1.0 ( 2.0 : 0)0.5 ( 1 : 0)1.0 ( 2 : 1)soleus 0 ( 1.0 : 0)1.0 ( 2.0 : 0)0 ( 1 : 0)1.0 ( 1.75 : 0.25)weakerhip adductors 0 ( 0.75 : 0)0 ( 1.0 : 0)0 ( 0 : 0)0 ( 1 : 0)quadriceps 0 ( 0.75 : 0)0 ( 1.75 to 0)0 ( 0 : 0)0.5 ( 1 : 0)hamstrings 0 ( 1.0 : 0)0 ( 1.75 : 0)0 ( 0 : 0.75)0.5 ( 1 : 0)soleus 0 ( 1.0 : 0)1.0 ( 1.75 : 0.25)0 ( 1 : 0)1.0 ( 1 : 0)ftsst ( s)1.18 ( 3.0 : 1.6)2.61 ( 5.3 : 1.1)3.28 ( 6.70 : 0.51)4.46 ( 8.09 : 2.41)pbs ( score)0 ( 0 : 1)0 ( 0 : 1)0 ( 2 : 2.75)2.5 ( 0 : 4)data are presented as the median difference ( quartile 1 : quartile 3 ) for all \n outcomes .", "p < 0.05 summarize the pre - and post - intervention measurement values of cg and eg . in \n the immediate effect of eg ,", "mas scores significantly decreased in all muscles of the \n stronger side and the soleus of the weaker side . in the immediate effect of cg", ", mas scores \n significantly decreased in the quadriceps , hamstrings and soleus muscles of the stronger \n side . in the comparison of the groups , only the mas score of the soleus of the weaker side \n was significantly improved by eg .", "after the 6-week treatment , all the evaluated items \n significantly improved in eg , and the mas scores of the hamstrings and soleus of the \n stronger side and ftsst improved in cg . in the comparison of the groups , the mas scores of \n the quadriceps and hamstrings of both sides , and soleus of the stronger side were more \n significantly decreased by eg than by cg .", "no significant differences between the groups were \n found for ftsst , pbs and mas of the hip adductors .", "p \n < 0.05 , p < 0.01 data are presented as the median difference ( quartile 1 : quartile 3 ) for all \n outcomes .", "the results show the combined treatment of pms and wbv had better effects than pms alone on \n the spasticity of children and adolescents with cp . immediately after one treatment , the \n combined treatment showed better improvement in the scores of the mas than pms alone .", "after \n the 6-week intervention , the combined treatment significantly decreased the scores of the \n mas when compared to pms alone . both treatments significantly reduced the children s \n performance times in the five times sit - to - stand test , while the combined treatment \n significantly increased the scores of the pediatric balance scale .", "thus , the results suggest \n that a 6-week intervention of a combination of prolonged pms and wbv may elicit beneficial \n effects for the spasticity and balance of patients with cerebral palsy", ". \n sustained passive muscle stretching for a long duration improves the range of movements , and \n reduces the spasticity of muscles11 , 12 .", "passive muscle stretching activates golgi \n tendon organs and inhibits the excitability of alpha motor neurons .", "current evidence supports the effectiveness of passive \n stretching of children with spastic cp , although the reduction in spasticity may not be \n linked with functional activities such as walking3 .", "a combination of exercise , massage therapy and therapeutic supports \n is an effective treatment for the release of muscle stiffness , decrease of pain , and \n improvement of physical functions14,15,16 . \n", "therefore , different therapeutic methods should be used to \n normalize muscle tone , maintain or increase soft - tissue extensibility , reduce contracture \n pain , and improve motor function18 .", "oscillation \n of the ankle joints with a vibrator helps to induce release of a stiff ankle joint . during \n vibration ,", "standing in a neutral ankle position with body weight bearing also stimulates the \n stretching of calf muscles .", "whole body vibration decreases the spasticity of muscles and \n increases the gross motor function of subjects with cp7 , 19 .", "whole body vibration \n improved muscle strength and power movement similar to conventional resistance training in \n older women20 and in adults with cp7 .", "the muscle spindle senses a very small \n change in muscle length when a skeletal muscle perceives vibration .", "this information is \n transmitted to the fibers of group ia or ii , eventually reaching the spinal cord . in the \n spinal cord", ", the information serves as presynaptic inhibition through an interstitial cell \n and suppresses the alpha motor neurons21 , 22 .", "the activation of these sensory receptors \n results in reflexive activation of motor units similar to the tonic vibration reflex23 .", "whole body vibration improved the performance of multiple \n sclerosis patients in the timed up & go test26 .", "this improvement in physical balance after whole body vibration \n may be associated with improvement of leg muscle strength27 .", "the activation of proprioreceptive spinal circuits can be induced \n by upright standing on a vibration platform .", "because reflexes are related to the \n time - differential activation of spindles in muscles and tendons28 , the improvement in balance might be positively related to \n muscle strength and proprioception after vibration training .", "whole body vibration may allow \n children and adolescents with cp to walk effectively with an improvement of balance .", "similar \n to our results , previous research has shown that whole body vibration improves the muscle \n tone , strength , balance and mobility of children with cp29,30,31 ; however , those studies used a vibration frequency with stepwise \n increment up to 18 hz compared to the constant vibration frequency of 20 hz used in the \n present study .", "a limitation of this study was the small number of subjects and the absence of \n double - blinding of subjects and physiotherapists .", "with regard to adherence to treatment , all \n subjects enjoyed whole body vibration therapy and engaged in regular interaction with the \n physiotherapists . therefore , whole body vibration should be added to the physiotherapy \n program . although improvements in outcomes were found in this study , the optimal \n dose - response vibration was not clear .", "thus , further studies should focus on the training \n protocol or whole body vibration that can provide the best results for children and \n adolescents with cp . in conclusion , this present study showed that 6 weeks of combined passive muscle stretching \n and whole body vibration could decrease the spasticity and increase the muscle strength and \n balance of children and adolescents with cp .", "whole body vibration could be an alternative \n additional treatment to passive muscle stretching for both clinical and home therapy \n programs for children and adolescents with cp ." ]

[ "<S> [ purpose ] this study evaluated the immediate and short - term effects of a combination of \n prolonged passive muscle stretching ( pms ) and whole body vibration ( wbv ) on the \n spasticity , strength and balance of children and adolescents with cerebral palsy . \n [ subjects and methods ] a randomized two - period crossover trial was designed . </S>", "<S> twelve \n subjects with cerebral palsy aged 10.6 2.4 years received both pms alone as a control \n group ( cg ) and a combination of pms and wbv as an experimental group ( eg ) . </S>", "<S> after random \n allocation to the trial schedules of either eg - cg or cg - eg , cg received prolonged pms \n while standing on a tilt - table for 40 minutes / day , and eg received prolonged pms for 30 \n minutes , followed by 10 minutes wbv . </S>", "<S> both cg and eg received the treatment 5 days / week for \n 6 weeks . </S>", "<S> [ results ] immediately after one treatment , eg resulted in better improvement in \n scores on the modified ashworth scale than cg . </S>", "<S> after the 6-week intervention , eg also \n showed significantly decreased scores on the modified ashworth scale compared to cg . </S>", "<S> both \n cg and eg showed significantly reduced the performance times in the five times sit to \n stand test , and eg also showed significantly increased scores on the pediatric balance \n scale . </S>", "<S> [ conclusion ] this study showed that 6 weeks of combined prolonged pms and wbv had \n beneficial effects on the spasticity , muscle strength and balance of children and \n adolescents with cp . </S>" ]

[ "INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "muscle dysfunction of the leg is very common in children with spastic diplegia cerebral \n palsy ( cp)1 .", "spasticity causes muscle \n stiffness and weakness , and decreases daily functional activities including standing and \n walking2 .", "passive muscle stretching is a \n common physical therapy for decreasing the spasticity of children and adults with cp \n spasticity .", "it has been reported that prolonged passive muscle stretching improves the range \n of movements and reduces spasticity3 . \n", "prolonged passive muscle stretching while standing on a tilt - table decreases the resistance \n to passive ankle joint movements in children with cp4 .", "therefore , it has been suggested as a treatment technique for \n children and adults with cp .", "whole body vibration has also been proposed as a treatment for \n children and adults with spastic cp .", "some studies have shown that whole body vibration decreases the \n spasticity of patients with stroke , spinal cord injury and multiple sclerosis5,6,7 .", "a whole body vibration intervention \n decreases spasticity and improves walking speed , muscle strength and gross motor function \n without any side effects in adults with cp7 . because both muscle stretching and vibration have underlying \n treatment efficacy in the alleviation of spasticity , and promote muscle function in adults \n with cp , a therapeutic program combining passive muscle stretching and whole body vibration \n may show better treatment effects in children and adolescents with spastic cp .", "however , no \n study has yet combined passive muscle stretching with whole body vibration for children and \n adolescents with spastic cp .", "therefore , the aim of this study was to investigate the effect \n of combining passive muscle stretching and whole body vibration on the spasticity , \n functional strength and balance of children and adolescents with spastic cp ." ], [ "twelve children and adolescents with spastic cp aged 618 years ( age , 10.58 2.35 years ; \n height , 127.25 9.72 cm ; body mass , 26.48 7.95 kg ; mean sd ) were recruited from khon \n kaen special school .", "subjects were classified for gross motor function at levels i , ii and \n iii according to the gross motor function classification system ( gmfcs ) , and were included \n in the study when their modified ashworth scale ( mas ) scores were greater than or equal to \n 1 .", "exclusion criteria for this study : were receiving pms more than 3 times / week ; medical \n treatment for spasticity such as a drugs , botulinum toxin injection , or orthopedic surgery \n during the last six months ; medical problems such as arthritis , congenital abnormality , \n cardiovascular or pulmonary diseases , or neuromuscular diseases ; musculoskeletal disorders ; \n or infectious disease .", "this study was approved by the local ethics committee for human \n research of khon kaen university .", "parents of the children and the children read and signed \n an informed consent form before the start of the study .", "the study was designed as a two - period cross - over trial in which all subjects received \n both , passive muscle stretching alone as a control group ( cg ) , and a combination of passive \n muscle stretching and whole body vibration as an experimental group ( eg ) , as shown in fig .", "cg : control group ; eg : experimental group ; pms : passive muscle stretching ; wbv : whole \n body vibration .", "both treatment groups were evaluated for immediate effect ( one treatment ) and for \n short - term effect ( 6 weeks treatment ) .", "the sample size was calculated based on a primary \n modified ashworth scale ( mas ) outcome which was determined as the predicted value ( the \n post - test value of mas decreased 20% ) .", "sampling \n randomization was stratified according to the gmfcs from levels i to iii , and ages of \n children ( 6 to < 10 years ) , and adolescents ( 1019 years ) .", "after the baseline assessment \n of all subjects , 12 children were randomly assigned to either eg - cg or cg - eg . in eg - cg , the \n number of subjects with gmfcs levels i , ii and iii was 1 , 1 and 4 , respectively , and 3 of \n the 6 subjects were under the age of 10 while in cg - eg , the number of subjects with gmfcs \n levels i , ii and iii was 1 , 2 and 3 , respectively and 2 of the 6 subjects were under the age \n of 10 .", "the gmfcs levels and ages were similar in both groups and each group was crossed over \n at the end of each phase was so that each group received the same treatment . in phase 1 , \n subjects were evaluated for outcome variables before and immediately after a one - time \n treatment and in phase 2 , the procedure was repeated after group crossover .", "all subjects \n rested for 2 days for a washout between phases 1 and 2 .", "after phase 2 had finished , subjects \n rested for the 2-day washout period , and in phase 3 , all individuals continuously received \n treatment for 6 weeks .", "after phase 3 , a washout period of 2 weeks was conducted before group \n crossover for phase 4 . pre - and post - treatment measurements were conducted in phases 3 and \n 4 .", "cg : control group ; eg : experimental group ; pms : passive muscle stretching ; wbv : whole \n body vibration for cg , passive muscle stretching was performed while the subjects stood on a tilt table \n for 40 minutes per session .", "the table was tilted to 7080 degrees relative to the horizontal \n plane , and two straps around the chest and knees helped to support the body . for eg , a \n combination of passive muscle stretching and whole body vibration was conducted .", "whole body \n vibration was applied at 20 hz on the oscillation platform ( aiko vibrator , etf-001cg , \n thailand ) .", "after passive muscle stretching for 30 minutes , a total of 10 minutes of \n one - minute vibration with one minute rest was performed while the subjects stood with equal \n weight - bearing on both feet .", "subjects stood in a standardized foot position on the platform \n as recommended by the manufacturer with center of the platform located between the legs \n placed shoulder - width apart .", "the amplitude of vibration becomes larger when the feet are \n placed further from the center line . for safety reasons , some subjects held a handle - bar \n during the vibration .", "both the cg and eg interventions were conducted for a total of 40 \n minutes treatment / day , 5 days / week for 6 weeks , in addition to the regular physiotherapy \n program at school of 1 session / week . before and after the single treatments and the 6-week \n treatments , subjects were evaluated using the modified ashworth scale ( mas ) , the five times \n sit - to - stand test ( ftsst ) and the pediatric balance scale ( pbs ) .", "mas is known as an essential \n clinical measure of muscle spasticity in people with neurological conditions8 .", "the topographic distribution of impairment was determined \n as the leg with lower or higher spasticity which represented the weaker or stronger side , \n respectively .", "mas scores were assigned numerical values for the evaluated mas scores ( 0 , 1 , \n 2 , 3 , 4 and 5 ) .", "a value of 0 indicates no increase in muscle tone ; 1 ( 1 point ) is a slight \n increase in muscle tone , manifested by a catch and release or by minimal resistance at the \n end of the range of motion when the affected part is moved in flexion or extension ; 1 + ( 2 \n points ) is a slight increase in muscle tone , manifested by a catch , followed by minimal \n resistance throughout the remainder ( less than half ) of the rom ( range of movement ) ; 2 ( 3 \n points ) is a more marked increase in muscle tone through most of the rom , but the affected \n part is easily moved ; 3 ( 4 points ) is a considerable increase in muscle tone passivity , with \n movement difficult ; 4 ( 5 points ) is an affected part rigid in flexion or extension .", "mas was \n assessed by the same evaluator throughout the experiment , and outcomes were tested for the \n intra - tester reliability . the five times sit - to - stand test ( ftsst ) is a reliable tool for evaluating lower limb \n muscle strength and balance ability9 . \n", "subjects sat on a chair with their arms crossed over the chest , and they were asked to stand \n up and sit down again as quickly as possible five times without using arm support .", "the test \n began with the word go and stopped when subjects sat after the fifth performance ; the time \n was recorded .", "the pediatric balance scale ( pbs ) is a modification of the berg balance scale , which was \n developed for the evaluation of the balance ability of school - age children with mild to \n moderate motor impairments .", "pbs has been demonstrated to have good test - retest and \n inter - rater reliabilities10 .", "there are 14 \n items and each item is scored on a five point ordinal scale from zero to four with a maximum \n score of 56 points . in order to demonstrate each task and give instruction , each child was \n allowed to practice each item once .", "if the child was unable to complete the task based on \n their inability to understand the directions , a second practice trial was allowed , and the \n best score was recorded11 .", "pbs took \n approximately 15 minutes to conduct , and if some children needed more time or felt fatigued \n during the test they were allowed to rest for a few minutes .", "the distribution of the data was examined using the shapiro - wilk s test to examine the \n assumption of normality .", "the \n differences of variables within and between the groups were assessed with the wilcoxon \n signed - ranks test and the mann - whitney u - test , respectively ." ], [ "tables 1table 1.pre-and post - intervention evaluation items of the control and experimental \n groupsimmediate effectshort - term effectcontrol groupexperimental groupcontrol groupexperimental groupbeforeafterbeforeafterbeforeafterbeforeaftermas scorestrongerhip adductors 2 ( 1:3.75)2 ( 1:2)2 ( 1:3)1 ( 1:2.75)2 ( 1:3)2 ( 1:2.8)2 ( 1:3)1 ( 1:1)quadriceps 2.5 ( 2:3)1 ( 1:2)2.5 ( 2:3)2 ( 1.25:2)3 ( 1.3:3)2 ( 1.3:2.8)3 ( 1.3:3)1 ( 1:2)hamstrings2 ( 1:3)1 ( 1:2)2 ( 1:3)1 ( 1:1)2.5 ( 2:3)2 ( 1.3:2.8)3 ( 2:3)1 ( 1:1.8)soleus2 ( 1.25:4.75)1.5 ( 1:3)2 ( 1.25:4.75)1 ( 1:2)3 ( 1.3:5)2 ( 1:4)3 ( 2:5)1.5 ( 1:2.8)weakerhip adductors 2 ( 1:3.75)1 ( 1:3)2 ( 1:3.75)1 ( 1:3)1.5 ( 1:2.8)1.5 ( 1:2.0)1.5 ( 1:2.8)1 ( 1:1)quadriceps 2 ( 1:2.75)2 ( 1:2)2 ( 1:2.75)1 ( 1:2)2 ( 1:2)2 ( 1:2)2 ( 1:2)1 ( 1:1.8)hamstrings 1.5 ( 1:2.75)1 ( 1:2)1.5 ( 1:2.75)1 ( 1:1)1.5 ( 1:2)2 ( 1:2.8)1.5 ( 1:2)1 ( 1:1)soleus2 ( 2:3.75)1.5 ( 1:3.75)2 ( 2:3.75)1 ( 1:2.5)2.5 ( 1:4)2 ( 1:3)2 ( 1:3.8)1 ( 1:2.8)ftsst ( s)16.59 ( 13.0:23.1)17.28 ( 10.6:22.7)16.93 ( 12.3:16.9)11.96 ( 10.9:19.9)13.34 ( 10.7:20.6)10.14 ( 9.6:12.7)13.55 ( 11.8:21.9)11.36 ( 8.1:13.4)pbs ( score)26.00 ( 7:42)25.00 ( 7.5:42.25)24.00 ( 8.5:42.25)26.50 ( 10:42.25)26.0 ( 7.3:45.8)27.5 ( 8.5:42.0)24.5 ( 8.5:42.0)31.0 ( 10.0:44.3)data are presented as median ( quartile1:quartile3 ) for all outcomes .", "p \n < 0.05 , p < 0.01 and 2table 2.comparison of the median differences between the control and experimental \n groupsimmediate effectshort - term effectcontrol groupexperimental groupcontrol groupexperimental groupmas scorestrongerhip adductors 0 ( 1.0 : 0)0 ( 1.0 : 0 ) 0 ( 1 : 0.75)0.5 ( 1 : 0)quadriceps 1.0 ( 1.75 : 0)1.0 ( 1 : 0 ) 0 ( 1 : 0)1.0 ( 1 : 1)hamstrings 0.5 ( 1.0 : 0)1.0 ( 2.0 : 0)0.5 ( 1 : 0)1.0 ( 2 : 1)soleus 0 ( 1.0 : 0)1.0 ( 2.0 : 0)0 ( 1 : 0)1.0 ( 1.75 : 0.25)weakerhip adductors 0 ( 0.75 : 0)0 ( 1.0 : 0)0 ( 0 : 0)0 ( 1 : 0)quadriceps 0 ( 0.75 : 0)0 ( 1.75 to 0)0 ( 0 : 0)0.5 ( 1 : 0)hamstrings 0 ( 1.0 : 0)0 ( 1.75 : 0)0 ( 0 : 0.75)0.5 ( 1 : 0)soleus 0 ( 1.0 : 0)1.0 ( 1.75 : 0.25)0 ( 1 : 0)1.0 ( 1 : 0)ftsst ( s)1.18 ( 3.0 : 1.6)2.61 ( 5.3 : 1.1)3.28 ( 6.70 : 0.51)4.46 ( 8.09 : 2.41)pbs ( score)0 ( 0 : 1)0 ( 0 : 1)0 ( 2 : 2.75)2.5 ( 0 : 4)data are presented as the median difference ( quartile 1 : quartile 3 ) for all \n outcomes .", "p < 0.05 summarize the pre - and post - intervention measurement values of cg and eg . in \n the immediate effect of eg ,", "mas scores significantly decreased in all muscles of the \n stronger side and the soleus of the weaker side . in the immediate effect of cg", ", mas scores \n significantly decreased in the quadriceps , hamstrings and soleus muscles of the stronger \n side . in the comparison of the groups , only the mas score of the soleus of the weaker side \n was significantly improved by eg .", "after the 6-week treatment , all the evaluated items \n significantly improved in eg , and the mas scores of the hamstrings and soleus of the \n stronger side and ftsst improved in cg . in the comparison of the groups , the mas scores of \n the quadriceps and hamstrings of both sides , and soleus of the stronger side were more \n significantly decreased by eg than by cg .", "no significant differences between the groups were \n found for ftsst , pbs and mas of the hip adductors .", "p \n < 0.05 , p < 0.01 data are presented as the median difference ( quartile 1 : quartile 3 ) for all \n outcomes ." ], [ "the results show the combined treatment of pms and wbv had better effects than pms alone on \n the spasticity of children and adolescents with cp . immediately after one treatment , the \n combined treatment showed better improvement in the scores of the mas than pms alone .", "after \n the 6-week intervention , the combined treatment significantly decreased the scores of the \n mas when compared to pms alone . both treatments significantly reduced the children s \n performance times in the five times sit - to - stand test , while the combined treatment \n significantly increased the scores of the pediatric balance scale .", "thus , the results suggest \n that a 6-week intervention of a combination of prolonged pms and wbv may elicit beneficial \n effects for the spasticity and balance of patients with cerebral palsy", ". \n sustained passive muscle stretching for a long duration improves the range of movements , and \n reduces the spasticity of muscles11 , 12 .", "passive muscle stretching activates golgi \n tendon organs and inhibits the excitability of alpha motor neurons .", "current evidence supports the effectiveness of passive \n stretching of children with spastic cp , although the reduction in spasticity may not be \n linked with functional activities such as walking3 .", "a combination of exercise , massage therapy and therapeutic supports \n is an effective treatment for the release of muscle stiffness , decrease of pain , and \n improvement of physical functions14,15,16 . \n", "therefore , different therapeutic methods should be used to \n normalize muscle tone , maintain or increase soft - tissue extensibility , reduce contracture \n pain , and improve motor function18 .", "oscillation \n of the ankle joints with a vibrator helps to induce release of a stiff ankle joint . during \n vibration ,", "standing in a neutral ankle position with body weight bearing also stimulates the \n stretching of calf muscles .", "whole body vibration decreases the spasticity of muscles and \n increases the gross motor function of subjects with cp7 , 19 .", "whole body vibration \n improved muscle strength and power movement similar to conventional resistance training in \n older women20 and in adults with cp7 .", "the muscle spindle senses a very small \n change in muscle length when a skeletal muscle perceives vibration .", "this information is \n transmitted to the fibers of group ia or ii , eventually reaching the spinal cord . in the \n spinal cord", ", the information serves as presynaptic inhibition through an interstitial cell \n and suppresses the alpha motor neurons21 , 22 .", "the activation of these sensory receptors \n results in reflexive activation of motor units similar to the tonic vibration reflex23 .", "whole body vibration improved the performance of multiple \n sclerosis patients in the timed up & go test26 .", "this improvement in physical balance after whole body vibration \n may be associated with improvement of leg muscle strength27 .", "the activation of proprioreceptive spinal circuits can be induced \n by upright standing on a vibration platform .", "because reflexes are related to the \n time - differential activation of spindles in muscles and tendons28 , the improvement in balance might be positively related to \n muscle strength and proprioception after vibration training .", "whole body vibration may allow \n children and adolescents with cp to walk effectively with an improvement of balance .", "similar \n to our results , previous research has shown that whole body vibration improves the muscle \n tone , strength , balance and mobility of children with cp29,30,31 ; however , those studies used a vibration frequency with stepwise \n increment up to 18 hz compared to the constant vibration frequency of 20 hz used in the \n present study .", "a limitation of this study was the small number of subjects and the absence of \n double - blinding of subjects and physiotherapists .", "with regard to adherence to treatment , all \n subjects enjoyed whole body vibration therapy and engaged in regular interaction with the \n physiotherapists . therefore , whole body vibration should be added to the physiotherapy \n program . although improvements in outcomes were found in this study , the optimal \n dose - response vibration was not clear .", "thus , further studies should focus on the training \n protocol or whole body vibration that can provide the best results for children and \n adolescents with cp . in conclusion , this present study showed that 6 weeks of combined passive muscle stretching \n and whole body vibration could decrease the spasticity and increase the muscle strength and \n balance of children and adolescents with cp .", "whole body vibration could be an alternative \n additional treatment to passive muscle stretching for both clinical and home therapy \n programs for children and adolescents with cp ." ] ]

[ "chirality is a hallmark of biological systems : proteins are composed of l - amino acids and dna and rna are composed of -d - nucleotides .", "it has , therefore , been widely accepted that enzymes that must correctly bind ligands and catalyze chemistry on their respective substrates , will invariably show high chiral selectivity .", "quite unexpectedly , however , it was demonstrated that it is the l - component of the racemic mixture ( )-bch-189 [ ( )--2,3-dideoxy-3-thiacytidine ] , which possesses significantly better anti - human immunodeficiency virus ( hiv ) properties than its d - enantiomeric counterpart ( 1,2 ) . since this discovery ,", "two non - physiologic l - nucleoside analogs [ ( )--2,3-dideoxy-3-thiacytidine ( 3tc ) , the l - enantiomer of bch-189 , and its 5-fluorinated analog ftc ] have been approved for treatment of human acquired immune deficiency syndrome ( aids ) .", "an additional six d - nucleoside analogs have been approved as aids therapeutics , and it is customary for patients to receive both l- and d - enantiomer anti - hiv agents simultaneously .", "the pharmacological potential of l - nucleoside analogs has been recently reviewed ( 35 ) .", "the dioxolane analog of 3tc , troxacitabine [ tro , ( )-l--2,3-dideoxy-3-oxacytidine ; figure 1a ] is currently in a phase ii / iii clinical trial for third - line treatment of acute myelogenous leukemia ( aml ) , a blood cancer .", "the l - nucleoside analogs 3tc and ftc are preferred as anti - virals over their d - analogs [ ( + ) -bch-189 and ( + ) -ftc , respectively ] , primarily because these hiv reverse transcriptase inhibitors do not affect cellular dna polymerases ( 4 ) , resulting in minimal toxicity .", "in contrast , tro , the first l - nucleoside analog to be used as a cytotoxic anti - cancer agent ( 6 ) , which differs from 3tc by only one atom ( s versus o in the five membered ring , figure 1a ) , has potent anti - viral activity yet displays substantial cellular toxicity due to inhibition of dna polymerases , and ( 7 ) .", "unlike the d - nucleoside analog anti - cancer agent cytarabine ( arac ) , tro is not a substrate for the drug - modifying enzyme cytidine deaminase ( 6 ) that confers cytarabine resistance in aml ( 8) . ( a ) schematic of the d- and l - nucleosides ( all in -form ) : 3tc , ( )-l-2,3-dideoxy-3-thiacytidine ( lamivudine , epivir ) ; tro , ( )-l-2,3-dideoxy-3-oxacytidine ( troxacitabine , troxatyl ) ; d - dc , d-2-deoxycytidine , and l - dc , l-2-deoxycytidine . experimental electron density map for the l - nucleoside analogs : ( b )", "the difference map ( |fobs| |fcalc| ) is contoured at 3 ( light blue ) and 8 ( orange ) .", "note the strong signal given by the sulfur atom in 3tc even at the high sigma cut - off .", "figures were generated using bobscript ( 26 ) and molscript ( 27 ) , and rendered with raster3d ( 28 ) .", "metabolic conversion of 3tc and tro to pharmacologically active drugs requires serial phosphorylation to their triphosphorylated forms .", "the essential first step in this process is catalyzed by deoxycytidine kinase ( dck ) ( 6,911 ) , which together with the two mitochondrial enzymes thymidine kinase ( tk2 ) and deoxyguanosine kinase ( dgk ) , constitute a family of three closely related non - enantioselective enzymes responsible for nucleoside phosphorylation ( 12,13 ) .", "tk2 phosphorylates both dc and deoxythymidine ( dt ) , and dgk phosphorylates both da and dg .", "the overlapping substrate specificity of these nucleoside kinases reflects their differing sub - cellular localization : dck is cytosolic , whereas tk2 and dgk are mitochondrial .", "all three enzymes are capable of phosphorylating both l- and d - enantiomers ( 1416 ) .", "the question that we set out to answer is what structural features endow certain enzymes , such as dck , the ability to accept substrates with non - physiological chirality ? answering this question is significant not only for our understanding of enzyme specificity , but also has medicinal ramifications as more l - nucleoside analogs enter clinical trials . to address this issue , we solved the crystal structures of human dck in complex with the two clinically relevant l - nucleoside analogs , 3tc and tro .", "this is the first report of an enzyme in complex with an l - nucleoside .", "initial attempts to crystallize wild - type dck with 3tc and tro were hampered by reproducibility .", "modification of all four surface - exposed cysteine residues yielded a significantly better behaved crystallization candidate , thereby making this study feasible .", "the dck variant containing the c9s / c45s / c59s / c146s mutations , designated c4s - dck , was created from the wild - type dck gene , using quickchange ( stratagene , inc . ) .", "the protein was produced in escherichia coli and purified to homogeneity ( e. sabini , s. hazra , m. konrad and a. lavie , manuscript in preparation ) in short , e.coli bl21(de3 ) harboring the pet14b expression plasmids were grown in 2yt media at 37c , induced with 0.1 mm isopropyl--d - thiogalactopyranoside ( iptg ) , and harvested by centrifugation after 4 h. after lysis by sonication and ultracentrifugation , the supernatant was loaded onto a histrap hp ni sepharose column ( ge healthcare ) and washed with buffer containing 50 mm hepes ( ph 7.5 ) , 500 mm nacl and 20 mm imidazole .", "the enzyme was eluted by the same washing buffer containing 200 mm imidazole . after protein concentration by centrifugation", ", the protein was injected onto a s-200 gel filtration column ( ge healthcare ) equilibrated with 20 mm hepes ( ph 7.5 ) , 200 mm sodium citrate and 2 mm edta .", "the fractions containing the enzyme were concentrated to 20 mg / ml and stored at 80c .", "ternary complexes were formed by mixing each nucleoside analog ( 3tc , obtained from the nih aids research and reference reagent program , division of aids ; tro , provided by sgx pharmaceuticals , inc . ) with adenosine diphosphate ( adp ) ( final concentrations 5 mm each ) and enzyme ( 1020 mg / ml ) in 5 mm mgcl2 .", "crystals were obtained under identical conditions as for wild - type dck ( 17 ) via hanging drop vapor diffusion against a reservoir containing 0.951.5 m sodium citrate and 100 mm hepes ( ph 7.5 ) .", "x - ray data were obtained under standard cryogenic conditions at the advanced photon source using sercat beamline bm-22 .", "diffraction data were indexed , scaled and merged using xds and xscale ( 18 ) ( see table 1 for data collection statistics ) .", "data collection and refinement statistics ternary complex structures were determined via molecular replacement [ molrep ( 19 ) ] using the structure of wild - type dck - d - dc / adp ( 17 ) [ pdb code : 1p60 ( 20 ) ] as the search model .", "refinement was carried out using refmac ( 21 ) and simulated annealing maps were calculated using cns ( 22 ) ( see table 1 for refinement statistics ) .", "a spectroscopic enzyme - coupled assay was used to determine enzyme activity as described previously ( 23 ) .", "this assay couples the production of adp or udp by dck to the phosphorylation of phosphoenolpyruvate ( pep ) by pyruvate kinase , followed by oxidation of nadh by lactate dehydrogenase .", "the decrease in nadh absorption is proportional to the adp / udp produced by dck .", "the extinction coefficient of nadh limits the precise measurement of km values below 3 m . for this reason ,", "substrates displaying a km value below 3 m are reported in table 2 to this higher limit .", "experiments were conducted at 37c in a kinetic buffer containing 50 mm tris hcl ( ph 7.5 ) , 100 mm kcl and 5 mm mgcl2 with dck : 0.35 m , adenosine triphosphate ( atp)-mg : 1 mm and uridine triphosphate ( utp)-mg : 1 mm .", "steady state kinetic data of wt and c4s - dck values shown are the averages of at least two experiments and sds are shown . to verify that the cysser mutations in c4s - dck did not perturb the structure , ternary complex crystals of the c4s variant", "were obtained in the presence of d - dc / adp ( pdb i d 2no1 ) and gemcitabine / adp ( pdb i d 2no0 ) and compared to structures previously determined with wild - type enzyme ( 17 ) .", "these control complexes crystallized in the same space group as the l - nucleoside complexes , and diffracted to 1.9 ( d - dc / adp complex ) and 1.8 ( gemcitabine / adp ) resolution ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) .", "no significant structural differences were noted between corresponding polypeptide chain structures [ root mean square deviation ( r.m.s.d . ) between 241 carbon atomic positions : wild - type dck - d - dc / adp versus c4s - dck - d - dc / adp = 0.16 ; dck - gemcitabine / adp versus c4s - dck - gemcitabine / adp = 0.60 ] or substrate positions . finally , no significant differences in catalytic efficiency were observed between wild - type and c4s - dck for two distinct phosphate donors and multiple acceptors ( table 2 ) .", "together , these control experiments validate the use of the c4s mutant for structure determination .", "when referring to structures of dck herein , we make no distinction between wild type and the c4s mutant , unless otherwise stated .", "initial attempts to crystallize wild - type dck with 3tc and tro were hampered by reproducibility .", "modification of all four surface - exposed cysteine residues yielded a significantly better behaved crystallization candidate , thereby making this study feasible .", "the dck variant containing the c9s / c45s / c59s / c146s mutations , designated c4s - dck , was created from the wild - type dck gene , using quickchange ( stratagene , inc . ) .", "the protein was produced in escherichia coli and purified to homogeneity ( e. sabini , s. hazra , m. konrad and a. lavie , manuscript in preparation ) in short , e.coli bl21(de3 ) harboring the pet14b expression plasmids were grown in 2yt media at 37c , induced with 0.1 mm isopropyl--d - thiogalactopyranoside ( iptg ) , and harvested by centrifugation after 4 h. after lysis by sonication and ultracentrifugation , the supernatant was loaded onto a histrap hp ni sepharose column ( ge healthcare ) and washed with buffer containing 50 mm hepes ( ph 7.5 ) , 500 mm nacl and 20 mm imidazole .", "the enzyme was eluted by the same washing buffer containing 200 mm imidazole . after protein concentration by centrifugation", ", the protein was injected onto a s-200 gel filtration column ( ge healthcare ) equilibrated with 20 mm hepes ( ph 7.5 ) , 200 mm sodium citrate and 2 mm edta .", "the fractions containing the enzyme were concentrated to 20 mg / ml and stored at 80c .", "ternary complexes were formed by mixing each nucleoside analog ( 3tc , obtained from the nih aids research and reference reagent program , division of aids ; tro , provided by sgx pharmaceuticals , inc . ) with adenosine diphosphate ( adp ) ( final concentrations 5 mm each ) and enzyme ( 1020 mg / ml ) in 5 mm mgcl2 .", "crystals were obtained under identical conditions as for wild - type dck ( 17 ) via hanging drop vapor diffusion against a reservoir containing 0.951.5 m sodium citrate and 100 mm hepes ( ph 7.5 ) .", "x - ray data were obtained under standard cryogenic conditions at the advanced photon source using sercat beamline bm-22 .", "diffraction data were indexed , scaled and merged using xds and xscale ( 18 ) ( see table 1 for data collection statistics ) .", "ternary complex structures were determined via molecular replacement [ molrep ( 19 ) ] using the structure of wild - type dck - d - dc / adp ( 17 ) [ pdb code : 1p60 ( 20 ) ] as the search model .", "refinement was carried out using refmac ( 21 ) and simulated annealing maps were calculated using cns ( 22 ) ( see table 1 for refinement statistics ) .", "a spectroscopic enzyme - coupled assay was used to determine enzyme activity as described previously ( 23 ) .", "this assay couples the production of adp or udp by dck to the phosphorylation of phosphoenolpyruvate ( pep ) by pyruvate kinase , followed by oxidation of nadh by lactate dehydrogenase .", "the decrease in nadh absorption is proportional to the adp / udp produced by dck .", "the extinction coefficient of nadh limits the precise measurement of km values below 3 m . for this reason ,", "substrates displaying a km value below 3 m are reported in table 2 to this higher limit .", "experiments were conducted at 37c in a kinetic buffer containing 50 mm tris hcl ( ph 7.5 ) , 100 mm kcl and 5 mm mgcl2 with dck : 0.35 m , adenosine triphosphate ( atp)-mg : 1 mm and uridine triphosphate ( utp)-mg : 1 mm .", "steady state kinetic data of wt and c4s - dck values shown are the averages of at least two experiments and sds are shown .", "to verify that the cysser mutations in c4s - dck did not perturb the structure , ternary complex crystals of the c4s variant were obtained in the presence of d - dc / adp ( pdb i d 2no1 ) and gemcitabine / adp ( pdb i d 2no0 ) and compared to structures previously determined with wild - type enzyme ( 17 ) .", "these control complexes crystallized in the same space group as the l - nucleoside complexes , and diffracted to 1.9 ( d - dc / adp complex ) and 1.8 ( gemcitabine / adp ) resolution ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) .", "no significant structural differences were noted between corresponding polypeptide chain structures [ root mean square deviation ( r.m.s.d . ) between 241 carbon atomic positions : wild - type dck - d - dc / adp versus c4s - dck - d - dc / adp = 0.16 ; dck - gemcitabine / adp versus c4s - dck - gemcitabine / adp = 0.60 ] or substrate positions . finally , no significant differences in catalytic efficiency were observed between wild - type and c4s - dck for two distinct phosphate donors and multiple acceptors ( table 2 ) .", "together , these control experiments validate the use of the c4s mutant for structure determination .", "when referring to structures of dck herein , we make no distinction between wild type and the c4s mutant , unless otherwise stated .", "the ternary complex structures of dck-3tc / adp and dck - tro / adp were determined at 1.8 and 2.15 resolution , respectively .", "excellent difference electron density maps were observed for the l - nucleoside analogs ( figure 1b and c ) .", "after several rounds of model building and refinement , in which the nucleotide adp and the respective l - nucleoside analogs were modeled into the density , and addition of water molecules , the final crystallographic r - factors ( rfrees ) converged to 19.2% ( 23.3% ) and 19.5% ( 25.5% ) for the 3tc and tro complexes , respectively ( table 1 ) . when compared to the ternary complex structures of dck - d - dc", "/ adp and dck - l - dc / adp ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) , binding of these l - nucleoside analogs does not affect the structure of the polypeptide chain r.m.s.d .", "for 239 common -carbon atoms among pairs of ternary complex structures = 0.180.30 ] nor the positions of the adp molecules ( figure 2a ) .", "in contrast , the mode of l - nucleoside / l - nucleoside analog binding differs from that of d - nucleosides ( figure 2b and c ) .", "( a ) shown are four superimposed structures of human dck : the c4s variant in complex with 3tc / adp ( yellow ) , tro / adp ( magenta ) and d - dc / adp ( blue ) ; the wt in complex with d - dc / adp ( gray ; pdb i d 1p5z ) .", "the structures in complex with d - dc / adp of the dck c4s variant and the wt dck are basically identical , apart from one loop involved in crystal contact .", "non - carbon atom color coding : oxygen - red , nitrogen - blue , phosphorous - green and sulfur - orange . the n- and c - terminal residues visible in the experimental electron density map are labeled n20 and c260 , respectively . (", "b ) stereo representation of the active site showing hydrogen bonding interactions ( some water molecules omitted , for clarity ) .", "( c ) stereo representation of the active site in an orientation rotated 90 relative to ( b ) depicting the cytosine base in the aromatic slot with leu82 and ile30 sandwiching the sugar .", "structures have been deposited in the pdb with accession i d 2noa for c4s-3tc / adp , 2no9 for c4s - tro / adp and 2no1 for c4s - d - dc / adp . the active site environment surrounding each l - nucleoside analog is shown in figure 3a , accompanied by similar views for dck - d - dc / adp and dck - l - dc / adp ( figure 3b ) .", "d - dc is the mirror image of l - dc ( chirality being determined by the location of the ch2oh group at the c4 chiral centre or ribose 4 position , figures 1a and 3b ) .", "3tc and tro closely resemble one another , and only differ from l - dc at the 3 position ( figure 1a ) .", "detailed comparison of the protein ligand interactions , presented schematically in figure 4 , reveals the structural basis for non - enantioselective phosphorylation by dck . for both 3tc and tro", ", the enzyme satisfies the same requirements for substrate recognition / catalysis that govern phosphorylation of its primary physiologic substrate , d - dc .", "first , hydrogen bonding interactions between the protein and the ligand permit the protein to detect the presence of a hydrogen bond donor ( nh2 ) and two hydrogen bond acceptors ( n ; o ) characteristic of the cytosine base ( figure 2b ) .", "second , the five - membered ring resembling the ribose is positioned so as to place the site of phosphorylation ( 5-oh ) in the appropriate location for phosphoryl transfer from atp .", "( a ) stereoview depicting the interactions made by the 5-oh groups of 3tc ( yellow ) and tro ( magenta ) .", "( b ) stereoview depicting the analogous interactions for d - dc ( blue ) and l - dc ( cyan ) .", "schematic representation of the ( a ) polar and ( b ) weakly polar and hydrophobic interactions made by 3tc and tro with the residues in the dck active site .", "distances ( ) are color - coded in ( a ) with 3tc - yellow and tro - magenta , while in ( b ) they are in black and correspond to the average closest contacts for both structures .", "water molecules are represented as cyan balls . despite the difference in chirality between the physiologic substrate d - dc and the l - nucleoside analogs 3tc / tro , the location of the cytosine base and its interactions with the enzyme are comparable . in all three ternary complex structures depicted in figure 2b , asp133 forms hydrogen bonds with the cytosine amino group", ". however , it is gln97 that serves as the primary cytosine recognition element contributing two hydrogen bonds between its side chain and the components of the base that normally engage in watson crick type hydrogen bonding with guanosine ( gln97 onh2 ; gln97 nh2n ; figures 2b and 4a ) .", "the fourth hydrogen bonding interaction between the protein and the base appears as a water bridge ( tyr204/tyr86h2oo ; figures 2b and 4a ) .", "analogous interactions with comparable hydrogen bond geometries occur between dck and the cytosine bases of d - dc and l - dc ( data not shown ) .", "the base itself sits deep in an aromatic slot , which is lined by the side chains of phe137 , phe96 and trp58 ( all conserved among dck , dgk and tk2 ) .", "phe137 engages in stacking against the cytosine base ( interplanar distance 3.5 ) . on the opposite face , phe96 and trp58 both engage in edge - to - face interactions that place electropositive aromatic hydrogen atoms close to the electronegative -electron cloud of the cytosine base ( figures 2c and 4b ) .", "quadrupole interactions characteristic of aromatic amino acid side chains in proteins as also seen in crystalline benzene ( 24 ) .", "the important systematic difference in the position of the cytosine bases of 3tc and tro versus that of d - dc is a 10 tilt ( figure 2c ) .", "we believe that this difference is a general feature of l - enantiomers , as the same base tilt is reproduced in the structure of l - dc bound to dck ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) .", "in contrast to the numerous interactions between the cytosine base and the aromatic slot , the five membered rings of 3tc and tro make only limited enzyme contacts ( figures 3a and 4 ) .", "critical for catalysis is correct positioning of the 5-oh , which receives the transferred phosphate group from atp . in the l - nucleoside analog structures depicted in figure 3a , the 5-oh group is hydrogen bonded to the side chain of arg128 .", "consequently in each case , the o5-hydroxyl is close to a side chain oxygen atom of glu53 ( 3.13.6 ) , the general base that activates 5-oh group attack of the -phosphate of atp ( see figures 2b and 3 ) . to achieve this critical interaction , concomitant with base tilting , the five - membered rings of 3tc and tro rotate 30 around the glycosidic bond ( , marked with an arrow in figure 2b ) relative to the dck - bound conformation of d - dc .", "a comparable rotation observed in the l - dc structure ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) provides additional evidence that our structural findings represent the way in which l - nucleosides and l - nucleoside analogs bind to dck .", "the four structures depicted in figure 3 document that positioning of the 5-oh group in the active site of dck is independent of chirality .", "this remarkable property derives from the internal symmetry and conformational flexibility ( i.e. rotation about the glycosidic torsion angle ) of nucleosides and nucleoside analogs . once the cytosine base is docked into the aromatic slot , the paucity of protein ligand interactions involving the five - membered ring and the conformational properties of the nucleoside permit appropriate positioning of the 5-oh group . within the aromatic slot ,", "possible glycosidic bond torsion angles are limited by the hydrophobic residues ile30 and leu82 ( figures 2c and 4 ) .", "an additional degree of freedom dictating the precise location of the hydroxyl is the pucker of the five - membered ring . for 3tc and tro ,", "the ring pucker is equivalent to c3 endo , whereas for l - dc and d - dc the sugar pucker is c3 exo .", "this difference can be attributed in part to the absence of a 3-oh group on 3tc and on tro , both of which can act as dna chain terminators . finally , a torsion angle rotation about the c4 and c5 bond , from gauche in the d - dc structure to trans in the complex structures of 3tc , tro and l - dc , direct the 5-oh group towards glu53 .", "although these features of 3tc and tro conformational flexibility bring the 5-oh group near to glu53 ( 3.13.6 ; figure 3a ) , they do not stabilize the very close , so - called catalytic , interactions seen with l - dc and d - dc ( 2.42.5 ; figure 3b ) .", "however , the fact that dck shows similar catalytic efficiency towards 3tc / tro and l - dc ( table 2 ) suggests that the slightly sub - optimal position observed for the 5-oh group in the 3tc / tro structures does not impede catalysis . in conclusion ,", "the structures reported herein document that dck phosphorylation of l - nucleosides and l - nucleoside analogs is due to both the nature of the enzyme active site and the nature of the substrates .", "we suggest that lack of enantiomeric selectivity may reflect the central role dck plays in nucleotide salvage , i.e. phosphorylating both the pyrimidine dc and the purines da and dg within the cytosolic compartment .", "similar arguments can be invoked to explain why the related enzymes dgk , which phosphorylates da and dg , and tk2 , which phosphorylates dt and dc , are not enantioselective .", "in contrast , human tk1 , which is restricted to d - dt phosphorylation , is exquisitely enantioselective ( 25 ) .", "we suggest that enzymes that evolved to catalyze more than one chemically distinct nucleoside substrate acquired less restrictive active sites , which in turn endowed such enzymes with hitherto unexpected catalytic activities for substrates with non - physiologic chirality ." ]

[ "<S> l - nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers . </S>", "<S> these pro - drugs achieve pharmacological activity only after enzyme - catalyzed conversion to their tri - phosphorylated forms . </S>", "<S> herein , we report the crystal structures of human deoxycytidine kinase ( dck ) in complex with the l - nucleosides ( )--2,3-dideoxy-3-thiacytidine ( 3tc)an approved anti - human immunodeficiency virus ( hiv ) agent and troxacitabine ( tro)an experimental anti - neoplastic agent . </S>", "<S> the first step in activating these agents is catalyzed by dck . </S>", "<S> our studies reveal how dck , which normally catalyzes phosphorylation of the natural d - nucleosides , can efficiently phosphorylate substrates with non - physiologic chirality . </S>", "<S> the capability of dck to phosphorylate both d- and l - nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves . </S>", "<S> first , the nucleoside - binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein - ligand interactions responsible for productive substrate positioning . </S>", "<S> second , the pseudo - symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the l- and d - enantiomeric forms to adopt similar shapes when bound to the enzyme . </S>", "<S> this is the first analysis of the structural basis for activation of l - nucleoside analogs , providing further impetus for discovery and clinical development of new agents in this molecular class . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Protein crystal preparation", "Data collection and processing", "Structure determination and refinement", "Steady-state kinetic assay", "Validation of choice of crystallization protein", "RESULTS AND DISCUSSION" ]

[ [ "chirality is a hallmark of biological systems : proteins are composed of l - amino acids and dna and rna are composed of -d - nucleotides .", "it has , therefore , been widely accepted that enzymes that must correctly bind ligands and catalyze chemistry on their respective substrates , will invariably show high chiral selectivity .", "quite unexpectedly , however , it was demonstrated that it is the l - component of the racemic mixture ( )-bch-189 [ ( )--2,3-dideoxy-3-thiacytidine ] , which possesses significantly better anti - human immunodeficiency virus ( hiv ) properties than its d - enantiomeric counterpart ( 1,2 ) . since this discovery ,", "two non - physiologic l - nucleoside analogs [ ( )--2,3-dideoxy-3-thiacytidine ( 3tc ) , the l - enantiomer of bch-189 , and its 5-fluorinated analog ftc ] have been approved for treatment of human acquired immune deficiency syndrome ( aids ) .", "an additional six d - nucleoside analogs have been approved as aids therapeutics , and it is customary for patients to receive both l- and d - enantiomer anti - hiv agents simultaneously .", "the pharmacological potential of l - nucleoside analogs has been recently reviewed ( 35 ) .", "the dioxolane analog of 3tc , troxacitabine [ tro , ( )-l--2,3-dideoxy-3-oxacytidine ; figure 1a ] is currently in a phase ii / iii clinical trial for third - line treatment of acute myelogenous leukemia ( aml ) , a blood cancer .", "the l - nucleoside analogs 3tc and ftc are preferred as anti - virals over their d - analogs [ ( + ) -bch-189 and ( + ) -ftc , respectively ] , primarily because these hiv reverse transcriptase inhibitors do not affect cellular dna polymerases ( 4 ) , resulting in minimal toxicity .", "in contrast , tro , the first l - nucleoside analog to be used as a cytotoxic anti - cancer agent ( 6 ) , which differs from 3tc by only one atom ( s versus o in the five membered ring , figure 1a ) , has potent anti - viral activity yet displays substantial cellular toxicity due to inhibition of dna polymerases , and ( 7 ) .", "unlike the d - nucleoside analog anti - cancer agent cytarabine ( arac ) , tro is not a substrate for the drug - modifying enzyme cytidine deaminase ( 6 ) that confers cytarabine resistance in aml ( 8) . ( a ) schematic of the d- and l - nucleosides ( all in -form ) : 3tc , ( )-l-2,3-dideoxy-3-thiacytidine ( lamivudine , epivir ) ; tro , ( )-l-2,3-dideoxy-3-oxacytidine ( troxacitabine , troxatyl ) ; d - dc , d-2-deoxycytidine , and l - dc , l-2-deoxycytidine . experimental electron density map for the l - nucleoside analogs : ( b )", "the difference map ( |fobs| |fcalc| ) is contoured at 3 ( light blue ) and 8 ( orange ) .", "note the strong signal given by the sulfur atom in 3tc even at the high sigma cut - off .", "figures were generated using bobscript ( 26 ) and molscript ( 27 ) , and rendered with raster3d ( 28 ) .", "metabolic conversion of 3tc and tro to pharmacologically active drugs requires serial phosphorylation to their triphosphorylated forms .", "the essential first step in this process is catalyzed by deoxycytidine kinase ( dck ) ( 6,911 ) , which together with the two mitochondrial enzymes thymidine kinase ( tk2 ) and deoxyguanosine kinase ( dgk ) , constitute a family of three closely related non - enantioselective enzymes responsible for nucleoside phosphorylation ( 12,13 ) .", "tk2 phosphorylates both dc and deoxythymidine ( dt ) , and dgk phosphorylates both da and dg .", "the overlapping substrate specificity of these nucleoside kinases reflects their differing sub - cellular localization : dck is cytosolic , whereas tk2 and dgk are mitochondrial .", "all three enzymes are capable of phosphorylating both l- and d - enantiomers ( 1416 ) .", "the question that we set out to answer is what structural features endow certain enzymes , such as dck , the ability to accept substrates with non - physiological chirality ? answering this question is significant not only for our understanding of enzyme specificity , but also has medicinal ramifications as more l - nucleoside analogs enter clinical trials . to address this issue , we solved the crystal structures of human dck in complex with the two clinically relevant l - nucleoside analogs , 3tc and tro .", "this is the first report of an enzyme in complex with an l - nucleoside ." ], [ "initial attempts to crystallize wild - type dck with 3tc and tro were hampered by reproducibility .", "modification of all four surface - exposed cysteine residues yielded a significantly better behaved crystallization candidate , thereby making this study feasible .", "the dck variant containing the c9s / c45s / c59s / c146s mutations , designated c4s - dck , was created from the wild - type dck gene , using quickchange ( stratagene , inc . ) .", "the protein was produced in escherichia coli and purified to homogeneity ( e. sabini , s. hazra , m. konrad and a. lavie , manuscript in preparation ) in short , e.coli bl21(de3 ) harboring the pet14b expression plasmids were grown in 2yt media at 37c , induced with 0.1 mm isopropyl--d - thiogalactopyranoside ( iptg ) , and harvested by centrifugation after 4 h. after lysis by sonication and ultracentrifugation , the supernatant was loaded onto a histrap hp ni sepharose column ( ge healthcare ) and washed with buffer containing 50 mm hepes ( ph 7.5 ) , 500 mm nacl and 20 mm imidazole .", "the enzyme was eluted by the same washing buffer containing 200 mm imidazole . after protein concentration by centrifugation", ", the protein was injected onto a s-200 gel filtration column ( ge healthcare ) equilibrated with 20 mm hepes ( ph 7.5 ) , 200 mm sodium citrate and 2 mm edta .", "the fractions containing the enzyme were concentrated to 20 mg / ml and stored at 80c .", "ternary complexes were formed by mixing each nucleoside analog ( 3tc , obtained from the nih aids research and reference reagent program , division of aids ; tro , provided by sgx pharmaceuticals , inc . ) with adenosine diphosphate ( adp ) ( final concentrations 5 mm each ) and enzyme ( 1020 mg / ml ) in 5 mm mgcl2 .", "crystals were obtained under identical conditions as for wild - type dck ( 17 ) via hanging drop vapor diffusion against a reservoir containing 0.951.5 m sodium citrate and 100 mm hepes ( ph 7.5 ) .", "x - ray data were obtained under standard cryogenic conditions at the advanced photon source using sercat beamline bm-22 .", "diffraction data were indexed , scaled and merged using xds and xscale ( 18 ) ( see table 1 for data collection statistics ) .", "data collection and refinement statistics ternary complex structures were determined via molecular replacement [ molrep ( 19 ) ] using the structure of wild - type dck - d - dc / adp ( 17 ) [ pdb code : 1p60 ( 20 ) ] as the search model .", "refinement was carried out using refmac ( 21 ) and simulated annealing maps were calculated using cns ( 22 ) ( see table 1 for refinement statistics ) .", "a spectroscopic enzyme - coupled assay was used to determine enzyme activity as described previously ( 23 ) .", "this assay couples the production of adp or udp by dck to the phosphorylation of phosphoenolpyruvate ( pep ) by pyruvate kinase , followed by oxidation of nadh by lactate dehydrogenase .", "the decrease in nadh absorption is proportional to the adp / udp produced by dck .", "the extinction coefficient of nadh limits the precise measurement of km values below 3 m . for this reason ,", "substrates displaying a km value below 3 m are reported in table 2 to this higher limit .", "experiments were conducted at 37c in a kinetic buffer containing 50 mm tris hcl ( ph 7.5 ) , 100 mm kcl and 5 mm mgcl2 with dck : 0.35 m , adenosine triphosphate ( atp)-mg : 1 mm and uridine triphosphate ( utp)-mg : 1 mm .", "steady state kinetic data of wt and c4s - dck values shown are the averages of at least two experiments and sds are shown . to verify that the cysser mutations in c4s - dck did not perturb the structure , ternary complex crystals of the c4s variant", "were obtained in the presence of d - dc / adp ( pdb i d 2no1 ) and gemcitabine / adp ( pdb i d 2no0 ) and compared to structures previously determined with wild - type enzyme ( 17 ) .", "these control complexes crystallized in the same space group as the l - nucleoside complexes , and diffracted to 1.9 ( d - dc / adp complex ) and 1.8 ( gemcitabine / adp ) resolution ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) .", "no significant structural differences were noted between corresponding polypeptide chain structures [ root mean square deviation ( r.m.s.d . ) between 241 carbon atomic positions : wild - type dck - d - dc / adp versus c4s - dck - d - dc / adp = 0.16 ; dck - gemcitabine / adp versus c4s - dck - gemcitabine / adp = 0.60 ] or substrate positions . finally , no significant differences in catalytic efficiency were observed between wild - type and c4s - dck for two distinct phosphate donors and multiple acceptors ( table 2 ) .", "together , these control experiments validate the use of the c4s mutant for structure determination .", "when referring to structures of dck herein , we make no distinction between wild type and the c4s mutant , unless otherwise stated ." ], [ "initial attempts to crystallize wild - type dck with 3tc and tro were hampered by reproducibility .", "modification of all four surface - exposed cysteine residues yielded a significantly better behaved crystallization candidate , thereby making this study feasible .", "the dck variant containing the c9s / c45s / c59s / c146s mutations , designated c4s - dck , was created from the wild - type dck gene , using quickchange ( stratagene , inc . ) .", "the protein was produced in escherichia coli and purified to homogeneity ( e. sabini , s. hazra , m. konrad and a. lavie , manuscript in preparation ) in short , e.coli bl21(de3 ) harboring the pet14b expression plasmids were grown in 2yt media at 37c , induced with 0.1 mm isopropyl--d - thiogalactopyranoside ( iptg ) , and harvested by centrifugation after 4 h. after lysis by sonication and ultracentrifugation , the supernatant was loaded onto a histrap hp ni sepharose column ( ge healthcare ) and washed with buffer containing 50 mm hepes ( ph 7.5 ) , 500 mm nacl and 20 mm imidazole .", "the enzyme was eluted by the same washing buffer containing 200 mm imidazole . after protein concentration by centrifugation", ", the protein was injected onto a s-200 gel filtration column ( ge healthcare ) equilibrated with 20 mm hepes ( ph 7.5 ) , 200 mm sodium citrate and 2 mm edta .", "the fractions containing the enzyme were concentrated to 20 mg / ml and stored at 80c .", "ternary complexes were formed by mixing each nucleoside analog ( 3tc , obtained from the nih aids research and reference reagent program , division of aids ; tro , provided by sgx pharmaceuticals , inc . ) with adenosine diphosphate ( adp ) ( final concentrations 5 mm each ) and enzyme ( 1020 mg / ml ) in 5 mm mgcl2 .", "crystals were obtained under identical conditions as for wild - type dck ( 17 ) via hanging drop vapor diffusion against a reservoir containing 0.951.5 m sodium citrate and 100 mm hepes ( ph 7.5 ) ." ], [ "x - ray data were obtained under standard cryogenic conditions at the advanced photon source using sercat beamline bm-22 .", "diffraction data were indexed , scaled and merged using xds and xscale ( 18 ) ( see table 1 for data collection statistics ) ." ], [ "ternary complex structures were determined via molecular replacement [ molrep ( 19 ) ] using the structure of wild - type dck - d - dc / adp ( 17 ) [ pdb code : 1p60 ( 20 ) ] as the search model .", "refinement was carried out using refmac ( 21 ) and simulated annealing maps were calculated using cns ( 22 ) ( see table 1 for refinement statistics ) ." ], [ "a spectroscopic enzyme - coupled assay was used to determine enzyme activity as described previously ( 23 ) .", "this assay couples the production of adp or udp by dck to the phosphorylation of phosphoenolpyruvate ( pep ) by pyruvate kinase , followed by oxidation of nadh by lactate dehydrogenase .", "the decrease in nadh absorption is proportional to the adp / udp produced by dck .", "the extinction coefficient of nadh limits the precise measurement of km values below 3 m . for this reason ,", "substrates displaying a km value below 3 m are reported in table 2 to this higher limit .", "experiments were conducted at 37c in a kinetic buffer containing 50 mm tris hcl ( ph 7.5 ) , 100 mm kcl and 5 mm mgcl2 with dck : 0.35 m , adenosine triphosphate ( atp)-mg : 1 mm and uridine triphosphate ( utp)-mg : 1 mm .", "steady state kinetic data of wt and c4s - dck values shown are the averages of at least two experiments and sds are shown ." ], [ "to verify that the cysser mutations in c4s - dck did not perturb the structure , ternary complex crystals of the c4s variant were obtained in the presence of d - dc / adp ( pdb i d 2no1 ) and gemcitabine / adp ( pdb i d 2no0 ) and compared to structures previously determined with wild - type enzyme ( 17 ) .", "these control complexes crystallized in the same space group as the l - nucleoside complexes , and diffracted to 1.9 ( d - dc / adp complex ) and 1.8 ( gemcitabine / adp ) resolution ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) .", "no significant structural differences were noted between corresponding polypeptide chain structures [ root mean square deviation ( r.m.s.d . ) between 241 carbon atomic positions : wild - type dck - d - dc / adp versus c4s - dck - d - dc / adp = 0.16 ; dck - gemcitabine / adp versus c4s - dck - gemcitabine / adp = 0.60 ] or substrate positions . finally , no significant differences in catalytic efficiency were observed between wild - type and c4s - dck for two distinct phosphate donors and multiple acceptors ( table 2 ) .", "together , these control experiments validate the use of the c4s mutant for structure determination .", "when referring to structures of dck herein , we make no distinction between wild type and the c4s mutant , unless otherwise stated ." ], [ "the ternary complex structures of dck-3tc / adp and dck - tro / adp were determined at 1.8 and 2.15 resolution , respectively .", "excellent difference electron density maps were observed for the l - nucleoside analogs ( figure 1b and c ) .", "after several rounds of model building and refinement , in which the nucleotide adp and the respective l - nucleoside analogs were modeled into the density , and addition of water molecules , the final crystallographic r - factors ( rfrees ) converged to 19.2% ( 23.3% ) and 19.5% ( 25.5% ) for the 3tc and tro complexes , respectively ( table 1 ) . when compared to the ternary complex structures of dck - d - dc", "/ adp and dck - l - dc / adp ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) , binding of these l - nucleoside analogs does not affect the structure of the polypeptide chain r.m.s.d .", "for 239 common -carbon atoms among pairs of ternary complex structures = 0.180.30 ] nor the positions of the adp molecules ( figure 2a ) .", "in contrast , the mode of l - nucleoside / l - nucleoside analog binding differs from that of d - nucleosides ( figure 2b and c ) .", "( a ) shown are four superimposed structures of human dck : the c4s variant in complex with 3tc / adp ( yellow ) , tro / adp ( magenta ) and d - dc / adp ( blue ) ; the wt in complex with d - dc / adp ( gray ; pdb i d 1p5z ) .", "the structures in complex with d - dc / adp of the dck c4s variant and the wt dck are basically identical , apart from one loop involved in crystal contact .", "non - carbon atom color coding : oxygen - red , nitrogen - blue , phosphorous - green and sulfur - orange . the n- and c - terminal residues visible in the experimental electron density map are labeled n20 and c260 , respectively . (", "b ) stereo representation of the active site showing hydrogen bonding interactions ( some water molecules omitted , for clarity ) .", "( c ) stereo representation of the active site in an orientation rotated 90 relative to ( b ) depicting the cytosine base in the aromatic slot with leu82 and ile30 sandwiching the sugar .", "structures have been deposited in the pdb with accession i d 2noa for c4s-3tc / adp , 2no9 for c4s - tro / adp and 2no1 for c4s - d - dc / adp . the active site environment surrounding each l - nucleoside analog is shown in figure 3a , accompanied by similar views for dck - d - dc / adp and dck - l - dc / adp ( figure 3b ) .", "d - dc is the mirror image of l - dc ( chirality being determined by the location of the ch2oh group at the c4 chiral centre or ribose 4 position , figures 1a and 3b ) .", "3tc and tro closely resemble one another , and only differ from l - dc at the 3 position ( figure 1a ) .", "detailed comparison of the protein ligand interactions , presented schematically in figure 4 , reveals the structural basis for non - enantioselective phosphorylation by dck . for both 3tc and tro", ", the enzyme satisfies the same requirements for substrate recognition / catalysis that govern phosphorylation of its primary physiologic substrate , d - dc .", "first , hydrogen bonding interactions between the protein and the ligand permit the protein to detect the presence of a hydrogen bond donor ( nh2 ) and two hydrogen bond acceptors ( n ; o ) characteristic of the cytosine base ( figure 2b ) .", "second , the five - membered ring resembling the ribose is positioned so as to place the site of phosphorylation ( 5-oh ) in the appropriate location for phosphoryl transfer from atp .", "( a ) stereoview depicting the interactions made by the 5-oh groups of 3tc ( yellow ) and tro ( magenta ) .", "( b ) stereoview depicting the analogous interactions for d - dc ( blue ) and l - dc ( cyan ) .", "schematic representation of the ( a ) polar and ( b ) weakly polar and hydrophobic interactions made by 3tc and tro with the residues in the dck active site .", "distances ( ) are color - coded in ( a ) with 3tc - yellow and tro - magenta , while in ( b ) they are in black and correspond to the average closest contacts for both structures .", "water molecules are represented as cyan balls . despite the difference in chirality between the physiologic substrate d - dc and the l - nucleoside analogs 3tc / tro , the location of the cytosine base and its interactions with the enzyme are comparable . in all three ternary complex structures depicted in figure 2b , asp133 forms hydrogen bonds with the cytosine amino group", ". however , it is gln97 that serves as the primary cytosine recognition element contributing two hydrogen bonds between its side chain and the components of the base that normally engage in watson crick type hydrogen bonding with guanosine ( gln97 onh2 ; gln97 nh2n ; figures 2b and 4a ) .", "the fourth hydrogen bonding interaction between the protein and the base appears as a water bridge ( tyr204/tyr86h2oo ; figures 2b and 4a ) .", "analogous interactions with comparable hydrogen bond geometries occur between dck and the cytosine bases of d - dc and l - dc ( data not shown ) .", "the base itself sits deep in an aromatic slot , which is lined by the side chains of phe137 , phe96 and trp58 ( all conserved among dck , dgk and tk2 ) .", "phe137 engages in stacking against the cytosine base ( interplanar distance 3.5 ) . on the opposite face , phe96 and trp58 both engage in edge - to - face interactions that place electropositive aromatic hydrogen atoms close to the electronegative -electron cloud of the cytosine base ( figures 2c and 4b ) .", "quadrupole interactions characteristic of aromatic amino acid side chains in proteins as also seen in crystalline benzene ( 24 ) .", "the important systematic difference in the position of the cytosine bases of 3tc and tro versus that of d - dc is a 10 tilt ( figure 2c ) .", "we believe that this difference is a general feature of l - enantiomers , as the same base tilt is reproduced in the structure of l - dc bound to dck ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) .", "in contrast to the numerous interactions between the cytosine base and the aromatic slot , the five membered rings of 3tc and tro make only limited enzyme contacts ( figures 3a and 4 ) .", "critical for catalysis is correct positioning of the 5-oh , which receives the transferred phosphate group from atp . in the l - nucleoside analog structures depicted in figure 3a , the 5-oh group is hydrogen bonded to the side chain of arg128 .", "consequently in each case , the o5-hydroxyl is close to a side chain oxygen atom of glu53 ( 3.13.6 ) , the general base that activates 5-oh group attack of the -phosphate of atp ( see figures 2b and 3 ) . to achieve this critical interaction , concomitant with base tilting , the five - membered rings of 3tc and tro rotate 30 around the glycosidic bond ( , marked with an arrow in figure 2b ) relative to the dck - bound conformation of d - dc .", "a comparable rotation observed in the l - dc structure ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) provides additional evidence that our structural findings represent the way in which l - nucleosides and l - nucleoside analogs bind to dck .", "the four structures depicted in figure 3 document that positioning of the 5-oh group in the active site of dck is independent of chirality .", "this remarkable property derives from the internal symmetry and conformational flexibility ( i.e. rotation about the glycosidic torsion angle ) of nucleosides and nucleoside analogs . once the cytosine base is docked into the aromatic slot , the paucity of protein ligand interactions involving the five - membered ring and the conformational properties of the nucleoside permit appropriate positioning of the 5-oh group . within the aromatic slot ,", "possible glycosidic bond torsion angles are limited by the hydrophobic residues ile30 and leu82 ( figures 2c and 4 ) .", "an additional degree of freedom dictating the precise location of the hydroxyl is the pucker of the five - membered ring . for 3tc and tro ,", "the ring pucker is equivalent to c3 endo , whereas for l - dc and d - dc the sugar pucker is c3 exo .", "this difference can be attributed in part to the absence of a 3-oh group on 3tc and on tro , both of which can act as dna chain terminators . finally , a torsion angle rotation about the c4 and c5 bond , from gauche in the d - dc structure to trans in the complex structures of 3tc , tro and l - dc , direct the 5-oh group towards glu53 .", "although these features of 3tc and tro conformational flexibility bring the 5-oh group near to glu53 ( 3.13.6 ; figure 3a ) , they do not stabilize the very close , so - called catalytic , interactions seen with l - dc and d - dc ( 2.42.5 ; figure 3b ) .", "however , the fact that dck shows similar catalytic efficiency towards 3tc / tro and l - dc ( table 2 ) suggests that the slightly sub - optimal position observed for the 5-oh group in the 3tc / tro structures does not impede catalysis . in conclusion ,", "the structures reported herein document that dck phosphorylation of l - nucleosides and l - nucleoside analogs is due to both the nature of the enzyme active site and the nature of the substrates .", "we suggest that lack of enantiomeric selectivity may reflect the central role dck plays in nucleotide salvage , i.e. phosphorylating both the pyrimidine dc and the purines da and dg within the cytosolic compartment .", "similar arguments can be invoked to explain why the related enzymes dgk , which phosphorylates da and dg , and tk2 , which phosphorylates dt and dc , are not enantioselective .", "in contrast , human tk1 , which is restricted to d - dt phosphorylation , is exquisitely enantioselective ( 25 ) .", "we suggest that enzymes that evolved to catalyze more than one chemically distinct nucleoside substrate acquired less restrictive active sites , which in turn endowed such enzymes with hitherto unexpected catalytic activities for substrates with non - physiologic chirality ." ] ]

[ "parasitic infections are increasing worldwide due to rapid urbanization of cities , global warming , and international traveling .", "the diagnosis of parasitic diseases of the respiratory system is challenging because clinical manifestation and radiological findings are not specific .", "the larvae can cause airway inflammation , whereas migration of the mature adult worm may cause mechanical obstruction .", "we present a case of interstitial pneumonitis caused by parasitic infestation , which was diagnosed on transbronchial lung biopsy ( tblb ) .", "a 54-year - old female presented with a history of exertional breathlessness and dry cough for two months .", "she was afebrile with spo2 of 91% on room air and bilateral crepitations on respiratory examination .", "l ( 46.4% ) , fvc of 1.45 l ( 45.2% ) , and fev1/fvc 83% with reduction in dlco 1.46 mmol / min / kpa ( 18% ) and corrected value with alveolar volume 0.49 mmol / min / kpa ( 30% ) .", "high - resolution computed tomography ( hrct ) of chest showed multiple ill - defined nodular opacities in both lung fields showing centrilobular distribution associated with ground glass opacities bilaterally .", "fine reticular densities were seen at places with segments of traction bronchiectasis [ figure 1 ] .", "total serum ige was 145 iu / ml and specific ige for pigeon droppings ( < 0.10", "tblb showed thickened alveolar septa with mild lymphoplasmacytic inflammatory infiltrate in the interstitium with cross - sections of calcified parasitic larvae with foreign body giant cell reaction around it [ figure 2 ] .", "travel history revealed frequent travels in the himalayan belts yearly for 20 years during which she had frequent episodes of diarrhea .", "she was given albendazole 400 mg for three days and oral corticosteroids for 12 weeks .", "bronchoalveolar lavage ( bal ) for tuberculosis culture at 6 weeks was negative . at 6 weeks", "high - resolution computed tomography chest showing multiple ill - defined centrilobular opacities with ground glass opacities bilaterally before treatment histopathology image of transbronchial lung biopsy showing calcified larva with thickened alveolar septa with mild lymphoplasmacytic inflammatory infiltrate in the interstitium high - resolution computed tomography chest showing remarkable improvement after treatment", "diffuse lung diseases can be further divided into transient pulmonary infiltrates and alveolar or interstitial lung diseases .", "ascariasis , anchylostomiasis , and toxocariasis usually cause transient pulmonary infiltrates , whereas schistosomiasis , strongyloidiasis , and tropical pulmonary eosinophilia can cause diffuse interstitial changes , as in the current case .", "strongyloidiasis causes reticulonodular opacities because of secondary infection , hemorrhage , inflammatory pneumonitis , and bacterial abscess formation .", "schistosomiasis eggs that are not passed into bladder or intestinal lumen are the main cause of chronic lung diseases causing granulomatous reaction and fibrosis .", "tropical pulmonary eosinophilia typically results from a hypersensitivity reaction to wuchereria bancrofti and brugia malayi .", "tropical pulmonary eosinophilia is an immunological response to microfilariae rather than acute infection , which usually present as reticulonodular opacities .", "the diagnosis of parasitic infections is difficult on chest radiography or hrct of chest because of nonspecific presentations .", "bronchoscopy may be helpful for diagnosis of parasitic infestation by variety of ways like direct visualization , bal , brushing , and tblb .", "parasitic lung diseases can also show microscopic pulmonary calcification on lung biopsy . in the current report ,", "transbronchial lung biopsy revealed showed thickened alveolar septa with mild lymphoplasmacytic inflammatory infiltrate in the interstitium with cross - sections of calcified parasitic larvae .", "tblb is performed for obtaining tissue specimen from peripheral lung masses and focal or diffuse lung infiltrates .", "the technique is useful in patients with suspected lung cancer , fungal and mycobacterial lung infections , unexplained infiltrates in immunocompromised hosts and in patients with suspected pulmonary sarcoidosis , lymphangitic carcinomatosis , and in selected cases of pulmonary langerhan 's cell histiocytosis , lymphangioleiomyomatosis , and cryptogenic organizing pneumonia .", "with increasing travels and worldwide migration , parasitic infections should be considered in differential diagnosis of interstitial lung diseases particularly in the evaluation of diffuse lung infiltrates .", "bronchoscopy and transbronchial lung biopsy can be useful in the diagnosis of diffuse lung infiltrates ." ]

[ "<S> parasite infections are increasing worldwide due to increasing migration and traveling . </S>", "<S> parasitic infections can affect lungs and present as a focal or diffuse lung diseases . </S>", "<S> high index of suspicion and detailed history are most important . </S>", "<S> we present a case of interstitial pneumonitis caused by parasite infestation , which was diagnosed on transbronchial lung biopsy . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest" ]

[ [ "parasitic infections are increasing worldwide due to rapid urbanization of cities , global warming , and international traveling .", "the diagnosis of parasitic diseases of the respiratory system is challenging because clinical manifestation and radiological findings are not specific .", "the larvae can cause airway inflammation , whereas migration of the mature adult worm may cause mechanical obstruction .", "we present a case of interstitial pneumonitis caused by parasitic infestation , which was diagnosed on transbronchial lung biopsy ( tblb ) ." ], [ "a 54-year - old female presented with a history of exertional breathlessness and dry cough for two months .", "she was afebrile with spo2 of 91% on room air and bilateral crepitations on respiratory examination .", "l ( 46.4% ) , fvc of 1.45 l ( 45.2% ) , and fev1/fvc 83% with reduction in dlco 1.46 mmol / min / kpa ( 18% ) and corrected value with alveolar volume 0.49 mmol / min / kpa ( 30% ) .", "high - resolution computed tomography ( hrct ) of chest showed multiple ill - defined nodular opacities in both lung fields showing centrilobular distribution associated with ground glass opacities bilaterally .", "fine reticular densities were seen at places with segments of traction bronchiectasis [ figure 1 ] .", "total serum ige was 145 iu / ml and specific ige for pigeon droppings ( < 0.10", "tblb showed thickened alveolar septa with mild lymphoplasmacytic inflammatory infiltrate in the interstitium with cross - sections of calcified parasitic larvae with foreign body giant cell reaction around it [ figure 2 ] .", "travel history revealed frequent travels in the himalayan belts yearly for 20 years during which she had frequent episodes of diarrhea .", "she was given albendazole 400 mg for three days and oral corticosteroids for 12 weeks .", "bronchoalveolar lavage ( bal ) for tuberculosis culture at 6 weeks was negative . at 6 weeks", "high - resolution computed tomography chest showing multiple ill - defined centrilobular opacities with ground glass opacities bilaterally before treatment histopathology image of transbronchial lung biopsy showing calcified larva with thickened alveolar septa with mild lymphoplasmacytic inflammatory infiltrate in the interstitium high - resolution computed tomography chest showing remarkable improvement after treatment" ], [ "diffuse lung diseases can be further divided into transient pulmonary infiltrates and alveolar or interstitial lung diseases .", "ascariasis , anchylostomiasis , and toxocariasis usually cause transient pulmonary infiltrates , whereas schistosomiasis , strongyloidiasis , and tropical pulmonary eosinophilia can cause diffuse interstitial changes , as in the current case .", "strongyloidiasis causes reticulonodular opacities because of secondary infection , hemorrhage , inflammatory pneumonitis , and bacterial abscess formation .", "schistosomiasis eggs that are not passed into bladder or intestinal lumen are the main cause of chronic lung diseases causing granulomatous reaction and fibrosis .", "tropical pulmonary eosinophilia typically results from a hypersensitivity reaction to wuchereria bancrofti and brugia malayi .", "tropical pulmonary eosinophilia is an immunological response to microfilariae rather than acute infection , which usually present as reticulonodular opacities .", "the diagnosis of parasitic infections is difficult on chest radiography or hrct of chest because of nonspecific presentations .", "bronchoscopy may be helpful for diagnosis of parasitic infestation by variety of ways like direct visualization , bal , brushing , and tblb .", "parasitic lung diseases can also show microscopic pulmonary calcification on lung biopsy . in the current report ,", "transbronchial lung biopsy revealed showed thickened alveolar septa with mild lymphoplasmacytic inflammatory infiltrate in the interstitium with cross - sections of calcified parasitic larvae .", "tblb is performed for obtaining tissue specimen from peripheral lung masses and focal or diffuse lung infiltrates .", "the technique is useful in patients with suspected lung cancer , fungal and mycobacterial lung infections , unexplained infiltrates in immunocompromised hosts and in patients with suspected pulmonary sarcoidosis , lymphangitic carcinomatosis , and in selected cases of pulmonary langerhan 's cell histiocytosis , lymphangioleiomyomatosis , and cryptogenic organizing pneumonia ." ], [ "with increasing travels and worldwide migration , parasitic infections should be considered in differential diagnosis of interstitial lung diseases particularly in the evaluation of diffuse lung infiltrates .", "bronchoscopy and transbronchial lung biopsy can be useful in the diagnosis of diffuse lung infiltrates ." ], [ "" ], [ "" ] ]

[ "muscle strength and power have been considered strong exogenous stimuli that are able to improve sports performance and promote therapeutic effects in several pathological conditions characterized by muscle wasting , such as cancer , disuse , sepsis , and sarcopenia [ 1 , 2 ] . in this context ,", "resistance exercise ( re ) has demonstrated significant effects regarding neural , metabolic , and functional adaptations in skeletal muscle .", "for this reason , recent studies in humans and rodents have focused on the mechanisms behind such responses promoted by re , especially molecular and phenotypic characterization [ 69 ] .", "although human studies provide strong evidence for practical application ( i.e. , greater external validity ) , it is not always possible to control the variables that may influence a biological response . furthermore , collection of muscle tissue samples in order to provide a significant body of cellular evidence [ 10 , 11 ] may limit mechanistic human studies .", "therefore , experimental research has been used to provide primary results that may support subsequent studies in humans . since the study of klitgaard", ", literature has described some experimental models of re for rodents that have presented interesting results concerning skeletal muscle adaptations [ 1316 ] .", "these studies have demonstrated that rodents are able to perform long- and short - term re with different re protocols and that their hindlimb muscle adapts similar to human muscles in terms of size , fiber type profile , and functionality .", "furthermore , molecular characterization of re effects have provided some insight in terms of cell signaling pathways ( transcription and translation ) involved in the control of skeletal muscle growth ( hypertrophy , atrophy , and inflammation ) and functionality ( strength capacity ) . however , these experimental models present limitations that may compromise the extrapolation of data to humans .", "there are many factors that can limit experimental models of re in comparison to those performed in humans and , among these , we can cite as the main one , the stimulus that is needed to encourage the animal to execute the exercise .", "indeed , environmental stress is required , but many models use fasting and electric shock as reward and punishment stimulus , respectively , which is beyond the physiological context observed in humans , and could be considered a limiting factor in experimental models characterized by appetite suppression and energy restriction .", "free in a given area and remain tied in the training apparatus during exercise session . regarding the exercise physiology context", ", these experimental models do not provide appropriate control of re variables : volume ( series and repetitions ) , intensity , and rest interval between repetitions and sets .", "this lead us to question whether the stimuli is quantitatively enough or not for muscle tissue in terms of volume and overload . ", "thus , the aim of the present report is to propose an experimental model of re for rodents that could mimic ( considering the limitation of the species ) the training carried out in humans .", "we developed an re model that does not use fasting nor electric shock as stimuli for exercise execution , allows operant conditioning so that the animal does not stay tied to the training apparatus , and promotes the control of re variables to perform specific muscle function tests . with this model", "it is possible to minimize environmental stress , to approach the animal in a physiological context , to study skeletal muscle adaptations in response to re in several experimental conditions , and to compare the responsiveness of rodents ' and humans ' muscles to different re protocols .", "the experiments were conducted in accordance with the national research council 's guidelines for the care and use of laboratory animals .", "six wistar male rats ( 400450 g ) were housed under controlled environmental conditions ( temperature , 22c ; 12-h dark period starting at 18 : 00 h ) .", "they were given free access to commercial laboratory chow and water before the experiments were performed . during periods of energy restriction ,", "the whole equipment consists of an re apparatus and an interface of control ( hardware ) on the rear panel of the equipment and was based on squat - type studies of klitgaard and wirth et al . .", "the re apparatus has its external structure built in acrylic , preferably dark , which holds all devices of the equipment and keeps the main tool of the experiment : the weight lifting cylinder .", "thus , its composition can be divided to the acrylic structure , rear panel , drawer , copper bars , cylinder lid , weight lifting cylinder , sensors , and actuators . \n", "acrylic structure ( figure 1 , item 1 ) : the acrylic structure is bicompartmentalized , that is , it has two divisions : the lower box , to house devices of communication , sensors , actuators , and electronic boards , and the higher box , to place the animal and the weight lifting cylinder .", "each compartment has distinct lids which have a slot for passage of sensor wires . \n", "rear panel ( figure 1 , item 2 ) : in the rear panel the connectors responsible for power supply and the hardware are allocated . \n", "drawer ( figure 1 , item 8 ; figure 2 , item 9 ) : it is an aluminum tray that lines the bottom of the higher box during the experiments , facilitating the removal of animal wasting .", "it can be washed with water and is usually covered with paper for easy cleaning . \n", "copper bars ( figure 1 , item 7 ; figure 2 , item 8) : responsible for supporting the weight lifting cylinder and can be exchanged for another metal material . \n", "cylinder lid : this device is attached on the top of the weight lifting cylinder to provide better fixation of the cylinder to the acrylic structure , avoiding fluctuations during the experiment due to the rise and fall of weights . the weight lifting cylinder is located inside the re apparatus and is composed by the following . \n acrylic cylinder ( figure 2 , item 1 ) : it is a holder that allows the animal to lift the weight as squat - type exercise . in this cylinder are coupled : weight plates , lifting ring , lifting bar , and the measurement axis . \n", "lifting ring ( figure 2 , item 6 ) : it is an acrylic piece which is located inside the acrylic cylinder , with one side attached to the measurement axis and the other to a vertical rail found in the body of the acrylic cylinder .", "the height of this device can be adjusted according to the size and species of the animal . \n", "lifting bar ( figure 2 , item 10 ) : it is a steel piece responsible for supporting weight plates for the lifting movement .", "weight plates must be placed at the predetermined distances relative to the rail found in the acrylic cylinder . \n", "weight plates : 50 g cylinders that should be placed in the lifting bar and fixed with the aid of elastics ( i.e. , orthodontic elastics ) .", "more than one weight plate can be placed in the lifting bar at a time , but they should stay together side by side .", "the point at which they are placed on the lifting bar must be considered as the midpoint of the set of weight plates on rail of the acrylic cylinder . \n", "axis measurement ( figure 2 , item 5 ) : it consists of a potentiometer and an acrylic base .", "this axis should enable the establishment of the lifting ring trough a cross - bolt to the measurement axis .", "it enables the measurement of angular displacement of the lifting bar through the angle sensor .", "support base ( figure 2 , item 7 ) : it is responsible for acrylic cylinder fixation to the copper bars .", "the support base consists of two symmetrical parts and must be put side by side below the acrylic cylinder .", "u shape on both sides of the support base allows its attachment to the copper bars .", "angle sensor : it is the sensor responsible for measuring the angle of the lifting bar .", "the angle sensor is attached to the weight lifting cylinder acting as the axis of the rotation of the lifting bar , allowing the software to calculate the angular displacement of the lifting bar when it is rotated . from this and other data", ", the software can also calculate the concentric and eccentric strength exerted by the animal as well as the length of each movement . \n", "nose poke ( figure 1 , item 5 ; figure 2 , item 12 ) : it is through this device that the system receives each nose poking signal of the animal .", "it has a circular hole in which the animal introduces the nose cutting an infrared light and triggering the device . at the bottom of the hole", "there is as light - emitting diode ( led ) that emits yellow light , in order to stimulate the animal when it is operating .", "the actuators , located inside the apparatus , are responsible for performing specific actions on the re apparatus .", "automatic feeder ( figure 1 , item 6 ; figure 2 , item 2 ) : it is installed inside the apparatus . a pellet at a time is released , requiring replacement of a new pellet into the feeder after the previous release .", "the release of pellets occurs through a door controlled by a motor servo which is controlled by the hardware in the rear panel of the equipment .", "light device ( figure 2 , item 11 ) : it is placed inside the apparatus in order to illuminate most of the distribution of acrylic structure through a lamp , preferably red and with low power . during the re session", "sound device ( figure 2 , item 4 ) : it is placed inside the apparatus and generates noise through a speaker that is also located inside the equipment and protected by a grid . during the exercise session ,", "the sound is activated as a conditioning tool ( encourage / disturb the animal ) . \n", "fan ( figure 2 , item 3 ) : it is installed into the upper compartment of the equipment and remains ventilating while the apparatus is on trial .", "it keeps the air circulation inside the equipment , produces a very low noise , and is not within the reach of the animal .", "the present equipment has all the sensors and devices described highly integrated into its structure and is highly resistant , supporting all actions and movements of the animal .", "moreover , it is not necessary to perform the assembly of each component individually prior to use and it is easy to operate and to clean .", "the phases of operant conditioning adopted in the present equipment are the same described by wirth et al . .", "however , we modified the logistics of such phases in order to optimize the exercise execution by the animal .", "as previously mentioned , we did not use fasting as a reward stimulus to execute the exercise , but for conditioning the animal .", "therefore , the environment where the experiment was conducted was extremely well - controlled : dark ( illuminated by a low - power red light ) , silent , and devoid of atypical odors .", "briefly , the phases of operant conditioning were separated by 48 h and carried out as follows . \n", "magazine : in this phase we only placed the animal inside the apparatus to adapt to the environment .", "after 12 h of energy restriction the animal remained inside the equipment for 1 hour . during this period , pellets of ~3040 mg were released ( a total of 30 pellets ) at regular intervals for the animal to identify the automatic feeder .", "nose - poke 1 : as shown in figure 2 , nose - poke is installed besides the automatic feeder .", "again , the animal was placed inside the equipment after 12 h of energy restriction .", "the automatic feeder was triggered each time the animal introduced its nose into the nose - poke .", "a total of 30 cycles was established for each experiment . based on our experience with the apparatus ,", "some food smell in the nose - poke was necessary to encourage the animal to introduce its nose inside it .", "nose - poke 2 : in the second phase of nose - poke , the sound device was integrated to the conditioning process .", "a constant noise was emitted and when the animal introduced its nose into the nose - poke , the automatic feeder was triggered and the noise stopped .", "the sound device was used to disturb the animal so that when the task ( poking ) was performed correctly , the animal was food - rewarded and the noise stopped disturbing .", "this phase introduced a new variable in order to replace the food reward as the only way to encourage the animal to perform the task proposed .", "a total of 30 interleaved cycles of pellets ( 15 with and without food ) release were performed .", "standing 1 : the nose - poke device was removed from the wall and placed inside the weight lifting cylinder , above the lifting ring , and a lid was placed in the nose - poke hole on the wall .", "this phase was conducted to encourage the animal to enter into the weight lifting cylinder , but without lifting the ring , and was performed twice . \n", "standing 2 : in the second phase of standing , food reward was completely removed and the animal had the constant sound as the only stimulus to perform the task .", "two sessions were required . \n lifting : in the last phase , the nose - poke device remained within the weight lifting cylinder , however , at an unreachable height to the animal . using the software ,", "a minimum lifting height was set depending on the species and size of the animal .", "we observed that a height of 4 cm was necessary for an animal of ~400 g to complete the movement ( full concentric contraction ) . during the lifting task , animals performed concentric muscle contractions being plantaris and soleus the main muscles involved in contraction .", "of note , the access of the lifting ring allowed the animal to pass only the head through the ring hole and its height was adjusted according to the animal 's characteristics .", "the sound was turned on and once the animal raised the lifting bar to the pre - determined height , the noise stopped .", "it was necessary to carry out one session without load and one session with light load .", "it is important to emphasize that the animal did not stay tied in the training apparatus .", "once conditioned , the animal underwent a muscle function test : maximal voluntary strength capacity ( mvsc ) as previously described by our group using the same apparatus and procedures described by klitgaard .", "the advantages of the present equipment are that the rest intervals between each repetition in the mvsc were optimized because of the sound stimulus , it was possible to calculate concentric and eccentric strengths of the animal during each muscular contraction and relativize the training intensity with better precision ( percent of mvsc , percent of body weight ) . additionally , the mvsc test can be performed during and at the end of the experimental protocol to assess the progression of muscle function . to configure the training protocol in the software it was necessary to enter the following data : training volume ( sets and repetitions ) , rest intervals between sets and repetitions , desired height of the lifting bar , and weight plate position in the lifting bar .", "the experiment could be interrupted at any time and all data were stored in a report file by the software .", "differences between nose - poke - phases and body weight values were tested by paired t - test . the level of significance was set at p < 0.05 .", "statistical analysis was performed using sas 8.2 ( sas institute inc . , cary , nc , usa ) .", "as previously described , to condition the animal to the experimental model it was necessary to carry out periods of energy restriction on the previous day of nose - poke 1 and 2 and standing 1 phases . however , as shown in figure 3 , energy restriction did not promote significant changes in body weight at the end of the conditioning process ( 414.1 4.8 g in pre versus 410.8 4.2 g in post period ; p > 0.05 ) . \n", "magazine phase is not described because it was carried out only to adapt the animal to the exercise environment . to complete the 30 cycles proposed in the nose - poke 1 phase", "interestingly , when the sound device was integrated to the conditioning ( nose - poke 2 phase ) , animals took 58.9% less time to complete the cycles ( 768.4 109.1 seconds ; p = 0.03 ) .", "it is important to emphasize that , although the animals were more conditioned to the poking task in nose - poke 2 phase , this factor did not contribute to reduce the time spent to complete the cycles .", "we repeated the nose - poke 1 phase ( without sound ) and observed reduction of time ( data not shown ) .", "standing phases time were not statistically different ( 1274 92.4 seconds in standing 1 versus 1234 113.3 seconds in standing 2 phases ; p > 0.05 ) .", "animals took 960.8 254.2 seconds to complete the lifting phases and lifted the bar to 4.1 0.1 cm . ", "height displacements during repetitions were quite similar to lifting phase and the result of the mvsc during the concentric phase of muscle contraction was 2.3 0.1 n ( figure 5 ) .", "we did not describe the eccentric strength performed because this experimental model is totally based on concentric muscle actions , since during the eccentric phase the animal does not counteract the external load .", "the rest interval between each repetition to perform a repetition ( contraction ) was 7.2 0.8 seconds ( figure 5 ) .", "thus , each time the sound stimulus was released , animals took approximately 7 seconds to perform the next repetition .", "the aim of the present report was to propose an experimental model of re for rodents with better conditioning ( no fasting and shocking ) and control of exercise variables ( volume , intensity and rest intervals ) .", "as previously described , animals were able to perform the conditioning phases within the logistics proposed .", "second , integrating the sound device into the conditioning process optimized the performance of the exercise learning .", "regarding exercise physiology , it was possible to optimize the encouragement of the animal to perform muscle contractions controlling repetitions and rest interval between repetitions .", "therefore , it was possible to control and optimize the variable of re rest interval . importantly ,", "although this experimental model uses energy restriction as the first stimulus to condition the animal to the exercise , it did not promote significant changes in body weight and , consequently , in muscle mass .", "this can be explained by the fact that the energy restriction was imposed only at the beginning and gradually abolished during the conditioning process .", "the applicability of such factor is quite important since models of skeletal muscle atrophy ( i.e. , glucocorticoids , fasting , cancer , and sepsis ) are usually characterized by reduction in food intake .", "furthermore , this experimental model demonstrates that it is not necessary to punish the animal ( shocking ) to encourage exercising . in this context , sound device proved to be a good disturbing ", "recently , we conducted an experiment to evaluate the effectiveness of this experimental model in counteracting dexamethasone - induced skeletal muscle atrophy , an experimental model characterized by severe muscle wasting and appetite suppression .", "even with loss of appetite , animals performed , during 7 days , 3 sessions of re composed by 3 sets of 10 repetitions separated by 48 hours .", "we demonstrated that , although re did not attenuate the atrophic response , it promoted significant phenotypic adaptations in plantaris muscles that in the long term may reflect structural remodeling .", "furthermore , re - trained animals presented higher concentric mvsc when compared with nontrained animals at the end of the experimental protocol .", "therefore , the experimental mode is effective in conditioning the animal to re and , according to the exercise protocol , to promote phenotypic , structural , and functional responses in skeletal muscle at least in dexamethasone - treated animals . ", "although the present equipment promotes good control of re variables , the main limitation concerns the strength data .", "concentric and eccentric measurements are calculated by estimative , that is , not directly measured by a strength platform as proposed by wirth et al . .", "furthermore , it proposes a more specific control of other re variables , especially rest interval between sets and repetitions .", "furthermore , it encourages the animal to exercise through short - term energy restriction and disturbing stimuli that do not promote alterations in body weight .", "therefore , we believe that this re apparatus is closer to the physiological context observed in humans . ", "future studies should evaluate the molecular characterization of distinct re protocols in order to understand the biological response of skeletal muscle and optimize the development of training methods .", "experimental models characterized by skeletal muscle wasting should also be investigated in order to propose re as a nonpharmacological tool for humans .", "additionally , this experimental model allows investigations regarding other stimulus associated with re , that is , nutritional , pharmacological , and so forth ." ]

[ "<S> this study aimed to develop an equipment and system of resistance exercise ( re ) , based on squat - type exercise for rodents , with control of training variables . </S>", "<S> we developed an operant conditioning system composed of sound , light and feeding devices that allowed optimized re performance by the animal . with this system , it is not necessary to impose fasting or electric shock for the animal to perform the task proposed ( muscle contraction ) . </S>", "<S> furthermore , it is possible to perform muscle function tests in vivo within the context of the exercise proposed and control variables such as intensity , volume ( sets and repetitions ) , and exercise session length , rest interval between sets and repetitions , and concentric strength . </S>", "<S> based on the experiments conducted , we demonstrated that the model proposed is able to perform more specific control of other re variables , especially rest interval between sets and repetitions , and encourages the animal to exercise through short - term energy restriction and disturbing stimulus that do not promote alterations in body weight . </S>", "<S> therefore , despite experimental limitations , we believe that this re apparatus is closer to the physiological context observed in humans . </S>" ]

[ "1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion" ]

[ [ "muscle strength and power have been considered strong exogenous stimuli that are able to improve sports performance and promote therapeutic effects in several pathological conditions characterized by muscle wasting , such as cancer , disuse , sepsis , and sarcopenia [ 1 , 2 ] . in this context ,", "resistance exercise ( re ) has demonstrated significant effects regarding neural , metabolic , and functional adaptations in skeletal muscle .", "for this reason , recent studies in humans and rodents have focused on the mechanisms behind such responses promoted by re , especially molecular and phenotypic characterization [ 69 ] .", "although human studies provide strong evidence for practical application ( i.e. , greater external validity ) , it is not always possible to control the variables that may influence a biological response . furthermore , collection of muscle tissue samples in order to provide a significant body of cellular evidence [ 10 , 11 ] may limit mechanistic human studies .", "therefore , experimental research has been used to provide primary results that may support subsequent studies in humans . since the study of klitgaard", ", literature has described some experimental models of re for rodents that have presented interesting results concerning skeletal muscle adaptations [ 1316 ] .", "these studies have demonstrated that rodents are able to perform long- and short - term re with different re protocols and that their hindlimb muscle adapts similar to human muscles in terms of size , fiber type profile , and functionality .", "furthermore , molecular characterization of re effects have provided some insight in terms of cell signaling pathways ( transcription and translation ) involved in the control of skeletal muscle growth ( hypertrophy , atrophy , and inflammation ) and functionality ( strength capacity ) . however , these experimental models present limitations that may compromise the extrapolation of data to humans .", "there are many factors that can limit experimental models of re in comparison to those performed in humans and , among these , we can cite as the main one , the stimulus that is needed to encourage the animal to execute the exercise .", "indeed , environmental stress is required , but many models use fasting and electric shock as reward and punishment stimulus , respectively , which is beyond the physiological context observed in humans , and could be considered a limiting factor in experimental models characterized by appetite suppression and energy restriction .", "free in a given area and remain tied in the training apparatus during exercise session . regarding the exercise physiology context", ", these experimental models do not provide appropriate control of re variables : volume ( series and repetitions ) , intensity , and rest interval between repetitions and sets .", "this lead us to question whether the stimuli is quantitatively enough or not for muscle tissue in terms of volume and overload . ", "thus , the aim of the present report is to propose an experimental model of re for rodents that could mimic ( considering the limitation of the species ) the training carried out in humans .", "we developed an re model that does not use fasting nor electric shock as stimuli for exercise execution , allows operant conditioning so that the animal does not stay tied to the training apparatus , and promotes the control of re variables to perform specific muscle function tests . with this model", "it is possible to minimize environmental stress , to approach the animal in a physiological context , to study skeletal muscle adaptations in response to re in several experimental conditions , and to compare the responsiveness of rodents ' and humans ' muscles to different re protocols ." ], [ "the experiments were conducted in accordance with the national research council 's guidelines for the care and use of laboratory animals .", "six wistar male rats ( 400450 g ) were housed under controlled environmental conditions ( temperature , 22c ; 12-h dark period starting at 18 : 00 h ) .", "they were given free access to commercial laboratory chow and water before the experiments were performed . during periods of energy restriction ,", "the whole equipment consists of an re apparatus and an interface of control ( hardware ) on the rear panel of the equipment and was based on squat - type studies of klitgaard and wirth et al . .", "the re apparatus has its external structure built in acrylic , preferably dark , which holds all devices of the equipment and keeps the main tool of the experiment : the weight lifting cylinder .", "thus , its composition can be divided to the acrylic structure , rear panel , drawer , copper bars , cylinder lid , weight lifting cylinder , sensors , and actuators . \n", "acrylic structure ( figure 1 , item 1 ) : the acrylic structure is bicompartmentalized , that is , it has two divisions : the lower box , to house devices of communication , sensors , actuators , and electronic boards , and the higher box , to place the animal and the weight lifting cylinder .", "each compartment has distinct lids which have a slot for passage of sensor wires . \n", "rear panel ( figure 1 , item 2 ) : in the rear panel the connectors responsible for power supply and the hardware are allocated . \n", "drawer ( figure 1 , item 8 ; figure 2 , item 9 ) : it is an aluminum tray that lines the bottom of the higher box during the experiments , facilitating the removal of animal wasting .", "it can be washed with water and is usually covered with paper for easy cleaning . \n", "copper bars ( figure 1 , item 7 ; figure 2 , item 8) : responsible for supporting the weight lifting cylinder and can be exchanged for another metal material . \n", "cylinder lid : this device is attached on the top of the weight lifting cylinder to provide better fixation of the cylinder to the acrylic structure , avoiding fluctuations during the experiment due to the rise and fall of weights . the weight lifting cylinder is located inside the re apparatus and is composed by the following . \n acrylic cylinder ( figure 2 , item 1 ) : it is a holder that allows the animal to lift the weight as squat - type exercise . in this cylinder are coupled : weight plates , lifting ring , lifting bar , and the measurement axis . \n", "lifting ring ( figure 2 , item 6 ) : it is an acrylic piece which is located inside the acrylic cylinder , with one side attached to the measurement axis and the other to a vertical rail found in the body of the acrylic cylinder .", "the height of this device can be adjusted according to the size and species of the animal . \n", "lifting bar ( figure 2 , item 10 ) : it is a steel piece responsible for supporting weight plates for the lifting movement .", "weight plates must be placed at the predetermined distances relative to the rail found in the acrylic cylinder . \n", "weight plates : 50 g cylinders that should be placed in the lifting bar and fixed with the aid of elastics ( i.e. , orthodontic elastics ) .", "more than one weight plate can be placed in the lifting bar at a time , but they should stay together side by side .", "the point at which they are placed on the lifting bar must be considered as the midpoint of the set of weight plates on rail of the acrylic cylinder . \n", "axis measurement ( figure 2 , item 5 ) : it consists of a potentiometer and an acrylic base .", "this axis should enable the establishment of the lifting ring trough a cross - bolt to the measurement axis .", "it enables the measurement of angular displacement of the lifting bar through the angle sensor .", "support base ( figure 2 , item 7 ) : it is responsible for acrylic cylinder fixation to the copper bars .", "the support base consists of two symmetrical parts and must be put side by side below the acrylic cylinder .", "u shape on both sides of the support base allows its attachment to the copper bars .", "angle sensor : it is the sensor responsible for measuring the angle of the lifting bar .", "the angle sensor is attached to the weight lifting cylinder acting as the axis of the rotation of the lifting bar , allowing the software to calculate the angular displacement of the lifting bar when it is rotated . from this and other data", ", the software can also calculate the concentric and eccentric strength exerted by the animal as well as the length of each movement . \n", "nose poke ( figure 1 , item 5 ; figure 2 , item 12 ) : it is through this device that the system receives each nose poking signal of the animal .", "it has a circular hole in which the animal introduces the nose cutting an infrared light and triggering the device . at the bottom of the hole", "there is as light - emitting diode ( led ) that emits yellow light , in order to stimulate the animal when it is operating .", "the actuators , located inside the apparatus , are responsible for performing specific actions on the re apparatus .", "automatic feeder ( figure 1 , item 6 ; figure 2 , item 2 ) : it is installed inside the apparatus . a pellet at a time is released , requiring replacement of a new pellet into the feeder after the previous release .", "the release of pellets occurs through a door controlled by a motor servo which is controlled by the hardware in the rear panel of the equipment .", "light device ( figure 2 , item 11 ) : it is placed inside the apparatus in order to illuminate most of the distribution of acrylic structure through a lamp , preferably red and with low power . during the re session", "sound device ( figure 2 , item 4 ) : it is placed inside the apparatus and generates noise through a speaker that is also located inside the equipment and protected by a grid . during the exercise session ,", "the sound is activated as a conditioning tool ( encourage / disturb the animal ) . \n", "fan ( figure 2 , item 3 ) : it is installed into the upper compartment of the equipment and remains ventilating while the apparatus is on trial .", "it keeps the air circulation inside the equipment , produces a very low noise , and is not within the reach of the animal .", "the present equipment has all the sensors and devices described highly integrated into its structure and is highly resistant , supporting all actions and movements of the animal .", "moreover , it is not necessary to perform the assembly of each component individually prior to use and it is easy to operate and to clean .", "the phases of operant conditioning adopted in the present equipment are the same described by wirth et al . .", "however , we modified the logistics of such phases in order to optimize the exercise execution by the animal .", "as previously mentioned , we did not use fasting as a reward stimulus to execute the exercise , but for conditioning the animal .", "therefore , the environment where the experiment was conducted was extremely well - controlled : dark ( illuminated by a low - power red light ) , silent , and devoid of atypical odors .", "briefly , the phases of operant conditioning were separated by 48 h and carried out as follows . \n", "magazine : in this phase we only placed the animal inside the apparatus to adapt to the environment .", "after 12 h of energy restriction the animal remained inside the equipment for 1 hour . during this period , pellets of ~3040 mg were released ( a total of 30 pellets ) at regular intervals for the animal to identify the automatic feeder .", "nose - poke 1 : as shown in figure 2 , nose - poke is installed besides the automatic feeder .", "again , the animal was placed inside the equipment after 12 h of energy restriction .", "the automatic feeder was triggered each time the animal introduced its nose into the nose - poke .", "a total of 30 cycles was established for each experiment . based on our experience with the apparatus ,", "some food smell in the nose - poke was necessary to encourage the animal to introduce its nose inside it .", "nose - poke 2 : in the second phase of nose - poke , the sound device was integrated to the conditioning process .", "a constant noise was emitted and when the animal introduced its nose into the nose - poke , the automatic feeder was triggered and the noise stopped .", "the sound device was used to disturb the animal so that when the task ( poking ) was performed correctly , the animal was food - rewarded and the noise stopped disturbing .", "this phase introduced a new variable in order to replace the food reward as the only way to encourage the animal to perform the task proposed .", "a total of 30 interleaved cycles of pellets ( 15 with and without food ) release were performed .", "standing 1 : the nose - poke device was removed from the wall and placed inside the weight lifting cylinder , above the lifting ring , and a lid was placed in the nose - poke hole on the wall .", "this phase was conducted to encourage the animal to enter into the weight lifting cylinder , but without lifting the ring , and was performed twice . \n", "standing 2 : in the second phase of standing , food reward was completely removed and the animal had the constant sound as the only stimulus to perform the task .", "two sessions were required . \n lifting : in the last phase , the nose - poke device remained within the weight lifting cylinder , however , at an unreachable height to the animal . using the software ,", "a minimum lifting height was set depending on the species and size of the animal .", "we observed that a height of 4 cm was necessary for an animal of ~400 g to complete the movement ( full concentric contraction ) . during the lifting task , animals performed concentric muscle contractions being plantaris and soleus the main muscles involved in contraction .", "of note , the access of the lifting ring allowed the animal to pass only the head through the ring hole and its height was adjusted according to the animal 's characteristics .", "the sound was turned on and once the animal raised the lifting bar to the pre - determined height , the noise stopped .", "it was necessary to carry out one session without load and one session with light load .", "it is important to emphasize that the animal did not stay tied in the training apparatus .", "once conditioned , the animal underwent a muscle function test : maximal voluntary strength capacity ( mvsc ) as previously described by our group using the same apparatus and procedures described by klitgaard .", "the advantages of the present equipment are that the rest intervals between each repetition in the mvsc were optimized because of the sound stimulus , it was possible to calculate concentric and eccentric strengths of the animal during each muscular contraction and relativize the training intensity with better precision ( percent of mvsc , percent of body weight ) . additionally , the mvsc test can be performed during and at the end of the experimental protocol to assess the progression of muscle function . to configure the training protocol in the software it was necessary to enter the following data : training volume ( sets and repetitions ) , rest intervals between sets and repetitions , desired height of the lifting bar , and weight plate position in the lifting bar .", "the experiment could be interrupted at any time and all data were stored in a report file by the software .", "differences between nose - poke - phases and body weight values were tested by paired t - test . the level of significance was set at p < 0.05 .", "statistical analysis was performed using sas 8.2 ( sas institute inc . , cary , nc , usa ) ." ], [ "as previously described , to condition the animal to the experimental model it was necessary to carry out periods of energy restriction on the previous day of nose - poke 1 and 2 and standing 1 phases . however , as shown in figure 3 , energy restriction did not promote significant changes in body weight at the end of the conditioning process ( 414.1 4.8 g in pre versus 410.8 4.2 g in post period ; p > 0.05 ) . \n", "magazine phase is not described because it was carried out only to adapt the animal to the exercise environment . to complete the 30 cycles proposed in the nose - poke 1 phase", "interestingly , when the sound device was integrated to the conditioning ( nose - poke 2 phase ) , animals took 58.9% less time to complete the cycles ( 768.4 109.1 seconds ; p = 0.03 ) .", "it is important to emphasize that , although the animals were more conditioned to the poking task in nose - poke 2 phase , this factor did not contribute to reduce the time spent to complete the cycles .", "we repeated the nose - poke 1 phase ( without sound ) and observed reduction of time ( data not shown ) .", "standing phases time were not statistically different ( 1274 92.4 seconds in standing 1 versus 1234 113.3 seconds in standing 2 phases ; p > 0.05 ) .", "animals took 960.8 254.2 seconds to complete the lifting phases and lifted the bar to 4.1 0.1 cm . ", "height displacements during repetitions were quite similar to lifting phase and the result of the mvsc during the concentric phase of muscle contraction was 2.3 0.1 n ( figure 5 ) .", "we did not describe the eccentric strength performed because this experimental model is totally based on concentric muscle actions , since during the eccentric phase the animal does not counteract the external load .", "the rest interval between each repetition to perform a repetition ( contraction ) was 7.2 0.8 seconds ( figure 5 ) .", "thus , each time the sound stimulus was released , animals took approximately 7 seconds to perform the next repetition ." ], [ "the aim of the present report was to propose an experimental model of re for rodents with better conditioning ( no fasting and shocking ) and control of exercise variables ( volume , intensity and rest intervals ) .", "as previously described , animals were able to perform the conditioning phases within the logistics proposed .", "second , integrating the sound device into the conditioning process optimized the performance of the exercise learning .", "regarding exercise physiology , it was possible to optimize the encouragement of the animal to perform muscle contractions controlling repetitions and rest interval between repetitions .", "therefore , it was possible to control and optimize the variable of re rest interval . importantly ,", "although this experimental model uses energy restriction as the first stimulus to condition the animal to the exercise , it did not promote significant changes in body weight and , consequently , in muscle mass .", "this can be explained by the fact that the energy restriction was imposed only at the beginning and gradually abolished during the conditioning process .", "the applicability of such factor is quite important since models of skeletal muscle atrophy ( i.e. , glucocorticoids , fasting , cancer , and sepsis ) are usually characterized by reduction in food intake .", "furthermore , this experimental model demonstrates that it is not necessary to punish the animal ( shocking ) to encourage exercising . in this context , sound device proved to be a good disturbing ", "recently , we conducted an experiment to evaluate the effectiveness of this experimental model in counteracting dexamethasone - induced skeletal muscle atrophy , an experimental model characterized by severe muscle wasting and appetite suppression .", "even with loss of appetite , animals performed , during 7 days , 3 sessions of re composed by 3 sets of 10 repetitions separated by 48 hours .", "we demonstrated that , although re did not attenuate the atrophic response , it promoted significant phenotypic adaptations in plantaris muscles that in the long term may reflect structural remodeling .", "furthermore , re - trained animals presented higher concentric mvsc when compared with nontrained animals at the end of the experimental protocol .", "therefore , the experimental mode is effective in conditioning the animal to re and , according to the exercise protocol , to promote phenotypic , structural , and functional responses in skeletal muscle at least in dexamethasone - treated animals . ", "although the present equipment promotes good control of re variables , the main limitation concerns the strength data .", "concentric and eccentric measurements are calculated by estimative , that is , not directly measured by a strength platform as proposed by wirth et al . .", "furthermore , it proposes a more specific control of other re variables , especially rest interval between sets and repetitions .", "furthermore , it encourages the animal to exercise through short - term energy restriction and disturbing stimuli that do not promote alterations in body weight .", "therefore , we believe that this re apparatus is closer to the physiological context observed in humans . ", "future studies should evaluate the molecular characterization of distinct re protocols in order to understand the biological response of skeletal muscle and optimize the development of training methods .", "experimental models characterized by skeletal muscle wasting should also be investigated in order to propose re as a nonpharmacological tool for humans .", "additionally , this experimental model allows investigations regarding other stimulus associated with re , that is , nutritional , pharmacological , and so forth ." ] ]

[ "this study was approved by king fahad medical city ( kfmc ) institutional review board ( irb ) in riyadh , kingdom of saudi arabia .", "all patients had a baseline brain mri , video - eeg recording , and a pre - surgical neuropsychological assessment composed of addenbrooke s cognitive examination - revised ( ace - r),5 handedness questionnaire,6 stanford - binet intelligence scale,7 hospital anxiety and depression scale ( hads),8 and daily memory questionnaire ( currently being tested in another study ) .", "these questionnaires were in arabic , given the language barrier , and were tested before being part of the standard neuropsychological evaluation of all patients admitted to the epilepsy monitoring unit ( emu ) in kfmc , especially if they were candidates for epilepsy surgery .", "potentially included patients are those who were diagnosed with tle and were candidates for temporal lobectomy / lobotomy .", "age range of the selected population for the procedure was between 18 and 65 years .", "patients were excluded if they had history of coagulopathy , carotid dissection , allergy to the contrast or anesthetic agent , elevated serum creatinine , were not fit for surgery ( for example , cardiopulmonary risk , major medical problems ) , or were unable to communicate efficiently during the procedure ( for example , deafness ) .", "patients were also excluded from the procedure if their ace - r score were less than 67 or if they were mentally retarded as per stanford - binet intelligence scale .", "patient selection was according to the helsinki declaration.9 all patients consented and received a detailed explanation on the nature , risks , and benefits of the procedure .", "since sa was not available , pf was chosen as an alternative , based on efficacy , safety profile , feasibility , potential side - effects , and several studies in the literature pertaining to the usage of pf in wada.10 - 17 the manufacturer of pf is astrazeneca , london , england , united kingdom .", "the procedure was carried out by a team that included an interventional neurologist , epileptologist , and a neuropsychologist . in each patient ,", "10 mg bolus of pf was injected into the internal carotid artery ( ica ) via a catheter inserted through the right femoral artery , except for one patient who received 15 mg bilaterally . neither continuous infusion nor maintenance doses were given .", "we began by injecting the right ica first , evaluated the left hemisphere , and then injected the left ica with a 30-minute interval between .", "prior to pf injection , the patient was asked to raise the contralateral arm to observe for weakness clinically , and count from one to 10 in numerical order to observe for speech arrest .", "in addition , patients were under continuous eeg monitoring to observe for any electrographic changes .", "the eeg demonstrated high amplitude theta and delta slowing of the ipsilateral hemisphere 5 - 18 seconds after the injection of propofol ( figure 1 ) .", "we assessed both hemispheres in the same session with 30-minutes interval between , during which baseline motor , speech , and electrographic activity were restored ( figure 2 ) .", "electroencephalogram paper speed 30 seconds per epoch , high amplitude theta and delta slowing starts on right hemispheric channels , seen 5 - 18 seconds after injection of propofol .", "electroencephalogram ( eeg ) paper speed 30 seconds per epoch , eeg starts to normalize , see reduction in amplitude of the slowing .", "the main elements of memory assessed were both recall ( verbal and non - verbal ) and recognition .", "the patient was shown 5 familiar objects and was asked if he / she recognizes them or not . during the test , the patient was shown 5 objects and 3 words and was asked to memorize them .", "the patient was asked to recall these objects and words as soon as the effect of pf wore - off .", "this study was approved by king fahad medical city ( kfmc ) institutional review board ( irb ) in riyadh , kingdom of saudi arabia .", "all patients had a baseline brain mri , video - eeg recording , and a pre - surgical neuropsychological assessment composed of addenbrooke s cognitive examination - revised ( ace - r),5 handedness questionnaire,6 stanford - binet intelligence scale,7 hospital anxiety and depression scale ( hads),8 and daily memory questionnaire ( currently being tested in another study ) .", "these questionnaires were in arabic , given the language barrier , and were tested before being part of the standard neuropsychological evaluation of all patients admitted to the epilepsy monitoring unit ( emu ) in kfmc , especially if they were candidates for epilepsy surgery .", "potentially included patients are those who were diagnosed with tle and were candidates for temporal lobectomy / lobotomy .", "age range of the selected population for the procedure was between 18 and 65 years .", "patients were excluded if they had history of coagulopathy , carotid dissection , allergy to the contrast or anesthetic agent , elevated serum creatinine , were not fit for surgery ( for example , cardiopulmonary risk , major medical problems ) , or were unable to communicate efficiently during the procedure ( for example , deafness ) .", "patients were also excluded from the procedure if their ace - r score were less than 67 or if they were mentally retarded as per stanford - binet intelligence scale .", "patient selection was according to the helsinki declaration.9 all patients consented and received a detailed explanation on the nature , risks , and benefits of the procedure .", "since sa was not available , pf was chosen as an alternative , based on efficacy , safety profile , feasibility , potential side - effects , and several studies in the literature pertaining to the usage of pf in wada.10 - 17 the manufacturer of pf is astrazeneca , london , england , united kingdom .", "the procedure was carried out by a team that included an interventional neurologist , epileptologist , and a neuropsychologist . in each patient ,", "10 mg bolus of pf was injected into the internal carotid artery ( ica ) via a catheter inserted through the right femoral artery , except for one patient who received 15 mg bilaterally . neither continuous infusion nor maintenance doses were given .", "we began by injecting the right ica first , evaluated the left hemisphere , and then injected the left ica with a 30-minute interval between .", "prior to pf injection , the patient was asked to raise the contralateral arm to observe for weakness clinically , and count from one to 10 in numerical order to observe for speech arrest .", "in addition , patients were under continuous eeg monitoring to observe for any electrographic changes .", "the eeg demonstrated high amplitude theta and delta slowing of the ipsilateral hemisphere 5 - 18 seconds after the injection of propofol ( figure 1 ) .", "we assessed both hemispheres in the same session with 30-minutes interval between , during which baseline motor , speech , and electrographic activity were restored ( figure 2 ) .", "electroencephalogram paper speed 30 seconds per epoch , high amplitude theta and delta slowing starts on right hemispheric channels , seen 5 - 18 seconds after injection of propofol .", "electroencephalogram ( eeg ) paper speed 30 seconds per epoch , eeg starts to normalize , see reduction in amplitude of the slowing .", "the main elements of memory assessed were both recall ( verbal and non - verbal ) and recognition .", "the patient was shown 5 familiar objects and was asked if he / she recognizes them or not . during the test , the patient was shown 5 objects and 3 words and was asked to memorize them .", "the patient was asked to recall these objects and words as soon as the effect of pf wore - off .", "once pf was injected , we noticed weakness of the contralateral side and aphasia if the involved hemisphere were the dominant one in which the patient stopped counting , could not obey commands , communicate with the neuropsychology team , recognize or memorize objects and words .", "both motor and electrographic changes reverted back to baseline within 10 - 12 minutes . out of 9 patients , with mean ( + sd ) age 26 ( + 5.8 ) years", "six of the right - handed patients had left tle , and 2 had right tle .", "all patients had mri findings : 3 patients were found to have left mesial temporal sclerosis ( mts ) , 2 had right mts , and 4 had altered hippocampal morphology .", "only one patient was documented to have abnormal cognitive functions ( addenbrooke s cognitive examination - revised 77/100 ) ( table 1 ) .", "we were able to lateralize speech in 8 patients , and memory in 6 patients .", "all right - handed patients had a left - sided representation of language . however , 2 out of 6 right - handed patients had bilateral representation of memory , while the rest had left - sided representation of memory .", "from the 3 patients who had right tle , 2 had left - sided representation of language while one had bilateral representation ( table 1 ) .", "in addition , 2 subjects experienced shivering during the procedure , and one subject complained of eye pain and facial pain ipsilateral to the site of the ica injection . the latter developed transient segmental spasm of the ica .", "this same patient required an additional 10 mg pf due to lack of effect with the first dose .", "the average blood pressure measured for all patients immediately after the procedure was 116/66 mm hg .", "all subjects were able to complete the procedure , testing both hemispheres in the same session . aside from the mentioned adverse events ,", "in addition , 2 subjects experienced shivering during the procedure , and one subject complained of eye pain and facial pain ipsilateral to the site of the ica injection . the latter developed transient segmental spasm of the ica .", "this same patient required an additional 10 mg pf due to lack of effect with the first dose .", "the average blood pressure measured for all patients immediately after the procedure was 116/66 mm hg .", "all subjects were able to complete the procedure , testing both hemispheres in the same session . aside from the mentioned adverse events ,", "propofol , an alkylphenol , is a lipid soluble anesthetic agent with rapid onset of action ( 30 seconds ) , and rapid rate of distribution ( 2 - 4 minutes).18,19 it is metabolized in the liver and excreted mainly through urine.18,19 propofol was first tried , by bazin et al20 in 1998 , and by silva et al21 in 2000 .", "both tried it on a single patient and recorded successful results . in terms of side - effects , the former reported perception of intense blue light , while the latter reported a hot sensation in the head .", "thereafter , several studies assessed the efficacy and adverse events of pf . with respect to adverse events , we encountered : shivering , facial pain , and eye pain .", "mikuni et al12 performed a case - series of 58 patients in which they studied the side - effects of pf in wada test .", "they classified the side - effects into 3 categories : grade 1 symptoms included pain , shivering , face contortion , lacrimation , laughing , and apathy .", "grade 3 symptoms included seizure - like activity described as increased muscle tone with twitching and rhythmic movements or tonic posturing .", "grade 1 and 2 symptoms were also seen with sa.12 some of these adverse events were also reported by takayama et al,13 mikati et al,11 and magee et al.22 it has been suggested that post - anesthetic shivering associated with pf , although not fully understood , is due to a thermoregulatory phenomenon associated with hypothermia.23,24 as for facial pain and eye pain , it has been proposed that they occur due to peripheral anastomosis and cross - filling between the internal carotid and external carotid arteries.12 a study10 showed that selective middle cerebral artery ( mca ) injection of pf was associated with a lower incidence of such side - effects ; thus , supporting the anastomosis hypothesis .", "although we did not encounter any of the grade 3 symptoms in our study , it has been reported that the frequency of grade 3 symptoms increased in patients older than 55 years , after a second injection of pf greater than 10 mg , and/or a total dose of pf greater than 20 mg.12 one of our subjects failed to elicit a response and developed transient segmental ica spasm .", "thereafter , we bypassed the segment of spasm and gave an additional 10 mg of pf , which was successful without any side - effects .", "thus , it is probable that vasospasm was secondary to the guide - wire or the catheter ( namely , iatrogenic ) .", "mechanically induced vasospasm has been reported in the literature as a complication of angiography , and is mostly self - limited.25,26 interestingly , a patient developed transient euphoria characterized by talkativeness , curiosity , and was asking many questions .", "however , there was an element of confusion to this picture and he was speaking incoherently rather than combativeness as reported in previous studies.11 - 13,22 it has been reported that iv pf might be associated with euphoria , and it might have an addictive effect.27,28 a possible explanation might be an increase in dopamine concentration in the nucleus accumbens , as seen in drug abusers , promoting drug seeking behavior.18,19 in terms of efficacy , it was not possible to compare pf to sa since it was not available", ". however , we were able to lateralize speech dominance in 8 patients , and memory dominance in 6 patients ( table 1 ) .", "we also found that the recovery time ( both clinically and electrophysiologically ) ranged between 10 - 12 minutes .", "takayama et al13 were able to lateralize language and memory in 12 and 9 out of 12 patients , as opposed to 52 and 41 out of 55 patients with sa .", "they also noticed that the recovery time of muscle power , after the injection , was shorter with pf , while time to verbal and non - verbal response was shorter with amobarbital .", "thus , they concluded that pf is a useful alternative to amobarbital in wada test .", "they noticed that the time to verbal and non - verbal responses and time to motor power 3/5 were not significantly different between the 2 groups .", "magee et al22 retrospectively reviewed the records of 129 patients who underwent wada test over an interval of 5 years .", "interestingly , they did not find a significant difference between pf and amobarbital in regards to length of time to end of hemiplegia , correlated with memory scores , pass / fail rates , and frequency and type of side - effects .", "it has been reported that left - handedness and left - hemispheric lesions increase the likelihood of bilateral representation of language.29 this patient was indeed left - handed and had bilateral mts .", "secobarbital has been shown to be effective and relatively safe in wada test.14 reported side - effects include visual changes and epileptic episodes.14 moreover , there have been some concerns regarding its availability.3 methohexital , a rapid - acting barbiturate , has proven to be a successful alternative to amobarbital in wada test.15 however , methohexital may have potential epileptogenic properties.15 a retrospective chart review study of 760 patients who underwent wada test by either amobarbital or methohexital concluded that patients with a previous history of epilepsy may be at higher risks of developing seizures after methohexital injection.5 pentabarbital , a short acting barbiturate , has shown to be equivalent to amobarbital for language and memory lateralization in wada test . unfortunately ,", "a patient experienced transient respiratory depression after hyperventilating for 3 minutes.16 etomidate - a rapid - acting , non - barbiturate , hypnotic agent - showed similar efficacy to amobarbital .", "its rapid duration of action necessitates an infusion pump , and maintenance doses.3 moreover , it has been reported to cause interictal epileptiform activity.17 in addition , it puts patients at risk of adrenal suppression especially if critically ill.30,31 propofol is a reasonable alternative for sa .", "its relatively short duration of action allows us to assess both hemispheres in one session without the need of maintenance doses .", "selective mca seems to decrease the incidence of adverse events , but requires a highly skilled neurointerventionalist .", "precautions should be taken since propofol may induce seizure - like activity , although not encountered in our study .", "transient ica spasm is probably secondary to guide - wire or catheter rather than being caused by pf .", "but if persistent , wada should be aborted to avoid cerebrovascular compromise . blood pressure should also be monitored .", "precautions should be exercised in patients with a documented history of bipolar disorder or are predisposed to experience such symptoms .", "interestingly , left - handedness and left - sided lesions increase the chance of bilateral representation of language , which carries a prognostic value , in terms of outcome of tle surgery", ". the limitations of our study are small sample size , the fact that it is an observational study , and the lack of a control group .", "a randomized controlled trial with larger sample size is needed to better assess the efficacy and adverse effects of propofol in comparison to sa . in summary", "the relatively short duration of action allowed us to examine both hemispheres in the same session .", "selective mca may decrease the incidence of these side - effects , but requires a skillful neurointerventionalist .", "in addition , we noticed 2 additional side - effects that were not mentioned in the previous studies;10 - 13,20 - 22 euphoria and transient ica spasm .", "transient ica spasm may be reversible , but if persistent , and severe , wada should be aborted .", "loring dw , meador kj , westerveld m. the wada test in the evaluation for epilepsy surgery ." ]

[ "<S> objective : to assess the tolerability of propofol ( pf ) in wada test in an arab population with temporal lobe epilepsy ( tle).methods : this observational study with consecutive sampling took place in king fahad medical city , riyadh , saudi arabia . </S>", "<S> nine consecutive patients with mean ( + sd ) age of 26 ( + 5.8 ) years , 6 males and 3 females , underwent wada test between january 2009 and december 2012 . </S>", "<S> six of them had left tle , and 3 had right tle . </S>", "<S> each patient received 10 mg of pf in the internal carotid artery ( ica ) . </S>", "<S> right hemispheric injection was followed by left hemisphere injection after 30 minutes . during the procedure , </S>", "<S> eeg monitoring showed changes within 5 - 18 seconds of injection as hemispheric delta slowing . </S>", "<S> neuropsychological tests were carried out for localization of memory and language.results:we were able to lateralize speech dominance in 8 patients and memory dominance in 6 patients . </S>", "<S> peri - procedural complications included transient euphoria ( n=1 ) , transient spasm of ica ( n=1 ) , eye pain ( n=1 ) , facial pain ( n=1 ) , and generalized tremulousness ( n=2 ) . </S>", "<S> none of the patients exhibited a symptomatic drop in blood pressure.conclusions:we found that pf is well tolerable for the wada test , with minimally significant complications , although blood pressure should be closely monitored . </S>" ]

[ "Methods", "Patient information", "Wada test", "Lateralization of the language and memory.", "Results", "Adverse effects", "Discussion", "None" ]

[ [ "this study was approved by king fahad medical city ( kfmc ) institutional review board ( irb ) in riyadh , kingdom of saudi arabia .", "all patients had a baseline brain mri , video - eeg recording , and a pre - surgical neuropsychological assessment composed of addenbrooke s cognitive examination - revised ( ace - r),5 handedness questionnaire,6 stanford - binet intelligence scale,7 hospital anxiety and depression scale ( hads),8 and daily memory questionnaire ( currently being tested in another study ) .", "these questionnaires were in arabic , given the language barrier , and were tested before being part of the standard neuropsychological evaluation of all patients admitted to the epilepsy monitoring unit ( emu ) in kfmc , especially if they were candidates for epilepsy surgery .", "potentially included patients are those who were diagnosed with tle and were candidates for temporal lobectomy / lobotomy .", "age range of the selected population for the procedure was between 18 and 65 years .", "patients were excluded if they had history of coagulopathy , carotid dissection , allergy to the contrast or anesthetic agent , elevated serum creatinine , were not fit for surgery ( for example , cardiopulmonary risk , major medical problems ) , or were unable to communicate efficiently during the procedure ( for example , deafness ) .", "patients were also excluded from the procedure if their ace - r score were less than 67 or if they were mentally retarded as per stanford - binet intelligence scale .", "patient selection was according to the helsinki declaration.9 all patients consented and received a detailed explanation on the nature , risks , and benefits of the procedure .", "since sa was not available , pf was chosen as an alternative , based on efficacy , safety profile , feasibility , potential side - effects , and several studies in the literature pertaining to the usage of pf in wada.10 - 17 the manufacturer of pf is astrazeneca , london , england , united kingdom .", "the procedure was carried out by a team that included an interventional neurologist , epileptologist , and a neuropsychologist . in each patient ,", "10 mg bolus of pf was injected into the internal carotid artery ( ica ) via a catheter inserted through the right femoral artery , except for one patient who received 15 mg bilaterally . neither continuous infusion nor maintenance doses were given .", "we began by injecting the right ica first , evaluated the left hemisphere , and then injected the left ica with a 30-minute interval between .", "prior to pf injection , the patient was asked to raise the contralateral arm to observe for weakness clinically , and count from one to 10 in numerical order to observe for speech arrest .", "in addition , patients were under continuous eeg monitoring to observe for any electrographic changes .", "the eeg demonstrated high amplitude theta and delta slowing of the ipsilateral hemisphere 5 - 18 seconds after the injection of propofol ( figure 1 ) .", "we assessed both hemispheres in the same session with 30-minutes interval between , during which baseline motor , speech , and electrographic activity were restored ( figure 2 ) .", "electroencephalogram paper speed 30 seconds per epoch , high amplitude theta and delta slowing starts on right hemispheric channels , seen 5 - 18 seconds after injection of propofol .", "electroencephalogram ( eeg ) paper speed 30 seconds per epoch , eeg starts to normalize , see reduction in amplitude of the slowing .", "the main elements of memory assessed were both recall ( verbal and non - verbal ) and recognition .", "the patient was shown 5 familiar objects and was asked if he / she recognizes them or not . during the test , the patient was shown 5 objects and 3 words and was asked to memorize them .", "the patient was asked to recall these objects and words as soon as the effect of pf wore - off ." ], [ "this study was approved by king fahad medical city ( kfmc ) institutional review board ( irb ) in riyadh , kingdom of saudi arabia .", "all patients had a baseline brain mri , video - eeg recording , and a pre - surgical neuropsychological assessment composed of addenbrooke s cognitive examination - revised ( ace - r),5 handedness questionnaire,6 stanford - binet intelligence scale,7 hospital anxiety and depression scale ( hads),8 and daily memory questionnaire ( currently being tested in another study ) .", "these questionnaires were in arabic , given the language barrier , and were tested before being part of the standard neuropsychological evaluation of all patients admitted to the epilepsy monitoring unit ( emu ) in kfmc , especially if they were candidates for epilepsy surgery .", "potentially included patients are those who were diagnosed with tle and were candidates for temporal lobectomy / lobotomy .", "age range of the selected population for the procedure was between 18 and 65 years .", "patients were excluded if they had history of coagulopathy , carotid dissection , allergy to the contrast or anesthetic agent , elevated serum creatinine , were not fit for surgery ( for example , cardiopulmonary risk , major medical problems ) , or were unable to communicate efficiently during the procedure ( for example , deafness ) .", "patients were also excluded from the procedure if their ace - r score were less than 67 or if they were mentally retarded as per stanford - binet intelligence scale .", "patient selection was according to the helsinki declaration.9 all patients consented and received a detailed explanation on the nature , risks , and benefits of the procedure ." ], [ "since sa was not available , pf was chosen as an alternative , based on efficacy , safety profile , feasibility , potential side - effects , and several studies in the literature pertaining to the usage of pf in wada.10 - 17 the manufacturer of pf is astrazeneca , london , england , united kingdom .", "the procedure was carried out by a team that included an interventional neurologist , epileptologist , and a neuropsychologist . in each patient ,", "10 mg bolus of pf was injected into the internal carotid artery ( ica ) via a catheter inserted through the right femoral artery , except for one patient who received 15 mg bilaterally . neither continuous infusion nor maintenance doses were given .", "we began by injecting the right ica first , evaluated the left hemisphere , and then injected the left ica with a 30-minute interval between .", "prior to pf injection , the patient was asked to raise the contralateral arm to observe for weakness clinically , and count from one to 10 in numerical order to observe for speech arrest .", "in addition , patients were under continuous eeg monitoring to observe for any electrographic changes .", "the eeg demonstrated high amplitude theta and delta slowing of the ipsilateral hemisphere 5 - 18 seconds after the injection of propofol ( figure 1 ) .", "we assessed both hemispheres in the same session with 30-minutes interval between , during which baseline motor , speech , and electrographic activity were restored ( figure 2 ) .", "electroencephalogram paper speed 30 seconds per epoch , high amplitude theta and delta slowing starts on right hemispheric channels , seen 5 - 18 seconds after injection of propofol .", "electroencephalogram ( eeg ) paper speed 30 seconds per epoch , eeg starts to normalize , see reduction in amplitude of the slowing ." ], [ "the main elements of memory assessed were both recall ( verbal and non - verbal ) and recognition .", "the patient was shown 5 familiar objects and was asked if he / she recognizes them or not . during the test , the patient was shown 5 objects and 3 words and was asked to memorize them .", "the patient was asked to recall these objects and words as soon as the effect of pf wore - off ." ], [ "once pf was injected , we noticed weakness of the contralateral side and aphasia if the involved hemisphere were the dominant one in which the patient stopped counting , could not obey commands , communicate with the neuropsychology team , recognize or memorize objects and words .", "both motor and electrographic changes reverted back to baseline within 10 - 12 minutes . out of 9 patients , with mean ( + sd ) age 26 ( + 5.8 ) years", "six of the right - handed patients had left tle , and 2 had right tle .", "all patients had mri findings : 3 patients were found to have left mesial temporal sclerosis ( mts ) , 2 had right mts , and 4 had altered hippocampal morphology .", "only one patient was documented to have abnormal cognitive functions ( addenbrooke s cognitive examination - revised 77/100 ) ( table 1 ) .", "we were able to lateralize speech in 8 patients , and memory in 6 patients .", "all right - handed patients had a left - sided representation of language . however , 2 out of 6 right - handed patients had bilateral representation of memory , while the rest had left - sided representation of memory .", "from the 3 patients who had right tle , 2 had left - sided representation of language while one had bilateral representation ( table 1 ) .", "in addition , 2 subjects experienced shivering during the procedure , and one subject complained of eye pain and facial pain ipsilateral to the site of the ica injection . the latter developed transient segmental spasm of the ica .", "this same patient required an additional 10 mg pf due to lack of effect with the first dose .", "the average blood pressure measured for all patients immediately after the procedure was 116/66 mm hg .", "all subjects were able to complete the procedure , testing both hemispheres in the same session . aside from the mentioned adverse events ," ], [ "in addition , 2 subjects experienced shivering during the procedure , and one subject complained of eye pain and facial pain ipsilateral to the site of the ica injection . the latter developed transient segmental spasm of the ica .", "this same patient required an additional 10 mg pf due to lack of effect with the first dose .", "the average blood pressure measured for all patients immediately after the procedure was 116/66 mm hg .", "all subjects were able to complete the procedure , testing both hemispheres in the same session . aside from the mentioned adverse events ," ], [ "propofol , an alkylphenol , is a lipid soluble anesthetic agent with rapid onset of action ( 30 seconds ) , and rapid rate of distribution ( 2 - 4 minutes).18,19 it is metabolized in the liver and excreted mainly through urine.18,19 propofol was first tried , by bazin et al20 in 1998 , and by silva et al21 in 2000 .", "both tried it on a single patient and recorded successful results . in terms of side - effects , the former reported perception of intense blue light , while the latter reported a hot sensation in the head .", "thereafter , several studies assessed the efficacy and adverse events of pf . with respect to adverse events , we encountered : shivering , facial pain , and eye pain .", "mikuni et al12 performed a case - series of 58 patients in which they studied the side - effects of pf in wada test .", "they classified the side - effects into 3 categories : grade 1 symptoms included pain , shivering , face contortion , lacrimation , laughing , and apathy .", "grade 3 symptoms included seizure - like activity described as increased muscle tone with twitching and rhythmic movements or tonic posturing .", "grade 1 and 2 symptoms were also seen with sa.12 some of these adverse events were also reported by takayama et al,13 mikati et al,11 and magee et al.22 it has been suggested that post - anesthetic shivering associated with pf , although not fully understood , is due to a thermoregulatory phenomenon associated with hypothermia.23,24 as for facial pain and eye pain , it has been proposed that they occur due to peripheral anastomosis and cross - filling between the internal carotid and external carotid arteries.12 a study10 showed that selective middle cerebral artery ( mca ) injection of pf was associated with a lower incidence of such side - effects ; thus , supporting the anastomosis hypothesis .", "although we did not encounter any of the grade 3 symptoms in our study , it has been reported that the frequency of grade 3 symptoms increased in patients older than 55 years , after a second injection of pf greater than 10 mg , and/or a total dose of pf greater than 20 mg.12 one of our subjects failed to elicit a response and developed transient segmental ica spasm .", "thereafter , we bypassed the segment of spasm and gave an additional 10 mg of pf , which was successful without any side - effects .", "thus , it is probable that vasospasm was secondary to the guide - wire or the catheter ( namely , iatrogenic ) .", "mechanically induced vasospasm has been reported in the literature as a complication of angiography , and is mostly self - limited.25,26 interestingly , a patient developed transient euphoria characterized by talkativeness , curiosity , and was asking many questions .", "however , there was an element of confusion to this picture and he was speaking incoherently rather than combativeness as reported in previous studies.11 - 13,22 it has been reported that iv pf might be associated with euphoria , and it might have an addictive effect.27,28 a possible explanation might be an increase in dopamine concentration in the nucleus accumbens , as seen in drug abusers , promoting drug seeking behavior.18,19 in terms of efficacy , it was not possible to compare pf to sa since it was not available", ". however , we were able to lateralize speech dominance in 8 patients , and memory dominance in 6 patients ( table 1 ) .", "we also found that the recovery time ( both clinically and electrophysiologically ) ranged between 10 - 12 minutes .", "takayama et al13 were able to lateralize language and memory in 12 and 9 out of 12 patients , as opposed to 52 and 41 out of 55 patients with sa .", "they also noticed that the recovery time of muscle power , after the injection , was shorter with pf , while time to verbal and non - verbal response was shorter with amobarbital .", "thus , they concluded that pf is a useful alternative to amobarbital in wada test .", "they noticed that the time to verbal and non - verbal responses and time to motor power 3/5 were not significantly different between the 2 groups .", "magee et al22 retrospectively reviewed the records of 129 patients who underwent wada test over an interval of 5 years .", "interestingly , they did not find a significant difference between pf and amobarbital in regards to length of time to end of hemiplegia , correlated with memory scores , pass / fail rates , and frequency and type of side - effects .", "it has been reported that left - handedness and left - hemispheric lesions increase the likelihood of bilateral representation of language.29 this patient was indeed left - handed and had bilateral mts .", "secobarbital has been shown to be effective and relatively safe in wada test.14 reported side - effects include visual changes and epileptic episodes.14 moreover , there have been some concerns regarding its availability.3 methohexital , a rapid - acting barbiturate , has proven to be a successful alternative to amobarbital in wada test.15 however , methohexital may have potential epileptogenic properties.15 a retrospective chart review study of 760 patients who underwent wada test by either amobarbital or methohexital concluded that patients with a previous history of epilepsy may be at higher risks of developing seizures after methohexital injection.5 pentabarbital , a short acting barbiturate , has shown to be equivalent to amobarbital for language and memory lateralization in wada test . unfortunately ,", "a patient experienced transient respiratory depression after hyperventilating for 3 minutes.16 etomidate - a rapid - acting , non - barbiturate , hypnotic agent - showed similar efficacy to amobarbital .", "its rapid duration of action necessitates an infusion pump , and maintenance doses.3 moreover , it has been reported to cause interictal epileptiform activity.17 in addition , it puts patients at risk of adrenal suppression especially if critically ill.30,31 propofol is a reasonable alternative for sa .", "its relatively short duration of action allows us to assess both hemispheres in one session without the need of maintenance doses .", "selective mca seems to decrease the incidence of adverse events , but requires a highly skilled neurointerventionalist .", "precautions should be taken since propofol may induce seizure - like activity , although not encountered in our study .", "transient ica spasm is probably secondary to guide - wire or catheter rather than being caused by pf .", "but if persistent , wada should be aborted to avoid cerebrovascular compromise . blood pressure should also be monitored .", "precautions should be exercised in patients with a documented history of bipolar disorder or are predisposed to experience such symptoms .", "interestingly , left - handedness and left - sided lesions increase the chance of bilateral representation of language , which carries a prognostic value , in terms of outcome of tle surgery", ". the limitations of our study are small sample size , the fact that it is an observational study , and the lack of a control group .", "a randomized controlled trial with larger sample size is needed to better assess the efficacy and adverse effects of propofol in comparison to sa . in summary", "the relatively short duration of action allowed us to examine both hemispheres in the same session .", "selective mca may decrease the incidence of these side - effects , but requires a skillful neurointerventionalist .", "in addition , we noticed 2 additional side - effects that were not mentioned in the previous studies;10 - 13,20 - 22 euphoria and transient ica spasm .", "transient ica spasm may be reversible , but if persistent , and severe , wada should be aborted ." ], [ "loring dw , meador kj , westerveld m. the wada test in the evaluation for epilepsy surgery ." ] ]

[ "ventriculoperitoneal shunt ( vps ) is a well - established cerebrospinal fluid ( csf ) diversion procedure in cases of hydrocephalus .", "there are a lot of complications associated with this procedure and shunt extrusion via different natural orifices is one of them .", "transanal and per abdomen shunt extrusion are well documented in literature while shunt extrusion via urinary bladder , vagina , uterus , gall bladder , urethra , and scrotum are sporadically reported in the literature .", "the distal end of shunt erodes through the wall of hollow viscera , approaches their lumen , and protrudes outside through natural orifices .", "the distal end of shunt protrusion depends on stiffness of shunt tip , thickness of the wall of hollow viscera , condition of the visceral wall , underlying infection , fixity of viscera , and the operative hand of the surgeon .", "the incidence of perforation is inversely related to the mobility of gut , and colon is the most frequently perforated viscus due to its immobility .", "a 10-month - old male child presented with protrusion of shunt tubing from the anus , incidentally noticed by his parents .", "the patient had a history of excessive cry along with extrusion of shunt tube through anal orifice [ figure 1a ] .", "the abdomen was soft , nontender , and bowel sounds were audible on abdominal auscultation .", "his developmental milestones were delayed , and rests of the neurological examinations were within normal limit .", "x - ray abdomen erect imaging showed no pneumoperitoneum [ figure 1b ] . computed tomography head showed dilated lateral and third ventricles .", "the patient was taken up for the removal of previous shunt assembly and placement of new one . at the time of distal end removal , lower end of shunt", "had retracted back into rectum ; hence , we planned to remove that via incision at abdominal insertion point .", "two incisions were given , one at the cranial end of shunt tubing , and another one at the abdominal insertion site .", "the shunt was divided at the abdominal insertion point , and cranial part was pulled out from cranial incision while abdominal part was pulled through the abdominal incision .", "the shunt tube had perforated the appendix and entering through the perforation [ figure 2a and b ] .", "an urgent gastrosurgery opinion was taken and urgent laparotomy via extension of same incision was performed by the gastrosurgeon .", "postoperative period was uneventful , and patient discharged from hospital after removal of stitches and is under follow - up .", "( a ) protrusion of the lower end of the shunt through anus , ( b ) a radiograph of a patient showing the course of the lower end of the shunt through a large intestine ( a ) an intraoperative photograph showing the appendix coming out of the abdominal incision while pulling the lower end of the shunt which was perforating the appendix at the tip ( black arrow ) , ( b ) the perforated appendix during appendicectomy", "vps is most commonly used and universally accepted surgical procedure for the management of hydrocephalus in children .", "a number of complications associated with this procedure are mentioned in literature with abdominal complications accounting for 1030% of all .", "bowel perforation by the tip of the catheter and extrusion of the shunt through external orifices is one of them . although this is a rare complication and have incidence rate of 0.10.7% only .", "the first case of anal extrusion of distal vps was reported by wilson and bertan in 1966 .", "the duration between shunt surgery and bowel perforation was found to be minimum in infants and was related with the age of the patient .", "the interval between shunt insertion to the protrusion of catheter from anus ranges from 2 to 20 months with an average of 6.1 months . in our case , this period was 6 months .", "nonenteric visceral perforation has also been sporadically reported in the literature and that includes urinary bladder , urethra , scrotum , vagina , gall bladder , and uterus .", "factors associated with perforation of gut are foreign body reaction , stiff tip of shunt , thin bowel wall in pediatric patients , abdominal infections , silicon allergy , use of trocar for insertion of peritoneal end , previous abdominal surgery , and chronic wear and tear produced by tip of the shunt .", "many patients do not present with significant abdominal symptoms because the fibrous tract formed at the perforated site usually seals the perforation and prevents spillage of fecal matter into the peritoneum , which would otherwise lead to peritonitis .", "hence , the correct diagnosis may be delayed until a very later stage at which gram - negative or anaerobic meningitis , encephalitis or ventriculitis has been fully established , leading to significant morbidity and/or mortality to the patients .", "mechanism of shunt extrusion is not well - established but most accepted hypothesis is that after bowel perforation shunt tubing propels outside with sequential peristaltic movement of the gut . in the cases of", "shunt extrusion through anal opening , most common site of bowel perforation is colon ( 70% ) while in cases of oral extrusion most common site is the stomach .", "early diagnosis , adequate clinical , radiological and biochemical evaluation , and prompt treatment are the key to successful treatment .", "the standard method of treatment is the removal of the extruded shunt system , control of infection , improvement of general condition followed by csf diversion procedure .", "the different available alternatives are laparotomy with the revision of the peritoneal end of shunt , conventional exploratory laparotomy and repair of bowel perforation , endoscopic localization of enterotomy site and removal of shunt , shunt removal , external diversion of csf and use of antibiotics , and later on replacement of vps when no infection are seen . in uncomplicated cases of", "shunt extrusion , shunt tubing can be removed via extruding orifice and bowel perforation can be managed conservatively while in complicated cases shunt removal is advised via a laparotomy .", "appendicitis after ventriculoperitoneal shunting is a known complication but to best of our knowledge shunt extrusion via appendicular perforation is not documented in literature yet . in such cases ,", "shunt removal via extruding orifice may be quite difficult . theoretically , there are high possibilities of associated appendicitis which can result in the development of frank peritonitis or appendicular abscess after shunt removal through extruding orifice .", "so in our opinion , such type of cases whether complicated or not should be placed in special category and shunt removal should be done via laparotomy and followed by prophylactic appendectomy .", ". such type of case should be consider in special category in which shunt removal without proper surgical exploration and added appendicectomy may be quite difficult and hazardous for the patient .", "optimal management plan in such type of cases either uncomplicated or complicated is laparotomy and removal of the shunt with added appendicectomy ." ]

[ "<S> perforation of abdominal viscera and protrusion of the distal end of ventriculoperitoneal shunt ( vps ) through natural orifice is well known but rare complication . we report a case of a transanal protrusion of distal end of vps through appendix perforation without any symptomatology of prior appendicitis . to the best of our knowledge , no case of such kind has been reported in literature yet . </S>", "<S> the management plan of these patients should be looked in a different way because they may have underlying inflammation of the appendix and distal end of shunt removal should be done by proper surgical exploration followed by added appendicectomy . </S>" ]

[ "Introduction", "Case Report", "Discussion", "Conclusion", "Financial support and sponsorship", "Conflicts of interest" ]

[ [ "ventriculoperitoneal shunt ( vps ) is a well - established cerebrospinal fluid ( csf ) diversion procedure in cases of hydrocephalus .", "there are a lot of complications associated with this procedure and shunt extrusion via different natural orifices is one of them .", "transanal and per abdomen shunt extrusion are well documented in literature while shunt extrusion via urinary bladder , vagina , uterus , gall bladder , urethra , and scrotum are sporadically reported in the literature .", "the distal end of shunt erodes through the wall of hollow viscera , approaches their lumen , and protrudes outside through natural orifices .", "the distal end of shunt protrusion depends on stiffness of shunt tip , thickness of the wall of hollow viscera , condition of the visceral wall , underlying infection , fixity of viscera , and the operative hand of the surgeon .", "the incidence of perforation is inversely related to the mobility of gut , and colon is the most frequently perforated viscus due to its immobility ." ], [ "a 10-month - old male child presented with protrusion of shunt tubing from the anus , incidentally noticed by his parents .", "the patient had a history of excessive cry along with extrusion of shunt tube through anal orifice [ figure 1a ] .", "the abdomen was soft , nontender , and bowel sounds were audible on abdominal auscultation .", "his developmental milestones were delayed , and rests of the neurological examinations were within normal limit .", "x - ray abdomen erect imaging showed no pneumoperitoneum [ figure 1b ] . computed tomography head showed dilated lateral and third ventricles .", "the patient was taken up for the removal of previous shunt assembly and placement of new one . at the time of distal end removal , lower end of shunt", "had retracted back into rectum ; hence , we planned to remove that via incision at abdominal insertion point .", "two incisions were given , one at the cranial end of shunt tubing , and another one at the abdominal insertion site .", "the shunt was divided at the abdominal insertion point , and cranial part was pulled out from cranial incision while abdominal part was pulled through the abdominal incision .", "the shunt tube had perforated the appendix and entering through the perforation [ figure 2a and b ] .", "an urgent gastrosurgery opinion was taken and urgent laparotomy via extension of same incision was performed by the gastrosurgeon .", "postoperative period was uneventful , and patient discharged from hospital after removal of stitches and is under follow - up .", "( a ) protrusion of the lower end of the shunt through anus , ( b ) a radiograph of a patient showing the course of the lower end of the shunt through a large intestine ( a ) an intraoperative photograph showing the appendix coming out of the abdominal incision while pulling the lower end of the shunt which was perforating the appendix at the tip ( black arrow ) , ( b ) the perforated appendix during appendicectomy" ], [ "vps is most commonly used and universally accepted surgical procedure for the management of hydrocephalus in children .", "a number of complications associated with this procedure are mentioned in literature with abdominal complications accounting for 1030% of all .", "bowel perforation by the tip of the catheter and extrusion of the shunt through external orifices is one of them . although this is a rare complication and have incidence rate of 0.10.7% only .", "the first case of anal extrusion of distal vps was reported by wilson and bertan in 1966 .", "the duration between shunt surgery and bowel perforation was found to be minimum in infants and was related with the age of the patient .", "the interval between shunt insertion to the protrusion of catheter from anus ranges from 2 to 20 months with an average of 6.1 months . in our case , this period was 6 months .", "nonenteric visceral perforation has also been sporadically reported in the literature and that includes urinary bladder , urethra , scrotum , vagina , gall bladder , and uterus .", "factors associated with perforation of gut are foreign body reaction , stiff tip of shunt , thin bowel wall in pediatric patients , abdominal infections , silicon allergy , use of trocar for insertion of peritoneal end , previous abdominal surgery , and chronic wear and tear produced by tip of the shunt .", "many patients do not present with significant abdominal symptoms because the fibrous tract formed at the perforated site usually seals the perforation and prevents spillage of fecal matter into the peritoneum , which would otherwise lead to peritonitis .", "hence , the correct diagnosis may be delayed until a very later stage at which gram - negative or anaerobic meningitis , encephalitis or ventriculitis has been fully established , leading to significant morbidity and/or mortality to the patients .", "mechanism of shunt extrusion is not well - established but most accepted hypothesis is that after bowel perforation shunt tubing propels outside with sequential peristaltic movement of the gut . in the cases of", "shunt extrusion through anal opening , most common site of bowel perforation is colon ( 70% ) while in cases of oral extrusion most common site is the stomach .", "early diagnosis , adequate clinical , radiological and biochemical evaluation , and prompt treatment are the key to successful treatment .", "the standard method of treatment is the removal of the extruded shunt system , control of infection , improvement of general condition followed by csf diversion procedure .", "the different available alternatives are laparotomy with the revision of the peritoneal end of shunt , conventional exploratory laparotomy and repair of bowel perforation , endoscopic localization of enterotomy site and removal of shunt , shunt removal , external diversion of csf and use of antibiotics , and later on replacement of vps when no infection are seen . in uncomplicated cases of", "shunt extrusion , shunt tubing can be removed via extruding orifice and bowel perforation can be managed conservatively while in complicated cases shunt removal is advised via a laparotomy .", "appendicitis after ventriculoperitoneal shunting is a known complication but to best of our knowledge shunt extrusion via appendicular perforation is not documented in literature yet . in such cases ,", "shunt removal via extruding orifice may be quite difficult . theoretically , there are high possibilities of associated appendicitis which can result in the development of frank peritonitis or appendicular abscess after shunt removal through extruding orifice .", "so in our opinion , such type of cases whether complicated or not should be placed in special category and shunt removal should be done via laparotomy and followed by prophylactic appendectomy ." ], [ ". such type of case should be consider in special category in which shunt removal without proper surgical exploration and added appendicectomy may be quite difficult and hazardous for the patient .", "optimal management plan in such type of cases either uncomplicated or complicated is laparotomy and removal of the shunt with added appendicectomy ." ], [ "" ], [ "" ] ]

[ "recently , new oral anticoagulants have been approved as alternatives to warfarin for patients with atrial fibrillation .", "rivaroxaban is one of the novel anticoagulants , which is an oxazolidinone derivative and inhibits both free factor xa and factor xa bound with the prothrombinase complex .", "it is a highly selective direct factor xa inhibitor with oral bioavailability and rapid onset of action .", "there are some advantages of the new agents compared with warfarin including rapid anticoagulation after an oral dose and lack of dietary or drug - drug interaction . however , there are no specific antidotes for the anticoagulant effect of rivaroxaban in the event of a major bleeding , unlike warfarin", ". we present a case of spontaneous rectus sheath hematoma ( rsh ) during rivaroxaban therapy for atrial fibrillation in an elderly female patient .", "a 75-year - old woman presented to the emergency department with the complaints of fatigue and abdominal pain after coughing .", "the patient had been started on new oral anticoagulant agent rivaroxaban therapy for nonvalvular atrial fibrillation for 3 days .", "the dose of 20 mg / day rivaroxaban was started because the creatinin clearance of the patient was above 50 ml / min .", "she had a blood pressure of 70/40 mmhg and an irregular heart rate of 115 beats / min on admission .", "the patient had no history of any trauma or surgery ; she reported that the symptoms started after vigorous coughing .", "blood analyses revealed leukocytosis ( 26.5 k / ul ) accompanied by severe anemia ( 5.4 g / dl ) .", "platelet counts were within normal ranges and her international normalized ratio ( inr ) was 1.48 .", "her abdominal x - ray was normal and the stool occult blood test was negative .", "after the first treatment , the patient was transferred to the intensive care unit ( icu ) .", "repeated abdominal examination in the icu revealed increased tenderness and a palpable mass on the left side of the umbilicus .", "noncontrast abdominal computerized tomography scan showed a left - sided rsh , 102 45 mm in size [ figure 1 ] . a specific antidote for rivaroxaban", "is not available then the patient was treated with fluid resuscitation and packed red blood cells .", "computed tomography scan of the abdomen shows a left - sided rectus sheath hematoma ( arrow )", "rivaroxaban is an oral anticoagulant agent that directly inhibits factor xa and interrupts both the intrinsic and extrinsic pathway of the coagulation cascade .", "rivaroxaban is currently indicated for use in patients for atrial fibrillation and prophylaxis of deep venous thrombosis .", "it does not require inr monitoring like warfarin . with the increasing use of the new anticoagulant agents like rivaroxaban in atrial fibrillation , bleeding complications due to these agents", "there are no specific antidotes for the anticoagulant effect of rivaroxaban and other new oral anticogulants unlike warfarin , thus the management of the bleeding complications include support and observation .", "currently , no available specific antidote exists for the management of rivaroxaban - associated bleeding events , but supporting therapy is useful which are likely to be effective for the majority of patients because of the short half - lives of these agents .", "recent studies showed that rivaroxaban was noninferior to warfarin for the primary endpoint of stroke and systemic embolism .", "there was no reduction in rates of mortality or ischemic stroke , but a significant reduction in hemorrhagic stroke and intracranial hemorrhage .", "the primary safety endpoint was the composite of major and clinically relevant nonmajor bleeding , which was not significantly different between rivaroxaban and warfarin but , with rivaroxaban , there was a significant reduction in fatal bleeding , as well as an increase in gastrointestinal bleeds and bleeds requiring transfusion .", "the main causes of the rsh include anticoagulant therapy , hematological disorders , trauma , excessive physical exercise , coughing , sneezing , and pregnancy . especially in elderly patients the risk of rsh may be increased due to the impaired functional status and weakened rectus muscle", ". early recognition , rapid assessment and treatment are important to reduce the complications such as hemodynamic instability , abdominal compartment syndrome , multiorgan dysfunction and even death .", "the treatment of such a hematoma includes transfusion with packed red blood cells and supporting therapy based on regularly monitoring of hemoglobin levels .", "rivaroxaban has a mean terminal half - life of 7 - 11 h so in bleeding events supporting therapies are likely to be effective for the majority of patients .", "several studies have shown that prothrombin complex concentrate may be useful in reversing the effects of rivaroxaban .", "other possible measures include the use of recombinant factor viia to reduce bleeding or the use of activated charcoal to reduce absorption in cases of overdose .", "several factors are reported which increase the risk of patients developing hemorrhage while receiving rivaroxaban , these include advanced age , hypertension , history of hepatic / renal disease , previous stroke , coagulopathy , concomitant use of antiplatelet agents and alcohol consumption .", "jaeger et al . have reported a 61-year - old female patient who developed a spontaneous spinal epidural hematoma after being treated by rivaroxaban .", "boland et al . also reported acute onset severe gastrointestinal tract hemorrhage in a postoperative patient taking rivaroxaban after total hip arthroplasty . in our patient", ", there was no other medication except rivaroxaban that could cause the hematoma . based on naranjo 's scale", ", a score of 7 showed that the rivaroxaban was the probable cause of the rsh .", "several case reports of muscle hematoma due to the antiplatelet and anticoagulant agents have been reported previously , but this is the first reported case of spontaneous rsh due to the rivaroxaban ." ]

[ "<S> rivaroxaban is an oral anticoagulant agent that directly inhibits factor xa and interrupts both the intrinsic and extrinsic pathway of the coagulation cascade and is currently indicated for use in patients for atrial fibrillation and prophylaxis of deep venous thrombosis . </S>", "<S> the present case reports of spontaneous rectus sheath hematoma during rivaroxaban therapy for atrial fibrillation in a 75-year - old woman . </S>" ]

[ "Introduction", "Case Report", "Discussion" ]

[ [ "recently , new oral anticoagulants have been approved as alternatives to warfarin for patients with atrial fibrillation .", "rivaroxaban is one of the novel anticoagulants , which is an oxazolidinone derivative and inhibits both free factor xa and factor xa bound with the prothrombinase complex .", "it is a highly selective direct factor xa inhibitor with oral bioavailability and rapid onset of action .", "there are some advantages of the new agents compared with warfarin including rapid anticoagulation after an oral dose and lack of dietary or drug - drug interaction . however , there are no specific antidotes for the anticoagulant effect of rivaroxaban in the event of a major bleeding , unlike warfarin", ". we present a case of spontaneous rectus sheath hematoma ( rsh ) during rivaroxaban therapy for atrial fibrillation in an elderly female patient ." ], [ "a 75-year - old woman presented to the emergency department with the complaints of fatigue and abdominal pain after coughing .", "the patient had been started on new oral anticoagulant agent rivaroxaban therapy for nonvalvular atrial fibrillation for 3 days .", "the dose of 20 mg / day rivaroxaban was started because the creatinin clearance of the patient was above 50 ml / min .", "she had a blood pressure of 70/40 mmhg and an irregular heart rate of 115 beats / min on admission .", "the patient had no history of any trauma or surgery ; she reported that the symptoms started after vigorous coughing .", "blood analyses revealed leukocytosis ( 26.5 k / ul ) accompanied by severe anemia ( 5.4 g / dl ) .", "platelet counts were within normal ranges and her international normalized ratio ( inr ) was 1.48 .", "her abdominal x - ray was normal and the stool occult blood test was negative .", "after the first treatment , the patient was transferred to the intensive care unit ( icu ) .", "repeated abdominal examination in the icu revealed increased tenderness and a palpable mass on the left side of the umbilicus .", "noncontrast abdominal computerized tomography scan showed a left - sided rsh , 102 45 mm in size [ figure 1 ] . a specific antidote for rivaroxaban", "is not available then the patient was treated with fluid resuscitation and packed red blood cells .", "computed tomography scan of the abdomen shows a left - sided rectus sheath hematoma ( arrow )" ], [ "rivaroxaban is an oral anticoagulant agent that directly inhibits factor xa and interrupts both the intrinsic and extrinsic pathway of the coagulation cascade .", "rivaroxaban is currently indicated for use in patients for atrial fibrillation and prophylaxis of deep venous thrombosis .", "it does not require inr monitoring like warfarin . with the increasing use of the new anticoagulant agents like rivaroxaban in atrial fibrillation , bleeding complications due to these agents", "there are no specific antidotes for the anticoagulant effect of rivaroxaban and other new oral anticogulants unlike warfarin , thus the management of the bleeding complications include support and observation .", "currently , no available specific antidote exists for the management of rivaroxaban - associated bleeding events , but supporting therapy is useful which are likely to be effective for the majority of patients because of the short half - lives of these agents .", "recent studies showed that rivaroxaban was noninferior to warfarin for the primary endpoint of stroke and systemic embolism .", "there was no reduction in rates of mortality or ischemic stroke , but a significant reduction in hemorrhagic stroke and intracranial hemorrhage .", "the primary safety endpoint was the composite of major and clinically relevant nonmajor bleeding , which was not significantly different between rivaroxaban and warfarin but , with rivaroxaban , there was a significant reduction in fatal bleeding , as well as an increase in gastrointestinal bleeds and bleeds requiring transfusion .", "the main causes of the rsh include anticoagulant therapy , hematological disorders , trauma , excessive physical exercise , coughing , sneezing , and pregnancy . especially in elderly patients the risk of rsh may be increased due to the impaired functional status and weakened rectus muscle", ". early recognition , rapid assessment and treatment are important to reduce the complications such as hemodynamic instability , abdominal compartment syndrome , multiorgan dysfunction and even death .", "the treatment of such a hematoma includes transfusion with packed red blood cells and supporting therapy based on regularly monitoring of hemoglobin levels .", "rivaroxaban has a mean terminal half - life of 7 - 11 h so in bleeding events supporting therapies are likely to be effective for the majority of patients .", "several studies have shown that prothrombin complex concentrate may be useful in reversing the effects of rivaroxaban .", "other possible measures include the use of recombinant factor viia to reduce bleeding or the use of activated charcoal to reduce absorption in cases of overdose .", "several factors are reported which increase the risk of patients developing hemorrhage while receiving rivaroxaban , these include advanced age , hypertension , history of hepatic / renal disease , previous stroke , coagulopathy , concomitant use of antiplatelet agents and alcohol consumption .", "jaeger et al . have reported a 61-year - old female patient who developed a spontaneous spinal epidural hematoma after being treated by rivaroxaban .", "boland et al . also reported acute onset severe gastrointestinal tract hemorrhage in a postoperative patient taking rivaroxaban after total hip arthroplasty . in our patient", ", there was no other medication except rivaroxaban that could cause the hematoma . based on naranjo 's scale", ", a score of 7 showed that the rivaroxaban was the probable cause of the rsh .", "several case reports of muscle hematoma due to the antiplatelet and anticoagulant agents have been reported previously , but this is the first reported case of spontaneous rsh due to the rivaroxaban ." ] ]

[ "all pcos patients met all three criteria of the revised 2003 rotterdam european society of human reproduction and embryology ( eshre)/american society of reproductive medicine ( asrm ) pcos consensus workshop group diagnostic criteria .", "the three criteria are 1 ) oligo- and/or anovulation , 2 ) clinical and/or biochemical signs of hyperandrogenism , and 3 ) polycystic ovaries ( 20 ) .", "furthermore , all subjects in the control arm had normal findings on pelvic ultrasound scan , regular periods , and no hirsutism / acne .", "exclusion criteria for the study included age > 40 years , known cardiovascular disease , thyroid disease , neoplasms , current smoking , diabetes , hypertension ( blood pressure > 140/90 mmhg ) , and renal impairment ( serum creatinine > 120 mol / l ) .", "none of these women were on any medications for at least 6 months prior to the study , including oral contraceptives , glucocorticoids , ovulation induction agents , antidiabetic and antiobesity drugs , and estrogenic , antiandrogenic , or antihypertensive medication .", "blood pressure was measured in a sitting position within a quiet and calm environment after a rest of at least 5 min .", "subjects were outpatients of the department of reproductive medicine and gynaecological endocrinology of magdeburg university and the department of obstetrics and gynaecology of martin - luther - university halle . the metabolic study was performed in the outpatient department of endocrinology and metabolism of magdeburg university .", "blood samples were collected between 0800 and 0900 h , after a 3-day normal carbohydrate diet and an overnight fast .", "blood samples were drawn into serum separator tubes that contain no additives or anticoagulants , allowed to clot , centrifuged ( 3,000 rpm for 10 min ) to separate sera , and stored at 80c . a treatment with metformin in an off - label use ", "was offered to all pcos women independently from the results of insulin sensitivity testing . in those pcos women that agreed ,", "therapy was initiated after basal assessment , and the dose of metformin was increased to a maintenance dose of 850 mg twice daily .", "carotid intima media thickness ( imt ) was measured in pcos subjects , before and after metformin ( see below ) .", "a total of 83 women of caucasian origin with pcos were recruited , 49 of which did not participate in the metformin arm of the study .", "the baseline characteristics of the 49 pcos women were comparable to the 34 pcos women who participated in the metformin arm of the study .", "furthermore , the 21 women who completed the metformin arm of the study were comparable to the 13 pcos women who did not complete the metformin arm of the study . for the purposes of elucidating the effects of metformin in pcos women ,", "the study design was approved by the local research ethics committee of the university of magdeburg , and written informed consent was obtained from all participants , in accordance with the guidelines in the declaration of helsinki 2000 .", "measurement of carotid imt is a widely accepted tool to provide information about preclinical vascular disease .", "b - mode sonography of the proximal part of the carotid bulb was conducted on both sides , and the segments of the common carotid arteries 1.0 cm proximal were scanned longitudinally with hitachi eub-5000 plus - g ( hitachi ltd . ,", "all women were investigated in a supine position with the head slightly hyperextended and turned away from the side being scanned .", "the image was focused on the posterior wall , and the resolution function was used to magnify the arterial far wall .", "when an optimal image was obtained , it was frozen , and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end - diastolic phase , to minimize variability during the cardiac cycle .", "five measurements were conducted on each side , and the mean imt was calculated as the average of these measurements .", "all measurements were performed by an experienced ultrasound sonographer ( intra - observer coefficient of variation was 6.8% ) who was blinded to the diagnosis of subjects .", "assays for glucose , insulin , cholesterol , triglycerides , luteinizing hormone , follicular stimulating hormone , testosterone , and rostenedione , dehydroepiandrosterone - sulfate , and sex hormone binding globulin were performed using an automated analyzer ( abbott architect , abbott laboratories , abbott park , usa ) .", "the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) score was calculated as previously described ( 21 ) .", "high - sensitivity c - reactive protein ( hs - crp ) was determined immunoturbidimetrically using a modular system random - access analyzer ( roche diagnostics ) .", "omentin-1 levels in sera were measured using a commercially available elisa kit ( axxora , nottingham , u.k . ) , according to manufacturer 's protocol , with an intra - assay coefficient of variation of < 6% .", "immunodepletion of serum omentin-1 and crp were performed using the immunoprecipitation protocol provided by dr .", "one ml of human serum was incubated with mouse - anti - human omentin-1 igg - protein g - sepharose conjugate ( 12 g/100500 g of total protein ) or control mouse igg - protein g - sepharose conjugate ; goat - anti - human crp iga - protein a - sepharose conjugate ( 10 g/100500 g of total protein ) or control goat iga - protein a - sepharose conjugate , mixed overnight at 4c and centrifuged to sediment the conjugated sepharose beads .", "supernatants and sera were subjected to immunoblotting for omentin-1 as previously described ( 13 ) .", "immunoblotting confirmed that these protocols decreased serum omentin-1 and crp by 80% ( data not shown ) , respectively .", "human microvascular endothelial cells ( hmec-1 ) were obtained from the center for disease control , atlanta , usa hmec-1 cells were cultured in mcdb medium ( sigma - aldrich , gillingham , u.k . ) supplemented with 10% fetal calf serum ( sigma - aldrich , gillingham , u.k . ) , 100 iu / ml penicillin ( sigma - aldrich , gillingham , u.k . ) , 100 g / ml streptomycin ( sigma - aldrich ) , 5 ml of 200 mmol / l l - glutamine/500 ml of media , hydrocortisone 2 mol / l , and epidermal growth factor 2 ng / ml ( invitrogen , paisley , u.k . ) at 37c in 5% co2/95% air . prior to each experiment , cells were fed with mcdb with addition of 1% fetal calf serum for 16 h. for endothelial cell signaling experiments , hmecs were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] at different time points ( 0 , 2 , 5 , 10 , 20 , 30 , and 60 min ) .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "angiogenesis was assessed by studying the formation of capillary tubelike structures by culturing hmec-1 cells on growth factor reduced matrigel ( bd biosciences , san jose , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera .", "trypsinized hmec-1 cells were then seeded onto the matrigel - coated plates at a density of 45 10/well in fresh media and incubated at 37c .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "capillary tube formation images were captured with a digital microscope camera system ( olympus , tokyo , japan ) .", "image pro plus software was used to quantify tube length formation ; the lengths of tubes in 34 randomly selected fields in each of the wells were measured ( 2326 ) .", "endothelial cell migration assay was performed in a modified boyden chamber using a protocol obtained from bd biocoat angiogenesis system ( bd biosciences , san jose , usa ) .", "endothelial cells were trypsinized ; a cell suspension of 4 10 cells / ml was prepared , 250 l of which was added to each of the trans - well inserts . starvation media ( 750 l )", "was added to the lower chamber and incubated overnight . after this , the cells were labeled by incubating with hank 's balanced salt solution medium ( sigma - aldrich , gillingham , u.k . ) containing 4 g / ml calcein - am ( bd biosciences , san jose , usa ) for 90 min .", "the cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation / emission wavelengths of 494/517 nm .", "only those labeled cells that have migrated through the pores of the membrane will be detected ( 2326 ) .", "we studied nf-b activation by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites ( pnf-b - luc ; stratagene , la jolla , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera for 2 h. cells were lysed , and luciferase activities were measured .", "experiments were also performed with tk plasmid ( promega , southampton , u.k . ) as negative control and tumor necrosis factor ( tnf- ) as positive control .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "endothelial cells were lysed with sds sample buffer ( 5 mol / l urea , 0.17 mol / l sds , 0.4 mol / l dithiothreitol , and 50 mmol / l tris - hcl , ph 8.0 ) , mixed , sonicated , boiled , centrifuged ( 5,000 rpm for 2 min ) , and then stored at 80c until use .", "eighty g of each sample , supernatants , and sera were subjected to sds - page ( 8% resolving gel ) and transferred to polyvinylidene fluoride ( pvdf ) membranes .", "pvdf membranes were blocked in tris - buffered saline containing 0.1% tween-20 and 5% bsa for 2 h. the pvdf membranes were then incubated with polyclonal primary rabbit - anti - human antibody for phospho - erk1/2 ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or polyclonal primary rabbit - anti - human antibody for phospho - akt ( ser473 ) ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or monoclonal primary goat - anti - human antibody for crp ( sigma - aldrich , gillingham , u.k . ) ( 1:1,000 dilution ) overnight at 4c . the membranes were washed thoroughly for 60 min with tris - buffered saline0.1% tween before incubation with the secondary anti - rabbit horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) or secondary anti - goat horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) for 1 h at room temperature .", "antibody complexes were visualized using chemiluminescence ( ecl+ ; ge healthcare , little chalfont , u.k . ) . for standardization ,", "the same membranes were stripped and reprobed using polyclonal primary rabbit - anti - human antibodies for total erk1/2 ( cell signaling technology inc .", "the densities were measured using a scanning densitometer coupled to scanning software scion image ( scion corporation , frederick , usa ) .", "standard curves were generated to ensure linearity of signal intensity over the range of protein amounts loaded into gel lanes .", "comparisons of densitometric signal intensities for phospho - erk1/2 , phospho - akt , total erk1/2 , and total akt were made only within this linearity range .", "whitney u test or kruskal wallis anova ( post hoc analysis : dunn 's test ) according to the number of groups compared .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 .", "measurement of carotid imt is a widely accepted tool to provide information about preclinical vascular disease .", "b - mode sonography of the proximal part of the carotid bulb was conducted on both sides , and the segments of the common carotid arteries 1.0 cm proximal were scanned longitudinally with hitachi eub-5000 plus - g ( hitachi ltd . ,", "all women were investigated in a supine position with the head slightly hyperextended and turned away from the side being scanned .", "the image was focused on the posterior wall , and the resolution function was used to magnify the arterial far wall .", "when an optimal image was obtained , it was frozen , and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end - diastolic phase , to minimize variability during the cardiac cycle .", "five measurements were conducted on each side , and the mean imt was calculated as the average of these measurements .", "all measurements were performed by an experienced ultrasound sonographer ( intra - observer coefficient of variation was 6.8% ) who was blinded to the diagnosis of subjects .", "assays for glucose , insulin , cholesterol , triglycerides , luteinizing hormone , follicular stimulating hormone , testosterone , and rostenedione , dehydroepiandrosterone - sulfate , and sex hormone binding globulin were performed using an automated analyzer ( abbott architect , abbott laboratories , abbott park , usa ) .", "the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) score was calculated as previously described ( 21 ) .", "high - sensitivity c - reactive protein ( hs - crp ) was determined immunoturbidimetrically using a modular system random - access analyzer ( roche diagnostics ) .", "omentin-1 levels in sera were measured using a commercially available elisa kit ( axxora , nottingham , u.k . ) , according to manufacturer 's protocol , with an intra - assay coefficient of variation of < 6% .", "immunodepletion of serum omentin-1 and crp were performed using the immunoprecipitation protocol provided by dr .", "one ml of human serum was incubated with mouse - anti - human omentin-1 igg - protein g - sepharose conjugate ( 12 g/100500 g of total protein ) or control mouse igg - protein g - sepharose conjugate ; goat - anti - human crp iga - protein a - sepharose conjugate ( 10 g/100500 g of total protein ) or control goat iga - protein a - sepharose conjugate , mixed overnight at 4c and centrifuged to sediment the conjugated sepharose beads .", "supernatants and sera were subjected to immunoblotting for omentin-1 as previously described ( 13 ) .", "immunoblotting confirmed that these protocols decreased serum omentin-1 and crp by 80% ( data not shown ) , respectively .", "human microvascular endothelial cells ( hmec-1 ) were obtained from the center for disease control , atlanta , usa hmec-1 cells were cultured in mcdb medium ( sigma - aldrich , gillingham , u.k . ) supplemented with 10% fetal calf serum ( sigma - aldrich , gillingham , u.k . ) , 100 iu / ml penicillin ( sigma - aldrich , gillingham , u.k . ) , 100 g / ml streptomycin ( sigma - aldrich ) , 5 ml of 200 mmol / l l - glutamine/500 ml of media , hydrocortisone 2 mol / l , and epidermal growth factor 2 ng / ml ( invitrogen , paisley , u.k . ) at 37c in 5% co2/95% air . prior to each experiment , cells were fed with mcdb with addition of 1% fetal calf serum for 16 h. for endothelial cell signaling experiments , hmecs were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] at different time points ( 0 , 2 , 5 , 10 , 20 , 30 , and 60 min ) .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "angiogenesis was assessed by studying the formation of capillary tubelike structures by culturing hmec-1 cells on growth factor reduced matrigel ( bd biosciences , san jose , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera . vascular endothelial growth factor ( vegf ) served as positive control .", "trypsinized hmec-1 cells were then seeded onto the matrigel - coated plates at a density of 45 10/well in fresh media and incubated at 37c .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "capillary tube formation images were captured with a digital microscope camera system ( olympus , tokyo , japan ) .", "image pro plus software was used to quantify tube length formation ; the lengths of tubes in 34 randomly selected fields in each of the wells were measured ( 2326 ) .", "endothelial cell migration assay was performed in a modified boyden chamber using a protocol obtained from bd biocoat angiogenesis system ( bd biosciences , san jose , usa ) .", "endothelial cells were trypsinized ; a cell suspension of 4 10 cells / ml was prepared , 250 l of which was added to each of the trans - well inserts . starvation media ( 750 l )", "after this , the cells were labeled by incubating with hank 's balanced salt solution medium ( sigma - aldrich , gillingham , u.k . ) containing 4 g / ml calcein - am ( bd biosciences , san jose , usa ) for 90 min .", "the cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation / emission wavelengths of 494/517 nm .", "only those labeled cells that have migrated through the pores of the membrane will be detected ( 2326 ) .", "we studied nf-b activation by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites ( pnf-b - luc ; stratagene , la jolla , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera for 2 h. cells were lysed , and luciferase activities were measured .", "experiments were also performed with tk plasmid ( promega , southampton , u.k . ) as negative control and tumor necrosis factor ( tnf- ) as positive control .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "endothelial cells were lysed with sds sample buffer ( 5 mol / l urea , 0.17 mol / l sds , 0.4 mol / l dithiothreitol , and 50 mmol / l tris - hcl , ph 8.0 ) , mixed , sonicated , boiled , centrifuged ( 5,000 rpm for 2 min ) , and then stored at 80c until use .", "eighty g of each sample , supernatants , and sera were subjected to sds - page ( 8% resolving gel ) and transferred to polyvinylidene fluoride ( pvdf ) membranes .", "pvdf membranes were blocked in tris - buffered saline containing 0.1% tween-20 and 5% bsa for 2 h. the pvdf membranes were then incubated with polyclonal primary rabbit - anti - human antibody for phospho - erk1/2 ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or polyclonal primary rabbit - anti - human antibody for phospho - akt ( ser473 ) ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or monoclonal primary goat - anti - human antibody for crp ( sigma - aldrich , gillingham , u.k . ) ( 1:1,000 dilution ) overnight at 4c .", "the membranes were washed thoroughly for 60 min with tris - buffered saline0.1% tween before incubation with the secondary anti - rabbit horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) or secondary anti - goat horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) for 1 h at room temperature .", "antibody complexes were visualized using chemiluminescence ( ecl+ ; ge healthcare , little chalfont , u.k . ) . for standardization ,", "the same membranes were stripped and reprobed using polyclonal primary rabbit - anti - human antibodies for total erk1/2 ( cell signaling technology inc .", "the densities were measured using a scanning densitometer coupled to scanning software scion image ( scion corporation , frederick , usa ) .", "standard curves were generated to ensure linearity of signal intensity over the range of protein amounts loaded into gel lanes .", "comparisons of densitometric signal intensities for phospho - erk1/2 , phospho - akt , total erk1/2 , and total akt were made only within this linearity range .", "whitney u test or kruskal wallis anova ( post hoc analysis : dunn 's test ) according to the number of groups compared .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 .", "insulin , homa - ir , cholesterol , triglycerides , luteinizing hormone , testosterone , free androgen index , systolic blood pressure , diastolic blood pressure , imt , and hs - crp were significantly higher whereas dehydroepiandrosterone - sulfate and sex hormone binding globulin were significantly lower in pcos women .", "clinical , hormonal , and metabolic features of women with pcos and control subjects data are median ( interquartile range ) .", "free androgen index ( fai ) = t ( nmol / l)/shbg ( nmol / l ) 100 .", "ns = not significant ; lh , luteinizing hormone ; fsh , follicular stimulating hormone ; dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; sbp , systolic blood pressure ; dbp , diastolic blood pressure .", "serum omentin-1 levels were significantly lower in pcos subjects compared with control subjects [ 23.7 ( 20.027.9 ) versus 27.6 ( 25.629.4 ) ng / ml ; p < 0.05 : table 1 ] .", "only 21 women completed the study and were investigated after 6 months of metformin treatment .", "reasons for subjects not completing study 2 were nausea and gastrointestinal side effects ( n = 4 ) , pregnancies ( n = 5 ) , incompliance ( n = 2 ) , and loss of contact ( n = 2 ) . clinical , hormonal , and metabolic features of women with pcos ( n = 21 ) before and after metformin treatment data are median ( interquartile range ) .", "free androgen index ( fai ) = t ( nmol / l)/shbg ( nmol / l ) 100 .", "ns = not significant ; dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; sbp , systolic blood pressure ; dbp , diastolic blood pressure .", "after 6 months of metformin treatment , there was a significant increase in serum omentin-1 [ 23.7 ( 20.027.9 ) versus 59.2 ( 52.160.6 ) ng / ml ; p < 0.01 : table 2 ] .", "also , there were significant decreases in waist - to - hip ratio ( whr ) , testosterone , glucose , homa - ir , and imt .", "spearman rank analyses demonstrated that serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp ( p < 0.05 ) .", "however , when subjected to multiple regression analysis , none of these variables were significantly negatively correlated with serum omentin-1 levels ( p > 0.05 ) . furthermore , we analyzed the correlation between the change in serum omentin-1 levels before and after metformin therapy ( omentin-1 ) and the changes ( ) in other covariates ( table 3 ) .", "we found that omentin-1 was significantly negatively associated with whr , glucose , homa - ir , and hs - crp .", "when subjected to multiple regression analysis , only hs - crp was significantly negatively correlated with omentin-1 ( = 0.526 ; p = 0.036 ) .", "linear regression analysis of variables associated with changes in serum omentin-1 levels ( before and after metformin treatment ) , omentin-1 , in pcos subjects ( n = 21 ) spearman rank correlation was used for calculation of associations between variables .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 .", "multiple regression analysis contained whr , glucose , homa - ir , and hs - crp .", "dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; fai , free androgen index ; sbp , systolic blood pressure ; dbp , diastolic blood pressure .", "given the above , we studied the effects of omentin-1 on in vitro migration and angiogenesis .", "capillary tube formation was optimal at 24 h , after which capillary tubes begin to disintegrate . in vitro migration and angiogenesis at 24 h", "was significantly increased when comparing sera of pcos women to normal control subjects ( figs . 1 and 2 ; p < 0.01 ) . also , in vitro migration and angiogenesis were significantly decreased in pcos women after 6 months of metformin treatment ( figs . 1 and 2 ; *", "* p < 0.01 ) and when omentin-1 ( 200 ng / ml ) or the pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) were added to sera ( figs . 1 and 2 ; * p < 0.05 , * * p < 0.01 ) . furthermore , crp ( 1ug / ml ) and vegf ( 10 ng / ml ) induced in vitro migration , and angiogenesis was significantly decreased by omentin-1 ( figs . 1 and 2 ; * p < 0.05 ) . in vitro tube formation by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . endothelial cell migration by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann - whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 .", "moreover , in vitro migration and angiogenesis were significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( figs . 1 and 2 ;", "nf-b was significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "nf-b was significantly activated by sera from pcos women compared with normal women ( fig . 3 ; * *", "also , nf-b activation was significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "3 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) was added to sera ( fig . 3 ;", "furthermore , crp ( 1 ug / ml ) and tnf- ( 10 ng / ml ) induced nf-b activation was significantly decreased by omentin-1 ( fig .", "* p < 0.05 ) . effects of serum from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and tnf- ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively , on nf-b activation in hmec-1 cells stably transfected with pnf-b - luciferase at 2 h. cells were lysed , and luciferase activities ( rlu ) were measured .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , nf-b activation was significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( fig .", "3 ; p > 0.05 , * p < 0.05 ) . in hmec-1 cells , erk1/2 and akt pathways", "were significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "erk1/2 and akt phosphorylation were significantly increased by sera from pcos women compared with normal women ( fig .", "also , erk1/2 and akt activation were significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "4a and b ; * p < 0.05 , * * p < 0.01 ) . furthermore , akt activation was significantly decreased when omentin-1 ( 200 ng / ml ) was added to sera from pcos women before metformin treatment ( fig .", "4a ) . the detected proteins for phosphorylated erk1/2 , total erk1/2 , phosphorylated akt , and total akthad apparent molecular weights of 44/42 , 44/42 , 60 , and 60 kda ,", "effects of serum from normal control subjects ( n = 39 ) , pcos women ( n = 21 ) , and pcos women after 6 months of metformin treatment with or without addition of omentin-1 ( 200 ng / ml ) ( n = 21 ) on erk1/2 and akt phosphorylation in hmec-1 cells at 2 and 5 min , respectively .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 .", "spearman rank analyses demonstrated that serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp ( p < 0.05 ) .", "however , when subjected to multiple regression analysis , none of these variables were significantly negatively correlated with serum omentin-1 levels ( p > 0.05 ) . furthermore , we analyzed the correlation between the change in serum omentin-1 levels before and after metformin therapy ( omentin-1 ) and the changes ( ) in other covariates ( table 3 ) .", "we found that omentin-1 was significantly negatively associated with whr , glucose , homa - ir , and hs - crp . when subjected to multiple regression analysis ,", "only hs - crp was significantly negatively correlated with omentin-1 ( = 0.526 ; p = 0.036 ) .", "linear regression analysis of variables associated with changes in serum omentin-1 levels ( before and after metformin treatment ) , omentin-1 , in pcos subjects ( n = 21 ) spearman rank correlation was used for calculation of associations between variables .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 .", "multiple regression analysis contained whr , glucose , homa - ir , and hs - crp .", "dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; fai , free androgen index ; sbp , systolic blood pressure ; dbp , diastolic blood pressure .", "given the above , we studied the effects of omentin-1 on in vitro migration and angiogenesis .", "capillary tube formation was optimal at 24 h , after which capillary tubes begin to disintegrate . in vitro migration and angiogenesis at 24 h", "was significantly increased when comparing sera of pcos women to normal control subjects ( figs . 1 and 2 ; p < 0.01 ) .", "also , in vitro migration and angiogenesis were significantly decreased in pcos women after 6 months of metformin treatment ( figs . 1 and 2 ;", "* * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) or the pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) were added to sera ( figs . 1 and 2 ; * p < 0.05 , * * p < 0.01 ) . furthermore , crp ( 1ug / ml ) and vegf ( 10 ng / ml ) induced in vitro migration , and angiogenesis was significantly decreased by omentin-1 ( figs . 1 and 2 ; * p < 0.05 ) . in vitro tube formation by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01", ". endothelial cell migration by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann - whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , in vitro migration and angiogenesis were significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( figs . 1 and 2 ; p > 0.05 , * p < 0.05 ) .", "in hmec-1 cells , nf-b was significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "nf-b was significantly activated by sera from pcos women compared with normal women ( fig . 3 ; * *", "also , nf-b activation was significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "3 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) was added to sera ( fig . 3 ;", "* p < 0.05 ) . furthermore , crp ( 1 ug / ml ) and tnf- ( 10 ng / ml ) induced nf-b activation was significantly decreased by omentin-1 ( fig", "effects of serum from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and tnf- ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively , on nf-b activation in hmec-1 cells stably transfected with pnf-b - luciferase at 2 h. cells were lysed , and luciferase activities ( rlu ) were measured .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , nf-b activation was significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( fig .", "in hmec-1 cells , erk1/2 and akt pathways were significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "erk1/2 and akt phosphorylation were significantly increased by sera from pcos women compared with normal women ( fig .", "also , erk1/2 and akt activation were significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "4a and b ; * p < 0.05 , * * p < 0.01 ) . furthermore , akt activation was significantly decreased when omentin-1 ( 200 ng / ml ) was added to sera from pcos women before metformin treatment ( fig .", "4a ) . the detected proteins for phosphorylated erk1/2 , total erk1/2 , phosphorylated akt , and total akthad apparent molecular weights of 44/42 , 44/42 , 60 , and 60 kda ,", "effects of serum from normal control subjects ( n = 39 ) , pcos women ( n = 21 ) , and pcos women after 6 months of metformin treatment with or without addition of omentin-1 ( 200 ng / ml ) ( n = 21 ) on erk1/2 and akt phosphorylation in hmec-1 cells at 2 and 5 min , respectively .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 .", "also , serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp .", "we report that metformin ( 6 months treatment ; 850 mg twice daily ) significantly increases serum omentin-1 levels with concomitant decreases in insulin resistance and carotid imt in pcos subjects .", "although the change in serum omentin-1 levels were significantly negatively associated with changes in whr , glucose , homa - ir , and hs - crp , only the change of hs - crp was significantly negatively correlated with the change of serum omentin-1 levels when subjected to multiple regression analysis .", "the possible implication is that changes in omentin-1 levels associated with metformin treatment observed in our pcos subjects are more robustly linked to changes in inflammatory rather than metabolic parameters . of relevance", ", there is evidence that inflammatory markers other than crp are elevated in pcos subjects ( 35 ) ; also , studies have indicated metformin 's potential anti - inflammatory effects via modulation of tnf- , il-6 , il-18 , and il-1- levels ( 2729 ) . in pcos women , metformin corrects the associated endocrine and metabolic abnormalities ( 30 ) ; metformin counters ir by inhibiting hepatic glucose production ( 3133 ) .", "therefore , glucose may regulate circulating omentin-1 levels ; however , it is possible that the effect of metformin on omentin-1 levels could be via modulation of other circulating factors .", "we , like others ( 34 ) , observed a significant decrease in testosterone levels after metformin treatment .", "however , treatments of omental adipose tissue explants with gonadal and adrenal steroids revealed no meaningful effects on omentin-1 levels ( 13 ) . it is therefore unlikely that the effects of metformin on serum omentin-1 levels observed in this study are attributable to altered gonadal and adrenal steroids .", "furthermore , functional assays revealed that in vitro migration and angiogenesis were significantly increased by sera from both normal and pcos women ; in pcos women , in vitro migration and angiogenesis was attenuated after 6 months of metformin treatment .", "addition of omentin-1 to sera from pcos women before metformin treatment significantly decreased in vitro migration and angiogenesis .", "omentin-1 also decreased the effects of crp and vegf induced in vitro migration and angiogenesis .", "however , in vitro migration and angiogenesis were significantly decreased in crp but not significantly altered by omentin-1 immunodepleted sera .", "it is important to consider that omentin-1 levels in tissues are unknown ; the levels are likely to be significantly higher than in sera as sequestration of omentin-1 in vascular tissue , in particular , within stromal - vascular cells , has been shown ( 10,35 ) .", "all of these effects appear to be associated with nf-b and akt signaling pathways , both well - established mediators of angiogenesis ( 3638 ) . taken together", ", increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women .", "it is important to bear in mind that metformin may also modulate angiogenesis via the vegf system . of relevance", "the link between angiogenesis and the pathogenesis of atherosclerosis has been well described ( 17 ) . given our data of an increase in circulating omentin-1 levels with concomitant decrease in imt , omentin-1 may have a potential role in the pathogenesis of atherosclerosis .", "omentin-1 , by promoting activation of the akt signaling pathway and in turn modulating the endothelial nitric oxide synthase in endothelial cells , may impact upon endothelial dysfunction , a step that is crucial in the pathogenesis of atherosclerosis ( 40,41 ) .", "our novel data of omentin-1 on angiogenesis in relation to the akt signaling pathway thus clarifies omentin-1 's potentially important role in the pathogenesis of cardiovascular disorders .", "a limitation of our study may relate to the lack of a lean pcos comparator group .", "only 3 of the 21 pcos subjects and 6 of the 39 control subjects had bmi < 25 kg / m ; thus , no meaningful result could be obtained .", "furthermore , given ethical constraints , that is , blood samples could and were only obtained at baseline and after 6 months of metformin treatment , it is therefore difficult to be certain as to whether changes in serum omentin-1 levels precede or follow changes in clinical and hormonal indexes as a consequence of metformin treatment .", "also , as diet and lifestyle modifications were only subjectively assessed , the changes in serum omentin-1 levels may be partially attributable to diet and lifestyle modifications .", "further studies are needed to elucidate this point . additionally , like others ( 42 ) , we used whr , a universally accepted , simple , and cost - effective measure of adiposity as a surrogate marker for central fat mass .", "however , cascella et al . has reported that whr does not distinguish between visceral and subcutaneous fat mass in pcos women ( 43 ) .", "therefore , more accurate and equally cost - effective methods of assessing central obesity are needed .", "finally , given that pcos is a proinflammatory state associated with clustering of cardiovascular risk factors , it would be of importance to study the effect of metformin therapy in the context of omentin-1 biology on reactive oxygen species in pcos women , as reactive oxygen species not only induces tissue damage and inflammation but also is increased in dysmetabolic states including pcos ( 44 ) . in conclusion , we provide evidence that increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women .", "our findings provide novel insights into the relationship between obesity , insulin resistance , and metformin therapy with dysregulated angiogenesis in pcos women in the context of omentin-1 biology ." ]

[ "<S> objectivepolycystic ovary syndrome ( pcos ) is associated with the metabolic syndrome . decreased omentin-1 levels are associated with obesity and diabetes . to study the effects of metformin treatment on omentin-1 levels in pcos subjects and effects of omentin-1 on in vitro migration and angiogenesis.research design and methodsserum omentin-1 </S>", "<S> was measured by elisa . </S>", "<S> angiogenesis was assessed by studying capillary tube formation in human microvascular endothelial cells ( hmec-1 ) on growth factor reduced matrigel . </S>", "<S> endothelial cell migration assay was performed in a modified boyden chamber . </S>", "<S> nuclear factor-b ( nf-b ) was studied by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites . </S>", "<S> akt phosphorylation was assessed by western blotting.resultsserum omentin-1 was significantly lower in pcos women ( p < 0.05 ) . </S>", "<S> after 6 months of metformin treatment , there was a significant increase in serum omentin-1 ( p < 0.01 ) . </S>", "<S> importantly , changes in hs - crp were significantly negatively correlated with changes in serum omentin-1 ( p = 0.036 ) . in vitro migration and angiogenesis </S>", "<S> were significantly increased in serum from pcos women ( p < 0.01 ) compared with matched control subjects ; these effects were significantly attenuated by metformin treatment ( p < 0.01 ) plausibly through the regulation of omentin-1 levels via nf-b and akt pathways . </S>", "<S> crp and vegf induced in vitro migration , and angiogenesis was significantly decreased by omentin-1.conclusionsincreases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women . </S>" ]

[ "RESEARCH DESIGN AND METHODS", "Intima media thickness.", "Biochemical and hormonal analysis.", "Immunodepletion of serum omentin-1 and CRP.", "Endothelial cell culture.", "In vitro angiogenesis assay.", "In vitro migration assay.", "NF-B activation.", "Western blotting.", "Statistics.", "RESULTS", "Correlation of omentin-1 with covariates.", "Effects of omentin-1 on in vitro migration and angiogenesis.", "Effects of omentin-1 on NF-B activation in human microvascular endothelial cells.", "Effects of omentin-1 on Erk1/2 and Akt signaling pathways in human microvascular endothelial cells.", "DISCUSSION" ]

[ [ "all pcos patients met all three criteria of the revised 2003 rotterdam european society of human reproduction and embryology ( eshre)/american society of reproductive medicine ( asrm ) pcos consensus workshop group diagnostic criteria .", "the three criteria are 1 ) oligo- and/or anovulation , 2 ) clinical and/or biochemical signs of hyperandrogenism , and 3 ) polycystic ovaries ( 20 ) .", "furthermore , all subjects in the control arm had normal findings on pelvic ultrasound scan , regular periods , and no hirsutism / acne .", "exclusion criteria for the study included age > 40 years , known cardiovascular disease , thyroid disease , neoplasms , current smoking , diabetes , hypertension ( blood pressure > 140/90 mmhg ) , and renal impairment ( serum creatinine > 120 mol / l ) .", "none of these women were on any medications for at least 6 months prior to the study , including oral contraceptives , glucocorticoids , ovulation induction agents , antidiabetic and antiobesity drugs , and estrogenic , antiandrogenic , or antihypertensive medication .", "blood pressure was measured in a sitting position within a quiet and calm environment after a rest of at least 5 min .", "subjects were outpatients of the department of reproductive medicine and gynaecological endocrinology of magdeburg university and the department of obstetrics and gynaecology of martin - luther - university halle . the metabolic study was performed in the outpatient department of endocrinology and metabolism of magdeburg university .", "blood samples were collected between 0800 and 0900 h , after a 3-day normal carbohydrate diet and an overnight fast .", "blood samples were drawn into serum separator tubes that contain no additives or anticoagulants , allowed to clot , centrifuged ( 3,000 rpm for 10 min ) to separate sera , and stored at 80c . a treatment with metformin in an off - label use ", "was offered to all pcos women independently from the results of insulin sensitivity testing . in those pcos women that agreed ,", "therapy was initiated after basal assessment , and the dose of metformin was increased to a maintenance dose of 850 mg twice daily .", "carotid intima media thickness ( imt ) was measured in pcos subjects , before and after metformin ( see below ) .", "a total of 83 women of caucasian origin with pcos were recruited , 49 of which did not participate in the metformin arm of the study .", "the baseline characteristics of the 49 pcos women were comparable to the 34 pcos women who participated in the metformin arm of the study .", "furthermore , the 21 women who completed the metformin arm of the study were comparable to the 13 pcos women who did not complete the metformin arm of the study . for the purposes of elucidating the effects of metformin in pcos women ,", "the study design was approved by the local research ethics committee of the university of magdeburg , and written informed consent was obtained from all participants , in accordance with the guidelines in the declaration of helsinki 2000 .", "measurement of carotid imt is a widely accepted tool to provide information about preclinical vascular disease .", "b - mode sonography of the proximal part of the carotid bulb was conducted on both sides , and the segments of the common carotid arteries 1.0 cm proximal were scanned longitudinally with hitachi eub-5000 plus - g ( hitachi ltd . ,", "all women were investigated in a supine position with the head slightly hyperextended and turned away from the side being scanned .", "the image was focused on the posterior wall , and the resolution function was used to magnify the arterial far wall .", "when an optimal image was obtained , it was frozen , and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end - diastolic phase , to minimize variability during the cardiac cycle .", "five measurements were conducted on each side , and the mean imt was calculated as the average of these measurements .", "all measurements were performed by an experienced ultrasound sonographer ( intra - observer coefficient of variation was 6.8% ) who was blinded to the diagnosis of subjects .", "assays for glucose , insulin , cholesterol , triglycerides , luteinizing hormone , follicular stimulating hormone , testosterone , and rostenedione , dehydroepiandrosterone - sulfate , and sex hormone binding globulin were performed using an automated analyzer ( abbott architect , abbott laboratories , abbott park , usa ) .", "the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) score was calculated as previously described ( 21 ) .", "high - sensitivity c - reactive protein ( hs - crp ) was determined immunoturbidimetrically using a modular system random - access analyzer ( roche diagnostics ) .", "omentin-1 levels in sera were measured using a commercially available elisa kit ( axxora , nottingham , u.k . ) , according to manufacturer 's protocol , with an intra - assay coefficient of variation of < 6% .", "immunodepletion of serum omentin-1 and crp were performed using the immunoprecipitation protocol provided by dr .", "one ml of human serum was incubated with mouse - anti - human omentin-1 igg - protein g - sepharose conjugate ( 12 g/100500 g of total protein ) or control mouse igg - protein g - sepharose conjugate ; goat - anti - human crp iga - protein a - sepharose conjugate ( 10 g/100500 g of total protein ) or control goat iga - protein a - sepharose conjugate , mixed overnight at 4c and centrifuged to sediment the conjugated sepharose beads .", "supernatants and sera were subjected to immunoblotting for omentin-1 as previously described ( 13 ) .", "immunoblotting confirmed that these protocols decreased serum omentin-1 and crp by 80% ( data not shown ) , respectively .", "human microvascular endothelial cells ( hmec-1 ) were obtained from the center for disease control , atlanta , usa hmec-1 cells were cultured in mcdb medium ( sigma - aldrich , gillingham , u.k . ) supplemented with 10% fetal calf serum ( sigma - aldrich , gillingham , u.k . ) , 100 iu / ml penicillin ( sigma - aldrich , gillingham , u.k . ) , 100 g / ml streptomycin ( sigma - aldrich ) , 5 ml of 200 mmol / l l - glutamine/500 ml of media , hydrocortisone 2 mol / l , and epidermal growth factor 2 ng / ml ( invitrogen , paisley , u.k . ) at 37c in 5% co2/95% air . prior to each experiment , cells were fed with mcdb with addition of 1% fetal calf serum for 16 h. for endothelial cell signaling experiments , hmecs were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] at different time points ( 0 , 2 , 5 , 10 , 20 , 30 , and 60 min ) .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "angiogenesis was assessed by studying the formation of capillary tubelike structures by culturing hmec-1 cells on growth factor reduced matrigel ( bd biosciences , san jose , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera .", "trypsinized hmec-1 cells were then seeded onto the matrigel - coated plates at a density of 45 10/well in fresh media and incubated at 37c .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "capillary tube formation images were captured with a digital microscope camera system ( olympus , tokyo , japan ) .", "image pro plus software was used to quantify tube length formation ; the lengths of tubes in 34 randomly selected fields in each of the wells were measured ( 2326 ) .", "endothelial cell migration assay was performed in a modified boyden chamber using a protocol obtained from bd biocoat angiogenesis system ( bd biosciences , san jose , usa ) .", "endothelial cells were trypsinized ; a cell suspension of 4 10 cells / ml was prepared , 250 l of which was added to each of the trans - well inserts . starvation media ( 750 l )", "was added to the lower chamber and incubated overnight . after this , the cells were labeled by incubating with hank 's balanced salt solution medium ( sigma - aldrich , gillingham , u.k . ) containing 4 g / ml calcein - am ( bd biosciences , san jose , usa ) for 90 min .", "the cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation / emission wavelengths of 494/517 nm .", "only those labeled cells that have migrated through the pores of the membrane will be detected ( 2326 ) .", "we studied nf-b activation by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites ( pnf-b - luc ; stratagene , la jolla , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera for 2 h. cells were lysed , and luciferase activities were measured .", "experiments were also performed with tk plasmid ( promega , southampton , u.k . ) as negative control and tumor necrosis factor ( tnf- ) as positive control .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "endothelial cells were lysed with sds sample buffer ( 5 mol / l urea , 0.17 mol / l sds , 0.4 mol / l dithiothreitol , and 50 mmol / l tris - hcl , ph 8.0 ) , mixed , sonicated , boiled , centrifuged ( 5,000 rpm for 2 min ) , and then stored at 80c until use .", "eighty g of each sample , supernatants , and sera were subjected to sds - page ( 8% resolving gel ) and transferred to polyvinylidene fluoride ( pvdf ) membranes .", "pvdf membranes were blocked in tris - buffered saline containing 0.1% tween-20 and 5% bsa for 2 h. the pvdf membranes were then incubated with polyclonal primary rabbit - anti - human antibody for phospho - erk1/2 ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or polyclonal primary rabbit - anti - human antibody for phospho - akt ( ser473 ) ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or monoclonal primary goat - anti - human antibody for crp ( sigma - aldrich , gillingham , u.k . ) ( 1:1,000 dilution ) overnight at 4c . the membranes were washed thoroughly for 60 min with tris - buffered saline0.1% tween before incubation with the secondary anti - rabbit horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) or secondary anti - goat horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) for 1 h at room temperature .", "antibody complexes were visualized using chemiluminescence ( ecl+ ; ge healthcare , little chalfont , u.k . ) . for standardization ,", "the same membranes were stripped and reprobed using polyclonal primary rabbit - anti - human antibodies for total erk1/2 ( cell signaling technology inc .", "the densities were measured using a scanning densitometer coupled to scanning software scion image ( scion corporation , frederick , usa ) .", "standard curves were generated to ensure linearity of signal intensity over the range of protein amounts loaded into gel lanes .", "comparisons of densitometric signal intensities for phospho - erk1/2 , phospho - akt , total erk1/2 , and total akt were made only within this linearity range .", "whitney u test or kruskal wallis anova ( post hoc analysis : dunn 's test ) according to the number of groups compared .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 ." ], [ "measurement of carotid imt is a widely accepted tool to provide information about preclinical vascular disease .", "b - mode sonography of the proximal part of the carotid bulb was conducted on both sides , and the segments of the common carotid arteries 1.0 cm proximal were scanned longitudinally with hitachi eub-5000 plus - g ( hitachi ltd . ,", "all women were investigated in a supine position with the head slightly hyperextended and turned away from the side being scanned .", "the image was focused on the posterior wall , and the resolution function was used to magnify the arterial far wall .", "when an optimal image was obtained , it was frozen , and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end - diastolic phase , to minimize variability during the cardiac cycle .", "five measurements were conducted on each side , and the mean imt was calculated as the average of these measurements .", "all measurements were performed by an experienced ultrasound sonographer ( intra - observer coefficient of variation was 6.8% ) who was blinded to the diagnosis of subjects ." ], [ "assays for glucose , insulin , cholesterol , triglycerides , luteinizing hormone , follicular stimulating hormone , testosterone , and rostenedione , dehydroepiandrosterone - sulfate , and sex hormone binding globulin were performed using an automated analyzer ( abbott architect , abbott laboratories , abbott park , usa ) .", "the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) score was calculated as previously described ( 21 ) .", "high - sensitivity c - reactive protein ( hs - crp ) was determined immunoturbidimetrically using a modular system random - access analyzer ( roche diagnostics ) .", "omentin-1 levels in sera were measured using a commercially available elisa kit ( axxora , nottingham , u.k . ) , according to manufacturer 's protocol , with an intra - assay coefficient of variation of < 6% ." ], [ "immunodepletion of serum omentin-1 and crp were performed using the immunoprecipitation protocol provided by dr .", "one ml of human serum was incubated with mouse - anti - human omentin-1 igg - protein g - sepharose conjugate ( 12 g/100500 g of total protein ) or control mouse igg - protein g - sepharose conjugate ; goat - anti - human crp iga - protein a - sepharose conjugate ( 10 g/100500 g of total protein ) or control goat iga - protein a - sepharose conjugate , mixed overnight at 4c and centrifuged to sediment the conjugated sepharose beads .", "supernatants and sera were subjected to immunoblotting for omentin-1 as previously described ( 13 ) .", "immunoblotting confirmed that these protocols decreased serum omentin-1 and crp by 80% ( data not shown ) , respectively ." ], [ "human microvascular endothelial cells ( hmec-1 ) were obtained from the center for disease control , atlanta , usa hmec-1 cells were cultured in mcdb medium ( sigma - aldrich , gillingham , u.k . ) supplemented with 10% fetal calf serum ( sigma - aldrich , gillingham , u.k . ) , 100 iu / ml penicillin ( sigma - aldrich , gillingham , u.k . ) , 100 g / ml streptomycin ( sigma - aldrich ) , 5 ml of 200 mmol / l l - glutamine/500 ml of media , hydrocortisone 2 mol / l , and epidermal growth factor 2 ng / ml ( invitrogen , paisley , u.k . ) at 37c in 5% co2/95% air . prior to each experiment , cells were fed with mcdb with addition of 1% fetal calf serum for 16 h. for endothelial cell signaling experiments , hmecs were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] at different time points ( 0 , 2 , 5 , 10 , 20 , 30 , and 60 min ) .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point ." ], [ "angiogenesis was assessed by studying the formation of capillary tubelike structures by culturing hmec-1 cells on growth factor reduced matrigel ( bd biosciences , san jose , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera . vascular endothelial growth factor ( vegf ) served as positive control .", "trypsinized hmec-1 cells were then seeded onto the matrigel - coated plates at a density of 45 10/well in fresh media and incubated at 37c .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "capillary tube formation images were captured with a digital microscope camera system ( olympus , tokyo , japan ) .", "image pro plus software was used to quantify tube length formation ; the lengths of tubes in 34 randomly selected fields in each of the wells were measured ( 2326 ) ." ], [ "endothelial cell migration assay was performed in a modified boyden chamber using a protocol obtained from bd biocoat angiogenesis system ( bd biosciences , san jose , usa ) .", "endothelial cells were trypsinized ; a cell suspension of 4 10 cells / ml was prepared , 250 l of which was added to each of the trans - well inserts . starvation media ( 750 l )", "after this , the cells were labeled by incubating with hank 's balanced salt solution medium ( sigma - aldrich , gillingham , u.k . ) containing 4 g / ml calcein - am ( bd biosciences , san jose , usa ) for 90 min .", "the cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point .", "fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation / emission wavelengths of 494/517 nm .", "only those labeled cells that have migrated through the pores of the membrane will be detected ( 2326 ) ." ], [ "we studied nf-b activation by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites ( pnf-b - luc ; stratagene , la jolla , usa ) .", "hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera for 2 h. cells were lysed , and luciferase activities were measured .", "experiments were also performed with tk plasmid ( promega , southampton , u.k . ) as negative control and tumor necrosis factor ( tnf- ) as positive control .", "dose- and time - dependent experiments were performed to determine the optimum concentration and time point ." ], [ "endothelial cells were lysed with sds sample buffer ( 5 mol / l urea , 0.17 mol / l sds , 0.4 mol / l dithiothreitol , and 50 mmol / l tris - hcl , ph 8.0 ) , mixed , sonicated , boiled , centrifuged ( 5,000 rpm for 2 min ) , and then stored at 80c until use .", "eighty g of each sample , supernatants , and sera were subjected to sds - page ( 8% resolving gel ) and transferred to polyvinylidene fluoride ( pvdf ) membranes .", "pvdf membranes were blocked in tris - buffered saline containing 0.1% tween-20 and 5% bsa for 2 h. the pvdf membranes were then incubated with polyclonal primary rabbit - anti - human antibody for phospho - erk1/2 ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or polyclonal primary rabbit - anti - human antibody for phospho - akt ( ser473 ) ( cell signaling technology inc .", ", beverly , usa ) ( 1:1,000 dilution ) or monoclonal primary goat - anti - human antibody for crp ( sigma - aldrich , gillingham , u.k . ) ( 1:1,000 dilution ) overnight at 4c .", "the membranes were washed thoroughly for 60 min with tris - buffered saline0.1% tween before incubation with the secondary anti - rabbit horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) or secondary anti - goat horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) for 1 h at room temperature .", "antibody complexes were visualized using chemiluminescence ( ecl+ ; ge healthcare , little chalfont , u.k . ) . for standardization ,", "the same membranes were stripped and reprobed using polyclonal primary rabbit - anti - human antibodies for total erk1/2 ( cell signaling technology inc .", "the densities were measured using a scanning densitometer coupled to scanning software scion image ( scion corporation , frederick , usa ) .", "standard curves were generated to ensure linearity of signal intensity over the range of protein amounts loaded into gel lanes .", "comparisons of densitometric signal intensities for phospho - erk1/2 , phospho - akt , total erk1/2 , and total akt were made only within this linearity range ." ], [ "whitney u test or kruskal wallis anova ( post hoc analysis : dunn 's test ) according to the number of groups compared .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 ." ], [ "insulin , homa - ir , cholesterol , triglycerides , luteinizing hormone , testosterone , free androgen index , systolic blood pressure , diastolic blood pressure , imt , and hs - crp were significantly higher whereas dehydroepiandrosterone - sulfate and sex hormone binding globulin were significantly lower in pcos women .", "clinical , hormonal , and metabolic features of women with pcos and control subjects data are median ( interquartile range ) .", "free androgen index ( fai ) = t ( nmol / l)/shbg ( nmol / l ) 100 .", "ns = not significant ; lh , luteinizing hormone ; fsh , follicular stimulating hormone ; dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; sbp , systolic blood pressure ; dbp , diastolic blood pressure .", "serum omentin-1 levels were significantly lower in pcos subjects compared with control subjects [ 23.7 ( 20.027.9 ) versus 27.6 ( 25.629.4 ) ng / ml ; p < 0.05 : table 1 ] .", "only 21 women completed the study and were investigated after 6 months of metformin treatment .", "reasons for subjects not completing study 2 were nausea and gastrointestinal side effects ( n = 4 ) , pregnancies ( n = 5 ) , incompliance ( n = 2 ) , and loss of contact ( n = 2 ) . clinical , hormonal , and metabolic features of women with pcos ( n = 21 ) before and after metformin treatment data are median ( interquartile range ) .", "free androgen index ( fai ) = t ( nmol / l)/shbg ( nmol / l ) 100 .", "ns = not significant ; dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; sbp , systolic blood pressure ; dbp , diastolic blood pressure .", "after 6 months of metformin treatment , there was a significant increase in serum omentin-1 [ 23.7 ( 20.027.9 ) versus 59.2 ( 52.160.6 ) ng / ml ; p < 0.01 : table 2 ] .", "also , there were significant decreases in waist - to - hip ratio ( whr ) , testosterone , glucose , homa - ir , and imt .", "spearman rank analyses demonstrated that serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp ( p < 0.05 ) .", "however , when subjected to multiple regression analysis , none of these variables were significantly negatively correlated with serum omentin-1 levels ( p > 0.05 ) . furthermore , we analyzed the correlation between the change in serum omentin-1 levels before and after metformin therapy ( omentin-1 ) and the changes ( ) in other covariates ( table 3 ) .", "we found that omentin-1 was significantly negatively associated with whr , glucose , homa - ir , and hs - crp .", "when subjected to multiple regression analysis , only hs - crp was significantly negatively correlated with omentin-1 ( = 0.526 ; p = 0.036 ) .", "linear regression analysis of variables associated with changes in serum omentin-1 levels ( before and after metformin treatment ) , omentin-1 , in pcos subjects ( n = 21 ) spearman rank correlation was used for calculation of associations between variables .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 .", "multiple regression analysis contained whr , glucose , homa - ir , and hs - crp .", "dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; fai , free androgen index ; sbp , systolic blood pressure ; dbp , diastolic blood pressure .", "given the above , we studied the effects of omentin-1 on in vitro migration and angiogenesis .", "capillary tube formation was optimal at 24 h , after which capillary tubes begin to disintegrate . in vitro migration and angiogenesis at 24 h", "was significantly increased when comparing sera of pcos women to normal control subjects ( figs . 1 and 2 ; p < 0.01 ) . also , in vitro migration and angiogenesis were significantly decreased in pcos women after 6 months of metformin treatment ( figs . 1 and 2 ; *", "* p < 0.01 ) and when omentin-1 ( 200 ng / ml ) or the pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) were added to sera ( figs . 1 and 2 ; * p < 0.05 , * * p < 0.01 ) . furthermore , crp ( 1ug / ml ) and vegf ( 10 ng / ml ) induced in vitro migration , and angiogenesis was significantly decreased by omentin-1 ( figs . 1 and 2 ; * p < 0.05 ) . in vitro tube formation by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . endothelial cell migration by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann - whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 .", "moreover , in vitro migration and angiogenesis were significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( figs . 1 and 2 ;", "nf-b was significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "nf-b was significantly activated by sera from pcos women compared with normal women ( fig . 3 ; * *", "also , nf-b activation was significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "3 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) was added to sera ( fig . 3 ;", "furthermore , crp ( 1 ug / ml ) and tnf- ( 10 ng / ml ) induced nf-b activation was significantly decreased by omentin-1 ( fig .", "* p < 0.05 ) . effects of serum from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and tnf- ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively , on nf-b activation in hmec-1 cells stably transfected with pnf-b - luciferase at 2 h. cells were lysed , and luciferase activities ( rlu ) were measured .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , nf-b activation was significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( fig .", "3 ; p > 0.05 , * p < 0.05 ) . in hmec-1 cells , erk1/2 and akt pathways", "were significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "erk1/2 and akt phosphorylation were significantly increased by sera from pcos women compared with normal women ( fig .", "also , erk1/2 and akt activation were significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "4a and b ; * p < 0.05 , * * p < 0.01 ) . furthermore , akt activation was significantly decreased when omentin-1 ( 200 ng / ml ) was added to sera from pcos women before metformin treatment ( fig .", "4a ) . the detected proteins for phosphorylated erk1/2 , total erk1/2 , phosphorylated akt , and total akthad apparent molecular weights of 44/42 , 44/42 , 60 , and 60 kda ,", "effects of serum from normal control subjects ( n = 39 ) , pcos women ( n = 21 ) , and pcos women after 6 months of metformin treatment with or without addition of omentin-1 ( 200 ng / ml ) ( n = 21 ) on erk1/2 and akt phosphorylation in hmec-1 cells at 2 and 5 min , respectively .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 ." ], [ "spearman rank analyses demonstrated that serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp ( p < 0.05 ) .", "however , when subjected to multiple regression analysis , none of these variables were significantly negatively correlated with serum omentin-1 levels ( p > 0.05 ) . furthermore , we analyzed the correlation between the change in serum omentin-1 levels before and after metformin therapy ( omentin-1 ) and the changes ( ) in other covariates ( table 3 ) .", "we found that omentin-1 was significantly negatively associated with whr , glucose , homa - ir , and hs - crp . when subjected to multiple regression analysis ,", "only hs - crp was significantly negatively correlated with omentin-1 ( = 0.526 ; p = 0.036 ) .", "linear regression analysis of variables associated with changes in serum omentin-1 levels ( before and after metformin treatment ) , omentin-1 , in pcos subjects ( n = 21 ) spearman rank correlation was used for calculation of associations between variables .", "if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 .", "multiple regression analysis contained whr , glucose , homa - ir , and hs - crp .", "dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone binding globulin ; fai , free androgen index ; sbp , systolic blood pressure ; dbp , diastolic blood pressure ." ], [ "given the above , we studied the effects of omentin-1 on in vitro migration and angiogenesis .", "capillary tube formation was optimal at 24 h , after which capillary tubes begin to disintegrate . in vitro migration and angiogenesis at 24 h", "was significantly increased when comparing sera of pcos women to normal control subjects ( figs . 1 and 2 ; p < 0.01 ) .", "also , in vitro migration and angiogenesis were significantly decreased in pcos women after 6 months of metformin treatment ( figs . 1 and 2 ;", "* * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) or the pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) were added to sera ( figs . 1 and 2 ; * p < 0.05 , * * p < 0.01 ) . furthermore , crp ( 1ug / ml ) and vegf ( 10 ng / ml ) induced in vitro migration , and angiogenesis was significantly decreased by omentin-1 ( figs . 1 and 2 ; * p < 0.05 ) . in vitro tube formation by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01", ". endothelial cell migration by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively .", "data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann - whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , in vitro migration and angiogenesis were significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( figs . 1 and 2 ; p > 0.05 , * p < 0.05 ) ." ], [ "in hmec-1 cells , nf-b was significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "nf-b was significantly activated by sera from pcos women compared with normal women ( fig . 3 ; * *", "also , nf-b activation was significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "3 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) was added to sera ( fig . 3 ;", "* p < 0.05 ) . furthermore , crp ( 1 ug / ml ) and tnf- ( 10 ng / ml ) induced nf-b activation was significantly decreased by omentin-1 ( fig", "effects of serum from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and tnf- ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively , on nf-b activation in hmec-1 cells stably transfected with pnf-b - luciferase at 2 h. cells were lysed , and luciferase activities ( rlu ) were measured .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , nf-b activation was significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( fig ." ], [ "in hmec-1 cells , erk1/2 and akt pathways were significantly activated by sera from normal and pcos women compared with control subjects ( fig .", "erk1/2 and akt phosphorylation were significantly increased by sera from pcos women compared with normal women ( fig .", "also , erk1/2 and akt activation were significantly decreased in pcos women after 6 months of metformin treatment ( fig .", "4a and b ; * p < 0.05 , * * p < 0.01 ) . furthermore , akt activation was significantly decreased when omentin-1 ( 200 ng / ml ) was added to sera from pcos women before metformin treatment ( fig .", "4a ) . the detected proteins for phosphorylated erk1/2 , total erk1/2 , phosphorylated akt , and total akthad apparent molecular weights of 44/42 , 44/42 , 60 , and 60 kda ,", "effects of serum from normal control subjects ( n = 39 ) , pcos women ( n = 21 ) , and pcos women after 6 months of metformin treatment with or without addition of omentin-1 ( 200 ng / ml ) ( n = 21 ) on erk1/2 and akt phosphorylation in hmec-1 cells at 2 and 5 min , respectively .", "data are expressed as % difference of median of basal [ medians ( interquartile range ) ] .", "wallis anova ( post hoc analysis : dunn 's test ) and mann whitney u test , respectively .", "p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 ." ], [ "also , serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp .", "we report that metformin ( 6 months treatment ; 850 mg twice daily ) significantly increases serum omentin-1 levels with concomitant decreases in insulin resistance and carotid imt in pcos subjects .", "although the change in serum omentin-1 levels were significantly negatively associated with changes in whr , glucose , homa - ir , and hs - crp , only the change of hs - crp was significantly negatively correlated with the change of serum omentin-1 levels when subjected to multiple regression analysis .", "the possible implication is that changes in omentin-1 levels associated with metformin treatment observed in our pcos subjects are more robustly linked to changes in inflammatory rather than metabolic parameters . of relevance", ", there is evidence that inflammatory markers other than crp are elevated in pcos subjects ( 35 ) ; also , studies have indicated metformin 's potential anti - inflammatory effects via modulation of tnf- , il-6 , il-18 , and il-1- levels ( 2729 ) . in pcos women , metformin corrects the associated endocrine and metabolic abnormalities ( 30 ) ; metformin counters ir by inhibiting hepatic glucose production ( 3133 ) .", "therefore , glucose may regulate circulating omentin-1 levels ; however , it is possible that the effect of metformin on omentin-1 levels could be via modulation of other circulating factors .", "we , like others ( 34 ) , observed a significant decrease in testosterone levels after metformin treatment .", "however , treatments of omental adipose tissue explants with gonadal and adrenal steroids revealed no meaningful effects on omentin-1 levels ( 13 ) . it is therefore unlikely that the effects of metformin on serum omentin-1 levels observed in this study are attributable to altered gonadal and adrenal steroids .", "furthermore , functional assays revealed that in vitro migration and angiogenesis were significantly increased by sera from both normal and pcos women ; in pcos women , in vitro migration and angiogenesis was attenuated after 6 months of metformin treatment .", "addition of omentin-1 to sera from pcos women before metformin treatment significantly decreased in vitro migration and angiogenesis .", "omentin-1 also decreased the effects of crp and vegf induced in vitro migration and angiogenesis .", "however , in vitro migration and angiogenesis were significantly decreased in crp but not significantly altered by omentin-1 immunodepleted sera .", "it is important to consider that omentin-1 levels in tissues are unknown ; the levels are likely to be significantly higher than in sera as sequestration of omentin-1 in vascular tissue , in particular , within stromal - vascular cells , has been shown ( 10,35 ) .", "all of these effects appear to be associated with nf-b and akt signaling pathways , both well - established mediators of angiogenesis ( 3638 ) . taken together", ", increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women .", "it is important to bear in mind that metformin may also modulate angiogenesis via the vegf system . of relevance", "the link between angiogenesis and the pathogenesis of atherosclerosis has been well described ( 17 ) . given our data of an increase in circulating omentin-1 levels with concomitant decrease in imt , omentin-1 may have a potential role in the pathogenesis of atherosclerosis .", "omentin-1 , by promoting activation of the akt signaling pathway and in turn modulating the endothelial nitric oxide synthase in endothelial cells , may impact upon endothelial dysfunction , a step that is crucial in the pathogenesis of atherosclerosis ( 40,41 ) .", "our novel data of omentin-1 on angiogenesis in relation to the akt signaling pathway thus clarifies omentin-1 's potentially important role in the pathogenesis of cardiovascular disorders .", "a limitation of our study may relate to the lack of a lean pcos comparator group .", "only 3 of the 21 pcos subjects and 6 of the 39 control subjects had bmi < 25 kg / m ; thus , no meaningful result could be obtained .", "furthermore , given ethical constraints , that is , blood samples could and were only obtained at baseline and after 6 months of metformin treatment , it is therefore difficult to be certain as to whether changes in serum omentin-1 levels precede or follow changes in clinical and hormonal indexes as a consequence of metformin treatment .", "also , as diet and lifestyle modifications were only subjectively assessed , the changes in serum omentin-1 levels may be partially attributable to diet and lifestyle modifications .", "further studies are needed to elucidate this point . additionally , like others ( 42 ) , we used whr , a universally accepted , simple , and cost - effective measure of adiposity as a surrogate marker for central fat mass .", "however , cascella et al . has reported that whr does not distinguish between visceral and subcutaneous fat mass in pcos women ( 43 ) .", "therefore , more accurate and equally cost - effective methods of assessing central obesity are needed .", "finally , given that pcos is a proinflammatory state associated with clustering of cardiovascular risk factors , it would be of importance to study the effect of metformin therapy in the context of omentin-1 biology on reactive oxygen species in pcos women , as reactive oxygen species not only induces tissue damage and inflammation but also is increased in dysmetabolic states including pcos ( 44 ) . in conclusion , we provide evidence that increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women .", "our findings provide novel insights into the relationship between obesity , insulin resistance , and metformin therapy with dysregulated angiogenesis in pcos women in the context of omentin-1 biology ." ] ]

[ "the term neuroendocrine reflects the origin of endocrine cells from the embryonic neural crest .", "neuroendocrine cells are characterized by their ability to produce and secrete different peptides and neuroamines .", "they aggregate in classical endocrine glands ( e.g. hypothalamus and the pituitary gland ) or in the diffuse neuroendocrine system distributed throughout the body , such as the gastrointestinal tract and lungs .", "gastroenteropancreatic neuroendocrine neoplasms ( gep - nens ) are a heterogeneous group of neoplasms defined as malignant transformation of neuroendocrine cells in the diffuse neuroendocrine system that regulates secretion , absorption and motility of the gastrointestinal tract .", "the incidence of gep - nens continues to exhibit a persistent increase ; the most recent epidemiological study from the us surveillance shows that the overall incidence of gep - nens increased from 1 to 3.65 cases per 100,000 .", "a genetic etiology of gep - nens has been well documented in around 5 - 10% of cases as part of hereditary syndromes such as multiple endocrine neoplasia type 1 , von hippel - lindau syndrome , neurofibromatosis 1 or tuberous sclerosis syndrome .", "these tumors commonly present with a wide spectrum of clinical manifestations according to site of origin and biological behavior .", "they can present with abdominal pain , weight loss , obstructive jaundice and intestinal obstruction .", "moreover , the symptoms related to hormone secretion include flushing , diarrhea , wheezing , hypoglycemia , cardiovascular and peptic ulcers disease .", "the diagnosis is based on histological features , including tumor differentiation and grade , in addition to immunohistochemical stains for general endocrine markers , including chromogranins , synaptophysin and neuron - specific enolase .", "tumor markers such as chromogranin a , pancreatic polypeptide , serum neuron - specific enolase and subunit of glycoprotein hormones have been used .", "it is useful for staging , prognosis and follow - up , since the serum concentration correlates with the tumor mass . in the presence of symptoms or syndrome suspicious of functional nens , biochemical testing for peptide hypersecretion", "should be ordered , e.g. gastrin level for patients with zollinger - ellison syndrome or insulin for hypoglycemic syndromes .", "imaging studies are an important tool in nen workup ; abdominal ultrasound , computed tomography and magnetic resonance imaging have a high sensitivity and specificity in detecting many types of primary nens .", "functional imaging such as in - pentetreotide ( octreoscan ) or i - metaiodobenzylguanidine ( mibg ) plays a critical role in the diagnosis and in establishing the eligibility for therapy . combining functional imaging with computed tomography or magnetic resonance imaging is superior for staging and detection of primary tumors of undetermined location .", "gep - nens can be classified according to the site of embryologic origin ( foregut , midgut and hindgut ) , grade , differentiation or the hormones produced .", "the grade refers to the biological aggressiveness of the neoplasm which is defined by the number of mitoses per 10 high - power microscopic fields or per 2 mm ( mitotic rate ) , or as the percentage of tumor cells that immunolabel for the ki-67 antigen ( ki-67 index ) . while the differentiation refers to the extent to which the neoplastic cells resemble their non - neoplastic counterparts , in 2010 , the world health organization proposed the most widely accepted classification : ( 1 ) neuroendocrine tumor grade 1 ( g1 ) , ( 2 ) neuroendocrine tumor grade 2 ( g2 ) , ( 3 ) neuroendocrine carcinoma ( nec ) , grade 3 ( g3 ) , and ( 4 ) mixed adenoneuroendocrine carcinoma .", "also , nens are divided into well - differentiated and poorly differentiated ( table 1 ) . in general ,", "gep - nens are staged according to the primary tumor ( t ) , lymph node involvement ( n ) and distant metastases ( m ) tnm system , similar to other types of carcinomas .", "medical therapy of inoperable or metastasized nens includes multimodality options such as long - acting somatostatin analogs , -interferon , systemic chemotherapy and ablation therapy .", "there is no established regimen due to heterogeneity of the studies , including various grades , sites and inconsistent response criteria .", "novel therapies like mammalian target of rapamycin ( mtor ) , tyrosine kinase and angiogenesis inhibitors have showing promising results in clinical trials . we herein report a case of esophageal large - cell necs and review the published literature on this rare disease .", "a 66-year - old saudi female housewife with a background of diabetes , hypertension and hypothyroidism was seen initially by a gastroenterologist .", "she was suffering from progressive difficulty in swallowing , mainly of solid food , with a feeling of food stuck in the middle of her chest for more than 6 months .", "she denied any painful swallowing , oral thrush , chocking attacks , food impaction or nasal regurgitation .", "she denied any abdominal pain , distension , rectal bleeding or change in bowel habits .", "she had lost about 3 kg from a baseline weight of 68 kg despite normal appetite .", "her history was unremarkable for esophageal disease , tuberculosis contact , neck swelling or radiation exposure .", "the patient underwent esophagogastroduodenoscopy ( egd ) which revealed a small esophageal nodule in an otherwise normal esophagus ( fig .", "1a ) , and nodule biopsy was consistent with papilloma . despite of the prescription of a proton pump inhibitor ( esomeprazole ) , her symptoms worsen so that she was referred to our institution in december 2014 for further evaluation and endoscopic ultrasound . upon evaluation she was well ,", "not pale or jaundiced , her weight was 64.8 kg and her body mass index was 26.6 .", "the abdomen was soft and non - tender , and no mass or lymph node was observed .", "her complete blood count showed white blood cell 16.9 10/l , hemoglobin 11.8 g / dl and platelets 306 10/l .", "c - reactive protein was < 3.5 mg / l and chest x - ray was normal .", "repeat egd and endoscopic ultrasound examination revealed an ulcerated esophageal mass 4 cm in diameter 23 - 27 cm from the incisor line ( fig .", "it was shown as a hypoechoic mass at the level of the third submucosal layer with no local lymph node or vascular infiltration ( fig .", "a computed tomography scan showed an eccentric 3.2 2.0 cm mass in the lower esophagus , inseparable from the esophageal wall .", "positron emission tomography / computed tomography revealed a hypermetabolic esophageal tumor with left cervical lymph node and midsacrum and right acetabulum metastasis ( fig .", "histopathological examination of the deep biopsies concluded that the mass was a nec of the large - cell type in view of irregular strands of large polygonal cells with an intermediate amount of pale oncophilic cytoplasm .", "the nuclei had occasional molding of salt and paper chromatin and visible nucleoli with extensive nuclear crushing artifact and immunohistochemical evidence of neuroendocrine differentiation .", "the patient was therefore staged as metastatic high - grade large - cell neuroendocrine tumor of the esophagus .", "chemotherapy was initiated by the oncologist and she received three cycles of cisplatin 90 mg / m in divided doses over 3 days and etoposide 100 mg / m per day on days 1 - 3 of each treatment cycle .", "large - cell nec of the esophagus is exceedingly rare ; few cases have been reported in the literature .", "the incidence of esophageal nens ranges between 0.05 and 2.4% in most of the recent studies .", "the literature clearly shows that nens in this anatomical location occur more frequently in males in the sixth and seventh decades of life , with a male : female ratio of 4:1 .", "similar to other esophageal tumors , patients usually present with vague symptoms , including constitutional symptoms , weight loss , chest pain or pressure and odynophagia . by far dysphagia", "is the most common presenting symptom and carcinoid symptoms are rare . from endoscopic findings , esophageal nens", "this is because neuroendocrine cells are mainly distributed in mucosal glands of the distal esophagus . among the categories of nens ,", "nec is a poorly differentiated , high - grade malignant neoplasm composed of small or large cells , sometimes with organoid features resembling well - differentiated neuroendocrine tumors , diffusely expressing the general markers of neuroendocrine differentiation ( diffuse expression of synaptophysin , faint or focal staining for chromogranin a ) , with marked nuclear atypia , multifocal necrosis and a high number of mitoses ( > 20 per 10 high - power fields ) ; high grade ( g3 ) is defined according to proliferation fraction and histology ( table 1 ) .", "large - cell tumors all consist of cells with solid nests or acinar structures , a low nuclear / cytoplasm ratio , often abundant cytoplasm , many mitoses , focal areas of necrosis , vesicular chromatin and visible nucleoli . for practical purposes ,", "esophageal necs can be staged according to the tnm / staging classification for esophageal carcinomas .", "in contrast to other gastrointestinal sites , there is no proposed tnm / staging classification for necs of the esophagus .", "however , proliferative activity , assessed by mitotic count or ki-67 immunostaining , was described as a significant prognostic factor in a previous study .", "there have been a few randomized , prospective and controlled therapeutic trials published thus far .", "unfortunately , in those trials several tumor entities ( e.g. tumors arising from the stomach , pancreas and colon ) were all included", ". moreover , there are not specific recommendations , given that doses and schedules were different in the various studies .", "high - grade necs of the esophagus are rare tumors exhibiting an overall aggressive behavior , and their prognosis is dismal .", "the majority are locally advanced or metastatic at presentation and are rarely associated with secretory hormonal syndromes .", "treatment regimens are extrapolated from more robust data published on pulmonary high - grade necs ." ]

[ "<S> neuroendocrine carcinomas of the esophagus are very rare , and the majority are high grade ( poorly differentiated ) . </S>", "<S> they occur most frequently in males in their sixth and seventh decades of life . </S>", "<S> there have been no concrete data published on clinical features or on prognosis . </S>", "<S> we report a case of large - cell neuroendocrine carcinoma of the esophagus in a 66-year - old saudi female with progressive dysphagia and weight loss . </S>", "<S> upper endoscopy revealed an esophageal ulcerated mass . </S>" ]

[ "Introduction", "Case Report", "Discussion", "Conclusion", "Statement of Ethics" ]

[ [ "the term neuroendocrine reflects the origin of endocrine cells from the embryonic neural crest .", "neuroendocrine cells are characterized by their ability to produce and secrete different peptides and neuroamines .", "they aggregate in classical endocrine glands ( e.g. hypothalamus and the pituitary gland ) or in the diffuse neuroendocrine system distributed throughout the body , such as the gastrointestinal tract and lungs .", "gastroenteropancreatic neuroendocrine neoplasms ( gep - nens ) are a heterogeneous group of neoplasms defined as malignant transformation of neuroendocrine cells in the diffuse neuroendocrine system that regulates secretion , absorption and motility of the gastrointestinal tract .", "the incidence of gep - nens continues to exhibit a persistent increase ; the most recent epidemiological study from the us surveillance shows that the overall incidence of gep - nens increased from 1 to 3.65 cases per 100,000 .", "a genetic etiology of gep - nens has been well documented in around 5 - 10% of cases as part of hereditary syndromes such as multiple endocrine neoplasia type 1 , von hippel - lindau syndrome , neurofibromatosis 1 or tuberous sclerosis syndrome .", "these tumors commonly present with a wide spectrum of clinical manifestations according to site of origin and biological behavior .", "they can present with abdominal pain , weight loss , obstructive jaundice and intestinal obstruction .", "moreover , the symptoms related to hormone secretion include flushing , diarrhea , wheezing , hypoglycemia , cardiovascular and peptic ulcers disease .", "the diagnosis is based on histological features , including tumor differentiation and grade , in addition to immunohistochemical stains for general endocrine markers , including chromogranins , synaptophysin and neuron - specific enolase .", "tumor markers such as chromogranin a , pancreatic polypeptide , serum neuron - specific enolase and subunit of glycoprotein hormones have been used .", "it is useful for staging , prognosis and follow - up , since the serum concentration correlates with the tumor mass . in the presence of symptoms or syndrome suspicious of functional nens , biochemical testing for peptide hypersecretion", "should be ordered , e.g. gastrin level for patients with zollinger - ellison syndrome or insulin for hypoglycemic syndromes .", "imaging studies are an important tool in nen workup ; abdominal ultrasound , computed tomography and magnetic resonance imaging have a high sensitivity and specificity in detecting many types of primary nens .", "functional imaging such as in - pentetreotide ( octreoscan ) or i - metaiodobenzylguanidine ( mibg ) plays a critical role in the diagnosis and in establishing the eligibility for therapy . combining functional imaging with computed tomography or magnetic resonance imaging is superior for staging and detection of primary tumors of undetermined location .", "gep - nens can be classified according to the site of embryologic origin ( foregut , midgut and hindgut ) , grade , differentiation or the hormones produced .", "the grade refers to the biological aggressiveness of the neoplasm which is defined by the number of mitoses per 10 high - power microscopic fields or per 2 mm ( mitotic rate ) , or as the percentage of tumor cells that immunolabel for the ki-67 antigen ( ki-67 index ) . while the differentiation refers to the extent to which the neoplastic cells resemble their non - neoplastic counterparts , in 2010 , the world health organization proposed the most widely accepted classification : ( 1 ) neuroendocrine tumor grade 1 ( g1 ) , ( 2 ) neuroendocrine tumor grade 2 ( g2 ) , ( 3 ) neuroendocrine carcinoma ( nec ) , grade 3 ( g3 ) , and ( 4 ) mixed adenoneuroendocrine carcinoma .", "also , nens are divided into well - differentiated and poorly differentiated ( table 1 ) . in general ,", "gep - nens are staged according to the primary tumor ( t ) , lymph node involvement ( n ) and distant metastases ( m ) tnm system , similar to other types of carcinomas .", "medical therapy of inoperable or metastasized nens includes multimodality options such as long - acting somatostatin analogs , -interferon , systemic chemotherapy and ablation therapy .", "there is no established regimen due to heterogeneity of the studies , including various grades , sites and inconsistent response criteria .", "novel therapies like mammalian target of rapamycin ( mtor ) , tyrosine kinase and angiogenesis inhibitors have showing promising results in clinical trials . we herein report a case of esophageal large - cell necs and review the published literature on this rare disease ." ], [ "a 66-year - old saudi female housewife with a background of diabetes , hypertension and hypothyroidism was seen initially by a gastroenterologist .", "she was suffering from progressive difficulty in swallowing , mainly of solid food , with a feeling of food stuck in the middle of her chest for more than 6 months .", "she denied any painful swallowing , oral thrush , chocking attacks , food impaction or nasal regurgitation .", "she denied any abdominal pain , distension , rectal bleeding or change in bowel habits .", "she had lost about 3 kg from a baseline weight of 68 kg despite normal appetite .", "her history was unremarkable for esophageal disease , tuberculosis contact , neck swelling or radiation exposure .", "the patient underwent esophagogastroduodenoscopy ( egd ) which revealed a small esophageal nodule in an otherwise normal esophagus ( fig .", "1a ) , and nodule biopsy was consistent with papilloma . despite of the prescription of a proton pump inhibitor ( esomeprazole ) , her symptoms worsen so that she was referred to our institution in december 2014 for further evaluation and endoscopic ultrasound . upon evaluation she was well ,", "not pale or jaundiced , her weight was 64.8 kg and her body mass index was 26.6 .", "the abdomen was soft and non - tender , and no mass or lymph node was observed .", "her complete blood count showed white blood cell 16.9 10/l , hemoglobin 11.8 g / dl and platelets 306 10/l .", "c - reactive protein was < 3.5 mg / l and chest x - ray was normal .", "repeat egd and endoscopic ultrasound examination revealed an ulcerated esophageal mass 4 cm in diameter 23 - 27 cm from the incisor line ( fig .", "it was shown as a hypoechoic mass at the level of the third submucosal layer with no local lymph node or vascular infiltration ( fig .", "a computed tomography scan showed an eccentric 3.2 2.0 cm mass in the lower esophagus , inseparable from the esophageal wall .", "positron emission tomography / computed tomography revealed a hypermetabolic esophageal tumor with left cervical lymph node and midsacrum and right acetabulum metastasis ( fig .", "histopathological examination of the deep biopsies concluded that the mass was a nec of the large - cell type in view of irregular strands of large polygonal cells with an intermediate amount of pale oncophilic cytoplasm .", "the nuclei had occasional molding of salt and paper chromatin and visible nucleoli with extensive nuclear crushing artifact and immunohistochemical evidence of neuroendocrine differentiation .", "the patient was therefore staged as metastatic high - grade large - cell neuroendocrine tumor of the esophagus .", "chemotherapy was initiated by the oncologist and she received three cycles of cisplatin 90 mg / m in divided doses over 3 days and etoposide 100 mg / m per day on days 1 - 3 of each treatment cycle ." ], [ "large - cell nec of the esophagus is exceedingly rare ; few cases have been reported in the literature .", "the incidence of esophageal nens ranges between 0.05 and 2.4% in most of the recent studies .", "the literature clearly shows that nens in this anatomical location occur more frequently in males in the sixth and seventh decades of life , with a male : female ratio of 4:1 .", "similar to other esophageal tumors , patients usually present with vague symptoms , including constitutional symptoms , weight loss , chest pain or pressure and odynophagia . by far dysphagia", "is the most common presenting symptom and carcinoid symptoms are rare . from endoscopic findings , esophageal nens", "this is because neuroendocrine cells are mainly distributed in mucosal glands of the distal esophagus . among the categories of nens ,", "nec is a poorly differentiated , high - grade malignant neoplasm composed of small or large cells , sometimes with organoid features resembling well - differentiated neuroendocrine tumors , diffusely expressing the general markers of neuroendocrine differentiation ( diffuse expression of synaptophysin , faint or focal staining for chromogranin a ) , with marked nuclear atypia , multifocal necrosis and a high number of mitoses ( > 20 per 10 high - power fields ) ; high grade ( g3 ) is defined according to proliferation fraction and histology ( table 1 ) .", "large - cell tumors all consist of cells with solid nests or acinar structures , a low nuclear / cytoplasm ratio , often abundant cytoplasm , many mitoses , focal areas of necrosis , vesicular chromatin and visible nucleoli . for practical purposes ,", "esophageal necs can be staged according to the tnm / staging classification for esophageal carcinomas .", "in contrast to other gastrointestinal sites , there is no proposed tnm / staging classification for necs of the esophagus .", "however , proliferative activity , assessed by mitotic count or ki-67 immunostaining , was described as a significant prognostic factor in a previous study .", "there have been a few randomized , prospective and controlled therapeutic trials published thus far .", "unfortunately , in those trials several tumor entities ( e.g. tumors arising from the stomach , pancreas and colon ) were all included", ". moreover , there are not specific recommendations , given that doses and schedules were different in the various studies ." ], [ "high - grade necs of the esophagus are rare tumors exhibiting an overall aggressive behavior , and their prognosis is dismal .", "the majority are locally advanced or metastatic at presentation and are rarely associated with secretory hormonal syndromes .", "treatment regimens are extrapolated from more robust data published on pulmonary high - grade necs ." ], [ "" ] ]

[ "toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds , causing toxoplasmosis .", "t. gondii is an important foodborne parasite that is primarily transmitted from animals to humans through the consumption of infected meat . in some countries ,", "pork is the most common meat consumed , and several ethnic groups consume raw pork .", "toxoplasmosis is a source of significant economic loss for swine farmers because of gross lesions in infected animals , which result in the carcass being condemned at the time of slaughter , the expense associated with treatment , and weight loss associated with clinical toxoplasmosis .", "the development of effective diagnostic reagents or vaccines is very important for worldwide public health and economic repercussions of t. gondii infection .", "the life cycle of t. gondii is relatively complex , and its antigenic component can change in specificity or makeup during different development stages ; therefore , the newly synthesized multiepitope antigen is one of the most promising antigens for the development of effective diagnostic reagents or vaccines [ 4 - 9 ] .", "however , the study of epitope - based vaccines and diagnostic reagents is highly dependent on the accurate identification of b - cell epitopes and t - cell epitopes .", "therefore , the identification of protein epitopes will be very important for diagnostic purposes and for the development of peptide vaccines [ 10 - 12 ] . among dense granule antigens ( gras ) ,", "gra6 was also demonstrated to be useful for designing novel and alternative diagnostic methods for toxoplasmosis or vaccines [ 13 - 17 ] .", "the gra6 gene does not contain any introns and is a single copy in the genome of t. gondii .", "gra6 localized in the dense granules and in the parasitophorous vacuole closely associated to the network .", "a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .", "the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .", "twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .", "the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa .", "t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3 and 5-atgcggccgcttaaaaatcaaactcattc-3 .", "the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .", "the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .", "the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .", "the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .", "positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr .", "the positive colonies identified by pcr were sequenced by takara biotechnology . to analyze the gra6 b cell epitopes ,", "the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .", "this subroutine uses the garnier - robson and chou - fasman algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle algorithm for predicting hydrophilicity , the karplus - schultz algorithm for predicting flexibility , the emini algorithm for predicting surface probability , and the jameson - wolf algorithm for predicting antigenicity .", "based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes .", "enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al . with minor modifications .", "the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .", "the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .", "non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c . the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .", "after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .", "after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .", "a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )", "was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory", "briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .", "the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody .", "a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .", "the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .", "twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .", "the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa .", "t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3 and 5-atgcggccgcttaaaaatcaaactcattc-3 .", "the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .", "the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .", "the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .", "the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .", "positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr .", "to analyze the gra6 b cell epitopes , the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .", "this subroutine uses the garnier - robson and chou - fasman algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle algorithm for predicting hydrophilicity , the karplus - schultz algorithm for predicting flexibility , the emini algorithm for predicting surface probability , and the jameson - wolf algorithm for predicting antigenicity .", "based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes .", "enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al . with minor modifications .", "the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .", "the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .", "non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c .", "the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .", "after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .", "after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .", "a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )", "was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory", "briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .", "the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody .", "the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .", "a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively ( fig .", "1 ) . based on the results obtained with these methods , potential b cell epitopes on gra6 were predicted , including 1 - 20 aa , 44 - 63 aa , 54 - 73 aa , 64 - 83 aa , 74 - 93 aa , 84 - 103 aa , 172 - 191 aa , 182 - 201 aa , 192 - 211 aa , and 202 - 221 aa .", "all of the 10 predicted epitope peptides were evaluated by elisa using pig sera from various time points after infection .", "the results of elisa for 3 peptides , p2 , p10 , and p11 , are shown in fig .", "no significant differences were found between the mean absorbances of the 3 groups ( g1 , g2 , and g3 ) as determined by anova .", "furthermore , no significant differences were found between the mean absorbances of the 3 peptides , p2 , p7 , and p9 .", "the other 7 peptides were recognized by selection of sera from various time points after infection ( fig .", "the number of positive samples / tested for each peptide was as follows : p1:8/51 , p3:25/51 , p4:18/51 , p5:9/51 , p6:27/51 , p8:20/51 , and p10:31/51 . to determine the specificity of the anti - peptide antibody , elisa using an irrelevant peptide", "4a ) . to compare the serological reactivity of the peptides with esa and recombinant gra6 , elisa using esa and recombinant gra6", "the adoption of immunoinformatics methods for the prediction of antigenic epitopes has become an indispensable tool for epitope localization .", "in addition , such techniques are economical and effective and can substantially reduce experimental costs .", "bioinformatics has been widely used in the analysis of protein epitopes [ 10 - 12 ] . in the present study ,", "the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .", "a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively .", "the existence of flexible regions , such as coil and turn region , provides powerful evidence for epitope identification . in the past ,", "several experimental techniques were developed for mapping antibody interacting residues on an antigen , including the identification of interacting residues from the structure of antibody - antigen complexes .", "many researchers have applied this technique to study epitopes [ 6,10 - 12,26,29 ] . using this technique and bioinformatics tools", ", we found that many regions of gra6 , particularly the regions represented by peptides p2 , p7 , and p9 , are involved in the pig antibody response , and strong reactivity with the t. gondii - infected pig sera was observed .", "the reactivity of these epitopes does not seem to be dependent upon the time of infection .", "the identification of b cell epitopes is important for understanding antigenic structure and parasite - antibody interactions at the molecular level and may assist in the design of vaccines and diagnostic reagents .", "conformational epitope selection relies on the determination of the tertiary structure of an antigen to identify residues that interact with antibodies .", "the experimental techniques required to determine the tertiary structure of the antigen , such as crystallography , are expensive and time - consuming , and the mapping of conformational epitopes has been severely hampered .", "the majority of methods and databases have focused on the identification of linear epitopes . in the present study", ", linear epitopes were analyzed using synthetic peptide techniques and bioinformatics tools , and 3 of the 10 predicted epitope peptides were confirmed by synthetic peptide techniques .", "the use of a molecular biology method in combination with a bioinformatics method is a useful method to screen for linear epitopes .", "we have precisely located the epitopes of t. gondii gra6 using pig sera collected at different time points after infection .", "the identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope - based vaccines and diagnostic methods .", "there are more linear and conformational b cell epitopes than previously predicted ; therefore , the number of identified epitopes should also increase with further studies ." ]

[ "<S> the study of antigenic epitopes from toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens , the reactions between antigens and antibodies , and many other aspects of immunology , but it also plays a significant role in the development of new diagnostic reagents and vaccines . in the present study , t. gondii gra6 epitopes were identified using bioinformatics tools and a synthetic peptide technique . </S>", "<S> the potential b cell epitopes of gra6 predicted by bioinformatics tools concentrated upon 3 regions of gra6 , 1 - 20 aa , 44 - 103 aa , and 172 - 221 aa . </S>", "<S> ten shorter peptides from the 3 regions were synthesized and assessed by elisa using pig sera from different time points after infection . </S>", "<S> three of the 10 peptides ( amino acids 44 - 63 , 172 - 191 , and 192 - 211 ) tested were recognized by all sera and determined to be immunodominant b - cell epitopes of gra6 . </S>", "<S> the results indicated that we precisely and accurately located the t. gondii gra6 epitopes using pig sera collected at different time points after infection . </S>", "<S> the identified epitopes may be very useful for further studies of epitope - based vaccines and diagnostic reagents . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Serum samples", "Amplification, cloning, and sequencing of the GRA6 gene", "Prediction of the epitopes", "ELISA analysis", "RESULTS", "DISCUSSION" ]

[ [ "toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds , causing toxoplasmosis .", "t. gondii is an important foodborne parasite that is primarily transmitted from animals to humans through the consumption of infected meat . in some countries ,", "pork is the most common meat consumed , and several ethnic groups consume raw pork .", "toxoplasmosis is a source of significant economic loss for swine farmers because of gross lesions in infected animals , which result in the carcass being condemned at the time of slaughter , the expense associated with treatment , and weight loss associated with clinical toxoplasmosis .", "the development of effective diagnostic reagents or vaccines is very important for worldwide public health and economic repercussions of t. gondii infection .", "the life cycle of t. gondii is relatively complex , and its antigenic component can change in specificity or makeup during different development stages ; therefore , the newly synthesized multiepitope antigen is one of the most promising antigens for the development of effective diagnostic reagents or vaccines [ 4 - 9 ] .", "however , the study of epitope - based vaccines and diagnostic reagents is highly dependent on the accurate identification of b - cell epitopes and t - cell epitopes .", "therefore , the identification of protein epitopes will be very important for diagnostic purposes and for the development of peptide vaccines [ 10 - 12 ] . among dense granule antigens ( gras ) ,", "gra6 was also demonstrated to be useful for designing novel and alternative diagnostic methods for toxoplasmosis or vaccines [ 13 - 17 ] .", "the gra6 gene does not contain any introns and is a single copy in the genome of t. gondii .", "gra6 localized in the dense granules and in the parasitophorous vacuole closely associated to the network ." ], [ "a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .", "the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .", "twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .", "the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa .", "t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3 and 5-atgcggccgcttaaaaatcaaactcattc-3 .", "the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .", "the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .", "the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .", "the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .", "positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr .", "the positive colonies identified by pcr were sequenced by takara biotechnology . to analyze the gra6 b cell epitopes ,", "the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .", "this subroutine uses the garnier - robson and chou - fasman algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle algorithm for predicting hydrophilicity , the karplus - schultz algorithm for predicting flexibility , the emini algorithm for predicting surface probability , and the jameson - wolf algorithm for predicting antigenicity .", "based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes .", "enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al . with minor modifications .", "the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .", "the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .", "non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c . the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .", "after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .", "after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .", "a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )", "was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory", "briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .", "the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody ." ], [ "a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .", "the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .", "twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .", "the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa ." ], [ "t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3 and 5-atgcggccgcttaaaaatcaaactcattc-3 .", "the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .", "the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .", "the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .", "the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .", "positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr ." ], [ "to analyze the gra6 b cell epitopes , the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .", "this subroutine uses the garnier - robson and chou - fasman algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle algorithm for predicting hydrophilicity , the karplus - schultz algorithm for predicting flexibility , the emini algorithm for predicting surface probability , and the jameson - wolf algorithm for predicting antigenicity .", "based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes ." ], [ "enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al . with minor modifications .", "the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .", "the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .", "non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c .", "the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .", "after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .", "after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .", "a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )", "was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory", "briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .", "the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody ." ], [ "the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .", "a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively ( fig .", "1 ) . based on the results obtained with these methods , potential b cell epitopes on gra6 were predicted , including 1 - 20 aa , 44 - 63 aa , 54 - 73 aa , 64 - 83 aa , 74 - 93 aa , 84 - 103 aa , 172 - 191 aa , 182 - 201 aa , 192 - 211 aa , and 202 - 221 aa .", "all of the 10 predicted epitope peptides were evaluated by elisa using pig sera from various time points after infection .", "the results of elisa for 3 peptides , p2 , p10 , and p11 , are shown in fig .", "no significant differences were found between the mean absorbances of the 3 groups ( g1 , g2 , and g3 ) as determined by anova .", "furthermore , no significant differences were found between the mean absorbances of the 3 peptides , p2 , p7 , and p9 .", "the other 7 peptides were recognized by selection of sera from various time points after infection ( fig .", "the number of positive samples / tested for each peptide was as follows : p1:8/51 , p3:25/51 , p4:18/51 , p5:9/51 , p6:27/51 , p8:20/51 , and p10:31/51 . to determine the specificity of the anti - peptide antibody , elisa using an irrelevant peptide", "4a ) . to compare the serological reactivity of the peptides with esa and recombinant gra6 , elisa using esa and recombinant gra6" ], [ "the adoption of immunoinformatics methods for the prediction of antigenic epitopes has become an indispensable tool for epitope localization .", "in addition , such techniques are economical and effective and can substantially reduce experimental costs .", "bioinformatics has been widely used in the analysis of protein epitopes [ 10 - 12 ] . in the present study ,", "the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .", "a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively .", "the existence of flexible regions , such as coil and turn region , provides powerful evidence for epitope identification . in the past ,", "several experimental techniques were developed for mapping antibody interacting residues on an antigen , including the identification of interacting residues from the structure of antibody - antigen complexes .", "many researchers have applied this technique to study epitopes [ 6,10 - 12,26,29 ] . using this technique and bioinformatics tools", ", we found that many regions of gra6 , particularly the regions represented by peptides p2 , p7 , and p9 , are involved in the pig antibody response , and strong reactivity with the t. gondii - infected pig sera was observed .", "the reactivity of these epitopes does not seem to be dependent upon the time of infection .", "the identification of b cell epitopes is important for understanding antigenic structure and parasite - antibody interactions at the molecular level and may assist in the design of vaccines and diagnostic reagents .", "conformational epitope selection relies on the determination of the tertiary structure of an antigen to identify residues that interact with antibodies .", "the experimental techniques required to determine the tertiary structure of the antigen , such as crystallography , are expensive and time - consuming , and the mapping of conformational epitopes has been severely hampered .", "the majority of methods and databases have focused on the identification of linear epitopes . in the present study", ", linear epitopes were analyzed using synthetic peptide techniques and bioinformatics tools , and 3 of the 10 predicted epitope peptides were confirmed by synthetic peptide techniques .", "the use of a molecular biology method in combination with a bioinformatics method is a useful method to screen for linear epitopes .", "we have precisely located the epitopes of t. gondii gra6 using pig sera collected at different time points after infection .", "the identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope - based vaccines and diagnostic methods .", "there are more linear and conformational b cell epitopes than previously predicted ; therefore , the number of identified epitopes should also increase with further studies ." ] ]

[ "spontaneous splenic rupture occurs usually secondary to abdominal trauma , most often in a spleen affected by infection or haematological malignancy .", "spontaneous splenic rupture is extremely rare , especially presenting in a patient subsequently diagnosed with splenic marginal cell lymphoma ( smzl ) , which itself is rare and as a result only one case has been described in the literature so far .", "we report a case of a 71-year - old woman with a past medical history of inflammatory myelitis , osteoporosis , asthma , coeliac disease and chronic back pain who was admitted as an emergency with new onset and severe back and abdominal pain .", "she described a 5-day history of persistent bilateral back pain radiating to the shoulder tips and groin worse when lying flat .", "she attended the emergency department 3 days before with a similar complaint but discharged home the same day .", "the inflammatory markers were following : crp 88 and white cell count 12.3 ; biochemistry and clotting screen was normal .", "laparotomy was performed and a large splenic haematoma with 1 l of blood in the abdomen and a splenectomy was performed .", "the postoperative period was complicated by a lower respiratory tract infection and some difficulty with mobilization but the patient made good recovery otherwise . during the post - operative period she developed a hypoglossal nerve palsy , which was assessed by the neurologist .", "figure 1ct chest / abdomen demonstrating a large splenic haematoma with hyper- and hypo - dense areas .", "ct chest / abdomen demonstrating a large splenic haematoma with hyper- and hypo - dense areas .", "high attenuation fluid is present within the pelvis consistent with blood . the excised spleen weighed 560 g with blood clots and was macroscopically normal .", "microscopically ( figs 24 ) , the histology and immunohistochemistry was consistent with a diagnosis of smzl .", "subsequently , a bone marrow biopsy and staging ct were performed and no distant spread of disease was demonstrated .", "cd79a highlights the presence of marginal expansion of the white pulp lymphoid mantle with satellitosis ( nodular infiltrates ) in the red pulp .", "the monomorphic population of medium - sized lymphoid cells also stained positive for cd20 , bcl2 , igm and igg consistent with smzl . \n", "figure 4marginal expansion of the white pulp lymphoid mantle with satellitosis in the red pulp .", "cd79a highlights the presence of marginal expansion of the white pulp lymphoid mantle with satellitosis ( nodular infiltrates ) in the red pulp .", "the monomorphic population of medium - sized lymphoid cells also stained positive for cd20 , bcl2 , igm and igg consistent with smzl .", "the term spontaneous rupture is poorly defined in the literature and distinguished from pathological rupture of the spleen . dating back to 1958", "peskin and orloff described true spontaneous rupture in cases where there is no trauma pre- or intraoperativly , the spleen is not affected by any disease , no perisplenic adhesions present indicating previous splenic trauma and on gross and histological examination of the spleen must be normal .", "however , in the current literature , spontaneous splenic rupture describes a splenic rupture occurring without trauma whether the spleen is involved in pathology or not .", "first , in haematological malignancies , the splenic parenchyma gets congested with blast cells causing increased intrasplenic tension , which exceeds the capacity of the non - distensible splenic capsule and eventually results in rupture .", "secondly , vascular occlusion secondary to reticular endothelial hyperplasia results in thrombosis and infarction , which may distort the splenic architecture making it weaker and thirdly deranged coagulation , which may be part of a systemic disease or caused by thrombocytopenia secondary to massive splenomegaly .", "splenic rupture is a life - threatening event and a differential diagnosis in a patient presenting with acute abdomen , although not all cases present with abdominal pain .", "haemodynamic instability and kehr 's sign , defined as left hypochondriac pain radiating to the left shoulder , occurs in 20% of cases although neither signs are sensitive nor specific .", "ultrasound scanning , which is usually readily available in emergency departments , is often a choice of initial imaging and is 7278% sensitive and 91100% specific .", "treatment of splenic rupture depends on the site and size of rupture , whether the patient is haemodynamically stable and the underlying pathology causing the rupture . in", "haemodynamically stable patients it is becoming more common to treat patients by splenic artery embolization , whilst unstable patients require surgical management [ 5 , 6 ] .", "splenic marginal zone lymphoma is classified under the non - hodgkin 's lymphomas ( nhl ) and account for < 1% of these .", "marginal cell lymphomas are indolent small b - cell lymphomas , which originate from the marginal zone of the lymphoid follicle , which consists of a germinal centre surrounded by a corona that is divided into the marginal zone and the mantle zone .", "the world health organization classifies the marginal zone - derived lymphomas into splenic ( smzl ) , lymph node ( nodal marginal zone lymphoma ) and extra - nodal mucosa - associated lymphoid tissue ( malt lymphoma ) despite having a common origin in the marginal zone of the b - follicle as they have distinct clinical and molecular characteristics [ 2 , 7 ] .", "smzl usually presents as massive splenomegaly and/or with abnormalities in the full blood count , especially anaemia and thrombocytopaenia usually secondary to splenic sequestration rather than bone marrow infiltration [ 5 , 7 ] .", "retrospective analysis of blood results dating back a number of years , revealed no abnormalities in the full blood count ; therefore , it appears that splenic rupture was the first presentation in this case and she is in remission at present after undergoing splenectomy as no distant disease was found .", "the treatment of smzl is not standardized and there are no reported prospective studies comparing outcome with immunotherapy , chemotherapy or splenectomy . in the past splenectomy was regarded as a choice of treatment until rituximab was introduced , which has shown responses of up to 100% .", "this trial concludes that splenectomy should not be regarded as first - line treatment but considered in patients who do not respond to immunotherapy or chemtotherapy .", "patients with splenomegaly may suffer abdominal discomfort , early satiety and cytopaenias where splenectomy is indicated for palliative purposes [ 7 , 8 ] . as this lady presented with spontaneous splenic rupture", ", it raises the question that when and if splenectomy should be performed prophylactically if picked up on a scan or whether surveillance is indicated but at present the literature does not address this question but ultimately comes down to assessing each individual case taking into account their overall health and stage of disease ." ]

[ "<S> rupture of the spleen is a potentially life - threatening condition , which most often occurs secondary to abdominal trauma . </S>", "<S> spontaneous rupture of the spleen is a much rarer event , usually occurring secondary to infections and less frequently secondary to haematological malignancies causing massive splenomegaly . </S>", "<S> we present a case of a 71-year - old woman who presented in the emergency department with acute abdominal and back pain and no history of trauma , with a ct scan diagnosis of splenic rupture . </S>", "<S> splenectomy was performed and the histological examination of the specimen revealed splenic marginal cell lymphoma ( smzl ) , which is classified under the non - hodgkins lymphomas ( nhl ) and accounts for < 1% of nhl . </S>", "<S> there is only one previously reported case of spontaneous splenic rupture smzl and this is the first recorded case of spontaneous splenic rupture in a patient without massive splenomegaly . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "spontaneous splenic rupture occurs usually secondary to abdominal trauma , most often in a spleen affected by infection or haematological malignancy .", "spontaneous splenic rupture is extremely rare , especially presenting in a patient subsequently diagnosed with splenic marginal cell lymphoma ( smzl ) , which itself is rare and as a result only one case has been described in the literature so far ." ], [ "we report a case of a 71-year - old woman with a past medical history of inflammatory myelitis , osteoporosis , asthma , coeliac disease and chronic back pain who was admitted as an emergency with new onset and severe back and abdominal pain .", "she described a 5-day history of persistent bilateral back pain radiating to the shoulder tips and groin worse when lying flat .", "she attended the emergency department 3 days before with a similar complaint but discharged home the same day .", "the inflammatory markers were following : crp 88 and white cell count 12.3 ; biochemistry and clotting screen was normal .", "laparotomy was performed and a large splenic haematoma with 1 l of blood in the abdomen and a splenectomy was performed .", "the postoperative period was complicated by a lower respiratory tract infection and some difficulty with mobilization but the patient made good recovery otherwise . during the post - operative period she developed a hypoglossal nerve palsy , which was assessed by the neurologist .", "figure 1ct chest / abdomen demonstrating a large splenic haematoma with hyper- and hypo - dense areas .", "ct chest / abdomen demonstrating a large splenic haematoma with hyper- and hypo - dense areas .", "high attenuation fluid is present within the pelvis consistent with blood . the excised spleen weighed 560 g with blood clots and was macroscopically normal .", "microscopically ( figs 24 ) , the histology and immunohistochemistry was consistent with a diagnosis of smzl .", "subsequently , a bone marrow biopsy and staging ct were performed and no distant spread of disease was demonstrated .", "cd79a highlights the presence of marginal expansion of the white pulp lymphoid mantle with satellitosis ( nodular infiltrates ) in the red pulp .", "the monomorphic population of medium - sized lymphoid cells also stained positive for cd20 , bcl2 , igm and igg consistent with smzl . \n", "figure 4marginal expansion of the white pulp lymphoid mantle with satellitosis in the red pulp .", "cd79a highlights the presence of marginal expansion of the white pulp lymphoid mantle with satellitosis ( nodular infiltrates ) in the red pulp .", "the monomorphic population of medium - sized lymphoid cells also stained positive for cd20 , bcl2 , igm and igg consistent with smzl ." ], [ "the term spontaneous rupture is poorly defined in the literature and distinguished from pathological rupture of the spleen . dating back to 1958", "peskin and orloff described true spontaneous rupture in cases where there is no trauma pre- or intraoperativly , the spleen is not affected by any disease , no perisplenic adhesions present indicating previous splenic trauma and on gross and histological examination of the spleen must be normal .", "however , in the current literature , spontaneous splenic rupture describes a splenic rupture occurring without trauma whether the spleen is involved in pathology or not .", "first , in haematological malignancies , the splenic parenchyma gets congested with blast cells causing increased intrasplenic tension , which exceeds the capacity of the non - distensible splenic capsule and eventually results in rupture .", "secondly , vascular occlusion secondary to reticular endothelial hyperplasia results in thrombosis and infarction , which may distort the splenic architecture making it weaker and thirdly deranged coagulation , which may be part of a systemic disease or caused by thrombocytopenia secondary to massive splenomegaly .", "splenic rupture is a life - threatening event and a differential diagnosis in a patient presenting with acute abdomen , although not all cases present with abdominal pain .", "haemodynamic instability and kehr 's sign , defined as left hypochondriac pain radiating to the left shoulder , occurs in 20% of cases although neither signs are sensitive nor specific .", "ultrasound scanning , which is usually readily available in emergency departments , is often a choice of initial imaging and is 7278% sensitive and 91100% specific .", "treatment of splenic rupture depends on the site and size of rupture , whether the patient is haemodynamically stable and the underlying pathology causing the rupture . in", "haemodynamically stable patients it is becoming more common to treat patients by splenic artery embolization , whilst unstable patients require surgical management [ 5 , 6 ] .", "splenic marginal zone lymphoma is classified under the non - hodgkin 's lymphomas ( nhl ) and account for < 1% of these .", "marginal cell lymphomas are indolent small b - cell lymphomas , which originate from the marginal zone of the lymphoid follicle , which consists of a germinal centre surrounded by a corona that is divided into the marginal zone and the mantle zone .", "the world health organization classifies the marginal zone - derived lymphomas into splenic ( smzl ) , lymph node ( nodal marginal zone lymphoma ) and extra - nodal mucosa - associated lymphoid tissue ( malt lymphoma ) despite having a common origin in the marginal zone of the b - follicle as they have distinct clinical and molecular characteristics [ 2 , 7 ] .", "smzl usually presents as massive splenomegaly and/or with abnormalities in the full blood count , especially anaemia and thrombocytopaenia usually secondary to splenic sequestration rather than bone marrow infiltration [ 5 , 7 ] .", "retrospective analysis of blood results dating back a number of years , revealed no abnormalities in the full blood count ; therefore , it appears that splenic rupture was the first presentation in this case and she is in remission at present after undergoing splenectomy as no distant disease was found .", "the treatment of smzl is not standardized and there are no reported prospective studies comparing outcome with immunotherapy , chemotherapy or splenectomy . in the past splenectomy was regarded as a choice of treatment until rituximab was introduced , which has shown responses of up to 100% .", "this trial concludes that splenectomy should not be regarded as first - line treatment but considered in patients who do not respond to immunotherapy or chemtotherapy .", "patients with splenomegaly may suffer abdominal discomfort , early satiety and cytopaenias where splenectomy is indicated for palliative purposes [ 7 , 8 ] . as this lady presented with spontaneous splenic rupture", ", it raises the question that when and if splenectomy should be performed prophylactically if picked up on a scan or whether surveillance is indicated but at present the literature does not address this question but ultimately comes down to assessing each individual case taking into account their overall health and stage of disease ." ] ]

[ "self - esteem is an important state of mind which allows individuals to accept themselves as they are and be self - confident .", "rosenberg ( 1965 ) considered self - esteem as the positive or negative attitude of an individual toward himself .", "if an individual has a positive attitude in self - assessment , his self - esteem would be high ; if he has a negative attitude , then his self - esteem would be low .", "self - esteem is a psychological phenomenon that has a definite effect on the emotional and cognitive dimensions of an individual .", "the individuals with a high self - esteem tend to perceive themselves worth being respected and approved , important and beneficial . on the other hand , the individuals who have a negative idea about themselves or who have low self - esteem tend to perceive themselves as not very important , lacking lovability , and with no comfort in themselves and their capabilities .", "high self - esteem is considered important because it is associated with higher levels of psychological health and functioning and low level of self - esteem is undesirable because it is associated with lower levels of psychological health and functioning .", "one of the major reasons for researchers interest on self - esteem is its effects on health .", "damaged self - esteem makes it impossible to endure the harsh conditions of everyday life and produces adverse physical and psychological outcomes .", "therefore , there is always the risk of appearance of mental disease in the postpartum period .", "the postpartum depression is a disorder of non - psychosis depression according to the study of hendrick et al . as per the american psychiatric association [ diagnostic and statistical manual of mental disorder - iv ( dsm - iv ) ] ,", "brown et al . reported that about 50% of women are affected by depression at the first 3 months after delivery , whereas one - third of them are affected by depression after 3 months of delivery .", "although several factors influence depression , the psychosocial factors play a strong role in prediction of this disorder .", "self - esteem is one of the variables that affects deleteriously on the pregnancy outcomes and postpartum period .", "'s study showed that self - esteem may be a useful predictor of disorders such as anxiety and depression .", "depression and self - esteem tend to be highly correlated with each other . it has been accepted that depressed people think in a negative manner and report lower self - esteem than non - depressed people .", "the robustness of the effect also strengthens the potential importance of self - esteem interventions , said sowislo .", "she believes that treatments aimed at reducing depression by way of improving self - esteem could provide not only short - term gains for clients but also long - term protection from depression for those most at risk .", "it was found that individuals who have effective and positive social problem - solving approach have a high self - esteem .", "the individuals with high self - esteem generally have more confidence in their initial approaches to the problems and , therefore , they seek less information before offering solutions and making decisions .", "problem - solving approach is a cognitive behavioral process . by means of problem - solving skills", ", people can identify and recommend the effective strategies to face stressful situations in everyday life . social problem solving", "can be viewed as an important conformity mechanism that serves to increase behavioral competence and it leads to decrease in psychological distress .", "dzurilla suggested that self - esteem may play an important role in the relationship between self - appraised social problem - solving ability and psychological adjustment .", "also , mccabe et al . found that social problem solving was predicted by self - esteem and depressive symptoms . in aylward 's study ,", "self - esteem emerged as the most important predictor of social problem - solving model .", "some evidence was found to link the relationship between self - esteem and problem - solving skills . in the study of mahmoudi rad ", "entitled the role of social problem solving and communication skills training on enhancing self - esteem level and its relationship to mental functioning and academic achievement of students , it was found that self - esteem scores of the two groups showed significant difference before and after intervention .", "the researchers stated that the findings suggest a direct effect and positive role of training of communication skills and social problem - solving abilities when a person faces social and personal problems .", "the study of abuhamzeh entitled the effect of problem solving training on increasing the self - esteem of students in school showed that training of problem solving significantly increases overall , academic , social , and family self - esteem in the intervention group than in the control group .", "this effect remains for up to 2months after the education . in the study of sharifi", ", it was found that problem - solving training positively affected the self - esteem of students with behavior problems .", "based on studies , we can conclude that with higher problem - solving skills and social problem solving , we can increase a person 's self - esteem and decrease the depressive symptoms .", "several studies showed that low self - esteem is associated with increased anxiety and depression .", "rees conducted an exploratory study of the effectiveness of guided imagery protocol with relaxation on reducing anxiety and depression and increasing self - esteem in primiparas during the first postpartum month .", "not only relaxation and imagery decrease stress , pain , and anxiety , but also they generate a more positive understanding and a more strong feeling about well - being . with relaxation", ", the person can attach importance to herself and she can perceive everything that she needs .", "this develops self - appraised process which is the first important step toward self - esteem .", "although there is little evidence about the effect of relaxation on self - esteem , its positive effect is somewhat proved .", "the relaxation training is simple technique and easy to practice at home ; it can increase the self - esteem and decrease psychological problems .", "relaxation and problem - solving training are behavioural cognitive interventions that help to decrease depressive symptoms and increase self - esteem ; also , the self - esteem of depressed women is low during postpartum period .", "hence , a study should be performed for determining the more effective and simpler technique .", "this study was done with the aim of comparing the effects of problem - solving skills training and relaxation on the self - esteem of women in postpartum period .", "this was a clinical trial with a control group in 39 weeks in postpartum period and was conducted after getting permission from the ethics committee of mashhad university of medical science .", "all women on days 1520 after delivery who referred to mashhad health centers from december 2009 to june 2010 participated in the study after completing the consent form .", "the inclusion criteria were : literate women , 1835 years old , with the term neonate , singleton , healthy infant , the history of lack of chronic medical illness , without history of previous depression , and no smoking .", "at first , edinburgh postnatal depression scale ( epds ) was completed by the subjects .", "women whose get score 10 or above , completed beck depression inventory ( bdi ) .", "those who obtained a score of 1428 and confirmed to be depressed by a clinical interview conducted by a psychologist ( based on dsm - iv criteria ) in the health centers were divided into three groups randomly : problem - solving training ( n = 26 ) , relaxation ( n = 26 ) , and control group ( n = 28 ) [ figure 1 ] .", "eysenck self - esteem scale which has seven items and a score of 140 was completed by the women at the onset of research .", "interventions in the two groups of problem solving and relaxation were performed by a researcher ( midwife ) .", "the researcher had been taught by a clinical psychologist and after gaining the skills , she received a certificate of performance of relaxation and problem solving skills .", "a pilot study had been done on depressed women in ebne - sina hospital in mashhad before conducting the main study .", "chart of sampling process intervention was performed in the problem - solving group , which consisted of six sessions weekly in the healthy centers .", "the researcher notified the appointment by telephone to the research units before the intervention . in the problem - solving group , at the first session which lasted 1 h , the participants were trained in five skills of problem solving ( general orientation , problem definition and formulation , generation of alternatives , decision making , and verification ) . in the next sessions which lasted 4550 min , depressive symptoms of women were studied according to the beck inventory ; different solutions were presented by women and the best solution was selected for performing until the next session .", "the recording checklist of problem - solving sessions was completed by the researcher in each session . in the relaxation group ,", "the first two sessions of relaxation were performed in the health center , and each session lasted about half an hour ; the other sessions took 1020 min . in the first session of relaxation training , the causes of postpartum depression and the ways of controlling it were discussed and the relaxation purposes were explained .", "also , women were trained about how to contract and relax their 16 groups of muscles similar to jacobson 's method , and the practice was demonstrated by the researcher", ". then the practice was repeated once in the presence of a researcher by a training mother . in the second session", ", progressive muscle relaxation technique was performed once again by a mother in the presence of the researcher and after correction and ensuring the accuracy of the technique , the cd of progressive muscle relaxation was given to the women to perform the exercise daily at home .", "in the next four sessions , in addition to the exercises of relaxation performed at home , the imagery techniques ( forest visualization , beach , pilgrimage sits , and green space with good climate ) were performed by the researcher .", "the relaxation exercise was performed by the women at home for about 20 min daily .", "the form of recording the exercises performed at home was given to women and it was emphasized to bring it along with them in each session . at the beginning of each session , the positive and negative experiences of women were considered and they asked about how they could perform relaxation techniques .", "the control group recoursed to health center weekly and they bring the recording checklistof postpartum depressive symptoms with themselves and routine care after delivery is performed for them according to the booklet of safe mother .", "nine weeks after delivery , each of the three groups completed the eysenck self - esteem scale again as a post - test . in this research ,", "the instruments of data collection included the edinburgh depression scale , beck depression inventory - ii , eysenck self - esteem scale , the socio - demographic questionnaire , recorded checklist of depressive symptoms , and recorded checklist of problem - solving sessions .", "the edinburgh scale is a standard instrument for screening depressive women in the postpartum period .", "this instrument assesses the feeling of women in the past 7 days and it has 10 items and each item has a score of 03 .", "it assesses the feeling of women in the past 1 week and its answers are scored between 0 and 3 .", "eysenck self - esteem scale has 7 items and each item has a score of 020 .", "the first two sections included 20 questions about personal characteristics , pregnancy , and recent childbirth , which were completed before implementing interventions and its two questions were completed on referring to patients files .", "the third section included four questions that collected information on the availability of someone to help at home , sleeplessness difficulties , and breastfeeding , and it was completed 9 weeks after delivery .", "the validity of beck depression test was confirmed by mohammad khani in tehran , and in this study , the alpha coefficient was 0.827 .", "the data obtained in this study were analyzed using descriptive statistics , analysis of one - way variance , chi - square test , and spearman correlation coefficient by spss software .", "intervention was performed in the problem - solving group , which consisted of six sessions weekly in the healthy centers .", "the researcher notified the appointment by telephone to the research units before the intervention . in the problem - solving group , at the first session which lasted 1 h , the participants were trained in five skills of problem solving ( general orientation , problem definition and formulation , generation of alternatives , decision making , and verification ) . in the next sessions which lasted 4550 min , depressive symptoms of women were studied according to the beck inventory ; different solutions were presented by women and the best solution was selected for performing until the next session .", "the recording checklist of problem - solving sessions was completed by the researcher in each session .", "in the relaxation group , the first two sessions of relaxation were performed in the health center , and each session lasted about half an hour ; the other sessions took 1020 min . in the first session of relaxation training , the causes of postpartum depression and the ways of controlling it were discussed and the relaxation purposes were explained .", "also , women were trained about how to contract and relax their 16 groups of muscles similar to jacobson 's method , and the practice was demonstrated by the researcher .", "then the practice was repeated once in the presence of a researcher by a training mother . in the second session", ", progressive muscle relaxation technique was performed once again by a mother in the presence of the researcher and after correction and ensuring the accuracy of the technique , the cd of progressive muscle relaxation was given to the women to perform the exercise daily at home . in the next four sessions , in addition to the exercises of relaxation performed at home , the imagery techniques ( forest visualization , beach , pilgrimage sits , and green space with good climate ) were performed by the researcher .", "the relaxation exercise was performed by the women at home for about 20 min daily .", "the form of recording the exercises performed at home was given to women and it was emphasized to bring it along with them in each session . at the beginning of each session , the positive and negative experiences of women were considered and they asked about how they could perform relaxation techniques .", "the control group recoursed to health center weekly and they bring the recording checklistof postpartum depressive symptoms with themselves and routine care after delivery is performed for them according to the booklet of safe mother .", "nine weeks after delivery , each of the three groups completed the eysenck self - esteem scale again as a post - test . in this research ,", "the instruments of data collection included the edinburgh depression scale , beck depression inventory - ii , eysenck self - esteem scale , the socio - demographic questionnaire , recorded checklist of depressive symptoms , and recorded checklist of problem - solving sessions .", "the edinburgh scale is a standard instrument for screening depressive women in the postpartum period .", "this instrument assesses the feeling of women in the past 7 days and it has 10 items and each item has a score of 03 .", "it assesses the feeling of women in the past 1 week and its answers are scored between 0 and 3 .", "eysenck self - esteem scale has 7 items and each item has a score of 020 .", "the first two sections included 20 questions about personal characteristics , pregnancy , and recent childbirth , which were completed before implementing interventions and its two questions were completed on referring to patients files .", "the third section included four questions that collected information on the availability of someone to help at home , sleeplessness difficulties , and breastfeeding , and it was completed 9 weeks after delivery .", "the validity of beck depression test was confirmed by mohammad khani in tehran , and in this study , the alpha coefficient was 0.827 .", "the data obtained in this study were analyzed using descriptive statistics , analysis of one - way variance , chi - square test , and spearman correlation coefficient by spss software .", "each of the three groups showed no significant statistical differences regarding their age ( p = 0.680 ) , husband support ( p = 1.000 ) , satisfaction in marital life ( question by likert scale ) ( p = 0.506 ) , unwanted pregnancy ( p = 0.392 ) , neonate sex ( p = 0.253 ) , social support ( cassidy social support scale ) ( p = 0.078 ) , self - esteem ( p = 0.145 ) , and the score of edinburgh depression scale ( p = 0.258 ) [ table 1 ] . in this study ,", "53.8% women had high , 42.5% had moderate , and 3.8% had low self - esteem .", "descriptive characteristics of problem - solving training , relaxation , and control groups before intervention the mean of self - esteem score was 103.7 21.6 before intervention in the problem - solving group that increased to 117.9 9.7 after the intervention . also , in the relaxation group , the mean of self - esteem score was 112.1 16.7 before the intervention , which increased to 117.0 11.8 after the intervention . in the control group ,", "the mean of self - esteem score was 111.8 13.1 in the beginning of the study and it changed to 113.5 10.4 at the end of the study . by means of self - esteem scale ,", "the self - esteem levels in each group were obtained . in our study , the self - esteem of relaxation group was moderate ( 42.3% ) and high ( 57.7% ) before the intervention and reached a moderate level in 23.1% and a high level in 76.9% of the cases after the intervention .", "results showed relaxation can increase moderate self - esteem to high self - esteem . in the present study ,", "the self - esteem score was low in 11.5% , moderate in 34.6% , and high in 53.8% of the problem - solving group before the intervention , which became moderate in 15.4% and high in 84.6% of the cases after the intervention . in the ninth week after delivery at which the intervention of relaxation and problem - solving training groups was completed , the statistical results of one - way variance analysis showed that the three groups had no significant differences at the end of study ( p = 0.288 ) . but according to the mean difference of self - esteem score before and after the intervention ( by one - way anova test ) , it was found that there was significant difference among the three groups [ table 2 ] .", "the tukey test showed that the two groups of relaxation and problem solving had significant difference ( p = 0.018 ) . also , the intervention groups had significant difference compared to the control group ( p = 0.000 ) [ table 3 ] .", "comparison of mean difference of self - esteem scores in three groups before and after intervention comparison of three groups based on tukey test", "generally , the three groups showed a significant difference regarding the mean difference of self - esteem score . in this study , both the interventions of relaxation and problem solving could increase the self - esteem score , while the control group showed no increase in self - esteem score .", "these findings indicate that both types of training ( problem solving and relaxation ) can produce a positive change in the overall self - esteem and improve the behavioral problems of women later in the postpartum period .", "findings and the methods used in the study of ress are similar to those of our study .", "unfortunately , no other study has been found that examined the effect of relaxation on self - esteem .", "aylward et al . found that self - esteem is the most important predictor of social problem - solving model and high self - esteem plays a major role in the relationship between problem - solving ability and psychological balance .", "the study of abuhamzeh showed that training of problem solving significantly increases the overall self - esteem in the intervention group than in the control group .", "although abuhamzeh worked on healthy students , the method of performing problem - solving skills is similar to our study .", "also , sharifi showed that problem - solving training could increase the self - esteem in students with behavioral problems .", "the present study showed similar results , but sharifi performed problem solving in 9 weeks , each session lasting 90 min , and we performed six sessions of 4560 min duration each .", "in addition , although the type of instrument used to evaluate self - esteem and the sex ( male and female ) and number of individuals were different in this study compared to our study , the results of both studies showed the effectiveness of this type of training in increasing the self - esteem score .", "we observed that the training of problem - solving skills increases the self - esteem score of depressed women more than relaxation training in the postpartum period .", "although problem solving had more effect on self - esteem , the relaxation method is simple and easily practiced at home .", "relaxation is accepted by most people and no need to haunt or many facilities . while in problem - solving method , an average of 214 min per patient visit with a psychiatrist or a general practitioner is required , and thus is time consuming .", "however , no study is found that compared the effect of relaxation and problem solving on self - esteem .", "although relaxation technique is simpler than the procedure of problem solving , the constitution of the classes and meetings for women can increase the self - esteem of subjects because women reach better understanding of the problem and solve it , while the relaxation technique was performed at home in a shorter time ( 20 min vs. 45 min sessions ) .", "in addition to the above studies , the study of stephens in southern connecticut state university shows problem - solving training can boost the self - esteem of the participants . in this study ,", "self - esteem scores were measured by a scale rating 21 questions before and 3 months after intervention .", "the average of self - esteem score increased after the intervention ( from 1.76 before intervention to 3.53 after intervention ) .", "the results of study showed that this type of training can increase the self - esteem scores in men , as well as in special occasions such as domestic violence and mental health problems such as postpartum depression .", "overall , the findings of the present study confirm that relaxation training and problem solving are successful as they can have an effect on a person 's self - concept and increase his / her self - esteem .", "also , women with postpartum depression often use dysfunctional problem - solving styles in stressful situations . the styles and techniques of treatment that are representative of the style of problem solving including cognitive behavioral therapy are fit and advantageous for the treatment of psychiatric and behavioral problems and can help women suffering from postpartum depression . because of the importance of self - esteem during pregnancy and postpartum period , it is recommended that these two types of training programs are performed in pregnancy and the postpartum period with longer follow - up period . since the midwife as a primary carer in the postpartum period could play a role in the prevention and promotion of self - esteem in women with depression , it is recommended to consider and apply both types of training programs in health centers by trained midwives .", "one of the study limitations was the time limit for performing this research in the pregnancy period .", "also , this study was not practical as a double - blind method ; the researcher herself was responsible for training the women .", "in addition , the relaxation technique was performed at home on a daily basis and so careful control by the researcher was not possible .", "so , we attempted to ensure that these techniques were performed on a daily basis only with repeated phone calls and by encouraging women , as well as by providing educational cds . also , it was not possible to use from clinical interview by psychiatrist due to high costs during two stages ( before and after the intervention ) ; therefore , such as similar studies , only depression was measured using a standard questionnaire .", "the strengths of this study is performance in three groups randomly and at various health centers . due to the lack of bias in the results", "consideration of postpartum depression using two questionnaires in the first step and confirmation by the clinical psychologist in the next phase of the study is another strong point .", "based on the results of this study and the relationship of self - esteem with depression and the major role of problem - solving skills in improving the self - esteem score , we suggest that this simple technique is performed in the health centers by midwives for increasing the self - esteem and decreasing the depressive symptoms , and thus improving the mental health . also , relaxation was effective in increasing self - esteem .", "therefore , relaxation is proposed as an auxiliary or replacement method along with other methods in increasing the self - esteem ." ]

[ "<S> background : self - esteem is a determinant factor of mental health . </S>", "<S> individuals with low self - esteem have depression , and low self - esteem is one of main symptoms of depression . </S>", "<S> aim of this study is to compare the effects of problem - solving skills and relaxation on the score of self - esteem in women with postpartum depression.materials and methods : this clinical trial was performed on 80 women . </S>", "<S> sampling was done in mashhad healthy centers from december 2009 to june 2010 . </S>", "<S> women were randomly divided and assigned to problem - solving skills ( n = 26 ) , relaxation ( n = 26 ) , and control groups ( n = 28 ) . </S>", "<S> interventions were implemented for 6 weeks and the subjects again completed eysenck self - esteem scale 9 weeks after delivery . </S>", "<S> data analysis was done by descriptive statistics , kruskal </S>", "<S> wallis test , and analysis of variance ( anova ) test by spss software.results:the findings showed that the mean of self - esteem scale scores was 117.9 9.7 after intervention in the problem - solving group , 117.0 11.8 in the relaxation group , and 113.5 10.4 in the control group and there was significant difference between the groups of relaxation and problem solving , and also between intervention groups and control group.conclusions:according to the results , problem - solving skills and relaxation can be used to prevent and recover from postpartum depression . </S>" ]

[ "I", "M", "Intervention in the problem-solving group", "Intervention in the relaxation group", "Control group", "R", "D", "C" ]

[ [ "self - esteem is an important state of mind which allows individuals to accept themselves as they are and be self - confident .", "rosenberg ( 1965 ) considered self - esteem as the positive or negative attitude of an individual toward himself .", "if an individual has a positive attitude in self - assessment , his self - esteem would be high ; if he has a negative attitude , then his self - esteem would be low .", "self - esteem is a psychological phenomenon that has a definite effect on the emotional and cognitive dimensions of an individual .", "the individuals with a high self - esteem tend to perceive themselves worth being respected and approved , important and beneficial . on the other hand , the individuals who have a negative idea about themselves or who have low self - esteem tend to perceive themselves as not very important , lacking lovability , and with no comfort in themselves and their capabilities .", "high self - esteem is considered important because it is associated with higher levels of psychological health and functioning and low level of self - esteem is undesirable because it is associated with lower levels of psychological health and functioning .", "one of the major reasons for researchers interest on self - esteem is its effects on health .", "damaged self - esteem makes it impossible to endure the harsh conditions of everyday life and produces adverse physical and psychological outcomes .", "therefore , there is always the risk of appearance of mental disease in the postpartum period .", "the postpartum depression is a disorder of non - psychosis depression according to the study of hendrick et al . as per the american psychiatric association [ diagnostic and statistical manual of mental disorder - iv ( dsm - iv ) ] ,", "brown et al . reported that about 50% of women are affected by depression at the first 3 months after delivery , whereas one - third of them are affected by depression after 3 months of delivery .", "although several factors influence depression , the psychosocial factors play a strong role in prediction of this disorder .", "self - esteem is one of the variables that affects deleteriously on the pregnancy outcomes and postpartum period .", "'s study showed that self - esteem may be a useful predictor of disorders such as anxiety and depression .", "depression and self - esteem tend to be highly correlated with each other . it has been accepted that depressed people think in a negative manner and report lower self - esteem than non - depressed people .", "the robustness of the effect also strengthens the potential importance of self - esteem interventions , said sowislo .", "she believes that treatments aimed at reducing depression by way of improving self - esteem could provide not only short - term gains for clients but also long - term protection from depression for those most at risk .", "it was found that individuals who have effective and positive social problem - solving approach have a high self - esteem .", "the individuals with high self - esteem generally have more confidence in their initial approaches to the problems and , therefore , they seek less information before offering solutions and making decisions .", "problem - solving approach is a cognitive behavioral process . by means of problem - solving skills", ", people can identify and recommend the effective strategies to face stressful situations in everyday life . social problem solving", "can be viewed as an important conformity mechanism that serves to increase behavioral competence and it leads to decrease in psychological distress .", "dzurilla suggested that self - esteem may play an important role in the relationship between self - appraised social problem - solving ability and psychological adjustment .", "also , mccabe et al . found that social problem solving was predicted by self - esteem and depressive symptoms . in aylward 's study ,", "self - esteem emerged as the most important predictor of social problem - solving model .", "some evidence was found to link the relationship between self - esteem and problem - solving skills . in the study of mahmoudi rad ", "entitled the role of social problem solving and communication skills training on enhancing self - esteem level and its relationship to mental functioning and academic achievement of students , it was found that self - esteem scores of the two groups showed significant difference before and after intervention .", "the researchers stated that the findings suggest a direct effect and positive role of training of communication skills and social problem - solving abilities when a person faces social and personal problems .", "the study of abuhamzeh entitled the effect of problem solving training on increasing the self - esteem of students in school showed that training of problem solving significantly increases overall , academic , social , and family self - esteem in the intervention group than in the control group .", "this effect remains for up to 2months after the education . in the study of sharifi", ", it was found that problem - solving training positively affected the self - esteem of students with behavior problems .", "based on studies , we can conclude that with higher problem - solving skills and social problem solving , we can increase a person 's self - esteem and decrease the depressive symptoms .", "several studies showed that low self - esteem is associated with increased anxiety and depression .", "rees conducted an exploratory study of the effectiveness of guided imagery protocol with relaxation on reducing anxiety and depression and increasing self - esteem in primiparas during the first postpartum month .", "not only relaxation and imagery decrease stress , pain , and anxiety , but also they generate a more positive understanding and a more strong feeling about well - being . with relaxation", ", the person can attach importance to herself and she can perceive everything that she needs .", "this develops self - appraised process which is the first important step toward self - esteem .", "although there is little evidence about the effect of relaxation on self - esteem , its positive effect is somewhat proved .", "the relaxation training is simple technique and easy to practice at home ; it can increase the self - esteem and decrease psychological problems .", "relaxation and problem - solving training are behavioural cognitive interventions that help to decrease depressive symptoms and increase self - esteem ; also , the self - esteem of depressed women is low during postpartum period .", "hence , a study should be performed for determining the more effective and simpler technique .", "this study was done with the aim of comparing the effects of problem - solving skills training and relaxation on the self - esteem of women in postpartum period ." ], [ "this was a clinical trial with a control group in 39 weeks in postpartum period and was conducted after getting permission from the ethics committee of mashhad university of medical science .", "all women on days 1520 after delivery who referred to mashhad health centers from december 2009 to june 2010 participated in the study after completing the consent form .", "the inclusion criteria were : literate women , 1835 years old , with the term neonate , singleton , healthy infant , the history of lack of chronic medical illness , without history of previous depression , and no smoking .", "at first , edinburgh postnatal depression scale ( epds ) was completed by the subjects .", "women whose get score 10 or above , completed beck depression inventory ( bdi ) .", "those who obtained a score of 1428 and confirmed to be depressed by a clinical interview conducted by a psychologist ( based on dsm - iv criteria ) in the health centers were divided into three groups randomly : problem - solving training ( n = 26 ) , relaxation ( n = 26 ) , and control group ( n = 28 ) [ figure 1 ] .", "eysenck self - esteem scale which has seven items and a score of 140 was completed by the women at the onset of research .", "interventions in the two groups of problem solving and relaxation were performed by a researcher ( midwife ) .", "the researcher had been taught by a clinical psychologist and after gaining the skills , she received a certificate of performance of relaxation and problem solving skills .", "a pilot study had been done on depressed women in ebne - sina hospital in mashhad before conducting the main study .", "chart of sampling process intervention was performed in the problem - solving group , which consisted of six sessions weekly in the healthy centers .", "the researcher notified the appointment by telephone to the research units before the intervention . in the problem - solving group , at the first session which lasted 1 h , the participants were trained in five skills of problem solving ( general orientation , problem definition and formulation , generation of alternatives , decision making , and verification ) . in the next sessions which lasted 4550 min , depressive symptoms of women were studied according to the beck inventory ; different solutions were presented by women and the best solution was selected for performing until the next session .", "the recording checklist of problem - solving sessions was completed by the researcher in each session . in the relaxation group ,", "the first two sessions of relaxation were performed in the health center , and each session lasted about half an hour ; the other sessions took 1020 min . in the first session of relaxation training , the causes of postpartum depression and the ways of controlling it were discussed and the relaxation purposes were explained .", "also , women were trained about how to contract and relax their 16 groups of muscles similar to jacobson 's method , and the practice was demonstrated by the researcher", ". then the practice was repeated once in the presence of a researcher by a training mother . in the second session", ", progressive muscle relaxation technique was performed once again by a mother in the presence of the researcher and after correction and ensuring the accuracy of the technique , the cd of progressive muscle relaxation was given to the women to perform the exercise daily at home .", "in the next four sessions , in addition to the exercises of relaxation performed at home , the imagery techniques ( forest visualization , beach , pilgrimage sits , and green space with good climate ) were performed by the researcher .", "the relaxation exercise was performed by the women at home for about 20 min daily .", "the form of recording the exercises performed at home was given to women and it was emphasized to bring it along with them in each session . at the beginning of each session , the positive and negative experiences of women were considered and they asked about how they could perform relaxation techniques .", "the control group recoursed to health center weekly and they bring the recording checklistof postpartum depressive symptoms with themselves and routine care after delivery is performed for them according to the booklet of safe mother .", "nine weeks after delivery , each of the three groups completed the eysenck self - esteem scale again as a post - test . in this research ,", "the instruments of data collection included the edinburgh depression scale , beck depression inventory - ii , eysenck self - esteem scale , the socio - demographic questionnaire , recorded checklist of depressive symptoms , and recorded checklist of problem - solving sessions .", "the edinburgh scale is a standard instrument for screening depressive women in the postpartum period .", "this instrument assesses the feeling of women in the past 7 days and it has 10 items and each item has a score of 03 .", "it assesses the feeling of women in the past 1 week and its answers are scored between 0 and 3 .", "eysenck self - esteem scale has 7 items and each item has a score of 020 .", "the first two sections included 20 questions about personal characteristics , pregnancy , and recent childbirth , which were completed before implementing interventions and its two questions were completed on referring to patients files .", "the third section included four questions that collected information on the availability of someone to help at home , sleeplessness difficulties , and breastfeeding , and it was completed 9 weeks after delivery .", "the validity of beck depression test was confirmed by mohammad khani in tehran , and in this study , the alpha coefficient was 0.827 .", "the data obtained in this study were analyzed using descriptive statistics , analysis of one - way variance , chi - square test , and spearman correlation coefficient by spss software ." ], [ "intervention was performed in the problem - solving group , which consisted of six sessions weekly in the healthy centers .", "the researcher notified the appointment by telephone to the research units before the intervention . in the problem - solving group , at the first session which lasted 1 h , the participants were trained in five skills of problem solving ( general orientation , problem definition and formulation , generation of alternatives , decision making , and verification ) . in the next sessions which lasted 4550 min , depressive symptoms of women were studied according to the beck inventory ; different solutions were presented by women and the best solution was selected for performing until the next session .", "the recording checklist of problem - solving sessions was completed by the researcher in each session ." ], [ "in the relaxation group , the first two sessions of relaxation were performed in the health center , and each session lasted about half an hour ; the other sessions took 1020 min . in the first session of relaxation training , the causes of postpartum depression and the ways of controlling it were discussed and the relaxation purposes were explained .", "also , women were trained about how to contract and relax their 16 groups of muscles similar to jacobson 's method , and the practice was demonstrated by the researcher .", "then the practice was repeated once in the presence of a researcher by a training mother . in the second session", ", progressive muscle relaxation technique was performed once again by a mother in the presence of the researcher and after correction and ensuring the accuracy of the technique , the cd of progressive muscle relaxation was given to the women to perform the exercise daily at home . in the next four sessions , in addition to the exercises of relaxation performed at home , the imagery techniques ( forest visualization , beach , pilgrimage sits , and green space with good climate ) were performed by the researcher .", "the relaxation exercise was performed by the women at home for about 20 min daily .", "the form of recording the exercises performed at home was given to women and it was emphasized to bring it along with them in each session . at the beginning of each session , the positive and negative experiences of women were considered and they asked about how they could perform relaxation techniques ." ], [ "the control group recoursed to health center weekly and they bring the recording checklistof postpartum depressive symptoms with themselves and routine care after delivery is performed for them according to the booklet of safe mother .", "nine weeks after delivery , each of the three groups completed the eysenck self - esteem scale again as a post - test . in this research ,", "the instruments of data collection included the edinburgh depression scale , beck depression inventory - ii , eysenck self - esteem scale , the socio - demographic questionnaire , recorded checklist of depressive symptoms , and recorded checklist of problem - solving sessions .", "the edinburgh scale is a standard instrument for screening depressive women in the postpartum period .", "this instrument assesses the feeling of women in the past 7 days and it has 10 items and each item has a score of 03 .", "it assesses the feeling of women in the past 1 week and its answers are scored between 0 and 3 .", "eysenck self - esteem scale has 7 items and each item has a score of 020 .", "the first two sections included 20 questions about personal characteristics , pregnancy , and recent childbirth , which were completed before implementing interventions and its two questions were completed on referring to patients files .", "the third section included four questions that collected information on the availability of someone to help at home , sleeplessness difficulties , and breastfeeding , and it was completed 9 weeks after delivery .", "the validity of beck depression test was confirmed by mohammad khani in tehran , and in this study , the alpha coefficient was 0.827 .", "the data obtained in this study were analyzed using descriptive statistics , analysis of one - way variance , chi - square test , and spearman correlation coefficient by spss software ." ], [ "each of the three groups showed no significant statistical differences regarding their age ( p = 0.680 ) , husband support ( p = 1.000 ) , satisfaction in marital life ( question by likert scale ) ( p = 0.506 ) , unwanted pregnancy ( p = 0.392 ) , neonate sex ( p = 0.253 ) , social support ( cassidy social support scale ) ( p = 0.078 ) , self - esteem ( p = 0.145 ) , and the score of edinburgh depression scale ( p = 0.258 ) [ table 1 ] . in this study ,", "53.8% women had high , 42.5% had moderate , and 3.8% had low self - esteem .", "descriptive characteristics of problem - solving training , relaxation , and control groups before intervention the mean of self - esteem score was 103.7 21.6 before intervention in the problem - solving group that increased to 117.9 9.7 after the intervention . also , in the relaxation group , the mean of self - esteem score was 112.1 16.7 before the intervention , which increased to 117.0 11.8 after the intervention . in the control group ,", "the mean of self - esteem score was 111.8 13.1 in the beginning of the study and it changed to 113.5 10.4 at the end of the study . by means of self - esteem scale ,", "the self - esteem levels in each group were obtained . in our study , the self - esteem of relaxation group was moderate ( 42.3% ) and high ( 57.7% ) before the intervention and reached a moderate level in 23.1% and a high level in 76.9% of the cases after the intervention .", "results showed relaxation can increase moderate self - esteem to high self - esteem . in the present study ,", "the self - esteem score was low in 11.5% , moderate in 34.6% , and high in 53.8% of the problem - solving group before the intervention , which became moderate in 15.4% and high in 84.6% of the cases after the intervention . in the ninth week after delivery at which the intervention of relaxation and problem - solving training groups was completed , the statistical results of one - way variance analysis showed that the three groups had no significant differences at the end of study ( p = 0.288 ) . but according to the mean difference of self - esteem score before and after the intervention ( by one - way anova test ) , it was found that there was significant difference among the three groups [ table 2 ] .", "the tukey test showed that the two groups of relaxation and problem solving had significant difference ( p = 0.018 ) . also , the intervention groups had significant difference compared to the control group ( p = 0.000 ) [ table 3 ] .", "comparison of mean difference of self - esteem scores in three groups before and after intervention comparison of three groups based on tukey test" ], [ "generally , the three groups showed a significant difference regarding the mean difference of self - esteem score . in this study , both the interventions of relaxation and problem solving could increase the self - esteem score , while the control group showed no increase in self - esteem score .", "these findings indicate that both types of training ( problem solving and relaxation ) can produce a positive change in the overall self - esteem and improve the behavioral problems of women later in the postpartum period .", "findings and the methods used in the study of ress are similar to those of our study .", "unfortunately , no other study has been found that examined the effect of relaxation on self - esteem .", "aylward et al . found that self - esteem is the most important predictor of social problem - solving model and high self - esteem plays a major role in the relationship between problem - solving ability and psychological balance .", "the study of abuhamzeh showed that training of problem solving significantly increases the overall self - esteem in the intervention group than in the control group .", "although abuhamzeh worked on healthy students , the method of performing problem - solving skills is similar to our study .", "also , sharifi showed that problem - solving training could increase the self - esteem in students with behavioral problems .", "the present study showed similar results , but sharifi performed problem solving in 9 weeks , each session lasting 90 min , and we performed six sessions of 4560 min duration each .", "in addition , although the type of instrument used to evaluate self - esteem and the sex ( male and female ) and number of individuals were different in this study compared to our study , the results of both studies showed the effectiveness of this type of training in increasing the self - esteem score .", "we observed that the training of problem - solving skills increases the self - esteem score of depressed women more than relaxation training in the postpartum period .", "although problem solving had more effect on self - esteem , the relaxation method is simple and easily practiced at home .", "relaxation is accepted by most people and no need to haunt or many facilities . while in problem - solving method , an average of 214 min per patient visit with a psychiatrist or a general practitioner is required , and thus is time consuming .", "however , no study is found that compared the effect of relaxation and problem solving on self - esteem .", "although relaxation technique is simpler than the procedure of problem solving , the constitution of the classes and meetings for women can increase the self - esteem of subjects because women reach better understanding of the problem and solve it , while the relaxation technique was performed at home in a shorter time ( 20 min vs. 45 min sessions ) .", "in addition to the above studies , the study of stephens in southern connecticut state university shows problem - solving training can boost the self - esteem of the participants . in this study ,", "self - esteem scores were measured by a scale rating 21 questions before and 3 months after intervention .", "the average of self - esteem score increased after the intervention ( from 1.76 before intervention to 3.53 after intervention ) .", "the results of study showed that this type of training can increase the self - esteem scores in men , as well as in special occasions such as domestic violence and mental health problems such as postpartum depression .", "overall , the findings of the present study confirm that relaxation training and problem solving are successful as they can have an effect on a person 's self - concept and increase his / her self - esteem .", "also , women with postpartum depression often use dysfunctional problem - solving styles in stressful situations . the styles and techniques of treatment that are representative of the style of problem solving including cognitive behavioral therapy are fit and advantageous for the treatment of psychiatric and behavioral problems and can help women suffering from postpartum depression . because of the importance of self - esteem during pregnancy and postpartum period , it is recommended that these two types of training programs are performed in pregnancy and the postpartum period with longer follow - up period . since the midwife as a primary carer in the postpartum period could play a role in the prevention and promotion of self - esteem in women with depression , it is recommended to consider and apply both types of training programs in health centers by trained midwives .", "one of the study limitations was the time limit for performing this research in the pregnancy period .", "also , this study was not practical as a double - blind method ; the researcher herself was responsible for training the women .", "in addition , the relaxation technique was performed at home on a daily basis and so careful control by the researcher was not possible .", "so , we attempted to ensure that these techniques were performed on a daily basis only with repeated phone calls and by encouraging women , as well as by providing educational cds . also , it was not possible to use from clinical interview by psychiatrist due to high costs during two stages ( before and after the intervention ) ; therefore , such as similar studies , only depression was measured using a standard questionnaire .", "the strengths of this study is performance in three groups randomly and at various health centers . due to the lack of bias in the results", "consideration of postpartum depression using two questionnaires in the first step and confirmation by the clinical psychologist in the next phase of the study is another strong point ." ], [ "based on the results of this study and the relationship of self - esteem with depression and the major role of problem - solving skills in improving the self - esteem score , we suggest that this simple technique is performed in the health centers by midwives for increasing the self - esteem and decreasing the depressive symptoms , and thus improving the mental health . also , relaxation was effective in increasing self - esteem .", "therefore , relaxation is proposed as an auxiliary or replacement method along with other methods in increasing the self - esteem ." ] ]

[ "neuromyelitis optica ( nmo ) or devic 's disease is a rare inflammatory , demyelinating disease of the central nervous system ( cns ) that predominantly targets the optic nerves and spinal cord 1 . the disease was first described in 1870 by albutt and 24 years later , devic described the clinical characteristics of nmo ", "the discovery of a specific nmo immunoglobulin ( nmoigg ) opened a new era in the classification and understanding of the pathogenesis of nmo 3 .", "nmoigg binds to aquaporin4 , which is the main channel that regulates water homeostasis in the cns .", "diagnostic criteria for nmo with aquaporin4 antibodies ( aqp4ab ) requires at least one core clinical characteristic , a positive test for aqp4ab using best available detection method ( cellbased assay recommended ) and exclusion of alternative diagnoses 1 .", "the core clinical characteristics are , for example , optic neuritis , acute myelitis , acute brainstem syndrome , symptomatic narcolepsy , or symptomatic cerebral syndrome with typical nmo brain lesions 1 .", "neuromyelitis optica must be distinguished from other demyelinating diseases , for example , multiple sclerosis .", "the presence of aqp4ab differentiates nmo from multiple sclerosis ( ms ) with high specificity 4 .", "in contrast to typical ms , the clinical events in nmo are usually more severe 5 , 6 .", "cerebrospinal fluid ( csf ) findings in nmo are also known to differ significantly from those in classical ms . csfrestricted oligoclonal igg bands are absent in most nmo patients .", "however , pleocytosis is usually mild , and frequently includes neutrophils , eosinophils , activated lymphocytes , and/or plasma cells 6 , 7 .", "studies carried out in europe , south east and southern asia , the caribbean and cuba suggest that the incidence and prevalence of nmo ranges from 0.050.4 to 0.524.4 per 100,000 , respectively 9 .", "the disease is mainly sporadic , although a few familial cases have been reported 10 .", "we describe a case of an unusual and severe course of nmo affecting almost the entire spinal cord and brain .", "examination on the day of admission revealed normal results as regards ecg , troponin i , and computed tomography ( ct ) of the chest and abdomen .", "the next day , the patient reported headache and neurological examination showed rightsided hemiparesis and afferent pupillary defect of the left eye suggesting an afferent optic nerve defect . within a few hours , the patient showed a rapid neurological deterioration with progressive tetraplegia and global decline .", "mri of the spinal cord showed myelitis in the spinal cord segments c2 to th5 ( fig .", "csf examination revealed polymorphonuclear pleocytosis ( leukocytes 1210 10/l , neutrophils 95% ) and an increased total protein concentration ( 2273 mg / l ) .", "due to spinal cord mri and csf findings , infectious transverse myelitis could not be excluded and the patient was treated with dexamethasone , acyclovir , ceftriaxone , ampicillin , and levofloxacin .", "( a ) sagittal t2fse mri of the spinal cord showing high signal changes . ( b )", "axial t2flair brain mri showing high signal changes in thalami , internal capsule , and corpus callosum .", "( c ) axial t2flair brain mri showing high signal changes in pons , medulla oblongata , cerebellum , and middle cerebellar peduncle .", "the next day , the patient 's condition worsened ; she became comatose and had a respiratory failure that required assisted ventilation ; this might be caused by bilateral phrenic nerve involvement as its roots originate from c3 to c5 where the lesion was also seen ( fig .", "brain mri showed high signal changes in thalami , internal capsule , and corpus callosum ( fig .", "1b ) and also similar changes in pons , medulla oblongata , cerebellum , and middle cerebellar peduncle ( fig .", "treatment with methylprednisolone and immunoglobulin was attempted and as serum aqp4ab was confirmed as positive ( indirect immunofluorescence assay was used , the titer was 19.85 , normal < 10 ) and the patient did not respond to the treatment , the patient was also treated by means of plasmapheresis .", "histological examination of the cns revealed extensive , sharply limited demyelination and axon defect ( fig . 2a and b ) .", "the spinal cord was almost entirely affected . only the lumbar area was partly spared .", "smaller demyelination foci were found in the periventricular area of the hippocampus and in the corpus callosum .", "demyelination was verified by showing both cd68positive macrophage infiltration and betaapp positivity as signs of axonal damage .", "( b ) the axon defect is shown by immunostaining ( brown staining ) of betaamyloid precursor protein ( app ) .", "this is the first case report of nmo described from finland from the aqp4ab era .", "there is only one older publication of a finnish nmo patient from the preaqp4antibody era 11 .", "the patient was referred to hospital with an acuteonset chest pain , which is an unusual first symptom of nmo .", "demyelination affected almost the entire spinal cord , sparing only partly the lumbar cord , which is unusual at first myelitis .", "lesions involving the lumbar or sacral spinal cord in addition to the cervical and thoracic portions have been reported only in 11% of patients at first myelitis .", "previous reports have revealed that 92% of the patients have at least one spinal cord lesion extending over three or more vertebral segments at their first myelitis .", "it has been reported that seropositive women have more severe clinical attacks than males , as evidenced by high lesion load in the spinal cord and other types of coexisting autoimmunity 6 .", "brain mri abnormalities are relatively common and may be relatively unique by virtue of localization and configuration 12 , as seen also in our patient .", "the histopathological findings in the cns , csf , and aqp4ab seropositivity are consistent with neuromyelitis opticatype demyelination , although the disease course of our case was not typical of nmo due to its rapid and severe course . according to hospitalbased observational studies , mortality of nmo ranges from 2.9% to 25% and", "reported that disease duration at the time of death ranged from 6 months to 23.6 years 6 .", "in addition , our patient 's histological examination revealed extensive , sharply limited demyelination of the spinal cord and brain in the acute phase of the disease . to the best of our knowledge ,", "the treatment in this particular case was targeted to multiple causes of the symptoms due to the unknown etiology in the beginning of the disease .", "acute attacks and relapses of nmo are generally treated with intravenous glucocorticoids followed by plasmapheresis for refractory or progressive symptoms 13 , 14 .", "however , there are no controlled trials evaluating the treatment of nmo , and recommendations are primarily supported by data from observational studies and by the clinical experience of experts . despite several forms of treatment , the patient did not survive ." ]

[ "<S> key clinical messageneuromyelitis optica is a rare inflammatory , demyelinating disease of the central nervous system that predominantly targets the optic nerves and spinal cord . </S>", "<S> our case represents an unusual and severe course of neuromyelitis optica . despite several forms of treatment </S>", "<S> , our patient died after a severe and shortterm attack . </S>" ]

[ "Background", "Clinical Details", "Discussion", "Conflict of Interest" ]

[ [ "neuromyelitis optica ( nmo ) or devic 's disease is a rare inflammatory , demyelinating disease of the central nervous system ( cns ) that predominantly targets the optic nerves and spinal cord 1 . the disease was first described in 1870 by albutt and 24 years later , devic described the clinical characteristics of nmo ", "the discovery of a specific nmo immunoglobulin ( nmoigg ) opened a new era in the classification and understanding of the pathogenesis of nmo 3 .", "nmoigg binds to aquaporin4 , which is the main channel that regulates water homeostasis in the cns .", "diagnostic criteria for nmo with aquaporin4 antibodies ( aqp4ab ) requires at least one core clinical characteristic , a positive test for aqp4ab using best available detection method ( cellbased assay recommended ) and exclusion of alternative diagnoses 1 .", "the core clinical characteristics are , for example , optic neuritis , acute myelitis , acute brainstem syndrome , symptomatic narcolepsy , or symptomatic cerebral syndrome with typical nmo brain lesions 1 .", "neuromyelitis optica must be distinguished from other demyelinating diseases , for example , multiple sclerosis .", "the presence of aqp4ab differentiates nmo from multiple sclerosis ( ms ) with high specificity 4 .", "in contrast to typical ms , the clinical events in nmo are usually more severe 5 , 6 .", "cerebrospinal fluid ( csf ) findings in nmo are also known to differ significantly from those in classical ms . csfrestricted oligoclonal igg bands are absent in most nmo patients .", "however , pleocytosis is usually mild , and frequently includes neutrophils , eosinophils , activated lymphocytes , and/or plasma cells 6 , 7 .", "studies carried out in europe , south east and southern asia , the caribbean and cuba suggest that the incidence and prevalence of nmo ranges from 0.050.4 to 0.524.4 per 100,000 , respectively 9 .", "the disease is mainly sporadic , although a few familial cases have been reported 10 .", "we describe a case of an unusual and severe course of nmo affecting almost the entire spinal cord and brain ." ], [ "examination on the day of admission revealed normal results as regards ecg , troponin i , and computed tomography ( ct ) of the chest and abdomen .", "the next day , the patient reported headache and neurological examination showed rightsided hemiparesis and afferent pupillary defect of the left eye suggesting an afferent optic nerve defect . within a few hours , the patient showed a rapid neurological deterioration with progressive tetraplegia and global decline .", "mri of the spinal cord showed myelitis in the spinal cord segments c2 to th5 ( fig .", "csf examination revealed polymorphonuclear pleocytosis ( leukocytes 1210 10/l , neutrophils 95% ) and an increased total protein concentration ( 2273 mg / l ) .", "due to spinal cord mri and csf findings , infectious transverse myelitis could not be excluded and the patient was treated with dexamethasone , acyclovir , ceftriaxone , ampicillin , and levofloxacin .", "( a ) sagittal t2fse mri of the spinal cord showing high signal changes . ( b )", "axial t2flair brain mri showing high signal changes in thalami , internal capsule , and corpus callosum .", "( c ) axial t2flair brain mri showing high signal changes in pons , medulla oblongata , cerebellum , and middle cerebellar peduncle .", "the next day , the patient 's condition worsened ; she became comatose and had a respiratory failure that required assisted ventilation ; this might be caused by bilateral phrenic nerve involvement as its roots originate from c3 to c5 where the lesion was also seen ( fig .", "brain mri showed high signal changes in thalami , internal capsule , and corpus callosum ( fig .", "1b ) and also similar changes in pons , medulla oblongata , cerebellum , and middle cerebellar peduncle ( fig .", "treatment with methylprednisolone and immunoglobulin was attempted and as serum aqp4ab was confirmed as positive ( indirect immunofluorescence assay was used , the titer was 19.85 , normal < 10 ) and the patient did not respond to the treatment , the patient was also treated by means of plasmapheresis .", "histological examination of the cns revealed extensive , sharply limited demyelination and axon defect ( fig . 2a and b ) .", "the spinal cord was almost entirely affected . only the lumbar area was partly spared .", "smaller demyelination foci were found in the periventricular area of the hippocampus and in the corpus callosum .", "demyelination was verified by showing both cd68positive macrophage infiltration and betaapp positivity as signs of axonal damage .", "( b ) the axon defect is shown by immunostaining ( brown staining ) of betaamyloid precursor protein ( app ) ." ], [ "this is the first case report of nmo described from finland from the aqp4ab era .", "there is only one older publication of a finnish nmo patient from the preaqp4antibody era 11 .", "the patient was referred to hospital with an acuteonset chest pain , which is an unusual first symptom of nmo .", "demyelination affected almost the entire spinal cord , sparing only partly the lumbar cord , which is unusual at first myelitis .", "lesions involving the lumbar or sacral spinal cord in addition to the cervical and thoracic portions have been reported only in 11% of patients at first myelitis .", "previous reports have revealed that 92% of the patients have at least one spinal cord lesion extending over three or more vertebral segments at their first myelitis .", "it has been reported that seropositive women have more severe clinical attacks than males , as evidenced by high lesion load in the spinal cord and other types of coexisting autoimmunity 6 .", "brain mri abnormalities are relatively common and may be relatively unique by virtue of localization and configuration 12 , as seen also in our patient .", "the histopathological findings in the cns , csf , and aqp4ab seropositivity are consistent with neuromyelitis opticatype demyelination , although the disease course of our case was not typical of nmo due to its rapid and severe course . according to hospitalbased observational studies , mortality of nmo ranges from 2.9% to 25% and", "reported that disease duration at the time of death ranged from 6 months to 23.6 years 6 .", "in addition , our patient 's histological examination revealed extensive , sharply limited demyelination of the spinal cord and brain in the acute phase of the disease . to the best of our knowledge ,", "the treatment in this particular case was targeted to multiple causes of the symptoms due to the unknown etiology in the beginning of the disease .", "acute attacks and relapses of nmo are generally treated with intravenous glucocorticoids followed by plasmapheresis for refractory or progressive symptoms 13 , 14 .", "however , there are no controlled trials evaluating the treatment of nmo , and recommendations are primarily supported by data from observational studies and by the clinical experience of experts . despite several forms of treatment , the patient did not survive ." ], [ "" ] ]

[ "a 16-month old , baby girl , weighing 9.5 kg and measuring 74 cm , visited the hospital because of a cough . in a chest radiograph", "done under the suspicion of an upper respiratory infection , a round foreign object with a diameter of 21 mm was detected in the upper esophagus ( fig .", "1 ) . in a sleep endoscopy , it was confirmed to be a circular battery ( fig .", "2 ) , and ingestion was estimated to occur three days prior to the hospital visit , and it was determined that endoscopic removal would not be easy .", "the infant had no underlying disease , and glycopyrrolate 0.05 mg was i m injected 30 minutes before arriving at the operating room as premedication for the anesthesia .", "the infant was monitored by ecg , non - invasive blood pressure ( nibp ) , and a pulse oximetry , and before anesthesia , her blood pressure was 85/61 mmhg ; her heart rate was 160 beats / min with 100% oxygen saturation .", "10 mg of ketamine was iv injected and after loss of consciousness , 5 mg of rocunium was iv injected .", "when the muscles were sufficiently relaxed , an uncuffed endotracheal tube with a 4 mm inside diameter was intubated .", "after intubation , both lungs were checked through a stethoscope and found to be normal , and the endotracheal tube was set to 11 cm in accordance with the top front teeth .", "anesthesia was maintained by 2 l / min of air , 2 l / min of o2 , and 2.0 - 3.5 vol% of sevoflurane .", "the tidal volume was 90 ml ; respiratory rate was 18/min through volume - controlled mechanical ventilation to maintain an end - tidal co2 ( etco2 ) pressure of 35 - 40 mmhg and the peak inspiratory pressure ( pip ) at this point was 19 cmh2o .", "an adult endoscope with a 9 mm outer diameter was used for the removal procedure because the hospital did not have a pediatric endoscope with forceps .", "2 minutes after inserting the endoscope , severe abdominal inflation was observed , and pip increased up to 28 - 30 cmh2o with the tidal volume decreasing to less than 50 ml .", "the battery had caused an inflammatory change and conglutinated onto surrounding tissue in the esophagus , and 3 minutes after inserting the endoscope , oxygen saturation decreased to 80% , and etco2 pressure rose to 70 mmhg therefore the surgery was stopped and the endoscope was removed . by manual ventilation using 100% oxygen , oxygen saturation was recovered to 100% and etco2 pressure to 40 mmhg , and thereafter , the endoscope was reinserted . in the second attempt of the procedure ,", "manual ventilation was done instead of mechanical ventilation . however , 1 minute after the insertion of the endoscope , a sudden strong resistance was felt in the reservoir bag , and the tidal volume was less than 20 ml despite applying more than 35 cmh2o for the pip .", "oxygen saturation dropped below 60% so the procedure could not be continued ; therefore , the endoscope was removed again . here , a pip of 25 - 30 cmh2o and a tidal volume of 50 ml were maintained .", "the endoscope was reinserted after recovering oxygen saturation to 100% , but due to the increased airway pressure , ventilation was not possible , and oxygen saturation dropped back down to less than 40% .", "the authors suspected the possibility of tef considering the period of intake and the excessive air volume seen in the stomach in the chest radiograph taken before the surgery .", "hence , the endotracheal tube was inserted until one lung ventilation was possible and then retreated while auscultating to secure onto the location where the pulmonary sound from both lungs became equal .", "the adjusted position of the endotracheal tube was 15 cm based on the top front teeth .", "a pip of 25 - 30 cmh2o and a tidal volume of 80 - 100 ml were maintained . in the next insertion of the endoscope ,", "a pip of 30 cmh2o and a tidal volume of 50 ml were maintained but abdominal inflation was excessive so air in the stomach was partially removed with the endoscope , and the operating doctor was required to use the minimal amount of air for the surgery to proceed .", "a pip of 25 - 30 cmh2o , a tidal volume of 50 - 80 ml , and oxygen saturation of 100% were maintained during the procedure , and the battery was removed . after the removal of the battery , the vital signs were blood pressure 96/69 mmhg , heart rate 152 beats / min , and 100% oxygen saturation .", "the patient was not extubated but moved to postoperative intensive care , and after pediatrics observed vital signs for about 3 hours to verify that there were no abnormalities , extubation was done .", "subsequent vital sign checks and physical exam were normal , and there were no abnormalities found in the blood test .", "moreover , the right main bronchus appeared on the screen in the postoperative esophagography ( fig .", "3 ) , and the chest ct scan found tef at 1.4 cm in the upper carina ( fig .", "4 ) . the guardian wanted to have surgery for tef at another hospital ; therefore , 3 days after the surgery , she was transferred .", "ingested batteries usually pass through the gastrointestinal tract and are defecated in a few days , but when it is caught in the esophagus , the moist environment of the esophagus allows for the discharge of substances inside the battery together with electrical discharges , which can cause tissue damage of the esophagus .", "in addition , necrosis can develop in the mucus membrane of the esophagus due to the pressure from the battery , and these can trigger complications such as esophageal burns , perforation , and tef .", "more severe complications arose in cases where the diameter of the battery was larger than 20 mm and the infant was younger than 4 years old .", "the main factors that cause severe complications in battery ingestion were the size ( larger than 20 - 30 mm ) and components of the battery .", "especially if the negative terminal of the battery is attached to the tissue , it can lead to more severe results .", "in addition , lithium batteries cause the most severe damages , and it was reported that in a dog 's esophagus , necrosis developed in the trachea within an hour . in this case study , the corroded battery and inflamed mucous membrane of the esophagus were confirmed in endoscopy before the surgery , but tef could not be tested and confirmed due to the urgency of the surgery .", "it is reported that esophageal burns start 4 hours after battery ingestion and perforations that form fistulas start in 6 hours . since the estimated ingestion period for this case was 3 days , it is enough time for tef to develop .", "the infant in this case study did not have respiratory difficulties before of the surgery , but ventilation failure during the surgery was because the endotracheal tube was fixed at 11 cm at first .", "the tef located approximately 2.6 cm under the end of the endotracheal tube allowed the inhalation gas for the anesthesia to flow into the stomach leading to abdominal inflation , and consecutively , the air injected for the endoscopic surgery flowed over to the respiratory tract to greatly increase the airway pressure .", "afterwards , when the endotracheal tube was inserted deeply into the carina , the tube was located past the tef so the influence of tef was eliminated for the decrease in airway pressure . when there is tef , it is important to position the endotracheal tube above the carina and below the tef to maintain anesthesia .", "this is because effective ventilation would not happen due to a loss of the tidal volume through the fistulous openings in the tef , the contents of the stomach being aspirated into the lungs , and excessive positive pressure ventilation when inducing anesthesia can cause abdominal inflation and cardiovascular suppression .", "however , in our case , ventilation was not completely recovered even when the endotracheal tube was inserted deeply to eliminate the influence of tef , and this is thought to be from accompanying ventilation failure from the endoscope .", "this ventilation failure can be caused by pressure on the pharynx and the trachea from the weight of the endoscope , or motor abnormality of the diaphragm due to abdominal inflation from the injected air .", "wengrower et al . reported that approximately 7% of infants who had endoscopic procedures under sedation or anesthesia exhibited temporary desaturation .", "there are reports that an infant with a similar height and weight developed ventilation failure from a tee probe with outer diameter of 10 8 mm . in our case , the fact that direct pressure on the respiratory tract was applied by using an adult endoscope with a 9 mm diameter and that air had to be injected continually since the battery had attached itself to the esophagus and was hard to remove could have been some other causes for the excessive airway pressure , ventilation failure , and hypoxemia .", "when upper gastrointestinal endoscopic procedures were done on 99 infants of 0.9 - 10.1 kg using an endoscope with a 5.2 mm outer diameter , only 1 infant with global developmental delay exhibited hypoxemia , and gryboski recommended using endoscopes with a 5 mm outer diameter on infants with esophageal diseases .", "it is important to use pediatric endoscopes with the smallest possible outer diameters to prevent hypoxemia during endoscopic procedures on infants . since the removal of foreign objects in the esophagus of infants", "are usually done under general anesthesia , anesthesiologists need to anticipate ventilation failure and hypoxemia from the physical pressure from the procedural equipment and the excessive injection of air and proceed with anesthesia under close cooperation with the operating surgeon .", "especially when a battery has been ingested , it is necessary to be aware that there could be complications such as tef in the location where the battery is depending on the battery 's components , size , and ingestion period .", "in addition , to more effectively block the mutual flow of air and anesthetic gas towards either the esophagus or the trachea in the case of a potential tef , a cuffed endotracheal tube should be chosen preferentially and special caution should be taken to fix the position of the tube and in selecting a suitable size for the endoscope used in the procedure ." ]

[ "<S> ingestion of disk batteries may have serious complications such as esophageal burn , perforation , and tracheoesophageal fistula , particularly when the battery is caught in the esophagus . </S>", "<S> proper placement of the tracheal tube is critical when tracheoesophageal fistula was occurred from esophageal impaction the battery . </S>", "<S> endoscopy of upper gastrointestinal tract in infants and children is an important and effective tool for the diagnosis and treatment of foreign body ingestion . but </S>", "<S> upper gastrointestinal endoscopy in infant and children has very high risk of tracheal compression and airway compromise . </S>", "<S> we present a case of ventilatory compromise during insertion of the upper gastrointestinal endoscopy in 16-month - old child with tracheoesophageal fistula secondary to disk battery ingestion . </S>" ]

[ "Case Report", "Discussion" ]

[ [ "a 16-month old , baby girl , weighing 9.5 kg and measuring 74 cm , visited the hospital because of a cough . in a chest radiograph", "done under the suspicion of an upper respiratory infection , a round foreign object with a diameter of 21 mm was detected in the upper esophagus ( fig .", "1 ) . in a sleep endoscopy , it was confirmed to be a circular battery ( fig .", "2 ) , and ingestion was estimated to occur three days prior to the hospital visit , and it was determined that endoscopic removal would not be easy .", "the infant had no underlying disease , and glycopyrrolate 0.05 mg was i m injected 30 minutes before arriving at the operating room as premedication for the anesthesia .", "the infant was monitored by ecg , non - invasive blood pressure ( nibp ) , and a pulse oximetry , and before anesthesia , her blood pressure was 85/61 mmhg ; her heart rate was 160 beats / min with 100% oxygen saturation .", "10 mg of ketamine was iv injected and after loss of consciousness , 5 mg of rocunium was iv injected .", "when the muscles were sufficiently relaxed , an uncuffed endotracheal tube with a 4 mm inside diameter was intubated .", "after intubation , both lungs were checked through a stethoscope and found to be normal , and the endotracheal tube was set to 11 cm in accordance with the top front teeth .", "anesthesia was maintained by 2 l / min of air , 2 l / min of o2 , and 2.0 - 3.5 vol% of sevoflurane .", "the tidal volume was 90 ml ; respiratory rate was 18/min through volume - controlled mechanical ventilation to maintain an end - tidal co2 ( etco2 ) pressure of 35 - 40 mmhg and the peak inspiratory pressure ( pip ) at this point was 19 cmh2o .", "an adult endoscope with a 9 mm outer diameter was used for the removal procedure because the hospital did not have a pediatric endoscope with forceps .", "2 minutes after inserting the endoscope , severe abdominal inflation was observed , and pip increased up to 28 - 30 cmh2o with the tidal volume decreasing to less than 50 ml .", "the battery had caused an inflammatory change and conglutinated onto surrounding tissue in the esophagus , and 3 minutes after inserting the endoscope , oxygen saturation decreased to 80% , and etco2 pressure rose to 70 mmhg therefore the surgery was stopped and the endoscope was removed . by manual ventilation using 100% oxygen , oxygen saturation was recovered to 100% and etco2 pressure to 40 mmhg , and thereafter , the endoscope was reinserted . in the second attempt of the procedure ,", "manual ventilation was done instead of mechanical ventilation . however , 1 minute after the insertion of the endoscope , a sudden strong resistance was felt in the reservoir bag , and the tidal volume was less than 20 ml despite applying more than 35 cmh2o for the pip .", "oxygen saturation dropped below 60% so the procedure could not be continued ; therefore , the endoscope was removed again . here , a pip of 25 - 30 cmh2o and a tidal volume of 50 ml were maintained .", "the endoscope was reinserted after recovering oxygen saturation to 100% , but due to the increased airway pressure , ventilation was not possible , and oxygen saturation dropped back down to less than 40% .", "the authors suspected the possibility of tef considering the period of intake and the excessive air volume seen in the stomach in the chest radiograph taken before the surgery .", "hence , the endotracheal tube was inserted until one lung ventilation was possible and then retreated while auscultating to secure onto the location where the pulmonary sound from both lungs became equal .", "the adjusted position of the endotracheal tube was 15 cm based on the top front teeth .", "a pip of 25 - 30 cmh2o and a tidal volume of 80 - 100 ml were maintained . in the next insertion of the endoscope ,", "a pip of 30 cmh2o and a tidal volume of 50 ml were maintained but abdominal inflation was excessive so air in the stomach was partially removed with the endoscope , and the operating doctor was required to use the minimal amount of air for the surgery to proceed .", "a pip of 25 - 30 cmh2o , a tidal volume of 50 - 80 ml , and oxygen saturation of 100% were maintained during the procedure , and the battery was removed . after the removal of the battery , the vital signs were blood pressure 96/69 mmhg , heart rate 152 beats / min , and 100% oxygen saturation .", "the patient was not extubated but moved to postoperative intensive care , and after pediatrics observed vital signs for about 3 hours to verify that there were no abnormalities , extubation was done .", "subsequent vital sign checks and physical exam were normal , and there were no abnormalities found in the blood test .", "moreover , the right main bronchus appeared on the screen in the postoperative esophagography ( fig .", "3 ) , and the chest ct scan found tef at 1.4 cm in the upper carina ( fig .", "4 ) . the guardian wanted to have surgery for tef at another hospital ; therefore , 3 days after the surgery , she was transferred ." ], [ "ingested batteries usually pass through the gastrointestinal tract and are defecated in a few days , but when it is caught in the esophagus , the moist environment of the esophagus allows for the discharge of substances inside the battery together with electrical discharges , which can cause tissue damage of the esophagus .", "in addition , necrosis can develop in the mucus membrane of the esophagus due to the pressure from the battery , and these can trigger complications such as esophageal burns , perforation , and tef .", "more severe complications arose in cases where the diameter of the battery was larger than 20 mm and the infant was younger than 4 years old .", "the main factors that cause severe complications in battery ingestion were the size ( larger than 20 - 30 mm ) and components of the battery .", "especially if the negative terminal of the battery is attached to the tissue , it can lead to more severe results .", "in addition , lithium batteries cause the most severe damages , and it was reported that in a dog 's esophagus , necrosis developed in the trachea within an hour . in this case study , the corroded battery and inflamed mucous membrane of the esophagus were confirmed in endoscopy before the surgery , but tef could not be tested and confirmed due to the urgency of the surgery .", "it is reported that esophageal burns start 4 hours after battery ingestion and perforations that form fistulas start in 6 hours . since the estimated ingestion period for this case was 3 days , it is enough time for tef to develop .", "the infant in this case study did not have respiratory difficulties before of the surgery , but ventilation failure during the surgery was because the endotracheal tube was fixed at 11 cm at first .", "the tef located approximately 2.6 cm under the end of the endotracheal tube allowed the inhalation gas for the anesthesia to flow into the stomach leading to abdominal inflation , and consecutively , the air injected for the endoscopic surgery flowed over to the respiratory tract to greatly increase the airway pressure .", "afterwards , when the endotracheal tube was inserted deeply into the carina , the tube was located past the tef so the influence of tef was eliminated for the decrease in airway pressure . when there is tef , it is important to position the endotracheal tube above the carina and below the tef to maintain anesthesia .", "this is because effective ventilation would not happen due to a loss of the tidal volume through the fistulous openings in the tef , the contents of the stomach being aspirated into the lungs , and excessive positive pressure ventilation when inducing anesthesia can cause abdominal inflation and cardiovascular suppression .", "however , in our case , ventilation was not completely recovered even when the endotracheal tube was inserted deeply to eliminate the influence of tef , and this is thought to be from accompanying ventilation failure from the endoscope .", "this ventilation failure can be caused by pressure on the pharynx and the trachea from the weight of the endoscope , or motor abnormality of the diaphragm due to abdominal inflation from the injected air .", "wengrower et al . reported that approximately 7% of infants who had endoscopic procedures under sedation or anesthesia exhibited temporary desaturation .", "there are reports that an infant with a similar height and weight developed ventilation failure from a tee probe with outer diameter of 10 8 mm . in our case , the fact that direct pressure on the respiratory tract was applied by using an adult endoscope with a 9 mm diameter and that air had to be injected continually since the battery had attached itself to the esophagus and was hard to remove could have been some other causes for the excessive airway pressure , ventilation failure , and hypoxemia .", "when upper gastrointestinal endoscopic procedures were done on 99 infants of 0.9 - 10.1 kg using an endoscope with a 5.2 mm outer diameter , only 1 infant with global developmental delay exhibited hypoxemia , and gryboski recommended using endoscopes with a 5 mm outer diameter on infants with esophageal diseases .", "it is important to use pediatric endoscopes with the smallest possible outer diameters to prevent hypoxemia during endoscopic procedures on infants . since the removal of foreign objects in the esophagus of infants", "are usually done under general anesthesia , anesthesiologists need to anticipate ventilation failure and hypoxemia from the physical pressure from the procedural equipment and the excessive injection of air and proceed with anesthesia under close cooperation with the operating surgeon .", "especially when a battery has been ingested , it is necessary to be aware that there could be complications such as tef in the location where the battery is depending on the battery 's components , size , and ingestion period .", "in addition , to more effectively block the mutual flow of air and anesthetic gas towards either the esophagus or the trachea in the case of a potential tef , a cuffed endotracheal tube should be chosen preferentially and special caution should be taken to fix the position of the tube and in selecting a suitable size for the endoscope used in the procedure ." ] ]

[ "lung cancer is the main cause of cancer deaths both in poland and in the world .", "non - small cell lung cancer accounts for about 85% of all lung cancers , and the progress in its treatment is still not very satisfactory . the therapeutic procedure of choice is surgery , but only every fifth patient qualifies for it .", "this is due to the fact that most patients at the time of diagnosis are in an advanced stage of the disease , usually accompanied with tumour metastases . in about 25% of such patients extension of life by a few month", "such disappointing results force us to search for new therapeutic options [ 2 , 3 ] .", "one of the new molecular targets for non - small cell lung cancer ( nsclc ) therapy is the epidermal growth factor receptor ( egfr ) .", "egfr ( her1 ) belongs to the erbb ( her ) receptor family . for its activation , not only", "a connection with a suitable ligand is necessary , but also homo- or heterodimerisation on the cell surface with other receptors of the her family ( her 2 - 4 ) .", "the intracellular domain of egfr has activity of tyrosine kinase and it is responsible for the phosphorylation of cellular proteins in the pi3k / akt signalling pathway .", "furthermore , this domain has regulatory functions . in many cancers , including nsclc , activating mutations or overexpression of the egfr gene are found . in some cases progression of the disease ", "uncontrolled cell proliferation , inhibition of apoptosis , and the ability to metastasise is the effect of excessive activation of intracellular pathways constantly stimulated by egfr .", "therefore , there is more and more interest in treatment associated with inhibition of egfr [ 46 ] .", "one of them is blocking of the extracellular domain of the receptor through monoclonal antibodies ( cetuximab , panitumumab ) , which prevents connection of the egfr ligand or receptor dimerisation . blocking of signal transduction from the cell membrane to the nucleus by small molecule inhibitors of tyrosine and serine - threonine kinase is still in the experimental phase . the biggest hopes for improving the prognosis in nsclc", "are associated with the introduction of small molecule , reversible tyrosine kinase inhibitors of egfr ( egfr tki ) gefitinib and erlotinib .", "the mechanism of their action is based on reversible binding to the intracellular tyrosine kinase domain of egfr and blocking of atp binding .", "selective connectivity of the inhibitor prevents phosphorylation of the tyrosine kinase domain , and in consequence activates the pathway of cellular signal transduction , and leads to cell cycle arrest in g1 phase and increase in apoptosis of tumour cells .", "the therapeutic effect of egfr tki depends on the amino acid structure of the tyrosine kinase domain conditioned upon the state of the egfr gene .", "appearance of the most frequent activating mutation deletion of 15 nucleotides in codons 746 - 750 in exon 19 and the l858r substitution in exon 21 of the egfr gene is associated with permanent stimulation of the receptor , but they also lead to an increase in both efficiency of reversible egfr tki and effectiveness of radiotherapy . activating mutations of the egfr gene have been reported in only about 10% of caucasians patients with nsclc , more often in non - smokers , women , and patients with adenocarcinoma .", "therefore , the first studies on the efficacy of erlotinib and gefitinib in second - line treatment ( br.21 , isel , interest ) have shown that an objective response to egfr tki treatment occurs in less than 10% of patients in the general population [ 1014 ] . in recent clinical trials ( ipass , optimal , eurtac ) conducted on carriers of activating mutations in the egfr gene , over 70% response and almost one year progression - free survival ( pfs )", "for this reason , both inhibitors were granted registration in the treatment of locally advanced and advanced nsclc in first - line of treatment , and are an alternative to standard chemotherapy in patients with activating mutations of the egfr gene .", "the method of qualification for egfr tki monotherapy in second- or third - line treatment in nsclc is still debatable and unclear [ 1016 ] . in the case of the wild type form of the egfr gene ,", "even if the receptor is overexpressed , egfr tki have not shown strong antitumor activity ( none or a few percent of objective response ) mainly for two reasons : lack of changes in the structure of the atp - binding pocket of tyrosine kinase domain and the small role of the normal egfr form in carcinogenesis of lung cancer .", "therefore , investigation of the expression of egfr on the cell surface ( by immunohistochemistry ) or amplification of the egfr gene detected by molecular probes and fluorescence in situ hybridization ( fish ) did not find a wider application in qualification for egfr tki therapy . in some cases ,", "it is possible to consider the usefulness of the determination of egfr gene amplification by the fish method , if examination of the mutation is unfeasible or the outcome is uncertain , bearing in mind the frequent co - existence of egfr gene mutations ( 10% of patients ) with an increase in copy number of the gene ( 40% of patients ) in tumour cells [ 7 , 8 ] .", "unfortunately , regardless of the applied line of egfr tki treatment , at some point of therapy , even in patients who carry an activating mutation in the egfr gene and show a response , there is a recurrence of the disease and the patient 's life is not prolonged [ 17 , 18 ] .", "some mechanisms associated with resistance to reversible egfr tki in nsclc are already known . the t790 m mutation in exon 20 of the egfr gene", "was detected for the first time in tumour cells of a patient with progression after a few months of efficacious therapy with gefitinib .", "that point mutation is caused by a single nucleotide substitution ( c- > t ) at position 2369 ( codon 790 ) in exon 20 of the egfr gene .", "the t790 m mutation leads to a substitution of threonine to methionine in the catalytic centre of egfr tyrosine kinase , which is located in the binding pocket of tki and atp .", "substitution of threonine at codon 790 into larger methionine probably results in blocking of the binding sites of erlotinib and gefitinib aromatic residues with their point of action . in vitro studies demonstrated that the effective concentration of erlotinib in tumour cells harbouring both the l858r and t790 m mutations has to be 300 times higher to induce apoptosis than in cells only with an activating mutation in codon 858 [ 19 , 20 ] .", "presence of a mutation in exon 20 of the egfr gene is a potential prognostic and predictive marker of egfr tki therapy and may play an important role in the planning of therapy .", "activating mutations in egfr were detected in cells obtained from primary tumour , circulating tumour cells in peripheral blood ( consistency of results 92% ) and in free circulating dna ( consistency of results ", "the authors also investigated presence of the t790 m mutation in similar samples , and they detected cells harbouring that mutation in 10 patients ( n = 26 ; 38.5% ) .", "median survival in patients with the t790 m mutation treated with reversible egfr tki was 7.7 months against 16.5 months in patients without that mutation ( p < 0.001 ) .", "mutations were detected using sarms real - time pcr technique . that investigation has also shown that mutation in exon 20 of the egfr gene leads to acquired resistance to treatment in patients not only with the wild type egfr gene , but also in patients with confirmed clinical sensitivity to egfr tyrosine kinase inhibitors .", "presence of t790 m substitution may be an indication for treatment with irreversible egfr tki , such as neratinib , keratinib and especially afatinib , which is currently in the third phase of clinical trials .", "the lux - lung 1 study has shown that afatinib induces an objective response and prolongation of progression - free survival ( pfs ) in nsclc patients who have been previously treated with egfr tki for at least 12 weeks .", "the rationale for this type of study is the fact that afatinib has a different molecular structure when compared to gefitinib and erlotinib .", "this structure causes that afatinib forms a covalent bond with cysteine at codon 733 of the egfr tyrosine kinase domain .", "therefore , inhibition of afatinib is only possible due to synthesis of new receptor proteins .", "inhibition of phosphorylation by egfr tyrosine kinase in tumour cells after afatinib exposure remains active for 48 hours ( after gefitinib only 8 hours ) , and phosphorylation activity of the kinase after that time ranges from 50 to 70% of the primary activity . in vitro studies and some case reports", "have shown that afatinib is effective ( cell apoptosis and disease stabilisation ) in cells harbouring the t790 m mutation in nsclc patients [ 19 , 2226 ] .", "the mechanism of mutation appearance in nsclc recurrence in egfr tki treated patients should be thoroughly examined .", "a resistant cell subpopulation harbouring t790 m can emerge in the tumour probably as a result of massive apoptosis of tumour cells harbouring an activating mutation associated with response to therapy .", "the authors examined presence of mutations in exons 18 - 24 of the egfr gene in patients treated with egfr tki in order to determine if additional mutations in the egfr gene are associated with progression of the disease . in patients with mutations identified in exon 19 or 21 additional mutations", "samples obtained from patients tumour cells before treatment were re - examined to exclude the possibility of missing changes in exon 20 of egfr . however , no evidence for the presence of the sought mutations was found .", "mutations associated with resistance to egfr tki were observed in three tumor recurrences that emerged after treatment .", "it was the t790 m mutation coexisting with deletion in exon 19 or l858r substitution in the egfr gene .", "results of the investigations suggest that the resistance to treatment is acquired during therapy with egfr tki , and mutations may form de novo .", "it is worth mentioning that tumour cell clones harbouring t790 m substitution may be rare , and the mutation may not be detected ( due to the small number of cells with the t790 m mutation in the whole sample ) .", "it is observed especially when the dna sequencing technique is employed , which is characterised by relatively low sensitivity and requires the presence of at least 50% of mutated tumour cells in the examined sample .", "currently it is believed that t790 m substitution occurs in over 50% of cases of acquired resistance to reversible egfr tki in nsclc patients and in approximately 5% of patients before egfr tki treatment .", "before the therapy with egfr tki , the t790 m mutation emerges along with activating mutations of the egfr gene , and secondarily it coexists with the primary activating mutation in egfr , especially l858r [ 17 , 2831 ] . with time", ", it was found that other mutations in exon 20 and 21 of egfr ( insertion - deletion , substitutions : d761y , t854a , etc . )", "these mutations account for less than 5% of all known mutations in the egfr gene .", "they are detected very rarely , so reports about their presence should be made carefully , and egfr tki treatment outcomes in patients harbouring these mutations should be confirmed on a larger group of patients .", "furthermore , it is necessary to understand the mechanism of resistance caused by these mutations .", "d761y and t854a mutations in the egfr gene were detected for the first time in patients with activating l858r mutation .", "probably substitution of aspartic acid with tyrosine at position 761 is responsible for acquisition of resistance to tki treatment of tumour cells with an activating mutation which are sensitive to the treatment ( this theory was confirmed in tumour cell lines in vitro ) .", "however , this resistance to egfr tki has been many times lower than the resistance in cells harbouring both the l858r and t790 m mutations . in case of occurrence of both l858r and t790 m mutations ,", "the concentration of erlotinib should be three times higher in order to achieve a therapeutic effect .", "analysis of the egfr structure in patients harbouring the t854a mutation suggests the presence of an additional side chain , localised near the egfr tki binding site .", "the role of rare mutations in exon 20 such as s768i and v769l in development of acquired resistance to egfr tki is still unknown .", "these substitutions were reported for the first time in nsclc patients with progression , bearing activating mutations , after gefitinib therapy . in vitro cultures of cell lines harbouring", "the s768i substitution showed slightly more resistance to egfr tki than cells with wild type egfr . in these cells permanent hyperphosphorylation of tyrosine at codon 1045 of the tyrosine kinase domain has been observed [ 17 , 3234 ] .", "insertion - deletion mutations in exon 20 of egfr are also associated with acquired resistance to egfr tki .", "these mutations , similarly to activating mutations in the egfr gene , are more often detectable in asians , non - smokers , women and in patients with adenocarcinoma . most of the pre - clinical investigations allowed observation of their impact on resistance to therapeutic doses of gefitinib or erlotinib , and some of the irreversible egfr tki , e.g. neratinib . however , in the most common mutations such as n771gy and a767-v769 duplication , even though lack of sensitivity to erlotinib has been reported , low sensitivity to irreversible egfr tki such as afatinib and pf-00299804 was observed .", "crystallographic analysis of epidermal growth factor receptor structure has shown that both mutations have an influence on the spatial structure of tyrosine kinase c - helix , which plays an important role in phosphorylation activity of that enzyme .", "other insertions in exon 20 of the egfr gene such as v738inskipvai , m766insasv , d770insnpg and d770insnph seem to be associated with that process , but their role in development of resistance to reversible egfr tki is still unknown [ 35 , 36 ] .", "many studies have demonstrated that the presence of mutations at codons 12 , 13 or 61 in the k - ras gene is not a predictive factor in treatment with egfr tki .", "these observations stem from the fact that mutations in the k - ras gene almost never coexist with mutations in the egfr gene .", "lack of mutations in egfr determines the inefficiency of egfr tki used in therapeutic doses , which means that the occurrence of mutations in the k - ras gene is no longer relevant . therefore there were no objective responses to treatment with egfr tki among carriers of k - ras mutations , which was initially considered as an independent , adverse predictive factor in egfr tki treatment .", "in fact , in vitro studies have shown that the destruction of tumour cells with the wild - type egfr and mutant k - ras gene is only possible with very high , non - therapeutic doses of egfr tki [ 3739 ] . however , in some individual case reports in which coexistence of both egfr and k - ras gene mutations have been observed , the efficacy of egfr tki was not entirely confirmed .", "the situation is presented in a different manner when monoclonal anti - egfr antibodies , such as cetuximab , are used in the treatment of colorectal cancer , head and neck cancer , and perhaps soon in nsclc with strong overexpression of egfr .", "mutations in the k - ras gene are a negative predictor here , and are routinely determined in qualification of patients for therapy . however , in the case of therapy with egfr tki the only test eligible for treatment is investigation of egfr gene mutations [ 37 , 41 , 42 ] .", "activation of an alternative signalling pathway from stimulated egfr protein leads to activation of ras protein and subsequently kinases associated with raf / mapk / erg proteins and transcription factors such as myc , fos and jun .", "mutations which are present in the k - ras gene lead to constant activation of ras protein , so the tumour cells become independent of the signals from the egfr , which explains the mechanism of resistance conferred by the egfr tki and monoclonal anti - egfr antibodies .", "mutations in the k - ras gene occur with a frequency of 1520% in caucasian nsclc patients .", "substitutions in codons 12 ( g12v , g12c , g12a , g12r , g12d , or g12s ) and 13 ( g13d ) in the k - ras gene are much more common than in codon 61 and they are mostly observed in smokers with adenocarcinoma ( about 30% of patients with this type of cancer ) [ 37 , 38 ] .", "egfr is not the only receptor on the cell surface responsible for initiation of signal transduction for cell activation and proliferation through pi3k / akt and ras / raf / mapk / erg pathways .", "insulin - like growth factor 1 receptor ( igf-1r ) and the c - met receptor for hepatocyte growth factor ( hgf ) also play an important role in this signal transduction .", "overexpression of these receptors and amplification of the genes encoding them often occur in nsclc cells .", "overexpression of c - met is associated with generation of an additional excitation signal ( without egfr ) in protein kinase of akt , which allows tumour cells to survive and proliferate and induces the formation of metalloproteinases responsible for invasion and metastasis .", "this mechanism is observed in 37% of patients who were not treated with egfr tki and may explain the initial resistance to this kind of treatment .", "however , approximately 20% of patients with egfr activating mutations develop met gene amplification as a result of the creation of acquired resistance to egfr tki . moreover", "blocking of c - met can restore the sensitivity of cancer cells to the egfr tki .", "unfortunately , more than half of patients with met gene amplification also presented the t790 m mutation in the egfr gene . despite the lack of unequivocal efficacy , simultaneous inhibition of c - met and egfr function seems to be an attractive alternative therapeutic option in resistance to reversible egfr tki .", "there are ongoing second and third phase trials of the c - met inhibitors tivantinib and pf-2341066 , used together with erlotinib in patients previously untreated with egfr tki or in case of progression after successful monotherapy with erlotinib [ 17 , 4348 ] . similarly to the c - met receptor , the igf-1r also leads to activation of the akt pathway without egfr in nsclc cells .", "development of effective small - molecule inhibitors of igf-1r encountered difficulties due to the similarity in the tyrosine kinase domain of insulin and igf-1 receptors .", "attempts to use such drugs ( e.g. linsitinib ) had ended with serious side effects in the form of disorders in carbohydrate and lipid metabolism .", "monoclonal anti - igf-1r antibodies , such as figitumumab , proved to be more selective for the igf-1r , but were also not devoid of side effects . as a result", ", attempts of the combined administration of figitumumab and erlotinib were discontinued in 2010 [ 4951 ] .", "mutations or amplifications of the her2 gene , whose product as well as egfr ( her1 ) belongs to the family of her receptors , plays an important role in the development of human tumours , such as breast , ovarian and stomach cancers .", "reactions between these receptors may play an important role in pathogenesis of nsclc . in the case of increased expression of her2 protein and amplification of its gene ( about 2030% of patients with nsclc , mainly patients with adenocarcinoma subtype )", "the dimerisation between her2 and egfr occurs more easily and the ability to increase proliferation of tumour cells is higher .", "mutations in the tyrosine kinase domain of her2 are reported in lung cancer as very rare ( about 24% , more often in adenocarcinoma , non - smokers and women ) .", "they occur as insertion - deletion changes , which change the reading frame in eight codons in exon 20 of the her2 gene .", "the most common disorders are insertion - deletion of 12 base pairs : a775yvma ( 66% of all discovered mutations in the her2 gene ) and m774ayvm .", "these mutations are analogous to a mutation in exon 20 of the egfr gene and cause changes in the tyrosine kinase domain of the her2 receptor . on the basis of this similarity , it is assumed that a mutation in the her2 gene is caused by similar factors as in the egfr gene and causes similar effects .", "narrowing of the atp binding gap leads to an increase in tyrosine kinase activity and excessive phosphorylation of signalling proteins and subsequent secondary resistance to the reversible egfr tki [ 53 , 54 ] .", "although the main mechanism of the decreased response to reversible egfr tki is caused by mutations in exon 20 ( t790 m ) of the egfr gene , equally strong resistance to reversible egfr tki may be caused by insertion - deletion changes in exon 20 of the her2 gene .", "however , studies of this phenomenon have been carried out only in cell cultures [ 5355 ] .", "egfr tki , which are effective for the t790 m mutation of the egfr gene , may also prove to be effective in patients with mutations in exon 20 of the her2 gene , but only if they have the ability to block the function of the tyrosine kinase associated with both egfr and her2 .", "a double reversible inhibitor of egfr and her2 receptors called lapatinib has not shown efficacy in this case .", "however , irreversible inhibitors , such as afatinib and neratinib , which additionally have a likely inhibition effect on the her4 tyrosine kinase , may be effective in the case of mutations in both genes .", "mutations in exon 20 of the her2 gene do not eliminate the possibility of afatinib binding to the tyrosine kinase domain , as the drug is covalently associated with cysteine at codon 805 of this domain . in in vitro cultures afatinib is characterised by about 10-fold greater ability to inhibit phosphorylation than gefitinib in case of mutations in exon 20 of egfr or her2 genes .", "also several cases have been reported where afatinib was effective in nsclc patients with mutations in exon 20 of the her2 gene .", "there are also premises about the effectiveness of combined use of afatinib and mtor inhibitors ( mammalian target of rapamycin ) if her2 mutation is present .", "signalling mtor protein is activated by the pi3k / akt cascade and provides the signal which regulates both mrna translation and cell growth .", "its inhibition by such agents as temsirolimus , everolimus or deforolimus can increase the potency of dual inhibitors of egfr and her2 and overcome the resistance to their use [ 25 , 5355 ] .", "the greatest hope for an extension of progression - free survival in patients treated with reversible egfr tki , in whom resistance has developed , is provided by test results on the effectiveness of irreversible egfr tki . in vitro studies", "have shown that these drugs exhibit activity against cells with the wild - type or mutated ( with activating mutation ) form of the egfr gene , as well as against cells which are resistant to them because of the presence of the t790 m mutation in the egfr gene and insertion- deletion changes in the her2 gene . however , in the case of wild - type egfr and mutations which cause resistance to drugs , the concentration of agents required to effectively induce apoptosis in tumour cells may exceed the tolerated dose level in therapy .", "currently , it is known that afatinib , which is in the most advanced trials , is effective in some patients who have benefited from pre - treatment with reversible egfr tki .", "therefore , the expansion of molecular tests with studies concerning mechanisms of resistance to reversible egfr tki is necessary .", "determination of presence of resistance mutations that cause insensitivity of cancer cells to reversible egfr tki , but which does not rule out the effectiveness of irreversible egfr tki , may be considered as a qualifying factor for changing the type of therapy [ 5557 ] .", "there are also investigations on the effectiveness of other irreversible egfr tki such as pf-00299802 , ekb-569 , canertinib ( ci-1033 ) and neratinib ( hki-272 ) .", "all of these agents are required in relatively high doses in order to overcome the resistance to egfr tki in cancer cells .", "however , an additional advantage of the irreversible egfr tki is their ability to suppress other receptors of the her group ( pan - erbb inhibitors ) , which are necessary for heterodimerisation with egfr .", "use of irreversible egfr tki and monoclonal anti - egfr ( cetuximab ) antibodies or concomitant administration of egfr and c - met inhibitors may present prospects for the future .", "the results of clinical trials of new generation drugs are promising and their usefulness in molecularly targeted therapies will probably be proven in the near future [ 5557 ] ." ]

[ "<S> abnormalities of epidermal growth factor receptor ( egfr ) in non - small - cell lung cancer ( nsclc ) patients consist of egfr overexpression and egfr ( her1 ) gene mutations . </S>", "<S> structural dysfunction of the tyrosine kinase domain of egfr is associated with the clinical response to tyrosine kinase inhibitors ( tki ) in patients with nsclc . </S>", "<S> the most common egfr gene mutations occur as either deletions in exon 19 or as substitution l858r in exon 21 and cause a clinically beneficial response to gefinitib or erlotinib treatment . </S>", "<S> unfortunately , the majority of patients finally develop resistance to these drugs . </S>", "<S> acquired resistance is linked to secondary mutations localised in the egfr gene , mainly substitution t790 m in exon 20 . through intense research </S>", "<S> a few different mechanisms of resistance to reversible tyrosine kinase inhibitors have been identified : amplification of met or igf-1r genes , abnormalities of pten and mtor proteins as well as rare mutations in egfr and her2 genes . </S>", "<S> extensively investigated new drugs could be of significant efficiency in nsclc patients with secondary resistance to reversible egfr tki . </S>" ]

[ "Introduction", "EGFR inhibitors in the treatment of non-small cell lung cancer", "Rare mutations in the ", "The importance of mutations in the ", "Activation of EGFR-independent signalling pathways in cancer cells", "Mutations in the ", "Directions for further studies", "None" ]

[ [ "lung cancer is the main cause of cancer deaths both in poland and in the world .", "non - small cell lung cancer accounts for about 85% of all lung cancers , and the progress in its treatment is still not very satisfactory . the therapeutic procedure of choice is surgery , but only every fifth patient qualifies for it .", "this is due to the fact that most patients at the time of diagnosis are in an advanced stage of the disease , usually accompanied with tumour metastases . in about 25% of such patients extension of life by a few month", "such disappointing results force us to search for new therapeutic options [ 2 , 3 ] .", "one of the new molecular targets for non - small cell lung cancer ( nsclc ) therapy is the epidermal growth factor receptor ( egfr ) .", "egfr ( her1 ) belongs to the erbb ( her ) receptor family . for its activation , not only", "a connection with a suitable ligand is necessary , but also homo- or heterodimerisation on the cell surface with other receptors of the her family ( her 2 - 4 ) .", "the intracellular domain of egfr has activity of tyrosine kinase and it is responsible for the phosphorylation of cellular proteins in the pi3k / akt signalling pathway .", "furthermore , this domain has regulatory functions . in many cancers , including nsclc , activating mutations or overexpression of the egfr gene are found . in some cases progression of the disease ", "uncontrolled cell proliferation , inhibition of apoptosis , and the ability to metastasise is the effect of excessive activation of intracellular pathways constantly stimulated by egfr .", "therefore , there is more and more interest in treatment associated with inhibition of egfr [ 46 ] ." ], [ "one of them is blocking of the extracellular domain of the receptor through monoclonal antibodies ( cetuximab , panitumumab ) , which prevents connection of the egfr ligand or receptor dimerisation . blocking of signal transduction from the cell membrane to the nucleus by small molecule inhibitors of tyrosine and serine - threonine kinase is still in the experimental phase . the biggest hopes for improving the prognosis in nsclc", "are associated with the introduction of small molecule , reversible tyrosine kinase inhibitors of egfr ( egfr tki ) gefitinib and erlotinib .", "the mechanism of their action is based on reversible binding to the intracellular tyrosine kinase domain of egfr and blocking of atp binding .", "selective connectivity of the inhibitor prevents phosphorylation of the tyrosine kinase domain , and in consequence activates the pathway of cellular signal transduction , and leads to cell cycle arrest in g1 phase and increase in apoptosis of tumour cells .", "the therapeutic effect of egfr tki depends on the amino acid structure of the tyrosine kinase domain conditioned upon the state of the egfr gene .", "appearance of the most frequent activating mutation deletion of 15 nucleotides in codons 746 - 750 in exon 19 and the l858r substitution in exon 21 of the egfr gene is associated with permanent stimulation of the receptor , but they also lead to an increase in both efficiency of reversible egfr tki and effectiveness of radiotherapy . activating mutations of the egfr gene have been reported in only about 10% of caucasians patients with nsclc , more often in non - smokers , women , and patients with adenocarcinoma .", "therefore , the first studies on the efficacy of erlotinib and gefitinib in second - line treatment ( br.21 , isel , interest ) have shown that an objective response to egfr tki treatment occurs in less than 10% of patients in the general population [ 1014 ] . in recent clinical trials ( ipass , optimal , eurtac ) conducted on carriers of activating mutations in the egfr gene , over 70% response and almost one year progression - free survival ( pfs )", "for this reason , both inhibitors were granted registration in the treatment of locally advanced and advanced nsclc in first - line of treatment , and are an alternative to standard chemotherapy in patients with activating mutations of the egfr gene .", "the method of qualification for egfr tki monotherapy in second- or third - line treatment in nsclc is still debatable and unclear [ 1016 ] . in the case of the wild type form of the egfr gene ,", "even if the receptor is overexpressed , egfr tki have not shown strong antitumor activity ( none or a few percent of objective response ) mainly for two reasons : lack of changes in the structure of the atp - binding pocket of tyrosine kinase domain and the small role of the normal egfr form in carcinogenesis of lung cancer .", "therefore , investigation of the expression of egfr on the cell surface ( by immunohistochemistry ) or amplification of the egfr gene detected by molecular probes and fluorescence in situ hybridization ( fish ) did not find a wider application in qualification for egfr tki therapy . in some cases ,", "it is possible to consider the usefulness of the determination of egfr gene amplification by the fish method , if examination of the mutation is unfeasible or the outcome is uncertain , bearing in mind the frequent co - existence of egfr gene mutations ( 10% of patients ) with an increase in copy number of the gene ( 40% of patients ) in tumour cells [ 7 , 8 ] .", "unfortunately , regardless of the applied line of egfr tki treatment , at some point of therapy , even in patients who carry an activating mutation in the egfr gene and show a response , there is a recurrence of the disease and the patient 's life is not prolonged [ 17 , 18 ] ." ], [ "some mechanisms associated with resistance to reversible egfr tki in nsclc are already known . the t790 m mutation in exon 20 of the egfr gene", "was detected for the first time in tumour cells of a patient with progression after a few months of efficacious therapy with gefitinib .", "that point mutation is caused by a single nucleotide substitution ( c- > t ) at position 2369 ( codon 790 ) in exon 20 of the egfr gene .", "the t790 m mutation leads to a substitution of threonine to methionine in the catalytic centre of egfr tyrosine kinase , which is located in the binding pocket of tki and atp .", "substitution of threonine at codon 790 into larger methionine probably results in blocking of the binding sites of erlotinib and gefitinib aromatic residues with their point of action . in vitro studies demonstrated that the effective concentration of erlotinib in tumour cells harbouring both the l858r and t790 m mutations has to be 300 times higher to induce apoptosis than in cells only with an activating mutation in codon 858 [ 19 , 20 ] .", "presence of a mutation in exon 20 of the egfr gene is a potential prognostic and predictive marker of egfr tki therapy and may play an important role in the planning of therapy .", "activating mutations in egfr were detected in cells obtained from primary tumour , circulating tumour cells in peripheral blood ( consistency of results 92% ) and in free circulating dna ( consistency of results ", "the authors also investigated presence of the t790 m mutation in similar samples , and they detected cells harbouring that mutation in 10 patients ( n = 26 ; 38.5% ) .", "median survival in patients with the t790 m mutation treated with reversible egfr tki was 7.7 months against 16.5 months in patients without that mutation ( p < 0.001 ) .", "mutations were detected using sarms real - time pcr technique . that investigation has also shown that mutation in exon 20 of the egfr gene leads to acquired resistance to treatment in patients not only with the wild type egfr gene , but also in patients with confirmed clinical sensitivity to egfr tyrosine kinase inhibitors .", "presence of t790 m substitution may be an indication for treatment with irreversible egfr tki , such as neratinib , keratinib and especially afatinib , which is currently in the third phase of clinical trials .", "the lux - lung 1 study has shown that afatinib induces an objective response and prolongation of progression - free survival ( pfs ) in nsclc patients who have been previously treated with egfr tki for at least 12 weeks .", "the rationale for this type of study is the fact that afatinib has a different molecular structure when compared to gefitinib and erlotinib .", "this structure causes that afatinib forms a covalent bond with cysteine at codon 733 of the egfr tyrosine kinase domain .", "therefore , inhibition of afatinib is only possible due to synthesis of new receptor proteins .", "inhibition of phosphorylation by egfr tyrosine kinase in tumour cells after afatinib exposure remains active for 48 hours ( after gefitinib only 8 hours ) , and phosphorylation activity of the kinase after that time ranges from 50 to 70% of the primary activity . in vitro studies and some case reports", "have shown that afatinib is effective ( cell apoptosis and disease stabilisation ) in cells harbouring the t790 m mutation in nsclc patients [ 19 , 2226 ] .", "the mechanism of mutation appearance in nsclc recurrence in egfr tki treated patients should be thoroughly examined .", "a resistant cell subpopulation harbouring t790 m can emerge in the tumour probably as a result of massive apoptosis of tumour cells harbouring an activating mutation associated with response to therapy .", "the authors examined presence of mutations in exons 18 - 24 of the egfr gene in patients treated with egfr tki in order to determine if additional mutations in the egfr gene are associated with progression of the disease . in patients with mutations identified in exon 19 or 21 additional mutations", "samples obtained from patients tumour cells before treatment were re - examined to exclude the possibility of missing changes in exon 20 of egfr . however , no evidence for the presence of the sought mutations was found .", "mutations associated with resistance to egfr tki were observed in three tumor recurrences that emerged after treatment .", "it was the t790 m mutation coexisting with deletion in exon 19 or l858r substitution in the egfr gene .", "results of the investigations suggest that the resistance to treatment is acquired during therapy with egfr tki , and mutations may form de novo .", "it is worth mentioning that tumour cell clones harbouring t790 m substitution may be rare , and the mutation may not be detected ( due to the small number of cells with the t790 m mutation in the whole sample ) .", "it is observed especially when the dna sequencing technique is employed , which is characterised by relatively low sensitivity and requires the presence of at least 50% of mutated tumour cells in the examined sample .", "currently it is believed that t790 m substitution occurs in over 50% of cases of acquired resistance to reversible egfr tki in nsclc patients and in approximately 5% of patients before egfr tki treatment .", "before the therapy with egfr tki , the t790 m mutation emerges along with activating mutations of the egfr gene , and secondarily it coexists with the primary activating mutation in egfr , especially l858r [ 17 , 2831 ] . with time", ", it was found that other mutations in exon 20 and 21 of egfr ( insertion - deletion , substitutions : d761y , t854a , etc . )", "these mutations account for less than 5% of all known mutations in the egfr gene .", "they are detected very rarely , so reports about their presence should be made carefully , and egfr tki treatment outcomes in patients harbouring these mutations should be confirmed on a larger group of patients .", "furthermore , it is necessary to understand the mechanism of resistance caused by these mutations .", "d761y and t854a mutations in the egfr gene were detected for the first time in patients with activating l858r mutation .", "probably substitution of aspartic acid with tyrosine at position 761 is responsible for acquisition of resistance to tki treatment of tumour cells with an activating mutation which are sensitive to the treatment ( this theory was confirmed in tumour cell lines in vitro ) .", "however , this resistance to egfr tki has been many times lower than the resistance in cells harbouring both the l858r and t790 m mutations . in case of occurrence of both l858r and t790 m mutations ,", "the concentration of erlotinib should be three times higher in order to achieve a therapeutic effect .", "analysis of the egfr structure in patients harbouring the t854a mutation suggests the presence of an additional side chain , localised near the egfr tki binding site .", "the role of rare mutations in exon 20 such as s768i and v769l in development of acquired resistance to egfr tki is still unknown .", "these substitutions were reported for the first time in nsclc patients with progression , bearing activating mutations , after gefitinib therapy . in vitro cultures of cell lines harbouring", "the s768i substitution showed slightly more resistance to egfr tki than cells with wild type egfr . in these cells permanent hyperphosphorylation of tyrosine at codon 1045 of the tyrosine kinase domain has been observed [ 17 , 3234 ] .", "insertion - deletion mutations in exon 20 of egfr are also associated with acquired resistance to egfr tki .", "these mutations , similarly to activating mutations in the egfr gene , are more often detectable in asians , non - smokers , women and in patients with adenocarcinoma . most of the pre - clinical investigations allowed observation of their impact on resistance to therapeutic doses of gefitinib or erlotinib , and some of the irreversible egfr tki , e.g. neratinib . however , in the most common mutations such as n771gy and a767-v769 duplication , even though lack of sensitivity to erlotinib has been reported , low sensitivity to irreversible egfr tki such as afatinib and pf-00299804 was observed .", "crystallographic analysis of epidermal growth factor receptor structure has shown that both mutations have an influence on the spatial structure of tyrosine kinase c - helix , which plays an important role in phosphorylation activity of that enzyme .", "other insertions in exon 20 of the egfr gene such as v738inskipvai , m766insasv , d770insnpg and d770insnph seem to be associated with that process , but their role in development of resistance to reversible egfr tki is still unknown [ 35 , 36 ] ." ], [ "many studies have demonstrated that the presence of mutations at codons 12 , 13 or 61 in the k - ras gene is not a predictive factor in treatment with egfr tki .", "these observations stem from the fact that mutations in the k - ras gene almost never coexist with mutations in the egfr gene .", "lack of mutations in egfr determines the inefficiency of egfr tki used in therapeutic doses , which means that the occurrence of mutations in the k - ras gene is no longer relevant . therefore there were no objective responses to treatment with egfr tki among carriers of k - ras mutations , which was initially considered as an independent , adverse predictive factor in egfr tki treatment .", "in fact , in vitro studies have shown that the destruction of tumour cells with the wild - type egfr and mutant k - ras gene is only possible with very high , non - therapeutic doses of egfr tki [ 3739 ] . however , in some individual case reports in which coexistence of both egfr and k - ras gene mutations have been observed , the efficacy of egfr tki was not entirely confirmed .", "the situation is presented in a different manner when monoclonal anti - egfr antibodies , such as cetuximab , are used in the treatment of colorectal cancer , head and neck cancer , and perhaps soon in nsclc with strong overexpression of egfr .", "mutations in the k - ras gene are a negative predictor here , and are routinely determined in qualification of patients for therapy . however , in the case of therapy with egfr tki the only test eligible for treatment is investigation of egfr gene mutations [ 37 , 41 , 42 ] .", "activation of an alternative signalling pathway from stimulated egfr protein leads to activation of ras protein and subsequently kinases associated with raf / mapk / erg proteins and transcription factors such as myc , fos and jun .", "mutations which are present in the k - ras gene lead to constant activation of ras protein , so the tumour cells become independent of the signals from the egfr , which explains the mechanism of resistance conferred by the egfr tki and monoclonal anti - egfr antibodies .", "mutations in the k - ras gene occur with a frequency of 1520% in caucasian nsclc patients .", "substitutions in codons 12 ( g12v , g12c , g12a , g12r , g12d , or g12s ) and 13 ( g13d ) in the k - ras gene are much more common than in codon 61 and they are mostly observed in smokers with adenocarcinoma ( about 30% of patients with this type of cancer ) [ 37 , 38 ] ." ], [ "egfr is not the only receptor on the cell surface responsible for initiation of signal transduction for cell activation and proliferation through pi3k / akt and ras / raf / mapk / erg pathways .", "insulin - like growth factor 1 receptor ( igf-1r ) and the c - met receptor for hepatocyte growth factor ( hgf ) also play an important role in this signal transduction .", "overexpression of these receptors and amplification of the genes encoding them often occur in nsclc cells .", "overexpression of c - met is associated with generation of an additional excitation signal ( without egfr ) in protein kinase of akt , which allows tumour cells to survive and proliferate and induces the formation of metalloproteinases responsible for invasion and metastasis .", "this mechanism is observed in 37% of patients who were not treated with egfr tki and may explain the initial resistance to this kind of treatment .", "however , approximately 20% of patients with egfr activating mutations develop met gene amplification as a result of the creation of acquired resistance to egfr tki . moreover", "blocking of c - met can restore the sensitivity of cancer cells to the egfr tki .", "unfortunately , more than half of patients with met gene amplification also presented the t790 m mutation in the egfr gene . despite the lack of unequivocal efficacy , simultaneous inhibition of c - met and egfr function seems to be an attractive alternative therapeutic option in resistance to reversible egfr tki .", "there are ongoing second and third phase trials of the c - met inhibitors tivantinib and pf-2341066 , used together with erlotinib in patients previously untreated with egfr tki or in case of progression after successful monotherapy with erlotinib [ 17 , 4348 ] . similarly to the c - met receptor , the igf-1r also leads to activation of the akt pathway without egfr in nsclc cells .", "development of effective small - molecule inhibitors of igf-1r encountered difficulties due to the similarity in the tyrosine kinase domain of insulin and igf-1 receptors .", "attempts to use such drugs ( e.g. linsitinib ) had ended with serious side effects in the form of disorders in carbohydrate and lipid metabolism .", "monoclonal anti - igf-1r antibodies , such as figitumumab , proved to be more selective for the igf-1r , but were also not devoid of side effects . as a result", ", attempts of the combined administration of figitumumab and erlotinib were discontinued in 2010 [ 4951 ] ." ], [ "mutations or amplifications of the her2 gene , whose product as well as egfr ( her1 ) belongs to the family of her receptors , plays an important role in the development of human tumours , such as breast , ovarian and stomach cancers .", "reactions between these receptors may play an important role in pathogenesis of nsclc . in the case of increased expression of her2 protein and amplification of its gene ( about 2030% of patients with nsclc , mainly patients with adenocarcinoma subtype )", "the dimerisation between her2 and egfr occurs more easily and the ability to increase proliferation of tumour cells is higher .", "mutations in the tyrosine kinase domain of her2 are reported in lung cancer as very rare ( about 24% , more often in adenocarcinoma , non - smokers and women ) .", "they occur as insertion - deletion changes , which change the reading frame in eight codons in exon 20 of the her2 gene .", "the most common disorders are insertion - deletion of 12 base pairs : a775yvma ( 66% of all discovered mutations in the her2 gene ) and m774ayvm .", "these mutations are analogous to a mutation in exon 20 of the egfr gene and cause changes in the tyrosine kinase domain of the her2 receptor . on the basis of this similarity , it is assumed that a mutation in the her2 gene is caused by similar factors as in the egfr gene and causes similar effects .", "narrowing of the atp binding gap leads to an increase in tyrosine kinase activity and excessive phosphorylation of signalling proteins and subsequent secondary resistance to the reversible egfr tki [ 53 , 54 ] .", "although the main mechanism of the decreased response to reversible egfr tki is caused by mutations in exon 20 ( t790 m ) of the egfr gene , equally strong resistance to reversible egfr tki may be caused by insertion - deletion changes in exon 20 of the her2 gene .", "however , studies of this phenomenon have been carried out only in cell cultures [ 5355 ] .", "egfr tki , which are effective for the t790 m mutation of the egfr gene , may also prove to be effective in patients with mutations in exon 20 of the her2 gene , but only if they have the ability to block the function of the tyrosine kinase associated with both egfr and her2 .", "a double reversible inhibitor of egfr and her2 receptors called lapatinib has not shown efficacy in this case .", "however , irreversible inhibitors , such as afatinib and neratinib , which additionally have a likely inhibition effect on the her4 tyrosine kinase , may be effective in the case of mutations in both genes .", "mutations in exon 20 of the her2 gene do not eliminate the possibility of afatinib binding to the tyrosine kinase domain , as the drug is covalently associated with cysteine at codon 805 of this domain . in in vitro cultures afatinib is characterised by about 10-fold greater ability to inhibit phosphorylation than gefitinib in case of mutations in exon 20 of egfr or her2 genes .", "also several cases have been reported where afatinib was effective in nsclc patients with mutations in exon 20 of the her2 gene .", "there are also premises about the effectiveness of combined use of afatinib and mtor inhibitors ( mammalian target of rapamycin ) if her2 mutation is present .", "signalling mtor protein is activated by the pi3k / akt cascade and provides the signal which regulates both mrna translation and cell growth .", "its inhibition by such agents as temsirolimus , everolimus or deforolimus can increase the potency of dual inhibitors of egfr and her2 and overcome the resistance to their use [ 25 , 5355 ] ." ], [ "the greatest hope for an extension of progression - free survival in patients treated with reversible egfr tki , in whom resistance has developed , is provided by test results on the effectiveness of irreversible egfr tki . in vitro studies", "have shown that these drugs exhibit activity against cells with the wild - type or mutated ( with activating mutation ) form of the egfr gene , as well as against cells which are resistant to them because of the presence of the t790 m mutation in the egfr gene and insertion- deletion changes in the her2 gene . however , in the case of wild - type egfr and mutations which cause resistance to drugs , the concentration of agents required to effectively induce apoptosis in tumour cells may exceed the tolerated dose level in therapy .", "currently , it is known that afatinib , which is in the most advanced trials , is effective in some patients who have benefited from pre - treatment with reversible egfr tki .", "therefore , the expansion of molecular tests with studies concerning mechanisms of resistance to reversible egfr tki is necessary .", "determination of presence of resistance mutations that cause insensitivity of cancer cells to reversible egfr tki , but which does not rule out the effectiveness of irreversible egfr tki , may be considered as a qualifying factor for changing the type of therapy [ 5557 ] .", "there are also investigations on the effectiveness of other irreversible egfr tki such as pf-00299802 , ekb-569 , canertinib ( ci-1033 ) and neratinib ( hki-272 ) .", "all of these agents are required in relatively high doses in order to overcome the resistance to egfr tki in cancer cells .", "however , an additional advantage of the irreversible egfr tki is their ability to suppress other receptors of the her group ( pan - erbb inhibitors ) , which are necessary for heterodimerisation with egfr .", "use of irreversible egfr tki and monoclonal anti - egfr ( cetuximab ) antibodies or concomitant administration of egfr and c - met inhibitors may present prospects for the future .", "the results of clinical trials of new generation drugs are promising and their usefulness in molecularly targeted therapies will probably be proven in the near future [ 5557 ] ." ], [ "" ] ]

[ "diffuse gliomas are the most common primary brain tumors in adults and encompass a heterogeneous group of tumors with different histopathological features , aggressiveness , imaging , response to treatment and survival .", "they are primarily defined by the histopathological tumor tissue analysis and subdivided by the world health organization ( who ) classification into grade ii ( low grade glioma ) , grade iii ( anaplastic glioma ) and grade iv ( glioblastoma multiforme , gbm ) . accurate distinction between different grades is of significant clinical importance because of its strong prognostic and treatment decision - making implications ( louis et al . , 2007 ) .", "however , histopathological criteria of the grading system present some limitations such as the lack of reproducibility and the prognostic heterogeneity within the same group ( ricard et al . , 2012 ) . to improve this classification , some molecular markers not only have diagnostic and prognostic implications , but also therapeutic efficacy prediction ( 1p/19q co - deletion , idh1/idh2 mutations and mgmt promoter methylation ) ( riemenschneider et al . , 2010 ) .", "however , these approaches require a resection or a biopsy and have limitations induced by intra - tumoral heterogeneity ( van den bent , 2010 ) . furthermore , there is a need to identify criteria of aggressiveness for inoperable patients .", "mri is a part of the daily management of the patients by providing anatomical and vascular information .", "the presence or the absence of t1 enhancement after gadolinium - chelate injection , necrosis , mass effect and surrounding tumor edema are established parameters used in characterizing tumor aggressiveness ( dean et al . ,", "the presence of contrast enhancement in diffuse glioma , related to the permeability of the blood brain - barrier ( bbb ) reflecting neovascularization , is often regarded as a sign of malignancy", ". however , it presents some limitations and does not allow for reliable separation between different grades .", "for example , although the absence of contrast enhancement is a commonly used criterion for identifying grade ii gliomas , it does not eliminate formally a malignant tumor ( ginsberg et al .", "in contrast , the presence of contrast enhancement and even necrosis , characteristic of gbm , can be observed in pure grade iii oligodendrogliomas . in this context ,", "multimodal imaging based on mri and pet parameters , by assessing tumor pathophysiology such as microvasculature , metabolism , proliferation or cellularity , could be of additional value .", "previous reports have described and compared the performance of glioma grading using advanced mri techniques such as relative cerebral blood volume ( rcbv ) , apparent diffusion coefficient ( adc ) , and metabolites of magnetic resonance spectroscopy ( mrs ) ( arvinda et al . , 2009 ; hilario et al . , 2012 ; law et al . , 2003 ; van den bent , 2010 ; yang et al . , 2002 ; zonari et al . , 2007 ;", "others focus specifically on glioma grading with the thymidine analog [ f]-fluoro - l - thymidine ( [ f]-flt ) , developed as a positron emission tomography ( pet ) tracer to assess the proliferation activity of tumors in vivo , particularly in brain tumors ( chalkidou et al .", ", 2012 ; chen et al . , 2005 ; hatakeyama et al . , 2008 ; jacobs et al . ,", "even if these multimodal imaging parameters are useful to distinguish grade ii from higher grades ( anaplastic and gbm ) , the differentiating criteria of grade iii from grade iv gliomas , two groups with different prognoses and management , are still poorly defined .", "the aim of this study was to compare the performance of these advanced mr techniques , namely perfusion , diffusion and proton mr spectroscopy ( h - mrs ) with that of [ f]-flt - pet , by pre - operatively assessing the diagnostic accuracy of each parameter in differentiating glioma grades .", "this study was based on a prospective clinical trial funded by inca ( institut national du cancer ) and approved by the local ethics committee and afssaps agreement ( clinicaltrials.gov identifier : nct00850278 ) .", "inclusion criteria were : a presumed diffuse glioma amenable to surgical resection or biopsy , age greater than or equal to 18 , kps greater than or equal to 50 , normal blood cell count , normal biological hepatic functions and a signed informed consent .", "the group sizes of the different tumor grades were the result of the incidence of each tumor in this prospective study .", "patients first underwent [ f]-flt - pet , followed by multiparametric mri / mrs within the same week and prior to surgery .", "single voxel mrs was localized in the area with maximum visible uptake of [ f]-flt .", "thereafter , patients underwent surgery , resection or biopsy depending on the location of the tumor , and the specimens were histopathologically evaluated by an experienced neuropathologist ( elz ) .", "majority of patients had image - guided total or subtotal surgical resection ( table 1 ) .", "for patients with biopsy only , the biopsy was guided by the area with maximum visible uptake of [ f]-flt . for grade iii tumors or for difficult cases , the diagnosis and grading were reviewed by the national multicentric network ", "prise en charge des oligodendrogliomes anaplasiques pola ( idbaih et al . , 2012 ) .", "after scout - view and coronal t2-weighted imaging , an axial flair ( fluid attenuated inversion recovery ) sequence was performed ( 24 slices , slice spacing 5.5 , pixel resolution 0.47 0.47 mm , tr / te = 9602/150 ms ) .", "diffusion weighted imaging ( dwi ) was performed using spin - echo - echo planar imaging ( se - epi ) ( 3 diffusion directions , 36 slices , slice spacing : 7 mm , pixel resolution : 1.09 1.09 mm , tr / te = 6000/96 ms , b 0 and b 1000 smm ) . for perfusion , dynamic t2 *", "-weighted epi images were acquired 30 s before , during the first pass of gd - dota ( dotarem , guerbet , france ) and 1 min after with a standard 0.1 mmol / kg body weight dose ( 14 slices , 35 repetitions , slice spacing : 7 mm , pixel resolution 2.19 2.19 mm , tr / te = 2280/60 ms ) .", "immediately thereafter , a 3dt1-weighted sequence , 3dt1w - gd ( 124 slices , slice spacing 1.5 , pixel resolution 1.01 1.01 mm , tr / te = 17/3 ms ) was performed to evaluate the tumor contrast enhancement .", "finally , mrs data were acquired in a single voxel ( 1 cm ) localized in the tumor in the area of maximal uptake of flt and in the normal - appearing contralateral side with press mrs sequence with multiple te ( 35 , 144 , 288 ms ) and water suppression .", "flt - pet [ f]-flt was produced by the ldm - tep group and synthesized at the cyceron tep center as previously described by jacobs et al .", "images of the brain were acquired 40 min after the intravenous injection of 5 mbq / kg of f - flt and lasted 10 min , to match a clinically feasible approach .", "the attenuation - corrected images were reconstructed with an osem 2d algorithm ( 9 subsets and 2 iterations ) and filtered in 3d with a butterworth filter .", "the adc ( apparent diffusion coefficient ) maps were calculated pixel - by - pixel using imagej software ( http://rsb.info.nih.gov/ij/ , 19972012 ) using the following equation : adc = [ln(s / s0)]/b where s is the signal ( averaged over the 3 directions ) acquired , b = 1000 , and s0 = the signal acquired without diffusion gradient .", "variations of the t2 * signal in the tissue , which is proportional to the concentration of the contrast agent , were calculated with in - house macros based on imagej software as : r(t ) = ln(s(t)/s0 ) , where s0 = the signal intensity before contrast agent injection , and s(t ) = the signal intensity over time .", "then , cbv maps were generated by integrating the area under the -variate fitted curves to avoid an effect of recirculation .", "images were then normalized by dividing cbv maps with the mean value of the normal - appearing contralateral side .", "coregistration : adc maps , rcbv maps , flair and pet - flt images were coregistered with trilinear interpolation , rigid matching and normalized mutual information on 3dt1w - gd images ( pmod 3.1 software ) .", "the hypersignal in flair images was manually delineated to define a region of interest ( roi ) in order to use a homogeneous method to define a pathological area ( tumor , peripheral edema and infiltration areas ) for the different grades , despite the presence or absence of t1 enhancement . when present , necrosis was excluded .", "tumor rois were reported for each modality ( rcbv , adc , t1 enhancement , flt - pet ) to extract the mean value for each modality , the maximum value for rcbv , ce and pet - flt , and the minimum value for adc . to avoid sensitivity to extreme values ,", "the minimum value was associated with the 10th percentile ( p10 ) and the maximum value with the 90th percentile ( p90 ) . in mrs , choline ( cho ) , creatine ( cr ) , n - acethyl - aspartate ( naa ) , phospholipids and lactate were quantified with sa / ge software ( ge ) to obtain peak amplitudes and areas ( proportional to concentration and relaxation ) from spectra with water resonance suppression , and expressed as a ratio .", "p - values of comparisons between grades were calculated using the non - parametric wilcoxon test .", "ordinal logistic regression was used to assess the degree of correlation ( r ) of each parameter with the grade . only r values with p - value < 0.05 were considered significant .", "this study was based on a prospective clinical trial funded by inca ( institut national du cancer ) and approved by the local ethics committee and afssaps agreement ( clinicaltrials.gov identifier : nct00850278 ) .", "inclusion criteria were : a presumed diffuse glioma amenable to surgical resection or biopsy , age greater than or equal to 18 , kps greater than or equal to 50 , normal blood cell count , normal biological hepatic functions and a signed informed consent .", "the group sizes of the different tumor grades were the result of the incidence of each tumor in this prospective study .", "patients first underwent [ f]-flt - pet , followed by multiparametric mri / mrs within the same week and prior to surgery .", "single voxel mrs was localized in the area with maximum visible uptake of [ f]-flt .", "thereafter , patients underwent surgery , resection or biopsy depending on the location of the tumor , and the specimens were histopathologically evaluated by an experienced neuropathologist ( elz ) .", "majority of patients had image - guided total or subtotal surgical resection ( table 1 ) .", "for patients with biopsy only , the biopsy was guided by the area with maximum visible uptake of [ f]-flt . for grade iii tumors or for difficult cases , the diagnosis and grading were reviewed by the national multicentric network ", "prise en charge des oligodendrogliomes anaplasiques pola ( idbaih et al . , 2012 ) .", "after scout - view and coronal t2-weighted imaging , an axial flair ( fluid attenuated inversion recovery ) sequence was performed ( 24 slices , slice spacing 5.5 , pixel resolution 0.47 0.47 mm , tr / te = 9602/150 ms ) .", "diffusion weighted imaging ( dwi ) was performed using spin - echo - echo planar imaging ( se - epi ) ( 3 diffusion directions , 36 slices , slice spacing : 7 mm , pixel resolution : 1.09 1.09 mm , tr / te = 6000/96 ms , b 0 and b 1000 smm ) . for perfusion , dynamic t2 *", "-weighted epi images were acquired 30 s before , during the first pass of gd - dota ( dotarem , guerbet , france ) and 1 min after with a standard 0.1 mmol / kg body weight dose ( 14 slices , 35 repetitions , slice spacing : 7 mm , pixel resolution 2.19 2.19 mm , tr / te = 2280/60 ms ) . immediately thereafter , a 3dt1-weighted sequence , 3dt1w - gd ( 124 slices , slice spacing 1.5 , pixel resolution 1.01 1.01 mm , tr / te = 17/3 ms ) was performed to evaluate the tumor contrast enhancement . finally , mrs data were acquired in a single voxel ( 1 cm ) localized in the tumor in the area of maximal uptake of flt and in the normal - appearing contralateral side with press mrs sequence with multiple te ( 35 , 144 , 288 ms ) and water suppression .", "flt - pet [ f]-flt was produced by the ldm - tep group and synthesized at the cyceron tep center as previously described by jacobs et al .", "images of the brain were acquired 40 min after the intravenous injection of 5 mbq / kg of f - flt and lasted 10 min , to match a clinically feasible approach .", "the attenuation - corrected images were reconstructed with an osem 2d algorithm ( 9 subsets and 2 iterations ) and filtered in 3d with a butterworth filter .", "the adc ( apparent diffusion coefficient ) maps were calculated pixel - by - pixel using imagej software ( http://rsb.info.nih.gov/ij/ , 19972012 ) using the following equation : adc = [ln(s / s0)]/b where s is the signal ( averaged over the 3 directions ) acquired , b = 1000 , and s0 = the signal acquired without diffusion gradient .", "variations of the t2 * signal in the tissue , which is proportional to the concentration of the contrast agent , were calculated with in - house macros based on imagej software as : r(t ) = ln(s(t)/s0 ) , where s0 = the signal intensity before contrast agent injection , and s(t ) = the signal intensity over time .", "then , cbv maps were generated by integrating the area under the -variate fitted curves to avoid an effect of recirculation .", "images were then normalized by dividing cbv maps with the mean value of the normal - appearing contralateral side .", "coregistration : adc maps , rcbv maps , flair and pet - flt images were coregistered with trilinear interpolation , rigid matching and normalized mutual information on 3dt1w - gd images ( pmod 3.1 software ) .", "the hypersignal in flair images was manually delineated to define a region of interest ( roi ) in order to use a homogeneous method to define a pathological area ( tumor , peripheral edema and infiltration areas ) for the different grades , despite the presence or absence of t1 enhancement . when present , necrosis was excluded .", "tumor rois were reported for each modality ( rcbv , adc , t1 enhancement , flt - pet ) to extract the mean value for each modality , the maximum value for rcbv , ce and pet - flt , and the minimum value for adc . to avoid sensitivity to extreme values ,", "the minimum value was associated with the 10th percentile ( p10 ) and the maximum value with the 90th percentile ( p90 ) . in mrs , choline ( cho ) , creatine ( cr ) , n - acethyl - aspartate ( naa ) , phospholipids and lactate were quantified with sa / ge software ( ge ) to obtain peak amplitudes and areas ( proportional to concentration and relaxation ) from spectra with water resonance suppression , and expressed as a ratio .", "p - values of comparisons between grades were calculated using the non - parametric wilcoxon test .", "ordinal logistic regression was used to assess the degree of correlation ( r ) of each parameter with the grade .", "among the 40 patients included in the study , 1 had an incomplete mri examination .", "of the 39 remaining patients , all presented histopathologically proven diffuse glioma based on who criteria and were eligible in the final analysis ( ranging from 21 to 79 years old , 17 women and 22 men ) .", "the diagnosis was established from biopsy specimens ( n = 9 ) or from resection ( 10 patients with a partial resection and 20 patients with a macroscopically complete resection ) yielding 7 patients with grade ii glioma , 9 patients with grade iii glioma and 23 patients with grade iv glioma .", "grade ii gliomas typically looked like a non - enhancing lesion that had no modification on cbv maps and no [ f]-flt uptake ( fig .", "most grade iii gliomas showed a slightly enhancing lesion with mild [ f]-flt uptake ( fig .", ", grade iv gliomas typically exhibited a strong contrast enhancement , elevated rcbv , and high [ f]-flt uptake ( fig .", "1 : grade iv ( a ) ) . interestingly , some grade iii glioma presented a profile indicative of a gbm , i.e. sustained contrast enhancement , strong modification in adc , elevated cho / cr and rcbv , appearance of lipids / lactate but [ f]-flt clearly demonstrated a weak tumor cell proliferation ( fig .", "a grade iv glioma presented non - objectivable modification in perfusion but an intense [ f]-flt uptake favoring the diagnosis of a gbm instead of a grade iii glioma ( fig .", "grade ii and grade iv gliomas rcbvmean , rcbvmax , fltmean , fltmax , cemean , cemax , cho / cr and cho / naa showed significant differences ( table 2 ) .", "2 , table 2 ) . between grade iii and grade iv gliomas , adcmean , adcmin , rcbvmean , rcbvmax , fltmean , fltmax , cemean , and", "the most relevant parameter was fltmax ( p < 0.0001 ) ( fig .", "cho / cr showed significant differences between grade ii and grade iii gliomas ( p = 0.03 ) .", "the parameter showing the best significant correlation with the grade was fltmax ( r = 0.5 , p < ", "among the 40 patients included in the study , 1 had an incomplete mri examination .", "of the 39 remaining patients , all presented histopathologically proven diffuse glioma based on who criteria and were eligible in the final analysis ( ranging from 21 to 79 years old , 17 women and 22 men ) .", "the diagnosis was established from biopsy specimens ( n = 9 ) or from resection ( 10 patients with a partial resection and 20 patients with a macroscopically complete resection ) yielding 7 patients with grade ii glioma , 9 patients with grade iii glioma and 23 patients with grade iv glioma .", "grade ii gliomas typically looked like a non - enhancing lesion that had no modification on cbv maps and no [ f]-flt uptake ( fig .", "most grade iii gliomas showed a slightly enhancing lesion with mild [ f]-flt uptake ( fig .", ", grade iv gliomas typically exhibited a strong contrast enhancement , elevated rcbv , and high [ f]-flt uptake ( fig .", "1 : grade iv ( a ) ) . interestingly , some grade iii glioma presented a profile indicative of a gbm , i.e. sustained contrast enhancement , strong modification in adc , elevated cho / cr and rcbv , appearance of lipids / lactate but [ f]-flt clearly demonstrated a weak tumor cell proliferation ( fig .", "a grade iv glioma presented non - objectivable modification in perfusion but an intense [ f]-flt uptake favoring the diagnosis of a gbm instead of a grade iii glioma ( fig .", "between grade ii and grade iv gliomas rcbvmean , rcbvmax , fltmean , fltmax , cemean , cemax , cho / cr and cho / naa showed significant differences ( table 2 ) .", "the most relevant parameter was fltmax ( p < 0.001 ) ( fig .", "2 , table 2 ) . between grade iii and grade iv gliomas , adcmean , adcmin , rcbvmean , rcbvmax , fltmean , fltmax , cemean , and", "the most relevant parameter was fltmax ( p < 0.0001 ) ( fig .", "cho / cr showed significant differences between grade ii and grade iii gliomas ( p = 0.03 ) .", "the parameter showing the best significant correlation with the grade was fltmax ( r = 0.5 , p < 0.0001 ) ( table 2 ) .", "the prognosis and management of patients with diffuse glioma are strongly dependent on the accurate grading of the tumor .", "mri plays a crucial role in the assessment of aggressiveness of gliomas before surgery but may remain insufficient to forecast prognosis of diffuse gliomas . therefore , multimodal imaging parameters with advanced mri ( cbv and adc ) , mrs and [ f]-flt - pet could identify relevant markers to better predict the grading of gliomas . among advanced technics ,", "this was also shown in our study , particularly between grade ii and grade iii gliomas ( p = 0.03 ) . indeed ,", "cho / cr is well known to be correlated with tumor cell proliferation ( herminghaus et al . , 2002 ) and usually presents significant differences between low grade and high grade gliomas ( law et al . , 2003 ; server et al . , 2011 ; yang et al", "our results suggest that the use of cho / cr ratio could be of interest in the management of grade ii and grade iii rather than [ f]-flt pet , that did not show significant differences between these grades .", "rcbv has been shown to be correlated with tumor aggressiveness and with grade in enhancing and in non - enhancing lesions ( aronen et al . , 1994 ;", "maia et al . , 2005 ; sadeghi et al . , 2008 ; shin et al . , 2002 ; sugahara et al . ,", "rcbv has a better diagnostic performance in differentiating between low grade and high grade gliomas than cho / cr and cho / naa ratios obtained with mrs ( law et al . , 2003 ) ,", "adc ( arvinda et al . , 2009 ) or ktrans ( law et al . , 2004 ) .", "while most studies compare high grade and low grade , our study suggests that cbvmean and cbvmax significantly differ between patients with grade iii and patients with grade iv . for zonari et al .", ", rcbv is the most important parameter for accurate tumor grading by differentiating grade ii from iii gliomas ( zonari et al . , 2007 ) .", "along with [ f]-fdg , numerous other pet radiotracers have been developed ( for review , see ( dhermain et al .", ", 2010 ; wester , 2007 ) ) to assess tumor aggressiveness such as 3-[f]fluoro-3-deoxy - l - thymidine ( [ f]-flt ) ( jacobs et al . , 2005 ) or amino acid metabolism by o-(2-[f]fluoroethyl)-l - tyrosine ( [ f]-fet ) ( hutterer et al . , 2011 ) or methyl-[c]-l - methionine ( [ c]-met ) ( derlon et al", "this tracer has already been used by us and others to assess grade ( ferdov et al . , 2015 ) , treatment efficacy ( corroyer - dulmont et al . , 2013 ; schwarzenberg et al . ,", "[ f]-flt has also been assessed and compared with other tracers as a marker for high grade gliomas and probably more adapted for the grading compared to [ c]-met or [ f]-fet in this setting ( chen et al . , 2005 ;", ", 2005 ; jeong and lim , 2012 ; miyake et al . , 2012 ) . in agreement with these reports , our study showed that [ f]-flt parameters were not sensitive enough to distinguish grade ii from grade iii gliomas , but was the best parameter to distinguish grade iv from lower grade gliomas .", "our results are in line of previous work that already showed the interest of using [ f]-flt to differentiate low and high grade gliomas ( yamamoto et al . , 2012 ) .", "we are also aware that pet imaging and more precisely flt pet are restricted , at the present time , to few hospital or research centers . in the present study", ", we propose that flt pet adds information in presence of suspicious patients where mri may be not sensitive enough before any treatment is decided but where biopsy examination may lose some very aggressive hot spot thanks to the great spatial heterogeneity . of note , we chose a static and delayed acquisition of [ f]-flt images since a dynamic acquisition ( e.g. 2 h of acquisition ) may not be realistic for routine clinical practice .", "it has been shown that [ f]-flt uptake occurs due to increased transport through the blood ", "when we focused on the condition for which the [ f]-flt tracer seemed to be relevant , i.e. the distinction between patients with grade iii with ce and those with grade iv , all the tumors had an alteration of the bbb . although the advanced mri and pet parameters show more and more interest and could become more accessible in the assessment of glioma ( heiss et al .", "2015 ) , studies have often evaluated independently the interest of using either mri or pet . in the present study ,", "we benefit from the ability to compare the parameters from both advanced mri and pet for each patient and illustrate their complementary role in glioma grading .", "in conclusion , this study shows that [ f]-flt could be of interest in glioma classification particularly in differentiating grade iv glioblastoma from grade iii or grade ii gliomas .", "our work also illustrates the added value to acquire both , advanced mri and [ f]-flt pet , to overcome the limitations of conventional imaging for doubtful patients .", "this study was funded by the institut national contre le cancer ( inca ) , the centre national de la recherche scientifique ( cnrs ) , the ministre de l'enseignement suprieur et de la recherche ( mesr ) , the universit de caen basse - normandie ( ucbn ) , the conseil rgional de basse - normandie ( crbn ) , the european union fonds europen de dveloppement rgional ( feder ) , l'europe s'engage en basse - normandie and the french national agency for research called ", "these sponsors were not involved in study design ; in the collection , analysis and interpretation of data ; in the writing of the report ; and in the decision to submit the article for publication ." ]

[ "<S> purposeconventional mri based on contrast enhancement is often not sufficient in differentiating grade ii from grade iii and grade iii from grade iv diffuse gliomas . </S>", "<S> we assessed advanced mri , mr spectroscopy and [ 18f]-fluoro - l - thymidine ( [ 18f]-flt ) pet as tools to overcome these limitations.methodsin this prospective study , thirty - nine patients with diffuse gliomas of grades ii , iii or iv underwent conventional mri , perfusion , diffusion , proton mr spectroscopy ( 1h - mrs ) and [ 18f]-flt - pet imaging before surgery . </S>", "<S> relative cerebral blood volume ( rcbv ) , apparent diffusion coefficient ( adc ) , cho / cr , naa / cr , cho / naa and flt - suv were compared between grades.resultscho/cr showed significant differences between grade ii and grade iii gliomas ( p = 0.03 ) . to discriminate grade ii from grade iv and grade iii from grade iv gliomas , </S>", "<S> the most relevant parameter was the maximum value of [ 18f]-flt uptake fltmax ( respectively , p < 0.001 and p < 0.0001 ) . </S>", "<S> the parameter showing the best correlation with the grade was the mean value of [ 18f]-flt uptake fltmean ( r2 = 0.36 , p < 0.0001 ) and fltmax ( r2 = 0.5 , p < 0.0001).conclusionwhereas advanced mri parameters give indications for the grading of gliomas , the addition of [ 18f]-flt - pet could be of interest for the accurate preoperative classification of diffuse gliomas , particularly for identification of doubtful grade iii and iv gliomas . </S>" ]

[ "Introduction", "Methods", "Patients", "Image acquisition", "Image analysis", "ROI segmentation", "Parameter extraction", "Statistical analysis", "Results", "Patients and tumor characteristics", "Interest of multimodal imaging at the individual level", "Comparisons of imaging parameters between grades", "Discussion", "Conclusion", "Conflicts of interest" ]

[ [ "diffuse gliomas are the most common primary brain tumors in adults and encompass a heterogeneous group of tumors with different histopathological features , aggressiveness , imaging , response to treatment and survival .", "they are primarily defined by the histopathological tumor tissue analysis and subdivided by the world health organization ( who ) classification into grade ii ( low grade glioma ) , grade iii ( anaplastic glioma ) and grade iv ( glioblastoma multiforme , gbm ) . accurate distinction between different grades is of significant clinical importance because of its strong prognostic and treatment decision - making implications ( louis et al . , 2007 ) .", "however , histopathological criteria of the grading system present some limitations such as the lack of reproducibility and the prognostic heterogeneity within the same group ( ricard et al . , 2012 ) . to improve this classification , some molecular markers not only have diagnostic and prognostic implications , but also therapeutic efficacy prediction ( 1p/19q co - deletion , idh1/idh2 mutations and mgmt promoter methylation ) ( riemenschneider et al . , 2010 ) .", "however , these approaches require a resection or a biopsy and have limitations induced by intra - tumoral heterogeneity ( van den bent , 2010 ) . furthermore , there is a need to identify criteria of aggressiveness for inoperable patients .", "mri is a part of the daily management of the patients by providing anatomical and vascular information .", "the presence or the absence of t1 enhancement after gadolinium - chelate injection , necrosis , mass effect and surrounding tumor edema are established parameters used in characterizing tumor aggressiveness ( dean et al . ,", "the presence of contrast enhancement in diffuse glioma , related to the permeability of the blood brain - barrier ( bbb ) reflecting neovascularization , is often regarded as a sign of malignancy", ". however , it presents some limitations and does not allow for reliable separation between different grades .", "for example , although the absence of contrast enhancement is a commonly used criterion for identifying grade ii gliomas , it does not eliminate formally a malignant tumor ( ginsberg et al .", "in contrast , the presence of contrast enhancement and even necrosis , characteristic of gbm , can be observed in pure grade iii oligodendrogliomas . in this context ,", "multimodal imaging based on mri and pet parameters , by assessing tumor pathophysiology such as microvasculature , metabolism , proliferation or cellularity , could be of additional value .", "previous reports have described and compared the performance of glioma grading using advanced mri techniques such as relative cerebral blood volume ( rcbv ) , apparent diffusion coefficient ( adc ) , and metabolites of magnetic resonance spectroscopy ( mrs ) ( arvinda et al . , 2009 ; hilario et al . , 2012 ; law et al . , 2003 ; van den bent , 2010 ; yang et al . , 2002 ; zonari et al . , 2007 ;", "others focus specifically on glioma grading with the thymidine analog [ f]-fluoro - l - thymidine ( [ f]-flt ) , developed as a positron emission tomography ( pet ) tracer to assess the proliferation activity of tumors in vivo , particularly in brain tumors ( chalkidou et al .", ", 2012 ; chen et al . , 2005 ; hatakeyama et al . , 2008 ; jacobs et al . ,", "even if these multimodal imaging parameters are useful to distinguish grade ii from higher grades ( anaplastic and gbm ) , the differentiating criteria of grade iii from grade iv gliomas , two groups with different prognoses and management , are still poorly defined .", "the aim of this study was to compare the performance of these advanced mr techniques , namely perfusion , diffusion and proton mr spectroscopy ( h - mrs ) with that of [ f]-flt - pet , by pre - operatively assessing the diagnostic accuracy of each parameter in differentiating glioma grades ." ], [ "this study was based on a prospective clinical trial funded by inca ( institut national du cancer ) and approved by the local ethics committee and afssaps agreement ( clinicaltrials.gov identifier : nct00850278 ) .", "inclusion criteria were : a presumed diffuse glioma amenable to surgical resection or biopsy , age greater than or equal to 18 , kps greater than or equal to 50 , normal blood cell count , normal biological hepatic functions and a signed informed consent .", "the group sizes of the different tumor grades were the result of the incidence of each tumor in this prospective study .", "patients first underwent [ f]-flt - pet , followed by multiparametric mri / mrs within the same week and prior to surgery .", "single voxel mrs was localized in the area with maximum visible uptake of [ f]-flt .", "thereafter , patients underwent surgery , resection or biopsy depending on the location of the tumor , and the specimens were histopathologically evaluated by an experienced neuropathologist ( elz ) .", "majority of patients had image - guided total or subtotal surgical resection ( table 1 ) .", "for patients with biopsy only , the biopsy was guided by the area with maximum visible uptake of [ f]-flt . for grade iii tumors or for difficult cases , the diagnosis and grading were reviewed by the national multicentric network ", "prise en charge des oligodendrogliomes anaplasiques pola ( idbaih et al . , 2012 ) .", "after scout - view and coronal t2-weighted imaging , an axial flair ( fluid attenuated inversion recovery ) sequence was performed ( 24 slices , slice spacing 5.5 , pixel resolution 0.47 0.47 mm , tr / te = 9602/150 ms ) .", "diffusion weighted imaging ( dwi ) was performed using spin - echo - echo planar imaging ( se - epi ) ( 3 diffusion directions , 36 slices , slice spacing : 7 mm , pixel resolution : 1.09 1.09 mm , tr / te = 6000/96 ms , b 0 and b 1000 smm ) . for perfusion , dynamic t2 *", "-weighted epi images were acquired 30 s before , during the first pass of gd - dota ( dotarem , guerbet , france ) and 1 min after with a standard 0.1 mmol / kg body weight dose ( 14 slices , 35 repetitions , slice spacing : 7 mm , pixel resolution 2.19 2.19 mm , tr / te = 2280/60 ms ) .", "immediately thereafter , a 3dt1-weighted sequence , 3dt1w - gd ( 124 slices , slice spacing 1.5 , pixel resolution 1.01 1.01 mm , tr / te = 17/3 ms ) was performed to evaluate the tumor contrast enhancement .", "finally , mrs data were acquired in a single voxel ( 1 cm ) localized in the tumor in the area of maximal uptake of flt and in the normal - appearing contralateral side with press mrs sequence with multiple te ( 35 , 144 , 288 ms ) and water suppression .", "flt - pet [ f]-flt was produced by the ldm - tep group and synthesized at the cyceron tep center as previously described by jacobs et al .", "images of the brain were acquired 40 min after the intravenous injection of 5 mbq / kg of f - flt and lasted 10 min , to match a clinically feasible approach .", "the attenuation - corrected images were reconstructed with an osem 2d algorithm ( 9 subsets and 2 iterations ) and filtered in 3d with a butterworth filter .", "the adc ( apparent diffusion coefficient ) maps were calculated pixel - by - pixel using imagej software ( http://rsb.info.nih.gov/ij/ , 19972012 ) using the following equation : adc = [ln(s / s0)]/b where s is the signal ( averaged over the 3 directions ) acquired , b = 1000 , and s0 = the signal acquired without diffusion gradient .", "variations of the t2 * signal in the tissue , which is proportional to the concentration of the contrast agent , were calculated with in - house macros based on imagej software as : r(t ) = ln(s(t)/s0 ) , where s0 = the signal intensity before contrast agent injection , and s(t ) = the signal intensity over time .", "then , cbv maps were generated by integrating the area under the -variate fitted curves to avoid an effect of recirculation .", "images were then normalized by dividing cbv maps with the mean value of the normal - appearing contralateral side .", "coregistration : adc maps , rcbv maps , flair and pet - flt images were coregistered with trilinear interpolation , rigid matching and normalized mutual information on 3dt1w - gd images ( pmod 3.1 software ) .", "the hypersignal in flair images was manually delineated to define a region of interest ( roi ) in order to use a homogeneous method to define a pathological area ( tumor , peripheral edema and infiltration areas ) for the different grades , despite the presence or absence of t1 enhancement . when present , necrosis was excluded .", "tumor rois were reported for each modality ( rcbv , adc , t1 enhancement , flt - pet ) to extract the mean value for each modality , the maximum value for rcbv , ce and pet - flt , and the minimum value for adc . to avoid sensitivity to extreme values ,", "the minimum value was associated with the 10th percentile ( p10 ) and the maximum value with the 90th percentile ( p90 ) . in mrs , choline ( cho ) , creatine ( cr ) , n - acethyl - aspartate ( naa ) , phospholipids and lactate were quantified with sa / ge software ( ge ) to obtain peak amplitudes and areas ( proportional to concentration and relaxation ) from spectra with water resonance suppression , and expressed as a ratio .", "p - values of comparisons between grades were calculated using the non - parametric wilcoxon test .", "ordinal logistic regression was used to assess the degree of correlation ( r ) of each parameter with the grade . only r values with p - value < 0.05 were considered significant ." ], [ "this study was based on a prospective clinical trial funded by inca ( institut national du cancer ) and approved by the local ethics committee and afssaps agreement ( clinicaltrials.gov identifier : nct00850278 ) .", "inclusion criteria were : a presumed diffuse glioma amenable to surgical resection or biopsy , age greater than or equal to 18 , kps greater than or equal to 50 , normal blood cell count , normal biological hepatic functions and a signed informed consent .", "the group sizes of the different tumor grades were the result of the incidence of each tumor in this prospective study .", "patients first underwent [ f]-flt - pet , followed by multiparametric mri / mrs within the same week and prior to surgery .", "single voxel mrs was localized in the area with maximum visible uptake of [ f]-flt .", "thereafter , patients underwent surgery , resection or biopsy depending on the location of the tumor , and the specimens were histopathologically evaluated by an experienced neuropathologist ( elz ) .", "majority of patients had image - guided total or subtotal surgical resection ( table 1 ) .", "for patients with biopsy only , the biopsy was guided by the area with maximum visible uptake of [ f]-flt . for grade iii tumors or for difficult cases , the diagnosis and grading were reviewed by the national multicentric network ", "prise en charge des oligodendrogliomes anaplasiques pola ( idbaih et al . , 2012 ) ." ], [ "after scout - view and coronal t2-weighted imaging , an axial flair ( fluid attenuated inversion recovery ) sequence was performed ( 24 slices , slice spacing 5.5 , pixel resolution 0.47 0.47 mm , tr / te = 9602/150 ms ) .", "diffusion weighted imaging ( dwi ) was performed using spin - echo - echo planar imaging ( se - epi ) ( 3 diffusion directions , 36 slices , slice spacing : 7 mm , pixel resolution : 1.09 1.09 mm , tr / te = 6000/96 ms , b 0 and b 1000 smm ) . for perfusion , dynamic t2 *", "-weighted epi images were acquired 30 s before , during the first pass of gd - dota ( dotarem , guerbet , france ) and 1 min after with a standard 0.1 mmol / kg body weight dose ( 14 slices , 35 repetitions , slice spacing : 7 mm , pixel resolution 2.19 2.19 mm , tr / te = 2280/60 ms ) . immediately thereafter , a 3dt1-weighted sequence , 3dt1w - gd ( 124 slices , slice spacing 1.5 , pixel resolution 1.01 1.01 mm , tr / te = 17/3 ms ) was performed to evaluate the tumor contrast enhancement . finally , mrs data were acquired in a single voxel ( 1 cm ) localized in the tumor in the area of maximal uptake of flt and in the normal - appearing contralateral side with press mrs sequence with multiple te ( 35 , 144 , 288 ms ) and water suppression .", "flt - pet [ f]-flt was produced by the ldm - tep group and synthesized at the cyceron tep center as previously described by jacobs et al .", "images of the brain were acquired 40 min after the intravenous injection of 5 mbq / kg of f - flt and lasted 10 min , to match a clinically feasible approach .", "the attenuation - corrected images were reconstructed with an osem 2d algorithm ( 9 subsets and 2 iterations ) and filtered in 3d with a butterworth filter ." ], [ "the adc ( apparent diffusion coefficient ) maps were calculated pixel - by - pixel using imagej software ( http://rsb.info.nih.gov/ij/ , 19972012 ) using the following equation : adc = [ln(s / s0)]/b where s is the signal ( averaged over the 3 directions ) acquired , b = 1000 , and s0 = the signal acquired without diffusion gradient .", "variations of the t2 * signal in the tissue , which is proportional to the concentration of the contrast agent , were calculated with in - house macros based on imagej software as : r(t ) = ln(s(t)/s0 ) , where s0 = the signal intensity before contrast agent injection , and s(t ) = the signal intensity over time .", "then , cbv maps were generated by integrating the area under the -variate fitted curves to avoid an effect of recirculation .", "images were then normalized by dividing cbv maps with the mean value of the normal - appearing contralateral side .", "coregistration : adc maps , rcbv maps , flair and pet - flt images were coregistered with trilinear interpolation , rigid matching and normalized mutual information on 3dt1w - gd images ( pmod 3.1 software ) ." ], [ "the hypersignal in flair images was manually delineated to define a region of interest ( roi ) in order to use a homogeneous method to define a pathological area ( tumor , peripheral edema and infiltration areas ) for the different grades , despite the presence or absence of t1 enhancement . when present , necrosis was excluded ." ], [ "tumor rois were reported for each modality ( rcbv , adc , t1 enhancement , flt - pet ) to extract the mean value for each modality , the maximum value for rcbv , ce and pet - flt , and the minimum value for adc . to avoid sensitivity to extreme values ,", "the minimum value was associated with the 10th percentile ( p10 ) and the maximum value with the 90th percentile ( p90 ) . in mrs , choline ( cho ) , creatine ( cr ) , n - acethyl - aspartate ( naa ) , phospholipids and lactate were quantified with sa / ge software ( ge ) to obtain peak amplitudes and areas ( proportional to concentration and relaxation ) from spectra with water resonance suppression , and expressed as a ratio ." ], [ "p - values of comparisons between grades were calculated using the non - parametric wilcoxon test .", "ordinal logistic regression was used to assess the degree of correlation ( r ) of each parameter with the grade ." ], [ "among the 40 patients included in the study , 1 had an incomplete mri examination .", "of the 39 remaining patients , all presented histopathologically proven diffuse glioma based on who criteria and were eligible in the final analysis ( ranging from 21 to 79 years old , 17 women and 22 men ) .", "the diagnosis was established from biopsy specimens ( n = 9 ) or from resection ( 10 patients with a partial resection and 20 patients with a macroscopically complete resection ) yielding 7 patients with grade ii glioma , 9 patients with grade iii glioma and 23 patients with grade iv glioma .", "grade ii gliomas typically looked like a non - enhancing lesion that had no modification on cbv maps and no [ f]-flt uptake ( fig .", "most grade iii gliomas showed a slightly enhancing lesion with mild [ f]-flt uptake ( fig .", ", grade iv gliomas typically exhibited a strong contrast enhancement , elevated rcbv , and high [ f]-flt uptake ( fig .", "1 : grade iv ( a ) ) . interestingly , some grade iii glioma presented a profile indicative of a gbm , i.e. sustained contrast enhancement , strong modification in adc , elevated cho / cr and rcbv , appearance of lipids / lactate but [ f]-flt clearly demonstrated a weak tumor cell proliferation ( fig .", "a grade iv glioma presented non - objectivable modification in perfusion but an intense [ f]-flt uptake favoring the diagnosis of a gbm instead of a grade iii glioma ( fig .", "grade ii and grade iv gliomas rcbvmean , rcbvmax , fltmean , fltmax , cemean , cemax , cho / cr and cho / naa showed significant differences ( table 2 ) .", "2 , table 2 ) . between grade iii and grade iv gliomas , adcmean , adcmin , rcbvmean , rcbvmax , fltmean , fltmax , cemean , and", "the most relevant parameter was fltmax ( p < 0.0001 ) ( fig .", "cho / cr showed significant differences between grade ii and grade iii gliomas ( p = 0.03 ) .", "the parameter showing the best significant correlation with the grade was fltmax ( r = 0.5 , p < " ], [ "among the 40 patients included in the study , 1 had an incomplete mri examination .", "of the 39 remaining patients , all presented histopathologically proven diffuse glioma based on who criteria and were eligible in the final analysis ( ranging from 21 to 79 years old , 17 women and 22 men ) .", "the diagnosis was established from biopsy specimens ( n = 9 ) or from resection ( 10 patients with a partial resection and 20 patients with a macroscopically complete resection ) yielding 7 patients with grade ii glioma , 9 patients with grade iii glioma and 23 patients with grade iv glioma ." ], [ "grade ii gliomas typically looked like a non - enhancing lesion that had no modification on cbv maps and no [ f]-flt uptake ( fig .", "most grade iii gliomas showed a slightly enhancing lesion with mild [ f]-flt uptake ( fig .", ", grade iv gliomas typically exhibited a strong contrast enhancement , elevated rcbv , and high [ f]-flt uptake ( fig .", "1 : grade iv ( a ) ) . interestingly , some grade iii glioma presented a profile indicative of a gbm , i.e. sustained contrast enhancement , strong modification in adc , elevated cho / cr and rcbv , appearance of lipids / lactate but [ f]-flt clearly demonstrated a weak tumor cell proliferation ( fig .", "a grade iv glioma presented non - objectivable modification in perfusion but an intense [ f]-flt uptake favoring the diagnosis of a gbm instead of a grade iii glioma ( fig ." ], [ "between grade ii and grade iv gliomas rcbvmean , rcbvmax , fltmean , fltmax , cemean , cemax , cho / cr and cho / naa showed significant differences ( table 2 ) .", "the most relevant parameter was fltmax ( p < 0.001 ) ( fig .", "2 , table 2 ) . between grade iii and grade iv gliomas , adcmean , adcmin , rcbvmean , rcbvmax , fltmean , fltmax , cemean , and", "the most relevant parameter was fltmax ( p < 0.0001 ) ( fig .", "cho / cr showed significant differences between grade ii and grade iii gliomas ( p = 0.03 ) .", "the parameter showing the best significant correlation with the grade was fltmax ( r = 0.5 , p < 0.0001 ) ( table 2 ) ." ], [ "the prognosis and management of patients with diffuse glioma are strongly dependent on the accurate grading of the tumor .", "mri plays a crucial role in the assessment of aggressiveness of gliomas before surgery but may remain insufficient to forecast prognosis of diffuse gliomas . therefore , multimodal imaging parameters with advanced mri ( cbv and adc ) , mrs and [ f]-flt - pet could identify relevant markers to better predict the grading of gliomas . among advanced technics ,", "this was also shown in our study , particularly between grade ii and grade iii gliomas ( p = 0.03 ) . indeed ,", "cho / cr is well known to be correlated with tumor cell proliferation ( herminghaus et al . , 2002 ) and usually presents significant differences between low grade and high grade gliomas ( law et al . , 2003 ; server et al . , 2011 ; yang et al", "our results suggest that the use of cho / cr ratio could be of interest in the management of grade ii and grade iii rather than [ f]-flt pet , that did not show significant differences between these grades .", "rcbv has been shown to be correlated with tumor aggressiveness and with grade in enhancing and in non - enhancing lesions ( aronen et al . , 1994 ;", "maia et al . , 2005 ; sadeghi et al . , 2008 ; shin et al . , 2002 ; sugahara et al . ,", "rcbv has a better diagnostic performance in differentiating between low grade and high grade gliomas than cho / cr and cho / naa ratios obtained with mrs ( law et al . , 2003 ) ,", "adc ( arvinda et al . , 2009 ) or ktrans ( law et al . , 2004 ) .", "while most studies compare high grade and low grade , our study suggests that cbvmean and cbvmax significantly differ between patients with grade iii and patients with grade iv . for zonari et al .", ", rcbv is the most important parameter for accurate tumor grading by differentiating grade ii from iii gliomas ( zonari et al . , 2007 ) .", "along with [ f]-fdg , numerous other pet radiotracers have been developed ( for review , see ( dhermain et al .", ", 2010 ; wester , 2007 ) ) to assess tumor aggressiveness such as 3-[f]fluoro-3-deoxy - l - thymidine ( [ f]-flt ) ( jacobs et al . , 2005 ) or amino acid metabolism by o-(2-[f]fluoroethyl)-l - tyrosine ( [ f]-fet ) ( hutterer et al . , 2011 ) or methyl-[c]-l - methionine ( [ c]-met ) ( derlon et al", "this tracer has already been used by us and others to assess grade ( ferdov et al . , 2015 ) , treatment efficacy ( corroyer - dulmont et al . , 2013 ; schwarzenberg et al . ,", "[ f]-flt has also been assessed and compared with other tracers as a marker for high grade gliomas and probably more adapted for the grading compared to [ c]-met or [ f]-fet in this setting ( chen et al . , 2005 ;", ", 2005 ; jeong and lim , 2012 ; miyake et al . , 2012 ) . in agreement with these reports , our study showed that [ f]-flt parameters were not sensitive enough to distinguish grade ii from grade iii gliomas , but was the best parameter to distinguish grade iv from lower grade gliomas .", "our results are in line of previous work that already showed the interest of using [ f]-flt to differentiate low and high grade gliomas ( yamamoto et al . , 2012 ) .", "we are also aware that pet imaging and more precisely flt pet are restricted , at the present time , to few hospital or research centers . in the present study", ", we propose that flt pet adds information in presence of suspicious patients where mri may be not sensitive enough before any treatment is decided but where biopsy examination may lose some very aggressive hot spot thanks to the great spatial heterogeneity . of note , we chose a static and delayed acquisition of [ f]-flt images since a dynamic acquisition ( e.g. 2 h of acquisition ) may not be realistic for routine clinical practice .", "it has been shown that [ f]-flt uptake occurs due to increased transport through the blood ", "when we focused on the condition for which the [ f]-flt tracer seemed to be relevant , i.e. the distinction between patients with grade iii with ce and those with grade iv , all the tumors had an alteration of the bbb . although the advanced mri and pet parameters show more and more interest and could become more accessible in the assessment of glioma ( heiss et al .", "2015 ) , studies have often evaluated independently the interest of using either mri or pet . in the present study ,", "we benefit from the ability to compare the parameters from both advanced mri and pet for each patient and illustrate their complementary role in glioma grading ." ], [ "in conclusion , this study shows that [ f]-flt could be of interest in glioma classification particularly in differentiating grade iv glioblastoma from grade iii or grade ii gliomas .", "our work also illustrates the added value to acquire both , advanced mri and [ f]-flt pet , to overcome the limitations of conventional imaging for doubtful patients ." ], [ "this study was funded by the institut national contre le cancer ( inca ) , the centre national de la recherche scientifique ( cnrs ) , the ministre de l'enseignement suprieur et de la recherche ( mesr ) , the universit de caen basse - normandie ( ucbn ) , the conseil rgional de basse - normandie ( crbn ) , the european union fonds europen de dveloppement rgional ( feder ) , l'europe s'engage en basse - normandie and the french national agency for research called ", "these sponsors were not involved in study design ; in the collection , analysis and interpretation of data ; in the writing of the report ; and in the decision to submit the article for publication ." ] ]

[ "computational probe mapping is frequently \n applied in structure - based \n drug design ( sbdd ) to identify potential binding pockets along a protein \n surface ( i.e. , hot spots ) . however , mapping is usually limited by the implementation \n of gas - phase minimizations , wherein protein flexibility and solvent \n competition are ignored .", "this leads to a rugged potential surface \n and many local energy minima , where druggable sites are indistinguishable \n from irrelevant minima .", "furthermore , predictions based on a static \n receptor structure often neglect important binding - site features , \n such as the presence of transient cavities or bridging water molecules . as a result , \n appropriately modeling the binding potential continues to be a challenge \n for sbdd .", "several computational and experimental models have \n emerged that \n address the limitations of traditional solvent mapping .", "the multiple \n protein structure ( mps ) method addressed the issue of protein flexibility \n by creating pharmacophore models from consensus mapping of conformational \n ensembles with solvent probes .", "while this technique has demonstrated success in \n mapping the binding sites of pharmaceutically relevant targets , it can not account for desolvation \n penalties or water - bridging contacts , which may be essential to accurate \n prediction .", "the multiple solvent crystal structure ( mscs ) technique uses x - ray crystallography to determine receptor structures \n in the presence of competing water and organic solvent .", "this paved \n the way for fragment - based methods by providing the first experimental \n identification of preferred sites for probe binding .", "mscs results \n have demonstrated high potential for use in drug development and have \n inspired several new computational approaches .", "simultaneous \n protein flexibility and solvent mapping was first \n implemented in 2009 when molecular dynamics ( md ) simulations were \n used as a tool to map hot spots .", "seco \n et al . performed md simulations with seven proteins that were solvated \n by a 20% volume / volume ( v / v ) solution of water and isopropanol ( ipa ) . \n", "although their method sought to detect binding sites and predict druggability , \n it did not address aromatic hotspots and was unable to reproduce some \n of the experimental binding sites .", "yang and wang have conducted similar \n studies that included phenol with the alcohol / water mix to address \n aromatic interactions .", "another md - based approach , site \n identification by ligand competitive saturation ( silcs ) , was implemented \n with a ternary - solvent box composed of 1 m benzene ( bnz ) , 1 m propane , \n and water to locate hot spots and to reproduce experimental binding \n sites .", "the authors introduced a dummy atom with \n a virtual repulsion term for both bnz and propane in order to solubilize \n the hydrophobic solvents and prevent probe ", "their fragment \n maps for bnz along the trypsin surface found abundant local minima \n that were mapped equally or better than the valid binding site .", "in addition , guvench and mackerell noted that \n the repulsive term may also prevent the mapping of secondary binding \n sites due to the unnatural physical interactions that are enforced \n between the hydrophobic probes .", "the most \n recent technique was developed by bakan et al . , which uses mixtures of water with multiple probes simultaneously \n ( ipa , acetamide , acetic acid , isopropylamine ) .", "although each of these \n md - based techniques have shown some success in identifying binding \n sites , they were limited in their ability to selectively map hot spots \n without the use of either a weighting or artificial repulsion term . \n", "many spurious minima were also identified . over the past few \n years , we have worked to develop mixed - solvent \n molecular dynamics ( mixmd ) using amber , with an aim of solvent mapping \n across a wide range of targets . instead of simply showing \n that probes could occupy regions of known binding sites", ", we sought \n to establish a stronger foundation that would allow for application \n of our method to target systems without requiring a priori knowledge of the binding sites .", "our initial study with mixmd used \n a 50% w / w solvent box composed of acetonitrile ( acn ) and water , and \n the results showed that mixmd reproduced binding sites from mscs studies \n with excellent convergence to the true hot spots and no spurious minima .", "we were interested in extending the probe set for mixmd to \n fully \n enable identification of hydrogen - bonding sites , hydrophobic contacts , \n protein protein interactions , and aromatic pockets .", "here , we \n show what happens when poor parameters are used in mapping a protein . \n", "we then use md simulations of water - probe boxes to examine which remain \n evenly mixed .", "it is important to identify additional functional sites , \n particularly aromatic sites , for mixmd to be most useful for sbdd . \n", "we also \n identify additional probe solvents that expand the ability of mixmd \n to locate important interaction profiles in protein ", "ligand \n binding : acetone ( ace ) , n - methylacetamide ( nma ) , \n imidazole ( imi ) , and pyrimidine ( 1p3 ) .", "we highlight the importance \n of developing a solid foundation for mapping with md , particularly \n as it relates to the validation of parameters through detailed evaluation \n of metrics for probe probe dispersion .", "solvent probes were selected to represent \n specific interaction types for use in mixmd ( table 1 ) .", "parameters for ace , acn , and nma were obtained \n from studies of liquid solvent with amber .", "therefore , \n we explored the suitability of opls parameters , which were developed \n to work with tip3p water and to reproduce \n the empirical data for pure liquids .", "the \n adaptation of nonbonded parameters from opls for use in amber simply \n required the conversion of to rmin , where 2 /2 = rmin .", "this is necessary because boss and amber use different combining rules \n for van der waals ( vdw ) parameters .", "a comprehensive list of the parameters \n used in our mixmd simulations is given in table s1 and figure s1 of \n the supporting information .", "pure solvent boxes of no fewer than \n 200 probe molecules were built using the tleap module \n in ambertools .", "md simulations were performed in amber10 through the sander module with \n shake and a 1 fs time step .", "each solvent box was subjected to 1000 steps \n of steepest descent followed by 49,000 cycles of conjugate gradient \n minimization and then heated over 20 ps from 10 to 300 k at constant \n volume .", "two nanoseconds of equilibration were followed by a 5 ns simulation \n at constant pressure with the temperature held at 300 k by a weak - coupling \n algorithm .", "the berendsen coupling method \n was chosen because it was used in the original publication of amber \n parameters for the solvent molecules used in this study .", "the behavior of the pure - probe boxes was used \n to confirm proper behavior in our setup .", "the final boxes of organic solvent were also used in the setup \n of the mixed boxes .", "mixed - solvent boxes were created in tleap by solvating a single probe molecule with a layer \n of tip3p water and then a layer of the \n equilibrated box of pure , organic solvent .", "the size of the outer box \n of probes is adjusted to achieve the desired solvent ratio .", "at least \n 2500 water molecules and the equivalent mass of probe molecules necessary \n to achieve a 50% w / w solution were used in order to ensure that realistic \n solvent behavior would be observed ( table s3 , supporting information ) .", "each layered mixed - solvent box underwent \n the same procedure for minimization , heating , equilibration , and production \n simulation as described for the pure - solvent boxes above .", "it \n should be noted that berendsen s coupling method can sometimes be problematic for simulations \n of mixtures because differential heating can occur for different components \n of the system .", "our use of the method \n with the small boxes could be considered a worst - case scenario , where \n probes may ( or may not ) have a small bias to aggregate .", "any mixture \n of water and probe solvent that displays homogeneous behavior under \n these circumstances is likely to be robust to a wide range of applications . \n in our examination of thermolysin with ipa+water", ", we chose the andersen \n coupling method to be consistent with our other published applications \n of mixmd .", "there is no bias from the temperature coupling \n that can cause the differences seen in the two parameter choices for \n ipa when used in mixmd of thermolysin .", "we characterized the \n distribution of probe density by computing \n atom atom radial distribution functions ( rdfs ) with the radial \n command in ptraj .", "the final 1 ns of production time \n was used to calculate the rdf with a bin size of 0.1 .", "when \n the solvent probes are fully solubilized into water , they are well \n dispersed throughout the solvent box , and the rdf converges toward \n unity at the vdw cutoff .", "when phase separation occurs in simulations \n of mixed solvent , too many probes and too little water occupy the \n local microenvironment around each probe .", "this makes the local environment \n too dense with organic probes , and the rdf will not converge to unity \n within the vdw cutoff .", "accordingly , we used these rdf descriptions \n of mixed vs aggregated solvent systems to evaluate the structure of \n each mixed - solvent box . the carbon", " carbon ( rcc ) , carbon oxygen \n ( rco ) , carbon nitrogen ( rcn ) , nitrogen nitrogen ( rnn ) , nitrogen oxygen ( rno ) , and oxygen oxygen ( roo ) distributions \n were assessed between probe probe , water water , and \n probe water as was relevant for each probe molecule . for brevity , \n", "cumulative \n density distributions of roo for water water \n distances in mixed solvent were also examined to determine the number \n density of water within the volume sphere . for the case of a 50% w / w \n mixed - solvent box , equitable distribution of solvent probes and water \n within the system will give a cumulative plot for roo of \n water", "the distribution of water molecules \n relative to one another is less than in pure solvent because obviously \n the presence of a miscible probe reduces the number of water molecules \n that can occupy the local microenvironment around each water .", "earlier papers ours included have \n used the behavior \n at the edges of the simulation box to prove even mixing of probes \n and water .", "the reasoning was that the box edges are far from the protein , \n outside the vdw cutoff , and should have minimal bias in the ratio \n of probe to water .", "if the edges of the box had the same ratio as the \n setup solvent box , then there was even mixing .", "however , taking a step \n back to these basic simulations of solvent boxes provided a better \n means of measuring mixing : the use of a rdf .", "rdfs of the solvents \n relative to one another can be calculated for simulations of proteins , \n and we recommend they be used in all mixed - solvent simulations to \n judge whether the behavior of the probes and waters are reasonable .", "the rdf , or pair correlation function g(r ) , can be used to describe the structure of the solvent \n based on atom / molecule pairs in a system .", "the function g(r ) enables a precise description of how likely \n a probe molecule will have another probe present at a separation distance \n of r. this function may be simplified for a liquid \n system in a solvent box , where g(r ) is proportional to the observed number density ( o ) divided by the expected number density ( e ) .", "the \n value o is the total number of atoms at a given \n distance , and e is the number expected at that same \n distance if the solvent were uniformly distributed .", "if the probe solvent \n and water are evenly mixed , the rdf will converge to 1.0 at long ranges . \n", "if it converges to a larger number , this indicates phase separation \n ( further discussion below ) .", "previously , mixmd was validated \n with acn as a probe , which specifies \n hot spots for amphipathic nitrogen - containing ligands .", "we applied amber parameters for acn from grabuleda \n et al . , and they accurately reproduce \n experimental data from mscs . for completeness , we ran simulations \n of acn+h2o boxes to confirm that the rdf demonstrated appropriate \n convergence to unity ( figure s2 , supporting information ) . in trying to reproduce simulations of thermolysin in ipa+h2o published by seco et al . , we \n found that the ipa phase - separated from water ( figure 1a ) .", "it is unclear whether the authors observed this same behavior , \n and they do not note which temperature - coupling method was used .", "they \n noted partial phase separation in the paper , and they \n rescaled the reference values for probe density to account for the \n solvent behavior .", "however , the bulk phase separation we observed signifies \n the implementation of inadequate parameters for liquid ipa , which \n were derived from the amber parameter files for threonine with charges \n assigned after resp calculations ( ipaseco ) .", "snapshots of mixmd simulations \n of thermolysin in ipa+h2o executed using the protocol outlined \n in seco et al .", "( a ) fully flexible simulations \n of thermolysin \n were performed with a mixed box of 50% w / w ipaseco+h2o .", "five independent runs were calculated , and every simulation \n resulted in the solvent layers separating between 4 and 5 ns and remaining \n separated when simulations were extended to 10 ns .", "( b ) \n the same simulation using ipaopls resulted in solvent remaining well distributed for the entire equilibration \n and 10 ns of production in all five independent simulations .", "we turned to the work of jorgensen \n et al . for alcohol parameters \n because they were developed to specifically reproduce a variety of \n pure - solvent behaviors and interactions with tip3p water . for small boxes without protein , we compared \n our simulations of ipaopls+h2o to experiments \n by langdon and keyes that determined the density for ipa+h2o systems at different temperatures .", "we found that our mixed box of ipaopls+h2o \n reproduced the experimental density at 308 k ( 0.830g / ml ) to within \n 0.06% .", "our ability to replicate this fundamental data confirmed the \n use of good parameters for our ipa+h2o systems .", "indeed , \n mixmd simulations of thermolysin resulted in proper mixing using the \n ipa parameters from jorgensen et al .", "the rdfs for the ipa probes ( roo and rcc ) and water ( roo ) in mixed - solvent boxes further justified \n the use of opls parameters . \n", "figure 2 compares the o o rdf for both \n mixed - solvent and pure - solvent boxes of ipa .", "the roo for ( ipa - ipa)opls converged to unity with \n an appropriately shorter peak than that of pure liquid alcohol .", "however , \n the roo for ( ipa - ipa)seco remained \n well above 1.0 at 8 and featured a large peak for the first \n solvent shell , the same as seen in the simulation of a pure ipa box . \n", "all simulations with ipaseco feature rdfs that indicate \n aggregation of the solvent molecules . for this first example , the \n count data behind the rdfs will be discussed in detail to further \n explain our interpretation , which is supported by viewing the boxes .", "water \n and ( b , c ) probe probe in a mixed - solvent environment with ipa . \n", "the impact of different parameters for ipa is demonstrated by the \n poor convergence to unity for o o in simulations of 50% w / w \n ipaseco ( b ) when compared to the appropriate convergence \n obtained for the same mixed solvent with ipaopls parameters \n ( c ) .", "ipa and the great majority of \n organic solvents chosen for this \n study are miscible with water ( table 3 ) .", "the \n definition of miscibility is that the two solutions are homogeneously \n distributed at all ratios of the two solvents .", "it might be reasonable \n to find some bias or local structure in the first and second solvent \n shells , but bulk separation should not be seen at long ranges .", "this \n has critical correspondence with one of the basic underlying assumptions \n of rdfs .", "rdfs are founded on a uniform distribution of solvent to \n describe e ( solvent count / box volume ) ; any deviations \n from uniformity ( 1.0 ) reflects the local structure of the solvent .", "in all the rdfs in \n figure 2 , water has its \n maximum g(r ) at 2.75 , and \n the ipa maxima are at 2.85 . for pure water at 2.75 , \n the maximum g(r )", "is 2.6 , and the \n cumulative count of water is 1.4 molecules ( table 4 ) .", "if we define the first solvent shell between 2.45 and 3.15 \n , it contains 3.9 neighboring water molecules .", "when simulated \n in a mixture with ipaopls , there are 1.1 waters within \n 2.75 and 2.9 waters within 3.15 ( table 4 ) . of course , both counts are reduced because some of the \n interactions are occasionally fulfilled by ipa .", "this is also incorporated \n into e , where the larger box volume reduces the \n expected density .", "it is that reduced expectation ( actually 0.244 at \n 2.75 ) that results in a g(r)of 4.6 .", "this is higher than the g(r ) maximum from the pure water simulation , but it does not reflect \n more interactions with water molecules . within a 7.95 radius \n sphere ,", "the observed and expected counts are nearly equal and g(r ) approaches 1.0 , showing that the molecules \n have a uniform distribution over larger scales .", "both o and e depend \n on the volume of the simulation box ; this drops out \n and makes each g(r ) = no(r)/ne(r ) .", "the values in this table have been rounded ; the g(r ) in the figures are the exact values . \n", "values for ne(r ) are based on the volume \n of the count \n sphere , the number of solvent in the whole box , and size of the whole \n box during the simulation . for the mixture of water and ipaseco ,", "the \n expected count of water was 34.0 , but the observed count was 51.6 \n water molecules ( table 4 ) . clearly , there are \n too many waters close to one another ! furthermore ,", "phase separation ( as in figure 1a ) is best \n quantified by high g(r ) at long r , but even the local changes reflected the phenomenon . \n within 2.75 and 3.15", " , there are 1.3 and 3.4 waters ( table 4 ) , respectively , which is much closer to the values \n shown in the pure - water boxes .", "the mixed box equilibrated to a slightly \n larger volume ( 1% change ) , which is reflected in the slight differences \n in the expected counts between ipaseco+water and ipaopls+water .", "however , the 1% change does not explain the maximum g(r ) of 5.1 , which is based on the increase \n in number of water molecules .", "the same patterns are seen in \n the rdfs of the ipa oxygens ( table 4 ) .", "pure \n ipa simulations show appropriate behavior \n for both sets of parameters ( black lines in figure 2bc , table s2 and text in supporting information ) .", "though the rdfs may imply ipaopls has slightly tighter \n solvent shells , they both sum to the same number of ipa molecules \n in the first solvent shell ( 2.1 for ipaopls and 2.0 for \n ipaseco ) . for mixed simulations ,", "both ipaseco and ipaopls show lower counts than pure - solvent boxes \n at their g(r ) maxima ( 2.85 ) \n and within their solvent shells ( 3.95 ) .", "influenced by the discrepancies in solvent behavior \n observed for ipaseco and ipaopls , we carefully \n examined the behavior of existing parameters for additional solvent \n probes to determine their applicability in mixmd .", "the inclusion of \n various probe types , including complicated molecular fragments , would \n facilitate the development of accurate pharmacophores for druggable \n hot spots , ligand binding sites , and protein protein interactions . \n", "the selection of additional probes for expanding the functional groups \n represented in mixmd was based on existing mscs data and common interaction \n types found in protein ligand systems .", "ipa maps both hydrogen \n bond - accepting and -donating locations , but additional hydrogen - bonding \n probes are needed to develop robust pharmacophore maps of binding \n sites .", "ace and nma were selected because each molecule represents \n a different hydrogen - bonding profile .", "ace locates where the protein \n donates hydrogen bonds , and nma represents protein protein \n or peptide binding .", "specific \n parameters for ace and nma had been developed for use with amber and opls .", "simulations of ace+h2o and nma+h2o were performed using each parameter set \n to assess their suitability .", "the g(r ) was calculated for probe probe , probe water , and \n water ", "analysis \n of the resulting rdf plots indicated that in each case all g(r ) converged to unity near 8 . \n", "we found that both the amber and opls parameters for liquid ace and \n nma demonstrated correct behavior according to rdf plots and visual \n inspection ( figure 3 ) .", "the o o rdf for \n water from the mixed - solvent simulations also illustrate that solvent \n mixing has occurred .", "analysis of the cumulative plots for the roo of water in solution with ace or nma showed \n that the number density of water within the local volume sphere was \n half the number density observed in pure water simulations , which \n is the expected result for a well - distributed system of 50% w / w probe \n and water .", "visual analysis of the trajectory snapshots served to corroborate \n the occurrence of proper solvent mixing .", "however , the rdf s \n deviation from unity at 8 was slightly higher for the opls \n descriptions of ace and nma ( table 2 ) . for \n this reason", ", we would recommend that the amber parameters be used \n for ace and nma in our mixmd method , especially when the amber parameters \n are also used to describe the protein .", "( a , d ) o o and \n ( b , e ) n n radial distribution functions \n for probe probe and o o rdfs for water water \n ( c , f ) in a mixed - solvent environment with ace and nma .", "the impact \n of optimized parameters from amber versus general solvent parameters \n from opls is shown in the slightly better convergence to unity for \n simulations using amber parameters ( d f ) compared to opls ( a c ) \n parameters .", "in particular , we wanted \n to find parameters for soluble heterocycles that could map aromatic \n hot spots without requiring an artificial repulsive term .", "no other \n md approach for probe mapping has successfully integrated aromatic \n probes without an artificial interaction term .", "futhermore , we were \n particularly interested in developing probes that matched common heterocycles \n used in modern pharmaceuticals . as a result ,", "a highly polar \n diazole , imi , is miscible with water and is present as a functional \n group in a wide range of biologically active molecules , including \n mercaptopurine ( anticancer ) , ketoconazole ( antifungal ) , and moxonidine \n ( antihypertensive ) .", "imi parameters were derived from jorgensen and \n mcdonald , and then simulations of pure \n imi and imi+h2o were conducted to assess the potential \n of imi as a probe for mixmd .", "the proper convergence of the rdf plots \n to 1.0 showed that imi was properly solubilized and well distributed \n within the binary - solvent system ( figure 4 ) . \n", "our pure - solvent rdfs were consistent with the results of jorgensen \n and co - workers and further validated our parameter \n choice and the accuracy of our protocol .", "also , visualization of simulation \n snapshots confirmed that the imi probe was well dispersed in the aqueous \n solution .", "( c ) water water in a mixed - solvent environment with ( a ) \n imi and ( b ) 1p3 .", "opls \n parameters for pyr , 1p2 , 1p3 , and 1p4 were each simulated in a binary \n water probe solution .", "visualization of the simulations of pyr+h2o , 1p2+h2o , and 1p4+h2o clearly showed \n that they underwent phase separation .", "this was proved by rdfs that \n converged to values well above 1.0 at 8 ( figure 5 ) .", "however , 1p3+h2o yielded a well - dispersed system \n with well - behaved rdf plots ( figure 4 ) .", "probe probe \n radial distribution functions for mixed solvent \n environments composed of ( a ) bnz+h2o , ( b ) pyr+h2o , ( c ) iph+h2o , ( d ) 1p2+h2o , and ( e ) 1p4+h2o clearly showed phase separation , with g(r ) values well above 1.0 at 8 .", "we also examined two nonpolar aromatic probes , \n bnz and iph , simply \n to confirm that they are not appropriate probes when an artificial \n repulsive term is not used .", "comparison of the resultant rdf plots \n to the reference rdf plot of pure water showed that the g(r ) values for bnz+h2o and iph+h2o do not converge to unity within 8 ( figure 5 ) .", "in fact , the g(r ) for bnz+h2o indicated convergence to a value of 2 . the \n simulations with iph as a probe molecule showed a converged g(r ) of approximately 1.45 at 8 . \n", "visualization of snapshots from these simulations showed significant \n aggregation of the probe molecules , verifying that these probes are \n not be soluble enough for use with mixmd without inclusion of a repulsive \n term . because bnz is one of the solvent probes used in silcs", ", \n we were \n able to compare the rdf results for our mixmd simulations of bnz+h2o to the published results from silcs mapping studies .", "the bnz probe used in silcs was parametrized \n based on the charmm force field and contained a repulsive term to \n correct for probe probe aggregation .", "although a nonbonded cutoff \n of 8 with a 58 switching term was applied in \n silcs , the probe probe distance for bnz converged to 1.0 at \n 13 ( solvent box size was 72 58 \n 43 ) . in comparison , the soluble aromatic probes applied \n in mixmd converged at approximately 67 .", "the first solvent \n shell of bnz in silcs was observed at 9 , while we observed \n the first peak at 45 .", "the presence of the second hydrophobic \n probe ( propane ) in silcs simulations may have influenced these differences \n in rdf .", "however , the use of a repulsive term to prevent probe probe \n aggregation does not correspond with the experimental properties of \n bnz , suggesting that silcs simulations with high concentrations of \n bnz may yield incorrect results . to determine whether bnz and \n iph would disperse more readily in \n the presence of an intermediary probe ,", "ternary mixed - solvent \n boxes were constructed to contain 1 m aromatic probe and 1 m ipa in \n water .", "we hypothesized that the introduction of the ipa molecule would \n enhance available interactions and aid in the dispersion of the aromatic \n probes into water .", "however , this was not observed in simulations of \n the ternary - solvent boxes . at 5", "ns of production time , the bnz molecules \n were predominantly clustered along one side of the solvent box , while \n the ipa molecules were dispersed throughout the water .", "an additional \n 20 ns of production time did not change the observed aggregation ( figure 6 ) .", "ternary - solvent simulations with iph indicated \n a similar result ; after 5 ns of production time , iph was separated \n with some dispersion toward the interior . elongating the run time \n out to 25 ns", "representative snapshots of md trajectory for solvent boxes of \n 1 m aromatic probe ( yellow ) and 1 m ipa ( purple ) in water ( cyan ) for \n ( a ) bnz+ipa+h2o at 25 ns and ( b ) iph+ipa+h2o \n at 25 ns .", "although \n all of the probes selected for \n use in our validation study had experimental data establishing their \n solubility in water , not all of these probes were soluble in simulation \n ( table 3 ) .", "the solvation conditions applied \n in a mixmd simulation included high probe concentrations , which may \n have affected probe solubility . to determine whether the outcomes \n we observed were justified based on experimental data , we considered \n their miscibility .", "the terms soluble and miscible are frequently used \n interchangeably ; however , they are two distinct properties .", "solubility \n is dependent upon temperature and pressure and refers to the ability \n of a solute ( solid , liquid , or gas ) to dissolve into a solvent ( solid , \n liquid , or gas ) , thereby forming a homogeneous mixture .", "miscibility \n refers to the ability of two liquids to form a homogeneous solution , \n independent of proportion .", "all probes that have been experimentally \n categorized as miscible were well dispersed in our computational simulations , \n with two exceptions ( table 3 ) .", "pyridine is \n a weak base with a nontrivial concentration of protonated pyridinium \n ions ( pka of 5.3 ) following solubilization \n into water .", "this effect was not simulated in our studies , and we suggest \n that the charged species may be responsible for solubilizing the uncharged \n pyridine molecules into water . a similar explanation may also be extended \n to pyridazine .", "one advantage of using silcs as a tool for solvent \n mapping has been that the ternary - solvent box can enable the development \n of a complete pharmacophore model from a single md run because the \n three probe types identify aromatic , hydrophobic , and hydrogen - bonding \n sites .", "using three of the probes with parameter \n sets we have validated for mixmd , we constructed a ternary system \n with imi ( aromatic probe ) , ipa ( hydrophobic and hydrogen - bonding probe ) , \n and water at a concentration of 33% w / w by probe .", "visualization of \n the trajectory data indicated appropriate solvent mixing , and our \n rdf results showed that both organic probes were fully dispersed into \n solution ( figure 7 ) .", "total run time required \n a comparable amount of simulation time to a binary mixmd simulation ; \n the total cost was an additional 4 or 8 h of production time on an \n 8-core cpu as compared to a binary ipa+h2o or imi+h2o simulation , respectively .", "( a ) final snapshot from the production \n simulation of a mixed - solvent \n box containing imi+ipa+h2o illustrates that the probes \n are well mixed within the system .", "( b d ) probe probe \n and water water radial distribution functions for imi and ipa \n establish convergence to unity within the trisolvent environment .", "we suggest that the use of this \n ternary mixmd model may offer several \n advantages over the silcs approach .", "we have shown that mixmd can be \n used to identify maximally occupied sites without recovering additional \n spurious minima , which is a feature that was not consistently seen \n in the silcs studies .", "in addition , instead \n of requiring water to act as a hydrogen - bonding probe , in mixmd , the \n druggable hydrogen - bonding sites can be identified in competition \n with water using ipa as the probe .", "this is important for ascertaining \n whether a drug - like molecule that contains a similar function group \n could displace water molecules at the proposed hydrogen - bonding site . \n", "a further advantage of using mixmd is the array of available probes , \n which can be tailored to an investigator s mapping needs . for \n example", ", either imi or 1p3 could be used in the ternary box to locate \n aromatic hotspots that are specific to the size of the ring .", "the ternary \n mixture of 33% w / w ace+acn+h2o was also simulated , and \n the results depicted a well - distributed solvent system for use in \n hot spot mapping with mixmd ( figure s3 , supporting \n information ) .", "we have examined solvent \n parameters for neat liquid from the literature \n for use in hot spot mapping through mixmd .", "our work highlights the \n importance of parameter validation and analysis of simulation data \n when performing computational solvent mapping in order to obtain appropriate \n behavior .", "influenced by the poor results obtained using a standard \n set of parameters used in the field for liquid isopropanol in water , \n we identified a set of functional group probes with specific parameters \n available for liquid simulation .", "we pursued validation of these parameters \n for neat and mixed - solvent simulations and identified six probes for \n use with tip3p in mixmd : acetonitrile , acetone , imidazole , isopropanol , n - methylacetamide , and pyrimidine .", "of course , these probes \n should be used with more proteins beyond thermolysin ( figure 1 ) to prove their applicability .", "we have previously \n applied opls ipa parameters to also examine elastase , hewl , hewl , \n p53 core , and rnase a. furthermore , we \n are currently using all of these probes to map 10 additional protein \n systems , but these are separate studies in their own right .", "the extent of ideal dispersion is most easily quantified by using \n the cumulative rdf of roo for water .", "although \n published results for pyridine and pyridazine indicated that they \n are miscible with water , these two probes were observed to separate \n from the aqueous phase during our simulations , rendering them unsuitable \n for solvent mapping in our context .", "we hypothesized that this disparity \n between experiment and computation was caused by the presence of the \n corresponding ionized species , which were not simulated . in order \n to avoid unphysical mapping or reduced accuracy ,", "our simulations did \n not employ the use of an artificial repulsion or weighted density \n term .", "we have successfully expanded the range of probes that can be \n incorporated into mixmd studies to allow for the mapping of druggable \n sites , including hydrogen - bonding regions , aromatic pockets , and protein ", ", a full pharmacophore \n model could be created from the simulation of a single protein+probes+water \n system , which would greatly reduce the computational costs of mixmd \n studies .", "this is a clear advantage of the silcs method that we would \n like to incorporate .", "ultimately , investigators could mix and \n match the probes used in mixmd to identify hotspots with a \n greater degree of specificity ." ]

[ "<S> probe mapping is a common approach \n for identifying potential binding \n sites in structure - based drug design ; however , it typically relies \n on energy minimizations of probes in the gas phase and a static protein \n structure . </S>", "<S> the mixed - solvent molecular dynamics ( mixmd ) approach was \n recently developed to account for full protein flexibility and solvation \n effects in hot - spot mapping . </S>", "<S> our first study used only acetonitrile \n as a probe , and here , we have augmented the set of functional group \n probes through careful testing and parameter validation . </S>", "<S> a diverse \n range of probes are needed in order to map complex binding interactions . \n </S>", "<S> a small variation in probe parameters can adversely effect mixed - solvent \n behavior , which we highlight with isopropanol . </S>", "<S> we tested 11 solvents \n to identify six with appropriate behavior in tip3p water to use as \n organic probes in the mixmd method . </S>", "<S> in addition to acetonitrile and \n isopropanol , we have identified acetone , n - methylacetamide , \n imidazole , and pyrimidine . </S>", "<S> these probe solvents will enable mixmd \n studies to recover hydrogen - bonding sites , hydrophobic pockets , protein </S>", "<S> protein \n interactions , and aromatic hotspots . also , we show that ternary - solvent \n systems can be incorporated within a single simulation . </S>", "<S> importantly , \n these binary and ternary solvents do not require artificial repulsion \n terms like other methods . within merely 5 ns , layered solvent boxes \n become evenly mixed for soluble probes . </S>", "<S> we used radial distribution \n functions to evaluate solvent behavior , determine adequate mixing , \n and confirm the absence of phase separation . </S>", "<S> we recommend that radial \n distribution functions should be used to assess adequate sampling \n in all mixed - solvent techniques rather than the current practice of \n examining the solvent ratios at the edges of the solvent box . </S>" ]

[ "Introduction", "Methods", "Results\nand Discussion", "Conclusion" ]

[ [ "computational probe mapping is frequently \n applied in structure - based \n drug design ( sbdd ) to identify potential binding pockets along a protein \n surface ( i.e. , hot spots ) . however , mapping is usually limited by the implementation \n of gas - phase minimizations , wherein protein flexibility and solvent \n competition are ignored .", "this leads to a rugged potential surface \n and many local energy minima , where druggable sites are indistinguishable \n from irrelevant minima .", "furthermore , predictions based on a static \n receptor structure often neglect important binding - site features , \n such as the presence of transient cavities or bridging water molecules . as a result , \n appropriately modeling the binding potential continues to be a challenge \n for sbdd .", "several computational and experimental models have \n emerged that \n address the limitations of traditional solvent mapping .", "the multiple \n protein structure ( mps ) method addressed the issue of protein flexibility \n by creating pharmacophore models from consensus mapping of conformational \n ensembles with solvent probes .", "while this technique has demonstrated success in \n mapping the binding sites of pharmaceutically relevant targets , it can not account for desolvation \n penalties or water - bridging contacts , which may be essential to accurate \n prediction .", "the multiple solvent crystal structure ( mscs ) technique uses x - ray crystallography to determine receptor structures \n in the presence of competing water and organic solvent .", "this paved \n the way for fragment - based methods by providing the first experimental \n identification of preferred sites for probe binding .", "mscs results \n have demonstrated high potential for use in drug development and have \n inspired several new computational approaches .", "simultaneous \n protein flexibility and solvent mapping was first \n implemented in 2009 when molecular dynamics ( md ) simulations were \n used as a tool to map hot spots .", "seco \n et al . performed md simulations with seven proteins that were solvated \n by a 20% volume / volume ( v / v ) solution of water and isopropanol ( ipa ) . \n", "although their method sought to detect binding sites and predict druggability , \n it did not address aromatic hotspots and was unable to reproduce some \n of the experimental binding sites .", "yang and wang have conducted similar \n studies that included phenol with the alcohol / water mix to address \n aromatic interactions .", "another md - based approach , site \n identification by ligand competitive saturation ( silcs ) , was implemented \n with a ternary - solvent box composed of 1 m benzene ( bnz ) , 1 m propane , \n and water to locate hot spots and to reproduce experimental binding \n sites .", "the authors introduced a dummy atom with \n a virtual repulsion term for both bnz and propane in order to solubilize \n the hydrophobic solvents and prevent probe ", "their fragment \n maps for bnz along the trypsin surface found abundant local minima \n that were mapped equally or better than the valid binding site .", "in addition , guvench and mackerell noted that \n the repulsive term may also prevent the mapping of secondary binding \n sites due to the unnatural physical interactions that are enforced \n between the hydrophobic probes .", "the most \n recent technique was developed by bakan et al . , which uses mixtures of water with multiple probes simultaneously \n ( ipa , acetamide , acetic acid , isopropylamine ) .", "although each of these \n md - based techniques have shown some success in identifying binding \n sites , they were limited in their ability to selectively map hot spots \n without the use of either a weighting or artificial repulsion term . \n", "many spurious minima were also identified . over the past few \n years , we have worked to develop mixed - solvent \n molecular dynamics ( mixmd ) using amber , with an aim of solvent mapping \n across a wide range of targets . instead of simply showing \n that probes could occupy regions of known binding sites", ", we sought \n to establish a stronger foundation that would allow for application \n of our method to target systems without requiring a priori knowledge of the binding sites .", "our initial study with mixmd used \n a 50% w / w solvent box composed of acetonitrile ( acn ) and water , and \n the results showed that mixmd reproduced binding sites from mscs studies \n with excellent convergence to the true hot spots and no spurious minima .", "we were interested in extending the probe set for mixmd to \n fully \n enable identification of hydrogen - bonding sites , hydrophobic contacts , \n protein protein interactions , and aromatic pockets .", "here , we \n show what happens when poor parameters are used in mapping a protein . \n", "we then use md simulations of water - probe boxes to examine which remain \n evenly mixed .", "it is important to identify additional functional sites , \n particularly aromatic sites , for mixmd to be most useful for sbdd . \n", "we also \n identify additional probe solvents that expand the ability of mixmd \n to locate important interaction profiles in protein ", "ligand \n binding : acetone ( ace ) , n - methylacetamide ( nma ) , \n imidazole ( imi ) , and pyrimidine ( 1p3 ) .", "we highlight the importance \n of developing a solid foundation for mapping with md , particularly \n as it relates to the validation of parameters through detailed evaluation \n of metrics for probe probe dispersion ." ], [ "solvent probes were selected to represent \n specific interaction types for use in mixmd ( table 1 ) .", "parameters for ace , acn , and nma were obtained \n from studies of liquid solvent with amber .", "therefore , \n we explored the suitability of opls parameters , which were developed \n to work with tip3p water and to reproduce \n the empirical data for pure liquids .", "the \n adaptation of nonbonded parameters from opls for use in amber simply \n required the conversion of to rmin , where 2 /2 = rmin .", "this is necessary because boss and amber use different combining rules \n for van der waals ( vdw ) parameters .", "a comprehensive list of the parameters \n used in our mixmd simulations is given in table s1 and figure s1 of \n the supporting information .", "pure solvent boxes of no fewer than \n 200 probe molecules were built using the tleap module \n in ambertools .", "md simulations were performed in amber10 through the sander module with \n shake and a 1 fs time step .", "each solvent box was subjected to 1000 steps \n of steepest descent followed by 49,000 cycles of conjugate gradient \n minimization and then heated over 20 ps from 10 to 300 k at constant \n volume .", "two nanoseconds of equilibration were followed by a 5 ns simulation \n at constant pressure with the temperature held at 300 k by a weak - coupling \n algorithm .", "the berendsen coupling method \n was chosen because it was used in the original publication of amber \n parameters for the solvent molecules used in this study .", "the behavior of the pure - probe boxes was used \n to confirm proper behavior in our setup .", "the final boxes of organic solvent were also used in the setup \n of the mixed boxes .", "mixed - solvent boxes were created in tleap by solvating a single probe molecule with a layer \n of tip3p water and then a layer of the \n equilibrated box of pure , organic solvent .", "the size of the outer box \n of probes is adjusted to achieve the desired solvent ratio .", "at least \n 2500 water molecules and the equivalent mass of probe molecules necessary \n to achieve a 50% w / w solution were used in order to ensure that realistic \n solvent behavior would be observed ( table s3 , supporting information ) .", "each layered mixed - solvent box underwent \n the same procedure for minimization , heating , equilibration , and production \n simulation as described for the pure - solvent boxes above .", "it \n should be noted that berendsen s coupling method can sometimes be problematic for simulations \n of mixtures because differential heating can occur for different components \n of the system .", "our use of the method \n with the small boxes could be considered a worst - case scenario , where \n probes may ( or may not ) have a small bias to aggregate .", "any mixture \n of water and probe solvent that displays homogeneous behavior under \n these circumstances is likely to be robust to a wide range of applications . \n in our examination of thermolysin with ipa+water", ", we chose the andersen \n coupling method to be consistent with our other published applications \n of mixmd .", "there is no bias from the temperature coupling \n that can cause the differences seen in the two parameter choices for \n ipa when used in mixmd of thermolysin .", "we characterized the \n distribution of probe density by computing \n atom atom radial distribution functions ( rdfs ) with the radial \n command in ptraj .", "the final 1 ns of production time \n was used to calculate the rdf with a bin size of 0.1 .", "when \n the solvent probes are fully solubilized into water , they are well \n dispersed throughout the solvent box , and the rdf converges toward \n unity at the vdw cutoff .", "when phase separation occurs in simulations \n of mixed solvent , too many probes and too little water occupy the \n local microenvironment around each probe .", "this makes the local environment \n too dense with organic probes , and the rdf will not converge to unity \n within the vdw cutoff .", "accordingly , we used these rdf descriptions \n of mixed vs aggregated solvent systems to evaluate the structure of \n each mixed - solvent box . the carbon", " carbon ( rcc ) , carbon oxygen \n ( rco ) , carbon nitrogen ( rcn ) , nitrogen nitrogen ( rnn ) , nitrogen oxygen ( rno ) , and oxygen oxygen ( roo ) distributions \n were assessed between probe probe , water water , and \n probe water as was relevant for each probe molecule . for brevity , \n", "cumulative \n density distributions of roo for water water \n distances in mixed solvent were also examined to determine the number \n density of water within the volume sphere . for the case of a 50% w / w \n mixed - solvent box , equitable distribution of solvent probes and water \n within the system will give a cumulative plot for roo of \n water", "the distribution of water molecules \n relative to one another is less than in pure solvent because obviously \n the presence of a miscible probe reduces the number of water molecules \n that can occupy the local microenvironment around each water ." ], [ "earlier papers ours included have \n used the behavior \n at the edges of the simulation box to prove even mixing of probes \n and water .", "the reasoning was that the box edges are far from the protein , \n outside the vdw cutoff , and should have minimal bias in the ratio \n of probe to water .", "if the edges of the box had the same ratio as the \n setup solvent box , then there was even mixing .", "however , taking a step \n back to these basic simulations of solvent boxes provided a better \n means of measuring mixing : the use of a rdf .", "rdfs of the solvents \n relative to one another can be calculated for simulations of proteins , \n and we recommend they be used in all mixed - solvent simulations to \n judge whether the behavior of the probes and waters are reasonable .", "the rdf , or pair correlation function g(r ) , can be used to describe the structure of the solvent \n based on atom / molecule pairs in a system .", "the function g(r ) enables a precise description of how likely \n a probe molecule will have another probe present at a separation distance \n of r. this function may be simplified for a liquid \n system in a solvent box , where g(r ) is proportional to the observed number density ( o ) divided by the expected number density ( e ) .", "the \n value o is the total number of atoms at a given \n distance , and e is the number expected at that same \n distance if the solvent were uniformly distributed .", "if the probe solvent \n and water are evenly mixed , the rdf will converge to 1.0 at long ranges . \n", "if it converges to a larger number , this indicates phase separation \n ( further discussion below ) .", "previously , mixmd was validated \n with acn as a probe , which specifies \n hot spots for amphipathic nitrogen - containing ligands .", "we applied amber parameters for acn from grabuleda \n et al . , and they accurately reproduce \n experimental data from mscs . for completeness , we ran simulations \n of acn+h2o boxes to confirm that the rdf demonstrated appropriate \n convergence to unity ( figure s2 , supporting information ) . in trying to reproduce simulations of thermolysin in ipa+h2o published by seco et al . , we \n found that the ipa phase - separated from water ( figure 1a ) .", "it is unclear whether the authors observed this same behavior , \n and they do not note which temperature - coupling method was used .", "they \n noted partial phase separation in the paper , and they \n rescaled the reference values for probe density to account for the \n solvent behavior .", "however , the bulk phase separation we observed signifies \n the implementation of inadequate parameters for liquid ipa , which \n were derived from the amber parameter files for threonine with charges \n assigned after resp calculations ( ipaseco ) .", "snapshots of mixmd simulations \n of thermolysin in ipa+h2o executed using the protocol outlined \n in seco et al .", "( a ) fully flexible simulations \n of thermolysin \n were performed with a mixed box of 50% w / w ipaseco+h2o .", "five independent runs were calculated , and every simulation \n resulted in the solvent layers separating between 4 and 5 ns and remaining \n separated when simulations were extended to 10 ns .", "( b ) \n the same simulation using ipaopls resulted in solvent remaining well distributed for the entire equilibration \n and 10 ns of production in all five independent simulations .", "we turned to the work of jorgensen \n et al . for alcohol parameters \n because they were developed to specifically reproduce a variety of \n pure - solvent behaviors and interactions with tip3p water . for small boxes without protein , we compared \n our simulations of ipaopls+h2o to experiments \n by langdon and keyes that determined the density for ipa+h2o systems at different temperatures .", "we found that our mixed box of ipaopls+h2o \n reproduced the experimental density at 308 k ( 0.830g / ml ) to within \n 0.06% .", "our ability to replicate this fundamental data confirmed the \n use of good parameters for our ipa+h2o systems .", "indeed , \n mixmd simulations of thermolysin resulted in proper mixing using the \n ipa parameters from jorgensen et al .", "the rdfs for the ipa probes ( roo and rcc ) and water ( roo ) in mixed - solvent boxes further justified \n the use of opls parameters . \n", "figure 2 compares the o o rdf for both \n mixed - solvent and pure - solvent boxes of ipa .", "the roo for ( ipa - ipa)opls converged to unity with \n an appropriately shorter peak than that of pure liquid alcohol .", "however , \n the roo for ( ipa - ipa)seco remained \n well above 1.0 at 8 and featured a large peak for the first \n solvent shell , the same as seen in the simulation of a pure ipa box . \n", "all simulations with ipaseco feature rdfs that indicate \n aggregation of the solvent molecules . for this first example , the \n count data behind the rdfs will be discussed in detail to further \n explain our interpretation , which is supported by viewing the boxes .", "water \n and ( b , c ) probe probe in a mixed - solvent environment with ipa . \n", "the impact of different parameters for ipa is demonstrated by the \n poor convergence to unity for o o in simulations of 50% w / w \n ipaseco ( b ) when compared to the appropriate convergence \n obtained for the same mixed solvent with ipaopls parameters \n ( c ) .", "ipa and the great majority of \n organic solvents chosen for this \n study are miscible with water ( table 3 ) .", "the \n definition of miscibility is that the two solutions are homogeneously \n distributed at all ratios of the two solvents .", "it might be reasonable \n to find some bias or local structure in the first and second solvent \n shells , but bulk separation should not be seen at long ranges .", "this \n has critical correspondence with one of the basic underlying assumptions \n of rdfs .", "rdfs are founded on a uniform distribution of solvent to \n describe e ( solvent count / box volume ) ; any deviations \n from uniformity ( 1.0 ) reflects the local structure of the solvent .", "in all the rdfs in \n figure 2 , water has its \n maximum g(r ) at 2.75 , and \n the ipa maxima are at 2.85 . for pure water at 2.75 , \n the maximum g(r )", "is 2.6 , and the \n cumulative count of water is 1.4 molecules ( table 4 ) .", "if we define the first solvent shell between 2.45 and 3.15 \n , it contains 3.9 neighboring water molecules .", "when simulated \n in a mixture with ipaopls , there are 1.1 waters within \n 2.75 and 2.9 waters within 3.15 ( table 4 ) . of course , both counts are reduced because some of the \n interactions are occasionally fulfilled by ipa .", "this is also incorporated \n into e , where the larger box volume reduces the \n expected density .", "it is that reduced expectation ( actually 0.244 at \n 2.75 ) that results in a g(r)of 4.6 .", "this is higher than the g(r ) maximum from the pure water simulation , but it does not reflect \n more interactions with water molecules . within a 7.95 radius \n sphere ,", "the observed and expected counts are nearly equal and g(r ) approaches 1.0 , showing that the molecules \n have a uniform distribution over larger scales .", "both o and e depend \n on the volume of the simulation box ; this drops out \n and makes each g(r ) = no(r)/ne(r ) .", "the values in this table have been rounded ; the g(r ) in the figures are the exact values . \n", "values for ne(r ) are based on the volume \n of the count \n sphere , the number of solvent in the whole box , and size of the whole \n box during the simulation . for the mixture of water and ipaseco ,", "the \n expected count of water was 34.0 , but the observed count was 51.6 \n water molecules ( table 4 ) . clearly , there are \n too many waters close to one another ! furthermore ,", "phase separation ( as in figure 1a ) is best \n quantified by high g(r ) at long r , but even the local changes reflected the phenomenon . \n within 2.75 and 3.15", " , there are 1.3 and 3.4 waters ( table 4 ) , respectively , which is much closer to the values \n shown in the pure - water boxes .", "the mixed box equilibrated to a slightly \n larger volume ( 1% change ) , which is reflected in the slight differences \n in the expected counts between ipaseco+water and ipaopls+water .", "however , the 1% change does not explain the maximum g(r ) of 5.1 , which is based on the increase \n in number of water molecules .", "the same patterns are seen in \n the rdfs of the ipa oxygens ( table 4 ) .", "pure \n ipa simulations show appropriate behavior \n for both sets of parameters ( black lines in figure 2bc , table s2 and text in supporting information ) .", "though the rdfs may imply ipaopls has slightly tighter \n solvent shells , they both sum to the same number of ipa molecules \n in the first solvent shell ( 2.1 for ipaopls and 2.0 for \n ipaseco ) . for mixed simulations ,", "both ipaseco and ipaopls show lower counts than pure - solvent boxes \n at their g(r ) maxima ( 2.85 ) \n and within their solvent shells ( 3.95 ) .", "influenced by the discrepancies in solvent behavior \n observed for ipaseco and ipaopls , we carefully \n examined the behavior of existing parameters for additional solvent \n probes to determine their applicability in mixmd .", "the inclusion of \n various probe types , including complicated molecular fragments , would \n facilitate the development of accurate pharmacophores for druggable \n hot spots , ligand binding sites , and protein protein interactions . \n", "the selection of additional probes for expanding the functional groups \n represented in mixmd was based on existing mscs data and common interaction \n types found in protein ligand systems .", "ipa maps both hydrogen \n bond - accepting and -donating locations , but additional hydrogen - bonding \n probes are needed to develop robust pharmacophore maps of binding \n sites .", "ace and nma were selected because each molecule represents \n a different hydrogen - bonding profile .", "ace locates where the protein \n donates hydrogen bonds , and nma represents protein protein \n or peptide binding .", "specific \n parameters for ace and nma had been developed for use with amber and opls .", "simulations of ace+h2o and nma+h2o were performed using each parameter set \n to assess their suitability .", "the g(r ) was calculated for probe probe , probe water , and \n water ", "analysis \n of the resulting rdf plots indicated that in each case all g(r ) converged to unity near 8 . \n", "we found that both the amber and opls parameters for liquid ace and \n nma demonstrated correct behavior according to rdf plots and visual \n inspection ( figure 3 ) .", "the o o rdf for \n water from the mixed - solvent simulations also illustrate that solvent \n mixing has occurred .", "analysis of the cumulative plots for the roo of water in solution with ace or nma showed \n that the number density of water within the local volume sphere was \n half the number density observed in pure water simulations , which \n is the expected result for a well - distributed system of 50% w / w probe \n and water .", "visual analysis of the trajectory snapshots served to corroborate \n the occurrence of proper solvent mixing .", "however , the rdf s \n deviation from unity at 8 was slightly higher for the opls \n descriptions of ace and nma ( table 2 ) . for \n this reason", ", we would recommend that the amber parameters be used \n for ace and nma in our mixmd method , especially when the amber parameters \n are also used to describe the protein .", "( a , d ) o o and \n ( b , e ) n n radial distribution functions \n for probe probe and o o rdfs for water water \n ( c , f ) in a mixed - solvent environment with ace and nma .", "the impact \n of optimized parameters from amber versus general solvent parameters \n from opls is shown in the slightly better convergence to unity for \n simulations using amber parameters ( d f ) compared to opls ( a c ) \n parameters .", "in particular , we wanted \n to find parameters for soluble heterocycles that could map aromatic \n hot spots without requiring an artificial repulsive term .", "no other \n md approach for probe mapping has successfully integrated aromatic \n probes without an artificial interaction term .", "futhermore , we were \n particularly interested in developing probes that matched common heterocycles \n used in modern pharmaceuticals . as a result ,", "a highly polar \n diazole , imi , is miscible with water and is present as a functional \n group in a wide range of biologically active molecules , including \n mercaptopurine ( anticancer ) , ketoconazole ( antifungal ) , and moxonidine \n ( antihypertensive ) .", "imi parameters were derived from jorgensen and \n mcdonald , and then simulations of pure \n imi and imi+h2o were conducted to assess the potential \n of imi as a probe for mixmd .", "the proper convergence of the rdf plots \n to 1.0 showed that imi was properly solubilized and well distributed \n within the binary - solvent system ( figure 4 ) . \n", "our pure - solvent rdfs were consistent with the results of jorgensen \n and co - workers and further validated our parameter \n choice and the accuracy of our protocol .", "also , visualization of simulation \n snapshots confirmed that the imi probe was well dispersed in the aqueous \n solution .", "( c ) water water in a mixed - solvent environment with ( a ) \n imi and ( b ) 1p3 .", "opls \n parameters for pyr , 1p2 , 1p3 , and 1p4 were each simulated in a binary \n water probe solution .", "visualization of the simulations of pyr+h2o , 1p2+h2o , and 1p4+h2o clearly showed \n that they underwent phase separation .", "this was proved by rdfs that \n converged to values well above 1.0 at 8 ( figure 5 ) .", "however , 1p3+h2o yielded a well - dispersed system \n with well - behaved rdf plots ( figure 4 ) .", "probe probe \n radial distribution functions for mixed solvent \n environments composed of ( a ) bnz+h2o , ( b ) pyr+h2o , ( c ) iph+h2o , ( d ) 1p2+h2o , and ( e ) 1p4+h2o clearly showed phase separation , with g(r ) values well above 1.0 at 8 .", "we also examined two nonpolar aromatic probes , \n bnz and iph , simply \n to confirm that they are not appropriate probes when an artificial \n repulsive term is not used .", "comparison of the resultant rdf plots \n to the reference rdf plot of pure water showed that the g(r ) values for bnz+h2o and iph+h2o do not converge to unity within 8 ( figure 5 ) .", "in fact , the g(r ) for bnz+h2o indicated convergence to a value of 2 . the \n simulations with iph as a probe molecule showed a converged g(r ) of approximately 1.45 at 8 . \n", "visualization of snapshots from these simulations showed significant \n aggregation of the probe molecules , verifying that these probes are \n not be soluble enough for use with mixmd without inclusion of a repulsive \n term . because bnz is one of the solvent probes used in silcs", ", \n we were \n able to compare the rdf results for our mixmd simulations of bnz+h2o to the published results from silcs mapping studies .", "the bnz probe used in silcs was parametrized \n based on the charmm force field and contained a repulsive term to \n correct for probe probe aggregation .", "although a nonbonded cutoff \n of 8 with a 58 switching term was applied in \n silcs , the probe probe distance for bnz converged to 1.0 at \n 13 ( solvent box size was 72 58 \n 43 ) . in comparison , the soluble aromatic probes applied \n in mixmd converged at approximately 67 .", "the first solvent \n shell of bnz in silcs was observed at 9 , while we observed \n the first peak at 45 .", "the presence of the second hydrophobic \n probe ( propane ) in silcs simulations may have influenced these differences \n in rdf .", "however , the use of a repulsive term to prevent probe probe \n aggregation does not correspond with the experimental properties of \n bnz , suggesting that silcs simulations with high concentrations of \n bnz may yield incorrect results . to determine whether bnz and \n iph would disperse more readily in \n the presence of an intermediary probe ,", "ternary mixed - solvent \n boxes were constructed to contain 1 m aromatic probe and 1 m ipa in \n water .", "we hypothesized that the introduction of the ipa molecule would \n enhance available interactions and aid in the dispersion of the aromatic \n probes into water .", "however , this was not observed in simulations of \n the ternary - solvent boxes . at 5", "ns of production time , the bnz molecules \n were predominantly clustered along one side of the solvent box , while \n the ipa molecules were dispersed throughout the water .", "an additional \n 20 ns of production time did not change the observed aggregation ( figure 6 ) .", "ternary - solvent simulations with iph indicated \n a similar result ; after 5 ns of production time , iph was separated \n with some dispersion toward the interior . elongating the run time \n out to 25 ns", "representative snapshots of md trajectory for solvent boxes of \n 1 m aromatic probe ( yellow ) and 1 m ipa ( purple ) in water ( cyan ) for \n ( a ) bnz+ipa+h2o at 25 ns and ( b ) iph+ipa+h2o \n at 25 ns .", "although \n all of the probes selected for \n use in our validation study had experimental data establishing their \n solubility in water , not all of these probes were soluble in simulation \n ( table 3 ) .", "the solvation conditions applied \n in a mixmd simulation included high probe concentrations , which may \n have affected probe solubility . to determine whether the outcomes \n we observed were justified based on experimental data , we considered \n their miscibility .", "the terms soluble and miscible are frequently used \n interchangeably ; however , they are two distinct properties .", "solubility \n is dependent upon temperature and pressure and refers to the ability \n of a solute ( solid , liquid , or gas ) to dissolve into a solvent ( solid , \n liquid , or gas ) , thereby forming a homogeneous mixture .", "miscibility \n refers to the ability of two liquids to form a homogeneous solution , \n independent of proportion .", "all probes that have been experimentally \n categorized as miscible were well dispersed in our computational simulations , \n with two exceptions ( table 3 ) .", "pyridine is \n a weak base with a nontrivial concentration of protonated pyridinium \n ions ( pka of 5.3 ) following solubilization \n into water .", "this effect was not simulated in our studies , and we suggest \n that the charged species may be responsible for solubilizing the uncharged \n pyridine molecules into water . a similar explanation may also be extended \n to pyridazine .", "one advantage of using silcs as a tool for solvent \n mapping has been that the ternary - solvent box can enable the development \n of a complete pharmacophore model from a single md run because the \n three probe types identify aromatic , hydrophobic , and hydrogen - bonding \n sites .", "using three of the probes with parameter \n sets we have validated for mixmd , we constructed a ternary system \n with imi ( aromatic probe ) , ipa ( hydrophobic and hydrogen - bonding probe ) , \n and water at a concentration of 33% w / w by probe .", "visualization of \n the trajectory data indicated appropriate solvent mixing , and our \n rdf results showed that both organic probes were fully dispersed into \n solution ( figure 7 ) .", "total run time required \n a comparable amount of simulation time to a binary mixmd simulation ; \n the total cost was an additional 4 or 8 h of production time on an \n 8-core cpu as compared to a binary ipa+h2o or imi+h2o simulation , respectively .", "( a ) final snapshot from the production \n simulation of a mixed - solvent \n box containing imi+ipa+h2o illustrates that the probes \n are well mixed within the system .", "( b d ) probe probe \n and water water radial distribution functions for imi and ipa \n establish convergence to unity within the trisolvent environment .", "we suggest that the use of this \n ternary mixmd model may offer several \n advantages over the silcs approach .", "we have shown that mixmd can be \n used to identify maximally occupied sites without recovering additional \n spurious minima , which is a feature that was not consistently seen \n in the silcs studies .", "in addition , instead \n of requiring water to act as a hydrogen - bonding probe , in mixmd , the \n druggable hydrogen - bonding sites can be identified in competition \n with water using ipa as the probe .", "this is important for ascertaining \n whether a drug - like molecule that contains a similar function group \n could displace water molecules at the proposed hydrogen - bonding site . \n", "a further advantage of using mixmd is the array of available probes , \n which can be tailored to an investigator s mapping needs . for \n example", ", either imi or 1p3 could be used in the ternary box to locate \n aromatic hotspots that are specific to the size of the ring .", "the ternary \n mixture of 33% w / w ace+acn+h2o was also simulated , and \n the results depicted a well - distributed solvent system for use in \n hot spot mapping with mixmd ( figure s3 , supporting \n information ) ." ], [ "we have examined solvent \n parameters for neat liquid from the literature \n for use in hot spot mapping through mixmd .", "our work highlights the \n importance of parameter validation and analysis of simulation data \n when performing computational solvent mapping in order to obtain appropriate \n behavior .", "influenced by the poor results obtained using a standard \n set of parameters used in the field for liquid isopropanol in water , \n we identified a set of functional group probes with specific parameters \n available for liquid simulation .", "we pursued validation of these parameters \n for neat and mixed - solvent simulations and identified six probes for \n use with tip3p in mixmd : acetonitrile , acetone , imidazole , isopropanol , n - methylacetamide , and pyrimidine .", "of course , these probes \n should be used with more proteins beyond thermolysin ( figure 1 ) to prove their applicability .", "we have previously \n applied opls ipa parameters to also examine elastase , hewl , hewl , \n p53 core , and rnase a. furthermore , we \n are currently using all of these probes to map 10 additional protein \n systems , but these are separate studies in their own right .", "the extent of ideal dispersion is most easily quantified by using \n the cumulative rdf of roo for water .", "although \n published results for pyridine and pyridazine indicated that they \n are miscible with water , these two probes were observed to separate \n from the aqueous phase during our simulations , rendering them unsuitable \n for solvent mapping in our context .", "we hypothesized that this disparity \n between experiment and computation was caused by the presence of the \n corresponding ionized species , which were not simulated . in order \n to avoid unphysical mapping or reduced accuracy ,", "our simulations did \n not employ the use of an artificial repulsion or weighted density \n term .", "we have successfully expanded the range of probes that can be \n incorporated into mixmd studies to allow for the mapping of druggable \n sites , including hydrogen - bonding regions , aromatic pockets , and protein ", ", a full pharmacophore \n model could be created from the simulation of a single protein+probes+water \n system , which would greatly reduce the computational costs of mixmd \n studies .", "this is a clear advantage of the silcs method that we would \n like to incorporate .", "ultimately , investigators could mix and \n match the probes used in mixmd to identify hotspots with a \n greater degree of specificity ." ] ]

[ "autoimmune hepatitis ( aih ) is characterized by chronic necroinflammation , manifesting as severe lobular necrosis and inflammation with broad areas of parenchymal collapse with no known cause .", "in fact , aih might be associated with a number of other autoimmune diseases,1 but the exact mechanisms of onset remain unknown . in aih ,", "the most common are antinuclear antibodies ( ana ) , smooth muscle antibodies ( sma ) , and antibodies to liver and kidney microsomes ( anti - lkm).1 evidence suggests that aih is induced by antibody - dependent cell - mediated cytotoxicity , which involves both antibody - mediated and cellular immunity against specific liver antigens on hepatocyte membranes.2 several studies have documented the involvement of genetic factors including human leukocyte antigen ( hla ) types dr3 and dr4.3 some reports have described aih cases in which a virus such as hepatitis a,4 hepatitis b,5 or epstein barr6 was the causative agent .", "sera from patients with multiple autoimmune diseases have been found to have elevated cytomegalovirus ( cmv ) igm titers , perhaps implying an as yet unidentified association between cmv and autoimmune diseases.7 we present a case of fluorescence ana - negative aih - associated primary biliary cirrhosis ( pbc ) that appears to have been triggered by reactivated cmv infection .", "in 2010 , a 63-year - old japanese woman was admitted to a local hospital , after which she was transferred to our hospital for investigation of liver dysfunction .", "one month prior , she had experienced fever . she had no history of medicine or drug use .", "the findings of physical examinations of admission to our hospital were the following : body temperature , 36.8c ; pulse rate , 69 beats / minute and regular ; blood pressure , 122/77 mmhg ; respiratory rate , 16/minute ; no sign of anemia at the conjunctiva palpebra , but an icteric finding was made at the conjunctiva bulbi .", "the results of urinalysis were normal , with the exception of a positive result for urobilinogen .", "the results of biochemical analysis of the blood were the following : total bilirubin , 6.6 mg / dl ( normal range : 0.21.2 ) ; direct bilirubin , 4.4 mg / dl ( normal range : 0.00.2 ) ; aspartate aminotransferase , 344 iu / l ( normal range : 1040 ) ; alanine aminotransferase , 238 iu / l ( normal range : 545 ) ; alkaline phosphatase , 572 iu / l ( normal range : 100325 ) ; -guanosine triphosphate , 129 iu / l ( normal value : < 30 ) ; igg , 2,498 mg / dl ( normal range : 8701,700 ) ; and igm , 279 mg / dl ( normal range : 46260 ) .", "cmvigm antibody was high ; igg antibody was above the normal ranges at admission ( table 1 ) .", "tests for hepatitis a igm antibody , hepatitis b surface antigen , and anti - hepatitis c virus antibody were negative .", "ana was negative by fluorescence assay ( < 40 ) , but it was positive as measured using enzyme - linked immunosorbent assay ( elisa ) ( 44.1 index ; normal range < 20 ) , anti - mitochondria m2 antibody level was 18.4 ( normal value : < 7.0 ) , anti - lkm antibody was negative , anti - sma was < 20 , and anti - dsdna antibody ( < 7.0 iu / ml ; normal value : < 20 iu / ml ) was negative .", "the level of anti - ribonucleoproteins antibody was 20.3 ( normal value : < 17 ) ( table 1 ) .", "histological examination showed severe lobular necrosis , inflammation with broad areas of parenchymal collapse , and both nondestructive and destructive cholangitis surrounded by many lymphocytes and plasma cells ( figure 2 ) .", "regarding results of electron microscopy , migrating lymphocytes were found more frequently in a medium - sized interlobular bile duct surrounded by many inflammatory cells .", "complete absence or partial loss of microvilli and microvillous bleb formation was visible in the same bile duct epithelial cell ( figure 3 ) .", "the clinical and pathological findings were highly suggestive of aih , the score of which was calculated according to the system proposed by the revised international aih group.8 the negative ana result assessed by fluorescent ana was used in the calculation .", "the clinical / pathological findings of anti - mitochondria m2 antibody and chronic nonsuppurative destructive cholangitis indicated pbc .", "her severe jaundice was improved , and her bilirubin and transaminase levels began to decline after administration of prednisolone for 3 days .", "the role of viruses in aih has been proposed repeatedly , but convincing evidence has linked two viruses hepatitis a and epstein barr virus to type 1 of the disease , only in rare cases where a genetic predisposition exists and the viral infection occurs at the right time . in spite of an impressive amount of information conclusively showing molecular mimicry between cytochrome p4502d6 sequences ( the target autoantigen of autoantibodies characteristic of aih type 2 ) and viral ( hepatitis c virus , herpes simplex virus 1 , cmv , human t lymphotropic viruses 1 and 2 ) or bacterial ( salmonella typhimurium ) antigens , no infectious agent has been clearly implicated for causing type 2 of the disease.9 at admission and at 2 , 4 , and 8 weeks after admission , the respective cmv igm indices ( normal value : < 0.8 ) were 2.17 , 0.93 , 1.08 , and 1.407 ; and igg indices ( normal value : < 2.0 ) were 10 , 34 , 89.4 , > 128 , and > 128 . consequently , serial cmv ig measurements demonstrated a slight increase of igm at admission which declined gradually over time , whereas igg increased with time to very high levels .", "these findings suggested cmv infection , consistent with a previous report that some patients with secondary cmv infection had long - lasting igm antibodies , and that all had igg antibodies to cmv.10 the present case is a rare case of negative fluorescence - ana and low - titer elisa - ana aih type 1 with coincident cmv infection .", "although the clinical features strongly suggest aih , calculation of aih score using the result did not recommend a diagnosis of aih from negative fluorescence - ana .", "immunofluorescence assay is the ana detection method used in the calculation of aih score.8 although immunofluorescence is a sensitive test , it is laborious when testing large quantities of patient samples and is subject to errors of human interpretation and variation in fluorescence microscopes.11 the enzyme immunoassay test system is an excellent alternative to the immunofluorescence assay system for screening patient serum for the presence of clinically significant anas .", "the enzyme immunoassay efficiently screens large quantities of patient samples and reduces risks of human error.12 both methods have different issues with respect to precision .", "our patient was positive for ama , and liver biopsy showed a mixed portal inflammatory infiltrate with destructive and nondestructive cholangitis ( figure 3c ) .", "the reported frequency of ama in patients with definitive aih was 18 in 206 ( 7.5% ) patients as assessed by immunoblotting.13 therefore , ama positivity is not a typical aih finding . in the present case ,", "the findings of ama positivity and destructive biliary injury resembling chronic nonsuppurative destructive cholangitis in the biopsy strongly suggest a diagnosis of pbc.14 although aih and pbc might share some common histological features , the patterns seen on liver biopsy typically differ .", "interface hepatitis or lobular hepatitis with infiltration of plasma cells is a characteristic of acute or chronic aih.15 nondestructive ductular reaction is detected more frequently in acute aih than in chronic disease , but the destructive cholangitis lesions that are typical of pbc are not present.15,16 moreover , for electron microscopy , the bile duct epithelial cell membrane facing the lymphocyte was disrupted .", "bile duct epithelial cell detachment is an important ultrastructural lesion associated with extensive bile duct destruction in pbc livers.17 cellular immune mechanisms involving t cell reaction were thought to be significantly involved in the formation of chronic nonsuppurative destructive cholangitis and bile duct loss.18 in our case , the presence of portal inflammation and mixed destructive and nondestructive cholangitis changes , supported by other biochemical changes , led to a final diagnosis of overlapping syndrome of acute exacerbation aih and pbc .", "this case is rare because these changes coincide or even precede cmv infection . in the present case ,", "elisa assay for ana and liver biopsy led to aih and early treatment with prednisolone , which produced a good clinical outcome ." ]

[ "<S> a 63-year - old woman , who presented with severe jaundice and elevated serum conjugated bilirubin level , denied alcohol and drug use and showed no evidence of viral hepatitis . </S>", "<S> based on clinical and laboratory features , she was diagnosed with autoimmune hepatitis with primary biliary cirrhosis . </S>", "<S> hematological and immunochemical assays , radiographic imaging , clinical examination , and liver biopsy were conducted . </S>", "<S> laboratory results were the following : negative for fluorescence antinuclear antibody , negative for antismooth muscle antibodies but positive for antinuclear antibody ( enzyme - linked immunosorbent assay ) and antimitochondrial m2 antibody , high titers of serum globulin , and positive for cytomegalovirus igm . </S>", "<S> liver biopsy showed submassive lobular necrosis , inflammation with broad areas of parenchymal collapse , and chronic nonsuppurative destructive cholangitis . </S>", "<S> the patient responded well to corticosteroid therapy . </S>", "<S> this case might illustrate an association between cytomegalovirus infection and the occurrence of autoimmune hepatitis . </S>" ]

[ "Introduction", "Case report", "Discussion" ]

[ [ "autoimmune hepatitis ( aih ) is characterized by chronic necroinflammation , manifesting as severe lobular necrosis and inflammation with broad areas of parenchymal collapse with no known cause .", "in fact , aih might be associated with a number of other autoimmune diseases,1 but the exact mechanisms of onset remain unknown . in aih ,", "the most common are antinuclear antibodies ( ana ) , smooth muscle antibodies ( sma ) , and antibodies to liver and kidney microsomes ( anti - lkm).1 evidence suggests that aih is induced by antibody - dependent cell - mediated cytotoxicity , which involves both antibody - mediated and cellular immunity against specific liver antigens on hepatocyte membranes.2 several studies have documented the involvement of genetic factors including human leukocyte antigen ( hla ) types dr3 and dr4.3 some reports have described aih cases in which a virus such as hepatitis a,4 hepatitis b,5 or epstein barr6 was the causative agent .", "sera from patients with multiple autoimmune diseases have been found to have elevated cytomegalovirus ( cmv ) igm titers , perhaps implying an as yet unidentified association between cmv and autoimmune diseases.7 we present a case of fluorescence ana - negative aih - associated primary biliary cirrhosis ( pbc ) that appears to have been triggered by reactivated cmv infection ." ], [ "in 2010 , a 63-year - old japanese woman was admitted to a local hospital , after which she was transferred to our hospital for investigation of liver dysfunction .", "one month prior , she had experienced fever . she had no history of medicine or drug use .", "the findings of physical examinations of admission to our hospital were the following : body temperature , 36.8c ; pulse rate , 69 beats / minute and regular ; blood pressure , 122/77 mmhg ; respiratory rate , 16/minute ; no sign of anemia at the conjunctiva palpebra , but an icteric finding was made at the conjunctiva bulbi .", "the results of urinalysis were normal , with the exception of a positive result for urobilinogen .", "the results of biochemical analysis of the blood were the following : total bilirubin , 6.6 mg / dl ( normal range : 0.21.2 ) ; direct bilirubin , 4.4 mg / dl ( normal range : 0.00.2 ) ; aspartate aminotransferase , 344 iu / l ( normal range : 1040 ) ; alanine aminotransferase , 238 iu / l ( normal range : 545 ) ; alkaline phosphatase , 572 iu / l ( normal range : 100325 ) ; -guanosine triphosphate , 129 iu / l ( normal value : < 30 ) ; igg , 2,498 mg / dl ( normal range : 8701,700 ) ; and igm , 279 mg / dl ( normal range : 46260 ) .", "cmvigm antibody was high ; igg antibody was above the normal ranges at admission ( table 1 ) .", "tests for hepatitis a igm antibody , hepatitis b surface antigen , and anti - hepatitis c virus antibody were negative .", "ana was negative by fluorescence assay ( < 40 ) , but it was positive as measured using enzyme - linked immunosorbent assay ( elisa ) ( 44.1 index ; normal range < 20 ) , anti - mitochondria m2 antibody level was 18.4 ( normal value : < 7.0 ) , anti - lkm antibody was negative , anti - sma was < 20 , and anti - dsdna antibody ( < 7.0 iu / ml ; normal value : < 20 iu / ml ) was negative .", "the level of anti - ribonucleoproteins antibody was 20.3 ( normal value : < 17 ) ( table 1 ) .", "histological examination showed severe lobular necrosis , inflammation with broad areas of parenchymal collapse , and both nondestructive and destructive cholangitis surrounded by many lymphocytes and plasma cells ( figure 2 ) .", "regarding results of electron microscopy , migrating lymphocytes were found more frequently in a medium - sized interlobular bile duct surrounded by many inflammatory cells .", "complete absence or partial loss of microvilli and microvillous bleb formation was visible in the same bile duct epithelial cell ( figure 3 ) .", "the clinical and pathological findings were highly suggestive of aih , the score of which was calculated according to the system proposed by the revised international aih group.8 the negative ana result assessed by fluorescent ana was used in the calculation .", "the clinical / pathological findings of anti - mitochondria m2 antibody and chronic nonsuppurative destructive cholangitis indicated pbc .", "her severe jaundice was improved , and her bilirubin and transaminase levels began to decline after administration of prednisolone for 3 days ." ], [ "the role of viruses in aih has been proposed repeatedly , but convincing evidence has linked two viruses hepatitis a and epstein barr virus to type 1 of the disease , only in rare cases where a genetic predisposition exists and the viral infection occurs at the right time . in spite of an impressive amount of information conclusively showing molecular mimicry between cytochrome p4502d6 sequences ( the target autoantigen of autoantibodies characteristic of aih type 2 ) and viral ( hepatitis c virus , herpes simplex virus 1 , cmv , human t lymphotropic viruses 1 and 2 ) or bacterial ( salmonella typhimurium ) antigens , no infectious agent has been clearly implicated for causing type 2 of the disease.9 at admission and at 2 , 4 , and 8 weeks after admission , the respective cmv igm indices ( normal value : < 0.8 ) were 2.17 , 0.93 , 1.08 , and 1.407 ; and igg indices ( normal value : < 2.0 ) were 10 , 34 , 89.4 , > 128 , and > 128 . consequently , serial cmv ig measurements demonstrated a slight increase of igm at admission which declined gradually over time , whereas igg increased with time to very high levels .", "these findings suggested cmv infection , consistent with a previous report that some patients with secondary cmv infection had long - lasting igm antibodies , and that all had igg antibodies to cmv.10 the present case is a rare case of negative fluorescence - ana and low - titer elisa - ana aih type 1 with coincident cmv infection .", "although the clinical features strongly suggest aih , calculation of aih score using the result did not recommend a diagnosis of aih from negative fluorescence - ana .", "immunofluorescence assay is the ana detection method used in the calculation of aih score.8 although immunofluorescence is a sensitive test , it is laborious when testing large quantities of patient samples and is subject to errors of human interpretation and variation in fluorescence microscopes.11 the enzyme immunoassay test system is an excellent alternative to the immunofluorescence assay system for screening patient serum for the presence of clinically significant anas .", "the enzyme immunoassay efficiently screens large quantities of patient samples and reduces risks of human error.12 both methods have different issues with respect to precision .", "our patient was positive for ama , and liver biopsy showed a mixed portal inflammatory infiltrate with destructive and nondestructive cholangitis ( figure 3c ) .", "the reported frequency of ama in patients with definitive aih was 18 in 206 ( 7.5% ) patients as assessed by immunoblotting.13 therefore , ama positivity is not a typical aih finding . in the present case ,", "the findings of ama positivity and destructive biliary injury resembling chronic nonsuppurative destructive cholangitis in the biopsy strongly suggest a diagnosis of pbc.14 although aih and pbc might share some common histological features , the patterns seen on liver biopsy typically differ .", "interface hepatitis or lobular hepatitis with infiltration of plasma cells is a characteristic of acute or chronic aih.15 nondestructive ductular reaction is detected more frequently in acute aih than in chronic disease , but the destructive cholangitis lesions that are typical of pbc are not present.15,16 moreover , for electron microscopy , the bile duct epithelial cell membrane facing the lymphocyte was disrupted .", "bile duct epithelial cell detachment is an important ultrastructural lesion associated with extensive bile duct destruction in pbc livers.17 cellular immune mechanisms involving t cell reaction were thought to be significantly involved in the formation of chronic nonsuppurative destructive cholangitis and bile duct loss.18 in our case , the presence of portal inflammation and mixed destructive and nondestructive cholangitis changes , supported by other biochemical changes , led to a final diagnosis of overlapping syndrome of acute exacerbation aih and pbc .", "this case is rare because these changes coincide or even precede cmv infection . in the present case ,", "elisa assay for ana and liver biopsy led to aih and early treatment with prednisolone , which produced a good clinical outcome ." ] ]

[ "european rabbits ( oryctolagus cuniculus ; figure 1 ) have played an important role as animal models in immunology for many decades . today , rabbits are still a major source for a wide variety of monoclonal antibodies ( mabs ) and polyclonal antibodies ( pabs ) with broad utility .", "pabs can be described as a set of different antibodies generated in response to a specific pathogen or antigen , generally targeting different epitopes .", "mabs , on the other hand , contain a defined antigen - binding site ( paratope ) that typically binds with high affinity and specificity to only one epitope . from a pharmaceutical point of view", ", mabs provide a molecularly defined and reproducible product , whereas pabs are traditionally an imprecise mixture of different antibodies .", "as is the case for mouse and human mabs , igg is the most common isotype of rabbit mabs ( figure 2 ) .", "rabbit pabs have been used extensively as analytical tools in biomedical research and especially for immunological techniques , such as immunohistochemistry ( ihc ) , western blotting and flow cytometry .", "atg is a mixture of purified polyclonal rabbit , horse or goat iggs against human t cells that has been used as an immunosuppressive drug for decades . in organ and", "allogeneic bone marrow transplantation , atg application causes rapid depletion of t cells , leading to a decreased risk for rejection and acute graft - versus - host disease . however , atg does not induce long - term tolerance .", "rabbit atg is one of the most commonly used atgs , due to its higher lymphocytotoxicity compared to horse atg .", ", cambridge , ma , usa ) was approved by the us food and drug administration ( fda ) in 1998 .", "more recently , an improved rabbit pabs cocktail targeting human leukocytes was reported as a potential immunosuppressive drug in xenogeneic ( for example , pig to human ) organ transplantation .", "in addition , one rabbit pab is currently approved by the fda as an in vitro diagnostic tool ( c - kit pharmdx ; agilent technologies , inc . , santa clara , ca , usa ) for the ihc - based detection of cd117 ( c - kit ) expression in gastrointestinal stromal tumors to aid treatment decisions .", "mabs and mab - derived antibody therapeutics are currently widely used to treat human diseases , such as cancer and autoimmune diseases .", "although no therapeutic rabbit mabs have been approved by the fda thus far , 11 rabbit mabs are fda - approved in vitro diagnostic tools in the clinic .", "ten of these mabs are being used to detect the expression of tumor - associated antigens , including her2 , estrogen receptors , progesterone receptors and pd - l1 .", "a rabbit mab to human androgen receptor splice variant 7 has emerged as a promising tool for the detection of circulating tumor cells by immunofluorescence and ihc in prostate cancer . in addition", ", several rabbit mab - derived therapeutics are currently being investigated in clinical trials registered at clinicaltrials.gov . in oncology , examples include sevacizumab ( simcere pharmaceutical group , inc . ,", "nanjing , china ) , a humanized rabbit anti - human vascular endothelial growth factor mab ( nct02453464 ) , apx005 m ( apexigen , inc . , san carlos , ca , usa ) , a humanized rabbit anti - human cd40 mab ( nct02482168 ) , and yyb101 , a humanized rabbit anti - human hgf mab ( yooyoung pharmaceutical co. , inc . ,", "further , chimeric antigen receptor t cells based on a rabbit anti - human ror1 mab have commenced clinical trials recently ( nct02706392 ) . in ophthalmology ,", "humanized rabbit anti - vascular endothelial growth factor mab brolucizumab ( alcon , inc . ,", "hnenberg , switzerland ) , a mab in scfv format administered intravitreally , is being investigated in advanced clinical trials ( nct02307682 and nct02434328 ) . following these examples , a number of rabbit mab - derived therapeutics are expected to transition from preclinical to clinical studies in the near future .", "currently , only a handful of companies develop rabbit or rabbit - derived mabs for laboratory research and for diagnostic and therapeutic applications .", "( cambridge , uk ; rabmab platform ; through acquisition of epitomics , inc . ,", "( through acquisition of esbatech , inc . , schlieren , switzerland in 2009 ) ; apexigen , inc .", "( san carlos , ca , usa ; spun out by epitomics , inc . in 2010 ) ; cell signaling technology , inc .", "( danvers , ma , usa ; currently listing > 4000 different rabbit mabs ) ; agilent technologies , inc . ; mab discovery , inc . ,", "neuried , germany ; lab vision corporation , inc . , fremont , ca , usa ; thermo fisher scientific , inc .", "( carlsbad , ca , usa ; invitrogen abfinity recombinant rabbit antibodies ) ; and ventana medical systems , inc .", "rabbits have been used to investigate immunological questions and to develop immunological techniques for > 100 years .", "thus , many standard procedures are established , published and validated , such as immunization and purification methods yielding high amounts of rabbit antibodies .", "in addition to these practical considerations , rabbits are characterized by a variety of natural features that make their antibody repertoire very attractive for the discussed applications .", "first , rabbits belong to the order lagomorpha , which is evolutionary distinct from the order rodentia , to which , for example , mice and rats belong .", "rabbit antibodies are able to recognize epitopes on human antigens that are not immunogenic in rodents , increasing the total number of targetable epitopes and facilitating the generation of antibodies that cross - react with mouse orthologs of human antigens .", "this is an important aspect for basic research and preclinical investigations with , for example , human tumor xenografts , where the presence or absence of on - target - off - tumor toxicities of therapeutic antibodies provides important information prior to clinical translation . in general ,", "rabbit anti - mouse reactivity is valuable in mouse models of human disease and has also been exploited in basic research , for example , on mouse stem cell antigens .", "second , it has been observed that rabbits elicit strong immune responses against small molecules and haptens , which is uncommon in rodents .", "a third important aspect is the scarcity of inbred rabbit strains , while most mouse strains are inbred .", "it is thought that inbred strains in general elicit less diverse immune responses , which makes it more difficult to raise strong and diverse binders .", "correspondingly , in many ihc studies that compared rabbit and mouse mabs to the same human antigens , rabbit mabs consistently revealed higher sensitivity . in a recent study ,", "1410 rabbit mabs raised against 15-amino - acid peptides representing 100 different antigens revealed a typical affinity range of 20200 pm ( median 66 pm ) , as determined by high - throughput surface plasmon resonance .", "a fraction of rabbit mabs even revealed higher affinities near the detection limit of 1 pm .", "however , the affinity range of 46 mouse mabs ( 30300 pm ; median 72 pm ) analyzed in parallel was not significantly different .", "more direct comparisons of rabbit and mouse mabs to a wider variety of antigens are warranted to determine a significant difference in affinity .", "fourth , most strategies to generate mabs are based on the recovery of b cells from spleen , bone marrow or blood , which are present in higher quantities in rabbits than in mice due to their overall larger body size .", "( the average body weight of a 3-month - old laboratory rabbit is 2.5 kg compared to 25 g for a 6-week - old laboratory mouse . ) for example , 50 times more spleen b cells can be recovered from rabbits compared to mice . in addition , the larger blood volume of rabbits compared to mice facilitates mass spectrometry analyses of bulk serum igg .", "fifth , as discussed in the next section , rabbits use different mechanisms to genetically generate and diversify their primary and secondary antibody repertoires compared to humans and mice , effectively creating a complementary set of binders for the discussed applications . the recent sequencing and annotation of the rabbit genome ( orycun2.0 assembly )", "the development of the rabbit b - cell repertoire significantly differs from that of other mammals .", "the current model proposes a three - step process ( figure 3 ) consisting of the neonatal b - cell repertoire generated by b lymphopoiesis in the fetal liver and omentum switching to bone marrow after birth , the primary pre - immune ' b - cell repertoire evolving during the first 2 months after birth in gut - associated lymphoid tissue ( galt ) and the secondary immune ' b - cell repertoire generated upon b - cell activation by immunogen binding .", "the neonatal b - cell repertoire starts developing between the second and third week of gestation .", "interestingly , b lymphopoiesis is very limited in the bone marrow of adult rabbits , indicating that b lymphopoiesis is mainly restricted to early development .", "in addition , in a rabbit - to - rabbit adoptive transfer model , it was shown that rabbit b cells were able to engraft into host stem cell niches , which led to the conclusion that rabbit b cells are long - lived and potentially self - renewing and may thus sustain the rabbit antibody repertoire throughout a rabbit 's lifetime .", "rabbits mainly rearrange their heavy chains first followed by their light chains and thus follow the primary b - cell development pathway proposed by ehlich et al . in brief , this model proposes the vh ", "jh recombination of the heavy chain , followed by its expression as a pre - b - cell receptor . upon stimulation of the pre - b - cell receptor , vl", "some evidence for non - templated nucleotide - addition ( also known as n - nucleotides ) has been observed in rabbit vj rearrangements in 1-day - old rabbits , suggesting that terminal deoxynucleotidyl transferase was recently expressed .", "although a potential pool of 200 vh genes are present for heavy - chain rearrangement , a majority of these are infrequently or not expressed .", "however , it is known that certain genes from these clusters are preferentially utilized : vh1 , the most d - proximal vh gene , is present in 8090% of all heavy - chain gene rearrangements .", "three different allotypes vha1 , vha2 and vha3 are known , resulting in the vha - positive serotype .", "this finding was somewhat surprising , because there is another functional , d - proximal vh sequence significantly closer to the vh1 segment .", "indeed , it could be shown that the vh4 segment is utilized in 8090% of the heavy - chain rearrangements in vh1-negative alicia strain rabbits .", "however , this heavy - chain rearrangement was very rare in adult rabbits , suggesting that the vh4 rearrangement is non - productive in most cases and thus lost in the course of development .", ". suggested negative self - antigen effects and non - efficient pairing with light chains and surrogate light chains during b - cell development as possible reasons .", "in addition , preferential utilization of d2a , d2b and d1 as well as jh4 could be shown .", "it is thought that rabbits compensate for this preferential utilization of certain vh , d and jh genes in part by n - nucleotide addition at the vh d and d jh junctions during recombination .", "using high - throughput sequencing of antibody repertoires from three new zealand white rabbits , lavinder et al . determined that the means.d .", "length of the third complementarity - determining region ( cdr ) of the heavy chain , which is known as hcdr3 , includes both junctions , and thus is the most hypervariable of all six cdrs , is 154 ( mode=13 ) amino acids compared to mouse and human hcdr3s with means.d .", "lengths of 112 ( mode=10 amino acids ) and 154 ( mode=15 ) amino acids , respectively .", "these findings confirmed that rabbit hcdr3s share more similarity with human hcdr3s than mouse hcdr3s and could be relevant for therapeutic applications .", "although there is limited heavy - chain usage in the neonatal rabbits ' b - cell repertoire , there may be compensation by usage of diverse light chains .", "there are two types of rabbit light chains and with the light chain present in two different isotypes called k1 and k2 .", "four k1 -light - chain allotypes b4 , b5 , b6 and b9have been observed in domesticated rabbits .", "notably , the constant domain sequences of these rabbit -light - chain allotypes have much higher amino - acid sequence diversity than human ig allotypes with pairwise amino - acid sequence differences of up to 40% .", "this high degree of polymorphism exceeds the allotypic diversity of the three heavy - chain allotypes , a1 , a2 and a3 ( ~20% amino - acid sequence differences ) , and even surpasses the allotypic diversity of major histocompatibility complex loci .", "k1 is by far the most abundant isotype and represents between 70 and 90% of all light chains , whereas the rest is usually a mixture of both k2 and -light chains .", "many k1 light chains are characterized by an intrachain disulfide bridge between cysteine 80 in v and cysteine 171 in c ( figure 2 ) .", "this additional disulfide bridge , which was discovered biochemically and later confirmed by x - ray crystallography , is not found in human or mouse light chains .", "it may contribute to the stability of rabbit antibodies and is one of many peculiarities of ig evolution in vertebrates .", "this disulfide bridge is absent from rabbits of the k1-negative basilea mutant strain that only expresses k2 and -light chains .", "some but not all of the k1 light chains of rabbits homozygous for the b9 -light - chain allotype have a cysteine 108 in j that is thought to create an alternative intrachain disulfide bridge with cysteine 171 in c. these allotypes play an important role for the generation of rabbit mabs by phage display and are discussed in more detail below .", "recently , using next - generation sequencing of rabbit antibody repertoires , kodangattil et al . demonstrated that light - chain rearrangements are significantly more diverse than heavy - chain rearrangements in rabbits .", "in addition , the imprecise junction of vl and jl genes in rabbits encompasses particularly long stretches of n - nucleotides , resulting in , on average , longer lcdr3s ( 122 amino acids ; mode=12 ) compared to mouse and human ( 91 amino acids ; mode=9 ; figure 4 ) .", "the longer rabbit lcdr3s are occasionally stabilized by disulfide bridges . taken together , by generating a more diverse neonatal light - chain repertoire followed by a comparable degree of further diversification into primary and secondary light - chain repertoires , the light chain can compensate for the limited diversity of heavy - chain repertoires in rabbits .", "this may also explain the dominance of the rabbit light chain in general and lcdr3 in particular in several of the antibody / antigen complexes for which three - dimensional structures have been determined by x - ray crystallography . the limited neonatal antibody repertoire is further diversified between week 4 and 8 after birth resulting in the primary antibody repertoire .", "this postnatal diversification is an unusual phenomenon and has so far only been observed in rabbits and pigs .", "the two main mechanisms are somatic gene conversion ( sgc ; templated ) and somatic hypermutation ( shm ; non - templated ; figures 3 and 4 ) .", "sgc , predominantly used only in chicken and rabbit ig gene diversification , leads to the replacement of large nucleic acid sequence stretches with dna fragments from non - utilized vh genes .", "thus , the non - expressible majority of vh genes play an important role for heavy - chain diversification . the predominance of this mechanism in rabbit ( 23% ) compared to human ( 2.5% ) and mouse ( 0.1% ) heavy chains , as determined by high - throughput sequencing , is thought to lead to decreased wastage : a substantial portion of vh d jh recombinations with n - nucleotides addition have out - of - frame junctions , whereas sgc , a homologous dna recombination event , tends to favor in - frame substitutions and extensions .", "in addition , shm is observed at considerable levels during this developmental stage . following rearrangement in the neonatal b - cell repertoire ,", "rabbit light - chain genes also undergo sgc and shm to diversify the primary b - cell repertoire ( figures 3 and 4 ) . further corroborating the importance of the light chain for generating diversity in rabbit antibody repertoires ,", "tract length ( 8648 nucleotides ) in rabbit -light chains compared to rabbit heavy chains .", "interestingly , the generation of the primary repertoire is highly galt - dependent . in short", ", galt involves the uptake of pathogens from the gastrointestinal track by specialized epithelial cells known as m cells and the presentation of antigens to b cells leading to b - cell stimulation , diversification and proliferation .", "surgical removal of galt tissues , such as the peyer 's patches in the small intestines , the sacculus rotundus and the appendix resulted in severely immunodeficient rabbits .", "the secondary b - cell repertoire is generated upon antigen - dependent b - cell stimulation .", "again , additional diversity is introduced by sgc and shm in both heavy and light chains ( figures 3 and 4 ) .", "these events further broaden the b - cell repertoire directed against a certain set of antigens associated with a specific pathogen .", "notably , rabbits only have one igg isotype ( one c gene ) compared to four igg isotypes in mice ( igg1 , igg2a , igg2b and igg3 ) and humans ( igg1 , igg2 , igg3 and igg4 ) . in contrast , rabbits have 13 c genes giving rise to at least 10 functional iga isotypes , the most diverse iga system known , compared to just one iga isotype in mice and two in humans . collectively , the unique features of b - cell ontogeny and antibody repertoire make rabbits a valuable source for the generation of antibodies of high affinity and specificity .", "the discovery and development of hybridoma technology for the generation of mabs by georges khler and csar milstein in 1975 has had a huge impact on biomedical research and its application to modern medicine .", "hybridoma technology is a method to generate stable cell lines that constantly secret a defined mab . for this purpose ,", "b cells derived from an immunized animal are fused with a myeloma cell line in the presence of polyethylene glycol .", "the hybridomas generated this way are cloned by limiting dilution , screened for favorable mab characteristics and then expanded in culture to obtain high amounts of the desired mab .", "generally , two hybridoma types can be distinguished : first , homo - hybridomas where both host b cells and fusion cell line emerged from the same species and , second , hetero - hybridomas derived from two different species .", "ever since their initial discovery , multiple aspects of the general technique have been modified in order to avoid certain problems related to fusion efficiency , hybridoma stability and mab titers .", "hybridoma technology has been used extensively to generate thousands of different mabs against a wide variety of antigens .", "in fact , the majority of fda - approved chimeric , humanized and human mabs originate from hybridoma technology . however ,", "all of these were derived from mouse b cells or transgenic mouse b cells with human ig genes . due to the favorable properties of rabbit antibodies", ". viral transformation of rabbit b cells to generate myeloma - like cell lines also proved to be difficult and rather inefficient . for these reasons ,", "unfortunately , all hetero - hybridomas generated in the early days of hybridoma technology revealed poor fusion efficiency , genetic instability and impaired functional rabbit heavy- and light - chain pairings . in 1988 , raybould et al .", "mouse hetero - hybridoma by polyethylene glycol - mediated fusion of rabbit spleen b cells with the mouse myeloma cell line sp2/0-ag14 . even though they observed stable rabbit igg expression for several months , other groups observed genetic instability and concomitant decrease of mab secretion .", "the first rabbit homo - hybridoma was developed in 1995 in the laboratory of katherine knight . in order to obtain a potential rabbit fusion cell line , transgenic rabbits were generated by single - cell zygote microinjection and mated to generate v - abl / c - myc double transgenic rabbits .", "this method led to the discovery of the first stable rabbit plasmacytoma cell line , 240e-1 , which could be used as an efficient fusion partner to generate rabbit homo - hybridomas .", "interestingly , spieker - polet et al . also showed in this publication that b cells derived from different rabbit tissues led to the generation of hybridomas secreting different ratios of igg , igm and iga .", "however , the stability of the obtained homo - hybridomas was still a major concern and igg secretion decreased over time .", "although this decay is frequently also observed for mouse homo - hybridomas , it appeared to be more drastic in the case of rabbit homo - hybridomas .", "for this reason , zhu and pytela attempted to further improve the initial 240e-1 cell line by iterative subcloning to screen for clones with higher fusion efficiency , yielding hybridomas with higher genetic stability and more stable rabbit igg secretion .", "the obtained fusion cell line 240e - w and its successors 240e - w2 and 240-w3 , which are characterized by higher fusion efficiency and the absence of endogenous rabbit heavy- and light - chain secretion ( us patent 7,429,487 ) , are part of the proprietary rabmab platform of epitomics , inc .", "( now abcam , inc . ) and were used to generate the therapeutic rabbit mabs sevacizumab and apx005 m discussed above . aside from therapeutic applications , rabbit mabs generated by hybridoma technology have become highly valuable reagents for diagnostic applications and for laboratory research .", "for example , highly specific rabbit mabs can detect activating mutations in the tyrosine kinase domain of egfr by ihc of lung cancer tissues .", "rabbit mabs against the hiv-1 protein gp120 were shown to mimic neutralizing human anti - hiv-1 gp120 mabs , promoting rabbit immunization as model for hiv-1 vaccine development .", "bacteriophage , also simply known as phage , are viruses that infect and replicate within bacteria .", "phage display was invented by george smith , who discovered that the minor coat protein ( piii ) of filamentous phage can be modified at its amino terminus to present peptide sequences without affecting phage infectivity .", "in addition , these phage particles contained the genomic information encoding the respectively modified coat protein , thus physically linking genotype and phenotype .", "a combination of phage selection and amplification could be used to efficiently enrich phage that contained certain peptide sequences .", "however , the incorporation of larger peptide sequences , protein domains or whole proteins represented a serious challenge due to decreasing infectivity .", "for this reason , two - component systems were developed that consisted of a phagemid encoding the piii fusion protein and a helper phage contributing the phage genome to encode all proteins necessary for generating infectious phage particles .", "usually , these helper phage contain a modified packaging signal , leading to the preferential assembly of phage particles containing the phagemid . in the early 1990s ,", "the first filamentous phage display antibody libraries based on piii fusion proteins were published using either scfv or fab fragments . to date , these formats are still dominating the selection of mabs by phage display .", "although phage display was established with mouse and human antibody libraries to mine immune , naive and synthetic antibody repertoires , the fact that rabbit mabs were difficult to generate by hybridoma technology for a number of years provided a strong incentive for exploring the accessibility of rabbit immune antibody repertoires by phage display . the first rabbit antibody library selected by phage display was reported by ridder et al . , using a scfv format as in subsequent independent studies .", "rabbit antibody libraries in fab format followed in short succession . due to the higher expression levels of human compared to rabbit constant domains in bacteria , a chimeric rabbit / human fab format consisting of rabbit variable domains vl and vh recombinantly fused to human constant domains cl and ch1 , respectively , proved particularly successful for the selection of rabbit mabs by phage display and their subsequent humanization ( figure 5 ) .", "however , the above - discussed intrachain disulfide bridge between cysteine 80 and cysteine 171 found in rabbit light chains of the dominating k1 isotype posed a challenge to the chimeric rabbit / human fab format as human ch1 does not harbor a cysteine 171 .", "in fact , only few fab originating from the k1 isotype were selected , indicating that the free thiol group of cysteine 80 is disfavored and that its presence diminishes the selectable diversity of chimeric rabbit / human fab . indeed , chimeric rabbit / human fab derived by phage display from immunized k1-negative basilea strain rabbits revealed higher sequence diversity and higher affinity compared to those from k1-positive new zealand white strain rabbits immunized with the same immunogens .", "interestingly , the same study by popkov et al . also compared rabbits homozygous for the b9 -light - chain allotype ( figure 1 ) with the presumed alternative intrachain disulfide bridge between cysteine 108 and cysteine 171 .", "fusion of the rabbit v and human c encoding sequences in the generation of the chimeric rabbit / human fab library removes cysteine 108 , thus avoiding the exposure of a free thiol group .", "consequently , immunized b9 allotype rabbits also revealed superior selectable diversity and were subsequently used in several additional studies .", "these include the generation of a chimeric rabbit / human fab against the hiv-1 protein rev .", "the fab was able to invert rev polymerization and allowed for the formation of fab - rev co - crystals that could be analyzed with x - ray crystallography providing the first three - dimensional structure of rev and the first three - dimensional structure of a rabbit mab .", "notably , the crystal structure revealed a dominant role for lcdr3 in the antigen - binding site ( figure 6 ) . a cyclic peptide derived from lcdr3", "was shown to bind hiv-1 rev with high affinity and to potently inhibit rev polymerization , corroborating the functional importance of the above - discussed high sequence diversity of rabbit light chains .", "a key advantage of using the natural fab format for phage display is its robust monomeric nature that permits affinity - driven selections .", "by contrast , the unnatural scfv format has a tendency to dimerize , trimerize and tetramerize , potentially causing avidity - driven selections .", "thus , we recommend using chimeric rabbit / human fab format - based phage display for applications that require rabbit mabs of high affinity . even if the application calls for scfv , such as for the generation of intrabodies , the chimeric rabbit / human fab format has been used for selection by phage display followed by conversion to the rabbit scfv format .", "in addition to fab and scfv formats , single - domain antibody formats , that is , vl or vh alone , which due to their smaller sizes have advantages for certain applications , such as the recognition of cryptic epitopes , have also been engineered from rabbit mabs .", "the noted dominance of the rabbit light chain may make phage display of rabbit vl libraries particularly attractive .", "as discussed , hybridoma technology and phage display have distinct virtues and shortcomings that have fueled the development of alternative techniques to generate mabs .", "unlike hybridoma technology , phage display relies on the proper transcription , translation , folding and assembly of light and heavy chains in bacteria .", "also , a potential disadvantage of phage display , as well as other display technologies , is their combinatorial nature , leading to the random pairing of light and heavy chains . as discussed above , limiting factors encountered during hybridoma technology , at least initially , included fusion efficiency and hybridoma stability . also , unlike mouse hybridoma technology ,", "rabbit hybridoma technology was patented , imposing intellectual property restrictions that have contributed to the incentive of developing alternative methods . in the generation of human mabs ,", "prominent alternative methods include the clonal expansion of b cells by , for example , epstein ", "barr virus immortalization , and single b - cell sorting followed by light- and heavy - chain - encoding dna amplification and sequencing .", "notably , like hybridoma technology but unlike phage display , both methods yield natural light- and heavy - chain pairs .", "single b - cell isolation based on antigen capture by fluorescence - activated cell sorting , magnetic beads , solid - phase panning or hemolytic plaques followed by light- and heavy - chain cloning has also been applied to the generation of rabbit mabs from peripheral b cells , which does not require killing of the animal and allows for multiple sampling points from primary and secondary lymphoid tissues .", "antigen capture - driven bulk spleen b - cell isolation from immunized rabbits followed by light- and heavy - chain - encoding dna amplification and their combinatorial assembly and expression in scfv fc or igg format in mammalian cells has also been reported . in the fluorescent foci method ,", "plasma cells from rabbit bone marrow are identified by fluorescent microscopy , isolated and subjected to light- and heavy - chain - encoding dna amplification and sequencing .", "high - throughput dna sequencing technologies have been applied to the in silico generation of mabs as well as to the analysis of naive and immune antibody repertoires in different species . in the rabbit ,", "high - throughput dna sequencing of b - cell repertoires along with mass spectrometry analysis of bulk serum igg has been used for the deconvolution of mabs .", "interestingly , this method revealed that the serum igg response to an invertebrate protein following hyperimmunization was oligoclonal rather than polyclonal , comprising 34 rabbit mabs belonging to 30 distinct clonotypes .", "the discovery and development of hybridoma technology for the generation of mabs by georges khler and csar milstein in 1975 has had a huge impact on biomedical research and its application to modern medicine .", "hybridoma technology is a method to generate stable cell lines that constantly secret a defined mab . for this purpose ,", "b cells derived from an immunized animal are fused with a myeloma cell line in the presence of polyethylene glycol .", "the hybridomas generated this way are cloned by limiting dilution , screened for favorable mab characteristics and then expanded in culture to obtain high amounts of the desired mab .", "generally , two hybridoma types can be distinguished : first , homo - hybridomas where both host b cells and fusion cell line emerged from the same species and , second , hetero - hybridomas derived from two different species .", "ever since their initial discovery , multiple aspects of the general technique have been modified in order to avoid certain problems related to fusion efficiency , hybridoma stability and mab titers .", "hybridoma technology has been used extensively to generate thousands of different mabs against a wide variety of antigens .", "in fact , the majority of fda - approved chimeric , humanized and human mabs originate from hybridoma technology . however ,", "all of these were derived from mouse b cells or transgenic mouse b cells with human ig genes . due to the favorable properties of rabbit antibodies", ". viral transformation of rabbit b cells to generate myeloma - like cell lines also proved to be difficult and rather inefficient . for these reasons ,", "unfortunately , all hetero - hybridomas generated in the early days of hybridoma technology revealed poor fusion efficiency , genetic instability and impaired functional rabbit heavy- and light - chain pairings . in 1988 , raybould et al .", "mouse hetero - hybridoma by polyethylene glycol - mediated fusion of rabbit spleen b cells with the mouse myeloma cell line sp2/0-ag14 . even though they observed stable rabbit igg expression for several months , other groups observed genetic instability and concomitant decrease of mab secretion .", "the first rabbit homo - hybridoma was developed in 1995 in the laboratory of katherine knight . in order to obtain a potential rabbit fusion cell line , transgenic rabbits were generated by single - cell zygote microinjection and mated to generate v - abl / c - myc double transgenic rabbits .", "this method led to the discovery of the first stable rabbit plasmacytoma cell line , 240e-1 , which could be used as an efficient fusion partner to generate rabbit homo - hybridomas .", "interestingly , spieker - polet et al . also showed in this publication that b cells derived from different rabbit tissues led to the generation of hybridomas secreting different ratios of igg , igm and iga .", "however , the stability of the obtained homo - hybridomas was still a major concern and igg secretion decreased over time .", "although this decay is frequently also observed for mouse homo - hybridomas , it appeared to be more drastic in the case of rabbit homo - hybridomas .", "for this reason , zhu and pytela attempted to further improve the initial 240e-1 cell line by iterative subcloning to screen for clones with higher fusion efficiency , yielding hybridomas with higher genetic stability and more stable rabbit igg secretion .", "the obtained fusion cell line 240e - w and its successors 240e - w2 and 240-w3 , which are characterized by higher fusion efficiency and the absence of endogenous rabbit heavy- and light - chain secretion ( us patent 7,429,487 ) , are part of the proprietary rabmab platform of epitomics , inc .", "( now abcam , inc . ) and were used to generate the therapeutic rabbit mabs sevacizumab and apx005 m discussed above . aside from therapeutic applications , rabbit mabs generated by hybridoma technology have become highly valuable reagents for diagnostic applications and for laboratory research .", "for example , highly specific rabbit mabs can detect activating mutations in the tyrosine kinase domain of egfr by ihc of lung cancer tissues .", "rabbit mabs against the hiv-1 protein gp120 were shown to mimic neutralizing human anti - hiv-1 gp120 mabs , promoting rabbit immunization as model for hiv-1 vaccine development .", "bacteriophage , also simply known as phage , are viruses that infect and replicate within bacteria .", "phage display was invented by george smith , who discovered that the minor coat protein ( piii ) of filamentous phage can be modified at its amino terminus to present peptide sequences without affecting phage infectivity .", "in addition , these phage particles contained the genomic information encoding the respectively modified coat protein , thus physically linking genotype and phenotype .", "a combination of phage selection and amplification could be used to efficiently enrich phage that contained certain peptide sequences .", "however , the incorporation of larger peptide sequences , protein domains or whole proteins represented a serious challenge due to decreasing infectivity .", "for this reason , two - component systems were developed that consisted of a phagemid encoding the piii fusion protein and a helper phage contributing the phage genome to encode all proteins necessary for generating infectious phage particles .", "usually , these helper phage contain a modified packaging signal , leading to the preferential assembly of phage particles containing the phagemid . in the early 1990s ,", "the first filamentous phage display antibody libraries based on piii fusion proteins were published using either scfv or fab fragments . to date", "although phage display was established with mouse and human antibody libraries to mine immune , naive and synthetic antibody repertoires , the fact that rabbit mabs were difficult to generate by hybridoma technology for a number of years provided a strong incentive for exploring the accessibility of rabbit immune antibody repertoires by phage display . the first rabbit antibody library selected by phage display was reported by ridder et al . , using a scfv format as in subsequent independent studies .", "rabbit antibody libraries in fab format followed in short succession . due to the higher expression levels of human compared to rabbit constant domains in bacteria , a chimeric rabbit / human fab format consisting of rabbit variable domains vl and vh recombinantly fused to human constant domains cl and ch1 , respectively , proved particularly successful for the selection of rabbit mabs by phage display and their subsequent humanization ( figure 5 ) .", "however , the above - discussed intrachain disulfide bridge between cysteine 80 and cysteine 171 found in rabbit light chains of the dominating k1 isotype posed a challenge to the chimeric rabbit / human fab format as human ch1 does not harbor a cysteine 171 .", "in fact , only few fab originating from the k1 isotype were selected , indicating that the free thiol group of cysteine 80 is disfavored and that its presence diminishes the selectable diversity of chimeric rabbit / human fab .", "indeed , chimeric rabbit / human fab derived by phage display from immunized k1-negative basilea strain rabbits revealed higher sequence diversity and higher affinity compared to those from k1-positive new zealand white strain rabbits immunized with the same immunogens .", "interestingly , the same study by popkov et al . also compared rabbits homozygous for the b9 -light - chain allotype ( figure 1 ) with the presumed alternative intrachain disulfide bridge between cysteine 108 and cysteine 171 .", "fusion of the rabbit v and human c encoding sequences in the generation of the chimeric rabbit / human fab library removes cysteine 108 , thus avoiding the exposure of a free thiol group .", "consequently , immunized b9 allotype rabbits also revealed superior selectable diversity and were subsequently used in several additional studies .", "these include the generation of a chimeric rabbit / human fab against the hiv-1 protein rev .", "the fab was able to invert rev polymerization and allowed for the formation of fab - rev co - crystals that could be analyzed with x - ray crystallography providing the first three - dimensional structure of rev and the first three - dimensional structure of a rabbit mab .", "notably , the crystal structure revealed a dominant role for lcdr3 in the antigen - binding site ( figure 6 ) . a cyclic peptide derived from lcdr3", "was shown to bind hiv-1 rev with high affinity and to potently inhibit rev polymerization , corroborating the functional importance of the above - discussed high sequence diversity of rabbit light chains .", "a key advantage of using the natural fab format for phage display is its robust monomeric nature that permits affinity - driven selections .", "by contrast , the unnatural scfv format has a tendency to dimerize , trimerize and tetramerize , potentially causing avidity - driven selections .", "thus , we recommend using chimeric rabbit / human fab format - based phage display for applications that require rabbit mabs of high affinity .", "even if the application calls for scfv , such as for the generation of intrabodies , the chimeric rabbit / human fab format has been used for selection by phage display followed by conversion to the rabbit scfv format .", "in addition to fab and scfv formats , single - domain antibody formats , that is , vl or vh alone , which due to their smaller sizes have advantages for certain applications , such as the recognition of cryptic epitopes , have also been engineered from rabbit mabs .", "the noted dominance of the rabbit light chain may make phage display of rabbit vl libraries particularly attractive .", "as discussed , hybridoma technology and phage display have distinct virtues and shortcomings that have fueled the development of alternative techniques to generate mabs .", "unlike hybridoma technology , phage display relies on the proper transcription , translation , folding and assembly of light and heavy chains in bacteria .", "also , a potential disadvantage of phage display , as well as other display technologies , is their combinatorial nature , leading to the random pairing of light and heavy chains . as discussed above , limiting factors encountered during hybridoma technology , at least initially , included fusion efficiency and hybridoma stability . also , unlike mouse hybridoma technology ,", "rabbit hybridoma technology was patented , imposing intellectual property restrictions that have contributed to the incentive of developing alternative methods . in the generation of human mabs ,", "prominent alternative methods include the clonal expansion of b cells by , for example , epstein ", "barr virus immortalization , and single b - cell sorting followed by light- and heavy - chain - encoding dna amplification and sequencing .", "notably , like hybridoma technology but unlike phage display , both methods yield natural light- and heavy - chain pairs .", "single b - cell isolation based on antigen capture by fluorescence - activated cell sorting , magnetic beads , solid - phase panning or hemolytic plaques followed by light- and heavy - chain cloning has also been applied to the generation of rabbit mabs from peripheral b cells , which does not require killing of the animal and allows for multiple sampling points from primary and secondary lymphoid tissues .", "antigen capture - driven bulk spleen b - cell isolation from immunized rabbits followed by light- and heavy - chain - encoding dna amplification and their combinatorial assembly and expression in scfv fc or igg format in mammalian cells has also been reported . in the fluorescent foci method ,", "plasma cells from rabbit bone marrow are identified by fluorescent microscopy , isolated and subjected to light- and heavy - chain - encoding dna amplification and sequencing .", "high - throughput dna sequencing technologies have been applied to the in silico generation of mabs as well as to the analysis of naive and immune antibody repertoires in different species . in the rabbit , high - throughput dna sequencing of b - cell repertoires along with mass spectrometry analysis of bulk", "interestingly , this method revealed that the serum igg response to an invertebrate protein following hyperimmunization was oligoclonal rather than polyclonal , comprising 34 rabbit mabs belonging to 30 distinct clonotypes .", "the human immune system is intended to recognize and selectively remove potentially pathogenic organisms and substances .", "the respective immune responses are characterized by high titers of human antibodies directed against these foreign antibody sequences .", "this has two main physiological consequences ; first of all , it leads to side effects comparable to allergic reactions of different levels of severity and , second , it leads to the rapid elimination of the administered mab , thus limiting its diagnostic or therapeutic efficacy . for these reasons ,", "nonhuman mabs are now routinely converted to chimeric mabs by combining the nonhuman variable domains with human constant domains and further to humanized mabs by grafting all or some of the cdrs of the nonhuman variable domains into human frameworks .", "the first chimeric and humanized rabbit mabs were reported by rader et al . in this study", ", chimeric rabbit / human fab selected by phage display were humanized by grafting the six rabbit cdrs , three from each light and heavy chain , into human frameworks that were diversified at certain positions to allow the selection of either human or rabbit residues by phage display .", "the resulting humanized rabbit mabs retained the high affinity and specificity of their parental mabs .", "as discussed above , aside from having higher utility for downstream clinical applications , using chimeric rabbit / human fab format for phage display has the added advantage of affording higher expression levels in bacteria and , concomitantly , higher display levels on phage . since this first report", ", a number of additional humanized rabbit mabs have been published and patented . in general ,", "humanization strategies that have worked for mouse mabs also work for rabbit mabs , whether it is by rational design , directed evolution or a combination of both .", "as is the case for mouse mabs , grafting of all six cdrs followed by iterative fine - tuning of framework residues is the most frequently used method for humanizing rabbit mabs .", "used a general acceptor framework for the humanization of rabbit scfv . in this study ,", "a certain human framework carrying five specific human - to - rabbit mutations was able to generate humanized rabbit mabs to two different antigens with conserved affinities and specificities , and improved biophysical properties .", "humanization strategies that confine the content of parental rabbit antibody sequences only to the most hypervariable of the cdrs , such as lcdr3 and hcdr3 , have also been successfully applied to rabbit mabs . thus far , there have been no reports that analyze the immunogenicity of clinically investigated humanized rabbit mabs .", "recent studies indicate that patients can still mount an immune response to humanized or even fully human mabs .", "these findings indicate that additional aspects , such as idiotype , allotype , glycosylation and aggregation , contribute to the immunogenicity of mabs .", "nonetheless , the occurrence of these unwanted side effects is significantly lower than previously observed for nonhuman antibodies .", "driven by the success of rabbit pabs on one hand and mouse mabs on the other hand , rabbit mabs have become highly successful reagents for laboratory research and for diagnostic and therapeutic applications .", "the unique ontogeny of rabbit b cells affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity , affinity and specificity .", "the ability to access rabbit antibody repertoires by hybridoma technology , phage display and alternative methods has fueled the increased use of rabbit mabs in many different applications .", "rabbit mabs generated by hybridoma technology are particularly attractive for ihc , with many studies demonstrating higher sensitivity compared to benchmark mouse mabs .", "rabbit mabs generated by phage display have found the use for applications ranging from the detection of uranium in water to chimeric antigen receptor t cell therapy of cancer .", "given that humanization strategies are now well established , the therapeutic utility of rabbit mabs is especially intriguing . with the first rabbit mab - derived therapeutics already in clinical trials , we predict that rabbit mabs will gain further traction in preclinical and clinical pipelines over the next decade ." ]

[ "<S> in this review , we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications . starting with the unique ontogeny of rabbit b cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity , affinity and specificity , </S>", "<S> we describe the generation of rabbit monoclonal antibodies by hybridoma technology , phage display and alternative methods , along with an account of successful humanization strategies . </S>" ]

[ "Introduction", "Ontogeny of rabbit B-cell and antibody repertoires", "Generation of rabbit mAbs", "Hybridoma technology", "Phage display technology", "Alternative methods", "Humanization of rabbit mAbs", "Summary and outlook" ]

[ [ "european rabbits ( oryctolagus cuniculus ; figure 1 ) have played an important role as animal models in immunology for many decades . today , rabbits are still a major source for a wide variety of monoclonal antibodies ( mabs ) and polyclonal antibodies ( pabs ) with broad utility .", "pabs can be described as a set of different antibodies generated in response to a specific pathogen or antigen , generally targeting different epitopes .", "mabs , on the other hand , contain a defined antigen - binding site ( paratope ) that typically binds with high affinity and specificity to only one epitope . from a pharmaceutical point of view", ", mabs provide a molecularly defined and reproducible product , whereas pabs are traditionally an imprecise mixture of different antibodies .", "as is the case for mouse and human mabs , igg is the most common isotype of rabbit mabs ( figure 2 ) .", "rabbit pabs have been used extensively as analytical tools in biomedical research and especially for immunological techniques , such as immunohistochemistry ( ihc ) , western blotting and flow cytometry .", "atg is a mixture of purified polyclonal rabbit , horse or goat iggs against human t cells that has been used as an immunosuppressive drug for decades . in organ and", "allogeneic bone marrow transplantation , atg application causes rapid depletion of t cells , leading to a decreased risk for rejection and acute graft - versus - host disease . however , atg does not induce long - term tolerance .", "rabbit atg is one of the most commonly used atgs , due to its higher lymphocytotoxicity compared to horse atg .", ", cambridge , ma , usa ) was approved by the us food and drug administration ( fda ) in 1998 .", "more recently , an improved rabbit pabs cocktail targeting human leukocytes was reported as a potential immunosuppressive drug in xenogeneic ( for example , pig to human ) organ transplantation .", "in addition , one rabbit pab is currently approved by the fda as an in vitro diagnostic tool ( c - kit pharmdx ; agilent technologies , inc . , santa clara , ca , usa ) for the ihc - based detection of cd117 ( c - kit ) expression in gastrointestinal stromal tumors to aid treatment decisions .", "mabs and mab - derived antibody therapeutics are currently widely used to treat human diseases , such as cancer and autoimmune diseases .", "although no therapeutic rabbit mabs have been approved by the fda thus far , 11 rabbit mabs are fda - approved in vitro diagnostic tools in the clinic .", "ten of these mabs are being used to detect the expression of tumor - associated antigens , including her2 , estrogen receptors , progesterone receptors and pd - l1 .", "a rabbit mab to human androgen receptor splice variant 7 has emerged as a promising tool for the detection of circulating tumor cells by immunofluorescence and ihc in prostate cancer . in addition", ", several rabbit mab - derived therapeutics are currently being investigated in clinical trials registered at clinicaltrials.gov . in oncology , examples include sevacizumab ( simcere pharmaceutical group , inc . ,", "nanjing , china ) , a humanized rabbit anti - human vascular endothelial growth factor mab ( nct02453464 ) , apx005 m ( apexigen , inc . , san carlos , ca , usa ) , a humanized rabbit anti - human cd40 mab ( nct02482168 ) , and yyb101 , a humanized rabbit anti - human hgf mab ( yooyoung pharmaceutical co. , inc . ,", "further , chimeric antigen receptor t cells based on a rabbit anti - human ror1 mab have commenced clinical trials recently ( nct02706392 ) . in ophthalmology ,", "humanized rabbit anti - vascular endothelial growth factor mab brolucizumab ( alcon , inc . ,", "hnenberg , switzerland ) , a mab in scfv format administered intravitreally , is being investigated in advanced clinical trials ( nct02307682 and nct02434328 ) . following these examples , a number of rabbit mab - derived therapeutics are expected to transition from preclinical to clinical studies in the near future .", "currently , only a handful of companies develop rabbit or rabbit - derived mabs for laboratory research and for diagnostic and therapeutic applications .", "( cambridge , uk ; rabmab platform ; through acquisition of epitomics , inc . ,", "( through acquisition of esbatech , inc . , schlieren , switzerland in 2009 ) ; apexigen , inc .", "( san carlos , ca , usa ; spun out by epitomics , inc . in 2010 ) ; cell signaling technology , inc .", "( danvers , ma , usa ; currently listing > 4000 different rabbit mabs ) ; agilent technologies , inc . ; mab discovery , inc . ,", "neuried , germany ; lab vision corporation , inc . , fremont , ca , usa ; thermo fisher scientific , inc .", "( carlsbad , ca , usa ; invitrogen abfinity recombinant rabbit antibodies ) ; and ventana medical systems , inc .", "rabbits have been used to investigate immunological questions and to develop immunological techniques for > 100 years .", "thus , many standard procedures are established , published and validated , such as immunization and purification methods yielding high amounts of rabbit antibodies .", "in addition to these practical considerations , rabbits are characterized by a variety of natural features that make their antibody repertoire very attractive for the discussed applications .", "first , rabbits belong to the order lagomorpha , which is evolutionary distinct from the order rodentia , to which , for example , mice and rats belong .", "rabbit antibodies are able to recognize epitopes on human antigens that are not immunogenic in rodents , increasing the total number of targetable epitopes and facilitating the generation of antibodies that cross - react with mouse orthologs of human antigens .", "this is an important aspect for basic research and preclinical investigations with , for example , human tumor xenografts , where the presence or absence of on - target - off - tumor toxicities of therapeutic antibodies provides important information prior to clinical translation . in general ,", "rabbit anti - mouse reactivity is valuable in mouse models of human disease and has also been exploited in basic research , for example , on mouse stem cell antigens .", "second , it has been observed that rabbits elicit strong immune responses against small molecules and haptens , which is uncommon in rodents .", "a third important aspect is the scarcity of inbred rabbit strains , while most mouse strains are inbred .", "it is thought that inbred strains in general elicit less diverse immune responses , which makes it more difficult to raise strong and diverse binders .", "correspondingly , in many ihc studies that compared rabbit and mouse mabs to the same human antigens , rabbit mabs consistently revealed higher sensitivity . in a recent study ,", "1410 rabbit mabs raised against 15-amino - acid peptides representing 100 different antigens revealed a typical affinity range of 20200 pm ( median 66 pm ) , as determined by high - throughput surface plasmon resonance .", "a fraction of rabbit mabs even revealed higher affinities near the detection limit of 1 pm .", "however , the affinity range of 46 mouse mabs ( 30300 pm ; median 72 pm ) analyzed in parallel was not significantly different .", "more direct comparisons of rabbit and mouse mabs to a wider variety of antigens are warranted to determine a significant difference in affinity .", "fourth , most strategies to generate mabs are based on the recovery of b cells from spleen , bone marrow or blood , which are present in higher quantities in rabbits than in mice due to their overall larger body size .", "( the average body weight of a 3-month - old laboratory rabbit is 2.5 kg compared to 25 g for a 6-week - old laboratory mouse . ) for example , 50 times more spleen b cells can be recovered from rabbits compared to mice . in addition , the larger blood volume of rabbits compared to mice facilitates mass spectrometry analyses of bulk serum igg .", "fifth , as discussed in the next section , rabbits use different mechanisms to genetically generate and diversify their primary and secondary antibody repertoires compared to humans and mice , effectively creating a complementary set of binders for the discussed applications . the recent sequencing and annotation of the rabbit genome ( orycun2.0 assembly )" ], [ "the development of the rabbit b - cell repertoire significantly differs from that of other mammals .", "the current model proposes a three - step process ( figure 3 ) consisting of the neonatal b - cell repertoire generated by b lymphopoiesis in the fetal liver and omentum switching to bone marrow after birth , the primary pre - immune ' b - cell repertoire evolving during the first 2 months after birth in gut - associated lymphoid tissue ( galt ) and the secondary immune ' b - cell repertoire generated upon b - cell activation by immunogen binding .", "the neonatal b - cell repertoire starts developing between the second and third week of gestation .", "interestingly , b lymphopoiesis is very limited in the bone marrow of adult rabbits , indicating that b lymphopoiesis is mainly restricted to early development .", "in addition , in a rabbit - to - rabbit adoptive transfer model , it was shown that rabbit b cells were able to engraft into host stem cell niches , which led to the conclusion that rabbit b cells are long - lived and potentially self - renewing and may thus sustain the rabbit antibody repertoire throughout a rabbit 's lifetime .", "rabbits mainly rearrange their heavy chains first followed by their light chains and thus follow the primary b - cell development pathway proposed by ehlich et al . in brief , this model proposes the vh ", "jh recombination of the heavy chain , followed by its expression as a pre - b - cell receptor . upon stimulation of the pre - b - cell receptor , vl", "some evidence for non - templated nucleotide - addition ( also known as n - nucleotides ) has been observed in rabbit vj rearrangements in 1-day - old rabbits , suggesting that terminal deoxynucleotidyl transferase was recently expressed .", "although a potential pool of 200 vh genes are present for heavy - chain rearrangement , a majority of these are infrequently or not expressed .", "however , it is known that certain genes from these clusters are preferentially utilized : vh1 , the most d - proximal vh gene , is present in 8090% of all heavy - chain gene rearrangements .", "three different allotypes vha1 , vha2 and vha3 are known , resulting in the vha - positive serotype .", "this finding was somewhat surprising , because there is another functional , d - proximal vh sequence significantly closer to the vh1 segment .", "indeed , it could be shown that the vh4 segment is utilized in 8090% of the heavy - chain rearrangements in vh1-negative alicia strain rabbits .", "however , this heavy - chain rearrangement was very rare in adult rabbits , suggesting that the vh4 rearrangement is non - productive in most cases and thus lost in the course of development .", ". suggested negative self - antigen effects and non - efficient pairing with light chains and surrogate light chains during b - cell development as possible reasons .", "in addition , preferential utilization of d2a , d2b and d1 as well as jh4 could be shown .", "it is thought that rabbits compensate for this preferential utilization of certain vh , d and jh genes in part by n - nucleotide addition at the vh d and d jh junctions during recombination .", "using high - throughput sequencing of antibody repertoires from three new zealand white rabbits , lavinder et al . determined that the means.d .", "length of the third complementarity - determining region ( cdr ) of the heavy chain , which is known as hcdr3 , includes both junctions , and thus is the most hypervariable of all six cdrs , is 154 ( mode=13 ) amino acids compared to mouse and human hcdr3s with means.d .", "lengths of 112 ( mode=10 amino acids ) and 154 ( mode=15 ) amino acids , respectively .", "these findings confirmed that rabbit hcdr3s share more similarity with human hcdr3s than mouse hcdr3s and could be relevant for therapeutic applications .", "although there is limited heavy - chain usage in the neonatal rabbits ' b - cell repertoire , there may be compensation by usage of diverse light chains .", "there are two types of rabbit light chains and with the light chain present in two different isotypes called k1 and k2 .", "four k1 -light - chain allotypes b4 , b5 , b6 and b9have been observed in domesticated rabbits .", "notably , the constant domain sequences of these rabbit -light - chain allotypes have much higher amino - acid sequence diversity than human ig allotypes with pairwise amino - acid sequence differences of up to 40% .", "this high degree of polymorphism exceeds the allotypic diversity of the three heavy - chain allotypes , a1 , a2 and a3 ( ~20% amino - acid sequence differences ) , and even surpasses the allotypic diversity of major histocompatibility complex loci .", "k1 is by far the most abundant isotype and represents between 70 and 90% of all light chains , whereas the rest is usually a mixture of both k2 and -light chains .", "many k1 light chains are characterized by an intrachain disulfide bridge between cysteine 80 in v and cysteine 171 in c ( figure 2 ) .", "this additional disulfide bridge , which was discovered biochemically and later confirmed by x - ray crystallography , is not found in human or mouse light chains .", "it may contribute to the stability of rabbit antibodies and is one of many peculiarities of ig evolution in vertebrates .", "this disulfide bridge is absent from rabbits of the k1-negative basilea mutant strain that only expresses k2 and -light chains .", "some but not all of the k1 light chains of rabbits homozygous for the b9 -light - chain allotype have a cysteine 108 in j that is thought to create an alternative intrachain disulfide bridge with cysteine 171 in c. these allotypes play an important role for the generation of rabbit mabs by phage display and are discussed in more detail below .", "recently , using next - generation sequencing of rabbit antibody repertoires , kodangattil et al . demonstrated that light - chain rearrangements are significantly more diverse than heavy - chain rearrangements in rabbits .", "in addition , the imprecise junction of vl and jl genes in rabbits encompasses particularly long stretches of n - nucleotides , resulting in , on average , longer lcdr3s ( 122 amino acids ; mode=12 ) compared to mouse and human ( 91 amino acids ; mode=9 ; figure 4 ) .", "the longer rabbit lcdr3s are occasionally stabilized by disulfide bridges . taken together , by generating a more diverse neonatal light - chain repertoire followed by a comparable degree of further diversification into primary and secondary light - chain repertoires , the light chain can compensate for the limited diversity of heavy - chain repertoires in rabbits .", "this may also explain the dominance of the rabbit light chain in general and lcdr3 in particular in several of the antibody / antigen complexes for which three - dimensional structures have been determined by x - ray crystallography . the limited neonatal antibody repertoire is further diversified between week 4 and 8 after birth resulting in the primary antibody repertoire .", "this postnatal diversification is an unusual phenomenon and has so far only been observed in rabbits and pigs .", "the two main mechanisms are somatic gene conversion ( sgc ; templated ) and somatic hypermutation ( shm ; non - templated ; figures 3 and 4 ) .", "sgc , predominantly used only in chicken and rabbit ig gene diversification , leads to the replacement of large nucleic acid sequence stretches with dna fragments from non - utilized vh genes .", "thus , the non - expressible majority of vh genes play an important role for heavy - chain diversification . the predominance of this mechanism in rabbit ( 23% ) compared to human ( 2.5% ) and mouse ( 0.1% ) heavy chains , as determined by high - throughput sequencing , is thought to lead to decreased wastage : a substantial portion of vh d jh recombinations with n - nucleotides addition have out - of - frame junctions , whereas sgc , a homologous dna recombination event , tends to favor in - frame substitutions and extensions .", "in addition , shm is observed at considerable levels during this developmental stage . following rearrangement in the neonatal b - cell repertoire ,", "rabbit light - chain genes also undergo sgc and shm to diversify the primary b - cell repertoire ( figures 3 and 4 ) . further corroborating the importance of the light chain for generating diversity in rabbit antibody repertoires ,", "tract length ( 8648 nucleotides ) in rabbit -light chains compared to rabbit heavy chains .", "interestingly , the generation of the primary repertoire is highly galt - dependent . in short", ", galt involves the uptake of pathogens from the gastrointestinal track by specialized epithelial cells known as m cells and the presentation of antigens to b cells leading to b - cell stimulation , diversification and proliferation .", "surgical removal of galt tissues , such as the peyer 's patches in the small intestines , the sacculus rotundus and the appendix resulted in severely immunodeficient rabbits .", "the secondary b - cell repertoire is generated upon antigen - dependent b - cell stimulation .", "again , additional diversity is introduced by sgc and shm in both heavy and light chains ( figures 3 and 4 ) .", "these events further broaden the b - cell repertoire directed against a certain set of antigens associated with a specific pathogen .", "notably , rabbits only have one igg isotype ( one c gene ) compared to four igg isotypes in mice ( igg1 , igg2a , igg2b and igg3 ) and humans ( igg1 , igg2 , igg3 and igg4 ) . in contrast , rabbits have 13 c genes giving rise to at least 10 functional iga isotypes , the most diverse iga system known , compared to just one iga isotype in mice and two in humans . collectively , the unique features of b - cell ontogeny and antibody repertoire make rabbits a valuable source for the generation of antibodies of high affinity and specificity ." ], [ "the discovery and development of hybridoma technology for the generation of mabs by georges khler and csar milstein in 1975 has had a huge impact on biomedical research and its application to modern medicine .", "hybridoma technology is a method to generate stable cell lines that constantly secret a defined mab . for this purpose ,", "b cells derived from an immunized animal are fused with a myeloma cell line in the presence of polyethylene glycol .", "the hybridomas generated this way are cloned by limiting dilution , screened for favorable mab characteristics and then expanded in culture to obtain high amounts of the desired mab .", "generally , two hybridoma types can be distinguished : first , homo - hybridomas where both host b cells and fusion cell line emerged from the same species and , second , hetero - hybridomas derived from two different species .", "ever since their initial discovery , multiple aspects of the general technique have been modified in order to avoid certain problems related to fusion efficiency , hybridoma stability and mab titers .", "hybridoma technology has been used extensively to generate thousands of different mabs against a wide variety of antigens .", "in fact , the majority of fda - approved chimeric , humanized and human mabs originate from hybridoma technology . however ,", "all of these were derived from mouse b cells or transgenic mouse b cells with human ig genes . due to the favorable properties of rabbit antibodies", ". viral transformation of rabbit b cells to generate myeloma - like cell lines also proved to be difficult and rather inefficient . for these reasons ,", "unfortunately , all hetero - hybridomas generated in the early days of hybridoma technology revealed poor fusion efficiency , genetic instability and impaired functional rabbit heavy- and light - chain pairings . in 1988 , raybould et al .", "mouse hetero - hybridoma by polyethylene glycol - mediated fusion of rabbit spleen b cells with the mouse myeloma cell line sp2/0-ag14 . even though they observed stable rabbit igg expression for several months , other groups observed genetic instability and concomitant decrease of mab secretion .", "the first rabbit homo - hybridoma was developed in 1995 in the laboratory of katherine knight . in order to obtain a potential rabbit fusion cell line , transgenic rabbits were generated by single - cell zygote microinjection and mated to generate v - abl / c - myc double transgenic rabbits .", "this method led to the discovery of the first stable rabbit plasmacytoma cell line , 240e-1 , which could be used as an efficient fusion partner to generate rabbit homo - hybridomas .", "interestingly , spieker - polet et al . also showed in this publication that b cells derived from different rabbit tissues led to the generation of hybridomas secreting different ratios of igg , igm and iga .", "however , the stability of the obtained homo - hybridomas was still a major concern and igg secretion decreased over time .", "although this decay is frequently also observed for mouse homo - hybridomas , it appeared to be more drastic in the case of rabbit homo - hybridomas .", "for this reason , zhu and pytela attempted to further improve the initial 240e-1 cell line by iterative subcloning to screen for clones with higher fusion efficiency , yielding hybridomas with higher genetic stability and more stable rabbit igg secretion .", "the obtained fusion cell line 240e - w and its successors 240e - w2 and 240-w3 , which are characterized by higher fusion efficiency and the absence of endogenous rabbit heavy- and light - chain secretion ( us patent 7,429,487 ) , are part of the proprietary rabmab platform of epitomics , inc .", "( now abcam , inc . ) and were used to generate the therapeutic rabbit mabs sevacizumab and apx005 m discussed above . aside from therapeutic applications , rabbit mabs generated by hybridoma technology have become highly valuable reagents for diagnostic applications and for laboratory research .", "for example , highly specific rabbit mabs can detect activating mutations in the tyrosine kinase domain of egfr by ihc of lung cancer tissues .", "rabbit mabs against the hiv-1 protein gp120 were shown to mimic neutralizing human anti - hiv-1 gp120 mabs , promoting rabbit immunization as model for hiv-1 vaccine development .", "bacteriophage , also simply known as phage , are viruses that infect and replicate within bacteria .", "phage display was invented by george smith , who discovered that the minor coat protein ( piii ) of filamentous phage can be modified at its amino terminus to present peptide sequences without affecting phage infectivity .", "in addition , these phage particles contained the genomic information encoding the respectively modified coat protein , thus physically linking genotype and phenotype .", "a combination of phage selection and amplification could be used to efficiently enrich phage that contained certain peptide sequences .", "however , the incorporation of larger peptide sequences , protein domains or whole proteins represented a serious challenge due to decreasing infectivity .", "for this reason , two - component systems were developed that consisted of a phagemid encoding the piii fusion protein and a helper phage contributing the phage genome to encode all proteins necessary for generating infectious phage particles .", "usually , these helper phage contain a modified packaging signal , leading to the preferential assembly of phage particles containing the phagemid . in the early 1990s ,", "the first filamentous phage display antibody libraries based on piii fusion proteins were published using either scfv or fab fragments . to date , these formats are still dominating the selection of mabs by phage display .", "although phage display was established with mouse and human antibody libraries to mine immune , naive and synthetic antibody repertoires , the fact that rabbit mabs were difficult to generate by hybridoma technology for a number of years provided a strong incentive for exploring the accessibility of rabbit immune antibody repertoires by phage display . the first rabbit antibody library selected by phage display was reported by ridder et al . , using a scfv format as in subsequent independent studies .", "rabbit antibody libraries in fab format followed in short succession . due to the higher expression levels of human compared to rabbit constant domains in bacteria , a chimeric rabbit / human fab format consisting of rabbit variable domains vl and vh recombinantly fused to human constant domains cl and ch1 , respectively , proved particularly successful for the selection of rabbit mabs by phage display and their subsequent humanization ( figure 5 ) .", "however , the above - discussed intrachain disulfide bridge between cysteine 80 and cysteine 171 found in rabbit light chains of the dominating k1 isotype posed a challenge to the chimeric rabbit / human fab format as human ch1 does not harbor a cysteine 171 .", "in fact , only few fab originating from the k1 isotype were selected , indicating that the free thiol group of cysteine 80 is disfavored and that its presence diminishes the selectable diversity of chimeric rabbit / human fab . indeed , chimeric rabbit / human fab derived by phage display from immunized k1-negative basilea strain rabbits revealed higher sequence diversity and higher affinity compared to those from k1-positive new zealand white strain rabbits immunized with the same immunogens .", "interestingly , the same study by popkov et al . also compared rabbits homozygous for the b9 -light - chain allotype ( figure 1 ) with the presumed alternative intrachain disulfide bridge between cysteine 108 and cysteine 171 .", "fusion of the rabbit v and human c encoding sequences in the generation of the chimeric rabbit / human fab library removes cysteine 108 , thus avoiding the exposure of a free thiol group .", "consequently , immunized b9 allotype rabbits also revealed superior selectable diversity and were subsequently used in several additional studies .", "these include the generation of a chimeric rabbit / human fab against the hiv-1 protein rev .", "the fab was able to invert rev polymerization and allowed for the formation of fab - rev co - crystals that could be analyzed with x - ray crystallography providing the first three - dimensional structure of rev and the first three - dimensional structure of a rabbit mab .", "notably , the crystal structure revealed a dominant role for lcdr3 in the antigen - binding site ( figure 6 ) . a cyclic peptide derived from lcdr3", "was shown to bind hiv-1 rev with high affinity and to potently inhibit rev polymerization , corroborating the functional importance of the above - discussed high sequence diversity of rabbit light chains .", "a key advantage of using the natural fab format for phage display is its robust monomeric nature that permits affinity - driven selections .", "by contrast , the unnatural scfv format has a tendency to dimerize , trimerize and tetramerize , potentially causing avidity - driven selections .", "thus , we recommend using chimeric rabbit / human fab format - based phage display for applications that require rabbit mabs of high affinity . even if the application calls for scfv , such as for the generation of intrabodies , the chimeric rabbit / human fab format has been used for selection by phage display followed by conversion to the rabbit scfv format .", "in addition to fab and scfv formats , single - domain antibody formats , that is , vl or vh alone , which due to their smaller sizes have advantages for certain applications , such as the recognition of cryptic epitopes , have also been engineered from rabbit mabs .", "the noted dominance of the rabbit light chain may make phage display of rabbit vl libraries particularly attractive .", "as discussed , hybridoma technology and phage display have distinct virtues and shortcomings that have fueled the development of alternative techniques to generate mabs .", "unlike hybridoma technology , phage display relies on the proper transcription , translation , folding and assembly of light and heavy chains in bacteria .", "also , a potential disadvantage of phage display , as well as other display technologies , is their combinatorial nature , leading to the random pairing of light and heavy chains . as discussed above , limiting factors encountered during hybridoma technology , at least initially , included fusion efficiency and hybridoma stability . also , unlike mouse hybridoma technology ,", "rabbit hybridoma technology was patented , imposing intellectual property restrictions that have contributed to the incentive of developing alternative methods . in the generation of human mabs ,", "prominent alternative methods include the clonal expansion of b cells by , for example , epstein ", "barr virus immortalization , and single b - cell sorting followed by light- and heavy - chain - encoding dna amplification and sequencing .", "notably , like hybridoma technology but unlike phage display , both methods yield natural light- and heavy - chain pairs .", "single b - cell isolation based on antigen capture by fluorescence - activated cell sorting , magnetic beads , solid - phase panning or hemolytic plaques followed by light- and heavy - chain cloning has also been applied to the generation of rabbit mabs from peripheral b cells , which does not require killing of the animal and allows for multiple sampling points from primary and secondary lymphoid tissues .", "antigen capture - driven bulk spleen b - cell isolation from immunized rabbits followed by light- and heavy - chain - encoding dna amplification and their combinatorial assembly and expression in scfv fc or igg format in mammalian cells has also been reported . in the fluorescent foci method ,", "plasma cells from rabbit bone marrow are identified by fluorescent microscopy , isolated and subjected to light- and heavy - chain - encoding dna amplification and sequencing .", "high - throughput dna sequencing technologies have been applied to the in silico generation of mabs as well as to the analysis of naive and immune antibody repertoires in different species . in the rabbit ,", "high - throughput dna sequencing of b - cell repertoires along with mass spectrometry analysis of bulk serum igg has been used for the deconvolution of mabs .", "interestingly , this method revealed that the serum igg response to an invertebrate protein following hyperimmunization was oligoclonal rather than polyclonal , comprising 34 rabbit mabs belonging to 30 distinct clonotypes ." ], [ "the discovery and development of hybridoma technology for the generation of mabs by georges khler and csar milstein in 1975 has had a huge impact on biomedical research and its application to modern medicine .", "hybridoma technology is a method to generate stable cell lines that constantly secret a defined mab . for this purpose ,", "b cells derived from an immunized animal are fused with a myeloma cell line in the presence of polyethylene glycol .", "the hybridomas generated this way are cloned by limiting dilution , screened for favorable mab characteristics and then expanded in culture to obtain high amounts of the desired mab .", "generally , two hybridoma types can be distinguished : first , homo - hybridomas where both host b cells and fusion cell line emerged from the same species and , second , hetero - hybridomas derived from two different species .", "ever since their initial discovery , multiple aspects of the general technique have been modified in order to avoid certain problems related to fusion efficiency , hybridoma stability and mab titers .", "hybridoma technology has been used extensively to generate thousands of different mabs against a wide variety of antigens .", "in fact , the majority of fda - approved chimeric , humanized and human mabs originate from hybridoma technology . however ,", "all of these were derived from mouse b cells or transgenic mouse b cells with human ig genes . due to the favorable properties of rabbit antibodies", ". viral transformation of rabbit b cells to generate myeloma - like cell lines also proved to be difficult and rather inefficient . for these reasons ,", "unfortunately , all hetero - hybridomas generated in the early days of hybridoma technology revealed poor fusion efficiency , genetic instability and impaired functional rabbit heavy- and light - chain pairings . in 1988 , raybould et al .", "mouse hetero - hybridoma by polyethylene glycol - mediated fusion of rabbit spleen b cells with the mouse myeloma cell line sp2/0-ag14 . even though they observed stable rabbit igg expression for several months , other groups observed genetic instability and concomitant decrease of mab secretion .", "the first rabbit homo - hybridoma was developed in 1995 in the laboratory of katherine knight . in order to obtain a potential rabbit fusion cell line , transgenic rabbits were generated by single - cell zygote microinjection and mated to generate v - abl / c - myc double transgenic rabbits .", "this method led to the discovery of the first stable rabbit plasmacytoma cell line , 240e-1 , which could be used as an efficient fusion partner to generate rabbit homo - hybridomas .", "interestingly , spieker - polet et al . also showed in this publication that b cells derived from different rabbit tissues led to the generation of hybridomas secreting different ratios of igg , igm and iga .", "however , the stability of the obtained homo - hybridomas was still a major concern and igg secretion decreased over time .", "although this decay is frequently also observed for mouse homo - hybridomas , it appeared to be more drastic in the case of rabbit homo - hybridomas .", "for this reason , zhu and pytela attempted to further improve the initial 240e-1 cell line by iterative subcloning to screen for clones with higher fusion efficiency , yielding hybridomas with higher genetic stability and more stable rabbit igg secretion .", "the obtained fusion cell line 240e - w and its successors 240e - w2 and 240-w3 , which are characterized by higher fusion efficiency and the absence of endogenous rabbit heavy- and light - chain secretion ( us patent 7,429,487 ) , are part of the proprietary rabmab platform of epitomics , inc .", "( now abcam , inc . ) and were used to generate the therapeutic rabbit mabs sevacizumab and apx005 m discussed above . aside from therapeutic applications , rabbit mabs generated by hybridoma technology have become highly valuable reagents for diagnostic applications and for laboratory research .", "for example , highly specific rabbit mabs can detect activating mutations in the tyrosine kinase domain of egfr by ihc of lung cancer tissues .", "rabbit mabs against the hiv-1 protein gp120 were shown to mimic neutralizing human anti - hiv-1 gp120 mabs , promoting rabbit immunization as model for hiv-1 vaccine development ." ], [ "bacteriophage , also simply known as phage , are viruses that infect and replicate within bacteria .", "phage display was invented by george smith , who discovered that the minor coat protein ( piii ) of filamentous phage can be modified at its amino terminus to present peptide sequences without affecting phage infectivity .", "in addition , these phage particles contained the genomic information encoding the respectively modified coat protein , thus physically linking genotype and phenotype .", "a combination of phage selection and amplification could be used to efficiently enrich phage that contained certain peptide sequences .", "however , the incorporation of larger peptide sequences , protein domains or whole proteins represented a serious challenge due to decreasing infectivity .", "for this reason , two - component systems were developed that consisted of a phagemid encoding the piii fusion protein and a helper phage contributing the phage genome to encode all proteins necessary for generating infectious phage particles .", "usually , these helper phage contain a modified packaging signal , leading to the preferential assembly of phage particles containing the phagemid . in the early 1990s ,", "the first filamentous phage display antibody libraries based on piii fusion proteins were published using either scfv or fab fragments . to date", "although phage display was established with mouse and human antibody libraries to mine immune , naive and synthetic antibody repertoires , the fact that rabbit mabs were difficult to generate by hybridoma technology for a number of years provided a strong incentive for exploring the accessibility of rabbit immune antibody repertoires by phage display . the first rabbit antibody library selected by phage display was reported by ridder et al . , using a scfv format as in subsequent independent studies .", "rabbit antibody libraries in fab format followed in short succession . due to the higher expression levels of human compared to rabbit constant domains in bacteria , a chimeric rabbit / human fab format consisting of rabbit variable domains vl and vh recombinantly fused to human constant domains cl and ch1 , respectively , proved particularly successful for the selection of rabbit mabs by phage display and their subsequent humanization ( figure 5 ) .", "however , the above - discussed intrachain disulfide bridge between cysteine 80 and cysteine 171 found in rabbit light chains of the dominating k1 isotype posed a challenge to the chimeric rabbit / human fab format as human ch1 does not harbor a cysteine 171 .", "in fact , only few fab originating from the k1 isotype were selected , indicating that the free thiol group of cysteine 80 is disfavored and that its presence diminishes the selectable diversity of chimeric rabbit / human fab .", "indeed , chimeric rabbit / human fab derived by phage display from immunized k1-negative basilea strain rabbits revealed higher sequence diversity and higher affinity compared to those from k1-positive new zealand white strain rabbits immunized with the same immunogens .", "interestingly , the same study by popkov et al . also compared rabbits homozygous for the b9 -light - chain allotype ( figure 1 ) with the presumed alternative intrachain disulfide bridge between cysteine 108 and cysteine 171 .", "fusion of the rabbit v and human c encoding sequences in the generation of the chimeric rabbit / human fab library removes cysteine 108 , thus avoiding the exposure of a free thiol group .", "consequently , immunized b9 allotype rabbits also revealed superior selectable diversity and were subsequently used in several additional studies .", "these include the generation of a chimeric rabbit / human fab against the hiv-1 protein rev .", "the fab was able to invert rev polymerization and allowed for the formation of fab - rev co - crystals that could be analyzed with x - ray crystallography providing the first three - dimensional structure of rev and the first three - dimensional structure of a rabbit mab .", "notably , the crystal structure revealed a dominant role for lcdr3 in the antigen - binding site ( figure 6 ) . a cyclic peptide derived from lcdr3", "was shown to bind hiv-1 rev with high affinity and to potently inhibit rev polymerization , corroborating the functional importance of the above - discussed high sequence diversity of rabbit light chains .", "a key advantage of using the natural fab format for phage display is its robust monomeric nature that permits affinity - driven selections .", "by contrast , the unnatural scfv format has a tendency to dimerize , trimerize and tetramerize , potentially causing avidity - driven selections .", "thus , we recommend using chimeric rabbit / human fab format - based phage display for applications that require rabbit mabs of high affinity .", "even if the application calls for scfv , such as for the generation of intrabodies , the chimeric rabbit / human fab format has been used for selection by phage display followed by conversion to the rabbit scfv format .", "in addition to fab and scfv formats , single - domain antibody formats , that is , vl or vh alone , which due to their smaller sizes have advantages for certain applications , such as the recognition of cryptic epitopes , have also been engineered from rabbit mabs .", "the noted dominance of the rabbit light chain may make phage display of rabbit vl libraries particularly attractive ." ], [ "as discussed , hybridoma technology and phage display have distinct virtues and shortcomings that have fueled the development of alternative techniques to generate mabs .", "unlike hybridoma technology , phage display relies on the proper transcription , translation , folding and assembly of light and heavy chains in bacteria .", "also , a potential disadvantage of phage display , as well as other display technologies , is their combinatorial nature , leading to the random pairing of light and heavy chains . as discussed above , limiting factors encountered during hybridoma technology , at least initially , included fusion efficiency and hybridoma stability . also , unlike mouse hybridoma technology ,", "rabbit hybridoma technology was patented , imposing intellectual property restrictions that have contributed to the incentive of developing alternative methods . in the generation of human mabs ,", "prominent alternative methods include the clonal expansion of b cells by , for example , epstein ", "barr virus immortalization , and single b - cell sorting followed by light- and heavy - chain - encoding dna amplification and sequencing .", "notably , like hybridoma technology but unlike phage display , both methods yield natural light- and heavy - chain pairs .", "single b - cell isolation based on antigen capture by fluorescence - activated cell sorting , magnetic beads , solid - phase panning or hemolytic plaques followed by light- and heavy - chain cloning has also been applied to the generation of rabbit mabs from peripheral b cells , which does not require killing of the animal and allows for multiple sampling points from primary and secondary lymphoid tissues .", "antigen capture - driven bulk spleen b - cell isolation from immunized rabbits followed by light- and heavy - chain - encoding dna amplification and their combinatorial assembly and expression in scfv fc or igg format in mammalian cells has also been reported . in the fluorescent foci method ,", "plasma cells from rabbit bone marrow are identified by fluorescent microscopy , isolated and subjected to light- and heavy - chain - encoding dna amplification and sequencing .", "high - throughput dna sequencing technologies have been applied to the in silico generation of mabs as well as to the analysis of naive and immune antibody repertoires in different species . in the rabbit , high - throughput dna sequencing of b - cell repertoires along with mass spectrometry analysis of bulk", "interestingly , this method revealed that the serum igg response to an invertebrate protein following hyperimmunization was oligoclonal rather than polyclonal , comprising 34 rabbit mabs belonging to 30 distinct clonotypes ." ], [ "the human immune system is intended to recognize and selectively remove potentially pathogenic organisms and substances .", "the respective immune responses are characterized by high titers of human antibodies directed against these foreign antibody sequences .", "this has two main physiological consequences ; first of all , it leads to side effects comparable to allergic reactions of different levels of severity and , second , it leads to the rapid elimination of the administered mab , thus limiting its diagnostic or therapeutic efficacy . for these reasons ,", "nonhuman mabs are now routinely converted to chimeric mabs by combining the nonhuman variable domains with human constant domains and further to humanized mabs by grafting all or some of the cdrs of the nonhuman variable domains into human frameworks .", "the first chimeric and humanized rabbit mabs were reported by rader et al . in this study", ", chimeric rabbit / human fab selected by phage display were humanized by grafting the six rabbit cdrs , three from each light and heavy chain , into human frameworks that were diversified at certain positions to allow the selection of either human or rabbit residues by phage display .", "the resulting humanized rabbit mabs retained the high affinity and specificity of their parental mabs .", "as discussed above , aside from having higher utility for downstream clinical applications , using chimeric rabbit / human fab format for phage display has the added advantage of affording higher expression levels in bacteria and , concomitantly , higher display levels on phage . since this first report", ", a number of additional humanized rabbit mabs have been published and patented . in general ,", "humanization strategies that have worked for mouse mabs also work for rabbit mabs , whether it is by rational design , directed evolution or a combination of both .", "as is the case for mouse mabs , grafting of all six cdrs followed by iterative fine - tuning of framework residues is the most frequently used method for humanizing rabbit mabs .", "used a general acceptor framework for the humanization of rabbit scfv . in this study ,", "a certain human framework carrying five specific human - to - rabbit mutations was able to generate humanized rabbit mabs to two different antigens with conserved affinities and specificities , and improved biophysical properties .", "humanization strategies that confine the content of parental rabbit antibody sequences only to the most hypervariable of the cdrs , such as lcdr3 and hcdr3 , have also been successfully applied to rabbit mabs . thus far , there have been no reports that analyze the immunogenicity of clinically investigated humanized rabbit mabs .", "recent studies indicate that patients can still mount an immune response to humanized or even fully human mabs .", "these findings indicate that additional aspects , such as idiotype , allotype , glycosylation and aggregation , contribute to the immunogenicity of mabs .", "nonetheless , the occurrence of these unwanted side effects is significantly lower than previously observed for nonhuman antibodies ." ], [ "driven by the success of rabbit pabs on one hand and mouse mabs on the other hand , rabbit mabs have become highly successful reagents for laboratory research and for diagnostic and therapeutic applications .", "the unique ontogeny of rabbit b cells affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity , affinity and specificity .", "the ability to access rabbit antibody repertoires by hybridoma technology , phage display and alternative methods has fueled the increased use of rabbit mabs in many different applications .", "rabbit mabs generated by hybridoma technology are particularly attractive for ihc , with many studies demonstrating higher sensitivity compared to benchmark mouse mabs .", "rabbit mabs generated by phage display have found the use for applications ranging from the detection of uranium in water to chimeric antigen receptor t cell therapy of cancer .", "given that humanization strategies are now well established , the therapeutic utility of rabbit mabs is especially intriguing . with the first rabbit mab - derived therapeutics already in clinical trials , we predict that rabbit mabs will gain further traction in preclinical and clinical pipelines over the next decade ." ] ]

[ "all animal procedures were performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the baylor scott & white institutional animal care and use committee . domestic male pigs ( 812 weeks old , 811 kg ) were purchased from real farms ( san antonio , tx , usa ) .", "diabetes ( i.e. , chronic hyperglycemia ) was induced by selective ablation of pancreatic -cells with intravenous injection of streptozocin ( stz ; zanosar , 150 mg / kg in saline ) via an ear vein ( 22 pigs ) as described in detail in our previous study .", "the control group was injected intravenously with saline instead of stz ( 14 pigs ) .", "an additional group of nondiabetic pigs without saline injection was used for the acute hyperglycemia study in vitro as described below ( 24 pigs ) .", "following stz or saline ( control ) injection , the animals were allowed free access to water and fed with syrup mixed with hog chow to prevent temporary hypoglycemia for 24 hours after stz injection . the animals were allowed free access to water and commercial diet thereafter .", "the general condition , body weight , and level of blood glucose were closely monitored in all pigs , and only those that had sustained hyperglycemia with a fasting blood glucose level between 250 and 540 mg / dl were included in the study .", "fasting blood glucose levels were obtained each day in the morning using a bayer contour glucometer ( bayer corporation , pittsburgh , pa , usa ) . following the 2-week time period , pigs were sedated with telazol ( 48 mg / kg , intramuscularly ) , anesthetized with 2% to 4% isoflurane , and intubated .", "the techniques used for identification , isolation , cannulation , pressurization , and visualization of the retinal vasculature have been described previously . in brief , the isolated retinal arterioles ( 4060 m in situ diameter ) were cannulated with a pair of glass micropipettes containing a physiologic salt solution ( pss ; in mm : nacl 145.0 , kcl 4.7 , cacl2 2.0 , mgso4 1.17 , nah2po4 1.2 , pyruvate 2.0 , edta 0.02 , and mops 3.0 ) with normal 5 mm d - glucose and 1% albumin .", "for some vessels , the d - glucose was increased to 25 mm in the pss .", "the increased osmolarity in this solution was balanced to 290 mosm by reducing the nacl concentration .", "the vessels then were pressurized to 55 cm h2o intraluminal pressure without flow by two independent pressure reservoir systems .", "cannulated , pressurized arterioles were bathed in pss - albumin at 36 to 37c to allow the development of basal tone . to evaluate the effect of chronic hyperglycemia on vasomotor function , endothelium - dependent vasodilation to bradykinin ( 1 pm to 1 nm ) and endothelium - independent vasodilation to sodium nitroprusside ( snp ; 1 nm to 10 m ) were established in vessels isolated from the 2-week diabetic and saline - control pigs .", "vessels were exposed to each concentration of agonist for 4 to 5 minutes until a stable diameter was maintained . to assess the involvement of stress - activated kinases in the chronic hyperglycemia - induced effect , the vasodilator responses were examined following intraluminal treatment of vessels with jnk inhibitor sp600125 ( 5 m ) , jnk - interacting protein-1 ( jip1 ) inhibitor bi-78d3 ( 7 m ; bio - techne / tocris , minneapolis , mn , usa ) , or p38 kinase inhibitor sb203580 ( 0.1 m ; emd millipore , billerica , ma , usa ) for 2 hours . for some vessels ,", "the contribution of no in the vasodilation to bradykinin was evaluated in the presence of nos inhibitor n - nitro - l - arginine methyl ester ( l - name ; 10 m , 40-minute incubation ) with or without stress - activated kinase inhibitor treatment .", "the acute effect of high glucose on vasodilations to bradykinin and snp was evaluated in vitro after intraluminal incubation of vessels from nondiabetic pigs with 25 mm d - glucose or 5 mm d - glucose for 2 hours .", "the impact of stress - activated kinases on the no - mediated vasodilator responses was evaluated following co - incubation of 25 mm glucose with sp600125 , bi-78d3 , or sb203580 for 2 hours . in some vessels ,", "the contribution of no was examined in the presence of a stress - activated kinase inhibitor by adding l - name ( 10 m , 40-minute incubation ) .", "bradykinin , snp , and l - name were dissolved in pss , whereas sp600125 , bi-78d3 , and sb203580 were dissolved in dimethyl sulfoxide ( dmso ) .", "the final concentrations of dmso in the vessel lumen did not exceed 0.07% by volume .", "the 0.07% dmso had no significant effect on vessel viability , vasodilator responses , or maintenance of tone ( data not shown ) .", "at the end of each experiment , the maximum diameter of the vessels was obtained at 0.1 mm snp in the presence of calcium - free pss with edta ( 1 mm ) .", "diameter changes in response to vasodilator agonists were normalized to this maximum vasodilation and expressed as percentage maximum dilation .", "data are reported as mean sem and n value represents the number of vessels ( 1 per pig per treatment group ) studied .", "student 's t - test or anova followed by bonferroni multiple - range test was used to determine the significance of experimental interventions , as appropriate . a value of p < 0.05 was considered significant .", "all animal procedures were performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the baylor scott & white institutional animal care and use committee . domestic male pigs ( 812 weeks old , 811 kg ) were purchased from real farms ( san antonio , tx , usa ) .", "diabetes ( i.e. , chronic hyperglycemia ) was induced by selective ablation of pancreatic -cells with intravenous injection of streptozocin ( stz ; zanosar , 150 mg / kg in saline ) via an ear vein ( 22 pigs ) as described in detail in our previous study .", "the control group was injected intravenously with saline instead of stz ( 14 pigs ) .", "an additional group of nondiabetic pigs without saline injection was used for the acute hyperglycemia study in vitro as described below ( 24 pigs ) .", "following stz or saline ( control ) injection , the animals were allowed free access to water and fed with syrup mixed with hog chow to prevent temporary hypoglycemia for 24 hours after stz injection . the animals were allowed free access to water and commercial diet thereafter .", "the general condition , body weight , and level of blood glucose were closely monitored in all pigs , and only those that had sustained hyperglycemia with a fasting blood glucose level between 250 and 540 mg / dl were included in the study .", "fasting blood glucose levels were obtained each day in the morning using a bayer contour glucometer ( bayer corporation , pittsburgh , pa , usa ) . following the 2-week time period , pigs were sedated with telazol ( 48 mg / kg , intramuscularly ) , anesthetized with 2% to 4% isoflurane , and intubated .", "the techniques used for identification , isolation , cannulation , pressurization , and visualization of the retinal vasculature have been described previously . in brief , the isolated retinal arterioles ( 4060 m in situ diameter ) were cannulated with a pair of glass micropipettes containing a physiologic salt solution ( pss ; in mm : nacl 145.0 , kcl 4.7 , cacl2 2.0 , mgso4 1.17 , nah2po4 1.2 , pyruvate 2.0 , edta 0.02 , and mops 3.0 ) with normal 5 mm d - glucose and 1% albumin .", "for some vessels , the d - glucose was increased to 25 mm in the pss .", "the increased osmolarity in this solution was balanced to 290 mosm by reducing the nacl concentration .", "the vessels then were pressurized to 55 cm h2o intraluminal pressure without flow by two independent pressure reservoir systems .", "cannulated , pressurized arterioles were bathed in pss - albumin at 36 to 37c to allow the development of basal tone . to evaluate the effect of chronic hyperglycemia on vasomotor function , endothelium - dependent vasodilation to bradykinin ( 1 pm to 1 nm ) and endothelium - independent vasodilation to sodium nitroprusside ( snp ; 1 nm to 10 m ) were established in vessels isolated from the 2-week diabetic and saline - control pigs .", "vessels were exposed to each concentration of agonist for 4 to 5 minutes until a stable diameter was maintained . to assess the involvement of stress - activated kinases in the chronic hyperglycemia - induced effect", ", the vasodilator responses were examined following intraluminal treatment of vessels with jnk inhibitor sp600125 ( 5 m ) , jnk - interacting protein-1 ( jip1 ) inhibitor bi-78d3 ( 7 m ; bio - techne / tocris , minneapolis , mn , usa ) , or p38 kinase inhibitor sb203580 ( 0.1 m ; emd millipore , billerica , ma , usa ) for 2 hours . for some vessels , the contribution of no in the vasodilation to bradykinin was evaluated in the presence of nos inhibitor n - nitro - l - arginine methyl ester ( l - name ; 10 m , 40-minute incubation ) with or without stress - activated kinase inhibitor treatment .", "the acute effect of high glucose on vasodilations to bradykinin and snp was evaluated in vitro after intraluminal incubation of vessels from nondiabetic pigs with 25 mm d - glucose or 5 mm d - glucose for 2 hours .", "the impact of stress - activated kinases on the no - mediated vasodilator responses was evaluated following co - incubation of 25 mm glucose with sp600125 , bi-78d3 , or sb203580 for 2 hours . in some vessels ,", "the contribution of no was examined in the presence of a stress - activated kinase inhibitor by adding l - name ( 10 m , 40-minute incubation ) .", "( st . louis , mo , usa ) except as specifically stated . bradykinin , snp , and l - name were dissolved in pss , whereas sp600125 , bi-78d3 , and sb203580 were dissolved in dimethyl sulfoxide ( dmso ) .", "the final concentrations of dmso in the vessel lumen did not exceed 0.07% by volume .", "the 0.07% dmso had no significant effect on vessel viability , vasodilator responses , or maintenance of tone ( data not shown ) .", "at the end of each experiment , the maximum diameter of the vessels was obtained at 0.1 mm snp in the presence of calcium - free pss with edta ( 1 mm ) .", "diameter changes in response to vasodilator agonists were normalized to this maximum vasodilation and expressed as percentage maximum dilation .", "data are reported as mean sem and n value represents the number of vessels ( 1 per pig per treatment group ) studied .", "student 's t - test or anova followed by bonferroni multiple - range test was used to determine the significance of experimental interventions , as appropriate . a value of p < 0.05 was considered significant .", "two weeks following stz injection , blood glucose levels in pigs elevated from 85 6 ( 4.7 0.3 mm ) to 471 23 ( 26.2 1.3 mm ) mg / dl , and the body weight increased from 11.1 0.4 to 14.7 0.6 kg .", "pigs injected with saline ( control ) had unaltered blood glucose levels after 2 weeks , 77 6 ( 4.3 0.4 mm ) vs. 79 5 ( 4.4 0.3 mm ) mg / dl , and the body weight increased from 10.3 0.6 to 17.1 1.4 kg .", "the weight gain was significantly greater for control than diabetic pigs ( control , 6.8 1.0 kg versus diabetes , 3.7 0.5 kg ) .", "retinal arterioles from control and diabetic pigs developed a comparable level of basal tone ( control , 49 2% of maximum diameter , 97 3 m versus diabetes , 48 1% of maximum diameter , 97 2 m ; p = 0.55 ) , but the concentration - dependent dilation to bradykinin was significantly less in arterioles from diabetic pigs ( fig .", "the maximum dilation to bradykinin at 1 nm was 37 4% in diabetic vessels and 71 5% in control vessels . in the presence of nos inhibitor l - name ,", "1 ) . the l - name treatment did not alter basal tone ( data not shown ) .", "concentration - dependent dilation of isolated retinal arterioles to bradykinin was examined in the absence or presence of nos inhibitor l - name after 2 weeks of euglycemia ( control ; n = 6 ) or hyperglycemia ( diabetes ; n = 9 ) in pigs . *", "incubation of diabetic vessels with jnk inhibitor sp600125 or with jip1 inhibitor bi-78d3 restored the vasodilator response to bradykinin ( fig .", "by contrast , p38 kinase inhibitor sb203580 did not affect the vasodilations to bradykinin ( fig .", "the restored bradykinin - induced vasodilations by sp600125 or bi-78d3 were significantly reduced by l - name ( fig .", "for control vessels , the sp600125 , bi-78d3 , and sb203580 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation reverses chronic hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2 weeks of euglycemia ( control ; n = 10 ) or hyperglycemia ( diabetes ; n = 18 ) in pigs in the absence or presence of jnk inhibitor sp600125 ( n = 9 ) , jip1 inhibitor bi-78d3 ( n = 6 ) , or p38 inhibitor sb203580 ( n = 6 ) .", "( b ) dilation of retinal arterioles to bradykinin from diabetic pigs was examined in the presence of sp600125 ( n = 4 ) or bi-78d3 ( n = 6 ) before and after treatment with l - name . * p < 0.05 versus diabetes / sp600125 or diabetes / bi-78d3 .", "the retinal arterioles isolated from the nondiabetic pigs developed a comparable level of basal tone after intraluminal exposure to normal glucose ( ng ; 5 mm ) or high glucose ( hg ; 25 mm ) for 2 hours ( ng , 50 2% of maximum diameter versus hg , 49 3% of maximum diameter , p = 0.85 ) .", "concentration - dependent dilation to bradykinin was significantly less in arterioles with intraluminal exposure to hg ( fig .", "the maximum dilation to bradykinin at 1 nm was 34 10% in hg - treated vessels and 69 7% in ng - treated vessels .", "these vasodilator responses in hg- and ng - treated vessels were almost completely eliminated in the presence of l - name ( fig .", "dilation of retinal arterioles to bradykinin was examined in the absence or presence of l - name after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 6 ) or hg ( 25 mm ; n = 6 ) . * p < 0.05 versus ng ( 5 mm ) ; # p < 0.05 versus ng ( 5 mm ) or hg ( 25 mm ) .", "incubation of hg - treated vessels with sp600125 or bi-78d3 but not sb203580 preserved the vasodilator response to bradykinin ( fig .", "4a ) without altering basal tone ( data not shown ) . these preserved vasodilations were attenuated in a similar manner by l - name ( fig .", ", the sp600125 and bi-78d3 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation prevents acute hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 19 ) or hg ( 25 mm ; n = 22 ) in the absence or presence of sp600125 ( n = 8) , bi-78d3 ( n = 11 ) , or sb203580 ( n = 5 ) . * p < 0.05 versus ng ( 5 mm ) .", "( b ) dilation of retinal arterioles to bradykinin was examined following exposure to hg in the presence of sp600125 ( n = 5 ) or bi-78d3 ( n = 5 ) before and after treatment with l - name . * p < 0.05 versus hg / sp600125 or hg / bi-78d3 .", "retinal arterioles from nondiabetic ( control ) and diabetic pigs dilated in a comparable manner to endothelium - independent no donor snp with maximum dilation of approximately 70% at 10 m ( fig . 5 ) .", "this vasodilator response also was similar between ng- and hg - treated vessels ( fig .", "concentration - dependent vasodilation to snp was examined after 2 weeks of euglycemia ( control ; n = 5 ) or hyperglycemia ( diabetes ; n = 5 ) in pigs or after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 5 ) or hg ( 25 mm ; n = 5 ) .", "two weeks following stz injection , blood glucose levels in pigs elevated from 85 6 ( 4.7 0.3 mm ) to 471 23 ( 26.2 1.3 mm ) mg / dl , and the body weight increased from 11.1 0.4 to 14.7 0.6 kg .", "pigs injected with saline ( control ) had unaltered blood glucose levels after 2 weeks , 77 6 ( 4.3 0.4 mm ) vs. 79 5 ( 4.4 0.3 mm ) mg / dl , and the body weight increased from 10.3 0.6 to 17.1 1.4 kg .", "the weight gain was significantly greater for control than diabetic pigs ( control , 6.8 1.0 kg versus diabetes , 3.7 0.5 kg ) .", "retinal arterioles from control and diabetic pigs developed a comparable level of basal tone ( control , 49 2% of maximum diameter , 97 3 m versus diabetes , 48 1% of maximum diameter , 97 2 m ; p = 0.55 ) , but the concentration - dependent dilation to bradykinin was significantly less in arterioles from diabetic pigs ( fig .", "the maximum dilation to bradykinin at 1 nm was 37 4% in diabetic vessels and 71 5% in control vessels . in the presence of nos inhibitor l - name ,", "1 ) . the l - name treatment did not alter basal tone ( data not shown ) .", "concentration - dependent dilation of isolated retinal arterioles to bradykinin was examined in the absence or presence of nos inhibitor l - name after 2 weeks of euglycemia ( control ; n = 6 ) or hyperglycemia ( diabetes ; n = 9 ) in pigs .", "* p < 0.05 versus control ; # p < 0.05 versus control or diabetes .", "incubation of diabetic vessels with jnk inhibitor sp600125 or with jip1 inhibitor bi-78d3 restored the vasodilator response to bradykinin ( fig .", "by contrast , p38 kinase inhibitor sb203580 did not affect the vasodilations to bradykinin ( fig .", "the restored bradykinin - induced vasodilations by sp600125 or bi-78d3 were significantly reduced by l - name ( fig .", ", the sp600125 , bi-78d3 , and sb203580 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation reverses chronic hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2 weeks of euglycemia ( control ; n = 10 ) or hyperglycemia ( diabetes ; n = 18 ) in pigs in the absence or presence of jnk inhibitor sp600125 ( n = 9 ) , jip1 inhibitor bi-78d3 ( n = 6 ) , or p38 inhibitor sb203580 ( n = 6 ) . * p < 0.05 versus control .", "( b ) dilation of retinal arterioles to bradykinin from diabetic pigs was examined in the presence of sp600125 ( n = 4 ) or bi-78d3 ( n = 6 ) before and after treatment with l - name .", "the retinal arterioles isolated from the nondiabetic pigs developed a comparable level of basal tone after intraluminal exposure to normal glucose ( ng ; 5 mm ) or high glucose ( hg ; 25 mm ) for 2 hours ( ng , 50 2% of maximum diameter versus hg , 49 3% of maximum diameter , p = 0.85 ) .", "concentration - dependent dilation to bradykinin was significantly less in arterioles with intraluminal exposure to hg ( fig .", "the maximum dilation to bradykinin at 1 nm was 34 10% in hg - treated vessels and 69 7% in ng - treated vessels .", "these vasodilator responses in hg- and ng - treated vessels were almost completely eliminated in the presence of l - name ( fig .", "dilation of retinal arterioles to bradykinin was examined in the absence or presence of l - name after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 6 ) or hg ( 25 mm ; n = 6 ) . *", "p < 0.05 versus ng ( 5 mm ) ; # p < 0.05 versus ng ( 5 mm ) or hg ( 25 mm ) .", "incubation of hg - treated vessels with sp600125 or bi-78d3 but not sb203580 preserved the vasodilator response to bradykinin ( fig .", "4a ) without altering basal tone ( data not shown ) . these preserved vasodilations were attenuated in a similar manner by l - name ( fig .", ", the sp600125 and bi-78d3 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation prevents acute hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 19 ) or hg ( 25 mm ; n = 22 ) in the absence or presence of sp600125 ( n = 8) , bi-78d3 ( n = 11 ) , or sb203580 ( n = 5 ) . * p < 0.05 versus ng ( 5 mm ) .", "( b ) dilation of retinal arterioles to bradykinin was examined following exposure to hg in the presence of sp600125 ( n = 5 ) or bi-78d3 ( n = 5 ) before and after treatment with l - name .", "retinal arterioles from nondiabetic ( control ) and diabetic pigs dilated in a comparable manner to endothelium - independent no donor snp with maximum dilation of approximately 70% at 10 m ( fig . 5 ) .", "this vasodilator response also was similar between ng- and hg - treated vessels ( fig .", "concentration - dependent vasodilation to snp was examined after 2 weeks of euglycemia ( control ; n = 5 ) or hyperglycemia ( diabetes ; n = 5 ) in pigs or after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 5 ) or hg ( 25 mm ; n = 5 ) .", "this study showed that exposure of porcine retinal arterioles to chronic hyperglycemia in vivo or acute hyperglycemia in vitro selectively diminished endothelium - dependent no - mediated dilation to bradykinin .", "pharmacologic inhibition of stress - activated kinase jnk and its upstream interaction with jip1 preserved retinal vasodilator function related to endothelial no .", "it appears that jip1/jnk signaling contributes directly to the impairment of endothelium - dependent no - mediated dilation of retinal arterioles following acute and chronic hyperglycemia .", "the current findings corroborated our previous report on selective impairment of endothelium - dependent dilation of retinal arterioles to bradykinin within 2 weeks of diabetes in pigs .", "this relatively rapid onset of endothelial vasodilator dysfunction in the pig model is consistent with previous evidence showing diminished increase in retinal blood flow following intravitreal administration of endothelial agonist acetylcholine in 2-week diabetic rats .", "however , these earlier studies did not investigate whether the endothelial impairment was a result of diminished nos activation .", "we tested this possibility by treating the isolated retinal arterioles with nonselective nos inhibitor l - name after 2 weeks of diabetes , and this series of experiments nearly abolished the remaining vasodilation to bradykinin . because l - name also completely blocked the dilation of retinal arterioles from 2-week control pigs , it is likely that chronic hyperglycemia in vivo attenuates the ability of the endothelium in these vessels to produce and/or release no via nos .", "the sources of no production within the retina include neuronal nos ( nnos ) and inducible nos ( inos ) in the perivascular tissue , and endothelial nos ( enos ) in blood vessels .", "all three isoforms of nos have been shown to contribute to regulation of retinal arteriolar tone under physiologic conditions .", "the activation of inos and nnos in the perivascular retina during exposure to acute hypoxia ( 5 minutes ) in pigs and flickering light stimulation in cats , respectively , promotes dilation of retinal arterioles .", "interestingly , these vasodilator responses have been shown to be diminished in diabetic patients with and without retinopathy .", "because bradykinin elicits endothelium - dependent dilation of porcine retinal arterioles and it predominantly activates enos in cultured endothelial cells , we believe that hyperglycemia can impair enos activation in the endothelium of arterioles .", "although our studies used an isolated vessel preparation devoid of perivascular retinal tissue , the results do not exclude the possible contribution of inos and nnos to the vascular impact of diabetes / hyperglycemia in vivo . to demonstrate whether the endothelium of retinal arterioles is sensitive to hyperglycemia per se , we exposed these vessels to high glucose intraluminally in vitro . within 2 hours of exposure to high glucose", ", the bradykinin - induced dilation of retinal arterioles was reduced to a similar level observed following 2 weeks of hyperglycemia in vivo .", "notably , the high glucose concentration of 25 mm was comparable to the average glucose level in the diabetic pigs .", "in addition , osmolarity was maintained at a normal level in the high glucose - treated vessels to obviate the potential impact of hyperosmolarity on endothelial vasodilator function and basal tone , as well as stress - activated kinases .", "the complete blockade of the dilation of retinal arterioles to bradykinin by l - name in the presence of high glucose indicates that short - term hyperglycemia can directly impair endothelial no function without triggering the activation of compensatory vasodilator mechanisms under these conditions . because endothelium - independent vasodilation to no donor snp was unaltered following the 2-hour high glucose exposure , the ability of the smooth muscle to relax in response to no appears to have remained intact .", "therefore , the detrimental effect of acute hyperglycemia was selective for the impairment of no synthesis / release from the endothelium . taken together , our findings directly support the notion that acute or chronic hyperglycemia for up to 2 weeks reduces the endothelium - dependent no - mediated dilation of retinal arterioles .", "accumulating evidence supports an association of inflammation and endothelial dysfunction within the retina during diabetes .", "interestingly , the inflammatory stress - activated kinases , jnk and p38 , have been shown to be activated in the neural retina and cultured retinal endothelium following diabetes or acute high glucose exposure .", "the potential impact of jnk and p38 activation on retinal arteriolar vasomotor function during hyperglycemia had not been explored .", "these kinases appeared to be reasonable targets because we have previously shown that inflammatory molecules c - reactive protein and tnf- directly activate p38 and jnk , respectively , in isolated arterioles . in the present study", ", we found that simultaneous treatment of retinal arterioles with high glucose and jnk inhibitor sp600125 but not p38 inhibitor sb203580 prevented the reduction in dilation to bradykinin .", "these findings suggested a role for jnk in the initiation of endothelial vasodilator dysfunction during acute hyperglycemia . to assess the potential clinical significance of jnk blockade to restore vasodilator function after prolonged exposure to hyperglycemia in vivo , isolated retinal arterioles were treated with sp600125 after 2 weeks of diabetes .", "the intraluminal treatment of diabetic vessels with sp600125 preserved the dilation to bradykinin . because these preserved vasodilator responses were sensitive to l - name , it is likely that sp600125 is able to effectively restore the ability of retinal arterioles to produce no via nos . on the other hand", ", sb203580 did not improve vasodilation to bradykinin following chronic hyperglycemia , suggesting that p38 kinase does not contribute to retinal endothelial dysfunction under these conditions .", "the concentration of sb203580 ( 0.1 m ) used in this study was sufficient because we have shown previously its efficacy in preventing endothelial vasodilator dysfunction in retinal arterioles induced by c - reactive protein .", "collectively , our current findings indicate that activation of jnk within retinal arterioles contributes to diabetes - induced endothelial vasodilator dysfunction .", "cellular activation of mapks , including jnk , involves a distinct protein kinase cascade , which is organized in signaling modules by scaffold proteins for precise regulation in response to various stress stimuli .", "a common feature of mapk cascades is the organization of 3 kinases in which a mapk kinase kinase ( map3k ) activates a map2k , which subsequently activates a mapk . in the mammalian jnk module ,", "the cytosolic jip group of scaffold proteins selectively enhance jnk signaling by interacting with and linking the upstream kinases to jnk activation .", "specifically , the jip1 isoform serves as a docking site for the binding of jnk , map3ks , and map2k7 , to facilitate sequential kinase activation of the jnk signaling pathway . the physiologic role for jip1 in jnk activation", "was supported by evidence that jip1 knockout mice lack the ability to elicit anoxic and excitotoxic stress - induced activation of jnk in hippocampal neurons . at the vascular level , in vitro studies showed that cultured rat retinal endothelial cells containing overexpression of glucose transporter-1 and elevated intracellular glucose concentrations exhibited an increased jip1 protein expression and jnk phosphorylation .", "our current study extended these earlier findings to determine whether jip1 influences retinal arteriolar vasodilator function during acute and chronic hyperglycemia .", "in 2-week diabetic pigs , we observed reversal of impaired retinal arteriolar dilation to bradykinin with the jip1 inhibitor bi-78d3 , which is a small molecule mimic of jip1 that competitively blocks the jip1 binding domain of jnk from interacting with its cognate substrates and endogenous jip1 .", "this jnk signaling inhibitor does not influence the atp - binding region of jnk but instead blocks protein ", "protein interaction at the jnk - binding site of jip1 . similarly , bradykinin - induced dilation of retinal arterioles during exposure to acute hyperglycemia was preserved in the presence of jip1 blockade . taken together", ", our findings with 2 structurally distinct jnk pathway inhibitors , sp600125 and bi-78d3 , suggest that the interaction of jip1 and jnk is important in initiating endothelial damage during hyperglycemia , and support the idea that this molecular event contributes to development of endothelial vasodilator dysfunction in retinal arterioles during the early stage of diabetes . at the mechanistic level it is reasonable to speculate that jnk activation may be linked with increased oxidative stress during diabetes in retinal arterioles with concomitant reduction in no bioavailability .", "this notion is supported by earlier work showing that pharmacologic blockade of superoxide production augmented the bradykinin - induced increase in retinal blood flow following 3 hours of hyperglycemia in cats in vivo .", "our previous study showed that activation of jnk by tnf- in porcine coronary arterioles leads to superoxide generation and subsequent reduction of no release via xanthine oxidase activation . on the other hand ,", "evidence also has been provided for superoxide - dependent activation of jnk in endothelial cells .", "future molecular studies are warranted to investigate whether oxidative stress in retinal arterioles during hyperglycemia is requisite for impairing nos - mediated vasodilation . in summary", ", we demonstrated that acute and chronic hyperglycemia promote activation of jip1/jnk signaling in retinal arterioles leading to impairment of endothelium - dependent no - mediated dilation .", "the ability of pharmacologic blockade of jip1 or jnk to fully restore arteriolar vasodilator function following 2 weeks of hyperglycemia suggests that these specific proteins may provide useful clinical targets to consider for retinal vascular treatment to ameliorate retinal blood flow during early diabetes ." ]

[ "<S> purposehyperglycemia , a hallmark of diabetes mellitus , is associated with retinal inflammation and impairment of endothelium - dependent nitric oxide ( no)mediated dilation of retinal arterioles . however , </S>", "<S> molecular mechanisms involved in this diminished endothelial vasodilator function remain unclear . </S>", "<S> we examined whether inflammatory stress - activated kinases , c - jun n - terminal kinase ( jnk ) and p38 , contribute to retinal arteriolar dysfunction during exposure to acute and chronic hyperglycemia.methodsretinal arterioles were isolated from streptozocin - induced diabetic pigs ( 2 weeks ; chronic hyperglycemia , 471 23 mg / dl ) or age - matched control pigs ( euglycemia , 79 5 mg / dl ) , and then cannulated and pressurized for vasoreactivity study . for acute hyperglycemia study , vessels from nondiabetic pigs were exposed intraluminally to high glucose ( 25 mm 450 mg / dl ) for 2 hours , and normal glucose ( 5 mm 90 mg / dl ) served as the control.resultsendothelium-dependent vasodilation to bradykinin was reduced in a similar manner after exposure to acute or chronic hyperglycemia . </S>", "<S> administration of no synthase inhibitor ng - nitro - l - arginine methyl ester ( l - name ) nearly abolished vasodilations either in control ( euglycemia and normal glucose ) or hyperglycemic ( acute and chronic ) vessels . </S>", "<S> treatment of either acute or chronic hyperglycemic vessels with jnk inhibitor sp600125 or jnk - interacting protein-1 ( jip1 ) inhibitor bi-78d3 , but not p38 inhibitor sb203580 , preserved bradykinin - induced dilation in an l - name sensitive manner . by contrast , </S>", "<S> endothelium - independent vasodilation to sodium nitroprusside was unaffected by acute or chronic hyperglycemia.conclusionsactivation of jip1/jnk signaling in retinal arterioles during exposure to acute or chronic hyperglycemia leads to selective impairment of endothelium - dependent no - mediated dilation . </S>", "<S> therapeutic targeting of the vascular jnk pathway may improve retinal endothelial vasodilator function during early diabetes . </S>" ]

[ "Methods", "Porcine Diabetes Model", "Isolation and Cannulation of Microvessels", "Study of Vasomotor Function", "Chemicals", "Data Analysis", "Results", "Animal Model of Diabetes Mellitus/Chronic Hyperglycemia", "Effect of Chronic Hyperglycemia on NOS-Mediated Vasodilation", "Roles of JNK, JIP1, and p38 in Chronic Hyperglycemia-Induced Vasodilator Dysfunction", "Effect of Acute Hyperglycemia on NOS-Mediated Vasodilation", "Roles of JNK, JIP1, and p38 in Acute Hyperglycemia-Induced Vasodilator Dysfunction", "Vasodilation to SNP Following Chronic and Acute Hyperglycemia", "Discussion" ]

[ [ "all animal procedures were performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the baylor scott & white institutional animal care and use committee . domestic male pigs ( 812 weeks old , 811 kg ) were purchased from real farms ( san antonio , tx , usa ) .", "diabetes ( i.e. , chronic hyperglycemia ) was induced by selective ablation of pancreatic -cells with intravenous injection of streptozocin ( stz ; zanosar , 150 mg / kg in saline ) via an ear vein ( 22 pigs ) as described in detail in our previous study .", "the control group was injected intravenously with saline instead of stz ( 14 pigs ) .", "an additional group of nondiabetic pigs without saline injection was used for the acute hyperglycemia study in vitro as described below ( 24 pigs ) .", "following stz or saline ( control ) injection , the animals were allowed free access to water and fed with syrup mixed with hog chow to prevent temporary hypoglycemia for 24 hours after stz injection . the animals were allowed free access to water and commercial diet thereafter .", "the general condition , body weight , and level of blood glucose were closely monitored in all pigs , and only those that had sustained hyperglycemia with a fasting blood glucose level between 250 and 540 mg / dl were included in the study .", "fasting blood glucose levels were obtained each day in the morning using a bayer contour glucometer ( bayer corporation , pittsburgh , pa , usa ) . following the 2-week time period , pigs were sedated with telazol ( 48 mg / kg , intramuscularly ) , anesthetized with 2% to 4% isoflurane , and intubated .", "the techniques used for identification , isolation , cannulation , pressurization , and visualization of the retinal vasculature have been described previously . in brief , the isolated retinal arterioles ( 4060 m in situ diameter ) were cannulated with a pair of glass micropipettes containing a physiologic salt solution ( pss ; in mm : nacl 145.0 , kcl 4.7 , cacl2 2.0 , mgso4 1.17 , nah2po4 1.2 , pyruvate 2.0 , edta 0.02 , and mops 3.0 ) with normal 5 mm d - glucose and 1% albumin .", "for some vessels , the d - glucose was increased to 25 mm in the pss .", "the increased osmolarity in this solution was balanced to 290 mosm by reducing the nacl concentration .", "the vessels then were pressurized to 55 cm h2o intraluminal pressure without flow by two independent pressure reservoir systems .", "cannulated , pressurized arterioles were bathed in pss - albumin at 36 to 37c to allow the development of basal tone . to evaluate the effect of chronic hyperglycemia on vasomotor function , endothelium - dependent vasodilation to bradykinin ( 1 pm to 1 nm ) and endothelium - independent vasodilation to sodium nitroprusside ( snp ; 1 nm to 10 m ) were established in vessels isolated from the 2-week diabetic and saline - control pigs .", "vessels were exposed to each concentration of agonist for 4 to 5 minutes until a stable diameter was maintained . to assess the involvement of stress - activated kinases in the chronic hyperglycemia - induced effect , the vasodilator responses were examined following intraluminal treatment of vessels with jnk inhibitor sp600125 ( 5 m ) , jnk - interacting protein-1 ( jip1 ) inhibitor bi-78d3 ( 7 m ; bio - techne / tocris , minneapolis , mn , usa ) , or p38 kinase inhibitor sb203580 ( 0.1 m ; emd millipore , billerica , ma , usa ) for 2 hours . for some vessels ,", "the contribution of no in the vasodilation to bradykinin was evaluated in the presence of nos inhibitor n - nitro - l - arginine methyl ester ( l - name ; 10 m , 40-minute incubation ) with or without stress - activated kinase inhibitor treatment .", "the acute effect of high glucose on vasodilations to bradykinin and snp was evaluated in vitro after intraluminal incubation of vessels from nondiabetic pigs with 25 mm d - glucose or 5 mm d - glucose for 2 hours .", "the impact of stress - activated kinases on the no - mediated vasodilator responses was evaluated following co - incubation of 25 mm glucose with sp600125 , bi-78d3 , or sb203580 for 2 hours . in some vessels ,", "the contribution of no was examined in the presence of a stress - activated kinase inhibitor by adding l - name ( 10 m , 40-minute incubation ) .", "bradykinin , snp , and l - name were dissolved in pss , whereas sp600125 , bi-78d3 , and sb203580 were dissolved in dimethyl sulfoxide ( dmso ) .", "the final concentrations of dmso in the vessel lumen did not exceed 0.07% by volume .", "the 0.07% dmso had no significant effect on vessel viability , vasodilator responses , or maintenance of tone ( data not shown ) .", "at the end of each experiment , the maximum diameter of the vessels was obtained at 0.1 mm snp in the presence of calcium - free pss with edta ( 1 mm ) .", "diameter changes in response to vasodilator agonists were normalized to this maximum vasodilation and expressed as percentage maximum dilation .", "data are reported as mean sem and n value represents the number of vessels ( 1 per pig per treatment group ) studied .", "student 's t - test or anova followed by bonferroni multiple - range test was used to determine the significance of experimental interventions , as appropriate . a value of p < 0.05 was considered significant ." ], [ "all animal procedures were performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the baylor scott & white institutional animal care and use committee . domestic male pigs ( 812 weeks old , 811 kg ) were purchased from real farms ( san antonio , tx , usa ) .", "diabetes ( i.e. , chronic hyperglycemia ) was induced by selective ablation of pancreatic -cells with intravenous injection of streptozocin ( stz ; zanosar , 150 mg / kg in saline ) via an ear vein ( 22 pigs ) as described in detail in our previous study .", "the control group was injected intravenously with saline instead of stz ( 14 pigs ) .", "an additional group of nondiabetic pigs without saline injection was used for the acute hyperglycemia study in vitro as described below ( 24 pigs ) .", "following stz or saline ( control ) injection , the animals were allowed free access to water and fed with syrup mixed with hog chow to prevent temporary hypoglycemia for 24 hours after stz injection . the animals were allowed free access to water and commercial diet thereafter .", "the general condition , body weight , and level of blood glucose were closely monitored in all pigs , and only those that had sustained hyperglycemia with a fasting blood glucose level between 250 and 540 mg / dl were included in the study .", "fasting blood glucose levels were obtained each day in the morning using a bayer contour glucometer ( bayer corporation , pittsburgh , pa , usa ) . following the 2-week time period , pigs were sedated with telazol ( 48 mg / kg , intramuscularly ) , anesthetized with 2% to 4% isoflurane , and intubated ." ], [ "the techniques used for identification , isolation , cannulation , pressurization , and visualization of the retinal vasculature have been described previously . in brief , the isolated retinal arterioles ( 4060 m in situ diameter ) were cannulated with a pair of glass micropipettes containing a physiologic salt solution ( pss ; in mm : nacl 145.0 , kcl 4.7 , cacl2 2.0 , mgso4 1.17 , nah2po4 1.2 , pyruvate 2.0 , edta 0.02 , and mops 3.0 ) with normal 5 mm d - glucose and 1% albumin .", "for some vessels , the d - glucose was increased to 25 mm in the pss .", "the increased osmolarity in this solution was balanced to 290 mosm by reducing the nacl concentration .", "the vessels then were pressurized to 55 cm h2o intraluminal pressure without flow by two independent pressure reservoir systems ." ], [ "cannulated , pressurized arterioles were bathed in pss - albumin at 36 to 37c to allow the development of basal tone . to evaluate the effect of chronic hyperglycemia on vasomotor function , endothelium - dependent vasodilation to bradykinin ( 1 pm to 1 nm ) and endothelium - independent vasodilation to sodium nitroprusside ( snp ; 1 nm to 10 m ) were established in vessels isolated from the 2-week diabetic and saline - control pigs .", "vessels were exposed to each concentration of agonist for 4 to 5 minutes until a stable diameter was maintained . to assess the involvement of stress - activated kinases in the chronic hyperglycemia - induced effect", ", the vasodilator responses were examined following intraluminal treatment of vessels with jnk inhibitor sp600125 ( 5 m ) , jnk - interacting protein-1 ( jip1 ) inhibitor bi-78d3 ( 7 m ; bio - techne / tocris , minneapolis , mn , usa ) , or p38 kinase inhibitor sb203580 ( 0.1 m ; emd millipore , billerica , ma , usa ) for 2 hours . for some vessels , the contribution of no in the vasodilation to bradykinin was evaluated in the presence of nos inhibitor n - nitro - l - arginine methyl ester ( l - name ; 10 m , 40-minute incubation ) with or without stress - activated kinase inhibitor treatment .", "the acute effect of high glucose on vasodilations to bradykinin and snp was evaluated in vitro after intraluminal incubation of vessels from nondiabetic pigs with 25 mm d - glucose or 5 mm d - glucose for 2 hours .", "the impact of stress - activated kinases on the no - mediated vasodilator responses was evaluated following co - incubation of 25 mm glucose with sp600125 , bi-78d3 , or sb203580 for 2 hours . in some vessels ,", "the contribution of no was examined in the presence of a stress - activated kinase inhibitor by adding l - name ( 10 m , 40-minute incubation ) ." ], [ "( st . louis , mo , usa ) except as specifically stated . bradykinin , snp , and l - name were dissolved in pss , whereas sp600125 , bi-78d3 , and sb203580 were dissolved in dimethyl sulfoxide ( dmso ) .", "the final concentrations of dmso in the vessel lumen did not exceed 0.07% by volume .", "the 0.07% dmso had no significant effect on vessel viability , vasodilator responses , or maintenance of tone ( data not shown ) ." ], [ "at the end of each experiment , the maximum diameter of the vessels was obtained at 0.1 mm snp in the presence of calcium - free pss with edta ( 1 mm ) .", "diameter changes in response to vasodilator agonists were normalized to this maximum vasodilation and expressed as percentage maximum dilation .", "data are reported as mean sem and n value represents the number of vessels ( 1 per pig per treatment group ) studied .", "student 's t - test or anova followed by bonferroni multiple - range test was used to determine the significance of experimental interventions , as appropriate . a value of p < 0.05 was considered significant ." ], [ "two weeks following stz injection , blood glucose levels in pigs elevated from 85 6 ( 4.7 0.3 mm ) to 471 23 ( 26.2 1.3 mm ) mg / dl , and the body weight increased from 11.1 0.4 to 14.7 0.6 kg .", "pigs injected with saline ( control ) had unaltered blood glucose levels after 2 weeks , 77 6 ( 4.3 0.4 mm ) vs. 79 5 ( 4.4 0.3 mm ) mg / dl , and the body weight increased from 10.3 0.6 to 17.1 1.4 kg .", "the weight gain was significantly greater for control than diabetic pigs ( control , 6.8 1.0 kg versus diabetes , 3.7 0.5 kg ) .", "retinal arterioles from control and diabetic pigs developed a comparable level of basal tone ( control , 49 2% of maximum diameter , 97 3 m versus diabetes , 48 1% of maximum diameter , 97 2 m ; p = 0.55 ) , but the concentration - dependent dilation to bradykinin was significantly less in arterioles from diabetic pigs ( fig .", "the maximum dilation to bradykinin at 1 nm was 37 4% in diabetic vessels and 71 5% in control vessels . in the presence of nos inhibitor l - name ,", "1 ) . the l - name treatment did not alter basal tone ( data not shown ) .", "concentration - dependent dilation of isolated retinal arterioles to bradykinin was examined in the absence or presence of nos inhibitor l - name after 2 weeks of euglycemia ( control ; n = 6 ) or hyperglycemia ( diabetes ; n = 9 ) in pigs . *", "incubation of diabetic vessels with jnk inhibitor sp600125 or with jip1 inhibitor bi-78d3 restored the vasodilator response to bradykinin ( fig .", "by contrast , p38 kinase inhibitor sb203580 did not affect the vasodilations to bradykinin ( fig .", "the restored bradykinin - induced vasodilations by sp600125 or bi-78d3 were significantly reduced by l - name ( fig .", "for control vessels , the sp600125 , bi-78d3 , and sb203580 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation reverses chronic hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2 weeks of euglycemia ( control ; n = 10 ) or hyperglycemia ( diabetes ; n = 18 ) in pigs in the absence or presence of jnk inhibitor sp600125 ( n = 9 ) , jip1 inhibitor bi-78d3 ( n = 6 ) , or p38 inhibitor sb203580 ( n = 6 ) .", "( b ) dilation of retinal arterioles to bradykinin from diabetic pigs was examined in the presence of sp600125 ( n = 4 ) or bi-78d3 ( n = 6 ) before and after treatment with l - name . * p < 0.05 versus diabetes / sp600125 or diabetes / bi-78d3 .", "the retinal arterioles isolated from the nondiabetic pigs developed a comparable level of basal tone after intraluminal exposure to normal glucose ( ng ; 5 mm ) or high glucose ( hg ; 25 mm ) for 2 hours ( ng , 50 2% of maximum diameter versus hg , 49 3% of maximum diameter , p = 0.85 ) .", "concentration - dependent dilation to bradykinin was significantly less in arterioles with intraluminal exposure to hg ( fig .", "the maximum dilation to bradykinin at 1 nm was 34 10% in hg - treated vessels and 69 7% in ng - treated vessels .", "these vasodilator responses in hg- and ng - treated vessels were almost completely eliminated in the presence of l - name ( fig .", "dilation of retinal arterioles to bradykinin was examined in the absence or presence of l - name after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 6 ) or hg ( 25 mm ; n = 6 ) . * p < 0.05 versus ng ( 5 mm ) ; # p < 0.05 versus ng ( 5 mm ) or hg ( 25 mm ) .", "incubation of hg - treated vessels with sp600125 or bi-78d3 but not sb203580 preserved the vasodilator response to bradykinin ( fig .", "4a ) without altering basal tone ( data not shown ) . these preserved vasodilations were attenuated in a similar manner by l - name ( fig .", ", the sp600125 and bi-78d3 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation prevents acute hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 19 ) or hg ( 25 mm ; n = 22 ) in the absence or presence of sp600125 ( n = 8) , bi-78d3 ( n = 11 ) , or sb203580 ( n = 5 ) . * p < 0.05 versus ng ( 5 mm ) .", "( b ) dilation of retinal arterioles to bradykinin was examined following exposure to hg in the presence of sp600125 ( n = 5 ) or bi-78d3 ( n = 5 ) before and after treatment with l - name . * p < 0.05 versus hg / sp600125 or hg / bi-78d3 .", "retinal arterioles from nondiabetic ( control ) and diabetic pigs dilated in a comparable manner to endothelium - independent no donor snp with maximum dilation of approximately 70% at 10 m ( fig . 5 ) .", "this vasodilator response also was similar between ng- and hg - treated vessels ( fig .", "concentration - dependent vasodilation to snp was examined after 2 weeks of euglycemia ( control ; n = 5 ) or hyperglycemia ( diabetes ; n = 5 ) in pigs or after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 5 ) or hg ( 25 mm ; n = 5 ) ." ], [ "two weeks following stz injection , blood glucose levels in pigs elevated from 85 6 ( 4.7 0.3 mm ) to 471 23 ( 26.2 1.3 mm ) mg / dl , and the body weight increased from 11.1 0.4 to 14.7 0.6 kg .", "pigs injected with saline ( control ) had unaltered blood glucose levels after 2 weeks , 77 6 ( 4.3 0.4 mm ) vs. 79 5 ( 4.4 0.3 mm ) mg / dl , and the body weight increased from 10.3 0.6 to 17.1 1.4 kg .", "the weight gain was significantly greater for control than diabetic pigs ( control , 6.8 1.0 kg versus diabetes , 3.7 0.5 kg ) ." ], [ "retinal arterioles from control and diabetic pigs developed a comparable level of basal tone ( control , 49 2% of maximum diameter , 97 3 m versus diabetes , 48 1% of maximum diameter , 97 2 m ; p = 0.55 ) , but the concentration - dependent dilation to bradykinin was significantly less in arterioles from diabetic pigs ( fig .", "the maximum dilation to bradykinin at 1 nm was 37 4% in diabetic vessels and 71 5% in control vessels . in the presence of nos inhibitor l - name ,", "1 ) . the l - name treatment did not alter basal tone ( data not shown ) .", "concentration - dependent dilation of isolated retinal arterioles to bradykinin was examined in the absence or presence of nos inhibitor l - name after 2 weeks of euglycemia ( control ; n = 6 ) or hyperglycemia ( diabetes ; n = 9 ) in pigs .", "* p < 0.05 versus control ; # p < 0.05 versus control or diabetes ." ], [ "incubation of diabetic vessels with jnk inhibitor sp600125 or with jip1 inhibitor bi-78d3 restored the vasodilator response to bradykinin ( fig .", "by contrast , p38 kinase inhibitor sb203580 did not affect the vasodilations to bradykinin ( fig .", "the restored bradykinin - induced vasodilations by sp600125 or bi-78d3 were significantly reduced by l - name ( fig .", ", the sp600125 , bi-78d3 , and sb203580 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation reverses chronic hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2 weeks of euglycemia ( control ; n = 10 ) or hyperglycemia ( diabetes ; n = 18 ) in pigs in the absence or presence of jnk inhibitor sp600125 ( n = 9 ) , jip1 inhibitor bi-78d3 ( n = 6 ) , or p38 inhibitor sb203580 ( n = 6 ) . * p < 0.05 versus control .", "( b ) dilation of retinal arterioles to bradykinin from diabetic pigs was examined in the presence of sp600125 ( n = 4 ) or bi-78d3 ( n = 6 ) before and after treatment with l - name ." ], [ "the retinal arterioles isolated from the nondiabetic pigs developed a comparable level of basal tone after intraluminal exposure to normal glucose ( ng ; 5 mm ) or high glucose ( hg ; 25 mm ) for 2 hours ( ng , 50 2% of maximum diameter versus hg , 49 3% of maximum diameter , p = 0.85 ) .", "concentration - dependent dilation to bradykinin was significantly less in arterioles with intraluminal exposure to hg ( fig .", "the maximum dilation to bradykinin at 1 nm was 34 10% in hg - treated vessels and 69 7% in ng - treated vessels .", "these vasodilator responses in hg- and ng - treated vessels were almost completely eliminated in the presence of l - name ( fig .", "dilation of retinal arterioles to bradykinin was examined in the absence or presence of l - name after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 6 ) or hg ( 25 mm ; n = 6 ) . *", "p < 0.05 versus ng ( 5 mm ) ; # p < 0.05 versus ng ( 5 mm ) or hg ( 25 mm ) ." ], [ "incubation of hg - treated vessels with sp600125 or bi-78d3 but not sb203580 preserved the vasodilator response to bradykinin ( fig .", "4a ) without altering basal tone ( data not shown ) . these preserved vasodilations were attenuated in a similar manner by l - name ( fig .", ", the sp600125 and bi-78d3 treatments did not alter the bradykinin - induced vasodilations ( data not shown ) .", "blockade of jnk activation prevents acute hyperglycemia - induced reduction of retinal arteriolar dilation to bradykinin .", "( a ) dilation of retinal arterioles to bradykinin was examined after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 19 ) or hg ( 25 mm ; n = 22 ) in the absence or presence of sp600125 ( n = 8) , bi-78d3 ( n = 11 ) , or sb203580 ( n = 5 ) . * p < 0.05 versus ng ( 5 mm ) .", "( b ) dilation of retinal arterioles to bradykinin was examined following exposure to hg in the presence of sp600125 ( n = 5 ) or bi-78d3 ( n = 5 ) before and after treatment with l - name ." ], [ "retinal arterioles from nondiabetic ( control ) and diabetic pigs dilated in a comparable manner to endothelium - independent no donor snp with maximum dilation of approximately 70% at 10 m ( fig . 5 ) .", "this vasodilator response also was similar between ng- and hg - treated vessels ( fig .", "concentration - dependent vasodilation to snp was examined after 2 weeks of euglycemia ( control ; n = 5 ) or hyperglycemia ( diabetes ; n = 5 ) in pigs or after 2-hour intraluminal exposure in vitro to ng ( 5 mm ; n = 5 ) or hg ( 25 mm ; n = 5 ) ." ], [ "this study showed that exposure of porcine retinal arterioles to chronic hyperglycemia in vivo or acute hyperglycemia in vitro selectively diminished endothelium - dependent no - mediated dilation to bradykinin .", "pharmacologic inhibition of stress - activated kinase jnk and its upstream interaction with jip1 preserved retinal vasodilator function related to endothelial no .", "it appears that jip1/jnk signaling contributes directly to the impairment of endothelium - dependent no - mediated dilation of retinal arterioles following acute and chronic hyperglycemia .", "the current findings corroborated our previous report on selective impairment of endothelium - dependent dilation of retinal arterioles to bradykinin within 2 weeks of diabetes in pigs .", "this relatively rapid onset of endothelial vasodilator dysfunction in the pig model is consistent with previous evidence showing diminished increase in retinal blood flow following intravitreal administration of endothelial agonist acetylcholine in 2-week diabetic rats .", "however , these earlier studies did not investigate whether the endothelial impairment was a result of diminished nos activation .", "we tested this possibility by treating the isolated retinal arterioles with nonselective nos inhibitor l - name after 2 weeks of diabetes , and this series of experiments nearly abolished the remaining vasodilation to bradykinin . because l - name also completely blocked the dilation of retinal arterioles from 2-week control pigs , it is likely that chronic hyperglycemia in vivo attenuates the ability of the endothelium in these vessels to produce and/or release no via nos .", "the sources of no production within the retina include neuronal nos ( nnos ) and inducible nos ( inos ) in the perivascular tissue , and endothelial nos ( enos ) in blood vessels .", "all three isoforms of nos have been shown to contribute to regulation of retinal arteriolar tone under physiologic conditions .", "the activation of inos and nnos in the perivascular retina during exposure to acute hypoxia ( 5 minutes ) in pigs and flickering light stimulation in cats , respectively , promotes dilation of retinal arterioles .", "interestingly , these vasodilator responses have been shown to be diminished in diabetic patients with and without retinopathy .", "because bradykinin elicits endothelium - dependent dilation of porcine retinal arterioles and it predominantly activates enos in cultured endothelial cells , we believe that hyperglycemia can impair enos activation in the endothelium of arterioles .", "although our studies used an isolated vessel preparation devoid of perivascular retinal tissue , the results do not exclude the possible contribution of inos and nnos to the vascular impact of diabetes / hyperglycemia in vivo . to demonstrate whether the endothelium of retinal arterioles is sensitive to hyperglycemia per se , we exposed these vessels to high glucose intraluminally in vitro . within 2 hours of exposure to high glucose", ", the bradykinin - induced dilation of retinal arterioles was reduced to a similar level observed following 2 weeks of hyperglycemia in vivo .", "notably , the high glucose concentration of 25 mm was comparable to the average glucose level in the diabetic pigs .", "in addition , osmolarity was maintained at a normal level in the high glucose - treated vessels to obviate the potential impact of hyperosmolarity on endothelial vasodilator function and basal tone , as well as stress - activated kinases .", "the complete blockade of the dilation of retinal arterioles to bradykinin by l - name in the presence of high glucose indicates that short - term hyperglycemia can directly impair endothelial no function without triggering the activation of compensatory vasodilator mechanisms under these conditions . because endothelium - independent vasodilation to no donor snp was unaltered following the 2-hour high glucose exposure , the ability of the smooth muscle to relax in response to no appears to have remained intact .", "therefore , the detrimental effect of acute hyperglycemia was selective for the impairment of no synthesis / release from the endothelium . taken together , our findings directly support the notion that acute or chronic hyperglycemia for up to 2 weeks reduces the endothelium - dependent no - mediated dilation of retinal arterioles .", "accumulating evidence supports an association of inflammation and endothelial dysfunction within the retina during diabetes .", "interestingly , the inflammatory stress - activated kinases , jnk and p38 , have been shown to be activated in the neural retina and cultured retinal endothelium following diabetes or acute high glucose exposure .", "the potential impact of jnk and p38 activation on retinal arteriolar vasomotor function during hyperglycemia had not been explored .", "these kinases appeared to be reasonable targets because we have previously shown that inflammatory molecules c - reactive protein and tnf- directly activate p38 and jnk , respectively , in isolated arterioles . in the present study", ", we found that simultaneous treatment of retinal arterioles with high glucose and jnk inhibitor sp600125 but not p38 inhibitor sb203580 prevented the reduction in dilation to bradykinin .", "these findings suggested a role for jnk in the initiation of endothelial vasodilator dysfunction during acute hyperglycemia . to assess the potential clinical significance of jnk blockade to restore vasodilator function after prolonged exposure to hyperglycemia in vivo , isolated retinal arterioles were treated with sp600125 after 2 weeks of diabetes .", "the intraluminal treatment of diabetic vessels with sp600125 preserved the dilation to bradykinin . because these preserved vasodilator responses were sensitive to l - name , it is likely that sp600125 is able to effectively restore the ability of retinal arterioles to produce no via nos . on the other hand", ", sb203580 did not improve vasodilation to bradykinin following chronic hyperglycemia , suggesting that p38 kinase does not contribute to retinal endothelial dysfunction under these conditions .", "the concentration of sb203580 ( 0.1 m ) used in this study was sufficient because we have shown previously its efficacy in preventing endothelial vasodilator dysfunction in retinal arterioles induced by c - reactive protein .", "collectively , our current findings indicate that activation of jnk within retinal arterioles contributes to diabetes - induced endothelial vasodilator dysfunction .", "cellular activation of mapks , including jnk , involves a distinct protein kinase cascade , which is organized in signaling modules by scaffold proteins for precise regulation in response to various stress stimuli .", "a common feature of mapk cascades is the organization of 3 kinases in which a mapk kinase kinase ( map3k ) activates a map2k , which subsequently activates a mapk . in the mammalian jnk module ,", "the cytosolic jip group of scaffold proteins selectively enhance jnk signaling by interacting with and linking the upstream kinases to jnk activation .", "specifically , the jip1 isoform serves as a docking site for the binding of jnk , map3ks , and map2k7 , to facilitate sequential kinase activation of the jnk signaling pathway . the physiologic role for jip1 in jnk activation", "was supported by evidence that jip1 knockout mice lack the ability to elicit anoxic and excitotoxic stress - induced activation of jnk in hippocampal neurons . at the vascular level , in vitro studies showed that cultured rat retinal endothelial cells containing overexpression of glucose transporter-1 and elevated intracellular glucose concentrations exhibited an increased jip1 protein expression and jnk phosphorylation .", "our current study extended these earlier findings to determine whether jip1 influences retinal arteriolar vasodilator function during acute and chronic hyperglycemia .", "in 2-week diabetic pigs , we observed reversal of impaired retinal arteriolar dilation to bradykinin with the jip1 inhibitor bi-78d3 , which is a small molecule mimic of jip1 that competitively blocks the jip1 binding domain of jnk from interacting with its cognate substrates and endogenous jip1 .", "this jnk signaling inhibitor does not influence the atp - binding region of jnk but instead blocks protein ", "protein interaction at the jnk - binding site of jip1 . similarly , bradykinin - induced dilation of retinal arterioles during exposure to acute hyperglycemia was preserved in the presence of jip1 blockade . taken together", ", our findings with 2 structurally distinct jnk pathway inhibitors , sp600125 and bi-78d3 , suggest that the interaction of jip1 and jnk is important in initiating endothelial damage during hyperglycemia , and support the idea that this molecular event contributes to development of endothelial vasodilator dysfunction in retinal arterioles during the early stage of diabetes . at the mechanistic level it is reasonable to speculate that jnk activation may be linked with increased oxidative stress during diabetes in retinal arterioles with concomitant reduction in no bioavailability .", "this notion is supported by earlier work showing that pharmacologic blockade of superoxide production augmented the bradykinin - induced increase in retinal blood flow following 3 hours of hyperglycemia in cats in vivo .", "our previous study showed that activation of jnk by tnf- in porcine coronary arterioles leads to superoxide generation and subsequent reduction of no release via xanthine oxidase activation . on the other hand ,", "evidence also has been provided for superoxide - dependent activation of jnk in endothelial cells .", "future molecular studies are warranted to investigate whether oxidative stress in retinal arterioles during hyperglycemia is requisite for impairing nos - mediated vasodilation . in summary", ", we demonstrated that acute and chronic hyperglycemia promote activation of jip1/jnk signaling in retinal arterioles leading to impairment of endothelium - dependent no - mediated dilation .", "the ability of pharmacologic blockade of jip1 or jnk to fully restore arteriolar vasodilator function following 2 weeks of hyperglycemia suggests that these specific proteins may provide useful clinical targets to consider for retinal vascular treatment to ameliorate retinal blood flow during early diabetes ." ] ]

[ "the processes that lead to the gradual atrophy of brain cells , and thus to dementia , are affected by many different factors .", "some researchers have called attention to the impact of activity over the lifespan , particularly in later periods of life , on the development of dementia .", "although the results of research on this problem have been equivocal , many studies have shown that greater activity involving cognitive functions reduces the risk of dementia [ 13 ] .", "activity manifests itself in diverse ways , in respect both to professional activity and to the realization of various personal interests . in studies conducted in the mid-1990s at the second clinic for mental illnesses at the gdansk academy of medicine , undertaken to identify risk factors for dementia of the alzheimer type ( dat ) , attention was called to reduced activity in the premorbid period as a factor that occurred significantly more often in persons with dementia .", "it turned out that persons who showed a heightened risk for dementia with primary degenerative processes had limited contact with other people and a lack of complex activity , e.g. , after retirement .", "other research conducted during the same time period also showed reduced activity in the preclinical phase of dementia [ 68 ] .", "in addition , our own research pointed to a correlation between reduced activity immediately after the first manifestation of symptoms and the rate of progression of cognitive symptoms .", "the clinical manifestation of dementia is often preceded by many years of slight cognitive decline .", "it can be very difficult to differentiate the initial phases in the development of a dementive illness from other mental states that can also present with slight deterioration of cognitive functions .", "it is generally accepted that the appearance of mci is associated with a serious risk of dementia developing within a few years .", "some studies have shown that within 34 years more than 50% of persons diagnosed with mci will develop full - blown dementia . on the other hand , this means that a significant number of persons diagnosed with mci will have no further deterioration of cognitive functions .", "research has been under way for some time now to identify the factors that would facilitate an accurate prognosis for persons diagnosed with mci .", "however , despite the number of such studies , the results have been equivocal ; therefore , it remains impossible at present to evaluate at an acceptable level of probability the degree of risk that mci will develop into dementia .", "given that mci can be treated in some cases as a preliminary phase of dementia , it is hardly a surprise that the risk factors and protective factors identified in earlier research on the preclinical stages of dementia have taken on increased importance .", "among all these factors , attention has also been drawn to the level of activity manifested by persons with mci as a possible predictive factor .", "although the results achieved to date have been rather inconsistent , comparative analysis of a number of studies devoted to this problem have indicated that at least some kinds of activity manifested by study participants seem to be significant . from the methodological point of view , it is no easy task to make an accurate evaluation of the level of activity displayed by research subjects .", "many studies have taken the average number of hours of activity in the course of a week as the measure of activity , without differentiating the types of activity .", "other studies , although they have distinguished between intellectual , physical , and social activity , have not really identified the constitutive elements of any of these types .", "moreover , it is not common practice to base the evaluation of activity on a measurement method that would make it possible to quantify the results .", "the goal of our study was to specify the relationship between the level of activity ( intellectual , physical , and social ) in persons diagnosed with mci and the further progression of cognitive dysfunction .", "the initial population consisted of persons who had reported to our mental health clinic in the period from 2005 to 2007 .", "it was assumed that these individuals would be under systematic psychiatric observation until dementia was diagnosed .", "subjects were qualified for the study according to the following inclusion criteria : informed consent to participate . a diagnosis of mci based on the criteria published by the working group on mild cognitive impairment , assuming that dementia has been ruled out ; i.e. , that there has been a perceptible decline in cognitive function ( based on self - evaluation or the reports of a caregiver , and then verified objectively by an examination confirming cognitive impairment and/or objective measures of a deterioration of cognitive functions over time ) , and that basic activities of daily living are preserved , with a slight or minimal level of impairment in carrying out complex activities . no psychiatric pharmacotherapy at the time of enrollment .", "the availability of an individual from the patient s own environment , someone who lives with the patient or at least visits several times a week , and who expresses a willingness to participate in the process of evaluating the patient s activity by providing information . a score on the global deterioration scale of reisberg et al . placing the patient on the third level ( slight impairment of cognitive functions )", "a score on the mini mental state examination ( mmse ) in the range from 24 to 30 points .", "the exclusion criteria were as follows : a diagnosis of dementia , regardless of its etiology .", "the presence at examination or in the history of affective disorder , schizophrenia , alcoholism , addiction to medications or drugs , epilepsy , parkinson s disease , or intellectual handicap .", "the presence at examination or during the evaluation period of disorders of consciousness or disturbances of locomotion , vision , or hearing that would impede compliance with instructions and procedures included in the clinical scales used in this study .", "all those who were qualified for enrollment were subjected to a complete examination , which included the mmse , the activity scale , and the instrumental activity of daily living ( iadl ) scale .", "point scores of the mmse can range from 0 ( severe dementia ) to 30 ( lack of cognitive impairment ) . due to the impact of age and education on the mmse score ,", "the raw scores were adjusted for these two factors , based on the equation proposed by mungas et al . and verified for polish conditions by jozwiak et al . .", "the activity scale was developed by christensen and mackinnon in 1993 , and consists of three subscales covering intellectual ( mental ) , physical , and social activity .", "the number of activities undertaken is evaluated ( the degree of diversification of activity ) , as well as the average number of hours spent in particular activities . however , in the course of pilot research involving 10 patients who met the enrollment criteria , both the subjects and their caregivers had considerable difficulty in specifying the number of hours occupied by the various activities .", "accordingly , we made a major modification of the activity scale by introducing a point scale to measure the intensity of the activity , ranging from 0 ( no activity ) to 3 ( active for most of the day ) , and applying one score ( 03 points ) for each type of activity , instead of the original three parameters ( number of differentiated activities , average number of hours spent on each activity , and the ratio of the one to the other ) used by christensen and mackinnon .", "the particular elements of the activity scale were divided into intellectual , physical , and social activity .", "intellectual activity was divided into five types : ( 1 ) reading books and magazines or doing crosswords ; ( 2 ) watching tv , listening to the radio , or using a computer on the internet ; ( 3 ) personal creative activity , such as keeping a diary , working on collections , or professional work ; ( 4 ) participation in discussions or lectures ; and ( 5 ) playing chess , cards , or logical games .", "physical activity was divided into three types : ( 1 ) participation in sports and recreation , such as bicycling , skiing , aerobics , or gymnastics ; ( 2 ) physical work around the home , including gardening and yard work ; and ( 3 ) going for walks , including the deliberate avoidance of mechanical transportation while attending to errands .", "social activity was divided into five types : ( 1 ) participation in social organizations ; ( 2 ) work for others , outside of institutional work ; ( 3 ) receiving guests and visiting others ; ( 4 ) informal meetings and conversations with other people , e.g. , while walking or shopping ; and ( 5 ) playing games or having a good time with others , or going on trips with others .", "all of the elements comprising one area ( intellectual , physical , or social ) were evaluated for average involvement during the preceding months on a scale from 0 ( no involvement ) to 1 ( sporadic involvement ) to 2 ( rather frequent involvement ) to 3 ( frequent involvement ) .", "the sum of the points for each of the elements constituted the score in each area of activity .", "the scores for intellectual and social activity could range from 0 to 15 points , while the physical activity score could range from 0 to 9 points .", "the iadl scale was developed by m. p. lawton and e. m. brody in 1969 .", "this scale serves to evaluate functioning in daily living , and covers such activities as using the telephone , shopping , preparing meals , cleaning house , laundry , transportation , taking medications , and personal finances .", "the scores for particular functions ranged from 1 ( full functionality ) to a maximum of 3 , 4 , or 5 , depending on which element was evaluated .", "the overall score ranged from 8 ( no dysfunction ) to 31 ( no possibility of independent functioning ) .", "the mmse was re - administered to all patients in the study group after approximately one year ( in practice , this occurred from 9 to 13 months after the baseline ) .", "the next administration of the mmse took place when the observation period ended : that is , either at the point when dementia was diagnosed or after seven years of observation .", "because all of these individuals were under the immediate care of the first and second authors of the present study , their mental state was monitored systematically , usually several times a year . at each successive psychiatric consultation ,", "if dementia was diagnosed , further tests were performed , including laboratory and radiological tests , in order to established the etiology .", "for statistical purposes , we used the two - tailed test for two independent means .", "only those test results that were equal to or less than 0.05 ( that is , p0.05 ) were regarded as statistically significant .", "the chi - squared test was used to verify the assumption of normal distribution in the research population ( using tests for two means ) , whereas the assumption of equal variance was verified with the test for two variances . according to the guidelines of the helsinki declaration ( 2008 ) ,", "subjects participating in the experiment were informed in detail about the test procedure and provided written consent for participation in the project .", "the study protocols received ethical approval from the ethical committee of the regional medical chamber nkbbn/279/2014 .", "according to the guidelines of the helsinki declaration ( 2008 ) , subjects participating in the experiment were informed in detail about the test procedure and provided written consent for participation in the project .", "the study protocols received ethical approval from the ethical committee of the regional medical chamber nkbbn/279/2014 .", "of the 193 persons initially enrolled for the study , 75 were available for the final analysis because they had either gone through the preset limit of seven years or had been diagnosed with dementia , regardless of how much time had passed since the baseline . during the observation period , dementia appeared in 34 subjects , including 16 with dat , 4 with vascular dementia , 2 with lewy - body dementia , 3 with fronto - temporal dementia , and 9 for whom there was either no basis for establishing the etiology at the time when the results were being analyzed , or a mixed dementia had been diagnosed .", "the types of dementia were not subjected to further analysis , due both to the small numbers and to the relatively large percentage of patients with no final ( etiological ) diagnosis . based on the final clinical diagnosis , in which dementia was either confirmed or precluded , we divided the subjects into two groups : those with mci that had converted to dementia at some point within the seven - year period ( n=34 ) , referred to hereinafter as mci - c .", "those whose mci was stable , i.e. , they had not developed dementia within that same period ( n=41 ) , hereinafter referred to as mci - s .", "table 1 gives the average values of the analyzed variables for those persons who completed the planned observation period ( n=75 ) .", "( mmse - c ) , are given for the baseline ( i ) and second examination after one year ( ii ) , with the differences between the baseline and second examinations given on the row below ; the results at the end of the study are given as mmse - c vii .", "it was found that there were no statistically significant differences in the baseline mmse between the persons with stable mci and the persons who had progressed to dementia .", "table 1 also provides the means from the two activity scales : the global score for the iadl and the scores for the three main areas of activity ( a - intellectual , a - physical , a - social ) and the global score ( a - global ) on the activity scale .", "table 2 presents the means for the analyzed variables in the two study groups : those with mci - s and those with mci - c , regardless of the etiology .", "there were statistically significant differences between the groups in the global scores from both instruments measuring activity .", "the patients from the two groups also had significantly different scores in the physical activity subscale and in four of the subscales of the iadl : telephone use ( iadl - tele ) , shopping ( iadl - shop ) , transportation ( iadl - tran ) , and personal finances ( iadl - pfin ) . in table 3 , the basis for dividing the subjects into two groups was the difference between the mmse scores at baseline and at the second examination ( after one year ) . based on the average difference ( 1.75 points ) , we divided the population into one group with a smaller difference ( mmse - c i ii : s ) and one group with a greater difference ( mmse - c i ii : g ) between the adjusted scores on the mmse - c i ii .", "table 4 shows the number of persons presenting a low level of physical activity ( 14 points ) and a high level of physical activity ( 510 points ) in two groups of patients : those with mci - c and with mci - s . the basis for distinguishing between a low level and a high level of physical activity was the average score in this area ( 4.68 points ) for the entire research group .", "the chi - squared result at df=1 was 13.38 ( p=0.0003 ) . with the yeats correction ,", "a diagnosis of mci carries with it a substantial risk that dementia will develop later . a significant number of persons with mci show symptoms of dementia within several years . on the other hand , many others will not experience any further loss of cognitive functions . in predicting what will happen", "in the course of mci , an evaluation of the level of activity can play an important role , as indicated by many previous studies of dementia . in our research , we attempted to evaluate the level of activity manifested by persons with mci in light of their further history in terms of cognitive functions .", "the basis for this evaluation was our modification of the activity scale developed by christensen and mackinnon , which enabled us to evaluate three areas of activity : intellectual , physical , and social .", "activity in these three areas was evaluated in respect to particular activities performed in the course of one month preceding each examination . on the one hand", ", this approach could cause some activities to be overlooked , but on the other , it enabled us to make , in our opinion , a more precise evaluation .", "the previous effort to estimate activity in a more global framework , and to express it in terms of the number of hours spent ( e.g. , in the course of a week ) , even if particular kinds of activity were taken into account ( intellectual , physical , social ) , seems to entail a greater risk of error due to the retrospective and highly subjective nature of the judgments involved , especially in regards to the various domains of activity .", "this scale evaluates the level of disturbances in the performance of basic social activities , such as shopping , transportation , etc . in research on the preclinical phase of dementia , reduced competence in activities of daily", "mci - c and mci - s , we did not find at baseline any major difficulties in activities of daily living that would impair social functioning , as indicated by the average iadl scores in both groups : 12.71 and 10.46 respectively , where the minimum score is 8 .", "however , despite the very slight difficulties experienced by persons with mci , the scores on the scale ( both the overall score and the subscores for some of its elements ) proved to be significant discriminating factors for persons with greater progression of cognitive symptoms , culminating in dementia with onset during the observation period . in our research , the elements of daily functioning in which difficulties appear sooner include using the telephone , shopping , using transportation , and personal finances . in other studies ,", "similar aspects of functioning have also been identified in the preclinical phase of dementia , where the earliest difficulties appear .", "it is generally recognized that losing the ability to use the telephone , avoiding the need to use transportation , poor management of personal finances , and problems with proper self - administration of medications can be predictive of the imminent onset of the disease .", "differences in scores ( shopping and taking medications ) seem rather to result from certain differences in functioning caused by social circumstances .", "for example , the persons qualified for some studies available in the literature most often did not prepare meals without assistance , which would make it more difficult to measure any possible problems in the area of shopping .", "an analysis of the result from the activity scale ( our own modification of the christensen and mackinnon scale ) points up a lower level of activity among those persons who declined into dementia during the seven - year observation period .", "although the persons from our mci - c group ( conversion to dementia ) had lower scores on the activity scale in each domain , this tendency still was confirmed statistically only in respect to physical activity .", "the lack of statistical significance in respect to intellectual activity can probably be explained on methodological grounds .", "the particular elements that contribute to the physical activity score consist of facts that are clearly delineated in time and differentiated from daily routine . by contrast , the particular activities that characterize intellectual activity may be harder to evaluate due to the difficulty inherent in distinguishing them from other activities being performed at the same time .", "one gets the general impression that in much of the research to date , more attention has been paid to intellectual activity , which can be related more directly to the concept of neurocognitive reserve .", "tasks that require considerable intellectual engagement have particularly been regarded as a protective factor against progressive loss of cognitive functions and the resultant decline into dementia .", "distinguishing the various domains of activity seems to have considerable importance because it very often points to different impacts on the process of cognitive decline . in view of the fact that the level of activity is typically evaluated retrospectively ,", "in one such study , one year s observation of persons subjected to systematic exercises involving both cognitive and physical activity demonstrated a positive clinical effect .", "a thorough analysis of the data obtained from studies of the neuroprotective effect of physical exercise ( or more generally physical activity per se ) shows a marked reduction of the risk of dementia .", "it is worth noting that this does not necessarily refer only to activity manifested just before onset , but rather to a level of activity in middle age , which is to say many years , for the most part , before the presentation of the first symptoms of cognitive decline . yet even among those persons who are already showing signs of such decline , physical activity can contribute to a milder course of the disease . some results have suggested that the activity is associated with greater volume of the hippocampus .", "other studies have suggested that physical activity can be associated with slowing the atrophy of gray matter .", "an evaluation of the neural network in functional magnetic resonance imaging tests has shown an increase in connectivity within the neural network after several months of systematic physical exercise .", "it seems that physical activity has an excitatory effect on the neurotrophic factors [ 2830 ] .", "physical activity has also been shown to have a significant impact on reducing the risk of a number of vascular factors that can also lead to dementia .", "it has also been observed that there is a rise in the level of the brain - derived neurotrophic factor in response to physical activity .", "we make a significant error , however , if we evaluate only the activity manifested by persons with mci without considering other factors as well .", "it is essential to remember that a number of important factors determine how active an individual will be , including education , physical health , physical fitness , and the status of the sensory organs .", "the level of activity an individual shows over the lifespan is significantly affected by certain personality traits , which are surprising seldom taken into account in the research .", "persons who display a more open and extraverted personality , with more activity , present with a higher level of cognitive ability later in life , as compared with persons who have other personality traits . in our own research , unfortunately , we were able to include only a few of the many factors that can affect the person s level of activity .", "our inclusion criteria eliminated from the study those persons who were afflicted by significant disorders affecting their daily functioning .", "we did not , however , perform a more detailed analysis of the sensory systems of our subjects ; moreover , their personality traits were not measured .", "the correlation between the level of activity over the lifespan and cognitive function , and thus the degree of impairment in the process of aging , can be explained at least partially by appealing to what is usually called the neurocognitive reserve .", "this concept pertains primarily to the degree of complexity of intersynaptic connections , which supports the performance of tasks that involve cognitive functions . the neurocognitive reserve is formed throughout the lifespan , but it would appear that the earlier periods of life are of key importance .", "good socioeconomic conditions , a low level of stress , and exposure to a diversified environment that requires creative adaptation , including the possibility of education all these factors seem to support the formation of a greater neurocognitive reserve , which indirectly reduces the clinical impact of the progression of cognitive decline in old age .", "although the younger years are of crucial importance for the formation of the neurocognitive reserve , a number of external factors can still have a positive or negative impact in every period of life .", "it has been demonstrated that any kind of activity that engages cognitive functions can have a protective effect .", "persons with a greater neurocognitive reserve can compensate for the sequelae of neurodegenerative processes for a much longer period of time , which causes any possible dementive illness to present much later in life , if at all .", "it would be an oversimplification , however , to consider the whole concept of mental reserve exclusively in terms of the neural network .", "the broader category of mental reserve should also include the whole set of both conscious and unconscious cognitive strategies , developed and enriched in the process of formation .", "thanks to a wide repertoire of such strategies , the individual can compensate for impaired functionality . in this way , complex cognitive functioning will have a protective impact , though it is important to clarify at this point that by protective activity we are referring to the delayed appearance of clinical signs of decline , and not to the stopping or slowing of the neurodegenerative process itself . in order to interpret properly the results reported here , some rather important limitations of the present study should be taken into account .", "there are certain objections that could be raised against the use of the mmse as the basis for the assessment of cognitive decline .", "we are fully aware that the mmse is not a very precise instrument for measurement , and does not enable the clinician to evaluate precisely any particular area of cognition .", "the purpose of our research , however , was not to make any such detailed analysis of cognitive domains , but only to assess overall functioning . moreover , and perhaps especially , the practical aspect has been dominant in research on mci .", "the tools used here are those that can be used in daily clinical practice involving a large number of subjects .", "although the mmse can be widely used in outpatient practice , many other more complex , elaborate instruments , especially those that are very time - consuming , can not be used on such a scale .", "even though we used a simplified instrument , it was possible to detect measurable differences within the first year of observation between persons who were well on their way to dementia and those who were not ( cf . , table 2 ) .", "another limitation , this one also resulting from the number of persons included in the research group , was the impossibility of taking treatment under consideration .", "although at the moment of enrollment none of the participants were taking psychotropic drugs , within the first year of observation ( the most essential period for the analysis of results ) some of them ( n=39 ) were already taking such drugs .", "the reasons for beginning treatment were typically anxiety , depressed mood , or sleep disturbances , but there were also instances of aggressive or impulsive behavior . whether or not it is actually possible to absolutely exclude the impact of such drugs on neurodegenerative mechanisms in the brain , their possible impact on the results of cognitive tests ( the mmse ) would seem to be an essential factor .", "our study was observational in character , which made it impossible to suspend treatment ( if there was any ) during the period preceding our assessment of cognitive functions .", "finally , the quantitative assessment of the level of activity displayed by individuals will always be a process subject to considerable simplification of the whole phenomenon , because a wide range of environmental factors and other factors with direct or indirect influence on activity scores [ 4042 ] are not taken into account .", "the use of quantifying instruments , though justified for methodological reasons , necessarily entails a narrowing of the field of observation . despite the importance of all these limitations ,", "the results obtained in our study seem to confirm the hypothesis ( not a new one , to be sure , but not a well - documented one , either ) that activity has a positive impact on the course of cognitive decline in advanced age . therefore , our results clearly point to the practical utility of the functional assessment of persons with mci , especially the level of activity they display , in establishing a prognosis .", "our results indicate that an assessment of the level of activity can be useful in establishing a prognosis for the future course of mci ." ]

[ "<S> backgroundour goal was to specify the relationship between the level of activity ( intellectual , physical , and social ) in persons diagnosed with mild cognitive impairment ( mci ) and the further progression of cognitive dysfunction.material/methodswe examined 193 patients diagnosed with mci ( according to the criteria of the working group on mild cognitive impairment ) and under treatment at our mental disorders clinic . it was assumed that these persons would remain under systematic psychiatric observation until dementia was diagnosed . </S>", "<S> the present study results from a seven - year observation period . the mini mental state examination ( mmse ) , the activity scale ( with the intellectual , physical , and social subscales ) , and the instrumental activities of daily living ( iadl ) scale were used to evaluate the participants status at baseline . </S>", "<S> the mmse was re - administered after one year and again at the end of the observation ( either upon diagnosis of dementia or after seven years ) . at each meeting with the participant , </S>", "<S> the clinical diagnosis was verified to determine if the patient had dementia or not . </S>", "<S> of the 193 people initially qualified for the study , 75 were available for the final analysis.resultsit was found that there was no statistically significant difference in the baseline mmse scores between the persons with stable mci and the persons who had progressed to dementia . </S>", "<S> however , statistically significant differences in the level of activity at baseline on both the global iadl scale and the activity scale between those with stable mci and those who had progressed to dementia were found . </S>", "<S> these differences were manifested in the iadl subscales for telephone use , shopping , transportation , and personal finances , and in the physical activity subscale.conclusionsan evaluation of intellectual , physical , and social activity can be useful in determining the prognosis for the future course of mci . </S>" ]

[ "Background", "Material and Methods", "Ethics statement", "Results", "Discussion", "Conclusions" ]

[ [ "the processes that lead to the gradual atrophy of brain cells , and thus to dementia , are affected by many different factors .", "some researchers have called attention to the impact of activity over the lifespan , particularly in later periods of life , on the development of dementia .", "although the results of research on this problem have been equivocal , many studies have shown that greater activity involving cognitive functions reduces the risk of dementia [ 13 ] .", "activity manifests itself in diverse ways , in respect both to professional activity and to the realization of various personal interests . in studies conducted in the mid-1990s at the second clinic for mental illnesses at the gdansk academy of medicine , undertaken to identify risk factors for dementia of the alzheimer type ( dat ) , attention was called to reduced activity in the premorbid period as a factor that occurred significantly more often in persons with dementia .", "it turned out that persons who showed a heightened risk for dementia with primary degenerative processes had limited contact with other people and a lack of complex activity , e.g. , after retirement .", "other research conducted during the same time period also showed reduced activity in the preclinical phase of dementia [ 68 ] .", "in addition , our own research pointed to a correlation between reduced activity immediately after the first manifestation of symptoms and the rate of progression of cognitive symptoms .", "the clinical manifestation of dementia is often preceded by many years of slight cognitive decline .", "it can be very difficult to differentiate the initial phases in the development of a dementive illness from other mental states that can also present with slight deterioration of cognitive functions .", "it is generally accepted that the appearance of mci is associated with a serious risk of dementia developing within a few years .", "some studies have shown that within 34 years more than 50% of persons diagnosed with mci will develop full - blown dementia . on the other hand , this means that a significant number of persons diagnosed with mci will have no further deterioration of cognitive functions .", "research has been under way for some time now to identify the factors that would facilitate an accurate prognosis for persons diagnosed with mci .", "however , despite the number of such studies , the results have been equivocal ; therefore , it remains impossible at present to evaluate at an acceptable level of probability the degree of risk that mci will develop into dementia .", "given that mci can be treated in some cases as a preliminary phase of dementia , it is hardly a surprise that the risk factors and protective factors identified in earlier research on the preclinical stages of dementia have taken on increased importance .", "among all these factors , attention has also been drawn to the level of activity manifested by persons with mci as a possible predictive factor .", "although the results achieved to date have been rather inconsistent , comparative analysis of a number of studies devoted to this problem have indicated that at least some kinds of activity manifested by study participants seem to be significant . from the methodological point of view , it is no easy task to make an accurate evaluation of the level of activity displayed by research subjects .", "many studies have taken the average number of hours of activity in the course of a week as the measure of activity , without differentiating the types of activity .", "other studies , although they have distinguished between intellectual , physical , and social activity , have not really identified the constitutive elements of any of these types .", "moreover , it is not common practice to base the evaluation of activity on a measurement method that would make it possible to quantify the results .", "the goal of our study was to specify the relationship between the level of activity ( intellectual , physical , and social ) in persons diagnosed with mci and the further progression of cognitive dysfunction ." ], [ "the initial population consisted of persons who had reported to our mental health clinic in the period from 2005 to 2007 .", "it was assumed that these individuals would be under systematic psychiatric observation until dementia was diagnosed .", "subjects were qualified for the study according to the following inclusion criteria : informed consent to participate . a diagnosis of mci based on the criteria published by the working group on mild cognitive impairment , assuming that dementia has been ruled out ; i.e. , that there has been a perceptible decline in cognitive function ( based on self - evaluation or the reports of a caregiver , and then verified objectively by an examination confirming cognitive impairment and/or objective measures of a deterioration of cognitive functions over time ) , and that basic activities of daily living are preserved , with a slight or minimal level of impairment in carrying out complex activities . no psychiatric pharmacotherapy at the time of enrollment .", "the availability of an individual from the patient s own environment , someone who lives with the patient or at least visits several times a week , and who expresses a willingness to participate in the process of evaluating the patient s activity by providing information . a score on the global deterioration scale of reisberg et al . placing the patient on the third level ( slight impairment of cognitive functions )", "a score on the mini mental state examination ( mmse ) in the range from 24 to 30 points .", "the exclusion criteria were as follows : a diagnosis of dementia , regardless of its etiology .", "the presence at examination or in the history of affective disorder , schizophrenia , alcoholism , addiction to medications or drugs , epilepsy , parkinson s disease , or intellectual handicap .", "the presence at examination or during the evaluation period of disorders of consciousness or disturbances of locomotion , vision , or hearing that would impede compliance with instructions and procedures included in the clinical scales used in this study .", "all those who were qualified for enrollment were subjected to a complete examination , which included the mmse , the activity scale , and the instrumental activity of daily living ( iadl ) scale .", "point scores of the mmse can range from 0 ( severe dementia ) to 30 ( lack of cognitive impairment ) . due to the impact of age and education on the mmse score ,", "the raw scores were adjusted for these two factors , based on the equation proposed by mungas et al . and verified for polish conditions by jozwiak et al . .", "the activity scale was developed by christensen and mackinnon in 1993 , and consists of three subscales covering intellectual ( mental ) , physical , and social activity .", "the number of activities undertaken is evaluated ( the degree of diversification of activity ) , as well as the average number of hours spent in particular activities . however , in the course of pilot research involving 10 patients who met the enrollment criteria , both the subjects and their caregivers had considerable difficulty in specifying the number of hours occupied by the various activities .", "accordingly , we made a major modification of the activity scale by introducing a point scale to measure the intensity of the activity , ranging from 0 ( no activity ) to 3 ( active for most of the day ) , and applying one score ( 03 points ) for each type of activity , instead of the original three parameters ( number of differentiated activities , average number of hours spent on each activity , and the ratio of the one to the other ) used by christensen and mackinnon .", "the particular elements of the activity scale were divided into intellectual , physical , and social activity .", "intellectual activity was divided into five types : ( 1 ) reading books and magazines or doing crosswords ; ( 2 ) watching tv , listening to the radio , or using a computer on the internet ; ( 3 ) personal creative activity , such as keeping a diary , working on collections , or professional work ; ( 4 ) participation in discussions or lectures ; and ( 5 ) playing chess , cards , or logical games .", "physical activity was divided into three types : ( 1 ) participation in sports and recreation , such as bicycling , skiing , aerobics , or gymnastics ; ( 2 ) physical work around the home , including gardening and yard work ; and ( 3 ) going for walks , including the deliberate avoidance of mechanical transportation while attending to errands .", "social activity was divided into five types : ( 1 ) participation in social organizations ; ( 2 ) work for others , outside of institutional work ; ( 3 ) receiving guests and visiting others ; ( 4 ) informal meetings and conversations with other people , e.g. , while walking or shopping ; and ( 5 ) playing games or having a good time with others , or going on trips with others .", "all of the elements comprising one area ( intellectual , physical , or social ) were evaluated for average involvement during the preceding months on a scale from 0 ( no involvement ) to 1 ( sporadic involvement ) to 2 ( rather frequent involvement ) to 3 ( frequent involvement ) .", "the sum of the points for each of the elements constituted the score in each area of activity .", "the scores for intellectual and social activity could range from 0 to 15 points , while the physical activity score could range from 0 to 9 points .", "the iadl scale was developed by m. p. lawton and e. m. brody in 1969 .", "this scale serves to evaluate functioning in daily living , and covers such activities as using the telephone , shopping , preparing meals , cleaning house , laundry , transportation , taking medications , and personal finances .", "the scores for particular functions ranged from 1 ( full functionality ) to a maximum of 3 , 4 , or 5 , depending on which element was evaluated .", "the overall score ranged from 8 ( no dysfunction ) to 31 ( no possibility of independent functioning ) .", "the mmse was re - administered to all patients in the study group after approximately one year ( in practice , this occurred from 9 to 13 months after the baseline ) .", "the next administration of the mmse took place when the observation period ended : that is , either at the point when dementia was diagnosed or after seven years of observation .", "because all of these individuals were under the immediate care of the first and second authors of the present study , their mental state was monitored systematically , usually several times a year . at each successive psychiatric consultation ,", "if dementia was diagnosed , further tests were performed , including laboratory and radiological tests , in order to established the etiology .", "for statistical purposes , we used the two - tailed test for two independent means .", "only those test results that were equal to or less than 0.05 ( that is , p0.05 ) were regarded as statistically significant .", "the chi - squared test was used to verify the assumption of normal distribution in the research population ( using tests for two means ) , whereas the assumption of equal variance was verified with the test for two variances . according to the guidelines of the helsinki declaration ( 2008 ) ,", "subjects participating in the experiment were informed in detail about the test procedure and provided written consent for participation in the project .", "the study protocols received ethical approval from the ethical committee of the regional medical chamber nkbbn/279/2014 ." ], [ "according to the guidelines of the helsinki declaration ( 2008 ) , subjects participating in the experiment were informed in detail about the test procedure and provided written consent for participation in the project .", "the study protocols received ethical approval from the ethical committee of the regional medical chamber nkbbn/279/2014 ." ], [ "of the 193 persons initially enrolled for the study , 75 were available for the final analysis because they had either gone through the preset limit of seven years or had been diagnosed with dementia , regardless of how much time had passed since the baseline . during the observation period , dementia appeared in 34 subjects , including 16 with dat , 4 with vascular dementia , 2 with lewy - body dementia , 3 with fronto - temporal dementia , and 9 for whom there was either no basis for establishing the etiology at the time when the results were being analyzed , or a mixed dementia had been diagnosed .", "the types of dementia were not subjected to further analysis , due both to the small numbers and to the relatively large percentage of patients with no final ( etiological ) diagnosis . based on the final clinical diagnosis , in which dementia was either confirmed or precluded , we divided the subjects into two groups : those with mci that had converted to dementia at some point within the seven - year period ( n=34 ) , referred to hereinafter as mci - c .", "those whose mci was stable , i.e. , they had not developed dementia within that same period ( n=41 ) , hereinafter referred to as mci - s .", "table 1 gives the average values of the analyzed variables for those persons who completed the planned observation period ( n=75 ) .", "( mmse - c ) , are given for the baseline ( i ) and second examination after one year ( ii ) , with the differences between the baseline and second examinations given on the row below ; the results at the end of the study are given as mmse - c vii .", "it was found that there were no statistically significant differences in the baseline mmse between the persons with stable mci and the persons who had progressed to dementia .", "table 1 also provides the means from the two activity scales : the global score for the iadl and the scores for the three main areas of activity ( a - intellectual , a - physical , a - social ) and the global score ( a - global ) on the activity scale .", "table 2 presents the means for the analyzed variables in the two study groups : those with mci - s and those with mci - c , regardless of the etiology .", "there were statistically significant differences between the groups in the global scores from both instruments measuring activity .", "the patients from the two groups also had significantly different scores in the physical activity subscale and in four of the subscales of the iadl : telephone use ( iadl - tele ) , shopping ( iadl - shop ) , transportation ( iadl - tran ) , and personal finances ( iadl - pfin ) . in table 3 , the basis for dividing the subjects into two groups was the difference between the mmse scores at baseline and at the second examination ( after one year ) . based on the average difference ( 1.75 points ) , we divided the population into one group with a smaller difference ( mmse - c i ii : s ) and one group with a greater difference ( mmse - c i ii : g ) between the adjusted scores on the mmse - c i ii .", "table 4 shows the number of persons presenting a low level of physical activity ( 14 points ) and a high level of physical activity ( 510 points ) in two groups of patients : those with mci - c and with mci - s . the basis for distinguishing between a low level and a high level of physical activity was the average score in this area ( 4.68 points ) for the entire research group .", "the chi - squared result at df=1 was 13.38 ( p=0.0003 ) . with the yeats correction ," ], [ "a diagnosis of mci carries with it a substantial risk that dementia will develop later . a significant number of persons with mci show symptoms of dementia within several years . on the other hand , many others will not experience any further loss of cognitive functions . in predicting what will happen", "in the course of mci , an evaluation of the level of activity can play an important role , as indicated by many previous studies of dementia . in our research , we attempted to evaluate the level of activity manifested by persons with mci in light of their further history in terms of cognitive functions .", "the basis for this evaluation was our modification of the activity scale developed by christensen and mackinnon , which enabled us to evaluate three areas of activity : intellectual , physical , and social .", "activity in these three areas was evaluated in respect to particular activities performed in the course of one month preceding each examination . on the one hand", ", this approach could cause some activities to be overlooked , but on the other , it enabled us to make , in our opinion , a more precise evaluation .", "the previous effort to estimate activity in a more global framework , and to express it in terms of the number of hours spent ( e.g. , in the course of a week ) , even if particular kinds of activity were taken into account ( intellectual , physical , social ) , seems to entail a greater risk of error due to the retrospective and highly subjective nature of the judgments involved , especially in regards to the various domains of activity .", "this scale evaluates the level of disturbances in the performance of basic social activities , such as shopping , transportation , etc . in research on the preclinical phase of dementia , reduced competence in activities of daily", "mci - c and mci - s , we did not find at baseline any major difficulties in activities of daily living that would impair social functioning , as indicated by the average iadl scores in both groups : 12.71 and 10.46 respectively , where the minimum score is 8 .", "however , despite the very slight difficulties experienced by persons with mci , the scores on the scale ( both the overall score and the subscores for some of its elements ) proved to be significant discriminating factors for persons with greater progression of cognitive symptoms , culminating in dementia with onset during the observation period . in our research , the elements of daily functioning in which difficulties appear sooner include using the telephone , shopping , using transportation , and personal finances . in other studies ,", "similar aspects of functioning have also been identified in the preclinical phase of dementia , where the earliest difficulties appear .", "it is generally recognized that losing the ability to use the telephone , avoiding the need to use transportation , poor management of personal finances , and problems with proper self - administration of medications can be predictive of the imminent onset of the disease .", "differences in scores ( shopping and taking medications ) seem rather to result from certain differences in functioning caused by social circumstances .", "for example , the persons qualified for some studies available in the literature most often did not prepare meals without assistance , which would make it more difficult to measure any possible problems in the area of shopping .", "an analysis of the result from the activity scale ( our own modification of the christensen and mackinnon scale ) points up a lower level of activity among those persons who declined into dementia during the seven - year observation period .", "although the persons from our mci - c group ( conversion to dementia ) had lower scores on the activity scale in each domain , this tendency still was confirmed statistically only in respect to physical activity .", "the lack of statistical significance in respect to intellectual activity can probably be explained on methodological grounds .", "the particular elements that contribute to the physical activity score consist of facts that are clearly delineated in time and differentiated from daily routine . by contrast , the particular activities that characterize intellectual activity may be harder to evaluate due to the difficulty inherent in distinguishing them from other activities being performed at the same time .", "one gets the general impression that in much of the research to date , more attention has been paid to intellectual activity , which can be related more directly to the concept of neurocognitive reserve .", "tasks that require considerable intellectual engagement have particularly been regarded as a protective factor against progressive loss of cognitive functions and the resultant decline into dementia .", "distinguishing the various domains of activity seems to have considerable importance because it very often points to different impacts on the process of cognitive decline . in view of the fact that the level of activity is typically evaluated retrospectively ,", "in one such study , one year s observation of persons subjected to systematic exercises involving both cognitive and physical activity demonstrated a positive clinical effect .", "a thorough analysis of the data obtained from studies of the neuroprotective effect of physical exercise ( or more generally physical activity per se ) shows a marked reduction of the risk of dementia .", "it is worth noting that this does not necessarily refer only to activity manifested just before onset , but rather to a level of activity in middle age , which is to say many years , for the most part , before the presentation of the first symptoms of cognitive decline . yet even among those persons who are already showing signs of such decline , physical activity can contribute to a milder course of the disease . some results have suggested that the activity is associated with greater volume of the hippocampus .", "other studies have suggested that physical activity can be associated with slowing the atrophy of gray matter .", "an evaluation of the neural network in functional magnetic resonance imaging tests has shown an increase in connectivity within the neural network after several months of systematic physical exercise .", "it seems that physical activity has an excitatory effect on the neurotrophic factors [ 2830 ] .", "physical activity has also been shown to have a significant impact on reducing the risk of a number of vascular factors that can also lead to dementia .", "it has also been observed that there is a rise in the level of the brain - derived neurotrophic factor in response to physical activity .", "we make a significant error , however , if we evaluate only the activity manifested by persons with mci without considering other factors as well .", "it is essential to remember that a number of important factors determine how active an individual will be , including education , physical health , physical fitness , and the status of the sensory organs .", "the level of activity an individual shows over the lifespan is significantly affected by certain personality traits , which are surprising seldom taken into account in the research .", "persons who display a more open and extraverted personality , with more activity , present with a higher level of cognitive ability later in life , as compared with persons who have other personality traits . in our own research , unfortunately , we were able to include only a few of the many factors that can affect the person s level of activity .", "our inclusion criteria eliminated from the study those persons who were afflicted by significant disorders affecting their daily functioning .", "we did not , however , perform a more detailed analysis of the sensory systems of our subjects ; moreover , their personality traits were not measured .", "the correlation between the level of activity over the lifespan and cognitive function , and thus the degree of impairment in the process of aging , can be explained at least partially by appealing to what is usually called the neurocognitive reserve .", "this concept pertains primarily to the degree of complexity of intersynaptic connections , which supports the performance of tasks that involve cognitive functions . the neurocognitive reserve is formed throughout the lifespan , but it would appear that the earlier periods of life are of key importance .", "good socioeconomic conditions , a low level of stress , and exposure to a diversified environment that requires creative adaptation , including the possibility of education all these factors seem to support the formation of a greater neurocognitive reserve , which indirectly reduces the clinical impact of the progression of cognitive decline in old age .", "although the younger years are of crucial importance for the formation of the neurocognitive reserve , a number of external factors can still have a positive or negative impact in every period of life .", "it has been demonstrated that any kind of activity that engages cognitive functions can have a protective effect .", "persons with a greater neurocognitive reserve can compensate for the sequelae of neurodegenerative processes for a much longer period of time , which causes any possible dementive illness to present much later in life , if at all .", "it would be an oversimplification , however , to consider the whole concept of mental reserve exclusively in terms of the neural network .", "the broader category of mental reserve should also include the whole set of both conscious and unconscious cognitive strategies , developed and enriched in the process of formation .", "thanks to a wide repertoire of such strategies , the individual can compensate for impaired functionality . in this way , complex cognitive functioning will have a protective impact , though it is important to clarify at this point that by protective activity we are referring to the delayed appearance of clinical signs of decline , and not to the stopping or slowing of the neurodegenerative process itself . in order to interpret properly the results reported here , some rather important limitations of the present study should be taken into account .", "there are certain objections that could be raised against the use of the mmse as the basis for the assessment of cognitive decline .", "we are fully aware that the mmse is not a very precise instrument for measurement , and does not enable the clinician to evaluate precisely any particular area of cognition .", "the purpose of our research , however , was not to make any such detailed analysis of cognitive domains , but only to assess overall functioning . moreover , and perhaps especially , the practical aspect has been dominant in research on mci .", "the tools used here are those that can be used in daily clinical practice involving a large number of subjects .", "although the mmse can be widely used in outpatient practice , many other more complex , elaborate instruments , especially those that are very time - consuming , can not be used on such a scale .", "even though we used a simplified instrument , it was possible to detect measurable differences within the first year of observation between persons who were well on their way to dementia and those who were not ( cf . , table 2 ) .", "another limitation , this one also resulting from the number of persons included in the research group , was the impossibility of taking treatment under consideration .", "although at the moment of enrollment none of the participants were taking psychotropic drugs , within the first year of observation ( the most essential period for the analysis of results ) some of them ( n=39 ) were already taking such drugs .", "the reasons for beginning treatment were typically anxiety , depressed mood , or sleep disturbances , but there were also instances of aggressive or impulsive behavior . whether or not it is actually possible to absolutely exclude the impact of such drugs on neurodegenerative mechanisms in the brain , their possible impact on the results of cognitive tests ( the mmse ) would seem to be an essential factor .", "our study was observational in character , which made it impossible to suspend treatment ( if there was any ) during the period preceding our assessment of cognitive functions .", "finally , the quantitative assessment of the level of activity displayed by individuals will always be a process subject to considerable simplification of the whole phenomenon , because a wide range of environmental factors and other factors with direct or indirect influence on activity scores [ 4042 ] are not taken into account .", "the use of quantifying instruments , though justified for methodological reasons , necessarily entails a narrowing of the field of observation . despite the importance of all these limitations ,", "the results obtained in our study seem to confirm the hypothesis ( not a new one , to be sure , but not a well - documented one , either ) that activity has a positive impact on the course of cognitive decline in advanced age . therefore , our results clearly point to the practical utility of the functional assessment of persons with mci , especially the level of activity they display , in establishing a prognosis ." ], [ "our results indicate that an assessment of the level of activity can be useful in establishing a prognosis for the future course of mci ." ] ]

[ "currently , virtually any psychiatric diagnosis is made through a combination of patient interviews , checklists , or self - report questionnaires , which rely on the symptoms coded in the diagnostic and statistical manual of mental disorders , either the fourth edition1 or the recently introduced fifth edition.2 yet , considerable debate exists about the actual validity of this sole symptom - based approach , thus sustaining the interest in objective biomarkers toward a better understanding of psychiatric disorders , including major depressive disorder ( mdd ) , and their pharmacological response.3 furthermore , a number of issues , including heterogeneous patients variables , high medical and psychiatric comorbidity rates , medications / triggers , inconsistency in specimen collection , storage and measurement protocols , and complexity of neuropsychiatric biological determinants of disorders , hinder the search for reliable biomarkers.4,5 ideally , a hassle - free biomarker reflecting the central nervous system ( cns ) functioning should provide a simplified , yet reliable , proxy measure of the activity of those neurotransmitters that are supposed to be involved in major psychiatric disturbances , to be flexibly applied both to clinical and healthy samples . among other approaches , the electroretinogram ( erg ) represents a relatively noninvasive , short , and cost - effective method developed to investigate the origin of a visual loss due to a retinal disease or injury , possibly representing a proxy of global cns activity of dopamine,6 a core monoamine in depression ,", "directly or indirectly modulated by a number of antidepressant drugs with different pharmacodynamics.7 specifically , since the retina is part of the cns due to its embryonic origin , the erg has been used to investigate not only mdd , but also seasonal affective disorder , schizophrenia and bipolar disorder , autism spectrum disorders , and drug addiction,8 both in clinical and healthy samples exposed to different pharmacological agents,817 as well as in animal samples.6,18 of note , dopamine modulates different retinal functions , including counterbalancing of the synthesis of melatonin as measured with the erg.1921 melatonergic activity is also often impaired in the course of depressive episodes with prominent circadian rhythm disturbances , even if there is no antidepressant effect firmly documented for the hormone melatonin , neither for its analogue , ramelteon.2225 this seems quite puzzling considering that the recently released pro - melatonergic antidepressant agomelatine has been approved in europe for the treatment of mdd , though not in the us.26,27 a potential explanation for the claimed antidepressant effect of agomelatine should nonetheless be represented by its 5-hydroxytryptamine receptor type 2b ( 5-ht2b ) and 5-ht2c serotonergic antagonism,28 which may also promote the dopaminergic firing at the ventral tegmental area , frontal cortex , hypothalamus , hippocampus , medulla and pons , and also the retina , which is part of the cns as well,29 via enhancement of norepinephrinergic activity at the locus coeruleus.30 yet , while there is no question about the impact of melatonergic modulation on circadian rhythms relevant for mood as well as other core functions , the discrimination of the most plausible antidepressant effect of agomelatine between 5-ht antagonism versus melatonergic receptor type 1 ( mt1 ) and type 2 ( mt2 ) agonism remains elusive , at least with regard to just the simplified paradigm of dopaminergic modulation .", "additionally , ago - melatine has been proposed to indirectly modulate even the release of glutamate from prefrontal and frontal cortex and hippocampus,31,32 which further hinders the identification of a sole putative neurobiological pathway accounting for the claimed antidepressant effect of the drug .", "therefore , the aim of this preliminary study carried out on healthy volunteers was to assess the impact of agomelatine on retinal dopaminergic activity measured using standard erg recording , to provide a dissertation on the potential clinical implications of a differential neuropsychopharmacological erg trend eventually observed for agomelatine versus the trend already documented for melatonin .", "the light reaches the photoreceptors ( either cones or rods ) hosted at the outer segment of the retina after crossing the anterior segment of the eye , then is absorbed by the photopigment of the photoreceptors , which ignites the phototransduction process . in dark conditions ,", "photoreceptors rest in a depolarized state , while the photon absorption leads to their hyperpolarization followed by the depolarization of the on bipolar cells .", "subsequently , the electric signal spreads to the ganglion cells , the axons of which reach the brain , mainly converging at the visual cortex .", "the retina also includes horizontal cells and amacrine cells , which are interneurons joining photoreceptors and bipolar cells , as well as the mller cells acting as glia.8 since dopamine represents a core retinal neurotransmitter involved in signal transduction , the erg should be a convenient technique by which to assess such dopaminergic activity both in light- and dark - adapted conditions,33 essentially by measurement of the implicit time and amplitude parameters of the b - wave,34 a trace component of the erg primarily determined by the bipolar cells.3537 any variation of the intensity or chromatic characteristic of the light stimulus could also be assessed by the erg , either for rods , cones , or mixed rod / cone photoreceptors functioning , thus allowing an objective quantitative measure of the retinal sensitivity to light.34 dopamine released by the amacrine and interplexiform cells38 interacts with the d1-like receptors ( namely , d1 and d5 subtypes ) at the horizontal , bipolar , amacrine , and ganglion cells , and with the d2-like ( d2 , d3 , and d4 ) receptors located at the retinal pigment epithelium cells , photoreceptors , and mller glial cells,3943 all of which are involved in the modulation of a number of retinal functions , including melatonin release.20 also , the daily synthesis and release of retinal dopamine , which is primarily influenced by lighting condition , follows a 24-hour rhythm,44 being influenced by the interaction between the amacrine and interplexiform dopaminergic neurons and photoreceptors .", "enhancement of dopamine activity via activation of d2-like receptors ( namely d4 ones ) located on photoreceptors inhibits the synthesis of melatonin , leading to subsequent inhibition of serotonergic n - acetyltransferase activity.45,46 in mammals , the d4 receptor accounts for the effects of the ligands on the whole d2 receptor family,42 either at the rods , cones , or retinal pigment epithelium,47 which , in turn , ultimately account for the impact of dopamine in the regulation of melatonin biosynthesis in vertebrate retina.48 substance p and dynorphin , which are expressed in d1 receptor - containing neurons , as well as preproenkephalin in d2 receptor - containing neurons , have also been used as monitors of dopaminergic activity in the cns,49 whereas there have been conflicting reports as to whether d1-like receptors are capable of increasing or decreasing the potassium efflux : d1-like agonists increase potassium current from chick retinal cells via an adenosine monophosphate ( amp)-independent mechanism , but inhibit this efflux in rat striatal neurons;50 as such , a conclusive understanding of the actual effect of d1 ( and possibly d5 ) stimulation or inhibition at the retina likewise remains elusive.51 melatonin suppresses the retinal release of dopamine via activation of its own melatonergic receptors ( primarily mt1 , but also mt2 and mt3 subtypes ) , widely distributed in the cns,52 including the retinal amacrine and interplexiform dopaminergic neurons.53,54 as mentioned previously , the b - wave response of the erg is influenced both by dopaminergic and melatonergic modulation .", "specifically , in daylight conditions , the b - wave response seems linked to cone dominance , possibly related to a shift toward a relative dominance of dopaminergic tone . on the contrary , under scotopic conditions , the b - wave response could be influenced by a relative dominance of rod tone,55 when the presence of circulating melatonin could account for rod dominance.56 erg studies carried out on healthy volunteers receiving melatonin , performed during conditions usually characterized by the absence of the hormone itself , showed a decline of cone response , either recorded as a decrease of the b - wave amplitude at 10 mg / day,57 or as a significant reduction of cones erg maximal response associated to a prolonged implicit time with 15 mg / day melatonin.58 concordant results were recently documented in animal samples exposed to high doses of melatonin during the day ( 90 mg / day in anaesthetized beagle dogs ) , showing a decrease of the photopic amplitude of both a- and b - waves , but no impact on the implicit time , which may indicate that the negative impact of melatonin on the photopic system may promote night vision.21 agomelatine shares the mt1 and mt2 agonism provided by melatonin , but not the agonism on mt3 receptors , with mt3 receptors having not been documented in mammalian samples.59 by blocking 5-ht2c receptors , agomelatine promotes the cns release of dopamine , possibly at the retina as well .", "however , at the time of writing , there are no available data whatsoever about the investigation of agomelatine using the flash erg in contrast to previous evidence for melatonin , this being the aim of this preliminary study .", "twenty - three healthy volunteers , both sexes , were enrolled at the department of neuroscience of the san martino hospital of genoa , genoa , italy , between september 2012 and march 2013 , upon approval by the local ethical committee and patient signatures on a valid informed consent form after procedures had been fully explained by the investigators ( either psychiatrists or neurologists ) .", "all procedures were carried out in accordance with the declaration of helsinki about human experimentation.60 no liver function monitoring was needed due to the prescription of just a single dose of agomelatine . in order to avoid any age - dependent effect affecting the b - wave amplitude,61 all subjects were aged between 22 and 35 years old .", "in addition , all subjects received the same single dose of agomelatine ( 25 mg / day ) , avoiding any concomitant medication to limit potential confounding biases as much as possible .", "similar considerations led to the inclusion of female subjects only during the luteal phase of the menstrual cycle ( objective measure of luteinizing hormone levels : range 120", "iu / l ) in order to avoid an additional confounding factor potentially affecting the b - wave amplitude.62 finally , the baseline erg assessments ( with each session requiring about 1 hour for completion ) were performed according to the following scheme : 1 ) first erg recording at 10 am ( baseline = t0 ) , immediately followed by oral intake of agomelatine 25 mg ( one pill ) ; 2 ) 1 hour s rest ; 3 ) second erg recording ; 4 ) 1 ( more ) hour s rest ; 5 ) third ( final ) erg measurement .", "finally , a pictorial description of the standard erg has been provided in figure 1 along with a synthetic flow of study procedures in figure 2 .", "the electric signal was recorded binocularly ( dilated pupils ) using 9 mm silver / silver chloride skin disc electrodes placed on the lower eyelid ; the reference electrodes were placed close to the outer canthus of the eye and the ground electrode was placed on the forehead .", "the electrode impedance was maintained below 5 k in order to provide consistent results independent of the response yielded by skin electrodes with lower amplitude and higher noise levels with regard to either the corneal or conjunctival electrodes .", "the band - pass filter was set between 0.1 hz and 500 hz , and the subjects were dark - adapted for 20 minutes before recording .", "a full - field ganzfeld stimulator was used throughout the study , with the subjects seated in front of the stimulator bowl fixating on a point incorporated in the stimulus dome during each session .", "the dark - adapted 0.01 erg ( rod response ) was recorded using a dim white flash of 0.01 cdsm2 .", "after 10 minutes of light adaptation , the light - adapted 3.0 erg ( single - flash cone response ) was recorded , using a 3 cdsm2 stimulus with a background luminance of 30 cd / m2 measured at the surface of the full - field stimulus bowl .", "b - wave amplitude and time to peak ( implicit time ) were measured ( ms ) for all ergs .", "the a - wave amplitude was measured from baseline to a - wave trough , while the b - wave amplitude was measured from a - wave trough to b - wave peak ( v ) ; the a - wave and b - wave implicit times were measured from the time of the flash to the peak of each wave .", "demographic and descriptive analyses were carried out using the ibm spss software package ( v 21 ) for microsoft windows ( v 7 ) ( ibm corporation , armonk , ny , usa ) . since data follow a nonparametric distribution , as assessed by the shapiro wilk test , friedman s analysis of variance was used in order to compare the median scores of the erg parameters .", "the light reaches the photoreceptors ( either cones or rods ) hosted at the outer segment of the retina after crossing the anterior segment of the eye , then is absorbed by the photopigment of the photoreceptors , which ignites the phototransduction process . in dark conditions ,", "photoreceptors rest in a depolarized state , while the photon absorption leads to their hyperpolarization followed by the depolarization of the on bipolar cells .", "subsequently , the electric signal spreads to the ganglion cells , the axons of which reach the brain , mainly converging at the visual cortex .", "the retina also includes horizontal cells and amacrine cells , which are interneurons joining photoreceptors and bipolar cells , as well as the mller cells acting as glia.8 since dopamine represents a core retinal neurotransmitter involved in signal transduction , the erg should be a convenient technique by which to assess such dopaminergic activity both in light- and dark - adapted conditions,33 essentially by measurement of the implicit time and amplitude parameters of the b - wave,34 a trace component of the erg primarily determined by the bipolar cells.3537 any variation of the intensity or chromatic characteristic of the light stimulus could also be assessed by the erg , either for rods , cones , or mixed rod / cone photoreceptors functioning , thus allowing an objective quantitative measure of the retinal sensitivity to light.34 dopamine released by the amacrine and interplexiform cells38 interacts with the d1-like receptors ( namely , d1 and d5 subtypes ) at the horizontal , bipolar , amacrine , and ganglion cells , and with the d2-like ( d2 , d3 , and d4 ) receptors located at the retinal pigment epithelium cells , photoreceptors , and mller glial cells,3943 all of which are involved in the modulation of a number of retinal functions , including melatonin release.20 also , the daily synthesis and release of retinal dopamine , which is primarily influenced by lighting condition , follows a 24-hour rhythm,44 being influenced by the interaction between the amacrine and interplexiform dopaminergic neurons and photoreceptors .", "enhancement of dopamine activity via activation of d2-like receptors ( namely d4 ones ) located on photoreceptors inhibits the synthesis of melatonin , leading to subsequent inhibition of serotonergic n - acetyltransferase activity.45,46 in mammals , the d4 receptor accounts for the effects of the ligands on the whole d2 receptor family,42 either at the rods , cones , or retinal pigment epithelium,47 which , in turn , ultimately account for the impact of dopamine in the regulation of melatonin biosynthesis in vertebrate retina.48 substance p and dynorphin , which are expressed in d1 receptor - containing neurons , as well as preproenkephalin in d2 receptor - containing neurons , have also been used as monitors of dopaminergic activity in the cns,49 whereas there have been conflicting reports as to whether d1-like receptors are capable of increasing or decreasing the potassium efflux : d1-like agonists increase potassium current from chick retinal cells via an adenosine monophosphate ( amp)-independent mechanism , but inhibit this efflux in rat striatal neurons;50 as such , a conclusive understanding of the actual effect of d1 ( and possibly d5 ) stimulation or inhibition at the retina likewise remains elusive.51 melatonin suppresses the retinal release of dopamine via activation of its own melatonergic receptors ( primarily mt1 , but also mt2 and mt3 subtypes ) , widely distributed in the cns,52 including the retinal amacrine and interplexiform dopaminergic neurons.53,54 as mentioned previously , the b - wave response of the erg is influenced both by dopaminergic and melatonergic modulation .", "specifically , in daylight conditions , the b - wave response seems linked to cone dominance , possibly related to a shift toward a relative dominance of dopaminergic tone . on the contrary , under scotopic conditions , the b - wave response could be influenced by a relative dominance of rod tone,55 when the presence of circulating melatonin could account for rod dominance.56 erg studies carried out on healthy volunteers receiving melatonin , performed during conditions usually characterized by the absence of the hormone itself , showed a decline of cone response , either recorded as a decrease of the b - wave amplitude at 10 mg / day,57 or as a significant reduction of cones erg maximal response associated to a prolonged implicit time with 15 mg / day melatonin.58 concordant results were recently documented in animal samples exposed to high doses of melatonin during the day ( 90 mg / day in anaesthetized beagle dogs ) , showing a decrease of the photopic amplitude of both a- and b - waves , but no impact on the implicit time , which may indicate that the negative impact of melatonin on the photopic system may promote night vision.21 agomelatine shares the mt1 and mt2 agonism provided by melatonin , but not the agonism on mt3 receptors , with mt3 receptors having not been documented in mammalian samples.59 by blocking 5-ht2c receptors , agomelatine promotes the cns release of dopamine , possibly at the retina as well .", "however , at the time of writing , there are no available data whatsoever about the investigation of agomelatine using the flash erg in contrast to previous evidence for melatonin , this being the aim of this preliminary study .", "twenty - three healthy volunteers , both sexes , were enrolled at the department of neuroscience of the san martino hospital of genoa , genoa , italy , between september 2012 and march 2013 , upon approval by the local ethical committee and patient signatures on a valid informed consent form after procedures had been fully explained by the investigators ( either psychiatrists or neurologists ) .", "all procedures were carried out in accordance with the declaration of helsinki about human experimentation.60 no liver function monitoring was needed due to the prescription of just a single dose of agomelatine . in order to avoid any age - dependent effect affecting the b - wave amplitude,61 all subjects were aged between 22 and 35 years old .", "in addition , all subjects received the same single dose of agomelatine ( 25 mg / day ) , avoiding any concomitant medication to limit potential confounding biases as much as possible .", "similar considerations led to the inclusion of female subjects only during the luteal phase of the menstrual cycle ( objective measure of luteinizing hormone levels : range 120", "iu / l ) in order to avoid an additional confounding factor potentially affecting the b - wave amplitude.62 finally , the baseline erg assessments ( with each session requiring about 1 hour for completion ) were performed according to the following scheme : 1 ) first erg recording at 10 am ( baseline = t0 ) , immediately followed by oral intake of agomelatine 25 mg ( one pill ) ; 2 ) 1 hour s rest ; 3 ) second erg recording ; 4 ) 1 ( more ) hour s rest ; 5 ) third ( final ) erg measurement .", "finally , a pictorial description of the standard erg has been provided in figure 1 along with a synthetic flow of study procedures in figure 2 .", "the electric signal was recorded binocularly ( dilated pupils ) using 9 mm silver / silver chloride skin disc electrodes placed on the lower eyelid ; the reference electrodes were placed close to the outer canthus of the eye and the ground electrode was placed on the forehead .", "the electrode impedance was maintained below 5 k in order to provide consistent results independent of the response yielded by skin electrodes with lower amplitude and higher noise levels with regard to either the corneal or conjunctival electrodes .", "the band - pass filter was set between 0.1 hz and 500 hz , and the subjects were dark - adapted for 20 minutes before recording .", "a full - field ganzfeld stimulator was used throughout the study , with the subjects seated in front of the stimulator bowl fixating on a point incorporated in the stimulus dome during each session .", "the dark - adapted 0.01 erg ( rod response ) was recorded using a dim white flash of 0.01 cdsm2 .", "after 10 minutes of light adaptation , the light - adapted 3.0 erg ( single - flash cone response ) was recorded , using a 3 cdsm2 stimulus with a background luminance of 30 cd / m2 measured at the surface of the full - field stimulus bowl .", "b - wave amplitude and time to peak ( implicit time ) were measured ( ms ) for all ergs .", "the a - wave amplitude was measured from baseline to a - wave trough , while the b - wave amplitude was measured from a - wave trough to b - wave peak ( v ) ; the a - wave and b - wave implicit times were measured from the time of the flash to the peak of each wave .", "demographic and descriptive analyses were carried out using the ibm spss software package ( v 21 ) for microsoft windows ( v 7 ) ( ibm corporation , armonk , ny , usa ) . since data follow a nonparametric distribution , as assessed by the shapiro wilk test , friedman s analysis of variance was used in order to compare the median scores of the erg parameters .", "twenty - three subjects ( females n=8 or 35% of the sample ; males n=15 or 65% ) were included in the study and completed all the scheduled appointments and procedures .", "notably , among different measures of the b - wave parameters for both eyes ( including latency and amplitude either for cones or rods , and for erg maximal response ) , the only statistically significant modifications observed across the study were those related to cones b - wave amplitude and latency , in both eyes ( table 1 ) .", "finally , no statistically significant difference was observed for such parameters in males versus females .", "the absence of a control group , including differential doses of agomelatine , placebo , or melatonin , or depressed controls , make the present results merely indicative at this time .", "moreover , while dopamine could also affect the erg , its effect was basically observed in terms of changes in the retinal sensitivity.63 therefore , in this study , the lack of a luminance response function to measure retinal sensitivity with the erg prompts for caution in the interpretation of our preliminary findings .", "oscillatory potentials most likely generated by the dopaminergic neurons of the amacrine ( or interplexiform ) cells , should also be accounted for in replication studies .", "similarly , we followed the international society for clinical electrophysiology of vision ( iscev ) standard64 to record the erg , which represents the minimum standard flashes to be used to investigate the retinal function ( for patients with an eye disease ) ; however , in terms of mere research purposes , assessing the dynamic of retinal functioning over a wide range of intensities should allow a more reliable assessment of the retinal sensitivity .", "specifically , this latter consideration may have hampered the validity of the present preliminary protocol in the investigation of a possible impact of the erg . in our sample , agomelatine induced a slight , yet statistically significant , increase of the cones b - wave amplitude and latency in both eyes . while it may be argued that the above mentioned change in amplitude", "may actually be within the normal variation of the measure , this is nonetheless in contrast to previous reports5658 from comparable erg studies involving healthy subjects receiving melatonin . while both melatonin and agomelatine act on melatonergic receptors , the presence of 5-ht2c ( and 5-ht2b ) antagonism for the antidepressant drug should be prudently hypothesized to account for the differential trend observed at erg , eventually observed to play a role in the therapeutic effect claimed in depressed samples .", "notably , a previous polysomnographic study carried out on a small sample of healthy volunteers with the same dose of agomelatine ( 25 mg / day ) raised doubts about the actual blockade of 5-ht2c receptors in humans , based on the observation that this agent failed to increase slow - wave sleep , whereas potent 5-ht2c antagonists ( eg , cyproheptadine 4 mg , ritanserin 5 mg , olanzapine 5 mg , or mirtazapine 30 mg ) did.65 furthermore , in a recent animal study , neither the long - term administration of melatonin ( 40 mg / kg / day ) nor the selective 5-ht2c receptor antagonist sb 242084 ( 0.5 mg / kg / day ) had an effect on the firing rate and burst parameters of serotonergic and dopaminergic neurons .", "the combination of these latter mechanisms , however , enhanced only the number of spontaneously active dopaminergic neurons at the ventral tegmental area , while leaving the firing of the serotonergic neurons unaltered at the raphe dorsal nucleus .", "finally , the addition of a specific 5-ht2b antagonist ( ly 266097 at 0.6 mg / kg / day ) , which proved by itself to be devoid of effect , to the previous dual regimen increased the number of bursts per minute of dopaminergic neurons and the percentage of spikes occurring in burst , which ultimately mimics the effect of the antidepressant agomelatine.66", "the absence of a control group , including differential doses of agomelatine , placebo , or melatonin , or depressed controls , make the present results merely indicative at this time .", "moreover , while dopamine could also affect the erg , its effect was basically observed in terms of changes in the retinal sensitivity.63 therefore , in this study , the lack of a luminance response function to measure retinal sensitivity with the erg prompts for caution in the interpretation of our preliminary findings .", "oscillatory potentials most likely generated by the dopaminergic neurons of the amacrine ( or interplexiform ) cells , should also be accounted for in replication studies .", "similarly , we followed the international society for clinical electrophysiology of vision ( iscev ) standard64 to record the erg , which represents the minimum standard flashes to be used to investigate the retinal function ( for patients with an eye disease ) ; however , in terms of mere research purposes , assessing the dynamic of retinal functioning over a wide range of intensities should allow a more reliable assessment of the retinal sensitivity .", "specifically , this latter consideration may have hampered the validity of the present preliminary protocol in the investigation of a possible impact of the erg .", "in our sample , agomelatine induced a slight , yet statistically significant , increase of the cones b - wave amplitude and latency in both eyes . while it may be argued that the above mentioned change in amplitude", "may actually be within the normal variation of the measure , this is nonetheless in contrast to previous reports5658 from comparable erg studies involving healthy subjects receiving melatonin .", "while both melatonin and agomelatine act on melatonergic receptors , the presence of 5-ht2c ( and 5-ht2b ) antagonism for the antidepressant drug should be prudently hypothesized to account for the differential trend observed at erg , eventually observed to play a role in the therapeutic effect claimed in depressed samples .", "notably , a previous polysomnographic study carried out on a small sample of healthy volunteers with the same dose of agomelatine ( 25 mg / day ) raised doubts about the actual blockade of 5-ht2c receptors in humans , based on the observation that this agent failed to increase slow - wave sleep , whereas potent 5-ht2c antagonists ( eg , cyproheptadine 4 mg , ritanserin 5 mg , olanzapine 5 mg , or mirtazapine 30 mg ) did.65 furthermore , in a recent animal study , neither the long - term administration of melatonin ( 40 mg / kg / day ) nor the selective 5-ht2c receptor antagonist sb 242084 ( 0.5 mg / kg / day ) had an effect on the firing rate and burst parameters of serotonergic and dopaminergic neurons . the combination of these latter mechanisms , however , enhanced only the number of spontaneously active dopaminergic neurons at the ventral tegmental area , while leaving the firing of the serotonergic neurons unaltered at the raphe dorsal nucleus .", "finally , the addition of a specific 5-ht2b antagonist ( ly 266097 at 0.6 mg / kg / day ) , which proved by itself to be devoid of effect , to the previous dual regimen increased the number of bursts per minute of dopaminergic neurons and the percentage of spikes occurring in burst , which ultimately mimics the effect of the antidepressant agomelatine.66", "agomelatine may differ from melatonin not only in the absence of mt3 agonism , but also in the presence of a dual antagonism on 5-ht2c and 5-ht2b , which may corroborate the claimed clinical antidepressant effect and account for the increase of the b - wave amplitude and latency of the cones ( in daylight conditions ) .", "nonetheless , along with the abovementioned limitations urging much prudence in the interpretation of our results and the acknowledgement that a decline in the central serotonin activity may itself affect the cone b - wave latency , it pays to note that the classical paradigm of monoamines ( including dopamine ) as a core pathway involved in depression has been debated , shifting more attention toward impaired circadian rhythms in depression.67 therefore , with the ultimate goal of shedding further light on the mechanism of action of novel antidepressants , with agomelatine being the case in point , additional , more methodologically accurate erg studies on the matter are warranted ." ]

[ "<S> backgroundagomelatine , the first melatonergic antidepressant , has been postulated to enhance the dopaminergic activity at the central nervous system by 5-hydroxytryptamine receptor type 2c ( 5-ht2c ) antagonism , yet the impact of melatonergic agonism on this pathway is unclear . </S>", "<S> previous studies employing simplified , yet reliable , proxy ( retinal ) measures of the central nervous system dopaminergic activity , namely the standard electroretinogram ( erg ) technique , suggested a reduction of the dopaminergic activity of the main erg parameter , the b - wave , by pure melatonin , notably a hormone devoid of any antidepressant activity . </S>", "<S> therefore , the antidepressant effects of the melatonergic antidepressant drug agomelatine should be reflected by a differential b - wave trend at erg versus the effect exerted by pure melatonin , which was eventually found to be due to a contrasting effect on central dopaminergic transmission between the two drugs.objective and methodsthe aim of the present preliminary erg study carried out on healthy volunteers ( n=23 ) receiving agomelatine was to explore the impact of this antidepressant drug on b - wave amplitude and latency of cones in daylight conditions using standard erg.resultsas postulated , agomelatine induced an enhancement of retinal dopaminergic activity , in contrast to what has been previously documented for melatonin.conclusiongiven the limits of this explorative study , especially the lack of a control group and that of a luminance response function to measure retinal sensitivity , further studies in clinical samples are recommended to allow more tenable conclusions about the potential role of erg in discriminating between 5-ht antagonism and melatonergic ( mt ) agonism in relationship to the claimed antidepressant effect of agomelatine . </S>" ]

[ "Introduction", "Methods", "The flash ERG: essential neuropsychopharmacological foundations", "Study subjects", "Stimulation and recording", "Statistical analysis", "Results", "Discussion", "Limitations of the study", "Clinical and neuropsychopharmacological implications of study results", "Conclusion" ]

[ [ "currently , virtually any psychiatric diagnosis is made through a combination of patient interviews , checklists , or self - report questionnaires , which rely on the symptoms coded in the diagnostic and statistical manual of mental disorders , either the fourth edition1 or the recently introduced fifth edition.2 yet , considerable debate exists about the actual validity of this sole symptom - based approach , thus sustaining the interest in objective biomarkers toward a better understanding of psychiatric disorders , including major depressive disorder ( mdd ) , and their pharmacological response.3 furthermore , a number of issues , including heterogeneous patients variables , high medical and psychiatric comorbidity rates , medications / triggers , inconsistency in specimen collection , storage and measurement protocols , and complexity of neuropsychiatric biological determinants of disorders , hinder the search for reliable biomarkers.4,5 ideally , a hassle - free biomarker reflecting the central nervous system ( cns ) functioning should provide a simplified , yet reliable , proxy measure of the activity of those neurotransmitters that are supposed to be involved in major psychiatric disturbances , to be flexibly applied both to clinical and healthy samples . among other approaches , the electroretinogram ( erg ) represents a relatively noninvasive , short , and cost - effective method developed to investigate the origin of a visual loss due to a retinal disease or injury , possibly representing a proxy of global cns activity of dopamine,6 a core monoamine in depression ,", "directly or indirectly modulated by a number of antidepressant drugs with different pharmacodynamics.7 specifically , since the retina is part of the cns due to its embryonic origin , the erg has been used to investigate not only mdd , but also seasonal affective disorder , schizophrenia and bipolar disorder , autism spectrum disorders , and drug addiction,8 both in clinical and healthy samples exposed to different pharmacological agents,817 as well as in animal samples.6,18 of note , dopamine modulates different retinal functions , including counterbalancing of the synthesis of melatonin as measured with the erg.1921 melatonergic activity is also often impaired in the course of depressive episodes with prominent circadian rhythm disturbances , even if there is no antidepressant effect firmly documented for the hormone melatonin , neither for its analogue , ramelteon.2225 this seems quite puzzling considering that the recently released pro - melatonergic antidepressant agomelatine has been approved in europe for the treatment of mdd , though not in the us.26,27 a potential explanation for the claimed antidepressant effect of agomelatine should nonetheless be represented by its 5-hydroxytryptamine receptor type 2b ( 5-ht2b ) and 5-ht2c serotonergic antagonism,28 which may also promote the dopaminergic firing at the ventral tegmental area , frontal cortex , hypothalamus , hippocampus , medulla and pons , and also the retina , which is part of the cns as well,29 via enhancement of norepinephrinergic activity at the locus coeruleus.30 yet , while there is no question about the impact of melatonergic modulation on circadian rhythms relevant for mood as well as other core functions , the discrimination of the most plausible antidepressant effect of agomelatine between 5-ht antagonism versus melatonergic receptor type 1 ( mt1 ) and type 2 ( mt2 ) agonism remains elusive , at least with regard to just the simplified paradigm of dopaminergic modulation .", "additionally , ago - melatine has been proposed to indirectly modulate even the release of glutamate from prefrontal and frontal cortex and hippocampus,31,32 which further hinders the identification of a sole putative neurobiological pathway accounting for the claimed antidepressant effect of the drug .", "therefore , the aim of this preliminary study carried out on healthy volunteers was to assess the impact of agomelatine on retinal dopaminergic activity measured using standard erg recording , to provide a dissertation on the potential clinical implications of a differential neuropsychopharmacological erg trend eventually observed for agomelatine versus the trend already documented for melatonin ." ], [ "the light reaches the photoreceptors ( either cones or rods ) hosted at the outer segment of the retina after crossing the anterior segment of the eye , then is absorbed by the photopigment of the photoreceptors , which ignites the phototransduction process . in dark conditions ,", "photoreceptors rest in a depolarized state , while the photon absorption leads to their hyperpolarization followed by the depolarization of the on bipolar cells .", "subsequently , the electric signal spreads to the ganglion cells , the axons of which reach the brain , mainly converging at the visual cortex .", "the retina also includes horizontal cells and amacrine cells , which are interneurons joining photoreceptors and bipolar cells , as well as the mller cells acting as glia.8 since dopamine represents a core retinal neurotransmitter involved in signal transduction , the erg should be a convenient technique by which to assess such dopaminergic activity both in light- and dark - adapted conditions,33 essentially by measurement of the implicit time and amplitude parameters of the b - wave,34 a trace component of the erg primarily determined by the bipolar cells.3537 any variation of the intensity or chromatic characteristic of the light stimulus could also be assessed by the erg , either for rods , cones , or mixed rod / cone photoreceptors functioning , thus allowing an objective quantitative measure of the retinal sensitivity to light.34 dopamine released by the amacrine and interplexiform cells38 interacts with the d1-like receptors ( namely , d1 and d5 subtypes ) at the horizontal , bipolar , amacrine , and ganglion cells , and with the d2-like ( d2 , d3 , and d4 ) receptors located at the retinal pigment epithelium cells , photoreceptors , and mller glial cells,3943 all of which are involved in the modulation of a number of retinal functions , including melatonin release.20 also , the daily synthesis and release of retinal dopamine , which is primarily influenced by lighting condition , follows a 24-hour rhythm,44 being influenced by the interaction between the amacrine and interplexiform dopaminergic neurons and photoreceptors .", "enhancement of dopamine activity via activation of d2-like receptors ( namely d4 ones ) located on photoreceptors inhibits the synthesis of melatonin , leading to subsequent inhibition of serotonergic n - acetyltransferase activity.45,46 in mammals , the d4 receptor accounts for the effects of the ligands on the whole d2 receptor family,42 either at the rods , cones , or retinal pigment epithelium,47 which , in turn , ultimately account for the impact of dopamine in the regulation of melatonin biosynthesis in vertebrate retina.48 substance p and dynorphin , which are expressed in d1 receptor - containing neurons , as well as preproenkephalin in d2 receptor - containing neurons , have also been used as monitors of dopaminergic activity in the cns,49 whereas there have been conflicting reports as to whether d1-like receptors are capable of increasing or decreasing the potassium efflux : d1-like agonists increase potassium current from chick retinal cells via an adenosine monophosphate ( amp)-independent mechanism , but inhibit this efflux in rat striatal neurons;50 as such , a conclusive understanding of the actual effect of d1 ( and possibly d5 ) stimulation or inhibition at the retina likewise remains elusive.51 melatonin suppresses the retinal release of dopamine via activation of its own melatonergic receptors ( primarily mt1 , but also mt2 and mt3 subtypes ) , widely distributed in the cns,52 including the retinal amacrine and interplexiform dopaminergic neurons.53,54 as mentioned previously , the b - wave response of the erg is influenced both by dopaminergic and melatonergic modulation .", "specifically , in daylight conditions , the b - wave response seems linked to cone dominance , possibly related to a shift toward a relative dominance of dopaminergic tone . on the contrary , under scotopic conditions , the b - wave response could be influenced by a relative dominance of rod tone,55 when the presence of circulating melatonin could account for rod dominance.56 erg studies carried out on healthy volunteers receiving melatonin , performed during conditions usually characterized by the absence of the hormone itself , showed a decline of cone response , either recorded as a decrease of the b - wave amplitude at 10 mg / day,57 or as a significant reduction of cones erg maximal response associated to a prolonged implicit time with 15 mg / day melatonin.58 concordant results were recently documented in animal samples exposed to high doses of melatonin during the day ( 90 mg / day in anaesthetized beagle dogs ) , showing a decrease of the photopic amplitude of both a- and b - waves , but no impact on the implicit time , which may indicate that the negative impact of melatonin on the photopic system may promote night vision.21 agomelatine shares the mt1 and mt2 agonism provided by melatonin , but not the agonism on mt3 receptors , with mt3 receptors having not been documented in mammalian samples.59 by blocking 5-ht2c receptors , agomelatine promotes the cns release of dopamine , possibly at the retina as well .", "however , at the time of writing , there are no available data whatsoever about the investigation of agomelatine using the flash erg in contrast to previous evidence for melatonin , this being the aim of this preliminary study .", "twenty - three healthy volunteers , both sexes , were enrolled at the department of neuroscience of the san martino hospital of genoa , genoa , italy , between september 2012 and march 2013 , upon approval by the local ethical committee and patient signatures on a valid informed consent form after procedures had been fully explained by the investigators ( either psychiatrists or neurologists ) .", "all procedures were carried out in accordance with the declaration of helsinki about human experimentation.60 no liver function monitoring was needed due to the prescription of just a single dose of agomelatine . in order to avoid any age - dependent effect affecting the b - wave amplitude,61 all subjects were aged between 22 and 35 years old .", "in addition , all subjects received the same single dose of agomelatine ( 25 mg / day ) , avoiding any concomitant medication to limit potential confounding biases as much as possible .", "similar considerations led to the inclusion of female subjects only during the luteal phase of the menstrual cycle ( objective measure of luteinizing hormone levels : range 120", "iu / l ) in order to avoid an additional confounding factor potentially affecting the b - wave amplitude.62 finally , the baseline erg assessments ( with each session requiring about 1 hour for completion ) were performed according to the following scheme : 1 ) first erg recording at 10 am ( baseline = t0 ) , immediately followed by oral intake of agomelatine 25 mg ( one pill ) ; 2 ) 1 hour s rest ; 3 ) second erg recording ; 4 ) 1 ( more ) hour s rest ; 5 ) third ( final ) erg measurement .", "finally , a pictorial description of the standard erg has been provided in figure 1 along with a synthetic flow of study procedures in figure 2 .", "the electric signal was recorded binocularly ( dilated pupils ) using 9 mm silver / silver chloride skin disc electrodes placed on the lower eyelid ; the reference electrodes were placed close to the outer canthus of the eye and the ground electrode was placed on the forehead .", "the electrode impedance was maintained below 5 k in order to provide consistent results independent of the response yielded by skin electrodes with lower amplitude and higher noise levels with regard to either the corneal or conjunctival electrodes .", "the band - pass filter was set between 0.1 hz and 500 hz , and the subjects were dark - adapted for 20 minutes before recording .", "a full - field ganzfeld stimulator was used throughout the study , with the subjects seated in front of the stimulator bowl fixating on a point incorporated in the stimulus dome during each session .", "the dark - adapted 0.01 erg ( rod response ) was recorded using a dim white flash of 0.01 cdsm2 .", "after 10 minutes of light adaptation , the light - adapted 3.0 erg ( single - flash cone response ) was recorded , using a 3 cdsm2 stimulus with a background luminance of 30 cd / m2 measured at the surface of the full - field stimulus bowl .", "b - wave amplitude and time to peak ( implicit time ) were measured ( ms ) for all ergs .", "the a - wave amplitude was measured from baseline to a - wave trough , while the b - wave amplitude was measured from a - wave trough to b - wave peak ( v ) ; the a - wave and b - wave implicit times were measured from the time of the flash to the peak of each wave .", "demographic and descriptive analyses were carried out using the ibm spss software package ( v 21 ) for microsoft windows ( v 7 ) ( ibm corporation , armonk , ny , usa ) . since data follow a nonparametric distribution , as assessed by the shapiro wilk test , friedman s analysis of variance was used in order to compare the median scores of the erg parameters ." ], [ "the light reaches the photoreceptors ( either cones or rods ) hosted at the outer segment of the retina after crossing the anterior segment of the eye , then is absorbed by the photopigment of the photoreceptors , which ignites the phototransduction process . in dark conditions ,", "photoreceptors rest in a depolarized state , while the photon absorption leads to their hyperpolarization followed by the depolarization of the on bipolar cells .", "subsequently , the electric signal spreads to the ganglion cells , the axons of which reach the brain , mainly converging at the visual cortex .", "the retina also includes horizontal cells and amacrine cells , which are interneurons joining photoreceptors and bipolar cells , as well as the mller cells acting as glia.8 since dopamine represents a core retinal neurotransmitter involved in signal transduction , the erg should be a convenient technique by which to assess such dopaminergic activity both in light- and dark - adapted conditions,33 essentially by measurement of the implicit time and amplitude parameters of the b - wave,34 a trace component of the erg primarily determined by the bipolar cells.3537 any variation of the intensity or chromatic characteristic of the light stimulus could also be assessed by the erg , either for rods , cones , or mixed rod / cone photoreceptors functioning , thus allowing an objective quantitative measure of the retinal sensitivity to light.34 dopamine released by the amacrine and interplexiform cells38 interacts with the d1-like receptors ( namely , d1 and d5 subtypes ) at the horizontal , bipolar , amacrine , and ganglion cells , and with the d2-like ( d2 , d3 , and d4 ) receptors located at the retinal pigment epithelium cells , photoreceptors , and mller glial cells,3943 all of which are involved in the modulation of a number of retinal functions , including melatonin release.20 also , the daily synthesis and release of retinal dopamine , which is primarily influenced by lighting condition , follows a 24-hour rhythm,44 being influenced by the interaction between the amacrine and interplexiform dopaminergic neurons and photoreceptors .", "enhancement of dopamine activity via activation of d2-like receptors ( namely d4 ones ) located on photoreceptors inhibits the synthesis of melatonin , leading to subsequent inhibition of serotonergic n - acetyltransferase activity.45,46 in mammals , the d4 receptor accounts for the effects of the ligands on the whole d2 receptor family,42 either at the rods , cones , or retinal pigment epithelium,47 which , in turn , ultimately account for the impact of dopamine in the regulation of melatonin biosynthesis in vertebrate retina.48 substance p and dynorphin , which are expressed in d1 receptor - containing neurons , as well as preproenkephalin in d2 receptor - containing neurons , have also been used as monitors of dopaminergic activity in the cns,49 whereas there have been conflicting reports as to whether d1-like receptors are capable of increasing or decreasing the potassium efflux : d1-like agonists increase potassium current from chick retinal cells via an adenosine monophosphate ( amp)-independent mechanism , but inhibit this efflux in rat striatal neurons;50 as such , a conclusive understanding of the actual effect of d1 ( and possibly d5 ) stimulation or inhibition at the retina likewise remains elusive.51 melatonin suppresses the retinal release of dopamine via activation of its own melatonergic receptors ( primarily mt1 , but also mt2 and mt3 subtypes ) , widely distributed in the cns,52 including the retinal amacrine and interplexiform dopaminergic neurons.53,54 as mentioned previously , the b - wave response of the erg is influenced both by dopaminergic and melatonergic modulation .", "specifically , in daylight conditions , the b - wave response seems linked to cone dominance , possibly related to a shift toward a relative dominance of dopaminergic tone . on the contrary , under scotopic conditions , the b - wave response could be influenced by a relative dominance of rod tone,55 when the presence of circulating melatonin could account for rod dominance.56 erg studies carried out on healthy volunteers receiving melatonin , performed during conditions usually characterized by the absence of the hormone itself , showed a decline of cone response , either recorded as a decrease of the b - wave amplitude at 10 mg / day,57 or as a significant reduction of cones erg maximal response associated to a prolonged implicit time with 15 mg / day melatonin.58 concordant results were recently documented in animal samples exposed to high doses of melatonin during the day ( 90 mg / day in anaesthetized beagle dogs ) , showing a decrease of the photopic amplitude of both a- and b - waves , but no impact on the implicit time , which may indicate that the negative impact of melatonin on the photopic system may promote night vision.21 agomelatine shares the mt1 and mt2 agonism provided by melatonin , but not the agonism on mt3 receptors , with mt3 receptors having not been documented in mammalian samples.59 by blocking 5-ht2c receptors , agomelatine promotes the cns release of dopamine , possibly at the retina as well .", "however , at the time of writing , there are no available data whatsoever about the investigation of agomelatine using the flash erg in contrast to previous evidence for melatonin , this being the aim of this preliminary study ." ], [ "twenty - three healthy volunteers , both sexes , were enrolled at the department of neuroscience of the san martino hospital of genoa , genoa , italy , between september 2012 and march 2013 , upon approval by the local ethical committee and patient signatures on a valid informed consent form after procedures had been fully explained by the investigators ( either psychiatrists or neurologists ) .", "all procedures were carried out in accordance with the declaration of helsinki about human experimentation.60 no liver function monitoring was needed due to the prescription of just a single dose of agomelatine . in order to avoid any age - dependent effect affecting the b - wave amplitude,61 all subjects were aged between 22 and 35 years old .", "in addition , all subjects received the same single dose of agomelatine ( 25 mg / day ) , avoiding any concomitant medication to limit potential confounding biases as much as possible .", "similar considerations led to the inclusion of female subjects only during the luteal phase of the menstrual cycle ( objective measure of luteinizing hormone levels : range 120", "iu / l ) in order to avoid an additional confounding factor potentially affecting the b - wave amplitude.62 finally , the baseline erg assessments ( with each session requiring about 1 hour for completion ) were performed according to the following scheme : 1 ) first erg recording at 10 am ( baseline = t0 ) , immediately followed by oral intake of agomelatine 25 mg ( one pill ) ; 2 ) 1 hour s rest ; 3 ) second erg recording ; 4 ) 1 ( more ) hour s rest ; 5 ) third ( final ) erg measurement .", "finally , a pictorial description of the standard erg has been provided in figure 1 along with a synthetic flow of study procedures in figure 2 ." ], [ "the electric signal was recorded binocularly ( dilated pupils ) using 9 mm silver / silver chloride skin disc electrodes placed on the lower eyelid ; the reference electrodes were placed close to the outer canthus of the eye and the ground electrode was placed on the forehead .", "the electrode impedance was maintained below 5 k in order to provide consistent results independent of the response yielded by skin electrodes with lower amplitude and higher noise levels with regard to either the corneal or conjunctival electrodes .", "the band - pass filter was set between 0.1 hz and 500 hz , and the subjects were dark - adapted for 20 minutes before recording .", "a full - field ganzfeld stimulator was used throughout the study , with the subjects seated in front of the stimulator bowl fixating on a point incorporated in the stimulus dome during each session .", "the dark - adapted 0.01 erg ( rod response ) was recorded using a dim white flash of 0.01 cdsm2 .", "after 10 minutes of light adaptation , the light - adapted 3.0 erg ( single - flash cone response ) was recorded , using a 3 cdsm2 stimulus with a background luminance of 30 cd / m2 measured at the surface of the full - field stimulus bowl .", "b - wave amplitude and time to peak ( implicit time ) were measured ( ms ) for all ergs .", "the a - wave amplitude was measured from baseline to a - wave trough , while the b - wave amplitude was measured from a - wave trough to b - wave peak ( v ) ; the a - wave and b - wave implicit times were measured from the time of the flash to the peak of each wave ." ], [ "demographic and descriptive analyses were carried out using the ibm spss software package ( v 21 ) for microsoft windows ( v 7 ) ( ibm corporation , armonk , ny , usa ) . since data follow a nonparametric distribution , as assessed by the shapiro wilk test , friedman s analysis of variance was used in order to compare the median scores of the erg parameters ." ], [ "twenty - three subjects ( females n=8 or 35% of the sample ; males n=15 or 65% ) were included in the study and completed all the scheduled appointments and procedures .", "notably , among different measures of the b - wave parameters for both eyes ( including latency and amplitude either for cones or rods , and for erg maximal response ) , the only statistically significant modifications observed across the study were those related to cones b - wave amplitude and latency , in both eyes ( table 1 ) .", "finally , no statistically significant difference was observed for such parameters in males versus females ." ], [ "the absence of a control group , including differential doses of agomelatine , placebo , or melatonin , or depressed controls , make the present results merely indicative at this time .", "moreover , while dopamine could also affect the erg , its effect was basically observed in terms of changes in the retinal sensitivity.63 therefore , in this study , the lack of a luminance response function to measure retinal sensitivity with the erg prompts for caution in the interpretation of our preliminary findings .", "oscillatory potentials most likely generated by the dopaminergic neurons of the amacrine ( or interplexiform ) cells , should also be accounted for in replication studies .", "similarly , we followed the international society for clinical electrophysiology of vision ( iscev ) standard64 to record the erg , which represents the minimum standard flashes to be used to investigate the retinal function ( for patients with an eye disease ) ; however , in terms of mere research purposes , assessing the dynamic of retinal functioning over a wide range of intensities should allow a more reliable assessment of the retinal sensitivity .", "specifically , this latter consideration may have hampered the validity of the present preliminary protocol in the investigation of a possible impact of the erg . in our sample , agomelatine induced a slight , yet statistically significant , increase of the cones b - wave amplitude and latency in both eyes . while it may be argued that the above mentioned change in amplitude", "may actually be within the normal variation of the measure , this is nonetheless in contrast to previous reports5658 from comparable erg studies involving healthy subjects receiving melatonin . while both melatonin and agomelatine act on melatonergic receptors , the presence of 5-ht2c ( and 5-ht2b ) antagonism for the antidepressant drug should be prudently hypothesized to account for the differential trend observed at erg , eventually observed to play a role in the therapeutic effect claimed in depressed samples .", "notably , a previous polysomnographic study carried out on a small sample of healthy volunteers with the same dose of agomelatine ( 25 mg / day ) raised doubts about the actual blockade of 5-ht2c receptors in humans , based on the observation that this agent failed to increase slow - wave sleep , whereas potent 5-ht2c antagonists ( eg , cyproheptadine 4 mg , ritanserin 5 mg , olanzapine 5 mg , or mirtazapine 30 mg ) did.65 furthermore , in a recent animal study , neither the long - term administration of melatonin ( 40 mg / kg / day ) nor the selective 5-ht2c receptor antagonist sb 242084 ( 0.5 mg / kg / day ) had an effect on the firing rate and burst parameters of serotonergic and dopaminergic neurons .", "the combination of these latter mechanisms , however , enhanced only the number of spontaneously active dopaminergic neurons at the ventral tegmental area , while leaving the firing of the serotonergic neurons unaltered at the raphe dorsal nucleus .", "finally , the addition of a specific 5-ht2b antagonist ( ly 266097 at 0.6 mg / kg / day ) , which proved by itself to be devoid of effect , to the previous dual regimen increased the number of bursts per minute of dopaminergic neurons and the percentage of spikes occurring in burst , which ultimately mimics the effect of the antidepressant agomelatine.66" ], [ "the absence of a control group , including differential doses of agomelatine , placebo , or melatonin , or depressed controls , make the present results merely indicative at this time .", "moreover , while dopamine could also affect the erg , its effect was basically observed in terms of changes in the retinal sensitivity.63 therefore , in this study , the lack of a luminance response function to measure retinal sensitivity with the erg prompts for caution in the interpretation of our preliminary findings .", "oscillatory potentials most likely generated by the dopaminergic neurons of the amacrine ( or interplexiform ) cells , should also be accounted for in replication studies .", "similarly , we followed the international society for clinical electrophysiology of vision ( iscev ) standard64 to record the erg , which represents the minimum standard flashes to be used to investigate the retinal function ( for patients with an eye disease ) ; however , in terms of mere research purposes , assessing the dynamic of retinal functioning over a wide range of intensities should allow a more reliable assessment of the retinal sensitivity .", "specifically , this latter consideration may have hampered the validity of the present preliminary protocol in the investigation of a possible impact of the erg ." ], [ "in our sample , agomelatine induced a slight , yet statistically significant , increase of the cones b - wave amplitude and latency in both eyes . while it may be argued that the above mentioned change in amplitude", "may actually be within the normal variation of the measure , this is nonetheless in contrast to previous reports5658 from comparable erg studies involving healthy subjects receiving melatonin .", "while both melatonin and agomelatine act on melatonergic receptors , the presence of 5-ht2c ( and 5-ht2b ) antagonism for the antidepressant drug should be prudently hypothesized to account for the differential trend observed at erg , eventually observed to play a role in the therapeutic effect claimed in depressed samples .", "notably , a previous polysomnographic study carried out on a small sample of healthy volunteers with the same dose of agomelatine ( 25 mg / day ) raised doubts about the actual blockade of 5-ht2c receptors in humans , based on the observation that this agent failed to increase slow - wave sleep , whereas potent 5-ht2c antagonists ( eg , cyproheptadine 4 mg , ritanserin 5 mg , olanzapine 5 mg , or mirtazapine 30 mg ) did.65 furthermore , in a recent animal study , neither the long - term administration of melatonin ( 40 mg / kg / day ) nor the selective 5-ht2c receptor antagonist sb 242084 ( 0.5 mg / kg / day ) had an effect on the firing rate and burst parameters of serotonergic and dopaminergic neurons . the combination of these latter mechanisms , however , enhanced only the number of spontaneously active dopaminergic neurons at the ventral tegmental area , while leaving the firing of the serotonergic neurons unaltered at the raphe dorsal nucleus .", "finally , the addition of a specific 5-ht2b antagonist ( ly 266097 at 0.6 mg / kg / day ) , which proved by itself to be devoid of effect , to the previous dual regimen increased the number of bursts per minute of dopaminergic neurons and the percentage of spikes occurring in burst , which ultimately mimics the effect of the antidepressant agomelatine.66" ], [ "agomelatine may differ from melatonin not only in the absence of mt3 agonism , but also in the presence of a dual antagonism on 5-ht2c and 5-ht2b , which may corroborate the claimed clinical antidepressant effect and account for the increase of the b - wave amplitude and latency of the cones ( in daylight conditions ) .", "nonetheless , along with the abovementioned limitations urging much prudence in the interpretation of our results and the acknowledgement that a decline in the central serotonin activity may itself affect the cone b - wave latency , it pays to note that the classical paradigm of monoamines ( including dopamine ) as a core pathway involved in depression has been debated , shifting more attention toward impaired circadian rhythms in depression.67 therefore , with the ultimate goal of shedding further light on the mechanism of action of novel antidepressants , with agomelatine being the case in point , additional , more methodologically accurate erg studies on the matter are warranted ." ] ]

[ "there was a time when numerous asthmatics had to be treated with systemic corticoids either on a regular basis or with repeated short courses in order to achieve asthma control . while inhaled 2 agonists became available in the seventies it took until late eighties to have corticoids administered by the inhaled route ( ics ) .", "it was then demonstrated that regular treatment with inhaled beclomethasone and budesonide was superior to regular inhalation of short acting 2 agonists in improving asthma symptoms and airway caliber and also reducing the use of rescue inhaled bronchodilators .", "it soon appeared at the population level that asthma mortality was decreasing in parallel to the increasing use of ics .", "in the mid - nineties it became clear that adding long acting 2 agonists ( laba ) to ics markedly improved airway caliber and symptoms , and in particular night symptoms , in those patients who remained uncontrolled with low dose of ics . due to their marked efficacy ,", "the combination therapy rapidly became the gold standard of asthma treatment , and given the high prevalence of asthma in the general population , this drug association became a blockbuster in western world countries .", "at the same time was launched cysteinyl - leukotriene receptor antagonist ( ltra ) as an alternative to ics for maintenance asthma treatment in those patients who did not tolerate or were afraid of taking ics . in randomized controlled trials ( rcts ) ,", "ltra proved to be less efficient than low dose of ics in improving airway caliber , reducing symptoms , and controlling airway eosinophilic inflammation in asthmatics selected on the basis of a high reversibility to 2 agonists .", "however , the superiority of ics disappeared when comparing the two classes of drugs in mild to moderate asthmatics in a real - life setting where ltra performed equally to ics to improve asthma control and quality of life , probably because of a better adherence to treatment given by the oral as compared to the inhaled route . at the turn of the century it was demonstrated that the combination of ics / laba was particularly effective in reducing asthma exacerbation in moderate to severe asthma , both ics and laba contributing to this important effect . the combination of ics / laba has shown not only efficacy in rcts but also effectiveness in real - life setting where patient selection criteria are less stringent , allowing a greater range of patients to participate .", "the technique of induced sputum has been pivotal in the emergence of inflammatory phenotype concept in asthma and it has become usual to classify asthmatics according to the proportion of eosinophils and neutrophils in the airways .", "it appeared that while eosinophilic asthmatics display a good clinical and functional response to a few - week treatment with ics , it was not the same for those patients without raised eosinophilic inflammation , and in particular in those with high sputum neutrophil counts , who really seemed to be insensitive to ics .", "the concept of severe asthma has recently been reshaped in a practical way that severe asthmatics are those patients in whom control may not be achieved with or requires a combination of high dose ics / laba .", "the first class of drug that was shown to bring clinical benefit is monoclonal antibody directed towards ige .", "omalizumab proved to be efficient in reducing exacerbation rate and improving quality of life in severe allergic asthmatics who remained uncontrolled despite a combination of high dose ics / laba .", "furthermore , the effectiveness of this medication has also been demonstrated in real life partly because of the magic and the regular follow - up imposed by the subcutaneous injection route .", "similar to mild to moderate asthma , severe asthma was found to be heterogeneous with respect to the type of airway inflammation .", "more than half of severe asthmatics display residual eosinophilic inflammation despite receiving high dose of ics and even oral corticoids by some of them . in those patients , increasing the dose of ics or even giving oral corticoids results in a significant reduction of exacerbation . in those severe eosinophilic asthmatics , mepolizumab , which is an antibody directed towards interleukin ( il)-5 , reduces exacerbation rate while allowing for a reduction and even a stop of oral corticoids in some patients .", "efficacy of anti - il-5 regarding exacerbation rate seems to be particularly related to the blood eosinophil count .", "it has recently been demonstrated that asthmatics who had both high blood and sputum eosinophil counts were more prone to exacerbation and poorer asthma control .", "the effects of anti - il-5 on day - to - day asthma control and lung function are more controversial but shown in some studies using reslizumab .", "it has recently been shown that the effect of omalizumab in reducing exacerbation rate was essentially limited to those patients with elevated feno ( fractional ex - haled nitric oxide ) and blood eosinophil counts .", "monoclonal antibodies directed towards other cytokines , and in particular towards tnf , have yielded disappointing results so far .", "some of them exhibit a marked increase in neutrophil counts in the airways . in those subjects , a few - week treatment with clarithromycin", "may reduce the neutrophil counts and slightly improve the quality of life but fails to improve day - to - day asthma control .", "long - term effect of such a treatment on asthma exacerbation remains unknown , but this point has to be clarified in the future . in this view", "it is worth noting that , in a pilot study , asthmatics uncontrolled with moderate to high doses of ics and displaying a low blood eosinophil count had their exacerbation rate reduced by chronic treatment with low dose azithromycin .", "effects of macrolides on severe neutrophilic asthma have to be confirmed in large scale rcts .", "we can however ask whether choosing neutrophils as the main target of treatment may be a double - edged sword , as these cells play a critical role in innate immunity operating in the airways .", "while ics have a potent effect on eosinophilic inflammation , their ability to oppose the airway remodeling is much more controversial .", "thermoplasty , a new technique that delivers high energy in the bronchi during an endoscopic procedure , may bring significant benefit there . applying this technique three times three weeks apart", "has proved to be safe and has resulted in a reduction of exacerbation and hospitalization together with improved quality of life in asthmatics uncontrolled by a combination of ics / laba .", "this treatment , approved by the food and drug administration in usa , has still to make its way in europe before becoming the official treatment for asthma .", "in particular it remains to determine in prospective studies which population of asthmatics may be the most suitable to benefit from this treatment procedure . as thermoplasty requires a technical expertise", "ics have dramatically changed the course of daily life in many asthmatics over the past decades and are responsible for the sharp reduction in asthma mortality observed since the nineties .", "nevertheless , this class of drugs may not be sufficient or even inefficient in some patients .", "the respiratory physician dealing with asthma , and in particular with severe asthma , has to proceed with detailed functional and inflammatory investigations to better phenotype his / her patient thereby allowing to choose the most appropriate treatment ." ]

[ "<S> inhaled corticoids ( ics ) made a dramatic breakthrough in the management of asthma in the late eighties resulting in a sharp reduction in morbidity and mortality in the following decades . soon after , the association between ics and long acting 2 agonists ( laba ) soon became the gold standard of maintenance asthma treatment . with the advent of sputum induction </S>", "<S> it has become clear that asthma could not be considered as a unique entity but rather a display of several inflammatory phenotypes . </S>", "<S> eosinophilic phenotype shows good response to ics while non - eosinophilic , and in particular the neutrophilic phenotype , seems to be more resistant . </S>", "<S> severe asthmatics show insufficient asthma control despite ics / laba . </S>", "<S> those who are allergic and eosinophilic may benefit from add - on treatment with anti - ige or anti - il-5 . </S>", "<S> severe neutrophilic asthma could benefit from maintenance treatment with macrolides while thermoplasty offers some promise to those in whom airway smooth muscle hypertrophy contributes to disease instability . </S>" ]

[ "INTRODUCTION", "THE REIGN OF THE COMBINATION ICS/LABA IN MAINTENANCE ASTHMA TREATMENT", "VARIABLE RESPONSE TO ICS ACCORDING TO INFLAMMATORY ASTHMA PHENOTYPE", "THE ADVENT OF MONOCLONAL ANTIBODIES IN SEVERE ALLERGIC AND EOSINOPHILIC ASTHMA", "MACROLIDES AS A POSSIBLE NEW TREATMENT STRATEGY IN SEVERE NEUTROPHILIC ASTHMA", "THERMOPLASTY AS A HOPE TO REVERSE AIRWAY REMODELING", "CONCLUSION" ]

[ [ "there was a time when numerous asthmatics had to be treated with systemic corticoids either on a regular basis or with repeated short courses in order to achieve asthma control . while inhaled 2 agonists became available in the seventies it took until late eighties to have corticoids administered by the inhaled route ( ics ) .", "it was then demonstrated that regular treatment with inhaled beclomethasone and budesonide was superior to regular inhalation of short acting 2 agonists in improving asthma symptoms and airway caliber and also reducing the use of rescue inhaled bronchodilators .", "it soon appeared at the population level that asthma mortality was decreasing in parallel to the increasing use of ics ." ], [ "in the mid - nineties it became clear that adding long acting 2 agonists ( laba ) to ics markedly improved airway caliber and symptoms , and in particular night symptoms , in those patients who remained uncontrolled with low dose of ics . due to their marked efficacy ,", "the combination therapy rapidly became the gold standard of asthma treatment , and given the high prevalence of asthma in the general population , this drug association became a blockbuster in western world countries .", "at the same time was launched cysteinyl - leukotriene receptor antagonist ( ltra ) as an alternative to ics for maintenance asthma treatment in those patients who did not tolerate or were afraid of taking ics . in randomized controlled trials ( rcts ) ,", "ltra proved to be less efficient than low dose of ics in improving airway caliber , reducing symptoms , and controlling airway eosinophilic inflammation in asthmatics selected on the basis of a high reversibility to 2 agonists .", "however , the superiority of ics disappeared when comparing the two classes of drugs in mild to moderate asthmatics in a real - life setting where ltra performed equally to ics to improve asthma control and quality of life , probably because of a better adherence to treatment given by the oral as compared to the inhaled route . at the turn of the century it was demonstrated that the combination of ics / laba was particularly effective in reducing asthma exacerbation in moderate to severe asthma , both ics and laba contributing to this important effect . the combination of ics / laba has shown not only efficacy in rcts but also effectiveness in real - life setting where patient selection criteria are less stringent , allowing a greater range of patients to participate ." ], [ "the technique of induced sputum has been pivotal in the emergence of inflammatory phenotype concept in asthma and it has become usual to classify asthmatics according to the proportion of eosinophils and neutrophils in the airways .", "it appeared that while eosinophilic asthmatics display a good clinical and functional response to a few - week treatment with ics , it was not the same for those patients without raised eosinophilic inflammation , and in particular in those with high sputum neutrophil counts , who really seemed to be insensitive to ics ." ], [ "the concept of severe asthma has recently been reshaped in a practical way that severe asthmatics are those patients in whom control may not be achieved with or requires a combination of high dose ics / laba .", "the first class of drug that was shown to bring clinical benefit is monoclonal antibody directed towards ige .", "omalizumab proved to be efficient in reducing exacerbation rate and improving quality of life in severe allergic asthmatics who remained uncontrolled despite a combination of high dose ics / laba .", "furthermore , the effectiveness of this medication has also been demonstrated in real life partly because of the magic and the regular follow - up imposed by the subcutaneous injection route .", "similar to mild to moderate asthma , severe asthma was found to be heterogeneous with respect to the type of airway inflammation .", "more than half of severe asthmatics display residual eosinophilic inflammation despite receiving high dose of ics and even oral corticoids by some of them . in those patients , increasing the dose of ics or even giving oral corticoids results in a significant reduction of exacerbation . in those severe eosinophilic asthmatics , mepolizumab , which is an antibody directed towards interleukin ( il)-5 , reduces exacerbation rate while allowing for a reduction and even a stop of oral corticoids in some patients .", "efficacy of anti - il-5 regarding exacerbation rate seems to be particularly related to the blood eosinophil count .", "it has recently been demonstrated that asthmatics who had both high blood and sputum eosinophil counts were more prone to exacerbation and poorer asthma control .", "the effects of anti - il-5 on day - to - day asthma control and lung function are more controversial but shown in some studies using reslizumab .", "it has recently been shown that the effect of omalizumab in reducing exacerbation rate was essentially limited to those patients with elevated feno ( fractional ex - haled nitric oxide ) and blood eosinophil counts .", "monoclonal antibodies directed towards other cytokines , and in particular towards tnf , have yielded disappointing results so far ." ], [ "some of them exhibit a marked increase in neutrophil counts in the airways . in those subjects , a few - week treatment with clarithromycin", "may reduce the neutrophil counts and slightly improve the quality of life but fails to improve day - to - day asthma control .", "long - term effect of such a treatment on asthma exacerbation remains unknown , but this point has to be clarified in the future . in this view", "it is worth noting that , in a pilot study , asthmatics uncontrolled with moderate to high doses of ics and displaying a low blood eosinophil count had their exacerbation rate reduced by chronic treatment with low dose azithromycin .", "effects of macrolides on severe neutrophilic asthma have to be confirmed in large scale rcts .", "we can however ask whether choosing neutrophils as the main target of treatment may be a double - edged sword , as these cells play a critical role in innate immunity operating in the airways ." ], [ "while ics have a potent effect on eosinophilic inflammation , their ability to oppose the airway remodeling is much more controversial .", "thermoplasty , a new technique that delivers high energy in the bronchi during an endoscopic procedure , may bring significant benefit there . applying this technique three times three weeks apart", "has proved to be safe and has resulted in a reduction of exacerbation and hospitalization together with improved quality of life in asthmatics uncontrolled by a combination of ics / laba .", "this treatment , approved by the food and drug administration in usa , has still to make its way in europe before becoming the official treatment for asthma .", "in particular it remains to determine in prospective studies which population of asthmatics may be the most suitable to benefit from this treatment procedure . as thermoplasty requires a technical expertise" ], [ "ics have dramatically changed the course of daily life in many asthmatics over the past decades and are responsible for the sharp reduction in asthma mortality observed since the nineties .", "nevertheless , this class of drugs may not be sufficient or even inefficient in some patients .", "the respiratory physician dealing with asthma , and in particular with severe asthma , has to proceed with detailed functional and inflammatory investigations to better phenotype his / her patient thereby allowing to choose the most appropriate treatment ." ] ]

[ "the search strategies used for this review involved literature searches of the medline and psychinfo electronic databases .", "the main heading terms included major depressive disorder , neurobiology , antidepressant , hippocampus , brain - derived neurotrophic growth factor , glucocorticoids and monoamines .", "as part of the research strategy , each article 's bibliography was reviewed for additional potential research findings relevant to these terms .", "major depressive disorder ( mdd ) is an illness with significant neurobiological consequences involving structural , functional and molecular alterations in several areas of the brain .", "clinicians are advised to intervene with mdd using an early , comprehensive treatment approach that has remission as the goal .", "major depressive disorder ( mdd ) remains one of the most frequently seen psychiatric illnesses in primary care settings ( 1 ) .", "although family and primary care physicians have greatly increased their recognition and treatment of this illness , mdd remains an unresolved treatment challenge for many physicians and patients ( 2 ) .", "increasing evidence has accrued in recent years regarding the impact of mdd on the structural and functional processes occurring in the brain . from the initial views", "chemical imbalance in the brain , this body of research has developed into a complex theory involving neuronal networks and plasticity ( 3 ) . the network model has also led to a greater understanding of the mechanisms of effective treatment interventions and their role in mitigating the deleterious effects of mdd ( 4 ) .", "the objectives of the present review were to summarise the key findings from the clinical literature regarding the neurobiology of mdd and their implications for maximising treatment outcomes .", "first , the evidence that mdd is not only a chronic and recurrent illness , but also a progressive illness will be presented .", "second , the impact of mdd on the primary neuroanatomical sites associated with mood regulation will be described at the structural and functional level .", "third , the molecular processes that have been implicated for mediating these structural and functional changes will be explored .", "fourth , the role of multiple neurotransmitter systems will be reviewed for their involvement in restoration and recovery from mdd .", "the last section will discuss the treatment guidelines for obtaining remission in the context of this neurobiological model .", "epidemiological studies have consistently shown that mdd is one of the most prevalent lifetime psychiatric disorders . in the national comorbidity replication survey , based on dsm - iv criteria for mdd ,", "the lifetime prevalence rate was 16.2% , with a 12-month estimate of 6.6% ( 5 ) .", "the presentation of mdd is heterogeneous with respect to both core and associated symptoms ( 6 ) . in the diagnostic and statistical manual of mental disorders fourth", "edition , text revision ( 7 ) , the diagnosis of mdd requires the experience of major depressive episodes that are defined by at least five of the following symptoms for at least 2 weeks duration : loss of interest , depressed mood , appetite / weight disturbance , sleep disturbance , psychomotor change , loss of energy , worthlessness / guilt , concentration difficulties / indecisiveness and thoughts of death / suicide .", "depressed mood or loss of interest must be one of the symptoms , but with the inclusion of compound criteria ( e.g. worthlessness or guilt ) , a diagnosis of mdd can be met by various permutations , and episodes may then be further qualified by other associated features ( e.g. postpartum , seasonal pattern , with melancholy or psychotic symptoms ) .", "even though mdd is characterised as an episodic illness , prospective studies have found that recurrence is the norm rather than the exception .", "for example , in a naturalistic , 15-year follow - up of a sample of 380 patients experiencing an index mdd episode , 73% experienced a recurrent episode ( 8) , with each subsequent episode increasing the probability of further episodes ( 9 ) .", "similarly , in the star*d project ( sequenced treatment alternatives to relieve depression ) that includes 1500 patients with mdd , 74% of patients had experienced more than one episode ( 10 ) .", "recurrence of mdd appears to be driven in part by neurobiological vulnerabilities . in the star*d project ,", "patients who experienced multiple episodes were more likely to have a positive family history of depressive illness and an earlier age of onset of their index depressive episode compared with patients who were in their first episode ( 10 ) .", "kindling hypothesis in which depressive episodes become more easily triggered over time ( 11 ) .", "as the number of depressive episodes increase , future episodes are predicted more by the number of prior episodes rather than by life stress ( 12 ) ( figure 1 ) .", "kindling can be described as a process which occurs by a lowering of the threshold for the impact of stressful life events ( i.e. sensitisation to minor events ) or by an increase in spontaneous dysregulation , both of which could indicate progressive effects of mdd ( 13 ) .", "an analysis of the risk of recurrence in a large study of twins also suggests genetic contributions as patients with a high genetic risk were ", "prekindled ; that is , they had a lower association between stressful life events and the onset of depressive episodes compared with patients having a low genetic risk ( 14 ) .", ", the risk for subsequent episodes is predicted more from the number of prior episodes and less from the occurrence of a recent life stress .", "( 14 ) early adverse experiences may also contribute to long - term neurobiological alterations associated with depression .", "in preclinical studies , maternal deprivation of rat pups during critical development periods resulted in subsequent hyper - reactivity to stress and marked behavioural changes in adult rats ( 15 ) . in children who had a history of early maltreatment", ", the risk for depressive symptoms was associated with an interaction between genotypes [ e.g. serotonin ( 5-ht ) transporter ] and history of maltreatment ( 16 ) .", "considering these findings , some researchers have suggested that greater neurobiological changes occur in patients with depression who have early adverse experiences compared with patients who are depressed but do not have such a history , indicating that these patients may represent an especially vulnerable subtype of depressive illness ( 17 ) .", "chronicity also suggests long - term neurobiological consequences associated with the mdd illness . in the star*d project , 25% of the patients ( with single or recurrent mdd )", "were identified as having a chronic episode of more than 2 years duration ( 10 ) . in another large multicentre treatment study ( n = 681 ) ,", "patients depression was classified using dsm - iv modifiers into four categories : chronic mdd ( episodes > 2 years ) , mdd with incomplete recovery ( partial response ) , mdd superimposed on dysthymia ( double depression ) and chronic mdd superimposed on dysthymia ( depressive symptoms > 4 years ) . despite multiple comparisons across a broad range of clinical and psychological variables , few differences were found among the four groups , resulting in the conclusion that various manifestations of chronic depression represent the same illness ( 18 ) .", "as the duration of depressive episodes increases , the probability of recovery substantially decreases over time . in a 5-year prospective study of outpatients with depression , approximately half recovered within the first 6 months , but afterwards the rate of recovery diminished substantially .", "for example , patients who had experienced depressive episodes of 1-year duration had a recovery rate of 16% compared with a 1% recovery rate for patients whose episodes persisted > 5 years ( 19 ) . similarly , in a prospective study of new onset depressive episodes , a longer duration ( > 12 weeks ) of previous episodes reduced the likelihood of recovery from the new onset episode by 37% ( 20 ) . even if patients no longer meet full criteria for an mdd episode , studies have found that a substantial subset of patients continue to experience residual symptoms and diminished functioning . in a 3-year longitudinal epidemiological study ,", "165 patients were assessed before and after an mdd episode . although mean values on functional measures returned to premorbid levels , 1540% of patients experienced a worsening in psychosocial functioning that persisted after the episode , and the overall functioning of the entire sample continued to be lower than that of a healthy cohort ( 21 ) . in a 10-year , naturalistic longitudinal study , patients who experienced subthreshold depressive symptoms following an mdd episode were at significantly greater risk for a recurrence , and they also had a much faster onset of their next episode compared with patients whose episode had fully remitted , suggesting that residual symptoms represent vulnerability because of an active disease state ( 22 ) .", "the recurrence and chronicity of mdd along with possible kindling effects have shifted the perspective of the appropriate treatment goal .", "the gold standard for treatment outcome has been raised from response ( reduction in symptoms ) to remission ( absence of symptoms ) or recovery ( extended period of remission ) ( 23 ) . however", ", obtaining recovery implies not only the remission of symptoms but also a restoration of the underlying physiology associated with the illness .", "therefore , further understanding of the neurobiological changes associated with mdd is necessary for identifying true recovery processes .", "although much information still needs to be attained , imaging and other methods have begun to elucidate the neurobiological abnormalities associated with mdd . in particular , several prefrontal and limbic structures and their interconnected circuits", "these neuroanatomical areas include the ventromedial prefrontal cortex ( vmpfc ) , lateral orbital prefrontal cortex ( lopfc ) , dorsolateral prefrontal cortex ( dlpfc ) , anterior cingulated cortex ( acc ) , ventral striatum ( including nucleus accumbens ) , amygdala and the hippocampus .", "abnormalities in these areas have been shown in patients with mdd compared with healthy controls and thus suggest a foundation for the symptomatic expression of mdd ( 24 , 25 ) .", "however , these deviations may be obscured or not present at the individual patient level and thus , these findings can not necessarily be considered pathognomic .", "major depressive disorder affects the dynamic connectivity among neuroanatomical structures involved in regulation of mood and stress response .", "limbic structures ( amygdala , hippocampus and nucleus accumbens ) have reciprocal connections with para - limbic cortical areas , subgenual anterior cingluate and ventromedial prefrontal cortex ( vmpfc ) .", "connectivity between limbic / para - limbic areas and rostral integrative prefrontal formations , results in compromised feedback regulation of limbic activity .", "consequently , dorsal cognitive / executive network is hypoactive while overly active limbic areas continue to stimulate the hypothalamus leading to neuroendocrine dysregulation and sympathetic hyperactivity as an integrated circuit , the prefrontal cortex , cingulate , amygdala , and hippocampus serves not only mood regulation , but also learning and contextual memory processes . within the prefrontal cortex , the vmpfc mediates pain , aggression , sexual functioning and eating behaviours whereas the lopfc assesses risk and modulates maladaptive and perseverative affective states ( behaviours ) .", "these two areas have a reciprocal pattern of activity with the dlfpc , which maintains executive function , effortful sustained attention , and working memory processes ( 26 ) .", "subdivisions within the acc assume diverse roles , with the dorsal acc being part of the cognitive / executive functioning network and the ventral acc being involved in assessing emotional and motivational information .", "the acc also monitors outcomes of behaviour and cognition and makes adjustments based on changing contingencies ( 27 , 28 ) . in patients with mdd ,", "regional blood flow studies suggest hyperactivity in the vmpfc and lopfc and hypoactivity in the dlfpc compared with controls ( 24 ) . given the functions of these regions , as previously described , this abnormal activity pattern may be responsible for the manifestations of symptoms associated with mdd .", "hyperactivity of the vmpfc is associated with enhanced sensitivity to pain , anxiety , depressive ruminations and tension whereas hypoactivity of the dlfpc may produce psychomotor retardation , apathy , and deficits in attention and working memory .", "using fmri paradigms , connectivity studies have also suggested a decrement in the communication between amygdala and acc regions ( 29 ) .", "a consequence of this loss of connectivity could be a failure of the acc to serve its inhibitory role in emotional regulation ( 30 ) , resulting in further motivational and affective disruption ( 31 ) . at the intersection of limbic , cognitive / executive and neuroendocrine regulatory circuits , including the hypothalamic - pituitary - adrenal axis ( hpa )", "imaging studies of hippocampal volume have been of particular interest . in a meta - analysis of 12 studies", ", hippocampal volume was found to be consistently and significantly reduced in patients with mdd compared with controls , and these reductions occurred bilaterally with a slightly greater decrement in right hippocampal volume ( 32 ) .", "other studies have shown that the degree of hippocampal reduction is directly proportional to the number and the duration of untreated depressive episodes ( 33 ) . among depressed inpatients , while controlling for the effect of age , hippocampal volume was significantly correlated with duration of illness prior to hospitalisation ( 34 ) .", "even after remission of an episode , patients with recurrent mdd have continued to show significantly smaller hippocampal volume compared with healthy controls ( 35 ) .", "differences in hippocampal volume between patients with depression and healthy controls may not be fully attributable to the disease state .", "heritability studies of hippocampal volume suggest both environmental and genetic contributions with heritability estimates of 54% in nonhuman primates and 40% in adult male twins ( 36 , 37 ) .", "several genomic imaging studies , comparing patients with mdd and healthy controls , have shown associations between hippocampal volume and specific genes that are implicated in mood disorders ( 38 , 39 ) . in a 1-year prospective study of 30 patients with mdd , hippocampal volume did not significantly change during the study period , but patients whose depression failed to remit had a significantly smaller hippocampus at baseline and at 1 year than did patients who did remit ( 40 ) . combining", "the evidence from these genetic , cross - sectional , and clinical treatment studies suggests that morphological differences in the hippocampus may be a predisposing factor in mdd , but changes can also accumulate in the course of the disease and thereby create an obstacle to full recovery .", "the alteration in the hippocampus signifies a potential outcome of injurious feedback that occurs via neuroendocrine dysregulation . a consistent finding in patients with mdd is a high level of the stress hormone cortisol , which may cause impairment in neuroplasticity and cellular resistance ( 41 ) .", "an imbalance between glucocorticoid and mineral corticoid receptors in mdd along with high - density glucocorticoid receptors ( grs ) may also contribute to the hippocampus susceptibility to neuronal damage ( 42 ) .", "subsequent hippocampal atrophy could result in further neuroendocrine dysfunction and hence a potential run - away system ( 43 ) .", "postmortem comparisons of brain tissue in patients with mdd and age - matched healthy controls have shown hippocampal shrinkage in depressed subjects that was caused by increased density of neuronal cells and a significant reduction in neuropil ( i.e. decreased dendridic branching and spine complexities ) ( 44 ) .", "a corollary of elevated glucocorticoids and compromised hippocampal functioning may also be the down - regulation of the gr sensitivity . under conditions of chronic stress ,", "decrease in gr sensitivity can have negative consequences as gr signalling becomes insufficient to turn off the initial responses to stress as part of a negative feedback process ( 45 , 46 ) ( figure 3 ) .", "subsequently , hpa hypothalamic overactivity , in conjunction with amygdala activation , leads to increased sympathetic tone , which promotes the release of cytokines from macrophages .", "increase in pro - inflammatory cytokines has been associated with loss of insulin and gr sensitivity , which further perpetuates metabolic and neuroendocrine disruption ( 47 ) .", "symptomatically , disruptions as a result of proinflammatory cytokines may be experienced as fatigue , loss of appetite and libido as well as hypersensitivity to pain ( 48 ) .", "stress results in release of glucocorticoids and corticotrophin releasing hormones ( crh ) and pro - inflammatory cytokines ( tnf , il-1 , il-6 ) . in depression ,", "disruption of serotonin ( 5-ht ) , norepinephrine ( ne ) and dopamine ( da ) transmission impair the regulatory feedback loops that ", ". they may also diminish central corticosteroid receptor sensitivity , leading to disruption of feedback control .", "( 46 ) proinflammatory cytokines may also diminish neurotrophic support and monoamine neurotransmission that can lead to neuronal apoptosis and glial damage .", "alterations in glia neuron relationships have been recently emphasised in the aetiology of neuropathic pain and mdd ( 47 , 49 ) .", "glia cells are involved in an intricate interaction with neurons in which astroglia and microglia maintain homeostasis of the neuronal environment by modulating electrolytes , neurotransmitters , cytokines and neurotrophic factors ( 50 ) .", "stress , depression and ensuing peripheral immune dysregulation lead to activation of microglia that then contribute to the existing immune disruption by additional release of inflammatory cytokines ( 51 ) .", "an integral part of maintaining the health of these glial neuron interactions may be mediated by brain - derived neurotrophic factor ( bdnf ) ( 52 ) . involved in neurogenesis , bdnf is the primary neurotrophin of the hippocampus . as a dimeric protein involved in cell maintenance , plasticity , growth and death ( apoptosis ) ,", "bdnf is structurally related to nerve growth factor and is distributed widely throughout the brain ( 53 ) .", "when bdnf interacts with tyrosine receptor kinase receptors ( trkb ) , it promotes cellular resilience and long - term potentiation .", "however , the precursor form of bdnf ( pro - bdnf ) can also precipitate reduction in dendritic spines and cell death when it binds with the p75 receptor .", "thus , depending upon its expression , bdnf can prune neural networks in an activity dependent manner that is regulated by various neurotransmitters [ glutamate , gaba , 5-ht , norepinephrine ( ne ) , acetylcholine , dopamine and hormones ] ( 54 ) .", "preclinical and clinical studies have suggested dysregulation in bdnf occurs under conditions of chronic stress and depression . in animal models ,", "similar results were also observed following administration of acute and chronic pain stimuli ( 55 ) . within humans , levels of serum bdnf", "has been found to be significantly lower in untreated patients with mdd compared with treated patients or healthy controls ( 56 ) .", "similarly , postmortem analyses of brains of persons who committed suicide showed that bdnf and another neurotrophin ( nt-3 ) were significantly reduced compared with non - suicide controls ( 57 ) . from the above observations ,", "the neurotrophic hypothesis has emerged as a major theory for the pathogenesis of major depression . in this model , stress and genetic vulnerability elevate glucocorticoid steroids and alter cellular plasticity via downregulation of growth factors and receptor sensitivity ( 4 ) .", "the reduction in growth factors , such as bdnf , impacts negatively on the structural and functional processes within the limbic system , especially for the hippocampus .", "chronic and recurrent mdd may result in subsequent atrophy and further disruptions in neurocircuitry . from this hypothesis , recovery and remission of mdd", "would be dependent upon a reversal of these processes , such as an increase in bdnf levels . complementing the neurotrophic hypothesis of mdd is the monoamine theory , which postulates that depression", "a recent imaging study of patients with untreated depression found a high global receptor density for the monoamine oxidase a ( mao - a ) , which nonspecifically metabolises these neurotransmitters . in this updated theory , long - term monoamine loss because of", "this global mao - a activity interacts with regional specific transporter densities ( i.e. 5-ht , ne ) , resulting in the expression of the depressive illness ( 58 ) . both 5-ht and ne ascending fibres originate from brainstem nuclei and innervate the limbic system , prefrontal cortex and associated structures involved in the regulation of mood .", "descending pathways project through the dorsolateral spinal column and are instrumental in the regulation of pain ( 59 , 60 ) .", "therefore , depending upon the specific transporter densities within these regions , various symptoms of depression ( mood , cognition and pain ) will be manifested within the context of the overall global reduction in monoamine levels ( 58 ) .", "therapeutically , selective serotonergic reuptake inhibitors ( ssris ) and ne reuptake inhibitors ( nris ) are known to increase their respective monoamine levels in the brain . chronic treatment with monoamine reuptake inhibitors increases activation of cyclic adenosine 3 - 5 monophosphatase ( camp ) , which in turn stimulates protein kinase a. activation of this protein enzyme regulates target genes leading to an increase in bdnf synthesis ( 52 ) .", "the antidepressant - induced camp activity can also enhance gr sensitivity and inhibit cytokine signalling , further assisting in the restoration of the neurocircuitry feedback loops ( 61 ) .", "the effect of increasing monoamine levels ( dopamine , 5-ht and ne ) on bdnf and growth factors may be one mechanism that produces the antidepressant response . preclinical study of rat brain cells has demonstrated that monoamenergic activity ( ne , 5-ht ) upregulates bdnf synthesis in astrocytes ( 62 ) .", "clinically , successful treatment with antidepressants results in normalisation of serum bdnf level , which is considered an indirect measure of cortical bdnf activity . support for the relationship between serum and cortical bdnf levels has been derived from correlations in animal studies as well as findings that serum bdnf passes the blood brain barrier and reflects stored and circulating bdnf in humans ( 63 , 64 ) . in a study of 10 patients who were treated for 12 weeks with a dual reuptake inhibitor ,", "improvement in depressive symptoms was correlated with increases in bdnf levels , and the bdnf levels of remitted patients had normalised to the same level observed in healthy controls ( 65 ) . response to various ssri and 5-ht noradrenalin reuptake inhibitors ( snri ) treatments has been similarly associated with restoration of normative bdnf values ( 66 ) ( figure 4 ) .", "postmortem analysis of brain tissue has shown that subjects who had been treated with an antidepressant at time of death had greater hippocampal bdnf expression as measured by immunoreactivity than did untreated subjects with mood disorders ( 67 ) .", "treatment with various selective serotonin antidepressant treatments and serotonergic noradrenergic reuptake inhibitors resulted in increases in serum brain - derived neurotrophic factor ( bdnf ) for patients with mdd to levels comparable that were observed with healthy controls .", "( 66 ) antidepressant therapeutic response is also associated with re - establishment of normative cortical activity .", "a study of 17 inpatients with mdd examined regional activity changes following 1 week and 6 week fluoxetine treatment . at 1 week , all patients showed increases in hippocampal activity and decreases in posterior cingulate and prefrontal cortex activity .", "after 6 weeks of treatment , patients who had responded to treatment showed a reversal of this pattern with decreased limbic activity and increased prefrontal cortical activity whereas non - responders continued to show the 1-week pattern ( 68 ) .", "normalisation in the amygdala and acc has also been associated with positive response to treatment . using a masking paradigm for subconscious activation ,", "patients with mdd showed a baseline hyper - reactivity of the left amygdala that attenuated following 8-week treatment with sertraline ( 69 ) .", "structural and functional mri assessments of patients with mdd who were treated with fluoxetine indicated the importance of acc grey matter volume for treatment response as there was a positive association among grey matter volume , normalisation of acc activity , and response to treatment ( 70 ) .", "conversely , in patients with mdd who failed to respond to antidepressant treatment , plasma levels of proinflammatory cytokines were elevated compared with healthy controls or euthymic patients with mdd ( 71 ) .", "symptomatically , improvements in specific mdd symptoms have been associated with regional improvements in brain metabolic activity . in 39 outpatients with mdd , improvement in cognitive symptoms", "was correlated with increases in dlpfc and improvements in fatigue / psychomotor retardation was associated with decreases in vmpfc activity .", "interestingly , these changes were seen in responders regardless of whether treatment was pharmacological or psychological ( 72 ) .", "restoration of the neurobiological regulation in mdd via neurotrophic factors and neurogenesis appears to be a common factor across various effective treatments for mdd , including pharmacological , psychological and somatic treatments , such as diet and exercise ( 73 ) .", "the neurobiological sequelae and repercussions of chronic or recurrent mdd indicate that interventions for mdd should be focused on achieving optimal treatment early .", "longitudinal studies have shown that one of the best predictors of remission status at 2 years was response to acute treatment , i.e. initial 6 weeks ( 74 ) .", "in addition , the adequacy of treatment may also have prognostic implications . for patients with late - life depression ,", "exposure to previous inadequate trials of antidepressants resulted in a reduced response rate to pharmacological intervention augmented by psychotherapy compared with treatment of naive patients , even after controlling for baseline severity ( 75 ) .", "similarly , in a large observational study of 996 patients with mdd , non - response or incomplete response to initial antidepressant treatment was a significant predictor of eventual treatment resistance ( 76 ) . on the positive side , an early response to antidepressants", "one way of maximising early response is to apply a comprehensive treatment that increases activity of multiple monoaminergic systems . in a double - blind , randomised treatment study , 39 inpatients with mdd received either fluoxetine ( a serotonergic intervention ) , desipramine ( a noradrenergic intervention ) or their combination .", "after 6 weeks of treatment , patients who had been given the combination treatment were more likely to achieve remission ( 53.8% ) than either intervention alone ( 0 % and 7.1% ) ( 78 ) .", "similarly , a recent large meta - analysis encompassing 93 trials and 17,036 patients compared efficacy outcomes of ssri with snri treatments for mdd that showed a modest but significant advantage in efficacy with snri treatments ( 79 ) .", "an earlier meta - analysis did not find a difference in efficacy between ssris and dual acting agents ( mostly tricyclic antidepressants ) , with the exception of the inpatient populations , where dual acting tricyclic antidepressants had an advantage ( 80 ) .", "thus , although current treatment algorithms for mdd usually are initiated with ssris , the role of combination treatment or dual reuptake inhibitors are increasingly being considered as a preferred option ( 81 ) .", "another advantage of targeting both of 5-ht and ne systems is improvement not only in the core features of mdd , but also in associated physical symptoms .", "painful physical symptoms are prevalent in patients with mdd , and these symptoms increase the illness burden and impair the ability to attain remission ( 82 , 83 ) . in a study of primary care patients with mdd who were treated with ssris for 9 months , mood symptoms continued to improve over time while painful physical symptoms persisted ( 84 ) .", "the occurrence of painful physical symptoms and mdd reflects the shared underlying pathophysiology between mood and pain regulation .", "importantly , there may be also a synergistic interaction between the 5-ht and ne systems to obtain analgesia . in an animal model of pain ,", "treatment with dual reuptake inhibitors or combination treatment ( 5-ht / ne ) appeared to enhance the effectiveness of pain alleviation ( 85 ) .", "clinically , patients with mdd who experienced a 50% or greater reduction in pain were more likely to achieve remission than patients whose pain reduction was < 50% ( 86 ) . with remission and recovery as the goal ,", "the treatment guidelines derived from the neurobiological model emphasise the need for not only early and comprehensive intervention , but also vigorous attention to residual symptoms . in a 2-year study of outpatients with mdd , patients who obtained only a partial remission of symptoms were more likely to relapse ( 67.5% ) than patients who had attained full remission ( 15.2% ) ( 87 ) .", "specific recommendations for the treatment of residual symptoms have not been determined empirically , but likely require additional augmentation with other pharmacological and psychological treatments ; in addition to reducing the risk of relapse , the treatment of residual symptoms may enhance compliance and long - term outcomes ( 88 ) .", "as the underlying neurobiological model of depression is increasingly understood , treatment providers are directed to recognise that the factors that may initiate a mdd episode and those that maintain the illness are likely to be very different .", "genetic and stress vulnerabilities interplay to initiate a cascade of neurobiological alterations that disrupt a dynamic system .", "progressive effects of recurrent and chronic mdd may then be potentiated by further structural and functional abnormalities . given these long - term consequences ,", "an essential objective of treatment must be to restore normative functioning and prevent further neurobiological structural alterations .", "increasing 5-ht and ne neurotransmission is likely to initiate true recovery with the restoration of neurotrophic support , glucocorticoid signalling and neuroendocrine regulation .", "the use of dual reuptake inhibitors enhances the probability of remission as it addresses the complex interplay of the emotional and physical symptoms of mdd .", "painful physical symptoms are increasingly recognised as having a significant impact on functioning and recovery ; thus , affirming the need for antidepressant treatments that can effectively reduce these symptoms as well . from the neurobiological model , the treatment guidelines of early", ", comprehensive and progressive treatment require a change in perspective for both patients and providers .", "a residual symptom may be interpreted as a proxy of an active disease state , with ensuing structural alterations and systemic consequences . with remission and recovery as the goal", ", patients will need to be educated about the benefits of long - term treatment rather than episodic or incomplete intervention .", "a biopsychosocial treatment model that incorporates cognitive - behavioural or interpersonal therapy along with pharmacological interventions serves to address both the initiation and maintenance factors and can reduce the risk of relapse ( 89 ) .", "once remission is attained , maintenance of effect may become the more appropriate term , rather than relapse prevention , to emphasise the necessity for an ongoing collaboration between patient and physician in order to maintain neurobiological homeostasis ." ]

[ "<S> aimsthe objectives of the present review were to summarise the key findings from the clinical literature regarding the neurobiology of major depressive disorder ( mdd ) and their implications for maximising treatment outcomes . </S>", "<S> several neuroanatomical structures in the prefrontal and limbic areas of the brain are involved in affective regulation . in patients with mdd , </S>", "<S> alterations in the dynamic patterns of activity among these structures have profound implications for the pathogenesis of this illness.discussionthe present work reviews the evidence for the progressive nature of mdd along with associated changes in neuroanatomical structure and function , especially for the hippocampus . </S>", "<S> the role of glucocorticoids , inflammatory cytokines and brain - derived growth factors are discussed as mediators of these pathological alterations . from this integrated model , the role of antidepressant therapy in restoring normative processes is examined along with additional treatment guidelines.conclusionmajor depressive disorder is an illness with significant neurobiological consequences involving structural , functional and molecular alterations in several areas of the brain . </S>", "<S> antidepressant pharmacotherapy is associated with restoration of the underlying physiology . </S>", "<S> clinicians are advised to intervene with mdd using an early , comprehensive treatment approach that has remission as the goal . </S>" ]

[ "Review Criteria", "Message for the Clinic", "Introduction", "Major depressive disorder as a progressive illness", "Functional and structural changes in MDD", "Molecular processes mediating neurobiological changes", "Role of neurotransmitters in recovery from MDD", "Treatment implications of the neurobiological model", "Conclusions" ]

[ [ "the search strategies used for this review involved literature searches of the medline and psychinfo electronic databases .", "the main heading terms included major depressive disorder , neurobiology , antidepressant , hippocampus , brain - derived neurotrophic growth factor , glucocorticoids and monoamines .", "as part of the research strategy , each article 's bibliography was reviewed for additional potential research findings relevant to these terms ." ], [ "major depressive disorder ( mdd ) is an illness with significant neurobiological consequences involving structural , functional and molecular alterations in several areas of the brain .", "clinicians are advised to intervene with mdd using an early , comprehensive treatment approach that has remission as the goal ." ], [ "major depressive disorder ( mdd ) remains one of the most frequently seen psychiatric illnesses in primary care settings ( 1 ) .", "although family and primary care physicians have greatly increased their recognition and treatment of this illness , mdd remains an unresolved treatment challenge for many physicians and patients ( 2 ) .", "increasing evidence has accrued in recent years regarding the impact of mdd on the structural and functional processes occurring in the brain . from the initial views", "chemical imbalance in the brain , this body of research has developed into a complex theory involving neuronal networks and plasticity ( 3 ) . the network model has also led to a greater understanding of the mechanisms of effective treatment interventions and their role in mitigating the deleterious effects of mdd ( 4 ) .", "the objectives of the present review were to summarise the key findings from the clinical literature regarding the neurobiology of mdd and their implications for maximising treatment outcomes .", "first , the evidence that mdd is not only a chronic and recurrent illness , but also a progressive illness will be presented .", "second , the impact of mdd on the primary neuroanatomical sites associated with mood regulation will be described at the structural and functional level .", "third , the molecular processes that have been implicated for mediating these structural and functional changes will be explored .", "fourth , the role of multiple neurotransmitter systems will be reviewed for their involvement in restoration and recovery from mdd .", "the last section will discuss the treatment guidelines for obtaining remission in the context of this neurobiological model ." ], [ "epidemiological studies have consistently shown that mdd is one of the most prevalent lifetime psychiatric disorders . in the national comorbidity replication survey , based on dsm - iv criteria for mdd ,", "the lifetime prevalence rate was 16.2% , with a 12-month estimate of 6.6% ( 5 ) .", "the presentation of mdd is heterogeneous with respect to both core and associated symptoms ( 6 ) . in the diagnostic and statistical manual of mental disorders fourth", "edition , text revision ( 7 ) , the diagnosis of mdd requires the experience of major depressive episodes that are defined by at least five of the following symptoms for at least 2 weeks duration : loss of interest , depressed mood , appetite / weight disturbance , sleep disturbance , psychomotor change , loss of energy , worthlessness / guilt , concentration difficulties / indecisiveness and thoughts of death / suicide .", "depressed mood or loss of interest must be one of the symptoms , but with the inclusion of compound criteria ( e.g. worthlessness or guilt ) , a diagnosis of mdd can be met by various permutations , and episodes may then be further qualified by other associated features ( e.g. postpartum , seasonal pattern , with melancholy or psychotic symptoms ) .", "even though mdd is characterised as an episodic illness , prospective studies have found that recurrence is the norm rather than the exception .", "for example , in a naturalistic , 15-year follow - up of a sample of 380 patients experiencing an index mdd episode , 73% experienced a recurrent episode ( 8) , with each subsequent episode increasing the probability of further episodes ( 9 ) .", "similarly , in the star*d project ( sequenced treatment alternatives to relieve depression ) that includes 1500 patients with mdd , 74% of patients had experienced more than one episode ( 10 ) .", "recurrence of mdd appears to be driven in part by neurobiological vulnerabilities . in the star*d project ,", "patients who experienced multiple episodes were more likely to have a positive family history of depressive illness and an earlier age of onset of their index depressive episode compared with patients who were in their first episode ( 10 ) .", "kindling hypothesis in which depressive episodes become more easily triggered over time ( 11 ) .", "as the number of depressive episodes increase , future episodes are predicted more by the number of prior episodes rather than by life stress ( 12 ) ( figure 1 ) .", "kindling can be described as a process which occurs by a lowering of the threshold for the impact of stressful life events ( i.e. sensitisation to minor events ) or by an increase in spontaneous dysregulation , both of which could indicate progressive effects of mdd ( 13 ) .", "an analysis of the risk of recurrence in a large study of twins also suggests genetic contributions as patients with a high genetic risk were ", "prekindled ; that is , they had a lower association between stressful life events and the onset of depressive episodes compared with patients having a low genetic risk ( 14 ) .", ", the risk for subsequent episodes is predicted more from the number of prior episodes and less from the occurrence of a recent life stress .", "( 14 ) early adverse experiences may also contribute to long - term neurobiological alterations associated with depression .", "in preclinical studies , maternal deprivation of rat pups during critical development periods resulted in subsequent hyper - reactivity to stress and marked behavioural changes in adult rats ( 15 ) . in children who had a history of early maltreatment", ", the risk for depressive symptoms was associated with an interaction between genotypes [ e.g. serotonin ( 5-ht ) transporter ] and history of maltreatment ( 16 ) .", "considering these findings , some researchers have suggested that greater neurobiological changes occur in patients with depression who have early adverse experiences compared with patients who are depressed but do not have such a history , indicating that these patients may represent an especially vulnerable subtype of depressive illness ( 17 ) .", "chronicity also suggests long - term neurobiological consequences associated with the mdd illness . in the star*d project , 25% of the patients ( with single or recurrent mdd )", "were identified as having a chronic episode of more than 2 years duration ( 10 ) . in another large multicentre treatment study ( n = 681 ) ,", "patients depression was classified using dsm - iv modifiers into four categories : chronic mdd ( episodes > 2 years ) , mdd with incomplete recovery ( partial response ) , mdd superimposed on dysthymia ( double depression ) and chronic mdd superimposed on dysthymia ( depressive symptoms > 4 years ) . despite multiple comparisons across a broad range of clinical and psychological variables , few differences were found among the four groups , resulting in the conclusion that various manifestations of chronic depression represent the same illness ( 18 ) .", "as the duration of depressive episodes increases , the probability of recovery substantially decreases over time . in a 5-year prospective study of outpatients with depression , approximately half recovered within the first 6 months , but afterwards the rate of recovery diminished substantially .", "for example , patients who had experienced depressive episodes of 1-year duration had a recovery rate of 16% compared with a 1% recovery rate for patients whose episodes persisted > 5 years ( 19 ) . similarly , in a prospective study of new onset depressive episodes , a longer duration ( > 12 weeks ) of previous episodes reduced the likelihood of recovery from the new onset episode by 37% ( 20 ) . even if patients no longer meet full criteria for an mdd episode , studies have found that a substantial subset of patients continue to experience residual symptoms and diminished functioning . in a 3-year longitudinal epidemiological study ,", "165 patients were assessed before and after an mdd episode . although mean values on functional measures returned to premorbid levels , 1540% of patients experienced a worsening in psychosocial functioning that persisted after the episode , and the overall functioning of the entire sample continued to be lower than that of a healthy cohort ( 21 ) . in a 10-year , naturalistic longitudinal study , patients who experienced subthreshold depressive symptoms following an mdd episode were at significantly greater risk for a recurrence , and they also had a much faster onset of their next episode compared with patients whose episode had fully remitted , suggesting that residual symptoms represent vulnerability because of an active disease state ( 22 ) .", "the recurrence and chronicity of mdd along with possible kindling effects have shifted the perspective of the appropriate treatment goal .", "the gold standard for treatment outcome has been raised from response ( reduction in symptoms ) to remission ( absence of symptoms ) or recovery ( extended period of remission ) ( 23 ) . however", ", obtaining recovery implies not only the remission of symptoms but also a restoration of the underlying physiology associated with the illness .", "therefore , further understanding of the neurobiological changes associated with mdd is necessary for identifying true recovery processes ." ], [ "although much information still needs to be attained , imaging and other methods have begun to elucidate the neurobiological abnormalities associated with mdd . in particular , several prefrontal and limbic structures and their interconnected circuits", "these neuroanatomical areas include the ventromedial prefrontal cortex ( vmpfc ) , lateral orbital prefrontal cortex ( lopfc ) , dorsolateral prefrontal cortex ( dlpfc ) , anterior cingulated cortex ( acc ) , ventral striatum ( including nucleus accumbens ) , amygdala and the hippocampus .", "abnormalities in these areas have been shown in patients with mdd compared with healthy controls and thus suggest a foundation for the symptomatic expression of mdd ( 24 , 25 ) .", "however , these deviations may be obscured or not present at the individual patient level and thus , these findings can not necessarily be considered pathognomic .", "major depressive disorder affects the dynamic connectivity among neuroanatomical structures involved in regulation of mood and stress response .", "limbic structures ( amygdala , hippocampus and nucleus accumbens ) have reciprocal connections with para - limbic cortical areas , subgenual anterior cingluate and ventromedial prefrontal cortex ( vmpfc ) .", "connectivity between limbic / para - limbic areas and rostral integrative prefrontal formations , results in compromised feedback regulation of limbic activity .", "consequently , dorsal cognitive / executive network is hypoactive while overly active limbic areas continue to stimulate the hypothalamus leading to neuroendocrine dysregulation and sympathetic hyperactivity as an integrated circuit , the prefrontal cortex , cingulate , amygdala , and hippocampus serves not only mood regulation , but also learning and contextual memory processes . within the prefrontal cortex , the vmpfc mediates pain , aggression , sexual functioning and eating behaviours whereas the lopfc assesses risk and modulates maladaptive and perseverative affective states ( behaviours ) .", "these two areas have a reciprocal pattern of activity with the dlfpc , which maintains executive function , effortful sustained attention , and working memory processes ( 26 ) .", "subdivisions within the acc assume diverse roles , with the dorsal acc being part of the cognitive / executive functioning network and the ventral acc being involved in assessing emotional and motivational information .", "the acc also monitors outcomes of behaviour and cognition and makes adjustments based on changing contingencies ( 27 , 28 ) . in patients with mdd ,", "regional blood flow studies suggest hyperactivity in the vmpfc and lopfc and hypoactivity in the dlfpc compared with controls ( 24 ) . given the functions of these regions , as previously described , this abnormal activity pattern may be responsible for the manifestations of symptoms associated with mdd .", "hyperactivity of the vmpfc is associated with enhanced sensitivity to pain , anxiety , depressive ruminations and tension whereas hypoactivity of the dlfpc may produce psychomotor retardation , apathy , and deficits in attention and working memory .", "using fmri paradigms , connectivity studies have also suggested a decrement in the communication between amygdala and acc regions ( 29 ) .", "a consequence of this loss of connectivity could be a failure of the acc to serve its inhibitory role in emotional regulation ( 30 ) , resulting in further motivational and affective disruption ( 31 ) . at the intersection of limbic , cognitive / executive and neuroendocrine regulatory circuits , including the hypothalamic - pituitary - adrenal axis ( hpa )", "imaging studies of hippocampal volume have been of particular interest . in a meta - analysis of 12 studies", ", hippocampal volume was found to be consistently and significantly reduced in patients with mdd compared with controls , and these reductions occurred bilaterally with a slightly greater decrement in right hippocampal volume ( 32 ) .", "other studies have shown that the degree of hippocampal reduction is directly proportional to the number and the duration of untreated depressive episodes ( 33 ) . among depressed inpatients , while controlling for the effect of age , hippocampal volume was significantly correlated with duration of illness prior to hospitalisation ( 34 ) .", "even after remission of an episode , patients with recurrent mdd have continued to show significantly smaller hippocampal volume compared with healthy controls ( 35 ) .", "differences in hippocampal volume between patients with depression and healthy controls may not be fully attributable to the disease state .", "heritability studies of hippocampal volume suggest both environmental and genetic contributions with heritability estimates of 54% in nonhuman primates and 40% in adult male twins ( 36 , 37 ) .", "several genomic imaging studies , comparing patients with mdd and healthy controls , have shown associations between hippocampal volume and specific genes that are implicated in mood disorders ( 38 , 39 ) . in a 1-year prospective study of 30 patients with mdd , hippocampal volume did not significantly change during the study period , but patients whose depression failed to remit had a significantly smaller hippocampus at baseline and at 1 year than did patients who did remit ( 40 ) . combining", "the evidence from these genetic , cross - sectional , and clinical treatment studies suggests that morphological differences in the hippocampus may be a predisposing factor in mdd , but changes can also accumulate in the course of the disease and thereby create an obstacle to full recovery ." ], [ "the alteration in the hippocampus signifies a potential outcome of injurious feedback that occurs via neuroendocrine dysregulation . a consistent finding in patients with mdd is a high level of the stress hormone cortisol , which may cause impairment in neuroplasticity and cellular resistance ( 41 ) .", "an imbalance between glucocorticoid and mineral corticoid receptors in mdd along with high - density glucocorticoid receptors ( grs ) may also contribute to the hippocampus susceptibility to neuronal damage ( 42 ) .", "subsequent hippocampal atrophy could result in further neuroendocrine dysfunction and hence a potential run - away system ( 43 ) .", "postmortem comparisons of brain tissue in patients with mdd and age - matched healthy controls have shown hippocampal shrinkage in depressed subjects that was caused by increased density of neuronal cells and a significant reduction in neuropil ( i.e. decreased dendridic branching and spine complexities ) ( 44 ) .", "a corollary of elevated glucocorticoids and compromised hippocampal functioning may also be the down - regulation of the gr sensitivity . under conditions of chronic stress ,", "decrease in gr sensitivity can have negative consequences as gr signalling becomes insufficient to turn off the initial responses to stress as part of a negative feedback process ( 45 , 46 ) ( figure 3 ) .", "subsequently , hpa hypothalamic overactivity , in conjunction with amygdala activation , leads to increased sympathetic tone , which promotes the release of cytokines from macrophages .", "increase in pro - inflammatory cytokines has been associated with loss of insulin and gr sensitivity , which further perpetuates metabolic and neuroendocrine disruption ( 47 ) .", "symptomatically , disruptions as a result of proinflammatory cytokines may be experienced as fatigue , loss of appetite and libido as well as hypersensitivity to pain ( 48 ) .", "stress results in release of glucocorticoids and corticotrophin releasing hormones ( crh ) and pro - inflammatory cytokines ( tnf , il-1 , il-6 ) . in depression ,", "disruption of serotonin ( 5-ht ) , norepinephrine ( ne ) and dopamine ( da ) transmission impair the regulatory feedback loops that ", ". they may also diminish central corticosteroid receptor sensitivity , leading to disruption of feedback control .", "( 46 ) proinflammatory cytokines may also diminish neurotrophic support and monoamine neurotransmission that can lead to neuronal apoptosis and glial damage .", "alterations in glia neuron relationships have been recently emphasised in the aetiology of neuropathic pain and mdd ( 47 , 49 ) .", "glia cells are involved in an intricate interaction with neurons in which astroglia and microglia maintain homeostasis of the neuronal environment by modulating electrolytes , neurotransmitters , cytokines and neurotrophic factors ( 50 ) .", "stress , depression and ensuing peripheral immune dysregulation lead to activation of microglia that then contribute to the existing immune disruption by additional release of inflammatory cytokines ( 51 ) .", "an integral part of maintaining the health of these glial neuron interactions may be mediated by brain - derived neurotrophic factor ( bdnf ) ( 52 ) . involved in neurogenesis , bdnf is the primary neurotrophin of the hippocampus . as a dimeric protein involved in cell maintenance , plasticity , growth and death ( apoptosis ) ,", "bdnf is structurally related to nerve growth factor and is distributed widely throughout the brain ( 53 ) .", "when bdnf interacts with tyrosine receptor kinase receptors ( trkb ) , it promotes cellular resilience and long - term potentiation .", "however , the precursor form of bdnf ( pro - bdnf ) can also precipitate reduction in dendritic spines and cell death when it binds with the p75 receptor .", "thus , depending upon its expression , bdnf can prune neural networks in an activity dependent manner that is regulated by various neurotransmitters [ glutamate , gaba , 5-ht , norepinephrine ( ne ) , acetylcholine , dopamine and hormones ] ( 54 ) .", "preclinical and clinical studies have suggested dysregulation in bdnf occurs under conditions of chronic stress and depression . in animal models ,", "similar results were also observed following administration of acute and chronic pain stimuli ( 55 ) . within humans , levels of serum bdnf", "has been found to be significantly lower in untreated patients with mdd compared with treated patients or healthy controls ( 56 ) .", "similarly , postmortem analyses of brains of persons who committed suicide showed that bdnf and another neurotrophin ( nt-3 ) were significantly reduced compared with non - suicide controls ( 57 ) . from the above observations ,", "the neurotrophic hypothesis has emerged as a major theory for the pathogenesis of major depression . in this model , stress and genetic vulnerability elevate glucocorticoid steroids and alter cellular plasticity via downregulation of growth factors and receptor sensitivity ( 4 ) .", "the reduction in growth factors , such as bdnf , impacts negatively on the structural and functional processes within the limbic system , especially for the hippocampus .", "chronic and recurrent mdd may result in subsequent atrophy and further disruptions in neurocircuitry . from this hypothesis , recovery and remission of mdd", "would be dependent upon a reversal of these processes , such as an increase in bdnf levels . complementing the neurotrophic hypothesis of mdd is the monoamine theory , which postulates that depression", "a recent imaging study of patients with untreated depression found a high global receptor density for the monoamine oxidase a ( mao - a ) , which nonspecifically metabolises these neurotransmitters . in this updated theory , long - term monoamine loss because of", "this global mao - a activity interacts with regional specific transporter densities ( i.e. 5-ht , ne ) , resulting in the expression of the depressive illness ( 58 ) . both 5-ht and ne ascending fibres originate from brainstem nuclei and innervate the limbic system , prefrontal cortex and associated structures involved in the regulation of mood .", "descending pathways project through the dorsolateral spinal column and are instrumental in the regulation of pain ( 59 , 60 ) .", "therefore , depending upon the specific transporter densities within these regions , various symptoms of depression ( mood , cognition and pain ) will be manifested within the context of the overall global reduction in monoamine levels ( 58 ) ." ], [ "therapeutically , selective serotonergic reuptake inhibitors ( ssris ) and ne reuptake inhibitors ( nris ) are known to increase their respective monoamine levels in the brain . chronic treatment with monoamine reuptake inhibitors increases activation of cyclic adenosine 3 - 5 monophosphatase ( camp ) , which in turn stimulates protein kinase a. activation of this protein enzyme regulates target genes leading to an increase in bdnf synthesis ( 52 ) .", "the antidepressant - induced camp activity can also enhance gr sensitivity and inhibit cytokine signalling , further assisting in the restoration of the neurocircuitry feedback loops ( 61 ) .", "the effect of increasing monoamine levels ( dopamine , 5-ht and ne ) on bdnf and growth factors may be one mechanism that produces the antidepressant response . preclinical study of rat brain cells has demonstrated that monoamenergic activity ( ne , 5-ht ) upregulates bdnf synthesis in astrocytes ( 62 ) .", "clinically , successful treatment with antidepressants results in normalisation of serum bdnf level , which is considered an indirect measure of cortical bdnf activity . support for the relationship between serum and cortical bdnf levels has been derived from correlations in animal studies as well as findings that serum bdnf passes the blood brain barrier and reflects stored and circulating bdnf in humans ( 63 , 64 ) . in a study of 10 patients who were treated for 12 weeks with a dual reuptake inhibitor ,", "improvement in depressive symptoms was correlated with increases in bdnf levels , and the bdnf levels of remitted patients had normalised to the same level observed in healthy controls ( 65 ) . response to various ssri and 5-ht noradrenalin reuptake inhibitors ( snri ) treatments has been similarly associated with restoration of normative bdnf values ( 66 ) ( figure 4 ) .", "postmortem analysis of brain tissue has shown that subjects who had been treated with an antidepressant at time of death had greater hippocampal bdnf expression as measured by immunoreactivity than did untreated subjects with mood disorders ( 67 ) .", "treatment with various selective serotonin antidepressant treatments and serotonergic noradrenergic reuptake inhibitors resulted in increases in serum brain - derived neurotrophic factor ( bdnf ) for patients with mdd to levels comparable that were observed with healthy controls .", "( 66 ) antidepressant therapeutic response is also associated with re - establishment of normative cortical activity .", "a study of 17 inpatients with mdd examined regional activity changes following 1 week and 6 week fluoxetine treatment . at 1 week , all patients showed increases in hippocampal activity and decreases in posterior cingulate and prefrontal cortex activity .", "after 6 weeks of treatment , patients who had responded to treatment showed a reversal of this pattern with decreased limbic activity and increased prefrontal cortical activity whereas non - responders continued to show the 1-week pattern ( 68 ) .", "normalisation in the amygdala and acc has also been associated with positive response to treatment . using a masking paradigm for subconscious activation ,", "patients with mdd showed a baseline hyper - reactivity of the left amygdala that attenuated following 8-week treatment with sertraline ( 69 ) .", "structural and functional mri assessments of patients with mdd who were treated with fluoxetine indicated the importance of acc grey matter volume for treatment response as there was a positive association among grey matter volume , normalisation of acc activity , and response to treatment ( 70 ) .", "conversely , in patients with mdd who failed to respond to antidepressant treatment , plasma levels of proinflammatory cytokines were elevated compared with healthy controls or euthymic patients with mdd ( 71 ) .", "symptomatically , improvements in specific mdd symptoms have been associated with regional improvements in brain metabolic activity . in 39 outpatients with mdd , improvement in cognitive symptoms", "was correlated with increases in dlpfc and improvements in fatigue / psychomotor retardation was associated with decreases in vmpfc activity .", "interestingly , these changes were seen in responders regardless of whether treatment was pharmacological or psychological ( 72 ) .", "restoration of the neurobiological regulation in mdd via neurotrophic factors and neurogenesis appears to be a common factor across various effective treatments for mdd , including pharmacological , psychological and somatic treatments , such as diet and exercise ( 73 ) ." ], [ "the neurobiological sequelae and repercussions of chronic or recurrent mdd indicate that interventions for mdd should be focused on achieving optimal treatment early .", "longitudinal studies have shown that one of the best predictors of remission status at 2 years was response to acute treatment , i.e. initial 6 weeks ( 74 ) .", "in addition , the adequacy of treatment may also have prognostic implications . for patients with late - life depression ,", "exposure to previous inadequate trials of antidepressants resulted in a reduced response rate to pharmacological intervention augmented by psychotherapy compared with treatment of naive patients , even after controlling for baseline severity ( 75 ) .", "similarly , in a large observational study of 996 patients with mdd , non - response or incomplete response to initial antidepressant treatment was a significant predictor of eventual treatment resistance ( 76 ) . on the positive side , an early response to antidepressants", "one way of maximising early response is to apply a comprehensive treatment that increases activity of multiple monoaminergic systems . in a double - blind , randomised treatment study , 39 inpatients with mdd received either fluoxetine ( a serotonergic intervention ) , desipramine ( a noradrenergic intervention ) or their combination .", "after 6 weeks of treatment , patients who had been given the combination treatment were more likely to achieve remission ( 53.8% ) than either intervention alone ( 0 % and 7.1% ) ( 78 ) .", "similarly , a recent large meta - analysis encompassing 93 trials and 17,036 patients compared efficacy outcomes of ssri with snri treatments for mdd that showed a modest but significant advantage in efficacy with snri treatments ( 79 ) .", "an earlier meta - analysis did not find a difference in efficacy between ssris and dual acting agents ( mostly tricyclic antidepressants ) , with the exception of the inpatient populations , where dual acting tricyclic antidepressants had an advantage ( 80 ) .", "thus , although current treatment algorithms for mdd usually are initiated with ssris , the role of combination treatment or dual reuptake inhibitors are increasingly being considered as a preferred option ( 81 ) .", "another advantage of targeting both of 5-ht and ne systems is improvement not only in the core features of mdd , but also in associated physical symptoms .", "painful physical symptoms are prevalent in patients with mdd , and these symptoms increase the illness burden and impair the ability to attain remission ( 82 , 83 ) . in a study of primary care patients with mdd who were treated with ssris for 9 months , mood symptoms continued to improve over time while painful physical symptoms persisted ( 84 ) .", "the occurrence of painful physical symptoms and mdd reflects the shared underlying pathophysiology between mood and pain regulation .", "importantly , there may be also a synergistic interaction between the 5-ht and ne systems to obtain analgesia . in an animal model of pain ,", "treatment with dual reuptake inhibitors or combination treatment ( 5-ht / ne ) appeared to enhance the effectiveness of pain alleviation ( 85 ) .", "clinically , patients with mdd who experienced a 50% or greater reduction in pain were more likely to achieve remission than patients whose pain reduction was < 50% ( 86 ) . with remission and recovery as the goal ,", "the treatment guidelines derived from the neurobiological model emphasise the need for not only early and comprehensive intervention , but also vigorous attention to residual symptoms . in a 2-year study of outpatients with mdd , patients who obtained only a partial remission of symptoms were more likely to relapse ( 67.5% ) than patients who had attained full remission ( 15.2% ) ( 87 ) .", "specific recommendations for the treatment of residual symptoms have not been determined empirically , but likely require additional augmentation with other pharmacological and psychological treatments ; in addition to reducing the risk of relapse , the treatment of residual symptoms may enhance compliance and long - term outcomes ( 88 ) ." ], [ "as the underlying neurobiological model of depression is increasingly understood , treatment providers are directed to recognise that the factors that may initiate a mdd episode and those that maintain the illness are likely to be very different .", "genetic and stress vulnerabilities interplay to initiate a cascade of neurobiological alterations that disrupt a dynamic system .", "progressive effects of recurrent and chronic mdd may then be potentiated by further structural and functional abnormalities . given these long - term consequences ,", "an essential objective of treatment must be to restore normative functioning and prevent further neurobiological structural alterations .", "increasing 5-ht and ne neurotransmission is likely to initiate true recovery with the restoration of neurotrophic support , glucocorticoid signalling and neuroendocrine regulation .", "the use of dual reuptake inhibitors enhances the probability of remission as it addresses the complex interplay of the emotional and physical symptoms of mdd .", "painful physical symptoms are increasingly recognised as having a significant impact on functioning and recovery ; thus , affirming the need for antidepressant treatments that can effectively reduce these symptoms as well . from the neurobiological model , the treatment guidelines of early", ", comprehensive and progressive treatment require a change in perspective for both patients and providers .", "a residual symptom may be interpreted as a proxy of an active disease state , with ensuing structural alterations and systemic consequences . with remission and recovery as the goal", ", patients will need to be educated about the benefits of long - term treatment rather than episodic or incomplete intervention .", "a biopsychosocial treatment model that incorporates cognitive - behavioural or interpersonal therapy along with pharmacological interventions serves to address both the initiation and maintenance factors and can reduce the risk of relapse ( 89 ) .", "once remission is attained , maintenance of effect may become the more appropriate term , rather than relapse prevention , to emphasise the necessity for an ongoing collaboration between patient and physician in order to maintain neurobiological homeostasis ." ] ]

[ "surface interactions of liquids in porous media are of great importance , particularly in the field of heterogeneous catalysis,[15 ] and the ability to understand surface interactions is essential for efficient and rational catalyst design . however ,", "probing liquid surface interactions in liquid - saturated porous media is particularly challenging . established techniques for probing the interaction of molecules at solid surfaces include isosteric heat of adsorption , temperature - programmed desorption ( tpd ) , infra - red ( ir ) spectroscopy , and nuclear magnetic resonance ( nmr ) chemical shift ( ) . however , all these measurements have limitations , and none are able to probe , non - destructively , the behaviour of molecules on catalyst surfaces at realistic reaction conditions .", "for example , whilst isosteric heat of adsorption measurements are non - invasive , they require the determination of adsorption isotherms at different temperatures , and are therefore extremely time - consuming ; characterisation of co - adsorbed systems may take in excess of well over ten hours .", "tpd is known to cause in situ reactions of some organic molecules leading to dehydration , isomerisation and decomposition , and is also limited in its ability to probe co - adsorption.[9 , 10 ] ir is well known to be limited to liquids and solids that are transparent in the frequency range of interest .", "finally , nmr chemical shift interpretation is robust only for monolayer surface coverage.[12 , 13 ] it has recently been reported that under certain conditions chemical shift measurements can be used to probe surface interactions over pvp - stabilised metal nanoparticles . in this example , the chemical shift of formic acid ( adsorbate ) on metal colloid catalysts , measured by c nmr spectroscopy in aqueous suspension , was used to infer the strength of surface interaction . however , to achieve this measurement it was necessary to avoid direct contact between the c atom of the adsorbate and the metal surface by inserting oxygen - atom spacers , in order to eliminate spectral line broadening . whilst this is clearly an elegant measurement ,", "it can not readily be transferred to measurements of liquid surface interactions in saturated porous materials of industrial relevance . in general , the use of nmr methods to probe adsorption is made challenging because of the broadening of the nmr lineshape associated with the adsorbed species , which arises because of the magnetic susceptibility differences between adsorbate and adsorbent . there exist many reports of nmr experiments probing different aspects of adsorption .", "c nmr is often the method of choice because of the wider chemical shift range , and hence spectral resolution attainable , relative to h observation . examples of c nmr to study adsorption include chemical shift studies to investigate the structure of the adsorbed species and the determination of acid site strength ( mostly in zeolites ) using suitable probe molecules such as acetonitrile or acetone.[16 , 17 ] c nmr approaches usually require the use of enriched c species due to the low signal - to - noise ratio of c signal at natural abundance .", "however , it is noted that c studies of adsorbed species at natural isotopic abundance using the distortionless enhancement by polarisation transfer ( dept ) technique have been reported , although the strength of interaction was not probed using this technique .", "h nmr has been used by various researchers to study acidic strength of hydroxyl groups in zeolites , using deuterated probe molecules such as acetonitrile and pyridine.[1719 ] for example , zheng et al .", "have conducted experimental and theoretical studies of deuterated pyridine adsorption to investigate acidic strength of solid acids and found a linear correlation between the h chemical shift of adsorbed pyridine and the proton affinity .", "thus , whilst various implementations of nmr spectroscopy have been used to study adsorption phenomena in porous media , a robust , generic technique readily applicable to co - adsorption and with the ability to study liquid - saturated pore spaces , as opposed to monolayer coverages , has not been presented .", "the method proposed in the present work exploits the modification of the nuclear spin relaxation time characteristics of the adsorbate that result from its interaction with the surface of the pore .", "the advantage of using so - called nmr relaxation measurements is that the characterisation of the adsorption interaction does not rely on the nmr lineshape and the ", "peak positionthe actual peak position associated with liquid confined within a porous medium , or chemical shift , may be influenced by factors other than the adsorbate ", "moreover , measurements based on chemical shift will be limited in terms of the systems that can be studied when multi - component liquid systems inside the pore space are of interest , because of the broad , overlapping , line shapes associated with each component .", "in contrast , the relaxation times of different chemical species in a multi - component system are often quite distinct even when their respective chemical shifts overlap .", "thus far[2124 ] it has been used as a qualitative probe yielding the relative strength of interactions between different adsorbate adsorbent systems .", "it is the purpose of this paper to demonstrate that the ratio of the spin - lattice to spin - spin ( or transverse ) relaxation time ( t1/t2 ) can be related directly to the activation energy of desorption characterising the strongest adsorption sites on the surface of the adsorbent , as determined by temperature programmed desorption ( tpd ) . in recent years", ", nmr relaxation has emerged as a non - invasive , chemically sensitive technique for studying surface interactions of liquids in saturated porous media . following radio - frequency ( rf ) excitation ,", "longitudinal t1 relaxation processes drive the longitudinal magnetisation to the equilibrium position , that is , aligned with the external magnetic field , whilst transverse t2 relaxation processes determine the rate of loss of phase coherence of the magnetisation in the transverse plane . reduced t1 and t2 relaxation times", "are observed when liquid molecules adsorb on a solid surface due to a change in the molecular mobility ; in bulk liquids , t1t2 . both t1 and", "t2 are affected by changes in the rotational correlation time of the adsorbate molecules . however , t2 is further influenced by a translational correlation time associated with surface diffusion.[27 , 28 ] consequently , when molecules adsorb on surfaces , changes in their translational and rotational dynamics influence t2 more than t1 , resulting in t1>t2 . in porous materials with a high surface - to - volume ratio ,", "s / v , the observed relaxation rates are proportional to s / v , so absolute t1 and t2 measurements can not be readily used to compare interactions between materials with differing pore geometry , pore size and density of adsorption sites . however , the ratio of relaxation times t1/t2 is ( to leading order ) independent of these characteristics .", "it has been observed empirically that the ratio t1/t2 provides an indication of the relative strength of surface interaction for different liquids in the same catalyst .", "for example , in the aforementioned work , it was observed that water imbibed in pd / al2o3 catalyst trilobes exhibits a much larger t1/t2 ratio ( stronger surface interaction ) than 2-butanone on the same surface ; water is known to poison this palladium catalyst in the hydrogenation of 2-butanone by preferential adsorption on the active sites .", "other studies , which exploit t1/t2 as a probe of surface interaction strength and the ability of these measurements to help understand catalytic performance , have recently been reported.[2123 ] it is important to note that besides the advantage of being independent of pore geometry , the use of t1 and t2 relaxation times is particularly advantageous if co - adsorption in porous materials is to be probed .", "this is because the method has the advantage that different species can often be separated based on their relaxation time values even when the two species can not be separated in the chemical shift domain , which is often the case in porous materials .", "this was shown when studying co - adsorption of various binary mixtures in porous al2o3 and sio2 supported catalysts .", "the aim of the present work is to confirm theoretically that the ratio t1/t2 can be used as a qualitative descriptor of surface affinity , and extend the measurement to provide a quantitative metric of the same .", "moreover , if this is the case , can the t1/t2 measurement be shown to correlate with an accepted laboratory characterisation of the adsorption interaction , namely tpd ?", "indeed , if the theoretical interpretation summarised below is appropriate , we might expect the value of t1/t2 to correlate with the strongest adsorption energies characterising a particular system , since nuclear spin relaxation is dominated by the strongest relaxation sinks present . in this section", "we establish proportionality between nmr relaxation and adsorption energy for a molecule interacting with the surface of a pore ; the relaxation of the adsorbed molecule will be modulated by the reduction in molecular mobility and the dipole ", "the present analysis develops from the established theory of surface relaxation , which assumes the pore surface contains paramagnetic impurities that act as relaxation sinks and it is dipole - electron coupling that determines t1/t2 . in recent work we demonstrated that the analysis of godefroy et al . for proton", "electron dipole coupling is extensible to proton proton dipole coupling , albeit with a much weaker coupling constant . in the absence of paramagnetic species , it is the active binding sites on the pore surface that are considered to be the relaxation sinks .", "molecules adsorbed onto a surface undergo two - dimensional ( 2d ) translational motion governed by the activation energy em .", "the diffusion coefficient associated with this surface motion is : in which dm0 is a temperature independent contribution to surface diffusion , r is the ideal gas constant , and t is the temperature . in liquid - saturated porous materials , adsorbed molecules", "the effective surface diffusion coefficient deff ( observed ) is modified from dm due to the finite residence time on the surface .", "therefore , the effective diffusion coefficient is : in which e = emes is an activation energy for surface diffusion .", "the diffusion coefficient has an associated correlation time m that describes the time for diffusion between active binding sites , such that deff(t)/(4m ) , where is the thickness of the adsorbed surface layer .", "thus , the surface correlation time is defined as : there will also be a surface residence time for adsorbed molecules , which is similarly defined as : the observed relaxation times for liquid saturated porous media are : in which 1,2 are the surface relaxivity values governing longitudinal and transverse relaxation processes at the pore surface , respectively , t1,2,bulk are the respective relaxation times of the bulk liquid , and p is the fraction of spins on the pore surface . for the catalyst supports of interest , the pore diameters are on the order of nanometres , such that the observed relaxation times are determined only by surface relaxation .", "the ratio of observed ( surface ) relaxation times is defined by the spectral density function j(0 ) as : at high field , this ratio is known to be insensitive to larmor frequency .", "based on typical measured values of m , s for liquids on an alumina surface , and given that 0 is large , we assume that ( m/s)(0m)1 . through a series of trivial algebraic steps", "suggest that t1/t2 is sensitive to small changes in ln(m ) but insensitive to large changes in ln(s/m ) as m , s are not independent variables .", "therefore , for the purpose of establishing a relationship between surface relaxation and adsorption energy it follows from equation ( 3 ) and the reduced form of equation ( 6 ) , given 0 is constant , that : we define a dimensionless surface interaction parameter esurf=t2/t1 for the purpose of interpreting surface relaxation as a surface energy . for the materials of interest in this study , we are concerned only with the dynamics of the adsorbed liquid that give rise to altered nmr relaxation times .", "however , it is important to consider the extent to which other properties of the sample will influence the observed relaxation times , and the t1/t2 ratio .", "the most important of these are 1 ) the presence of paramagnetic species on the pore surface , and 2 ) the presence of magnetic susceptibility contrast between the solid and liquid .", "first , considering the case of presence of paramagnetic species on the pore surface ; such species will alter the sensitivity of the relaxation mechanism to m and s .", "empirical evidence suggests the ratio t1/t2 remains independent of the density of surface paramagnetic species ( such as fe ) when the concentration of the paramagnetic impurity is less than 5000 ppm,[36 , 37 ] even though the individual values of t1 and t2 are affected by impurities at concentrations greater than 200 ppm . at paramagnetic impurity contents > 5000 ppm , re - calibration of the correlation between esurf ( nmr ) and emax ( tpd )", ", the presence of magnetic susceptibility contrast between the solid and liquid will distort the magnetic field such that the diffusion - sensitive t2 measurement is no longer determined by surface adsorption .", "we have shown recently that the correct t2 relaxation time can be extracted in the presence of pore - scale magnetic field inhomogeneities .", "neither of these conditions apply to the materials studied here , so the observed t1/t2 ratios can be interpreted directly in terms of a surface adsorption energy .", "in this section we establish proportionality between nmr relaxation and adsorption energy for a molecule interacting with the surface of a pore ; the relaxation of the adsorbed molecule will be modulated by the reduction in molecular mobility and the dipole dipole coupling between protons in the adsorbed molecule and those bound to the surface .", "the present analysis develops from the established theory of surface relaxation , which assumes the pore surface contains paramagnetic impurities that act as relaxation sinks and it is dipole - electron coupling that determines t1/t2 . in recent work we demonstrated that the analysis of godefroy et al . for proton", " electron dipole coupling is extensible to proton proton dipole coupling , albeit with a much weaker coupling constant . in the absence of paramagnetic species , it is the active binding sites on the pore surface that are considered to be the relaxation sinks .", "molecules adsorbed onto a surface undergo two - dimensional ( 2d ) translational motion governed by the activation energy em .", "the diffusion coefficient associated with this surface motion is : in which dm0 is a temperature independent contribution to surface diffusion , r is the ideal gas constant , and t is the temperature . in liquid - saturated porous materials , adsorbed molecules exchange with molecules not directly interacting with the pore surface .", "the effective surface diffusion coefficient deff ( observed ) is modified from dm due to the finite residence time on the surface .", "therefore , the effective diffusion coefficient is : in which e = emes is an activation energy for surface diffusion .", "the diffusion coefficient has an associated correlation time m that describes the time for diffusion between active binding sites , such that deff(t)/(4m ) , where is the thickness of the adsorbed surface layer .", "thus , the surface correlation time is defined as : there will also be a surface residence time for adsorbed molecules , which is similarly defined as : the observed relaxation times for liquid saturated porous media are : in which 1,2 are the surface relaxivity values governing longitudinal and transverse relaxation processes at the pore surface , respectively , t1,2,bulk are the respective relaxation times of the bulk liquid , and p is the fraction of spins on the pore surface . for the catalyst supports of interest , the pore diameters are on the order of nanometres , such that the observed relaxation times are determined only by surface relaxation .", "the ratio of observed ( surface ) relaxation times is defined by the spectral density function j(0 ) as : at high field , this ratio is known to be insensitive to larmor frequency .", "based on typical measured values of m , s for liquids on an alumina surface , and given that 0 is large , we assume that ( m/s)(0m)1 . through a series of trivial algebraic steps it can be shown that t1/t2 is proportional to ln(s/m)/ln(0m ) .", "theoretical calculations previously reported by mcdonald et al . suggest that t1/t2 is sensitive to small changes in ln(m ) but insensitive to large changes in ln(s/m ) as m , s are not independent variables .", "therefore , for the purpose of establishing a relationship between surface relaxation and adsorption energy it follows from equation ( 3 ) and the reduced form of equation ( 6 ) , given 0 is constant , that : we define a dimensionless surface interaction parameter esurf=t2/t1 for the purpose of interpreting surface relaxation as a surface energy . for the materials of interest in this study ,", "we are concerned only with the dynamics of the adsorbed liquid that give rise to altered nmr relaxation times .", "however , it is important to consider the extent to which other properties of the sample will influence the observed relaxation times , and the t1/t2 ratio .", "the most important of these are 1 ) the presence of paramagnetic species on the pore surface , and 2 ) the presence of magnetic susceptibility contrast between the solid and liquid .", "first , considering the case of presence of paramagnetic species on the pore surface ; such species will alter the sensitivity of the relaxation mechanism to m and s .", "empirical evidence suggests the ratio t1/t2 remains independent of the density of surface paramagnetic species ( such as fe ) when the concentration of the paramagnetic impurity is less than 5000 ppm,[36 , 37 ] even though the individual values of t1 and t2 are affected by impurities at concentrations greater than 200 ppm . at paramagnetic impurity contents > 5000 ppm , re - calibration of the correlation between esurf ( nmr ) and emax ( tpd )", ", the presence of magnetic susceptibility contrast between the solid and liquid will distort the magnetic field such that the diffusion - sensitive t2 measurement is no longer determined by surface adsorption .", "we have shown recently that the correct t2 relaxation time can be extracted in the presence of pore - scale magnetic field inhomogeneities .", "neither of these conditions apply to the materials studied here , so the observed t1/t2 ratios can be interpreted directly in terms of a surface adsorption energy .", "zirconia ( zro2 ) , -alumina ( -al2o3 ) , -alumina ( -al2o3 ) and silica ( sio2 ) were supplied by johnson matthey plc ; anatase titania ( tio2-a ) and rutile titania ( tio2-r ) were supplied by evonik - degussa .", "all materials were used as - received in the form of 34 mm diameter cylindrical and 45 mm length extrudate pellets .", "samples for nmr relaxation and tpd measurements were prepared by impregnating the different porous materials in deionised water for at least 24 h. excess surface water was removed from the pellets by gently contacting them on a pre - soaked filter paper .", "the suppliers of the alumina , silica and zirconia samples do not report any measureable paramagnetic impurities .", "the average pore diameter determined was as follows : tio2-a ( 23 nm ) ; tio2-r ( 38 nm ) ; -al2o3 ( 15 nm ) ; -al2o3 ( 7 nm ) ; sio2 ( 5 nm ) and zro2 ( 9 nm ) .", "nmr experiments were carried out on a bruker biospec ( horizontal bore ) av 85 mhz spectrometer .", "samples were prepared by soaking the different solid materials in the liquid for at least 24 h ; the pellets of solid material were dried on a pre - soaked filter paper , in order to remove any excess liquid on the external surface , and finally transferred into 20 mm diameter glass vials , which were then sealed .", "a standard t1t2 pulse sequence was used to acquire 2d correlation data . the specific sequence of rf pulses is illustrated in figure 1 .", "a 180 rf inversion pulse rotates the spin magnetisation , initially at equilibrium along the z axis , onto the z axis .", "longitudinal t1 relaxation then occurs for a time 1 ; during this time the spin system recovers along the z axis and is aligned with the static magnetic field of magnitude b0 . after this recovery time , a 90 rf excitation pulse is applied and rotates the recovered spin magnetisation into the xy plane .", "a series of 180 rf refocusing pulses are then applied to generate a train of n spin echoes , each separated in time by te=22 .", "the amplitude of each echo is recorded as a single point , and all echo amplitudes are recorded in a single scan ( no chemical resolution ) .", "the amplitude of the initial echo is determined by the degree of recovery during 1 ; the envelope of the echo train is described by the transverse t2 relaxation of the spin ensemble . by repeating the experiment for different 1 recovery times , a 2d data matrix is constructed . here ,", "16 t1 recovery delays were used , ranging from 1=1 ms to 10 s ; n=1024 spin echoes were acquired in a single shot with an echo time spacing te=0.8 ms .", "a recycle delay of 10 s was included between each scan to ensure maximum signal was obtained at all times .", "the total experiment duration was 1.5 h , and included 32 repeat scans to accommodate the rf phase cycle and provide signal averaging to improve the signal - to - noise ratio ( snr ) of the data .", "the t1t2 pulse sequence , showing the rf pulses as thin ( 90 ) and thick ( 180 ) vertical bars .", "the amplitude of each of the n spin echoes is recorded as a single datum .", "the 2d data are inverted numerically to form a 2d distribution of t2 correlated against t1 .", "the nmr data are described by the first kind fredholm integral equation : in which the nmr signal is b , is the experimental error ( noise ) , f(t1,t2 ) is the required 2d correlation , and the kernel function k(1,t1,nte , t2 ) represents the expected form of the data so that : in which the first exponent describes the t1 relaxation and the second exponent describes the t2 relaxation . as the two exponents do not share a common time base , the kernel function is separable , that is , the expected behaviour of the signal amplitude for a given set of ( 1,t1 ) can be determined separately from the expected behaviour of the signal amplitude for a given set of ( nte , t2 ) .", "this separation allows us to solve the fredholm integral equation in ( 9 ) efficiently in vector ", "the result is biased to be positive , smooth , and bounded by pre - determined limits in t1 and t2 .", "a stable distribution , obtained in the presence of noise , is found using tikhonov regularisation with the smoothing parameter chosen using the generalised cross validation method ; a review of data inversion techniques is presented elsewhere .", "the inversion is highly susceptible to noise fluctuations and the degree of smoothing increases as the snr decreases .", "variations in distribution shape of less than an order of magnitude on the relaxation time axes are therefore considered to be determined by the signal quality and not the sample .", "tpd experiments were performed on a catlab - pcs ( hiden analytical ) , comprising a microreactor module with integrated mass spectrometer .", "pellets saturated with water were placed into the glass microreactor in a high purity helium flow at a constant rate of 40 ml min and left for one hour at 45 c until all the physisorbed water was removed .", "tpd curves of water ( m=18 amu ) were recorded in the range 451000 c with a heating rate of 20 c min .", "each tpd measurement lasted 3 h. the amount of water desorbed from each porous material during the tpd experiments was calculated by using a calibration factor , which was obtained by measuring the area under the tpd profile corresponding to a known amount of desorbed water from a calibration sample .", "the analysis of the tpd curves was carried out according to the condensation approximation method reported by barrie in order to obtain the distribution function of the activation energy of desorption .", "having derived equation ( 7 ) , we now demonstrate that t1/t2 for the systems studied can indeed be related to the strongest relaxation sinks present ( i.e. , the strongest adsorption sites present)the strength of the strongest adsorption sites being determined by tpd analysis .", "we determine the strength of water adsorption in six porous oxides ( extruded pellets ) used as supports and catalysts : tio2-a ( anatase ) , tio2-r ( rutile ) ( supplied by evonik - degussa ) , zro2 , -al2o3 , -al2o3 and sio2 . we chose water as the adsorbate because it is central to aqueous - phase heterogeneous catalytic processes and , in the context of this work , has the additional advantages of not decomposing during thermally driven desorption .", "the tpd energy distributions for water desorbing from the porous oxides are shown in figure 2 . due to the uncertainty associated with the tail of the tpd curves ,", "we determine the maximum activation energy of desorption ( emax ) where the integral area of the desorption curves is 95 % of the total , see figure 2 .", "energy distribution functions obtained from tpd analysis of water in a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the vertical dashed lines indicate the activation energy of desorption ( emax ) at which 95 % of species have desorbed .", "it is clearly seen that the shape of the tpd curves differs widely between the oxides studied . ignoring any detailed structure in these plots", ", we see that the tio2 materials ( figure 2a , b ) are associated with significantly lower maximum desorption energies than the other materials studied .", "based on the values of emax determined from the tpd the strength of the interaction of water with the porous solid decreases in the order : the t1t2 correlations for water in the porous oxides are shown in figure 3 . in each case , a single relaxation time component is observed ; the relaxation times are dominated by surface adsorbed species due to the small pore size and high surface area in these oxides .", "the ratio t1/t2 is obtained from the logarithmic mean of the individual t1,2 dimensions , which corresponds almost exactly with the maximum intensity of the 2d peak in these mono - modal distributions . in these porous materials with narrow , mono - modal pore size distributions , details of the peak shape are determined predominantly by the raw data quality ( degree of smoothing on inversion ) and are not considered representative of physical sample properties .", "the 2d correlations provide a straightforward visual comparison and are required to interpret relaxation results obtained from complicated systems with multiple liquids or diffusive exchange.[24 , 29 ] t1t2 correlation plots for water in : a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the solid diagonal line indicates t1=t2 ; the dashed line indicates t1/t2 at the maximum of the peak .", "a comparison between the nmr relaxation analysis and tpd is achieved by converting the t1/t2 ratio into an effective surface interaction parameter , esurf , using equation ( 7 ) .", "the comparison between emax ( tpd ) and esurf ( nmr ) is given in figure 4 , in which an excellent correlation is obtained between the two measurements .", "notably , the tio2 samples have a markedly lower esurf value compared to the other oxides , consistent with their tpd spectra being significantly different to those of the other oxides . elsewhere ,", "a comparison between zro2 and tio2 surfaces has been carried out by ignatchenko and co - workers , who concluded , through isotopic exchange experiments and density functional theory ( dft ) calculations , that zro2 has a greater affinity for water than tio2 .", "our results agree with these previous findings , and the high value of esurf obtained for water on zro2 correlates with the known strong affinity of water for this oxide surface .", "values of the isosteric heat of adsorption for water on sio2[47 , 48 ] have been reported to be comparable to those measured over -al2o3 .", "again , this is in line with the finding of this work , which shows very similar esurf values for sio2 and -al2o3 , with sio2 showing a slightly higher value .", "overall , our results show that esurf for a given liquid adsorbed within a range of chemically similar materials does correlate directly with the maximum activation energy of desorption obtained by tpd analysis , as shown in figure 4 .", "solid systems under investigation can be obtained , the esurf values can be converted to an estimate of the absolute maximum desorption energies characteristic of that system .", "comparison of esurf ( nmr ) against emax ( tpd ) for water on oxides .", "symbols correspond to ( left to right ) tio2-a , tio2-r , -al2o3 , sio2 , -al2o3 and zro2 .", "error bars represent the uncertainty in determining the peak position in the t1t2 distributions ( esurf ) and 5 % integral area under the tpd curves ( emax ) .", "the adsorption site densities , calculated from the area under the tpd curve according to the procedure described in the experimental section , were : 0.11 ( tio2-a and tio2-r ) , 0.84 ( -al2o3 ) , 1.38 ( sio2 ) , 2.95 ( -al2o3 ) , and 0.60 mmol m ( zro2 ) .", "no strong correlation is found between these values and the t1/t2 ratios , as expected from the earlier discussion[29 , 30 ] regarding the relatively low sensitivity of t1/t2 to site density compared to the changing nature of interactions between liquids and surfaces of different chemistry .", "zirconia ( zro2 ) , -alumina ( -al2o3 ) , -alumina ( -al2o3 ) and silica ( sio2 ) were supplied by johnson matthey plc ; anatase titania ( tio2-a ) and rutile titania ( tio2-r ) were supplied by evonik - degussa .", "all materials were used as - received in the form of 34 mm diameter cylindrical and 45 mm length extrudate pellets .", "samples for nmr relaxation and tpd measurements were prepared by impregnating the different porous materials in deionised water for at least 24 h. excess surface water was removed from the pellets by gently contacting them on a pre - soaked filter paper .", "the suppliers of the alumina , silica and zirconia samples do not report any measureable paramagnetic impurities .", "the average pore diameter determined was as follows : tio2-a ( 23 nm ) ; tio2-r ( 38 nm ) ; -al2o3 ( 15 nm ) ; -al2o3 ( 7 nm ) ; sio2 ( 5 nm ) and zro2 ( 9 nm ) .", "nmr experiments were carried out on a bruker biospec ( horizontal bore ) av 85 mhz spectrometer .", "samples were prepared by soaking the different solid materials in the liquid for at least 24 h ; the pellets of solid material were dried on a pre - soaked filter paper , in order to remove any excess liquid on the external surface , and finally transferred into 20 mm diameter glass vials , which were then sealed . a standard t1t2 pulse sequence was used to acquire 2d correlation data .", "a 180 rf inversion pulse rotates the spin magnetisation , initially at equilibrium along the z axis , onto the z axis .", "longitudinal t1 relaxation then occurs for a time 1 ; during this time the spin system recovers along the z axis and is aligned with the static magnetic field of magnitude b0 . after this recovery time , a 90 rf excitation pulse is applied and rotates the recovered spin magnetisation into the xy plane . a series of 180 rf refocusing pulses", "are then applied to generate a train of n spin echoes , each separated in time by te=22 .", "the amplitude of each echo is recorded as a single point , and all echo amplitudes are recorded in a single scan ( no chemical resolution ) .", "the amplitude of the initial echo is determined by the degree of recovery during 1 ; the envelope of the echo train is described by the transverse t2 relaxation of the spin ensemble . by repeating the experiment for different 1 recovery times , a 2d data matrix is constructed . here , 16 t1 recovery delays were used , ranging from 1=1 ms to 10 s ; n=1024 spin echoes were acquired in a single shot with an echo time spacing te=0.8 ms .", "a recycle delay of 10 s was included between each scan to ensure maximum signal was obtained at all times .", "the total experiment duration was 1.5 h , and included 32 repeat scans to accommodate the rf phase cycle and provide signal averaging to improve the signal - to - noise ratio ( snr ) of the data .", "the t1t2 pulse sequence , showing the rf pulses as thin ( 90 ) and thick ( 180 ) vertical bars .", "the amplitude of each of the n spin echoes is recorded as a single datum .", "the 2d data are inverted numerically to form a 2d distribution of t2 correlated against t1 .", "the nmr data are described by the first kind fredholm integral equation : in which the nmr signal is b , is the experimental error ( noise ) , f(t1,t2 ) is the required 2d correlation , and the kernel function k(1,t1,nte , t2 ) represents the expected form of the data so that : in which the first exponent describes the t1 relaxation and the second exponent describes the t2 relaxation . as the two exponents do not share a common time base , the kernel function is separable , that is , the expected behaviour of the signal amplitude for a given set of ( 1,t1 ) can be determined separately from the expected behaviour of the signal amplitude for a given set of ( nte , t2 ) .", "this separation allows us to solve the fredholm integral equation in ( 9 ) efficiently in vector ", "the result is biased to be positive , smooth , and bounded by pre - determined limits in t1 and t2 .", "a stable distribution , obtained in the presence of noise , is found using tikhonov regularisation with the smoothing parameter chosen using the generalised cross validation method ; a review of data inversion techniques is presented elsewhere .", "the inversion is highly susceptible to noise fluctuations and the degree of smoothing increases as the snr decreases .", "variations in distribution shape of less than an order of magnitude on the relaxation time axes are therefore considered to be determined by the signal quality and not the sample .", "tpd experiments were performed on a catlab - pcs ( hiden analytical ) , comprising a microreactor module with integrated mass spectrometer .", "pellets saturated with water were placed into the glass microreactor in a high purity helium flow at a constant rate of 40 ml min and left for one hour at 45 c until all the physisorbed water was removed .", "tpd curves of water ( m=18 amu ) were recorded in the range 451000 c with a heating rate of 20 c min .", "each tpd measurement lasted 3 h. the amount of water desorbed from each porous material during the tpd experiments was calculated by using a calibration factor , which was obtained by measuring the area under the tpd profile corresponding to a known amount of desorbed water from a calibration sample .", "the analysis of the tpd curves was carried out according to the condensation approximation method reported by barrie in order to obtain the distribution function of the activation energy of desorption .", "having derived equation ( 7 ) , we now demonstrate that t1/t2 for the systems studied can indeed be related to the strongest relaxation sinks present ( i.e. , the strongest adsorption sites present)the strength of the strongest adsorption sites being determined by tpd analysis .", "we determine the strength of water adsorption in six porous oxides ( extruded pellets ) used as supports and catalysts : tio2-a ( anatase ) , tio2-r ( rutile ) ( supplied by evonik - degussa ) , zro2 , -al2o3 , -al2o3 and sio2 .", "we chose water as the adsorbate because it is central to aqueous - phase heterogeneous catalytic processes and , in the context of this work , has the additional advantages of not decomposing during thermally driven desorption .", "the tpd energy distributions for water desorbing from the porous oxides are shown in figure 2 . due to the uncertainty associated with the tail of the tpd curves ,", "we determine the maximum activation energy of desorption ( emax ) where the integral area of the desorption curves is 95 % of the total , see figure 2 .", "energy distribution functions obtained from tpd analysis of water in a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the vertical dashed lines indicate the activation energy of desorption ( emax ) at which 95 % of species have desorbed .", "it is clearly seen that the shape of the tpd curves differs widely between the oxides studied . ignoring any detailed structure in these plots", ", we see that the tio2 materials ( figure 2a , b ) are associated with significantly lower maximum desorption energies than the other materials studied . based on the values of emax determined from the tpd the strength of the interaction of water with the porous solid decreases in the order : the t1t2 correlations for water in the porous oxides are shown in figure 3 . in each case , a single relaxation time component is observed ; the relaxation times are dominated by surface adsorbed species due to the small pore size and high surface area in these oxides .", "the ratio t1/t2 is obtained from the logarithmic mean of the individual t1,2 dimensions , which corresponds almost exactly with the maximum intensity of the 2d peak in these mono - modal distributions . in these porous materials with narrow , mono - modal pore size distributions , details of the peak shape are determined predominantly by the raw data quality ( degree of smoothing on inversion ) and are not considered representative of physical sample properties .", "the 2d correlations provide a straightforward visual comparison and are required to interpret relaxation results obtained from complicated systems with multiple liquids or diffusive exchange.[24 , 29 ] t1t2 correlation plots for water in : a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the solid diagonal line indicates t1=t2 ; the dashed line indicates t1/t2 at the maximum of the peak .", "a comparison between the nmr relaxation analysis and tpd is achieved by converting the t1/t2 ratio into an effective surface interaction parameter , esurf , using equation ( 7 ) .", "the comparison between emax ( tpd ) and esurf ( nmr ) is given in figure 4 , in which an excellent correlation is obtained between the two measurements .", "notably , the tio2 samples have a markedly lower esurf value compared to the other oxides , consistent with their tpd spectra being significantly different to those of the other oxides . elsewhere ,", "a comparison between zro2 and tio2 surfaces has been carried out by ignatchenko and co - workers , who concluded , through isotopic exchange experiments and density functional theory ( dft ) calculations , that zro2 has a greater affinity for water than tio2 .", "our results agree with these previous findings , and the high value of esurf obtained for water on zro2 correlates with the known strong affinity of water for this oxide surface . measurements of isosteric heat of adsorption have also been used to study porous oxides .", "values of the isosteric heat of adsorption for water on sio2[47 , 48 ] have been reported to be comparable to those measured over -al2o3 .", "again , this is in line with the finding of this work , which shows very similar esurf values for sio2 and -al2o3 , with sio2 showing a slightly higher value .", "overall , our results show that esurf for a given liquid adsorbed within a range of chemically similar materials does correlate directly with the maximum activation energy of desorption obtained by tpd analysis , as shown in figure 4 .", "solid systems under investigation can be obtained , the esurf values can be converted to an estimate of the absolute maximum desorption energies characteristic of that system .", "comparison of esurf ( nmr ) against emax ( tpd ) for water on oxides .", "symbols correspond to ( left to right ) tio2-a , tio2-r , -al2o3 , sio2 , -al2o3 and zro2 .", "error bars represent the uncertainty in determining the peak position in the t1t2 distributions ( esurf ) and 5 % integral area under the tpd curves ( emax ) .", "the adsorption site densities , calculated from the area under the tpd curve according to the procedure described in the experimental section , were : 0.11 ( tio2-a and tio2-r ) , 0.84 ( -al2o3 ) , 1.38 ( sio2 ) , 2.95 ( -al2o3 ) , and 0.60 mmol m ( zro2 ) .", "no strong correlation is found between these values and the t1/t2 ratios , as expected from the earlier discussion[29 , 30 ] regarding the relatively low sensitivity of t1/t2 to site density compared to the changing nature of interactions between liquids and surfaces of different chemistry .", "in this work we have presented a theoretical analysis to show that the ratio of nmr relaxation times , t1/t2 , can be related directly to the adsorbate ", "adsorbent interaction energy characterising adsorption in a porous material , and we introduce a quantitative metric esurf ( based on the relaxation time ratio ) characterising the strength of this surface interaction .", "we confirm that the t1/t2 ratio is insensitive to pore geometry , allowing adsorption of liquids to be compared between different materials .", "given that the relaxation time characteristics of the nuclear spin system will be dominated by the strongest adsorption sites , the hypothesis that t2/t1 should correlate directly with the maximum activation energy of desorption observed in a temperature - programmed desorption experiment was tested ; the data were found to support this hypothesis . given this result", ", we now have a physical interpretation of the nmr relaxation data in terms of a surface activation energy of desorption .", "it follows from the analysis that for a series of materials for which at least two calibration values are obtainable by tpd , the esurf parameter yields a direct estimate of the maximum activation energy of desorption from the surface .", "although the method has been demonstrated for water , the nmr relaxation analysis can be extended to probe non - invasively any adsorbed species , notably organic molecules .", "further , given that nmr relaxation is readily extended to measurements at elevated conditions of temperature and pressure , these results suggest that the technique might be considered a useful addition to the toolkit of techniques employed to characterise adsorption in porous media used for applications in catalysis and separations processes . beyond these applications , demonstrating that t1/t2 , when expressed as esurf , is a quantitative measure of interaction strength has implications for determining surface wettability in oil - field reservoir rocks , an important parameter for predicting oil recovery .", "an improved correlation between relaxation time and surface adsorption energy could be obtained by measuring t1/t2 as a function of temperature or field strength , although such procedures for providing measurements of surface interaction strength would be considerably more time consuming .", "nmr relaxation has been validated as a robust and versatile tool for quantitatively comparing liquid ", "solid interactions in porous materials . in principle , the method is applicable to the characterisation of both liquid and gas phase adsorption , although signal - to - noise considerations may limit its applicability to gas solid interactions .", "this limitation and the consideration of chemically different surfaces ( e.g. , carbon - based ) , or materials with much smaller pore size ( e.g. , zeolites ) are the subject of further consideration ." ]

[ "<S> nuclear magnetic resonance ( nmr ) relaxation times are shown to provide a unique probe of adsorbate adsorbent interactions in liquid - saturated porous materials . </S>", "<S> a short theoretical analysis is presented , which shows that the ratio of the longitudinal to transverse relaxation times ( t1/t2 ) is related to an adsorbate adsorbent interaction energy , and we introduce a quantitative metric esurf ( based on the relaxation time ratio ) characterising the strength of this surface interaction . </S>", "<S> we then consider the interaction of water with a range of oxide surfaces ( tio2 anatase , tio2 rutile , -al2o3 , sio2 , -al2o3 and zro2 ) and show that esurf correlates with the strongest adsorption sites present , as determined by temperature programmed desorption ( tpd ) . </S>", "<S> thus we demonstrate that nmr relaxation measurements have a direct physical interpretation in terms of the characterisation of activation energy of desorption from the surface . </S>", "<S> further , for a series of chemically similar solid materials , in this case a range of oxide materials , for which at least two calibration values are obtainable by tpd , the esurf parameter yields a direct estimate of the maximum activation energy of desorption from the surface . </S>", "<S> the results suggest that t1/t2 measurements may become a useful addition to the methods available to characterise liquid - phase adsorption in porous materials . </S>", "<S> the particular motivation for this work is to characterise adsorbate </S>", "<S> surface interactions in liquid - phase catalysis . </S>" ]

[ "Introduction", "Surface relaxation and adsorption", "Experimental Section", "Materials", "NMR experiments and data analysis", "TPD experiments", "Experimental results and discussion", "Conclusion" ]

[ [ "surface interactions of liquids in porous media are of great importance , particularly in the field of heterogeneous catalysis,[15 ] and the ability to understand surface interactions is essential for efficient and rational catalyst design . however ,", "probing liquid surface interactions in liquid - saturated porous media is particularly challenging . established techniques for probing the interaction of molecules at solid surfaces include isosteric heat of adsorption , temperature - programmed desorption ( tpd ) , infra - red ( ir ) spectroscopy , and nuclear magnetic resonance ( nmr ) chemical shift ( ) . however , all these measurements have limitations , and none are able to probe , non - destructively , the behaviour of molecules on catalyst surfaces at realistic reaction conditions .", "for example , whilst isosteric heat of adsorption measurements are non - invasive , they require the determination of adsorption isotherms at different temperatures , and are therefore extremely time - consuming ; characterisation of co - adsorbed systems may take in excess of well over ten hours .", "tpd is known to cause in situ reactions of some organic molecules leading to dehydration , isomerisation and decomposition , and is also limited in its ability to probe co - adsorption.[9 , 10 ] ir is well known to be limited to liquids and solids that are transparent in the frequency range of interest .", "finally , nmr chemical shift interpretation is robust only for monolayer surface coverage.[12 , 13 ] it has recently been reported that under certain conditions chemical shift measurements can be used to probe surface interactions over pvp - stabilised metal nanoparticles . in this example , the chemical shift of formic acid ( adsorbate ) on metal colloid catalysts , measured by c nmr spectroscopy in aqueous suspension , was used to infer the strength of surface interaction . however , to achieve this measurement it was necessary to avoid direct contact between the c atom of the adsorbate and the metal surface by inserting oxygen - atom spacers , in order to eliminate spectral line broadening . whilst this is clearly an elegant measurement ,", "it can not readily be transferred to measurements of liquid surface interactions in saturated porous materials of industrial relevance . in general , the use of nmr methods to probe adsorption is made challenging because of the broadening of the nmr lineshape associated with the adsorbed species , which arises because of the magnetic susceptibility differences between adsorbate and adsorbent . there exist many reports of nmr experiments probing different aspects of adsorption .", "c nmr is often the method of choice because of the wider chemical shift range , and hence spectral resolution attainable , relative to h observation . examples of c nmr to study adsorption include chemical shift studies to investigate the structure of the adsorbed species and the determination of acid site strength ( mostly in zeolites ) using suitable probe molecules such as acetonitrile or acetone.[16 , 17 ] c nmr approaches usually require the use of enriched c species due to the low signal - to - noise ratio of c signal at natural abundance .", "however , it is noted that c studies of adsorbed species at natural isotopic abundance using the distortionless enhancement by polarisation transfer ( dept ) technique have been reported , although the strength of interaction was not probed using this technique .", "h nmr has been used by various researchers to study acidic strength of hydroxyl groups in zeolites , using deuterated probe molecules such as acetonitrile and pyridine.[1719 ] for example , zheng et al .", "have conducted experimental and theoretical studies of deuterated pyridine adsorption to investigate acidic strength of solid acids and found a linear correlation between the h chemical shift of adsorbed pyridine and the proton affinity .", "thus , whilst various implementations of nmr spectroscopy have been used to study adsorption phenomena in porous media , a robust , generic technique readily applicable to co - adsorption and with the ability to study liquid - saturated pore spaces , as opposed to monolayer coverages , has not been presented .", "the method proposed in the present work exploits the modification of the nuclear spin relaxation time characteristics of the adsorbate that result from its interaction with the surface of the pore .", "the advantage of using so - called nmr relaxation measurements is that the characterisation of the adsorption interaction does not rely on the nmr lineshape and the ", "peak positionthe actual peak position associated with liquid confined within a porous medium , or chemical shift , may be influenced by factors other than the adsorbate ", "moreover , measurements based on chemical shift will be limited in terms of the systems that can be studied when multi - component liquid systems inside the pore space are of interest , because of the broad , overlapping , line shapes associated with each component .", "in contrast , the relaxation times of different chemical species in a multi - component system are often quite distinct even when their respective chemical shifts overlap .", "thus far[2124 ] it has been used as a qualitative probe yielding the relative strength of interactions between different adsorbate adsorbent systems .", "it is the purpose of this paper to demonstrate that the ratio of the spin - lattice to spin - spin ( or transverse ) relaxation time ( t1/t2 ) can be related directly to the activation energy of desorption characterising the strongest adsorption sites on the surface of the adsorbent , as determined by temperature programmed desorption ( tpd ) . in recent years", ", nmr relaxation has emerged as a non - invasive , chemically sensitive technique for studying surface interactions of liquids in saturated porous media . following radio - frequency ( rf ) excitation ,", "longitudinal t1 relaxation processes drive the longitudinal magnetisation to the equilibrium position , that is , aligned with the external magnetic field , whilst transverse t2 relaxation processes determine the rate of loss of phase coherence of the magnetisation in the transverse plane . reduced t1 and t2 relaxation times", "are observed when liquid molecules adsorb on a solid surface due to a change in the molecular mobility ; in bulk liquids , t1t2 . both t1 and", "t2 are affected by changes in the rotational correlation time of the adsorbate molecules . however , t2 is further influenced by a translational correlation time associated with surface diffusion.[27 , 28 ] consequently , when molecules adsorb on surfaces , changes in their translational and rotational dynamics influence t2 more than t1 , resulting in t1>t2 . in porous materials with a high surface - to - volume ratio ,", "s / v , the observed relaxation rates are proportional to s / v , so absolute t1 and t2 measurements can not be readily used to compare interactions between materials with differing pore geometry , pore size and density of adsorption sites . however , the ratio of relaxation times t1/t2 is ( to leading order ) independent of these characteristics .", "it has been observed empirically that the ratio t1/t2 provides an indication of the relative strength of surface interaction for different liquids in the same catalyst .", "for example , in the aforementioned work , it was observed that water imbibed in pd / al2o3 catalyst trilobes exhibits a much larger t1/t2 ratio ( stronger surface interaction ) than 2-butanone on the same surface ; water is known to poison this palladium catalyst in the hydrogenation of 2-butanone by preferential adsorption on the active sites .", "other studies , which exploit t1/t2 as a probe of surface interaction strength and the ability of these measurements to help understand catalytic performance , have recently been reported.[2123 ] it is important to note that besides the advantage of being independent of pore geometry , the use of t1 and t2 relaxation times is particularly advantageous if co - adsorption in porous materials is to be probed .", "this is because the method has the advantage that different species can often be separated based on their relaxation time values even when the two species can not be separated in the chemical shift domain , which is often the case in porous materials .", "this was shown when studying co - adsorption of various binary mixtures in porous al2o3 and sio2 supported catalysts .", "the aim of the present work is to confirm theoretically that the ratio t1/t2 can be used as a qualitative descriptor of surface affinity , and extend the measurement to provide a quantitative metric of the same .", "moreover , if this is the case , can the t1/t2 measurement be shown to correlate with an accepted laboratory characterisation of the adsorption interaction , namely tpd ?", "indeed , if the theoretical interpretation summarised below is appropriate , we might expect the value of t1/t2 to correlate with the strongest adsorption energies characterising a particular system , since nuclear spin relaxation is dominated by the strongest relaxation sinks present . in this section", "we establish proportionality between nmr relaxation and adsorption energy for a molecule interacting with the surface of a pore ; the relaxation of the adsorbed molecule will be modulated by the reduction in molecular mobility and the dipole ", "the present analysis develops from the established theory of surface relaxation , which assumes the pore surface contains paramagnetic impurities that act as relaxation sinks and it is dipole - electron coupling that determines t1/t2 . in recent work we demonstrated that the analysis of godefroy et al . for proton", "electron dipole coupling is extensible to proton proton dipole coupling , albeit with a much weaker coupling constant . in the absence of paramagnetic species , it is the active binding sites on the pore surface that are considered to be the relaxation sinks .", "molecules adsorbed onto a surface undergo two - dimensional ( 2d ) translational motion governed by the activation energy em .", "the diffusion coefficient associated with this surface motion is : in which dm0 is a temperature independent contribution to surface diffusion , r is the ideal gas constant , and t is the temperature . in liquid - saturated porous materials , adsorbed molecules", "the effective surface diffusion coefficient deff ( observed ) is modified from dm due to the finite residence time on the surface .", "therefore , the effective diffusion coefficient is : in which e = emes is an activation energy for surface diffusion .", "the diffusion coefficient has an associated correlation time m that describes the time for diffusion between active binding sites , such that deff(t)/(4m ) , where is the thickness of the adsorbed surface layer .", "thus , the surface correlation time is defined as : there will also be a surface residence time for adsorbed molecules , which is similarly defined as : the observed relaxation times for liquid saturated porous media are : in which 1,2 are the surface relaxivity values governing longitudinal and transverse relaxation processes at the pore surface , respectively , t1,2,bulk are the respective relaxation times of the bulk liquid , and p is the fraction of spins on the pore surface . for the catalyst supports of interest , the pore diameters are on the order of nanometres , such that the observed relaxation times are determined only by surface relaxation .", "the ratio of observed ( surface ) relaxation times is defined by the spectral density function j(0 ) as : at high field , this ratio is known to be insensitive to larmor frequency .", "based on typical measured values of m , s for liquids on an alumina surface , and given that 0 is large , we assume that ( m/s)(0m)1 . through a series of trivial algebraic steps", "suggest that t1/t2 is sensitive to small changes in ln(m ) but insensitive to large changes in ln(s/m ) as m , s are not independent variables .", "therefore , for the purpose of establishing a relationship between surface relaxation and adsorption energy it follows from equation ( 3 ) and the reduced form of equation ( 6 ) , given 0 is constant , that : we define a dimensionless surface interaction parameter esurf=t2/t1 for the purpose of interpreting surface relaxation as a surface energy . for the materials of interest in this study , we are concerned only with the dynamics of the adsorbed liquid that give rise to altered nmr relaxation times .", "however , it is important to consider the extent to which other properties of the sample will influence the observed relaxation times , and the t1/t2 ratio .", "the most important of these are 1 ) the presence of paramagnetic species on the pore surface , and 2 ) the presence of magnetic susceptibility contrast between the solid and liquid .", "first , considering the case of presence of paramagnetic species on the pore surface ; such species will alter the sensitivity of the relaxation mechanism to m and s .", "empirical evidence suggests the ratio t1/t2 remains independent of the density of surface paramagnetic species ( such as fe ) when the concentration of the paramagnetic impurity is less than 5000 ppm,[36 , 37 ] even though the individual values of t1 and t2 are affected by impurities at concentrations greater than 200 ppm . at paramagnetic impurity contents > 5000 ppm , re - calibration of the correlation between esurf ( nmr ) and emax ( tpd )", ", the presence of magnetic susceptibility contrast between the solid and liquid will distort the magnetic field such that the diffusion - sensitive t2 measurement is no longer determined by surface adsorption .", "we have shown recently that the correct t2 relaxation time can be extracted in the presence of pore - scale magnetic field inhomogeneities .", "neither of these conditions apply to the materials studied here , so the observed t1/t2 ratios can be interpreted directly in terms of a surface adsorption energy ." ], [ "in this section we establish proportionality between nmr relaxation and adsorption energy for a molecule interacting with the surface of a pore ; the relaxation of the adsorbed molecule will be modulated by the reduction in molecular mobility and the dipole dipole coupling between protons in the adsorbed molecule and those bound to the surface .", "the present analysis develops from the established theory of surface relaxation , which assumes the pore surface contains paramagnetic impurities that act as relaxation sinks and it is dipole - electron coupling that determines t1/t2 . in recent work we demonstrated that the analysis of godefroy et al . for proton", " electron dipole coupling is extensible to proton proton dipole coupling , albeit with a much weaker coupling constant . in the absence of paramagnetic species , it is the active binding sites on the pore surface that are considered to be the relaxation sinks .", "molecules adsorbed onto a surface undergo two - dimensional ( 2d ) translational motion governed by the activation energy em .", "the diffusion coefficient associated with this surface motion is : in which dm0 is a temperature independent contribution to surface diffusion , r is the ideal gas constant , and t is the temperature . in liquid - saturated porous materials , adsorbed molecules exchange with molecules not directly interacting with the pore surface .", "the effective surface diffusion coefficient deff ( observed ) is modified from dm due to the finite residence time on the surface .", "therefore , the effective diffusion coefficient is : in which e = emes is an activation energy for surface diffusion .", "the diffusion coefficient has an associated correlation time m that describes the time for diffusion between active binding sites , such that deff(t)/(4m ) , where is the thickness of the adsorbed surface layer .", "thus , the surface correlation time is defined as : there will also be a surface residence time for adsorbed molecules , which is similarly defined as : the observed relaxation times for liquid saturated porous media are : in which 1,2 are the surface relaxivity values governing longitudinal and transverse relaxation processes at the pore surface , respectively , t1,2,bulk are the respective relaxation times of the bulk liquid , and p is the fraction of spins on the pore surface . for the catalyst supports of interest , the pore diameters are on the order of nanometres , such that the observed relaxation times are determined only by surface relaxation .", "the ratio of observed ( surface ) relaxation times is defined by the spectral density function j(0 ) as : at high field , this ratio is known to be insensitive to larmor frequency .", "based on typical measured values of m , s for liquids on an alumina surface , and given that 0 is large , we assume that ( m/s)(0m)1 . through a series of trivial algebraic steps it can be shown that t1/t2 is proportional to ln(s/m)/ln(0m ) .", "theoretical calculations previously reported by mcdonald et al . suggest that t1/t2 is sensitive to small changes in ln(m ) but insensitive to large changes in ln(s/m ) as m , s are not independent variables .", "therefore , for the purpose of establishing a relationship between surface relaxation and adsorption energy it follows from equation ( 3 ) and the reduced form of equation ( 6 ) , given 0 is constant , that : we define a dimensionless surface interaction parameter esurf=t2/t1 for the purpose of interpreting surface relaxation as a surface energy . for the materials of interest in this study ,", "we are concerned only with the dynamics of the adsorbed liquid that give rise to altered nmr relaxation times .", "however , it is important to consider the extent to which other properties of the sample will influence the observed relaxation times , and the t1/t2 ratio .", "the most important of these are 1 ) the presence of paramagnetic species on the pore surface , and 2 ) the presence of magnetic susceptibility contrast between the solid and liquid .", "first , considering the case of presence of paramagnetic species on the pore surface ; such species will alter the sensitivity of the relaxation mechanism to m and s .", "empirical evidence suggests the ratio t1/t2 remains independent of the density of surface paramagnetic species ( such as fe ) when the concentration of the paramagnetic impurity is less than 5000 ppm,[36 , 37 ] even though the individual values of t1 and t2 are affected by impurities at concentrations greater than 200 ppm . at paramagnetic impurity contents > 5000 ppm , re - calibration of the correlation between esurf ( nmr ) and emax ( tpd )", ", the presence of magnetic susceptibility contrast between the solid and liquid will distort the magnetic field such that the diffusion - sensitive t2 measurement is no longer determined by surface adsorption .", "we have shown recently that the correct t2 relaxation time can be extracted in the presence of pore - scale magnetic field inhomogeneities .", "neither of these conditions apply to the materials studied here , so the observed t1/t2 ratios can be interpreted directly in terms of a surface adsorption energy ." ], [ "zirconia ( zro2 ) , -alumina ( -al2o3 ) , -alumina ( -al2o3 ) and silica ( sio2 ) were supplied by johnson matthey plc ; anatase titania ( tio2-a ) and rutile titania ( tio2-r ) were supplied by evonik - degussa .", "all materials were used as - received in the form of 34 mm diameter cylindrical and 45 mm length extrudate pellets .", "samples for nmr relaxation and tpd measurements were prepared by impregnating the different porous materials in deionised water for at least 24 h. excess surface water was removed from the pellets by gently contacting them on a pre - soaked filter paper .", "the suppliers of the alumina , silica and zirconia samples do not report any measureable paramagnetic impurities .", "the average pore diameter determined was as follows : tio2-a ( 23 nm ) ; tio2-r ( 38 nm ) ; -al2o3 ( 15 nm ) ; -al2o3 ( 7 nm ) ; sio2 ( 5 nm ) and zro2 ( 9 nm ) .", "nmr experiments were carried out on a bruker biospec ( horizontal bore ) av 85 mhz spectrometer .", "samples were prepared by soaking the different solid materials in the liquid for at least 24 h ; the pellets of solid material were dried on a pre - soaked filter paper , in order to remove any excess liquid on the external surface , and finally transferred into 20 mm diameter glass vials , which were then sealed .", "a standard t1t2 pulse sequence was used to acquire 2d correlation data . the specific sequence of rf pulses is illustrated in figure 1 .", "a 180 rf inversion pulse rotates the spin magnetisation , initially at equilibrium along the z axis , onto the z axis .", "longitudinal t1 relaxation then occurs for a time 1 ; during this time the spin system recovers along the z axis and is aligned with the static magnetic field of magnitude b0 . after this recovery time , a 90 rf excitation pulse is applied and rotates the recovered spin magnetisation into the xy plane .", "a series of 180 rf refocusing pulses are then applied to generate a train of n spin echoes , each separated in time by te=22 .", "the amplitude of each echo is recorded as a single point , and all echo amplitudes are recorded in a single scan ( no chemical resolution ) .", "the amplitude of the initial echo is determined by the degree of recovery during 1 ; the envelope of the echo train is described by the transverse t2 relaxation of the spin ensemble . by repeating the experiment for different 1 recovery times , a 2d data matrix is constructed . here ,", "16 t1 recovery delays were used , ranging from 1=1 ms to 10 s ; n=1024 spin echoes were acquired in a single shot with an echo time spacing te=0.8 ms .", "a recycle delay of 10 s was included between each scan to ensure maximum signal was obtained at all times .", "the total experiment duration was 1.5 h , and included 32 repeat scans to accommodate the rf phase cycle and provide signal averaging to improve the signal - to - noise ratio ( snr ) of the data .", "the t1t2 pulse sequence , showing the rf pulses as thin ( 90 ) and thick ( 180 ) vertical bars .", "the amplitude of each of the n spin echoes is recorded as a single datum .", "the 2d data are inverted numerically to form a 2d distribution of t2 correlated against t1 .", "the nmr data are described by the first kind fredholm integral equation : in which the nmr signal is b , is the experimental error ( noise ) , f(t1,t2 ) is the required 2d correlation , and the kernel function k(1,t1,nte , t2 ) represents the expected form of the data so that : in which the first exponent describes the t1 relaxation and the second exponent describes the t2 relaxation . as the two exponents do not share a common time base , the kernel function is separable , that is , the expected behaviour of the signal amplitude for a given set of ( 1,t1 ) can be determined separately from the expected behaviour of the signal amplitude for a given set of ( nte , t2 ) .", "this separation allows us to solve the fredholm integral equation in ( 9 ) efficiently in vector ", "the result is biased to be positive , smooth , and bounded by pre - determined limits in t1 and t2 .", "a stable distribution , obtained in the presence of noise , is found using tikhonov regularisation with the smoothing parameter chosen using the generalised cross validation method ; a review of data inversion techniques is presented elsewhere .", "the inversion is highly susceptible to noise fluctuations and the degree of smoothing increases as the snr decreases .", "variations in distribution shape of less than an order of magnitude on the relaxation time axes are therefore considered to be determined by the signal quality and not the sample .", "tpd experiments were performed on a catlab - pcs ( hiden analytical ) , comprising a microreactor module with integrated mass spectrometer .", "pellets saturated with water were placed into the glass microreactor in a high purity helium flow at a constant rate of 40 ml min and left for one hour at 45 c until all the physisorbed water was removed .", "tpd curves of water ( m=18 amu ) were recorded in the range 451000 c with a heating rate of 20 c min .", "each tpd measurement lasted 3 h. the amount of water desorbed from each porous material during the tpd experiments was calculated by using a calibration factor , which was obtained by measuring the area under the tpd profile corresponding to a known amount of desorbed water from a calibration sample .", "the analysis of the tpd curves was carried out according to the condensation approximation method reported by barrie in order to obtain the distribution function of the activation energy of desorption .", "having derived equation ( 7 ) , we now demonstrate that t1/t2 for the systems studied can indeed be related to the strongest relaxation sinks present ( i.e. , the strongest adsorption sites present)the strength of the strongest adsorption sites being determined by tpd analysis .", "we determine the strength of water adsorption in six porous oxides ( extruded pellets ) used as supports and catalysts : tio2-a ( anatase ) , tio2-r ( rutile ) ( supplied by evonik - degussa ) , zro2 , -al2o3 , -al2o3 and sio2 . we chose water as the adsorbate because it is central to aqueous - phase heterogeneous catalytic processes and , in the context of this work , has the additional advantages of not decomposing during thermally driven desorption .", "the tpd energy distributions for water desorbing from the porous oxides are shown in figure 2 . due to the uncertainty associated with the tail of the tpd curves ,", "we determine the maximum activation energy of desorption ( emax ) where the integral area of the desorption curves is 95 % of the total , see figure 2 .", "energy distribution functions obtained from tpd analysis of water in a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the vertical dashed lines indicate the activation energy of desorption ( emax ) at which 95 % of species have desorbed .", "it is clearly seen that the shape of the tpd curves differs widely between the oxides studied . ignoring any detailed structure in these plots", ", we see that the tio2 materials ( figure 2a , b ) are associated with significantly lower maximum desorption energies than the other materials studied .", "based on the values of emax determined from the tpd the strength of the interaction of water with the porous solid decreases in the order : the t1t2 correlations for water in the porous oxides are shown in figure 3 . in each case , a single relaxation time component is observed ; the relaxation times are dominated by surface adsorbed species due to the small pore size and high surface area in these oxides .", "the ratio t1/t2 is obtained from the logarithmic mean of the individual t1,2 dimensions , which corresponds almost exactly with the maximum intensity of the 2d peak in these mono - modal distributions . in these porous materials with narrow , mono - modal pore size distributions , details of the peak shape are determined predominantly by the raw data quality ( degree of smoothing on inversion ) and are not considered representative of physical sample properties .", "the 2d correlations provide a straightforward visual comparison and are required to interpret relaxation results obtained from complicated systems with multiple liquids or diffusive exchange.[24 , 29 ] t1t2 correlation plots for water in : a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the solid diagonal line indicates t1=t2 ; the dashed line indicates t1/t2 at the maximum of the peak .", "a comparison between the nmr relaxation analysis and tpd is achieved by converting the t1/t2 ratio into an effective surface interaction parameter , esurf , using equation ( 7 ) .", "the comparison between emax ( tpd ) and esurf ( nmr ) is given in figure 4 , in which an excellent correlation is obtained between the two measurements .", "notably , the tio2 samples have a markedly lower esurf value compared to the other oxides , consistent with their tpd spectra being significantly different to those of the other oxides . elsewhere ,", "a comparison between zro2 and tio2 surfaces has been carried out by ignatchenko and co - workers , who concluded , through isotopic exchange experiments and density functional theory ( dft ) calculations , that zro2 has a greater affinity for water than tio2 .", "our results agree with these previous findings , and the high value of esurf obtained for water on zro2 correlates with the known strong affinity of water for this oxide surface .", "values of the isosteric heat of adsorption for water on sio2[47 , 48 ] have been reported to be comparable to those measured over -al2o3 .", "again , this is in line with the finding of this work , which shows very similar esurf values for sio2 and -al2o3 , with sio2 showing a slightly higher value .", "overall , our results show that esurf for a given liquid adsorbed within a range of chemically similar materials does correlate directly with the maximum activation energy of desorption obtained by tpd analysis , as shown in figure 4 .", "solid systems under investigation can be obtained , the esurf values can be converted to an estimate of the absolute maximum desorption energies characteristic of that system .", "comparison of esurf ( nmr ) against emax ( tpd ) for water on oxides .", "symbols correspond to ( left to right ) tio2-a , tio2-r , -al2o3 , sio2 , -al2o3 and zro2 .", "error bars represent the uncertainty in determining the peak position in the t1t2 distributions ( esurf ) and 5 % integral area under the tpd curves ( emax ) .", "the adsorption site densities , calculated from the area under the tpd curve according to the procedure described in the experimental section , were : 0.11 ( tio2-a and tio2-r ) , 0.84 ( -al2o3 ) , 1.38 ( sio2 ) , 2.95 ( -al2o3 ) , and 0.60 mmol m ( zro2 ) .", "no strong correlation is found between these values and the t1/t2 ratios , as expected from the earlier discussion[29 , 30 ] regarding the relatively low sensitivity of t1/t2 to site density compared to the changing nature of interactions between liquids and surfaces of different chemistry ." ], [ "zirconia ( zro2 ) , -alumina ( -al2o3 ) , -alumina ( -al2o3 ) and silica ( sio2 ) were supplied by johnson matthey plc ; anatase titania ( tio2-a ) and rutile titania ( tio2-r ) were supplied by evonik - degussa .", "all materials were used as - received in the form of 34 mm diameter cylindrical and 45 mm length extrudate pellets .", "samples for nmr relaxation and tpd measurements were prepared by impregnating the different porous materials in deionised water for at least 24 h. excess surface water was removed from the pellets by gently contacting them on a pre - soaked filter paper .", "the suppliers of the alumina , silica and zirconia samples do not report any measureable paramagnetic impurities .", "the average pore diameter determined was as follows : tio2-a ( 23 nm ) ; tio2-r ( 38 nm ) ; -al2o3 ( 15 nm ) ; -al2o3 ( 7 nm ) ; sio2 ( 5 nm ) and zro2 ( 9 nm ) ." ], [ "nmr experiments were carried out on a bruker biospec ( horizontal bore ) av 85 mhz spectrometer .", "samples were prepared by soaking the different solid materials in the liquid for at least 24 h ; the pellets of solid material were dried on a pre - soaked filter paper , in order to remove any excess liquid on the external surface , and finally transferred into 20 mm diameter glass vials , which were then sealed . a standard t1t2 pulse sequence was used to acquire 2d correlation data .", "a 180 rf inversion pulse rotates the spin magnetisation , initially at equilibrium along the z axis , onto the z axis .", "longitudinal t1 relaxation then occurs for a time 1 ; during this time the spin system recovers along the z axis and is aligned with the static magnetic field of magnitude b0 . after this recovery time , a 90 rf excitation pulse is applied and rotates the recovered spin magnetisation into the xy plane . a series of 180 rf refocusing pulses", "are then applied to generate a train of n spin echoes , each separated in time by te=22 .", "the amplitude of each echo is recorded as a single point , and all echo amplitudes are recorded in a single scan ( no chemical resolution ) .", "the amplitude of the initial echo is determined by the degree of recovery during 1 ; the envelope of the echo train is described by the transverse t2 relaxation of the spin ensemble . by repeating the experiment for different 1 recovery times , a 2d data matrix is constructed . here , 16 t1 recovery delays were used , ranging from 1=1 ms to 10 s ; n=1024 spin echoes were acquired in a single shot with an echo time spacing te=0.8 ms .", "a recycle delay of 10 s was included between each scan to ensure maximum signal was obtained at all times .", "the total experiment duration was 1.5 h , and included 32 repeat scans to accommodate the rf phase cycle and provide signal averaging to improve the signal - to - noise ratio ( snr ) of the data .", "the t1t2 pulse sequence , showing the rf pulses as thin ( 90 ) and thick ( 180 ) vertical bars .", "the amplitude of each of the n spin echoes is recorded as a single datum .", "the 2d data are inverted numerically to form a 2d distribution of t2 correlated against t1 .", "the nmr data are described by the first kind fredholm integral equation : in which the nmr signal is b , is the experimental error ( noise ) , f(t1,t2 ) is the required 2d correlation , and the kernel function k(1,t1,nte , t2 ) represents the expected form of the data so that : in which the first exponent describes the t1 relaxation and the second exponent describes the t2 relaxation . as the two exponents do not share a common time base , the kernel function is separable , that is , the expected behaviour of the signal amplitude for a given set of ( 1,t1 ) can be determined separately from the expected behaviour of the signal amplitude for a given set of ( nte , t2 ) .", "this separation allows us to solve the fredholm integral equation in ( 9 ) efficiently in vector ", "the result is biased to be positive , smooth , and bounded by pre - determined limits in t1 and t2 .", "a stable distribution , obtained in the presence of noise , is found using tikhonov regularisation with the smoothing parameter chosen using the generalised cross validation method ; a review of data inversion techniques is presented elsewhere .", "the inversion is highly susceptible to noise fluctuations and the degree of smoothing increases as the snr decreases .", "variations in distribution shape of less than an order of magnitude on the relaxation time axes are therefore considered to be determined by the signal quality and not the sample ." ], [ "tpd experiments were performed on a catlab - pcs ( hiden analytical ) , comprising a microreactor module with integrated mass spectrometer .", "pellets saturated with water were placed into the glass microreactor in a high purity helium flow at a constant rate of 40 ml min and left for one hour at 45 c until all the physisorbed water was removed .", "tpd curves of water ( m=18 amu ) were recorded in the range 451000 c with a heating rate of 20 c min .", "each tpd measurement lasted 3 h. the amount of water desorbed from each porous material during the tpd experiments was calculated by using a calibration factor , which was obtained by measuring the area under the tpd profile corresponding to a known amount of desorbed water from a calibration sample .", "the analysis of the tpd curves was carried out according to the condensation approximation method reported by barrie in order to obtain the distribution function of the activation energy of desorption ." ], [ "having derived equation ( 7 ) , we now demonstrate that t1/t2 for the systems studied can indeed be related to the strongest relaxation sinks present ( i.e. , the strongest adsorption sites present)the strength of the strongest adsorption sites being determined by tpd analysis .", "we determine the strength of water adsorption in six porous oxides ( extruded pellets ) used as supports and catalysts : tio2-a ( anatase ) , tio2-r ( rutile ) ( supplied by evonik - degussa ) , zro2 , -al2o3 , -al2o3 and sio2 .", "we chose water as the adsorbate because it is central to aqueous - phase heterogeneous catalytic processes and , in the context of this work , has the additional advantages of not decomposing during thermally driven desorption .", "the tpd energy distributions for water desorbing from the porous oxides are shown in figure 2 . due to the uncertainty associated with the tail of the tpd curves ,", "we determine the maximum activation energy of desorption ( emax ) where the integral area of the desorption curves is 95 % of the total , see figure 2 .", "energy distribution functions obtained from tpd analysis of water in a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the vertical dashed lines indicate the activation energy of desorption ( emax ) at which 95 % of species have desorbed .", "it is clearly seen that the shape of the tpd curves differs widely between the oxides studied . ignoring any detailed structure in these plots", ", we see that the tio2 materials ( figure 2a , b ) are associated with significantly lower maximum desorption energies than the other materials studied . based on the values of emax determined from the tpd the strength of the interaction of water with the porous solid decreases in the order : the t1t2 correlations for water in the porous oxides are shown in figure 3 . in each case , a single relaxation time component is observed ; the relaxation times are dominated by surface adsorbed species due to the small pore size and high surface area in these oxides .", "the ratio t1/t2 is obtained from the logarithmic mean of the individual t1,2 dimensions , which corresponds almost exactly with the maximum intensity of the 2d peak in these mono - modal distributions . in these porous materials with narrow , mono - modal pore size distributions , details of the peak shape are determined predominantly by the raw data quality ( degree of smoothing on inversion ) and are not considered representative of physical sample properties .", "the 2d correlations provide a straightforward visual comparison and are required to interpret relaxation results obtained from complicated systems with multiple liquids or diffusive exchange.[24 , 29 ] t1t2 correlation plots for water in : a ) tio2-a , b ) tio2-r , c ) -al2o3 , d ) sio2 , e ) -al2o3 and f ) zro2 .", "the solid diagonal line indicates t1=t2 ; the dashed line indicates t1/t2 at the maximum of the peak .", "a comparison between the nmr relaxation analysis and tpd is achieved by converting the t1/t2 ratio into an effective surface interaction parameter , esurf , using equation ( 7 ) .", "the comparison between emax ( tpd ) and esurf ( nmr ) is given in figure 4 , in which an excellent correlation is obtained between the two measurements .", "notably , the tio2 samples have a markedly lower esurf value compared to the other oxides , consistent with their tpd spectra being significantly different to those of the other oxides . elsewhere ,", "a comparison between zro2 and tio2 surfaces has been carried out by ignatchenko and co - workers , who concluded , through isotopic exchange experiments and density functional theory ( dft ) calculations , that zro2 has a greater affinity for water than tio2 .", "our results agree with these previous findings , and the high value of esurf obtained for water on zro2 correlates with the known strong affinity of water for this oxide surface . measurements of isosteric heat of adsorption have also been used to study porous oxides .", "values of the isosteric heat of adsorption for water on sio2[47 , 48 ] have been reported to be comparable to those measured over -al2o3 .", "again , this is in line with the finding of this work , which shows very similar esurf values for sio2 and -al2o3 , with sio2 showing a slightly higher value .", "overall , our results show that esurf for a given liquid adsorbed within a range of chemically similar materials does correlate directly with the maximum activation energy of desorption obtained by tpd analysis , as shown in figure 4 .", "solid systems under investigation can be obtained , the esurf values can be converted to an estimate of the absolute maximum desorption energies characteristic of that system .", "comparison of esurf ( nmr ) against emax ( tpd ) for water on oxides .", "symbols correspond to ( left to right ) tio2-a , tio2-r , -al2o3 , sio2 , -al2o3 and zro2 .", "error bars represent the uncertainty in determining the peak position in the t1t2 distributions ( esurf ) and 5 % integral area under the tpd curves ( emax ) .", "the adsorption site densities , calculated from the area under the tpd curve according to the procedure described in the experimental section , were : 0.11 ( tio2-a and tio2-r ) , 0.84 ( -al2o3 ) , 1.38 ( sio2 ) , 2.95 ( -al2o3 ) , and 0.60 mmol m ( zro2 ) .", "no strong correlation is found between these values and the t1/t2 ratios , as expected from the earlier discussion[29 , 30 ] regarding the relatively low sensitivity of t1/t2 to site density compared to the changing nature of interactions between liquids and surfaces of different chemistry ." ], [ "in this work we have presented a theoretical analysis to show that the ratio of nmr relaxation times , t1/t2 , can be related directly to the adsorbate ", "adsorbent interaction energy characterising adsorption in a porous material , and we introduce a quantitative metric esurf ( based on the relaxation time ratio ) characterising the strength of this surface interaction .", "we confirm that the t1/t2 ratio is insensitive to pore geometry , allowing adsorption of liquids to be compared between different materials .", "given that the relaxation time characteristics of the nuclear spin system will be dominated by the strongest adsorption sites , the hypothesis that t2/t1 should correlate directly with the maximum activation energy of desorption observed in a temperature - programmed desorption experiment was tested ; the data were found to support this hypothesis . given this result", ", we now have a physical interpretation of the nmr relaxation data in terms of a surface activation energy of desorption .", "it follows from the analysis that for a series of materials for which at least two calibration values are obtainable by tpd , the esurf parameter yields a direct estimate of the maximum activation energy of desorption from the surface .", "although the method has been demonstrated for water , the nmr relaxation analysis can be extended to probe non - invasively any adsorbed species , notably organic molecules .", "further , given that nmr relaxation is readily extended to measurements at elevated conditions of temperature and pressure , these results suggest that the technique might be considered a useful addition to the toolkit of techniques employed to characterise adsorption in porous media used for applications in catalysis and separations processes . beyond these applications , demonstrating that t1/t2 , when expressed as esurf , is a quantitative measure of interaction strength has implications for determining surface wettability in oil - field reservoir rocks , an important parameter for predicting oil recovery .", "an improved correlation between relaxation time and surface adsorption energy could be obtained by measuring t1/t2 as a function of temperature or field strength , although such procedures for providing measurements of surface interaction strength would be considerably more time consuming .", "nmr relaxation has been validated as a robust and versatile tool for quantitatively comparing liquid ", "solid interactions in porous materials . in principle , the method is applicable to the characterisation of both liquid and gas phase adsorption , although signal - to - noise considerations may limit its applicability to gas solid interactions .", "this limitation and the consideration of chemically different surfaces ( e.g. , carbon - based ) , or materials with much smaller pore size ( e.g. , zeolites ) are the subject of further consideration ." ] ]

[ "juvenile polyposis syndrome , a rare disorder in children , is characterized with multiple hamartomatous polyps in alimentary tract .", "a variety of manifestations include bleeding , intussusception , or polyp prolapse . in this study , we present an 8-month - old male infant of juvenile polyposis syndrome initially presenting with chronic anemia . to the best of our knowledge ,", "we report a rare case of an 8-month - old male infant who presented with chronic anemia and gastrointestinal bleeding initially .", "panendoscopy and abdominal computed tomography showed multiple polyposis throughout the entire alimentary tract leading to intussusception .", "technetium-99m - labeled red blood cell ( rbc ) bleeding scan revealed the possibility of gastrointestinal tract bleeding in the jejunum .", "histopathological examination on biopsy samples showed peutz - jeghers syndrome was excluded , whereas the diagnosis of juvenile polyposis syndrome was established .", "however , polyps recurred and occupied the majority of the gastrointestinal tract in 6 months .", "juvenile polyposis syndrome is an inherited disease , so it is not possible to prevent it .", "concerning of its poor outcome and high mortality rate , it is important that we should increase awareness and education of the parents at its earliest stages .", "it has been shown that affected children are susceptible to cancers and fatal medical conditions .", "the common presentations include anemia , recurrent gastrointestinal bleeding , diarrhea , rectal prolapse , intussusception , protein - losing enteropathy , starvation , and malnutrition .", "an 8-month - old male infant presented melena and iron deficiency anemia with hematocrit of 10.6% and hemoglobin of 2.9 g / dl .", "panendoscopy revealed several polypoid lesions with ulceration over the body and the prepyloric area , which were bleeding to touch ( fig .", "computed tomography depicted numerous nodules throughout the entire alimentary tract , indicating intestinal polyposis ( fig .", "panendoscopy : at least 6 polypoid lesions over gastric body and prepyloric area with ulceration and touch bleeding .", "abdominal computed tomography showed multiple nodules within the stomach , small intestine ( including duodenum , jejunum , and ileum ) , and descending colon leading to intussusception . owing to uncorrectable anemia as well as sonography constantly demonstrating intussusceptions ( figs . 3 and 4 ) , laparoscopy - assisted enteroscopic polypectomy and reduction of intussusceptions were performed ( fig .", "the pathology report described hamartomatous polyps with elongation , tortuosity , and dilatation of the gastric foveae and intestinal mucosal glands .", "the underlying stroma is characterized by broadband smooth muscle fibers , intermingling with the glands ( fig .", "technetium-99m - labeled rbc bleeding scan : the possibility of gastrointestinal tract bleeding in the jejunum .", "-1 polypectomy and reduction of intussusceptions : ( a ) 2 intussusceptions were found , 1 at 20 cm distal to treitz ligament and another 1 at jejunoileal junction .", "( b , d ) numerous polyps were found in whole small bowel , especially in jejunum .", "microscopically , hamartomatous polyps composed of elongation , tortuosity , and dilatation of the gastric foveolae and intestinal mucosal glands .", "the underlying stroma is characterized by broadband smooth muscle fibers , intermingling with the glands .", "thereafter , the patient suffered from multiple episodes of anemia , gastrointestinal hemorrhage , rectal prolapsed polyps , symptomatic colic - colic intussusceptions requiring radiologic reductions , protein - losing enteropathy , and immunodeficiency .", "it was our impression that the immunodeficiency was secondary to the remarkable protein - losing enteropathy and malnutrition .", "human immunodeficiency virus ( hiv ) infection was excluded due to negative maternal hiv testing during prenatal checkups .", "it is not a routine in our institution to conduct another hiv examination before 24 months of age in infants with prior negative virologic tests .", "the serology study showed notably hypoglobulinemia with igg of 115 mg / dl and igm of 33 mg / dl .", "complement levels were also significantly below the normal limits with c3 of 51 mg / dl and c4 of 7 mg / dl .", "intravenous immunoglobulin was administered . in 6 months , polyps recurred and occupied the majority of the gastrointestinal tract .", "juvenile polyps are mostly solitary , influencing approximately 1% of preschool and school - aged children .", "juvenile polyposis syndrome may extensively affect a large portion of alimentary tract , and be usually related to malignant potential .", "it has been known with 3 subtypes : diffuse juvenile polyposis of infancy ( < 6 months of age ) , diffuse juvenile polyposis ( 6 months5 years of age ) , and juvenile polyposis coli ( 515 years of age ) .", "histologically , the polyps in juvenile polyposis syndrome are composed of mucous filled , dilated glands that are often associated with inflammatory cell infiltration . unlike those in peutz - jeghers syndrome , smooth muscle proliferation is rarely seen .", "nevertheless , the polyps consisted of smooth muscle bands in stroma in this case . peutz - jeghers syndrome was initially suspected . however , the patient had neither relevant family history nor oral pigmented lesions that were highly associated with peutz - jeghers syndrome .", "peutz - jeghers syndrome was excluded , whereas the diagnosis of juvenile polyposis syndrome was established .", "it is featured with widespread hamartomatous polyps in the entire gastrointestinal tract in infants < 6 months of age .", "the initial manifestations of anemia and gastrointestinal bleeding were present at 8 months of age .", "we presumed that this patient should be categorized to juvenile polyposis of infancy . with regard to treatment , surgery is the mainstay to remove polyps .", "however , repeated operations are usually required because recurrence is not uncommon . in our case ,", "redo enteroscopic polypectomy was technically difficult as well as might bear much higher risks , so it was abandoned . as nearly the entire small bowel is involved , resection of affected bowels was less considered because it would lead to prominent intestinal failure .", "the decision was made to perform supportive treatment . to the best of our knowledge ," ]

[ "<S> abstractbackground : juvenile polyposis syndrome , a rare disorder in children , is characterized with multiple hamartomatous polyps in alimentary tract . </S>", "<S> a variety of manifestations include bleeding , intussusception , or polyp prolapse . in this study , we present an 8-month - old male infant of juvenile polyposis syndrome initially presenting with chronic anemia . to the best of our knowledge , </S>", "<S> this is the youngest case reported in the literature.methods:we report a rare case of an 8-month - old male infant who presented with chronic anemia and gastrointestinal bleeding initially . </S>", "<S> panendoscopy and abdominal computed tomography showed multiple polyposis throughout the entire alimentary tract leading to intussusception . </S>", "<S> technetium-99m - labeled red blood cell ( rbc ) bleeding scan revealed the possibility of gastrointestinal tract bleeding in the jejunum . </S>", "<S> histopathological examination on biopsy samples showed peutz - jeghers syndrome was excluded , whereas the diagnosis of juvenile polyposis syndrome was established.results:enteroscopic polypectomy is the mainstay of the treatment . </S>", "<S> however , polyps recurred and occupied the majority of the gastrointestinal tract in 6 months . </S>", "<S> supportive management was given . </S>", "<S> the patient expired for severe sepsis at the age of 18 months.conclusion:juvenile polyposis syndrome is an inherited disease , so it is not possible to prevent it . </S>", "<S> concerning of its poor outcome and high mortality rate , it is important that we should increase awareness and education of the parents at its earliest stages . </S>" ]

[ "Background:", "Methods:", "Results:", "Conclusion:", "Introduction", "Case report", "Discussion" ]

[ [ "juvenile polyposis syndrome , a rare disorder in children , is characterized with multiple hamartomatous polyps in alimentary tract .", "a variety of manifestations include bleeding , intussusception , or polyp prolapse . in this study , we present an 8-month - old male infant of juvenile polyposis syndrome initially presenting with chronic anemia . to the best of our knowledge ," ], [ "we report a rare case of an 8-month - old male infant who presented with chronic anemia and gastrointestinal bleeding initially .", "panendoscopy and abdominal computed tomography showed multiple polyposis throughout the entire alimentary tract leading to intussusception .", "technetium-99m - labeled red blood cell ( rbc ) bleeding scan revealed the possibility of gastrointestinal tract bleeding in the jejunum .", "histopathological examination on biopsy samples showed peutz - jeghers syndrome was excluded , whereas the diagnosis of juvenile polyposis syndrome was established ." ], [ "however , polyps recurred and occupied the majority of the gastrointestinal tract in 6 months ." ], [ "juvenile polyposis syndrome is an inherited disease , so it is not possible to prevent it .", "concerning of its poor outcome and high mortality rate , it is important that we should increase awareness and education of the parents at its earliest stages ." ], [ "it has been shown that affected children are susceptible to cancers and fatal medical conditions .", "the common presentations include anemia , recurrent gastrointestinal bleeding , diarrhea , rectal prolapse , intussusception , protein - losing enteropathy , starvation , and malnutrition ." ], [ "an 8-month - old male infant presented melena and iron deficiency anemia with hematocrit of 10.6% and hemoglobin of 2.9 g / dl .", "panendoscopy revealed several polypoid lesions with ulceration over the body and the prepyloric area , which were bleeding to touch ( fig .", "computed tomography depicted numerous nodules throughout the entire alimentary tract , indicating intestinal polyposis ( fig .", "panendoscopy : at least 6 polypoid lesions over gastric body and prepyloric area with ulceration and touch bleeding .", "abdominal computed tomography showed multiple nodules within the stomach , small intestine ( including duodenum , jejunum , and ileum ) , and descending colon leading to intussusception . owing to uncorrectable anemia as well as sonography constantly demonstrating intussusceptions ( figs . 3 and 4 ) , laparoscopy - assisted enteroscopic polypectomy and reduction of intussusceptions were performed ( fig .", "the pathology report described hamartomatous polyps with elongation , tortuosity , and dilatation of the gastric foveae and intestinal mucosal glands .", "the underlying stroma is characterized by broadband smooth muscle fibers , intermingling with the glands ( fig .", "technetium-99m - labeled rbc bleeding scan : the possibility of gastrointestinal tract bleeding in the jejunum .", "-1 polypectomy and reduction of intussusceptions : ( a ) 2 intussusceptions were found , 1 at 20 cm distal to treitz ligament and another 1 at jejunoileal junction .", "( b , d ) numerous polyps were found in whole small bowel , especially in jejunum .", "microscopically , hamartomatous polyps composed of elongation , tortuosity , and dilatation of the gastric foveolae and intestinal mucosal glands .", "the underlying stroma is characterized by broadband smooth muscle fibers , intermingling with the glands .", "thereafter , the patient suffered from multiple episodes of anemia , gastrointestinal hemorrhage , rectal prolapsed polyps , symptomatic colic - colic intussusceptions requiring radiologic reductions , protein - losing enteropathy , and immunodeficiency .", "it was our impression that the immunodeficiency was secondary to the remarkable protein - losing enteropathy and malnutrition .", "human immunodeficiency virus ( hiv ) infection was excluded due to negative maternal hiv testing during prenatal checkups .", "it is not a routine in our institution to conduct another hiv examination before 24 months of age in infants with prior negative virologic tests .", "the serology study showed notably hypoglobulinemia with igg of 115 mg / dl and igm of 33 mg / dl .", "complement levels were also significantly below the normal limits with c3 of 51 mg / dl and c4 of 7 mg / dl .", "intravenous immunoglobulin was administered . in 6 months , polyps recurred and occupied the majority of the gastrointestinal tract ." ], [ "juvenile polyps are mostly solitary , influencing approximately 1% of preschool and school - aged children .", "juvenile polyposis syndrome may extensively affect a large portion of alimentary tract , and be usually related to malignant potential .", "it has been known with 3 subtypes : diffuse juvenile polyposis of infancy ( < 6 months of age ) , diffuse juvenile polyposis ( 6 months5 years of age ) , and juvenile polyposis coli ( 515 years of age ) .", "histologically , the polyps in juvenile polyposis syndrome are composed of mucous filled , dilated glands that are often associated with inflammatory cell infiltration . unlike those in peutz - jeghers syndrome , smooth muscle proliferation is rarely seen .", "nevertheless , the polyps consisted of smooth muscle bands in stroma in this case . peutz - jeghers syndrome was initially suspected . however , the patient had neither relevant family history nor oral pigmented lesions that were highly associated with peutz - jeghers syndrome .", "peutz - jeghers syndrome was excluded , whereas the diagnosis of juvenile polyposis syndrome was established .", "it is featured with widespread hamartomatous polyps in the entire gastrointestinal tract in infants < 6 months of age .", "the initial manifestations of anemia and gastrointestinal bleeding were present at 8 months of age .", "we presumed that this patient should be categorized to juvenile polyposis of infancy . with regard to treatment , surgery is the mainstay to remove polyps .", "however , repeated operations are usually required because recurrence is not uncommon . in our case ,", "redo enteroscopic polypectomy was technically difficult as well as might bear much higher risks , so it was abandoned . as nearly the entire small bowel is involved , resection of affected bowels was less considered because it would lead to prominent intestinal failure .", "the decision was made to perform supportive treatment . to the best of our knowledge ," ] ]

[ "the phylum apicomplexa includes a large group of protozoan parasites responsible for a wide range of animal and human diseases . among the human pathogens are plasmodium falciparum and plasmodium vivax , the major causative agents of human malaria , as well as cryptosporidium parvum and toxoplasma gondii , which are particularly pathogenic in immunocompromised patients .", "apicomplexa are intracellular obligatory parasites that multiply in a so - called parasitophorous vacuole . although the alterations of the host cell harboring toxoplasma or plasmodium parasites have been extensively documented at the cellular level , still little is known about how the parasite manipulates the host cell at the molecular level , with the notable exception of plasmodium infection of host erythrocytes .", "so far , most molecular studies on the host - parasite interface have focused on the role of parasite factors that are secreted by the invading parasite , as well as by the resident intracellular parasite , into the host cell .", "these intracellular parasites are expected to profoundly reorganize the host cell for their own needs to ensure safe growth and persistence , and presumably to deploy the most sophisticated mechanisms to this end .", "emerging evidence indicates that viruses and bacteria manipulate the microrna ( mirna ) pathways of the host cells they infect .", "mirnas are the most abundant class of small , non - coding , single - stranded rnas and are involved in regulating gene expression at the post - transcriptional level . in silico target prediction", "suggests that mirnas may control up to 30% of the translation of the human transcriptome . as such", ", they govern a variety of fundamental cell functions , including cell proliferation and apoptosis , and are key regulators of cell metabolism . when homeostatic conditions are disrupted - for example , when cells encounter micro - organisms - these regulatory pathways might also contribute to host cell responses / defenses ( i.e. , the inflammatory response ) against the foreign bodies .", "for example , cell infection by mammalian viruses might be counteracted by cellular mirnas that target either the virus itself , as in the case of the rhabdoviral vesicular stomatitis virus , or a host factor critical to the virus , as for the lentiviral hiv .", "conversely , mirnas can also act in favor of the micro - organism , either when it is pathogen - encoded ( e.g. , mammalian virus - encoded mirnas ) or when the micro - organism subverts host mirnas to its own benefit .", "effectors from the bacteria pseudomonas syringae have been recently shown to suppress transcriptional activation of some mirnas generated upon sensing of pamps ( pathogen - associated molecular patterns ) by arabidopsis .", "recent data have begun to show how two apicomplexan parasites , cryptosporidium and toxoplasma , are able to target mirnas in the host cell to alter the cellular environment in ways that favor their intracellular development .", "cryptosporidium is able to trigger the down - regulation of let-7i ( a mirna with complementarity to toll - like receptor ( tlr)-4 mrna ) in the host cell , leading to the up - regulation of tlr4 , a key pathogen recognition molecule that plays a central role in epithelial innate immunity to cryptosporidium infection .", "various studies have further substantiated the ability of cryptosporidium to alter mirna expression in cholangiocytes [ 9 - 11 ] .", "it is emerging from these studies that following cryptosporidium infection , specific mirna cluster genes are activated by the binding of the nf-b ( nuclear factor - kappa b ) p65 subunit to their promoter , and that inhibition of these mirnas increases parasite burden .", "these results mirror those showing differential alterations in mature mirna expression profiles in primary human fibroblast cells following toxoplasma infection .", "zeiner et al . showed that toxoplasma infection specifically increased the transcription of the mir-17/92 loci by two- to three - fold in human fibroblasts .", "the effect is apparently a specific response to toxoplasma infection since levels of the mature mir-17/92-derived mirnas remained unchanged upon infection by the closely related parasite neospora caninum .", "microarray data comparing the mirna profiles of cells infected by toxoplasma or cryptosporidium or treated with lipopolysaccharide ( lps ) have revealed several important findings .", "for example , mir-155/bic is up - regulated upon toxoplasma infection but remains unaffected or is down - regulated when exposed to cryptosporidium or lps , respectively . of note", ", mir-155 has an important role in the mammalian immune system , regulating , at least in part , cytokine production .", "two other mirnas , mir-198 and mir-320 , are both up - regulated upon toxoplasma infection whereas they are down - regulated after cryptosporidium infection and unaffected after lps stimulation .", "these data point to specific modifications of host mirna profiles upon cell infection by apicomplexa parasites .", "obviously , any change in the host cell mirna pattern might indicate either a defense mechanism by the cell or a subversion strategy by the parasite , two processes that can be differentiated by evaluating the consequences on parasite growth of disruption or over - expression of the target mirna pathway .", "given the propensity of apicomplexan parasites to co - opt cellular pathways and activities for their benefit , it is perhaps not surprising that these parasites could also reshape their cellular environment by reprogramming the host s rna interference machinery .", "specific host mirnas could either counteract the intracellular growth of parasites or facilitate it , the two possibilities being not mutually exclusive and depending on the physiological context .", "these findings open an exciting opportunity to pursue a deep understanding of how the host proteome can be reprogrammed dynamically and reversibly upon apicomplexa infection .", "an additional line of research should explore the upstream regulatory mechanisms , that is , how the parasites directly interfere with rna silencing pathways and , more specifically , the parasite effectors that are involved in the process . as discussed above , in response to cryptosporidium and toxoplasma infections , the expression of specific mirna genes is altered at the transcriptional level .", "mirna are generated through the concerted action of multi - subunit complexes that promote the sequential cleavage , export , and loading of mirna into silencing complexes .", "an increasing number of reports suggest that , beyond the transcriptional control of genes that code cluster mirnas , each of these steps serves as a potential point of regulation , and therefore adds additional complexity to mirna - dependent gene regulation", ". could parasite regulators of host mirnas be ribonucleic acids ? unlike in cryptosporidium and plasmodium species , the toxoplasma genome encodes elaborate rna silencing machinery that generates endogenous small silencing rnas , including specific mirnas .", "thus , an attractive hypothesis is that toxoplasma has the potential to secrete its own mirnas to hijack the host cell mirna defense pathway , similar to what some viruses are able to do .", "it is known that apicomplexan parasites inject various molecules into the host cell resulting in extensive remodeling of the host cell gene expression profile and metabolic pathways [ 17 - 19 ] .", "the expression of host mirnas can also be altered in response to parasite recognition by cell surface tlrs .", "both intrinsic and extrinsic acting factors could then interfere with target host mirnas , at any point of the processing of the pri - mirnas and biogenesis of the mirnas - transcription , processing , or export ." ]

[ "<S> rna silencing plays an important role in development through the action of micrornas , which fine tune the expression of a large portion of the genome . </S>", "<S> it is also very important in innate immune responses , especially in antiviral and antibacterial defenses in plants , insects , and animals . </S>", "<S> two recent papers now indicate that apicomplexan parasites display the ability to interfere with host microrna populations . </S>" ]

[ "Introduction and context", "Major recent advances", "Future directions", "Competing Interests" ]

[ [ "the phylum apicomplexa includes a large group of protozoan parasites responsible for a wide range of animal and human diseases . among the human pathogens are plasmodium falciparum and plasmodium vivax , the major causative agents of human malaria , as well as cryptosporidium parvum and toxoplasma gondii , which are particularly pathogenic in immunocompromised patients .", "apicomplexa are intracellular obligatory parasites that multiply in a so - called parasitophorous vacuole . although the alterations of the host cell harboring toxoplasma or plasmodium parasites have been extensively documented at the cellular level , still little is known about how the parasite manipulates the host cell at the molecular level , with the notable exception of plasmodium infection of host erythrocytes .", "so far , most molecular studies on the host - parasite interface have focused on the role of parasite factors that are secreted by the invading parasite , as well as by the resident intracellular parasite , into the host cell .", "these intracellular parasites are expected to profoundly reorganize the host cell for their own needs to ensure safe growth and persistence , and presumably to deploy the most sophisticated mechanisms to this end .", "emerging evidence indicates that viruses and bacteria manipulate the microrna ( mirna ) pathways of the host cells they infect .", "mirnas are the most abundant class of small , non - coding , single - stranded rnas and are involved in regulating gene expression at the post - transcriptional level . in silico target prediction", "suggests that mirnas may control up to 30% of the translation of the human transcriptome . as such", ", they govern a variety of fundamental cell functions , including cell proliferation and apoptosis , and are key regulators of cell metabolism . when homeostatic conditions are disrupted - for example , when cells encounter micro - organisms - these regulatory pathways might also contribute to host cell responses / defenses ( i.e. , the inflammatory response ) against the foreign bodies .", "for example , cell infection by mammalian viruses might be counteracted by cellular mirnas that target either the virus itself , as in the case of the rhabdoviral vesicular stomatitis virus , or a host factor critical to the virus , as for the lentiviral hiv .", "conversely , mirnas can also act in favor of the micro - organism , either when it is pathogen - encoded ( e.g. , mammalian virus - encoded mirnas ) or when the micro - organism subverts host mirnas to its own benefit .", "effectors from the bacteria pseudomonas syringae have been recently shown to suppress transcriptional activation of some mirnas generated upon sensing of pamps ( pathogen - associated molecular patterns ) by arabidopsis ." ], [ "recent data have begun to show how two apicomplexan parasites , cryptosporidium and toxoplasma , are able to target mirnas in the host cell to alter the cellular environment in ways that favor their intracellular development .", "cryptosporidium is able to trigger the down - regulation of let-7i ( a mirna with complementarity to toll - like receptor ( tlr)-4 mrna ) in the host cell , leading to the up - regulation of tlr4 , a key pathogen recognition molecule that plays a central role in epithelial innate immunity to cryptosporidium infection .", "various studies have further substantiated the ability of cryptosporidium to alter mirna expression in cholangiocytes [ 9 - 11 ] .", "it is emerging from these studies that following cryptosporidium infection , specific mirna cluster genes are activated by the binding of the nf-b ( nuclear factor - kappa b ) p65 subunit to their promoter , and that inhibition of these mirnas increases parasite burden .", "these results mirror those showing differential alterations in mature mirna expression profiles in primary human fibroblast cells following toxoplasma infection .", "zeiner et al . showed that toxoplasma infection specifically increased the transcription of the mir-17/92 loci by two- to three - fold in human fibroblasts .", "the effect is apparently a specific response to toxoplasma infection since levels of the mature mir-17/92-derived mirnas remained unchanged upon infection by the closely related parasite neospora caninum .", "microarray data comparing the mirna profiles of cells infected by toxoplasma or cryptosporidium or treated with lipopolysaccharide ( lps ) have revealed several important findings .", "for example , mir-155/bic is up - regulated upon toxoplasma infection but remains unaffected or is down - regulated when exposed to cryptosporidium or lps , respectively . of note", ", mir-155 has an important role in the mammalian immune system , regulating , at least in part , cytokine production .", "two other mirnas , mir-198 and mir-320 , are both up - regulated upon toxoplasma infection whereas they are down - regulated after cryptosporidium infection and unaffected after lps stimulation .", "these data point to specific modifications of host mirna profiles upon cell infection by apicomplexa parasites .", "obviously , any change in the host cell mirna pattern might indicate either a defense mechanism by the cell or a subversion strategy by the parasite , two processes that can be differentiated by evaluating the consequences on parasite growth of disruption or over - expression of the target mirna pathway ." ], [ "given the propensity of apicomplexan parasites to co - opt cellular pathways and activities for their benefit , it is perhaps not surprising that these parasites could also reshape their cellular environment by reprogramming the host s rna interference machinery .", "specific host mirnas could either counteract the intracellular growth of parasites or facilitate it , the two possibilities being not mutually exclusive and depending on the physiological context .", "these findings open an exciting opportunity to pursue a deep understanding of how the host proteome can be reprogrammed dynamically and reversibly upon apicomplexa infection .", "an additional line of research should explore the upstream regulatory mechanisms , that is , how the parasites directly interfere with rna silencing pathways and , more specifically , the parasite effectors that are involved in the process . as discussed above , in response to cryptosporidium and toxoplasma infections , the expression of specific mirna genes is altered at the transcriptional level .", "mirna are generated through the concerted action of multi - subunit complexes that promote the sequential cleavage , export , and loading of mirna into silencing complexes .", "an increasing number of reports suggest that , beyond the transcriptional control of genes that code cluster mirnas , each of these steps serves as a potential point of regulation , and therefore adds additional complexity to mirna - dependent gene regulation", ". could parasite regulators of host mirnas be ribonucleic acids ? unlike in cryptosporidium and plasmodium species , the toxoplasma genome encodes elaborate rna silencing machinery that generates endogenous small silencing rnas , including specific mirnas .", "thus , an attractive hypothesis is that toxoplasma has the potential to secrete its own mirnas to hijack the host cell mirna defense pathway , similar to what some viruses are able to do .", "it is known that apicomplexan parasites inject various molecules into the host cell resulting in extensive remodeling of the host cell gene expression profile and metabolic pathways [ 17 - 19 ] .", "the expression of host mirnas can also be altered in response to parasite recognition by cell surface tlrs .", "both intrinsic and extrinsic acting factors could then interfere with target host mirnas , at any point of the processing of the pri - mirnas and biogenesis of the mirnas - transcription , processing , or export ." ], [ "" ] ]

[ "we have observed a case of renal involvement complicated by granular corneal dystrophy type ii ( gcd2 ) .", "gcd2 , also known as avellino corneal dystrophy ( cd ) , is an autosomal dominant disorder caused by a mutation in the transforming growth factor--induced ( tgfbi ) gene .", "this mutation can be found in several distinct autosomal dominant genetically determined cases of cd ; however , it is not known whether this mutation produces other clinical manifestations other than cd .", "tgfbi proteins ( tgfbip ) interact with several extracellular matrix ( ecm ) components [ 3 , 4 ] .", "we believe that our study was a type of oculorenal syndrome associated with a tgfbi mutation , which remains to be acknowledged .", "the following clinical laboratory values were noted : serum urea nitrogen ( bun ) , 14.9 mg / dl ; creatinine ( cre ) , 0.79 mg / dl ; total cholesterol , 189 mg / d ; total protein , 6.4 g / dl ; and albumin , 3.9 g / dl .", "the levels of c - reactive protein , immunoglobulins ( ig ) , and total complement , c3 , c4 , and c1q were all normal .", "tests for antinuclear antibody , hepatitis b virus surface antigen , hepatitis c virus antibody , and cryoglobulins were all negative .", "renal ultrasound and computed tomography revealed normal kidneys . a kidney biopsy , performed using light microscopy , revealed 11 glomeruli , 1 of which was obsolete or sclerosed ( fig 1a ) .", "light microscopy did not demonstrate any remarkable changes in the glomeruli ( fig 1b ) .", "focal tubular atrophy with dilation of peritubular capillaries and focal infiltration of small round cells were observed .", "after 7 years , the patient developed mild hypertension and began taking 4 mg / day of losartan potassium .", "the patient 's mild proteinuria ( 11.5 g / g cre ) continued , and her renal function was mildly decreased .", "after 10 years , the patient was re - admitted for additional evaluation of proteinuria .", "laboratory testing revealed the following : urinary protein level of 1.5 g / day , bun level of 15.0 mg / dl , and cre level of 0.94 mg / dl .", "approximately 2 years before her second admission , the patient complained of mild blurred vision and was diagnosed with cd .", "slit - lamp examination revealed a large number of gray - white central granular and linear opacities in both eyes ( fig 2 ) ; therefore , we diagnosed her condition as gcd2 .", "a second kidney biopsy was performed under light microscopy , revealing 18 glomeruli , 6 of which were obsolete or sclerosed ( fig 3a ) .", "segmental double contours of the glomerular capillary walls were also observed ( fig 3c ) .", "focal tubular atrophy with mild interstitial inflammation , dilation of peritubular capillaries , and segmental thickening of tubular basement membranes ( tbm ) were observed .", "the subendothelial space was widened , and irregularity of the glomerular basement membrane ( gbm ) was segmentally observed .", "segmental irregular thinning , basket - waving , duplication , lamellation , and reticulation of gbm and tbm were observed partially and slightly ( fig 3e ", "immunostaining of the -5 chains of type iv collagen was normal . upon her renal pathological findings", ", we assumed the existence of a genetic cause . after obtaining informed consent , we collected dna from the patient .", "the genome dna was extracted from the whole blood , and targeted next - generation sequencing of candidate genes for inherited renal diseases was negative ( online suppl .", ". real - time polymerase chain reaction using a simple buccal swab ( avellino labs universal test ; alut ) revealed tgfbi heteromutation ( r124h ) .", "we speculated that this condition was a novel case of oculorenal syndrome associated with tgfbi mutation .", "her 74-year - old father had the same mutation of tgfbi ( r124h ) and was diagnosed with relatively mild gcd2 .", "her 77-year - old mother and 26-year - old daughter , however , did not have mutated tgfbi ( r124h ) or gcd2 .", "her father had no proteinuria but had a slightly elevated level of urinary n - acetyl--d - glucosaminidase ( 3.0 u / g cre ) ; however , his renal function was normal ( serum cre , 0.79 mg / dl ) .", "subsequently , the patient was again treated in our outpatient clinic with 4 mg / day of losartan potassium but with no immunosuppressive agents .", "these two conditions may coincidentally coexist ; however , findings demonstrating an association between renal involvement and gcd2 have been presented .", "the tgfbip ( also known as ig - h3 , keratoepithelin ) is a 68-kda ecm protein with four evolutionary conserved fasciclin-1 domains and a carboxy - terminal arg - gly - asp sequence .", "this protein participates in many physiological processes , including morphogenesis , adhesion / migration , tumorigenesis , angiogenesis , wound healing , and inflammation .", "tgfbip is found in ecm of several human tissues and is abundant in the cornea .", "mutations of the human tgfbi gene have been linked to several autosomal dominant multiple types of cd , including gcd2 .", "almost all cases of gcd2 are caused by tgfbi gene mutations ( 5q31 ) , particularly p.arg124his ( r124h ) . in a previous study ,", "tgfbi gene mutation was estimated to have a prevalence of at least 11.5 affected people per 10,000 individuals in korea . according to embryonic expression studies using a mouse knock - out model of tgfbi , schorderet et al . ", "mutations in adhesion and ecm molecules , such as integrins and laminin-2 , play an important role in the pathogenesis of focal segmental glomerulosclerosis ; however , thus far , the relationship between tgfbi mutation and kidney disease has not been established . in an autopsy patient with tgfbi - related cd , pathologic deposits caused by tgfbip accumulation", "were only observed in the cornea and in no other tissue or organ , including the kidney ; however , an electromicroscopic examination was not performed in that report .", "tgfbip was present in the capsule and tbm of the developing kidney and was predominantly localized in the epithelial cells of the collecting ducts as well as the distal proximal tubules .", "tgfbip is secreted into the extracellular space and may bind to fibronectin , laminin , and type i , ii , and iv collagens as well as integrins [ 12 , 13 ] .", "proteomic analysis revealed that tgfbip is a component of glomerular ecm [ 3 , 4 ] , and it exhibits protein - protein interactions between the following ecm proteins : -2 macroglobulin ; -1 , -2 chain type i collagen ; -1 chain type ii collagen ; -1 , -2 , -3 , and -4 chain type iv collagen ; fibronectin ; and fibrillin-1 .", "proteoglycans directly bind to tgfbip and affect collagen vi aggregation and possibly the interaction between integrin and collagen vi .", "binding allows tgfbip , including ecm proteins , to play an important role in cell - collagen signaling interactions that comprise bm , bone formation , and development as well as cell migration and growth . in our patient ,", "various pathological findings of gbm and tbm and widening of the subendothelial space of gbm were observed by electron microscopy .", "a negative genetic analysis of well - known monogenic kidney diseases prompted us to consider that tgfbi mutation could affect the bm of the developing kidneys and produce such bm findings .", "the pathophysiological mechanisms and the incidence of this condition or genotype - phenotype correlation for tgfbi mutations are not obvious .", "if detailed examination of the corneas is not performed , gcd2 may not be observed until middle - age and older . it should be noted that detailed ocular examinations , including cornea assessments , are valuable when diagnosing nephropathy associated with tgfbi mutations .", "presently , alut testing is beneficial for laser - assisted in situ keratomileusis to protect patients from accelerated vision loss . as demonstrated with our patient , this test is easy and safe to perform when diagnosing patients with nephropathy associated with tgfbi mutations . in conclusion ,", "we have reported the first case of a unique nephropathy complicated by tgfbi - related cd .", "tgfbi - related nephropathy remains unknown and is difficult to diagnose without electron microscopic examination .", "further reports should be accumulated to determine whether the incidence of renal diseases associated with this mutation may presently be more frequent .", "patients with tgfbi - related cds , including gcd2 , should be examined for renal abnormalities .", "the authors have no ethical conflicts to disclose , and the patient provided informed consent ." ]

[ "<S> many types of inherited renal diseases have ocular features that occasionally support a diagnosis . </S>", "<S> the following study describes an unusual example of a 40-year - old woman with granular corneal dystrophy type ii complicated by renal involvement . </S>", "<S> these two conditions may coincidentally coexist ; however , there are some reports that demonstrate an association between renal involvement and granular corneal dystrophy type ii . </S>", "<S> granular corneal dystrophy type ii is caused by a mutation in the transforming growth factor--induced ( tgfbi ) gene . </S>", "<S> the patient was referred to us because of the presence of mild proteinuria without hematuria that was subsequently suggested to be granular corneal dystrophy type ii . </S>", "<S> a kidney biopsy revealed various glomerular and tubular basement membrane changes and widening of the subendothelial space of the glomerular basement membrane by electron microscopy . </S>", "<S> however , next - generation sequencing revealed that she had no mutation in a gene that is known to be associated with monogenic kidney diseases . </S>", "<S> conversely , real - time polymerase chain reaction , using a simple buccal swab , revealed tgfbi heteromutation ( r124h ) . </S>", "<S> the tgfbi protein plays an important role in cell - collagen signaling interactions , including extracellular matrix proteins which compose the renal basement membrane . </S>", "<S> this mutation can present not only as corneal dystrophy but also as renal disease . </S>", "<S> tgfbi - related oculorenal syndrome may have been unrecognized . </S>", "<S> it is difficult to diagnose this condition without renal electron microscopic studies . to the best of our knowledge , </S>", "<S> this is the first detailed report of nephropathy associated with a tgfbi mutation . </S>" ]

[ "Introduction", "Case Report", "Discussion", "Statement of Ethics", "Disclosure Statement" ]

[ [ "we have observed a case of renal involvement complicated by granular corneal dystrophy type ii ( gcd2 ) .", "gcd2 , also known as avellino corneal dystrophy ( cd ) , is an autosomal dominant disorder caused by a mutation in the transforming growth factor--induced ( tgfbi ) gene .", "this mutation can be found in several distinct autosomal dominant genetically determined cases of cd ; however , it is not known whether this mutation produces other clinical manifestations other than cd .", "tgfbi proteins ( tgfbip ) interact with several extracellular matrix ( ecm ) components [ 3 , 4 ] .", "we believe that our study was a type of oculorenal syndrome associated with a tgfbi mutation , which remains to be acknowledged ." ], [ "the following clinical laboratory values were noted : serum urea nitrogen ( bun ) , 14.9 mg / dl ; creatinine ( cre ) , 0.79 mg / dl ; total cholesterol , 189 mg / d ; total protein , 6.4 g / dl ; and albumin , 3.9 g / dl .", "the levels of c - reactive protein , immunoglobulins ( ig ) , and total complement , c3 , c4 , and c1q were all normal .", "tests for antinuclear antibody , hepatitis b virus surface antigen , hepatitis c virus antibody , and cryoglobulins were all negative .", "renal ultrasound and computed tomography revealed normal kidneys . a kidney biopsy , performed using light microscopy , revealed 11 glomeruli , 1 of which was obsolete or sclerosed ( fig 1a ) .", "light microscopy did not demonstrate any remarkable changes in the glomeruli ( fig 1b ) .", "focal tubular atrophy with dilation of peritubular capillaries and focal infiltration of small round cells were observed .", "after 7 years , the patient developed mild hypertension and began taking 4 mg / day of losartan potassium .", "the patient 's mild proteinuria ( 11.5 g / g cre ) continued , and her renal function was mildly decreased .", "after 10 years , the patient was re - admitted for additional evaluation of proteinuria .", "laboratory testing revealed the following : urinary protein level of 1.5 g / day , bun level of 15.0 mg / dl , and cre level of 0.94 mg / dl .", "approximately 2 years before her second admission , the patient complained of mild blurred vision and was diagnosed with cd .", "slit - lamp examination revealed a large number of gray - white central granular and linear opacities in both eyes ( fig 2 ) ; therefore , we diagnosed her condition as gcd2 .", "a second kidney biopsy was performed under light microscopy , revealing 18 glomeruli , 6 of which were obsolete or sclerosed ( fig 3a ) .", "segmental double contours of the glomerular capillary walls were also observed ( fig 3c ) .", "focal tubular atrophy with mild interstitial inflammation , dilation of peritubular capillaries , and segmental thickening of tubular basement membranes ( tbm ) were observed .", "the subendothelial space was widened , and irregularity of the glomerular basement membrane ( gbm ) was segmentally observed .", "segmental irregular thinning , basket - waving , duplication , lamellation , and reticulation of gbm and tbm were observed partially and slightly ( fig 3e ", "immunostaining of the -5 chains of type iv collagen was normal . upon her renal pathological findings", ", we assumed the existence of a genetic cause . after obtaining informed consent , we collected dna from the patient .", "the genome dna was extracted from the whole blood , and targeted next - generation sequencing of candidate genes for inherited renal diseases was negative ( online suppl .", ". real - time polymerase chain reaction using a simple buccal swab ( avellino labs universal test ; alut ) revealed tgfbi heteromutation ( r124h ) .", "we speculated that this condition was a novel case of oculorenal syndrome associated with tgfbi mutation .", "her 74-year - old father had the same mutation of tgfbi ( r124h ) and was diagnosed with relatively mild gcd2 .", "her 77-year - old mother and 26-year - old daughter , however , did not have mutated tgfbi ( r124h ) or gcd2 .", "her father had no proteinuria but had a slightly elevated level of urinary n - acetyl--d - glucosaminidase ( 3.0 u / g cre ) ; however , his renal function was normal ( serum cre , 0.79 mg / dl ) .", "subsequently , the patient was again treated in our outpatient clinic with 4 mg / day of losartan potassium but with no immunosuppressive agents ." ], [ "these two conditions may coincidentally coexist ; however , findings demonstrating an association between renal involvement and gcd2 have been presented .", "the tgfbip ( also known as ig - h3 , keratoepithelin ) is a 68-kda ecm protein with four evolutionary conserved fasciclin-1 domains and a carboxy - terminal arg - gly - asp sequence .", "this protein participates in many physiological processes , including morphogenesis , adhesion / migration , tumorigenesis , angiogenesis , wound healing , and inflammation .", "tgfbip is found in ecm of several human tissues and is abundant in the cornea .", "mutations of the human tgfbi gene have been linked to several autosomal dominant multiple types of cd , including gcd2 .", "almost all cases of gcd2 are caused by tgfbi gene mutations ( 5q31 ) , particularly p.arg124his ( r124h ) . in a previous study ,", "tgfbi gene mutation was estimated to have a prevalence of at least 11.5 affected people per 10,000 individuals in korea . according to embryonic expression studies using a mouse knock - out model of tgfbi , schorderet et al . ", "mutations in adhesion and ecm molecules , such as integrins and laminin-2 , play an important role in the pathogenesis of focal segmental glomerulosclerosis ; however , thus far , the relationship between tgfbi mutation and kidney disease has not been established . in an autopsy patient with tgfbi - related cd , pathologic deposits caused by tgfbip accumulation", "were only observed in the cornea and in no other tissue or organ , including the kidney ; however , an electromicroscopic examination was not performed in that report .", "tgfbip was present in the capsule and tbm of the developing kidney and was predominantly localized in the epithelial cells of the collecting ducts as well as the distal proximal tubules .", "tgfbip is secreted into the extracellular space and may bind to fibronectin , laminin , and type i , ii , and iv collagens as well as integrins [ 12 , 13 ] .", "proteomic analysis revealed that tgfbip is a component of glomerular ecm [ 3 , 4 ] , and it exhibits protein - protein interactions between the following ecm proteins : -2 macroglobulin ; -1 , -2 chain type i collagen ; -1 chain type ii collagen ; -1 , -2 , -3 , and -4 chain type iv collagen ; fibronectin ; and fibrillin-1 .", "proteoglycans directly bind to tgfbip and affect collagen vi aggregation and possibly the interaction between integrin and collagen vi .", "binding allows tgfbip , including ecm proteins , to play an important role in cell - collagen signaling interactions that comprise bm , bone formation , and development as well as cell migration and growth . in our patient ,", "various pathological findings of gbm and tbm and widening of the subendothelial space of gbm were observed by electron microscopy .", "a negative genetic analysis of well - known monogenic kidney diseases prompted us to consider that tgfbi mutation could affect the bm of the developing kidneys and produce such bm findings .", "the pathophysiological mechanisms and the incidence of this condition or genotype - phenotype correlation for tgfbi mutations are not obvious .", "if detailed examination of the corneas is not performed , gcd2 may not be observed until middle - age and older . it should be noted that detailed ocular examinations , including cornea assessments , are valuable when diagnosing nephropathy associated with tgfbi mutations .", "presently , alut testing is beneficial for laser - assisted in situ keratomileusis to protect patients from accelerated vision loss . as demonstrated with our patient , this test is easy and safe to perform when diagnosing patients with nephropathy associated with tgfbi mutations . in conclusion ,", "we have reported the first case of a unique nephropathy complicated by tgfbi - related cd .", "tgfbi - related nephropathy remains unknown and is difficult to diagnose without electron microscopic examination .", "further reports should be accumulated to determine whether the incidence of renal diseases associated with this mutation may presently be more frequent .", "patients with tgfbi - related cds , including gcd2 , should be examined for renal abnormalities ." ], [ "the authors have no ethical conflicts to disclose , and the patient provided informed consent ." ], [ "" ] ]

[ "a 60-year - old male patient , with a known history of arterial hypertension and type-2 diabetes mellitus , was referred to the outpatient echocardiography laboratory to undergo stress echocardiography because of recent episodes of chest pain occurring on mild exertion .", "he was on drug therapy with angiotensin - receptor blocker , thiazide diuretic , nondihydropyridine calcium antagonist , and dronedarone , which had been started because of recent episodes of paroxysmal atrial fibrillation and discontinued 2 days before the stress test .", "the patient received a standard protocol of high dipyridamole infusion in two doses ( 0.56 mg / kg and 0.28 mg / kg ) followed by atropine administration ( 1 mg in four 0.25 mg doses ) . at rest , no ischemic abnormalities were observed on electrocardiogram ( ecg ) and transthoracic echocardiography ( tte ) [ figure 1 and videos 13 ] .", "after completion of dipyridamole infusion , the patient complained a mild chest discomfort , without any significant ecg changes and any apparent wall - motion abnormalities on tte [ videos 46 ] .", "after atropine injection , a worsening of the anginal symptoms combined with a descending st - depression in v3 occurred ; despite the absence of relevant echocardiographic changes , two - dimensional ( 2d ) strain analysis showed lower longitudinal strain of the anterior interventricular septum from rest to peak dose [ figure 2 ] .", "afterward , as recommended , aminophylline was administered ; interestingly , a more pronounced st - depression and deep inverted t - waves in v2v4 appeared [ figure 3 ] .", "the patient was admitted to the cardiology department and underwent coronary angiography from the radial access , which revealed a long myocardial bridge ( mb ) of the left anterior descending ( lad ) artery with systolic milking [ figures 4 , 5 and videos 79 ] .", "the patient was , thereafter , treated with a beta - blocker and discharged without symptoms and ischemic abnormalities on rest ecg .", "electrocardiogram at rest two - dimensional strain analysis showing global longitudinal strain at rest ( upper panel ) and at peak dose of dipyridamole ( lower panel ) electrocardiogram after injection of atropine and aminophylline coronary angiography images in diastole ( left ) and systole ( right ) showing myocardial bridge of the left anterior descending artery with systolic milking angiographic images did not change after intracoronary nitroglycerine administration", "this case depicts a clinical scenario of positive dipyridamole stress test in a patient affected by mb of the lad .", "although mb has been classically deemed a benign coronary artery abnormality , it has been recently related to acute myocardial infarction and sudden cardiac death .", "moreover , mb has been also associated with endothelial dysfunction , early atherosclerosis , and coronary vasospasm . in particular ,", "a worsening of systolic coronary narrowing of mb has been found when using vasodilator agents , such as nitroglycerine , which are usually not administered in these patients . in our case ,", "dipyridamole provoked chest pain associated with minor ecg ischemic changes , such as an only one - lead ( v3 ) st - depression ; these abnormalities worsened after the administration of atropine and later , aminophylline , likely because of drug - induced positive inotropic and chronotropic effects .", "atropine might also have determined myocardial ischemia through a paradoxical coronary vasoconstriction induced by acetylcholine as observed in the presence of endothelial dysfunction and mb .", "this case has the following interesting implications : ( 1 ) the ability to detect mb also using a vasodilator stress test ( and not only dobutamine echocardiography or exercise test ) , particularly with the addition of atropine injection , ( 2 ) the utility of 2d strain analysis in confirming subtle regional wall - motion abnormalities , and ( 3 ) peculiar diagnostic features , on stress echocardiography , suggesting mb rather than obstructive coronary artery disease . in particular , the observed ecg and echocardiographic ischemic changes , although suggestive of a lad disease , appeared to be late occurring and less extended than usually observed in patients with stable hemodynamic lad obstruction , which should have determined more pronounced ecg and wall - motion abnormalities .", "indeed mb , differently from a fixed coronary obstruction , is a dynamic stenotic lesion , requiring a consistent increase in heart rate and myocardial contractility to provoke myocardial ischemia .", "hence , mb could have been clearly unmasked only whenever positive inotropic and chronotropic agents had been added to the vasodilator stress caused by dipyridamole .", "thus , the finding of worsening ischemic abnormalities after the administration of atropine ad aminophylline , during dipyridamole stress echocardiography , may represent a particular diagnostic feature of mb ." ]

[ "<S> a 60-year - old male patient was submitted to dipyridamole - atropine stress echocardiography ( dse ) for chest pain during exertion . at rest , no electrocardiographic ( ecg ) and transthoracic echocardiographic ( tte ) abnormalities were observed . </S>", "<S> after dipyridamole infusion , the patient complained a mild chest discomfort , without ecg changes and tte wall - motion abnormalities . </S>", "<S> subsequently , worsening of the anginal symptoms combined with descending st - depression and t - negative waves occurred after atropine and unexpectedly , aminophylline administration . </S>", "<S> coronary angiography was performed showing a myocardial bridge ( mb ) of the left anterior descending artery . </S>", "<S> the occurrence , during dse , of worsening ischemic abnormalities after atropine and aminophylline administration may be a particular diagnostic feature of mb . </S>" ]

[ "CASE REPORT", "DISCUSSION", "Financial support and sponsorship", "Conflicts of interest", "Vidoes Available on: " ]

[ [ "a 60-year - old male patient , with a known history of arterial hypertension and type-2 diabetes mellitus , was referred to the outpatient echocardiography laboratory to undergo stress echocardiography because of recent episodes of chest pain occurring on mild exertion .", "he was on drug therapy with angiotensin - receptor blocker , thiazide diuretic , nondihydropyridine calcium antagonist , and dronedarone , which had been started because of recent episodes of paroxysmal atrial fibrillation and discontinued 2 days before the stress test .", "the patient received a standard protocol of high dipyridamole infusion in two doses ( 0.56 mg / kg and 0.28 mg / kg ) followed by atropine administration ( 1 mg in four 0.25 mg doses ) . at rest , no ischemic abnormalities were observed on electrocardiogram ( ecg ) and transthoracic echocardiography ( tte ) [ figure 1 and videos 13 ] .", "after completion of dipyridamole infusion , the patient complained a mild chest discomfort , without any significant ecg changes and any apparent wall - motion abnormalities on tte [ videos 46 ] .", "after atropine injection , a worsening of the anginal symptoms combined with a descending st - depression in v3 occurred ; despite the absence of relevant echocardiographic changes , two - dimensional ( 2d ) strain analysis showed lower longitudinal strain of the anterior interventricular septum from rest to peak dose [ figure 2 ] .", "afterward , as recommended , aminophylline was administered ; interestingly , a more pronounced st - depression and deep inverted t - waves in v2v4 appeared [ figure 3 ] .", "the patient was admitted to the cardiology department and underwent coronary angiography from the radial access , which revealed a long myocardial bridge ( mb ) of the left anterior descending ( lad ) artery with systolic milking [ figures 4 , 5 and videos 79 ] .", "the patient was , thereafter , treated with a beta - blocker and discharged without symptoms and ischemic abnormalities on rest ecg .", "electrocardiogram at rest two - dimensional strain analysis showing global longitudinal strain at rest ( upper panel ) and at peak dose of dipyridamole ( lower panel ) electrocardiogram after injection of atropine and aminophylline coronary angiography images in diastole ( left ) and systole ( right ) showing myocardial bridge of the left anterior descending artery with systolic milking angiographic images did not change after intracoronary nitroglycerine administration" ], [ "this case depicts a clinical scenario of positive dipyridamole stress test in a patient affected by mb of the lad .", "although mb has been classically deemed a benign coronary artery abnormality , it has been recently related to acute myocardial infarction and sudden cardiac death .", "moreover , mb has been also associated with endothelial dysfunction , early atherosclerosis , and coronary vasospasm . in particular ,", "a worsening of systolic coronary narrowing of mb has been found when using vasodilator agents , such as nitroglycerine , which are usually not administered in these patients . in our case ,", "dipyridamole provoked chest pain associated with minor ecg ischemic changes , such as an only one - lead ( v3 ) st - depression ; these abnormalities worsened after the administration of atropine and later , aminophylline , likely because of drug - induced positive inotropic and chronotropic effects .", "atropine might also have determined myocardial ischemia through a paradoxical coronary vasoconstriction induced by acetylcholine as observed in the presence of endothelial dysfunction and mb .", "this case has the following interesting implications : ( 1 ) the ability to detect mb also using a vasodilator stress test ( and not only dobutamine echocardiography or exercise test ) , particularly with the addition of atropine injection , ( 2 ) the utility of 2d strain analysis in confirming subtle regional wall - motion abnormalities , and ( 3 ) peculiar diagnostic features , on stress echocardiography , suggesting mb rather than obstructive coronary artery disease . in particular , the observed ecg and echocardiographic ischemic changes , although suggestive of a lad disease , appeared to be late occurring and less extended than usually observed in patients with stable hemodynamic lad obstruction , which should have determined more pronounced ecg and wall - motion abnormalities .", "indeed mb , differently from a fixed coronary obstruction , is a dynamic stenotic lesion , requiring a consistent increase in heart rate and myocardial contractility to provoke myocardial ischemia .", "hence , mb could have been clearly unmasked only whenever positive inotropic and chronotropic agents had been added to the vasodilator stress caused by dipyridamole .", "thus , the finding of worsening ischemic abnormalities after the administration of atropine ad aminophylline , during dipyridamole stress echocardiography , may represent a particular diagnostic feature of mb ." ], [ "" ], [ "" ], [ "" ] ]

[ "natriuretic peptides are a family of hormones , sharing similar chemical structure ( a characteristic 17-amino acid ring structure , stabilized by a cysteine bridge , which contains several invariant amino acids and variable c- and n - terminal tails ) and biological function , with relevant effects in cardiovascular physiology and pathology .", "the study of natriuretic peptides began 50 years ago , when electron microscopy revealed the presence of secretory granules in cardiac atrial cells containing atrial natriuretic peptide ( anp ) as demonstrated by de bold and coworkers in the early 80s . from 1988 to 1990 , two more members of the natriuretic peptide family , the brain natriuretic peptide ( bnp ) and c - type natriuretic peptide ( cnp ) were identified in porcine brain [ 2 , 3 ] . more recently a new peptide that shares structural and functional characteristics with the previous natriuretic peptides was identified in the venom of the green mamba ( dendroaspis angusticeps ) and received the denomination of dendroaspis natriuretic peptide ( dnp ) .", "in addition , other peptides with similar cardiovascular effects have been identified in mammalians , including urodilatin , a peptide derived from alternative cleavage of pro - anp in the distal tubules of the kidney , where it exerts its natriuretic actions , or the intestinal epithelium derived peptides , guanylin and uroguanylin , which participate in water absorption .", "the classic physiological role of natriuretic peptides includes promotion of renal excretion of sodium ( natriuresis ) and water ( diuresis ) .", "natriuretic peptides also exert autocrine and paracrine effects within circulation , such as vasodilatation by relaxing vascular smooth muscle cells , regulation of renin , progesterone , endothelin and vasopressin secretion .", "anp and bnp are 28- and 32-amino acid peptides , respectively , mainly synthesized and released by atrial ( in the case of anp ) and ventricular cardiomyocytes ( for bnp ) in response to blood pressure and volume loading [ 8 , 9 ] . in addition ,", "anp and bnp secretion from the ventricles increases associated to a number of ventricular dysfunctions , and the extent of anp and bnp release under these circumstances is in relation to the severity of the pathology , that has led to the use of both natriuretic peptides as diagnostic tests for heart failure [ 10 , 11 ] .", "anp and bnp are synthesized as larger molecules that are subsequently cleaved to yield the active peptide hormone and the biologically inactive n - terminal peptide fragment [ 12 , 13 ] . both natriuretic peptides circulate in the blood reducing vascular tone and promoting diuresis / natriuresis to lower blood volume and pressure .", "anp and bnp are removed from the circulation by two different mechanisms : receptor - mediated internalization and proteolytic degradation by neutral endopeptidase , that has been shown to take place in the kidneys , vascular endothelium , lungs and heart .", "circulating half - life of anp is approximately 35 min , whereas the half - life of bnp is significantly greater , about 23 min . ,", "and even greater is that of the inactive terminal fragment of bnp , nt - probnp , from 60 to 120 min , which is relevant to their value as diagnostic tests .", "cnp , the third natriuretic peptide identified , is mainly expressed in the nervous system and vascular endothelial cells [ 14 , 15 ] , where cnp exerts autocrine and paracrine actions on vascular tone and muscle cell growth [ 15 , 16 ] .", "the cardiovascular effects of cnp are more likely mediated by its vascular local effects or by central actions on vasopressin and adrenocorticotropine release than to its natriuretic and diuretic effects , that are weaker than that of anp and bnp [ 16 , 17 ] .", "cnp gene expression is stimulated by several vasoactive mediators , such as interleukin 1 , vascular endothelial growth factor , transforming growth factor , tumour necrosis factor - a and insulin [ 15 , 1820 ] .", "dnp is a recently isolated 38-amino acid peptide that shows structural and functional properties of the previously identified members of the natriuretic peptide family .", "although dnp purification from human blood has not yet been achieved , dnp immunoreactivity has been reported in human plasma .", "dnplike immunoreactivity has been found in rat aorta , carotid artery and kidney [ 22 , 23 ] , where it has been found to induce vasorelaxation and inhibition of vascular smooth muscle cell proliferation .", "natriuretic peptides regulate cardiovascular homeostasis by the occupation of three membrane receptors ; two are guanylyl cyclase - coupled receptors , known as natriuretic peptide receptor ( npr)-a and npr - b , while npr - c lacks enzymatic activity and among other functions acts as a clearance receptor [ 24 , 25 ] .", "npr - a is activated by anp , bnp and dnp [ 25 , 26 ] , npr - b shows high affinity and is activated by cnp and , finally , the npr - c binds all natriuretic peptides .", "npr - a and npr - b are single - transmembrane receptors of approximately 120 kd with a similar basic structure that consist of a variable extracellular natriuretic peptide - binding region , a conserved intracellular kinase homology domain and a guanylyl cyclase domain with enzyme activity .", "occupation of npr - a and npr - b induces cellular responses through the elevation of intracellular cgmp levels , which , in turn , leads to the activation of a cgmpdependent protein kinase that phosphorylates a target protein in serine of threonine residues and mediates the specific physiological function .", "signal transduction activated by these receptors is terminated by cgmp phosphodiesterases that modulate the intracellular concentrations of cgmp and the duration and magnitude of the responses .", "npr - c is a transmembrane receptor with an extracellular domain containing the natriuretic peptide - binding region , a transmembrane domain and a cytosolic domain .", "two different subtypes of npr - c , of approximately 67 and 77 kd in size , have been identified [ 28 , 29 ] .", "npr - c has been involved in peptide clearance , removing natriuretic peptides from the circulation and in the mediation of natriuretic peptide - induced inhibition of camp synthesis , an effect that requires the involvement of a heterotrimeric g protein [ 7 , 31 , 32 ] .", "we have provided evidence that the 77 kd npr - c - like protein is involved in peptide internalization whereas the 67 kd npr - c - like protein is likely involved in adenylyl cyclase inhibition .", "in the past decade , much attention has been given to the investigation of natriuretic peptides testing in the evaluation of patients with different pathologies , such as congestive heart failure ( chf ) .", "studies in patients with chf showed that both anp and bnp secretion from ventricular myocytes increases in relation to the rigor of dysfunction .", "these findings led to investigate whether the plasma levels of anp and bnp may assist in the diagnosis of patients with heart failure . since half - life of bnp is greater than that of anp comparison of the diagnostic value of anp and bnp", "as reported above , the inactive n - terminal fragment of bnp , ntprobnp , has an even greater half - life than bnp ; thus , most of the clinical investigations concerning natriuretic peptides as biochemical markers for chf has focused on bnp or nt - probnp .", "basal plasma levels of bnp have been established between 5 and 50 pg / ml , while the corresponding values for nt - probnp are from 7 to 160 pg / ml . the approved cut - off levels to consider an abnormal range are 100 pg / ml for bnp and 125 pg / ml for ntprobnp ( 450 pg / ml for individuals older than 75 years old ) , although they vary according to age , sex and estimated glomerular filtration rate .", "levels of both bnp and nt - probnp are elevated under a number of pathological situations including cardiac dysfunctions , such as chf , diastolic dysfunction , valvular heart disease , atrial fibrillation and non - cardiac pathologies , such as acute pulmonary embolism , pulmonary hypertension , sepsis or hyperthyroidism [ 11 , 3436 ] .", "four major applications of bnp and nt - probnp testing in patients with chf have been presented in the last few years , including diagnosis , screening , prognosis and monitoring of therapy .", "different studies have reported the efficacy of bnp and ntprobnp tests increasing the diagnostic accuracy of heart failure [ 37 , 38 ] .", "the sensitivity and specificity of the test is especially high at a cut - off of 76 pg / ml bnp , and nt - probnp has been proved to be particularly important in decreasing the overdiagnosis of heart failure that occurs in primary care . for ntprobnp , levels over 450 pg / ml ( over 900 pg / ml for patients older than 50 years old ) are sensitive and specific for heart failure ; however , if the value is below 300 pg / ml , heart failure is highly unlikely with a negative predictive value of 99% .", "in addition , recent studies have reported that bnp is a significant prognostic indicator in patients diagnosed with heart failure and in asymptomatic patients .", "nt - probnp has been found to be a stronger risk biomarker for cardiovascular disease and death than c - reactive protein .", "high - risk subject with cardiovascular risk , which would be a great advance prompting more aggressive primary prevention .", "however , a number of limitations have been reported about the prognostic value of bnp and nt - probnp . in patients with advanced chf , atrial fibrillation", "bnp or nt - probnp can also be used as a guide for therapy in heart failure .", "for this use , it should be taken into account that therapies that reduce clinical disorders in heart failure also reduce bnp levels and those that improve heart failure conditions act primarily through mechanisms that are linked with changes in natriuretic peptide levels .", "in addition , bnp and nt - probnp levels do not decrease as rapidly in response to therapy as might be expected from their short half - lives , suggesting that the natriuretic peptide system need some time to autoregulate . considering this", ", bnp and nt - probnp levels might guide the clinician to adjust the treatment in order to achieve a plasmatic level of these agents below a critical value .", "sepsis and septic shock is associated with an elevation of cardiac troponin levels that has been shown to indicate left ventricular dysfunction and a poor prognosis . whereas cardiac troponins are a good biomarker for myocardial dysfunction in patients with severe sepsis or septic shock the use of natriuretic peptides , including bnp , as an indicator of prognosis remains controversial .", "renal insufficiency has also been shown to affect the plasmatic levels of both bnp and nt - probnp both in children and adult patients .", "the levels of bnp are correlated with renal function , so that an increase reaching about 200 pg / ml have been reported in patients with reduced creatinine clearance ( below 60 ml ", "similar observations have been reported with nt - probnp and a recommended reference value of 1200 pg / ml has been shown for patients with reduced creatinine clearance .", "it has been shown that peritoneal dialysis does not alter plasma nt - bnp or bnp levels in patients with renal failure , whereas both blood urea nitrogen and creatinine levels declined as expected , which should be taken into account if plasmatic levels of these hormones are used as a guide to the management of patients with renal failure .", "nt - pro - bnp and bnp have been recently presented as useful biomarkers for coronary artery disease and left ventricular hypertrophy in patients with chronic kidney disease , where detection of coronary artery disease and left ventricular hypertrophy in these patients has remained elusive despite the greater prevalence in patients with chronic kidney disease .", "natriuretic peptides , especially anp , have long been reported to have the potential to restore renal function after ischaemic injury , and have been shown to counteract renal sympathetic nerve activity in renal function .", "anp overexpression has been shown to exhibit protection against gentamycin - induced nephrotoxicity , as demonstrated by daily subcutaneous administration of gentamycin , for 10 days , in sprague dawley rats either treated with an intravenous injection of adenovirus ( ad.rsv-anp ) , carrying the human anp gene , the first day of gentamycin administration , or not treated ( control ) for comparison .", "this finding raises the possibility of using anp gene therapy for the treatment of drug - induced renal failure .", "in addition , anp has been reported to exert a protective effect on the outcome of acute renal failure in animals when infused over short periods of time , while it has been shown that prolonged infusion of anp does not alter the course of acute renal failure . on the other hand , infusion of wistar rats with", "anp exerted little renoprotective effect against endotoxin - induced acute renal failure , since anp infusion did not improve the hyponatriuresis and oliguria induced by endotoxin administration .", "recent studies have pointed out that continuous infusion of synthetic human anp is effective for preventing acute renal failure , which is a major problem occurring immediately after liver transplantation and requiring haemodialysis .", "this is the case of a model of transgenic mice overexpressing bnp that show reduced glomerular injury than control mice during the development of diabetes mellitus .", "glomerular hyperfiltration is an early haemodynamic alteration and one of the key mechanisms of the pathogenesis of diabetic nephropathy .", "this observation suggests that renoprotective effects of natriuretic peptides may prevent the progression of diabetic nephropathy , although further studies are necessary to establish therapeutic strategies to palliate renal complications associated to diabetes mellitus .", "further evidences for the renoprotective role of natriuretic peptides come from studies where the peptide hydrolysis is inhibited .", "neutral endopeptidase is an endothelial cell surface zinc metallopeptidase and the major pathway involved in the degradation of the natriuretic peptides .", "inhibition of neutral endopeptidase increases levels of anp , bnp and cnp , and has been shown to offer a therapeutic advantage in the treatment of hypertension , heart failure and endothelial dysfunction .", "endopeptidase inhibitors reduce vasoconstriction and improve sodium / water balance ; as a result these inhibitors decrease peripheral vascular resistance and blood pressure and improve local blood flow .", "endopeptidase inhibitors also reduce the activity of the angiotensin - converting enzyme ( ace ) , leading to a reduction of vasoconstrictor and proliferative mediators , such as angiotensin ii and increase local levels of bradykinin .", "recent studies have reported that anp reduces angiotensin ii - induced renomedullary interstitial cells proliferation and extracellular matrix synthesis in diabetic subjects more efficiently in neutral endopeptidase - deficient mice , which provide evidence for the beneficial effect of inhibition of neutral endopeptidase in attenuating abnormal cell growth associated with diabetic nephropathy .", "in addition to the renoprotective effect , neutral endopeptidase inhibition , in combination with inhibition of the ace activity , has been shown to reduce blood pressure in spontaneously hypertensive rats .", "this is the case of omapatrilat , a potent vasopeptidase inhibitor that exerts anti - hypertensive effects by inhibition of the neutral endopeptidase and ace at the tissue level , which results in beneficial effects on the cardiovascular structure .", "the dual metalloprotease inhibitors of ace and neutral endopeptidases , called vasopeptidase inhibitors , provide an advance over individual ace or neutral endopeptidase inhibitors , and might represent a new and attractive therapeutic strategy for the treatment of cardiovascular disease .", "nesiritide is a recombinant form of human bnp and its amino acid sequence is identical to that of endogenous human bnp .", "administration of nesiritide results in venous , arterial and coronary vasodilatation , reducing cardiac the pre - load and after - load , which increase cardiac output without direct inotropic effects [ 6062 ] .", "in addition , nesiritide increases glomerular filtration rate and filtration fraction , suppresses the renin - angiotensin - aldosterone axis , and enhances diuresis and natriuresis .", "nesiritide is currently used in the treatment of acute decompensated heart failure , where it has been shown to decrease pulmonary capillary wedge pressure , pulmonary artery pressure , right atrial pressure and systemic vascular resistance , as well as increase cardiac index and stroke volume index [ 6466 ] . in comparison with nitroglycerin ,", "treatment with nesiritide did not induce the appearance of tachyphylaxis and other adverse effects . unlike inotropes", ", the beneficial haemodynamic effects produced by nesiritide do not cause an increase in myocardial oxygen consumption , an important consideration for patients with acutely decompensated heart failure . since nesiritide is not an inotrope , it does not affect myocardial contractility , as does the agonists of -adrenergic receptors or the inhibitors of phosphodiesterase iii . as a result", "studies aimed to determine the impact of early initiation of acute decompensated heart failure therapy with nesiritide on subsequent outcomes have confirmed its beneficial effects reducing the severity of the associated complications , reducing the mean total hospital length of stay and the requirement of transfer to the intensive care unit . in comparison with dobutamine and milrinone ,", "nesiritide therapy was associated with a lower in - hospital mortality rate and shorter length of stay .", "in addition , total health care costs with nesiritide were decreased compared with the other drugs , since although the acquisition cost of nesiritide was higher than that of milrinone and dobutamine , nesiritide has been shown as a more cost - effective treatment option for patients with acute decompensated heart failure [ 69 , 70 ] . despite its beneficial effects on the cardiovascular system ,", "recent studies have raised the question of safety with nesiritide therapy . in three randomized controlled trials sackner - bernstein and coworkers expressed concern of possible short - term ( 30-day ) risk of death after nesiritide use for the treatment of acute decompensated heart failure . as mentioned by the authors ,", "first , the three randomized controlled trials used the nesiritide study group efficacy trial ( nsget ) , the vasodilation in the management of acute congestive heart failure ( vmac ) , and the prospective randomized outcomes study of acutely decompensated chf treated initially in outpatients with natrecor ( proaction ) were not designed to determine whether nesiritide is associated with risk of death .", "in addition , there was not complete information concerning the use of additional medications or procedures through the 30-day period of the study .", "the same group has reported that nesiritide significantly increases the risk of worsening renal function in patients with acute decompensated heart failure , although whether renal dysfunction reflects haemodynamic effect or renal injury is unclear .", "concerning this issue , more recent studies have reported that the effect of nesiritide on renal function strongly depends on the infusion times , so that nesiritide infusion time 24 hrs is associated with elevated markers of worsening renal function in patients with acutely decompensated heart failure compare with infusion of less than 24 hrs .", "several studies have reported that an increase in serum creatinine levels , such as that observed with nesiritide , predicts a higher risk of death even when that increase is transient [ 74 , 75 ] . unlike these previous studies ,", "a more recent meta - analysis has reported that nesiritide is not associated with a higher 30- or 180-day mortality .", "given the limitations of the meta - analyses , without randomized controlled trials powered to evaluate mortality , arora and coworkers suggest that it is premature to abandon the use of nesiritide . a summary of the current information concerning the benefits and deleterious effects of the use of nesiritide is provided in table 1 .", "future clinical trials are necessary to address the concerns raised and provide a better understanding of the actions of nesiritide in the management of acute decompensated heart failure .", "in addition , recent studies have reported that npr - b , not npr - a , which is the receptor of nesiritide , is the predominant npr in the failing heart , suggesting that drugs directed towards both nprs might provide a greater benefit than nesiritide per se .", "beneficial and deleterious effects of nesiritide in cardiovascular and renal systems the clinical role of natriuretic peptides either as biomarkers for diagnosis , prognosis or monitoring of therapy , or as therapeutic strategies for cardiovascular and renal disorders has gained acceptance over the last decade . despite specific basic studies and clinical trials necessary to better understand the possibilities of natriuretic peptides in therapeutic interventions , the use of these peptides to treat cardiovascular dysfunction seems to be most promising ." ]

[ "<S> abstractthe natriuretic peptides are a family of related hormones that play a crucial role in cardiovascular and renal homeostasis . </S>", "<S> they have recently emerged as potentially important clinical biomarkers in heart failure . </S>", "<S> natriuretic peptides , particularly brain natriuretic peptide ( bnp ) and the inactive n - terminal fragment of bnp , nt - probnp , that has an even greater half - life than bnp , are elevated in heart failure and therefore considered to be excellent predictors of disease outcome . </S>", "<S> nesiritide , a recombinant human bnp , has been shown to provide symptomatic and haemodynamic improvement in acute decompensated heart failure , although recent reports have suggested an increased short - term risk of death with nesiritide use . </S>", "<S> this review article describes : the current use of bnp and its inactive precursor nt - probnp in diagnosis , screening , prognosis and monitoring of therapy for congestive heart failure , the renoprotective actions of natriuretic peptides after renal failure and the controversy around the therapeutic use of the recombinant human bnp nesiritide . </S>" ]

[ "Introduction", "Natriuretic peptides as a biomarker of congestive heart failure and acute renal failure/insufficiency", "Renoprotective effect of natriuretic peptides after acute renal failure", "Nesiritide current benefits/hazards of drug usage" ]

[ [ "natriuretic peptides are a family of hormones , sharing similar chemical structure ( a characteristic 17-amino acid ring structure , stabilized by a cysteine bridge , which contains several invariant amino acids and variable c- and n - terminal tails ) and biological function , with relevant effects in cardiovascular physiology and pathology .", "the study of natriuretic peptides began 50 years ago , when electron microscopy revealed the presence of secretory granules in cardiac atrial cells containing atrial natriuretic peptide ( anp ) as demonstrated by de bold and coworkers in the early 80s . from 1988 to 1990 , two more members of the natriuretic peptide family , the brain natriuretic peptide ( bnp ) and c - type natriuretic peptide ( cnp ) were identified in porcine brain [ 2 , 3 ] . more recently a new peptide that shares structural and functional characteristics with the previous natriuretic peptides was identified in the venom of the green mamba ( dendroaspis angusticeps ) and received the denomination of dendroaspis natriuretic peptide ( dnp ) .", "in addition , other peptides with similar cardiovascular effects have been identified in mammalians , including urodilatin , a peptide derived from alternative cleavage of pro - anp in the distal tubules of the kidney , where it exerts its natriuretic actions , or the intestinal epithelium derived peptides , guanylin and uroguanylin , which participate in water absorption .", "the classic physiological role of natriuretic peptides includes promotion of renal excretion of sodium ( natriuresis ) and water ( diuresis ) .", "natriuretic peptides also exert autocrine and paracrine effects within circulation , such as vasodilatation by relaxing vascular smooth muscle cells , regulation of renin , progesterone , endothelin and vasopressin secretion .", "anp and bnp are 28- and 32-amino acid peptides , respectively , mainly synthesized and released by atrial ( in the case of anp ) and ventricular cardiomyocytes ( for bnp ) in response to blood pressure and volume loading [ 8 , 9 ] . in addition ,", "anp and bnp secretion from the ventricles increases associated to a number of ventricular dysfunctions , and the extent of anp and bnp release under these circumstances is in relation to the severity of the pathology , that has led to the use of both natriuretic peptides as diagnostic tests for heart failure [ 10 , 11 ] .", "anp and bnp are synthesized as larger molecules that are subsequently cleaved to yield the active peptide hormone and the biologically inactive n - terminal peptide fragment [ 12 , 13 ] . both natriuretic peptides circulate in the blood reducing vascular tone and promoting diuresis / natriuresis to lower blood volume and pressure .", "anp and bnp are removed from the circulation by two different mechanisms : receptor - mediated internalization and proteolytic degradation by neutral endopeptidase , that has been shown to take place in the kidneys , vascular endothelium , lungs and heart .", "circulating half - life of anp is approximately 35 min , whereas the half - life of bnp is significantly greater , about 23 min . ,", "and even greater is that of the inactive terminal fragment of bnp , nt - probnp , from 60 to 120 min , which is relevant to their value as diagnostic tests .", "cnp , the third natriuretic peptide identified , is mainly expressed in the nervous system and vascular endothelial cells [ 14 , 15 ] , where cnp exerts autocrine and paracrine actions on vascular tone and muscle cell growth [ 15 , 16 ] .", "the cardiovascular effects of cnp are more likely mediated by its vascular local effects or by central actions on vasopressin and adrenocorticotropine release than to its natriuretic and diuretic effects , that are weaker than that of anp and bnp [ 16 , 17 ] .", "cnp gene expression is stimulated by several vasoactive mediators , such as interleukin 1 , vascular endothelial growth factor , transforming growth factor , tumour necrosis factor - a and insulin [ 15 , 1820 ] .", "dnp is a recently isolated 38-amino acid peptide that shows structural and functional properties of the previously identified members of the natriuretic peptide family .", "although dnp purification from human blood has not yet been achieved , dnp immunoreactivity has been reported in human plasma .", "dnplike immunoreactivity has been found in rat aorta , carotid artery and kidney [ 22 , 23 ] , where it has been found to induce vasorelaxation and inhibition of vascular smooth muscle cell proliferation .", "natriuretic peptides regulate cardiovascular homeostasis by the occupation of three membrane receptors ; two are guanylyl cyclase - coupled receptors , known as natriuretic peptide receptor ( npr)-a and npr - b , while npr - c lacks enzymatic activity and among other functions acts as a clearance receptor [ 24 , 25 ] .", "npr - a is activated by anp , bnp and dnp [ 25 , 26 ] , npr - b shows high affinity and is activated by cnp and , finally , the npr - c binds all natriuretic peptides .", "npr - a and npr - b are single - transmembrane receptors of approximately 120 kd with a similar basic structure that consist of a variable extracellular natriuretic peptide - binding region , a conserved intracellular kinase homology domain and a guanylyl cyclase domain with enzyme activity .", "occupation of npr - a and npr - b induces cellular responses through the elevation of intracellular cgmp levels , which , in turn , leads to the activation of a cgmpdependent protein kinase that phosphorylates a target protein in serine of threonine residues and mediates the specific physiological function .", "signal transduction activated by these receptors is terminated by cgmp phosphodiesterases that modulate the intracellular concentrations of cgmp and the duration and magnitude of the responses .", "npr - c is a transmembrane receptor with an extracellular domain containing the natriuretic peptide - binding region , a transmembrane domain and a cytosolic domain .", "two different subtypes of npr - c , of approximately 67 and 77 kd in size , have been identified [ 28 , 29 ] .", "npr - c has been involved in peptide clearance , removing natriuretic peptides from the circulation and in the mediation of natriuretic peptide - induced inhibition of camp synthesis , an effect that requires the involvement of a heterotrimeric g protein [ 7 , 31 , 32 ] .", "we have provided evidence that the 77 kd npr - c - like protein is involved in peptide internalization whereas the 67 kd npr - c - like protein is likely involved in adenylyl cyclase inhibition ." ], [ "in the past decade , much attention has been given to the investigation of natriuretic peptides testing in the evaluation of patients with different pathologies , such as congestive heart failure ( chf ) .", "studies in patients with chf showed that both anp and bnp secretion from ventricular myocytes increases in relation to the rigor of dysfunction .", "these findings led to investigate whether the plasma levels of anp and bnp may assist in the diagnosis of patients with heart failure . since half - life of bnp is greater than that of anp comparison of the diagnostic value of anp and bnp", "as reported above , the inactive n - terminal fragment of bnp , ntprobnp , has an even greater half - life than bnp ; thus , most of the clinical investigations concerning natriuretic peptides as biochemical markers for chf has focused on bnp or nt - probnp .", "basal plasma levels of bnp have been established between 5 and 50 pg / ml , while the corresponding values for nt - probnp are from 7 to 160 pg / ml . the approved cut - off levels to consider an abnormal range are 100 pg / ml for bnp and 125 pg / ml for ntprobnp ( 450 pg / ml for individuals older than 75 years old ) , although they vary according to age , sex and estimated glomerular filtration rate .", "levels of both bnp and nt - probnp are elevated under a number of pathological situations including cardiac dysfunctions , such as chf , diastolic dysfunction , valvular heart disease , atrial fibrillation and non - cardiac pathologies , such as acute pulmonary embolism , pulmonary hypertension , sepsis or hyperthyroidism [ 11 , 3436 ] .", "four major applications of bnp and nt - probnp testing in patients with chf have been presented in the last few years , including diagnosis , screening , prognosis and monitoring of therapy .", "different studies have reported the efficacy of bnp and ntprobnp tests increasing the diagnostic accuracy of heart failure [ 37 , 38 ] .", "the sensitivity and specificity of the test is especially high at a cut - off of 76 pg / ml bnp , and nt - probnp has been proved to be particularly important in decreasing the overdiagnosis of heart failure that occurs in primary care . for ntprobnp , levels over 450 pg / ml ( over 900 pg / ml for patients older than 50 years old ) are sensitive and specific for heart failure ; however , if the value is below 300 pg / ml , heart failure is highly unlikely with a negative predictive value of 99% .", "in addition , recent studies have reported that bnp is a significant prognostic indicator in patients diagnosed with heart failure and in asymptomatic patients .", "nt - probnp has been found to be a stronger risk biomarker for cardiovascular disease and death than c - reactive protein .", "high - risk subject with cardiovascular risk , which would be a great advance prompting more aggressive primary prevention .", "however , a number of limitations have been reported about the prognostic value of bnp and nt - probnp . in patients with advanced chf , atrial fibrillation", "bnp or nt - probnp can also be used as a guide for therapy in heart failure .", "for this use , it should be taken into account that therapies that reduce clinical disorders in heart failure also reduce bnp levels and those that improve heart failure conditions act primarily through mechanisms that are linked with changes in natriuretic peptide levels .", "in addition , bnp and nt - probnp levels do not decrease as rapidly in response to therapy as might be expected from their short half - lives , suggesting that the natriuretic peptide system need some time to autoregulate . considering this", ", bnp and nt - probnp levels might guide the clinician to adjust the treatment in order to achieve a plasmatic level of these agents below a critical value .", "sepsis and septic shock is associated with an elevation of cardiac troponin levels that has been shown to indicate left ventricular dysfunction and a poor prognosis . whereas cardiac troponins are a good biomarker for myocardial dysfunction in patients with severe sepsis or septic shock the use of natriuretic peptides , including bnp , as an indicator of prognosis remains controversial .", "renal insufficiency has also been shown to affect the plasmatic levels of both bnp and nt - probnp both in children and adult patients .", "the levels of bnp are correlated with renal function , so that an increase reaching about 200 pg / ml have been reported in patients with reduced creatinine clearance ( below 60 ml ", "similar observations have been reported with nt - probnp and a recommended reference value of 1200 pg / ml has been shown for patients with reduced creatinine clearance .", "it has been shown that peritoneal dialysis does not alter plasma nt - bnp or bnp levels in patients with renal failure , whereas both blood urea nitrogen and creatinine levels declined as expected , which should be taken into account if plasmatic levels of these hormones are used as a guide to the management of patients with renal failure .", "nt - pro - bnp and bnp have been recently presented as useful biomarkers for coronary artery disease and left ventricular hypertrophy in patients with chronic kidney disease , where detection of coronary artery disease and left ventricular hypertrophy in these patients has remained elusive despite the greater prevalence in patients with chronic kidney disease ." ], [ "natriuretic peptides , especially anp , have long been reported to have the potential to restore renal function after ischaemic injury , and have been shown to counteract renal sympathetic nerve activity in renal function .", "anp overexpression has been shown to exhibit protection against gentamycin - induced nephrotoxicity , as demonstrated by daily subcutaneous administration of gentamycin , for 10 days , in sprague dawley rats either treated with an intravenous injection of adenovirus ( ad.rsv-anp ) , carrying the human anp gene , the first day of gentamycin administration , or not treated ( control ) for comparison .", "this finding raises the possibility of using anp gene therapy for the treatment of drug - induced renal failure .", "in addition , anp has been reported to exert a protective effect on the outcome of acute renal failure in animals when infused over short periods of time , while it has been shown that prolonged infusion of anp does not alter the course of acute renal failure . on the other hand , infusion of wistar rats with", "anp exerted little renoprotective effect against endotoxin - induced acute renal failure , since anp infusion did not improve the hyponatriuresis and oliguria induced by endotoxin administration .", "recent studies have pointed out that continuous infusion of synthetic human anp is effective for preventing acute renal failure , which is a major problem occurring immediately after liver transplantation and requiring haemodialysis .", "this is the case of a model of transgenic mice overexpressing bnp that show reduced glomerular injury than control mice during the development of diabetes mellitus .", "glomerular hyperfiltration is an early haemodynamic alteration and one of the key mechanisms of the pathogenesis of diabetic nephropathy .", "this observation suggests that renoprotective effects of natriuretic peptides may prevent the progression of diabetic nephropathy , although further studies are necessary to establish therapeutic strategies to palliate renal complications associated to diabetes mellitus .", "further evidences for the renoprotective role of natriuretic peptides come from studies where the peptide hydrolysis is inhibited .", "neutral endopeptidase is an endothelial cell surface zinc metallopeptidase and the major pathway involved in the degradation of the natriuretic peptides .", "inhibition of neutral endopeptidase increases levels of anp , bnp and cnp , and has been shown to offer a therapeutic advantage in the treatment of hypertension , heart failure and endothelial dysfunction .", "endopeptidase inhibitors reduce vasoconstriction and improve sodium / water balance ; as a result these inhibitors decrease peripheral vascular resistance and blood pressure and improve local blood flow .", "endopeptidase inhibitors also reduce the activity of the angiotensin - converting enzyme ( ace ) , leading to a reduction of vasoconstrictor and proliferative mediators , such as angiotensin ii and increase local levels of bradykinin .", "recent studies have reported that anp reduces angiotensin ii - induced renomedullary interstitial cells proliferation and extracellular matrix synthesis in diabetic subjects more efficiently in neutral endopeptidase - deficient mice , which provide evidence for the beneficial effect of inhibition of neutral endopeptidase in attenuating abnormal cell growth associated with diabetic nephropathy .", "in addition to the renoprotective effect , neutral endopeptidase inhibition , in combination with inhibition of the ace activity , has been shown to reduce blood pressure in spontaneously hypertensive rats .", "this is the case of omapatrilat , a potent vasopeptidase inhibitor that exerts anti - hypertensive effects by inhibition of the neutral endopeptidase and ace at the tissue level , which results in beneficial effects on the cardiovascular structure .", "the dual metalloprotease inhibitors of ace and neutral endopeptidases , called vasopeptidase inhibitors , provide an advance over individual ace or neutral endopeptidase inhibitors , and might represent a new and attractive therapeutic strategy for the treatment of cardiovascular disease ." ], [ "nesiritide is a recombinant form of human bnp and its amino acid sequence is identical to that of endogenous human bnp .", "administration of nesiritide results in venous , arterial and coronary vasodilatation , reducing cardiac the pre - load and after - load , which increase cardiac output without direct inotropic effects [ 6062 ] .", "in addition , nesiritide increases glomerular filtration rate and filtration fraction , suppresses the renin - angiotensin - aldosterone axis , and enhances diuresis and natriuresis .", "nesiritide is currently used in the treatment of acute decompensated heart failure , where it has been shown to decrease pulmonary capillary wedge pressure , pulmonary artery pressure , right atrial pressure and systemic vascular resistance , as well as increase cardiac index and stroke volume index [ 6466 ] . in comparison with nitroglycerin ,", "treatment with nesiritide did not induce the appearance of tachyphylaxis and other adverse effects . unlike inotropes", ", the beneficial haemodynamic effects produced by nesiritide do not cause an increase in myocardial oxygen consumption , an important consideration for patients with acutely decompensated heart failure . since nesiritide is not an inotrope , it does not affect myocardial contractility , as does the agonists of -adrenergic receptors or the inhibitors of phosphodiesterase iii . as a result", "studies aimed to determine the impact of early initiation of acute decompensated heart failure therapy with nesiritide on subsequent outcomes have confirmed its beneficial effects reducing the severity of the associated complications , reducing the mean total hospital length of stay and the requirement of transfer to the intensive care unit . in comparison with dobutamine and milrinone ,", "nesiritide therapy was associated with a lower in - hospital mortality rate and shorter length of stay .", "in addition , total health care costs with nesiritide were decreased compared with the other drugs , since although the acquisition cost of nesiritide was higher than that of milrinone and dobutamine , nesiritide has been shown as a more cost - effective treatment option for patients with acute decompensated heart failure [ 69 , 70 ] . despite its beneficial effects on the cardiovascular system ,", "recent studies have raised the question of safety with nesiritide therapy . in three randomized controlled trials sackner - bernstein and coworkers expressed concern of possible short - term ( 30-day ) risk of death after nesiritide use for the treatment of acute decompensated heart failure . as mentioned by the authors ,", "first , the three randomized controlled trials used the nesiritide study group efficacy trial ( nsget ) , the vasodilation in the management of acute congestive heart failure ( vmac ) , and the prospective randomized outcomes study of acutely decompensated chf treated initially in outpatients with natrecor ( proaction ) were not designed to determine whether nesiritide is associated with risk of death .", "in addition , there was not complete information concerning the use of additional medications or procedures through the 30-day period of the study .", "the same group has reported that nesiritide significantly increases the risk of worsening renal function in patients with acute decompensated heart failure , although whether renal dysfunction reflects haemodynamic effect or renal injury is unclear .", "concerning this issue , more recent studies have reported that the effect of nesiritide on renal function strongly depends on the infusion times , so that nesiritide infusion time 24 hrs is associated with elevated markers of worsening renal function in patients with acutely decompensated heart failure compare with infusion of less than 24 hrs .", "several studies have reported that an increase in serum creatinine levels , such as that observed with nesiritide , predicts a higher risk of death even when that increase is transient [ 74 , 75 ] . unlike these previous studies ,", "a more recent meta - analysis has reported that nesiritide is not associated with a higher 30- or 180-day mortality .", "given the limitations of the meta - analyses , without randomized controlled trials powered to evaluate mortality , arora and coworkers suggest that it is premature to abandon the use of nesiritide . a summary of the current information concerning the benefits and deleterious effects of the use of nesiritide is provided in table 1 .", "future clinical trials are necessary to address the concerns raised and provide a better understanding of the actions of nesiritide in the management of acute decompensated heart failure .", "in addition , recent studies have reported that npr - b , not npr - a , which is the receptor of nesiritide , is the predominant npr in the failing heart , suggesting that drugs directed towards both nprs might provide a greater benefit than nesiritide per se .", "beneficial and deleterious effects of nesiritide in cardiovascular and renal systems the clinical role of natriuretic peptides either as biomarkers for diagnosis , prognosis or monitoring of therapy , or as therapeutic strategies for cardiovascular and renal disorders has gained acceptance over the last decade . despite specific basic studies and clinical trials necessary to better understand the possibilities of natriuretic peptides in therapeutic interventions , the use of these peptides to treat cardiovascular dysfunction seems to be most promising ." ] ]

[ "forty - five - year - old male shelter resident , chronic alcoholic , chronic smoker , and marijuana abuser presented to emergency department ( ed ) with complaints of intermittent fever with night sweats and productive - cough for 10 days with yellowish sputum associated with pleuritic chest pain .", "he complained of generalized weakness and decreased appetite and claimed to have lost approximately 10 pounds in 2 weeks .", "laboratory results showed leukocytosis of 17,000/mm with neutrophil differential of 11,200/mm ( 65% ) with band neutrophils of 10% .", "chest radiograph was reported as right middle lobe consolidation but careful observation revealed a foreign body ( fb ) , likely a tooth in the right bronchus intermedius ( fig .", "ct scan of chest showed a well calcified fb likely a tooth at the level of carina obstructing right intermedius bronchus and consolidation in right lower lobe indicating post - obstructive pneumonia ( fig .", "2 ) . patient was admitted and prescribed intravenous antibiotics targeting gram - negative organisms and anaerobes .", "deep conscious sedation was achieved by propofol infusion in the presence of an anesthesiologist and cardiothoracic surgery backup .", "bronchoscopy showed tooth in right intermediate bronchus with ~75% occlusion , and purulent secretions from the right main stem ( figs .", "the fb was removed successfully with rat tooth forceps ( figs . 7 and 8) .", "post bronchoscopy inspection showed right main stem , bronchus intermedius , and right middle and lower lobe bronchi to be well patent .", "closer view of the foreign body , identifiable as tooth in right main stem bronchus .", "the aspirated tooth removed by rat tooth forceps . upon further questioning , the patient said that he did not remember having aspirated the tooth and hence the duration of aspiration could not be known .", "he was discharged home on oral antibiotics a couple of days after the bronchoscopy and counseled to quit alcohol and drugs .", "fba is more common in children than adults with about 80% occurring in children aged less than 15 years .", "it is the fourth most common cause of unintentional injury deaths in the united states . while choking is a hazard for all ages , choking deaths peaked at age 84 in 2011 as per national safety council injury facts 2015 ( 1 ) .", "fba in adults is often overlooked as a potential cause of airway obstruction especially if there is no asphyxiation .", "adults with risk factors such as alcoholism , drug abuse , mental retardation , and neuromuscular conditions are predisposed to aspiration .", "however , accidental aspiration in adults without the aforementioned risk factors has been described ( 25 ) . in adults ,", "most common foreign bodies aspirated are food and broken fragments of teeth ( 68 ) .", "ct scan is more sensitive and specific than chest radiograph in diagnosing radiolucent foreign bodies and for characterizing the attenuation of a suspected fb ( 9 ) .", "unlike in children , less than half of fbs are lodged in the proximal airways in adults .", "most of the fbs are lodged in the right bronchial tree , whereas in children no significant difference was seen between right and left bronchial tree ( 6 ) .", "bronchoscopy for the removal of fb was introduced by gustav killian , an otolaryngologist in 1897 .", "animal studies performed by zavala and rhodes ( 11 ) showed that fbr could be used to retrieve different kinds of fbs by using grasping forceps through the bronchoscope .", "fbr is considered as the diagnostic test of choice for initial diagnosis of fb in adults .", "the advantages of fbr over rigid bronchoscopy are that it can be performed under local anesthesia , visualization of smaller peripheral airway is better with relatively easier manipulation , and can be performed in patients with deformities of c - spine and pharynx .", "it is also a relatively easy and safer procedure in experienced - hands ( 10 , 12 ) .", "rigid bronchoscopy is recommended if fbr fails , if the fb is centrally located , if firmly embedded in scar tissue , and for removal of sharp objects which require maneuvering to minimize mucosal trauma ( 13 ) .", "there have been several case series which have reported successful removal of foreign bodies by fbr .", "cunanan ( 12 ) reported a success rate of 89% in using fbr for the removal of fb in 300 mentally retarded and physically handicapped patients .", "they reported a decrease in mortality from 12 to 1% with the use of fbr over rigid bronchoscope .", "they , however , attribute to the increased mortality from rigid bronchoscopy to complications of general anesthesia in patients with comorbidities .", "limper and prakash had a success rate of 60% with fbr , whereas rigid bronchoscopy showed a success rate of 98% . in their experience , fbr is advantageous in distally lodged fb , in cases with cervical instability , mechanically ventilated patients , and also in removal of small fbs , which can be securely grasped with a fiberoptic bronchoscope ( 14 ) .", "( 15 ) report a success rate of 97% for fb removal via fbr with low mortality and morbidity .", "they also reported that granulation tissue can be removed without much bleeding ; thus , removal of fb can be achieved in a repeat fbr a week later .", "( 13 ) indicate that fbr can be used to remove foreign bodies especially if they are small and peripheral and that fbr may be superior to rigid bronchoscopy for grasping tiny and far - reaching foreign bodies .", "rigid bronchoscopy is recommended if fbr fails and especially if the fb is large and located in central bronchi or trachea .", "however , in children who may have central , asphyxiating foreign bodies , the rigid bronchoscope under general anesthesia is preferentially indicated .", "team - based approach with designated roles for team members improves the chances of a successful procedure .", "anesthesia and ct surgery backup is essential . team - based analysis of the nature , location of the fb , and appropriate accessory instruments is equally necessary .", "steroids can be given to minimize inflammatory changes as they may lead to difficulty in fb extraction .", "orotracheal approach is preferred over nasotracheal , as it avoids fb from being lost in the nasal passages if accidently dislodged .", "care should be taken to keep fb in the center of visual field and to avoid pushing fb distally into the airways .", "once fb has been secured , bronchoscope , accessory instrument , and the secured fb are all withdrawn from the airway simultaneously .", "rare complications of fbr are likelihood of asphyxiation from losing the fb in the subglottic area and hemoptysis ( 10 ) .", "another approach is to reinsert flexible bronchoscope to push the fb into more peripheral airway thus resolving the central airway obstruction . in case of hemoptysis ,", "airway can be better managed by rigid bronchoscope , but this complication is very rare if performed by trained operators .", "incidence of massive hemoptysis even in patients with significant granulation tissue around the fb is extremely low ( 15 ) .", "organic materials can cause severe inflammation in a short period of time and tend to absorb water with development of airway obstruction relatively earlier .", "in contrast , inorganic fbs are inert , and so patients might be asymptomatic for a prolonged period ( 16 ) . grasping forceps , baskets , magnet extractors ,", "yag laser , and cryoprobes are available as accessories for fb removal via flexible bronchoscope ( 10 ) .", "rat tooth forceps is a type of a grasping forceps , which is particularly useful for removing hard , flat , or thin organic or inorganic foreign bodies .", "these are used primarily when the fb is hard as a firm grip can be achieved .", "these can not be used for the retrieval of organic friable objects which tend to fracture due to the firmness of the grip provided by these forceps ( 10 ) .", "zero - tip basket is used in urological procedures like retrieval of ureteral stones . the tipless design allows close approximation to fb .", "they are used to retrieve fbs lodged in distal bronchi and mobile fbs ( 16 , 17 ) .", "fish net basket , a modified polypectomy snare , is useful in removing bulky fbs ( 10 ) .", "magnet extractors contain flexible probe tipped with a magnet , useful for retrieval of metallic fbs ( 10 ) .", "fbs embedded in granulation tissue can be removed by yag laser , which vaporizes the surrounding granulation tissue .", "cryoprobe is used to freeze friable organic and small inorganic materials . freezing the foreign bodies", "high index of suspicion is needed especially for high - risk patients presenting with compatible symptoms .", "fbr is the initial diagnostic and therapeutic procedure recommended with low complication rates in experienced hands .", "rigid bronchoscopy is performed if the flexible bronchoscopy fails or if the fb is large and centrally located , in case of significant hemoptysis and if significant granulation tissue is present with a fb which is deeply embedded ." ]

[ "<S> foreign body aspiration ( fba ) is more common in children than adults with about 80% occurring in children aged less than 15 years . </S>", "<S> fba in adults is often overlooked as a potential cause of airway obstruction especially if there is no asphyxiation . </S>", "<S> we present a case of a 45-year - old male with alcohol abuse who presented with post - obstructive pneumonia secondary to aspiration of tooth of unknown duration . </S>", "<S> the tooth was removed via flexible bronchoscopy ( fbr ) and we will discuss the use of fbr for foreign body ( fb ) removal , which fb can be easily removed by fbr , and the different techniques and devices used for fb removal via fbr . </S>" ]

[ "Case report", "Discussion", "Conclusions", "Conflict of interest and funding" ]

[ [ "forty - five - year - old male shelter resident , chronic alcoholic , chronic smoker , and marijuana abuser presented to emergency department ( ed ) with complaints of intermittent fever with night sweats and productive - cough for 10 days with yellowish sputum associated with pleuritic chest pain .", "he complained of generalized weakness and decreased appetite and claimed to have lost approximately 10 pounds in 2 weeks .", "laboratory results showed leukocytosis of 17,000/mm with neutrophil differential of 11,200/mm ( 65% ) with band neutrophils of 10% .", "chest radiograph was reported as right middle lobe consolidation but careful observation revealed a foreign body ( fb ) , likely a tooth in the right bronchus intermedius ( fig .", "ct scan of chest showed a well calcified fb likely a tooth at the level of carina obstructing right intermedius bronchus and consolidation in right lower lobe indicating post - obstructive pneumonia ( fig .", "2 ) . patient was admitted and prescribed intravenous antibiotics targeting gram - negative organisms and anaerobes .", "deep conscious sedation was achieved by propofol infusion in the presence of an anesthesiologist and cardiothoracic surgery backup .", "bronchoscopy showed tooth in right intermediate bronchus with ~75% occlusion , and purulent secretions from the right main stem ( figs .", "the fb was removed successfully with rat tooth forceps ( figs . 7 and 8) .", "post bronchoscopy inspection showed right main stem , bronchus intermedius , and right middle and lower lobe bronchi to be well patent .", "closer view of the foreign body , identifiable as tooth in right main stem bronchus .", "the aspirated tooth removed by rat tooth forceps . upon further questioning , the patient said that he did not remember having aspirated the tooth and hence the duration of aspiration could not be known .", "he was discharged home on oral antibiotics a couple of days after the bronchoscopy and counseled to quit alcohol and drugs ." ], [ "fba is more common in children than adults with about 80% occurring in children aged less than 15 years .", "it is the fourth most common cause of unintentional injury deaths in the united states . while choking is a hazard for all ages , choking deaths peaked at age 84 in 2011 as per national safety council injury facts 2015 ( 1 ) .", "fba in adults is often overlooked as a potential cause of airway obstruction especially if there is no asphyxiation .", "adults with risk factors such as alcoholism , drug abuse , mental retardation , and neuromuscular conditions are predisposed to aspiration .", "however , accidental aspiration in adults without the aforementioned risk factors has been described ( 25 ) . in adults ,", "most common foreign bodies aspirated are food and broken fragments of teeth ( 68 ) .", "ct scan is more sensitive and specific than chest radiograph in diagnosing radiolucent foreign bodies and for characterizing the attenuation of a suspected fb ( 9 ) .", "unlike in children , less than half of fbs are lodged in the proximal airways in adults .", "most of the fbs are lodged in the right bronchial tree , whereas in children no significant difference was seen between right and left bronchial tree ( 6 ) .", "bronchoscopy for the removal of fb was introduced by gustav killian , an otolaryngologist in 1897 .", "animal studies performed by zavala and rhodes ( 11 ) showed that fbr could be used to retrieve different kinds of fbs by using grasping forceps through the bronchoscope .", "fbr is considered as the diagnostic test of choice for initial diagnosis of fb in adults .", "the advantages of fbr over rigid bronchoscopy are that it can be performed under local anesthesia , visualization of smaller peripheral airway is better with relatively easier manipulation , and can be performed in patients with deformities of c - spine and pharynx .", "it is also a relatively easy and safer procedure in experienced - hands ( 10 , 12 ) .", "rigid bronchoscopy is recommended if fbr fails , if the fb is centrally located , if firmly embedded in scar tissue , and for removal of sharp objects which require maneuvering to minimize mucosal trauma ( 13 ) .", "there have been several case series which have reported successful removal of foreign bodies by fbr .", "cunanan ( 12 ) reported a success rate of 89% in using fbr for the removal of fb in 300 mentally retarded and physically handicapped patients .", "they reported a decrease in mortality from 12 to 1% with the use of fbr over rigid bronchoscope .", "they , however , attribute to the increased mortality from rigid bronchoscopy to complications of general anesthesia in patients with comorbidities .", "limper and prakash had a success rate of 60% with fbr , whereas rigid bronchoscopy showed a success rate of 98% . in their experience , fbr is advantageous in distally lodged fb , in cases with cervical instability , mechanically ventilated patients , and also in removal of small fbs , which can be securely grasped with a fiberoptic bronchoscope ( 14 ) .", "( 15 ) report a success rate of 97% for fb removal via fbr with low mortality and morbidity .", "they also reported that granulation tissue can be removed without much bleeding ; thus , removal of fb can be achieved in a repeat fbr a week later .", "( 13 ) indicate that fbr can be used to remove foreign bodies especially if they are small and peripheral and that fbr may be superior to rigid bronchoscopy for grasping tiny and far - reaching foreign bodies .", "rigid bronchoscopy is recommended if fbr fails and especially if the fb is large and located in central bronchi or trachea .", "however , in children who may have central , asphyxiating foreign bodies , the rigid bronchoscope under general anesthesia is preferentially indicated .", "team - based approach with designated roles for team members improves the chances of a successful procedure .", "anesthesia and ct surgery backup is essential . team - based analysis of the nature , location of the fb , and appropriate accessory instruments is equally necessary .", "steroids can be given to minimize inflammatory changes as they may lead to difficulty in fb extraction .", "orotracheal approach is preferred over nasotracheal , as it avoids fb from being lost in the nasal passages if accidently dislodged .", "care should be taken to keep fb in the center of visual field and to avoid pushing fb distally into the airways .", "once fb has been secured , bronchoscope , accessory instrument , and the secured fb are all withdrawn from the airway simultaneously .", "rare complications of fbr are likelihood of asphyxiation from losing the fb in the subglottic area and hemoptysis ( 10 ) .", "another approach is to reinsert flexible bronchoscope to push the fb into more peripheral airway thus resolving the central airway obstruction . in case of hemoptysis ,", "airway can be better managed by rigid bronchoscope , but this complication is very rare if performed by trained operators .", "incidence of massive hemoptysis even in patients with significant granulation tissue around the fb is extremely low ( 15 ) .", "organic materials can cause severe inflammation in a short period of time and tend to absorb water with development of airway obstruction relatively earlier .", "in contrast , inorganic fbs are inert , and so patients might be asymptomatic for a prolonged period ( 16 ) . grasping forceps , baskets , magnet extractors ,", "yag laser , and cryoprobes are available as accessories for fb removal via flexible bronchoscope ( 10 ) .", "rat tooth forceps is a type of a grasping forceps , which is particularly useful for removing hard , flat , or thin organic or inorganic foreign bodies .", "these are used primarily when the fb is hard as a firm grip can be achieved .", "these can not be used for the retrieval of organic friable objects which tend to fracture due to the firmness of the grip provided by these forceps ( 10 ) .", "zero - tip basket is used in urological procedures like retrieval of ureteral stones . the tipless design allows close approximation to fb .", "they are used to retrieve fbs lodged in distal bronchi and mobile fbs ( 16 , 17 ) .", "fish net basket , a modified polypectomy snare , is useful in removing bulky fbs ( 10 ) .", "magnet extractors contain flexible probe tipped with a magnet , useful for retrieval of metallic fbs ( 10 ) .", "fbs embedded in granulation tissue can be removed by yag laser , which vaporizes the surrounding granulation tissue .", "cryoprobe is used to freeze friable organic and small inorganic materials . freezing the foreign bodies" ], [ "high index of suspicion is needed especially for high - risk patients presenting with compatible symptoms .", "fbr is the initial diagnostic and therapeutic procedure recommended with low complication rates in experienced hands .", "rigid bronchoscopy is performed if the flexible bronchoscopy fails or if the fb is large and centrally located , in case of significant hemoptysis and if significant granulation tissue is present with a fb which is deeply embedded ." ], [ "" ] ]

[ "porcine reproductive and respiratory syndrome virus ( prrsv ) was first reported in north america in 1987 , and is one of the most economically important diseases in the swine industry .", "infection with prrsv is characterized by two clinical signs : reproductive failure in sows during the early - to - late stage of gestation and respiratory problems in piglets at any age .", "numerous studies have been conducted to determine the localization of the virus in experimental pigs . in boars , prrsv appears to persist in lymphoid tissues , particularly in tonsils , rather than the reproductive system .", "a study of stillborn and live birth piglets from intranasally inoculated sows indicated that prrsv replicates primarily in lymphoid tissues before the virus affects internal organs such as the lungs , heart , liver , spleen , and kidneys . in experimentally infected sows", ", prrsv was detected in the tonsils , heart , uterus , kidneys , and lymph nodes .", "however , little information is available on localization of prrsv in sows in the field .", "there are several situations in which oral fluid sampling may be advantageous for a breeding herd since this method can be performed more frequently at any time during gestation without jeopardizing the welfare of the animals .", "although it has been reported that oral fluid has the potential for replacing serum to detect prrsv in experimental pigs , the source of prrsv in oral fluid has not been determined . in breeding herds , gilt introduction is a very important factor for prrsv control .", "gilts are susceptible to prrsv infection and are a potential reservoir of the virus in the herd if they become viremic during the breeding via horizontal as well as vertical viral transmission .", "the goal of a prrsv acclimatization program is to expose gilts to the same strain of virus to which the herd is resistant .", "this allows sufficient time to permit full recovery of the gilts before their introduction into the breeding herd .", "information on natural prrsv infection in replacement gilts during the acclimatization period would be useful for establishing a suitable acclimatization program and prrsv elimination strategy .", "therefore , the goals of the present study were to clarify the localization of prrsv in infected swine and to evaluate the use of oral fluid specimens for prrsv detection in the field .", "pigs were collected from a 400-sow herd isolated from other pig farms in miyazaki , japan .", "reduction in the farrowing rate and increased incidences of abortion were observed among the sows .", "the herd was considered to be endemically infected with prrsv based on diagnostic history and on - going surveillance .", "two 3-year - old pregnant sows ( a and b ) that naturally aborted after 76 and 92 days of gestation along with seven 2-month - old grower pigs with respiratory problems were euthanized with mafropane ( ds pharma animal health , japan ) and subjected to necropsy . one hour prior to euthanasia , serum and oral fluid were collected .", "samples of lungs , brain , heart , liver , spleen , kidneys , ileum , and tissues from the oral cavity ( i.e. , tonsils , salivary glands , submandibular lymph nodes , and oral mucosa ) were taken from each animal .", "eight fetuses were randomly selected from the sow litters to survey for prrsv with nested reverse transcription pcr ( nrt - pcr ) . in order to assess prrsv infection during the acclimatization period ,", "70 6-month - old prrsv - free gilts were purchased from zen - noh livestock ( japan ) and housed in seven pens ( 10 gilts / pen ) in an isolated acclimation building at the farthest corner of the farm .", "disinfectant footbaths and changing coveralls ( kenis , japan ) were used prior to entering each pen . during the 3-week period after arrival , the technician worked in other pig buildings before going to the acclimation zone without changing coveralls in order to introduce gilts to the current prrsv strain circulating in the farm .", "the gilts were vaccinated with a prrsv modified live vaccine 2 ml dose ( ingelvac prrs mlv ; boehringer ingelheim vetmedica , usa ) three times 1 day after arrival , 4 weeks after the first injection , and 3 weeks before first round of insemination . in the breeding herd ,", "oral fluids were collected using a sterile cotton rope ( 100% cotton rope ; kenis ) to absorb the fluid in the oral cavity from pigs in each pen at 1 week intervals for 11 weeks .", "serum samples were collected from individual gilts by using serum separator tube ( venoject ii ; terumo medical corporation , usa ) on the first and eighth weeks of the acclimatization period .", "prrsv antibody in serum was identified by an enzyme - linked immunosorbent assay ( elisa ) kit ( anigen prrs ab elisa kit ; bionote , korean ) according to the manufacturer 's instructions .", "rna from the tissue samples was extracted with trizol ( trizol reagent ; invitrogen , usa ) while that in sera and oral fluid was extracted with viral rna extraction kit ( qiaamp viral rna mini kit ; qiagen , germany ) according to the manufacturer 's instructions .", "the first pcr primers were 13586f ( 5'-gtggtatttggcaatg tgtc-3')/14652r ( 5'-ctccaggtttctatggctga-3 ' ) , which amplified 1067 bp of the open reading frame ( orf ) 4 - 6 region of prrsv . the second primers , p420f ( 5'-ccattctgttggcaatttga-3')/p620r ( 5'-ggcatatatcatcactggcg-3 ' ) , which amplified 713 bp of the first rt - pcr product .", "the first rt - pcr was performed with accessquick rt - pcr system ( promega , usa ) and the second pcr was performed with gotaq green master mix ( promega ) .", "the first rt - pcr reaction was carried out for one cycle of reverse transcription at 45 for 45 min , inactivation at 94 in 2 min , and 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and final incubation at 72 for 5 min .", "the second pcr reaction was performed as first denaturation at 94 for 2 min , on 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and following the final incubation at 72 for 5 min .", "multiplex pcr and multiplex rt - pcr were also performed as previously described to screen for other pathogens including porcine circovirus type 2 ( pcv2 ) , suid herpesvirus 1 , porcine parvovirus , getah virus , and japanese encephalitis virus .", "the north american ( na)-type prrsv open reading frame ( orf ) 5 gene was sequenced with abi prism bigdye terminator v3.1 cycle sequencing kits ( applied biosystems , usa ) using primer p420f / p620r ( sigma - aldrich , usa ) .", "alignment was performed with isolates in this study , 3 previous isolates from the farm ( farm10/2011 , farm 11/2011 , and farm 12/2011 ) , and the inoculated vaccine strain .", "a phylogenetic tree was constructed using the neighbor - joining method and a bootstrap analysis was carried out with 1,000 replications .", "tissue samples from the sows and grower pigs were fixed in 4% paraformaldehyde for one day then embedded in paraffin by standard histologic procedures .", "two - micrometer - thick tissue sections were made for hematoxylin and eosin ( he ) , immunohistochemistry ( ihc ) , in situ hybridization ( ish ) , double ihc - ish , and double immunofluorescence stain .", "for ihc , antigens were retrieved by incubation with 20 g / ml proteinase k ( wako , japan ) at 37 in 10 min .", "endogenous peroxidase activity was suppressed by 3% hydrogen peroxide in methanol for 30 min at room temperature .", "non - specific binding was inhibited with a blocking reagent ( blocking one ; nacalai tesque , japan ) for 30 min at 37 in moist chamber .", "the tissue samples were then incubated at 4 overnight with a monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 in phosphate - buffered saline [ pbs ] ; rural technologies , usa ) .", "next , the sections were incubated with envision ( dakocytomation , japan ) as a secondary antibody for 30 min at 37. after each incubation period , the sections were washed three times with pbs .", "finally , the sections were stained using a peroxidase stain diaminobenzidine ( dab ) kit ( nacalai tesque ) . for ish ,", "antisense crna probes specific for prrsv rna were synthesized from the orf 7 gene sequence as previously described .", "digoxigenin ( dig)-labeled crna probes were prepared using a dig rna labeling kit ( roche diagnostics , germany ) .", "tissues positive for prrsv according to ihc and/or ish were further subjected to double staining to confirm whether macrophages were the target cells for prrsv . for double ihc - ish , the tissue sections were first processed for ihc staining using lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ; dako , denmark ) as a macrophage marker .", "sections were incubated with an universal immnuno - alkaline - phosphatase polymer , anti - mouse and -rabbit ( histofine simple stain ap ( multi ) ; nichirei , japan ) for 30 min at 37. immnunoreactivity was visualized by fast red ii ( nichirei ) .", "consequently , sections were process for ish using prrsv rna probe . finally , color was developed using the dab substrate ( liquid dab+substrate chromogen system ; dakocytomation ) and counterstained with hematoxylin .", "double immunofluorescence labeling was performed with lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ) and fluorescein isothiocyanate ( fitc)-conjugated polyclonal swine anti - rabbit igg ( dilute 1 : 40 in pbs ; dakocytomation ) .", "the sections were subsequently incubated with the monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 ) followed by alexa fluor 594 ( dilute 1 : 200 , molecular probes , usa ) at room temperature in dark moist chamber for 60 min each antibody .", "finally , the tissue sections were mounted using vectashield with 4',6-diamidino-2-phenylindole ( vector laboratories , usa ) .", "confocal microscope ( a1 confocal microscope ; nikon , japan ) was used to view and analyze the stained cells .", "pigs were collected from a 400-sow herd isolated from other pig farms in miyazaki , japan .", "reduction in the farrowing rate and increased incidences of abortion were observed among the sows .", "the herd was considered to be endemically infected with prrsv based on diagnostic history and on - going surveillance .", "two 3-year - old pregnant sows ( a and b ) that naturally aborted after 76 and 92 days of gestation along with seven 2-month - old grower pigs with respiratory problems were euthanized with mafropane ( ds pharma animal health , japan ) and subjected to necropsy . one hour prior to euthanasia , serum and oral fluid were collected .", "samples of lungs , brain , heart , liver , spleen , kidneys , ileum , and tissues from the oral cavity ( i.e. , tonsils , salivary glands , submandibular lymph nodes , and oral mucosa ) were taken from each animal .", "eight fetuses were randomly selected from the sow litters to survey for prrsv with nested reverse transcription pcr ( nrt - pcr ) . in order to assess prrsv infection during the acclimatization period ,", "70 6-month - old prrsv - free gilts were purchased from zen - noh livestock ( japan ) and housed in seven pens ( 10 gilts / pen ) in an isolated acclimation building at the farthest corner of the farm .", "disinfectant footbaths and changing coveralls ( kenis , japan ) were used prior to entering each pen . during the 3-week period after arrival , the technician worked in other pig buildings before going to the acclimation zone without changing coveralls in order to introduce gilts to the current prrsv strain circulating in the farm .", "the gilts were vaccinated with a prrsv modified live vaccine 2 ml dose ( ingelvac prrs mlv ; boehringer ingelheim vetmedica , usa ) three times 1 day after arrival , 4 weeks after the first injection , and 3 weeks before first round of insemination . in the breeding herd ,", "oral fluids were collected using a sterile cotton rope ( 100% cotton rope ; kenis ) to absorb the fluid in the oral cavity from pigs in each pen at 1 week intervals for 11 weeks .", "serum samples were collected from individual gilts by using serum separator tube ( venoject ii ; terumo medical corporation , usa ) on the first and eighth weeks of the acclimatization period .", "prrsv antibody in serum was identified by an enzyme - linked immunosorbent assay ( elisa ) kit ( anigen prrs ab elisa kit ; bionote , korean ) according to the manufacturer 's instructions .", "rna from the tissue samples was extracted with trizol ( trizol reagent ; invitrogen , usa ) while that in sera and oral fluid was extracted with viral rna extraction kit ( qiaamp viral rna mini kit ; qiagen , germany ) according to the manufacturer 's instructions .", "the first pcr primers were 13586f ( 5'-gtggtatttggcaatg tgtc-3')/14652r ( 5'-ctccaggtttctatggctga-3 ' ) , which amplified 1067 bp of the open reading frame ( orf ) 4 - 6 region of prrsv . the second primers , p420f ( 5'-ccattctgttggcaatttga-3')/p620r ( 5'-ggcatatatcatcactggcg-3 ' ) , which amplified 713 bp of the first rt - pcr product .", "the first rt - pcr was performed with accessquick rt - pcr system ( promega , usa ) and the second pcr was performed with gotaq green master mix ( promega ) .", "the first rt - pcr reaction was carried out for one cycle of reverse transcription at 45 for 45 min , inactivation at 94 in 2 min , and 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and final incubation at 72 for 5 min .", "the second pcr reaction was performed as first denaturation at 94 for 2 min , on 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and following the final incubation at 72 for 5 min .", "multiplex pcr and multiplex rt - pcr were also performed as previously described to screen for other pathogens including porcine circovirus type 2 ( pcv2 ) , suid herpesvirus 1 , porcine parvovirus , getah virus , and japanese encephalitis virus .", "the north american ( na)-type prrsv open reading frame ( orf ) 5 gene was sequenced with abi prism bigdye terminator v3.1 cycle sequencing kits ( applied biosystems , usa ) using primer p420f / p620r ( sigma - aldrich , usa ) .", "alignment was performed with isolates in this study , 3 previous isolates from the farm ( farm10/2011 , farm 11/2011 , and farm 12/2011 ) , and the inoculated vaccine strain .", "a phylogenetic tree was constructed using the neighbor - joining method and a bootstrap analysis was carried out with 1,000 replications .", "tissue samples from the sows and grower pigs were fixed in 4% paraformaldehyde for one day then embedded in paraffin by standard histologic procedures .", "two - micrometer - thick tissue sections were made for hematoxylin and eosin ( he ) , immunohistochemistry ( ihc ) , in situ hybridization ( ish ) , double ihc - ish , and double immunofluorescence stain .", "for ihc , antigens were retrieved by incubation with 20 g / ml proteinase k ( wako , japan ) at 37 in 10 min .", "endogenous peroxidase activity was suppressed by 3% hydrogen peroxide in methanol for 30 min at room temperature .", "non - specific binding was inhibited with a blocking reagent ( blocking one ; nacalai tesque , japan ) for 30 min at 37 in moist chamber .", "the tissue samples were then incubated at 4 overnight with a monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 in phosphate - buffered saline [ pbs ] ; rural technologies , usa ) .", "next , the sections were incubated with envision ( dakocytomation , japan ) as a secondary antibody for 30 min at 37. after each incubation period , the sections were washed three times with pbs .", "finally , the sections were stained using a peroxidase stain diaminobenzidine ( dab ) kit ( nacalai tesque ) . for ish ,", "antisense crna probes specific for prrsv rna were synthesized from the orf 7 gene sequence as previously described .", "digoxigenin ( dig)-labeled crna probes were prepared using a dig rna labeling kit ( roche diagnostics , germany ) .", "tissues positive for prrsv according to ihc and/or ish were further subjected to double staining to confirm whether macrophages were the target cells for prrsv . for double ihc - ish , the tissue sections were first processed for ihc staining using lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ; dako , denmark ) as a macrophage marker .", "sections were incubated with an universal immnuno - alkaline - phosphatase polymer , anti - mouse and -rabbit ( histofine simple stain ap ( multi ) ; nichirei , japan ) for 30 min at 37. immnunoreactivity was visualized by fast red ii ( nichirei ) .", "consequently , sections were process for ish using prrsv rna probe . finally , color was developed using the dab substrate ( liquid dab+substrate chromogen system ; dakocytomation ) and counterstained with hematoxylin .", "double immunofluorescence labeling was performed with lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ) and fluorescein isothiocyanate ( fitc)-conjugated polyclonal swine anti - rabbit igg ( dilute 1 : 40 in pbs ; dakocytomation ) .", "the sections were subsequently incubated with the monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 ) followed by alexa fluor 594 ( dilute 1 : 200 , molecular probes , usa ) at room temperature in dark moist chamber for 60 min each antibody .", "finally , the tissue sections were mounted using vectashield with 4',6-diamidino-2-phenylindole ( vector laboratories , usa ) .", "confocal microscope ( a1 confocal microscope ; nikon , japan ) was used to view and analyze the stained cells .", "prrsv rna was identified in the oral fluid and tonsils of both sows but not in the sera , salivary glands , uterus , or other organs ( table 1 ) .", "viral rna was detected in lungs and placentas of three out of eight fetuses from these sows .", "histologically , tonsils from the sows were characterized by mild infiltration of macrophages , neutrophils , lymphocytes , and cryptal epithelial cells in the dilated tonsillar crypts .", "with ish , prrsv rna was detected in the cytoplasm of large oval cells that possessed morphological features similar to those of macrophages or dendritic cells near tonsillar crypts .", "prrsv antigens , however , were not identified by ihc in formalin - fixed tissues from the sows . in all seven grower pigs , lesions", "were mainly observed in cranioventral lung lobes that were characterized by consolidation , discoloration , and failure to collapse when the thorax was opened .", "microscopically , all seven animals were found to have interstitial pneumonia based on evaluation of the lung samples .", "alveolar walls were thickened due to the infiltration of macrophages , lymphocytes , and neutrophils .", "tonsillitis in the grower pigs was characterized by erosion of the crypt epithelia and dilated crypt lumina filled with macrophages , neutrophils , lymphocytes , and cryptal epithelial cells ( a and b in fig .", "1 ) . a few prrsv - positive cells were found in the tonsillar cryptal lumen by ish ( c and d in fig .", "prrsv was consistently detected in the tonsils , oral fluid , salivary glands , and lungs of all seven grower pigs along with the oral mucosae and submandibular lymph nodes of six grower pigs .", "in contrast , only two serum samples were positive for the virus as detected by nrt - pcr .", "viral antigen and nucleic acids were detected in the lungs ( grower pigs 1 and 6 ) and tonsils ( grower pigs 1 and 3 ) by both ihc and ish ( table 1 ) .", "none of the samples from the brain , heart , liver , spleen , kidney , or ileum obtained from these grower pigs was positive for prrsv .", "no other viruses , including pcv2 , suid herpesvirus 1 , porcine parvovirus , getah virus , and japanese encephalitis virus , were detected in any samples from the sows or grower pigs .", "sections of tonsils from the sows and grower pigs were double stained to identify target cells of prrsv . in the double labeling ihc - ish experiment , macrophages and prrsv rna were stained red or brown , respectively .", "double ihc - ish findings revealed that prrsv rna was distributed in the cytoplasm of macrophages within and adjacent to the crypt epithelium ( fig .", "double immunofluorescence produced positive signals for prrsv antigen and lysozymes that appeared as red or green , respectively .", "prrsv antigen that colocalized with lysozymes was visualized as red and green or orange signals in the cytoplasm of macrophages near the tonsillar crypts ( fig .", "the course of prrsv infection in replacement gilts during acclimatization is shown in table 2 . at the first week following introduction , oral fluid was collected from animals in three out of seven pens .", "after the first collection , the pigs became more familiar with the rope and oral fluid samples from all animals could be collected .", "prrsv rna was detected in the serum of one gilt 1 week after the live vaccine was injected .", "three weeks post - introduction , the gilts were anorexic and prrsv was detected in oral fluid from the animals in three pens . by week 4 , gilts in all seven pens were infected by the virus .", "the periods during which the virus could be detected in oral fluid differed from pen to pen and ranged from 3 to 6 weeks .", "virus was detected in oral fluid from the pigs in five pens until week 8 , but was not found in any serum samples taken from individual gilts .", "prrsv was not identified in oral fluid samples obtained between week 9 and week 11 . at week 1 , all gilts were seronegative with sample : positive ( s : p ) ratios 0.4 . by week 8 , the anti - prrsv antibody levels had increased with mean s : p ratios 2.6 indicating that all gilts had seroconverted .", "prrsv isolates from tonsils of the sows and grower pigs or oral fluids from the gilts were sequenced .", "percent identity of orf 5 nucleotide sequences among virus from the sows , grower pigs , gilts , and previous isolates from the farm ( farm10/2011 , farm 11/2011 , and farm 12/2011 ) ranged from 95% to 100% .", "in contrast , all sequence from this farm shared an 87% to 89% nucleotide identity with the inoculated vaccine strain .", "thus , it was concluded that the replacement gilts were infected with the same virus strain that exist in the farm ( fig .", "in order to determine the localization of prrsv , sera and tissue samples from two sows that had naturally aborted and seven grower pigs were examined . in sows , viral nucleic acids were detected in the absence of gross and microscopic lesions with the exception of mild tonsillitis .", "similar to previous studies , abortion associated with prrsv infection was not associated with the appearance of lesions .", "an acceptable explanation for these observations may be that the present study was conducted on naturally infected sows whereas previous investigations examined experimentally infected sows .", "one possible reason is that the sows were vaccinated four times per year whereas grower pigs were not vaccinated .", "therefore , immune responses to prrsv in the sows might have been stronger than that in grower pigs .", "previous studies demonstrated that prrsv primarily replicates in tonsils and then spreads to other organs .", "therefore , tonsils may also be an important site for prrsv replication in naturally infected sows and grower pigs .", "traditionally , serum has been the specimen of choice for detecting various pathogens including prrsv .", "oral fluids are increasingly being used for analysis in the swine industry since they are easily collected from individual pigs and are suitable for pen - based sampling . in the current study ,", "prrsv was examined in oral fluid and serum from prrsv - free gilts during acclimatization .", "one week following inoculation , the virus was detected in the serum of one gilt .", "unfortunately , sequencing could not be performed for this sample to confirm whether the gilt was infected by the vaccine strain or field strain due to a low rna concentration . because a modified live prrsv vaccine was used", "one week post - vaccination , the gilt may have been in the acute phase of infection . in agreement with findings from a previous study ,", "the virus was detected in the serum of gilts 3 days post - vaccination . during acute infection", ", serum was found to be superior to oral fluid for early detection of prrsv . however , prrsv was identified week 8 post - arrival in five out of seven oral fluid samples from the gilts but none of the serum samples from all 70 swine .", "prrsv was detected in all oral fluid samples from the grower pigs but in only two out of seven serum samples .", "for the sows , prrsv rna was detectable in both oral fluid specimens but was not found in serum", ". therefore , analysis of oral fluid specimens could be used as an alternative to serum sampling to identify prrsv in cases of persistent infection . early in the acclimatization period", ", gilts can be exposed to viruses through vaccination or live virus injection as well as exposure to viremic nursery pigs .", "live virus injection is associated with potential risks such as the spread of other pathogens and increased mortality . in the current study , gilts were introduced to a farm where prrsv was endemic and a modified live vaccine was used to exposure the gilts to the virus .", "however , the gilts were infected by the prrsv field strain 3 weeks post - introduction .", "thus , the gilts may have been infected before the vaccine - induced immune response to the virus was initiated . moreover , nucleotide sequencing data indicated that the gilts were infected by the field strain , which shared only a 87% to 89% nucleotide identity with the vaccine strain .", "a previous investigation demonstrated that the degree of protection conferred by vaccination depends on the degree of similarity between the prrsv vaccine strain and field strain to which the pigs are exposed .", "therefore , the current vaccine that contains a single strain of prrsv might not be effective for protecting the animals against infection with genetically different strains of prrsv .", "the source of infection in this study may have involved indirect routes of transmission such as ones associated with contaminated fomites .", "indirect transmission might be a useful method for exposing replacement gilts to the current prrsv circulating in the farm and inducing an immune response before entering the prrsv - positive breeding herd .", "this technique may also reduce the risk of transmission of other pathogens unlike exposure to viremic pigs or live virus injection .", "further studies are necessary to confirm the effect of indirect transmission on replacement gilt immunization . in conclusion ," ]

[ "<S> the objectives of the present study were to evaluate the anatomic localization of porcine reproductive and respiratory syndrome virus ( prrsv ) in naturally infected pigs and to determine whether oral fluid could be used to detect the virus in infected animals . </S>", "<S> two sows , seven 2-month - old grower pigs , and 70 6-month - old gilts were included in this study . </S>", "<S> prrsv in sera and oral fluid were identified by nested reverse transcription pcr ( nrt - pcr ) while lung , tonsil , and tissue associated with oral cavity were subjected to nrt - pcr , immunohistochemistry , and in situ hybridization . in sows </S>", "<S> , prrsv was identified in oral fluid and tonsils . </S>", "<S> prrsv was also detected in oral fluid , tonsils , salivary glands , oral mucosa , and lungs of all seven grower pigs . </S>", "<S> however , viremia was observed in only two grower pigs . </S>", "<S> double staining revealed that prrsv was distributed in macrophages within and adjacent to the tonsillar crypt epithelium . in gilts , the north american type prrsv field strain was detected 3 to 8 weeks after introducing these animals onto the farm . </S>", "<S> these results confirm previous findings that prrsv primarily replicates in tonsils and is then shed into oral fluid . </S>", "<S> therefore , oral fluid sampling may be effective for the surveillance of prrsv in breeding herds . </S>" ]

[ "Introduction", "Materials and Methods", "Animals", "nRT-PCR", "PRRSV ORF 5 sequencing", "Histopathological examinations", "Results", "Discussion" ]

[ [ "porcine reproductive and respiratory syndrome virus ( prrsv ) was first reported in north america in 1987 , and is one of the most economically important diseases in the swine industry .", "infection with prrsv is characterized by two clinical signs : reproductive failure in sows during the early - to - late stage of gestation and respiratory problems in piglets at any age .", "numerous studies have been conducted to determine the localization of the virus in experimental pigs . in boars , prrsv appears to persist in lymphoid tissues , particularly in tonsils , rather than the reproductive system .", "a study of stillborn and live birth piglets from intranasally inoculated sows indicated that prrsv replicates primarily in lymphoid tissues before the virus affects internal organs such as the lungs , heart , liver , spleen , and kidneys . in experimentally infected sows", ", prrsv was detected in the tonsils , heart , uterus , kidneys , and lymph nodes .", "however , little information is available on localization of prrsv in sows in the field .", "there are several situations in which oral fluid sampling may be advantageous for a breeding herd since this method can be performed more frequently at any time during gestation without jeopardizing the welfare of the animals .", "although it has been reported that oral fluid has the potential for replacing serum to detect prrsv in experimental pigs , the source of prrsv in oral fluid has not been determined . in breeding herds , gilt introduction is a very important factor for prrsv control .", "gilts are susceptible to prrsv infection and are a potential reservoir of the virus in the herd if they become viremic during the breeding via horizontal as well as vertical viral transmission .", "the goal of a prrsv acclimatization program is to expose gilts to the same strain of virus to which the herd is resistant .", "this allows sufficient time to permit full recovery of the gilts before their introduction into the breeding herd .", "information on natural prrsv infection in replacement gilts during the acclimatization period would be useful for establishing a suitable acclimatization program and prrsv elimination strategy .", "therefore , the goals of the present study were to clarify the localization of prrsv in infected swine and to evaluate the use of oral fluid specimens for prrsv detection in the field ." ], [ "pigs were collected from a 400-sow herd isolated from other pig farms in miyazaki , japan .", "reduction in the farrowing rate and increased incidences of abortion were observed among the sows .", "the herd was considered to be endemically infected with prrsv based on diagnostic history and on - going surveillance .", "two 3-year - old pregnant sows ( a and b ) that naturally aborted after 76 and 92 days of gestation along with seven 2-month - old grower pigs with respiratory problems were euthanized with mafropane ( ds pharma animal health , japan ) and subjected to necropsy . one hour prior to euthanasia , serum and oral fluid were collected .", "samples of lungs , brain , heart , liver , spleen , kidneys , ileum , and tissues from the oral cavity ( i.e. , tonsils , salivary glands , submandibular lymph nodes , and oral mucosa ) were taken from each animal .", "eight fetuses were randomly selected from the sow litters to survey for prrsv with nested reverse transcription pcr ( nrt - pcr ) . in order to assess prrsv infection during the acclimatization period ,", "70 6-month - old prrsv - free gilts were purchased from zen - noh livestock ( japan ) and housed in seven pens ( 10 gilts / pen ) in an isolated acclimation building at the farthest corner of the farm .", "disinfectant footbaths and changing coveralls ( kenis , japan ) were used prior to entering each pen . during the 3-week period after arrival , the technician worked in other pig buildings before going to the acclimation zone without changing coveralls in order to introduce gilts to the current prrsv strain circulating in the farm .", "the gilts were vaccinated with a prrsv modified live vaccine 2 ml dose ( ingelvac prrs mlv ; boehringer ingelheim vetmedica , usa ) three times 1 day after arrival , 4 weeks after the first injection , and 3 weeks before first round of insemination . in the breeding herd ,", "oral fluids were collected using a sterile cotton rope ( 100% cotton rope ; kenis ) to absorb the fluid in the oral cavity from pigs in each pen at 1 week intervals for 11 weeks .", "serum samples were collected from individual gilts by using serum separator tube ( venoject ii ; terumo medical corporation , usa ) on the first and eighth weeks of the acclimatization period .", "prrsv antibody in serum was identified by an enzyme - linked immunosorbent assay ( elisa ) kit ( anigen prrs ab elisa kit ; bionote , korean ) according to the manufacturer 's instructions .", "rna from the tissue samples was extracted with trizol ( trizol reagent ; invitrogen , usa ) while that in sera and oral fluid was extracted with viral rna extraction kit ( qiaamp viral rna mini kit ; qiagen , germany ) according to the manufacturer 's instructions .", "the first pcr primers were 13586f ( 5'-gtggtatttggcaatg tgtc-3')/14652r ( 5'-ctccaggtttctatggctga-3 ' ) , which amplified 1067 bp of the open reading frame ( orf ) 4 - 6 region of prrsv . the second primers , p420f ( 5'-ccattctgttggcaatttga-3')/p620r ( 5'-ggcatatatcatcactggcg-3 ' ) , which amplified 713 bp of the first rt - pcr product .", "the first rt - pcr was performed with accessquick rt - pcr system ( promega , usa ) and the second pcr was performed with gotaq green master mix ( promega ) .", "the first rt - pcr reaction was carried out for one cycle of reverse transcription at 45 for 45 min , inactivation at 94 in 2 min , and 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and final incubation at 72 for 5 min .", "the second pcr reaction was performed as first denaturation at 94 for 2 min , on 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and following the final incubation at 72 for 5 min .", "multiplex pcr and multiplex rt - pcr were also performed as previously described to screen for other pathogens including porcine circovirus type 2 ( pcv2 ) , suid herpesvirus 1 , porcine parvovirus , getah virus , and japanese encephalitis virus .", "the north american ( na)-type prrsv open reading frame ( orf ) 5 gene was sequenced with abi prism bigdye terminator v3.1 cycle sequencing kits ( applied biosystems , usa ) using primer p420f / p620r ( sigma - aldrich , usa ) .", "alignment was performed with isolates in this study , 3 previous isolates from the farm ( farm10/2011 , farm 11/2011 , and farm 12/2011 ) , and the inoculated vaccine strain .", "a phylogenetic tree was constructed using the neighbor - joining method and a bootstrap analysis was carried out with 1,000 replications .", "tissue samples from the sows and grower pigs were fixed in 4% paraformaldehyde for one day then embedded in paraffin by standard histologic procedures .", "two - micrometer - thick tissue sections were made for hematoxylin and eosin ( he ) , immunohistochemistry ( ihc ) , in situ hybridization ( ish ) , double ihc - ish , and double immunofluorescence stain .", "for ihc , antigens were retrieved by incubation with 20 g / ml proteinase k ( wako , japan ) at 37 in 10 min .", "endogenous peroxidase activity was suppressed by 3% hydrogen peroxide in methanol for 30 min at room temperature .", "non - specific binding was inhibited with a blocking reagent ( blocking one ; nacalai tesque , japan ) for 30 min at 37 in moist chamber .", "the tissue samples were then incubated at 4 overnight with a monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 in phosphate - buffered saline [ pbs ] ; rural technologies , usa ) .", "next , the sections were incubated with envision ( dakocytomation , japan ) as a secondary antibody for 30 min at 37. after each incubation period , the sections were washed three times with pbs .", "finally , the sections were stained using a peroxidase stain diaminobenzidine ( dab ) kit ( nacalai tesque ) . for ish ,", "antisense crna probes specific for prrsv rna were synthesized from the orf 7 gene sequence as previously described .", "digoxigenin ( dig)-labeled crna probes were prepared using a dig rna labeling kit ( roche diagnostics , germany ) .", "tissues positive for prrsv according to ihc and/or ish were further subjected to double staining to confirm whether macrophages were the target cells for prrsv . for double ihc - ish , the tissue sections were first processed for ihc staining using lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ; dako , denmark ) as a macrophage marker .", "sections were incubated with an universal immnuno - alkaline - phosphatase polymer , anti - mouse and -rabbit ( histofine simple stain ap ( multi ) ; nichirei , japan ) for 30 min at 37. immnunoreactivity was visualized by fast red ii ( nichirei ) .", "consequently , sections were process for ish using prrsv rna probe . finally , color was developed using the dab substrate ( liquid dab+substrate chromogen system ; dakocytomation ) and counterstained with hematoxylin .", "double immunofluorescence labeling was performed with lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ) and fluorescein isothiocyanate ( fitc)-conjugated polyclonal swine anti - rabbit igg ( dilute 1 : 40 in pbs ; dakocytomation ) .", "the sections were subsequently incubated with the monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 ) followed by alexa fluor 594 ( dilute 1 : 200 , molecular probes , usa ) at room temperature in dark moist chamber for 60 min each antibody .", "finally , the tissue sections were mounted using vectashield with 4',6-diamidino-2-phenylindole ( vector laboratories , usa ) .", "confocal microscope ( a1 confocal microscope ; nikon , japan ) was used to view and analyze the stained cells ." ], [ "pigs were collected from a 400-sow herd isolated from other pig farms in miyazaki , japan .", "reduction in the farrowing rate and increased incidences of abortion were observed among the sows .", "the herd was considered to be endemically infected with prrsv based on diagnostic history and on - going surveillance .", "two 3-year - old pregnant sows ( a and b ) that naturally aborted after 76 and 92 days of gestation along with seven 2-month - old grower pigs with respiratory problems were euthanized with mafropane ( ds pharma animal health , japan ) and subjected to necropsy . one hour prior to euthanasia , serum and oral fluid were collected .", "samples of lungs , brain , heart , liver , spleen , kidneys , ileum , and tissues from the oral cavity ( i.e. , tonsils , salivary glands , submandibular lymph nodes , and oral mucosa ) were taken from each animal .", "eight fetuses were randomly selected from the sow litters to survey for prrsv with nested reverse transcription pcr ( nrt - pcr ) . in order to assess prrsv infection during the acclimatization period ,", "70 6-month - old prrsv - free gilts were purchased from zen - noh livestock ( japan ) and housed in seven pens ( 10 gilts / pen ) in an isolated acclimation building at the farthest corner of the farm .", "disinfectant footbaths and changing coveralls ( kenis , japan ) were used prior to entering each pen . during the 3-week period after arrival , the technician worked in other pig buildings before going to the acclimation zone without changing coveralls in order to introduce gilts to the current prrsv strain circulating in the farm .", "the gilts were vaccinated with a prrsv modified live vaccine 2 ml dose ( ingelvac prrs mlv ; boehringer ingelheim vetmedica , usa ) three times 1 day after arrival , 4 weeks after the first injection , and 3 weeks before first round of insemination . in the breeding herd ,", "oral fluids were collected using a sterile cotton rope ( 100% cotton rope ; kenis ) to absorb the fluid in the oral cavity from pigs in each pen at 1 week intervals for 11 weeks .", "serum samples were collected from individual gilts by using serum separator tube ( venoject ii ; terumo medical corporation , usa ) on the first and eighth weeks of the acclimatization period .", "prrsv antibody in serum was identified by an enzyme - linked immunosorbent assay ( elisa ) kit ( anigen prrs ab elisa kit ; bionote , korean ) according to the manufacturer 's instructions ." ], [ "rna from the tissue samples was extracted with trizol ( trizol reagent ; invitrogen , usa ) while that in sera and oral fluid was extracted with viral rna extraction kit ( qiaamp viral rna mini kit ; qiagen , germany ) according to the manufacturer 's instructions .", "the first pcr primers were 13586f ( 5'-gtggtatttggcaatg tgtc-3')/14652r ( 5'-ctccaggtttctatggctga-3 ' ) , which amplified 1067 bp of the open reading frame ( orf ) 4 - 6 region of prrsv . the second primers , p420f ( 5'-ccattctgttggcaatttga-3')/p620r ( 5'-ggcatatatcatcactggcg-3 ' ) , which amplified 713 bp of the first rt - pcr product .", "the first rt - pcr was performed with accessquick rt - pcr system ( promega , usa ) and the second pcr was performed with gotaq green master mix ( promega ) .", "the first rt - pcr reaction was carried out for one cycle of reverse transcription at 45 for 45 min , inactivation at 94 in 2 min , and 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and final incubation at 72 for 5 min .", "the second pcr reaction was performed as first denaturation at 94 for 2 min , on 40 cycles of denaturation at 94 for 15 sec , annealing at 55 for 30 sec , extension at 68 for 1 min , and following the final incubation at 72 for 5 min .", "multiplex pcr and multiplex rt - pcr were also performed as previously described to screen for other pathogens including porcine circovirus type 2 ( pcv2 ) , suid herpesvirus 1 , porcine parvovirus , getah virus , and japanese encephalitis virus ." ], [ "the north american ( na)-type prrsv open reading frame ( orf ) 5 gene was sequenced with abi prism bigdye terminator v3.1 cycle sequencing kits ( applied biosystems , usa ) using primer p420f / p620r ( sigma - aldrich , usa ) .", "alignment was performed with isolates in this study , 3 previous isolates from the farm ( farm10/2011 , farm 11/2011 , and farm 12/2011 ) , and the inoculated vaccine strain .", "a phylogenetic tree was constructed using the neighbor - joining method and a bootstrap analysis was carried out with 1,000 replications ." ], [ "tissue samples from the sows and grower pigs were fixed in 4% paraformaldehyde for one day then embedded in paraffin by standard histologic procedures .", "two - micrometer - thick tissue sections were made for hematoxylin and eosin ( he ) , immunohistochemistry ( ihc ) , in situ hybridization ( ish ) , double ihc - ish , and double immunofluorescence stain .", "for ihc , antigens were retrieved by incubation with 20 g / ml proteinase k ( wako , japan ) at 37 in 10 min .", "endogenous peroxidase activity was suppressed by 3% hydrogen peroxide in methanol for 30 min at room temperature .", "non - specific binding was inhibited with a blocking reagent ( blocking one ; nacalai tesque , japan ) for 30 min at 37 in moist chamber .", "the tissue samples were then incubated at 4 overnight with a monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 in phosphate - buffered saline [ pbs ] ; rural technologies , usa ) .", "next , the sections were incubated with envision ( dakocytomation , japan ) as a secondary antibody for 30 min at 37. after each incubation period , the sections were washed three times with pbs .", "finally , the sections were stained using a peroxidase stain diaminobenzidine ( dab ) kit ( nacalai tesque ) . for ish ,", "antisense crna probes specific for prrsv rna were synthesized from the orf 7 gene sequence as previously described .", "digoxigenin ( dig)-labeled crna probes were prepared using a dig rna labeling kit ( roche diagnostics , germany ) .", "tissues positive for prrsv according to ihc and/or ish were further subjected to double staining to confirm whether macrophages were the target cells for prrsv . for double ihc - ish , the tissue sections were first processed for ihc staining using lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ; dako , denmark ) as a macrophage marker .", "sections were incubated with an universal immnuno - alkaline - phosphatase polymer , anti - mouse and -rabbit ( histofine simple stain ap ( multi ) ; nichirei , japan ) for 30 min at 37. immnunoreactivity was visualized by fast red ii ( nichirei ) .", "consequently , sections were process for ish using prrsv rna probe . finally , color was developed using the dab substrate ( liquid dab+substrate chromogen system ; dakocytomation ) and counterstained with hematoxylin .", "double immunofluorescence labeling was performed with lysozyme ec 3.2.1.17 ( dilute 1 : 1,000 in pbs ) and fluorescein isothiocyanate ( fitc)-conjugated polyclonal swine anti - rabbit igg ( dilute 1 : 40 in pbs ; dakocytomation ) .", "the sections were subsequently incubated with the monoclonal anti - prrsv antibody sr30 ( dilute 1 : 10,000 ) followed by alexa fluor 594 ( dilute 1 : 200 , molecular probes , usa ) at room temperature in dark moist chamber for 60 min each antibody .", "finally , the tissue sections were mounted using vectashield with 4',6-diamidino-2-phenylindole ( vector laboratories , usa ) .", "confocal microscope ( a1 confocal microscope ; nikon , japan ) was used to view and analyze the stained cells ." ], [ "prrsv rna was identified in the oral fluid and tonsils of both sows but not in the sera , salivary glands , uterus , or other organs ( table 1 ) .", "viral rna was detected in lungs and placentas of three out of eight fetuses from these sows .", "histologically , tonsils from the sows were characterized by mild infiltration of macrophages , neutrophils , lymphocytes , and cryptal epithelial cells in the dilated tonsillar crypts .", "with ish , prrsv rna was detected in the cytoplasm of large oval cells that possessed morphological features similar to those of macrophages or dendritic cells near tonsillar crypts .", "prrsv antigens , however , were not identified by ihc in formalin - fixed tissues from the sows . in all seven grower pigs , lesions", "were mainly observed in cranioventral lung lobes that were characterized by consolidation , discoloration , and failure to collapse when the thorax was opened .", "microscopically , all seven animals were found to have interstitial pneumonia based on evaluation of the lung samples .", "alveolar walls were thickened due to the infiltration of macrophages , lymphocytes , and neutrophils .", "tonsillitis in the grower pigs was characterized by erosion of the crypt epithelia and dilated crypt lumina filled with macrophages , neutrophils , lymphocytes , and cryptal epithelial cells ( a and b in fig .", "1 ) . a few prrsv - positive cells were found in the tonsillar cryptal lumen by ish ( c and d in fig .", "prrsv was consistently detected in the tonsils , oral fluid , salivary glands , and lungs of all seven grower pigs along with the oral mucosae and submandibular lymph nodes of six grower pigs .", "in contrast , only two serum samples were positive for the virus as detected by nrt - pcr .", "viral antigen and nucleic acids were detected in the lungs ( grower pigs 1 and 6 ) and tonsils ( grower pigs 1 and 3 ) by both ihc and ish ( table 1 ) .", "none of the samples from the brain , heart , liver , spleen , kidney , or ileum obtained from these grower pigs was positive for prrsv .", "no other viruses , including pcv2 , suid herpesvirus 1 , porcine parvovirus , getah virus , and japanese encephalitis virus , were detected in any samples from the sows or grower pigs .", "sections of tonsils from the sows and grower pigs were double stained to identify target cells of prrsv . in the double labeling ihc - ish experiment , macrophages and prrsv rna were stained red or brown , respectively .", "double ihc - ish findings revealed that prrsv rna was distributed in the cytoplasm of macrophages within and adjacent to the crypt epithelium ( fig .", "double immunofluorescence produced positive signals for prrsv antigen and lysozymes that appeared as red or green , respectively .", "prrsv antigen that colocalized with lysozymes was visualized as red and green or orange signals in the cytoplasm of macrophages near the tonsillar crypts ( fig .", "the course of prrsv infection in replacement gilts during acclimatization is shown in table 2 . at the first week following introduction , oral fluid was collected from animals in three out of seven pens .", "after the first collection , the pigs became more familiar with the rope and oral fluid samples from all animals could be collected .", "prrsv rna was detected in the serum of one gilt 1 week after the live vaccine was injected .", "three weeks post - introduction , the gilts were anorexic and prrsv was detected in oral fluid from the animals in three pens . by week 4 , gilts in all seven pens were infected by the virus .", "the periods during which the virus could be detected in oral fluid differed from pen to pen and ranged from 3 to 6 weeks .", "virus was detected in oral fluid from the pigs in five pens until week 8 , but was not found in any serum samples taken from individual gilts .", "prrsv was not identified in oral fluid samples obtained between week 9 and week 11 . at week 1 , all gilts were seronegative with sample : positive ( s : p ) ratios 0.4 . by week 8 , the anti - prrsv antibody levels had increased with mean s : p ratios 2.6 indicating that all gilts had seroconverted .", "prrsv isolates from tonsils of the sows and grower pigs or oral fluids from the gilts were sequenced .", "percent identity of orf 5 nucleotide sequences among virus from the sows , grower pigs , gilts , and previous isolates from the farm ( farm10/2011 , farm 11/2011 , and farm 12/2011 ) ranged from 95% to 100% .", "in contrast , all sequence from this farm shared an 87% to 89% nucleotide identity with the inoculated vaccine strain .", "thus , it was concluded that the replacement gilts were infected with the same virus strain that exist in the farm ( fig ." ], [ "in order to determine the localization of prrsv , sera and tissue samples from two sows that had naturally aborted and seven grower pigs were examined . in sows , viral nucleic acids were detected in the absence of gross and microscopic lesions with the exception of mild tonsillitis .", "similar to previous studies , abortion associated with prrsv infection was not associated with the appearance of lesions .", "an acceptable explanation for these observations may be that the present study was conducted on naturally infected sows whereas previous investigations examined experimentally infected sows .", "one possible reason is that the sows were vaccinated four times per year whereas grower pigs were not vaccinated .", "therefore , immune responses to prrsv in the sows might have been stronger than that in grower pigs .", "previous studies demonstrated that prrsv primarily replicates in tonsils and then spreads to other organs .", "therefore , tonsils may also be an important site for prrsv replication in naturally infected sows and grower pigs .", "traditionally , serum has been the specimen of choice for detecting various pathogens including prrsv .", "oral fluids are increasingly being used for analysis in the swine industry since they are easily collected from individual pigs and are suitable for pen - based sampling . in the current study ,", "prrsv was examined in oral fluid and serum from prrsv - free gilts during acclimatization .", "one week following inoculation , the virus was detected in the serum of one gilt .", "unfortunately , sequencing could not be performed for this sample to confirm whether the gilt was infected by the vaccine strain or field strain due to a low rna concentration . because a modified live prrsv vaccine was used", "one week post - vaccination , the gilt may have been in the acute phase of infection . in agreement with findings from a previous study ,", "the virus was detected in the serum of gilts 3 days post - vaccination . during acute infection", ", serum was found to be superior to oral fluid for early detection of prrsv . however , prrsv was identified week 8 post - arrival in five out of seven oral fluid samples from the gilts but none of the serum samples from all 70 swine .", "prrsv was detected in all oral fluid samples from the grower pigs but in only two out of seven serum samples .", "for the sows , prrsv rna was detectable in both oral fluid specimens but was not found in serum", ". therefore , analysis of oral fluid specimens could be used as an alternative to serum sampling to identify prrsv in cases of persistent infection . early in the acclimatization period", ", gilts can be exposed to viruses through vaccination or live virus injection as well as exposure to viremic nursery pigs .", "live virus injection is associated with potential risks such as the spread of other pathogens and increased mortality . in the current study , gilts were introduced to a farm where prrsv was endemic and a modified live vaccine was used to exposure the gilts to the virus .", "however , the gilts were infected by the prrsv field strain 3 weeks post - introduction .", "thus , the gilts may have been infected before the vaccine - induced immune response to the virus was initiated . moreover , nucleotide sequencing data indicated that the gilts were infected by the field strain , which shared only a 87% to 89% nucleotide identity with the vaccine strain .", "a previous investigation demonstrated that the degree of protection conferred by vaccination depends on the degree of similarity between the prrsv vaccine strain and field strain to which the pigs are exposed .", "therefore , the current vaccine that contains a single strain of prrsv might not be effective for protecting the animals against infection with genetically different strains of prrsv .", "the source of infection in this study may have involved indirect routes of transmission such as ones associated with contaminated fomites .", "indirect transmission might be a useful method for exposing replacement gilts to the current prrsv circulating in the farm and inducing an immune response before entering the prrsv - positive breeding herd .", "this technique may also reduce the risk of transmission of other pathogens unlike exposure to viremic pigs or live virus injection .", "further studies are necessary to confirm the effect of indirect transmission on replacement gilt immunization . in conclusion ," ] ]

[ "tooth wear ( tw ) , also known as tooth surface loss ( tsl ) , is an insidious and cumulative multifactorial process involving destruction of enamel and dentine which can threaten tooth survival and the oral health related quality of life of affected individuals [ 1 , 2 ] . despite the overall trends towards improved oral health and reduced dental caries incidence over the last decades", ", epidemiological evidence supports the contention that tw is increasing in severity and prevalence , not only amongst older people who are living longer and retaining more teeth , but also amongst those in the early decades of their adult life [ 3 , 4 ] .", "greater understanding of the pathophysiology of tw has driven advances in dental materials and techniques for the benefit of affected patients .", "these advances have led to biologically based prosthodontic strategies that challenge many traditional or currently prevailing concepts of tw management .", "will ensure that as much good as possible is achieved for the patient ( beneficence ) whilst avoiding harm ( nonmaleficience ) and upholding the patients ' rights to have the reasonable treatment undertaken that most closely matches their wishes and expectations ( autonomy ) .", "traditional concepts must now be reassessed in order to achieve a radical paradigm shift in the philosophies behind tw management .", "this paper will therefore , review the fundamental principles that should be considered when deciding how to manage patients with tw .", "the current state of knowledge of the aetiology and differential diagnoses of tw will be discussed , followed by an analysis of patient wishes and expectations when seeking sensible solutions for their tw problems .", "the relative risks and benefits of the possible management options will then be weighed up with reference to current available evidence .", "possible solutions which aim to put patients ' long - term interests first will be outlined with reference to ethically sound healthcare principles and using some case examples .", "there are three main , or widely recognized , aetiologies of tw , namely , erosion , attrition , and abrasion .", "there is a fourth aetiological factor which has been recognized by some but is certainly not universally accepted , namely , abfraction .", "each of these has many different clinical presentations which can be challenging to accurately diagnose , because the aetiology is usually multifactorial .", "erosion ( loss of tooth tissue by the chemical dissolution of enamel or dentine by the action of nonbacterial acids from dietary or gastric sources ) initially appears as silky - glazed dull enamel surfaces , with loss of enamel characterization such as perikymata . in moderate cases , the buccal and lingual surfaces of maxillary anterior teeth appear smooth and shiny with the loss of some anatomical features ( figure 1(a ) ) . in advanced cases , there is complete loss of the enamel , and dentine is exposed .", "often an intact ring of enamel is spared and remains present around the gingival area of the teeth , as seen in figure 1(b ) .", "this enamel ring is probably due to the neutralisation of the acid by the gingival crevicular fluid .", "as the lesion advances , multiple hollowed or cupped out areas form on the occlusal surfaces , as seen in ( c ) of figure 1 . in gastric erosion ,", "the palatal surfaces of maxillary anterior teeth are initially affected , as the tongue and the buccal mucosa protect the other surfaces from exposure to gastric acid . however , as the condition progresses , the protective effect is often lost , and the erosive tw becomes more widespread [ 11 , 12 ] .", "in contrast , dietary erosion presents as widespread cupped out lesions with the specific pattern being dependent upon the specific habits and diet of the patient .", "modern sociocultural standards of the ideal body image have resulted in the ubiquitous use in the media and fashion industries of very slim models to portray the supposedly ideal female body shape and size .", "increased exposure to such media has been shown to be psychologically detrimental to the well - being and perceived self - image of many young women .", "the emphasis on thinness may cause impressionable young people to adopt obsessive dieting and/or exercise behaviours , and there is evidence that increased exposure to such media may lead to an increased risk of eating disorders such as anorexia nervosa .", "one unfortunate outcome of these pressures to be slim can be excessive oral exposure to both dietary and gastric acids which may partly explain the increased prevalence of tw in young adults in the uk between 1998 and 2009 .", "refrigerated transportation and increased soft drinks consumption are also likely to contribute to the increase in dietary dental erosion , as fruit and fruit juices are available all year round rather than being limited by seasons .", "if fresh fruits ( particularly fruits containing citric or malic acid with high titratable acidity ) are consumed regularly , then the frequency of acid contact episodes increase as does the risk of dental erosion .", "the quantity and quality of saliva , salivary pellicle , physiological soft - tissue movements , and tooth anatomy and position in relation to the soft tissues will also influence the development and progression of erosive tw .", "behavioural factors , such as style and frequency of eating and drinking , have important consequences .", "sluiced in the mouth before swallowing will increase the contact time of the solution with the tooth surface and therefore the risk of dissolution of the hard tissues increases .", "frequent sipping of small quantities of acidic drinks will also increase their erosive potential . ", "figure 2 shows localized tooth wear due to frequent sipping of carbonated drinks in a 15 year old .", "the area of erosion corresponds to the ring pull ( v shaped ) area of the can , which explains the localisation of the erosion to the central incisors only and the relative sparing of the lateral incisors and canines .", "attrition ( wear of dental hard tissue as a result of tooth - to - tooth contact with no foreign substance intervening ) usually affects the incisal / occlusal surfaces of teeth in such a way that the opposing occluding surfaces of mandibular and maxillary teeth interrelate .", "there is usually symmetry with an antagonist tooth but this is often in one of the border positions and frequently not in their habitual intercuspal position .", "the specific pattern of wear coincides with how and where the patient bruxes or rubs their teeth forcibly against one another during their parafunction . ", "figure 3 illustrates a pattern of attritional tw which shows even wear of the maxillary and mandibular teeth .", "although there is considerable evidence of bruxism , there is a marked absence of buccocervical wear lesions , which further refutes the role of stress - related wear ( abfraction ) as a plausible cause of buccocervical tw .", "abrasion ( mechanical wear of dental hard tissue not involving tooth - to - tooth contact ) often presents in the cervical region of teeth , especially when associated with tooth brushing habits removing acid softened enamel and dentine in areas where gingival recession has occurred .", "figures 4(a ) and 4(b ) show the extensive abrasion of her third full mouth reconstruction in five years .", "the patient was referred for psychiatric help with her destructive habits . as the dentition has previously been extensively prepared for conventional porcelain crowns ,", "the dentition was subsequently restored , as shown in ( c)-(d ) , using conventional materials and metal on occluding surfaces .", "dietary , medical , social , and dental histories are very important to help to distinguish between the various clinical presentations .", "it is especially important to distinguish erosion from attrition so that the tw can be managed appropriately , taking into account the very different aetiologies and their consequences .", "for instance , if the chemical dissolution from dietary acids is the main aetiological factor , then the restorative material primarily needs resistance to acid attack in order to protect the remaining sound tooth tissue from further acid dissolution .", "diagnosis has been made and the patient concerns have been determined , the main aims of biologically sensible management are the following : the preservation of the remaining tooth tissue , a pragmatic improvement in aesthetics , the restoration of patient confidence ( both in terms of their ability to manage their own condition and the likelihood of their remaining tooth tissue lasting for the rest of their lifetime ) .", "the preservation of tooth structure is of the greatest concern . in cases of gastric erosion ,", "it is of real importance to prevent further exposure of the eroded teeth to the damaging gastric contents .", "the management strategy may include , for example , referring the patient to a gastroenterologist for medical management of their gastro - oesophageal reflux or to a psychologist or psychiatrist for behavioural and/or psychological management of an eating disorder .", "effectively managing gastro - oesophageal reflux has been shown to reduce enamel erosion even within a six week period of effective treatment . however , with the exception of management of gastric erosion , there is , currently , no high - level evidence about the clinical effectiveness of any other preventative measures such as monitoring periods and/or the use of oral care products in tw . epidemiological data from industrialized countries such as the uk show that increasingly more patients will retain many of their teeth for their lifetime .", "therefore , any dental material used to manage tw must ensure the survival of the structural strength of the underlying remaining tooth tissue .", "it follows , therefore , that the survival of the tooth is of paramount importance , and by way of comparison , the survival of the restoration is of far less consequence .", "in fact , because modern restorative materials are now considered expendable , reparable , and renewable , the traditional full mouth rehabilitation approach as a rationale for restoring a worn dentition must now change and focus instead on protecting the remaining sound tooth structure .", "studies into the use of dentine bonding agents as a management strategy have found that the coating was retained for a short period of time only . ", "figure 5 shows an example of directly applied resin composite bonded at an increased vertical dimension in order to provide long - term protection to ( a ) the eroded palatal surface of a maxillary incisor and ( b ) the occlusal surfaces of mandibular molars ( compare with figure 1 )", ". the social and psychological impact of dental disease have been well documented , and it has been shown that poor oral health has a detrimental effect on one 's ability to live comfortably , be successful in employment , enjoy life , experience relationships , and possess a positive self - image . when seeking a solution for the management of patients with tw , it is important to determine what specific aspects of the problems are of most concern to the patient , for example , lack of visibility or sharpness of teeth , sensitivity to thermal changes , or the colour of their teeth or shape problems .", "this brings patient 's wishes and expectations to the forefront by considering how they wish to have their problem managed .", "when the biologically sound concept of protecting their remaining sound tooth tissue is explained to patients , not only do they readily understand the value and rationale of such an approach , but also they usually actively seek to avoid destructive treatments which would remove more of their sound tooth tissue , and involve possible pulpal damage , loss of vitality , further endodontic complications , or ultimately loss of aggressively treated teeth .", "it is vital to remember that the survival of the tooth and dentinopulpal complex is of paramount importance , and therefore , the focus should be on the survival of the tooth or teeth rather than on the success or survival of the restorations .", "this biologically sensible approach accepts a lifetime of repair and renewal of restorations , in lieu of , further loss of sound tooth tissue . when choosing the appropriate material to manage tw , the daughter test in elective aesthetic dentistry is of pertinence .", "this test proposes that whenever elective intervention is contemplated , the following question should be asked : knowing what i know about dentistry and the effects of this elective treatment on the health and structure of these teeth in the long - term , would i carry out this treatment on my own daughter ? . if , in answering this question honestly , dentists would be unwilling to carry out electively destructive treatment on their own daughter ( or son , younger sister / brother , mother / father , husband / wife ) , then why would they ever consider carrying out such dental treatment on one of their trusting patients ?", "there is evidence that when patients are adequately informed , most prefer more conservative options such as resin composite rather than destructive options such as porcelain .", "patients do not perceive porcelain restorations to be necessarily more aesthetic than resin composite restorations . using resin composite to manage", "tw is conservative , predictable , and usually aesthetically acceptable to patients ( figure 5 ) .", "the use of resin composite is also safe with minimal long - term pulpal or structural complications being reported when this is applied to the external aspects of teeth in thick section .", "long - term follow - up studies show that the main complications are repairable and retrievable with no loss of tooth vitality or need for loss of further teeth .", "in marked contrast , the use of dental porcelain veneered onto various copings requires much more of the remaining sound tooth tissue to be removed from the already worn teeth in order to gain adequate space for the brittle porcelain .", "few patients with tooth wear are told that in order to have an all - ceramic or ceramic - bonded - to - metal approach to manage their wear that much more of their already reduced teeth will be further destroyed .", "edelhoff and sorensen demonstrated that when teeth are prepared for metal - ceramic or all - ceramic crown between 63%72% of coronal tooth structure is removed .", "the long - term biological costs of this amount of elective structural and pulpal damage can be potentially huge .", "ethical clinicians are under a duty of care to ensure that informed consent has been obtained for any elective destructive procedures .", "informed consent means that the patient must fully understand that alternative safer or more biologically sound treatment options actually do exist for them .", "managing worn incisors using a conservative approach is a critical part of the overall approach to biologically sound tw management .", "incisors are relatively small teeth , and therefore , what little structure they have left following significant wear needs to be protected and preserved . \n", "figure 6 shows photographic images of a female patient with tw , resulting in short maxillary anterior clinical crown height .", "the aetiology was erosion from intrinsic acid as a result of bulimia , to which the patient readily admitted on direct questioning .", "she had previously been managed on a watch and wait basis by the making of impressions to provide stone casts of her teeth on a yearly basis for seven years .", "this approach proved futile and costly in terms of both valuable sound tooth tissue and expensive clinical time . a resin composite", " mock up on the unetched , but dried enamel was used to show the patient what could be done to change her dental appearance .", "as the patient liked that appearance , the temporary mock up resin composite was removed , the affected teeth were then etched , and a three bottle bonding system was used prior to placing resin composite freehand directly onto just the anterior eroded teeth , at an increased anterior occlusal vertical dimension .", "this was done without damaging the already eroded teeth in any way or involving the mainly intact posterior teeth . pragmatic ", "one - hit direct resin composite bonding has been shown to be clinically successful with minimal or no - long - term iatrogenic effects [ 29 , 3133 ] , and the use of three - step ( etch , primer , and bond ) bonding system is still the gold standard for mixed enamel / dentine bonding . ", " mocked up with directly applied hybrid resin composite at an increased occlusal vertical dimension .", "this resulted in improved aesthetics with minimal biological costs , as the residual eroded tooth tissue was protected in one appointment without any treatment of the posterior dentition , which returned to full occlusal contact within 3 months .", "increasing the anterior occlusion only is a predictable and safe procedure with a mean time to re - establishing posterior occlusal contacts of 7 months [ 29 , 31 ] .", "it should be explained to patients to be treated with this approach that they will need time to adapt to the changes in their occlusion .", "the resin composite acts as a direct fixed orthodontic device and the teeth are protected by proprioception in the periodontal ligaments while the patient adapts .", "confidence in this procedure is based on work originally undertaken by anderson in 1962 showing that patients readily adapt to changes in their occlusion [ 3235 ] .", "these original studies have now been backed up by 30 years of follow - up data showing minimal or no - long - term biological costs occur from this increase in the occlusal vertical dimension . \n", "figure 8 shows some of the clinical procedures used to treat a patient using this approach .", "management of generalised tw can be facilitated by raising the occlusal vertical dimension using direct composite resin bonding techniques in the incisor and canine areas , thus opening the posterior occlusion .", "if the tw is generalised , this space can be secured by temporarily placing fast setting , contrasting colour , glass ionomer cement onto the molar teeth at the increased anterior vertical dimension .", "the premolars or molars in between these newly bonded teeth can then be definitively restored at leisure during subsequent appointments with an appropriate approach and materials , all depending on the degree of wear or the existing restorations in the posterior teeth . ", "figure 9 shows photographs of gold adhesive onlays used to restore worn posterior teeth at 15 year followup .", "if the premolar or molar teeth have existing large restorations and need conventional crowns or onlays , then the longer preparations that are made possible , due to the fact that no further occlusal reduction is required , mean that these longer castings made possible in this biologically sensible way will be better retained with either conventional or resin - based cements as indicated .", "these biologically based approaches advocated in this paper are worthy of equal , if not greater , financial reward than traditional destructive approaches using conventional crown and bridgework .", "dentists who are able to practice and hone their skills using additive techniques with resin composite and not subtractive techniques with a dental bur usually have greater artistic craft skills and a profounder appreciation of the biologic long term value of their patients remaining sound enamel and dentine .", "these valuable skills are more likely to be sought after by sensible patients with tw , and when they understand the real issues they will usually be willing to pay a fair fee for this biologically sound approach which ensures that they retain their remaining sound tooth tissue with their intact and healthy pulps .", "medium destructive approaches and only then consider the highly destructive tooth preparations required for full mouth rehabilitations . in the authors ' view", ", the philosophy of full mouth reconstruction using conventional fixed prosthodontics has very limited biological advantages .", "the preparation of multiple sound or minimally worn teeth for indirect all - porcelain or porcelain - fused - to - metal restorations is not justifiable , simply because teeth elsewhere in the dentition have been affected by tw , or because it is easier , traditional , or more lucrative , for the clinician to aggressively treat the tw in this manner .", "regretfully , many remuneration systems and prosthodontic training courses have retained an emphasis on teaching highly destructive management strategies which have a very poor fallback position ( i.e. , the ability to repair , retrieve , and retain the teeth and place new sound restorations ) when problems inevitably occur .", "this paper has argued that a paradigm shift is now required in relation to managing tooth wear . for far too long , the emphasis in managing tooth wear has been on the wrong aspects , namely , the length of time that the dental restoration is successful or survives .", "the emphasis has to change to a more biologically sensible , patient - centred approach , involving minimal destruction of their worn teeth and an acceptance that the materials used to repair or protect the worn tooth tissue will need to be repaired , repolished , renewed , or recycled as required .", "damage or chipping of the resin composite itself is of minimal consequence , as it can be readily repaired , whereas the residual load - bearing sound tooth structure and pulpal health are invaluable .", "the focus for restorative management of tooth wear should , therefore , be on using additive , constructive prosthodontic skills rather than relying on the destructive approaches and skills involved in the traditional full mouth reconstruction using full - ceramic or cast - ceramic veneered restorations . in biologic terms , the destruction of sound tooth structure or the hazarding of dental pulps can no longer be condoned by sensible , caring dentists , whose trusting patients depend on when seeking help with their tooth wear ." ]

[ "<S> the prevalence and severity of tooth wear is increasing in industrialised nations . yet </S>", "<S> , there is no high - level evidence to support or refute any therapeutic intervention . in the absence of such evidence , many currently prevailing management strategies for tooth wear may be failing in their duty of care to first and foremost improve the oral health of patients with this disease . </S>", "<S> this paper promotes biologically sound approaches to the management of tooth wear on the basis of current best evidence of the aetiology and clinical features of this disease . </S>", "<S> the relative risks and benefits of the varying approaches to managing tooth wear are discussed with reference to long - term follow - up studies . using reference to ethical standards such as the daughter test </S>", "<S> , this paper presents case reports of patients with moderate - to - severe levels of tooth wear managed in line with these biologically sound principles . </S>" ]

[ "1. Introduction", "2. Aetiology and Differential Diagnoses of Tooth Wear (TW)", "3. Principles of Management", "4. Conclusion" ]

[ [ "tooth wear ( tw ) , also known as tooth surface loss ( tsl ) , is an insidious and cumulative multifactorial process involving destruction of enamel and dentine which can threaten tooth survival and the oral health related quality of life of affected individuals [ 1 , 2 ] . despite the overall trends towards improved oral health and reduced dental caries incidence over the last decades", ", epidemiological evidence supports the contention that tw is increasing in severity and prevalence , not only amongst older people who are living longer and retaining more teeth , but also amongst those in the early decades of their adult life [ 3 , 4 ] .", "greater understanding of the pathophysiology of tw has driven advances in dental materials and techniques for the benefit of affected patients .", "these advances have led to biologically based prosthodontic strategies that challenge many traditional or currently prevailing concepts of tw management .", "will ensure that as much good as possible is achieved for the patient ( beneficence ) whilst avoiding harm ( nonmaleficience ) and upholding the patients ' rights to have the reasonable treatment undertaken that most closely matches their wishes and expectations ( autonomy ) .", "traditional concepts must now be reassessed in order to achieve a radical paradigm shift in the philosophies behind tw management .", "this paper will therefore , review the fundamental principles that should be considered when deciding how to manage patients with tw .", "the current state of knowledge of the aetiology and differential diagnoses of tw will be discussed , followed by an analysis of patient wishes and expectations when seeking sensible solutions for their tw problems .", "the relative risks and benefits of the possible management options will then be weighed up with reference to current available evidence .", "possible solutions which aim to put patients ' long - term interests first will be outlined with reference to ethically sound healthcare principles and using some case examples ." ], [ "there are three main , or widely recognized , aetiologies of tw , namely , erosion , attrition , and abrasion .", "there is a fourth aetiological factor which has been recognized by some but is certainly not universally accepted , namely , abfraction .", "each of these has many different clinical presentations which can be challenging to accurately diagnose , because the aetiology is usually multifactorial .", "erosion ( loss of tooth tissue by the chemical dissolution of enamel or dentine by the action of nonbacterial acids from dietary or gastric sources ) initially appears as silky - glazed dull enamel surfaces , with loss of enamel characterization such as perikymata . in moderate cases , the buccal and lingual surfaces of maxillary anterior teeth appear smooth and shiny with the loss of some anatomical features ( figure 1(a ) ) . in advanced cases , there is complete loss of the enamel , and dentine is exposed .", "often an intact ring of enamel is spared and remains present around the gingival area of the teeth , as seen in figure 1(b ) .", "this enamel ring is probably due to the neutralisation of the acid by the gingival crevicular fluid .", "as the lesion advances , multiple hollowed or cupped out areas form on the occlusal surfaces , as seen in ( c ) of figure 1 . in gastric erosion ,", "the palatal surfaces of maxillary anterior teeth are initially affected , as the tongue and the buccal mucosa protect the other surfaces from exposure to gastric acid . however , as the condition progresses , the protective effect is often lost , and the erosive tw becomes more widespread [ 11 , 12 ] .", "in contrast , dietary erosion presents as widespread cupped out lesions with the specific pattern being dependent upon the specific habits and diet of the patient .", "modern sociocultural standards of the ideal body image have resulted in the ubiquitous use in the media and fashion industries of very slim models to portray the supposedly ideal female body shape and size .", "increased exposure to such media has been shown to be psychologically detrimental to the well - being and perceived self - image of many young women .", "the emphasis on thinness may cause impressionable young people to adopt obsessive dieting and/or exercise behaviours , and there is evidence that increased exposure to such media may lead to an increased risk of eating disorders such as anorexia nervosa .", "one unfortunate outcome of these pressures to be slim can be excessive oral exposure to both dietary and gastric acids which may partly explain the increased prevalence of tw in young adults in the uk between 1998 and 2009 .", "refrigerated transportation and increased soft drinks consumption are also likely to contribute to the increase in dietary dental erosion , as fruit and fruit juices are available all year round rather than being limited by seasons .", "if fresh fruits ( particularly fruits containing citric or malic acid with high titratable acidity ) are consumed regularly , then the frequency of acid contact episodes increase as does the risk of dental erosion .", "the quantity and quality of saliva , salivary pellicle , physiological soft - tissue movements , and tooth anatomy and position in relation to the soft tissues will also influence the development and progression of erosive tw .", "behavioural factors , such as style and frequency of eating and drinking , have important consequences .", "sluiced in the mouth before swallowing will increase the contact time of the solution with the tooth surface and therefore the risk of dissolution of the hard tissues increases .", "frequent sipping of small quantities of acidic drinks will also increase their erosive potential . ", "figure 2 shows localized tooth wear due to frequent sipping of carbonated drinks in a 15 year old .", "the area of erosion corresponds to the ring pull ( v shaped ) area of the can , which explains the localisation of the erosion to the central incisors only and the relative sparing of the lateral incisors and canines .", "attrition ( wear of dental hard tissue as a result of tooth - to - tooth contact with no foreign substance intervening ) usually affects the incisal / occlusal surfaces of teeth in such a way that the opposing occluding surfaces of mandibular and maxillary teeth interrelate .", "there is usually symmetry with an antagonist tooth but this is often in one of the border positions and frequently not in their habitual intercuspal position .", "the specific pattern of wear coincides with how and where the patient bruxes or rubs their teeth forcibly against one another during their parafunction . ", "figure 3 illustrates a pattern of attritional tw which shows even wear of the maxillary and mandibular teeth .", "although there is considerable evidence of bruxism , there is a marked absence of buccocervical wear lesions , which further refutes the role of stress - related wear ( abfraction ) as a plausible cause of buccocervical tw .", "abrasion ( mechanical wear of dental hard tissue not involving tooth - to - tooth contact ) often presents in the cervical region of teeth , especially when associated with tooth brushing habits removing acid softened enamel and dentine in areas where gingival recession has occurred .", "figures 4(a ) and 4(b ) show the extensive abrasion of her third full mouth reconstruction in five years .", "the patient was referred for psychiatric help with her destructive habits . as the dentition has previously been extensively prepared for conventional porcelain crowns ,", "the dentition was subsequently restored , as shown in ( c)-(d ) , using conventional materials and metal on occluding surfaces ." ], [ "dietary , medical , social , and dental histories are very important to help to distinguish between the various clinical presentations .", "it is especially important to distinguish erosion from attrition so that the tw can be managed appropriately , taking into account the very different aetiologies and their consequences .", "for instance , if the chemical dissolution from dietary acids is the main aetiological factor , then the restorative material primarily needs resistance to acid attack in order to protect the remaining sound tooth tissue from further acid dissolution .", "diagnosis has been made and the patient concerns have been determined , the main aims of biologically sensible management are the following : the preservation of the remaining tooth tissue , a pragmatic improvement in aesthetics , the restoration of patient confidence ( both in terms of their ability to manage their own condition and the likelihood of their remaining tooth tissue lasting for the rest of their lifetime ) .", "the preservation of tooth structure is of the greatest concern . in cases of gastric erosion ,", "it is of real importance to prevent further exposure of the eroded teeth to the damaging gastric contents .", "the management strategy may include , for example , referring the patient to a gastroenterologist for medical management of their gastro - oesophageal reflux or to a psychologist or psychiatrist for behavioural and/or psychological management of an eating disorder .", "effectively managing gastro - oesophageal reflux has been shown to reduce enamel erosion even within a six week period of effective treatment . however , with the exception of management of gastric erosion , there is , currently , no high - level evidence about the clinical effectiveness of any other preventative measures such as monitoring periods and/or the use of oral care products in tw . epidemiological data from industrialized countries such as the uk show that increasingly more patients will retain many of their teeth for their lifetime .", "therefore , any dental material used to manage tw must ensure the survival of the structural strength of the underlying remaining tooth tissue .", "it follows , therefore , that the survival of the tooth is of paramount importance , and by way of comparison , the survival of the restoration is of far less consequence .", "in fact , because modern restorative materials are now considered expendable , reparable , and renewable , the traditional full mouth rehabilitation approach as a rationale for restoring a worn dentition must now change and focus instead on protecting the remaining sound tooth structure .", "studies into the use of dentine bonding agents as a management strategy have found that the coating was retained for a short period of time only . ", "figure 5 shows an example of directly applied resin composite bonded at an increased vertical dimension in order to provide long - term protection to ( a ) the eroded palatal surface of a maxillary incisor and ( b ) the occlusal surfaces of mandibular molars ( compare with figure 1 )", ". the social and psychological impact of dental disease have been well documented , and it has been shown that poor oral health has a detrimental effect on one 's ability to live comfortably , be successful in employment , enjoy life , experience relationships , and possess a positive self - image . when seeking a solution for the management of patients with tw , it is important to determine what specific aspects of the problems are of most concern to the patient , for example , lack of visibility or sharpness of teeth , sensitivity to thermal changes , or the colour of their teeth or shape problems .", "this brings patient 's wishes and expectations to the forefront by considering how they wish to have their problem managed .", "when the biologically sound concept of protecting their remaining sound tooth tissue is explained to patients , not only do they readily understand the value and rationale of such an approach , but also they usually actively seek to avoid destructive treatments which would remove more of their sound tooth tissue , and involve possible pulpal damage , loss of vitality , further endodontic complications , or ultimately loss of aggressively treated teeth .", "it is vital to remember that the survival of the tooth and dentinopulpal complex is of paramount importance , and therefore , the focus should be on the survival of the tooth or teeth rather than on the success or survival of the restorations .", "this biologically sensible approach accepts a lifetime of repair and renewal of restorations , in lieu of , further loss of sound tooth tissue . when choosing the appropriate material to manage tw , the daughter test in elective aesthetic dentistry is of pertinence .", "this test proposes that whenever elective intervention is contemplated , the following question should be asked : knowing what i know about dentistry and the effects of this elective treatment on the health and structure of these teeth in the long - term , would i carry out this treatment on my own daughter ? . if , in answering this question honestly , dentists would be unwilling to carry out electively destructive treatment on their own daughter ( or son , younger sister / brother , mother / father , husband / wife ) , then why would they ever consider carrying out such dental treatment on one of their trusting patients ?", "there is evidence that when patients are adequately informed , most prefer more conservative options such as resin composite rather than destructive options such as porcelain .", "patients do not perceive porcelain restorations to be necessarily more aesthetic than resin composite restorations . using resin composite to manage", "tw is conservative , predictable , and usually aesthetically acceptable to patients ( figure 5 ) .", "the use of resin composite is also safe with minimal long - term pulpal or structural complications being reported when this is applied to the external aspects of teeth in thick section .", "long - term follow - up studies show that the main complications are repairable and retrievable with no loss of tooth vitality or need for loss of further teeth .", "in marked contrast , the use of dental porcelain veneered onto various copings requires much more of the remaining sound tooth tissue to be removed from the already worn teeth in order to gain adequate space for the brittle porcelain .", "few patients with tooth wear are told that in order to have an all - ceramic or ceramic - bonded - to - metal approach to manage their wear that much more of their already reduced teeth will be further destroyed .", "edelhoff and sorensen demonstrated that when teeth are prepared for metal - ceramic or all - ceramic crown between 63%72% of coronal tooth structure is removed .", "the long - term biological costs of this amount of elective structural and pulpal damage can be potentially huge .", "ethical clinicians are under a duty of care to ensure that informed consent has been obtained for any elective destructive procedures .", "informed consent means that the patient must fully understand that alternative safer or more biologically sound treatment options actually do exist for them .", "managing worn incisors using a conservative approach is a critical part of the overall approach to biologically sound tw management .", "incisors are relatively small teeth , and therefore , what little structure they have left following significant wear needs to be protected and preserved . \n", "figure 6 shows photographic images of a female patient with tw , resulting in short maxillary anterior clinical crown height .", "the aetiology was erosion from intrinsic acid as a result of bulimia , to which the patient readily admitted on direct questioning .", "she had previously been managed on a watch and wait basis by the making of impressions to provide stone casts of her teeth on a yearly basis for seven years .", "this approach proved futile and costly in terms of both valuable sound tooth tissue and expensive clinical time . a resin composite", " mock up on the unetched , but dried enamel was used to show the patient what could be done to change her dental appearance .", "as the patient liked that appearance , the temporary mock up resin composite was removed , the affected teeth were then etched , and a three bottle bonding system was used prior to placing resin composite freehand directly onto just the anterior eroded teeth , at an increased anterior occlusal vertical dimension .", "this was done without damaging the already eroded teeth in any way or involving the mainly intact posterior teeth . pragmatic ", "one - hit direct resin composite bonding has been shown to be clinically successful with minimal or no - long - term iatrogenic effects [ 29 , 3133 ] , and the use of three - step ( etch , primer , and bond ) bonding system is still the gold standard for mixed enamel / dentine bonding . ", " mocked up with directly applied hybrid resin composite at an increased occlusal vertical dimension .", "this resulted in improved aesthetics with minimal biological costs , as the residual eroded tooth tissue was protected in one appointment without any treatment of the posterior dentition , which returned to full occlusal contact within 3 months .", "increasing the anterior occlusion only is a predictable and safe procedure with a mean time to re - establishing posterior occlusal contacts of 7 months [ 29 , 31 ] .", "it should be explained to patients to be treated with this approach that they will need time to adapt to the changes in their occlusion .", "the resin composite acts as a direct fixed orthodontic device and the teeth are protected by proprioception in the periodontal ligaments while the patient adapts .", "confidence in this procedure is based on work originally undertaken by anderson in 1962 showing that patients readily adapt to changes in their occlusion [ 3235 ] .", "these original studies have now been backed up by 30 years of follow - up data showing minimal or no - long - term biological costs occur from this increase in the occlusal vertical dimension . \n", "figure 8 shows some of the clinical procedures used to treat a patient using this approach .", "management of generalised tw can be facilitated by raising the occlusal vertical dimension using direct composite resin bonding techniques in the incisor and canine areas , thus opening the posterior occlusion .", "if the tw is generalised , this space can be secured by temporarily placing fast setting , contrasting colour , glass ionomer cement onto the molar teeth at the increased anterior vertical dimension .", "the premolars or molars in between these newly bonded teeth can then be definitively restored at leisure during subsequent appointments with an appropriate approach and materials , all depending on the degree of wear or the existing restorations in the posterior teeth . ", "figure 9 shows photographs of gold adhesive onlays used to restore worn posterior teeth at 15 year followup .", "if the premolar or molar teeth have existing large restorations and need conventional crowns or onlays , then the longer preparations that are made possible , due to the fact that no further occlusal reduction is required , mean that these longer castings made possible in this biologically sensible way will be better retained with either conventional or resin - based cements as indicated .", "these biologically based approaches advocated in this paper are worthy of equal , if not greater , financial reward than traditional destructive approaches using conventional crown and bridgework .", "dentists who are able to practice and hone their skills using additive techniques with resin composite and not subtractive techniques with a dental bur usually have greater artistic craft skills and a profounder appreciation of the biologic long term value of their patients remaining sound enamel and dentine .", "these valuable skills are more likely to be sought after by sensible patients with tw , and when they understand the real issues they will usually be willing to pay a fair fee for this biologically sound approach which ensures that they retain their remaining sound tooth tissue with their intact and healthy pulps .", "medium destructive approaches and only then consider the highly destructive tooth preparations required for full mouth rehabilitations . in the authors ' view", ", the philosophy of full mouth reconstruction using conventional fixed prosthodontics has very limited biological advantages .", "the preparation of multiple sound or minimally worn teeth for indirect all - porcelain or porcelain - fused - to - metal restorations is not justifiable , simply because teeth elsewhere in the dentition have been affected by tw , or because it is easier , traditional , or more lucrative , for the clinician to aggressively treat the tw in this manner .", "regretfully , many remuneration systems and prosthodontic training courses have retained an emphasis on teaching highly destructive management strategies which have a very poor fallback position ( i.e. , the ability to repair , retrieve , and retain the teeth and place new sound restorations ) when problems inevitably occur ." ], [ "this paper has argued that a paradigm shift is now required in relation to managing tooth wear . for far too long , the emphasis in managing tooth wear has been on the wrong aspects , namely , the length of time that the dental restoration is successful or survives .", "the emphasis has to change to a more biologically sensible , patient - centred approach , involving minimal destruction of their worn teeth and an acceptance that the materials used to repair or protect the worn tooth tissue will need to be repaired , repolished , renewed , or recycled as required .", "damage or chipping of the resin composite itself is of minimal consequence , as it can be readily repaired , whereas the residual load - bearing sound tooth structure and pulpal health are invaluable .", "the focus for restorative management of tooth wear should , therefore , be on using additive , constructive prosthodontic skills rather than relying on the destructive approaches and skills involved in the traditional full mouth reconstruction using full - ceramic or cast - ceramic veneered restorations . in biologic terms , the destruction of sound tooth structure or the hazarding of dental pulps can no longer be condoned by sensible , caring dentists , whose trusting patients depend on when seeking help with their tooth wear ." ] ]

[ "there is extensive evidence that benzodiazepines , the most widely prescribed psychotropic drug class ( greenblatt et al 1983 ) , induce anterograde amnesia in both humans and animals ( lister 1985 ; thiebot 1985 ) . the findings of several experiments suggest that the anxiolytic properties of benzodiazepines involve effects mediated by the amygdala .", "intra - amygdala injections of benzodiazepines produce anxiolytic effects comparable to those induced by systemic injections ( nagy et al 1979 ; scheel - krger and petersen 1982 ; shibata et al 1982 ; niehoff and kuhar 1983 ; petersen et al 1985 ) .", "furthermore , intra - amygdala injections of the benzodiazepine antagonist flumazenil attenuate the anxiolytic effects of systemically administered benzodiazepines ( hodges et al 1987 ) .", "recent findings by izquierdo and colleagues ( 1990 ) suggest that benzodiazepine impairment of memory involves gabaergic type a receptors in the amygdala .", "post - training intra - amygdala injection of flumazenil causes memory facilitation comparable to that found with systemic injections , and systemic injection of flumazenil before training attenuates the amnestic effects of post - training intra - amygdala injection of muscimol .", "studies examining the memory - modulating effects of drug treatments have provided evidence that memory can be modulated by systemic as well as intra - amygdala gabaergic compounds . when administered shortly after training , gabaergic agonists ( eg , muscimol and baclofen ) impair memory retention , while gabaergic antagonists ( eg , picrotoxin and bicuculline ) enhance retention ( breen and mcgaugh 1961 ; brioni and mcgaugh 1988 ; brioni et al 1989 ; castellano et al 1989 ; ammassari - teule et al 1991 ) . on the other hand , there is extensive evidence indicating a key role for gaba neurotransmission in the modulation of fearful / defensive behaviors .", "it has been found that systemic or intra - amygdala injections of gaba agonists reduce and gaba antagonists enhance experimental fear and anxiety ( graeff 1990 ) .", "furthermore , lesions of the amygdala attenuate the antianxiety as well as the memory - modulating effects of gabaergic drugs ( shibata et al 1989 ; ammassari - teule et al 1991 ) . a recent study with magnetic resonance spectroscopy revealed low gaba levels in the occipital cortex of depressed patients ,", "cortical benzodiazepine binding to gaba(a ) receptors has been measured with i - labeled flumazenil and single photon emission computed tomography in unmedicated patients with major depression and healthy volunteers ( kugaya et al 2003 ) .", "depression is currently seen as a chronic medical disorder that produces as much functional limitation and morbidity as chronic diseases such as hypertension and diabetes ( angst 1999 ) .", "one of the most frequent and neuropsychologically well investigated symptoms in depression is reduced memory capacity ( burt et al 1995 ; ilsley et al 1995 ) .", "the condition is thought to result from dysfunctions in monoaminergic systems affecting norepinephrine , serotonin , and dopamine at several effector sites .", "disturbances of the limbic hypothalamic pituitary adrenal axis and the serotonin system were found , and changes in adrenoceptors associated with the pituitary adrenal axis function strongly implicate a disorder in central noradrenergic transmission ( leonard 2000 ) .", "the effect of corticotropin - releasing factor in modulating the activity of noradrenergic neurons in the locus ceruleus may provide a link between environmental trigger factors and central noradrenergic dysfunction ( leonard 2000 ) .", "we hypothesized that depression may affect the amygdala noradrenergic modulation of memory and , through the noradrenergic / gabaergic connection , may modify the anterograde amnestic effect of benzodiazepines .", "furthermore , the action of benzodiazepines may block the negative tendency on emotional memory tasks described for depressed patients .", "the purpose of the present study was evaluation of the effect of diazepam on explicit memory ( emotional and non - emotional tasks ) in patients with major depression who were not previously receiving benzodiazepines .", "a double - blind , placebo - controlled experiment with diazepam 10 mg ( novaquim - sigma ) was carried out with dsm - iv major depression patients ( by certificated psychiatrists ) during the first 24 hours after admission in the psychiatric unit of a general university hospital .", "exclusion criteria were psychotic symptoms , other psychiatric comorbidity ( eg , alcohol and drug abuse ) , long - term use of benzodiazepines ( last 30 days ) , cognitive deficit , use of tricyclic antidepressants , and previous ect .", "cognitive status was checked with the mini - mental state examination ( folstein et al 1975 ) with cut - offs 24 and 17 for education of > 4 and 4 years , respectively ( baseline mini - mental ) . during this period ,", "drug groups did not differ in age , education , and sex distributions ( table 1 ) .", "demographic variables and baseline mini - mental ( mm ) of the studied groups ; mean and standard deviation ( range ) chi - square ( fisher s test ) .", "all patients signed a written consent after the nature of procedures and objectives of the study were explained .", "only patients who were not taking benzodiazepines were selected for the study ( before and after hospitalization ) .", "there were no group differences ( diazepam versus placebo ) in the dose or type of antidepressant being taken .", "patients who presented severe insomnia received promethazine 25 mg , whereas anxiety was behaviorally handled .", "all patients were given either placebo ( 50 mg of starch ) or diazepam ( 10 mg ) on the morning of the fourth day ( figure 1 ) .", "the drugs were given orally in enteric capsules , and patients were assigned to the two groups by a double - blind design .", "the medications were specially processed for the experiment in the hospital pharmacy , where they were assembled in identical capsules .", "only one member of the team carried out the randomization and kept the codes of identification of the medications .", "those who applied tests and gave medication to patients did not know which was being administered to each patient , and patients did not know which medication they were receiving .", "thirty minutes and 6 hours after drug or placebo administration , patients were exposed to an emotionally neutral word list , and to two closely matched and emotionally arousing lists negative and positive ( ceitlin et al 1995 ) .", "also administered were the logical memory ( subtest of the wechsler memory scale ; a neutral short story ) ( wechsler 1973 ) and the non - verbal silhouette test ( black images of universally recognized buildings ) ( rosat et al 1990 ) .", "two sets of equivalent tests ( word lists , short story , and silhouettes ) were used in the study .", "these tests for explicit memory were selected because they evaluate immediate and delayed ( short - term ) memory , recognition , and semantic memory in visual or auditory modality and were well studied in the population from which the sample was recruited .", "the brazilian portuguese words were obtained in two studies ( ceitlin et al 1995 ) for the development of emotionally negative , positive , and neutral lists .", "the emotional content of the words , effect on other words in mixed lists , and effect of age , education , and symptoms of depression were evaluated before the final lists were achieved .", "the portuguese idiom in brazil still lacks population studies on quantitative linguistics ( such as word frequency and age of acquisition ) .", "therefore , the ceitlin study covered most of these factors for generation of the present word lists .", "the effect of the emotionally arousing word lists on a patient s subjective states ( mood and anxiety ) was evaluated by the visual analog mood scale ( vams ) ( norris 1971 ) , which consists of 16 analog items composed of two adjectives with opposite feelings , separated by a 10-cm line on which the subject has to mark the point which best describes his / her feelings at the time .", "these items were combined into four factors ( anxiety : calm excited , relaxed ", "feeble ; mental sedation : alert drowsy , attentive dreamy ; other feelings and attitudes : interested bored , amicable antagonistic , happy sad , contented discontented ) according to a factorial analysis performed on a brazilian sample ( zuardi et al 1993 ) .", "all procedures were executed by the statistical package for the social sciences ( spss / pc plus ) and epi - info 6.4 .", "parametric data were analyzed by student s t - test for independent samples and by multivariate procedures of manova .", "categories were tested by analysis of association ( chi - square test with yates correction or fisher s exact test ) .", "neuropsychological scores were submitted to a pegboard test ( normal probability plot ) before being analyzed by manova ( either between or within effects ) .", "the univariate analysis within the multivariate procedure was family - wise controlled for alpha values , and two - tailed significance values were chosen . an index of variation ( rate ) for the word lists was calculated from the recalls at 30 minutes and 6 hours , based on the calculation for rate of forgetfulness ( isaac and mayes 1999 ) : where xi is the score of recall 30 minutes after drug intake and xii of recall at 6 hours .", "this rate may enhance the effect of the intervention because both retrievals occurred after exposure to the list ( auditory task ) ; consequently , second recall was more likely to be higher owing to the learning effect .", "in the case of a positive index , first retrieval was higher than second , expressing a greater effect of the drug than of practice .", "all procedures were executed by the statistical package for the social sciences ( spss / pc plus ) and epi - info 6.4 .", "parametric data were analyzed by student s t - test for independent samples and by multivariate procedures of manova .", "categories were tested by analysis of association ( chi - square test with yates correction or fisher s exact test ) .", "neuropsychological scores were submitted to a pegboard test ( normal probability plot ) before being analyzed by manova ( either between or within effects ) .", "the univariate analysis within the multivariate procedure was family - wise controlled for alpha values , and two - tailed significance values were chosen .", "an index of variation ( rate ) for the word lists was calculated from the recalls at 30 minutes and 6 hours , based on the calculation for rate of forgetfulness ( isaac and mayes 1999 ) : where xi is the score of recall 30 minutes after drug intake and xii of recall at 6 hours .", "this rate may enhance the effect of the intervention because both retrievals occurred after exposure to the list ( auditory task ) ; consequently , second recall was more likely to be higher owing to the learning effect . in the case of a positive index ,", "first retrieval was higher than second , expressing a greater effect of the drug than of practice .", "performance on word lists with positive emotional content showed a statistically significant score gradient from 30 minutes to 6 hours after drug administration only in the group of patients who received diazepam ( figure 2 ) .", "patients who received diazepam presented higher and more positive indexes for positive words , while negative and neutral words presented negative rates .", "although not statistically significant , the index negativity was lower for negative and higher for neutral words .", "we observed a very large standard deviation for recall of neutral words ; however , individual scores of this word list were all negative within the diazepam group and both negative and positive within the placebo group .", "for positive words , the gradient showed the least variation among the diazepam group , significantly less than in placebo patients .", "these findings may suggest a facilitatory effect of diazepam ( 10 mg ) on the recall of positive words ( 30 minutes after administration ) . at the 6-hour evaluation , performance on the positively loaded words decreased to a similar level to that of the placebo group . mean and standard deviation of rate of recall of the word lists emotionally toned ( positive , negative , neutral ) of patients on diazepam and on placebo .", "the scores of the vams were not different within each group ( p > 0.05 ) or between groups ( p > 0.05 ) .", "therefore , the significant rate of recall for positively loaded words among diazepam patients could not be explained by lower levels of anxiety or other symptoms .", "patients , either diazepam or placebo , showed similar levels of symptoms at the two time - points .", "no parallel improvement on other immediate recall was seen at 30 minutes relative to 6 hours after dosing ( diazepam group ) .", "the score of the immediate recall of the short history at 30 minutes was lower than at 6 hours ( p = 0.035 ) ( table 2 ) .", "there was no difference between the immediate and delayed retrievals of this test at 30 minutes after medication ( p = 0.361 ) .", "however , at the 6-hour evaluation , immediate recall was higher than delayed ( p = 0.028 ) . mean and standard deviation ( range ) of scores of tests without emotional content and with immediate ( i ) and delayed ( d ) recalls within - group comparison .", "p = 0.028 ( immediate > delayed recall short story at 6 hours ) . for the positive word list , the score during the effect of diazepam was higher than 6 hours later ( p = 0.043 ) .", "the visual recognition task ( silhouette test ) showed higher performance at 6 hours than at 30 minutes for the immediate ( p = 0.018 ) and delayed ( p = 0.028 ) retrievals ( table 2 ) .", "however , no significant difference was observed for the comparison of the immediate and delayed recalls 30 minutes and 6 hours after drug intake ( p = 0.139 and p = 0.234 , respectively ) . in the placebo group ,", "the score of immediate recall of the short history at 6 hours after medication was higher than that at 30 minutes ( p = 0.012 ) , as was the delayed retrieval ( p = 0.036 ) .", "there was no significant difference between the immediate and delayed retrievals either 30 minutes or 6 hours after medication ( p = 0.753 and p = 0.655 , respectively ) .", "immediate performance in the visual recognition task ( silhouette test ) after 6 hours was higher than at the 30-minute session ( p = 0.030 ) .", "the comparison of performances between immediate and delayed at 30 minutes and at 6 hours ( p = 0.484 and p = 0.675 , respectively ) and between the delayed recognitions ( p = 0.183 ) showed no statistical differences ( table 2 ) .", "the enhancement of emotionally positive tasks in the diazepam group relative to the placebo group may suggest improvement of the retrieval of information by diazepam .", "improvement of retrieval by benzodiazepines has been observed ( izquierdo and chaves 1988 ; chaves et al 1990 ) , and it was hypothesized that the phenomenon would not be a true facilitation of retrieval processes , but the result of reduced interference from items presented after drug administration and thus a secondary consequence of drug - induced amnesia ( retroactive interference ) ( loftus and palmer 1974 ; chaves et al 1990 ) . because of the small sample size and the relatively large number of comparisons that were carried out , consideration of our results should take these limitations into account .", "for that reason , selection of the statistical techniques ( analyses and alpha control ) was especially careful .", "however , our results are provocative and raise an interesting hypothesis . comparisons between diazepam and placebo groups showed no difference on non - emotional memory tests .", "patients did not present anterograde amnesia following administration of diazepam ; however , this effect has been demonstrated in normal volunteers and animals ( sutton et al 1988 ; zuardi et al 1993 ) .", "benzodiazepine effects are mediated through the gaba(a ) complex by enhancing gaba - induced synaptic inhibition .", "as the gabaergic system in the amygdaloid complex is a site of action for the anxiolytic effects of benzodiazepines , it has been suggested that benzodiazepines may also influence memory through the amygdala .", "lesions in the amygdaloid complex can block diazepam - induced retention deficits , and central and lateral , but not basolateral , amygdala nuclei lesions impaired retention ( tomaz et al 1993 ) .", "the amygdala is a key structure in the brain s integration of emotional meaning with perception and experience and has been implicated in the pathophysiology of major depression ( drevets 1999 ; bremner et al 2000 ) .", "there is growing interest in understanding brain mechanisms of memory formation for emotionally arousing events , a development closely related to renewed interest in the concept of memory consolidation .", "there is little doubt that memory for emotionally arousing events is better than for neutral stimuli .", "this is clearly adaptive , because emotional stimuli , whether pleasant or aversive , are generally more important to species survival ( hamann et al 1999 ) .", "most current evidence indicates that the amygdala is not a site of storage of memory processes but plays a key role in modulation of emotional memory for both human ( mcgaugh et al 1996 ; cahill and mcgaugh 1998 ) and nonhuman subjects ( barros et al 1999 ; bianchin et al 1999 ) .", "long - term , but not short - term , memory has been shown to be enhanced by emotional arousal ( quevedo et al 2003 ) .", "depression may affect the amygdala noradrenergic modulation of memory and , through the noradrenergic / gabaergic connection , may modify the anterograde amnestic effect of benzodiazepines .", "functional neuro - imaging studies of the anatomical correlates of familial major depressive disorder and bipolar disorder have identified abnormalities of resting blood flow and glucose metabolism in depression in the amygdala and the orbital and medial prefrontal cortical areas that are extensively connected with the amygdala ( drevets 1999 ) .", "the amygdala metabolism in major depression and bipolar disorder is positively correlated with both depression severity and stressed plasma cortisol concentrations measured during scanning .", "thus , a neural model of a dysfunction of limbic prefrontal cortical structures impairing modulation of the amygdala in major depression , leading to abnormal processing of emotional stimuli , may be considered .", "consequently , the action of diazepam on the amygdala , which has been proposed to be the basis of its anxiolytic action , might be altered , modifying the modulation of memory in our patients .", "substantial evidence from animal and human subject studies converges on the view that memory for emotionally arousing events is modulated by an endogenous memory - modulating system consisting , at a minimum , of stress hormones and the amygdaloid complex . within the normal range of emotions experienced", ", this system is viewed as an evolutionarily adaptive method of creating memory strength that is , in general , proportional to memory importance ( cahill 1997 ) ." ]

[ "<S> with the hypothesis that depression affects memory through a mechanism other than that of the benzodiazepines , the present study evaluated the acute effect of diazepam 10 mg upon explicit memory in patients with major depression . </S>", "<S> a double - blind , placebo ( starch 50 mg ) controlled experiment was carried out with 19 patients randomly divided into diazepam ( n = 10 ) and placebo ( n = 9 ) groups . </S>", "<S> they were evaluated by the mini - mental state examination , and tests were conducted for immediate and delayed ( short - term ) memory with emotionally toned stimuli ( negative , positive , neutral ) , recognition , and semantic memory in visual or auditory modality . </S>", "<S> the visual analog mood scale ( vams ) was applied to measure anxiety and mood changes after the administration of drugs ( 30 minutes and 6 hours ) . </S>", "<S> higher scores in the positively toned list among patients who received diazepam were observed , at the 30-minute compared with the 6-hour evaluation . </S>", "<S> the recall index of positive words in the diazepam group was positive and significantly different from the index of the placebo group . </S>", "<S> no anterograde amnesia following diazepam was observed . the neural model of a dysfunction of limbic prefrontal cortical structures that impairs the modulation of the amygdala in major depression may explain the present results . </S>", "<S> consequently , the action of diazepam on the amygdala , which has been proposed to be the basis of its anxiolytic action , might be altered , modifying the modulation of memory in our patients . </S>" ]

[ "Introduction", "Methods", "Statistical analysis", "Results", "Discussion" ]

[ [ "there is extensive evidence that benzodiazepines , the most widely prescribed psychotropic drug class ( greenblatt et al 1983 ) , induce anterograde amnesia in both humans and animals ( lister 1985 ; thiebot 1985 ) . the findings of several experiments suggest that the anxiolytic properties of benzodiazepines involve effects mediated by the amygdala .", "intra - amygdala injections of benzodiazepines produce anxiolytic effects comparable to those induced by systemic injections ( nagy et al 1979 ; scheel - krger and petersen 1982 ; shibata et al 1982 ; niehoff and kuhar 1983 ; petersen et al 1985 ) .", "furthermore , intra - amygdala injections of the benzodiazepine antagonist flumazenil attenuate the anxiolytic effects of systemically administered benzodiazepines ( hodges et al 1987 ) .", "recent findings by izquierdo and colleagues ( 1990 ) suggest that benzodiazepine impairment of memory involves gabaergic type a receptors in the amygdala .", "post - training intra - amygdala injection of flumazenil causes memory facilitation comparable to that found with systemic injections , and systemic injection of flumazenil before training attenuates the amnestic effects of post - training intra - amygdala injection of muscimol .", "studies examining the memory - modulating effects of drug treatments have provided evidence that memory can be modulated by systemic as well as intra - amygdala gabaergic compounds . when administered shortly after training , gabaergic agonists ( eg , muscimol and baclofen ) impair memory retention , while gabaergic antagonists ( eg , picrotoxin and bicuculline ) enhance retention ( breen and mcgaugh 1961 ; brioni and mcgaugh 1988 ; brioni et al 1989 ; castellano et al 1989 ; ammassari - teule et al 1991 ) . on the other hand , there is extensive evidence indicating a key role for gaba neurotransmission in the modulation of fearful / defensive behaviors .", "it has been found that systemic or intra - amygdala injections of gaba agonists reduce and gaba antagonists enhance experimental fear and anxiety ( graeff 1990 ) .", "furthermore , lesions of the amygdala attenuate the antianxiety as well as the memory - modulating effects of gabaergic drugs ( shibata et al 1989 ; ammassari - teule et al 1991 ) . a recent study with magnetic resonance spectroscopy revealed low gaba levels in the occipital cortex of depressed patients ,", "cortical benzodiazepine binding to gaba(a ) receptors has been measured with i - labeled flumazenil and single photon emission computed tomography in unmedicated patients with major depression and healthy volunteers ( kugaya et al 2003 ) .", "depression is currently seen as a chronic medical disorder that produces as much functional limitation and morbidity as chronic diseases such as hypertension and diabetes ( angst 1999 ) .", "one of the most frequent and neuropsychologically well investigated symptoms in depression is reduced memory capacity ( burt et al 1995 ; ilsley et al 1995 ) .", "the condition is thought to result from dysfunctions in monoaminergic systems affecting norepinephrine , serotonin , and dopamine at several effector sites .", "disturbances of the limbic hypothalamic pituitary adrenal axis and the serotonin system were found , and changes in adrenoceptors associated with the pituitary adrenal axis function strongly implicate a disorder in central noradrenergic transmission ( leonard 2000 ) .", "the effect of corticotropin - releasing factor in modulating the activity of noradrenergic neurons in the locus ceruleus may provide a link between environmental trigger factors and central noradrenergic dysfunction ( leonard 2000 ) .", "we hypothesized that depression may affect the amygdala noradrenergic modulation of memory and , through the noradrenergic / gabaergic connection , may modify the anterograde amnestic effect of benzodiazepines .", "furthermore , the action of benzodiazepines may block the negative tendency on emotional memory tasks described for depressed patients .", "the purpose of the present study was evaluation of the effect of diazepam on explicit memory ( emotional and non - emotional tasks ) in patients with major depression who were not previously receiving benzodiazepines ." ], [ "a double - blind , placebo - controlled experiment with diazepam 10 mg ( novaquim - sigma ) was carried out with dsm - iv major depression patients ( by certificated psychiatrists ) during the first 24 hours after admission in the psychiatric unit of a general university hospital .", "exclusion criteria were psychotic symptoms , other psychiatric comorbidity ( eg , alcohol and drug abuse ) , long - term use of benzodiazepines ( last 30 days ) , cognitive deficit , use of tricyclic antidepressants , and previous ect .", "cognitive status was checked with the mini - mental state examination ( folstein et al 1975 ) with cut - offs 24 and 17 for education of > 4 and 4 years , respectively ( baseline mini - mental ) . during this period ,", "drug groups did not differ in age , education , and sex distributions ( table 1 ) .", "demographic variables and baseline mini - mental ( mm ) of the studied groups ; mean and standard deviation ( range ) chi - square ( fisher s test ) .", "all patients signed a written consent after the nature of procedures and objectives of the study were explained .", "only patients who were not taking benzodiazepines were selected for the study ( before and after hospitalization ) .", "there were no group differences ( diazepam versus placebo ) in the dose or type of antidepressant being taken .", "patients who presented severe insomnia received promethazine 25 mg , whereas anxiety was behaviorally handled .", "all patients were given either placebo ( 50 mg of starch ) or diazepam ( 10 mg ) on the morning of the fourth day ( figure 1 ) .", "the drugs were given orally in enteric capsules , and patients were assigned to the two groups by a double - blind design .", "the medications were specially processed for the experiment in the hospital pharmacy , where they were assembled in identical capsules .", "only one member of the team carried out the randomization and kept the codes of identification of the medications .", "those who applied tests and gave medication to patients did not know which was being administered to each patient , and patients did not know which medication they were receiving .", "thirty minutes and 6 hours after drug or placebo administration , patients were exposed to an emotionally neutral word list , and to two closely matched and emotionally arousing lists negative and positive ( ceitlin et al 1995 ) .", "also administered were the logical memory ( subtest of the wechsler memory scale ; a neutral short story ) ( wechsler 1973 ) and the non - verbal silhouette test ( black images of universally recognized buildings ) ( rosat et al 1990 ) .", "two sets of equivalent tests ( word lists , short story , and silhouettes ) were used in the study .", "these tests for explicit memory were selected because they evaluate immediate and delayed ( short - term ) memory , recognition , and semantic memory in visual or auditory modality and were well studied in the population from which the sample was recruited .", "the brazilian portuguese words were obtained in two studies ( ceitlin et al 1995 ) for the development of emotionally negative , positive , and neutral lists .", "the emotional content of the words , effect on other words in mixed lists , and effect of age , education , and symptoms of depression were evaluated before the final lists were achieved .", "the portuguese idiom in brazil still lacks population studies on quantitative linguistics ( such as word frequency and age of acquisition ) .", "therefore , the ceitlin study covered most of these factors for generation of the present word lists .", "the effect of the emotionally arousing word lists on a patient s subjective states ( mood and anxiety ) was evaluated by the visual analog mood scale ( vams ) ( norris 1971 ) , which consists of 16 analog items composed of two adjectives with opposite feelings , separated by a 10-cm line on which the subject has to mark the point which best describes his / her feelings at the time .", "these items were combined into four factors ( anxiety : calm excited , relaxed ", "feeble ; mental sedation : alert drowsy , attentive dreamy ; other feelings and attitudes : interested bored , amicable antagonistic , happy sad , contented discontented ) according to a factorial analysis performed on a brazilian sample ( zuardi et al 1993 ) .", "all procedures were executed by the statistical package for the social sciences ( spss / pc plus ) and epi - info 6.4 .", "parametric data were analyzed by student s t - test for independent samples and by multivariate procedures of manova .", "categories were tested by analysis of association ( chi - square test with yates correction or fisher s exact test ) .", "neuropsychological scores were submitted to a pegboard test ( normal probability plot ) before being analyzed by manova ( either between or within effects ) .", "the univariate analysis within the multivariate procedure was family - wise controlled for alpha values , and two - tailed significance values were chosen . an index of variation ( rate ) for the word lists was calculated from the recalls at 30 minutes and 6 hours , based on the calculation for rate of forgetfulness ( isaac and mayes 1999 ) : where xi is the score of recall 30 minutes after drug intake and xii of recall at 6 hours .", "this rate may enhance the effect of the intervention because both retrievals occurred after exposure to the list ( auditory task ) ; consequently , second recall was more likely to be higher owing to the learning effect .", "in the case of a positive index , first retrieval was higher than second , expressing a greater effect of the drug than of practice ." ], [ "all procedures were executed by the statistical package for the social sciences ( spss / pc plus ) and epi - info 6.4 .", "parametric data were analyzed by student s t - test for independent samples and by multivariate procedures of manova .", "categories were tested by analysis of association ( chi - square test with yates correction or fisher s exact test ) .", "neuropsychological scores were submitted to a pegboard test ( normal probability plot ) before being analyzed by manova ( either between or within effects ) .", "the univariate analysis within the multivariate procedure was family - wise controlled for alpha values , and two - tailed significance values were chosen .", "an index of variation ( rate ) for the word lists was calculated from the recalls at 30 minutes and 6 hours , based on the calculation for rate of forgetfulness ( isaac and mayes 1999 ) : where xi is the score of recall 30 minutes after drug intake and xii of recall at 6 hours .", "this rate may enhance the effect of the intervention because both retrievals occurred after exposure to the list ( auditory task ) ; consequently , second recall was more likely to be higher owing to the learning effect . in the case of a positive index ,", "first retrieval was higher than second , expressing a greater effect of the drug than of practice ." ], [ "performance on word lists with positive emotional content showed a statistically significant score gradient from 30 minutes to 6 hours after drug administration only in the group of patients who received diazepam ( figure 2 ) .", "patients who received diazepam presented higher and more positive indexes for positive words , while negative and neutral words presented negative rates .", "although not statistically significant , the index negativity was lower for negative and higher for neutral words .", "we observed a very large standard deviation for recall of neutral words ; however , individual scores of this word list were all negative within the diazepam group and both negative and positive within the placebo group .", "for positive words , the gradient showed the least variation among the diazepam group , significantly less than in placebo patients .", "these findings may suggest a facilitatory effect of diazepam ( 10 mg ) on the recall of positive words ( 30 minutes after administration ) . at the 6-hour evaluation , performance on the positively loaded words decreased to a similar level to that of the placebo group . mean and standard deviation of rate of recall of the word lists emotionally toned ( positive , negative , neutral ) of patients on diazepam and on placebo .", "the scores of the vams were not different within each group ( p > 0.05 ) or between groups ( p > 0.05 ) .", "therefore , the significant rate of recall for positively loaded words among diazepam patients could not be explained by lower levels of anxiety or other symptoms .", "patients , either diazepam or placebo , showed similar levels of symptoms at the two time - points .", "no parallel improvement on other immediate recall was seen at 30 minutes relative to 6 hours after dosing ( diazepam group ) .", "the score of the immediate recall of the short history at 30 minutes was lower than at 6 hours ( p = 0.035 ) ( table 2 ) .", "there was no difference between the immediate and delayed retrievals of this test at 30 minutes after medication ( p = 0.361 ) .", "however , at the 6-hour evaluation , immediate recall was higher than delayed ( p = 0.028 ) . mean and standard deviation ( range ) of scores of tests without emotional content and with immediate ( i ) and delayed ( d ) recalls within - group comparison .", "p = 0.028 ( immediate > delayed recall short story at 6 hours ) . for the positive word list , the score during the effect of diazepam was higher than 6 hours later ( p = 0.043 ) .", "the visual recognition task ( silhouette test ) showed higher performance at 6 hours than at 30 minutes for the immediate ( p = 0.018 ) and delayed ( p = 0.028 ) retrievals ( table 2 ) .", "however , no significant difference was observed for the comparison of the immediate and delayed recalls 30 minutes and 6 hours after drug intake ( p = 0.139 and p = 0.234 , respectively ) . in the placebo group ,", "the score of immediate recall of the short history at 6 hours after medication was higher than that at 30 minutes ( p = 0.012 ) , as was the delayed retrieval ( p = 0.036 ) .", "there was no significant difference between the immediate and delayed retrievals either 30 minutes or 6 hours after medication ( p = 0.753 and p = 0.655 , respectively ) .", "immediate performance in the visual recognition task ( silhouette test ) after 6 hours was higher than at the 30-minute session ( p = 0.030 ) .", "the comparison of performances between immediate and delayed at 30 minutes and at 6 hours ( p = 0.484 and p = 0.675 , respectively ) and between the delayed recognitions ( p = 0.183 ) showed no statistical differences ( table 2 ) ." ], [ "the enhancement of emotionally positive tasks in the diazepam group relative to the placebo group may suggest improvement of the retrieval of information by diazepam .", "improvement of retrieval by benzodiazepines has been observed ( izquierdo and chaves 1988 ; chaves et al 1990 ) , and it was hypothesized that the phenomenon would not be a true facilitation of retrieval processes , but the result of reduced interference from items presented after drug administration and thus a secondary consequence of drug - induced amnesia ( retroactive interference ) ( loftus and palmer 1974 ; chaves et al 1990 ) . because of the small sample size and the relatively large number of comparisons that were carried out , consideration of our results should take these limitations into account .", "for that reason , selection of the statistical techniques ( analyses and alpha control ) was especially careful .", "however , our results are provocative and raise an interesting hypothesis . comparisons between diazepam and placebo groups showed no difference on non - emotional memory tests .", "patients did not present anterograde amnesia following administration of diazepam ; however , this effect has been demonstrated in normal volunteers and animals ( sutton et al 1988 ; zuardi et al 1993 ) .", "benzodiazepine effects are mediated through the gaba(a ) complex by enhancing gaba - induced synaptic inhibition .", "as the gabaergic system in the amygdaloid complex is a site of action for the anxiolytic effects of benzodiazepines , it has been suggested that benzodiazepines may also influence memory through the amygdala .", "lesions in the amygdaloid complex can block diazepam - induced retention deficits , and central and lateral , but not basolateral , amygdala nuclei lesions impaired retention ( tomaz et al 1993 ) .", "the amygdala is a key structure in the brain s integration of emotional meaning with perception and experience and has been implicated in the pathophysiology of major depression ( drevets 1999 ; bremner et al 2000 ) .", "there is growing interest in understanding brain mechanisms of memory formation for emotionally arousing events , a development closely related to renewed interest in the concept of memory consolidation .", "there is little doubt that memory for emotionally arousing events is better than for neutral stimuli .", "this is clearly adaptive , because emotional stimuli , whether pleasant or aversive , are generally more important to species survival ( hamann et al 1999 ) .", "most current evidence indicates that the amygdala is not a site of storage of memory processes but plays a key role in modulation of emotional memory for both human ( mcgaugh et al 1996 ; cahill and mcgaugh 1998 ) and nonhuman subjects ( barros et al 1999 ; bianchin et al 1999 ) .", "long - term , but not short - term , memory has been shown to be enhanced by emotional arousal ( quevedo et al 2003 ) .", "depression may affect the amygdala noradrenergic modulation of memory and , through the noradrenergic / gabaergic connection , may modify the anterograde amnestic effect of benzodiazepines .", "functional neuro - imaging studies of the anatomical correlates of familial major depressive disorder and bipolar disorder have identified abnormalities of resting blood flow and glucose metabolism in depression in the amygdala and the orbital and medial prefrontal cortical areas that are extensively connected with the amygdala ( drevets 1999 ) .", "the amygdala metabolism in major depression and bipolar disorder is positively correlated with both depression severity and stressed plasma cortisol concentrations measured during scanning .", "thus , a neural model of a dysfunction of limbic prefrontal cortical structures impairing modulation of the amygdala in major depression , leading to abnormal processing of emotional stimuli , may be considered .", "consequently , the action of diazepam on the amygdala , which has been proposed to be the basis of its anxiolytic action , might be altered , modifying the modulation of memory in our patients .", "substantial evidence from animal and human subject studies converges on the view that memory for emotionally arousing events is modulated by an endogenous memory - modulating system consisting , at a minimum , of stress hormones and the amygdaloid complex . within the normal range of emotions experienced", ", this system is viewed as an evolutionarily adaptive method of creating memory strength that is , in general , proportional to memory importance ( cahill 1997 ) ." ] ]

[ "syphilis is a chronic systemic infectious disease caused by treponema pallidum , a microaerophilic spirochete , whose transmission occurs mainly by sexual contact .", "its course is characterized by a multistage evolution , in a total of 4 distinct clinical stages in cases of non - treated disease , in which symptomatic phases alternate with periods of latency .", "the myriad of possible clinical manifestations often represent a great challenge , a fact that led sir william osler to label it as the great imitator. in this report , we present 2 cases in which the clinical manifestations of the patients initially simulated other dermatologic diseases , giving the impression of mucha - habermann disease ( pityriasis lichenoides et varioliformis acuta ) , in the first case and reiter 's syndrome ( reactive arthritis ) , in the second .", "male patient , 18 years old , white , single , student , born in and resident of rio de janeiro .", "he presented with a 1-month history of cutaneous lesions , jaundice , darkened urine and acholic stools .", "there was lesional polymorphism with lesions varying from erythematous papules and pustules to ulceronecrotic lesions with disseminated crusts on the face , trunk and limbs ( fig .", "1 ) . physical examination revealed jaundice ( + + /4 + ) and bilateral inguinal lymphadenopathy .", "the exams showed positive serology for hepatitis b and alteration of the hepatic function tests .", "the initial impression of cutaneous vasculitis was not confirmed and clinical data , serologic tests and histologic findings led to the diagnosis of early malignant syphilis in an immunocompetent patient .", "male patient , 19 years old , single , student , born in and resident of rio de janeiro , presenting with conjunctival hyperemia and eyelid swelling on the right side for almost 2 years , which initially improved with steroid eye drops .", "later , there was progressive loss of visual acuity and onset of erythematous scaly lesions on the buttocks , palms and soles , besides erosions on the glans and inside the oral cavity ( fig .", "3 ) . during the 6-month period preceding the medical consultation , arthralgia appeared on the knees , left sacroiliac articulation and elbows , accompanied by headache .", "the ophthalmological examination revealed panuveitis and the dermatological examination revealed keratoderma palmoplantaris , diffuse erythema on the trunk , scrotum erythema and erosion , circular residual violaceous erythema on the glans , anonychia and onychodystrophy , which hinted at the diagnosis of reactive arthritis .", "male patient , 18 years old , white , single , student , born in and resident of rio de janeiro .", "he presented with a 1-month history of cutaneous lesions , jaundice , darkened urine and acholic stools .", "there was lesional polymorphism with lesions varying from erythematous papules and pustules to ulceronecrotic lesions with disseminated crusts on the face , trunk and limbs ( fig .", "1 ) . physical examination revealed jaundice ( + + /4 + ) and bilateral inguinal lymphadenopathy .", "the exams showed positive serology for hepatitis b and alteration of the hepatic function tests .", "the initial impression of cutaneous vasculitis was not confirmed and clinical data , serologic tests and histologic findings led to the diagnosis of early malignant syphilis in an immunocompetent patient .", "male patient , 19 years old , single , student , born in and resident of rio de janeiro , presenting with conjunctival hyperemia and eyelid swelling on the right side for almost 2 years , which initially improved with steroid eye drops .", "later , there was progressive loss of visual acuity and onset of erythematous scaly lesions on the buttocks , palms and soles , besides erosions on the glans and inside the oral cavity ( fig .", "3 ) . during the 6-month period preceding the medical consultation , arthralgia appeared on the knees , left sacroiliac articulation and elbows , accompanied by headache . the ophthalmological examination revealed panuveitis and the dermatological examination revealed keratoderma palmoplantaris , diffuse erythema on the trunk , scrotum erythema and erosion , circular residual violaceous erythema on the glans , anonychia and onychodystrophy , which hinted at the diagnosis of reactive arthritis .", "syphilis is characterized not only for evolving in distinct clinical stages , but also for having the ability to mimic a wide variety of diseases , in each of its phases . in primary syphilis ,", "localized erythema develops at the site of inoculation , which evolves into hardened and painless papules .", "classically , after the surface necrosis , the typical well - circumscribed ulceration develops , with hardened borders and clear base the chancre associated with regional adenopathy .", "primary syphilis , however , may present atypical morphology , location and symptoms , causing diagnostic difficulties , which results in only 3040% of patients being diagnosed in the primary stage .", "extragenital chancres can occur in any mucocutaneous surface exposed to the infection , but are more common in the oral cavity and anal region .", "any ulcerated nodular lesion associated to lymph adenopathy should lead to the suspicion of primary syphilis .", "differential diagnosis must be made with other infections , including tularemia ; cat - scratch disease ; sporotrichosis ; mycobacteriosis ; leishmaniasis and staphylococcal lymphangitis ; and with granulomatous diseases and neoplasms with nodal metastasis . in the anal region , it is rarely restricted only to a nodular hardening and can be accompanied by fissures , which leads to confusion with hemorrhoids , anal fissures or even neoplasms .", "features vary according to the number of inoculated spirochetes , concomitant use of antibiotics , presence of co - infections , and the patient 's immune state [ 1 , 4 ] .", "the secondary stage results from the hematogenous and lymphatic dissemination and multiplication of the microorganism in different tissues .", "these may be subtle , transient and pass unnoticed ; or so severe as to require hospitalization .", "however , over 90% of patients present rash , which is almost always characteristic . at the beginning", ", there is a macular rash with discrete pink , non - scaling , oval lesions the syphilitic roseola , predominantly on the trunk and upper flexor areas of limbs .", "then , the lesions may develop into a papular - macular , papular - desquamative , lenticular , corymbose , nodular , annular , follicular , pustular or impetiginous aspect .", "secondary syphilis polymorphism depends entirely on the intensity of the inflammatory infiltrate , level of cutaneous vascular involvement and the resulting ischemia . when papular lesions are pruritic and lichenoid , it may be difficult to differentiate from lichen planus .", "annular lesions may also resemble annular granuloma , pityriasis rosea and dermatophytosis . where there is a hyper keratotic component , the resulting lesions are indistinguishable from psoriasis [ 5 , 8 ] .", "hyperkeratotic plaques on the soles give the impression of calluses and , when desquamative , can mimic tinea pedis [ 1 , 5 ] .", "the differential diagnosis of nodular syphilis includes systemic mycosis , kaposi 's sarcoma , bacillary angiomatosis , foreign body granuloma type , lymphoma , pseudolymphoma , leprosy , sarcoidosis , and halogenoderma .", "secondary syphilis with pustular lesions can also lead to the erroneous diagnosis of pustular acne [ 8 , 9 ] .", "mucosal involvement can be part of the condition , consisting mainly of mucous patches , pharyngitis and condyloma lata .", "hair can be affected on the scalp , eyebrows and beard , and diffuse pattern alopecia increases the index of suspicion for the disease .", "the latter may be similar to alopecia areata , neoplastic alopecia , tinea capitis or trichotillomania .", "ungual disorders include fissures , beau lines , onycholysis , onychomadesis , onychogryphosis , nail pitting and others [ 1 , 6 ] . completing the picture", ", general symptoms may occur and rarely hepatitis , periostitis , arthritis , gastritis , meningitis and uveitis . in the second case reported here , the ocular and articular involvements associated with the mucocutaneous lesions led to a first diagnostic impression of reactive arthritis .", "this syndrome is characterized by the classical triad of arthritis , urethritis and conjunctivitis , after urethral or gastrointestinal infection . despite conjunctivitis", "being classically described in the syndrome , there are other associated ocular involvements , such as iritis and uveitis , the latter being diagnosed in our patient .", "that young man also presented , at the dermatological examination , keratoderma palmoplantaris and erosions on the glans , simulating , respectively , keratoderma blenorrhagica and circinate balanitis , and erosive lesions of the oral cavity .", "all these findings have been described in up to 20% of patients with reactive arthritis .", "a variant of secondary syphilis known as early malignant syphilis is characterized by a fast progression of papules , plaques , nodules , and polymorphic vesicular pustules with tendency to ulceration , covered with several layers of thick crusts , acquiring a rupioid aspect .", "however , at first glance , the acute onset and the exuberant features of the lesions led to the diagnostic hypothesis of pityriasis lichenoides et varioliformis acuta .", "also known as mucha - habermann disease , this disease is characterized by the appearance of generalized erythematous - purpuric papules that evolve with necrosis .", "finally , untreated syphilis evolves into a third form , very rare nowadays , with a variety of manifestations that occur months to years after the beginning of the infection .", "this stage is characterized by the presence of a small number of treponema , but with high cellular immune reactivity against this agent .", "microorganisms can invade different systems and lead to injury by a delayed type of hypersensitivity reaction .", "syphilis presents a highly variable clinical course and wide diversity of manifestations , which has made it known as the great mimicker .", "its ability to simulate various other diseases often causes its diagnosis to be a challenge .", "however , the lack of diagnosis of this quite prevalent disease can entail serious consequences for the patient .", "thus , it is of considerable importance that dermatologists and other specialists be familiarized with the many manifestations of syphilis and start considering it more often among their diagnostic hypotheses ." ]

[ "<S> the authors present two cases of syphilis : one mimicking reactive arthritis and the other mucha - habermann disease . </S>", "<S> both reports illustrate syphilis as the great imitator , a description given by sir william osler , and call attention to the strong need for awareness among physicians of all specialties , especially the younger ones , who are not used to seeing this increasingly prevalent disease , as it once was in the past . </S>" ]

[ "Introduction", "Case Reports", "Patient 1", "Patient 2", "Discussion", "Conclusion" ]

[ [ "syphilis is a chronic systemic infectious disease caused by treponema pallidum , a microaerophilic spirochete , whose transmission occurs mainly by sexual contact .", "its course is characterized by a multistage evolution , in a total of 4 distinct clinical stages in cases of non - treated disease , in which symptomatic phases alternate with periods of latency .", "the myriad of possible clinical manifestations often represent a great challenge , a fact that led sir william osler to label it as the great imitator. in this report , we present 2 cases in which the clinical manifestations of the patients initially simulated other dermatologic diseases , giving the impression of mucha - habermann disease ( pityriasis lichenoides et varioliformis acuta ) , in the first case and reiter 's syndrome ( reactive arthritis ) , in the second ." ], [ "male patient , 18 years old , white , single , student , born in and resident of rio de janeiro .", "he presented with a 1-month history of cutaneous lesions , jaundice , darkened urine and acholic stools .", "there was lesional polymorphism with lesions varying from erythematous papules and pustules to ulceronecrotic lesions with disseminated crusts on the face , trunk and limbs ( fig .", "1 ) . physical examination revealed jaundice ( + + /4 + ) and bilateral inguinal lymphadenopathy .", "the exams showed positive serology for hepatitis b and alteration of the hepatic function tests .", "the initial impression of cutaneous vasculitis was not confirmed and clinical data , serologic tests and histologic findings led to the diagnosis of early malignant syphilis in an immunocompetent patient .", "male patient , 19 years old , single , student , born in and resident of rio de janeiro , presenting with conjunctival hyperemia and eyelid swelling on the right side for almost 2 years , which initially improved with steroid eye drops .", "later , there was progressive loss of visual acuity and onset of erythematous scaly lesions on the buttocks , palms and soles , besides erosions on the glans and inside the oral cavity ( fig .", "3 ) . during the 6-month period preceding the medical consultation , arthralgia appeared on the knees , left sacroiliac articulation and elbows , accompanied by headache .", "the ophthalmological examination revealed panuveitis and the dermatological examination revealed keratoderma palmoplantaris , diffuse erythema on the trunk , scrotum erythema and erosion , circular residual violaceous erythema on the glans , anonychia and onychodystrophy , which hinted at the diagnosis of reactive arthritis ." ], [ "male patient , 18 years old , white , single , student , born in and resident of rio de janeiro .", "he presented with a 1-month history of cutaneous lesions , jaundice , darkened urine and acholic stools .", "there was lesional polymorphism with lesions varying from erythematous papules and pustules to ulceronecrotic lesions with disseminated crusts on the face , trunk and limbs ( fig .", "1 ) . physical examination revealed jaundice ( + + /4 + ) and bilateral inguinal lymphadenopathy .", "the exams showed positive serology for hepatitis b and alteration of the hepatic function tests .", "the initial impression of cutaneous vasculitis was not confirmed and clinical data , serologic tests and histologic findings led to the diagnosis of early malignant syphilis in an immunocompetent patient ." ], [ "male patient , 19 years old , single , student , born in and resident of rio de janeiro , presenting with conjunctival hyperemia and eyelid swelling on the right side for almost 2 years , which initially improved with steroid eye drops .", "later , there was progressive loss of visual acuity and onset of erythematous scaly lesions on the buttocks , palms and soles , besides erosions on the glans and inside the oral cavity ( fig .", "3 ) . during the 6-month period preceding the medical consultation , arthralgia appeared on the knees , left sacroiliac articulation and elbows , accompanied by headache . the ophthalmological examination revealed panuveitis and the dermatological examination revealed keratoderma palmoplantaris , diffuse erythema on the trunk , scrotum erythema and erosion , circular residual violaceous erythema on the glans , anonychia and onychodystrophy , which hinted at the diagnosis of reactive arthritis ." ], [ "syphilis is characterized not only for evolving in distinct clinical stages , but also for having the ability to mimic a wide variety of diseases , in each of its phases . in primary syphilis ,", "localized erythema develops at the site of inoculation , which evolves into hardened and painless papules .", "classically , after the surface necrosis , the typical well - circumscribed ulceration develops , with hardened borders and clear base the chancre associated with regional adenopathy .", "primary syphilis , however , may present atypical morphology , location and symptoms , causing diagnostic difficulties , which results in only 3040% of patients being diagnosed in the primary stage .", "extragenital chancres can occur in any mucocutaneous surface exposed to the infection , but are more common in the oral cavity and anal region .", "any ulcerated nodular lesion associated to lymph adenopathy should lead to the suspicion of primary syphilis .", "differential diagnosis must be made with other infections , including tularemia ; cat - scratch disease ; sporotrichosis ; mycobacteriosis ; leishmaniasis and staphylococcal lymphangitis ; and with granulomatous diseases and neoplasms with nodal metastasis . in the anal region , it is rarely restricted only to a nodular hardening and can be accompanied by fissures , which leads to confusion with hemorrhoids , anal fissures or even neoplasms .", "features vary according to the number of inoculated spirochetes , concomitant use of antibiotics , presence of co - infections , and the patient 's immune state [ 1 , 4 ] .", "the secondary stage results from the hematogenous and lymphatic dissemination and multiplication of the microorganism in different tissues .", "these may be subtle , transient and pass unnoticed ; or so severe as to require hospitalization .", "however , over 90% of patients present rash , which is almost always characteristic . at the beginning", ", there is a macular rash with discrete pink , non - scaling , oval lesions the syphilitic roseola , predominantly on the trunk and upper flexor areas of limbs .", "then , the lesions may develop into a papular - macular , papular - desquamative , lenticular , corymbose , nodular , annular , follicular , pustular or impetiginous aspect .", "secondary syphilis polymorphism depends entirely on the intensity of the inflammatory infiltrate , level of cutaneous vascular involvement and the resulting ischemia . when papular lesions are pruritic and lichenoid , it may be difficult to differentiate from lichen planus .", "annular lesions may also resemble annular granuloma , pityriasis rosea and dermatophytosis . where there is a hyper keratotic component , the resulting lesions are indistinguishable from psoriasis [ 5 , 8 ] .", "hyperkeratotic plaques on the soles give the impression of calluses and , when desquamative , can mimic tinea pedis [ 1 , 5 ] .", "the differential diagnosis of nodular syphilis includes systemic mycosis , kaposi 's sarcoma , bacillary angiomatosis , foreign body granuloma type , lymphoma , pseudolymphoma , leprosy , sarcoidosis , and halogenoderma .", "secondary syphilis with pustular lesions can also lead to the erroneous diagnosis of pustular acne [ 8 , 9 ] .", "mucosal involvement can be part of the condition , consisting mainly of mucous patches , pharyngitis and condyloma lata .", "hair can be affected on the scalp , eyebrows and beard , and diffuse pattern alopecia increases the index of suspicion for the disease .", "the latter may be similar to alopecia areata , neoplastic alopecia , tinea capitis or trichotillomania .", "ungual disorders include fissures , beau lines , onycholysis , onychomadesis , onychogryphosis , nail pitting and others [ 1 , 6 ] . completing the picture", ", general symptoms may occur and rarely hepatitis , periostitis , arthritis , gastritis , meningitis and uveitis . in the second case reported here , the ocular and articular involvements associated with the mucocutaneous lesions led to a first diagnostic impression of reactive arthritis .", "this syndrome is characterized by the classical triad of arthritis , urethritis and conjunctivitis , after urethral or gastrointestinal infection . despite conjunctivitis", "being classically described in the syndrome , there are other associated ocular involvements , such as iritis and uveitis , the latter being diagnosed in our patient .", "that young man also presented , at the dermatological examination , keratoderma palmoplantaris and erosions on the glans , simulating , respectively , keratoderma blenorrhagica and circinate balanitis , and erosive lesions of the oral cavity .", "all these findings have been described in up to 20% of patients with reactive arthritis .", "a variant of secondary syphilis known as early malignant syphilis is characterized by a fast progression of papules , plaques , nodules , and polymorphic vesicular pustules with tendency to ulceration , covered with several layers of thick crusts , acquiring a rupioid aspect .", "however , at first glance , the acute onset and the exuberant features of the lesions led to the diagnostic hypothesis of pityriasis lichenoides et varioliformis acuta .", "also known as mucha - habermann disease , this disease is characterized by the appearance of generalized erythematous - purpuric papules that evolve with necrosis .", "finally , untreated syphilis evolves into a third form , very rare nowadays , with a variety of manifestations that occur months to years after the beginning of the infection .", "this stage is characterized by the presence of a small number of treponema , but with high cellular immune reactivity against this agent .", "microorganisms can invade different systems and lead to injury by a delayed type of hypersensitivity reaction ." ], [ "syphilis presents a highly variable clinical course and wide diversity of manifestations , which has made it known as the great mimicker .", "its ability to simulate various other diseases often causes its diagnosis to be a challenge .", "however , the lack of diagnosis of this quite prevalent disease can entail serious consequences for the patient .", "thus , it is of considerable importance that dermatologists and other specialists be familiarized with the many manifestations of syphilis and start considering it more often among their diagnostic hypotheses ." ] ]

[ "inflammatory bowel disease ( ibd ) is a common diagnostic and therapeutic problem , affecting patients at increasingly young ages .", "a standard diagnostic method includes macroscopic assessment of the intestinal mucosa during colonoscopy and histopathological evaluation of the obtained biopsies . according to porto criteria ,", "the diagnosis of ibd is based on the clinical presentation , intestinal endoscopic and histological features , laboratory tests , and radiological examination .", "recently , reports on the usefulness of a noninvasive examination , that is , faecal calprotectin measurement in ibd diagnosis , have been published .", "evaluation of faecal calprotectin ( fc ) seems to be a screening test selecting patients requiring further invasive diagnostics .", "furthermore , there have been reports on using calprotectin assays in monitoring the treatment of crohn 's disease and ulcerative colitis in adults and children [ 14 ] . to our knowledge ,", "there are no published data on the usefulness of faecal calprotectin assays in the diagnosis of other atypical forms of bowel inflammation , such as eosinophilic , lymphocytic , or nonspecific colitis .", "the aim of the study is to assess the usefulness of faecal calprotectin measurement in children with various types of ibd and to evaluate fc concentration in children with crohn 's disease and ulcerative colitis in relation to disease activity .", "91 patients were included in the analysis , including 49 boys ( 53.85% ) and 42 girls ( 46.15% ) , ranging from 6 to 18 years of age ( the mean age was 13.38 years ) .", "the study group comprised 71 children with various types of ibd , who were subsequently divided into six subgroups : b124 ( 33.8% ) children with crohn 's disease ( cd ) , b216 ( 22.5% ) with ulcerative colitis ( uc ) , b37 ( 9.8% ) with eosinophilic colitis ( ec ) , b48 ( 11.26% ) with lymphocytic colitis ( lc ) , and b516 ( 22.5% ) with nonspecific colitis ( nc , colitis indeterminata ", "the control group ( k ) comprised 20 healthy , age- and sex - matched subjects .", "patients with ibd underwent following procedures : anamnesis , physical examination , laboratory tests ( inflammatory state markers , biochemical parameters of liver , pancreas , and kidney function , sweat test , coprological tests , and immunoassays ) , diagnostic imaging ( abdominal ultrasound ) , and endoscopy with histopathological evaluation . in all patients ,", "faecal calprotectin was measured by means of elisa method , using phical test ( calpro ) .", "calprotectin concentrations ranging from 0 to 50 mg / kg were considered to be normal reference values .", "the results were evaluated by using the following tests : komogorow - smirnow , t - student , u mann - whitney , fisher , and yates ; the analysis of correlation was based on the spearman 's rank correlation coefficient . a p value of < 0.05 was considered statistically significant .", "all patients and their caregivers gave informed consent to participate in the study , which was approved by the bioethics committee of the medical university of silesia in katowice ( consent no .", "a statistically significant increase in the mean concentrations of faecal calprotectin was observed in the group of children with cd and uc , as compared to the control group .", "concentrations of fc were also higher in children with uc than in patients with cd .", "faecal calprotectin concentrations were within the normal limits in patients with eosinophilic , lymphocytic , and nonspecific colitis , similarly to the healthy subjects ( table 1 , figure 1 ) in children with cd , faecal calprotectin concentrations positively correlated with the disease severity assessed according to the pcdai scale . in patients suffering from uc , faecal calprotectin", "also positively correlated with the truelove - witts scale and the rachmilewitz endoscopic index ( figures 2 , 3 , and 4 ) . a significant increase in faecal calprotectin concentrations", "was observed in children suffering from cd , with lesions located in both small and large intestine , and in patients presenting with inflammatory changes in 5 or more sections of the intestine ( figure 5 ) .", "faecal calprotectin is a promising , noninvasive screening method for diagnosing patients suffering from gastrointestinal disorders , such as abdominal pain or diarrhea , which are also typical for ibd [ 57 ] .", "so far , many authors have considered calprotectin as a useful marker in differential diagnosis of ibd and functional gastrointestinal disorders ( e.g. , irritable bowel syndrome ) [ 8 , 9 ] .", "many authors evaluated faecal calprotectin concentrations in patients with suspected inflammatory process of the large intestine .", "demonstrated in the group of paediatric patients that this assay is characterised by 95% sensitivity and 93% specificity , and high calprotectin concentrations show strong positive correlation with the presence of inflammatory lesions in the large intestine . according to these authors", "so far , only limited studies , based on very small groups of patients , evaluated faecal calprotectin concentrations in patients with other types of bowel inflammations less common than cd and uc , such as lymphocytic , eosinophilic , and nonspecific colitis , which seem to be a considerable clinical problem in everyday pediatric practice . in studies conducted by bunn et al . , two children with nonspecific colitis and 3 children with allergic colitis were included in the analysis . in both cases ,", "calprotectin concentrations were found to be within the normal ranges . in our study involving 31 children : 7 with eosinophilic colitis , 8 with lymphocytic colitis , and 16 with nonspecific colitis , respectively ,", "these results support the hypothesis that in the aforementioned types of bowel inflammation , histopathological examination does not reveal infiltrations of neutrophil cells , whose cytosols contain calprotectin . therefore , its concentration in faeces is directly related to the number of neutrophils in the large intestine lumen [ 11 , 12 ] . in paediatric patients , the diagnosis of nonspecific colitis ( indeterminata colitis ) remains unchanged in approximately 36% . over time in some patients , the diagnosis may be changed into ulcerative colitis ( in approximately 3372.5% ) or crohn 's disease ( in approximately 1727.5% ) . in our group of 6 cd patients , previously diagnosed conditions included single cases of ulcerative colitis , lymphocytic colitis , and nonspecific colitis , whereas eosinophilic colitis was found in 3 subjects .", "increased calprotectin concentrations may be useful when making a decision on extending diagnostic procedures in patients with less frequent types of bowel inflammation . in our study ,", "the mean calprotectin concentration in the examined patients with ibd was higher than it was observed by bremner et al . ; however , patients in remission were also enrolled in the latter research . in the presented material , a significant correlation was demonstrated between calprotectin activity and the disease severity assessed by the modified pcdai scale for cd and the modified truelove - witts scale and the rachmilewitz endoscopic index for uc .", ", demonstrating a strong positive correlation between calprotectin concentrations and the disease severity according to the modified truelove - witts scale only in uc . analyses performed in children assessed a correlation between fc concentrations and intensity of macroscopic and microscopic inflammatory lesions in the large intestine , observed in the course of ibd .", "studies carried out by fagerberg et al . included 39 children with ibd , in whom calprotectin concentrations strongly correlated with intensity and extent of micro- and macroscopic abnormalities .", "norwegian researchers also confirmed such a correlation and , moreover , suggested that intensity of inflammatory lesions rather than their extent influences faecal calprotectin concentration .", "this suggestion can be supported by data obtained from our study , indicating that in patients with uc calprotectin concentration depends on disease activity and not on the extent of lesions , in contrast to patients suffering from cd . in the latter group , calprotectin concentrations correlated with the disease activity and", "were significantly higher in children with inflammatory lesions present in both small and large intestine and located in 5 or more sections of the large intestine .", "these results support conclusions from the studies on clinical expression of the disease , which demonstrated that in children and adolescents the disease is more severe and cd lesions are located in the small intestine [ 17 , 18 ] . so far", "faecal calprotectin measurement seems a promising test to evaluate disease activity and a tool for monitoring ibd treatment . in everyday practice ,", "a disadvantage of this method is its low specificity , which is connected with the fact that in patients with increased calprotectin concentration many organic diseases should be excluded .", "it seems that measurement of faecal calprotectin concentration can be a useful , safe , and noninvasive test in children suspected for ibd , since it is found to be increased in children with cd and uc as compared to patients with other inflammatory diseases ( eosinophilic , lymphocytic , and nonspecific colitis ) and also in the reference to healthy subjects . when the faecal calprotectin concentration is increased in children with less common types of bowel inflammation , a further follow - up of such patients", "faecal calprotectin concentration correlates positively with the disease severity in cd and uc patients ; thus , it may be useful when choosing or modifying the appropriate treatment regimen ." ]

[ "<S> \n introduction . </S>", "<S> the aim of the study was to assess the usefulness of the fc measurement in children with various types of ibd and relation to the disease activity . </S>", "<S> patients and methods . </S>", "<S> 91 patients ( 49 boys : 53.85% and 42 girls : 46.15% , mean age : 13.38 years , range 618 years ) were included in the analysis . </S>", "<S> patients were divided into the groups : b124 children with cd , b216 patients with uc , and a group comprising 31 children with other types of colitis ; the control group ( k ) comprised 20 healthy children . </S>", "<S> fc was assayed by elisa method , using phical test ( calpro ) . </S>", "<S> results . </S>", "<S> the mean faecal calprotectin concentrations were higher in children with cd and uc as compared to healthy controls , patients with eosinophilic , lymphocytic , and nonspecific colitis . </S>", "<S> a positive correlation was observed between fc concentrations and the disease activity ( the pcdai scale , the truelove - witts scale , and the endoscopic rachmilewitz index ) . </S>", "<S> conclusion . </S>", "<S> it seems that the fc concentrations can be a useful , safe , and noninvasive test in children suspected for ibd , since fc concentration is higher in children with cd and uc than in patients with other inflammatory diseases . \n </S>" ]

[ "1. Introduction", "2. Aim of Study", "3. Patients and Methods", "4. Results", "5. Discussion", "6. Conclusion" ]

[ [ "inflammatory bowel disease ( ibd ) is a common diagnostic and therapeutic problem , affecting patients at increasingly young ages .", "a standard diagnostic method includes macroscopic assessment of the intestinal mucosa during colonoscopy and histopathological evaluation of the obtained biopsies . according to porto criteria ,", "the diagnosis of ibd is based on the clinical presentation , intestinal endoscopic and histological features , laboratory tests , and radiological examination .", "recently , reports on the usefulness of a noninvasive examination , that is , faecal calprotectin measurement in ibd diagnosis , have been published .", "evaluation of faecal calprotectin ( fc ) seems to be a screening test selecting patients requiring further invasive diagnostics .", "furthermore , there have been reports on using calprotectin assays in monitoring the treatment of crohn 's disease and ulcerative colitis in adults and children [ 14 ] . to our knowledge ,", "there are no published data on the usefulness of faecal calprotectin assays in the diagnosis of other atypical forms of bowel inflammation , such as eosinophilic , lymphocytic , or nonspecific colitis ." ], [ "the aim of the study is to assess the usefulness of faecal calprotectin measurement in children with various types of ibd and to evaluate fc concentration in children with crohn 's disease and ulcerative colitis in relation to disease activity ." ], [ "91 patients were included in the analysis , including 49 boys ( 53.85% ) and 42 girls ( 46.15% ) , ranging from 6 to 18 years of age ( the mean age was 13.38 years ) .", "the study group comprised 71 children with various types of ibd , who were subsequently divided into six subgroups : b124 ( 33.8% ) children with crohn 's disease ( cd ) , b216 ( 22.5% ) with ulcerative colitis ( uc ) , b37 ( 9.8% ) with eosinophilic colitis ( ec ) , b48 ( 11.26% ) with lymphocytic colitis ( lc ) , and b516 ( 22.5% ) with nonspecific colitis ( nc , colitis indeterminata ", "the control group ( k ) comprised 20 healthy , age- and sex - matched subjects .", "patients with ibd underwent following procedures : anamnesis , physical examination , laboratory tests ( inflammatory state markers , biochemical parameters of liver , pancreas , and kidney function , sweat test , coprological tests , and immunoassays ) , diagnostic imaging ( abdominal ultrasound ) , and endoscopy with histopathological evaluation . in all patients ,", "faecal calprotectin was measured by means of elisa method , using phical test ( calpro ) .", "calprotectin concentrations ranging from 0 to 50 mg / kg were considered to be normal reference values .", "the results were evaluated by using the following tests : komogorow - smirnow , t - student , u mann - whitney , fisher , and yates ; the analysis of correlation was based on the spearman 's rank correlation coefficient . a p value of < 0.05 was considered statistically significant .", "all patients and their caregivers gave informed consent to participate in the study , which was approved by the bioethics committee of the medical university of silesia in katowice ( consent no ." ], [ "a statistically significant increase in the mean concentrations of faecal calprotectin was observed in the group of children with cd and uc , as compared to the control group .", "concentrations of fc were also higher in children with uc than in patients with cd .", "faecal calprotectin concentrations were within the normal limits in patients with eosinophilic , lymphocytic , and nonspecific colitis , similarly to the healthy subjects ( table 1 , figure 1 ) in children with cd , faecal calprotectin concentrations positively correlated with the disease severity assessed according to the pcdai scale . in patients suffering from uc , faecal calprotectin", "also positively correlated with the truelove - witts scale and the rachmilewitz endoscopic index ( figures 2 , 3 , and 4 ) . a significant increase in faecal calprotectin concentrations", "was observed in children suffering from cd , with lesions located in both small and large intestine , and in patients presenting with inflammatory changes in 5 or more sections of the intestine ( figure 5 ) ." ], [ "faecal calprotectin is a promising , noninvasive screening method for diagnosing patients suffering from gastrointestinal disorders , such as abdominal pain or diarrhea , which are also typical for ibd [ 57 ] .", "so far , many authors have considered calprotectin as a useful marker in differential diagnosis of ibd and functional gastrointestinal disorders ( e.g. , irritable bowel syndrome ) [ 8 , 9 ] .", "many authors evaluated faecal calprotectin concentrations in patients with suspected inflammatory process of the large intestine .", "demonstrated in the group of paediatric patients that this assay is characterised by 95% sensitivity and 93% specificity , and high calprotectin concentrations show strong positive correlation with the presence of inflammatory lesions in the large intestine . according to these authors", "so far , only limited studies , based on very small groups of patients , evaluated faecal calprotectin concentrations in patients with other types of bowel inflammations less common than cd and uc , such as lymphocytic , eosinophilic , and nonspecific colitis , which seem to be a considerable clinical problem in everyday pediatric practice . in studies conducted by bunn et al . , two children with nonspecific colitis and 3 children with allergic colitis were included in the analysis . in both cases ,", "calprotectin concentrations were found to be within the normal ranges . in our study involving 31 children : 7 with eosinophilic colitis , 8 with lymphocytic colitis , and 16 with nonspecific colitis , respectively ,", "these results support the hypothesis that in the aforementioned types of bowel inflammation , histopathological examination does not reveal infiltrations of neutrophil cells , whose cytosols contain calprotectin . therefore , its concentration in faeces is directly related to the number of neutrophils in the large intestine lumen [ 11 , 12 ] . in paediatric patients , the diagnosis of nonspecific colitis ( indeterminata colitis ) remains unchanged in approximately 36% . over time in some patients , the diagnosis may be changed into ulcerative colitis ( in approximately 3372.5% ) or crohn 's disease ( in approximately 1727.5% ) . in our group of 6 cd patients , previously diagnosed conditions included single cases of ulcerative colitis , lymphocytic colitis , and nonspecific colitis , whereas eosinophilic colitis was found in 3 subjects .", "increased calprotectin concentrations may be useful when making a decision on extending diagnostic procedures in patients with less frequent types of bowel inflammation . in our study ,", "the mean calprotectin concentration in the examined patients with ibd was higher than it was observed by bremner et al . ; however , patients in remission were also enrolled in the latter research . in the presented material , a significant correlation was demonstrated between calprotectin activity and the disease severity assessed by the modified pcdai scale for cd and the modified truelove - witts scale and the rachmilewitz endoscopic index for uc .", ", demonstrating a strong positive correlation between calprotectin concentrations and the disease severity according to the modified truelove - witts scale only in uc . analyses performed in children assessed a correlation between fc concentrations and intensity of macroscopic and microscopic inflammatory lesions in the large intestine , observed in the course of ibd .", "studies carried out by fagerberg et al . included 39 children with ibd , in whom calprotectin concentrations strongly correlated with intensity and extent of micro- and macroscopic abnormalities .", "norwegian researchers also confirmed such a correlation and , moreover , suggested that intensity of inflammatory lesions rather than their extent influences faecal calprotectin concentration .", "this suggestion can be supported by data obtained from our study , indicating that in patients with uc calprotectin concentration depends on disease activity and not on the extent of lesions , in contrast to patients suffering from cd . in the latter group , calprotectin concentrations correlated with the disease activity and", "were significantly higher in children with inflammatory lesions present in both small and large intestine and located in 5 or more sections of the large intestine .", "these results support conclusions from the studies on clinical expression of the disease , which demonstrated that in children and adolescents the disease is more severe and cd lesions are located in the small intestine [ 17 , 18 ] . so far", "faecal calprotectin measurement seems a promising test to evaluate disease activity and a tool for monitoring ibd treatment . in everyday practice ,", "a disadvantage of this method is its low specificity , which is connected with the fact that in patients with increased calprotectin concentration many organic diseases should be excluded ." ], [ "it seems that measurement of faecal calprotectin concentration can be a useful , safe , and noninvasive test in children suspected for ibd , since it is found to be increased in children with cd and uc as compared to patients with other inflammatory diseases ( eosinophilic , lymphocytic , and nonspecific colitis ) and also in the reference to healthy subjects . when the faecal calprotectin concentration is increased in children with less common types of bowel inflammation , a further follow - up of such patients", "faecal calprotectin concentration correlates positively with the disease severity in cd and uc patients ; thus , it may be useful when choosing or modifying the appropriate treatment regimen ." ] ]

[ "periodontal diseases ( pd ) are chronic inflammatory disorders encompassing destructive \n and nondestructive diseases of the periodontal supporting tissues of the teeth . \n", "aggressive periodontitis is characterized by severe and rapid loss of periodontal \n attachment often commencing at or after puberty .", "even though the \n diagnosis and classification of pd is essentially based on clinical parameters , new \n auxiliary diagnostic tools based on the analysis of saliva and gingival crevicular fluid \n have been studied and developed . in \n particular , saliva has been extensively studied in relation to pd since it can be easily \n collected and analyzed .", "saliva is necessary for the maintenance of oral health , and the unique properties of \n this fluid are derived in large part from the proteins that are present . in the oral \n cavity , salivary proteins participate in formation of the acquired enamel pellicle , \n occur in the biofilm covering oral surfaces , initiate digestion , promote agglutination \n and clearance of bacteria , and protect oral tissues against noxious compounds produced \n by various microorganisms .", "in particular , mucin glycoprotein-2 ( mg2 ) was reported to be present in pellicle formed \n on cementum surfaces , to exhibit \n candidacidal activity , and to kill \n the periodontal pathogen aggregatibacter \n actinomycetemcomitans .", "salivary lactoferrin removes free iron in fluids decreasing the \n availability of this metal to microbes .", "in addition , lactoferrin exhibits antibacterial , antimycotic , \n antiviral , and anti - inflammatory activity .", "thus , mg2 and lactoferrin are active components of the innate \n immune system in the oral environment .", "several studies reported that the levels of distinct salivary proteins are altered in \n individuals with pd .", "therefore , the present study was \n undertaken to investigate and compare the pattern of secretion and the expression of mg2 \n and lactoferrin in individuals with aggressive periodontitis , chronic periodontitis and \n without periodontitis .", "during a 12-month period , individuals that were referred to the authors ' department \n for treatment and volunteered to participate in this study were screened .", "exclusion \n criteria for the study included : pregnancy ; use of antibiotics within the past 3 \n months ; individuals with systemic debilitating diseases or conditions that could \n affect periodontal status ( e.g. diabetes ) ; smokers and former smokers ; recent trauma . \n", "additionally , individuals that had aggressive or chronic periodontitis and had \n received periodontal treatment within the previous year were excluded in order to \n observe the expression of the salivary glycoproteins mg2 and lactoferrin in the \n inflammatory context of periodontitis .", "individuals meeting inclusion criteria received a complete periodontal exam that \n included assessment of probing depth , probing attachment level and recession , \n evaluation of bleeding and/or suppuration on probing , radiographic examination and \n complete anamnesis .", "probing depths were measured at six sites per \n tooth ( mesio- , mid- , disto - vestibular / palatal or lingual ) .", "the specific inclusion \n criteria used to differentiate the 3 groups were based on previous \n publications including : aggressive periodontitis group ( apg ) : ( i ) localized aggressive periodontitis - \n periodontal damage being localized to permanent first molars and incisors ; \n generalized aggressive periodontitis : ( ii ) generalized interproximal attachment loss \n affecting at least 3 permanent teeth other than the permanent first molars and \n incisors ; ( iii ) clinical attachment loss ( cal ) 5 mm ; probing depths 6 mm . \n generalized chronic periodontitis group ( cpg ) : ( i ) more than 30% of sites involved ; \n moderate : cal 3 - 4 mm ; probing depths 4 - 6 mm ; severe : cal 5 mm ; probing depths 6 mm . \n", "each group had 5 individuals ( 2 males and 3 females ) with age ranges of 19 - 28 years \n for apg , 36 - 50 years for cpg , 21 - 50 years for cg and as expected individuals from apg exhibited a \n lower age range .", "this study was independently reviewed and approved by the local \n research ethics committee , informed consent was obtained from all individuals prior \n to their participation and subjects ' rights were protected at all times .", "non - stimulated and stimulated submandibular and sublingual saliva ( smsl ) was \n collected from subjects between 10:00 a.m. and 11:00 a.m. subjects were asked to \n refrain from eating or drinking two h prior to collection .", "subjects rinsed with \n water , and smsl samples were collected with a custom - fitted device as described \n previously .", "the first 4 min of non - stimulated smsl were discarded in order to avoid possible \n interference of the device dead volume .", "four consecutive two - minute samples collected \n in separate 1.5 ml centrifuge tubes and subsequently four consecutive two - minute \n samples of stimulated smsl were collected in separate 15 ml falcon screw cap tubes . \n", "gustatory stimulation of stimulated samples was induced by placing lemon fruit \n flavored candies on the tongue .", "tubes of non - stimulated smsl were labeled t1 , t2 , t3 \n and t4 .", "all samples were \n kept on ice during the collection procedure , the flow rate from each subject was \n recorded and samples were frozen at -20c until used .", "smsl samples were thawed , and for each subject , equal volumes ( 50 l ) of \n non - stimulated and stimulated smsl were lyophilized , taken up in 15 l of sample \n buffer , heated at 95c for 5 min and subjected to sodium dodecylsulfate polyacrylamide \n gel electrophoresis ( sds - page ) on 7.5% gels .", "proteins in gels were electrophoretically transferred to nitrocellulose membranes \n ( protran , schleicher and schuell , keene , nh , usa ) in 25 mm tris - hcl , ph 8.3 , \n containing 192 mm glycine and 20% methanol at 100 volts for 1 h at room temperature . \n", "briefly , blots were equilibrated in 10 mm tris - hcl , ph 8.0 containing \n 150 mm nacl and 0.1% tween-20 ( tbst ) for 5 min and blocked with 5% non - fat , dried \n milk in tbst overnight at room temperature . after blocking ,", "the specificity of the anti - mg2 antibody has been \n demonstrated in previous studies , and this antibody was diluted \n 1:1000 .", "antibodies were diluted in 1% milk / tbst and incubated for 1 h at room \n temperature .", "after washing , blots were incubated with the second antibody , which was \n anti - rabbit igg coupled to alkaline phosphatase ( promega , madison , wi , usa ) diluted \n 1:7,500 in 1% milk / tbst for 1 h at room temperature .", "blots were washed three times in \n tbst for 5 min and color development was carried out with \n 5-bromo-4-chloro-3-indolyl - phosphate ( bcip ) and nitro blue tetrazolium ( nbt ) \n according to the manufacturer 's ( promega ) instructions .", "comparison of the effect of non - stimulated and stimulated conditions on smsl flow \n rate was tested for statistical significance ( =0.05 ) by a two - way repeated - measures \n analysis of variance ( anova ; post hoc analysis - fisher 's exact \n test ) .", "the statistical analysis was performed using statview 4.5 ( abacus concepts \n inc . , berkeley , ca , usa ) .", "during a 12-month period , individuals that were referred to the authors ' department \n for treatment and volunteered to participate in this study were screened .", "exclusion \n criteria for the study included : pregnancy ; use of antibiotics within the past 3 \n months ; individuals with systemic debilitating diseases or conditions that could \n affect periodontal status ( e.g. diabetes ) ; smokers and former smokers ; recent trauma . \n", "additionally , individuals that had aggressive or chronic periodontitis and had \n received periodontal treatment within the previous year were excluded in order to \n observe the expression of the salivary glycoproteins mg2 and lactoferrin in the \n inflammatory context of periodontitis .", "individuals meeting inclusion criteria received a complete periodontal exam that \n included assessment of probing depth , probing attachment level and recession , \n evaluation of bleeding and/or suppuration on probing , radiographic examination and \n complete anamnesis .", "probing depths were measured at six sites per \n tooth ( mesio- , mid- , disto - vestibular / palatal or lingual ) .", "the specific inclusion \n criteria used to differentiate the 3 groups were based on previous \n publications including : aggressive periodontitis group ( apg ) : ( i ) localized aggressive periodontitis - \n periodontal damage being localized to permanent first molars and incisors ; \n generalized aggressive periodontitis : ( ii ) generalized interproximal attachment loss \n affecting at least 3 permanent teeth other than the permanent first molars and \n incisors ; ( iii ) clinical attachment loss ( cal ) 5 mm ; probing depths 6 mm . \n generalized chronic periodontitis group ( cpg ) : ( i ) more than 30% of sites involved ; \n moderate : cal 3 - 4 mm ; probing depths 4 - 6 mm ; severe : cal 5 mm ; probing depths 6 mm . \n", "each group had 5 individuals ( 2 males and 3 females ) with age ranges of 19 - 28 years \n for apg , 36 - 50 years for cpg , 21 - 50 years for cg and as expected individuals from apg exhibited a \n lower age range .", "this study was independently reviewed and approved by the local \n research ethics committee , informed consent was obtained from all individuals prior \n to their participation and subjects ' rights were protected at all times .", "non - stimulated and stimulated submandibular and sublingual saliva ( smsl ) was \n collected from subjects between 10:00 a.m. and 11:00 a.m. subjects were asked to \n refrain from eating or drinking two h prior to collection .", "subjects rinsed with \n water , and smsl samples were collected with a custom - fitted device as described \n previously .", "the first 4 min of non - stimulated smsl were discarded in order to avoid possible \n interference of the device dead volume .", "four consecutive two - minute samples collected \n in separate 1.5 ml centrifuge tubes and subsequently four consecutive two - minute \n samples of stimulated smsl were collected in separate 15 ml falcon screw cap tubes . \n", "gustatory stimulation of stimulated samples was induced by placing lemon fruit \n flavored candies on the tongue .", "tubes of non - stimulated smsl were labeled t1 , t2 , t3 \n and t4 .", "all samples were \n kept on ice during the collection procedure , the flow rate from each subject was \n recorded and samples were frozen at -20c until used .", "smsl samples were thawed , and for each subject , equal volumes ( 50 l ) of \n non - stimulated and stimulated smsl were lyophilized , taken up in 15 l of sample \n buffer , heated at 95c for 5 min and subjected to sodium dodecylsulfate polyacrylamide \n gel electrophoresis ( sds - page ) on 7.5% gels .", "proteins in gels were electrophoretically transferred to nitrocellulose membranes \n ( protran , schleicher and schuell , keene , nh , usa ) in 25 mm tris - hcl , ph 8.3 , \n containing 192 mm glycine and 20% methanol at 100 volts for 1 h at room temperature . \n", "briefly , blots were equilibrated in 10 mm tris - hcl , ph 8.0 containing \n 150 mm nacl and 0.1% tween-20 ( tbst ) for 5 min and blocked with 5% non - fat , dried \n milk in tbst overnight at room temperature . after blocking ,", "the specificity of the anti - mg2 antibody has been \n demonstrated in previous studies , and this antibody was diluted \n 1:1000 . the anti - lactoferrin antibody ( sigma , st .", "antibodies were diluted in 1% milk / tbst and incubated for 1 h at room \n temperature .", "after washing , blots were incubated with the second antibody , which was \n anti - rabbit igg coupled to alkaline phosphatase ( promega , madison , wi , usa ) diluted \n 1:7,500 in 1% milk / tbst for 1 h at room temperature .", "blots were washed three times in \n tbst for 5 min and color development was carried out with \n 5-bromo-4-chloro-3-indolyl - phosphate ( bcip ) and nitro blue tetrazolium ( nbt ) \n according to the manufacturer 's ( promega ) instructions .", "comparison of the effect of non - stimulated and stimulated conditions on smsl flow \n rate was tested for statistical significance ( =0.05 ) by a two - way repeated - measures \n analysis of variance ( anova ; post hoc analysis - fisher 's exact \n test ) .", "as expected , the observed flow rate values obtained at rest ( t1-t4 ) were lower than \n stimulated ( t5-t8 ) conditions in all groups .", "the mean value for flow rates under \n non - stimulated conditions was 0.460.18 ml / min for apg , 0.300.20 for cpg and 0.470.31 \n for cg . under stimulated conditions", ", it was 1.790.92 ml / min for apg , 1.470.38 ml / min \n for cpg and 1.870.99 ml / min for cg .", "stimulation significantly raised salivary volumes \n in all groups ( apg - p=0.01 ; cpg - p=0.03 ; cg - p=0.01 ) ; no significant time effect \n was observed for non - stimulated or stimulated conditions , and the observed flow rates \n under both conditions did not differ among groups .", "mg2 immunoreactive bands from apg , cpg and cg exhibited a weak immunoreactive signal \n under non - stimulated conditions whereas upon gustatory stimulation , the intensity of \n the immunoreactive signal increased ( figure \n 1a ) . blots", "probed with anti - lactoferrin antibodies revealed a difference in \n banding pattern among groups ( figure 1b ) ; \n individuals from apg and cpg had an increase in expression upon gustatory \n stimulation , whereas individuals from cg exhibited a decrease .", "a - representative western \n blots of mg2 pattern observed in all individuals from each group .", "b - \n representative western blots of lactoferrin pattern observed in all individuals \n from each group . t1t4", "( non - stimulated submandibular and sublingual \n saliva - smsl ) and t5t8 ( stimulated submandibular and sublingual saliva - smsl ) . \n", "apg - aggressive periodontitis group ; cpg - generalized chronic periodontitis \n group ; cg - control group comparison of expression among groups under non - stimulated conditions revealed that \n cg exhibited the highest expression of mg2 and lactoferrin ( figures 2a and 2b ) .", "apg \n showed the lowest levels of mg2 , and cpg of lactoferrin ( figures 2a and 2b ) . \n", "comparison of expression among groups under stimulated conditions showed that cg \n exhibited the highest expression of mg2 , whereas apg the highest of lactoferrin \n ( figures 2c and 2d ) .", "again , apg showed the lowest levels of mg2 , and cpg of \n lactoferrin ( figures 2c and 2d ) .", "comparison of glycoproteins expression under non - stimulated ( a , b ) or \n stimulated ( c , d ) conditions .", "a - mg2 western blot ; b - lactoferrin western blot ; \n c - mg2 western blot ; d - lactoferrin western blot ; apgaggressive periodontitis \n group ; cpg - generalized chronic periodontitis group ; cg - control group", "as expected , the observed flow rate values obtained at rest ( t1-t4 ) were lower than \n stimulated ( t5-t8 ) conditions in all groups .", "the mean value for flow rates under \n non - stimulated conditions was 0.460.18 ml / min for apg , 0.300.20 for cpg and 0.470.31 \n for cg . under stimulated conditions", ", it was 1.790.92 ml / min for apg , 1.470.38 ml / min \n for cpg and 1.870.99 ml / min for cg .", "stimulation significantly raised salivary volumes \n in all groups ( apg - p=0.01 ; cpg - p=0.03 ; cg - p=0.01 ) ; no significant time effect \n was observed for non - stimulated or stimulated conditions , and the observed flow rates \n under both conditions did not differ among groups .", "mg2 immunoreactive bands from apg , cpg and cg exhibited a weak immunoreactive signal \n under non - stimulated conditions whereas upon gustatory stimulation , the intensity of \n the immunoreactive signal increased ( figure \n 1a ) . blots", "probed with anti - lactoferrin antibodies revealed a difference in \n banding pattern among groups ( figure 1b ) ; \n individuals from apg and cpg had an increase in expression upon gustatory \n stimulation , whereas individuals from cg exhibited a decrease . pattern of secretion of salivary glycoproteins .", "a - representative western \n blots of mg2 pattern observed in all individuals from each group .", "b - \n representative western blots of lactoferrin pattern observed in all individuals \n from each group . t1t4", "( non - stimulated submandibular and sublingual \n saliva - smsl ) and t5t8 ( stimulated submandibular and sublingual saliva - smsl ) . \n", "apg - aggressive periodontitis group ; cpg - generalized chronic periodontitis \n group ; cg - control group", "comparison of expression among groups under non - stimulated conditions revealed that \n cg exhibited the highest expression of mg2 and lactoferrin ( figures 2a and 2b ) .", "apg \n showed the lowest levels of mg2 , and cpg of lactoferrin ( figures 2a and 2b ) . \n", "comparison of expression among groups under stimulated conditions showed that cg \n exhibited the highest expression of mg2 , whereas apg the highest of lactoferrin \n ( figures 2c and 2d ) .", "again , apg showed the lowest levels of mg2 , and cpg of \n lactoferrin ( figures 2c and 2d ) .", "comparison of glycoproteins expression under non - stimulated ( a , b ) or \n stimulated ( c , d ) conditions .", "a - mg2 western blot ; b - lactoferrin western blot ; \n c - mg2 western blot ; d - lactoferrin western blot ; apgaggressive periodontitis \n group ; cpg - generalized chronic periodontitis group ; cg - control group", "the infectious nature of the periodontitis is related to specific microbiota as well as \n to the expression of particular inflammatory markers .", "additionally , initiation and progression of periodontal \n infections is clearly modified by local and systemic conditions ( risk factors ) , such as \n diabetes mellitus and cigarette smoking , as well as potentially important risk \n indicators including stress . \n", "salivary proteins are not included in the category of risk indicators , but this concept \n is being explored .", "investigation of salivary proteins in individuals with periodontitis may be useful to \n enhance the knowledge of their role in this population .", "statistical analysis did not \n reveal flow rate differences among groups , stimulated or non - stimulated .", "therefore , we \n decided to analyze equal volumes ( 50 l ) of smsl by immunoblotting , instead of equal \n amounts of protein . ", "it has been previously reported that mg2 can agglutinate several pathogens from the oral \n cavity , and in \n addition , interact and kill the periodontal pathogen a. \n actinomycetemcomitans . \n in a previous study", ", the concentration of mg2 was determined in stimulated whole saliva \n of subjects suffering from a. actinomycetemcomitans associated \n pd .", "it has been reported that \n mg2 output in the diseased subjects was decreased at least by a factor three in \n comparison to periodontally healthy subjects and that this low concentration of mg2 \n suggested a decline in mucin defense and consequently , a higher susceptibility to oral \n infections . in the present study ,", "blotting analysis revealed that all groups showed an increase of \n mg2 expression upon stimulation revealing a similar pattern ( figures 1a and 1b ) .", "a previous \n study that analyzed the pattern of secretion of mg2 in samples from individuals without \n periodontitis reported that pas \n stained gels did not reveal differences in mg2 secretion patterns upon stimulation . \n however , western blots indicated that mg2 expression was enhanced after stimulation ; a \n result that is in agreement with the observed pattern in the present study for cg .", "it was also observed that individuals without periodontitis exhibited the highest \n expression of this small salivary mucin under specific conditions ( figures 2a and 2c ) .", "the \n reduced output of this salivary mucin in individuals with aggressive periodontitis \n observed on blots ( figures 2a and 2c ) is similar to the one described . since in chronic periodontitis", "the \n destruction of the periodontal ligament and loss of the adjacent supporting bone usually \n displays a slow to moderate rate of progression , we hypothesize that the intermediate \n expression of mg2 observed in individuals belonging to this group ( figures 2a and 2c ) could in \n part contribute to decrease the disease progression rate in comparison to aggressive \n periodontitis .", "salivary lactoferrin is a single chain iron - binding glycoprotein that binds ferric ions \n and possesses antibacterial , antimycotic , antiviral and anti - inflammatory \n properties .", "the impact of a surgical periodontal treatment in the \n concentration of this glycoprotein in non - stimulated and stimulated whole saliva samples \n from individuals with chronic periodontitis was analyzed previously .", "it was reported that the concentrations \n decreased significantly in both non - stimulated and stimulated samples after the surgical \n treatment , suggesting that lactoferrin may be suitable for monitoring periodontal \n treatment results .", "another study evaluated lactoferrin levels from individuals with localized aggressive \n periodontitis before and after periodontal therapy . among other samples", ", \n paraffin - stimulated whole saliva was obtained from these individuals and from \n periodontally healthy controls .", "differences between groups and with respect to \n periodontal therapy were not observed in lactoferrin concentrations in whole \n saliva .", "concentrations and output of lactoferrin were also determined in whole saliva samples \n from subjects suffering from a. actinomycetemcomitans - associated \n pd .", "the output of lactoferrin \n was not significantly different in healthy and diseased subjects although a higher \n iron - saturation of lactoferrin in subjects with the disease was described .", "lactoferrin expression was examined in whole and parotid saliva from individuals with \n localized aggressive periodontitis and compared to age- , gender- , and race - matched \n controls .", "it was reported that \n whole saliva from the test group had higher levels of the glycoprotein although the \n levels of bound iron were significantly reduced , suggesting that lactoferrin from \n individuals with localized aggressive periodontitis might interfere in the disease \n progression . in the present study , it was observed that individuals with or without periodontitis \n exhibited a similar pattern of lactoferrin secretion ( figure 1b ) .", "comparison of expression among groups revealed that under \n non - stimulated conditions individuals without periodontitis ( cg ) showed the highest \n expression of lactoferrin ( figure 2b ) whereas \n under stimulated conditions individuals with periodontitis ( apg and cpg ) exhibited a \n more intense immunoreactive signal ( figure \n 2d ) .", "the higher expression of this glycoprotein in individuals with aggressive periodontitis \n is in agreement with a previous report , but is in contrast to others .", "the differences in \n these two studies from the present could be at least in part explained by variations in \n samples that were analyzed ( i.e. smsl versus whole saliva ) since lactoferrin derived \n from the gingival crevicular fluid will be present in samples from whole saliva .", "the increase in the amount of lactoferrin in stimulated samples from smsl of individuals \n with generalized chronic periodontitis ( figure 2d ) \n found in the present study is in contrast to another report in which the levels of lactoferrin in samples from \n non - stimulated whole saliva were higher than in samples of stimulated whole saliva . \n", "again , the differences in this study from the present could also be in part explained by \n smsl versus whole saliva .", "exclusion of individuals that had aggressive or chronic periodontitis and received \n periodontal treatment during the last year had a direct impact in our sample size , since \n it was difficult to select individuals that met this specific criterion .", "nevertheless , \n this is the first study that compares the expression of salivary proteins among \n individuals with aggressive and chronic periodontitis at the same time , and that \n evaluates the influence of stimulation over time in the expression of mg2 and \n lactoferrin in individuals with periodontitis .", "analysis of these salivary constituents \n appears to have potential to be used as markers for aggressive or chronic periodontitis , \n and since both mg2 and lactoferrin possess antimicrobial activity , specific domains \n could be considered as possible candidates for the development of new drugs that in \n principle could be of benefit to individuals with periodontitis .", "the biological plausibility of the differences observed in smsl physiology in our study \n could be derived by periodontal bacterial components triggering the host - immune \n response , and causing inflammation and activation of pro - inflammatory mediators .", "it has \n been described that these molecules traveling via blood to other organs and tissues \n might influence a variety of mechanisms .", "it is important to mention that the observed results show that \n future studies should purposely collect non - stimulated and/or stimulated saliva samples \n since protein expression under these circumstances varies .", "the reduced expression of \n these glycoproteins observed in apg and cpg particularly under non - stimulated \n conditions , decrease the oral cavity innate defense mechanisms provided by these \n salivary components in these individuals .", "it remains unclear to what extent the reduced output of mg2 and lactoferrin might impact \n the etiopathogenesis of aggressive and chronic periodontitis .", "however , the data suggest \n an active role of these glycoproteins in the innate immune regulation of periodontal \n bacterial colonization and disease progression ." ]

[ "<S> objectives the aim of this study was to compare the pattern of secretion and the expression \n of mucin glycoprotein-2 ( mg2 ) and lactoferrin in individuals with or without \n periodontitis . </S>", "<S> material and methods five individuals with aggressive periodontitis ( apg ) , 5 with generalized chronic \n periodontitis ( cpg ) and 5 without periodontitis ( cg ) were enrolled after informed \n consent . non - stimulated and stimulated submandibular and sublingual </S>", "<S> saliva was \n collected and samples analyzed by western blot probed with specific antibodies . \n </S>", "<S> results stimulated and non - stimulated salivary flow rates did not differ among groups . \n </S>", "<S> western blot analysis revealed that stimulation led to : an increase in mg2 \n expression in all groups , and to lactoferrin expression in apg and cpg . in \n non - stimulated saliva </S>", "<S> , cg exhibited the highest expression of both glycoproteins . \n in stimulated saliva </S>", "<S> , cg exhibited the highest expression of mg2 , whereas apg the \n highest of lactoferrin . </S>", "<S> conclusions the pattern of secretion of mg2 and lactoferrin in health and disease is complex . \n although the present study analyzed samples from a limited number of participants , \n the reduced expression of mg2 and lactoferrin in apg and cpg under non - stimulated \n condition , the predominant circumstance of salivary secretion during the day , \n suggests that these salivary constituents may play a role in the etiopathogenesis \n of these diseases . </S>" ]

[ "INTRODUCTION", "MATERIAL AND METHODS", "Participant selection", "Saliva collection", "Gel electrophoresis", "Western blots", "Statistical analysis", "RESULTS", "SMSL flow rate", "Analysis of the secretion patterns ", "Comparison of glycoprotein expression", "DISCUSSION", "CONCLUSIONS" ]

[ [ "periodontal diseases ( pd ) are chronic inflammatory disorders encompassing destructive \n and nondestructive diseases of the periodontal supporting tissues of the teeth . \n", "aggressive periodontitis is characterized by severe and rapid loss of periodontal \n attachment often commencing at or after puberty .", "even though the \n diagnosis and classification of pd is essentially based on clinical parameters , new \n auxiliary diagnostic tools based on the analysis of saliva and gingival crevicular fluid \n have been studied and developed . in \n particular , saliva has been extensively studied in relation to pd since it can be easily \n collected and analyzed .", "saliva is necessary for the maintenance of oral health , and the unique properties of \n this fluid are derived in large part from the proteins that are present . in the oral \n cavity , salivary proteins participate in formation of the acquired enamel pellicle , \n occur in the biofilm covering oral surfaces , initiate digestion , promote agglutination \n and clearance of bacteria , and protect oral tissues against noxious compounds produced \n by various microorganisms .", "in particular , mucin glycoprotein-2 ( mg2 ) was reported to be present in pellicle formed \n on cementum surfaces , to exhibit \n candidacidal activity , and to kill \n the periodontal pathogen aggregatibacter \n actinomycetemcomitans .", "salivary lactoferrin removes free iron in fluids decreasing the \n availability of this metal to microbes .", "in addition , lactoferrin exhibits antibacterial , antimycotic , \n antiviral , and anti - inflammatory activity .", "thus , mg2 and lactoferrin are active components of the innate \n immune system in the oral environment .", "several studies reported that the levels of distinct salivary proteins are altered in \n individuals with pd .", "therefore , the present study was \n undertaken to investigate and compare the pattern of secretion and the expression of mg2 \n and lactoferrin in individuals with aggressive periodontitis , chronic periodontitis and \n without periodontitis ." ], [ "during a 12-month period , individuals that were referred to the authors ' department \n for treatment and volunteered to participate in this study were screened .", "exclusion \n criteria for the study included : pregnancy ; use of antibiotics within the past 3 \n months ; individuals with systemic debilitating diseases or conditions that could \n affect periodontal status ( e.g. diabetes ) ; smokers and former smokers ; recent trauma . \n", "additionally , individuals that had aggressive or chronic periodontitis and had \n received periodontal treatment within the previous year were excluded in order to \n observe the expression of the salivary glycoproteins mg2 and lactoferrin in the \n inflammatory context of periodontitis .", "individuals meeting inclusion criteria received a complete periodontal exam that \n included assessment of probing depth , probing attachment level and recession , \n evaluation of bleeding and/or suppuration on probing , radiographic examination and \n complete anamnesis .", "probing depths were measured at six sites per \n tooth ( mesio- , mid- , disto - vestibular / palatal or lingual ) .", "the specific inclusion \n criteria used to differentiate the 3 groups were based on previous \n publications including : aggressive periodontitis group ( apg ) : ( i ) localized aggressive periodontitis - \n periodontal damage being localized to permanent first molars and incisors ; \n generalized aggressive periodontitis : ( ii ) generalized interproximal attachment loss \n affecting at least 3 permanent teeth other than the permanent first molars and \n incisors ; ( iii ) clinical attachment loss ( cal ) 5 mm ; probing depths 6 mm . \n generalized chronic periodontitis group ( cpg ) : ( i ) more than 30% of sites involved ; \n moderate : cal 3 - 4 mm ; probing depths 4 - 6 mm ; severe : cal 5 mm ; probing depths 6 mm . \n", "each group had 5 individuals ( 2 males and 3 females ) with age ranges of 19 - 28 years \n for apg , 36 - 50 years for cpg , 21 - 50 years for cg and as expected individuals from apg exhibited a \n lower age range .", "this study was independently reviewed and approved by the local \n research ethics committee , informed consent was obtained from all individuals prior \n to their participation and subjects ' rights were protected at all times .", "non - stimulated and stimulated submandibular and sublingual saliva ( smsl ) was \n collected from subjects between 10:00 a.m. and 11:00 a.m. subjects were asked to \n refrain from eating or drinking two h prior to collection .", "subjects rinsed with \n water , and smsl samples were collected with a custom - fitted device as described \n previously .", "the first 4 min of non - stimulated smsl were discarded in order to avoid possible \n interference of the device dead volume .", "four consecutive two - minute samples collected \n in separate 1.5 ml centrifuge tubes and subsequently four consecutive two - minute \n samples of stimulated smsl were collected in separate 15 ml falcon screw cap tubes . \n", "gustatory stimulation of stimulated samples was induced by placing lemon fruit \n flavored candies on the tongue .", "tubes of non - stimulated smsl were labeled t1 , t2 , t3 \n and t4 .", "all samples were \n kept on ice during the collection procedure , the flow rate from each subject was \n recorded and samples were frozen at -20c until used .", "smsl samples were thawed , and for each subject , equal volumes ( 50 l ) of \n non - stimulated and stimulated smsl were lyophilized , taken up in 15 l of sample \n buffer , heated at 95c for 5 min and subjected to sodium dodecylsulfate polyacrylamide \n gel electrophoresis ( sds - page ) on 7.5% gels .", "proteins in gels were electrophoretically transferred to nitrocellulose membranes \n ( protran , schleicher and schuell , keene , nh , usa ) in 25 mm tris - hcl , ph 8.3 , \n containing 192 mm glycine and 20% methanol at 100 volts for 1 h at room temperature . \n", "briefly , blots were equilibrated in 10 mm tris - hcl , ph 8.0 containing \n 150 mm nacl and 0.1% tween-20 ( tbst ) for 5 min and blocked with 5% non - fat , dried \n milk in tbst overnight at room temperature . after blocking ,", "the specificity of the anti - mg2 antibody has been \n demonstrated in previous studies , and this antibody was diluted \n 1:1000 .", "antibodies were diluted in 1% milk / tbst and incubated for 1 h at room \n temperature .", "after washing , blots were incubated with the second antibody , which was \n anti - rabbit igg coupled to alkaline phosphatase ( promega , madison , wi , usa ) diluted \n 1:7,500 in 1% milk / tbst for 1 h at room temperature .", "blots were washed three times in \n tbst for 5 min and color development was carried out with \n 5-bromo-4-chloro-3-indolyl - phosphate ( bcip ) and nitro blue tetrazolium ( nbt ) \n according to the manufacturer 's ( promega ) instructions .", "comparison of the effect of non - stimulated and stimulated conditions on smsl flow \n rate was tested for statistical significance ( =0.05 ) by a two - way repeated - measures \n analysis of variance ( anova ; post hoc analysis - fisher 's exact \n test ) .", "the statistical analysis was performed using statview 4.5 ( abacus concepts \n inc . , berkeley , ca , usa ) ." ], [ "during a 12-month period , individuals that were referred to the authors ' department \n for treatment and volunteered to participate in this study were screened .", "exclusion \n criteria for the study included : pregnancy ; use of antibiotics within the past 3 \n months ; individuals with systemic debilitating diseases or conditions that could \n affect periodontal status ( e.g. diabetes ) ; smokers and former smokers ; recent trauma . \n", "additionally , individuals that had aggressive or chronic periodontitis and had \n received periodontal treatment within the previous year were excluded in order to \n observe the expression of the salivary glycoproteins mg2 and lactoferrin in the \n inflammatory context of periodontitis .", "individuals meeting inclusion criteria received a complete periodontal exam that \n included assessment of probing depth , probing attachment level and recession , \n evaluation of bleeding and/or suppuration on probing , radiographic examination and \n complete anamnesis .", "probing depths were measured at six sites per \n tooth ( mesio- , mid- , disto - vestibular / palatal or lingual ) .", "the specific inclusion \n criteria used to differentiate the 3 groups were based on previous \n publications including : aggressive periodontitis group ( apg ) : ( i ) localized aggressive periodontitis - \n periodontal damage being localized to permanent first molars and incisors ; \n generalized aggressive periodontitis : ( ii ) generalized interproximal attachment loss \n affecting at least 3 permanent teeth other than the permanent first molars and \n incisors ; ( iii ) clinical attachment loss ( cal ) 5 mm ; probing depths 6 mm . \n generalized chronic periodontitis group ( cpg ) : ( i ) more than 30% of sites involved ; \n moderate : cal 3 - 4 mm ; probing depths 4 - 6 mm ; severe : cal 5 mm ; probing depths 6 mm . \n", "each group had 5 individuals ( 2 males and 3 females ) with age ranges of 19 - 28 years \n for apg , 36 - 50 years for cpg , 21 - 50 years for cg and as expected individuals from apg exhibited a \n lower age range .", "this study was independently reviewed and approved by the local \n research ethics committee , informed consent was obtained from all individuals prior \n to their participation and subjects ' rights were protected at all times ." ], [ "non - stimulated and stimulated submandibular and sublingual saliva ( smsl ) was \n collected from subjects between 10:00 a.m. and 11:00 a.m. subjects were asked to \n refrain from eating or drinking two h prior to collection .", "subjects rinsed with \n water , and smsl samples were collected with a custom - fitted device as described \n previously .", "the first 4 min of non - stimulated smsl were discarded in order to avoid possible \n interference of the device dead volume .", "four consecutive two - minute samples collected \n in separate 1.5 ml centrifuge tubes and subsequently four consecutive two - minute \n samples of stimulated smsl were collected in separate 15 ml falcon screw cap tubes . \n", "gustatory stimulation of stimulated samples was induced by placing lemon fruit \n flavored candies on the tongue .", "tubes of non - stimulated smsl were labeled t1 , t2 , t3 \n and t4 .", "all samples were \n kept on ice during the collection procedure , the flow rate from each subject was \n recorded and samples were frozen at -20c until used ." ], [ "smsl samples were thawed , and for each subject , equal volumes ( 50 l ) of \n non - stimulated and stimulated smsl were lyophilized , taken up in 15 l of sample \n buffer , heated at 95c for 5 min and subjected to sodium dodecylsulfate polyacrylamide \n gel electrophoresis ( sds - page ) on 7.5% gels ." ], [ "proteins in gels were electrophoretically transferred to nitrocellulose membranes \n ( protran , schleicher and schuell , keene , nh , usa ) in 25 mm tris - hcl , ph 8.3 , \n containing 192 mm glycine and 20% methanol at 100 volts for 1 h at room temperature . \n", "briefly , blots were equilibrated in 10 mm tris - hcl , ph 8.0 containing \n 150 mm nacl and 0.1% tween-20 ( tbst ) for 5 min and blocked with 5% non - fat , dried \n milk in tbst overnight at room temperature . after blocking ,", "the specificity of the anti - mg2 antibody has been \n demonstrated in previous studies , and this antibody was diluted \n 1:1000 . the anti - lactoferrin antibody ( sigma , st .", "antibodies were diluted in 1% milk / tbst and incubated for 1 h at room \n temperature .", "after washing , blots were incubated with the second antibody , which was \n anti - rabbit igg coupled to alkaline phosphatase ( promega , madison , wi , usa ) diluted \n 1:7,500 in 1% milk / tbst for 1 h at room temperature .", "blots were washed three times in \n tbst for 5 min and color development was carried out with \n 5-bromo-4-chloro-3-indolyl - phosphate ( bcip ) and nitro blue tetrazolium ( nbt ) \n according to the manufacturer 's ( promega ) instructions ." ], [ "comparison of the effect of non - stimulated and stimulated conditions on smsl flow \n rate was tested for statistical significance ( =0.05 ) by a two - way repeated - measures \n analysis of variance ( anova ; post hoc analysis - fisher 's exact \n test ) ." ], [ "as expected , the observed flow rate values obtained at rest ( t1-t4 ) were lower than \n stimulated ( t5-t8 ) conditions in all groups .", "the mean value for flow rates under \n non - stimulated conditions was 0.460.18 ml / min for apg , 0.300.20 for cpg and 0.470.31 \n for cg . under stimulated conditions", ", it was 1.790.92 ml / min for apg , 1.470.38 ml / min \n for cpg and 1.870.99 ml / min for cg .", "stimulation significantly raised salivary volumes \n in all groups ( apg - p=0.01 ; cpg - p=0.03 ; cg - p=0.01 ) ; no significant time effect \n was observed for non - stimulated or stimulated conditions , and the observed flow rates \n under both conditions did not differ among groups .", "mg2 immunoreactive bands from apg , cpg and cg exhibited a weak immunoreactive signal \n under non - stimulated conditions whereas upon gustatory stimulation , the intensity of \n the immunoreactive signal increased ( figure \n 1a ) . blots", "probed with anti - lactoferrin antibodies revealed a difference in \n banding pattern among groups ( figure 1b ) ; \n individuals from apg and cpg had an increase in expression upon gustatory \n stimulation , whereas individuals from cg exhibited a decrease .", "a - representative western \n blots of mg2 pattern observed in all individuals from each group .", "b - \n representative western blots of lactoferrin pattern observed in all individuals \n from each group . t1t4", "( non - stimulated submandibular and sublingual \n saliva - smsl ) and t5t8 ( stimulated submandibular and sublingual saliva - smsl ) . \n", "apg - aggressive periodontitis group ; cpg - generalized chronic periodontitis \n group ; cg - control group comparison of expression among groups under non - stimulated conditions revealed that \n cg exhibited the highest expression of mg2 and lactoferrin ( figures 2a and 2b ) .", "apg \n showed the lowest levels of mg2 , and cpg of lactoferrin ( figures 2a and 2b ) . \n", "comparison of expression among groups under stimulated conditions showed that cg \n exhibited the highest expression of mg2 , whereas apg the highest of lactoferrin \n ( figures 2c and 2d ) .", "again , apg showed the lowest levels of mg2 , and cpg of \n lactoferrin ( figures 2c and 2d ) .", "comparison of glycoproteins expression under non - stimulated ( a , b ) or \n stimulated ( c , d ) conditions .", "a - mg2 western blot ; b - lactoferrin western blot ; \n c - mg2 western blot ; d - lactoferrin western blot ; apgaggressive periodontitis \n group ; cpg - generalized chronic periodontitis group ; cg - control group" ], [ "as expected , the observed flow rate values obtained at rest ( t1-t4 ) were lower than \n stimulated ( t5-t8 ) conditions in all groups .", "the mean value for flow rates under \n non - stimulated conditions was 0.460.18 ml / min for apg , 0.300.20 for cpg and 0.470.31 \n for cg . under stimulated conditions", ", it was 1.790.92 ml / min for apg , 1.470.38 ml / min \n for cpg and 1.870.99 ml / min for cg .", "stimulation significantly raised salivary volumes \n in all groups ( apg - p=0.01 ; cpg - p=0.03 ; cg - p=0.01 ) ; no significant time effect \n was observed for non - stimulated or stimulated conditions , and the observed flow rates \n under both conditions did not differ among groups ." ], [ "mg2 immunoreactive bands from apg , cpg and cg exhibited a weak immunoreactive signal \n under non - stimulated conditions whereas upon gustatory stimulation , the intensity of \n the immunoreactive signal increased ( figure \n 1a ) . blots", "probed with anti - lactoferrin antibodies revealed a difference in \n banding pattern among groups ( figure 1b ) ; \n individuals from apg and cpg had an increase in expression upon gustatory \n stimulation , whereas individuals from cg exhibited a decrease . pattern of secretion of salivary glycoproteins .", "a - representative western \n blots of mg2 pattern observed in all individuals from each group .", "b - \n representative western blots of lactoferrin pattern observed in all individuals \n from each group . t1t4", "( non - stimulated submandibular and sublingual \n saliva - smsl ) and t5t8 ( stimulated submandibular and sublingual saliva - smsl ) . \n", "apg - aggressive periodontitis group ; cpg - generalized chronic periodontitis \n group ; cg - control group" ], [ "comparison of expression among groups under non - stimulated conditions revealed that \n cg exhibited the highest expression of mg2 and lactoferrin ( figures 2a and 2b ) .", "apg \n showed the lowest levels of mg2 , and cpg of lactoferrin ( figures 2a and 2b ) . \n", "comparison of expression among groups under stimulated conditions showed that cg \n exhibited the highest expression of mg2 , whereas apg the highest of lactoferrin \n ( figures 2c and 2d ) .", "again , apg showed the lowest levels of mg2 , and cpg of \n lactoferrin ( figures 2c and 2d ) .", "comparison of glycoproteins expression under non - stimulated ( a , b ) or \n stimulated ( c , d ) conditions .", "a - mg2 western blot ; b - lactoferrin western blot ; \n c - mg2 western blot ; d - lactoferrin western blot ; apgaggressive periodontitis \n group ; cpg - generalized chronic periodontitis group ; cg - control group" ], [ "the infectious nature of the periodontitis is related to specific microbiota as well as \n to the expression of particular inflammatory markers .", "additionally , initiation and progression of periodontal \n infections is clearly modified by local and systemic conditions ( risk factors ) , such as \n diabetes mellitus and cigarette smoking , as well as potentially important risk \n indicators including stress . \n", "salivary proteins are not included in the category of risk indicators , but this concept \n is being explored .", "investigation of salivary proteins in individuals with periodontitis may be useful to \n enhance the knowledge of their role in this population .", "statistical analysis did not \n reveal flow rate differences among groups , stimulated or non - stimulated .", "therefore , we \n decided to analyze equal volumes ( 50 l ) of smsl by immunoblotting , instead of equal \n amounts of protein . ", "it has been previously reported that mg2 can agglutinate several pathogens from the oral \n cavity , and in \n addition , interact and kill the periodontal pathogen a. \n actinomycetemcomitans . \n in a previous study", ", the concentration of mg2 was determined in stimulated whole saliva \n of subjects suffering from a. actinomycetemcomitans associated \n pd .", "it has been reported that \n mg2 output in the diseased subjects was decreased at least by a factor three in \n comparison to periodontally healthy subjects and that this low concentration of mg2 \n suggested a decline in mucin defense and consequently , a higher susceptibility to oral \n infections . in the present study ,", "blotting analysis revealed that all groups showed an increase of \n mg2 expression upon stimulation revealing a similar pattern ( figures 1a and 1b ) .", "a previous \n study that analyzed the pattern of secretion of mg2 in samples from individuals without \n periodontitis reported that pas \n stained gels did not reveal differences in mg2 secretion patterns upon stimulation . \n however , western blots indicated that mg2 expression was enhanced after stimulation ; a \n result that is in agreement with the observed pattern in the present study for cg .", "it was also observed that individuals without periodontitis exhibited the highest \n expression of this small salivary mucin under specific conditions ( figures 2a and 2c ) .", "the \n reduced output of this salivary mucin in individuals with aggressive periodontitis \n observed on blots ( figures 2a and 2c ) is similar to the one described . since in chronic periodontitis", "the \n destruction of the periodontal ligament and loss of the adjacent supporting bone usually \n displays a slow to moderate rate of progression , we hypothesize that the intermediate \n expression of mg2 observed in individuals belonging to this group ( figures 2a and 2c ) could in \n part contribute to decrease the disease progression rate in comparison to aggressive \n periodontitis .", "salivary lactoferrin is a single chain iron - binding glycoprotein that binds ferric ions \n and possesses antibacterial , antimycotic , antiviral and anti - inflammatory \n properties .", "the impact of a surgical periodontal treatment in the \n concentration of this glycoprotein in non - stimulated and stimulated whole saliva samples \n from individuals with chronic periodontitis was analyzed previously .", "it was reported that the concentrations \n decreased significantly in both non - stimulated and stimulated samples after the surgical \n treatment , suggesting that lactoferrin may be suitable for monitoring periodontal \n treatment results .", "another study evaluated lactoferrin levels from individuals with localized aggressive \n periodontitis before and after periodontal therapy . among other samples", ", \n paraffin - stimulated whole saliva was obtained from these individuals and from \n periodontally healthy controls .", "differences between groups and with respect to \n periodontal therapy were not observed in lactoferrin concentrations in whole \n saliva .", "concentrations and output of lactoferrin were also determined in whole saliva samples \n from subjects suffering from a. actinomycetemcomitans - associated \n pd .", "the output of lactoferrin \n was not significantly different in healthy and diseased subjects although a higher \n iron - saturation of lactoferrin in subjects with the disease was described .", "lactoferrin expression was examined in whole and parotid saliva from individuals with \n localized aggressive periodontitis and compared to age- , gender- , and race - matched \n controls .", "it was reported that \n whole saliva from the test group had higher levels of the glycoprotein although the \n levels of bound iron were significantly reduced , suggesting that lactoferrin from \n individuals with localized aggressive periodontitis might interfere in the disease \n progression . in the present study , it was observed that individuals with or without periodontitis \n exhibited a similar pattern of lactoferrin secretion ( figure 1b ) .", "comparison of expression among groups revealed that under \n non - stimulated conditions individuals without periodontitis ( cg ) showed the highest \n expression of lactoferrin ( figure 2b ) whereas \n under stimulated conditions individuals with periodontitis ( apg and cpg ) exhibited a \n more intense immunoreactive signal ( figure \n 2d ) .", "the higher expression of this glycoprotein in individuals with aggressive periodontitis \n is in agreement with a previous report , but is in contrast to others .", "the differences in \n these two studies from the present could be at least in part explained by variations in \n samples that were analyzed ( i.e. smsl versus whole saliva ) since lactoferrin derived \n from the gingival crevicular fluid will be present in samples from whole saliva .", "the increase in the amount of lactoferrin in stimulated samples from smsl of individuals \n with generalized chronic periodontitis ( figure 2d ) \n found in the present study is in contrast to another report in which the levels of lactoferrin in samples from \n non - stimulated whole saliva were higher than in samples of stimulated whole saliva . \n", "again , the differences in this study from the present could also be in part explained by \n smsl versus whole saliva .", "exclusion of individuals that had aggressive or chronic periodontitis and received \n periodontal treatment during the last year had a direct impact in our sample size , since \n it was difficult to select individuals that met this specific criterion .", "nevertheless , \n this is the first study that compares the expression of salivary proteins among \n individuals with aggressive and chronic periodontitis at the same time , and that \n evaluates the influence of stimulation over time in the expression of mg2 and \n lactoferrin in individuals with periodontitis .", "analysis of these salivary constituents \n appears to have potential to be used as markers for aggressive or chronic periodontitis , \n and since both mg2 and lactoferrin possess antimicrobial activity , specific domains \n could be considered as possible candidates for the development of new drugs that in \n principle could be of benefit to individuals with periodontitis .", "the biological plausibility of the differences observed in smsl physiology in our study \n could be derived by periodontal bacterial components triggering the host - immune \n response , and causing inflammation and activation of pro - inflammatory mediators .", "it has \n been described that these molecules traveling via blood to other organs and tissues \n might influence a variety of mechanisms .", "it is important to mention that the observed results show that \n future studies should purposely collect non - stimulated and/or stimulated saliva samples \n since protein expression under these circumstances varies .", "the reduced expression of \n these glycoproteins observed in apg and cpg particularly under non - stimulated \n conditions , decrease the oral cavity innate defense mechanisms provided by these \n salivary components in these individuals ." ], [ "it remains unclear to what extent the reduced output of mg2 and lactoferrin might impact \n the etiopathogenesis of aggressive and chronic periodontitis .", "however , the data suggest \n an active role of these glycoproteins in the innate immune regulation of periodontal \n bacterial colonization and disease progression ." ] ]

[ "parasitic protozoans of the phylum apicomplexa are the most prevalent and successful pathogens known to humankind .", "today , half of the world 's population is at risk of malaria caused by four plasmodium species , and more than 50 billion livestock reared for food production suffer from debilitating intestinal diseases caused by many species of eimeria , theileria , and babesia , amongst others .", "eimeria is the cause of coccidiosis in chickens , a parasite that infects the intestinal mucosa of the infected bird leading to severe weight loss and even death of the host .", "cryptosporidiosis is caused by cryptosporidium species and , like eimeria , is transmitted by accidental ingestion of highly resistant and environmentally stable oocysts that contaminate the food and water .", "the disease is marked by self - limiting diarrhoea in immunocompetent individuals , but in immunocompromised patients , the disease can be fatal .", "babesia is related to the malaria parasite in that it infects the reticulocytes of the infected cow and causes severe pathology and can cause death as well .", "toxoplasma is the cause of toxoplasmosis in humans , a disease characterized by mild flu - like symptoms in healthy hosts .", "however , immunocompromised individuals , such as hiv / aids patients and organ transplant recipients , often suffer from ocular toxoplasmosis or even encephalitis .", "theileria , an important cattle parasite transmitted by ticks , is characterized by anaemia and high mortality rate especially in pregnant cows . \n", "plasmodium infects red blood cells and is the cause of malaria in humans as well as in several other vertebrate and bird species .", "nearly one million human deaths are attributed to malaria each year , meaning that every 30 seconds a child dies of this disease in africa .", "this high toll in human and animal life and wellbeing has been further exacerbated by the inappropriate use of antimicrobial compounds over the years .", "thus , widespread resistance to most ( if not all ) drugs used to date makes control of these parasites extremely difficult [ 3 , 4 ] .", "the novel artemisinin - based therapies are considered to be the new hope for malaria control and have proved to be successful in interrupting the maturation of the infectious stages ( oocysts ) in the related parasite , eimeria , thereby reducing or blocking the transmission and spread of the parasite [ 57 ] . however", ", there are fears that overuse of these novel compounds will also facilitate the selection of even more potent strains . over the past three decades", ", a number of putative protective antigens from several members of the phylum has attracted a great deal of research and commercial interest in the hope to develop vaccines and alleviate the burden on public health and world economy imposed by this class of parasites [ 817 ] .", "the first antiprotozoan subunit vaccine developed to date , coxabic , which contains antigens isolated from the sexual stages of development of eimeria is a proof of principle for transmission - blocking immunity and an example of a strategy that has been proved successful in helping to tackle one of these important apicomplexan diseases [ 1821 ] . despite the enormous efforts to characterise the apicomplexan immunostimulatory antigens and genes encoding them , the fine cellular and molecular details of the effector mechanisms crucial for parasite inhibition and stimulation of protective immunity are still not fully understood .", "it is hoped that unravelling the proteomes and genome sequences of these protozoan pathogens will facilitate our understanding of the mechanisms involved in the infectious process and lead to the design of new effective control strategies .", "early studies concerning the developmental biology and immunology of the apicomplexans have provided valuable insights into the immune mechanisms responsible for the inhibition of parasite growth and development and in the establishment of host resistance to infection [ 2327 ] .", "efforts by research laboratories across the globe have demonstrated that , in order to control the infection , both the innate and adaptive arms of the immune system are crucial for resistance and cross - protection .", "this paper provides an overview of apicomplexan biology and focuses on the protective immune response against the various types of apicomplexan parasites , from eimeria to plasmodium including toxoplasma , cryptosporidium , theileria , and babesia .", "it also addresses the evolutionary relationship of these parasites and their hosts and the modulation of the host immune response that are critical in determining the outcome of an infection .", "the apicomplexan life cycle includes both asexual multiplication ( schizogony , merogony ) and sexual reproduction ( gametogony ) [ 25 , 29 , 30 ] .", "while some members of the phylum require an intermediate host and a variety of cell types to complete their developmental life cycle ( i.e. , plasmodium , babesia , theileria , toxoplasma ) , others lead a monoxenous life style with the asexual and sexual stages of development restricted to specific tissues of a single host ( i.e. , eimeria and cryptosporidium species ) .", "thus , the possibility of culturing asexual stages in vitro , as well as the feasibility of isolating relatively large numbers of sexual forms ( gametocytes ) , has granted eimeria species a status of an attractive and a relatively simple model for investigating parasite - host interactions , as well as applying transmission - blocking immunity . ", "apicomplexan parasites affect all classes of vertebrates , including fish , amphibians , reptiles , birds and mammals .", "the apparently long coevolutionary history of the apicomplexans means that targeting the metabolic reactions and pathways of the parasite also harms the host , making the identification of therapeutic targets extremely difficult .", "recent advancements in molecular biology have shed new light into the coevolutionary history of the apicomplexa and their hosts .", "this is based on the finding that the branching of the evolutionary tree of at least some of these parasites coincided with the evolution of the vertebrate host .", "in addition , it was found that several enzymes involved in a variety of metabolic pathways are highly conserved between the parasite and its host .", "furthermore , comparative studies of whole genome nucleotide sequences in several members of the phylum have revealed that surface proteins , unlike the house - keeping proteins and enzymes , have evolved rapidly over the past 500 million years due to functional and immune selective pressure .", "this is especially evident in the molecules comprising the apical complex , the invasion machinery mediating physical recognition , cytoadherence , and penetration of the host , which are parasite specific . ", "the successful invasion of the host cell by the apicomplexan parasite is dependent upon the sequential secretion of proteins and other molecules from the rhoptries , micronemes , and dense granules and results in the formation of the parasitophorous vacuole ( pv ) ( i.e. , toxoplasma , cryptosporidium , plasmodium , and eimeria spp . ) .", "this in turn provides access to intracellular nutrients and protection from the host 's immune system .", "although the pv shields the parasite from the host defences , at the same time it restricts access to nutrients in the host 's cytoplasm .", "thus , the apicomplexa has adopted different tactics to circumvent this problem , including biochemical modification of the pv making it permeable to essential nutrients .", "in contrast , some parasites , such as theileria spp . , do not form the pv and proliferate freely in the cytoplasm with direct access to host nutrients . ", "lateral gene transfer , best known for its role in antibiotic resistance in bacteria , has been proposed as a mechanism by which these opportunistic organisms acquire new genes that confer parasite fitness [ 3638 ] .", "for example , the apical complex , actin - myosin - powered motor , evolved as a result of the nuclear transfer of genes acquired during the secondary endosymbiotic event .", "it is believed that origin of the apicoplast can be attributed to endosymbiotic partnership in which the plastid - containing eukaryote was engulfed by a second eukaryotic cell . in addition , sexual replication appears to be another contributing factor to the diverse functions of the apicomplexan surface proteins and the adaptations to genera - specific niches [ 3941 ] .", "therefore , it is not surprising that plasmodium and babesia species share evolutionarily conserved mechanisms of erythrocyte invasion .", "moreover , transmembrane proteins ( thrombospondin - related anonymous proteins trap ) bridging the apical complex to the host cell in plasmodium , eimeria , and toxoplasma also share a high degree of homology [ 34 , 4244 ] , suggesting that apicomplexa use the same molecular machinery to invade a wide variety of cells . a great deal of research has also been carried out to study the role of surface antigens in parasite growth , development , and survival .", "passive transfer of polyclonal and monoclonal antibodies raised to asexual stage surface antigens of t. gondii are capable of conferring resistance against lethal challenge with this parasite [ 45 , 46 ] . similarly , eimeria and plasmodium antisporozoite and antimerozoite antibodies that recognize surface antigens , namely , glycosyl - phosphatidylinositol- ( gpi- ) anchored antigens in e. tenella ( etsag1 ) and p. falciparum ( msp1 ) , respectively , are able to induce a strong inhibitory response and provide protection against infection [ 47 , 48 ] .", "in addition , the antigens found on the surface of sporozoites have been implicated in the recognition and invasion of the hepatocytes in malaria and , therefore , represent promising targets for vaccine developers [ 10 , 42 , 49 , 50 ] ( a detailed description of apicomplexan invasion and egress has been reviewed recently by westwood and colleagues with a special emphasis on elements of the apical complex and their potential role as vaccine targets ) .", "thus , both asexual - stage surface proteins and the molecules associated with the apical complex have been proposed as potential candidates for vaccine development . ", "another conserved feature of the phylum is the transmissible , environmentally durable oocyst / cyst , the zygote stage of the coccidia ( i.e. , toxoplasma , eimeria , cryptosporidium , and neospora species ) [ 29 , 52 ] .", "it is intriguing that asexual reproduction in eimeria is tightly regulated , and although signals initiating the start of sexual reproduction have not yet been identified , the 2nd , 3rd , and 4th generation merozoites ( depending upon which eimeria species infects the host chicken ) differentiate into male ( micro- ) and female ( macro- ) gametes in a synchronised manner .", "the trademark of apicomplexan gametogenesis is the synthesis of numerous lipid bodies and polysaccharide granules , believed to be acquired from the host cell , serving as an energy source for the developing zygote .", "moreover , increased dna synthesis and rna transcription to produce multinucleated microgametocytes and heightened protein synthesis in the macrogametocytes to produce wall forming bodies ( wfbs ) are characteristics of sexual stage development at the molecular level [ 5356 ] .", "gametogenesis is completed by the formation of environmentally resilient oocysts in the mucosae lining the gastrointestinal tract of chickens , humans , and cats , respectively .", "the oocysts are then excreted with the faeces where they mature ( sporulate ) becoming infectious .", "cryptosporidium is slightly different from eimeria and toxoplasma in that the cryptosporidium oocysts can also release sporozoites in the gut which are capable of infecting new epithelial cells ( i.e. , autoreinfection ) . in all three cases ,", "transmission is primarily by the accidental ingestion of sporulated oocysts . unlike the intestinal parasites described above , gametogenesis of plasmodium and babesia spp .", "is completed in the gut of their arthropod hosts . during development , after fertilization of macrogametes by the microgametes and invasion of the gut by the ookinete , the zygotes encase themselves in a protective single - layered cyst wall to form an oocyst .", "sporozoites then exit the oocyst and migrate from the midgut to the salivary gland of their arthropod host .", "from there , they are injected into the blood and subcutaneous tissue of the next vertebrate host the arthropod bites .", "studies to elucidate the mechanism(s ) of the protective immune response to apicomplexan parasites have been limited mainly due to the lack of being able to carry out such studies in the definitive host such as cattle or human beings .", "thus , much of our understanding of the protective immune response to apicomplexan infections has been derived from murine models which can be genetically modified [ 26 , 57 ] .", "generally speaking , parasite replication in the host eventually leads to host cell lysis and parasite egress .", "it is now widely accepted that cells of the innate immune system and the molecules they produce and/or secrete are important controlling factors of parasite infectivity and in limiting the extent of parasitemia [ 5861 ] .", "it is not clear exactly how these molecules , particularly in the case of parasites infecting host erythrocytes ( i.e. , plasmodium and babesia spp . ) , interfere with parasite development . ", "what does appear to be the case is that this inhibition is accomplished by the production of gamma interferon ( ifn-),by natural killer cells ( nk ) , and tumour necrosis factor alpha ( tnf- ) , nitric oxide ( no ) and reactive oxygen species ( ros ) by macrophages [ 6264 ] .", "despite the fact that the mechanisms by which ifn- mediates protection are not completely understood , studies using ifn- and inos knock - out mice infected with t. gondii indicated that activation of p47 guanosine triphosphatases ( gtpases ) leads to degradation of the pv in infected cells . while the immune response in healthy hosts is often but not always able to control parasite replication and limit the disease , immunocompromised individuals fail to stop parasite growth , and clinical disease develops in nearly all cases .", "this is especially evident in toxoplasmosis where immunocompetent hosts control parasite replication causing tachyzoites ( the rapidly replicating asexual stages ) to migrate to muscle and brain tissues where they differentiate into bradyzoite cysts ( the slow - replicating form ) and persist throughout the host 's life . although the tissue cysts can become reactivated periodically , most healthy hosts never develop clinical disease . in contrast ,", "immunocompromised patients , such as those suffering from aids , remain chronically infected , whereby reactivation of the tissue cysts can lead to toxoplasmic encephalitis with severe pathological consequences . ", "the ability to clear acute and chronic infections with the apicomplexa seems to correlate with host cd4 + t - cell levels [ 62 , 66 , 67 ] .", "thus , studies involving athymic animals have shown that t cells play a crucial role in the infectious process [ 62 , 68 ] . ", "the population of t cells coexpressing markers appears to be important for host defence against apicomplexan infections [ 69 , 70 ] .", "thus , it was demonstrated that tcr-deficient mice developed severe disease when compared to controls .", "furthermore , mice lacking major histocompatibility complex class ii expression ( i.e. , cd4 + t - cell deficient ) appeared more susceptible to apicomplexan infections [ 62 , 67 ] . cd8 + t cells also appear to play a role in parasite growth and dissemination because they provide sporozoite transport from their initial infection site , as is the case for eimeria and toxoplasma infections [ 69 , 71 ] .", "during the course of primary infection with eimeria , cd8 + t cells appear to play a role in parasite growth and dissemination because they provide sporozoite transport from their initial infection site to the crypt cells . on the other hand", ", reports have also shown that increased numbers of cd8 + t cells in the crypt epithelium act as cytotoxic killer cells facilitating the clearance of the parasite - infected cells [ 72 , 73 ] .", "furthermore , studies in infected chickens have shown that the contribution of both cd4 + and cd8 + t - cell populations differs according to the infective species used . nevertheless , during avian coccidiosis and babesiosis", ", cd4 + t - cell subsets were found to be elevated in animals following challenge infections [ 67 , 70 , 72 ] .", "an intriguing question arises from all of these observations : why are some species or strains of parasites extremely ", "immunogenic and induce protective immunity , while others seem to be invisible to the immune system ? studies on naturally acquired immunity to malaria have shown that adequate protective immunity to p. falciparum , the etiological agent of the most severe malaria in humans , usually required repeated infections . thus , protection against this particular strain appeared to be acquired more slowly than against the less pathogenic p. vivax or p. malariae .", "moreover , numerous studies have shown that immunity appeared to be species specific and did not confer protection against challenge with heterologous species . however , it was reported that heavy exposure to parasites induces the development of antigenic memory .", "although the molecular and cellular mechanisms driving the onset of protective host immunity against malaria or any other pathogenic protozoan are not entirely understood , it is believed that susceptibility to infection is driven by extrinsic factors such as antigenic variation and also by intrinsically inappropriate immune responses .", "this is particularly evident in theileria infections of immunocompromised cattle , which often results in the death of the host animal .", "it is theorised that the ability of theileria to interfere with the host 's apoptotic pathways is the crucial factor contributing to mortality [ 35 , 76 ] . once inside the host cell", ", theileria resides free in the cytoplasm and induces uncontrolled proliferation of the infected cell .", "thus , it appears that during theileria infection the immune system fails to control this proliferation in time , in turn resulting in potent , nonspecific lysis of both infected and noninfected cells . ", "studies using immunocompetent animals have shown that , in addition to innate host resistance , ifn- plays a key role in the development of adaptive immunity and clearing of apicomplexa infections .", "for example , cryptosporidium - infected mice , with faulty ifn- gene expression , suffered from severe mucosal destruction , and as a result they also secreted more oocysts [ 60 , 77 ] . furthermore", ", mopping - up the secreted ifn- by antibodies in immunocompromised animals seemed to worsen c. parvum infection .", "in addition , it is now widely accepted that il-12 , known to increase host ifn- production , can reduce the severity and even prevent infection by apicomplexan parasites .", "although believed to be mediated by an ifn--dependent mechanism , the pathways or downstream molecules crucial in this process have not been well defined to date .", "although viruses , which entirely depend on the host machinery for replication and assembly of new viral particles , are the experts in host cell manipulation , the apicomplexa are considered to be the masters of disguise .", "this is because they have evolved to evade the host immune system to aid in their own survival .", "antigenic variation has been proposed as a key factor in this process . unlike allelic polymorphism , which results in different phenotypes or so - called parasite strains ,", "antigenic variation is the tightly regulated expression of different genes of a clonal population of parasites over the natural course of infection .", "antigenic variation amongst malarial and babesia parasites is a prime example of sophistication apicomplexans employed to avoid antibody - mediated inhibition [ 7981 ] .", "p. falciparum achieves this by secreting a single type of a variant molecule ( parasite - derived erythrocyte membrane protein 1 - pfemp1 ) on the surface of the infected erythrocyte at any one time .", "the pfemp1 surface proteins are encoded by a family of genes , called var genes , and each individual parasite expresses only a single var gene , keeping all other members of var gene family in a transcriptionally silent state [ 8284 ] .", "this strategy in turn induces adhesion of the parasite - infected erythrocytes to the blood vessels to avoid reaching the spleen , whose main function is to rid the body of damaged and/or infected blood cells . similarly ,", "sequestration of babesia - infected erythrocytes in the microvasculature enables the babesia to persist within the host maximizing its chances of transmission .", "this cytoadherence in babesia is mediated by constant gene conversion of ves family genes encoding the variant erythrocyte surface antigen 1 ( vesa1 ) . ", "a puzzling question arises from these observations : if infected erythrocytes pass through the body unchecked since they lack major histocompatibility complex ( mhc ) expression , overwhelming proliferation of the parasites may cause premature death of the host prior to successful transmission to an arthropod vector ?", "interestingly , in spite of the fact that malaria parasites sequentially express variant surface molecules exposing the immunodominant antigens to the host immune defences , infection is actually prolonged .", "thus , the parasite must undergo antigenic variation and rates of growth that enable the host to control infection while allowing for transmission of the parasite prior to its death .", "due to coating of the merozoite surface with glycosyl - phosphatidyl - anchored proteins crucial for initial attachment to the host erythrocyte surface , they are targeted by host - protective antibodies .", "these surface - anchored proteins ( variable merozoite surface antigens - vmsa ) exhibit varying degrees of intra - species antigenic polymorphisms allowing these parasites to evade the host immune system at the population level .", "nevertheless , studies involving african children have shown that variant specific immunity , namely , secretion of igg antibodies directed against p. falciparum variant surface antigens ( vsa ) , has been correlated with protection from clinical malaria in ghana , kenya , and tanzania [ 79 , 87 , 88 ] .", "thus , vsa antigens have been proposed as excellent candidates for malaria and babesia vaccine development .", "although b cells have been regarded as minor contributors to protective immunity and resistance to primary infections with apicomplexa , numerous studies have shown that hosts infected with these parasites are capable of producing protective , parasite - specific immunoglobulins ( ig ) of all major classes after an episode of infection and recovery [ 57 , 8992 ] .", "thus , early work by rose and colleagues has shown that humoral antibodies , induced by live eimeria infection , can provide excellent passive protection against challenge infections with the same parasite [ 93 , 94 ] .", "likewise , studies on mice infected with t. gondii have shown that intestinal iga antibodies to major surface protein sag-1 ( p30 ) were produced after peroral infection and found to inhibit infection of murine enterocytes by directly blocking the parasite entry .", "in addition , precigout et al . have demonstrated an inhibitory effect of antibodies directed against a 17-kda merozoites membrane protein on b. divergens parasite growth . ", "furthermore , studies on invasion of red blood cells by p. falciparum merozoites have revealed that since rbcs do not express the mhc complex , parasite killing by t lymphocytes is not important . instead , antibodies specific to merozoite surface molecules ( msp-1 ) and proteins externalised from the apical complex play a major role in immunity to asexual blood stages . ", "the plasmodium merozoite surface protein 1 ( msp-1 ) is a 200 kda multicomponent precursor complex derived by proteolytic processing during erythrocyte invasion .", "the 42 kda c - terminal component is cleaved ( i.e. , secondary processing ) to produce soluble 33 kda and 19 kda fragments that remain on the merozoites surface .", "studies have shown that anti - merozoite antibodies are capable of neutralizing parasites by fc - dependent mechanisms involving macrophages , thus reducing the parasitemia and clinical disease [ 87 , 97 , 98 ] .", "in addition , a number of recent studies have shown that children naturally infected with malaria secrete anti - msp-1 antibodies ( msp-119 mab ) that block the binding of plasmodium merozoites to the surface of the red blood cells and also inhibit secondary processing of msp-1 .", "in addition , studies investigating the protective properties of maternally derived igg and igm antibodies to the 19 kda domain of msp-1 of p. falciparum have shown that mothers who have tested positive for anti - msp-1 ( 19 kda fragment ) igg antibodies conferred protection against placental infection and infection in their infants . ", "it has been shown that , in babesiosis infection , igg antibodies produced as a result of live infection can prevent infection of erythrocytes by binding and neutralizing sporozoites before they invade their target cells .", "similar observations were reported in chickens where antisporozoite antibodies specific to glycosyl - phosphatidylinositol - anchored e. tenella surface antigen 1 ( etsag1 ) appeared to inhibit parasite binding and invasion of the host cell .", "however , it seems that the protective role of these antibodies is limited since it can only neutralize sporozoites from the time the parasites egress and the time they gain access to new cells .", "thus , it is hoped that genome - wide fingerprinting techniques will aid in the identification of additional immunoprotective antigens that can be used in combination to induce the maximal inhibitory humoral immune response . ", "in addition to antigen - specific polyclonal and monoclonal antibodies capable of inhibiting asexual stages , antibodies raised to antigens localized exclusively to gametocyte / zygote stages were also found to be highly immunogenic and capable of providing passive protection in vivo [ 20 , 101 , 102 ] .", "early experiments involving immunisation with purified sexual - stage gametes of p. gallinaceum in chickens showed that effective transmission - blocking immunity can be achieved by reducing the infectivity of gametocytes and oocyst development [ 103 , 104 ] .", "thus , pfs25 and pvs25 proteins expressed on the surface of ookinetes in the mosquito stage of p. falciparum and p. vivax have been used extensively as candidates for malaria transmission - blocking vaccines , since lowering the density of circulating parasites would not produce sterilizing immunity , instead it would allow individuals to develop long - lasting , naturally acquired immunity to malaria [ 12 , 105 , 106 ] . ", "work by wallach and coworkers , aimed at applying transmission - blocking immunity to control infections caused by eimeria , hypothesised that antibodies raised against the gametocyte / zygote stages of development can act to inhibit oocyst development and thereby provide a block in parasite transmission ( see figure 1 ) .", "a method was developed for purifying e. maxima gametocytes from the infected chicken gut mucosa and immunodominant gametocyte antigens , namely , emgam56", ", emgam82 , and emgam230 localized to the wfbs and the oocyst wall of the maturing zygote , were extracted [ 53 , 102 , 108 ] .", "passive immunisation experiments showed that there was a good correlation between the intensity of igg and igm antibodies binding to gametocyte antigens by western and elisa with the ability of those sera to provide passive protection in vivo .", "the mechanisms by which these antibodies inhibit oocyst maturation are still obscure ; however , it is hypothesised that antibodies raised to the immunodominant antigens retard zygote development by interfering with the processing of wall proteins or the wall - hardening processes [ 53 , 110 ] .", "in addition , a protective monoclonal antibody raised against emgam56 localised to the wfb2 ( 1e11 - 11 ) , as well as the inner layer of the oocyst wall , was also found to react strongly with the stieda body of the sporulated oocysts ( m. wallach , unpublished data ) .", "similar results were reported by krcken et al . using a monoclonal antibody e2e5 raised to wfb2s of e. tenella .", "the in vitro excystation inhibition assay showed that the antibody e2e5 can significantly interfere with parasite development by impairing sporozoite excystation .", "it is tempting to speculate that the 1e11 - 11 monoclonal antibody inhibits or blocks excystation of the sporocyst in a similar manner , thereby reducing the number of infectious sporozoites released in the intestine of infected birds allowing them to develop protective immunity induced by exposure to low doses of parasites . ", "jenkins and colleagues have shown that ruminants immunized with a dna vaccine expressing a gene isolated from c. parvum encoding a sporozoite antigen ( cp 15/60 ) were capable of inducing antigen - specific antibodies [ 111 , 112 ] . in that study , it was found that using various routes of vaccination resulted in differing antibody responses and titres .", "the authors , therefore , suggested that the route of antigen delivery of any protozoan vaccine requires careful formulation and optimisation of delivery systems .", "finally , in studies carried out by wallach and co - workers on eimeria , it was found that in order to achieve protective immunity using parasite extracts requires the inclusion of the correct antigens and exclusion of the irrelevant ones .", "their results indicated that while some parasite - specific antigens induce protective immunity , others actually induce an exacerbation of the infection .", "therefore , in the design of any parasitic vaccine , it is crucial that the combination of various antigens maximizes their inhibitory effect on parasite growth and development .", "one of the main defence mechanisms employed by host cells is programmed cell death ( apoptosis ) ensuring regulated removal of damaged and infected cells . but", "because the survival and development of intracellular apicomplexan parasites is dependent upon the continuous supply of host cell nutrients and protection from immune attack , the parasites have adapted to extend the life of the infected cells by inhibiting the host cell apoptotic machinery through interference with the intracellular signalling molecules , notably phosphatidylinositol 3-kinase ( pi3-k ) .", "pi3-k is involved in a variety of functions including cell growth , proliferation , and intracellular trafficking , amongst others [ 61 , 114117 ] .", "p. falciparum is a good example of how parasite secreted proteins prevent host cell death to ensure its own development and survival .", "sporozoites of plasmodium species are stealthy invaders that first travel to the liver ( hepatocyte ) cells , where the growth and development of the daughter cells , hepatic merozoites , takes place .", "recent results have shown that prior to the establishment of the pv , sporozoites of p. falciparum transmigrate through a number of hepatocytes before they anchor to and invade the suitable cell via exocytosis of proteins contained within the apical complex .", "it has been shown that the thrombospondin - related adhesive - protein- ( trap- ) like protein plays a role in this process . additionally , the wounding of the hepatocyte induced by invading sporozoites releases growth factors which in turn appear to inhibit pi3-k and block the signalling pathways destined for apoptosis .", "leirio and colleagues have shown that once the parasite is established in the hepatocyte , it secretes hgf / met signalling molecules into the host cell cytoplasm , thereby conferring resistance to apoptosis to ensure survival and maturation of the daughter cells . however , which signalling upstream of pi3-k occurs during plasmodium infection is yet to be determined .", "interestingly , upon maturation of merozoites , plasmodium seems to be able to induce host cell death to liberate the motile progeny .", "have shown that this process involves cysteine proteases [ 49 , 50 ] . moreover ,", "similar mechanisms were found to play a role in release of sporozoites from the oocysts .", "although work is ongoing to try and elucidate the mechanisms involved in these processes , it appears that the apicomplexa have learned to inhibit host cell death during parasite development and subsequently activate it , liberating thousands of new progeny . \n", "t. gondii has also evolved a broad spectrum of adaptations to challenges presented by its life style .", "chronic toxoplasmosis is the trademark of the parasite 's success and is induced by the slow - replicating bradyzoites safely tucked away in the remodelled pv , the tissue cyst . like plasmodium ,", "t. gondii modulates host cell apoptosis by both inhibiting and triggering the programmed cell death .", "chen et al . have shown that fas / fasl ligand - dependent mechanisms mediate the inflammatory responses induced by the apicomplexan infection [ 115 , 121 ] .", "but the parasites have evolved to neutralize granzyme / perforin - mediated killing of infected t cells and natural killer cells ( nk ) by modifying transcription and posttranscriptional modification of ifn--regulated genes , the major mediators of resistance to t. gondii infections . likewise , del cacho et al .", "have demonstrated that e. tenella and e. necatrix second - generation schizonts first induce nf- activation to protect the transformed cells from apoptosis , allowing the schizonts to mature and later cause nf- inhibition to trigger host cell apoptosis to facilitate the release of merozoites . ", "the apicomplexa have evolved to live in synergy with their infected hosts because they completely depend on it for survival ; however , some apicomplexan infections induce a great deal of immunopathology and can lead to host cell death .", "for example , eimeria and cyclospora both interfere with the absorption of nutrients across the intestinal mucosa and can cause death due to malaise , diarrhoea , and dehydration . because apicomplexans increase in numbers while , in their hosts , the severity of infection is proportional to the parasite density the smaller the number , the greater the chance of asymptomatic infection and the greater the chances of the parasite survival .", "however , the immunological defence of a host can also cause extensive tissue damage and clinical symptoms .", "patients with cerebral malaria usually have elevated levels of tumour necrosis factor alpha ( tnf- ) and ige considered to be responsible for fever and tissue lesions to an extent where vital functions of the host fail leading to a coma .", "despite a great deal of effort and technological advancements in biotechnology , molecular biology , genetics , immunology , and vaccinology , there are no vaccines for humans against malaria and toxoplasmosis at the present time , and it seems that we are losing the battle in the fight against pathogenic protozoans . the current failure to develop a practical vaccine may well be attributed to our inadequate understanding of the mechanisms underlying ( 1 ) the naturally acquired immunity against apicomplexans , ( 2 ) acquired parasite resistance to most ( if not all ) antimicrobial compounds used to date , and ( 3 ) in the case of arthropod transmitted protozoans , failure to implement adequate vector control programs in tropical and subtropical regions . the life cycles of the apicomplexa are complex , thus , it is hoped that a multivalent , multistage vaccine will alleviate the problems caused by these pathogenic protozoans .", "although this approach has attracted a great deal of commercial and research interest , the critical issues to be addressed include the identification of stage - specific antigens capable of inducing protective immunity and the delivery methods in a form that will stimulate an adequate protective immune response .", "the main impediment in the search and selection for immunostimulatory antigens is the lack of in vitro assays to analyse and predict immune responses .", "the transmission blocking assays , relying on counting the number of oocysts produced , and the inhibition of sporozoite invasion assays have both been used extensively to evaluate parasite inhibition induced by neutralizing antibodies . ", "although in vivo experimentation is extremely difficult for malaria , other model systems can be used to dissect the fine details and the effect of neutralizing antibodies .", "it is very possible that , in the development of an antiprotozoan vaccine capable of inducing only partial immunity , resistant mutants would be selected that are even more pathogenic than existing strains . in the malaria scenario", ", this could be catastrophic since the parasite would undergo recycling and be transmitted throughout the community leading to an increase in morbidity and mortality .", "it is , therefore , of great hope that in the battle against these pathogenic protozoan parasites , including plasmodium , cryptosporidium , and toxoplasma , the completion of their genomes and proteomes may provide information needed to design vaccines , assess the effects of immunization on parasite pathogenicity and the selection of unwanted mutants , and in the final analysis control the diseases caused by this class of parasites ." ]

[ "<S> members of the phylum apicomplexa , which includes the species plasmodium , eimeria , toxoplasma , and babesia amongst others , are the most successful intracellular pathogens known to humankind . </S>", "<S> the widespread acquisition of antimicrobial resistance to most drugs used to date has sparked a great deal of research and commercial interest in the development of vaccines as alternative control strategies . </S>", "<S> a few antigens from the asexual and sexual stages of apicomplexan development have been identified and their genes characterised ; however , the fine cellular and molecular details of the effector mechanisms crucial for parasite inhibition and stimulation of protective immunity are still not entirely understood . </S>", "<S> this paper provides an overview of what is currently known about the protective immune response against the various types of apicomplexan parasites and focuses mainly on the similarities of these pathogens and their host interaction . finally , the evolutionary relationships of these parasites and their hosts , as well as the modulation of immune functions that are critical in determining the outcome of the infection by these pathogenic organisms , are discussed . </S>" ]

[ "1. Introduction", "2. Apicomplexan Life Cycle and Parasite-Host Relationship", "3. Naturally Acquired Immunity to Apicomplexan Parasites Is Exclusively Dependent upon Cell-Mediated Immunity", "4. Masters of Disguise", "5. Humoral Immunity Protects against Challenge Infections", "6. The Apicomplexa Are the Manipulators of Host Defence Mechanisms", "7. Are We Losing the Battle against Apicomplexan Parasites?" ]

[ [ "parasitic protozoans of the phylum apicomplexa are the most prevalent and successful pathogens known to humankind .", "today , half of the world 's population is at risk of malaria caused by four plasmodium species , and more than 50 billion livestock reared for food production suffer from debilitating intestinal diseases caused by many species of eimeria , theileria , and babesia , amongst others .", "eimeria is the cause of coccidiosis in chickens , a parasite that infects the intestinal mucosa of the infected bird leading to severe weight loss and even death of the host .", "cryptosporidiosis is caused by cryptosporidium species and , like eimeria , is transmitted by accidental ingestion of highly resistant and environmentally stable oocysts that contaminate the food and water .", "the disease is marked by self - limiting diarrhoea in immunocompetent individuals , but in immunocompromised patients , the disease can be fatal .", "babesia is related to the malaria parasite in that it infects the reticulocytes of the infected cow and causes severe pathology and can cause death as well .", "toxoplasma is the cause of toxoplasmosis in humans , a disease characterized by mild flu - like symptoms in healthy hosts .", "however , immunocompromised individuals , such as hiv / aids patients and organ transplant recipients , often suffer from ocular toxoplasmosis or even encephalitis .", "theileria , an important cattle parasite transmitted by ticks , is characterized by anaemia and high mortality rate especially in pregnant cows . \n", "plasmodium infects red blood cells and is the cause of malaria in humans as well as in several other vertebrate and bird species .", "nearly one million human deaths are attributed to malaria each year , meaning that every 30 seconds a child dies of this disease in africa .", "this high toll in human and animal life and wellbeing has been further exacerbated by the inappropriate use of antimicrobial compounds over the years .", "thus , widespread resistance to most ( if not all ) drugs used to date makes control of these parasites extremely difficult [ 3 , 4 ] .", "the novel artemisinin - based therapies are considered to be the new hope for malaria control and have proved to be successful in interrupting the maturation of the infectious stages ( oocysts ) in the related parasite , eimeria , thereby reducing or blocking the transmission and spread of the parasite [ 57 ] . however", ", there are fears that overuse of these novel compounds will also facilitate the selection of even more potent strains . over the past three decades", ", a number of putative protective antigens from several members of the phylum has attracted a great deal of research and commercial interest in the hope to develop vaccines and alleviate the burden on public health and world economy imposed by this class of parasites [ 817 ] .", "the first antiprotozoan subunit vaccine developed to date , coxabic , which contains antigens isolated from the sexual stages of development of eimeria is a proof of principle for transmission - blocking immunity and an example of a strategy that has been proved successful in helping to tackle one of these important apicomplexan diseases [ 1821 ] . despite the enormous efforts to characterise the apicomplexan immunostimulatory antigens and genes encoding them , the fine cellular and molecular details of the effector mechanisms crucial for parasite inhibition and stimulation of protective immunity are still not fully understood .", "it is hoped that unravelling the proteomes and genome sequences of these protozoan pathogens will facilitate our understanding of the mechanisms involved in the infectious process and lead to the design of new effective control strategies .", "early studies concerning the developmental biology and immunology of the apicomplexans have provided valuable insights into the immune mechanisms responsible for the inhibition of parasite growth and development and in the establishment of host resistance to infection [ 2327 ] .", "efforts by research laboratories across the globe have demonstrated that , in order to control the infection , both the innate and adaptive arms of the immune system are crucial for resistance and cross - protection .", "this paper provides an overview of apicomplexan biology and focuses on the protective immune response against the various types of apicomplexan parasites , from eimeria to plasmodium including toxoplasma , cryptosporidium , theileria , and babesia .", "it also addresses the evolutionary relationship of these parasites and their hosts and the modulation of the host immune response that are critical in determining the outcome of an infection ." ], [ "the apicomplexan life cycle includes both asexual multiplication ( schizogony , merogony ) and sexual reproduction ( gametogony ) [ 25 , 29 , 30 ] .", "while some members of the phylum require an intermediate host and a variety of cell types to complete their developmental life cycle ( i.e. , plasmodium , babesia , theileria , toxoplasma ) , others lead a monoxenous life style with the asexual and sexual stages of development restricted to specific tissues of a single host ( i.e. , eimeria and cryptosporidium species ) .", "thus , the possibility of culturing asexual stages in vitro , as well as the feasibility of isolating relatively large numbers of sexual forms ( gametocytes ) , has granted eimeria species a status of an attractive and a relatively simple model for investigating parasite - host interactions , as well as applying transmission - blocking immunity . ", "apicomplexan parasites affect all classes of vertebrates , including fish , amphibians , reptiles , birds and mammals .", "the apparently long coevolutionary history of the apicomplexans means that targeting the metabolic reactions and pathways of the parasite also harms the host , making the identification of therapeutic targets extremely difficult .", "recent advancements in molecular biology have shed new light into the coevolutionary history of the apicomplexa and their hosts .", "this is based on the finding that the branching of the evolutionary tree of at least some of these parasites coincided with the evolution of the vertebrate host .", "in addition , it was found that several enzymes involved in a variety of metabolic pathways are highly conserved between the parasite and its host .", "furthermore , comparative studies of whole genome nucleotide sequences in several members of the phylum have revealed that surface proteins , unlike the house - keeping proteins and enzymes , have evolved rapidly over the past 500 million years due to functional and immune selective pressure .", "this is especially evident in the molecules comprising the apical complex , the invasion machinery mediating physical recognition , cytoadherence , and penetration of the host , which are parasite specific . ", "the successful invasion of the host cell by the apicomplexan parasite is dependent upon the sequential secretion of proteins and other molecules from the rhoptries , micronemes , and dense granules and results in the formation of the parasitophorous vacuole ( pv ) ( i.e. , toxoplasma , cryptosporidium , plasmodium , and eimeria spp . ) .", "this in turn provides access to intracellular nutrients and protection from the host 's immune system .", "although the pv shields the parasite from the host defences , at the same time it restricts access to nutrients in the host 's cytoplasm .", "thus , the apicomplexa has adopted different tactics to circumvent this problem , including biochemical modification of the pv making it permeable to essential nutrients .", "in contrast , some parasites , such as theileria spp . , do not form the pv and proliferate freely in the cytoplasm with direct access to host nutrients . ", "lateral gene transfer , best known for its role in antibiotic resistance in bacteria , has been proposed as a mechanism by which these opportunistic organisms acquire new genes that confer parasite fitness [ 3638 ] .", "for example , the apical complex , actin - myosin - powered motor , evolved as a result of the nuclear transfer of genes acquired during the secondary endosymbiotic event .", "it is believed that origin of the apicoplast can be attributed to endosymbiotic partnership in which the plastid - containing eukaryote was engulfed by a second eukaryotic cell . in addition , sexual replication appears to be another contributing factor to the diverse functions of the apicomplexan surface proteins and the adaptations to genera - specific niches [ 3941 ] .", "therefore , it is not surprising that plasmodium and babesia species share evolutionarily conserved mechanisms of erythrocyte invasion .", "moreover , transmembrane proteins ( thrombospondin - related anonymous proteins trap ) bridging the apical complex to the host cell in plasmodium , eimeria , and toxoplasma also share a high degree of homology [ 34 , 4244 ] , suggesting that apicomplexa use the same molecular machinery to invade a wide variety of cells . a great deal of research has also been carried out to study the role of surface antigens in parasite growth , development , and survival .", "passive transfer of polyclonal and monoclonal antibodies raised to asexual stage surface antigens of t. gondii are capable of conferring resistance against lethal challenge with this parasite [ 45 , 46 ] . similarly , eimeria and plasmodium antisporozoite and antimerozoite antibodies that recognize surface antigens , namely , glycosyl - phosphatidylinositol- ( gpi- ) anchored antigens in e. tenella ( etsag1 ) and p. falciparum ( msp1 ) , respectively , are able to induce a strong inhibitory response and provide protection against infection [ 47 , 48 ] .", "in addition , the antigens found on the surface of sporozoites have been implicated in the recognition and invasion of the hepatocytes in malaria and , therefore , represent promising targets for vaccine developers [ 10 , 42 , 49 , 50 ] ( a detailed description of apicomplexan invasion and egress has been reviewed recently by westwood and colleagues with a special emphasis on elements of the apical complex and their potential role as vaccine targets ) .", "thus , both asexual - stage surface proteins and the molecules associated with the apical complex have been proposed as potential candidates for vaccine development . ", "another conserved feature of the phylum is the transmissible , environmentally durable oocyst / cyst , the zygote stage of the coccidia ( i.e. , toxoplasma , eimeria , cryptosporidium , and neospora species ) [ 29 , 52 ] .", "it is intriguing that asexual reproduction in eimeria is tightly regulated , and although signals initiating the start of sexual reproduction have not yet been identified , the 2nd , 3rd , and 4th generation merozoites ( depending upon which eimeria species infects the host chicken ) differentiate into male ( micro- ) and female ( macro- ) gametes in a synchronised manner .", "the trademark of apicomplexan gametogenesis is the synthesis of numerous lipid bodies and polysaccharide granules , believed to be acquired from the host cell , serving as an energy source for the developing zygote .", "moreover , increased dna synthesis and rna transcription to produce multinucleated microgametocytes and heightened protein synthesis in the macrogametocytes to produce wall forming bodies ( wfbs ) are characteristics of sexual stage development at the molecular level [ 5356 ] .", "gametogenesis is completed by the formation of environmentally resilient oocysts in the mucosae lining the gastrointestinal tract of chickens , humans , and cats , respectively .", "the oocysts are then excreted with the faeces where they mature ( sporulate ) becoming infectious .", "cryptosporidium is slightly different from eimeria and toxoplasma in that the cryptosporidium oocysts can also release sporozoites in the gut which are capable of infecting new epithelial cells ( i.e. , autoreinfection ) . in all three cases ,", "transmission is primarily by the accidental ingestion of sporulated oocysts . unlike the intestinal parasites described above , gametogenesis of plasmodium and babesia spp .", "is completed in the gut of their arthropod hosts . during development , after fertilization of macrogametes by the microgametes and invasion of the gut by the ookinete , the zygotes encase themselves in a protective single - layered cyst wall to form an oocyst .", "sporozoites then exit the oocyst and migrate from the midgut to the salivary gland of their arthropod host .", "from there , they are injected into the blood and subcutaneous tissue of the next vertebrate host the arthropod bites ." ], [ "studies to elucidate the mechanism(s ) of the protective immune response to apicomplexan parasites have been limited mainly due to the lack of being able to carry out such studies in the definitive host such as cattle or human beings .", "thus , much of our understanding of the protective immune response to apicomplexan infections has been derived from murine models which can be genetically modified [ 26 , 57 ] .", "generally speaking , parasite replication in the host eventually leads to host cell lysis and parasite egress .", "it is now widely accepted that cells of the innate immune system and the molecules they produce and/or secrete are important controlling factors of parasite infectivity and in limiting the extent of parasitemia [ 5861 ] .", "it is not clear exactly how these molecules , particularly in the case of parasites infecting host erythrocytes ( i.e. , plasmodium and babesia spp . ) , interfere with parasite development . ", "what does appear to be the case is that this inhibition is accomplished by the production of gamma interferon ( ifn-),by natural killer cells ( nk ) , and tumour necrosis factor alpha ( tnf- ) , nitric oxide ( no ) and reactive oxygen species ( ros ) by macrophages [ 6264 ] .", "despite the fact that the mechanisms by which ifn- mediates protection are not completely understood , studies using ifn- and inos knock - out mice infected with t. gondii indicated that activation of p47 guanosine triphosphatases ( gtpases ) leads to degradation of the pv in infected cells . while the immune response in healthy hosts is often but not always able to control parasite replication and limit the disease , immunocompromised individuals fail to stop parasite growth , and clinical disease develops in nearly all cases .", "this is especially evident in toxoplasmosis where immunocompetent hosts control parasite replication causing tachyzoites ( the rapidly replicating asexual stages ) to migrate to muscle and brain tissues where they differentiate into bradyzoite cysts ( the slow - replicating form ) and persist throughout the host 's life . although the tissue cysts can become reactivated periodically , most healthy hosts never develop clinical disease . in contrast ,", "immunocompromised patients , such as those suffering from aids , remain chronically infected , whereby reactivation of the tissue cysts can lead to toxoplasmic encephalitis with severe pathological consequences . ", "the ability to clear acute and chronic infections with the apicomplexa seems to correlate with host cd4 + t - cell levels [ 62 , 66 , 67 ] .", "thus , studies involving athymic animals have shown that t cells play a crucial role in the infectious process [ 62 , 68 ] . ", "the population of t cells coexpressing markers appears to be important for host defence against apicomplexan infections [ 69 , 70 ] .", "thus , it was demonstrated that tcr-deficient mice developed severe disease when compared to controls .", "furthermore , mice lacking major histocompatibility complex class ii expression ( i.e. , cd4 + t - cell deficient ) appeared more susceptible to apicomplexan infections [ 62 , 67 ] . cd8 + t cells also appear to play a role in parasite growth and dissemination because they provide sporozoite transport from their initial infection site , as is the case for eimeria and toxoplasma infections [ 69 , 71 ] .", "during the course of primary infection with eimeria , cd8 + t cells appear to play a role in parasite growth and dissemination because they provide sporozoite transport from their initial infection site to the crypt cells . on the other hand", ", reports have also shown that increased numbers of cd8 + t cells in the crypt epithelium act as cytotoxic killer cells facilitating the clearance of the parasite - infected cells [ 72 , 73 ] .", "furthermore , studies in infected chickens have shown that the contribution of both cd4 + and cd8 + t - cell populations differs according to the infective species used . nevertheless , during avian coccidiosis and babesiosis", ", cd4 + t - cell subsets were found to be elevated in animals following challenge infections [ 67 , 70 , 72 ] .", "an intriguing question arises from all of these observations : why are some species or strains of parasites extremely ", "immunogenic and induce protective immunity , while others seem to be invisible to the immune system ? studies on naturally acquired immunity to malaria have shown that adequate protective immunity to p. falciparum , the etiological agent of the most severe malaria in humans , usually required repeated infections . thus , protection against this particular strain appeared to be acquired more slowly than against the less pathogenic p. vivax or p. malariae .", "moreover , numerous studies have shown that immunity appeared to be species specific and did not confer protection against challenge with heterologous species . however , it was reported that heavy exposure to parasites induces the development of antigenic memory .", "although the molecular and cellular mechanisms driving the onset of protective host immunity against malaria or any other pathogenic protozoan are not entirely understood , it is believed that susceptibility to infection is driven by extrinsic factors such as antigenic variation and also by intrinsically inappropriate immune responses .", "this is particularly evident in theileria infections of immunocompromised cattle , which often results in the death of the host animal .", "it is theorised that the ability of theileria to interfere with the host 's apoptotic pathways is the crucial factor contributing to mortality [ 35 , 76 ] . once inside the host cell", ", theileria resides free in the cytoplasm and induces uncontrolled proliferation of the infected cell .", "thus , it appears that during theileria infection the immune system fails to control this proliferation in time , in turn resulting in potent , nonspecific lysis of both infected and noninfected cells . ", "studies using immunocompetent animals have shown that , in addition to innate host resistance , ifn- plays a key role in the development of adaptive immunity and clearing of apicomplexa infections .", "for example , cryptosporidium - infected mice , with faulty ifn- gene expression , suffered from severe mucosal destruction , and as a result they also secreted more oocysts [ 60 , 77 ] . furthermore", ", mopping - up the secreted ifn- by antibodies in immunocompromised animals seemed to worsen c. parvum infection .", "in addition , it is now widely accepted that il-12 , known to increase host ifn- production , can reduce the severity and even prevent infection by apicomplexan parasites .", "although believed to be mediated by an ifn--dependent mechanism , the pathways or downstream molecules crucial in this process have not been well defined to date ." ], [ "although viruses , which entirely depend on the host machinery for replication and assembly of new viral particles , are the experts in host cell manipulation , the apicomplexa are considered to be the masters of disguise .", "this is because they have evolved to evade the host immune system to aid in their own survival .", "antigenic variation has been proposed as a key factor in this process . unlike allelic polymorphism , which results in different phenotypes or so - called parasite strains ,", "antigenic variation is the tightly regulated expression of different genes of a clonal population of parasites over the natural course of infection .", "antigenic variation amongst malarial and babesia parasites is a prime example of sophistication apicomplexans employed to avoid antibody - mediated inhibition [ 7981 ] .", "p. falciparum achieves this by secreting a single type of a variant molecule ( parasite - derived erythrocyte membrane protein 1 - pfemp1 ) on the surface of the infected erythrocyte at any one time .", "the pfemp1 surface proteins are encoded by a family of genes , called var genes , and each individual parasite expresses only a single var gene , keeping all other members of var gene family in a transcriptionally silent state [ 8284 ] .", "this strategy in turn induces adhesion of the parasite - infected erythrocytes to the blood vessels to avoid reaching the spleen , whose main function is to rid the body of damaged and/or infected blood cells . similarly ,", "sequestration of babesia - infected erythrocytes in the microvasculature enables the babesia to persist within the host maximizing its chances of transmission .", "this cytoadherence in babesia is mediated by constant gene conversion of ves family genes encoding the variant erythrocyte surface antigen 1 ( vesa1 ) . ", "a puzzling question arises from these observations : if infected erythrocytes pass through the body unchecked since they lack major histocompatibility complex ( mhc ) expression , overwhelming proliferation of the parasites may cause premature death of the host prior to successful transmission to an arthropod vector ?", "interestingly , in spite of the fact that malaria parasites sequentially express variant surface molecules exposing the immunodominant antigens to the host immune defences , infection is actually prolonged .", "thus , the parasite must undergo antigenic variation and rates of growth that enable the host to control infection while allowing for transmission of the parasite prior to its death .", "due to coating of the merozoite surface with glycosyl - phosphatidyl - anchored proteins crucial for initial attachment to the host erythrocyte surface , they are targeted by host - protective antibodies .", "these surface - anchored proteins ( variable merozoite surface antigens - vmsa ) exhibit varying degrees of intra - species antigenic polymorphisms allowing these parasites to evade the host immune system at the population level .", "nevertheless , studies involving african children have shown that variant specific immunity , namely , secretion of igg antibodies directed against p. falciparum variant surface antigens ( vsa ) , has been correlated with protection from clinical malaria in ghana , kenya , and tanzania [ 79 , 87 , 88 ] .", "thus , vsa antigens have been proposed as excellent candidates for malaria and babesia vaccine development ." ], [ "although b cells have been regarded as minor contributors to protective immunity and resistance to primary infections with apicomplexa , numerous studies have shown that hosts infected with these parasites are capable of producing protective , parasite - specific immunoglobulins ( ig ) of all major classes after an episode of infection and recovery [ 57 , 8992 ] .", "thus , early work by rose and colleagues has shown that humoral antibodies , induced by live eimeria infection , can provide excellent passive protection against challenge infections with the same parasite [ 93 , 94 ] .", "likewise , studies on mice infected with t. gondii have shown that intestinal iga antibodies to major surface protein sag-1 ( p30 ) were produced after peroral infection and found to inhibit infection of murine enterocytes by directly blocking the parasite entry .", "in addition , precigout et al . have demonstrated an inhibitory effect of antibodies directed against a 17-kda merozoites membrane protein on b. divergens parasite growth . ", "furthermore , studies on invasion of red blood cells by p. falciparum merozoites have revealed that since rbcs do not express the mhc complex , parasite killing by t lymphocytes is not important . instead , antibodies specific to merozoite surface molecules ( msp-1 ) and proteins externalised from the apical complex play a major role in immunity to asexual blood stages . ", "the plasmodium merozoite surface protein 1 ( msp-1 ) is a 200 kda multicomponent precursor complex derived by proteolytic processing during erythrocyte invasion .", "the 42 kda c - terminal component is cleaved ( i.e. , secondary processing ) to produce soluble 33 kda and 19 kda fragments that remain on the merozoites surface .", "studies have shown that anti - merozoite antibodies are capable of neutralizing parasites by fc - dependent mechanisms involving macrophages , thus reducing the parasitemia and clinical disease [ 87 , 97 , 98 ] .", "in addition , a number of recent studies have shown that children naturally infected with malaria secrete anti - msp-1 antibodies ( msp-119 mab ) that block the binding of plasmodium merozoites to the surface of the red blood cells and also inhibit secondary processing of msp-1 .", "in addition , studies investigating the protective properties of maternally derived igg and igm antibodies to the 19 kda domain of msp-1 of p. falciparum have shown that mothers who have tested positive for anti - msp-1 ( 19 kda fragment ) igg antibodies conferred protection against placental infection and infection in their infants . ", "it has been shown that , in babesiosis infection , igg antibodies produced as a result of live infection can prevent infection of erythrocytes by binding and neutralizing sporozoites before they invade their target cells .", "similar observations were reported in chickens where antisporozoite antibodies specific to glycosyl - phosphatidylinositol - anchored e. tenella surface antigen 1 ( etsag1 ) appeared to inhibit parasite binding and invasion of the host cell .", "however , it seems that the protective role of these antibodies is limited since it can only neutralize sporozoites from the time the parasites egress and the time they gain access to new cells .", "thus , it is hoped that genome - wide fingerprinting techniques will aid in the identification of additional immunoprotective antigens that can be used in combination to induce the maximal inhibitory humoral immune response . ", "in addition to antigen - specific polyclonal and monoclonal antibodies capable of inhibiting asexual stages , antibodies raised to antigens localized exclusively to gametocyte / zygote stages were also found to be highly immunogenic and capable of providing passive protection in vivo [ 20 , 101 , 102 ] .", "early experiments involving immunisation with purified sexual - stage gametes of p. gallinaceum in chickens showed that effective transmission - blocking immunity can be achieved by reducing the infectivity of gametocytes and oocyst development [ 103 , 104 ] .", "thus , pfs25 and pvs25 proteins expressed on the surface of ookinetes in the mosquito stage of p. falciparum and p. vivax have been used extensively as candidates for malaria transmission - blocking vaccines , since lowering the density of circulating parasites would not produce sterilizing immunity , instead it would allow individuals to develop long - lasting , naturally acquired immunity to malaria [ 12 , 105 , 106 ] . ", "work by wallach and coworkers , aimed at applying transmission - blocking immunity to control infections caused by eimeria , hypothesised that antibodies raised against the gametocyte / zygote stages of development can act to inhibit oocyst development and thereby provide a block in parasite transmission ( see figure 1 ) .", "a method was developed for purifying e. maxima gametocytes from the infected chicken gut mucosa and immunodominant gametocyte antigens , namely , emgam56", ", emgam82 , and emgam230 localized to the wfbs and the oocyst wall of the maturing zygote , were extracted [ 53 , 102 , 108 ] .", "passive immunisation experiments showed that there was a good correlation between the intensity of igg and igm antibodies binding to gametocyte antigens by western and elisa with the ability of those sera to provide passive protection in vivo .", "the mechanisms by which these antibodies inhibit oocyst maturation are still obscure ; however , it is hypothesised that antibodies raised to the immunodominant antigens retard zygote development by interfering with the processing of wall proteins or the wall - hardening processes [ 53 , 110 ] .", "in addition , a protective monoclonal antibody raised against emgam56 localised to the wfb2 ( 1e11 - 11 ) , as well as the inner layer of the oocyst wall , was also found to react strongly with the stieda body of the sporulated oocysts ( m. wallach , unpublished data ) .", "similar results were reported by krcken et al . using a monoclonal antibody e2e5 raised to wfb2s of e. tenella .", "the in vitro excystation inhibition assay showed that the antibody e2e5 can significantly interfere with parasite development by impairing sporozoite excystation .", "it is tempting to speculate that the 1e11 - 11 monoclonal antibody inhibits or blocks excystation of the sporocyst in a similar manner , thereby reducing the number of infectious sporozoites released in the intestine of infected birds allowing them to develop protective immunity induced by exposure to low doses of parasites . ", "jenkins and colleagues have shown that ruminants immunized with a dna vaccine expressing a gene isolated from c. parvum encoding a sporozoite antigen ( cp 15/60 ) were capable of inducing antigen - specific antibodies [ 111 , 112 ] . in that study , it was found that using various routes of vaccination resulted in differing antibody responses and titres .", "the authors , therefore , suggested that the route of antigen delivery of any protozoan vaccine requires careful formulation and optimisation of delivery systems .", "finally , in studies carried out by wallach and co - workers on eimeria , it was found that in order to achieve protective immunity using parasite extracts requires the inclusion of the correct antigens and exclusion of the irrelevant ones .", "their results indicated that while some parasite - specific antigens induce protective immunity , others actually induce an exacerbation of the infection .", "therefore , in the design of any parasitic vaccine , it is crucial that the combination of various antigens maximizes their inhibitory effect on parasite growth and development ." ], [ "one of the main defence mechanisms employed by host cells is programmed cell death ( apoptosis ) ensuring regulated removal of damaged and infected cells . but", "because the survival and development of intracellular apicomplexan parasites is dependent upon the continuous supply of host cell nutrients and protection from immune attack , the parasites have adapted to extend the life of the infected cells by inhibiting the host cell apoptotic machinery through interference with the intracellular signalling molecules , notably phosphatidylinositol 3-kinase ( pi3-k ) .", "pi3-k is involved in a variety of functions including cell growth , proliferation , and intracellular trafficking , amongst others [ 61 , 114117 ] .", "p. falciparum is a good example of how parasite secreted proteins prevent host cell death to ensure its own development and survival .", "sporozoites of plasmodium species are stealthy invaders that first travel to the liver ( hepatocyte ) cells , where the growth and development of the daughter cells , hepatic merozoites , takes place .", "recent results have shown that prior to the establishment of the pv , sporozoites of p. falciparum transmigrate through a number of hepatocytes before they anchor to and invade the suitable cell via exocytosis of proteins contained within the apical complex .", "it has been shown that the thrombospondin - related adhesive - protein- ( trap- ) like protein plays a role in this process . additionally , the wounding of the hepatocyte induced by invading sporozoites releases growth factors which in turn appear to inhibit pi3-k and block the signalling pathways destined for apoptosis .", "leirio and colleagues have shown that once the parasite is established in the hepatocyte , it secretes hgf / met signalling molecules into the host cell cytoplasm , thereby conferring resistance to apoptosis to ensure survival and maturation of the daughter cells . however , which signalling upstream of pi3-k occurs during plasmodium infection is yet to be determined .", "interestingly , upon maturation of merozoites , plasmodium seems to be able to induce host cell death to liberate the motile progeny .", "have shown that this process involves cysteine proteases [ 49 , 50 ] . moreover ,", "similar mechanisms were found to play a role in release of sporozoites from the oocysts .", "although work is ongoing to try and elucidate the mechanisms involved in these processes , it appears that the apicomplexa have learned to inhibit host cell death during parasite development and subsequently activate it , liberating thousands of new progeny . \n", "t. gondii has also evolved a broad spectrum of adaptations to challenges presented by its life style .", "chronic toxoplasmosis is the trademark of the parasite 's success and is induced by the slow - replicating bradyzoites safely tucked away in the remodelled pv , the tissue cyst . like plasmodium ,", "t. gondii modulates host cell apoptosis by both inhibiting and triggering the programmed cell death .", "chen et al . have shown that fas / fasl ligand - dependent mechanisms mediate the inflammatory responses induced by the apicomplexan infection [ 115 , 121 ] .", "but the parasites have evolved to neutralize granzyme / perforin - mediated killing of infected t cells and natural killer cells ( nk ) by modifying transcription and posttranscriptional modification of ifn--regulated genes , the major mediators of resistance to t. gondii infections . likewise , del cacho et al .", "have demonstrated that e. tenella and e. necatrix second - generation schizonts first induce nf- activation to protect the transformed cells from apoptosis , allowing the schizonts to mature and later cause nf- inhibition to trigger host cell apoptosis to facilitate the release of merozoites . ", "the apicomplexa have evolved to live in synergy with their infected hosts because they completely depend on it for survival ; however , some apicomplexan infections induce a great deal of immunopathology and can lead to host cell death .", "for example , eimeria and cyclospora both interfere with the absorption of nutrients across the intestinal mucosa and can cause death due to malaise , diarrhoea , and dehydration . because apicomplexans increase in numbers while , in their hosts , the severity of infection is proportional to the parasite density the smaller the number , the greater the chance of asymptomatic infection and the greater the chances of the parasite survival .", "however , the immunological defence of a host can also cause extensive tissue damage and clinical symptoms .", "patients with cerebral malaria usually have elevated levels of tumour necrosis factor alpha ( tnf- ) and ige considered to be responsible for fever and tissue lesions to an extent where vital functions of the host fail leading to a coma ." ], [ "despite a great deal of effort and technological advancements in biotechnology , molecular biology , genetics , immunology , and vaccinology , there are no vaccines for humans against malaria and toxoplasmosis at the present time , and it seems that we are losing the battle in the fight against pathogenic protozoans . the current failure to develop a practical vaccine may well be attributed to our inadequate understanding of the mechanisms underlying ( 1 ) the naturally acquired immunity against apicomplexans , ( 2 ) acquired parasite resistance to most ( if not all ) antimicrobial compounds used to date , and ( 3 ) in the case of arthropod transmitted protozoans , failure to implement adequate vector control programs in tropical and subtropical regions . the life cycles of the apicomplexa are complex , thus , it is hoped that a multivalent , multistage vaccine will alleviate the problems caused by these pathogenic protozoans .", "although this approach has attracted a great deal of commercial and research interest , the critical issues to be addressed include the identification of stage - specific antigens capable of inducing protective immunity and the delivery methods in a form that will stimulate an adequate protective immune response .", "the main impediment in the search and selection for immunostimulatory antigens is the lack of in vitro assays to analyse and predict immune responses .", "the transmission blocking assays , relying on counting the number of oocysts produced , and the inhibition of sporozoite invasion assays have both been used extensively to evaluate parasite inhibition induced by neutralizing antibodies . ", "although in vivo experimentation is extremely difficult for malaria , other model systems can be used to dissect the fine details and the effect of neutralizing antibodies .", "it is very possible that , in the development of an antiprotozoan vaccine capable of inducing only partial immunity , resistant mutants would be selected that are even more pathogenic than existing strains . in the malaria scenario", ", this could be catastrophic since the parasite would undergo recycling and be transmitted throughout the community leading to an increase in morbidity and mortality .", "it is , therefore , of great hope that in the battle against these pathogenic protozoan parasites , including plasmodium , cryptosporidium , and toxoplasma , the completion of their genomes and proteomes may provide information needed to design vaccines , assess the effects of immunization on parasite pathogenicity and the selection of unwanted mutants , and in the final analysis control the diseases caused by this class of parasites ." ] ]

[ "giant cell tumour is the commonest benign bone tumour arising at the epiphyseometaphyseal regions of long bones . around the knee", "there are very few similar cases reported worldwide and it is the purpose of this report to describe the management of such a case .", "a 17 year old girl presented with swelling of ankle and pain while walking for six months .", "radiographs were suggestive of a giant cell tumour , computerised tomography revealed cortical break , en block resection was done with ipsilateral proximal fibula used in reconstruction of ankle mortise .", "giant cell tumour of long bones are common but those involving the distal fibula are exceedingly rare .", "the management of such tumours with high recurrence rates can be easily accomplished by en block resection and reconstruction of the ankle mortise with proximal fibula ensuring good range of motion of the joint post operatively .", "first described by sir astley cooper in the year 1818 , giant cell tumour of bone or osteoclastoma is the commonest benign bone tumour encountered by an orthopaedic surgeon .", "it is characterised radiographically as a lytic lesion occurring in the ends of bones and has well known propensity for local recurrence after surgical management .", "current treatment modalities including a meticulous curettage with extension of tumour removal using high speed burrs and adjuvant local therapy has significantly lowered the recurrence rates to less than 10% from 60% in the past with curettage alone .", "the commonest age is the 3rd or the 4th decade with a slight female predominance .", "the other less common infrequent sites are sacrum , distal tibia , proximal humerus , proximal femur and proximal fibula .", "involvement of distal fibula by benign aggressive and malignant tumors usually necessitates resection of the involved segment of fibula .", "the incidence of giant cell tumour of distal fibula was found to be less than 1% of 1182 cases .", "schajowicz , in his series of 362 cases has reported only a single case affecting the lower end of the fibula ( 0.28% ) .", "a seventeen year old girl presented with swelling around the right ankle for six months associated with pain while walking and restriction on squatting .", "the swelling was six by four by two centimetres in size , firm to hard in consistency , no tenderness on deep palpation .", "[ fig 1 ] clinical picture showing swelling and radiographs showing expansile lesion with soap bubble appearance .", "anteroposterior and lateral radiographs were taken which showed single epiphyseal expansile lesion with soap bubble appearance .", "magnetic resonance imaging could not be done as the facility was not available then in our government hospital and patient s financial background prevented us getting an imaging from private centres .", "all routine haematological investigations were found to be normal and chest radiograph was also found to be normal .", "an excisional biopsy was planned with reconstruction using the proximal end of the ipsilateral fibula . under pneumatic tourniquet without exsanguination an en bloc excision of the lateral malleolus with lower third of the fibula", "the level of resection of distal fibula was determined by the computerised tomography , clinical intra operative findings and by pre operative radiographs .", "the proximal fibula was reversed with head of fibula incorporating into the ankle mortise and fixed to the remaining fibula using plate and screws .", "meticulous haemostasis was achieved after release of the tourniquet , and the wounds were closed in layers .", "patient kept non weight bearing for three months and full weight bearing at six months after the removal of screws .", "patient was followed up and at the end of one year patient had full range of motions with mild restriction of dorsiflexion of the affected ankle [ figs 3 ] .", "b- fixation of the reversed proximal end of fibula and incorporation into the ankle mortise .", "d - photomicrograph of the specimen showing multinucleate giant cells suggestive of giant cell tumor six month follow up showing good radiological union ( a ) and clinical photograph showing good dorsiflexion and plantar flexion ( b , c ) .", "the proximal fibula can be sacrificed for the purposes of reconstruction as is recommended for lower end fibula and distal radius .", "giant cell tumour of the bone has an unpredictable behaviour , not always related to radiographic or histological appearance .", "many earlier studies had shown very high local recurrence rates after curettage and bone grafting . the use of modern imaging techniques and extended curettage through the use of power burrs and local adjuvants have improved outcome with reduced recurrence rates .", "phenol , liquid nitrogen , bone cement , hydrogen peroxide , zinc chloride and more recently , argon beam cauterization have been employed as local adjuvants .", "chemical or physical agents work by inducing an additional circumferential area of necrosis to extend the curettage . in distal fibular resection without reconstruction ,", "soft - tissue reinforcement , even when it is possible , can not fully compensate for the loss of stability .", "resection of the lateral ankle can cause a varus instability or a collapse into valgus .", "this technique of ankle resection and reconstruction has provided good oncological and functional results and recommended in young active patients requiring resection of distal fibula .", "giant cell tumour of long bones are common but those involving the distal fibula are exceedingly rare .", "the management of such tumours with high recurrence rates can be easily accomplished by en block resection and reconstruction of the ankle mortise with proximal fibula ensuring good range of motion of the joint post operatively .", "resection arthrodesis which was the method primarily employed for bone tumours involving ankle can now be replaced with ankle reconstruction .", "distal fibula gct being an extremely rare entity and its management not been described , reconstruction of ankle with proximal giant cell tumour of distal fibula are extremely rare and such benign tumours with high recurrence rates with the evidence of medial cortical break should be managed by an en block resection and reconstruction of the ankle mortise and the preferable method would be by the usage of proximal fibula graft .", "this method produced no recurrence and ensured good range of motions and can effectively replace resection arthrodesis as management in cases which require resection of lateral malleolus ." ]

[ "<S> introduction : giant cell tumour is the commonest benign bone tumour arising at the epiphyseometaphyseal regions of long bones . around the knee </S>", "<S> is commonest site followed by distal radius . </S>", "<S> a giant cell tumour of the distal fibula is extremely rare . </S>", "<S> we report here a case of giant cell tumour of distal fibula . </S>", "<S> there are very few similar cases reported worldwide and it is the purpose of this report to describe the management of such a case.case report : a 17 year old girl presented with swelling of ankle and pain while walking for six months . </S>", "<S> radiographs were suggestive of a giant cell tumour , computerised tomography revealed cortical break , en block resection was done with ipsilateral proximal fibula used in reconstruction of ankle mortise.conclusion:giant cell tumour of long bones are common but those involving the distal fibula are exceedingly rare . </S>", "<S> the management of such tumours with high recurrence rates can be easily accomplished by en block resection and reconstruction of the ankle mortise with proximal fibula ensuring good range of motion of the joint post operatively . </S>" ]

[ "Introduction:", "Case Report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion" ]

[ [ "giant cell tumour is the commonest benign bone tumour arising at the epiphyseometaphyseal regions of long bones . around the knee", "there are very few similar cases reported worldwide and it is the purpose of this report to describe the management of such a case ." ], [ "a 17 year old girl presented with swelling of ankle and pain while walking for six months .", "radiographs were suggestive of a giant cell tumour , computerised tomography revealed cortical break , en block resection was done with ipsilateral proximal fibula used in reconstruction of ankle mortise ." ], [ "giant cell tumour of long bones are common but those involving the distal fibula are exceedingly rare .", "the management of such tumours with high recurrence rates can be easily accomplished by en block resection and reconstruction of the ankle mortise with proximal fibula ensuring good range of motion of the joint post operatively ." ], [ "first described by sir astley cooper in the year 1818 , giant cell tumour of bone or osteoclastoma is the commonest benign bone tumour encountered by an orthopaedic surgeon .", "it is characterised radiographically as a lytic lesion occurring in the ends of bones and has well known propensity for local recurrence after surgical management .", "current treatment modalities including a meticulous curettage with extension of tumour removal using high speed burrs and adjuvant local therapy has significantly lowered the recurrence rates to less than 10% from 60% in the past with curettage alone .", "the commonest age is the 3rd or the 4th decade with a slight female predominance .", "the other less common infrequent sites are sacrum , distal tibia , proximal humerus , proximal femur and proximal fibula .", "involvement of distal fibula by benign aggressive and malignant tumors usually necessitates resection of the involved segment of fibula .", "the incidence of giant cell tumour of distal fibula was found to be less than 1% of 1182 cases .", "schajowicz , in his series of 362 cases has reported only a single case affecting the lower end of the fibula ( 0.28% ) ." ], [ "a seventeen year old girl presented with swelling around the right ankle for six months associated with pain while walking and restriction on squatting .", "the swelling was six by four by two centimetres in size , firm to hard in consistency , no tenderness on deep palpation .", "[ fig 1 ] clinical picture showing swelling and radiographs showing expansile lesion with soap bubble appearance .", "anteroposterior and lateral radiographs were taken which showed single epiphyseal expansile lesion with soap bubble appearance .", "magnetic resonance imaging could not be done as the facility was not available then in our government hospital and patient s financial background prevented us getting an imaging from private centres .", "all routine haematological investigations were found to be normal and chest radiograph was also found to be normal .", "an excisional biopsy was planned with reconstruction using the proximal end of the ipsilateral fibula . under pneumatic tourniquet without exsanguination an en bloc excision of the lateral malleolus with lower third of the fibula", "the level of resection of distal fibula was determined by the computerised tomography , clinical intra operative findings and by pre operative radiographs .", "the proximal fibula was reversed with head of fibula incorporating into the ankle mortise and fixed to the remaining fibula using plate and screws .", "meticulous haemostasis was achieved after release of the tourniquet , and the wounds were closed in layers .", "patient kept non weight bearing for three months and full weight bearing at six months after the removal of screws .", "patient was followed up and at the end of one year patient had full range of motions with mild restriction of dorsiflexion of the affected ankle [ figs 3 ] .", "b- fixation of the reversed proximal end of fibula and incorporation into the ankle mortise .", "d - photomicrograph of the specimen showing multinucleate giant cells suggestive of giant cell tumor six month follow up showing good radiological union ( a ) and clinical photograph showing good dorsiflexion and plantar flexion ( b , c ) ." ], [ "the proximal fibula can be sacrificed for the purposes of reconstruction as is recommended for lower end fibula and distal radius .", "giant cell tumour of the bone has an unpredictable behaviour , not always related to radiographic or histological appearance .", "many earlier studies had shown very high local recurrence rates after curettage and bone grafting . the use of modern imaging techniques and extended curettage through the use of power burrs and local adjuvants have improved outcome with reduced recurrence rates .", "phenol , liquid nitrogen , bone cement , hydrogen peroxide , zinc chloride and more recently , argon beam cauterization have been employed as local adjuvants .", "chemical or physical agents work by inducing an additional circumferential area of necrosis to extend the curettage . in distal fibular resection without reconstruction ,", "soft - tissue reinforcement , even when it is possible , can not fully compensate for the loss of stability .", "resection of the lateral ankle can cause a varus instability or a collapse into valgus .", "this technique of ankle resection and reconstruction has provided good oncological and functional results and recommended in young active patients requiring resection of distal fibula ." ], [ "giant cell tumour of long bones are common but those involving the distal fibula are exceedingly rare .", "the management of such tumours with high recurrence rates can be easily accomplished by en block resection and reconstruction of the ankle mortise with proximal fibula ensuring good range of motion of the joint post operatively .", "resection arthrodesis which was the method primarily employed for bone tumours involving ankle can now be replaced with ankle reconstruction .", "distal fibula gct being an extremely rare entity and its management not been described , reconstruction of ankle with proximal giant cell tumour of distal fibula are extremely rare and such benign tumours with high recurrence rates with the evidence of medial cortical break should be managed by an en block resection and reconstruction of the ankle mortise and the preferable method would be by the usage of proximal fibula graft .", "this method produced no recurrence and ensured good range of motions and can effectively replace resection arthrodesis as management in cases which require resection of lateral malleolus ." ] ]

[ "diagnostic hysteroscopy for direct assessment of the uterine cavity has been a relatively recent acquisition in the armamentarium of the gynecologists . in an outpatient setting", ", the entire evaluatory workup takes at least two to three visits prior to arriving at a definitive diagnosis and commencement of appropriate therapy . in the current scenario of increasing cost awareness and an ever - increasing litigious environment", ", a balance has to be achieved between the practice of blanket medicine aiming at performance of all investigations possibly contributing to a diagnosis versus a condition appropriate approach .", "the study presented was undertaken to evaluate the sensitivity and specificity of embx and transvaginal ultrasound tvs for detection of intracavitary uterine lesions compared with results of office hysteroscopy ( oh ) , which were taken as the gold standard .", "a secondary objective of the study was to evaluate the protocol for assessment of the common problem of aub .", "appropriate management could thus be instituted at the earliest possible opportunity , minimizing the number of unnecessary investigations as well as the numerical patient visits to the clinic , without compromising the quality of patient care .", "prospective observational study conducted at the department of gynecology at massachusetts general hospital ( mgh ) between january , 1995 , and september , 1996 .", "patients attending the gynecology department at mgh with complaints of abnormal uterine bleeding were evaluated for inclusion in the study .", "the catchment population was from the resident managed gynecology clinic and from practices of two attending physicians with a special interest in reproductive endocrinology .", "aub was defined as either cyclic excessive bleeding , irregular menstrual cycles , postmenopausal bleeding , and excessive or unscheduled withdrawal bleeding on hormone replacement therapy ( hrt ) .", "each patient presenting underwent a preliminary assessment by history and clinical examination ; pap smear status was evaluated and updated if indicated .", "the uterine size was assessed clinically and determined as normal or enlarged . the study protocol aimed at obtaining an outpatient endometrial biopsy ( embx ) usually at the initial presentation , followed by a transvaginal pelvic ultrasound ( tvs ) .", "other than oh , the other investigations were performed without consideration of the phase of the menstrual cycle ; oh was scheduled in the early proliferative phase of the menstrual cycle in patients complaining of regular aub .", "aseptic precautions were employed by cleansing the cervix with betadine prior to insertion of the catheter .", "antibiotic prophylaxis was given if indicated , using doxycycline 100 mg bid for 5 days .", "the tvs was performed in an office setting by one of the two attendings in gynecology .", "the uterine anatomy and the adnexae were visualized using a 7.5 mhz vaginal probe transducer ( general electric , milwaukee , rt 3200 advantage ii real time sector scanner ) .", "appearance of the endometrial stripe was commented upon as either normal or abnormal ; a specific note was made of any focal lesion seen in terms of impression of an endometrial polyp , submucous fibroid , intramural fibroid , suspicion of hyperplasia or endometrial carcinoma .", "the contour of the endometrial stripe was assessed in the midline sagittal plane and the point of maximum thickness of the stripe ( et ) was measured on a frozen image at 1.5 magnification .", "office hysteroscopy ( oh ) was performed using a 3.6 mm single channel flexible hysteroscope ( hyf - p olympus america , ny ) with a fiberoptic cold light source ; normal saline was used as the distending medium and the procedure was performed under direct video monitoring .", "a total of 54 patients completed the study and underwent embx , tvs and oh . in none of the oh procedures was cervical dilatation required , nor was any form of local anesthetic used .", "baseline laboratory investigations included a complete blood count , results for which were available for 47 patients .", "the results of oh were taken as the gold standard for detection of intracavitary pathology as a contributing factor to the clinical presentation .", "sensitivity and specificity of the embx and tvs in detecting the intracavitary lesions were calculated .", "the sensitivity of clinical finding of an enlarged uterus and the pattern of bleeding in predicting an intracavitary lesion were also estimated , as was the association of significant anemia ( hb < 11 gm% ) and the incidence of concomitant intrauterine lesions .", "an age - related prevalence of intracavitary lesions ( submucous fibroids / polyps / endometrial hyperplasia / chronic endometritis / endometrial cancer ) was calculated in the study population . statistical analysis was performed using unpaired t tests using the statistical package graphpad in stat version 1.01 .", "thirteen patients were postmenopausal . fifty - two percent of the patients presenting with abnormal uterine bleeding had evidence of an intracavitary lesion as detected by office hysteroscopy .", "congenital uterine anomalies detected incidentally on oh included one case each of a unicornuate , and bicornuate and two cases of septate uterus .", "( tvs ) performed detected a distortion of the endometrial cavity suggestive of a focal pathology in 20 patients .", "a subsequent oh confirmed the diagnosis in 17/20 cases where a focal lesion was suspected ( table 1 ) .", "however , of the 34 patients in whom tvs showed a normal endometrium , oh confirmed 11 false negative cases .", "the sensitivity of an abnormal transvaginal ultrasound scan when compared to office hysteroscopy for detection of an intracavitary lesion was thus calculated as 0.60 .", "of the 11 false negative tvs results , oh demonstrated five cases of endometrial polyp and six submucous myomas ; in 5/6 of the latter there was of the 54 patients who underwent tvs , a specific mention of the thickness of the endometrial stripe was available in 28 , the thickness of the endometrium ( et ) ranging from 1.8 mm to 25 mm . when correlating the thickness in mm with the presence of intracavitary lesions confirmed by oh , it was noted that the et 6 mm had a negative predictive value of 92% ; et of 10 mm had a positive predictive value of 89% .", "detection of intracavitary uterine lesions : comparison of results of office hysteroscopy to transvaginal ultrasound .", "tvs : transvaginal ultrasound the highest prevalence of intracavitary lesions was seen in the age group of 36 - 40 years ( 71% ) ; in the < 30 years age group , the incidence was 50% , the difference in the prevalence between the different ages not being of statistical significance ( table 2 ) .", "submucous myomas were the most common lesions seen in the younger patients ; an increasing incidence of endometrial polyps was seen with increasing age , polyps being the most frequently diagnosed pathology in the postmenopausal women , as shown in earlier studies .", "age related prevalence of intracavitary lesions determined by oh . the endometrial biopsy ( embx ) specimens were considered adequate for reporting in 49/54 cases ; two samples had inadequate tissue and three specimens showed menstrual blood , making interpretation difficult .", "no evidence of pathology was seen in 44/49 specimens . of the patients with a normal embx ,", "19 were true negative and 25 were false negative , when compared to results of oh ( table 3 ) .", "the negative predictive value of a normal embx was calculated as 51% . in 5/49 biopsy specimens ,", "a pathology was detected and included fragments of endometrial polyp in two cases ; office hysteroscopy confirmed the presence of a residual polyp in one of these patients .", "there was evidence of chronic endometritis in one patient , in whom an endometrial polyp was detected on hysteroscopy , and focal hyperplasia within a secretory endometrium was shown in the fourth patient , who had a normal appearing endometrial cavity on oh . the sensitivity of endometrial biopsy was 0.04 with a specificity of 0.83 .", "no pathological evidence of endometrial carcinoma was detected in the embx specimens available , which included 14 specimens from postmenopausal patients .", "the incidence of inconclusive sampling was 9% and that of focal endometrial hyperplasia was 2% .", "detection of intracavitary uterine lesions : comparison of results \n of office hysteroscopy to endometrial biopsy .", "difference between a and b is statistically significant p<0.05 of the 13 postmenopausal patients , the age range was 47 - 74 years ; seven patients were on some form of hrt for a period of at least three months prior to the presentation .", "the prevalence of intracavitary lesions in the post - menopausal group was 31% ( 4/13 ) ; two of the lesions were confirmed to be benign endometrial polyps on histology and the third was a submucous myoma ; in the fourth patient the biopsy of an irregular focal area of the endometrium provided a benign proliferative histological specimen .", "results of tvs specifically noted endometrial thickness in 10 postmenopausal patients , the thickness ranging from 1.8 mm -15 mm . in 7/10 patients ,", "the et was < 6 mm and 6/7 had a normal uterine cavity of oh ; the seventh patient , a 72-year - old woman , was noted to have an area of endometrial irregularity on the anterior uterine wall and a directed biopsy performed showed histological evidence of proliferative endometrium with no evidence of malignancy .", "of the three postmenopausal patients in whom a tvs showed an endometrial thickness of greater than 6 mm , all were found to have an endometrial polyp on oh . thus taking the et of 6 mm as the cut - off in the postmenopausal women , the positive predictive value for detection of intracavitary lesions for et > 6 mm was deter - mined to be 100% , whereas the negative predictive value for an et of < 6 mm was also calculated as 100% . of the 54 patients included in the evaluation , 26 underwent definitive surgical management for the presenting problem ; two patients had a normal uterine cavity and opted to undergo endometrial ablation , whereas 24 had operative hysteroscopic resection of the focal lesion .", "the most common histological diagnosis was a submucous myoma , seen in 65% , followed by benign endometrial polyps in 26% of the resected lesions ; in 5% , a combination of the two existed .", "the uterus was classified as normal in size on clinical examination in 63% of the patients ( 36/54 ) .", "of the 18 enlarged uteri , 12 showed an intracavitary lesion on oh , giving a ppv of 67% . in eight of the 47 patients in whom a cbc was available ,", "the hemoglobin was less than 11 gm% ; 6/8 ( 75% ) of these patients had an abnormal finding on oh . nineteen of", "the 54 patients were experiencing heavy , though regular menstrual flow ; of these , 14 ( 74% ) had an abnormal oh compared to 14/35 ( 40% ) of those presenting with irregular bleeding , the difference in the prevalence of lesions being statistically significant ( p=0.008 ) .", "the high incidence of intrauterine lesions ( 52% ) in our patient population presenting with abnormal uterine bleeding ( aub ) is consistent with results of earlier studies.1 the traditional approach to evaluation of aub in an outpatient setting has included an endometrial sampling following a preliminary assessment based on the history and clinical examination .", "the patient commonly makes an average of two to three clinic visits , depending on the urgency of presentation , before being commenced on a specific treatment protocol . in the present era of cost containment and capping of procedure related reimbursements ,", "the physicians should be acquainted not only with the relative informative yield but also the cost per investigation , so as to channelize their diagnostic approach .", "the aim of management is to thus minimize the cost incurred per patient , while adhering to the principals of the standard of care .", "diagnostic hysteroscopy , though being increasingly employed for evaluation of aub , is still underutilized .", "since the introduction of the hysteroscopic technique , the procedure has undergone significant modifications , contributing to an increase in patient acceptance .", "introduction of fiberoptics , reduction in the caliber of the endoscopes , use of simpler distending media and availability of safer local infiltrative anesthetics have all contributed to an increasing utilization of this technique in evaluation of the uterine cavity .", "baskett et al . in a recent commentary on the efficiency of a one - stop menstrual clinic", "have demonstrated the cost and clinical effectiveness of utilizing hysteroscopy as a preliminary investigation performed at the initial visit in selected patients .", "more than 50% of all diagnostic hysteroscopies , however , are still being performed in the operating room ( or ) , and this trend could be attributed to a combination of a lack of awareness on the part of the physician and perhaps nonavailability of smaller caliber endoscopes .", "compared to tvs , hysteroscopy allows for a direct visualization of the endometrial cavity and hence detection of any focal lesion .", "it offers the additional opportunity of obtaining a directed biopsy in the same setting if indicated , thus obviating the need for a separate scheduling of the procedure .", "studies have demonstrated a superior yield of directed biopsies compared to d&c in providing representative histological specimens . in terms of cost containment ,", "the procedure being performed by the investigating gynecologist precludes the involvement , and hence need for reimbursement of an additional specialist .", "furthermore , it dictates the need for a histological specimen if indicated , and allows for a directed biopsy , ensuring provision of a representative specimen of the focal pathology for evaluation .", "performing an oh early in the evaluation in appropriately selected patients would render utilization of any further investigation like embx or tvs unnecessary .", "the net result would ensure cost containment by reduction in the number of patient visits for investigational purposes , and by enabling the physician to embark on an appropriate management plan at the earliest opportunity , contribute significantly to patient satisfaction .", "the significant incidence of intrauterine lesions in the postmenopausal patients evaluated ( 31% ) is comparable to other studies and underscores the importance of direct visualization of the uterine cavity in this subgroup . since this represents the patient population at the highest risk for significant uterine pathology , i.e. endometrial carcinoma , and because the hysteroscopic appearances of early lesions are well recognized , the argument is further strengthened in favor for an early visualization of the cavity .", "an insight into the clinical staging of an existing endometrial carcinoma would be an additional benefit contributing to the final management plan .", "the high incidence of intracavitary lesions seen in the patients with clinically enlarged uteri ( 62% ) and in those with significant anemia ( 80% ) underscores the importance of proceeding with oh early in the evaluatory process in this subcategory of patients .", "the rationale for attempting to evaluate the endometrial specimen histologically , obtained by either d&c or outpatient biopsy , is to provide an early diagnosis of a significant pathology , namely endometrial cancer and/or endometrial hyperplasia .", "while the risk of such an occurrence is high in a subset of patients , i.e. postmenopausal women with abnormal bleeding ( 10% ) , anovulatory patients and those with a history of prior endometrial hyperplasia , the yield of endometrial sampling from the rest of the patient population in terms of obtaining a pathological diagnosis is negligible .", "this is consistent with our data and questions the almost universal practice of biopsying the endometrium in any patient presenting with aub . in our experience", ", an expenditure of approximately $ 250 for an embx could be easily avoided in the majority of premenopausal patients with aub .", "an embx , however , has a definite place in the diagnostic workup of postmenopausal patients as well as those premenopausal patients who are at high risk for endometrial hyperplasia .", "the high propensity of missing focal intrauterine lesions like submucous myomas and polyps with d&c is well documented in recent literature . ", "moreover , the alleged therapeutic effect of d&c in the management of aub remains questionable .", "most of the data available on the diagnostic accuracy of tvs in evaluation of abnormal uterine bleeding is relevant only for the postmenopausal patients .", "the general consensus of opinion is that an endometrial thickness of less than 5 - 6 mm in a patient presenting with postmenopausal bleeding does not warrant an extensive workup , as the risk of endometrial carcinoma and/or hyperplasia is negligible.11,13 the sensitivity and specificity of tvs in detecting focal intrauterine lesions in the study presented is comparable to the results shown by tombin et al . ( 0.54 and 0.40 , respectively ) .", "scheduling the ultrasound evaluation in the follicular phase of the menstrual cycle in the premenopausal patients may enhance the sensitivity since the hyperechoic secretory endometrium may mask the endometrial polyps .", "presence of intramural myomas may obscure the endometrial stripe , thus contributing to a significant incidence of false negative interpretations , as shown in our series .", "the majority of pelvic ultrasound scans are still being performed by the radiologists ; the cost of a tvs ranges from $ 250-$400 , depending on the involvement of the radiology department .", "furthermore , an impression of an intracavitary lesion directs the management towards performance of a hysteroscopic procedure for the ultimate diagnosis and management .", "there is a high incidence of intracavitary uterine pathology in patients presenting with abnormal uterine bleeding .", "this is especially true when considering the 35 - 50 years age group who present with heavy regular bleeding , clinically enlarged uteri and significant anemia .", "the relatively poor sensitivity of both endometrial biopsy and transvaginal ultrasound in the detection of intrauterine focal pathology encourage us to propose that oh be utilized as a first line investigation in these patient evaluations . the cost - benefit analysis of investigations like embx and tvs as well as the financial burden of clinic visits during the entire evaluatory process" ]

[ "<S> this is a prospective , observational study that utilizes hysteroscopy as the gold standard for evaluation of transvaginal ultrasound and endometrial biopsy in the detection of intrauterine pathology . </S>", "<S> the sensitivity and specificity for detecting focal intrauterine lesions are examined in order to suggest the most cost - effective approach in patients with abnormal uterine bleeding . </S>" ]

[ "INTRODUCTION", "DESIGN", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION" ]

[ [ "evaluation of the problem has included investigations ranging from the traditional diagnostic dilatation and curettage ( d&c ) , office based endometrial biopsy ( embx ) , to pelvic ultrasonography for evaluation of endometrial / myometrial pathology contributing to the presentation .", "diagnostic hysteroscopy for direct assessment of the uterine cavity has been a relatively recent acquisition in the armamentarium of the gynecologists . in an outpatient setting", ", the entire evaluatory workup takes at least two to three visits prior to arriving at a definitive diagnosis and commencement of appropriate therapy . in the current scenario of increasing cost awareness and an ever - increasing litigious environment", ", a balance has to be achieved between the practice of blanket medicine aiming at performance of all investigations possibly contributing to a diagnosis versus a condition appropriate approach .", "the study presented was undertaken to evaluate the sensitivity and specificity of embx and transvaginal ultrasound tvs for detection of intracavitary uterine lesions compared with results of office hysteroscopy ( oh ) , which were taken as the gold standard .", "a secondary objective of the study was to evaluate the protocol for assessment of the common problem of aub .", "appropriate management could thus be instituted at the earliest possible opportunity , minimizing the number of unnecessary investigations as well as the numerical patient visits to the clinic , without compromising the quality of patient care ." ], [ "prospective observational study conducted at the department of gynecology at massachusetts general hospital ( mgh ) between january , 1995 , and september , 1996 ." ], [ "patients attending the gynecology department at mgh with complaints of abnormal uterine bleeding were evaluated for inclusion in the study .", "the catchment population was from the resident managed gynecology clinic and from practices of two attending physicians with a special interest in reproductive endocrinology .", "aub was defined as either cyclic excessive bleeding , irregular menstrual cycles , postmenopausal bleeding , and excessive or unscheduled withdrawal bleeding on hormone replacement therapy ( hrt ) .", "each patient presenting underwent a preliminary assessment by history and clinical examination ; pap smear status was evaluated and updated if indicated .", "the uterine size was assessed clinically and determined as normal or enlarged . the study protocol aimed at obtaining an outpatient endometrial biopsy ( embx ) usually at the initial presentation , followed by a transvaginal pelvic ultrasound ( tvs ) .", "other than oh , the other investigations were performed without consideration of the phase of the menstrual cycle ; oh was scheduled in the early proliferative phase of the menstrual cycle in patients complaining of regular aub .", "aseptic precautions were employed by cleansing the cervix with betadine prior to insertion of the catheter .", "antibiotic prophylaxis was given if indicated , using doxycycline 100 mg bid for 5 days .", "the tvs was performed in an office setting by one of the two attendings in gynecology .", "the uterine anatomy and the adnexae were visualized using a 7.5 mhz vaginal probe transducer ( general electric , milwaukee , rt 3200 advantage ii real time sector scanner ) .", "appearance of the endometrial stripe was commented upon as either normal or abnormal ; a specific note was made of any focal lesion seen in terms of impression of an endometrial polyp , submucous fibroid , intramural fibroid , suspicion of hyperplasia or endometrial carcinoma .", "the contour of the endometrial stripe was assessed in the midline sagittal plane and the point of maximum thickness of the stripe ( et ) was measured on a frozen image at 1.5 magnification .", "office hysteroscopy ( oh ) was performed using a 3.6 mm single channel flexible hysteroscope ( hyf - p olympus america , ny ) with a fiberoptic cold light source ; normal saline was used as the distending medium and the procedure was performed under direct video monitoring .", "a total of 54 patients completed the study and underwent embx , tvs and oh . in none of the oh procedures was cervical dilatation required , nor was any form of local anesthetic used .", "baseline laboratory investigations included a complete blood count , results for which were available for 47 patients .", "the results of oh were taken as the gold standard for detection of intracavitary pathology as a contributing factor to the clinical presentation .", "sensitivity and specificity of the embx and tvs in detecting the intracavitary lesions were calculated .", "the sensitivity of clinical finding of an enlarged uterus and the pattern of bleeding in predicting an intracavitary lesion were also estimated , as was the association of significant anemia ( hb < 11 gm% ) and the incidence of concomitant intrauterine lesions .", "an age - related prevalence of intracavitary lesions ( submucous fibroids / polyps / endometrial hyperplasia / chronic endometritis / endometrial cancer ) was calculated in the study population . statistical analysis was performed using unpaired t tests using the statistical package graphpad in stat version 1.01 ." ], [ "thirteen patients were postmenopausal . fifty - two percent of the patients presenting with abnormal uterine bleeding had evidence of an intracavitary lesion as detected by office hysteroscopy .", "congenital uterine anomalies detected incidentally on oh included one case each of a unicornuate , and bicornuate and two cases of septate uterus .", "( tvs ) performed detected a distortion of the endometrial cavity suggestive of a focal pathology in 20 patients .", "a subsequent oh confirmed the diagnosis in 17/20 cases where a focal lesion was suspected ( table 1 ) .", "however , of the 34 patients in whom tvs showed a normal endometrium , oh confirmed 11 false negative cases .", "the sensitivity of an abnormal transvaginal ultrasound scan when compared to office hysteroscopy for detection of an intracavitary lesion was thus calculated as 0.60 .", "of the 11 false negative tvs results , oh demonstrated five cases of endometrial polyp and six submucous myomas ; in 5/6 of the latter there was of the 54 patients who underwent tvs , a specific mention of the thickness of the endometrial stripe was available in 28 , the thickness of the endometrium ( et ) ranging from 1.8 mm to 25 mm . when correlating the thickness in mm with the presence of intracavitary lesions confirmed by oh , it was noted that the et 6 mm had a negative predictive value of 92% ; et of 10 mm had a positive predictive value of 89% .", "detection of intracavitary uterine lesions : comparison of results of office hysteroscopy to transvaginal ultrasound .", "tvs : transvaginal ultrasound the highest prevalence of intracavitary lesions was seen in the age group of 36 - 40 years ( 71% ) ; in the < 30 years age group , the incidence was 50% , the difference in the prevalence between the different ages not being of statistical significance ( table 2 ) .", "submucous myomas were the most common lesions seen in the younger patients ; an increasing incidence of endometrial polyps was seen with increasing age , polyps being the most frequently diagnosed pathology in the postmenopausal women , as shown in earlier studies .", "age related prevalence of intracavitary lesions determined by oh . the endometrial biopsy ( embx ) specimens were considered adequate for reporting in 49/54 cases ; two samples had inadequate tissue and three specimens showed menstrual blood , making interpretation difficult .", "no evidence of pathology was seen in 44/49 specimens . of the patients with a normal embx ,", "19 were true negative and 25 were false negative , when compared to results of oh ( table 3 ) .", "the negative predictive value of a normal embx was calculated as 51% . in 5/49 biopsy specimens ,", "a pathology was detected and included fragments of endometrial polyp in two cases ; office hysteroscopy confirmed the presence of a residual polyp in one of these patients .", "there was evidence of chronic endometritis in one patient , in whom an endometrial polyp was detected on hysteroscopy , and focal hyperplasia within a secretory endometrium was shown in the fourth patient , who had a normal appearing endometrial cavity on oh . the sensitivity of endometrial biopsy was 0.04 with a specificity of 0.83 .", "no pathological evidence of endometrial carcinoma was detected in the embx specimens available , which included 14 specimens from postmenopausal patients .", "the incidence of inconclusive sampling was 9% and that of focal endometrial hyperplasia was 2% .", "detection of intracavitary uterine lesions : comparison of results \n of office hysteroscopy to endometrial biopsy .", "difference between a and b is statistically significant p<0.05 of the 13 postmenopausal patients , the age range was 47 - 74 years ; seven patients were on some form of hrt for a period of at least three months prior to the presentation .", "the prevalence of intracavitary lesions in the post - menopausal group was 31% ( 4/13 ) ; two of the lesions were confirmed to be benign endometrial polyps on histology and the third was a submucous myoma ; in the fourth patient the biopsy of an irregular focal area of the endometrium provided a benign proliferative histological specimen .", "results of tvs specifically noted endometrial thickness in 10 postmenopausal patients , the thickness ranging from 1.8 mm -15 mm . in 7/10 patients ,", "the et was < 6 mm and 6/7 had a normal uterine cavity of oh ; the seventh patient , a 72-year - old woman , was noted to have an area of endometrial irregularity on the anterior uterine wall and a directed biopsy performed showed histological evidence of proliferative endometrium with no evidence of malignancy .", "of the three postmenopausal patients in whom a tvs showed an endometrial thickness of greater than 6 mm , all were found to have an endometrial polyp on oh . thus taking the et of 6 mm as the cut - off in the postmenopausal women , the positive predictive value for detection of intracavitary lesions for et > 6 mm was deter - mined to be 100% , whereas the negative predictive value for an et of < 6 mm was also calculated as 100% . of the 54 patients included in the evaluation , 26 underwent definitive surgical management for the presenting problem ; two patients had a normal uterine cavity and opted to undergo endometrial ablation , whereas 24 had operative hysteroscopic resection of the focal lesion .", "the most common histological diagnosis was a submucous myoma , seen in 65% , followed by benign endometrial polyps in 26% of the resected lesions ; in 5% , a combination of the two existed .", "the uterus was classified as normal in size on clinical examination in 63% of the patients ( 36/54 ) .", "of the 18 enlarged uteri , 12 showed an intracavitary lesion on oh , giving a ppv of 67% . in eight of the 47 patients in whom a cbc was available ,", "the hemoglobin was less than 11 gm% ; 6/8 ( 75% ) of these patients had an abnormal finding on oh . nineteen of", "the 54 patients were experiencing heavy , though regular menstrual flow ; of these , 14 ( 74% ) had an abnormal oh compared to 14/35 ( 40% ) of those presenting with irregular bleeding , the difference in the prevalence of lesions being statistically significant ( p=0.008 ) ." ], [ "the high incidence of intrauterine lesions ( 52% ) in our patient population presenting with abnormal uterine bleeding ( aub ) is consistent with results of earlier studies.1 the traditional approach to evaluation of aub in an outpatient setting has included an endometrial sampling following a preliminary assessment based on the history and clinical examination .", "the patient commonly makes an average of two to three clinic visits , depending on the urgency of presentation , before being commenced on a specific treatment protocol . in the present era of cost containment and capping of procedure related reimbursements ,", "the physicians should be acquainted not only with the relative informative yield but also the cost per investigation , so as to channelize their diagnostic approach .", "the aim of management is to thus minimize the cost incurred per patient , while adhering to the principals of the standard of care .", "diagnostic hysteroscopy , though being increasingly employed for evaluation of aub , is still underutilized .", "since the introduction of the hysteroscopic technique , the procedure has undergone significant modifications , contributing to an increase in patient acceptance .", "introduction of fiberoptics , reduction in the caliber of the endoscopes , use of simpler distending media and availability of safer local infiltrative anesthetics have all contributed to an increasing utilization of this technique in evaluation of the uterine cavity .", "baskett et al . in a recent commentary on the efficiency of a one - stop menstrual clinic", "have demonstrated the cost and clinical effectiveness of utilizing hysteroscopy as a preliminary investigation performed at the initial visit in selected patients .", "more than 50% of all diagnostic hysteroscopies , however , are still being performed in the operating room ( or ) , and this trend could be attributed to a combination of a lack of awareness on the part of the physician and perhaps nonavailability of smaller caliber endoscopes .", "compared to tvs , hysteroscopy allows for a direct visualization of the endometrial cavity and hence detection of any focal lesion .", "it offers the additional opportunity of obtaining a directed biopsy in the same setting if indicated , thus obviating the need for a separate scheduling of the procedure .", "studies have demonstrated a superior yield of directed biopsies compared to d&c in providing representative histological specimens . in terms of cost containment ,", "the procedure being performed by the investigating gynecologist precludes the involvement , and hence need for reimbursement of an additional specialist .", "furthermore , it dictates the need for a histological specimen if indicated , and allows for a directed biopsy , ensuring provision of a representative specimen of the focal pathology for evaluation .", "performing an oh early in the evaluation in appropriately selected patients would render utilization of any further investigation like embx or tvs unnecessary .", "the net result would ensure cost containment by reduction in the number of patient visits for investigational purposes , and by enabling the physician to embark on an appropriate management plan at the earliest opportunity , contribute significantly to patient satisfaction .", "the significant incidence of intrauterine lesions in the postmenopausal patients evaluated ( 31% ) is comparable to other studies and underscores the importance of direct visualization of the uterine cavity in this subgroup . since this represents the patient population at the highest risk for significant uterine pathology , i.e. endometrial carcinoma , and because the hysteroscopic appearances of early lesions are well recognized , the argument is further strengthened in favor for an early visualization of the cavity .", "an insight into the clinical staging of an existing endometrial carcinoma would be an additional benefit contributing to the final management plan .", "the high incidence of intracavitary lesions seen in the patients with clinically enlarged uteri ( 62% ) and in those with significant anemia ( 80% ) underscores the importance of proceeding with oh early in the evaluatory process in this subcategory of patients .", "the rationale for attempting to evaluate the endometrial specimen histologically , obtained by either d&c or outpatient biopsy , is to provide an early diagnosis of a significant pathology , namely endometrial cancer and/or endometrial hyperplasia .", "while the risk of such an occurrence is high in a subset of patients , i.e. postmenopausal women with abnormal bleeding ( 10% ) , anovulatory patients and those with a history of prior endometrial hyperplasia , the yield of endometrial sampling from the rest of the patient population in terms of obtaining a pathological diagnosis is negligible .", "this is consistent with our data and questions the almost universal practice of biopsying the endometrium in any patient presenting with aub . in our experience", ", an expenditure of approximately $ 250 for an embx could be easily avoided in the majority of premenopausal patients with aub .", "an embx , however , has a definite place in the diagnostic workup of postmenopausal patients as well as those premenopausal patients who are at high risk for endometrial hyperplasia .", "the high propensity of missing focal intrauterine lesions like submucous myomas and polyps with d&c is well documented in recent literature . ", "moreover , the alleged therapeutic effect of d&c in the management of aub remains questionable .", "most of the data available on the diagnostic accuracy of tvs in evaluation of abnormal uterine bleeding is relevant only for the postmenopausal patients .", "the general consensus of opinion is that an endometrial thickness of less than 5 - 6 mm in a patient presenting with postmenopausal bleeding does not warrant an extensive workup , as the risk of endometrial carcinoma and/or hyperplasia is negligible.11,13 the sensitivity and specificity of tvs in detecting focal intrauterine lesions in the study presented is comparable to the results shown by tombin et al . ( 0.54 and 0.40 , respectively ) .", "scheduling the ultrasound evaluation in the follicular phase of the menstrual cycle in the premenopausal patients may enhance the sensitivity since the hyperechoic secretory endometrium may mask the endometrial polyps .", "presence of intramural myomas may obscure the endometrial stripe , thus contributing to a significant incidence of false negative interpretations , as shown in our series .", "the majority of pelvic ultrasound scans are still being performed by the radiologists ; the cost of a tvs ranges from $ 250-$400 , depending on the involvement of the radiology department .", "furthermore , an impression of an intracavitary lesion directs the management towards performance of a hysteroscopic procedure for the ultimate diagnosis and management ." ], [ "there is a high incidence of intracavitary uterine pathology in patients presenting with abnormal uterine bleeding .", "this is especially true when considering the 35 - 50 years age group who present with heavy regular bleeding , clinically enlarged uteri and significant anemia .", "the relatively poor sensitivity of both endometrial biopsy and transvaginal ultrasound in the detection of intrauterine focal pathology encourage us to propose that oh be utilized as a first line investigation in these patient evaluations . the cost - benefit analysis of investigations like embx and tvs as well as the financial burden of clinic visits during the entire evaluatory process" ] ]

[ "endodontically treated teeth presenting partial or total destruction of crowns require \n reconstruction to create a core to provide mechanical conditions for the indirect \n restoration to be fixed and remain in function for a long period .", "the materials most \n employed for that purpose are composite resins and cast metallic posts .", "resin - based cements have been widely employed for cementation of metaloceramic or \n ceramic crowns due to their adhesive capacity to both tooth structure and restoration , \n combined with esthetic and mechanical properties .", "since the success of cementation depends on the \n achievement of a strong and long - lasting bond among cement , restoration and tooth \n structure , the strength of \n such adhesion procedure is directly proportional to the adequate curing of the cement \n and is crucial to achieve optimal physical and mechanical properties and satisfactory \n clinical performance . several factors may influence the curing of the cement used for luting ceramic crowns . \n", "these include : the composition , thickness , opacity and shade of the ceramic , which may \n reduce the light transmission and consequently affect the light - curing of the \n cement ; characteristics of substrates and luting \n agent ; incompatibility of simplified adhesive systems with self- or \n dual - cured resin cements ; and permeability of simplified adhesives , which ultimately \n compromises the bonding between the cement and the adhesive . considering the lack of information in the literature on the possible influence of the \n type of material used as coronal reconstruction on the physical properties of the cement \n used for luting ceramic crowns", ", this study evaluated the microhardness of the resin \n cement variolink ii along the cement line in the cervical , medium and \n occlusal thirds on the buccal and lingual aspects , and on the occlusal surface , when \n used for luting ceramic crowns on different substrates ( dentin , metal , and composite \n resin ) , after 7 days and 3 months of water storage .", "the null hypotheses tested were that \n microhardness would not be influenced by the region of prepared surfaces , coronal \n substrate , and water storage .", "thirty human third molars were embedded with plaster in plastic cylinders with the \n cementoenamel junction approximately 3 mm above the top of the cylinder .", "the teeth were \n prepared with diamond burs for full - ceramic crowns with a shoulder of 1.2 mm with \n internal rounded angles , and axial reduction of 1.5 mm with 6 to 10 convergence angle \n was performed .", "occlusal reduction was performed resulting in an axial height of 4.0 mm \n ( figure 1a ) .", "they were randomly divided into 3 \n groups ( n=10 ) as follows : group d- the prepared crown surface was kept in dentin ; group \n m- the crowns were sectioned at the level of the cementoenamel junction and the core was \n modeled in acrylic resin duralay ( reliance dental mfg .", "co. worth , illinois , usa ) , cast \n in aluminum - copper alloy , and luted with zinc phosphate ( s. s. white artigos dentrios \n ltda . , rio de janeiro , rj , brasil ) ; group r- the crowns were sectioned as in group m , \n and filling of the cores was performed with composite resin filtek z250 ( 3 m \n espe , st paul , mn , usa ) by the incremental technique .", "light irradiation was obtained \n from a quartz tungsten halogen ( qth ) device v.i.p .", "junior ( bisco , schaumburg , il , usa , \n 500 mw / cm ) for 20 s for each increment , and 40 s for the last one .", "the reduction , \n convergence and height of axial walls followed the same principles described above for \n group d. scheme of experimental stages : a ) prepared tooth ; b ) wax relief ; c ) cemented \n crown ; d ) sectioned crown exhibiting microhardness measurements along the cement \n line and ceramic thicknesses silicon molds were made of all sound tooth crowns before preparation .", "these were then \n used to guide the construction of the ceramic crowns to a thickness of 1.5 mm on the \n axial walls and 2.0 mm on the occlusal surfaces . on the center of buccal , occlusal and lingual aspects of the prepared surfaces ,", "a relief \n was made in wax with approximate thickness of 0.25 mm and width of 2.0 mm to purposely \n allow for a thicker cement line and permit the microhardness tests ( figure 1b ) .", "impressions of the preparations were taken with \n polyvinyl siloxane express ( 3 m espe , st paul , mn , usa ) and cast with type iv \n plaster .", "the crowns were fabricated from the type iv models with monolithic ceramic ips \n e.max press lt ( ivoclar vivadent , schaan , liechtenstein ) , shade a2 , \n following the manufacturer 's instructions . after fitting adjustments", ", all crowns were luted with variolink ii cement ( figure 2 ) , following the procedures described in \n figure 3 , and submitted to a static load of 5 \n kg during light - curing process .", "after removal of the excess of cement , light curing was \n performed on the buccal , lingual and occlusal surfaces , for 40 s on each surface ( figure 1c ) .", "the specimens were then stored in \n lightproof flasks , immersed in deionized water and kept at 37c .", "the specimens were \n randomly divided in subgroups of 5 for each substrate and sectioned either after 7 days \n or 3 months of water storage .", "chemical composition of the resin cement and adhesive system the teeth were removed from the embedding cups and transversely sectioned below the \n crown margins using a diamond disc ( buehler , lake bluff , il , usa ) under constant \n irrigation .", "the crowns were then sectioned in a buccolingual direction , at the center \n of the relief area , to expose the cement line . to facilitate positioning of the \n specimen in the microhardness tester , a second parallel section limited to ceramic \n was made to keep the surface to be analyzed perpendicular to the indenter .", "the cut surface was sequentially polished with 600- and 1200-grit sic paper , followed \n by 1-m diamond paste on a cloth , under constant irrigation . between each polishing \n step ,", "the specimens were rinsed with water for 30 s and ultrasonicated in deionized \n water for 2 min .", "the polished crown sections were kept in moist gauze in lightproof \n flasks until tested .", "measurements were performed on a shimadzu microhardness tester hmv-2,000 ( shimadzu \n corporation - kyoto , japan ) with knoop indenter under a static load of 50 g for 10 s. \n indentations were made from cervical to occlusal surface in 0.5 mm intervals along \n the cement line ( figure 1d ) .", "the hardness was \n expressed as a knoop hardness number ( khn ) , and at the end of measurements the \n average microhardness values were obtained for the cervical , medium and occlusal \n thirds and occlusal surface .", "data were analyzed by three - way anova ( substrates , thirds / occlusal surface , and \n storage ) , and two - way anova was applied ( substrate / storage ) .", "the teeth were removed from the embedding cups and transversely sectioned below the \n crown margins using a diamond disc ( buehler , lake bluff , il , usa ) under constant \n irrigation .", "the crowns were then sectioned in a buccolingual direction , at the center \n of the relief area , to expose the cement line . to facilitate positioning of the \n specimen in the microhardness tester , a second parallel section limited to ceramic \n was made to keep the surface to be analyzed perpendicular to the indenter .", "the cut surface was sequentially polished with 600- and 1200-grit sic paper , followed \n by 1-m diamond paste on a cloth , under constant irrigation . between each polishing \n step ,", "the specimens were rinsed with water for 30 s and ultrasonicated in deionized \n water for 2 min .", "the polished crown sections were kept in moist gauze in lightproof \n flasks until tested .", "measurements were performed on a shimadzu microhardness tester hmv-2,000 ( shimadzu \n corporation - kyoto , japan ) with knoop indenter under a static load of 50 g for 10 s. \n indentations were made from cervical to occlusal surface in 0.5 mm intervals along \n the cement line ( figure 1d ) .", "the hardness was \n expressed as a knoop hardness number ( khn ) , and at the end of measurements the \n average microhardness values were obtained for the cervical , medium and occlusal \n thirds and occlusal surface .", "data were analyzed by three - way anova ( substrates , thirds / occlusal surface , and \n storage ) , and two - way anova was applied ( substrate / storage ) .", "data were analyzed by three - way anova ( substrates , thirds , and storage ) .", "considering \n each factor independently , the substrates showed significant differences ( p=0.000 ) , \n without differences for factors storage ( p=0.573 ) or thirds ( p=0.231 ) ( table 1 )", "since the analysis did not reveal \n significant differences between thirds , the values of substrate and storage were \n submitted to two - way anova , which showed significant effects of core materials \n ( p<0.001 ) .", "hardness values were significantly lower when crowns were cemented on \n resin cores and measured after 7 days of storage ( p=0.007 ) ( table 2 , figure 4 ) .", "knoop hardness number ( standard deviation ) at cervical , middle , occlusal thirds \n and occlusal surface according to the conditions substrate / time storage n=5 specimens in each group .", "equal capital letters indicate lack of \n statistically significant difference ( analysis of storage ) ; equal lowercase \n letters indicate lack of statistically significant difference ( analysis of \n substrate ) ; equal symbols indicate lack of statistically significant difference \n ( analysis of thirds ) .", "p<0.05 knoop hardness number ( standard deviation ) of samples according to the conditions \n substrate / time storage n=5 specimens in each group .", "equal capital letters indicate lack of \n statistically significant difference ( analysis by row ) ; equal lowercase letters \n indicate lack of statistically significant difference ( analysis by column ) . \n p<0.05 box plots : a ) results after 7 days in water storage ; b ) results after 3 months in \n water storage", "the fact that there were no differences among thirds and faces is probably due to the \n ceramic thickness employed ( 1.5 mm on axial walls and 2.0 mm on the occlusal aspect ) and \n the composition of ceramic ips e.max press , a vitreous ceramic composed of \n lithium disilicate , which may have \n allowed sufficient light transmission throughout the crown extent .", "( 2008 ) also found uniform \n microhardness values along the cement layer for the cement variolink ii used to cement \n leucite - based ceramic crowns with 1.4 mm and 2.0 mm of thickness . concerning the type of substrate , only the composite resin core , after storage in water \n for 7 days , resulted in significantly lower hardness values when compared with the other \n groups and storage conditions .", "when full crowns are cemented on metal or fiber - reinforced resin posts or cores , the \n permeability of the simplified adhesive will not be in effect .", "but when the substrate is \n hydrated dentin , the permeability might be more relevant and harmful than the chemical \n incompatibility in relation to metal and composite resin substrates .", "this occurs because the fluid \n transudation through the adhesive may result in water accumulation at the interface \n between adhesive and cement , causing significant reductions in bond strength .", "this water \n accumulation is originated from the hydrated dentin , and the negative effect of this \n permeability on the adhesive resistance of resin cements was confirmed by in \n vitro studies .", "when \n crowns are cemented on core substrates other than dentin , the permeability is absent or \n reduced , but the chemical incompatibility persists between the acidity of simplified \n adhesives and the components of the chemical curing route of resins .", "the reduction of mechanical properties \n of composites stored in water is predominantly related to water absorption by the \n polymer , which is softened by the tumescence of polymeric chains and reduction of \n frictional strength of these chains .", "once saturated in water , the polymeric \n chains are stabilized and there is no further reduction of material properties .", "the effects of humidity on the \n mechanical properties of resin cements have been extensively investigated , and there is \n consensus that the action of solvents on the polymeric chain is deleterious to the \n mechanical properties of the cement . as previously mentioned , it has been reported that resin monomers originated from \n two - step conventional and one - step self - etching adhesives may impair the co - curing and \n consequent bond between these types of adhesives and composites , whose curing reaction \n is initiated by a redox reaction between the tertiary amine and benzoyl peroxide . as a \n consequence ,", "low adhesive strength values are reported when these materials are \n combined . in an attempt to avoid this chemical incompatibility \n and enhance the adhesive strength ,", "manufacturers have been adding co - initiators in \n adhesive systems that react with acidic resin monomers and produce phenyl or \n benzenesulphonic radicals that initiate the curing reaction in dual resin cements or \n when there is no adequate light exposure .", "the microbrush of the adhesive system used in this study contains initiators that are \n fundamental for the self - cure mechanism .", "additionally , this mechanism requires other \n initiators that are originated from a composite that also presents a self - cure \n mechanism and may come from a dual or chemically cured \n reconstruction composite ( when the adhesive is used for reconstruction ) or dual or \n chemically cured cement ( in cases of luting ) .", "that is to say , the self - cure mechanism of \n dsc adhesive may not occur if it does not get in contact with dual or \n self - cured resin .", "for this reason , if the dsc adhesive is exclusively \n used with a light - cured resin , it should necessarily be light - cured before placement of \n composite resin . based on this assumption , it is understood that there clearly is a chemical reaction \n between the dsc adhesive and self - cured or dual - cured resins .", "this reaction should favor \n both the adhesive and cement curing . in this context ,", "the dsc adhesive layer applied on \n the substrates , though light - cured , contained initiators of chemical reaction ready to \n react with initiators present in the cement variolink ii and provide fast \n consolidation of curing of the dsc adhesive , also favoring the cement curing , especially \n in areas less accessible to light , thus characterizing a \" collaboration \" reaction of \n initiators of the adhesive and cement to enhance the curing of both . considering that \n the quantity of such initiators in the adhesive is limited to the applied layer , when \n the adhesive was applied on dentin , all radicals were free to react with the cement . \n", "however , when the adhesive was applied on the core resin , it is speculated that some \n initiators of the adhesive reacted with uncured free radicals of the core resin and \n consequently reduced the availability of initiators to react with the cement .", "the \n consequence was that the cement cure and certainly also the adhesive cure were delayed , \n resulting in lower microhardness values in the initial storage period ( 7 days ) .", "analysis \n of table 1 reveals that , although not \n statistically significant , the microhardness of group r after 7 days of storage \n decreased from the cervical margin to the occlusal aspect , suggesting that in areas \n close to margins , in which the light acts on the cement curing with greater intensity , \n the conflict of utilization of initiators between core resin and cement is surpassed by \n the curing achieved by light - curing .", "since the effects of this conflict are temporary , \n causing only a delay in the curing process , the same phenomenon is not observed after 3 \n months of storage .", "the composite resin , when used as filling material for cores , provides advantages as \n easy handling , fast curing , good translucency shade , which does not interfere with the \n ceramic shade .", "however , in the routine clinical practice , the resin is often in contact \n with saliva for a considerable time . it is not known to which extent the lower hardness \n outcomes observed when cementing over the composite resin core may cause any relevant \n clinical problem .", "further studies should be conducted to enhance the understanding on \n the reactions occurring between this substrate and the resin cement .", "based on the results , the following could be concluded : there was no significant difference in the microhardness results between the cervical , \n medium , and occlusal thirds and occlusal surface ; there was significant difference in the microhardness results between substrates . for \n the 7-day storage period , the results of the composite resin substrate were lower than \n dentin and metal .", "after 3 months of storage , the results were similar for the 3 \n substrates ; the type of material employed for coronal reconstruction of preparations for prosthetic \n purposes may influence the cement properties ." ]

[ "<S> composite resin and metallic posts are the materials most employed for reconstruction \n of teeth presenting partial or total destruction of crowns . </S>", "<S> resin - based cements have \n been widely used for cementation of ceramic crowns . </S>", "<S> the success of cementation \n depends on the achievement of adequate cement curing.objectivesto evaluate the microhardness of variolink ii ( ivoclar vivadent , \n schaan , liechtenstein ) , used for cementing ceramic crowns onto three different \n coronal substrate preparations ( dentin , metal , and composite resin ) , after 7 days \n and 3 months of water storage . </S>", "<S> the evaluation was performed along the cement line \n in the cervical , medium and occlusal thirds on the buccal and lingual aspects , and \n on the occlusal surface . </S>", "<S> material and methodsthirty molars were distributed in three groups ( n=10 ) according to the type of \n coronal substrate : group d- the prepared surfaces were kept in dentin ; groups m \n ( metal ) and r ( resin)- the crowns were sectioned at the level of the cementoenamel \n junction and restored with metallic cast posts or resin build - up cores , \n respectively . </S>", "<S> the crowns were fabricated in ceramic ips e.max press \n ( ivoclar vivadent , schaan , liechtenstein ) and luted with variolink ii . </S>", "<S> after 7 \n days of water storage , 5 specimens of each group were sectioned in buccolingual \n direction for microhardness measurements . </S>", "<S> the other specimens ( n=5 ) were kept \n stored in deionized water at 37c for three months , followed by sectioning and \n microhardness measurements . </S>", "<S> resultsdata were first analyzed by three - way anova that did not reveal significant \n differences between thirds and occlusal surface ( p=0.231 ) . </S>", "<S> two - way anova showed \n significant effect of substrates ( p<0.001 ) and the tukey test revealed that \n microhardness was significantly lower when crowns were cemented on resin cores and \n tested after 7 days of water storage ( p=0.007 ) . </S>", "<S> conclusionthe type of material employed for coronal reconstruction of preparations for \n prosthetic purposes may influence the cement properties . </S>" ]

[ "INTRODUCTION", "MATERIAL AND METHODS", "Sectioning and Knoop hardness (KHN) measurements", "Data treatment", "RESULTS", "DISCUSSION", "CONCLUSIONS" ]

[ [ "endodontically treated teeth presenting partial or total destruction of crowns require \n reconstruction to create a core to provide mechanical conditions for the indirect \n restoration to be fixed and remain in function for a long period .", "the materials most \n employed for that purpose are composite resins and cast metallic posts .", "resin - based cements have been widely employed for cementation of metaloceramic or \n ceramic crowns due to their adhesive capacity to both tooth structure and restoration , \n combined with esthetic and mechanical properties .", "since the success of cementation depends on the \n achievement of a strong and long - lasting bond among cement , restoration and tooth \n structure , the strength of \n such adhesion procedure is directly proportional to the adequate curing of the cement \n and is crucial to achieve optimal physical and mechanical properties and satisfactory \n clinical performance . several factors may influence the curing of the cement used for luting ceramic crowns . \n", "these include : the composition , thickness , opacity and shade of the ceramic , which may \n reduce the light transmission and consequently affect the light - curing of the \n cement ; characteristics of substrates and luting \n agent ; incompatibility of simplified adhesive systems with self- or \n dual - cured resin cements ; and permeability of simplified adhesives , which ultimately \n compromises the bonding between the cement and the adhesive . considering the lack of information in the literature on the possible influence of the \n type of material used as coronal reconstruction on the physical properties of the cement \n used for luting ceramic crowns", ", this study evaluated the microhardness of the resin \n cement variolink ii along the cement line in the cervical , medium and \n occlusal thirds on the buccal and lingual aspects , and on the occlusal surface , when \n used for luting ceramic crowns on different substrates ( dentin , metal , and composite \n resin ) , after 7 days and 3 months of water storage .", "the null hypotheses tested were that \n microhardness would not be influenced by the region of prepared surfaces , coronal \n substrate , and water storage ." ], [ "thirty human third molars were embedded with plaster in plastic cylinders with the \n cementoenamel junction approximately 3 mm above the top of the cylinder .", "the teeth were \n prepared with diamond burs for full - ceramic crowns with a shoulder of 1.2 mm with \n internal rounded angles , and axial reduction of 1.5 mm with 6 to 10 convergence angle \n was performed .", "occlusal reduction was performed resulting in an axial height of 4.0 mm \n ( figure 1a ) .", "they were randomly divided into 3 \n groups ( n=10 ) as follows : group d- the prepared crown surface was kept in dentin ; group \n m- the crowns were sectioned at the level of the cementoenamel junction and the core was \n modeled in acrylic resin duralay ( reliance dental mfg .", "co. worth , illinois , usa ) , cast \n in aluminum - copper alloy , and luted with zinc phosphate ( s. s. white artigos dentrios \n ltda . , rio de janeiro , rj , brasil ) ; group r- the crowns were sectioned as in group m , \n and filling of the cores was performed with composite resin filtek z250 ( 3 m \n espe , st paul , mn , usa ) by the incremental technique .", "light irradiation was obtained \n from a quartz tungsten halogen ( qth ) device v.i.p .", "junior ( bisco , schaumburg , il , usa , \n 500 mw / cm ) for 20 s for each increment , and 40 s for the last one .", "the reduction , \n convergence and height of axial walls followed the same principles described above for \n group d. scheme of experimental stages : a ) prepared tooth ; b ) wax relief ; c ) cemented \n crown ; d ) sectioned crown exhibiting microhardness measurements along the cement \n line and ceramic thicknesses silicon molds were made of all sound tooth crowns before preparation .", "these were then \n used to guide the construction of the ceramic crowns to a thickness of 1.5 mm on the \n axial walls and 2.0 mm on the occlusal surfaces . on the center of buccal , occlusal and lingual aspects of the prepared surfaces ,", "a relief \n was made in wax with approximate thickness of 0.25 mm and width of 2.0 mm to purposely \n allow for a thicker cement line and permit the microhardness tests ( figure 1b ) .", "impressions of the preparations were taken with \n polyvinyl siloxane express ( 3 m espe , st paul , mn , usa ) and cast with type iv \n plaster .", "the crowns were fabricated from the type iv models with monolithic ceramic ips \n e.max press lt ( ivoclar vivadent , schaan , liechtenstein ) , shade a2 , \n following the manufacturer 's instructions . after fitting adjustments", ", all crowns were luted with variolink ii cement ( figure 2 ) , following the procedures described in \n figure 3 , and submitted to a static load of 5 \n kg during light - curing process .", "after removal of the excess of cement , light curing was \n performed on the buccal , lingual and occlusal surfaces , for 40 s on each surface ( figure 1c ) .", "the specimens were then stored in \n lightproof flasks , immersed in deionized water and kept at 37c .", "the specimens were \n randomly divided in subgroups of 5 for each substrate and sectioned either after 7 days \n or 3 months of water storage .", "chemical composition of the resin cement and adhesive system the teeth were removed from the embedding cups and transversely sectioned below the \n crown margins using a diamond disc ( buehler , lake bluff , il , usa ) under constant \n irrigation .", "the crowns were then sectioned in a buccolingual direction , at the center \n of the relief area , to expose the cement line . to facilitate positioning of the \n specimen in the microhardness tester , a second parallel section limited to ceramic \n was made to keep the surface to be analyzed perpendicular to the indenter .", "the cut surface was sequentially polished with 600- and 1200-grit sic paper , followed \n by 1-m diamond paste on a cloth , under constant irrigation . between each polishing \n step ,", "the specimens were rinsed with water for 30 s and ultrasonicated in deionized \n water for 2 min .", "the polished crown sections were kept in moist gauze in lightproof \n flasks until tested .", "measurements were performed on a shimadzu microhardness tester hmv-2,000 ( shimadzu \n corporation - kyoto , japan ) with knoop indenter under a static load of 50 g for 10 s. \n indentations were made from cervical to occlusal surface in 0.5 mm intervals along \n the cement line ( figure 1d ) .", "the hardness was \n expressed as a knoop hardness number ( khn ) , and at the end of measurements the \n average microhardness values were obtained for the cervical , medium and occlusal \n thirds and occlusal surface .", "data were analyzed by three - way anova ( substrates , thirds / occlusal surface , and \n storage ) , and two - way anova was applied ( substrate / storage ) ." ], [ "the teeth were removed from the embedding cups and transversely sectioned below the \n crown margins using a diamond disc ( buehler , lake bluff , il , usa ) under constant \n irrigation .", "the crowns were then sectioned in a buccolingual direction , at the center \n of the relief area , to expose the cement line . to facilitate positioning of the \n specimen in the microhardness tester , a second parallel section limited to ceramic \n was made to keep the surface to be analyzed perpendicular to the indenter .", "the cut surface was sequentially polished with 600- and 1200-grit sic paper , followed \n by 1-m diamond paste on a cloth , under constant irrigation . between each polishing \n step ,", "the specimens were rinsed with water for 30 s and ultrasonicated in deionized \n water for 2 min .", "the polished crown sections were kept in moist gauze in lightproof \n flasks until tested .", "measurements were performed on a shimadzu microhardness tester hmv-2,000 ( shimadzu \n corporation - kyoto , japan ) with knoop indenter under a static load of 50 g for 10 s. \n indentations were made from cervical to occlusal surface in 0.5 mm intervals along \n the cement line ( figure 1d ) .", "the hardness was \n expressed as a knoop hardness number ( khn ) , and at the end of measurements the \n average microhardness values were obtained for the cervical , medium and occlusal \n thirds and occlusal surface ." ], [ "data were analyzed by three - way anova ( substrates , thirds / occlusal surface , and \n storage ) , and two - way anova was applied ( substrate / storage ) ." ], [ "data were analyzed by three - way anova ( substrates , thirds , and storage ) .", "considering \n each factor independently , the substrates showed significant differences ( p=0.000 ) , \n without differences for factors storage ( p=0.573 ) or thirds ( p=0.231 ) ( table 1 )", "since the analysis did not reveal \n significant differences between thirds , the values of substrate and storage were \n submitted to two - way anova , which showed significant effects of core materials \n ( p<0.001 ) .", "hardness values were significantly lower when crowns were cemented on \n resin cores and measured after 7 days of storage ( p=0.007 ) ( table 2 , figure 4 ) .", "knoop hardness number ( standard deviation ) at cervical , middle , occlusal thirds \n and occlusal surface according to the conditions substrate / time storage n=5 specimens in each group .", "equal capital letters indicate lack of \n statistically significant difference ( analysis of storage ) ; equal lowercase \n letters indicate lack of statistically significant difference ( analysis of \n substrate ) ; equal symbols indicate lack of statistically significant difference \n ( analysis of thirds ) .", "p<0.05 knoop hardness number ( standard deviation ) of samples according to the conditions \n substrate / time storage n=5 specimens in each group .", "equal capital letters indicate lack of \n statistically significant difference ( analysis by row ) ; equal lowercase letters \n indicate lack of statistically significant difference ( analysis by column ) . \n p<0.05 box plots : a ) results after 7 days in water storage ; b ) results after 3 months in \n water storage" ], [ "the fact that there were no differences among thirds and faces is probably due to the \n ceramic thickness employed ( 1.5 mm on axial walls and 2.0 mm on the occlusal aspect ) and \n the composition of ceramic ips e.max press , a vitreous ceramic composed of \n lithium disilicate , which may have \n allowed sufficient light transmission throughout the crown extent .", "( 2008 ) also found uniform \n microhardness values along the cement layer for the cement variolink ii used to cement \n leucite - based ceramic crowns with 1.4 mm and 2.0 mm of thickness . concerning the type of substrate , only the composite resin core , after storage in water \n for 7 days , resulted in significantly lower hardness values when compared with the other \n groups and storage conditions .", "when full crowns are cemented on metal or fiber - reinforced resin posts or cores , the \n permeability of the simplified adhesive will not be in effect .", "but when the substrate is \n hydrated dentin , the permeability might be more relevant and harmful than the chemical \n incompatibility in relation to metal and composite resin substrates .", "this occurs because the fluid \n transudation through the adhesive may result in water accumulation at the interface \n between adhesive and cement , causing significant reductions in bond strength .", "this water \n accumulation is originated from the hydrated dentin , and the negative effect of this \n permeability on the adhesive resistance of resin cements was confirmed by in \n vitro studies .", "when \n crowns are cemented on core substrates other than dentin , the permeability is absent or \n reduced , but the chemical incompatibility persists between the acidity of simplified \n adhesives and the components of the chemical curing route of resins .", "the reduction of mechanical properties \n of composites stored in water is predominantly related to water absorption by the \n polymer , which is softened by the tumescence of polymeric chains and reduction of \n frictional strength of these chains .", "once saturated in water , the polymeric \n chains are stabilized and there is no further reduction of material properties .", "the effects of humidity on the \n mechanical properties of resin cements have been extensively investigated , and there is \n consensus that the action of solvents on the polymeric chain is deleterious to the \n mechanical properties of the cement . as previously mentioned , it has been reported that resin monomers originated from \n two - step conventional and one - step self - etching adhesives may impair the co - curing and \n consequent bond between these types of adhesives and composites , whose curing reaction \n is initiated by a redox reaction between the tertiary amine and benzoyl peroxide . as a \n consequence ,", "low adhesive strength values are reported when these materials are \n combined . in an attempt to avoid this chemical incompatibility \n and enhance the adhesive strength ,", "manufacturers have been adding co - initiators in \n adhesive systems that react with acidic resin monomers and produce phenyl or \n benzenesulphonic radicals that initiate the curing reaction in dual resin cements or \n when there is no adequate light exposure .", "the microbrush of the adhesive system used in this study contains initiators that are \n fundamental for the self - cure mechanism .", "additionally , this mechanism requires other \n initiators that are originated from a composite that also presents a self - cure \n mechanism and may come from a dual or chemically cured \n reconstruction composite ( when the adhesive is used for reconstruction ) or dual or \n chemically cured cement ( in cases of luting ) .", "that is to say , the self - cure mechanism of \n dsc adhesive may not occur if it does not get in contact with dual or \n self - cured resin .", "for this reason , if the dsc adhesive is exclusively \n used with a light - cured resin , it should necessarily be light - cured before placement of \n composite resin . based on this assumption , it is understood that there clearly is a chemical reaction \n between the dsc adhesive and self - cured or dual - cured resins .", "this reaction should favor \n both the adhesive and cement curing . in this context ,", "the dsc adhesive layer applied on \n the substrates , though light - cured , contained initiators of chemical reaction ready to \n react with initiators present in the cement variolink ii and provide fast \n consolidation of curing of the dsc adhesive , also favoring the cement curing , especially \n in areas less accessible to light , thus characterizing a \" collaboration \" reaction of \n initiators of the adhesive and cement to enhance the curing of both . considering that \n the quantity of such initiators in the adhesive is limited to the applied layer , when \n the adhesive was applied on dentin , all radicals were free to react with the cement . \n", "however , when the adhesive was applied on the core resin , it is speculated that some \n initiators of the adhesive reacted with uncured free radicals of the core resin and \n consequently reduced the availability of initiators to react with the cement .", "the \n consequence was that the cement cure and certainly also the adhesive cure were delayed , \n resulting in lower microhardness values in the initial storage period ( 7 days ) .", "analysis \n of table 1 reveals that , although not \n statistically significant , the microhardness of group r after 7 days of storage \n decreased from the cervical margin to the occlusal aspect , suggesting that in areas \n close to margins , in which the light acts on the cement curing with greater intensity , \n the conflict of utilization of initiators between core resin and cement is surpassed by \n the curing achieved by light - curing .", "since the effects of this conflict are temporary , \n causing only a delay in the curing process , the same phenomenon is not observed after 3 \n months of storage .", "the composite resin , when used as filling material for cores , provides advantages as \n easy handling , fast curing , good translucency shade , which does not interfere with the \n ceramic shade .", "however , in the routine clinical practice , the resin is often in contact \n with saliva for a considerable time . it is not known to which extent the lower hardness \n outcomes observed when cementing over the composite resin core may cause any relevant \n clinical problem .", "further studies should be conducted to enhance the understanding on \n the reactions occurring between this substrate and the resin cement ." ], [ "based on the results , the following could be concluded : there was no significant difference in the microhardness results between the cervical , \n medium , and occlusal thirds and occlusal surface ; there was significant difference in the microhardness results between substrates . for \n the 7-day storage period , the results of the composite resin substrate were lower than \n dentin and metal .", "after 3 months of storage , the results were similar for the 3 \n substrates ; the type of material employed for coronal reconstruction of preparations for prosthetic \n purposes may influence the cement properties ." ] ]

[ "lung transplantation is an established therapeutic option for patients with advanced cystic fibrosis ( cf ) .", "the success of the first transplant for a patient with cf in 1983 spurred further refinement of the management and selection criteria of patients , leading to significant survival benefit .", "however , patients with advanced cf present a unique microbiological challenge , with disease characterised by bronchiectasis , severe airflow obstruction , high bacterial loads , and recurrent lower respiratory tract infections [ 46 ] . unsurprisingly", ", some centres turn down patients on the basis of colonisation with multiresistant bacteria . the most common respiratory pathogen that colonises patients with cf is pseudomonas aeruginosa , with up to 80% of patients being culture - positive for this organism .", "as the presence of this microorganism has negative prognostic implications , it is disturbing to note that levels of resistance to frontline antipseudomonal agents are very high .", "the presence of multi- and pan - resistant p. aeruginosa renders methods of single - agent antibiotic susceptibility testing suboptimal .", "the conventional manner of managing patients who are colonised with these microorganisms is to empirically treat them with combinations of antibiotics in the peritransplant period .", "unfortunately , an empirical approach might lead to inadequate bactericidal levels and the prescription of antibiotics that antagonise each other .", "this approach is undesirable , as patients with pan - resistant bacteria have shorter follow - up periods and decreased survival rates after transplant . ", "an alternative approach is to utilise multiple combination bactericidal testing ( mcbt ) , a technique that had previously been used to systematically test bacterial isolates against multiple combinations of antibiotics to determine susceptibility patterns and identify optimal combinations for potential treatment .", "previous studies have demonstrated that using combinations of antimicrobials may generate higher levels of in vitro bactericidal activity against p. aeruginosa and burkholderia cepacia complex . ", "our centre currently advocates the use of mcbt ( with modified antimicrobial concentrations ) to determine appropriate prophylactic regimens in patients about to undergo lung transplantation for cf and other lung pathologies that are colonised with antibiotic - resistant gram - negative bacteria .", "to evaluate the effectiveness of our strategy , we undertook a retrospective analysis to compare the rates of posttransplant infection in patients whose peritransplant antimicrobial regimens were determined using the mcbt versus those who had their antibiotics chosen via conventional sensitivity testing .", "we performed a retrospective analysis of all patients who underwent lung transplantation for cf between january 2000 and august 2010 .", "patients were included in the review if they were colonized pretransplant with p. aeruginosa ( as demonstrated by sequential sputum cultures ) and were excluded if they were colonized with b. cepacia complex .", "data was collected from patients ' case notes and clinical charts ; looking specifically at incidences of septicaemia at 30 days , posttransplant wound infection at 30 days , empyema at 30 days , all - cause mortality at 30 days , and all - cause mortality at one year .", "these were mapped against antibiotic resistance , method of sensitivity testing , and choice of antibiotics that were administered .", "infections were defined by combining positive laboratory culture from tissue source ( blood , surgical wound , and pleural fluid ) with at least two of the following four parameters : tachycardia ( heart rate > 90 beats per minute),hypotension ( systolic blood pressure < 90 mmhg),body temperature < 36c or > 38c , abnormal inflammatory markers ( white cell count < 4 10 cells / l or > 12 10 cells / l , c - reactive protein > 10 mg / l ) . tachycardia ( heart rate > 90 beats per minute ) , hypotension ( systolic blood pressure < 90 mmhg ) , body temperature < 36c or > 38c , abnormal inflammatory markers ( white cell count < 4 10 cells / l or > 12 10 cells / l , c - reactive protein > 10 mg", "prophylactic antimicrobials were chosen based on disc susceptibility testing using british society for antimicrobial chemotherapy ( bsac ) breakpoints .", "if sputum was culture positive for pan - resistant p. aeruginosa or recent culture results were unavailable , patients were treated empirically with aztreonam , an antistaphylococcal agent ( either flucloxacillin or clindamycin ) , and metronidazole .", "patients who did not receive peritransplant antibiotics chosen via the mcbt method were deemed to have received antibiotics using conventional means .", "perioperative antibiotics were continued until the patient was extubated and could demonstrate a good cough , ( two - three days ) to a maximum of seven days .", "this was used in conjunction with conventional means of choosing peritransplant antibiotics , but , since 2008 , mcbt became the default method for the determination of bactericidal agents for all patients colonised with antibiotic - resistant gram - negative nonfermenters .", "bactericidal activity was determined by testing at least 12 antimicrobials individually and in combination with each other , leading to 66 different combinations .", "several morphotypes of p. aeruginosa from at least two pretransplant sputa were selected for testing . ", "each antimicrobial or combination of antimicrobials was tested in isosensitest broth using systemic breakpoint concentrations as specified by the bsac .", "after 48 hours incubation at 37c , the turbidity of each broth was measured at 620 nm .", "broths without detectable bacterial growth were subcultured onto blood agar to calculate 99.9% bacterial kill .", "peritransplant antibiotic regimens were then chosen based on mcbt results and patients ' allergy history . any other gram - negative species ( including nonfermenters and/or enterobacteriaceae ) that may have isolated alongside p. aeruginosa", "were also tested using the mcbt , and antibiotic cocktails were chosen that showed bactericidal activity against such mixtures of species . ", "our centre used a three - day induction protocol with antithymocyte globulin ( titrated by flow cytometric analysis of peripheral blood t lymphocytes ) and intravenous methylprednisolone at a dose of 2 mg / kg .", "alternatives were used in the context of an international clinical trial ( mycophenolate ) or in cases of ciclosporin intolerance ( tacrolimus was used ) .", "up to five days of intravenous ciclosporin was given in the context of poor ciclosporin absorption in patients with cf .", "between january 27 , 2000 and august 23 , 2010 , 163 lung transplants were performed on patients with cf .", "this number included patients who were previously turned down by other centres on the basis of their microbiology .", "a total of 129 patients were colonized with p. aeruginosa and not colonized with b. cepacia complex .", "fifty patients were given antibiotics that were chosen based on the mcbt , and there were 79 patients in the conventional group .", "our patients were colonized with strains of p. aeruginosa with varying degrees of antibiotic resistance .", "we defined pan - resistance as resistance to antipseudomonal quinolones , -lactams , aminoglycosides , and colomycin . in this cohort", "these organisms were resistant to all single agents tested but bactericidal combinations with colomycin and another agent were identified .", "seventy - one patients were colonised with multiresistant p. aeruginosa , and nine patients were colonised with fully susceptible strains .", "forty - seven patients were colonised with organisms that were resistant to one or two groups of antimicrobials . \n", "figure 1 shows the relative rates of infectious complications after lung transplantation in both groups .", "two patients ( 4% ) who were given antibiotics based on mcbt developed septicaemia compared to 13 ( 16.5% ) in the conventional group ( p 0.05 ) .", "the occurrence of gram - negative sepsis was statistically lower in the mcbt group , and p. aeruginosa was responsible for only one case of septicaemia compared with seven in the conventional therapy group ( see table 2 ) .", "p. aeruginosa was recovered from the posttransplant pleural fluid of one patient ( 2% ) in the mcbt group , as opposed to six ( 7.6% ) in the conventional group ( p = 0.25 ) .", "there were no statistically significant differences in the rates of surgical wound infection ( 6% in the mcbt group , 3.8% in the conventional group ) .", "there were no statistically significant differences in all - cause mortality rate at 30 days , with a 10% mortality rate in the mcbt cohort and 6.33% in the conventional group .", "this lack of statistical significance was replicated in all - cause mortality rate at one year ( 22% in the mcbt group , 19% in the conventional group ) .", "lung transplantation for cf accounts for approximately one - third of all single sequential lung transplants performed at our centre .", "the complex microbiology involved in this cohort of patients may lead to anxiety when listing patients with multi- and pan - resistant p. aeruginosa , but this paper demonstrates for the first time that the mcbt may have a significant role in altering posttransplant infective complications for patients with cf .", "the data indicates that whilst there may not be evidence for an effect on all - cause mortality , patients who had antibiotics chosen using mcbt had lower rates of morbidity .", "the presence of pleural infection and septicaemia not only negatively impacts the patients ' transplant journey but prolongs the hospital length - of - stay and adds substantial economic burden . we had excluded patients who were colonised with b. cepacia complex for the purposes of this retrospective analysis .", "this is due to the tendency of these patients to succumb to overwhelming sepsis with one - year mortality rates of between 50% and 100% [ 18 , 19 ] .", "our group had also previously noted that infection with burkholderia cenocepacia in particular led to even poorer mortality outcomes . unfortunately , we were unable to look at exact causes of death ( septicaemia in particular ) in our patient cohort as causes of death were not readily identifiable in all cases .", "this is a reflection of the wide geographical referral area that is covered by our centre as well as the degree of shared care with referring centres ( covering the north of england , scotland , northern ireland , and the republic of ireland ) .", "a previous analysis of all patients who underwent lung transplantation for cf at our centre had identified sepsis as the cause of death in 18 cases ( 26% of all recorded cf transplantation recipient deaths ) .", "b. cepacia complex was implicated in seven of these cases . in an additional three cases , clinical sepsis", "prospective studies would play an important role in determining if the use of mcbt can significantly decrease the rate of early mortality due to septicaemia . ", "aaron et al . had compared the efficacy of using antimicrobial combinations derived from mcbt with those derived from conventional susceptibility testing for treatment of acute pulmonary exacerbations of cf .", "they concluded that regimens based on mcbt results did not result in a better clinical or bacteriological outcome .", "it is difficult to make comparisons between our findings and theirs for both clinical and technical reasons . on the clinical side , the patient populations ( post - lung - transplant cf versus non - lung - transplant cf ) and outcome measures were different .", "there are also major differences in the antimicrobial concentrations used in our modified mcbt test and the test as originally described [ 13 , 14 ] . in our study ,", "systemic breakpoint concentrations specified by the bsac to define susceptibility were used in a modified mcbt whereas antimicrobial concentrations in the original mcbt were chosen on the basis of published estimates of the average peak levels seen in serum after standard single - dose intravenous administration . as a result ,", "the antimicrobial concentrations used in our modified mcbt were typically two- to eightfold lower than those previously described .", "as a consequence , fewer isolates would be likely to be classified as susceptible in our study . ", "it is impossible to conclude whether the use of lower breakpoint concentrations of antimicrobials would have led to more favourable outcomes in the study of aaron et al . . ", "as this is a retrospective study , we readily acknowledge that there are inherent limitations to our findings .", "the ten - year period covered by the study has seen numerous changes with regards to developments in lung transplantation .", "these include changes in immunosuppression regimen and the natural progression with regards to peritransplant management as the members of our centre gain more experience . given the nonrandomized nature of this study and temporal confounding factors , we can not definitely conclude that mcbt should be given at induction to all patients colonized with p. aeruginosa .", "however , the fact that we are dealing with a unique cohort of patients with significant microbiological challenges might render it unethical to conduct a full randomized control trial .", "furthermore , it is unlikely that sufficient numbers are recruited to adequately power such a study .", "the data presented is the first indication that patients given antibiotics based on mcbt results had significantly lower rates of septicaemia and lower rates of positive microbiological cultures in their pleural effusions .", "this is an encouraging finding , lending credence to the need for multi - centre prospective studies to be performed that will ideally lead to no patients being turned down for a lung transplant on the basis of colonisation with resistant microorganisms ." ]

[ "<S> early infection is a recognised complication after lung transplantation in patients with cystic fibrosis ( cf ) . </S>", "<S> our centre uses multiple combination bactericidal testing ( mcbt ) when determining appropriate peritransplant prophylactic regimens . to evaluate our strategy , we compared the incidence of posttransplant infection in patients whose peritransplant antimicrobial regimens were determined using mcbt versus standard sensitivity testing . \n </S>", "<S> patients with cf who were infected with pseudomonas aeruginosa and underwent lung transplantations between 2000 and 2010 were included . </S>", "<S> data was collected from clinical records and our microbiology database . </S>", "<S> microorganisms cultured were mapped against antibiotic resistance , method of sensitivity testing , and antibiotics administered peritransplant . \n </S>", "<S> 129 patients were identified ( mean age 28 , male : female , 63 : 66 ) . </S>", "<S> fifty patients ( 38.8% ) had antibiotics determined by mcbt . </S>", "<S> two patients in the mcbt group developed septicaemia , 13 in the conventional group ( p 0.05 , 2-tailed fisher 's test ) . </S>", "<S> sepsis was attributable to p. aeruginosa in one patient from the mcbt group and seven patients in the conventional group ( p = 0.15 ) . </S>", "<S> p. aeruginosa was recovered from the posttransplant pleural fluid of one patient who received mcbt - guided prophylaxis , six patients in the conventional group ( p = 0.25 ) . </S>", "<S> patients given antibiotics based on mcbt had significantly lower rates of septicaemia and lower rates of empyema . \n </S>" ]

[ "1. Introduction", "2. Methods", "3. Results ", "4. Discussion" ]

[ [ "lung transplantation is an established therapeutic option for patients with advanced cystic fibrosis ( cf ) .", "the success of the first transplant for a patient with cf in 1983 spurred further refinement of the management and selection criteria of patients , leading to significant survival benefit .", "however , patients with advanced cf present a unique microbiological challenge , with disease characterised by bronchiectasis , severe airflow obstruction , high bacterial loads , and recurrent lower respiratory tract infections [ 46 ] . unsurprisingly", ", some centres turn down patients on the basis of colonisation with multiresistant bacteria . the most common respiratory pathogen that colonises patients with cf is pseudomonas aeruginosa , with up to 80% of patients being culture - positive for this organism .", "as the presence of this microorganism has negative prognostic implications , it is disturbing to note that levels of resistance to frontline antipseudomonal agents are very high .", "the presence of multi- and pan - resistant p. aeruginosa renders methods of single - agent antibiotic susceptibility testing suboptimal .", "the conventional manner of managing patients who are colonised with these microorganisms is to empirically treat them with combinations of antibiotics in the peritransplant period .", "unfortunately , an empirical approach might lead to inadequate bactericidal levels and the prescription of antibiotics that antagonise each other .", "this approach is undesirable , as patients with pan - resistant bacteria have shorter follow - up periods and decreased survival rates after transplant . ", "an alternative approach is to utilise multiple combination bactericidal testing ( mcbt ) , a technique that had previously been used to systematically test bacterial isolates against multiple combinations of antibiotics to determine susceptibility patterns and identify optimal combinations for potential treatment .", "previous studies have demonstrated that using combinations of antimicrobials may generate higher levels of in vitro bactericidal activity against p. aeruginosa and burkholderia cepacia complex . ", "our centre currently advocates the use of mcbt ( with modified antimicrobial concentrations ) to determine appropriate prophylactic regimens in patients about to undergo lung transplantation for cf and other lung pathologies that are colonised with antibiotic - resistant gram - negative bacteria .", "to evaluate the effectiveness of our strategy , we undertook a retrospective analysis to compare the rates of posttransplant infection in patients whose peritransplant antimicrobial regimens were determined using the mcbt versus those who had their antibiotics chosen via conventional sensitivity testing ." ], [ "we performed a retrospective analysis of all patients who underwent lung transplantation for cf between january 2000 and august 2010 .", "patients were included in the review if they were colonized pretransplant with p. aeruginosa ( as demonstrated by sequential sputum cultures ) and were excluded if they were colonized with b. cepacia complex .", "data was collected from patients ' case notes and clinical charts ; looking specifically at incidences of septicaemia at 30 days , posttransplant wound infection at 30 days , empyema at 30 days , all - cause mortality at 30 days , and all - cause mortality at one year .", "these were mapped against antibiotic resistance , method of sensitivity testing , and choice of antibiotics that were administered .", "infections were defined by combining positive laboratory culture from tissue source ( blood , surgical wound , and pleural fluid ) with at least two of the following four parameters : tachycardia ( heart rate > 90 beats per minute),hypotension ( systolic blood pressure < 90 mmhg),body temperature < 36c or > 38c , abnormal inflammatory markers ( white cell count < 4 10 cells / l or > 12 10 cells / l , c - reactive protein > 10 mg / l ) . tachycardia ( heart rate > 90 beats per minute ) , hypotension ( systolic blood pressure < 90 mmhg ) , body temperature < 36c or > 38c , abnormal inflammatory markers ( white cell count < 4 10 cells / l or > 12 10 cells / l , c - reactive protein > 10 mg", "prophylactic antimicrobials were chosen based on disc susceptibility testing using british society for antimicrobial chemotherapy ( bsac ) breakpoints .", "if sputum was culture positive for pan - resistant p. aeruginosa or recent culture results were unavailable , patients were treated empirically with aztreonam , an antistaphylococcal agent ( either flucloxacillin or clindamycin ) , and metronidazole .", "patients who did not receive peritransplant antibiotics chosen via the mcbt method were deemed to have received antibiotics using conventional means .", "perioperative antibiotics were continued until the patient was extubated and could demonstrate a good cough , ( two - three days ) to a maximum of seven days .", "this was used in conjunction with conventional means of choosing peritransplant antibiotics , but , since 2008 , mcbt became the default method for the determination of bactericidal agents for all patients colonised with antibiotic - resistant gram - negative nonfermenters .", "bactericidal activity was determined by testing at least 12 antimicrobials individually and in combination with each other , leading to 66 different combinations .", "several morphotypes of p. aeruginosa from at least two pretransplant sputa were selected for testing . ", "each antimicrobial or combination of antimicrobials was tested in isosensitest broth using systemic breakpoint concentrations as specified by the bsac .", "after 48 hours incubation at 37c , the turbidity of each broth was measured at 620 nm .", "broths without detectable bacterial growth were subcultured onto blood agar to calculate 99.9% bacterial kill .", "peritransplant antibiotic regimens were then chosen based on mcbt results and patients ' allergy history . any other gram - negative species ( including nonfermenters and/or enterobacteriaceae ) that may have isolated alongside p. aeruginosa", "were also tested using the mcbt , and antibiotic cocktails were chosen that showed bactericidal activity against such mixtures of species . ", "our centre used a three - day induction protocol with antithymocyte globulin ( titrated by flow cytometric analysis of peripheral blood t lymphocytes ) and intravenous methylprednisolone at a dose of 2 mg / kg .", "alternatives were used in the context of an international clinical trial ( mycophenolate ) or in cases of ciclosporin intolerance ( tacrolimus was used ) .", "up to five days of intravenous ciclosporin was given in the context of poor ciclosporin absorption in patients with cf ." ], [ "between january 27 , 2000 and august 23 , 2010 , 163 lung transplants were performed on patients with cf .", "this number included patients who were previously turned down by other centres on the basis of their microbiology .", "a total of 129 patients were colonized with p. aeruginosa and not colonized with b. cepacia complex .", "fifty patients were given antibiotics that were chosen based on the mcbt , and there were 79 patients in the conventional group .", "our patients were colonized with strains of p. aeruginosa with varying degrees of antibiotic resistance .", "we defined pan - resistance as resistance to antipseudomonal quinolones , -lactams , aminoglycosides , and colomycin . in this cohort", "these organisms were resistant to all single agents tested but bactericidal combinations with colomycin and another agent were identified .", "seventy - one patients were colonised with multiresistant p. aeruginosa , and nine patients were colonised with fully susceptible strains .", "forty - seven patients were colonised with organisms that were resistant to one or two groups of antimicrobials . \n", "figure 1 shows the relative rates of infectious complications after lung transplantation in both groups .", "two patients ( 4% ) who were given antibiotics based on mcbt developed septicaemia compared to 13 ( 16.5% ) in the conventional group ( p 0.05 ) .", "the occurrence of gram - negative sepsis was statistically lower in the mcbt group , and p. aeruginosa was responsible for only one case of septicaemia compared with seven in the conventional therapy group ( see table 2 ) .", "p. aeruginosa was recovered from the posttransplant pleural fluid of one patient ( 2% ) in the mcbt group , as opposed to six ( 7.6% ) in the conventional group ( p = 0.25 ) .", "there were no statistically significant differences in the rates of surgical wound infection ( 6% in the mcbt group , 3.8% in the conventional group ) .", "there were no statistically significant differences in all - cause mortality rate at 30 days , with a 10% mortality rate in the mcbt cohort and 6.33% in the conventional group .", "this lack of statistical significance was replicated in all - cause mortality rate at one year ( 22% in the mcbt group , 19% in the conventional group ) ." ], [ "lung transplantation for cf accounts for approximately one - third of all single sequential lung transplants performed at our centre .", "the complex microbiology involved in this cohort of patients may lead to anxiety when listing patients with multi- and pan - resistant p. aeruginosa , but this paper demonstrates for the first time that the mcbt may have a significant role in altering posttransplant infective complications for patients with cf .", "the data indicates that whilst there may not be evidence for an effect on all - cause mortality , patients who had antibiotics chosen using mcbt had lower rates of morbidity .", "the presence of pleural infection and septicaemia not only negatively impacts the patients ' transplant journey but prolongs the hospital length - of - stay and adds substantial economic burden . we had excluded patients who were colonised with b. cepacia complex for the purposes of this retrospective analysis .", "this is due to the tendency of these patients to succumb to overwhelming sepsis with one - year mortality rates of between 50% and 100% [ 18 , 19 ] .", "our group had also previously noted that infection with burkholderia cenocepacia in particular led to even poorer mortality outcomes . unfortunately , we were unable to look at exact causes of death ( septicaemia in particular ) in our patient cohort as causes of death were not readily identifiable in all cases .", "this is a reflection of the wide geographical referral area that is covered by our centre as well as the degree of shared care with referring centres ( covering the north of england , scotland , northern ireland , and the republic of ireland ) .", "a previous analysis of all patients who underwent lung transplantation for cf at our centre had identified sepsis as the cause of death in 18 cases ( 26% of all recorded cf transplantation recipient deaths ) .", "b. cepacia complex was implicated in seven of these cases . in an additional three cases , clinical sepsis", "prospective studies would play an important role in determining if the use of mcbt can significantly decrease the rate of early mortality due to septicaemia . ", "aaron et al . had compared the efficacy of using antimicrobial combinations derived from mcbt with those derived from conventional susceptibility testing for treatment of acute pulmonary exacerbations of cf .", "they concluded that regimens based on mcbt results did not result in a better clinical or bacteriological outcome .", "it is difficult to make comparisons between our findings and theirs for both clinical and technical reasons . on the clinical side , the patient populations ( post - lung - transplant cf versus non - lung - transplant cf ) and outcome measures were different .", "there are also major differences in the antimicrobial concentrations used in our modified mcbt test and the test as originally described [ 13 , 14 ] . in our study ,", "systemic breakpoint concentrations specified by the bsac to define susceptibility were used in a modified mcbt whereas antimicrobial concentrations in the original mcbt were chosen on the basis of published estimates of the average peak levels seen in serum after standard single - dose intravenous administration . as a result ,", "the antimicrobial concentrations used in our modified mcbt were typically two- to eightfold lower than those previously described .", "as a consequence , fewer isolates would be likely to be classified as susceptible in our study . ", "it is impossible to conclude whether the use of lower breakpoint concentrations of antimicrobials would have led to more favourable outcomes in the study of aaron et al . . ", "as this is a retrospective study , we readily acknowledge that there are inherent limitations to our findings .", "the ten - year period covered by the study has seen numerous changes with regards to developments in lung transplantation .", "these include changes in immunosuppression regimen and the natural progression with regards to peritransplant management as the members of our centre gain more experience . given the nonrandomized nature of this study and temporal confounding factors , we can not definitely conclude that mcbt should be given at induction to all patients colonized with p. aeruginosa .", "however , the fact that we are dealing with a unique cohort of patients with significant microbiological challenges might render it unethical to conduct a full randomized control trial .", "furthermore , it is unlikely that sufficient numbers are recruited to adequately power such a study .", "the data presented is the first indication that patients given antibiotics based on mcbt results had significantly lower rates of septicaemia and lower rates of positive microbiological cultures in their pleural effusions .", "this is an encouraging finding , lending credence to the need for multi - centre prospective studies to be performed that will ideally lead to no patients being turned down for a lung transplant on the basis of colonisation with resistant microorganisms ." ] ]

[ "acute promyelocytic leukemia ( apl ) is one of the most characteristic subtypes of aml in which abnormal promyelocytes predominate within peripheral blood or bone marrow .", "also , t(15;17)(q22;q21 ) shows a characteristic chromosomal translocation in apl , observable in 70 - 90% of apl patients .", "owing to all trans - retinoic acid ( atra ) combined with chemotherapy , apl has one of the highest cure rates of all types of aml .", "seventy to eighty percent of newly diagnosed apl patients with the pml - rara rearrangement are cured or under long - term remission , yet some of them have a poor prognosis [ 2 - 5 ] . because cytogenetics is one of the most powerful prognostic factors for the outcome of aml , cytogenetic abnormalities can cause change in treatment response , relapse , and clinicopathological characteristics .", "incidence of secondary cytogenetic abnormalities has been observed in ~40% of apl cases , but their prognostic significance is still unclear [ 5 - 7 ] .", "about 1% of the reported secondary cytogenetic abnormalities in apl patients are ider(17)(q10)t(15;17)(q22;q12 ) , an infrequent type of additional recurrent chromosomal abnormality , according to a recent study . however , ider(17)(q10)t(15;17 ) associated with the pml - rara rearrangement in microgranular variant apl is even more rare . as far as we know ,", "only 2 cases of the ider(17)(q10)t(15;17 ) abnormality in microgranular apl have been previously reported [ 8 , 9 ] . here", ", we describe an unusual microgranular apl case associated with ider(17)(q10)t(15;17 ) , identified by both conventional cytogenetics and fish analyses at the initial diagnosis .", "a 59-yr - old woman who had previously been diagnosed with cerebral infarction was brought to our hospital due to right side weakness in november 2007 .", "the initial complete blood count showed pancytopenia , hb level of 9.9 g / dl ( reference range 12 - 16 g / dl ) , platelet count of 83,000/l ( reference range 150,000 - 350,000/l ) , and white blood cell count of 1,000/l ( reference range 4,000 - 10,000/l ) .", "bone marrow aspiration showed a hypercellular marrow replaced by increased promyelocytes with a paucity or absence of granules , accounting for 36% of all nucleated cells ( fig .", "the results of special staining of bone marrow specimens were as follows : myeloperoxidase , positive ; periodic acid schiff , negative ; nonspecific esterase , negative .", "flow cytometric analysis was conducted and showed that the blasts were positive for cd13 ( 91.1% ) , cd33 ( 83.9% ) , cd117 ( 59.2% ) , cd2 ( 43.9% ) , and cd45 ( 25.4% ) , and negative for hla - dr ( 3.4% ) , cd3 ( 1.3% ) , cd7 ( 0.6% ) , cd10 ( 1.8% ) , cd14 ( 2.4% ) , cd19 ( 5.1% ) , cd34 ( 1.4% ) , cd41 ( 2.9% ) , cd56 ( 1.2% ) , and tdt ( 0.9% ) .", "fish signals from pml - rara probes ( abbott molecular / vysis , des plaines , il , usa ) yielded the results of nuc ish(pml , rara)4(rara con pml3)[24/138 ] , ( pml , rara)6(rara con pml5)[14/138 ] , ( pml , rara)3(rara con pml2)[13/138 ] , consistent with the abnormal fusion signal patterns seen in 37% of the nuclei examined ( fig .", "the patient was diagnosed with apl and treated with induction chemotherapy consisting of daunorubicin , cytosine arabinoside , and atra . after completing induction chemotherapy ,", "follow up bone marrow examination in january 2008 showed no evidence of morphologically visible residual leukemia .", "the concurrent karyotype analysis result was 46,xx in all analyzed cells ; and pml - rara fish showed \" nuc ish ( pml , rara)2 \" in which the abnormal signal pattern was not observed .", "there was no evidence of a pml - rara fusion gene in the reverse transcriptase - pcr ( rt - pcr ) analysis .", "as indicated by follow - up bone marrow biopsies conducted until september 2008 , the patient remained in complete remission . during this period", ", the rt - pcr analysis did not show any signs of the pml - rara fusion gene while other cytogenetic studies also indicated normal findings .", "apl is a distinct subtype of aml and constitutes about 5 - 8% of all cases of aml diagnosis . according to the 2008 who classification", ", apl can be diagnosed when there is a t(15;17 ) or a pml - rara rearrangement , even if peripheral blood or bone marrow studies show less than 20% promyelocytes . as recently reported by manola et al . and", "our study group , the ider(17)(q10)t(15;17 ) , an isochromosomal abnormality that occurs on the long arm of ider(17)t(15;17 ) after reciprocal translocation of t(15;17 ) , is a relatively rare type of an additional recurrent cytogenetic abnormality that has been reported in 62 apl patients worldwide [ 8 - 13 ] . according to these studies , the influence of ider(17)(q10)t(15 ; 17 ) on the prognosis of adult apl patients", "indeed , 4 previously reported apl cases in children were all related to poor prognosis [ 8 , 13 - 15 ] , inferring that a more close and careful interpretation is necessary for childhood apl cases .", "what is interesting is that so far , reports of ider(17)(q10)t(15;17 ) from microgranular variant ( aml - m3v ) type are extremely rare .", "out of 62 total cases , information on apl morphology type were available in 42 cases , and most of these cases ( 40/42 ) were of the hypergranular apl type , except for 2 cases that clearly indicated aml - m3v ( table 1 ) [ 8 , 9 ] .", "therefore , further research is required to determine whether ider(17)(q10 ) and aml - m3v have a low association , and more careful observation should be conducted to prevent underestimating aml - m3v patients among ider(17)(q10)t(15;17 ) cases .", "furthermore , double ider(17 ) ( q10)t(15;17 ) is so rare in the international public databases that only 2 cases of apl patients indicating double ider(17)(q10)t(15;17 ) chromosomal abnormalities have been reported ( table 2 ) [ 16 , 17 ] . in double ider(17)(q10)t(15;17 ) , a gene dosage effect is observed owing to chromosomal abnormalities such as the pml - rara fusion gene on chromosome 17 or the quadruplication of der(17q ) . in addition , since the deletion of the tumor suppressor gene tp53 occurs by the loss of 17p , further research is necessary to resolve the adverse prognosis of the apl group related to such copy number variations .", "owing to the limited amount of clinical data in the literature , the relatedness between double ider(17)(q10)t(15;17 ) and an adverse prognosis is still unclear [ 16 , 17 ] . in the case of our patient", ", it was hard to determine a strong association between the additional genetic aberration and prognosis because of the small clonal size of the \" double ider(17)(q10)t(15;17 ) \" abnormality .", "nevertheless , at least from a diagnostic perspective and as indicated in the authors ' recent studies [ 13 , 18 ] , minimal residual disease detection using such multiple abnormal fusion signals through the pml - rara fish analysis in apl patients associated with ider(17)(q10)t(15;17 ) or double ider(17)(q10)t(15;17 ) would be considered to be a useful follow - up marker in clinical laboratories or hospitals .", "additional study would contribute toward a better understanding of the influence of ider(17)(q10)t(15;17 ) on the prognosis , survival , and treatment response of such apl cases in adults or children . to the best of our knowledge", ", however , this is the third case report of microgranular variant apl associated with ider(17)(q10)t(15;17 ) ." ]

[ "<S> we present a rare case of microgranular variant acute promyelocytic leukemia ( apl ) associated with ider(17)(q10)t(15;17)(q22;q12 ) of an old - age patient . </S>", "<S> the initial chromosome study showed a 46,xx , del(6)(?q21q25),der(15)t(15;17)(q22;q12),ider(17)(q10)t(15;17)[10]/47,sl,+ider(17)(q10)t(15;17)[3]/46,xx[16 ] . </S>", "<S> fish signals from a dual color dual fusion translocation pml - rara probe were consistent with the results of conventional cytogenetics . </S>", "<S> because of the rarity of ider(17)(q10)t(15;17 ) in microgranular apl , further studies on both gene dosage effect of this chromosomal abnormality and the influence of ider(17)(q10)t(15;17 ) on clinical features such as prognosis , survival , and treatment response of apl cases are recommended . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "acute promyelocytic leukemia ( apl ) is one of the most characteristic subtypes of aml in which abnormal promyelocytes predominate within peripheral blood or bone marrow .", "also , t(15;17)(q22;q21 ) shows a characteristic chromosomal translocation in apl , observable in 70 - 90% of apl patients .", "owing to all trans - retinoic acid ( atra ) combined with chemotherapy , apl has one of the highest cure rates of all types of aml .", "seventy to eighty percent of newly diagnosed apl patients with the pml - rara rearrangement are cured or under long - term remission , yet some of them have a poor prognosis [ 2 - 5 ] . because cytogenetics is one of the most powerful prognostic factors for the outcome of aml , cytogenetic abnormalities can cause change in treatment response , relapse , and clinicopathological characteristics .", "incidence of secondary cytogenetic abnormalities has been observed in ~40% of apl cases , but their prognostic significance is still unclear [ 5 - 7 ] .", "about 1% of the reported secondary cytogenetic abnormalities in apl patients are ider(17)(q10)t(15;17)(q22;q12 ) , an infrequent type of additional recurrent chromosomal abnormality , according to a recent study . however , ider(17)(q10)t(15;17 ) associated with the pml - rara rearrangement in microgranular variant apl is even more rare . as far as we know ,", "only 2 cases of the ider(17)(q10)t(15;17 ) abnormality in microgranular apl have been previously reported [ 8 , 9 ] . here", ", we describe an unusual microgranular apl case associated with ider(17)(q10)t(15;17 ) , identified by both conventional cytogenetics and fish analyses at the initial diagnosis ." ], [ "a 59-yr - old woman who had previously been diagnosed with cerebral infarction was brought to our hospital due to right side weakness in november 2007 .", "the initial complete blood count showed pancytopenia , hb level of 9.9 g / dl ( reference range 12 - 16 g / dl ) , platelet count of 83,000/l ( reference range 150,000 - 350,000/l ) , and white blood cell count of 1,000/l ( reference range 4,000 - 10,000/l ) .", "bone marrow aspiration showed a hypercellular marrow replaced by increased promyelocytes with a paucity or absence of granules , accounting for 36% of all nucleated cells ( fig .", "the results of special staining of bone marrow specimens were as follows : myeloperoxidase , positive ; periodic acid schiff , negative ; nonspecific esterase , negative .", "flow cytometric analysis was conducted and showed that the blasts were positive for cd13 ( 91.1% ) , cd33 ( 83.9% ) , cd117 ( 59.2% ) , cd2 ( 43.9% ) , and cd45 ( 25.4% ) , and negative for hla - dr ( 3.4% ) , cd3 ( 1.3% ) , cd7 ( 0.6% ) , cd10 ( 1.8% ) , cd14 ( 2.4% ) , cd19 ( 5.1% ) , cd34 ( 1.4% ) , cd41 ( 2.9% ) , cd56 ( 1.2% ) , and tdt ( 0.9% ) .", "fish signals from pml - rara probes ( abbott molecular / vysis , des plaines , il , usa ) yielded the results of nuc ish(pml , rara)4(rara con pml3)[24/138 ] , ( pml , rara)6(rara con pml5)[14/138 ] , ( pml , rara)3(rara con pml2)[13/138 ] , consistent with the abnormal fusion signal patterns seen in 37% of the nuclei examined ( fig .", "the patient was diagnosed with apl and treated with induction chemotherapy consisting of daunorubicin , cytosine arabinoside , and atra . after completing induction chemotherapy ,", "follow up bone marrow examination in january 2008 showed no evidence of morphologically visible residual leukemia .", "the concurrent karyotype analysis result was 46,xx in all analyzed cells ; and pml - rara fish showed \" nuc ish ( pml , rara)2 \" in which the abnormal signal pattern was not observed .", "there was no evidence of a pml - rara fusion gene in the reverse transcriptase - pcr ( rt - pcr ) analysis .", "as indicated by follow - up bone marrow biopsies conducted until september 2008 , the patient remained in complete remission . during this period", ", the rt - pcr analysis did not show any signs of the pml - rara fusion gene while other cytogenetic studies also indicated normal findings ." ], [ "apl is a distinct subtype of aml and constitutes about 5 - 8% of all cases of aml diagnosis . according to the 2008 who classification", ", apl can be diagnosed when there is a t(15;17 ) or a pml - rara rearrangement , even if peripheral blood or bone marrow studies show less than 20% promyelocytes . as recently reported by manola et al . and", "our study group , the ider(17)(q10)t(15;17 ) , an isochromosomal abnormality that occurs on the long arm of ider(17)t(15;17 ) after reciprocal translocation of t(15;17 ) , is a relatively rare type of an additional recurrent cytogenetic abnormality that has been reported in 62 apl patients worldwide [ 8 - 13 ] . according to these studies , the influence of ider(17)(q10)t(15 ; 17 ) on the prognosis of adult apl patients", "indeed , 4 previously reported apl cases in children were all related to poor prognosis [ 8 , 13 - 15 ] , inferring that a more close and careful interpretation is necessary for childhood apl cases .", "what is interesting is that so far , reports of ider(17)(q10)t(15;17 ) from microgranular variant ( aml - m3v ) type are extremely rare .", "out of 62 total cases , information on apl morphology type were available in 42 cases , and most of these cases ( 40/42 ) were of the hypergranular apl type , except for 2 cases that clearly indicated aml - m3v ( table 1 ) [ 8 , 9 ] .", "therefore , further research is required to determine whether ider(17)(q10 ) and aml - m3v have a low association , and more careful observation should be conducted to prevent underestimating aml - m3v patients among ider(17)(q10)t(15;17 ) cases .", "furthermore , double ider(17 ) ( q10)t(15;17 ) is so rare in the international public databases that only 2 cases of apl patients indicating double ider(17)(q10)t(15;17 ) chromosomal abnormalities have been reported ( table 2 ) [ 16 , 17 ] . in double ider(17)(q10)t(15;17 ) , a gene dosage effect is observed owing to chromosomal abnormalities such as the pml - rara fusion gene on chromosome 17 or the quadruplication of der(17q ) . in addition , since the deletion of the tumor suppressor gene tp53 occurs by the loss of 17p , further research is necessary to resolve the adverse prognosis of the apl group related to such copy number variations .", "owing to the limited amount of clinical data in the literature , the relatedness between double ider(17)(q10)t(15;17 ) and an adverse prognosis is still unclear [ 16 , 17 ] . in the case of our patient", ", it was hard to determine a strong association between the additional genetic aberration and prognosis because of the small clonal size of the \" double ider(17)(q10)t(15;17 ) \" abnormality .", "nevertheless , at least from a diagnostic perspective and as indicated in the authors ' recent studies [ 13 , 18 ] , minimal residual disease detection using such multiple abnormal fusion signals through the pml - rara fish analysis in apl patients associated with ider(17)(q10)t(15;17 ) or double ider(17)(q10)t(15;17 ) would be considered to be a useful follow - up marker in clinical laboratories or hospitals .", "additional study would contribute toward a better understanding of the influence of ider(17)(q10)t(15;17 ) on the prognosis , survival , and treatment response of such apl cases in adults or children . to the best of our knowledge", ", however , this is the third case report of microgranular variant apl associated with ider(17)(q10)t(15;17 ) ." ] ]

[ "the three most commonly encountered forms of mercury are metal vapor ( hg ) , ionic compounds ( hg ) and organic mercury , principally methyl mercury ( hgch3 ) .", "organic mercury is the most deadly of the mercury compounds , probably due to its ability to penetrate cells . within the cell it can destroy various components selectively or in total by releasing lysosomes , damaging dna and rupturing the cell membrane .", "the toxicity of high levels of mercury contamination was brought to public attention in the 1950s as a result of mercury dumping in the minamata bay in japan 8 9 ] .", "the general population is primarily exposed to mercury via food , fish being a major source of methyl mercury exposure .", "mercury is incorporated into the food chain as methyl mercury , primarily through the action of bacteria and other microbes transforming elemental or inorganic forms .", "humans are also exposed to mercury via thimerosal ( a preservative added to vaccines and many other pharmaceuticals ) , and amalgam or mercury - based dental fillings . mercury vapor released from mercury dental fillings", "is absorbed very rapidly and thoroughly by the body , mainly through inhalation and swallowing .", "adverse health effects , particularly of a neurological nature , have resulted from low exposure levels , especially to the fetus in pregnant women .", "using young rats as a model of mercury toxicity , results from previous studies and from our group have demonstrated that exposure to hgcl2 ( 25 mg / kg ) for only five consecutive days causes cerebral and whole body weight losses , increases in kidney weight , mercury accumulation in tissues , inhibition of porphobilinogen synthase activity[1517 ] , alterations in renal and hepatic functions and glycemia .", "metal intoxication is usually treated with chelating agents , which are not specific and are often more toxic than the metal itself .", "because chelation therapy for heavy metals intoxication can also be toxic , researchers are looking for natural preventive and therapeutic agents for heavy metal toxicity .", "kumis , also known as koumiss , is an alcoholic beverage made from fermented mare 's milk .", "mare 's milk is usually not consumed raw , because it tends to have a strong laxative effect , although this effect is sometimes used medically . instead ,", "fermentation destroys lactose in milk , converting it into lactic acid , ethanol , and carbon dioxide .", "fermentation takes place in 3 - 8 hours and is carried out by a mixture of indigenous yeasts and lactic acid bacteria .", "currently , kumis is manufactured at an industrial level in some countries using pure cultures of yeasts and lactic acid bacteria .", "kumis is rich in ferments , trace elements , antibiotics , vitamins a , b1 , b2 , b12 , d , e , c , ethyl alcohol , lactic acid and carbonic acid .", "kumis drinking has a healing influence on the gastrointestinal tract , metabolism , cardiovascular and nervous systems , and kidneys ; it aids the development of immunity ; and has been used to treat weight loss and anemia . adding soluble fiber to the diet has also been shown to have positive effects on health .", "various studies have shown a reduction in colon cancer risk and enhanced health by consuming inulin and oligo fructose in ratios ranging from 2% to 10% .", "probiotics include viable lactic acid bacteria and other bacilli that are able to survive in the intestine .", "prebiotics include carbohydrates that are not digested in the upper part of the gastrointestinal tract , but selectively fermented by bacteria in the colon .", "this selective fermentation affects the composition of the intestinal microflora by stimulating bifidobacteria and lactobacilli , both in humans and in animals , where these bacteria have health promoting properties .", "we have combined these three health - promoting materials to produce a functional food composed of fermented mare 's milk plus a high level ( 6% ) of soluble fiber and probiotics .", "the current study was designed to determine if feeding the high fiber probiotic fermented mare 's milk could alleviate the toxic effects of mercury in rat model .", "starter cultures of streptococcus thermophilus , lactobacillus acidophilus , and bifidobacterium bifidum were obtained from hansen 's laboratory , copenhagen , denmark .", "dandelion root ( taraxacum officinale ) was purchased from haraz herbal market , cairo , egypt .", "fresh mare 's milk was obtained from lactating and healthy arabian mares in the experimental animal unit , college of agriculture and veterinary medicine , qassim university , saudi arabia .", "briefly , fresh mare 's milk ( 11% total solid ) was heated at 85 c for 15 minutes , cooled to 40 c , then inoculated with the 1.0 10 probiotic bacteria streptococcus thermophilus , lactobacillus acidophilus and bifidobacterium bifidum and was then incubated for 4 - 8 hours at 42 c. after coagulation , the curd was tested for ph and stored refrigerated ( 4 - 6 c ) .", "a hot water extract of soluble dandelion root fiber was prepared according the methods described by abdel - salam et al .. dandelion roots were cut into small pieces and boiled for 10 minutes in distilled water .", "the mixture was filtered twice : first through cheesecloth ( 50% cotton/50% polyester ) , and then through filter paper ( whatman no.2 ) .", "the solutions obtained were preserved in sterile dark bottles in a cool environment ( 4 c ) until use .", "the high fiber probiotic mare 's milk was prepared by mixing the concentrated hot water extract of dandelion root with the fermented mare 's milk at 6% total solids .", "a sensory evaluation test of the fermented milk products was adopted from the national aeronautics and space administration ( n.a.s.a . ) .", "the evaluation was performed by a panel composed of the staff and students in the department of food science and human nutrition .", "the evaluation rated the appearance , color , odor , flavor , and overall impression of the product , and results were expressed as very good ( + + + ) , good ( + + ) , acceptable ( + ) , or unacceptable ( - ) .", "antioxidant capacity was measured with a 1,1-diphenyl-2-picrylhydrazyl ( dpph ) assay according to the method of brand - williams et al . with some modifications .", "a stock solution was prepared by dissolving 24 mg of dpph in 100 ml methanol .", "the working solution was obtained by mixing 10 ml stock solution with 45 ml methanol to obtain an absorbance of 1.1 0.02 units at 515 nm using a spectrophotometer .", "milk extracts ( 750 l ) were allowed to react with 1500 l of the dpph solution for 5 minutes in the dark .", "trolox is a water - soluble derivative of vitamin e it is an antioxidant , like vitamin e , and is used in biological or biochemical applications to reduce oxidative stress or damage .", "trolox equivalent antioxidant activity ( teac ) is a measurement of antioxidant strength based on trolox , measured in units called trolox equivalents ( te ) , trolox equivalency is used as a benchmark for the antioxidant capacity of such a mixture .", "thirty male adult swiss albino rats weighing 200 - 250 g 40 g were used .", "the animals were housed in standard cages and were assigned to a specific treatment diet .", "the composition of the basal diet was as follows : milk protein ( 12% ) , sucrose ( 5% ) , fat ( 10% ) , vitamin mixtures ( 1% ) , salt mixtures ( 4% ) , fiber ( 4% ) and starch ( 64 % ) .", "the positive control group was fed the basal diet and received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water .", "the third group was fed the basal diet , received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water , and was fed the high fiber fermented mare 's milk solution containing probiotics .", "the weights of the rats were measured at the beginning and end of the experiment .", "sera were harvested , labeled and stored frozen ( -20 c ) pending analysis . in sera the following parameters were analyzed : urea was determined according to the methods of tietz .", "glutathione - s - transferase ( gst ) activity was determined according to habig et.al .. lactate dehydrogenase ( ldh ) activity was determined according to bergmeyer .", "samples of brain , cerebellum , and kidneys were taken , and fixed in 10% neutral buffered formalin for 24 hours .", "paraffin sections 6 m thick were prepared and stained with hematoxylin and eosin ( h & e ) for the examination of tissue and cellular changes by light microscopy . mean and standard deviation of the data from each experimental group were calculated and", "briefly , fresh mare 's milk ( 11% total solid ) was heated at 85 c for 15 minutes , cooled to 40 c , then inoculated with the 1.0 10 probiotic bacteria streptococcus thermophilus , lactobacillus acidophilus and bifidobacterium bifidum and was then incubated for 4 - 8 hours at 42 c. after coagulation , the curd was tested for ph and stored refrigerated ( 4 - 6 c ) .", "a hot water extract of soluble dandelion root fiber was prepared according the methods described by abdel - salam et al .. dandelion roots were cut into small pieces and boiled for 10 minutes in distilled water .", "the mixture was filtered twice : first through cheesecloth ( 50% cotton/50% polyester ) , and then through filter paper ( whatman no.2 ) .", "the solutions obtained were preserved in sterile dark bottles in a cool environment ( 4 c ) until use .", "the high fiber probiotic mare 's milk was prepared by mixing the concentrated hot water extract of dandelion root with the fermented mare 's milk at 6% total solids .", "a sensory evaluation test of the fermented milk products was adopted from the national aeronautics and space administration ( n.a.s.a . ) .", "the evaluation was performed by a panel composed of the staff and students in the department of food science and human nutrition .", "the evaluation rated the appearance , color , odor , flavor , and overall impression of the product , and results were expressed as very good ( + + + ) , good ( + + ) , acceptable ( + ) , or unacceptable ( - ) .", "antioxidant capacity was measured with a 1,1-diphenyl-2-picrylhydrazyl ( dpph ) assay according to the method of brand - williams et al . with some modifications .", "a stock solution was prepared by dissolving 24 mg of dpph in 100 ml methanol .", "the working solution was obtained by mixing 10 ml stock solution with 45 ml methanol to obtain an absorbance of 1.1 0.02 units at 515 nm using a spectrophotometer .", "milk extracts ( 750 l ) were allowed to react with 1500 l of the dpph solution for 5 minutes in the dark .", "trolox is a water - soluble derivative of vitamin e it is an antioxidant , like vitamin e , and is used in biological or biochemical applications to reduce oxidative stress or damage .", "trolox equivalent antioxidant activity ( teac ) is a measurement of antioxidant strength based on trolox , measured in units called trolox equivalents ( te ) , trolox equivalency is used as a benchmark for the antioxidant capacity of such a mixture .", "thirty male adult swiss albino rats weighing 200 - 250 g 40 g were used .", "the animals were housed in standard cages and were assigned to a specific treatment diet .", "the composition of the basal diet was as follows : milk protein ( 12% ) , sucrose ( 5% ) , fat ( 10% ) , vitamin mixtures ( 1% ) , salt mixtures ( 4% ) , fiber ( 4% ) and starch ( 64 % ) .", "the positive control group was fed the basal diet and received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water .", "the third group was fed the basal diet , received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water , and was fed the high fiber fermented mare 's milk solution containing probiotics .", "the weights of the rats were measured at the beginning and end of the experiment .", "sera were harvested , labeled and stored frozen ( -20 c ) pending analysis . in sera", "the following parameters were analyzed : urea was determined according to the methods of tietz .", "glutathione - s - transferase ( gst ) activity was determined according to habig et.al .. lactate dehydrogenase ( ldh ) activity was determined according to bergmeyer .", "samples of brain , cerebellum , and kidneys were taken , and fixed in 10% neutral buffered formalin for 24 hours .", "paraffin sections 6 m thick were prepared and stained with hematoxylin and eosin ( h & e ) for the examination of tissue and cellular changes by light microscopy .", "mean and standard deviation of the data from each experimental group were calculated and according the method described by miller and miller .", "briefly , fresh mare 's milk ( 11% total solid ) was heated at 85 c for 15 minutes , cooled to 40 c , then inoculated with the 1.0 10 probiotic bacteria streptococcus thermophilus , lactobacillus acidophilus and bifidobacterium bifidum and was then incubated for 4 - 8 hours at 42 c. after coagulation , the curd was tested for ph and stored refrigerated ( 4 - 6 c ) .", "a hot water extract of soluble dandelion root fiber was prepared according the methods described by abdel - salam et al .. dandelion roots were cut into small pieces and boiled for 10 minutes in distilled water .", "the mixture was filtered twice : first through cheesecloth ( 50% cotton/50% polyester ) , and then through filter paper ( whatman no.2 ) .", "the solutions obtained were preserved in sterile dark bottles in a cool environment ( 4 c ) until use .", "the high fiber probiotic mare 's milk was prepared by mixing the concentrated hot water extract of dandelion root with the fermented mare 's milk at 6% total solids .", "a sensory evaluation test of the fermented milk products was adopted from the national aeronautics and space administration ( n.a.s.a . ) .", "the evaluation was performed by a panel composed of the staff and students in the department of food science and human nutrition .", "the evaluation rated the appearance , color , odor , flavor , and overall impression of the product , and results were expressed as very good ( + + + ) , good ( + + ) , acceptable ( + ) , or unacceptable ( - ) .", "antioxidant capacity was measured with a 1,1-diphenyl-2-picrylhydrazyl ( dpph ) assay according to the method of brand - williams et al . with some modifications .", "a stock solution was prepared by dissolving 24 mg of dpph in 100 ml methanol .", "the working solution was obtained by mixing 10 ml stock solution with 45 ml methanol to obtain an absorbance of 1.1 0.02 units at 515 nm using a spectrophotometer .", "milk extracts ( 750 l ) were allowed to react with 1500 l of the dpph solution for 5 minutes in the dark .", "trolox is a water - soluble derivative of vitamin e it is an antioxidant , like vitamin e , and is used in biological or biochemical applications to reduce oxidative stress or damage .", "trolox equivalent antioxidant activity ( teac ) is a measurement of antioxidant strength based on trolox , measured in units called trolox equivalents ( te ) , trolox equivalency is used as a benchmark for the antioxidant capacity of such a mixture .", "thirty male adult swiss albino rats weighing 200 - 250 g 40 g were used .", "the animals were housed in standard cages and were assigned to a specific treatment diet .", "the composition of the basal diet was as follows : milk protein ( 12% ) , sucrose ( 5% ) , fat ( 10% ) , vitamin mixtures ( 1% ) , salt mixtures ( 4% ) , fiber ( 4% ) and starch ( 64 % ) .", "the positive control group was fed the basal diet and received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water .", "the third group was fed the basal diet , received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water , and was fed the high fiber fermented mare 's milk solution containing probiotics .", "the weights of the rats were measured at the beginning and end of the experiment .", "in sera the following parameters were analyzed : urea was determined according to the methods of tietz .", "glutathione - s - transferase ( gst ) activity was determined according to habig et.al .. lactate dehydrogenase ( ldh ) activity was determined according to bergmeyer .", "samples of brain , cerebellum , and kidneys were taken , and fixed in 10% neutral buffered formalin for 24 hours .", "paraffin sections 6 m thick were prepared and stained with hematoxylin and eosin ( h & e ) for the examination of tissue and cellular changes by light microscopy .", "mean and standard deviation of the data from each experimental group were calculated and according the method described by miller and miller .", "the results of the sensory evaluation of the high fiber fermented mare 's milk containing probiotics showed that it 's appearance and color were rated ", "good , and it 's odor , flavor , and overall impression were rated ", "table 1 shows a comparison of the composition and antioxidant capacity of fresh , fermented , and high fiber fermented mare 's milk containing probiotics .", "although the composition of all 3 was similar , the antioxidant capacity was highest in the high fiber probiotic fermented mare 's milk compared to fresh or fermented mare 's milk . chemical compositions and antioxidant capacity of fresh , fermented , and high fiber fermented mare 's milk as shown in table 2 , the untreated ( negative control ) rats gained weight during this experiment , while there was a large decrease in the weight of the rats in the positive control group .", "the weight of the rats consuming the high fiber probiotic fermented mare 's milk treated group increased rather than decreased during the course of this experiment .", "body weight changes following different treatments treatment with mercuric chloride ( positive control)resulted in an increase in the rat 's kidney weight compared to the untreated negative control rats ( table 3 ) .", "less kidney weight increase was observed in rats treated with mercury and high fiber probiotic fermented mare 's milk , compared to the positive control animals .", "kidney weight following different treatments the values of several biochemical markers measured in serum are shown in table 4 .", "there was a decrease in the activity of glutathione - s- transferase ( gst ) in the sera of the positive control rats compared to the untreated negative control rats .", "however , gst activity increased in the rats receiving the high fiber probiotic fermented mare 's milk in addition to mercury .", "the activity of lactate dehydrogenase ( ldh ) increased in the group treated with mercuric chloride only ; however , ldh activity decreased in the group that received mercuric chloride and high fiber probiotic fermented mare 's milk .", "serum creatinine levels in the positive control group decreased compared to the negative control . however , treatment with high fiber probiotic fermented mare 's milk resulted in serum creatinine levels returning to normal values . there was a decrease in the concentration of serum triglycerides in the control positive group receiving mercury only , but the concentration of serum triglycerides was almost normal in the group that received mercury together with high fiber probiotic fermented mare 's milk .", "there was a significant increase in the level of uric acid and urea in serum of the positive control group compared to the negative controls .", "however , uric acid and urea were lower in the group that ingested high fiber probiotic fermented mare 's milk with mercury than in the negative control group .", "biochemical markers of kidney function measured in serum histopathological changes in the brain and kidney sections stained with h&e were evaluated by light microscopy .", "there was generalized and localized edema in the white matter of cerebrum and cerebellum in the positive control rats .", "the positive controls also showed congested blood vessels in both cerebrum and cerebellum which may occur prior to edema .", "these sections showed neuronal necrosis in cerebrum , necrosis and chromatolysis of purkinje cells in the cerebellum , and demyelination in the axons of the neuropil .", "histopathological changes in rat brain and kidney following different treatments the histopathological changes in kidneys of rats were also evaluated . in the positive control group , the renal epithelium suffered vacuolar and hydropic degeneration in most tubules .", "necrotic debris occasionally formed acidophil casts within the tubuli . in some cases , necrosis of the whole tubules was observed .", "a few tubules showed hyaline droplet degeneration in their epithelium , together with some hyaline casts within tubular lumen .", "perivascular and interstitial inflammatory cellular infiltrate with pyknosis of the nucleus was observed , as was severe cloudy swelling of renal tubular epithelium with moderate necrosis and hydropic degeneration but no significant histological alterations were shown in the glomeruli .", "administration of high fiber probiotic fermented mare 's milk partially protected the kidneys from the histopathological changes produced by mercury .", "the histopathological changes were severe with mercury alone but they were moderate to mild when mercury was administered together with high fiber probiotic fermented mare 's milk .", "rats treated with high fiber probiotic fermented mare 's milk showed no histopathological changes in the brain .", "mild changes were observed in kidneys as manifested by hydrophobic degeneration , necrosis , and hemorrhage , but no inflammatory cells or cloudy swelling were observed .", "these results indicate that high fiber probiotic fermented mare 's milk protected the rats against the adverse effects produced by mercury in brain and kidney .", "in recent years the popularity of complementary medicine , such as the use of probiotics and functional foods has increased .", "knowledge is accumulating that microbiota modulates gut physiology , immunological functions , and may produce other beneficial effects .", "this has led scientists to investigate the efficacy of probiotics , prebiotics and synbiotics in the treatment of diseases and toxicities .", "increasing the variety of functional foods is a challenge , and the promotion and formulation of functional foods and may enhance human health .", "it is well documented that hgcl2 exposure promotes hg accumulation in all tissues , with the highest levels in the liver , followed by the kidney and blood .", "the increased blood urea and creatinine are in agreement with the results obtained by rana et al . in male rats treated with mercury .", "elevated blood urea is known to be a function of or related to increased protein catabolism in mammals and/or the conversion of ammonia to urea as a result of increased synthesis of arginase enzyme involved in urea production . in agreement with this , the present results show that body weights decreased in animals treated with mercury indicating body wasting and increased protein catabolism .", "the increase in plasma urea and creatinine concentrations in the present experiment may be due to kidney dysfunction , as suggested by the increased weight of the kidneys .", "the decrease in the activity of gst in serum of rats receiving mercury is similar to the findings reported by el - missiry and shalaby , who found that the heavy metal cadmium decreased the activity of gst in brain and testes of male rats .", "el - demerdash also reported that cadmium caused a decrease in gst activity in fish .", "gst catalyses the reaction of alkylating agents with the thiol ( -sh ) group of glutathione , thereby neutralizing an electrophilic site and rendering the products more water soluble .", "the decline in serum gst activity may be due to the effect of mercury on glutathione .", "mercury has a high affinity to this molecule where a sulfhydryl , an amino and two carboxylic acid groups , as well as two peptide linkages , represents reactive sites for metals .", "reactions of metals with glutathione may lead to either the formation of complexes or the oxidation of glutathione .", "high fiber probiotics fermented mare 's milk was found to have a high antioxidant capacity ( table 1 ) .", "this high antioxidant capacity may contribute in ameliorating the oxidative stress effects and damage produced by mercury .", "our results were similar to those of hussain et al , who found that the nervous system is affected by mercuric chloride toxicity . in our study ,", "cellular and tissue changes were noticed in the positive control , including generalized and localized edema in the white mater of cerebrum and cerebellum .", "edema is a clear sign of compromised blood - brain barrier to mercuric chloride intoxication .", "the positive control also showed congested blood vessels in both cerebrum and cerebellum which could be prior to edema .", "the neuronal changes could be due to oxidative stress secondary to mercuric chloride toxicity . in the rats", "fed the high fiber fermented mare 's milk containing probiotic , the histological picture improved ; this could be due to antioxidative stress effect of the high fiber fermented mare 's milk containing probiotic .", "oxidative stress may induce peroxidation injury in the membrane of lipids and protein as well as dna fragmentation , which can result in disruption of nerve cell function and integrity .", "it is well known that brain is more sensitive tissue to oxidative damage because of its high concentration of unsaturated lipids and its high rate of oxidative metabolism .", "we conclude that high fiber fermented mare 's milk containing probiotic had a protective effect against brain and kidney alterations produced by mercury toxicity ." ]

[ "<S> background : since the advent of the industrial revolution in the late 19th century , we have all been unfortunately exposed to an increasingly toxic and polluted world . among the most dangerous of these pollutants is mercury , which is considered to be the most toxic non - radioactive heavy metal . </S>", "<S> fermented foods may help cleanse the body of heavy metals . </S>", "<S> fermentation breaks down the nutrients in foods by the action of beneficial microorganisms and creates natural chelators that are available to bind toxins and remove them from the body.aims:the current study was designed to determine the impact of feeding a high fiber probiotic fermented mare 's milk on the biological effects of mercury toxicity in rat model.methods and materials : the high fiber fermented mare 's milk containing probiotics was prepared and its sensory properties , chemical composition , and antioxidant activity were determined . </S>", "<S> a rat model of mercury toxicity was used . </S>", "<S> the effect of feeding the high fiber probiotic fermented mare 's milk to rats , along with mercury ingestion , was determined by the analysis of several biochemical markers in serum and histopathological examinations of brain and kidney.results:the high fiber fermented mare 's milk containing probiotics was found to be acceptable by all test panels and volunteers . </S>", "<S> mercury ingestion was found to cause biochemical and histopathological alterations in rat serum and tissues . </S>", "<S> the mercury - treated rats showed a decrease in body weight and an increase in kidney weight . </S>", "<S> sera of the mercury treated rats showed alterations in biochemical parameters , and histopathological changes in brain and kidney . </S>", "<S> however , the rats fed high fiber fermented mare`s milk along with mercury ingestion showed improved histopathology of kidney and brain , and there was restoration of the biochemical parameters in serum to almost normal values.conclusions:feeding high fiber fermented mare`s milk may reduce the toxic effects of mercury . </S>" ]

[ "Introduction", "Materials and Methods", "None", "Preparation of probiotic fermented mare's milk", "Preparation of dandelion root fiber", "Preparation of high fiber probiotic fermented mare's milk", "Sensory evaluation of the product", "Antioxidant capacity assay: of fresh, fermented and high fiber fermented mare's milk", "Animals and experimental feeding groups", "Biochemical Analysis", "Histopathological examination", "Statistical analysis", "Results", "Discussion", "Conclusion" ]

[ [ "mercury , a toxic divalent heavy metal without any biological function , causes several deleterious effects in adults and developing organisms .", "the three most commonly encountered forms of mercury are metal vapor ( hg ) , ionic compounds ( hg ) and organic mercury , principally methyl mercury ( hgch3 ) .", "organic mercury is the most deadly of the mercury compounds , probably due to its ability to penetrate cells . within the cell it can destroy various components selectively or in total by releasing lysosomes , damaging dna and rupturing the cell membrane .", "the toxicity of high levels of mercury contamination was brought to public attention in the 1950s as a result of mercury dumping in the minamata bay in japan 8 9 ] .", "the general population is primarily exposed to mercury via food , fish being a major source of methyl mercury exposure .", "mercury is incorporated into the food chain as methyl mercury , primarily through the action of bacteria and other microbes transforming elemental or inorganic forms .", "humans are also exposed to mercury via thimerosal ( a preservative added to vaccines and many other pharmaceuticals ) , and amalgam or mercury - based dental fillings . mercury vapor released from mercury dental fillings", "is absorbed very rapidly and thoroughly by the body , mainly through inhalation and swallowing .", "adverse health effects , particularly of a neurological nature , have resulted from low exposure levels , especially to the fetus in pregnant women .", "using young rats as a model of mercury toxicity , results from previous studies and from our group have demonstrated that exposure to hgcl2 ( 25 mg / kg ) for only five consecutive days causes cerebral and whole body weight losses , increases in kidney weight , mercury accumulation in tissues , inhibition of porphobilinogen synthase activity[1517 ] , alterations in renal and hepatic functions and glycemia .", "metal intoxication is usually treated with chelating agents , which are not specific and are often more toxic than the metal itself .", "because chelation therapy for heavy metals intoxication can also be toxic , researchers are looking for natural preventive and therapeutic agents for heavy metal toxicity .", "kumis , also known as koumiss , is an alcoholic beverage made from fermented mare 's milk .", "mare 's milk is usually not consumed raw , because it tends to have a strong laxative effect , although this effect is sometimes used medically . instead ,", "fermentation destroys lactose in milk , converting it into lactic acid , ethanol , and carbon dioxide .", "fermentation takes place in 3 - 8 hours and is carried out by a mixture of indigenous yeasts and lactic acid bacteria .", "currently , kumis is manufactured at an industrial level in some countries using pure cultures of yeasts and lactic acid bacteria .", "kumis is rich in ferments , trace elements , antibiotics , vitamins a , b1 , b2 , b12 , d , e , c , ethyl alcohol , lactic acid and carbonic acid .", "kumis drinking has a healing influence on the gastrointestinal tract , metabolism , cardiovascular and nervous systems , and kidneys ; it aids the development of immunity ; and has been used to treat weight loss and anemia . adding soluble fiber to the diet has also been shown to have positive effects on health .", "various studies have shown a reduction in colon cancer risk and enhanced health by consuming inulin and oligo fructose in ratios ranging from 2% to 10% .", "probiotics include viable lactic acid bacteria and other bacilli that are able to survive in the intestine .", "prebiotics include carbohydrates that are not digested in the upper part of the gastrointestinal tract , but selectively fermented by bacteria in the colon .", "this selective fermentation affects the composition of the intestinal microflora by stimulating bifidobacteria and lactobacilli , both in humans and in animals , where these bacteria have health promoting properties .", "we have combined these three health - promoting materials to produce a functional food composed of fermented mare 's milk plus a high level ( 6% ) of soluble fiber and probiotics .", "the current study was designed to determine if feeding the high fiber probiotic fermented mare 's milk could alleviate the toxic effects of mercury in rat model ." ], [ "starter cultures of streptococcus thermophilus , lactobacillus acidophilus , and bifidobacterium bifidum were obtained from hansen 's laboratory , copenhagen , denmark .", "dandelion root ( taraxacum officinale ) was purchased from haraz herbal market , cairo , egypt .", "fresh mare 's milk was obtained from lactating and healthy arabian mares in the experimental animal unit , college of agriculture and veterinary medicine , qassim university , saudi arabia .", "briefly , fresh mare 's milk ( 11% total solid ) was heated at 85 c for 15 minutes , cooled to 40 c , then inoculated with the 1.0 10 probiotic bacteria streptococcus thermophilus , lactobacillus acidophilus and bifidobacterium bifidum and was then incubated for 4 - 8 hours at 42 c. after coagulation , the curd was tested for ph and stored refrigerated ( 4 - 6 c ) .", "a hot water extract of soluble dandelion root fiber was prepared according the methods described by abdel - salam et al .. dandelion roots were cut into small pieces and boiled for 10 minutes in distilled water .", "the mixture was filtered twice : first through cheesecloth ( 50% cotton/50% polyester ) , and then through filter paper ( whatman no.2 ) .", "the solutions obtained were preserved in sterile dark bottles in a cool environment ( 4 c ) until use .", "the high fiber probiotic mare 's milk was prepared by mixing the concentrated hot water extract of dandelion root with the fermented mare 's milk at 6% total solids .", "a sensory evaluation test of the fermented milk products was adopted from the national aeronautics and space administration ( n.a.s.a . ) .", "the evaluation was performed by a panel composed of the staff and students in the department of food science and human nutrition .", "the evaluation rated the appearance , color , odor , flavor , and overall impression of the product , and results were expressed as very good ( + + + ) , good ( + + ) , acceptable ( + ) , or unacceptable ( - ) .", "antioxidant capacity was measured with a 1,1-diphenyl-2-picrylhydrazyl ( dpph ) assay according to the method of brand - williams et al . with some modifications .", "a stock solution was prepared by dissolving 24 mg of dpph in 100 ml methanol .", "the working solution was obtained by mixing 10 ml stock solution with 45 ml methanol to obtain an absorbance of 1.1 0.02 units at 515 nm using a spectrophotometer .", "milk extracts ( 750 l ) were allowed to react with 1500 l of the dpph solution for 5 minutes in the dark .", "trolox is a water - soluble derivative of vitamin e it is an antioxidant , like vitamin e , and is used in biological or biochemical applications to reduce oxidative stress or damage .", "trolox equivalent antioxidant activity ( teac ) is a measurement of antioxidant strength based on trolox , measured in units called trolox equivalents ( te ) , trolox equivalency is used as a benchmark for the antioxidant capacity of such a mixture .", "thirty male adult swiss albino rats weighing 200 - 250 g 40 g were used .", "the animals were housed in standard cages and were assigned to a specific treatment diet .", "the composition of the basal diet was as follows : milk protein ( 12% ) , sucrose ( 5% ) , fat ( 10% ) , vitamin mixtures ( 1% ) , salt mixtures ( 4% ) , fiber ( 4% ) and starch ( 64 % ) .", "the positive control group was fed the basal diet and received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water .", "the third group was fed the basal diet , received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water , and was fed the high fiber fermented mare 's milk solution containing probiotics .", "the weights of the rats were measured at the beginning and end of the experiment .", "sera were harvested , labeled and stored frozen ( -20 c ) pending analysis . in sera the following parameters were analyzed : urea was determined according to the methods of tietz .", "glutathione - s - transferase ( gst ) activity was determined according to habig et.al .. lactate dehydrogenase ( ldh ) activity was determined according to bergmeyer .", "samples of brain , cerebellum , and kidneys were taken , and fixed in 10% neutral buffered formalin for 24 hours .", "paraffin sections 6 m thick were prepared and stained with hematoxylin and eosin ( h & e ) for the examination of tissue and cellular changes by light microscopy . mean and standard deviation of the data from each experimental group were calculated and" ], [ "briefly , fresh mare 's milk ( 11% total solid ) was heated at 85 c for 15 minutes , cooled to 40 c , then inoculated with the 1.0 10 probiotic bacteria streptococcus thermophilus , lactobacillus acidophilus and bifidobacterium bifidum and was then incubated for 4 - 8 hours at 42 c. after coagulation , the curd was tested for ph and stored refrigerated ( 4 - 6 c ) .", "a hot water extract of soluble dandelion root fiber was prepared according the methods described by abdel - salam et al .. dandelion roots were cut into small pieces and boiled for 10 minutes in distilled water .", "the mixture was filtered twice : first through cheesecloth ( 50% cotton/50% polyester ) , and then through filter paper ( whatman no.2 ) .", "the solutions obtained were preserved in sterile dark bottles in a cool environment ( 4 c ) until use .", "the high fiber probiotic mare 's milk was prepared by mixing the concentrated hot water extract of dandelion root with the fermented mare 's milk at 6% total solids .", "a sensory evaluation test of the fermented milk products was adopted from the national aeronautics and space administration ( n.a.s.a . ) .", "the evaluation was performed by a panel composed of the staff and students in the department of food science and human nutrition .", "the evaluation rated the appearance , color , odor , flavor , and overall impression of the product , and results were expressed as very good ( + + + ) , good ( + + ) , acceptable ( + ) , or unacceptable ( - ) .", "antioxidant capacity was measured with a 1,1-diphenyl-2-picrylhydrazyl ( dpph ) assay according to the method of brand - williams et al . with some modifications .", "a stock solution was prepared by dissolving 24 mg of dpph in 100 ml methanol .", "the working solution was obtained by mixing 10 ml stock solution with 45 ml methanol to obtain an absorbance of 1.1 0.02 units at 515 nm using a spectrophotometer .", "milk extracts ( 750 l ) were allowed to react with 1500 l of the dpph solution for 5 minutes in the dark .", "trolox is a water - soluble derivative of vitamin e it is an antioxidant , like vitamin e , and is used in biological or biochemical applications to reduce oxidative stress or damage .", "trolox equivalent antioxidant activity ( teac ) is a measurement of antioxidant strength based on trolox , measured in units called trolox equivalents ( te ) , trolox equivalency is used as a benchmark for the antioxidant capacity of such a mixture .", "thirty male adult swiss albino rats weighing 200 - 250 g 40 g were used .", "the animals were housed in standard cages and were assigned to a specific treatment diet .", "the composition of the basal diet was as follows : milk protein ( 12% ) , sucrose ( 5% ) , fat ( 10% ) , vitamin mixtures ( 1% ) , salt mixtures ( 4% ) , fiber ( 4% ) and starch ( 64 % ) .", "the positive control group was fed the basal diet and received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water .", "the third group was fed the basal diet , received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water , and was fed the high fiber fermented mare 's milk solution containing probiotics .", "the weights of the rats were measured at the beginning and end of the experiment .", "sera were harvested , labeled and stored frozen ( -20 c ) pending analysis . in sera", "the following parameters were analyzed : urea was determined according to the methods of tietz .", "glutathione - s - transferase ( gst ) activity was determined according to habig et.al .. lactate dehydrogenase ( ldh ) activity was determined according to bergmeyer .", "samples of brain , cerebellum , and kidneys were taken , and fixed in 10% neutral buffered formalin for 24 hours .", "paraffin sections 6 m thick were prepared and stained with hematoxylin and eosin ( h & e ) for the examination of tissue and cellular changes by light microscopy .", "mean and standard deviation of the data from each experimental group were calculated and according the method described by miller and miller ." ], [ "briefly , fresh mare 's milk ( 11% total solid ) was heated at 85 c for 15 minutes , cooled to 40 c , then inoculated with the 1.0 10 probiotic bacteria streptococcus thermophilus , lactobacillus acidophilus and bifidobacterium bifidum and was then incubated for 4 - 8 hours at 42 c. after coagulation , the curd was tested for ph and stored refrigerated ( 4 - 6 c ) ." ], [ "a hot water extract of soluble dandelion root fiber was prepared according the methods described by abdel - salam et al .. dandelion roots were cut into small pieces and boiled for 10 minutes in distilled water .", "the mixture was filtered twice : first through cheesecloth ( 50% cotton/50% polyester ) , and then through filter paper ( whatman no.2 ) .", "the solutions obtained were preserved in sterile dark bottles in a cool environment ( 4 c ) until use ." ], [ "the high fiber probiotic mare 's milk was prepared by mixing the concentrated hot water extract of dandelion root with the fermented mare 's milk at 6% total solids ." ], [ "a sensory evaluation test of the fermented milk products was adopted from the national aeronautics and space administration ( n.a.s.a . ) .", "the evaluation was performed by a panel composed of the staff and students in the department of food science and human nutrition .", "the evaluation rated the appearance , color , odor , flavor , and overall impression of the product , and results were expressed as very good ( + + + ) , good ( + + ) , acceptable ( + ) , or unacceptable ( - ) ." ], [ "antioxidant capacity was measured with a 1,1-diphenyl-2-picrylhydrazyl ( dpph ) assay according to the method of brand - williams et al . with some modifications .", "a stock solution was prepared by dissolving 24 mg of dpph in 100 ml methanol .", "the working solution was obtained by mixing 10 ml stock solution with 45 ml methanol to obtain an absorbance of 1.1 0.02 units at 515 nm using a spectrophotometer .", "milk extracts ( 750 l ) were allowed to react with 1500 l of the dpph solution for 5 minutes in the dark .", "trolox is a water - soluble derivative of vitamin e it is an antioxidant , like vitamin e , and is used in biological or biochemical applications to reduce oxidative stress or damage .", "trolox equivalent antioxidant activity ( teac ) is a measurement of antioxidant strength based on trolox , measured in units called trolox equivalents ( te ) , trolox equivalency is used as a benchmark for the antioxidant capacity of such a mixture ." ], [ "thirty male adult swiss albino rats weighing 200 - 250 g 40 g were used .", "the animals were housed in standard cages and were assigned to a specific treatment diet .", "the composition of the basal diet was as follows : milk protein ( 12% ) , sucrose ( 5% ) , fat ( 10% ) , vitamin mixtures ( 1% ) , salt mixtures ( 4% ) , fiber ( 4% ) and starch ( 64 % ) .", "the positive control group was fed the basal diet and received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water .", "the third group was fed the basal diet , received 25 ppm mercury as mercuric chloride ( hgcl2 ) in drinking water , and was fed the high fiber fermented mare 's milk solution containing probiotics .", "the weights of the rats were measured at the beginning and end of the experiment ." ], [ "in sera the following parameters were analyzed : urea was determined according to the methods of tietz .", "glutathione - s - transferase ( gst ) activity was determined according to habig et.al .. lactate dehydrogenase ( ldh ) activity was determined according to bergmeyer ." ], [ "samples of brain , cerebellum , and kidneys were taken , and fixed in 10% neutral buffered formalin for 24 hours .", "paraffin sections 6 m thick were prepared and stained with hematoxylin and eosin ( h & e ) for the examination of tissue and cellular changes by light microscopy ." ], [ "mean and standard deviation of the data from each experimental group were calculated and according the method described by miller and miller ." ], [ "the results of the sensory evaluation of the high fiber fermented mare 's milk containing probiotics showed that it 's appearance and color were rated ", "good , and it 's odor , flavor , and overall impression were rated ", "table 1 shows a comparison of the composition and antioxidant capacity of fresh , fermented , and high fiber fermented mare 's milk containing probiotics .", "although the composition of all 3 was similar , the antioxidant capacity was highest in the high fiber probiotic fermented mare 's milk compared to fresh or fermented mare 's milk . chemical compositions and antioxidant capacity of fresh , fermented , and high fiber fermented mare 's milk as shown in table 2 , the untreated ( negative control ) rats gained weight during this experiment , while there was a large decrease in the weight of the rats in the positive control group .", "the weight of the rats consuming the high fiber probiotic fermented mare 's milk treated group increased rather than decreased during the course of this experiment .", "body weight changes following different treatments treatment with mercuric chloride ( positive control)resulted in an increase in the rat 's kidney weight compared to the untreated negative control rats ( table 3 ) .", "less kidney weight increase was observed in rats treated with mercury and high fiber probiotic fermented mare 's milk , compared to the positive control animals .", "kidney weight following different treatments the values of several biochemical markers measured in serum are shown in table 4 .", "there was a decrease in the activity of glutathione - s- transferase ( gst ) in the sera of the positive control rats compared to the untreated negative control rats .", "however , gst activity increased in the rats receiving the high fiber probiotic fermented mare 's milk in addition to mercury .", "the activity of lactate dehydrogenase ( ldh ) increased in the group treated with mercuric chloride only ; however , ldh activity decreased in the group that received mercuric chloride and high fiber probiotic fermented mare 's milk .", "serum creatinine levels in the positive control group decreased compared to the negative control . however , treatment with high fiber probiotic fermented mare 's milk resulted in serum creatinine levels returning to normal values . there was a decrease in the concentration of serum triglycerides in the control positive group receiving mercury only , but the concentration of serum triglycerides was almost normal in the group that received mercury together with high fiber probiotic fermented mare 's milk .", "there was a significant increase in the level of uric acid and urea in serum of the positive control group compared to the negative controls .", "however , uric acid and urea were lower in the group that ingested high fiber probiotic fermented mare 's milk with mercury than in the negative control group .", "biochemical markers of kidney function measured in serum histopathological changes in the brain and kidney sections stained with h&e were evaluated by light microscopy .", "there was generalized and localized edema in the white matter of cerebrum and cerebellum in the positive control rats .", "the positive controls also showed congested blood vessels in both cerebrum and cerebellum which may occur prior to edema .", "these sections showed neuronal necrosis in cerebrum , necrosis and chromatolysis of purkinje cells in the cerebellum , and demyelination in the axons of the neuropil .", "histopathological changes in rat brain and kidney following different treatments the histopathological changes in kidneys of rats were also evaluated . in the positive control group , the renal epithelium suffered vacuolar and hydropic degeneration in most tubules .", "necrotic debris occasionally formed acidophil casts within the tubuli . in some cases , necrosis of the whole tubules was observed .", "a few tubules showed hyaline droplet degeneration in their epithelium , together with some hyaline casts within tubular lumen .", "perivascular and interstitial inflammatory cellular infiltrate with pyknosis of the nucleus was observed , as was severe cloudy swelling of renal tubular epithelium with moderate necrosis and hydropic degeneration but no significant histological alterations were shown in the glomeruli .", "administration of high fiber probiotic fermented mare 's milk partially protected the kidneys from the histopathological changes produced by mercury .", "the histopathological changes were severe with mercury alone but they were moderate to mild when mercury was administered together with high fiber probiotic fermented mare 's milk .", "rats treated with high fiber probiotic fermented mare 's milk showed no histopathological changes in the brain .", "mild changes were observed in kidneys as manifested by hydrophobic degeneration , necrosis , and hemorrhage , but no inflammatory cells or cloudy swelling were observed .", "these results indicate that high fiber probiotic fermented mare 's milk protected the rats against the adverse effects produced by mercury in brain and kidney ." ], [ "in recent years the popularity of complementary medicine , such as the use of probiotics and functional foods has increased .", "knowledge is accumulating that microbiota modulates gut physiology , immunological functions , and may produce other beneficial effects .", "this has led scientists to investigate the efficacy of probiotics , prebiotics and synbiotics in the treatment of diseases and toxicities .", "increasing the variety of functional foods is a challenge , and the promotion and formulation of functional foods and may enhance human health .", "it is well documented that hgcl2 exposure promotes hg accumulation in all tissues , with the highest levels in the liver , followed by the kidney and blood .", "the increased blood urea and creatinine are in agreement with the results obtained by rana et al . in male rats treated with mercury .", "elevated blood urea is known to be a function of or related to increased protein catabolism in mammals and/or the conversion of ammonia to urea as a result of increased synthesis of arginase enzyme involved in urea production . in agreement with this , the present results show that body weights decreased in animals treated with mercury indicating body wasting and increased protein catabolism .", "the increase in plasma urea and creatinine concentrations in the present experiment may be due to kidney dysfunction , as suggested by the increased weight of the kidneys .", "the decrease in the activity of gst in serum of rats receiving mercury is similar to the findings reported by el - missiry and shalaby , who found that the heavy metal cadmium decreased the activity of gst in brain and testes of male rats .", "el - demerdash also reported that cadmium caused a decrease in gst activity in fish .", "gst catalyses the reaction of alkylating agents with the thiol ( -sh ) group of glutathione , thereby neutralizing an electrophilic site and rendering the products more water soluble .", "the decline in serum gst activity may be due to the effect of mercury on glutathione .", "mercury has a high affinity to this molecule where a sulfhydryl , an amino and two carboxylic acid groups , as well as two peptide linkages , represents reactive sites for metals .", "reactions of metals with glutathione may lead to either the formation of complexes or the oxidation of glutathione .", "high fiber probiotics fermented mare 's milk was found to have a high antioxidant capacity ( table 1 ) .", "this high antioxidant capacity may contribute in ameliorating the oxidative stress effects and damage produced by mercury .", "our results were similar to those of hussain et al , who found that the nervous system is affected by mercuric chloride toxicity . in our study ,", "cellular and tissue changes were noticed in the positive control , including generalized and localized edema in the white mater of cerebrum and cerebellum .", "edema is a clear sign of compromised blood - brain barrier to mercuric chloride intoxication .", "the positive control also showed congested blood vessels in both cerebrum and cerebellum which could be prior to edema .", "the neuronal changes could be due to oxidative stress secondary to mercuric chloride toxicity . in the rats", "fed the high fiber fermented mare 's milk containing probiotic , the histological picture improved ; this could be due to antioxidative stress effect of the high fiber fermented mare 's milk containing probiotic .", "oxidative stress may induce peroxidation injury in the membrane of lipids and protein as well as dna fragmentation , which can result in disruption of nerve cell function and integrity .", "it is well known that brain is more sensitive tissue to oxidative damage because of its high concentration of unsaturated lipids and its high rate of oxidative metabolism ." ], [ "we conclude that high fiber fermented mare 's milk containing probiotic had a protective effect against brain and kidney alterations produced by mercury toxicity ." ] ]