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any form of communication requires the existence of three obligatory components : a sender , a message , and a receiver . the process starts with the release of a message by a sender and ends with the understanding of the message by a receiver . this type of cycle has been developed with different degrees of complexity from prokaryote to higher eukaryotes optimizing fitness and adaptation for individual members and populations . the nature and mode of action of communication is as diverse as the response to the information it carries . inter- and intraspecies communication has been widely studied analyzing the exchange of information between fungi and bacteria or fungi and plant cells [ 1 , 2 ] . this review will focus predominantly on intraspecies fungal communication addressing key biological functions including mating , growth , morphological switching , or the regulation of virulence factor expression ( figure 1 ) . we will show that in the fungal kingdom most of these mechanisms are controlled by a variety of messengers including small peptides , alcohols , lipids , and volatile compounds . pheromones have been known to act as an informative molecule since 1959 and were reported to be involved in the sexual cycle of fungi in 1974 . in the fungal kingdom , they are involved in the reconnaissance of compatible sexual partner to promote plasmogamy and karyogamy between two opposite mating types followed by meiosis . taking the example of the extensively described sexual cycle of saccharomyces cerevisiae , pheromones are diffusible peptides called a - factor ( 12 aa ) when produced by a cells , and -factor ( 13 aa ) when produced by cells . each mating type responds to the opposite factor , and is able to produce only one of the two peptide pheromones depending on the alleles present at the mat locus . indeed , mata or mat controls the expression of a and specific genes , respectively , such as genes encoding the prepro - factor and the pheromone receptor ( for a comprehensive description of the mat locus , see review ) . in the example of cells , mf1 encodes the pheromone precursor , prepro--factor , which undergoes several proteolytic reactions in the classical secretory pathway before releasing the mature pheromone . contrary to the -factor , the abc transporter ste6p is required to secrete a - factor . these peptides are subsequently recognized by a 7 transmembrane receptor present on the surface of cells : ste2p on a cells binds the -factor and ste3p on cells binds the a - factor . ste2p and ste3p are g - protein coupled receptors ( gpcr ) and the binding of pheromone induces the separation of the associated heterotrimeric g - protein into a monomeric subunit gtpase ( gpa1p ) and a dimer ( ste4p ste18p ) . this mechanism results in the recruitment of ste5p by ste4p to the membrane , which activates a protein kinase cascade ultimately resulting in the phosphorylation of the map kinases fus3p and kss1p . once phosphorylated fus3p migrates to the nucleus were it activates the transcriptional factor ste12p leading to the expression of pheromone responsive genes . phenotypically , the morphological response of cells to opposite mating pheromone is the development of a shmoo , that is , directional cell growth in response to the pheromone gradient . as each opposite cell develops a shmoo , the plasmogamy between the two cells occurs when the shmoos establish contact , starting the first step of the sexual cycle . contrary to s. cerevisiae , where karyogamy is followed by meiosis to end the sexual cycle , candida albicans has not yet been described to undergo meiosis . this yeast displays only an imperfect sexual cycle , where karyogamy results in the formation of tetraploid cells that restore natural diploidy via random loss of chromosomes . this process occurs only after c. albicans has undergone a so - called white - to - opaque switch ( see review ) , where opaque cells are the sole mating competent form of this yeast . the opaque cells morphologically respond to pheromone by producing a shmoo , like in s. cerevisiae , but the mating incompetent form , white cells , is also sensitive to pheromone . indeed , c. albicans-factor but also a - factor promotes the formation of biofilm by white cells via enhancing their adhesiveness . a process which uses the same receptor ( ste2p or ste3p ) and transduction pathway as the response of opaque cells to pheromone . the formation of fungal biomass by white cells facilitates the establishment of a pheromone gradient in a population of individual cells and assists the mating process of opaque cells . this process involves another molecule , farnesol , as the production of this molecule under aerobic conditions induces the death of the mating competent opaque cells . the mechanism of pheromone communication has a broad significance in diverse fungi including ascomycetes like histoplasma capsulatum or aspergillus fumigatus [ 15 , 16 ] , to basidiomycetes such as cryptococcus neoformans and ustilago maydis , which possess a tetrapolar mating system . pheromone communication appears to be a critical mechanism for fungi as it supports the exchange of genetic material between cells and by extension the ability of the organism to evolve in response to their environment . quorum sensing is a mechanism of communication based on the accumulation of a messenger molecule in the medium of culture . as the production of messenger molecules increases with cell number , this system reflects population size . initially discovered in bacteria , quorum sensing in fungi became relevant for the control of virulence factor expression in c. albicans . in 1979 , hazen and cutler showed that the supernatant from a 48 h culture of c. albicans prevents the yeast to hyphae switch of a fresh culture . the quorum sensing molecule ( qsm ) responsible for this effect has since been identified as an acyclic sesquiterpene alcohol called farnesol . c. albicans produces farnesol at a rate of 0.120.133 mg / g of cells dry weight from an intermediate of the mevalonate pathway ( sterol biosynthesis ) , farnesyl pyrophosphate . at concentrations of 10250 m farnesol inhibits the formation of hyphae when induced with proline , n - acetylglucosamine , and serum , but does not suppress further elongation of preexisting hyphae . farnesol - dependent quorum sensing involves the histidine kinase chk1p and the ras1-cyr1 pathway but the receptor for farnesol remains to be identified . farnesol regulates the expression of several genes and induces tup1 , a transcriptional cofactor repressing filamentation , while repressing cph1 and hst7 expression , which are both activators of the morphological switch . the oxidized form of farnesol , farnesoic acid , has also been reported to inhibit hyphal growth by acting via pho81 . however , morphological inhibition is stronger with farnesol , although farnesoic acid is less toxic at high concentration , it displays only 3% of farnesols qsm activity . while the function of farnesol as a cell density regulator remains to be established , farnesol has been described to inhibit c. albicans biofilm formation due to its repressing function on the morphological switch . additionally it has been shown to increase resistance to oxidative stress by suppressing the ras1-camp pathway . notably , farnesol also acts as an interspecies qsm that impacts on growth of other candida species including candida tropicalis or candida parapsilosis as well as s. cerevisiae or the mould aspergillus nidulans and a. fumigatus [ 3538 ] . in the case of a. fumigatus , farnesol has been described to alter the localization of afrho1p and afrho3p , proteins involved in the cell wall integrity ( cwi ) pathway and cytoskeleton regulation . this phenotype is explained by the fact that farnesyl derivatives interfere with prenylated proteins such as the two rho gtpases [ 39 , 40 ] . the cwi pathway implies the activation of afpkca by afrho1p , which leads on to the map kinase cascade and subsequent afmpka phosphorylation . showed that in the presence of only 40 m farnesol , phosphorylation of afmpka in response to congo red was completely inhibited . in s. cerevisiae , farnesol prevents growth via a different mechanism , which involves an increase of mitochondrial reactive oxygen species ( ros ) . the latter observation was also reported for a. nidulans where ros augmentation induced cellular apoptosis but had no role on hyphal morphogenesis . two proteins have been identified in this response ; the g subunit fada of a heterotrimeric g protein , where hyperactivation leads to a strong increase in farnesol sensitivity , and the kinase pkca . mutation of pkca increases resistance to farnesol while overexpression results in a higher rate of cell death in response to the qsm . finally , farnesol has also been described to induce apoptosis of cancerous cells in vivo ( see review ) , as well as increasing antibiotic sensitivity of staphylococcus aureus . thus , farnesol appears to function as both an intraspecies and inter - species communication molecule . tyrosol , an aromatic alcohol , is produced from aromatic amino acids undergoing the processes of transamination ( aro8 , aro9 ) , decarboxylation ( aro10 ) , and reduction by alcohol dehydrogenase ( adh ) . this synthesis pathway is strongly dependant on growth conditions including environmental ph , availability of aromatic amino acids , oxygen levels , or presence of ammonium salts . similar to farnesol , fungal responses to tyrosol include the induction of germ tubes in planktonically growing cells and during the early stages of biofilm formation , as well as a reduction in the lag phase of c. albicans growth following dilution of a highly concentrated culture to fresh minimal medium [ 45 , 46 ] . the latter phenotype occurs predominantly at low concentrations of cells ( 5 10 cell / ml ) by promoting the expression of genes involved in dna replication , chromosome segregation , and cell cycle processes . aromatic alcohol synthesis is not exclusive to c. albicans but can also be found in s. cerevisiae , which produces phenylethanol and tryptophol via a similar pathway involving aros genes . both molecules stimulate diploid pseudohyphal growth at concentrations above 20 m on low - ammonium agar ( slad ) by inducing the pka pathway resulting in flo11 induction . recently , response to phenylethanol and tryptophol has been proposed to involve two main transcriptional regulators : cat8p and mig1p . however , the messenger molecule is not yet characterized [ 50 , 51 ] . at low density , h. capsulatum cells have low amounts of -(1,3)-glucan in their cell walls and addition of supernatant from a stationary phase culture induces -(1,3)-glucan incorporation into the cell wall . similarly addition of c. ulmi spent medium to a fresh culture promotes a switch from hyphae to yeast growth . oxylipins are oxygenated fatty acids used as cell messengers and have been intensely studied in plants and mammalian cells ( see review ) . a. nidulans was reported to produce one of the first oxylipins called psi factor ( precocious sexual inducer ) , which is composed of a series of different oxylipin derivates from oleic acid ( c18:1 ) , linoleic acid ( c18:2 ) , and linolenic acid ( c18:3 ) called psia , psib , and psic , respectively . the genes involved in the production of psi factor are called ppos ( for psi - producing oxygenases ) . in the case of a. nidulans , ppoa is involved in psib synthesis and ppob and ppoc contribute to psib biogenesis [ 54 , 55 ] . inactivation of these genes results in perturbations not only of psi factor production but also mycotoxins production , as well as in the ratio between the development of sexual and asexual ascospores [ 54 , 55 ] . the latter phenotype is due to the fact that oxylipins control the expression of nsdd and brla , transcription factors required for meiotic and mitotic sporulation , respectively [ 54 , 56 ] . overexpression or addition of psib or psic to the culture medium stimulates sexual sporulation and represses asexual spore development while an opposite effect is observed for psia and psib . secondary metabolite mycotoxin sterigmatocystin ( st ) and antibiotic penicillin ( pn ) production are also dependent on oxylipin . indeed , inactivation of the ppos genes results in the inability to secrete st as a result of downregulation of st biosynthesis genes including aflr and stcu , and the overproduction of pn through induction of the gene involved in its biosynthesis : ipna . interestingly , the exact opposite observations are found when fada , an subunit of a gpcr , is constitutively activated due to a g42r mutation , a reaction which is mediated by the pkaa enzyme . this result , in addition to the fact that oxylipins in mammalian cells are sensed via gpcr complexes led to the hypothesis that fungi use the same system to detect oxylipin , ultimately activating the camp / pka pathway . the ppo and gpcr encoding genes have been identified in the genomes of several filamentous fungi predicting a broader role of oxylipin in fungal biology . in fact , inactivation of the ppo genes in aspergillus flavus and fusarium sporotrichioides has already been shown to perturb mycotoxin and spore production [ 61 , 62 ] . finally , using confocal laser scanning microscopy , oxylipins have been described to accumulate in the capsule of c. neoformans before being released into the external medium under the form of hydrophobic droplets that are transported via tubular protuberances . recently , nigam et al . have described a 3(r)-hydroxy - tetradecanoic acid , a derivate of linoleic acid as a novel qsm of c. albicans . previously known to be produced during the sexual phase of dipodascopsis uninucleata , this oxylipin increases filamentation in c. albicans in response to n - acetylglucosamine at a concentration of 1 m . although the receptor of 3(r)-hydroxy - tetradecanoic is not known , this qsm induces hwp1 and cap1 mrna transcripts . interestingly , 3(r)-hydroxy - tetradecanoic is metabolized inside cells to generate two new compounds that could also act as messenger molecules . another family of oxylipin are the eicosanoids , which are molecules containing a 20 carbon backbone . pge2 is produced by c. albicans from exogenous arachidonic acid via enzymes not yet characterized . pge2 is also produced by humans , similar to other prostaglandins ( pg ) , and it appears that fungal pge2 can enter in competition with human pg impacting on the host 's immune response . indeed , pge2 is known to balance th1/th2 differentiation as this molecule decreases the expression of il-12r and inactivates th1 differentiation while activating the th2-related immune responses . in addition to releasing mediators into solution or onto solid growth media , organisms also exchange information via the liberation of messenger molecules into air . for example , insects have been thoroughly studied for their secretion of pheromones into air to attract mating partners . in the fungal kingdom , as early as in the 1970s , volatile compounds from fungi and others organism have been described to impact on fungal growth ( review [ 71 , 72 ] ) . more recently , palkova et al . observed that s. cerevisiae colonies grown on complex agar form a turbid path in the vicinity of another colony . subsequently , they discovered that this reaction is induced by the small volatile messenger molecule , later described as ammonia , which also required amino - acid uptake for its production . indeed , inactivation of shr3 , which is responsible for the correct localization of several amino - acid permeases , disrupts the turbid path between colonies . trichoderma species have been described to produce the volatile molecule 6-pentyl--pyrone , a secondary metabolite with antifungal activity . however , more recently the induction of conidiation in trichoderma species , which is known to be regulated by a circadian cycle , has also been shown to be controlled via a volatile agent . solid - phase microextraction linked with gas chromatography and mass spectrometry has allowed the identification of the chemical profiles of volatile molecules produced from nonconidiated colonies grown in darkness and conidiating colonies grown in light . comparison of the two profiles identified production of the 8-carbon compounds molecules 1-octen-3-ol , 3-octanol and 3-octanone specifically during conidiation . each of these three compounds induces conidiation in colonies placed in the dark . this regulation could involve a calcium - dependant signaling pathway as it has been shown that high concentration of calcium can induce conidiation of penicillium isariaeform in darkness . 1-octen-3-ol is the most efficient molecule being active at concentrations of only 0.1 m . interestingly , concentrations above 500 m of any of the three compounds suppress conidiation and growth of trichoderma species . these observations are consistent with a previously described putative fungistatic and fungicidal role of the molecules [ 77 , 78 ] . notably , the same compounds have previously been shown to function as insect attractants improving fungal spore dispersal [ 77 , 78 ] , and inter - species communication has already been described between epichlo species and the female botanophila flies . fungi are not only responsive to volatile compounds that they produce but also , in at least one example , to a gas liberated during respiration : carbon dioxide ( co2 ) . as early as 1961 , vakil et al . demonstrated that the optimum co2 concentration for the germination of aspergillus niger conidiospores is reached not under normal atmospheric concentrations of co2 ( 0.033% ) but at 0.5% . since then several additional phenotypes in fungi have been attributed to changes in the concentration of environmental co2 including the sporulation of alternaria crassa and alternaria cassiae , conidiation of neurospora crassa , or capsule formation and mating in c. neoformans [ 83 , 84 ] . recently , significant advances have been made in the understanding of co2 sensing in fungi . it was already known that the yeast to hyphae morphological switch in c. albicans is triggered by elevated environmental co2 . furthermore , the frequency of white - to - opaque switching can be increased 16-fold in hypercapnic conditions as opposed to atmospheric co2 . two different studies show that both phenotypes involve the c. albicans adenylyl cyclase cyr1 , first fungal co2 sensor . this enzyme generates the secondary messenger camp , which in the context of the camp / pka signaling pathway has a fundamental impact on c. albicans morphogenesis [ 86 , 87 ] . co2 activation of cyr1p depends on the concentration of bicarbonate , the hydrated form of co2 . co2 hydration occurs naturally at a very low rate , but is enhanced by the enzyme carbonic anhydrase . inactivation of cyr1 results in a loss of filamentation and white to opaque switching frequency in response to hypercapnia [ 86 , 87 ] . have now demonstrated that lysine 1373 of the cyr1 catalytic domain is essential for co2 sensing in c. albicans as mutation of this amino acid leads to a loss of filamentation in response to co2 but not to serum , another morphological cue . these data show that in fungi environmental co2 is sensed via the adenylyl cyclase , which transduces the message via the regulation of the camp / pka pathway . hall et al . also showed that hypercapnia is not a condition solely encountered inside the host but can also establish itself as a population event , such as the center of a colony grown under normal atmospheric conditions . another study demonstrated that c. albicans produces co2 via the conversion of arginine to urea . urea is ultimately degraded to generate co2 by the enzyme urea amidolyase ( dur1,2 ) . inactivation of the latter interferes with c. albicans filamentation in response to arginine and urea compared to the control strain but not to elevated co2 . control of the white to opaque switch - frequency in c. albicans by environmental co2 also involves the gtpase ras1 and the transcriptional factor wor1 . indeed , ras1 , cyr1 , and wor1 are critical for increasing the white to opaque switch in response to concentrations of co2 at 1% , but ras1 and cyr1 become optional for the induction at higher concentrations ( 20% ) . however , wor1 remains essential for the switch even at high co2 . these results imply that an alternative co2 sensing pathway is involved in the regulation of wor1 at high co2 in c. albicans . however , it is important to note that under this condition a significant increase of the internal ph may occur which could also be a component of this alternative co2 sensing pathway . acetaldehyde , an organic compound involved in several cellular pathways , has been shown to impact on cell - density - dependent glycolytic oscillations of s. cerevisiae . in 1964 , chance et al . described that the level of nadh in yeast oscillated when starved cells endure a pulse of glucose after a switch to anaerobic conditions . since then other metabolites have been described to oscillate in yeast including glucose-6-phosphate , fructose-6-phosphate , fructose-1,6-biphosphate , amp , adp , and atp ( for a comprehensive review see ) . interestingly , at a population level these oscillations are not chaotic but appear to be subject to synchronization . the most striking observation was achieved when mixing two populations with a 180 out - of - phase oscillation showing that within minutes the oscillation of the new population were synchronized . acetaldehyde was identified as the active molecule in the synchronization of these oscillations , as the use of acetaldehyde traps induced the oscillation to be damped and addition of acetaldehyde to the medium produced a phase shift in the oscillation . no specific target for acetaldehyde is known ; however , this compound has an important impact on the nad / nadh balance . acetaldehyde is also a volatile molecule , a property used to study inter and intraspecies communication in a synthetic ecosystem . by engineering sender cells that liberate volatile acetaldehyde and receiver cells that contain a construct under an acetaldehyde - inducible promoter , it was possible to study volatile cell communication in a controlled environment . using mammalian ( cho - k1 ) , bacterial ( escherichia coli ) , yeast ( s. cerevisiae ) , or plant ( lepidium sativum ) cells , all combination of sender / receiver for inter and intraspecies resulted in a positive communication between cells . clearly such models could bring new insight in the understanding of communication in complex living systems . . many essential compounds of the communication process have been identified , the sender ( in our case fungi ) , the message ( protein , alcohol , lipid , gas ) , and the receiver ( bacteria , fungi , plant , mammalian ) . however , in most cases the actual molecular mechanism of such communication remains for most parts unknown . the determination of these pathways is of substantial significance as molecular messengers control the expression of fungal virulence determinants including the yeast - to - hyphae switch and biofilm formation in c. albicans , capsule formation in c. neoformans , or mycotoxin synthesis in a. nidulans , but also the propagation of these organisms via the regulation of their sexual and asexual cycle . a better knowledge of fungal communication is now required to permit the development of innovative strategies aiming to control disease or toxin production of these organisms . fungi have already taken advantage of the different communication processes and particularly inter - species communication to gain competitive advantages over other species . good examples are the production of pollinators attracting insects to give phytopathogenic fungi a better chance for dispersal of their spores . additionally , synthesis of pge2 by the human pathogens c. albicans and c. neoformans modify the host immune response and may enhance fungal survival . such mechanisms reveal the close coevolution of fungi with their environmental partner and give insights into multispecies communication . the remarkable versatility of communication in the fungal kingdom also raises the question how these organisms integrate intra- and inter - species messages that can have opposing effects . as the molecular mechanisms of fungal communication unravel further , they will promote our understanding of the highly attractive but challenging topic of the fungal communicome .

we will discuss fungal communication in the context of fundamental biological functions including mating , growth , morphogenesis , and the regulation of fungal virulence determinants . we will address intraspecies but also interkingdom signaling by systematically discussing the sender of the message , the molecular message , and receiver . analyzing communication shows the close coevolution of fungi with organisms present in their environment giving insights into multispecies communication . a better understanding of the molecular mechanisms underlying microbial communication will promote our understanding of the fungal communicome .

1. Introduction 2. Peptides: Pheromones 3. Alcohols: Quorum Sensing 4. Lipids: Oxylipins 5. Volatile Compounds and Gas 6. Small Molecule: Acetaldehyde 7. Concluding Remarks and Outlooks

any form of communication requires the existence of three obligatory components : a sender , a message , and a receiver . the process starts with the release of a message by a sender and ends with the understanding of the message by a receiver . this type of cycle has been developed with different degrees of complexity from prokaryote to higher eukaryotes optimizing fitness and adaptation for individual members and populations . inter- and intraspecies communication has been widely studied analyzing the exchange of information between fungi and bacteria or fungi and plant cells [ 1 , 2 ] . this review will focus predominantly on intraspecies fungal communication addressing key biological functions including mating , growth , morphological switching , or the regulation of virulence factor expression ( figure 1 ) . we will show that in the fungal kingdom most of these mechanisms are controlled by a variety of messengers including small peptides , alcohols , lipids , and volatile compounds . pheromones have been known to act as an informative molecule since 1959 and were reported to be involved in the sexual cycle of fungi in 1974 . in the fungal kingdom , they are involved in the reconnaissance of compatible sexual partner to promote plasmogamy and karyogamy between two opposite mating types followed by meiosis . taking the example of the extensively described sexual cycle of saccharomyces cerevisiae , pheromones are diffusible peptides called a - factor ( 12 aa ) when produced by a cells , and -factor ( 13 aa ) when produced by cells . each mating type responds to the opposite factor , and is able to produce only one of the two peptide pheromones depending on the alleles present at the mat locus . indeed , mata or mat controls the expression of a and specific genes , respectively , such as genes encoding the prepro - factor and the pheromone receptor ( for a comprehensive description of the mat locus , see review ) . in the example of cells , mf1 encodes the pheromone precursor , prepro--factor , which undergoes several proteolytic reactions in the classical secretory pathway before releasing the mature pheromone . contrary to the -factor , the abc transporter ste6p is required to secrete a - factor . ste2p and ste3p are g - protein coupled receptors ( gpcr ) and the binding of pheromone induces the separation of the associated heterotrimeric g - protein into a monomeric subunit gtpase ( gpa1p ) and a dimer ( ste4p ste18p ) . this mechanism results in the recruitment of ste5p by ste4p to the membrane , which activates a protein kinase cascade ultimately resulting in the phosphorylation of the map kinases fus3p and kss1p . phenotypically , the morphological response of cells to opposite mating pheromone is the development of a shmoo , that is , directional cell growth in response to the pheromone gradient . as each opposite cell develops a shmoo , the plasmogamy between the two cells occurs when the shmoos establish contact , starting the first step of the sexual cycle . this yeast displays only an imperfect sexual cycle , where karyogamy results in the formation of tetraploid cells that restore natural diploidy via random loss of chromosomes . the opaque cells morphologically respond to pheromone by producing a shmoo , like in s. cerevisiae , but the mating incompetent form , white cells , is also sensitive to pheromone . indeed , c. albicans-factor but also a - factor promotes the formation of biofilm by white cells via enhancing their adhesiveness . the formation of fungal biomass by white cells facilitates the establishment of a pheromone gradient in a population of individual cells and assists the mating process of opaque cells . this process involves another molecule , farnesol , as the production of this molecule under aerobic conditions induces the death of the mating competent opaque cells . pheromone communication appears to be a critical mechanism for fungi as it supports the exchange of genetic material between cells and by extension the ability of the organism to evolve in response to their environment . quorum sensing is a mechanism of communication based on the accumulation of a messenger molecule in the medium of culture . initially discovered in bacteria , quorum sensing in fungi became relevant for the control of virulence factor expression in c. albicans . c. albicans produces farnesol at a rate of 0.120.133 mg / g of cells dry weight from an intermediate of the mevalonate pathway ( sterol biosynthesis ) , farnesyl pyrophosphate . at concentrations of 10250 m farnesol inhibits the formation of hyphae when induced with proline , n - acetylglucosamine , and serum , but does not suppress further elongation of preexisting hyphae . farnesol - dependent quorum sensing involves the histidine kinase chk1p and the ras1-cyr1 pathway but the receptor for farnesol remains to be identified . farnesol regulates the expression of several genes and induces tup1 , a transcriptional cofactor repressing filamentation , while repressing cph1 and hst7 expression , which are both activators of the morphological switch . however , morphological inhibition is stronger with farnesol , although farnesoic acid is less toxic at high concentration , it displays only 3% of farnesols qsm activity . in the case of a. fumigatus , farnesol has been described to alter the localization of afrho1p and afrho3p , proteins involved in the cell wall integrity ( cwi ) pathway and cytoskeleton regulation . this phenotype is explained by the fact that farnesyl derivatives interfere with prenylated proteins such as the two rho gtpases [ 39 , 40 ] . the cwi pathway implies the activation of afpkca by afrho1p , which leads on to the map kinase cascade and subsequent afmpka phosphorylation . showed that in the presence of only 40 m farnesol , phosphorylation of afmpka in response to congo red was completely inhibited . two proteins have been identified in this response ; the g subunit fada of a heterotrimeric g protein , where hyperactivation leads to a strong increase in farnesol sensitivity , and the kinase pkca . mutation of pkca increases resistance to farnesol while overexpression results in a higher rate of cell death in response to the qsm . finally , farnesol has also been described to induce apoptosis of cancerous cells in vivo ( see review ) , as well as increasing antibiotic sensitivity of staphylococcus aureus . tyrosol , an aromatic alcohol , is produced from aromatic amino acids undergoing the processes of transamination ( aro8 , aro9 ) , decarboxylation ( aro10 ) , and reduction by alcohol dehydrogenase ( adh ) . similar to farnesol , fungal responses to tyrosol include the induction of germ tubes in planktonically growing cells and during the early stages of biofilm formation , as well as a reduction in the lag phase of c. albicans growth following dilution of a highly concentrated culture to fresh minimal medium [ 45 , 46 ] . the latter phenotype occurs predominantly at low concentrations of cells ( 5 10 cell / ml ) by promoting the expression of genes involved in dna replication , chromosome segregation , and cell cycle processes . however , the messenger molecule is not yet characterized [ 50 , 51 ] . at low density , h. capsulatum cells have low amounts of -(1,3)-glucan in their cell walls and addition of supernatant from a stationary phase culture induces -(1,3)-glucan incorporation into the cell wall . similarly addition of c. ulmi spent medium to a fresh culture promotes a switch from hyphae to yeast growth . oxylipins are oxygenated fatty acids used as cell messengers and have been intensely studied in plants and mammalian cells ( see review ) . a. nidulans was reported to produce one of the first oxylipins called psi factor ( precocious sexual inducer ) , which is composed of a series of different oxylipin derivates from oleic acid ( c18:1 ) , linoleic acid ( c18:2 ) , and linolenic acid ( c18:3 ) called psia , psib , and psic , respectively . the genes involved in the production of psi factor are called ppos ( for psi - producing oxygenases ) . in the case of a. nidulans , ppoa is involved in psib synthesis and ppob and ppoc contribute to psib biogenesis [ 54 , 55 ] . inactivation of these genes results in perturbations not only of psi factor production but also mycotoxins production , as well as in the ratio between the development of sexual and asexual ascospores [ 54 , 55 ] . the latter phenotype is due to the fact that oxylipins control the expression of nsdd and brla , transcription factors required for meiotic and mitotic sporulation , respectively [ 54 , 56 ] . indeed , inactivation of the ppos genes results in the inability to secrete st as a result of downregulation of st biosynthesis genes including aflr and stcu , and the overproduction of pn through induction of the gene involved in its biosynthesis : ipna . interestingly , the exact opposite observations are found when fada , an subunit of a gpcr , is constitutively activated due to a g42r mutation , a reaction which is mediated by the pkaa enzyme . the ppo and gpcr encoding genes have been identified in the genomes of several filamentous fungi predicting a broader role of oxylipin in fungal biology . in fact , inactivation of the ppo genes in aspergillus flavus and fusarium sporotrichioides has already been shown to perturb mycotoxin and spore production [ 61 , 62 ] . finally , using confocal laser scanning microscopy , oxylipins have been described to accumulate in the capsule of c. neoformans before being released into the external medium under the form of hydrophobic droplets that are transported via tubular protuberances . interestingly , 3(r)-hydroxy - tetradecanoic is metabolized inside cells to generate two new compounds that could also act as messenger molecules . pge2 is produced by c. albicans from exogenous arachidonic acid via enzymes not yet characterized . pge2 is also produced by humans , similar to other prostaglandins ( pg ) , and it appears that fungal pge2 can enter in competition with human pg impacting on the host 's immune response . indeed , pge2 is known to balance th1/th2 differentiation as this molecule decreases the expression of il-12r and inactivates th1 differentiation while activating the th2-related immune responses . for example , insects have been thoroughly studied for their secretion of pheromones into air to attract mating partners . in the fungal kingdom , as early as in the 1970s , volatile compounds from fungi and others organism have been described to impact on fungal growth ( review [ 71 , 72 ] ) . observed that s. cerevisiae colonies grown on complex agar form a turbid path in the vicinity of another colony . subsequently , they discovered that this reaction is induced by the small volatile messenger molecule , later described as ammonia , which also required amino - acid uptake for its production . indeed , inactivation of shr3 , which is responsible for the correct localization of several amino - acid permeases , disrupts the turbid path between colonies . trichoderma species have been described to produce the volatile molecule 6-pentyl--pyrone , a secondary metabolite with antifungal activity . however , more recently the induction of conidiation in trichoderma species , which is known to be regulated by a circadian cycle , has also been shown to be controlled via a volatile agent . solid - phase microextraction linked with gas chromatography and mass spectrometry has allowed the identification of the chemical profiles of volatile molecules produced from nonconidiated colonies grown in darkness and conidiating colonies grown in light . comparison of the two profiles identified production of the 8-carbon compounds molecules 1-octen-3-ol , 3-octanol and 3-octanone specifically during conidiation . each of these three compounds induces conidiation in colonies placed in the dark . interestingly , concentrations above 500 m of any of the three compounds suppress conidiation and growth of trichoderma species . these observations are consistent with a previously described putative fungistatic and fungicidal role of the molecules [ 77 , 78 ] . notably , the same compounds have previously been shown to function as insect attractants improving fungal spore dispersal [ 77 , 78 ] , and inter - species communication has already been described between epichlo species and the female botanophila flies . fungi are not only responsive to volatile compounds that they produce but also , in at least one example , to a gas liberated during respiration : carbon dioxide ( co2 ) . as early as 1961 , vakil et al . demonstrated that the optimum co2 concentration for the germination of aspergillus niger conidiospores is reached not under normal atmospheric concentrations of co2 ( 0.033% ) but at 0.5% . since then several additional phenotypes in fungi have been attributed to changes in the concentration of environmental co2 including the sporulation of alternaria crassa and alternaria cassiae , conidiation of neurospora crassa , or capsule formation and mating in c. neoformans [ 83 , 84 ] . recently , significant advances have been made in the understanding of co2 sensing in fungi . furthermore , the frequency of white - to - opaque switching can be increased 16-fold in hypercapnic conditions as opposed to atmospheric co2 . this enzyme generates the secondary messenger camp , which in the context of the camp / pka signaling pathway has a fundamental impact on c. albicans morphogenesis [ 86 , 87 ] . co2 activation of cyr1p depends on the concentration of bicarbonate , the hydrated form of co2 . co2 hydration occurs naturally at a very low rate , but is enhanced by the enzyme carbonic anhydrase . inactivation of cyr1 results in a loss of filamentation and white to opaque switching frequency in response to hypercapnia [ 86 , 87 ] . have now demonstrated that lysine 1373 of the cyr1 catalytic domain is essential for co2 sensing in c. albicans as mutation of this amino acid leads to a loss of filamentation in response to co2 but not to serum , another morphological cue . these data show that in fungi environmental co2 is sensed via the adenylyl cyclase , which transduces the message via the regulation of the camp / pka pathway . also showed that hypercapnia is not a condition solely encountered inside the host but can also establish itself as a population event , such as the center of a colony grown under normal atmospheric conditions . urea is ultimately degraded to generate co2 by the enzyme urea amidolyase ( dur1,2 ) . inactivation of the latter interferes with c. albicans filamentation in response to arginine and urea compared to the control strain but not to elevated co2 . control of the white to opaque switch - frequency in c. albicans by environmental co2 also involves the gtpase ras1 and the transcriptional factor wor1 . indeed , ras1 , cyr1 , and wor1 are critical for increasing the white to opaque switch in response to concentrations of co2 at 1% , but ras1 and cyr1 become optional for the induction at higher concentrations ( 20% ) . however , wor1 remains essential for the switch even at high co2 . these results imply that an alternative co2 sensing pathway is involved in the regulation of wor1 at high co2 in c. albicans . however , it is important to note that under this condition a significant increase of the internal ph may occur which could also be a component of this alternative co2 sensing pathway . acetaldehyde , an organic compound involved in several cellular pathways , has been shown to impact on cell - density - dependent glycolytic oscillations of s. cerevisiae . described that the level of nadh in yeast oscillated when starved cells endure a pulse of glucose after a switch to anaerobic conditions . since then other metabolites have been described to oscillate in yeast including glucose-6-phosphate , fructose-6-phosphate , fructose-1,6-biphosphate , amp , adp , and atp ( for a comprehensive review see ) . interestingly , at a population level these oscillations are not chaotic but appear to be subject to synchronization . the most striking observation was achieved when mixing two populations with a 180 out - of - phase oscillation showing that within minutes the oscillation of the new population were synchronized . acetaldehyde was identified as the active molecule in the synchronization of these oscillations , as the use of acetaldehyde traps induced the oscillation to be damped and addition of acetaldehyde to the medium produced a phase shift in the oscillation . acetaldehyde is also a volatile molecule , a property used to study inter and intraspecies communication in a synthetic ecosystem . by engineering sender cells that liberate volatile acetaldehyde and receiver cells that contain a construct under an acetaldehyde - inducible promoter , it was possible to study volatile cell communication in a controlled environment . clearly such models could bring new insight in the understanding of communication in complex living systems . . many essential compounds of the communication process have been identified , the sender ( in our case fungi ) , the message ( protein , alcohol , lipid , gas ) , and the receiver ( bacteria , fungi , plant , mammalian ) . however , in most cases the actual molecular mechanism of such communication remains for most parts unknown . the determination of these pathways is of substantial significance as molecular messengers control the expression of fungal virulence determinants including the yeast - to - hyphae switch and biofilm formation in c. albicans , capsule formation in c. neoformans , or mycotoxin synthesis in a. nidulans , but also the propagation of these organisms via the regulation of their sexual and asexual cycle . a better knowledge of fungal communication is now required to permit the development of innovative strategies aiming to control disease or toxin production of these organisms . fungi have already taken advantage of the different communication processes and particularly inter - species communication to gain competitive advantages over other species . good examples are the production of pollinators attracting insects to give phytopathogenic fungi a better chance for dispersal of their spores . such mechanisms reveal the close coevolution of fungi with their environmental partner and give insights into multispecies communication . the remarkable versatility of communication in the fungal kingdom also raises the question how these organisms integrate intra- and inter - species messages that can have opposing effects . as the molecular mechanisms of fungal communication unravel further , they will promote our understanding of the highly attractive but challenging topic of the fungal communicome .

ghrelin is a gut / brain peptide comprising 28 amino acids and is originally identified as the endogenous ligand of the growth hormone secretagogue receptor ( ghsr ) ( 1 , 2 ) . this peptide is also expressed in the brain , primarily in the hypothalamic arcuate nucleus , an important region for controlling energy balance and reproduction ( 1 , 3 ) . in addition , the presence of ghrelin and its receptors has been reported in gnrh neurons ( 4 ) and gonadotrophs of pituitary ( 57 ) . also , ghrelin inhibits luteinizing hormone ( lh ) release from the pituitary in vivo and in vitro ( 811 ) . the gnrh neurons are the central core of the hypothalamic - pituitary - gonadal ( hpg ) axis in all vertebrate species ; therefore , according to the localization patterns of ghrelin , this peptide has a role in the regulation of hpg axis function ( 8) . moreover , this peptide has also a suppressive role in the release of testosterone in male mammals ( 10 , 12 ) . on the other hand , ghrelin has been shown to have effects on all three tissues of the hpg axis ( 10 , 12 ) . several studies have demonstrated the facilitative effects of hormones secreted from hpg axis on reproductive behavior in male and female animals ( 1317 ) . it has been previously shown that gnrh - immunoreactive fibers are present in regions of the brain known to regulate sexual behavior including the bed nucleus of the stria terminalis ( bnst ) , the medial amygdala ( mag ) and most notably , the medial preoptic area ( mpoa ) ( 18 ) . according to previous studies , administration of gonadotropins and androgens facilitated the sexual behavior in male mammals ( 1315 ) . in addition , there are immunohistochemical evidences for the endocrine / paracrine role for ghrelin in the reproductive tissues of mammals ( 8) . these evidences led us to postulate that ghrelin may also reduce sexual activity in addition to inhibiting the hpg axis . to test this hypothesis , the effects of centrally administered ghrelin on sexual behavior in adult male rats were examined . this is the first study for analyzing the effects of ghrelin on all aspects of male sexual behavior in any mammalian species . moreover , to test whether the locomotion prospects have a role in changes of sexual behavior , another experiment was developed to analyze rats locomotor activity evaluated by an open field . alongside the behavioral study , the aim of this assessment was to evaluate the ghrelin s effect on synthesis of lh as one of the indices of hpg axis activity . additionally , an attempt was made to examine whether the possible effects of ghrelin on sexual behavior and lh beta - subunit gene expression depends on an interaction with ghsr-1a ( 57 ) . the broad range of biological processes exerted by ghrelin seem to be mediated by the interaction with specific receptors ( 57 ) ; most of these processes have been abolished by pretreatment with [ d - lys]-ghrp-6 ( dls ) , a selective ghs - r1a antagonist ( 1922 ) . hence , dls was used to determine the role of the ghs - r1a , and its antagonistic actions on ghrelin - induced effects . the aim of present study was to investigate the effects of intracerebroventricular injection of ghrelin , dls or co - administration of these peptides on sexual behavior and luteinizing hormone beta - subunit gene expression in male rats to understand the role of ghrelin as the starvation hormone on attenuation of the sexual behavior indices and lh synthesis in hpg axis . one hundred and twenty - eight adult male wistar rats weighing 20020 g were used in this study . the animals were group - housed in polycarbonate cages . rats were maintained in a temperature controlled room ( 222c ) under a 12 hr/12 hr light / dark cycle ( lights on 08:00 ) . all procedures for the maintenance and use of experimental animals were pre - approved by the local institutional committee for the ethical use of animals . all rats were anesthetized by intraperitoneal injection of a mixture of ketamine ( 80 mg / kg bw ) and xylazine ( 20 mg / kg bw ) . for central injections , a guide cannula was stereotaxically implanted in the third cerebral ventricle according to the stereotaxic coordinates ( ap=2.3 , ml=0.0 , dv=6.5 ) published in the atlas of paxinos and watson ( 1989 ) . the guide cannula consisted of a 22-gauge stainless steel needle secured to the skull with three stainless steel screws and dental cement . one 28-guage stainless steel removable obturator was inserted into the guide cannula to ensure that the cannula remained patent ( 23 ) . following the surgery , after the eight - day recovery period , these animals were divided into two groups . each group ( n=64 ) was further subdivided into eight subgroups ( n=8 rats / subgroup ) . sixty four rats in 8 groups intracerebroventricularly received saline ( 3 l ) , ghrelin ( 2 , 4 or 8 nmol/3 l ) , dls ( 5 or 10 nmol/3 l ) , ghrelin ( 4 nmol/1.5 l)+dls ( 5 or 10 nmol/1.5 l ) in order to study the sexual behavior , and the other sixty four rats in 8 groups intracerebroventricularly received the same treatments for studying the locomotor activity and gene expression . doses were chosen based on previous studies , which had established their stimulatory or inhibitory effects on hpg axis or feeding behavior . ghrelin and dls ( phoenix pharmaceutical inc . , ca , usa ) were dissolved in saline . solutions were freshly prepared just before use and were injected by a hamilton microsyringe ( hamilton inc . this experiment was conducted in a testing arena ( 563232 cm ) with a frontal glass . each nave male rat was individually placed in the arena 10 min after drug administration and allowed to habituate to testing chamber for 5 min ; then a sexually active female rat was introduced and the sexual behavior test was performed in 40 min period . the female rats were tested for receptivity before being placed with the males ; females that presented lordosis after the mount of a male rat were selected for studying the sexual behavior of nave administrated males . all tests were performed during the late photo phase ( 15:0019:00 ) and the following parameters for each male were recorded : the latencies of first mount ( male places its forequarters on hindquarters of female from behind ) ( ml ) , first intromission ( mount with vaginal insertion ) ( il ) , and first ejaculation ( el ) ; total number of mounts ( nm ) , intromissions ( ni ) and ejaculations ( ne ) in 40 min ; number of mounts ( nm ) and intromissions ( ni ) until the first ejaculation , and post ejaculatory interval ( pei ) . additionally , other derived measures were calculated using mentioned parameters including : sum of the nm , ni and ne ; copulatory efficiency ( ce ) , and sexual activity index ( sai ) based on agmo et al . ( 24 ) : ce=(ni+ne / nm+ni+ne)100 sai = log ( 1/mlt)+log ( 1/ilt)+log ( 1/elt) ( nm+ni)+y the time of the observation was indicated with t , and y means 4 when an animal ejaculated and 0 when it did not . all latencies were calculated in seconds . ten minutes after injection , all rats were individually placed in a square arena ( 454535 cm ) divided in to nine equal sectors on the floor . the total number of sectors crossing with all four paws was recorded for 5 min ( 25 ) . all administrated male rats were decapitated 2 hr after injections ; pituitaries were dissected and frozen in liquid nitrogen and stored at 80c until processed for reverse transcriptase polymerase chain reaction ( rt - pcr ) . at first , total rna was extracted using rnx - plus solution ( cinnagen , iran ) . briefly , pituitaries were separately transferred to 1 ml ice cold rnx - plus solution and homogenized . chloroform ( 200 l ) was added to each sample and after vigorous mixing , centrifuged at 12000 rpm at 4c for 15 min ; the supernatant was gently mixed with an equal volume of cold isopropanol for 10 min ; then the mixture was centrifuged at 12000 rpm at 4c for 10 min . the aqueous phase was discarded and the rna pellet was washed with 1 ml of 75% ethanol . the rna pellet was quickly dried and reconstituted in 30 l diethylpyrocarbonate ( depc ) water . the purified total rna was quantified by the nano - drop spectrophotometer ( nano - drop technologies , usa ) . then , the synthesis of complementary dna and the amplification of cdna templates were carried out using the 2-step rt - pcr kit ( vivantis technologies , malaysia ) , according to kit s recommended protocol . for each gene of interest , cdna template amplification was carried out in a final volume of 25 l ; the mixture consisted of 2.5 l of synthesized cdna solution , 1 u of taq dna polymerase , 2.5 l of 10x pcr buffer ( 10x vi buffer a ) , 0.75 l of 50 mm mgcl2 , 0.5 l of 10 mm dntps mix , and 0.75 l of forward and reverse primer mixture . nm_012858 ) forward : aga gaa tga gtt ctg ccc agt ctg , reverse : agg tca ttg gtt gag tcc tgg g ( amplicon length 274 bp ) ; and gapdh ( accession no . bc_013915 ) forward : aag aag gtg gtg aag cag gca tc , reverse : cga agg tgg aag agt ggg agt tg ( amplicon length 112 bp ) . the expression levels of lh were normalized by dividing by gapdh mrna expression levels . pcr cycling conditions were as follows : initial denaturation and enzyme activation at 94c for 2 min , followed by 35 cycles of denaturation at 94c for 30 s , annealing at 59c for 30 s ( lh ) , or 58c for 30 s ( gapdh ) , and extension at 72c for 30 s. as a final step , samples were kept at 72c for 7 min . after the thermal cycling steps , pcr - amplified products were analyzed by electrophoresis on a 1.5% safe red solution mixed with agarose gel . the molecular size marker , a 100 bp plus , ( cinnagen , iran ) was run concurrently . the intensity of each band was quantified using imagej software ( version 1.41 , usa ) and the ratio of lh to gapdh was determined . multiple comparisons were performed using one - way analysis of variance ( anova ) followed by tukey - hsd test by spss software ( version 19.0 , spss inc . one hundred and twenty - eight adult male wistar rats weighing 20020 g were used in this study . the animals were group - housed in polycarbonate cages . rats were maintained in a temperature controlled room ( 222c ) under a 12 hr/12 hr light / dark cycle ( lights on 08:00 ) . all procedures for the maintenance and use of experimental animals were pre - approved by the local institutional committee for the ethical use of animals . all rats were anesthetized by intraperitoneal injection of a mixture of ketamine ( 80 mg / kg bw ) and xylazine ( 20 mg / kg bw ) . for central injections , a guide cannula was stereotaxically implanted in the third cerebral ventricle according to the stereotaxic coordinates ( ap=2.3 , ml=0.0 , dv=6.5 ) published in the atlas of paxinos and watson ( 1989 ) . the guide cannula consisted of a 22-gauge stainless steel needle secured to the skull with three stainless steel screws and dental cement . one 28-guage stainless steel removable obturator was inserted into the guide cannula to ensure that the cannula remained patent ( 23 ) . after the eight - day recovery period , these animals were divided into two groups . each group ( n=64 ) was further subdivided into eight subgroups ( n=8 rats / subgroup ) . sixty four rats in 8 groups intracerebroventricularly received saline ( 3 l ) , ghrelin ( 2 , 4 or 8 nmol/3 l ) , dls ( 5 or 10 nmol/3 l ) , ghrelin ( 4 nmol/1.5 l)+dls ( 5 or 10 nmol/1.5 l ) in order to study the sexual behavior , and the other sixty four rats in 8 groups intracerebroventricularly received the same treatments for studying the locomotor activity and gene expression . doses were chosen based on previous studies , which had established their stimulatory or inhibitory effects on hpg axis or feeding behavior . solutions were freshly prepared just before use and were injected by a hamilton microsyringe ( hamilton inc . this experiment was conducted in a testing arena ( 563232 cm ) with a frontal glass . each nave male rat was individually placed in the arena 10 min after drug administration and allowed to habituate to testing chamber for 5 min ; then a sexually active female rat was introduced and the sexual behavior test was performed in 40 min period . the female rats were tested for receptivity before being placed with the males ; females that presented lordosis after the mount of a male rat were selected for studying the sexual behavior of nave administrated males . all tests were performed during the late photo phase ( 15:0019:00 ) and the following parameters for each male were recorded : the latencies of first mount ( male places its forequarters on hindquarters of female from behind ) ( ml ) , first intromission ( mount with vaginal insertion ) ( il ) , and first ejaculation ( el ) ; total number of mounts ( nm ) , intromissions ( ni ) and ejaculations ( ne ) in 40 min ; number of mounts ( nm ) and intromissions ( ni ) until the first ejaculation , and post ejaculatory interval ( pei ) . additionally , other derived measures were calculated using mentioned parameters including : sum of the nm , ni and ne ; copulatory efficiency ( ce ) , and sexual activity index ( sai ) based on agmo et al . ( 24 ) : ce=(ni+ne / nm+ni+ne)100 sai = log ( 1/mlt)+log ( 1/ilt)+log ( 1/elt) ( nm+ni)+y the time of the observation was indicated with t , and y means 4 when an animal ejaculated and 0 when it did not . all latencies were calculated in seconds . ten minutes after injection , all rats were individually placed in a square arena ( 454535 cm ) divided in to nine equal sectors on the floor . the total number of sectors crossing with all four paws was recorded for 5 min ( 25 ) . all administrated male rats were decapitated 2 hr after injections ; pituitaries were dissected and frozen in liquid nitrogen and stored at 80c until processed for reverse transcriptase polymerase chain reaction ( rt - pcr ) . at first , total rna was extracted using rnx - plus solution ( cinnagen , iran ) . briefly , pituitaries were separately transferred to 1 ml ice cold rnx - plus solution and homogenized . chloroform ( 200 l ) was added to each sample and after vigorous mixing , centrifuged at 12000 rpm at 4c for 15 min ; the supernatant was gently mixed with an equal volume of cold isopropanol for 10 min ; then the mixture was centrifuged at 12000 rpm at 4c for 10 min . the aqueous phase was discarded and the rna pellet was washed with 1 ml of 75% ethanol . the rna pellet was quickly dried and reconstituted in 30 l diethylpyrocarbonate ( depc ) water . the purified total rna was quantified by the nano - drop spectrophotometer ( nano - drop technologies , usa ) . then , the synthesis of complementary dna and the amplification of cdna templates were carried out using the 2-step rt - pcr kit ( vivantis technologies , malaysia ) , according to kit s recommended protocol . for each gene of interest , cdna template amplification was carried out in a final volume of 25 l ; the mixture consisted of 2.5 l of synthesized cdna solution , 1 u of taq dna polymerase , 2.5 l of 10x pcr buffer ( 10x vi buffer a ) , 0.75 l of 50 mm mgcl2 , 0.5 l of 10 mm dntps mix , and 0.75 l of forward and reverse primer mixture . nm_012858 ) forward : aga gaa tga gtt ctg ccc agt ctg , reverse : agg tca ttg gtt gag tcc tgg g ( amplicon length 274 bp ) ; and gapdh ( accession no . bc_013915 ) forward : aag aag gtg gtg aag cag gca tc , reverse : cga agg tgg aag agt ggg agt tg ( amplicon length 112 bp ) . pcr cycling conditions were as follows : initial denaturation and enzyme activation at 94c for 2 min , followed by 35 cycles of denaturation at 94c for 30 s , annealing at 59c for 30 s ( lh ) , or 58c for 30 s ( gapdh ) , and extension at 72c for 30 s. as a final step , samples were kept at 72c for 7 min . after the thermal cycling steps , pcr - amplified products were analyzed by electrophoresis on a 1.5% safe red solution mixed with agarose gel . the molecular size marker , a 100 bp plus , ( cinnagen , iran ) was run concurrently . the intensity of each band was quantified using imagej software ( version 1.41 , usa ) and the ratio of lh to gapdh was determined . multiple comparisons were performed using one - way analysis of variance ( anova ) followed by tukey - hsd test by spss software ( version 19.0 , spss inc . figure 1 shows the sexual behavior latencies of ghrelin- or dls - treated male rats . in relation to saline group , 4 or 8 no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on ml , il and el ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - injection of ghrelin ( 4 nmol ) and dls ( 5 or 10 nmol ) significantly decreased the ml , il and el compared to ghrelin ( 4 nmol ) group , but there was no significant difference between the effects of ghrelin ( 4 nmol)+dls ( 5 or 10 nmol ) groups and saline group on ml , il and el ( figures 1a , 1b , 1c ) . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on mount ; a : intromission ; b : and ejaculation ; c : latencies in male wistar rats . data are represented as meansem ; * p<0.05 ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.05 , p<0.01 ; p<0.001 vs. ghrelin ( 4 nmol ) group according to figures 2a and 2c , the total number of mount significantly increased and the total number of ejaculation significantly decreased in 40 min following the ghrelin ( 4 or 8 nmol ) injection . no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on nm and ne ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - administration of ghrelin ( 4 nmol ) and dls ( 10 nmol ) significantly decreased the nm and significantly increased the ne compared to ghrelin ( 4 nmol ) group , but a significant difference was not observed between the effects of ghrelin ( 4 nmol)+ dls ( 5 nmol ) and ghrelin ( 4 nmol ) group on nm and ne ( figures 2a , 2c ) . according to figure 2b , there were no significant differences among the groups on number of intromission in 40 min . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on total number of mounts ; a : intromissions ; b : and ejaculations ; c : in 40 min in male wistar rats . data are represented as meansem ; * p < 0.05 , * * p<0.01 vs. saline group ; p<0.05 ; p<0.01 vs. ghrelin ( 4 nmol ) group table 1 shows the other parameters of sexual behavior . the post - ejaculatory interval and the parameters related to motor activity ( mount and intromission frequencies until the first ejaculation ) increased in the ghrelin - received animals compared to control group ; co - administration of ghrelin and dls decreased the mentioned parameters to control range . however , the sum of the total mount , intromission and ejaculation frequencies did not alter under several treatments . the sexual activity index ( sai ) and the copulatory efficiency ( ce ) decreased following the ghrelin injection ; dls pretreatment abolished the inhibitory effect of ghrelin on ce in both 5 and 10 nmol and on sai at the high dose ( 10 nmol ) . different p - values depended on different doses and are shown in table 1 . sexual behavior parameters of male wistar rats following ghrelin or [ d - lys]-ghrp-6 ( dls ) injection nm = number of mounts until the first ejaculation ; ni = number of intromissions until the first ejaculation ; pei = post ejaculatory interval ; nm+ni+ne = sum of the total number of mount , intromission and ejaculation in 40 min ; sai = sexual activity index ; ce = copulatory efficiency . data are represented as meansem ; p<0.001 vs. saline group ; p<0.001 vs. ghrelin ( 4 nmol ) group figure 3 shows the locomotor activity on administrated rats in 5 min . total number of sectors crossing significantly increased in ghrelin injected rats as compared to control group , while the injection of high dose of dls ( 10 nmol ) significantly decreased the locomotor activity as compared to saline received rats . co - administration of ghrelin ( 4 nmol ) and dls ( 5 and 10 nmol ) significantly decreased locomotion as compared to ghrelin received animals . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on locomotor activity in 5 min in male wistar rats . data are represented as meansem ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.001 vs. ghrelin ( 4 nmol ) group expression of lh and gapdh mrna in the pituitary of male rats is shown in figures 4a and 4b , respectively . the ratio of lh to gapdh was determined after the quantification of each band using imagej software and is shown in figure 4c . relative lh mrna expression significantly ( p<0.001 ) decreased following the ghrelin ( 4 or 8 nmol ) injection , while the administration of dls ( 5 or 10 nmol ) had no significant effect on gene expression levels . however , co - injection of the lowest effective dose of ghrelin ( 4 nmol ) and the highest non - effective dose of dls ( 10 nmol ) significantly ( p<0.01 ) increased the relative expression of lh mrna compared to ghrelin ( 4 and 8 nmol ) groups . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on amplification products of pcr ; lh subunit ; a : gapdh ; b : gene expression levels and the relative expression levels of lh to gapdh ; c : in male wistar rats . data are represented as meansem ; * * * p < 0.001 vs. saline group ; p<0.01 and p<0.05 vs. ghrelin ( 4 nmol ) group figure 1 shows the sexual behavior latencies of ghrelin- or dls - treated male rats . in relation to saline group , 4 or 8 no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on ml , il and el ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - injection of ghrelin ( 4 nmol ) and dls ( 5 or 10 nmol ) significantly decreased the ml , il and el compared to ghrelin ( 4 nmol ) group , but there was no significant difference between the effects of ghrelin ( 4 nmol)+dls ( 5 or 10 nmol ) groups and saline group on ml , il and el ( figures 1a , 1b , 1c ) . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on mount ; a : intromission ; b : and ejaculation ; c : latencies in male wistar rats . data are represented as meansem ; * p<0.05 ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.05 , p<0.01 ; p<0.001 vs. ghrelin ( 4 nmol ) group according to figures 2a and 2c , the total number of mount significantly increased and the total number of ejaculation significantly decreased in 40 min following the ghrelin ( 4 or 8 nmol ) injection . no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on nm and ne ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - administration of ghrelin ( 4 nmol ) and dls ( 10 nmol ) significantly decreased the nm and significantly increased the ne compared to ghrelin ( 4 nmol ) group , but a significant difference was not observed between the effects of ghrelin ( 4 nmol)+ dls ( 5 nmol ) and ghrelin ( 4 nmol ) group on nm and ne ( figures 2a , 2c ) . according to figure 2b , there were no significant differences among the groups on number of intromission in 40 min . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on total number of mounts ; a : intromissions ; b : and ejaculations ; c : in 40 min in male wistar rats . data are represented as meansem ; * p < 0.05 , * * p<0.01 vs. saline group ; p<0.05 ; p<0.01 vs. ghrelin ( 4 nmol ) group table 1 shows the other parameters of sexual behavior . the post - ejaculatory interval and the parameters related to motor activity ( mount and intromission frequencies until the first ejaculation ) increased in the ghrelin - received animals compared to control group ; co - administration of ghrelin and dls decreased the mentioned parameters to control range . however , the sum of the total mount , intromission and ejaculation frequencies did not alter under several treatments . the sexual activity index ( sai ) and the copulatory efficiency ( ce ) decreased following the ghrelin injection ; dls pretreatment abolished the inhibitory effect of ghrelin on ce in both 5 and 10 nmol and on sai at the high dose ( 10 nmol ) . different p - values depended on different doses and are shown in table 1 . sexual behavior parameters of male wistar rats following ghrelin or [ d - lys]-ghrp-6 ( dls ) injection nm = number of mounts until the first ejaculation ; ni = number of intromissions until the first ejaculation ; pei = post ejaculatory interval ; nm+ni+ne = sum of the total number of mount , intromission and ejaculation in 40 min ; sai = sexual activity index ; ce = copulatory efficiency . data are represented as meansem ; p<0.001 vs. saline group ; p<0.001 vs. ghrelin ( 4 nmol ) group total number of sectors crossing significantly increased in ghrelin injected rats as compared to control group , while the injection of high dose of dls ( 10 nmol ) significantly decreased the locomotor activity as compared to saline received rats . co - administration of ghrelin ( 4 nmol ) and dls ( 5 and 10 nmol ) significantly decreased locomotion as compared to ghrelin received animals . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on locomotor activity in 5 min in male wistar rats . data are represented as meansem ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.001 vs. ghrelin ( 4 nmol ) group expression of lh and gapdh mrna in the pituitary of male rats is shown in figures 4a and 4b , respectively . the ratio of lh to gapdh was determined after the quantification of each band using imagej software and is shown in figure 4c . relative lh mrna expression significantly ( p<0.001 ) decreased following the ghrelin ( 4 or 8 nmol ) injection , while the administration of dls ( 5 or 10 nmol ) had no significant effect on gene expression levels . however , co - injection of the lowest effective dose of ghrelin ( 4 nmol ) and the highest non - effective dose of dls ( 10 nmol ) significantly ( p<0.01 ) increased the relative expression of lh mrna compared to ghrelin ( 4 and 8 nmol ) groups . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on amplification products of pcr ; lh subunit ; a : gapdh ; b : gene expression levels and the relative expression levels of lh to gapdh ; c : in male wistar rats . data are represented as meansem ; * * * p < 0.001 vs. saline group ; p<0.01 and p<0.05 vs. ghrelin ( 4 nmol ) group identification of ghrelin and its receptors in hypothalamus , pituitary and gonads ( 38 ) and the subsequent demonstration of the facilitative effects of hormones secreted from these tissues on reproductive behavior ( 10 , 1316 , 18 ) led us to hypothesize that in addition to affecting on hpg axis , ghrelin may also alter the sexual behavior . this is the first study to analyze the effects of ghrelin and dls ( [ d - lys]-ghrp-6 ) on male sexual behavior in any mammalian species . present results show that ghrelin injection distorted some aspects of sexual behavior and reduced lh subunit gene expression while enhanced loco - motor activity of male rats . in this study , it was found that ghrelin has similar inhibitory effects on sexual behavior in male rats as previously reported for female mice in a dose - dependent manner ( 26 ) . these latencies are commonly used for evaluating male sexual motivation ( 27 ) characterized by a synchronization of sexual desire arising in the brain and its transmission to the periphery , resulting in penile erection ( 16 , 17 ) . it has been suggested that testosterone positively control both the initiation and the end of the penile erection and that it is a main synchronizer of sexual activity ( 16 , 17 ) . in the absence of testosterone , the sexual desire arising is delayed and uncoordinated with penile erection ( 16 , 17 ) . previous studies have shown the suppressive role of ghrelin in the release of testosterone in male mammals ( 10 , 12 ) . , as was observed , pretreatment with dls reduced the latencies of studied rats . in this study , despite the decreased number of intromission and ejaculation in ghrelin - received animals , the number of mounts increased in these rats . nevertheless , there was no difference in sum of the total number of mounts , intromissions and ejaculations among different groups . it suggests that the decrease in the number of intromission and ejaculation after ghrelin injection was compensated by an increase in the number of mounts . in this study , it was demonstrated that ghrelin increased the locomotor activity in the open field test . presumably , increasing the number of mounts in ghrelin - received animals due to the effects of this peptide on locomotor activity may be a compensatory mechanism to prevent the decreasing of locomotor activity during the intercourse . previously , a number of studies have been conducted into the effect of ghrelin on locomotion indicating the facilitative effects of this peptide on locomotor activity ( 28 , 29 ) . nevertheless , there is controversy over the increasing effect of ghrelin on locomotor activity . these discrepancies among studies may be due to the differences in the animal species used , rout or dose of peptide administrated or the photoperiodic condition of experiments ( 30 , 31 ) . also , few studies showed that dls treatment or ghrelin receptor deficiency reduced the locomotor activity ( 29 , 32 , 33 ) . these findings are compatible with our observations showing that dls attenuated the enhanced ghrelin - induced locomotor activity and mount frequency . altogether , it is concluded that the damage in sexual behavior following the ghrelin injection might be the result of other variables rather than locomotion prospects . in other words , this possibility is rejected that reduced sexual behavior after ghrelin injection may result from decreased motor activity . considerable decrease in the number of ejaculation as the main factor of success in reproduction process caused a significant decrease in copulatory efficiency in ghrelin - received animals . this peptide led to a decrease in the body s ability to perform the ejaculation presumably through the mechanisms regulating energy balance . nowadays , it is proved that negative energy balance and many food intake - enhancing hormones such as ghrelin have inhibitory effects on the reproductive axis ( 10 , 12 , 34 , 35 ) . accordingly , our findings indicating the inhibitory effect of ghrelin on sexual behavior is compatible with the fact that starvation conditions and negative energy balance prevent the loss of energy in the reproductive pathways . moreover , ghrelin also increased the interval between the copulatory cycles ( pei ) , probably due to the delay in the recovery of energy for the next copulation . calculation of sexual activity index ( sai ) in the studied animals confirmed the suppressive effects of ghrelin on reproductive behavior . however , pretreatment with dls increased the number of ejaculations and in turn improved the mating efficiencies . the potential implications of these findings are clear ; ghrelin has a deleterious effect on male rat s sexual behavior . up to now , there is no report about the involvement of ghrelin in regulation of the male sexual behavior . however , bertoldi et al . demonstrated that ghrelin was able to inhibit the sexual behavior of female mice and that it was involved in receptivity reduction after food scarcity ( 26 ) . also , shah and nyby showed that ghrelin quickly suppressed the androgen - dependent behaviors including the ultrasonic mating calls to a female and the latency to attack a stimulus male through its direct effects on the brain in male house mice , mus musculus ( 36 ) . additionally , in this study dls , the ghsr-1a antagonist , abolished the prejudicial effects of ghrelin on sexual behavior . the sexual behavior aspects under dls treatment alone had no significant differences with those in saline - treated rats , while dls modified the behavioral effects of ghrelin in co - administrated animals . these data suggest that the inhibitory effect of ghrelin on sexual behavior is mediated by ghsr-1a . previously , dls has been investigated in food intake ( 19 , 20 ) , inflammatory pain ( 37 ) , ischemia - reperfusion ( 21 ) , hormone secretion ( 22 ) and some other studies in which the several effects of ghrelin were or were not attenuated in animals pretreated with dls . however , this is the first study evaluating the effects of dls on male sexual behavior . it was also revealed that ghrelin and dls had regulatory effects on lh subunit gene expression in male rats . in this study , previous neuroanatomical analysis indicated that many ghrelin fibers have been projected to lh contained neurons ( 68 ) and ghrelin secretion decreased fos induction in lh neurons and suppressed the lh secretion ( 8 , 10 , 11 ) . therefore , our findings showing the inhibitory effects of ghrelin on lh synthesis are in line with previous studies based on reducing effects of ghrelin on lh release . notwithstanding the original reports , where ghrelin decreased lh release in several species ( 8 , 10 , 11 ) , few studies to date had addressed the suppressive role of ghrelin in lh subunit gene expression . moreover , to see whether the effect of ghrelin on lh expression is mediated through the ghs - r1a or not , dls was used in this study . notably , blocking the ghrelin receptor ( ghs - r1a ) with the antagonist dls prevented the ghrelin - induced effects on lh expression . several lines of evidence suggested the direct and indirect stimulatory effect of luteinizing hormone on regions of the brain which have a role in regulating the male sexual behavior ( 10 , 18 , 38 ) . according to these evidences , inhibiting all in all , our data suggest that ghrelin reduces the lh synthesis and in turn decreases sexual behavior . it is worth mentioning that more studies are required to clarify the direct effects of ghrelin on regions of the brain known to regulate male sexual behavior . furthermore , the dose dependent effects of ghrelin were observed on both sexual behavior and locomotor activity in male rats . therefore , ghrelin in the lowest dose had no significant effect on most of the aspects of sexual behavior while at the same dose , it significantly affected the locomotor activity . it is suggested that the sensitivity of ghrelin receptors in locomotor activity regulating system is more than the ones in the brain regions which control the sexual behavior . presumably , ghrelin as a starvation hormone in low physiological doses has a negligible effect on mating behavior but continuity of ghrelin release has a remarkable effect on reproductive behavior . therefore , the negative energy balance that causes the surge of ghrelin may affect reproduction and sexual activities . in this study , copulatory actions of ghrelin and its specific antagonist , dls , were examined . to do so , behavioral tests and lh subunit gene expression assessment were implemented in male rats after injection of different doses of ghrelin or its antagonist through the central rout of administration . overall , our results suggest that ghrelin in a dose - dependent manner decreased the sexual desire and ability , and postponed the beginning of the intercourse . dls antagonizes the putative inhibitory role of ghrelin in the regulation of sexual behavior , which involves indirect actions through inhibition of lh synthesis . additional studies are required about the possible direct effects of ghrelin on brain regions which regulate the male sexual behavior and also about the efficacy of ghrelin following chronic administration .

background : the hormones of hypothalamo - pituitary - gonadal ( hpg ) axis have facilitative effects on reproductive behavior in mammals . ghrelin as a starvation hormone has an inhibitory effect on hpg axis function . hence , it is postulated that ghrelin may reduce the sexual behavior through inhibiting of hpg axis . the aim of this study was to examine the effects of ghrelin and its antagonist , [ d - lys3 ] -ghrp-6 , on sexual behavior and lh beta - subunit gene expression in male rats.methods:in this experimental study , 128 male wistar rats were divided into two groups . each group was further subdivided into eight subgroups ( n=8 rats / subgroup ) including the animals that received saline , ghrelin ( 2 , 4 or 8 nmol ) , [ d - lys3 ] -ghrp-6 ( 5 or 10 nmol ) or co - administration of ghrelin ( 4 nmol ) and [ d - lys3 ] -ghrp-6 ( 5 or 10 nmol ) through the stereotaxically implanted cannula into the third cerebral ventricle . the sexual behavior of male rats encountering with females and the hypo - physeal lh beta - subunit gene expression were evaluated at two different groups . data were analyzed by anova and p<0.05 was considered statistically significant.results:ghrelin injection ( 4 and 8 nmol ) significantly ( p<0.01 ) increased the latencies to the first mount , intromission and ejaculation as well as the post - ejaculatory interval . also , 4 and 8 nmol ghrelin significantly ( p<0.05 ) increased the number of mount and decreased the number of ejaculation . in co - administrated groups , [ d - lys3 ] -ghrp-6 antagonized the effects of ghrelin . ghrelin injection ( 4 and 8 nmol ) reduced the lh beta - subunit gene expression while pretreatment with [ d - lys3 ] -ghrp-6 improved the gene expression.conclusion:ghrelin decreased the sexual behavior and lh beta - subunit gene expression in male rats , whereas [ d - lys3 ] -ghrp-6 antagonizes these effects .

Introduction Methods Animals: Surgery and Injections: Sexual behavior test: Locomotor activity trial: Gene expression assay: Statistical analysis: Results Sexual behavior test: Locomotor activity trial: Gene expression assay: Discussion Conclusion

in addition , the presence of ghrelin and its receptors has been reported in gnrh neurons ( 4 ) and gonadotrophs of pituitary ( 57 ) . the gnrh neurons are the central core of the hypothalamic - pituitary - gonadal ( hpg ) axis in all vertebrate species ; therefore , according to the localization patterns of ghrelin , this peptide has a role in the regulation of hpg axis function ( 8) . several studies have demonstrated the facilitative effects of hormones secreted from hpg axis on reproductive behavior in male and female animals ( 1317 ) . to test this hypothesis , the effects of centrally administered ghrelin on sexual behavior in adult male rats were examined . this is the first study for analyzing the effects of ghrelin on all aspects of male sexual behavior in any mammalian species . alongside the behavioral study , the aim of this assessment was to evaluate the ghrelin s effect on synthesis of lh as one of the indices of hpg axis activity . additionally , an attempt was made to examine whether the possible effects of ghrelin on sexual behavior and lh beta - subunit gene expression depends on an interaction with ghsr-1a ( 57 ) . the broad range of biological processes exerted by ghrelin seem to be mediated by the interaction with specific receptors ( 57 ) ; most of these processes have been abolished by pretreatment with [ d - lys]-ghrp-6 ( dls ) , a selective ghs - r1a antagonist ( 1922 ) . the aim of present study was to investigate the effects of intracerebroventricular injection of ghrelin , dls or co - administration of these peptides on sexual behavior and luteinizing hormone beta - subunit gene expression in male rats to understand the role of ghrelin as the starvation hormone on attenuation of the sexual behavior indices and lh synthesis in hpg axis . each group ( n=64 ) was further subdivided into eight subgroups ( n=8 rats / subgroup ) . sixty four rats in 8 groups intracerebroventricularly received saline ( 3 l ) , ghrelin ( 2 , 4 or 8 nmol/3 l ) , dls ( 5 or 10 nmol/3 l ) , ghrelin ( 4 nmol/1.5 l)+dls ( 5 or 10 nmol/1.5 l ) in order to study the sexual behavior , and the other sixty four rats in 8 groups intracerebroventricularly received the same treatments for studying the locomotor activity and gene expression . all tests were performed during the late photo phase ( 15:0019:00 ) and the following parameters for each male were recorded : the latencies of first mount ( male places its forequarters on hindquarters of female from behind ) ( ml ) , first intromission ( mount with vaginal insertion ) ( il ) , and first ejaculation ( el ) ; total number of mounts ( nm ) , intromissions ( ni ) and ejaculations ( ne ) in 40 min ; number of mounts ( nm ) and intromissions ( ni ) until the first ejaculation , and post ejaculatory interval ( pei ) . for central injections , a guide cannula was stereotaxically implanted in the third cerebral ventricle according to the stereotaxic coordinates ( ap=2.3 , ml=0.0 , dv=6.5 ) published in the atlas of paxinos and watson ( 1989 ) . each group ( n=64 ) was further subdivided into eight subgroups ( n=8 rats / subgroup ) . sixty four rats in 8 groups intracerebroventricularly received saline ( 3 l ) , ghrelin ( 2 , 4 or 8 nmol/3 l ) , dls ( 5 or 10 nmol/3 l ) , ghrelin ( 4 nmol/1.5 l)+dls ( 5 or 10 nmol/1.5 l ) in order to study the sexual behavior , and the other sixty four rats in 8 groups intracerebroventricularly received the same treatments for studying the locomotor activity and gene expression . all tests were performed during the late photo phase ( 15:0019:00 ) and the following parameters for each male were recorded : the latencies of first mount ( male places its forequarters on hindquarters of female from behind ) ( ml ) , first intromission ( mount with vaginal insertion ) ( il ) , and first ejaculation ( el ) ; total number of mounts ( nm ) , intromissions ( ni ) and ejaculations ( ne ) in 40 min ; number of mounts ( nm ) and intromissions ( ni ) until the first ejaculation , and post ejaculatory interval ( pei ) . in relation to saline group , 4 or 8 no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on ml , il and el ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - injection of ghrelin ( 4 nmol ) and dls ( 5 or 10 nmol ) significantly decreased the ml , il and el compared to ghrelin ( 4 nmol ) group , but there was no significant difference between the effects of ghrelin ( 4 nmol)+dls ( 5 or 10 nmol ) groups and saline group on ml , il and el ( figures 1a , 1b , 1c ) . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on mount ; a : intromission ; b : and ejaculation ; c : latencies in male wistar rats . data are represented as meansem ; * p<0.05 ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.05 , p<0.01 ; p<0.001 vs. ghrelin ( 4 nmol ) group according to figures 2a and 2c , the total number of mount significantly increased and the total number of ejaculation significantly decreased in 40 min following the ghrelin ( 4 or 8 nmol ) injection . no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on nm and ne ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - administration of ghrelin ( 4 nmol ) and dls ( 10 nmol ) significantly decreased the nm and significantly increased the ne compared to ghrelin ( 4 nmol ) group , but a significant difference was not observed between the effects of ghrelin ( 4 nmol)+ dls ( 5 nmol ) and ghrelin ( 4 nmol ) group on nm and ne ( figures 2a , 2c ) . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on total number of mounts ; a : intromissions ; b : and ejaculations ; c : in 40 min in male wistar rats . data are represented as meansem ; * p < 0.05 , * * p<0.01 vs. saline group ; p<0.05 ; p<0.01 vs. ghrelin ( 4 nmol ) group table 1 shows the other parameters of sexual behavior . the post - ejaculatory interval and the parameters related to motor activity ( mount and intromission frequencies until the first ejaculation ) increased in the ghrelin - received animals compared to control group ; co - administration of ghrelin and dls decreased the mentioned parameters to control range . the sexual activity index ( sai ) and the copulatory efficiency ( ce ) decreased following the ghrelin injection ; dls pretreatment abolished the inhibitory effect of ghrelin on ce in both 5 and 10 nmol and on sai at the high dose ( 10 nmol ) . sexual behavior parameters of male wistar rats following ghrelin or [ d - lys]-ghrp-6 ( dls ) injection nm = number of mounts until the first ejaculation ; ni = number of intromissions until the first ejaculation ; pei = post ejaculatory interval ; nm+ni+ne = sum of the total number of mount , intromission and ejaculation in 40 min ; sai = sexual activity index ; ce = copulatory efficiency . total number of sectors crossing significantly increased in ghrelin injected rats as compared to control group , while the injection of high dose of dls ( 10 nmol ) significantly decreased the locomotor activity as compared to saline received rats . co - administration of ghrelin ( 4 nmol ) and dls ( 5 and 10 nmol ) significantly decreased locomotion as compared to ghrelin received animals . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on locomotor activity in 5 min in male wistar rats . data are represented as meansem ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.001 vs. ghrelin ( 4 nmol ) group expression of lh and gapdh mrna in the pituitary of male rats is shown in figures 4a and 4b , respectively . relative lh mrna expression significantly ( p<0.001 ) decreased following the ghrelin ( 4 or 8 nmol ) injection , while the administration of dls ( 5 or 10 nmol ) had no significant effect on gene expression levels . however , co - injection of the lowest effective dose of ghrelin ( 4 nmol ) and the highest non - effective dose of dls ( 10 nmol ) significantly ( p<0.01 ) increased the relative expression of lh mrna compared to ghrelin ( 4 and 8 nmol ) groups . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on amplification products of pcr ; lh subunit ; a : gapdh ; b : gene expression levels and the relative expression levels of lh to gapdh ; c : in male wistar rats . data are represented as meansem ; * * * p < 0.001 vs. saline group ; p<0.01 and p<0.05 vs. ghrelin ( 4 nmol ) group figure 1 shows the sexual behavior latencies of ghrelin- or dls - treated male rats . in relation to saline group , 4 or 8 no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on ml , il and el ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - injection of ghrelin ( 4 nmol ) and dls ( 5 or 10 nmol ) significantly decreased the ml , il and el compared to ghrelin ( 4 nmol ) group , but there was no significant difference between the effects of ghrelin ( 4 nmol)+dls ( 5 or 10 nmol ) groups and saline group on ml , il and el ( figures 1a , 1b , 1c ) . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on mount ; a : intromission ; b : and ejaculation ; c : latencies in male wistar rats . data are represented as meansem ; * p<0.05 ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.05 , p<0.01 ; p<0.001 vs. ghrelin ( 4 nmol ) group according to figures 2a and 2c , the total number of mount significantly increased and the total number of ejaculation significantly decreased in 40 min following the ghrelin ( 4 or 8 nmol ) injection . no significant difference was observed between the effects of 5 or 10 nmol dls and saline group on nm and ne ; whereas , a significant difference was observed between the 5 or 10 nmol dls and 4 nmol ghrelin group . co - administration of ghrelin ( 4 nmol ) and dls ( 10 nmol ) significantly decreased the nm and significantly increased the ne compared to ghrelin ( 4 nmol ) group , but a significant difference was not observed between the effects of ghrelin ( 4 nmol)+ dls ( 5 nmol ) and ghrelin ( 4 nmol ) group on nm and ne ( figures 2a , 2c ) . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on total number of mounts ; a : intromissions ; b : and ejaculations ; c : in 40 min in male wistar rats . data are represented as meansem ; * p < 0.05 , * * p<0.01 vs. saline group ; p<0.05 ; p<0.01 vs. ghrelin ( 4 nmol ) group table 1 shows the other parameters of sexual behavior . the post - ejaculatory interval and the parameters related to motor activity ( mount and intromission frequencies until the first ejaculation ) increased in the ghrelin - received animals compared to control group ; co - administration of ghrelin and dls decreased the mentioned parameters to control range . the sexual activity index ( sai ) and the copulatory efficiency ( ce ) decreased following the ghrelin injection ; dls pretreatment abolished the inhibitory effect of ghrelin on ce in both 5 and 10 nmol and on sai at the high dose ( 10 nmol ) . sexual behavior parameters of male wistar rats following ghrelin or [ d - lys]-ghrp-6 ( dls ) injection nm = number of mounts until the first ejaculation ; ni = number of intromissions until the first ejaculation ; pei = post ejaculatory interval ; nm+ni+ne = sum of the total number of mount , intromission and ejaculation in 40 min ; sai = sexual activity index ; ce = copulatory efficiency . data are represented as meansem ; p<0.001 vs. saline group ; p<0.001 vs. ghrelin ( 4 nmol ) group total number of sectors crossing significantly increased in ghrelin injected rats as compared to control group , while the injection of high dose of dls ( 10 nmol ) significantly decreased the locomotor activity as compared to saline received rats . co - administration of ghrelin ( 4 nmol ) and dls ( 5 and 10 nmol ) significantly decreased locomotion as compared to ghrelin received animals . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on locomotor activity in 5 min in male wistar rats . data are represented as meansem ; * * p<0.01 ; * * * p<0.001 vs. saline group ; p<0.001 vs. ghrelin ( 4 nmol ) group expression of lh and gapdh mrna in the pituitary of male rats is shown in figures 4a and 4b , respectively . relative lh mrna expression significantly ( p<0.001 ) decreased following the ghrelin ( 4 or 8 nmol ) injection , while the administration of dls ( 5 or 10 nmol ) had no significant effect on gene expression levels . however , co - injection of the lowest effective dose of ghrelin ( 4 nmol ) and the highest non - effective dose of dls ( 10 nmol ) significantly ( p<0.01 ) increased the relative expression of lh mrna compared to ghrelin ( 4 and 8 nmol ) groups . effects of ghrelin , [ d - lys3]-ghrp-6 ( dls ) or co - administration of ghrelin and dls on amplification products of pcr ; lh subunit ; a : gapdh ; b : gene expression levels and the relative expression levels of lh to gapdh ; c : in male wistar rats . data are represented as meansem ; * * * p < 0.001 vs. saline group ; p<0.01 and p<0.05 vs. ghrelin ( 4 nmol ) group identification of ghrelin and its receptors in hypothalamus , pituitary and gonads ( 38 ) and the subsequent demonstration of the facilitative effects of hormones secreted from these tissues on reproductive behavior ( 10 , 1316 , 18 ) led us to hypothesize that in addition to affecting on hpg axis , ghrelin may also alter the sexual behavior . this is the first study to analyze the effects of ghrelin and dls ( [ d - lys]-ghrp-6 ) on male sexual behavior in any mammalian species . present results show that ghrelin injection distorted some aspects of sexual behavior and reduced lh subunit gene expression while enhanced loco - motor activity of male rats . in this study , it was found that ghrelin has similar inhibitory effects on sexual behavior in male rats as previously reported for female mice in a dose - dependent manner ( 26 ) . in this study , despite the decreased number of intromission and ejaculation in ghrelin - received animals , the number of mounts increased in these rats . presumably , increasing the number of mounts in ghrelin - received animals due to the effects of this peptide on locomotor activity may be a compensatory mechanism to prevent the decreasing of locomotor activity during the intercourse . previously , a number of studies have been conducted into the effect of ghrelin on locomotion indicating the facilitative effects of this peptide on locomotor activity ( 28 , 29 ) . considerable decrease in the number of ejaculation as the main factor of success in reproduction process caused a significant decrease in copulatory efficiency in ghrelin - received animals . also , shah and nyby showed that ghrelin quickly suppressed the androgen - dependent behaviors including the ultrasonic mating calls to a female and the latency to attack a stimulus male through its direct effects on the brain in male house mice , mus musculus ( 36 ) . additionally , in this study dls , the ghsr-1a antagonist , abolished the prejudicial effects of ghrelin on sexual behavior . the sexual behavior aspects under dls treatment alone had no significant differences with those in saline - treated rats , while dls modified the behavioral effects of ghrelin in co - administrated animals . it was also revealed that ghrelin and dls had regulatory effects on lh subunit gene expression in male rats . furthermore , the dose dependent effects of ghrelin were observed on both sexual behavior and locomotor activity in male rats . presumably , ghrelin as a starvation hormone in low physiological doses has a negligible effect on mating behavior but continuity of ghrelin release has a remarkable effect on reproductive behavior . in this study , copulatory actions of ghrelin and its specific antagonist , dls , were examined . to do so , behavioral tests and lh subunit gene expression assessment were implemented in male rats after injection of different doses of ghrelin or its antagonist through the central rout of administration .

four - month - old whole - body ampk2 mice ( 29 ) backcrossed to c57bl/6j mice for nine generations , and wild - type littermate controls were fed on either chow , chf , or chf+f diet for nine weeks . body weight and food consumption were recorded , and edta - plasma and tissues were collected for various analyses as described in the online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1716/dc1 . male mice were used for all the experiments , except for the measurements of hepatic ampk activity , which were performed on female mice . the experiments were conducted under the guidelines for the use and care of laboratory animals of the institute of physiology and followed the principles of laboratory animal care ( national institutes of health publication no . 85 - 23 , revised 1985 ) . nonesterified fatty acids ( nefas ) , triglycerides , and total cholesterol were determined in plasma using the following enzymatic photometric tests : nefa - c ( wako chemicals , neuss , germany ) , triacylglycerols liquid , and cholesterol liquid ( pliva - lachema diagnostika , brno , czech republic ) , respectively . plasma insulin was measured using the sensitive rat insulin ria kit ( linco research , st . total adiponectin levels and adiponectin multimeric complexes were determined using western blotting ( 31 ) . the tissue content of triglycerides was estimated in ethanolic koh tissue lysates as described before ( 8) . the content and fatty acid composition of the phospholipid , diacylglycerol , triglyceride , and ceramide fractions were assessed in tissue lipid extracts ; gene expression was evaluated using real - time rt - pcr ( see online appendix ) . livers were collected by freeze - clamping , ampk was immunoprecipitated from tissue extracts , and the activity was assayed using a peptide substrate ( 32 ) ; see the online appendix . five days before the experiment , an indwelling catheter was placed into the left femoral vein under anesthesia ( 33 ) . mice were allowed to recover for 57 days , followed by a 6-h fast ( 8:00 a.m.2:00 p.m. ) prior to the experiment . the whole - body glucose turnover was determined under basal ( nonstimulated ) and insulin - stimulated conditions ( hyperinsulinemic - euglycemic clamp ) , using separate groups of mice . insulin ( actrapid , novo nordisk pharma , denmark ) was infused at a constant rate of 4.8 mu / kgmin for 3 h , while d-[3-h]glucose ( perkin elmer , boston , ma ) was infused at a rate of 15.9 kbq / min . throughout the infusion , glucose concentration and d-[3-h]glucose specific activity ( during the last hour of infusion ) mmol / l ) was maintained by periodically adjusting a variable infusion of 33% glucose ( 33 ) . at the end of a 3-h infusion period , mice were first anesthetized by diethylether , exsanguinated through the cervical incision , and then killed by cervical dislocation , and tissues ( liver and quadriceps muscle ) and edta - plasma were collected for biochemical analyses ( see supplementary table 1 and research design and methods of the online appendix for details on basic clamp parameters and methodology ) . hepatocytes were isolated from livers of fed mice by a modification of the collagenase method ( 34 ) and seeded at a density of 0.5 10 cells per each 35-mm petri dish . rates of basal and insulin - stimulated de novo lipogenesis and aicar - stimulated fatty acid oxidation were measured using [ 1-c ] acetate and [ 1-c ] palmitate , respectively ( see online appendix ) . nonesterified fatty acids ( nefas ) , triglycerides , and total cholesterol were determined in plasma using the following enzymatic photometric tests : nefa - c ( wako chemicals , neuss , germany ) , triacylglycerols liquid , and cholesterol liquid ( pliva - lachema diagnostika , brno , czech republic ) , respectively . plasma insulin was measured using the sensitive rat insulin ria kit ( linco research , st . total adiponectin levels and adiponectin multimeric complexes were determined using western blotting ( 31 ) . the tissue content of triglycerides was estimated in ethanolic koh tissue lysates as described before ( 8) . the content and fatty acid composition of the phospholipid , diacylglycerol , triglyceride , and ceramide fractions were assessed in tissue lipid extracts ; gene expression was evaluated using real - time rt - pcr ( see online appendix ) . livers were collected by freeze - clamping , ampk was immunoprecipitated from tissue extracts , and the activity was assayed using a peptide substrate ( 32 ) ; see the online appendix . five days before the experiment , an indwelling catheter was placed into the left femoral vein under anesthesia ( 33 ) . mice were allowed to recover for 57 days , followed by a 6-h fast ( 8:00 a.m.2:00 p.m. ) prior to the experiment . the whole - body glucose turnover was determined under basal ( nonstimulated ) and insulin - stimulated conditions ( hyperinsulinemic - euglycemic clamp ) , using separate groups of mice . insulin ( actrapid , novo nordisk pharma , denmark ) was infused at a constant rate of 4.8 mu / kgmin for 3 h , while d-[3-h]glucose ( perkin elmer , boston , ma ) was infused at a rate of 15.9 kbq / min . throughout the infusion , glucose concentration and d-[3-h]glucose specific activity ( during the last hour of infusion ) mmol / l ) was maintained by periodically adjusting a variable infusion of 33% glucose ( 33 ) . at the end of a 3-h infusion period , mice were first anesthetized by diethylether , exsanguinated through the cervical incision , and then killed by cervical dislocation , and tissues ( liver and quadriceps muscle ) and edta - plasma were collected for biochemical analyses ( see supplementary table 1 and research design and methods of the online appendix for details on basic clamp parameters and methodology ) . hepatocytes were isolated from livers of fed mice by a modification of the collagenase method ( 34 ) and seeded at a density of 0.5 10 cells per each 35-mm petri dish . rates of basal and insulin - stimulated de novo lipogenesis and aicar - stimulated fatty acid oxidation were measured using [ 1-c ] acetate and [ 1-c ] palmitate , respectively ( see online appendix ) . specific activities of ampk1 and ampk2 were evaluated in the liver of ad libitum - fed mice after nine weeks of the differential dietary treatment ( fig . no significant effect of either diet ( chow , chf , and chf+f ) or genotype ( wild - type versus ampk2 ) on ampk1-specific activity was observed , although the ampk1 activity tended to be higher in the ampk2 mice ( fig . in contrast , ampk2 activity was stimulated by n-3 lc - pufas ( chf+f diet ; fig . no changes were detected in the activity of ampk1 and ampk2 in the quadriceps muscle in response to n-3 lc - pufas ( not shown ) . liver ampk1 ( a ) and ampk2 ( b ) activity in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks . the data are the means se ( n = 58 ) . in the ampk2 mice , * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf . at four months of age , at the beginning of dietary treatments , wild - type and ampk2 mice fed the chow diet exhibited similar body weights ( table 1 ) . in mice of both genotypes , chf - feeding for nine weeks resulted in greater body weight gain compared with the chow - fed mice . however , this effect was less pronounced in ampk2 mice ( table 1 ) . in both wild - type and ampk2 mice , the chf+f diet induced smaller body weight gain than the chf diet ( table 1 and supplementary figure 1 ) . none of the differences in body weight gain could be explained by caloric intake , which was similar in all experimental groups ( table 1 ) . the weight of fat depots increased in response to chf feeding , while the chf+f diet partially prevented this increase ( table 1 ) . triglycerides and nefa levels in plasma of ad libitum - fed mice were similar in the chow- and chf - fed mice , while cholesterol levels were markedly and significantly elevated by the chf diet . triglycerides and nefa levels were strongly reduced , even below the levels observed in the chow - fed mice ( table 1 ) . metabolic and plasma parameters in wild - type and ampk2 mice data are means se of 2730 mice for metabolic parameters and 1315 mice for other measures . ampk2 and wild - type mice were fed either a chow diet , chf , or chf+f for nine weeks . body weight gain ( see supplementary fig . 1 ) and plasma parameters were assessed in ad libitum - fed mice after nine weeks . plasma insulin levels were also assessed in fasted mice after eight weeks . at , adipose tissue ; a.u . , arbitary units ; hmw , total adiponectin , ratio of high molecular weight to total adiponectin ( for levels of all molecular weight forms of adiponectin ; nd , no data ; see supplementary fig . 2 ) ; * p < 0.05 vs. genotype chow ; p < 0.05 vs. genotype chf ; p < 0.05 vs. wild - type on respective diet . after nine weeks of dietary treatment , no change was observed in blood glucose , but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes . however , the increase in plasma insulin levels was less pronounced in ampk2 mice , closely reflecting the genotype - dependent differences in body weight gain ( table 1 ) . a similar pattern of changes in insulin levels was also observed in fasted mice , in which n-3 lc - pufas significantly reduced insulin levels only in wild - type animals ( table 1 ) . as expected , plasma levels of total as well as high molecular weight form of adiponectin , an adipokine associated with increased insulin sensitivity ( 35 ) , were increased 1.4- and 1.2-fold , respectively , in wild - type mice in response to n-3 lc - pufa supplementation ( table 1 and supplementary fig . 2 , available in an online appendix ) ; however , no significant increase of plasma adiponectin by n-3 lc - pufas was observed in ampk2 mice . in further experiments , under basal conditions , glucose turnover rate ( gto ; i.e. , glucose uptake in peripheral tissues ) was similar in all groups of mice ( supplementary table 1 ) . under insulin - stimulated conditions ( fig . f ) , the amount of exogenous glucose required to maintain euglycemia during the clamp , i.e. , the glucose infusion rate ( gir ) , was 1.3-fold lower in ampk2 than in wild - type mice fed the chow diet ( fig . gir was decreased by the chf diet to a similar level in mice of both genotypes , manifesting diet - induced insulin resistance . this was attributed to a decreased gto and , in particular , to an impaired suppression of hepatic glucose production ( hgp ) by insulin , with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice ( fig . 2c ) . in wild - type mice , chf+f diet feeding increased gir and gto ( 1.9- and 1.2-fold increase , respectively ) as compared with chf - fed mice , while hgp was lowered to a similar level as in the chow - fed mice . these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice , namely , at the level of hgp . in contrast , none of these beneficial effects of n-3 lc - pufas was observed in ampk2 mice , in which neither the gir ( fig . 2b ) differed between the chf+f - fed and the chf - fed mice , whereas the rate of hgp was even higher in the chf+f - fed than in the chf - fed ampk2 mice ( fig . 2c ) . 2d ) , the rate of whole - body glycogen synthesis , which reflects insulin sensitivity of muscle glucose metabolism , was dependent on both diet and genotype ( fig . 2e ) . in the chow - fed mice , the rate of whole - body glycogen synthesis tended to be higher in wild - type mice than in ampk2 mice . only in the former mice thus , in wild - type mice , glycogen synthesis was decreased 2.4-fold in response to the chf diet , while n-3 lc - pufas provided a partial protection from this decrease ( fig . a similar pattern of changes in the glycogen synthesis rate in response to n-3 lc - pufas was observed when measured directly in the skeletal muscle ( fig . thus , in accordance with the previous study ( 26 ) , the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice . however , ampk2 mice seemed to be partially protected against chf - induced insulin resistance , while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding . gir ( a ) , gto ( b ) , hgp ( c ) , whole - body glycolysis ( gl - wb ; d ) , whole - body glycogen synthesis ( gs - wb ; e ) ; and glycogen synthesis in quadriceps muscle ( gs - qm ; f ) were measured in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks . the data are the means se ( n = 58 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . in addition to the ad libitum - fed mice ( table 1 ) , plasma lipid levels were also measured in fasted mice , as well as in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 2 ) . in contrast to the ad libitum - fed state , chf+f diet did not affect either triglyceride or nefa levels under fasting conditions . under the hyperinsulinemic - euglycemic conditions , both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice ( 1.6-fold and 1.4-fold difference , respectively ) , but no such difference between the diets was observed in ampk2 mice . cholesterol levels were consistently decreased by n-3 lc - pufas independently of both the metabolic state and genotype ( supplementary table 2 ) . in ad libitum - fed mice of both genotypes , the hepatic triglyceride content was increased twofold by chf compared with the chow diet , while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes , documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas ( fig . 3a ) . under hyperinsulinemic - euglycemic conditions , n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides . however , this effect was absent in ampk2 mice ( fig . 3b ) . moreover , a strong correlation was found between plasma nefa levels and hepatic triglyceride content assessed under the clamp conditions in the chf+f - fed ampk2 mice ( r = 0.43 , p < 0.05 ) but not in wild - type mice ( r = 0.08 , p = 0.40 ) . triglyceride concentration in the livers of ad libitum - fed mice ( a ) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp ( b ) . wild - type and ampk2 mice were fed either a chow diet , chf , or chf+f for 9 weeks . the data are the means se ( a , n = 1315 ; b , n = 814 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . for the detailed fatty acid composition of triglyceride fractions in the livers of ad libitum - fed mice , see supplementary table 4 . we sought to determine whether the differential effect of n-3 lc - pufas on accumulation of liver triglycerides in wild - type and ampk2 mice under the clamp conditions could be explained by hepatic lipid metabolism . in cultured hepatocytes isolated from mice following the different dietary treatments , activities of both fatty acid oxidation and de novo fatty acid synthesis were evaluated . the stimulatory effects of 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ( aicar ) , an ampk activator , and insulin on fatty acid oxidation and synthesis are shown in fig . 4a and b , respectively ( for corresponding basal metabolic activities , see supplementary table 3 , available in an online appendix ) . in hepatocytes from both chow and chf diet - fed mice , the absence of ampk2 was associated with a trend for lower aicar - stimulated fatty acid oxidation . although hepatocytes from chf - fed mice showed reduced stimulatory effect of aicar irrespective of the genotype , chf+f feeding normalized this defect in wild - type but not in ampk2 hepatocytes ( fig . 5a ) , suggesting ampk - dependent induction of capacity for fatty acid oxidation by n-3 lc - pufas in the liver . the stimulatory effect of insulin on de novo fatty acid synthesis was reduced in hepatocytes from chf - fed wild - type mice , whereas it was retained in the hepatocytes from chf - fed ampk2 mice ( fig . chf+f feeding tended to restore the stimulatory effect of insulin only in wild - type hepatocytes ( fig . the effect of differential dietary treatment on the regulation of metabolic fluxes in the liver . aicar - stimulated fatty acid oxidation ( a ) and insulin - stimulated de novo fatty acid synthesis ( b ) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet , chf , or chf+f . for basal the expression of scd-1 ( c ) and srebp-1c ( d ) genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp following the differential dietary treatment for 9 weeks . the data are means se ( isolated hepatocytes , n = 3 in triplets ; hepatic gene expression , n = 58 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . , arbitrary units . the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids ( tfas ; a ) , pufas ( b ) , monounsaturated fatty acids ( mufas ; c ) , and saturated fatty acids ( sfas ; d ) . animals were fed either a chow diet , chf , or chf+f for 9 weeks . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . for the detailed fatty acid composition of diacylglycerol fractions in the livers of ad libitum fed mice , see supplementary table 4 . to further characterize hepatic effects of differential dietary treatment , the expression of selected genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp ( fig . 4c and d ) . feeding chf diet suppressed expression of lipogenic genes stearoyl - coa desaturase ( scd-1 ) and srebp-1c in all groups ( except for srebp-1c in ampk2 mice ) . this suppression was partially counteracted by chf+f diet in wild - type but not ampk2 mice ( fig . 4c and d ) . together with the de novo fatty acid synthesis data , these results further support the ampk2-dependent improvement of liver insulin sensitivity by n-3 lc - pufas . to identify factors predisposing animals to insulin resistance in an ampk2-dependent manner , no major genotype - dependent differences in the contents of either ceramides or phospholipids were observed ( supplementary table 4 ) . in contrast , hepatic content of diacylglycerols was affected in a genotype- and diet - dependent manner ( fig . wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice , while this effect of the chf+f diet was not observed in ampk2 mice . moreover , the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids ( fig . the increase in the pufa content tended to be smaller in the wild - type compared with ampk2 mice ( 1.7-fold and 2.2-fold , respectively ) . administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice , whereas their level in the ampk2 animals , although decreased , was still significantly higher compared with genotype - matched chow - fed mice ( fig . -linolenic acid ( 18:3n-3 ) appeared to be by far the most differentially regulated pufa in the diacylglycerol fraction in the two genotypes ( supplementary table 4 ) . in addition , chf+f diet markedly reduced hepatic content of monounsaturated diacylglycerols in wild - type but not in knockout animals ( fig . hepatic diacylglycerol levels and their fatty acid composition were also analyzed in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 5 and supplementary fig . no significant differences among the groups were observed in total diacylglycerols content or in their saturated or monounsaturated fatty acid fractions ( supplementary fig . specific activities of ampk1 and ampk2 were evaluated in the liver of ad libitum - fed mice after nine weeks of the differential dietary treatment ( fig . no significant effect of either diet ( chow , chf , and chf+f ) or genotype ( wild - type versus ampk2 ) on ampk1-specific activity was observed , although the ampk1 activity tended to be higher in the ampk2 mice ( fig . in contrast , ampk2 activity was stimulated by n-3 lc - pufas ( chf+f diet ; fig . no changes were detected in the activity of ampk1 and ampk2 in the quadriceps muscle in response to n-3 lc - pufas ( not shown ) . liver ampk1 ( a ) and ampk2 ( b ) activity in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks . the data are the means se ( n = 58 ) . in the ampk2 mice , at four months of age , at the beginning of dietary treatments , wild - type and ampk2 mice fed the chow diet exhibited similar body weights ( table 1 ) . in mice of both genotypes , chf - feeding for nine weeks resulted in greater body weight gain compared with the chow - fed mice . however , this effect was less pronounced in ampk2 mice ( table 1 ) . in both wild - type and ampk2 mice , the chf+f diet induced smaller body weight gain than the chf diet ( table 1 and supplementary figure 1 ) . none of the differences in body weight gain could be explained by caloric intake , which was similar in all experimental groups ( table 1 ) . the weight of fat depots increased in response to chf feeding , while the chf+f diet partially prevented this increase ( table 1 ) . triglycerides and nefa levels in plasma of ad libitum - fed mice were similar in the chow- and chf - fed mice , while cholesterol levels were markedly and significantly elevated by the chf diet . triglycerides and nefa levels were strongly reduced , even below the levels observed in the chow - fed mice ( table 1 ) . metabolic and plasma parameters in wild - type and ampk2 mice data are means se of 2730 mice for metabolic parameters and 1315 mice for other measures . ampk2 and wild - type mice were fed either a chow diet , chf , or chf+f for nine weeks . body weight gain ( see supplementary fig . 1 ) and plasma parameters were assessed in ad libitum - fed mice after nine weeks . plasma insulin levels were also assessed in fasted mice after eight weeks . at , adipose tissue ; a.u . , arbitary units ; hmw , total adiponectin , ratio of high molecular weight to total adiponectin ( for levels of all molecular weight forms of adiponectin ; nd , no data ; see supplementary fig . 2 ) ; * p < 0.05 vs. genotype chow ; p < 0.05 vs. genotype chf ; p < 0.05 vs. wild - type on respective diet . after nine weeks of dietary treatment , no change was observed in blood glucose , but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes . however , the increase in plasma insulin levels was less pronounced in ampk2 mice , closely reflecting the genotype - dependent differences in body weight gain ( table 1 ) . a similar pattern of changes in insulin levels was also observed in fasted mice , in which n-3 lc - pufas significantly reduced insulin levels only in wild - type animals ( table 1 ) . as expected , plasma levels of total as well as high molecular weight form of adiponectin , an adipokine associated with increased insulin sensitivity ( 35 ) , were increased 1.4- and 1.2-fold , respectively , in wild - type mice in response to n-3 lc - pufa supplementation ( table 1 and supplementary fig . 2 , available in an online appendix ) ; however , no significant increase of plasma adiponectin by n-3 lc - pufas was observed in ampk2 mice . in further experiments , hyperinsulinemic - euglycemic clamps were performed to evaluate whole - body insulin sensitivity . under basal conditions , glucose turnover rate ( gto ; i.e. , glucose uptake in peripheral tissues ) was similar in all groups of mice ( supplementary table 1 ) . under insulin - stimulated conditions ( fig . f ) , the amount of exogenous glucose required to maintain euglycemia during the clamp , i.e. , the glucose infusion rate ( gir ) , was 1.3-fold lower in ampk2 than in wild - type mice fed the chow diet ( fig gir was decreased by the chf diet to a similar level in mice of both genotypes , manifesting diet - induced insulin resistance . this was attributed to a decreased gto and , in particular , to an impaired suppression of hepatic glucose production ( hgp ) by insulin , with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice ( fig . 2c ) . in wild - type mice , chf+f diet feeding increased gir and gto ( 1.9- and 1.2-fold increase , respectively ) as compared with chf - fed mice , while hgp was lowered to a similar level as in the chow - fed mice . these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice , namely , at the level of hgp . in contrast , none of these beneficial effects of n-3 lc - pufas was observed in ampk2 mice , in which neither the gir ( fig . 2b ) differed between the chf+f - fed and the chf - fed mice , whereas the rate of hgp was even higher in the chf+f - fed than in the chf - fed ampk2 mice ( fig . 2c ) . 2d ) , the rate of whole - body glycogen synthesis , which reflects insulin sensitivity of muscle glucose metabolism , was dependent on both diet and genotype ( fig . the rate of whole - body glycogen synthesis tended to be higher in wild - type mice than in ampk2 mice . only in the former mice was it significantly affected by dietary treatment . thus , in wild - type mice , glycogen synthesis was decreased 2.4-fold in response to the chf diet , while n-3 lc - pufas provided a partial protection from this decrease ( fig . a similar pattern of changes in the glycogen synthesis rate in response to n-3 lc - pufas was observed when measured directly in the skeletal muscle ( fig . thus , in accordance with the previous study ( 26 ) , the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice . however , ampk2 mice seemed to be partially protected against chf - induced insulin resistance , while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding . gir ( a ) , gto ( b ) , hgp ( c ) , whole - body glycolysis ( gl - wb ; d ) , whole - body glycogen synthesis ( gs - wb ; e ) ; and glycogen synthesis in quadriceps muscle ( gs - qm ; f ) were measured in wild - type and ampk2 mice fed either a chow diet , chf , or chf+f for 9 weeks . p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . in addition to the ad libitum - fed mice ( table 1 ) , plasma lipid levels were also measured in fasted mice , as well as in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 2 ) . in contrast to the ad libitum - fed state , chf+f diet did not affect either triglyceride or nefa levels under fasting conditions . under the hyperinsulinemic - euglycemic conditions , both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice ( 1.6-fold and 1.4-fold difference , respectively ) , but cholesterol levels were consistently decreased by n-3 lc - pufas independently of both the metabolic state and genotype ( supplementary table 2 ) . in ad libitum - fed mice of both genotypes , the hepatic triglyceride content was increased twofold by chf compared with the chow diet , while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes , documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas ( fig . 3a ) . under hyperinsulinemic - euglycemic conditions , n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides . however 3b ) . moreover , a strong correlation was found between plasma nefa levels and hepatic triglyceride content assessed under the clamp conditions in the chf+f - fed ampk2 mice ( r = 0.43 , p < 0.05 ) but not in wild - type mice ( r = 0.08 , p = 0.40 ) . triglyceride concentration in the livers of ad libitum - fed mice ( a ) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp ( b ) . wild - type and ampk2 mice were fed either a chow diet , chf , or chf+f for 9 weeks . the data are the means se ( a , n = 1315 ; b , n = 814 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . for the detailed fatty acid composition of triglyceride fractions in the livers of ad libitum - fed mice , see supplementary table 4 . we sought to determine whether the differential effect of n-3 lc - pufas on accumulation of liver triglycerides in wild - type and ampk2 mice under the clamp conditions could be explained by hepatic lipid metabolism . in cultured hepatocytes isolated from mice following the different dietary treatments , activities of both fatty acid oxidation and de novo fatty acid synthesis were evaluated . the stimulatory effects of 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ( aicar ) , an ampk activator , and insulin on fatty acid oxidation and synthesis are shown in fig . 4a and b , respectively ( for corresponding basal metabolic activities , see supplementary table 3 , available in an online appendix ) . in hepatocytes from both chow and chf diet - fed mice , the absence of ampk2 was associated with a trend for lower aicar - stimulated fatty acid oxidation . although hepatocytes from chf - fed mice showed reduced stimulatory effect of aicar irrespective of the genotype , chf+f feeding normalized this defect in wild - type but not in ampk2 hepatocytes ( fig . 5a ) , suggesting ampk - dependent induction of capacity for fatty acid oxidation by n-3 lc - pufas in the liver . the stimulatory effect of insulin on de novo fatty acid synthesis was reduced in hepatocytes from chf - fed wild - type mice , whereas it was retained in the hepatocytes from chf - fed ampk2 mice ( fig . chf+f feeding tended to restore the stimulatory effect of insulin only in wild - type hepatocytes ( fig . the effect of differential dietary treatment on the regulation of metabolic fluxes in the liver . aicar - stimulated fatty acid oxidation ( a ) and insulin - stimulated de novo fatty acid synthesis ( b ) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet , chf , or chf+f . for basal the expression of scd-1 ( c ) and srebp-1c ( d ) genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp following the differential dietary treatment for 9 weeks . the data are means se ( isolated hepatocytes , n = 3 in triplets ; hepatic gene expression , n = 58 ) . * p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . a.u . , arbitrary units . the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids ( tfas ; a ) , pufas ( b ) , monounsaturated fatty acids ( mufas ; c ) , and saturated fatty acids ( sfas ; d ) . animals were fed either a chow diet , chf , or chf+f for 9 weeks . < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet . for the detailed fatty acid composition of diacylglycerol fractions in the livers of ad libitum fed mice , see supplementary table 4 . to further characterize hepatic effects of differential dietary treatment , the expression of selected genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp ( fig . feeding chf diet suppressed expression of lipogenic genes stearoyl - coa desaturase ( scd-1 ) and srebp-1c in all groups ( except for srebp-1c in ampk2 mice ) . this suppression was partially counteracted by chf+f diet in wild - type but not ampk2 mice ( fig . together with the de novo fatty acid synthesis data , these results further support the ampk2-dependent improvement of liver insulin sensitivity by n-3 lc - pufas . to identify factors predisposing animals to insulin resistance in an ampk2-dependent manner , a detailed analysis of hepatic lipids in ad libitum - fed mice was performed . no major genotype - dependent differences in the contents of either ceramides or phospholipids were observed ( supplementary table 4 ) . in contrast , hepatic content of diacylglycerols was affected in a genotype- and diet - dependent manner ( fig . wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice , while this effect of the chf+f diet was not observed in ampk2 mice . moreover , the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids ( fig . the increase in the pufa content tended to be smaller in the wild - type compared with ampk2 mice ( 1.7-fold and 2.2-fold , respectively ) . administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice , whereas their level in the ampk2 animals , although decreased , was still significantly higher compared with genotype - matched chow - fed mice ( fig . , -linolenic acid ( 18:3n-3 ) appeared to be by far the most differentially regulated pufa in the diacylglycerol fraction in the two genotypes ( supplementary table 4 ) . in addition , chf+f diet markedly reduced hepatic content of monounsaturated diacylglycerols in wild - type but not in knockout animals ( fig . hepatic diacylglycerol levels and their fatty acid composition were also analyzed in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 5 and supplementary fig . no significant differences among the groups were observed in total diacylglycerols content or in their saturated or monounsaturated fatty acid fractions ( supplementary fig . previous animal studies demonstrated that n-3 lc - pufas could counteract the development of both hepatic steatosis ( 8,18,36,37 ) and hepatic insulin resistance ( 8,9,16 ) , while suppressing lipogenesis and augmenting lipid catabolism in the liver ( 8,13,19,21 ) . using mice with a whole - body deletion of ampk2 and high - fat feeding , we show for the first time that ampk2 is required for the effect of n-3 lc - pufas to preserve whole - body , muscle , and especially hepatic insulin sensitivity , as well as to suppress hepatic and plasma triglycerides as well as nefa levels under hyperinsulinemic - euglycemic clamp conditions . in contrast , ampk2 was not required for protection by n-3 lc - pufas from hepatic lipid accumulation and dyslipidemia in ad libitum - fed mice . in addition to ampk2 , ppar was previously identified as an important determinant of n-3 lc - pufa 's effect on lipid metabolism , especially short - term modulation of hepatic gene expression ( 14 ) and insulin sensitivity ( 16 ) . thus , the reduction in hepatic triglyceride concentrations by fish oil feeding did not rescue insulin action in ppar-null mice , while hepatic diacylglycerol concentrations were decreased by fish oil in a ppar-dependent manner and were associated with a preserved hepatic insulin sensitivity ( 16 ) . it is generally accepted that 1 ) diacylglycerols rather than triglycerides or ceramides mediate hepatic insulin resistance in mice fed a high - fat diet ( 19,38,39 ) , 2 ) diacylglycerol - induced insulin resistance depends on activation of protein kinase c , and 3 ) that polyunsaturated diacylglycerols in particular are better protein kinase c activators than saturated diacylglycerol species [ reviewed in refs ( 38,39 ) ] . also our results showed that chf diet - induced insulin resistance was associated primarily with the accumulation of pufa in hepatic diacylglycerols and that n-3 lc - pufa completely prevented chf diet - induced increase in pufa diacylglycerols in wild - type mice , whereas in ampk2 animals , the content of these lipids was still significantly higher compared with the control . moreover , it was only in ad libitum - fed mice but not in mice subjected to hyperinsulinemic - euglycemic clamps that the levels of hepatic diacylglycerols and their fatty acid compositions were associated with hepatic insulin sensitivity . it is possible that under clamp conditions ampk2-dependent effects of n-3 lc - pufas on liver diacylglycerols were masked by metabolic changes occurring during a 3-h infusion of insulin and glucose . the failure of n-3 lc - pufas to decrease hepatic lipids in ampk2 mice under clamp conditions could be due to primary alterations in metabolic fluxes in the liver , reflecting 1 ) increased de novo fatty acid synthesis , 2 ) decreased secretion of vldl triglycerides , or 3 ) reduced fatty acid oxidation . de novo fatty acid synthesis was not the responsible factor , because hepatocytes of the n-3 lc - pufa - fed ampk2 mice showed decreased insulin - stimulated de novo fatty acid synthesis and reduced expression of srebp-1c and scd-1 , as compared with hepatocytes isolated from n-3 lc - pufa - fed wild - type mice , reflecting probably low insulin sensitivity of the liver in ampk2 mice . moreover , aicar - stimulated fatty acid oxidation in hepatocytes from n-3 lc - pufa - fed ampk2 mice was markedly reduced as compared with those from wild - type mice , suggesting decreased hepatic capacity for fatty acid oxidation in the absence of ampk2 , which could contribute to enhanced lipid accumulation . therefore , these experiments supported a major role of hepatic ampk2 in the regulation of both insulin sensitivity and lipid metabolism by n-3 lc - pufas . however , the differential modulation of lipid accumulation by n-3 lc - pufas in the livers of wild - type and ampk2 mice under clamp conditions could also be secondary to the ampk2-dependent effects of n-3 lc - pufas in other tissues , resulting in a relatively high hepatic uptake of circulating nefa in ampk2 mice . this is supported by persistently elevated plasma levels of nefa in ampk2 mice , as well as by a significant correlation between plasma nefa levels and hepatic triglyceride content observed under clamp conditions in ampk2 but not wild - type mice fed n-3 lc - pufa - containing diet . moreover , it has been shown in humans with nonalcoholic fatty liver disease that most of hepatic triglycerides arise from circulating nefa ( 40 ) . that plasma nefa levels under clamp conditions were reduced only in wild - type but not in ampk2 mice fed n-3 lc - pufas may reflect a role of ampk2 in muscle lipid uptake mediated by lipoprotein lipase ( 41 ) , as well as the antilipolytic effect of ampk in adipose tissue , documented for ampk1 ( 42 ) . in any case , decreased fatty acid oxidation in situ in the liver and , possibly even more importantly , abundant supply of circulating nefa could be responsible for the lack of the antisteatotic effect of n-3 lc - pufas in ampk2 mice under clamp conditions ( fig . . putative involvement of ampk2 in antisteatotic action of n-3 lc - pufas in the liver . dietary intake of n-3 lc - pufas increases the capacity of hepatocytes to oxidize fatty acids in wild - type ( left panels ) but not in ampk2 mice ( right panels ) . when insulin and glucose levels are high , such as during hyperinsulinemic - euglycemic clamp , wild - type mice fed n-3 lc - pufas exhibit improved hepatic insulin sensitivity and decreased plasma levels of nefas as compared with high - fat diet - fed controls . this is associated with increased expression of lipogenic genes such as srebp-1c and scd-1 and increased drive for de novo fatty acid synthesis . despite the elevated lipogenic drive under clamp conditions , the livers of wild - type mice fed n-3 lc - pufas show reduced accumulation of triglycerides . however , in ampk2 mice fed n-3 lc - pufas , hepatic triglyceride content is markedly elevated despite reduced rates of de novo fatty acid synthesis . this effect could be secondary to persisting elevated nefa levels in circulation and thus better substrate availability in ampk2 mice under clamp conditions . previous studies reported contradictory results , showing either 1 ) activation of ampk in rat liver ( 22 ) and murine adipose tissue ( 24 ) or 2 ) no changes in ampk activity in the liver , skeletal muscle , and heart of mice ( 43 ) in response to dietary n-3 lc - pufas . these discrepancies could be related to differences in dietary n-3 lc - pufa intake , nutritional state of animals , and other parameters . in accordance with the involvement of ampk2 in various effects of n-3 lc - pufas , our results document activation of ampk2 ( but not ampk1 ) in the liver of mice by long - term n-3 lc - pufa treatment , in the absence of significant changes in either the amp to atp ratio assessed in whole liver extracts [ not shown and ref ( 44 ) ] or the phosphorylation status of lkb1 , an upstream kinase for ampk [ not shown and ref ( 45 ) ] . in addition , no effect on ampk activity in either cultured hepatocytes ( 21 ) or embryonic kidney cells ( not shown ) of n-3 lc - pufas added to the cell culture medium could be detected . therefore , the activation of ampk2 by n-3 lc - pufas probably does not depend on a direct interaction between n-3 lc - pufas and ampk . on the other hand , induction of adiponectin by n-3 lc - pufas [ results of this study and refs ( 46,47 ) ] could be involved , because adiponectin activates ampk in both the liver and skeletal muscle ( 35 ) . adiponectin is also required for the activation of ampk upon administration of ppar agonists thiazolidinediones , whereas mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to these compounds ( 48 ) . thus , absence of ampk2 may blunt adiponectin - mediated effects of n-3 lc - pufas . in accordance with the previous study ( 49 ) moreover , the induction of adiponectin by n-3 lc - pufas in ampk2 mice was compromised ( table 1 and supplementary figure 2 ) . ampk1 and ampk2 contribute equally to total ampk activity in the liver ( 50 ) . in mice with liver - specific ablation of ampk2 ( 27 ) , hepatic ampk2 was essential for suppressing hepatic glucose production and maintaining fasting blood glucose levels ; however , the absence of ampk2 did not affect inhibitory action of insulin on hepatic glucose production . in our study , although fasting blood glucose levels were unaltered by whole - body ablation of ampk2 , the beneficial effect of dietary n-3 lc - pufas on hepatic insulin sensitivity was clearly ampk2-dependent . differential regulation of glucose homeostasis in the above transgenic models likely reflects the complexity of whole - body ( 26 ) versus liver - specific ( 27 ) deletion of ampk2 . in contrast to the previous report , showing induction of adiposity and adipocyte hypertrophy in ampk2 mice fed a lard - based high - fat diet ( 49 ) , our study documented a relatively low weight gain , low adiposity , and smaller fat cells in ampk2 mice fed a corn - oil based high - fat diet . this discrepancy could be related to the differences in the composition of experimental high - fat diets ; however , our results are consistent with the elevated sympathetic tonus of ampk2 mice ( 26 ) , which may stimulate energy dissipation in these animals . in any case , lower body weight of chf - fed ampk2 mice as compared with their wild - type counterparts could be related to better insulin sensitivity of the former mice , as suggested by the differences in insulinemia , results of hyperinsulinemic - euglycemic clamp , stimulatory effect of insulin on lipogenesis , and expression of lipogenic genes in the liver . in conclusion , the preservation of hepatic insulin sensitivity by n-3 lc - pufas in mice fed a high - fat diet depends on ampk2 . the accumulation of diacylglycerols , which is regulated in an ampk2-dependent manner , could contribute to the modulation of hepatic insulin sensitivity in response to dietary n-3 lc - pufas . on the other hand , the ampk2-dependent acute changes in lipid metabolism and hepatic triglyceride accumulation , which are unmasked under insulin - stimulated conditions such as during hyperinsulinemic - euglycemic clamp , largely reflect the extrahepatic action of n-3 lc - pufas . our results are relevant for the development of novel strategies for prevention and treatment of the metabolic syndrome .

objectivethe induction of obesity , dyslipidemia , and insulin resistance by high - fat diet in rodents can be prevented by n-3 long - chain polyunsaturated fatty acids ( lc - pufas ) . we tested a hypothesis whether amp - activated protein kinase ( ampk ) has a role in the beneficial effects of n-3 lc-pufas.research design and methodsmice with a whole - body deletion of the 2 catalytic subunit of ampk ( ampk2/ ) and their wild - type littermates were fed on either a low - fat chow , or a corn oil - based high - fat diet ( chf ) , or a chf diet with 15% lipids replaced by n-3 lc - pufa concentrate ( chf+f).resultsfeeding a chf diet induced obesity , dyslipidemia , hepatic steatosis , and whole - body insulin resistance in mice of both genotypes . although chf+f feeding increased hepatic ampk2 activity , the body weight gain , dyslipidemia , and the accumulation of hepatic triglycerides were prevented by the chf+f diet to a similar degree in both ampk2/ and wild - type mice in ad libitum - fed state . however , preservation of hepatic insulin sensitivity by n-3 lc - pufas required functional ampk2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content . under hyperinsulinemic - euglycemic conditions , ampk2 was essential for preserving low levels of both hepatic and plasma triglycerides , as well as plasma free fatty acids , in response to the n-3 lc - pufa treatment.conclusionsour results show that n-3 lc - pufas prevent hepatic insulin resistance in an ampk2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity . ampk2 is also essential for hypolipidemic and antisteatotic effects of n-3 lc - pufa under insulin - stimulated conditions .

RESEARCH DESIGN AND METHODS Blood and plasma parameters. Lipid content and gene expression in the liver. Activity of 1 and 2 AMPK isoforms. Hyperinsulinemic-euglycemic clamp. Primary cultures of hepatocytes. Statistics. RESULTS Enhancement of hepatic AMPK2 activity by n-3 LC-PUFAs. AMPK2 is not required for antiobesity and hypolipidemic effects of n-3 LC-PUFAs in ad libitum-fed mice. AMPK2 is essential for the preservation of insulin sensitivity in response to n-3 LC-PUFAs. Unmasking the role of AMPK2 in the lipid-lowering effect of n-3 LC-PUFAs under hyperinsulinemic-euglycemic conditions. Dietary n-3 LC-PUFAs increase 5-aminoimidazole-4-carboxamide-1-- Changes in hepatic diacylglycerol levels are associated with insulin-sensitizing effects of n-3 LC-PUFAs. DISCUSSION Supplementary Material

four - month - old whole - body ampk2 mice ( 29 ) backcrossed to c57bl/6j mice for nine generations , and wild - type littermate controls were fed on either chow , chf , or chf+f diet for nine weeks . the whole - body glucose turnover was determined under basal ( nonstimulated ) and insulin - stimulated conditions ( hyperinsulinemic - euglycemic clamp ) , using separate groups of mice . the whole - body glucose turnover was determined under basal ( nonstimulated ) and insulin - stimulated conditions ( hyperinsulinemic - euglycemic clamp ) , using separate groups of mice . in mice of both genotypes , chf - feeding for nine weeks resulted in greater body weight gain compared with the chow - fed mice . in both wild - type and ampk2 mice , the chf+f diet induced smaller body weight gain than the chf diet ( table 1 and supplementary figure 1 ) . after nine weeks of dietary treatment , no change was observed in blood glucose , but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes . as expected , plasma levels of total as well as high molecular weight form of adiponectin , an adipokine associated with increased insulin sensitivity ( 35 ) , were increased 1.4- and 1.2-fold , respectively , in wild - type mice in response to n-3 lc - pufa supplementation ( table 1 and supplementary fig . gir was decreased by the chf diet to a similar level in mice of both genotypes , manifesting diet - induced insulin resistance . this was attributed to a decreased gto and , in particular , to an impaired suppression of hepatic glucose production ( hgp ) by insulin , with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice ( fig . in wild - type mice , chf+f diet feeding increased gir and gto ( 1.9- and 1.2-fold increase , respectively ) as compared with chf - fed mice , while hgp was lowered to a similar level as in the chow - fed mice . these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice , namely , at the level of hgp . only in the former mice thus , in wild - type mice , glycogen synthesis was decreased 2.4-fold in response to the chf diet , while n-3 lc - pufas provided a partial protection from this decrease ( fig . thus , in accordance with the previous study ( 26 ) , the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice . however , ampk2 mice seemed to be partially protected against chf - induced insulin resistance , while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding . in addition to the ad libitum - fed mice ( table 1 ) , plasma lipid levels were also measured in fasted mice , as well as in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 2 ) . under the hyperinsulinemic - euglycemic conditions , both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice ( 1.6-fold and 1.4-fold difference , respectively ) , but no such difference between the diets was observed in ampk2 mice . in ad libitum - fed mice of both genotypes , the hepatic triglyceride content was increased twofold by chf compared with the chow diet , while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes , documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas ( fig . under hyperinsulinemic - euglycemic conditions , n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides . triglyceride concentration in the livers of ad libitum - fed mice ( a ) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp ( b ) . aicar - stimulated fatty acid oxidation ( a ) and insulin - stimulated de novo fatty acid synthesis ( b ) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet , chf , or chf+f . the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids ( tfas ; a ) , pufas ( b ) , monounsaturated fatty acids ( mufas ; c ) , and saturated fatty acids ( sfas ; d ) . wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice , while this effect of the chf+f diet was not observed in ampk2 mice . moreover , the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids ( fig . administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice , whereas their level in the ampk2 animals , although decreased , was still significantly higher compared with genotype - matched chow - fed mice ( fig . in both wild - type and ampk2 mice , the chf+f diet induced smaller body weight gain than the chf diet ( table 1 and supplementary figure 1 ) . after nine weeks of dietary treatment , no change was observed in blood glucose , but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes . as expected , plasma levels of total as well as high molecular weight form of adiponectin , an adipokine associated with increased insulin sensitivity ( 35 ) , were increased 1.4- and 1.2-fold , respectively , in wild - type mice in response to n-3 lc - pufa supplementation ( table 1 and supplementary fig . , the glucose infusion rate ( gir ) , was 1.3-fold lower in ampk2 than in wild - type mice fed the chow diet ( fig gir was decreased by the chf diet to a similar level in mice of both genotypes , manifesting diet - induced insulin resistance . this was attributed to a decreased gto and , in particular , to an impaired suppression of hepatic glucose production ( hgp ) by insulin , with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice ( fig . in wild - type mice , chf+f diet feeding increased gir and gto ( 1.9- and 1.2-fold increase , respectively ) as compared with chf - fed mice , while hgp was lowered to a similar level as in the chow - fed mice . these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice , namely , at the level of hgp . thus , in wild - type mice , glycogen synthesis was decreased 2.4-fold in response to the chf diet , while n-3 lc - pufas provided a partial protection from this decrease ( fig . thus , in accordance with the previous study ( 26 ) , the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice . however , ampk2 mice seemed to be partially protected against chf - induced insulin resistance , while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding . in addition to the ad libitum - fed mice ( table 1 ) , plasma lipid levels were also measured in fasted mice , as well as in mice subjected to hyperinsulinemic - euglycemic clamp ( supplementary table 2 ) . under the hyperinsulinemic - euglycemic conditions , both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice ( 1.6-fold and 1.4-fold difference , respectively ) , but cholesterol levels were consistently decreased by n-3 lc - pufas independently of both the metabolic state and genotype ( supplementary table 2 ) . in ad libitum - fed mice of both genotypes , the hepatic triglyceride content was increased twofold by chf compared with the chow diet , while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes , documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas ( fig . under hyperinsulinemic - euglycemic conditions , n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides . triglyceride concentration in the livers of ad libitum - fed mice ( a ) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp ( b ) . aicar - stimulated fatty acid oxidation ( a ) and insulin - stimulated de novo fatty acid synthesis ( b ) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet , chf , or chf+f . the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids ( tfas ; a ) , pufas ( b ) , monounsaturated fatty acids ( mufas ; c ) , and saturated fatty acids ( sfas ; d ) . together with the de novo fatty acid synthesis data , these results further support the ampk2-dependent improvement of liver insulin sensitivity by n-3 lc - pufas . to identify factors predisposing animals to insulin resistance in an ampk2-dependent manner , a detailed analysis of hepatic lipids in ad libitum - fed mice was performed . wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice , while this effect of the chf+f diet was not observed in ampk2 mice . moreover , the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids ( fig . administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice , whereas their level in the ampk2 animals , although decreased , was still significantly higher compared with genotype - matched chow - fed mice ( fig . previous animal studies demonstrated that n-3 lc - pufas could counteract the development of both hepatic steatosis ( 8,18,36,37 ) and hepatic insulin resistance ( 8,9,16 ) , while suppressing lipogenesis and augmenting lipid catabolism in the liver ( 8,13,19,21 ) . using mice with a whole - body deletion of ampk2 and high - fat feeding , we show for the first time that ampk2 is required for the effect of n-3 lc - pufas to preserve whole - body , muscle , and especially hepatic insulin sensitivity , as well as to suppress hepatic and plasma triglycerides as well as nefa levels under hyperinsulinemic - euglycemic clamp conditions . in contrast , ampk2 was not required for protection by n-3 lc - pufas from hepatic lipid accumulation and dyslipidemia in ad libitum - fed mice . in addition to ampk2 , ppar was previously identified as an important determinant of n-3 lc - pufa 's effect on lipid metabolism , especially short - term modulation of hepatic gene expression ( 14 ) and insulin sensitivity ( 16 ) . it is generally accepted that 1 ) diacylglycerols rather than triglycerides or ceramides mediate hepatic insulin resistance in mice fed a high - fat diet ( 19,38,39 ) , 2 ) diacylglycerol - induced insulin resistance depends on activation of protein kinase c , and 3 ) that polyunsaturated diacylglycerols in particular are better protein kinase c activators than saturated diacylglycerol species [ reviewed in refs ( 38,39 ) ] . also our results showed that chf diet - induced insulin resistance was associated primarily with the accumulation of pufa in hepatic diacylglycerols and that n-3 lc - pufa completely prevented chf diet - induced increase in pufa diacylglycerols in wild - type mice , whereas in ampk2 animals , the content of these lipids was still significantly higher compared with the control . moreover , it was only in ad libitum - fed mice but not in mice subjected to hyperinsulinemic - euglycemic clamps that the levels of hepatic diacylglycerols and their fatty acid compositions were associated with hepatic insulin sensitivity . de novo fatty acid synthesis was not the responsible factor , because hepatocytes of the n-3 lc - pufa - fed ampk2 mice showed decreased insulin - stimulated de novo fatty acid synthesis and reduced expression of srebp-1c and scd-1 , as compared with hepatocytes isolated from n-3 lc - pufa - fed wild - type mice , reflecting probably low insulin sensitivity of the liver in ampk2 mice . moreover , aicar - stimulated fatty acid oxidation in hepatocytes from n-3 lc - pufa - fed ampk2 mice was markedly reduced as compared with those from wild - type mice , suggesting decreased hepatic capacity for fatty acid oxidation in the absence of ampk2 , which could contribute to enhanced lipid accumulation . therefore , these experiments supported a major role of hepatic ampk2 in the regulation of both insulin sensitivity and lipid metabolism by n-3 lc - pufas . however , the differential modulation of lipid accumulation by n-3 lc - pufas in the livers of wild - type and ampk2 mice under clamp conditions could also be secondary to the ampk2-dependent effects of n-3 lc - pufas in other tissues , resulting in a relatively high hepatic uptake of circulating nefa in ampk2 mice . this is supported by persistently elevated plasma levels of nefa in ampk2 mice , as well as by a significant correlation between plasma nefa levels and hepatic triglyceride content observed under clamp conditions in ampk2 but not wild - type mice fed n-3 lc - pufa - containing diet . that plasma nefa levels under clamp conditions were reduced only in wild - type but not in ampk2 mice fed n-3 lc - pufas may reflect a role of ampk2 in muscle lipid uptake mediated by lipoprotein lipase ( 41 ) , as well as the antilipolytic effect of ampk in adipose tissue , documented for ampk1 ( 42 ) . when insulin and glucose levels are high , such as during hyperinsulinemic - euglycemic clamp , wild - type mice fed n-3 lc - pufas exhibit improved hepatic insulin sensitivity and decreased plasma levels of nefas as compared with high - fat diet - fed controls . despite the elevated lipogenic drive under clamp conditions , the livers of wild - type mice fed n-3 lc - pufas show reduced accumulation of triglycerides . previous studies reported contradictory results , showing either 1 ) activation of ampk in rat liver ( 22 ) and murine adipose tissue ( 24 ) or 2 ) no changes in ampk activity in the liver , skeletal muscle , and heart of mice ( 43 ) in response to dietary n-3 lc - pufas . in accordance with the involvement of ampk2 in various effects of n-3 lc - pufas , our results document activation of ampk2 ( but not ampk1 ) in the liver of mice by long - term n-3 lc - pufa treatment , in the absence of significant changes in either the amp to atp ratio assessed in whole liver extracts [ not shown and ref ( 44 ) ] or the phosphorylation status of lkb1 , an upstream kinase for ampk [ not shown and ref ( 45 ) ] . in accordance with the previous study ( 49 ) moreover , the induction of adiponectin by n-3 lc - pufas in ampk2 mice was compromised ( table 1 and supplementary figure 2 ) . in mice with liver - specific ablation of ampk2 ( 27 ) , hepatic ampk2 was essential for suppressing hepatic glucose production and maintaining fasting blood glucose levels ; however , the absence of ampk2 did not affect inhibitory action of insulin on hepatic glucose production . in our study , although fasting blood glucose levels were unaltered by whole - body ablation of ampk2 , the beneficial effect of dietary n-3 lc - pufas on hepatic insulin sensitivity was clearly ampk2-dependent . in contrast to the previous report , showing induction of adiposity and adipocyte hypertrophy in ampk2 mice fed a lard - based high - fat diet ( 49 ) , our study documented a relatively low weight gain , low adiposity , and smaller fat cells in ampk2 mice fed a corn - oil based high - fat diet . in any case , lower body weight of chf - fed ampk2 mice as compared with their wild - type counterparts could be related to better insulin sensitivity of the former mice , as suggested by the differences in insulinemia , results of hyperinsulinemic - euglycemic clamp , stimulatory effect of insulin on lipogenesis , and expression of lipogenic genes in the liver . in conclusion , the preservation of hepatic insulin sensitivity by n-3 lc - pufas in mice fed a high - fat diet depends on ampk2 . the accumulation of diacylglycerols , which is regulated in an ampk2-dependent manner , could contribute to the modulation of hepatic insulin sensitivity in response to dietary n-3 lc - pufas . on the other hand , the ampk2-dependent acute changes in lipid metabolism and hepatic triglyceride accumulation , which are unmasked under insulin - stimulated conditions such as during hyperinsulinemic - euglycemic clamp , largely reflect the extrahepatic action of n-3 lc - pufas .

avaliar , em um modelo pulmonar simulando um paciente sob ventilao mecnica , a eficincia e a segurana da manobra de hiperinsuflao manual ( hm ) com o intuito de remover secreo pulmonar . oito fisioterapeutas utilizaram um ressuscitador manual autoinflvel para realizar hm com o objetivo de remover secrees , em duas condies : conforme rotineiramente aplicada durante sua prtica clnica , e aps receberem instrues verbais baseadas em recomendaes de especialistas . trs cenrios clnicos foram simulados : funo pulmonar normal , doena pulmonar restritiva e doena pulmonar obstrutiva . antes da instruo , o uso de duas compresses sequenciais do ressuscitador era comum , e a presso proximal ( pprox ) foi mais alta em relao obtida aps a instruo . entretanto , a presso alveolar ( palv ) nunca excedeu 42,5 cmh2o ( mediana , 16,1 ; intervalo interquartil [ iq ] , 11,7 - 24,5 ) , mesmo com valores de pprox de at 96,6 cmh2o ( mediana , 36,7 ; iq , 22,9 - 49,4 ) . o volume corrente ( vc ) gerado foi relativamente pequeno ( mediana , 640 ml ; iq , 505 - 735 ) e o pico de fluxo inspiratrio ( pfi ) geralmente excedeu o pico de fluxo expiratrio ( pfe ) : 1,37 l / s ( iq , 0,99 - 1,90 ) e 1,01 l / s ( iq , 0,55 - 1,28 ) , respectivamente . uma relao pfi / pfe < 0,9 ( que teoricamente favorece a migrao do muco em direo s vias areas centrais ) foi obtida em somente 16,7% das manobras . nas condies testadas , a hm gerou valores seguros de palv mesmo com altas pprox . entretanto , a hm foi comumente realizada de um modo que no favorecia a remoo de secreo ( pfi excedendo pfe ) mesmo aps a instruo . a relao pfi / pfe desfavorvel foi explicada pelas insuflaes rpidas e o baixo vc . manual hyperinflation ( mh ) is a proposed technique that is purported to promote secretion clearance and to re - expand areas of atelectasis , thereby improving lung compliance and oxygenation in patients on mechanical ventilation . despite a lack of scientific evidence confirming its benefits on clinical outcomes , mh the maneuver is widely accepted as effective and , in brazil , is largely embraced as a means of removing retained secretions . the rationale for the use of the technique as an aid for secretion removal is that it simulates a cough . when applied before suctioning , it theoretically moves secretions toward the central airways , thus increasing the efficacy of the suctioning procedure . it has been shown that the efficiency of the mh maneuver is affected by the operator and the type of manual resuscitator used , as well as by the resistance and compliance of the respiratory system , largely affecting the pressures and flows generated in the respiratory system . under certain conditions , the use of this technique can generate high peak airway pressures , increasing the risk of barotrauma . according to expert recommendations , in order to promote secretion clearance , mh should consist of a slow , deep inspiration , an inspiratory pause , and a quick release of the resuscitation bag to promote passive exhalation with high expiratory flow rates . however , the way in which the mh maneuver is performed can vary from country to country , impeding the understanding of its effects . since the 1980s , there has been increasing evidence that the peak expiratory flow to peak inspiratory flow ( pef / pif ) ratio is a critical factor for the removal of lung secretions , especially in heavily sedated or paralyzed patients . in fact , more than their ratio , the difference between the two , in terms of their absolute values , seems to be the major determinant : above a given threshold , whenever the pif exceeds the pef , secretions migrate deeper into the lung . intuitively , respiratory therapists ( rts ) have been promoting maneuvers to increase expiratory flows , analogous to those observed during coughing . however , relatively little attention has been given to their inspiratory counterpart , despite expert recommendations to apply a slow , deep inspiration during mh . the inspiratory phase of mh can be performed in many different ways , depending on the personal experience of the operator , and there is little evidence that the mh maneuver generates an adequate flow bias ( i.e. , with pef exceeding pif ) or that the ultimate goal of the maneuver ( i.e. , improved secretion clearance ) is achieved . the aim of this study was to evaluate the efficiency and safety of mh ( as performed by rts in a lung model simulating a mechanically ventilated patient ) as a means of increasing secretion clearance under two different conditions : mh performed in accordance with the routine clinical practice of rts ; and mh performed in accordance with expert recommendations . the efficiency of mh was determined through analysis of the volume and flow patterns generated in relation to the anticipated effects on secretion removal . the safety of mh was evaluated on the basis of the inspiratory pressures achieved ( i.e. , whether those pressures remained within safe limits during the maneuver ) . our ultimate objective was to obtain a qualitative analysis of how mh is performed by experienced professionals and not to address how it is performed in brazil . this was an experimental study conducted in the laboratory for medical research 09 , specializing in pulmonology , at the university of so paulo school of medicine , in the city of so paulo , brazil . the study was approved by the research ethics committee of the university of so paulo school of medicine hospital das clnicas . the study comprised two phases , evaluating the effects of mh , as performed by rts , in promoting the clearance of pulmonary secretions . in the first phase ( the pre - instruction phase ) , the rts were given no explicit verbal instructions on how to apply the maneuver . in the second phase , the rts were given instructions based on expert recommendations , as detailed below . the two phases are hereafter referred to as the pre- and post - instruction phases . all maneuvers were performed with self - inflating manual resuscitator ( spur ; ambu , ballerup , denmark ) , with a capacity of 1,500 ml . the maneuver was applied by eight experienced rts ( four males and four females ) , with an average of 2.6 years in practice ( range , 2 - 4 years ) in icus in the city of so paulo . five of the eight had graduated from universities located within the state of so paulo , and all had completed one of the one - year postgraduate training programs in respiratory therapy offered by university hospitals . the maneuver was applied to a mechanical model of the respiratory system known as a training and test lung ( ttl 2600 ; michigan instruments , grand rapids , mi ) , connected to a tracheal tube ( 8.5 mm ) . the airway flow was measured proximally ( between the manual resuscitator and proximal pressure sensor ) by a pneumotach . two pressure transducers were connected to the model : one to measure proximal pressure ( pprox - at the airway , between the flow sensor and tracheal tube ) ; and another to record alveolar pressure ( palv ) . study phases , we altered the resistance and compliance of the lung model in order to simulate three different clinical scenarios : a patient with obstructive lung disease ( resistance at 20 cmh2o / l per second and compliance at 0.08 l / cmh2o ) ; a normal patient ( resistance at 5 cmh2o / l per second and compliance at 0.05 l / cmh2o ) ; and a patient with restrictive lung disease ( resistance at 5 cmh2o / l per second and compliance at 0.025 l / cmh2o ) . in the pre - instruction phase ( as previously mentioned ) , the rts were instructed to perform mh to promote pulmonary secretion clearance in accordance with their routine clinical practice . the lung model was set according to those three clinical scenarios explained above and covered with a bed sheet , allowing rts to sense the movement of the bellows while remaining blinded to the clinical scenario selected . after approximately eight cycles of mh , the settings were changed to simulate the next scenario ( in the sequence described above ) until all three scenarios had been run . in the post - instruction phase , each rt received brief verbal instructions on how to perform mh according to expert recommendations , and all of the steps performed in the pre - instruction phase were repeated . the verbal instructions were given within the space of approximately 2 min , and the rts were not trained to follow a given pattern of insufflation . the instructions were always given by the same researcher , who instructed each rt as follows : " now you should perform mh with a slow inflation and a 2-s inspiratory pause , followed by rapid release of the bag " . while giving the instructions , the researcher demonstrated the maneuver with the manual resuscitator used in the study . the analog signals from the flow and pressure transducers were amplified , digitized , and recorded at 200 hz , using a data acquisition and off - line analysis system developed within the software laboratory virtual instrumentation and engineering workbench ( labview ; national instruments , austin , tx , usa ) . for each experimental condition , the beginning of the inspiratory phase was defined as zero flow immediately before bagging , and the end of the respiratory cycle was defined as zero flow at the end of exhalation . the pprox was defined as the peak pressure value during the inspiratory phase , measured by the proximal pressure transducer . the pif was defined as the peak flow value during the inspiratory phase , measured by the flow transducer , and the pef was defined as the peak flow during expiratory phase . the palv was defined as the peak pressure value during the inspiratory phase , measured by the alveolar pressure transducer . inspiratory time ( ti ) was defined as the duration of the inflation plus the inspiratory pause . repeated measures anova was used to evaluate , for each variable , the following within - subject factors : the three clinical scenarios ; and the two phases ( before and after instructions ) . inspiratory versus expiratory flow and alveolar versus proximal pressures were also tested as within - subject factors to compare peak flow and peak pressure variables , respectively . for each of the six experimental conditions ( three clinical scenarios in each study phase ) , we analyzed three respiratory cycles , corresponding to eighteen respiratory cycles for each of the eight rts . therefore , we analyzed a total of 144 respiratory cycles . among all of the respiratory cycles analyzed , the maximum palv observed was 42.5 cmh2o ( median , 16.1 cmh2o ; iqr , 11.7 - 24.5 ) , despite the much higher pprox values ( maximum , 96.6 cmh2o - median , 36.7 ; iqr , 22.9 - 49.4 ) . the vt values were relatively low ( maximum , 955 ml - median , 640 ; iqr , 505 - 735 ) , and the ti was short ( median , 1.29 s ; iqr , 0.95 - 1.72 ) . a pif / pef ratio < 0.9 , which theoretically favors mucus migration toward the central airways , was achieved in only 24 ( 16.7% ) of the 144 maneuvers evaluated . this favorable ratio occurred primarily in the post - instruction phase ( in 18 of the 24 maneuvers ) and was strongly associated with just one of the rts tested ( who was responsible for 12 of the 24 maneuvers ) . during the pre - instruction phase , six of the eight rts performed mh using two compressions of the resuscitator bag and produced a pif higher than that produced in the post - instruction phase ( figure 2 ) . figure 2differences in proximal pressures achieved by two different respiratory therapists ( a and b ) before and after explicit verbal instruction ( routine clinical practice vs. expert recommendations - dashed lines and solid lines , respectively ) . table 1 shows a comparison between the pre- and post - instruction phases , in terms of the mechanical variables evaluated . after instruction , mh was performed in a slower manner , with a longer ti and a lower pif , producing a lower pprox . there were no statistically significant differences between the two phases in terms of the palv , vt and pef . figure 2 illustrates the difference between the two study phases and between two different rts . table 1mechanical variables before and after verbal instruction ( routine clinical practice vs. expert recommendations).variablepre - instructionpost - instructionp proximal pressure ( cmh2o)44.5 ( 33.4 - 63.4)26.8 ( 18.6 - 37.5)0.004 alveolar pressure ( cmh2o)16.3 ( 11.6 - 25.8)14.8 ( 11.7 - 23.8)0.93 peak inspiratory flow ( l / s)1.84 ( 1.28 - 2.19)1.14 ( 0.87 - 1.44)0.001 peak expiratory flow ( l / s)1.04 ( 0.57 - 1.33)0.99 ( 0.55 - 1.27)0.28 tidal volume ( ml)628 ( 497 - 699)647 ( 518 - 746)0.63 inspiratory time ( s)0.95 ( 0.78 - 1.19)1.71 ( 1.44 - 2.13)<0.001 pif / pef ratio1.80 ( 1.29 - 2.34)1.15 ( 0.87 - 1.80)0.004values are expressed as median ( 25 - 75% interquartile range)pif : peak inspiratory flowpef : peak expiratory flow values are expressed as median ( 25 - 75% interquartile range ) : peak inspiratory flow : peak expiratory flow comparing all three clinical scenarios tested and the two phases of the study , we found that pprox was markedly higher than was palv in all instances , with the exception of the restrictive lung disease scenario in the post - instruction phase ( figure 3 ) . in the pre - instruction phase , in the post - instruction phase , the pif / pef ratio was closer to 1:1 . the only situation in which that ratio was consistently unfavorable ( pif still far exceeding pef ) was the obstructive lung disease scenario ( figure 4 ) . figure 3median values of proximal and alveolar pressures ( interquartile ranges as error bars ) obtained in the pre - instruction ( routine clinical practice ) and post - instruction ( in accordance with expert recommendations ) phases of the study . the dashed line indicates 30 cmh2o . * p < 0.01 ( difference between pre - instruction and post - instruction ) . # p < 0.05 ( difference between proximal and alveolar pressure ) . figure 4median values for peak inspiratory and expiratory flows ( interquartile ranges as error bars ) obtained in the pre - instruction ( routine clinical practice ) and post - instruction ( in accordance with expert recommendations ) phases of the study . p < 0.01 ( difference between pre - instruction and postinstruction ) . # p < 0.05 ( difference between peak inspiratory and expiratory flows ) . the major finding of the present study was that , even after receiving explicit instructions , the rts evaluated here performed mh in a way that probably would not aid secretion removal , with pif commonly exceeding pef . compressing the resuscitator bag multiple times , in rapid succession , resulted in high values of pif and pprox . however , this finding was typically associated with a relatively low vt , probably because of short bag - compression times . therefore , the palv was often low at the start of exhalation , resulting in a low pef . both factors ( rapid , multiple compressions and low palv ) appear to be responsible for the unfavorable relationship between pif and pef . finally , although the mh maneuver might not promote secretion clearance , our results suggest that this maneuver , as performed in our study , is unlikely to cause barotrauma , a concern expressed by other authors because of the high pprox it generates . in the present study , however , the palv values - roughly represented by plateau pressures , which correlate better with barotrauma than do pprox values - were within the safe range . the mh maneuver was originally described as consisting of a slow , deep inspiration , with an inspiratory pause and rapid release of the resuscitator bag . since then , many different mh techniques have been described , including one providing a vt that is greater than the baseline vt for the patient in question ; one providing a vt that is 50% greater than that delivered by the ventilator ; and one consisting of a slow ( 3-s ) inspiration to achieve a peak airway pressure of 40 cmh2o . in contrast , we found that rts working in the city of so paulo have customized the maneuver according to personal practice and bias , frequently applying two compressions of the resuscitator bag , without an inspiratory pause , resulting in pif being higher than pef . one possible explanation is that the maneuver applied in that way stimulates coughing and , consequently , improves secretion clearance , or at least gives the rt that impression . as observed in this study , however , applying the maneuver in that way might make it ineffective , with unfavorable relationships between inspiratory and expiratory flows , especially if the patient has a depressed cough reflex or is unable to cough efficiently . according to previous studies , above a given flow threshold , the direction of mucus transport ( in or out of the respiratory system ) is governed by the highest peak flow : a pef 10% higher than the pif ( i.e. , a pif / pef ratio < 0.9 ) will favor secretion movement from the distal to the central airways . a recent study evaluating the transport of artificial mucus in a test lung system reported that mucus transport is better explained by the absolute difference between pif and pef than by the pif / pef ratio . when pef is higher than pif , a greater difference between the two translates to better mucus transport . in the present study , the pif / pef ratio was below 0.9 in only 24 ( 16.7% ) of the 144 maneuvers evaluated , most of those 24 being performed in the post - instruction phase . consequently , the median pif was much higher than was the median pef , which is a cause for great concern . this disappointing result is in agreement with the findings of some other studies in which self - inflating resuscitators were also used . the manual resuscitator tested in the present study had a self - inflating bag ( the type of manual resuscitator most widely used in icus in brazil ) . such resuscitators usually generate a lower vt than that achieved with resuscitators that have flow - inflating bags . even after receiving explicit instructions , the rts produced vt values that were lower than those reported in other studies . that difference might be related solely to the size of the bag employed : 1.5 l in the present study , compared with 1.6 - 2.0 liters in the other studies cited . much more favorable pif / pef ratios have been reported when flow - inflating devices were used . the verbal instruction on how to perform the mh improved the performance of the rts in that the post - instruction maneuvers generated lower pifs and longer tis . however , those differences did little to improve the efficiency of the technique , because the pefs were still quite low . that is likely attributable to the fact that the palv was also quite low at the start of exhalation , which resulted in a low driving pressure for expiratory flow . other authors have reported great variability in the practical implementation of the mh maneuver , the execution of which rarely follows its original description . this makes it practically impossible to compare the effectiveness of mh across clinical studies . if we accept the concept that the relationship between pif and pef is responsible for the direction in which secretions move , we should question the use of an inspiratory pause on physiological grounds . because of stress relaxation , the effective driving pressure for flow after a pause will be always lower than immediately after end - inspiration . therefore the use of an inspiratory pause could decrease the pef and , consequently , impair the efficiency of the mh maneuver . the small number of rts participating in this study prevents us from generalizing our data for application in clinical practice . however , we believe that this qualitative analysis , performed with representative operators ( of both genders , recruited from different hospitals from within the same city ) , illustrated scenarios that commonly occur in some icus . another limitation of this study was the use of a lung model , which can not be extrapolated to the complexity of human lungs . nevertheless , we believe that similar results - high pif / pef ratios , low vt , and high pprox - would also be obtained in human patients on mechanical ventilation , especially in those that are heavily sedated , and this should draw the attention of the rts to the way in which they perform the mh maneuver . whether applying high pif during mh will enhance secretion clearance in patients with preserved cough is a question that merits further investigation . that notwithstanding , the key message here is that performing mh with high pif / pef ratios in patients who are unable to cough will not aid secretion removal and might even contribute to mucus retention . another point that should be mentioned is that the mh maneuver can be performed in combination with expiratory chest compression in order to maximize the increase in the pef ; however we were not able to investigate the use of that combination , because of the experimental system used . in addition , as previously mentioned , we can not discard the possibility that the use of a different resuscitator device ( a self - inflating resuscitator with larger internal volume bags or a flow - inflating resuscitator ) might produce better results . given that , in brazil , mh is typically performed without pprox monitoring , the option of performing the maneuver with a mechanical ventilator , so that inspiratory flow , vt and pressure can be easily monitored and adjusted , should be considered . in studies comparing mh with a manual resuscitator and mh with a mechanical ventilator , no differences were found between the two modalities in terms of the amount of secretion removed , although the advantages of better monitoring with the mechanical ventilator were acknowledged . it is important to note that , although the mh maneuver was originally designed to reduce lung collapse and to improve oxygenation or lung compliance , those potential benefits were not tested here . in fact , we believe that mh should be used only as a secretion clearance technique . if the technique is applied to recruit collapsed lungs , its beneficial effects are going to be offset , at least in part , by the fact that the patient must be disconnected from the ventilator , thus exposing the lung to the lower pressure of the ambient air at the end of each compression . finally , we found no evidence that the mh maneuver , as performed here , increases the risk of barotrauma . the palv value is a result of the insufflating volume and lung compliance , pprox values that are higher than the palv although there have been anecdotal reports of high pprox during mh in adults , with volumes < 1 l , there is no hard evidence that the maneuver is dangerous . in conclusion , when asked to apply mh in accordance with their routine clinical practice , this small sample of rts performed the maneuver quite differently from what is recommended by experts , producing a concerning pattern of ventilation in terms of secretion clearance . the repetition of the maneuver after explicit instructions reduced the pprox but did not help much . alveolar pressures were usually low ( because of the small generated vt ) despite high proximal pressures and high inspiratory peak flows . as a result , pef was also low , far lower than the preceding pif , a condition theoretically impacting the secretions deeper into the lung . further studies are necessary in this area , especially focusing on the use of flow - inflating devices delivering higher tidal volumes .

objective : to evaluate , in a lung model simulating a mechanically ventilated patient , the efficiency and safety of the manual hyperinflation ( mh ) maneuver as a means of removing pulmonary secretions . methods : eight respiratory therapists ( rts ) were asked to use a self - inflating manual resuscitator on a lung model to perform mh as if to remove secretions , under two conditions : as routinely applied during their clinical practice ; and after receiving verbal instructions based on expert recommendations . in both conditions , three clinical scenarios were simulated : normal lung function , restrictive lung disease , and obstructive lung disease . results : before instruction , it was common for an rt to compress the resuscitator bag two times , in rapid succession . proximal pressure ( pprox ) was higher before instruction than after . however , alveolar pressure ( palv ) never exceeded 42.5 cmh2o ( median , 16.1 ; interquartile range [ iqr ] , 11.7 - 24.5 ) , despite pprox values as high as 96.6 cmh2o ( median , 36.7 ; iqr , 22.9 - 49.4 ) . the tidal volume ( vt ) generated was relatively low ( median , 640 ml ; iqr , 505 - 735 ) , and peak inspiratory flow ( pif ) often exceeded peak expiratory flow ( pef ) , the median values being 1.37 l / s ( iqr , 0.99 - 1.90 ) and 1.01 l / s ( iqr , 0.55 - 1.28 ) , respectively . a pif / pef ratio < 0.9 ( which theoretically favors mucus migration toward the central airways ) was achieved in only 16.7% of the maneuvers . conclusions : under the conditions tested , mh produced safe palv levels despite high pprox . however , the mh maneuver was often performed in a way that did not favor secretion removal ( pif exceeding pef ) , even after instruction . the unfavorable pif / pef ratio was attributable to overly rapid inflations and low vt .

OBJETIVO: MTODOS: RESULTADOS: CONCLUSES: Introduction Methods Results Discussion

antes da instruo , o uso de duas compresses sequenciais do ressuscitador era comum , e a presso proximal ( pprox ) foi mais alta em relao obtida aps a instruo . entretanto , a presso alveolar ( palv ) nunca excedeu 42,5 cmh2o ( mediana , 16,1 ; intervalo interquartil [ iq ] , 11,7 - 24,5 ) , mesmo com valores de pprox de at 96,6 cmh2o ( mediana , 36,7 ; iq , 22,9 - 49,4 ) . o volume corrente ( vc ) gerado foi relativamente pequeno ( mediana , 640 ml ; iq , 505 - 735 ) e o pico de fluxo inspiratrio ( pfi ) geralmente excedeu o pico de fluxo expiratrio ( pfe ) : 1,37 l / s ( iq , 0,99 - 1,90 ) e 1,01 l / s ( iq , 0,55 - 1,28 ) , respectivamente . manual hyperinflation ( mh ) is a proposed technique that is purported to promote secretion clearance and to re - expand areas of atelectasis , thereby improving lung compliance and oxygenation in patients on mechanical ventilation . despite a lack of scientific evidence confirming its benefits on clinical outcomes , mh the maneuver is widely accepted as effective and , in brazil , is largely embraced as a means of removing retained secretions . the rationale for the use of the technique as an aid for secretion removal is that it simulates a cough . when applied before suctioning , it theoretically moves secretions toward the central airways , thus increasing the efficacy of the suctioning procedure . it has been shown that the efficiency of the mh maneuver is affected by the operator and the type of manual resuscitator used , as well as by the resistance and compliance of the respiratory system , largely affecting the pressures and flows generated in the respiratory system . under certain conditions , the use of this technique can generate high peak airway pressures , increasing the risk of barotrauma . according to expert recommendations , in order to promote secretion clearance , mh should consist of a slow , deep inspiration , an inspiratory pause , and a quick release of the resuscitation bag to promote passive exhalation with high expiratory flow rates . however , the way in which the mh maneuver is performed can vary from country to country , impeding the understanding of its effects . since the 1980s , there has been increasing evidence that the peak expiratory flow to peak inspiratory flow ( pef / pif ) ratio is a critical factor for the removal of lung secretions , especially in heavily sedated or paralyzed patients . in fact , more than their ratio , the difference between the two , in terms of their absolute values , seems to be the major determinant : above a given threshold , whenever the pif exceeds the pef , secretions migrate deeper into the lung . intuitively , respiratory therapists ( rts ) have been promoting maneuvers to increase expiratory flows , analogous to those observed during coughing . however , relatively little attention has been given to their inspiratory counterpart , despite expert recommendations to apply a slow , deep inspiration during mh . the inspiratory phase of mh can be performed in many different ways , depending on the personal experience of the operator , and there is little evidence that the mh maneuver generates an adequate flow bias ( i.e. , with pef exceeding pif ) or that the ultimate goal of the maneuver ( i.e. the aim of this study was to evaluate the efficiency and safety of mh ( as performed by rts in a lung model simulating a mechanically ventilated patient ) as a means of increasing secretion clearance under two different conditions : mh performed in accordance with the routine clinical practice of rts ; and mh performed in accordance with expert recommendations . the efficiency of mh was determined through analysis of the volume and flow patterns generated in relation to the anticipated effects on secretion removal . the safety of mh was evaluated on the basis of the inspiratory pressures achieved ( i.e. the study comprised two phases , evaluating the effects of mh , as performed by rts , in promoting the clearance of pulmonary secretions . in the first phase ( the pre - instruction phase ) , the rts were given no explicit verbal instructions on how to apply the maneuver . in the second phase , the rts were given instructions based on expert recommendations , as detailed below . all maneuvers were performed with self - inflating manual resuscitator ( spur ; ambu , ballerup , denmark ) , with a capacity of 1,500 ml . the maneuver was applied by eight experienced rts ( four males and four females ) , with an average of 2.6 years in practice ( range , 2 - 4 years ) in icus in the city of so paulo . five of the eight had graduated from universities located within the state of so paulo , and all had completed one of the one - year postgraduate training programs in respiratory therapy offered by university hospitals . the maneuver was applied to a mechanical model of the respiratory system known as a training and test lung ( ttl 2600 ; michigan instruments , grand rapids , mi ) , connected to a tracheal tube ( 8.5 mm ) . the airway flow was measured proximally ( between the manual resuscitator and proximal pressure sensor ) by a pneumotach . two pressure transducers were connected to the model : one to measure proximal pressure ( pprox - at the airway , between the flow sensor and tracheal tube ) ; and another to record alveolar pressure ( palv ) . study phases , we altered the resistance and compliance of the lung model in order to simulate three different clinical scenarios : a patient with obstructive lung disease ( resistance at 20 cmh2o / l per second and compliance at 0.08 l / cmh2o ) ; a normal patient ( resistance at 5 cmh2o / l per second and compliance at 0.05 l / cmh2o ) ; and a patient with restrictive lung disease ( resistance at 5 cmh2o / l per second and compliance at 0.025 l / cmh2o ) . in the pre - instruction phase ( as previously mentioned ) , the rts were instructed to perform mh to promote pulmonary secretion clearance in accordance with their routine clinical practice . the lung model was set according to those three clinical scenarios explained above and covered with a bed sheet , allowing rts to sense the movement of the bellows while remaining blinded to the clinical scenario selected . in the post - instruction phase , each rt received brief verbal instructions on how to perform mh according to expert recommendations , and all of the steps performed in the pre - instruction phase were repeated . the verbal instructions were given within the space of approximately 2 min , and the rts were not trained to follow a given pattern of insufflation . the instructions were always given by the same researcher , who instructed each rt as follows : " now you should perform mh with a slow inflation and a 2-s inspiratory pause , followed by rapid release of the bag " . while giving the instructions , the researcher demonstrated the maneuver with the manual resuscitator used in the study . for each experimental condition , the beginning of the inspiratory phase was defined as zero flow immediately before bagging , and the end of the respiratory cycle was defined as zero flow at the end of exhalation . inspiratory time ( ti ) was defined as the duration of the inflation plus the inspiratory pause . repeated measures anova was used to evaluate , for each variable , the following within - subject factors : the three clinical scenarios ; and the two phases ( before and after instructions ) . inspiratory versus expiratory flow and alveolar versus proximal pressures were also tested as within - subject factors to compare peak flow and peak pressure variables , respectively . for each of the six experimental conditions ( three clinical scenarios in each study phase ) , we analyzed three respiratory cycles , corresponding to eighteen respiratory cycles for each of the eight rts . among all of the respiratory cycles analyzed , the maximum palv observed was 42.5 cmh2o ( median , 16.1 cmh2o ; iqr , 11.7 - 24.5 ) , despite the much higher pprox values ( maximum , 96.6 cmh2o - median , 36.7 ; iqr , 22.9 - 49.4 ) . the vt values were relatively low ( maximum , 955 ml - median , 640 ; iqr , 505 - 735 ) , and the ti was short ( median , 1.29 s ; iqr , 0.95 - 1.72 ) . a pif / pef ratio < 0.9 , which theoretically favors mucus migration toward the central airways , was achieved in only 24 ( 16.7% ) of the 144 maneuvers evaluated . this favorable ratio occurred primarily in the post - instruction phase ( in 18 of the 24 maneuvers ) and was strongly associated with just one of the rts tested ( who was responsible for 12 of the 24 maneuvers ) . during the pre - instruction phase , six of the eight rts performed mh using two compressions of the resuscitator bag and produced a pif higher than that produced in the post - instruction phase ( figure 2 ) . figure 2differences in proximal pressures achieved by two different respiratory therapists ( a and b ) before and after explicit verbal instruction ( routine clinical practice vs. expert recommendations - dashed lines and solid lines , respectively ) . table 1 shows a comparison between the pre- and post - instruction phases , in terms of the mechanical variables evaluated . after instruction , mh was performed in a slower manner , with a longer ti and a lower pif , producing a lower pprox . table 1mechanical variables before and after verbal instruction ( routine clinical practice vs. expert recommendations).variablepre - instructionpost - instructionp proximal pressure ( cmh2o)44.5 ( 33.4 - 63.4)26.8 ( 18.6 - 37.5)0.004 alveolar pressure ( cmh2o)16.3 ( 11.6 - 25.8)14.8 ( 11.7 - 23.8)0.93 peak inspiratory flow ( l / s)1.84 ( 1.28 - 2.19)1.14 ( 0.87 - 1.44)0.001 peak expiratory flow ( l / s)1.04 ( 0.57 - 1.33)0.99 ( 0.55 - 1.27)0.28 tidal volume ( ml)628 ( 497 - 699)647 ( 518 - 746)0.63 inspiratory time ( s)0.95 ( 0.78 - 1.19)1.71 ( 1.44 - 2.13)<0.001 pif / pef ratio1.80 ( 1.29 - 2.34)1.15 ( 0.87 - 1.80)0.004values are expressed as median ( 25 - 75% interquartile range)pif : peak inspiratory flowpef : peak expiratory flow values are expressed as median ( 25 - 75% interquartile range ) : peak inspiratory flow : peak expiratory flow comparing all three clinical scenarios tested and the two phases of the study , we found that pprox was markedly higher than was palv in all instances , with the exception of the restrictive lung disease scenario in the post - instruction phase ( figure 3 ) . in the pre - instruction phase , in the post - instruction phase , the pif / pef ratio was closer to 1:1 . the only situation in which that ratio was consistently unfavorable ( pif still far exceeding pef ) was the obstructive lung disease scenario ( figure 4 ) . figure 3median values of proximal and alveolar pressures ( interquartile ranges as error bars ) obtained in the pre - instruction ( routine clinical practice ) and post - instruction ( in accordance with expert recommendations ) phases of the study . # p < 0.05 ( difference between proximal and alveolar pressure ) . figure 4median values for peak inspiratory and expiratory flows ( interquartile ranges as error bars ) obtained in the pre - instruction ( routine clinical practice ) and post - instruction ( in accordance with expert recommendations ) phases of the study . # p < 0.05 ( difference between peak inspiratory and expiratory flows ) . the major finding of the present study was that , even after receiving explicit instructions , the rts evaluated here performed mh in a way that probably would not aid secretion removal , with pif commonly exceeding pef . compressing the resuscitator bag multiple times , in rapid succession , resulted in high values of pif and pprox . however , this finding was typically associated with a relatively low vt , probably because of short bag - compression times . therefore , the palv was often low at the start of exhalation , resulting in a low pef . both factors ( rapid , multiple compressions and low palv ) appear to be responsible for the unfavorable relationship between pif and pef . finally , although the mh maneuver might not promote secretion clearance , our results suggest that this maneuver , as performed in our study , is unlikely to cause barotrauma , a concern expressed by other authors because of the high pprox it generates . in the present study , however , the palv values - roughly represented by plateau pressures , which correlate better with barotrauma than do pprox values - were within the safe range . the mh maneuver was originally described as consisting of a slow , deep inspiration , with an inspiratory pause and rapid release of the resuscitator bag . in contrast , we found that rts working in the city of so paulo have customized the maneuver according to personal practice and bias , frequently applying two compressions of the resuscitator bag , without an inspiratory pause , resulting in pif being higher than pef . according to previous studies , above a given flow threshold , the direction of mucus transport ( in or out of the respiratory system ) is governed by the highest peak flow : a pef 10% higher than the pif ( i.e. , a pif / pef ratio < 0.9 ) will favor secretion movement from the distal to the central airways . a recent study evaluating the transport of artificial mucus in a test lung system reported that mucus transport is better explained by the absolute difference between pif and pef than by the pif / pef ratio . in the present study , the pif / pef ratio was below 0.9 in only 24 ( 16.7% ) of the 144 maneuvers evaluated , most of those 24 being performed in the post - instruction phase . this disappointing result is in agreement with the findings of some other studies in which self - inflating resuscitators were also used . the manual resuscitator tested in the present study had a self - inflating bag ( the type of manual resuscitator most widely used in icus in brazil ) . even after receiving explicit instructions , the rts produced vt values that were lower than those reported in other studies . much more favorable pif / pef ratios have been reported when flow - inflating devices were used . the verbal instruction on how to perform the mh improved the performance of the rts in that the post - instruction maneuvers generated lower pifs and longer tis . however , those differences did little to improve the efficiency of the technique , because the pefs were still quite low . that is likely attributable to the fact that the palv was also quite low at the start of exhalation , which resulted in a low driving pressure for expiratory flow . other authors have reported great variability in the practical implementation of the mh maneuver , the execution of which rarely follows its original description . therefore the use of an inspiratory pause could decrease the pef and , consequently , impair the efficiency of the mh maneuver . however , we believe that this qualitative analysis , performed with representative operators ( of both genders , recruited from different hospitals from within the same city ) , illustrated scenarios that commonly occur in some icus . another limitation of this study was the use of a lung model , which can not be extrapolated to the complexity of human lungs . nevertheless , we believe that similar results - high pif / pef ratios , low vt , and high pprox - would also be obtained in human patients on mechanical ventilation , especially in those that are heavily sedated , and this should draw the attention of the rts to the way in which they perform the mh maneuver . that notwithstanding , the key message here is that performing mh with high pif / pef ratios in patients who are unable to cough will not aid secretion removal and might even contribute to mucus retention . another point that should be mentioned is that the mh maneuver can be performed in combination with expiratory chest compression in order to maximize the increase in the pef ; however we were not able to investigate the use of that combination , because of the experimental system used . in addition , as previously mentioned , we can not discard the possibility that the use of a different resuscitator device ( a self - inflating resuscitator with larger internal volume bags or a flow - inflating resuscitator ) might produce better results . given that , in brazil , mh is typically performed without pprox monitoring , the option of performing the maneuver with a mechanical ventilator , so that inspiratory flow , vt and pressure can be easily monitored and adjusted , should be considered . in studies comparing mh with a manual resuscitator and mh with a mechanical ventilator , no differences were found between the two modalities in terms of the amount of secretion removed , although the advantages of better monitoring with the mechanical ventilator were acknowledged . it is important to note that , although the mh maneuver was originally designed to reduce lung collapse and to improve oxygenation or lung compliance , those potential benefits were not tested here . finally , we found no evidence that the mh maneuver , as performed here , increases the risk of barotrauma . the palv value is a result of the insufflating volume and lung compliance , pprox values that are higher than the palv although there have been anecdotal reports of high pprox during mh in adults , with volumes < 1 l , there is no hard evidence that the maneuver is dangerous . in conclusion , when asked to apply mh in accordance with their routine clinical practice , this small sample of rts performed the maneuver quite differently from what is recommended by experts , producing a concerning pattern of ventilation in terms of secretion clearance . the repetition of the maneuver after explicit instructions reduced the pprox but did not help much . alveolar pressures were usually low ( because of the small generated vt ) despite high proximal pressures and high inspiratory peak flows .

like many countries , the united states is struggling with the rising costs of cancer care . nowhere is this more apparent than within medicare , the primary payer of health services for those of age 65 years , where the rise in spending on cancer drugs over the past 15 years has been singled out as a leading cause of increased spending overall . presently , medicare 's ability to control rising spending on cancer drugs is limited , and it is also well known that medicare does not formally consider cost or cost - effectiveness as a criterion for coverage and reimbursement . since most new drugs cost considerably more than older drugs [ 2 , 3 ] , and since many are added to rather than substituted for drugs in existing regimens , the net cost impact of new drugs will be determined primarily by the cost of the drug itself , and secondarily by the extent to which those costs are augmented or offset by its clinical benefits . in cancer care , clinical benefits that have cost implications include reducing the rate of disease progression or relapse after first - line therapy and improving survival . it is standard practice to include all of these factors in cost - effectiveness analyses of new cancer drugs . however , such analyses often are conducted early in the commercial life - cycle of the drug , and in the absence of comprehensive data from routine clinical practice , usually they are based on models that incorporate data from multiple sources , including drug regimens and clinical outcomes from phase iii trials . while these analyses provide important insight into the potential cost and cost - effectiveness of new drugs in routine clinical practice , and may have a high level of internal validity due to the sources of clinical data used , invariably they will lack some external validity . this is due to differences in the types of patients treated , for example , patient age , cancer stage , the approaches to managing disease progression or relapse , and the net cost impact of improving overall survival in patients with higher comorbidity burdens . in 1997 , rituximab became the first therapeutic antibody approved for the treatment of cancer . initially , it was approved for treating relapsed or refractory , cd20 + , b - cell low grade , non - hodgkin 's lymphoma ( nhl ) . since then , the indication has expanded several times to include first - line therapy , follicular lymphoma ( fl ) , chronic lymphocytic leukemia ( cll ) , and rheumatoid arthritis . for instance , addition of rituximab to first - line chemotherapy has been shown in clinical trials of dlbcl [ 58 ] to improve progression - free and overall survival in elderly patients , where reported risk - ratios for all - cause mortality range from 0.53 ( 95% confidence interval ( ci ) 0.370.77 ) to 0.72 ( 95% ci 0.521.00 ) , and in younger patients . the efficacy of rituximab as first - line therapy for advanced fl was established in several randomized trials [ 912 ] . the german low grade lymphoma study group compared chop ( cyclophosphamide ( c ) , doxorubicin , vincristine ( v ) , and prednisone ( p ) ) ( n = 205 ) to r - chop ( n = 223 ) in a cohort of patients with untreated , advanced - stage iii / iv fl . they showed a statistically significant improvement in overall survival , with 6 deaths in the r - chop group compared to 17 deaths in the chop group within the first three years . another phase iii study compared cvp alone ( n = 162 ) to r - cvp ( n = 159 ) in a similar cohort of untreated fl patients . in that study , the percent of patients ; surviving four years was significantly higher in the r - cvp ( 83% ) versus the cvp - alone group ( 77% ) ( p = 0.029 ) . the effectiveness of rituximab in routine clinical practice has been evaluated in several studies using the national cancer institute 's ( nci ) surveillance , epidemiology , and end results ( seer ) medicare database [ 1315 ] . they indicate that the survival benefits observed in clinical trials extend to elderly patients treated in routine clinical practice , and to mantle cell lymphoma ( mcl ) where survival benefits have not been demonstrated in clinical trials . they also show that even though rituximab was not approved initially for first - line treatment , its use as part of first - line chemotherapy increased rapidly after medicare began coverage in 1998 , and now the majority of seer - medicare patients who receive chemotherapy as first - line therapy for fl , mcl , and cll also receive rituximab [ 1315 ] . efficacy and effectiveness notwithstanding , rituximab is an expensive addition to first - line therapy for nhl with an estimate of $ 24,034 in fl and $ 13,900 in dlbcl based on the projections of cost - effectiveness models , and in part dependent on the year of the analysis and number of administrations of rituximab . the current medicare allowed amount ( including 20% patient copayment ) for rituximab is $ 611 per 100 mg vial . at a recommended dose of 375 mg / m body surface area ( bsa ) , and assuming an average bsa of 1.72 m and 68 cycles per first - line course of rituximab , the current cost to medicare could be $ 20,530$27,373 per course , an estimate that excludes the 20% copayment . a disproportionate number of patients diagnosed with nhl are aged 65 years , including 50% of the estimated 129,500 prevalent and 59% of the 21,800 incident dlbcl patients and 52% of fl patients [ 21 , 22 ] . consequently , medicare is an important payer for therapy and other cancer care in nhl , and rituximab is an important line item for medicare . in 2009 , rituximab accounted for the largest percent of medicare part b expenditures ( 7.8% ) of any drug administered in physicians ' offices or furnished by suppliers . given the efficacy and cost of rituximab , there has been considerable interest in evaluating its net cost impact and cost - effectiveness , and studies have been conducted in dlbcl [ 17 , 2427 ] and fl [ 16 , 28 , 29 ] . several studies of adding first - line rituximab to chemotherapy in dlbcl projected cost offsets [ 17 , 2427 ] , and in one instance savings , associated with lower rates of progression / relapse and improved survival . two cost - effectiveness analyses of rituximab in combination with chemotherapy have been conducted in fl , one in the us , and one in the united kingdom [ 16 , 28 ] . both were based on computer models , and incorporated data from a number of sources including clinical data from randomized trials . in the us , hornberger and colleagues projected adding rituximab to cvp would result in a lifetime cost difference of + $ 26,439 , the majority of which was due to rituximab itself ( $ 24,034 ) , a difference in mean overall survival of + 1.51 years , and a cost per quality - adjusted life - year gained of $ 28,565 . the objective of the present study was to estimate the cost and cost - effectiveness of rituximab added to first - line chemotherapy for fl , using a single source of data representative of routine clinical practice in elderly patients in the us . it differs from previous economic studies of rituximab in nhl , the majority of which were based on models , incorporated data from multiple sources including clinical trials , and projected cost and survival beyond the available data . the source of data for this study was the national cancer institute 's ( nci ) surveillance , epidemiology , and end results ( seer ) cancer registry linked to medicare administrative and claims data . presently , seer contains cancer incidence and survival data from 17 population - based cancer registries throughout the united states covering approximately 28% of the population . the registries routinely collect data on patient demographics , primary tumor site , tumor morphology and stage at diagnosis , first course of treatment , and followup for vital status . in seer - medicare , cancer registry data are linked to medicare enrollment and claims data , which are available for 93% of those aged 65 years in the seer registry . at the time this study was performed , the seer - medicare linkage included all medicare - eligible persons appearing in the seer data through 2005 and their medicare claims through 2007 . medicare claims files linked to seer consist of the following : medicare provider analysis and review ( medpar)all hospital ( part a ) short stay , long stay , and skilled nursing facility bills ; national claims history ( nch)all physician / supplier ( part b ) bills ; outpatient , which includes all bills from institutional outpatient ( part b ) providers ; home health agency ( hha)all claims for home health services ; hospice ; and durable medical equipment ( dme ) . the medicare benefit for oral drugs ( part d ) began on january 1 , 2006 , and claims for oral drugs without an intravenous equivalent were not available for our study . patients were included if they were diagnosed with fl between january 1 , 1999 , and december 31 , 2005 , fl was the first primary cancer diagnosed , they began infused chop ( cyclophosphamide ( c ) , doxorubicin , vincristine ( v ) , and prednisone ( p ) ) or cvp chemotherapy with or without rituximab within 90 days following diagnosis , and they survived at least 60 days after first claim indicating the beginning of therapy . we required a minimum 60 days survival after the first claim to classify the initial treatment regimen . in addition , to ensure complete claims history for purposes of calculating an nci comorbidity index score [ 34 , 35 ] , patients had to have been enrolled in both medicare parts a and b , with no health maintenance organization ( hmo ) coverage , for 12 months prior to fl diagnosis . to preserve patient confidentiality , seer reports only the calendar month of diagnosis . therefore , we set the date of diagnosis to the first day of that month . identification of fl was made using the world health organization ( who ) international classification of diseases for oncology , 3rd edition ( icd - o-3 ) histology codes 9695 ( fl grade 1 ) , 9691 ( fl grade 2 ) , 9698 ( fl grade 3 ) , and 9690 ( fl not otherwise specified ) . patients were followed for up to four years from the day first - line therapy began , or until death , the end of their claims ( december 31 , 2007 ) , or the end of their medicare part a and/or part b coverage , whichever came first . the date of death was assigned by using the medicare date because it is considered more current than the seer date . patients were described according to their demographic , clinical , and socioeconomic characteristics . requiring eligible patients to have at least one year of medicare enrollment prior to diagnosis meant that the minimum age in the cohort was 66 years . patients were classified by stage and extranodal involvement ( yes / no ) at fl diagnosis . in the absence of hemoglobin level , serum lactate dehydrogenase ( ldh ) level , or the number of nodal areas , three of the five prognostic factors for mortality in the follicular lymphoma international prognostic index ( flipi ) , we used medicare claims to identify several predictors of poor performance status , including the use of oxygen and related respiratory therapy supplies , wheel chair and supplies , home health agency , and skilled nursing facility , all from 12 months before until 30 days after fl diagnosis . individual services were combined into a score of 0 ( none ) or 1 ( use of any service ) . medicare claims also were used to identify anemia , and to calculate an nci comorbidity index score [ 34 , 35 ] for each patient . seer - medicare contains information from the 2000 census , reported at the tract level in which the patient lives , for the percent of the population living in poverty and the percent of those aged 25 years or older with some college . we used these as indicators of the socioeconomic status of individual patients in the cohort . we searched the medicare outpatient and nch files to identify claims containing healthcare common procedure coding system ( hcpcs ) j codes for cyclophosphamide ( j8530 , j9070 , j9080 , j9090j9097 ) , doxorubicin ( j9000 , j9001 ) , and vincristine ( j9370 , j9375 , j9380 ) . all such claims within 60 days after the first were used to classify the patient 's first - line regimen as rituximab plus chemotherapy ( chop or cvp ) or chemotherapy alone . in the absence of data on oral therapy , prednisone was assumed to have been included in the regimen when the other three chemotherapy agents were present . the total direct medical cost to medicare , from the beginning of first - line therapy until the end of the observation period ( maximum four years ) , was estimated based on medicare paid amounts obtained from bills within the claims files . diagnosis and procedure codes within claims were used to identify three mutually exclusive categories of costs : immunochemotherapy , which included the costs of infused cancer therapy agents provided during first - line and any subsequent treatment , other cancer care ( any claim with an international classification of diseases , 9th revision , clinical modification [ icd-9-cm ] diagnosis code 140.xx208.xx [ malignant neoplasms ] , but no code for immunochemotherapy ) , and noncancer care ( any other claim ) . all paid amounts were inflated to 2009 us dollars based on the hospital input price index for part a claims and the medical expenditure index for part b claims . since seer - medicare data are limited in time , our approach to estimating population mean costs accounted for censoring in the data using inverse probability weighting ( ipw ) [ 3945 ] . conventional approaches to analyzing adjusted differences in survival between alternative treatments are based primarily on proportional hazards regression . however , this approach does not produce an estimate of the adjusted difference in overall survival between two treatment groups at specific time - points , as required for performing cost - effectiveness analysis directly from observational data . in this study , we used inverse probability of treatment weighting ( iptw ) for survival data to estimate the adjusted difference in cumulative survival between the two treatment groups . first , we estimated the conditional probability of assignment to the rituximab plus chemotherapy group for each patient using logistic regression with treatment group as the dependent variable and all other independent variables from the cost model as predictor variables . then , the inverse of this conditional probability estimate was assigned as a weight to each patient , and it was used to adjust the kaplan - meier cumulative survival estimate for each treatment group . chi - square analysis was used to test for the independence between patient characteristics specified as categorical variables and the two first - line treatment groups . we performed partitioned ipw least squares regression analysis to examine adjusted associations between cumulative costs over four years and patient demographic , clinical , and treatment factors . coefficients for each patient factor were summed across the 48 partitions to obtain the cumulative , incremental cost associated with that factor . confidence intervals ( ci ) for the cumulative cost coefficients were calculated by a bootstrap approach , in which the process of performing 48 partitioned regression analyses and summing coefficients across partitions was repeated 1,000 times using sampling with replacement from the original cohort . this process was applied to total costs and each of the three cost categories , for a total of 192,000 regressions . using the adjusted kaplan - meier estimator based on iptw , we estimated the adjusted difference in survival between rituximab plus chemotherapy and chemotherapy alone in each of 48 monthly partitions following the beginning of first - line therapy . since patients in this study were required to have survived at least 60 days after the beginning of first - line therapy , by definition the difference in survival was zero during the first two months . we obtained 95% cis for the partitioned incremental survival estimates from the weighted log rank statistic . cost effectiveness ratios were calculated in months 348 ( ignoring months 1 - 2 due to the inclusion criteria ; there was no mortality during this period ) after the beginning of first - line therapy , by dividing the incremental cumulative cost of rituximab by the cumulative survival difference . confidence intervals for the cost - effectiveness ratio were calculated by dividing the upper bound of the 95% ci for cost by the lower bound of the 95% ci for survival , and then repeating the process with the lower cost and upper survival bounds . there were 1,117 patients in the final cohort : 750 ( 67% ) received rituximab plus chemotherapy , and 367 ( 33% ) received chemotherapy alone ( table 1 ) . the median age at diagnosis was 73 years ( minimum 66 years ) , 56% had stage iii / iv disease , 42% had extranodal involvement , and 15% had 1 indicator of poor performance status . rituximab patients were diagnosed later in the study period , and were more likely to have higher - grade histology . there were no differences in stage at diagnosis , extranodal involvement , or nci comorbidity index score between the two groups . overall , 67% ( 754 ) received chop , and the remaining 33% ( 363 ) received cvp . rituximab patients were more likely to receive chop ( 72% versus 58% for chemotherapy alone ; p < 0.0001 ) . among those who received rituximab , the average number of rituximab administrations during first - line therapy was 6.3 ( median 6 ; interquartile range [ iqr ] 57 ) , and the average number of units was 7.3 100 mg vials ( median 7 ; iqr 68 ) . the mean unadjusted ipw cumulative cost was $ 111,815 ( 95% ci $ 104,455$119,466 ) in the rituximab group compared to $ 80,826 ( 95% ci $ 74,006$88,113 ) for chemotherapy alone ( figure 1 ) . the mean cost of first - line rituximab was $ 15,640 ( median $ 14,786 ; iqr $ 10,831$18,560 ) , and the average cost per administration was $ 2,836 ( median $ 2,747 ; iqr $ 2,617$3,113 ) . in the multivariate analyses , rituximab was associated with statistically significantly higher incremental total ( $ 18,695 ; 95% confidence interval [ ci ] $ 9,302$28,643 ) and immunochemotherapy ( $ 13,336 ; 95% ci $ 9,364$17,552 ) costs after four years , but not other cancer ( $ 4,816 ; 95% ci $ 1,899$11,303 ) or noncancer ( $ 351 ; 95% ci $ 3,481$4,202 ) costs ( table 2 ) . most of the separation in total and immunochemotherapy costs between the two treatment groups occurred during the first 6 months after the beginning of first - line therapy ( figure 2 ) . thereafter , there was very little separation in total or immunochemotherapy costs . during the first 36 months , incremental costs of other cancer services were statistically significantly higher in the rituximab group . however , costs of other cancer services declined in later months , and by month 48 the difference between groups was not significant . there was no difference in non - cancer costs at any time during the observation period . other factors associated with higher costs were stage ( immunochemotherapy cost ) , extranodal involvement ( total and other cancer costs ) , nci comorbidity index ( predominately non - cancer cost ) , and 1 indicator of poor performance status ( total and other cancer costs ) . factors associated with lower costs were age > 80 years ( immunochemotherapy cost ) , other race / ethnicity ( total and immunochemotherapy costs ) , and urban / rural ( compared to metropolitan ) area ( total and other cancer cost ) . results of the multivariate analysis of total cost that included only patients treated with chop were similar to those that included all patients ( table 3 ) . overall , unadjusted ( crude ) cumulative survival was 83% at 24 months and 73% at 48 months ; 85% and 77% , respectively , for rituximab plus chemotherapy ; 79% and 66% , respectively , for chemotherapy alone . the incremental cumulative adjusted survival ( years ) in the rituximab group was 0.05 ( 95% ci 0.020.09 ) after two years , 0.11 ( 95% ci 0.050.16 ) after three years , and 0.18 ( 95% ci 0.100.27 ) after four years of observation ( figure 3 ) . the cost per life year gained in the rituximab group was $ 382,642 ( 95% ci $ 164,900$1,360,559 ) after two years , $ 193,859 ( 95% ci $ 77,314$609,607 ) after three years , and $ 102,142 ( 95% ci $ 34,531$296,337 ) after four years of observation ( figure 4 ) . at no time point during the observation period did the 95% ci for the cost - effectiveness ratio contain a value indicating that rituximab plus chemotherapy was both more costly and less effective ( dominated ) , or less costly and more effective ( dominates ) than chemotherapy alone . in this study , we identified a cohort of elderly fl patients who received first - line chemotherapy with or without rituximab . we followed this cohort for up to four years after the beginning of therapy to estimate the net cost impact and cost - effectiveness of adding rituximab to first - line therapy . our findings indicate that over this time horizon , the initial cost of rituximab accounted for the majority of the cost difference between the two treatment groups . the mean cost of first - line rituximab in our study ( $ 15,640 ) was substantially lower than expected based on the projections from a cost - effectiveness model in fl ( $ 24,034 ) , and our own calculations based on recommended dosing and current medicare reimbursement . patients in our study received an average of 6.3 administrations and 7.3 100 mg vials per administration during first - line therapy . hornberger and colleagues assumed 8 administrations , 700 mg per administration , and $ 5.82/mg in estimating the cost of first - line rituximab . part of the difference may be explained by the fact that patients in our study received almost 25% fewer administrations . another reason for the difference is that our calculations were made using medicare paid amounts , which exclude the 20% patient copayment . finally , although we inflated our costs to us$2009 , it is possible that our inflation factors did not adequately compensate for inflation in the price of rituximab during the study period . dividing the mean cost of rituximab by the average number of milligrams delivered during the course of therapy ( 4,599 ) , the average cost per milligram was only $ 3.40 in our study . it is noteworthy that in the rituximab group the cumulative incremental cost of immunochemotherapy during the four - year period ( $ 13,336 ) was lower than the cost of first - line rituximab ( $ 15,640 ) . this suggests the initial costs of rituximab were offset to some extent by higher treatment costs in the chemotherapy group due to higher rates of nonremitting and/or relapsing disease . the cumulative incremental cost of other cancer care also was significantly higher in the rituximab group through much of the study period . most of the difference between the two groups appeared during first - line therapy , which suggests there may have been differences in supportive care and other cancer services during first - line therapy that were not captured in the immunochemotherapy category . after first - line therapy , the cumulative incremental cost of other cancer care in the rituximab group declined , and by year three it was no longer statistically significant . this suggests there were some cost offsets possibly associated with lower rates of relapse in the rituximab group . there were no differences in the costs of noncancer services at any time during the observation period , suggesting that improved survival in the rituximab group had no appreciable impact on the costs of managing other diseases . those with more advanced stage cancer and higher comorbidity burden also had higher costs , which is consistent with a priori expectations that increased patient complexity requires a higher level of care . the cost findings also suggest that the very elderly receive less first - line and subsequent immunochemotherapy . the cumulative survival benefit of rituximab continued to accrue , and was statistically significant , throughout the observation period , and since the cumulative cost impact was negligible after the end of first - line therapy , the cost per life year gained declined ( became more favorable for rituximab ) rapidly during the observation period . although we did not attempt to extrapolate our findings to a longer time period , it is likely that following these patients for a longer period would result in a lower cost - effectiveness ratio . also , as we noted above , our study inclusion criteria required patients to have survived at least 60 days after the first claim for immunochemotherapy . consequently , we did not account in our survival and cost - effectiveness analyses for patients who died during the first two months after starting first - line therapy . neither pivotal phase iii trial of first - line rituximab showed significant separation in the overall survival curves within the first several months after beginning therapy [ 9 , 11 ] . however , it is possible we would have observed larger survival differences , and hence smaller cost effectiveness ratios , had we included patients who died within the first two months of therapy . seer - medicare does not include information on patient quality of life ( qol ) or utility . therefore , we did not adjust the survival estimates for qol prior to calculating the cost - effectiveness ratios . in their cost - effectiveness analysis based on a simulation model , hornberger and colleagues projected that adding rituximab to cvp would increase mean overall survival by 1.51 years and quality - adjusted life - years by 0.93 years , which suggests that had we adjusted our survival estimates for qol , the cost - effectiveness ratios may have been larger than those based on unadjusted survival . the main threat to the validity of our findings is selection bias , in which unobserved factors influence both treatment selection and the outcome of interest . for example , seer - medicare does not contain data on hemoglobin level , ldh level , or the number of nodal areas , three of the five prognostic factors for mortality in the flipi . we did , however , include age ( all patients were older than the age threshold for excess risk of mortality in flipi [ 60 ] ) , stage ( iii - iv is associated with increased risk in flipi ) , and whether the patient had extranodal involvement . also , we included a claims - based indicator of poor performance status . in the multivariate analysis , 1 indicator of poor performance , extranodal involvement , and later stage diagnosis it is important to note that any variable constructed using claims for medical services , such as performance status and nci comorbidity index [ 34 , 35 ] , that is then included in a multivariate cost analysis , has a greater likelihood of being statistically significant simply by virtue of the fact that most claims have a cost attached . in this study , patients diagnosed in later years were more likely to receive rituximab . this may have introduced several possible sources of bias including differential observation and censoring , and secular trends in both the assignment of patients to different chemotherapy regimens and in the use of supportive care . in the survival and cost analyses we used iptw in the estimation of cumulative incremental survival . in the cost analysis , we used ipw and established separate weights for the two treatment groups to account for differential censoring between the two groups . also , we included year of diagnosis as a covariable in the survival analysis and all the cost models to adjust for temporal differences in patterns of care . when we restricted the cohort to those who received chop , the cost coefficients for the rituximab group were similar to those in the primary analyses . therefore , we used medicare paid amounts reported in claims to estimate total direct medical costs to medicare . however , it is important to note that medicare pays only 80% of part b outpatient costs . therefore , total direct medical costs to all payers including patients and other insurers could be up to 20% higher than reported in this study . also , medicare did not pay for most oral drugs for the majority of the period in which our study was conducted , which is another reason why the costs reported in this study may underestimate the total direct medical costs of care to all payers . also , it is likely that use of rituximab as maintenance therapy has increased in the years since the period covered by our data . finally , the confidence intervals for the cost - effectiveness ratio were calculated by dividing the upper bound of the 95% ci for cost by the lower bound of the 95% ci for survival , and then repeating the process with the lower - cost and upper - survival bounds . in theory , however , to our knowledge such an approach has not been developed and reported in the literature . in sum , adding rituximab to first - line chemotherapy for elderly patients with fl results in higher direct medical costs to medicare and longer overall survival after four years . the net cost impact is due primarily to the treatment cost of first - line rituximab , although there may be small cost offsets associated with lower progression and/or relapse . the cost - effectiveness ratio we estimated is substantially higher than projected in a previous analysis based on a model . extending the time horizon for our study , and including patients who died during the first two months of therapy our findings suggest even highly effective cancer therapies will do little to offset the rising costs of cancer care in the united states , and that additional public policies will be required to address this problem .

rituximab improves survival in follicular lymphoma ( fl ) , but is considerably more expensive than conventional chemotherapy . we estimated the total direct medical costs , cumulative survival , and cost - effectiveness of adding rituximab to first - line chemotherapy for fl , based on a single source of data representing routine practice in the elderly . using surveillance , epidemiology , and end results ( seer ) registry data plus medicare claims , we identified 1,117 fl patients who received first - line chop ( cyclophosphamide ( c ) , doxorubicin , vincristine ( v ) , and prednisone ( p ) ) or cvp + / rituximab . multivariate regression was used to estimate adjusted cumulative cost and survival differences between the two groups over four years after beginning treatment . the median age was 73 years ( minimum 66 years ) , 56% had stage iii - iv disease , and 67% received rituximab . adding rituximab to first - line chemotherapy was associated with higher adjusted incremental total cost ( $ 18,695 ; 95% confidence interval ( ci ) $ 9,302$28,643 ) and longer adjusted cumulative survival ( 0.18 years ; 95% ci 0.100.27 ) over four years of followup . the expected cost - effectiveness was $ 102,142 ( 95% ci $ 34,531296,337 ) per life - year gained . in routine clinical practice , adding rituximab to first - line chemotherapy for elderly patients with fl results in higher direct medical costs to medicare and longer cumulative survival after four years .

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

presently , medicare 's ability to control rising spending on cancer drugs is limited , and it is also well known that medicare does not formally consider cost or cost - effectiveness as a criterion for coverage and reimbursement . however , such analyses often are conducted early in the commercial life - cycle of the drug , and in the absence of comprehensive data from routine clinical practice , usually they are based on models that incorporate data from multiple sources , including drug regimens and clinical outcomes from phase iii trials . while these analyses provide important insight into the potential cost and cost - effectiveness of new drugs in routine clinical practice , and may have a high level of internal validity due to the sources of clinical data used , invariably they will lack some external validity . this is due to differences in the types of patients treated , for example , patient age , cancer stage , the approaches to managing disease progression or relapse , and the net cost impact of improving overall survival in patients with higher comorbidity burdens . since then , the indication has expanded several times to include first - line therapy , follicular lymphoma ( fl ) , chronic lymphocytic leukemia ( cll ) , and rheumatoid arthritis . for instance , addition of rituximab to first - line chemotherapy has been shown in clinical trials of dlbcl [ 58 ] to improve progression - free and overall survival in elderly patients , where reported risk - ratios for all - cause mortality range from 0.53 ( 95% confidence interval ( ci ) 0.370.77 ) to 0.72 ( 95% ci 0.521.00 ) , and in younger patients . the german low grade lymphoma study group compared chop ( cyclophosphamide ( c ) , doxorubicin , vincristine ( v ) , and prednisone ( p ) ) ( n = 205 ) to r - chop ( n = 223 ) in a cohort of patients with untreated , advanced - stage iii / iv fl . they showed a statistically significant improvement in overall survival , with 6 deaths in the r - chop group compared to 17 deaths in the chop group within the first three years . in that study , the percent of patients ; surviving four years was significantly higher in the r - cvp ( 83% ) versus the cvp - alone group ( 77% ) ( p = 0.029 ) . the effectiveness of rituximab in routine clinical practice has been evaluated in several studies using the national cancer institute 's ( nci ) surveillance , epidemiology , and end results ( seer ) medicare database [ 1315 ] . they indicate that the survival benefits observed in clinical trials extend to elderly patients treated in routine clinical practice , and to mantle cell lymphoma ( mcl ) where survival benefits have not been demonstrated in clinical trials . they also show that even though rituximab was not approved initially for first - line treatment , its use as part of first - line chemotherapy increased rapidly after medicare began coverage in 1998 , and now the majority of seer - medicare patients who receive chemotherapy as first - line therapy for fl , mcl , and cll also receive rituximab [ 1315 ] . efficacy and effectiveness notwithstanding , rituximab is an expensive addition to first - line therapy for nhl with an estimate of $ 24,034 in fl and $ 13,900 in dlbcl based on the projections of cost - effectiveness models , and in part dependent on the year of the analysis and number of administrations of rituximab . at a recommended dose of 375 mg / m body surface area ( bsa ) , and assuming an average bsa of 1.72 m and 68 cycles per first - line course of rituximab , the current cost to medicare could be $ 20,530$27,373 per course , an estimate that excludes the 20% copayment . given the efficacy and cost of rituximab , there has been considerable interest in evaluating its net cost impact and cost - effectiveness , and studies have been conducted in dlbcl [ 17 , 2427 ] and fl [ 16 , 28 , 29 ] . several studies of adding first - line rituximab to chemotherapy in dlbcl projected cost offsets [ 17 , 2427 ] , and in one instance savings , associated with lower rates of progression / relapse and improved survival . two cost - effectiveness analyses of rituximab in combination with chemotherapy have been conducted in fl , one in the us , and one in the united kingdom [ 16 , 28 ] . in the us , hornberger and colleagues projected adding rituximab to cvp would result in a lifetime cost difference of + $ 26,439 , the majority of which was due to rituximab itself ( $ 24,034 ) , a difference in mean overall survival of + 1.51 years , and a cost per quality - adjusted life - year gained of $ 28,565 . the objective of the present study was to estimate the cost and cost - effectiveness of rituximab added to first - line chemotherapy for fl , using a single source of data representative of routine clinical practice in elderly patients in the us . it differs from previous economic studies of rituximab in nhl , the majority of which were based on models , incorporated data from multiple sources including clinical trials , and projected cost and survival beyond the available data . the source of data for this study was the national cancer institute 's ( nci ) surveillance , epidemiology , and end results ( seer ) cancer registry linked to medicare administrative and claims data . in seer - medicare , cancer registry data are linked to medicare enrollment and claims data , which are available for 93% of those aged 65 years in the seer registry . patients were included if they were diagnosed with fl between january 1 , 1999 , and december 31 , 2005 , fl was the first primary cancer diagnosed , they began infused chop ( cyclophosphamide ( c ) , doxorubicin , vincristine ( v ) , and prednisone ( p ) ) or cvp chemotherapy with or without rituximab within 90 days following diagnosis , and they survived at least 60 days after first claim indicating the beginning of therapy . identification of fl was made using the world health organization ( who ) international classification of diseases for oncology , 3rd edition ( icd - o-3 ) histology codes 9695 ( fl grade 1 ) , 9691 ( fl grade 2 ) , 9698 ( fl grade 3 ) , and 9690 ( fl not otherwise specified ) . patients were followed for up to four years from the day first - line therapy began , or until death , the end of their claims ( december 31 , 2007 ) , or the end of their medicare part a and/or part b coverage , whichever came first . in the absence of hemoglobin level , serum lactate dehydrogenase ( ldh ) level , or the number of nodal areas , three of the five prognostic factors for mortality in the follicular lymphoma international prognostic index ( flipi ) , we used medicare claims to identify several predictors of poor performance status , including the use of oxygen and related respiratory therapy supplies , wheel chair and supplies , home health agency , and skilled nursing facility , all from 12 months before until 30 days after fl diagnosis . medicare claims also were used to identify anemia , and to calculate an nci comorbidity index score [ 34 , 35 ] for each patient . we searched the medicare outpatient and nch files to identify claims containing healthcare common procedure coding system ( hcpcs ) j codes for cyclophosphamide ( j8530 , j9070 , j9080 , j9090j9097 ) , doxorubicin ( j9000 , j9001 ) , and vincristine ( j9370 , j9375 , j9380 ) . all such claims within 60 days after the first were used to classify the patient 's first - line regimen as rituximab plus chemotherapy ( chop or cvp ) or chemotherapy alone . the total direct medical cost to medicare , from the beginning of first - line therapy until the end of the observation period ( maximum four years ) , was estimated based on medicare paid amounts obtained from bills within the claims files . diagnosis and procedure codes within claims were used to identify three mutually exclusive categories of costs : immunochemotherapy , which included the costs of infused cancer therapy agents provided during first - line and any subsequent treatment , other cancer care ( any claim with an international classification of diseases , 9th revision , clinical modification [ icd-9-cm ] diagnosis code 140.xx208.xx [ malignant neoplasms ] , but no code for immunochemotherapy ) , and noncancer care ( any other claim ) . in this study , we used inverse probability of treatment weighting ( iptw ) for survival data to estimate the adjusted difference in cumulative survival between the two treatment groups . first , we estimated the conditional probability of assignment to the rituximab plus chemotherapy group for each patient using logistic regression with treatment group as the dependent variable and all other independent variables from the cost model as predictor variables . then , the inverse of this conditional probability estimate was assigned as a weight to each patient , and it was used to adjust the kaplan - meier cumulative survival estimate for each treatment group . chi - square analysis was used to test for the independence between patient characteristics specified as categorical variables and the two first - line treatment groups . we performed partitioned ipw least squares regression analysis to examine adjusted associations between cumulative costs over four years and patient demographic , clinical , and treatment factors . confidence intervals ( ci ) for the cumulative cost coefficients were calculated by a bootstrap approach , in which the process of performing 48 partitioned regression analyses and summing coefficients across partitions was repeated 1,000 times using sampling with replacement from the original cohort . using the adjusted kaplan - meier estimator based on iptw , we estimated the adjusted difference in survival between rituximab plus chemotherapy and chemotherapy alone in each of 48 monthly partitions following the beginning of first - line therapy . cost effectiveness ratios were calculated in months 348 ( ignoring months 1 - 2 due to the inclusion criteria ; there was no mortality during this period ) after the beginning of first - line therapy , by dividing the incremental cumulative cost of rituximab by the cumulative survival difference . confidence intervals for the cost - effectiveness ratio were calculated by dividing the upper bound of the 95% ci for cost by the lower bound of the 95% ci for survival , and then repeating the process with the lower cost and upper survival bounds . there were 1,117 patients in the final cohort : 750 ( 67% ) received rituximab plus chemotherapy , and 367 ( 33% ) received chemotherapy alone ( table 1 ) . the median age at diagnosis was 73 years ( minimum 66 years ) , 56% had stage iii / iv disease , 42% had extranodal involvement , and 15% had 1 indicator of poor performance status . there were no differences in stage at diagnosis , extranodal involvement , or nci comorbidity index score between the two groups . among those who received rituximab , the average number of rituximab administrations during first - line therapy was 6.3 ( median 6 ; interquartile range [ iqr ] 57 ) , and the average number of units was 7.3 100 mg vials ( median 7 ; iqr 68 ) . the mean unadjusted ipw cumulative cost was $ 111,815 ( 95% ci $ 104,455$119,466 ) in the rituximab group compared to $ 80,826 ( 95% ci $ 74,006$88,113 ) for chemotherapy alone ( figure 1 ) . the mean cost of first - line rituximab was $ 15,640 ( median $ 14,786 ; iqr $ 10,831$18,560 ) , and the average cost per administration was $ 2,836 ( median $ 2,747 ; iqr $ 2,617$3,113 ) . in the multivariate analyses , rituximab was associated with statistically significantly higher incremental total ( $ 18,695 ; 95% confidence interval [ ci ] $ 9,302$28,643 ) and immunochemotherapy ( $ 13,336 ; 95% ci $ 9,364$17,552 ) costs after four years , but not other cancer ( $ 4,816 ; 95% ci $ 1,899$11,303 ) or noncancer ( $ 351 ; 95% ci $ 3,481$4,202 ) costs ( table 2 ) . most of the separation in total and immunochemotherapy costs between the two treatment groups occurred during the first 6 months after the beginning of first - line therapy ( figure 2 ) . other factors associated with higher costs were stage ( immunochemotherapy cost ) , extranodal involvement ( total and other cancer costs ) , nci comorbidity index ( predominately non - cancer cost ) , and 1 indicator of poor performance status ( total and other cancer costs ) . factors associated with lower costs were age > 80 years ( immunochemotherapy cost ) , other race / ethnicity ( total and immunochemotherapy costs ) , and urban / rural ( compared to metropolitan ) area ( total and other cancer cost ) . the incremental cumulative adjusted survival ( years ) in the rituximab group was 0.05 ( 95% ci 0.020.09 ) after two years , 0.11 ( 95% ci 0.050.16 ) after three years , and 0.18 ( 95% ci 0.100.27 ) after four years of observation ( figure 3 ) . the cost per life year gained in the rituximab group was $ 382,642 ( 95% ci $ 164,900$1,360,559 ) after two years , $ 193,859 ( 95% ci $ 77,314$609,607 ) after three years , and $ 102,142 ( 95% ci $ 34,531$296,337 ) after four years of observation ( figure 4 ) . at no time point during the observation period did the 95% ci for the cost - effectiveness ratio contain a value indicating that rituximab plus chemotherapy was both more costly and less effective ( dominated ) , or less costly and more effective ( dominates ) than chemotherapy alone . in this study , we identified a cohort of elderly fl patients who received first - line chemotherapy with or without rituximab . we followed this cohort for up to four years after the beginning of therapy to estimate the net cost impact and cost - effectiveness of adding rituximab to first - line therapy . the mean cost of first - line rituximab in our study ( $ 15,640 ) was substantially lower than expected based on the projections from a cost - effectiveness model in fl ( $ 24,034 ) , and our own calculations based on recommended dosing and current medicare reimbursement . hornberger and colleagues assumed 8 administrations , 700 mg per administration , and $ 5.82/mg in estimating the cost of first - line rituximab . it is noteworthy that in the rituximab group the cumulative incremental cost of immunochemotherapy during the four - year period ( $ 13,336 ) was lower than the cost of first - line rituximab ( $ 15,640 ) . most of the difference between the two groups appeared during first - line therapy , which suggests there may have been differences in supportive care and other cancer services during first - line therapy that were not captured in the immunochemotherapy category . after first - line therapy , the cumulative incremental cost of other cancer care in the rituximab group declined , and by year three it was no longer statistically significant . the cumulative survival benefit of rituximab continued to accrue , and was statistically significant , throughout the observation period , and since the cumulative cost impact was negligible after the end of first - line therapy , the cost per life year gained declined ( became more favorable for rituximab ) rapidly during the observation period . consequently , we did not account in our survival and cost - effectiveness analyses for patients who died during the first two months after starting first - line therapy . neither pivotal phase iii trial of first - line rituximab showed significant separation in the overall survival curves within the first several months after beginning therapy [ 9 , 11 ] . however , it is possible we would have observed larger survival differences , and hence smaller cost effectiveness ratios , had we included patients who died within the first two months of therapy . therefore , we did not adjust the survival estimates for qol prior to calculating the cost - effectiveness ratios . in their cost - effectiveness analysis based on a simulation model , hornberger and colleagues projected that adding rituximab to cvp would increase mean overall survival by 1.51 years and quality - adjusted life - years by 0.93 years , which suggests that had we adjusted our survival estimates for qol , the cost - effectiveness ratios may have been larger than those based on unadjusted survival . we did , however , include age ( all patients were older than the age threshold for excess risk of mortality in flipi [ 60 ] ) , stage ( iii - iv is associated with increased risk in flipi ) , and whether the patient had extranodal involvement . in the multivariate analysis , 1 indicator of poor performance , extranodal involvement , and later stage diagnosis it is important to note that any variable constructed using claims for medical services , such as performance status and nci comorbidity index [ 34 , 35 ] , that is then included in a multivariate cost analysis , has a greater likelihood of being statistically significant simply by virtue of the fact that most claims have a cost attached . in the cost analysis , we used ipw and established separate weights for the two treatment groups to account for differential censoring between the two groups . when we restricted the cohort to those who received chop , the cost coefficients for the rituximab group were similar to those in the primary analyses . therefore , we used medicare paid amounts reported in claims to estimate total direct medical costs to medicare . therefore , total direct medical costs to all payers including patients and other insurers could be up to 20% higher than reported in this study . also , medicare did not pay for most oral drugs for the majority of the period in which our study was conducted , which is another reason why the costs reported in this study may underestimate the total direct medical costs of care to all payers . finally , the confidence intervals for the cost - effectiveness ratio were calculated by dividing the upper bound of the 95% ci for cost by the lower bound of the 95% ci for survival , and then repeating the process with the lower - cost and upper - survival bounds . in sum , adding rituximab to first - line chemotherapy for elderly patients with fl results in higher direct medical costs to medicare and longer overall survival after four years . the net cost impact is due primarily to the treatment cost of first - line rituximab , although there may be small cost offsets associated with lower progression and/or relapse . the cost - effectiveness ratio we estimated is substantially higher than projected in a previous analysis based on a model . extending the time horizon for our study , and including patients who died during the first two months of therapy our findings suggest even highly effective cancer therapies will do little to offset the rising costs of cancer care in the united states , and that additional public policies will be required to address this problem .

the toxicological properties of mercury have been attributed to both its thiophilicity and its selenophilicity . with respect to the latter , selenium is an important component of antioxidants , and the interaction between hg(ii ) and selenium compounds may reduce the bioavailability of selenium via the formation of insoluble mercury selenide species . furthermore , mercury may bind to the active sites of selenoenzymes and thereby inhibit their functions . for example , selenium is a component of a variety of enzymes that incorporate the amino acids selenocysteine and selenomethionine ( figure 1 ) , as illustrated by glutathione peroxidases , thioredoxin reductases , glycine reductases , formate dehydrogenases , and selenoprotein p. other examples of selenium - containing biomolecules include the amino acid derivatives selenoneine and se - methylselenoneine ( figure 1 ) , of which the latter was identified in human urine and blood . it has recently been shown that selenoamino acids ( namely l - selenocysteine , l - selenoglutathione , d , l - selenopenicillamine , and l - selenomethionine ) complex readily to methylmercury species and that cleavage of the hg c bond may be achieved under physiologically relevant conditions to yield mercury selenide via ( mehg)2se . insoluble mercury selenide particles have also been observed in the brains of humans exposed to methylmercury species , and these particles are considered to be much less toxic than mobile , soluble methylmercury species such as cyshgme . this observation provides evidence of the neuroprotective effects of selenium with respect to the prevention of mercury - induced damage to the central nervous system . additionally , recent in vitro studies have shown that selenoneine may assist cells in removal of cyshgme . however , the interactions between mercury and selenium in biological systems are complex , and animal studies have produced contradictory results . for example , it has been observed that co - administration of diphenyl diselenide compounds with methylmercury chloride partially ameliorated methylmercury - induced oxidative damage to proteins in the livers and brains of intoxicated mice ; on the other hand , rats simultaneously dosed with methylmercury chloride and diphenyl diselenide were shown to suffer more severe neurological symptoms , such as motor deficits and weight loss , than rats dosed with methylmercury chloride alone . a detailed understanding of the impact of mercury on the biochemical roles of selenium would , therefore , benefit considerably from the development of the chemistry of mercury in a coordination environment that features selenium . therefore , we describe here the reactivity of 1-methyl-1,3-dihydro-2h - benzimidazole-2-selone ( figure 2 ) , h(sebenzim ) , a structural analogue of selenoneine , towards mercury , including the protolytic cleavage of mercury 1-r - imidazole-2-thiones , h(mim ) , of which the methyl derivative is the well - known antithyroid drug , methimazole ( tapazole ) , are a widely studied class of molecules that can bind to a variety of metals , including mercury . however , in contrast to the numerous studies pertaining to 1-r - imidazole-2-thiones , there are few corresponding investigations of 1-r - imidazole-2-selones , h(seim ) . for example , only h(seim ) and h(seim ) , and the benzannulated derivatives , h(sebenzim ) and h(sebenzim evidence for the ability of the imidazole-2-selone , h(sebenzim ) , to coordinate to the mercury centers of hgx2 ( x = cl , br , i ) in solution ( scheme 1 ) is provided by a combination of h , se{h } , and hg{h } nmr spectroscopies . for example , the hg ( table 1 ) chemical shift changes progressively upon addition of h(sebenzim ) to a solution of hgcl2 in dmso - d6 . correspondingly , the se ( table 1 ) and h ( table 2 and figure 3 ) chemical shifts associated with h(sebenzim ) also progressively shift upon addition to hgcl2 . in addition to providing evidence for coordination of h(sebenzim ) to mercury , the observation of a single resonance in both the se{h } and hg{h } nmr spectra for each concentration ratio , and also a single set of resonances in the h nmr spectra , indicates that the coordination is reversible and that the process is facile on the nmr time scale at room temperature . furthermore , low temperature ( 40 c ) spectra in dmf - d7 likewise show single resonances , thereby demonstrating that the exchange is still rapid at this temperature ( data not shown ) . variation of h nmr chemical shift of the methyl group of h(sebenzim ) in the presence of hgx2 as a function of the molar ratio . although the fluxionality prevents identification of the precise solution composition ( scheme 1 ) , the tetrakis , tris , and bis complexes , [ h(sebenzim)]4hgcl2 , [ h(sebenzim)]3hgcl2 , and [ h(sebenzim)]2hgcl2 , may be obtained by crystallization from a solution that contains the respective number of equivalents of h(sebenzim ) . the molecular structures of [ h(sebenzim)]3hgcl2 and [ h(sebenzim)]4hgcl2 have been determined by x - ray diffraction , as illustrated in figures 4 and 5 , respectively . of these , the latter compound is particularly important because there are no structurally characterized mononuclear mercury compounds with four dative l - type selenium donors currently listed in the cambridge structural database ( csd ) . furthermore , efforts to synthesize a tetrakis selone complex of mercury ( other than for unsubstituted selenourea ) have been reported to be unsuccessful . for example , treatment of hgcl2 with 4 equiv of n , n - dimethylselenourea ( dmseu ) was reported to yield only the bis complex , ( dmseu)2hgcl 2 . molecular structure of [ h(sebenzim)]3hgcl2 , which is more appropriately represented as the ion pair , { [ h(sebenzim)]3hgcl}[cl ] . molecular structure of the cation { [ h(sebenzim)]4hg } of { [ h(sebenzim)]4hg}[cl]2 ( only one of the independent molecules is shown ) . in addition to [ h(sebenzim)]4hgcl2 being of significance because its existence demonstrates that a mercury center can accommodate four selenium l - type donor ligands , the tris complex , [ h(sebenzim)]3hgcl2 , is of interest because structurally characterized mercury compounds with three l - type selenium donors are also uncommon . thus , compounds with a hgse3 motif are typically polynuclear selenide or selenolate derivatives ; there are , nevertheless a few structurally characterized mononuclear compounds that contain mercury coordinated to three dative l - type selenium ligands , of which [ ( meimse)3hgcl]cl , { [ n(ch2ch2seph)3hg(-no3)}(no3 ) , and { [ cpfe(co)2p(opr)2se]3hg}(clo4)2 are illustrative . comparison of the molecular structures of [ h(sebenzim)]3hgcl2 ( figure 4 ) and [ h(sebenzim)]4hgcl2 ( figure 5 ) with that of [ h(sebenzim)]2hgcl2 reveals interesting structural variations as a function of composition , as summarized in figure 6 . first , there is a progressive increase in the hg cl distances in the sequence [ h(sebenzim)]2hgcl2 thus , whereas the two hg cl bond lengths in the bis complex [ h(sebenzim)]2hgcl2 [ 2.4942(7 ) and 2.5727(8 ) ] are comparable to the mean value of 2.43 for structurally characterized four - coordinate mercury compounds listed in the csd , the shortest hgcl distance in the tetrakis complex , [ h(sebenzim)]4hgcl2 , is 3.913 , such that the compound may be better represented as { [ h(sebenzim)]4hg}[cl]2 . the hg cl distances in the tris complex , [ h(sebenzim)]3hgcl2 , are intermediate between those of [ h(sebenzim)]2hgcl2 and [ h(sebenzim)]4hgcl2 , with values of 2.7506(10 ) and 3.2397(9 ) . while the latter value is sufficiently large that it can not be considered to correspond to a hg cl covalent bond , the shorter distance of 2.7506(10 ) is only 0.32 longer than the csd average ( vide supra ) and may therefore be viewed as corresponding to a weak covalent interaction , such that the compound can be formulated as { [ h(sebenzim)]3hgcl}[cl ] . in accord with the long hg cl bond distance , the coordination geometry of { [ h(sebenzim)]3hgcl } deviates significantly from tetrahedral . thus , the four - coordinate 4 index ( table 4 ) of { [ h(sebenzim)]3hgcl } ( 0.78 ) is close to that for an idealized trigonal monopyramid ( 0.85 ) in which chlorine occupies an axial position ; in the extreme that the axial chlorine is considered to serve the role of a counterion , the mercury would be described as approximately trigonal planar . comparison of the mercury coordination environments of [ h(sebenzim)]2hgcl2 ( top ) , [ h(sebenzim)]3hgcl2 ( center ) , and [ h(sebenzim)]2hgcl2 ( bottom ) . data taken from ref ( 31a ) . by comparison to the large variation in hg cl interactions within [ h(sebenzim)]xhgcl2 , the average hg se bond lengths exhibit little variation , increasing only slightly as a function of x , i.e. , bis ( 2.591 ) < tris ( 2.611 ) < tetrakis ( 2.671 ) . these hg se bond lengths are comparable to the mean value of 2.643 for compounds listed in the csd , but are longer than those in compounds such as hg(seph)2 [ 2.480 ] and [ tm a common feature of all [ h(sebenzim)]xhgcl2 structures is that each chloride , regardless of whether it is attached covalently to the mercury center , participates in hydrogen bonding interactions with the imidazole n h moieties . there is , nevertheless , an interesting difference with respect to the nature of the hydrogen bonding interactions . specifically , each chlorine that is covalently bound to mercury participates in an intramolecular n hcl interaction , whereas each outer - sphere chloride anion participates in a n hclh n interaction that serves to link together two h(sebenzim ) moieties , as summarized in figure 7 . n hcl interactions and is a discrete mononuclear species , [ h(sebenzim)]3hgcl2 and [ h(sebenzim)]4hgcl2 also exhibit intermolecular n n interactions that bridge two molecules , thereby creating a dimeric structure ( figure 8) , while [ h(sebenzim)]4hgcl2 exhibits an intramolecular n hclh the various hydrogen bonding ncl distances in [ h(sebenzim)]xhgcl2 are in the range 3.031(7)3.227(2 ) and are comparable to the values for other compounds with n hcl interactions listed in the csd [ dav(ncl ) = 3.181 ] . n interactions that link together pairs of molecules are characterized by ncln angles in the range 100.1119.7 , which are comparable to the average value of 99.9 for compounds listed in the csd that feature n hclh n interactions wherein the chloride ion is not covalently bonded to any other atoms . n hydrogen bonding creates a chain of { [ h(sebenzim)]4hg}[cl ] } moieties , bridged by cl ions . the various hydrogen bonding networks in [ h(sebenzim)]xhgcl2 may be described by the graph set notations that are summarized in table 5 . for example , the hydrogen - bonded dimer of [ h(sebenzim)]3hgcl2 forms a 20-membered ring that is described by the unitary graph set dds(6 ) and the binary graph set r42(20 ) . coordination of h(sebenzim ) to hgcl2 is accompanied by only relatively small increases in the lengths of the c se bonds . thus , the c se bond lengths of [ h(sebenzim)]2hgcl2 [ 1.862(3 ) and 1.864(3 ) ] , [ h(sebenzim)]3hgcl2 [ 1.868(3 ) , 1.859(3 ) , and 1.857(3 ) ] , and [ h(sebenzim)]4hgcl2 [ 1.854(12 ) , 1.896(11 ) , 1.851(9 ) , 1.851(9 ) , 1.857(11 ) , 1.854(11 ) , 1.869(9 ) , and 1.856(9 ) ] are only slightly longer than that of free h(sebenzim ) [ 1.838(2 ) ] . despite these minor metrical changes , however , it is interesting to note that both the c ( see experimental section and ref ( 31a ) ) and se nmr ( table 1 ) chemical shifts of the [ cse ] moiety are sensitive towards the changes induced by coordination to mercury . similar spectroscopic trends have been observed in related systems , and also for thione counterparts . nmr spectroscopic studies also demonstrate that h(sebenzim ) binds reversibly to hgbr2 and hgi2 in dmso - d6 , and that the processes are facile on the nmr time scale , as indicated by the observation of single sets h nmr chemical shifts for the h(sebenzim ) signals ( table 2 and figure 3 ) . interestingly , the se nmr chemical shift of the h(sebenzim ) moiety is more sensitive towards coordination of hgcl2 than to coordination of either hgbr2 or hgi2 . for example , the se nmr chemical shifts of 2:1 mixtures of h(sebenzim ) and hgx2 move upfield from the value of pure h(sebenzim ) by values of 68 ppm ( x = cl ) , 54 ppm ( x = br ) , and 35 ppm ( x = i ) . despite the reversibility of coordination of h(sebenzim ) , the bis complex , [ h(sebenzim)]2hgi2 , may , nevertheless , be isolated from reactions performed in either acetonitrile or benzene . interestingly , the crystals of [ h(sebenzim)]2hgi2 obtained from the two different reaction solvents are not isomorphous , and the molecules adopt different geometries , as illustrated in figures 10 and 11 . specifically , the h(sebenzim ) ligands are oriented in different directions relative to both each other and the iodide ligands . accompanying these variations in conformation are differences in the mercury coordination environments . for example , whereas the orthorhombic form of [ h(sebenzim)]2hgi2 obtained from acetonitrile ( figure 10 ) , with a 4 index of 0.94 , is close to tetrahedral ( 4 = 1.00 ) , monoclinic [ h(sebenzim)]2hgi2 obtained from benzene ( figure 11 ) , with a 4 index of 0.88 , is distorted towards trigonal monopyramidal ( 4 = 0.85 ) . in addition to these angular variations , there are small differences in hg se and hg thus , while the average hg i bond length of orthorhombic [ h(sebenzim)]2hgi2 ( 2.792 ) is longer than that of the monoclinic version ( 2.737 ) , the average hg se bond length of orthorhombic [ h(sebenzim)]2hgi2 ( 2.627 ) is shorter than that of the monoclinic version ( 2.692 ) . similarly to hgcl2 , coordination of h(sebenzim ) to hgi2 is accompanied by only small increases in the lengths of the c se bonds . the c se bond lengths in [ h(sebenzim)]2hgi2 [ 1.852(9 ) and 1.858(9 ) for the orthorhombic form and 1.871(3 ) and 1.863(3 ) for the monoclinic form ] are comparable to those observed in [ h(sebenzim)]xhgcl2 , which range from 1.851(9 ) to 1.896(11 ) . molecular structure of orthorhombic [ h(sebenzim)]2hgi2 obtained from acetonitrile solution . the most striking differences in the structures of orthorhombic and monoclinic [ h(sebenzim)]2hgi2 do not , however , pertain to the mercury coordination environment . rather , the differences are associated with the distinct hydrogen bonding motifs ( figures 12 and 13 ) . furthermore , these hydrogen bonding patterns are also different from that of the chloride counterpart , [ h(sebenzim)]2hgcl2 ( vide supra ) , as illustrated in figure 14 . hydrogen bonding network for orthorhombic [ h(sebenzim)]2hgi2 obtained from acetonitrile solution , illustrating intramolecular and intermolecular n hi interactions . hydrogen bonding network for monoclinic [ h(sebenzim)]2hgi2 obtained from benzene solution , illustrating head - to - head for example , whereas [ h(sebenzim)]2hgcl2 is observed to have two intramolecular n hcl interactions , the orthorhombic form of [ h(sebenzim)]2hgi2 possesses one intramolecular and one intermolecular n hi interaction , thereby creating a hydrogen - bonded helical chain of [ h(sebenzim)]2hgi2 molecules ( figure 12 ) . in contrast to [ h(sebenzim)]2hgcl2 and orthorhombic [ h(sebenzim)]2hgi2 , however , the monoclinic form of [ h(sebenzim)]2hgi2 possesses no intramolecular or intermolecular n hi interactions . rather , the n h groups of the h(sebenzim ) ligands participate in pairs of centrosymmetric hse interactions that link adjacent molecules together in a manner similar to that observed for certain h(seim ) derivatives in the absence of metal coordination ( figure 13 ) . interestingly , h(sebenzim ) itself does not adopt this head - to - head motif , but rather adopts a polymeric head - to - tail structure . as such , coordination of the selenium to a metal promotes centrosymmetric n hse interactions in this system , with there being no comparable structures currently listed in the csd . the existence of this motif is undoubtedly a consequence of the fact that iodide is , by comparison to chloride , a poor hydrogen bond acceptor , such that n as would be expected , the hydrogen bonding ni interactions in orthorhombic [ h(sebenzim)]2hgi2 [ 3.486(7 ) and 3.589(7 ) ] are substantially longer than the analogous ncl interactions in [ h(sebenzim)]2hgcl2 . thus , while the mean ncl distance in [ h(sebenzim)]2hgcl2 is 3.182 , the mean ni distance in orthorhombic [ h(sebenzim)]2hgi2 is 3.541 . for reference , the mean ncl distance for compounds listed in the csd with n hcl interactions involving a terminal metal chloride is 3.332 , while the analogous ni distance is 3.707 . in view of the fact that the protolytic cleavage of the hg c bond is a critical step in detoxification of organomercurials , and recognizing that h(sebenzim ) is an analogue of selenoneine , we have also investigated the reactivity of h(sebenzim ) towards methylmercury halides . significantly , we have observed that h(sebenzim ) not only coordinates to the mercury center , as observed for hgx2 , but it is also capable of cleaving the hg for example , h(sebenzim ) reacts with mehgi at 100 c to liberate ch4 ( as observed by h nmr spectroscopy ) and afford [ h(sebenzim)2]hgi ( scheme 2 ) . the importance of this observation is underscored by the fact that selenoneine , of which h(sebenzim ) is a structural analogue , has recently been shown to achieve demethylation of cyshgme . the molecular structure of [ h(sebenzim)2]hgi has been determined by x - ray diffraction , as illustrated in figure 15 , which demonstrates that it features mercury in an approximately trigonal planar environment , with a pyramidality ( p ) value of only 0.2. the bond angles at mercury , however , deviate from 120 [ se hg i = 114.87(2) and 104.02(2) ] , such that the geometry is distorted towards t - shaped , which is not uncommon for mercury . molecular structure of the monomeric unit , [ h(sebenzim)2]hgi . the most interesting feature of [ h(sebenzim)2]hgi , however , pertains to the fact that the h(sebenzim ) and ( sebenzim ) moieties are linked by n hn hydrogen bonding interactions , with a nn distance of 2.720(6 ) . as such , the combined fragment , [ h(sebenzim)2 ] , may be viewed as an lx - type ligand . in this regard , the two hg se bond lengths present in [ h(sebenzim)2]hgi [ 2.5466(6 ) and 2.5748(6 ) ] are very similar . while the primary coordination environment about mercury is trigonal planar , it is evident that there are additional intermolecular hgse interactions [ 3.0904(6 ) and 3.3215(6 ) ] that are substantially longer than those within [ h(sebenzim)2]hgi [ 2.5466(6 ) and 2.5748(6 ) ] , and which serve to link together adjacent molecules , as illustrated in figure 16 . in this regard , the extended coordination geometry of mercury may be viewed as five - coordinate and , with a 5 index of 0.51 , is intermediate between the idealized values for square pyramidal ( 5 = 0 ) and trigonal bipyramidal ( 5 = 1 ) geometries . extended structure of { [ h(sebenzim)2]hgi}x . in view of the kinetic stability of two - coordinate rhgx complexes towards protolytic cleavage , it is likely that the mechanism for formation of [ h(sebenzim)2]hgi involves the initial formation of an adduct , [ h(sebenzim)]xhg(me)i , which undergoes either intramolecular protolytic cleavage of the hg me bond , or cleavage in an intermolecular manner to afford a mercury c bond of mehgcl , although the reaction follows a different course than that of mehgi . specifically , reaction of mehgcl with h(sebenzim ) at 100 c results in evolution of methane , as observed by h nmr spectroscopy , and the formation of a mixture of [ h(sebenzim)]4hgcl2 ( vide supra ) and [ h(sebenzim)2]2hg ( scheme 3 ) . the formation of [ h(sebenzim)]4hgcl2 and [ h(sebenzim)2]2hg upon treatment of mehgcl with h(sebenzim ) is indicative of a ligand redistribution process . for example , one possibility is that incipient { [ h(sebenzim)2]hgcl } , the counterpart of the above iodide derivative , could redistribute to give [ h(sebenzim)2]2hg and hgcl2 , of which the latter would be trapped by h(sebenzim ) to afford [ h(sebenzim)]4hgcl2 . the molecular structure of [ h(sebenzim)2]2hg has been determined by x - ray diffraction ( figure 17 ) , which demonstrates that pairs of h(sebenzim ) and ( sebenzim ) ligands are linked together via hydrogen bonding interactions to produce the combined lx - type ligand , [ h(sebenzim)2 ] , in a manner akin to that observed for [ h(sebenzim)2]hgi . however , while the nn distances within [ h(sebenzim)2]2hg [ 2.724(14 ) and 2.732(14 ) ] are comparable to that observed for [ h(sebenzim)2]hgi [ 2.720(6 ) ] , the angles between the h(sebenzim ) and ( sebenzim ) planes ( 76.6 and 76.5 ) are distinctly larger than that in [ h(sebenzim)2]hgi ( 47.3 ) . thus , it is evident that the hydrogen - bonded [ h(sebenzim)2 ] ligand is quite flexible with respect to the twist angles of the benzimidazole ring systems . the coordination geometry about mercury in [ h(sebenzim)2]2hg is distorted tetrahedral ( 4 = 0.88 ) , with hg se bond lengths in a narrow range of 2.6228(12)2.6367(13 ) . molecular structure of [ h(sebenzim)2]2hg . in summary , 1-methyl-1,3-dihydro-2h - benzimidazole-2-selone , h(sebenzim ) , is a structural analogue of selenoneine and coordinates reversibly to the metal centers of hgx2 ( x = cl , br , i ) . furthermore , h(sebenzim ) is also capable of cleaving the hg c bond of methylmercury halides , thereby mimicking the role of selenoneine in demethylating cyshgme . x - ray diffraction studies demonstrate that while two equivalents of h(sebenzim ) simply coordinate to mercury centers of hgx2 ( x = cl , i ) , the third and fourth equivalents result in displacement of the chloride ligands . thus , [ h(sebenzim)]3hgcl2 and [ h(sebenzim)]4hgcl2 are better represented as ion pairs , namely { [ h(sebenzim)]3hgcl}[cl ] and { [ h(sebenzim)]4hg}[cl]2 , of which the latter is the first example of a structurally characterized tetrahedral mercury compound that features four l - type selenium donors . a common feature of all [ h(sebenzim)]xhgcl2 structures is that each chloride , regardless of whether it is attached covalently to the mercury center or serves as a counterion , participates in hydrogen bonding interactions with the imidazole n h moieties . the nature of the network , however , depends critically on the number of h(sebenzim ) donors . for example , whereas [ h(sebenzim)]2hgcl2 exhibits only intramolecular n hcl interactions and is a discrete mononuclear species , [ h(sebenzim)]3hgcl2 exhibits an intramolecular n hcl interaction and intermolecular n interactions that bridge two molecules , resulting in a dimeric structure , while [ h(sebenzim)]4hgcl2 exhibits an intramolecular n hclh this investigation demonstrates that not only is h(sebenzim ) a good ligand for mercury , capable of displacing halide ligands , but is also capable of protolytically cleaving mercury carbon bonds , a result that is of relevance to the role of selenium compounds in the detoxification of mercury compounds . h nmr spectra are reported in ppm relative to sime4 ( = 0 ) and were referenced internally with respect to the protio solvent impurity ( 7.16 for c6d5h and 2.50 for dmso - d5).c nmr spectra are reported in ppm relative to sime4 ( = 0 ) and were referenced internally with respect to the solvent ( 128.06 for c6d6 and 39.52 for dmso - d6).se nmr spectra are reported in ppm relative to neat me2se ( = 0 ) and were referenced using a solution of ph2se2 in c6d6 ( = 460 ) as an external standard.hg nmr spectra are reported in ppm relative to neat me2hg ( = 0 ) and were referenced using a 1.0 m solution of hgi2 in dmso - d6 ( = 3106 ) as an external standard . ir spectra were recorded as kbr pellets on a nicolet is10 ft - ir spectrometer ( thermoscientific ) , and the data are reported in reciprocal centimeters . 1-methyl-1,3-dihydro-2h - benzimidazole-2-selone was obtained by a literature method , and all other chemicals were purchased from sigma - aldrich . caution ! all mercury compounds are toxic , and appropriate safety precautions must be taken in handling these compounds . single - crystal x - ray diffraction data were collected on a bruker apex ii diffractometer , and crystal data , data collection , and refinement parameters are summarized in table 6 . the structures were solved using direct methods and standard difference map techniques , and were refined by full - matrix least - squares procedures on f with shelxtl ( version 2013/4 ) . a suspension of h(sebenzim ) ( 46 mg , 0.22 mmol ) and hgi2 ( 50 mg , 0.11 mmol ) in c6d6 ( 2 ml ) in an nmr tube equipped with a j. young valve was heated overnight at 100 c . over this period , yellow , x - ray - quality crystals of [ h(sebenzim)]2hgi2 ( 54 mg , 56% yield ) were deposited and isolated by decanting the solution . calcd for c16h16i2hgn4se2 : c , 21.9 ; h , 1.8 ; n , 6.4 . found : c , 22.0 ; h , 1.6 ; n , 6.4 . h nmr ( dmso - d6 ) : 3.90 [ s , 6h of ch3 ] , 7.42 [ m , 4h of c6h4 ] , 7.51 [ m , 2h of c6h4 ] , 7.71 [ m , 2h of c6h4 ] , not observed [ nh ] . c{h } nmr ( dmso - d6 ) : 33.3 [ ch3 ] , 111.8 [ ch of c6h4 ] , 112.1 [ ch of c6h4 ] , 124.4 [ ch of c6h4 ] , 125.0 [ ch of c6h4 ] , 131.8 [ c of c6h4 ] , 133.6 [ c of c6h4 ] , 152.7 [ cse ] . se{h } nmr ( dmso - d6 ) : 48 ppm . hg{h } nmr ( dmso - d6 ) : not observed . ir data ( kbr pellet , cm ) : 3172 ( m ) , 3114 ( m ) , 3056 ( m ) , 2986 ( w ) , 2929 ( w ) , 1619 ( w ) , 1498 ( m ) , 1486 ( m ) , 1447 ( vs ) , 1391 ( w ) , 1364 ( w ) , 1346 ( s ) , 1333 ( m ) , 1246 ( w ) , 1226 ( w ) , 1159 ( w ) , 1132 ( m ) , 1091 ( m ) , 1008 ( w ) , 902 ( vw ) , 804 ( w ) , 748 ( vs ) , 727 ( w ) , 664 ( w ) . a solution of hgcl2 ( 17 mg , 0.06 mmol ) in ch3cn ( 1 ml ) was added to a solution of h(sebenzim ) ( 40 mg , 0.19 mmol ) in chcl3 ( 2 ml ) . the pale yellow solution was allowed to stand at room temperature for 4 days at room temperature , over which period colorless crystals were deposited as the solution evaporated . x - ray - quality crystals of [ h(sebenzim)]3hgcl2(ch3cn ) were isolated by decanting the mother liquor and dried in vacuo ( 39 mg , 66% yield ) . calcd for c26h27cl2hgn7se3 : c , 33.0 ; h , 2.9 ; n , 10.4 . found : c , 33.6 ; h , 2.3 ; n , 9.9 . h nmr ( dmso - d6 ) : 3.83 [ s , 9h of ch3 ] , 7.35 [ m , 6h of c6h4 ] , 7.40 [ m , 3h of c6h4 ] , 7.62 [ m , 3h of c6h4 ] , 13.93 [ br , n - h ] . c{h } nmr ( dmso - d6 ) : 32.7 [ ch3 ] , 111.3 [ ch of c6h4 ] , 111.4 [ ch of c6h4 ] , 123.9 [ ch of c6h4 ] , 124.5 [ ch of c6h4 ] , 131.6 [ c of c6h4 ] , 133.4 [ c of c6h4 ] , 154.6 [ cse ] . se{h } nmr ( dmso - d6 ) : 35 ppm . hg{h } nmr ( dmso - d6 ) : 1020 ppm . ir data ( kbr pellet , cm ) : 3448 ( w ) , 3032 ( m ) , 2969 ( m ) , 2918 ( m ) , 2850 ( m ) , 2804 ( m ) , 2740 ( m ) , 2693 ( m ) , 2588 ( w ) , 2514 ( w ) , 1618 ( w ) , 1502 ( s ) , 1449 ( vs ) , 1399 ( m ) , 1360 ( m ) , 1348 ( s ) , 1334 ( s ) , 1258 ( m ) , 1242 ( m ) , 1154 ( w ) , 1132 ( w ) , 1096 ( s ) , 1008 ( w ) , 805 ( w ) , 740 ( vs ) . a suspension of h(sebenzim ) ( 85 mg , 0.40 mmol ) and hgcl2 ( 27 mg , 0.10 mmol ) in cd3cn ( 2 ml ) in an nmr tube equipped with a j. young valve was heated overnight at 100 c . over this period , pale yellow , x - ray - quality crystals of [ h(sebenzim)]4hgcl2 ( 94 mg , 84% yield ) were deposited and isolated by decanting the solution . calcd for c32h32cl2hgn8se4 : c , 34.4 ; h , 2.9 ; n , 10.0 . found : c , 34.7 ; h , 2.6 ; n , 10.0 . h nmr ( dmso - d6 ) : 3.80 [ s , 12h of ch3 ] , 7.33 [ m , 12h of c6h4 ] , 7.56 [ m , 4h of c6h4 ] , 13.72 [ br , n - h ] . c{h } nmr ( dmso - d6 ) : 32.5 [ ch3 ] , 111.0 [ ch of c6h4 ] , 111.1 [ ch of c6h4 ] , 123.6 [ ch of c6h4 ] , 124.2 [ ch of c6h4 ] , 131.7 [ ring junction c of c6h4 ] , 133.5 [ ring junction c of c6h4 ] , 156.6 [ cse ] . se{h } nmr ( dmso - d6 ) : 44 ppm . hg{h } nmr ( dmso - d6 ) : 1012 ppm . ir data ( kbr pellet , cm ) : 3424 ( w ) , 3032 ( m ) , 2971 ( m ) , 2919 ( m ) , 2849 ( m ) , 2727 ( w ) , 2668 ( w ) , 1618 ( w ) , 1498 ( m ) , 1447 ( vs ) , 1390 ( w ) , 1346 ( s ) , 1333 ( m ) , 1247 ( w ) , 1156 ( w ) , 1134 ( w ) , 1097 ( m ) , 1009 ( w ) , 901 ( vw ) , 804 ( w ) , 756 ( m ) , 747 ( s ) . a suspension of h(sebenzim ) ( 40 mg , 0.19 mmol ) and ph2hg ( 17 mg , 0.05 mmol ) in cd3cn ( 0.7 ml ) in an nmr tube equipped with a j. young valve was heated overnight at 100 c . over this period , very pale yellow , x - ray - quality crystals of [ h(sebenzim)2]2hg ( 32 mg , 65% yield ) were deposited and isolated by decanting the solution . calcd for c32h30hgn8se4 : c , 36.9 ; h , 2.9 ; n , 10.7 . found : c , 36.3 ; h , 2.9 ; n , 10.4 . h nmr ( dmso - d6 ) : 3.72 [ s , 12h of ch3 ] , 7.17 [ m , 8h of c6h4 ] , 7.31 [ m , 4h of c6h4 ] , 7.40 [ m , 4h of c6h4 ] , not observed [ nh ] . c{h } nmr ( dmso - d6 ) : 32.0 [ ch3 ] , 109.7 [ ch of c6h4 ] , 113.4 [ ch of c6h4 ] , 121.9 [ ch of c6h4 ] , 122.2 [ ch of c6h4 ] , 136.4 [ c of c6h4 ] , 156.0 [ cse ] . se{h } nmr ( dmso - d6 ) : 74 ppm . hg{h } nmr ( dmso - d6 ) : not observed . ir data ( kbr pellet , cm ) : 3450 ( vw ) , 3054 ( w ) , 2932 ( w ) , 2461 ( w ) , 1904 ( w ) , 1619 ( w ) , 1514 ( m ) , 1466 ( vs ) , 1432 ( vs ) , 1392 ( s ) , 1359 ( s ) , 1332 ( vs ) , 1277 ( vs ) , 1236 ( m ) , 1150 ( w ) , 1113 ( w ) , 1086 ( s ) , 1007 ( m ) , 912 ( w ) , 838 ( vw ) , 806 ( w ) , 736 ( vs ) , 728 ( vs ) , 662 ( vw ) . a suspension of h(sebenzim ) ( 64 mg , 0.30 mmol ) and mehgi ( 52 mg , 0.15 mmol ) in c6d6 ( 2 ml ) in an nmr tube equipped with a j. young valve was heated overnight at 100 c . over this period , pale yellow , x - ray - quality crystals of [ h(sebenzim)2]hgi0.5(benzene ) ( 67 mg , 56% yield ) were deposited and isolated by decanting the solution . calcd for c19h18hgin4se2 : c , 29.0 ; h , 2.3 ; n , 7.1 . found : c , 29.1 ; h , 2.4 ; n , 7.1 . h nmr ( dmso - d6 ) : 3.75 [ s , 6h of ch3 ] , 7.21 [ m , 4h of c6h4 ] , 7.41 [ m , 2h of c6h4 ] , 7.45 [ m , 2h of c6h4 ] , not observed [ nh ] . c{h } nmr ( dmso - d6 ) : 32.7 [ ch3 ] , 110.6 [ ch of c6h4 ] , 113.8 [ ch of c6h4 ] , 122.9 [ ch of c6h4 ] , 123.2 [ ch of c6h4 ] , 134.6 [ c of c6h4 ] , 136.6 [ c of c6h4 ] , 151.2 [ cse , jse hg{h } nmr ( dmso - d6 ) : not observed . ir data ( kbr pellet , cm ) : 3453 ( vw ) , 3053 ( w ) , 2932 ( w ) , 2387 ( w ) , 1901 ( w ) , 1872 ( w ) , 1863 ( w ) , 1610 ( w ) , 1523 ( w ) , 1466 ( vs ) , 1432 ( vs ) , 1395 ( s ) , 1336 ( vs ) , 1277 ( vs ) , 1236 ( m ) , 1156 ( w ) , 1112 ( w ) , 1087 ( s ) , 1007 ( m ) , 910 ( w ) , 846 ( vw ) , 807 ( w ) , 743 ( vs ) , 736 ( vs ) , 661 ( vw ) . a suspension of h(sebenzim ) ( 85 mg , 0.40 mmol ) and mehgcl ( 25 mg , 0.10 mmol ) in cd3cn ( 2 ml ) in an nmr tube equipped with a j. young valve was heated overnight at 100 c . over this period , colorless plates of [ h(sebenzim)2]2hg and large , yellow blocks of [ h(sebenzim)]4hgcl2 were deposited and were isolated by decanting the solution . the crystals were separated manually under a microscope for purposes of performing x - ray diffraction experiments . a solution of hgx2 ( x = cl , br , i ; 0.05 mmol ) in dmso - d6 ( 0.6 ml ) was treated with aliquots ( 40 l ) of a solution of h(sebenzim ) ( 126.7 mg , 0.6 mmol ) in dmso - d6 ( 0.48 ml ) and monitored by h nmr spectroscopy . a solution of hgcl2 ( 17.0 mg , 0.063 mmol ) in dmso - d6 ( 0.6 ml ) was treated with four aliquots of h(sebenzim ) ( 13.2 mg , 0.063 mmol ) and monitored by se{h } and hg{h } nmr spectroscopy . a solution of hgx2 ( x = cl , br , i ) in dmso - d6 ( 0.05 mmol in 0.6 ml ) was treated with 200 l of a solution of h(sebenzim ) in dmso - d6 ( 0.5 mmol in 1.00 ml ) and was monitored by se{h } nmr spectroscopy .

multinuclear ( 1h , 77se , and 199hg ) nmr spectroscopy demonstrates that 1-methyl-1,3-dihydro-2h - benzimidazole-2-selone , h(sebenzimme ) , a structural analogue of the selenoamino acid , selenoneine , binds rapidly and reversibly to the mercury centers of hgx2 ( x = cl , br , i ) , while x - ray diffraction studies provide evidence for the existence of adducts of composition [ h(sebenzimme)]xhgx2 ( x = cl , x = 2 , 3 , 4 ; x = i , x = 2 ) in the solid state . h(sebenzimme ) also reacts with methylmercury halides , but the reaction is accompanied by elimination of methane resulting from protolytic cleavage of the hg c bond , an observation that is of relevance to the report that selenoneine demethylates cyshgme , thereby providing a mechanism for mercury detoxification . interestingly , the structures of [ h(sebenzimme)]xhgx2 exhibit a variety of different hydrogen bonding patterns resulting from the ability of the n h groups to form hydrogen bonds with chlorine , iodine , and selenium .

Introduction Results and Discussion Conclusions Experimental Section

for example , selenium is a component of a variety of enzymes that incorporate the amino acids selenocysteine and selenomethionine ( figure 1 ) , as illustrated by glutathione peroxidases , thioredoxin reductases , glycine reductases , formate dehydrogenases , and selenoprotein p. other examples of selenium - containing biomolecules include the amino acid derivatives selenoneine and se - methylselenoneine ( figure 1 ) , of which the latter was identified in human urine and blood . it has recently been shown that selenoamino acids ( namely l - selenocysteine , l - selenoglutathione , d , l - selenopenicillamine , and l - selenomethionine ) complex readily to methylmercury species and that cleavage of the hg c bond may be achieved under physiologically relevant conditions to yield mercury selenide via ( mehg)2se . therefore , we describe here the reactivity of 1-methyl-1,3-dihydro-2h - benzimidazole-2-selone ( figure 2 ) , h(sebenzim ) , a structural analogue of selenoneine , towards mercury , including the protolytic cleavage of mercury 1-r - imidazole-2-thiones , h(mim ) , of which the methyl derivative is the well - known antithyroid drug , methimazole ( tapazole ) , are a widely studied class of molecules that can bind to a variety of metals , including mercury . for example , only h(seim ) and h(seim ) , and the benzannulated derivatives , h(sebenzim ) and h(sebenzim evidence for the ability of the imidazole-2-selone , h(sebenzim ) , to coordinate to the mercury centers of hgx2 ( x = cl , br , i ) in solution ( scheme 1 ) is provided by a combination of h , se{h } , and hg{h } nmr spectroscopies . in addition to providing evidence for coordination of h(sebenzim ) to mercury , the observation of a single resonance in both the se{h } and hg{h } nmr spectra for each concentration ratio , and also a single set of resonances in the h nmr spectra , indicates that the coordination is reversible and that the process is facile on the nmr time scale at room temperature . variation of h nmr chemical shift of the methyl group of h(sebenzim ) in the presence of hgx2 as a function of the molar ratio . although the fluxionality prevents identification of the precise solution composition ( scheme 1 ) , the tetrakis , tris , and bis complexes , [ h(sebenzim)]4hgcl2 , [ h(sebenzim)]3hgcl2 , and [ h(sebenzim)]2hgcl2 , may be obtained by crystallization from a solution that contains the respective number of equivalents of h(sebenzim ) . the molecular structures of [ h(sebenzim)]3hgcl2 and [ h(sebenzim)]4hgcl2 have been determined by x - ray diffraction , as illustrated in figures 4 and 5 , respectively . comparison of the molecular structures of [ h(sebenzim)]3hgcl2 ( figure 4 ) and [ h(sebenzim)]4hgcl2 ( figure 5 ) with that of [ h(sebenzim)]2hgcl2 reveals interesting structural variations as a function of composition , as summarized in figure 6 . first , there is a progressive increase in the hg cl distances in the sequence [ h(sebenzim)]2hgcl2 thus , whereas the two hg cl bond lengths in the bis complex [ h(sebenzim)]2hgcl2 [ 2.4942(7 ) and 2.5727(8 ) ] are comparable to the mean value of 2.43 for structurally characterized four - coordinate mercury compounds listed in the csd , the shortest hgcl distance in the tetrakis complex , [ h(sebenzim)]4hgcl2 , is 3.913 , such that the compound may be better represented as { [ h(sebenzim)]4hg}[cl]2 . thus , the four - coordinate 4 index ( table 4 ) of { [ h(sebenzim)]3hgcl } ( 0.78 ) is close to that for an idealized trigonal monopyramid ( 0.85 ) in which chlorine occupies an axial position ; in the extreme that the axial chlorine is considered to serve the role of a counterion , the mercury would be described as approximately trigonal planar . comparison of the mercury coordination environments of [ h(sebenzim)]2hgcl2 ( top ) , [ h(sebenzim)]3hgcl2 ( center ) , and [ h(sebenzim)]2hgcl2 ( bottom ) . these hg se bond lengths are comparable to the mean value of 2.643 for compounds listed in the csd , but are longer than those in compounds such as hg(seph)2 [ 2.480 ] and [ tm a common feature of all [ h(sebenzim)]xhgcl2 structures is that each chloride , regardless of whether it is attached covalently to the mercury center , participates in hydrogen bonding interactions with the imidazole n h moieties . there is , nevertheless , an interesting difference with respect to the nature of the hydrogen bonding interactions . n hcl interactions and is a discrete mononuclear species , [ h(sebenzim)]3hgcl2 and [ h(sebenzim)]4hgcl2 also exhibit intermolecular n n interactions that bridge two molecules , thereby creating a dimeric structure ( figure 8) , while [ h(sebenzim)]4hgcl2 exhibits an intramolecular n hclh the various hydrogen bonding ncl distances in [ h(sebenzim)]xhgcl2 are in the range 3.031(7)3.227(2 ) and are comparable to the values for other compounds with n hcl interactions listed in the csd [ dav(ncl ) = 3.181 ] . coordination of h(sebenzim ) to hgcl2 is accompanied by only relatively small increases in the lengths of the c se bonds . thus , the c se bond lengths of [ h(sebenzim)]2hgcl2 [ 1.862(3 ) and 1.864(3 ) ] , [ h(sebenzim)]3hgcl2 [ 1.868(3 ) , 1.859(3 ) , and 1.857(3 ) ] , and [ h(sebenzim)]4hgcl2 [ 1.854(12 ) , 1.896(11 ) , 1.851(9 ) , 1.851(9 ) , 1.857(11 ) , 1.854(11 ) , 1.869(9 ) , and 1.856(9 ) ] are only slightly longer than that of free h(sebenzim ) [ 1.838(2 ) ] . nmr spectroscopic studies also demonstrate that h(sebenzim ) binds reversibly to hgbr2 and hgi2 in dmso - d6 , and that the processes are facile on the nmr time scale , as indicated by the observation of single sets h nmr chemical shifts for the h(sebenzim ) signals ( table 2 and figure 3 ) . for example , the se nmr chemical shifts of 2:1 mixtures of h(sebenzim ) and hgx2 move upfield from the value of pure h(sebenzim ) by values of 68 ppm ( x = cl ) , 54 ppm ( x = br ) , and 35 ppm ( x = i ) . interestingly , the crystals of [ h(sebenzim)]2hgi2 obtained from the two different reaction solvents are not isomorphous , and the molecules adopt different geometries , as illustrated in figures 10 and 11 . in addition to these angular variations , there are small differences in hg se and hg thus , while the average hg i bond length of orthorhombic [ h(sebenzim)]2hgi2 ( 2.792 ) is longer than that of the monoclinic version ( 2.737 ) , the average hg se bond length of orthorhombic [ h(sebenzim)]2hgi2 ( 2.627 ) is shorter than that of the monoclinic version ( 2.692 ) . similarly to hgcl2 , coordination of h(sebenzim ) to hgi2 is accompanied by only small increases in the lengths of the c se bonds . the most striking differences in the structures of orthorhombic and monoclinic [ h(sebenzim)]2hgi2 do not , however , pertain to the mercury coordination environment . furthermore , these hydrogen bonding patterns are also different from that of the chloride counterpart , [ h(sebenzim)]2hgcl2 ( vide supra ) , as illustrated in figure 14 . hydrogen bonding network for monoclinic [ h(sebenzim)]2hgi2 obtained from benzene solution , illustrating head - to - head for example , whereas [ h(sebenzim)]2hgcl2 is observed to have two intramolecular n hcl interactions , the orthorhombic form of [ h(sebenzim)]2hgi2 possesses one intramolecular and one intermolecular n hi interaction , thereby creating a hydrogen - bonded helical chain of [ h(sebenzim)]2hgi2 molecules ( figure 12 ) . rather , the n h groups of the h(sebenzim ) ligands participate in pairs of centrosymmetric hse interactions that link adjacent molecules together in a manner similar to that observed for certain h(seim ) derivatives in the absence of metal coordination ( figure 13 ) . the existence of this motif is undoubtedly a consequence of the fact that iodide is , by comparison to chloride , a poor hydrogen bond acceptor , such that n as would be expected , the hydrogen bonding ni interactions in orthorhombic [ h(sebenzim)]2hgi2 [ 3.486(7 ) and 3.589(7 ) ] are substantially longer than the analogous ncl interactions in [ h(sebenzim)]2hgcl2 . in view of the fact that the protolytic cleavage of the hg c bond is a critical step in detoxification of organomercurials , and recognizing that h(sebenzim ) is an analogue of selenoneine , we have also investigated the reactivity of h(sebenzim ) towards methylmercury halides . significantly , we have observed that h(sebenzim ) not only coordinates to the mercury center , as observed for hgx2 , but it is also capable of cleaving the hg for example , h(sebenzim ) reacts with mehgi at 100 c to liberate ch4 ( as observed by h nmr spectroscopy ) and afford [ h(sebenzim)2]hgi ( scheme 2 ) . the importance of this observation is underscored by the fact that selenoneine , of which h(sebenzim ) is a structural analogue , has recently been shown to achieve demethylation of cyshgme . the molecular structure of [ h(sebenzim)2]hgi has been determined by x - ray diffraction , as illustrated in figure 15 , which demonstrates that it features mercury in an approximately trigonal planar environment , with a pyramidality ( p ) value of only 0.2. the bond angles at mercury , however , deviate from 120 [ se hg i = 114.87(2) and 104.02(2) ] , such that the geometry is distorted towards t - shaped , which is not uncommon for mercury . in view of the kinetic stability of two - coordinate rhgx complexes towards protolytic cleavage , it is likely that the mechanism for formation of [ h(sebenzim)2]hgi involves the initial formation of an adduct , [ h(sebenzim)]xhg(me)i , which undergoes either intramolecular protolytic cleavage of the hg me bond , or cleavage in an intermolecular manner to afford a mercury c bond of mehgcl , although the reaction follows a different course than that of mehgi . specifically , reaction of mehgcl with h(sebenzim ) at 100 c results in evolution of methane , as observed by h nmr spectroscopy , and the formation of a mixture of [ h(sebenzim)]4hgcl2 ( vide supra ) and [ h(sebenzim)2]2hg ( scheme 3 ) . the molecular structure of [ h(sebenzim)2]2hg has been determined by x - ray diffraction ( figure 17 ) , which demonstrates that pairs of h(sebenzim ) and ( sebenzim ) ligands are linked together via hydrogen bonding interactions to produce the combined lx - type ligand , [ h(sebenzim)2 ] , in a manner akin to that observed for [ h(sebenzim)2]hgi . in summary , 1-methyl-1,3-dihydro-2h - benzimidazole-2-selone , h(sebenzim ) , is a structural analogue of selenoneine and coordinates reversibly to the metal centers of hgx2 ( x = cl , br , i ) . furthermore , h(sebenzim ) is also capable of cleaving the hg c bond of methylmercury halides , thereby mimicking the role of selenoneine in demethylating cyshgme . x - ray diffraction studies demonstrate that while two equivalents of h(sebenzim ) simply coordinate to mercury centers of hgx2 ( x = cl , i ) , the third and fourth equivalents result in displacement of the chloride ligands . a common feature of all [ h(sebenzim)]xhgcl2 structures is that each chloride , regardless of whether it is attached covalently to the mercury center or serves as a counterion , participates in hydrogen bonding interactions with the imidazole n h moieties . for example , whereas [ h(sebenzim)]2hgcl2 exhibits only intramolecular n hcl interactions and is a discrete mononuclear species , [ h(sebenzim)]3hgcl2 exhibits an intramolecular n hcl interaction and intermolecular n interactions that bridge two molecules , resulting in a dimeric structure , while [ h(sebenzim)]4hgcl2 exhibits an intramolecular n hclh this investigation demonstrates that not only is h(sebenzim ) a good ligand for mercury , capable of displacing halide ligands , but is also capable of protolytically cleaving mercury carbon bonds , a result that is of relevance to the role of selenium compounds in the detoxification of mercury compounds . single - crystal x - ray diffraction data were collected on a bruker apex ii diffractometer , and crystal data , data collection , and refinement parameters are summarized in table 6 . over this period , yellow , x - ray - quality crystals of [ h(sebenzim)]2hgi2 ( 54 mg , 56% yield ) were deposited and isolated by decanting the solution . over this period , pale yellow , x - ray - quality crystals of [ h(sebenzim)]4hgcl2 ( 94 mg , 84% yield ) were deposited and isolated by decanting the solution . over this period , very pale yellow , x - ray - quality crystals of [ h(sebenzim)2]2hg ( 32 mg , 65% yield ) were deposited and isolated by decanting the solution . over this period , pale yellow , x - ray - quality crystals of [ h(sebenzim)2]hgi0.5(benzene ) ( 67 mg , 56% yield ) were deposited and isolated by decanting the solution . a solution of hgx2 ( x = cl , br , i ; 0.05 mmol ) in dmso - d6 ( 0.6 ml ) was treated with aliquots ( 40 l ) of a solution of h(sebenzim ) ( 126.7 mg , 0.6 mmol ) in dmso - d6 ( 0.48 ml ) and monitored by h nmr spectroscopy . a solution of hgx2 ( x = cl , br , i ) in dmso - d6 ( 0.05 mmol in 0.6 ml ) was treated with 200 l of a solution of h(sebenzim ) in dmso - d6 ( 0.5 mmol in 1.00 ml ) and was monitored by se{h } nmr spectroscopy .

alcohol dependence ( ad ; mendelian inheritance in man , ( mim ) % 1037800 ) , a chronic relapsing disorder , is a serious health concern globally . it is characterised by loss of sensitivity and development of tolerance to , and withdrawal symptoms and craving for alcohol . in addition , ad also leads to a plethora of disabling complications , such as hepatitis , hepatic cirrhosis , chronic pancreatitis , testicular atrophy and avascular necrosis of the hip joint . the morbidity , mortality and high cost associated with the treatment of alcoholism and its related complications makes ad a major socio - economic burden on society . alcohol abuse disorders result from the interaction between an individual 's genetic and environmental susceptibility and repeated intake of alcohol over time . it is not possible to become alcoholic without repeatedly consuming alcohol , but only a small percentage of all drinkers become alcoholic . epidemiological studies suggest that ethnicity and genetic susceptibility are crucial in the genesis of ad [ 4 - 10 ] , with american indians , native hawaiians , african americans and hispanics being highly susceptible to developing ad . although the prevalence of ad in the asian population is low , a sizable number of people in india are suffering from alcohol use disorders . genetic predisposition to ad , conferred by various candidate genes , differs across ethnic groups . this differential contribution leads to inconsistent genetic association and non - replication of association between different populations . linkage and association studies to determine genetic factors predisposing to ad have been carried out in several western , african , jewish , korean , japanese and chinese populations . although india represents about one - sixth of the world 's population , there are very few data on genes / polymorphisms that confer susceptibility to ad in this population . genetic polymorphisms , particularly those of the alcohol metabolising enzymes alcohol dehydrogenase ( adh ) and aldehyde dehydrogenase ( aldh ) , have been largely implicated in the development of ad [ 16 - 18 ] . in order to identify specific genes affecting vulnerability or resistance , long et al . performed a whole - autosomal genome scan for genetic linkage to ad in a southwestern american indian tribe . a highly suggestive piece of evidence for the linkage of three loci in the adh gene cluster on chromosome 4q was observed . these findings were confirmed by the collaborative study on the genetics of alcoholism ( coga ) study . on the basis of the genotype frequencies of adh1b and aldh2 polymorphisms , the risk for alcoholism in the japanese population was accurately estimated by higuchi et al . they concluded that the japanese population may be protected from alcoholism by inactive aldh2 and higher frequencies of atypical adh1b . functional polymorphisms in the genes encoding these enzymes influence the rate of synthesis and metabolism of acetaldehyde , the toxic metabolite of ethanol . the presence / absence of these polymorphisms in the population could modulate the probability of the development and relapse of ad , and confer protection / susceptibility to alcohol abuse and alcohol - related complications . the most widely studied functional polymorphisms in the ethanol metabolic pathway are the adh1b arg47his and aldh2 glu487lys polymorphisms . the adh1b*2 ( adh1b*47his ) allele codes for a higher activity enzyme as compared with the adh1b*1 ( adh1b*47arg ) allele and is thus known to influence drinking behaviour , resulting in protection from alcoholism . chen et al . , in a case control analysis , found that individuals carrying one or two copies of the adh1b*2 allele and a single copy of aldh2 * 2 had the lowest risk ( odds ratios 0.04 - 0.05 ) for alcoholism , as compared with the adh1b*1/*1 and aldh2 * 1/*1 genotype . further , the authors concluded that the disease risk associated with the adh1b*2/*2 -aldh2 * 1/*1 genotype is about half of that associated with the adh1b * 1/*2 - aldh2 * 1/*1 genotype . their result suggests that the protection afforded by the adh1b*2 allele may be independent of that afforded by aldh2 . a considerable variation in the allele frequency of this polymorphism has been observed among different ethnic groups . the adh1b*2 allele is found to be more common in non - alcoholic than in alcoholic groups in populations from east asia , taiwan , spain , new zealand and in jews from israel and the usa . due to the very low frequency of the adh1b*2 allele in the caucasian population reports on the adh1b*2 allele ( arg47his polymorphism ) frequency in the indian population are inconsistent . reported a 9.9 per cent adh1b*2 allele frequency in a heterogeneous sample from the indian population , whereas another study from india ( on the kachari population ) reported a 6.6 per cent frequency . recently , in a robust study involving 28 indian tribal populations , the polymorphism has been found to be monomorphic ( adh1b*1/*1 ) , with complete absence of the adh1b*2 allele . based on their findings , those authors argued that indians ( mainly those comprising the lower caste and working class ) could have developed tolerance to the adverse effects of alcohol , and thus the protection - conferring adh1b*2 allele has been selected out from the population . sensitivity to ethanol is highly associated with a functional polymorphism , glu487lys , and the 487lys ( aldh2 * 2 ) allele is responsible for a deficiency in aldh2 activity . high concentrations of acetaldehyde , due to the presence of the aldh2 * 2 allele , lead to adverse reactions to alcohol , which reduce the probability of heavy drinking vis - - vis ad and other alcohol - related problems . clinical and pharmacokinetic studies have indicated that individuals who show initial sensitivity to alcohol by virtue of their genetic make - up ( ie the presence ofthe aldh2 * 2 allele ) are discouraged from drinking excessive amounts of alcohol . . found that aldh2 * 2/*2 subjects are at minimum risk for developing ad and alcohol - related organ damage , compared with those with the aldh2 * 1/*2 and aldh2 * 1/*1 genotypes . a significant variation in the allele frequency of the glu487lys polymorphism has been observed across different populations worldwide , with almost complete absence of the aldh2 * 2 allele in caucasians , africans and americans ( single nucleotide polymorphisms [ snp ] database ; national center for biotechnology information ) and a relatively higher frequency in east asians . studies on chinese and korean populations found that alcoholics are less likely to have the aldh2 * 2 allele than controls . pertinent to the east asian population , approximately 2 - 12 per cent of chinese and korean individuals are homozygous for the aldh2 * 2 allele ( aldh2 * 2/*2 genotype ) and appear to be almost completely protected . the glu487lys polymorphism , however , is found to be monomorphic in indian populations from madhya pradesh , maharashtra and andhra pradesh . due to the paucity of indian data with regard to adh1b/ aldh2 gene polymorphisms , the present study aimed to characterise the arg47his and glu487lys polymorphisms in indian subjects with ad and to establish the genotype / phenotype correlation ( if any ) . in this study , 174 alcohol - dependent male subjects satisfying dsm 1v criteria derived from the diagnostic interview for genetic study ( digs ) , and aged between 18 and 60 years of age , from the national drug dependence treatment center at the all india institute of medical sciences , were recruited . the study was carried out in accordance with the declaration of helsinki ( 2000 ) of the world medical association . clinical details , including ethnicity , family history , age at first use of alcohol , quantity of alcohol consumed ( g / day ) , duration of alcohol use , duration of ad , age at onset of dependence , presence / absence of delirium and any other psychiatric or physical illness were assessed and recorded . serum proteins ( albumin , bilirubin , glutamic oxaloacetic transaminase [ sgot ] and glutamic pyruvic transaminase [ sgpt ] ) were estimated on an autoanalyser , using biochemical kits from boehringer mannheim ( mannheim , germany ) . peripheral blood ( 6 ml ) was collected in sterile ethylene diamine tetra - acetic acid ( edta)-coated vacutainers and stored at 4c until processing for dna extraction was carried out . genomic dna was extracted from peripheral blood leukocytes using the standard phenol - chloroform method . snp genotyping was done using the polymerase chain reaction ( pcr)-restriction fragment length polymorphism ( rflp ) approach . forward and reverse primer sequences used for the amplification of the adh1b arg47his snp were 5'-aatcttttctgaa tctgaacag-3 ' and 5'-gaaggggggtcacca ggttgc-3 ' , respectively ; and primer pairs for aldh2 glu487lys were 5'-caaattacagggt caagggct-3 ' ( forward ) and 5'-ccacactca cagttttctctt-3 ' ( reverse ) . after amplification , the pcr product ( 20 l ) was divided into two equal parts . 10 l of the amplified product was incubated at 37c for two hours with nmuci and mbo ii restriction enzymes ( re ) for adh1b and aldh2 , respectively . dna fragments in the re digested and undigested mixture were analysed on 10 per cent polyacrylamide gel electrophoresis ( page ) using one per cent tris - borate - edta ( tbe ) buffer and sized with the help of known dna markers . for the adh1b polymorphism , the presence of 95 base pair ( bp ) and 65 bp restriction fragments indicated the presence of wild - type arg and mutant his alleles , respectively . the presence of 134 bp and 123 bp fragments indicated the presence of wild - type glu and mutant lys alleles , respectively for the aldh2 snp . genotyping for approximately 10 per cent of the samples was repeated at random for quality control , and complete agreement was found . comparison of all clinical variables between subjects with different genotypic profiles for adh1b and aldh2 gene polymorphisms were carried out using the t - test or mann whitney 's u test , as appropriate ; p values < 0.05 were considered significant . in this study , 174 alcohol - dependent male subjects satisfying dsm 1v criteria derived from the diagnostic interview for genetic study ( digs ) , and aged between 18 and 60 years of age , from the national drug dependence treatment center at the all india institute of medical sciences , were recruited . the study was carried out in accordance with the declaration of helsinki ( 2000 ) of the world medical association . clinical details , including ethnicity , family history , age at first use of alcohol , quantity of alcohol consumed ( g / day ) , duration of alcohol use , duration of ad , age at onset of dependence , presence / absence of delirium and any other psychiatric or physical illness were assessed and recorded . serum proteins ( albumin , bilirubin , glutamic oxaloacetic transaminase [ sgot ] and glutamic pyruvic transaminase [ sgpt ] ) were estimated on an autoanalyser , using biochemical kits from boehringer mannheim ( mannheim , germany ) . peripheral blood ( 6 ml ) was collected in sterile ethylene diamine tetra - acetic acid ( edta)-coated vacutainers and stored at 4c until processing for dna extraction was carried out . genomic dna was extracted from peripheral blood leukocytes using the standard phenol - chloroform method . snp genotyping was done using the polymerase chain reaction ( pcr)-restriction fragment length polymorphism ( rflp ) approach . forward and reverse primer sequences used for the amplification of the adh1b arg47his snp were 5'-aatcttttctgaa tctgaacag-3 ' and 5'-gaaggggggtcacca ggttgc-3 ' , respectively ; and primer pairs for aldh2 glu487lys were 5'-caaattacagggt caagggct-3 ' ( forward ) and 5'-ccacactca cagttttctctt-3 ' ( reverse ) . after amplification , the pcr product ( 20 l ) was divided into two equal parts . 10 l of the amplified product was incubated at 37c for two hours with nmuci and mbo ii restriction enzymes ( re ) for adh1b and aldh2 , respectively . dna fragments in the re digested and undigested mixture were analysed on 10 per cent polyacrylamide gel electrophoresis ( page ) using one per cent tris - borate - edta ( tbe ) buffer and sized with the help of known dna markers . for the adh1b polymorphism , the presence of 95 base pair ( bp ) and 65 bp restriction fragments indicated the presence of wild - type arg and mutant his alleles , respectively . the presence of 134 bp and 123 bp fragments indicated the presence of wild - type glu and mutant lys alleles , respectively for the aldh2 snp . genotyping for approximately 10 per cent of the samples was repeated at random for quality control , and complete agreement was found . comparison of all clinical variables between subjects with different genotypic profiles for adh1b and aldh2 gene polymorphisms were carried out using the t - test or mann whitney 's u test , as appropriate ; p values < 0.05 were considered significant . the population was divided into three groups based on patients ' genotypic profile for aldh2 ( glu487lys ) gene polymorphism , and the distribution of clinical variables was evaluated . adh1b gene polymorphism was found to be largely monomorphic , with a minor allele frequency ( adh1b*2 ) of 0.0014 . for the aldh2 glu487lys snp , the genotypic frequencies were 0.73 ( 2 * 1/*1 ) , 0.16 ( 2 * 1/*2 ) and 0.11 ( 2 * 2/*2 ) , with a minor allele frequency ( aldh2 * 2 ) of 0.19 . the significance of association of various clinical parameters with aldh2 polymorphism is included in table 1 . about 80 per cent of subjects with the aldh2 * 1/*2 genotype , and 38 per cent with the aldh2 * 1/*1 genotype reported a history of severe alcohol withdrawal ( delirium ) during their alcohol drinking years . about 39 per cent of those with the aldh2 * 1/2 * 1 and 29 per cent of those with the aldh2 * 1/*2 genotype drank almost every day . subjects drinking excessive amounts ( > 60g / day ) , however , were more likely to have the aldh2 * 1/*1 or the aldh2 * 1/*2 genotype than the aldh2 * 2/*2 genotype . clinical details of the study population significant at p < 0.05 . although the role of adh1b and aldh2 genes in susceptibility to alcoholism were discovered individually , they have been shown to act additively when they co - occur . the adh1b*2 and aldh2 * 2 alleles raise the levels of acetaldehyde by increasing the rate of synthesis and decreasing the rate of metabolism respectively , thus interacting additively but not synergistically . at the protein level , the allelic series for adh1b is generated by variation at two different sites at the genomic level . the adh1b*1 allele is composed of 47arg and 369arg ; the adh1b*2 allele is composed of 47his and 369arg ; and the adh1b*3 allele is composed of 47arg and 369cys . caucasians are largely monomorphic for the adh1b*1 allele , with a very low frequency of the aldh2 * 2 allele . the adh1b*2 allele , which is known to confer protection against alcoholism and is relatively common in east asian populations , was found to be present at very low frequency ( < 0.001 ) in our study . our observation is similar to that reported by reddy et al . in tribal populations from southern india . since the adh1b*3 ( adh1b*369cys ) allele has been documented to be essentially specific to the african population , we did not genotype adh1b*3 in our study population . . , the very low frequency of the adh1b*2 allele found in our population is surprising , since it is present at a substantial frequency in the neighbouring east asian population and confers protection against alcoholism . a very low frequency of adh1b*2 could indicate a selection pressure operating against the adh1b*2 allele in our population due to its inability / redundancy to protect the indian population from ad . commenting on the alcohol - consuming practice of the service / working and lower caste of indian society , reddy et al . suggested that , due to the heavy amount of alcohol consumption on a regular basis , most of these subjects might have developed a genetic adaptation to withstand drinking alcohol ( unlike most east asian populations with known sensitivity to alcohol consumption ) , thereby possibly requiring no protective mechanism and hence resulting in the absence of the adh1b*2 allele . the present study was conducted at a government facility , where treatment is available free of charge and is accessed mainly by people from low income strata belonging to the working class of indian society . it is well known that socio - economically underprivileged groups consume alcohol for mental and physical relaxation . therefore , the explanation given by reddy et al . could also support a very low frequency of the adh1b * 2 allele in the present study . a stronger influence of social setting and age than the effect of the adh1b*2 allele on alcohol drinking has also been found in a study in a jewish population , which supports the above - mentioned hypothesis of an environmental influence on genetic constitution with regard to the adh1b gene in the indian population . the aldh2 gene is located on chromosome 12q24 . the single bp difference ( g > a ; glu487lys ) in exon 12 causes the normal allele , aldh2 * 1 , to become a non - functional ( aldh2 * 2 ) allele , which codes for the inactive enzyme . the aldh2 * 2 allele , which is prevalent in asian populations but is extremely rare in non - asians , has the strongest protective association with ad . asian subjects homozygous for aldh2 * 2 have an almost zero risk of developing ad , whereas heterozygotes ( aldh2 * 1/2 * 2 ) are about one - third as likely to be alcoholic , compared with those without this allele . reported that the homozygous aldh2 * 2/*2 genotype is present at a very low frequency ( 0 - 0.3 per cent ) in alcoholic patients , compared with 3 - 12 per cent among non - alcoholic subjects . the heterozygous aldh2 * 1/*2 genotype has been found to be present in 6 - 17 per cent of alcohol - dependent subjects , compared with 30 - 45 per cent of non - alcoholic subjects . this difference in aldh2 * 2 allele frequency between alcoholic and non - alcoholic subjects has been postulated to be responsible for the phenomenon of adverse responses to alcohol consumption , such as facial flushing , nausea and tachycardia in individuals with the inactive form of the enzyme . in contrast to the reports from the neighbouring east asian population , the present study found comparatively higher allele ( aldh2 * 2 = 0.19 ) and genotypic ( aldh2 * 2/*2 = 0.11 , aldh2 * 1/*2 = 0.16 ) frequencies in alcohol - dependent individuals . a recent study from india is equally exciting , however , as it found the snp to be monomorphic ( aldh2 * 1/2 * 1 ) in healthy control subjects . in light of the findings of that recent report , as well as that of goedde et al . , the higher frequency of the inactive aldh2 allele observed in our indian alcohol - dependent subjects , compared with the control population , provides evidence for a direct role of acetaldehyde in the development of alcohol addiction ( despite its toxic actions ) . further , the amount of alcohol consumed by aldh2 * 2/*2 individuals is significantly lower than those with aldh2 * 1/*1 and aldh2 * 1/*2 genotypes ( table 1 ) . it should be noted that , although subjects with the aldh2 * 2/*2 genotype in this study are classified as alcohol dependent by dsm iv criteria , these individuals are only drinking three to five restaurant glasses per day ( of indian liquor , with 8 g alcohol / drink ) , which is not enough to produce ethanol intoxication , but rather acetaldehyde intoxication . when we compared our findings in alcohol - dependent subjects with those of an indian control population ( see bhaskar et al . , where the pleasurable effects of acetaldehyde led to higher positive expectancies in individuals with the aldh2 * 1/*2 compared with the aldh2 * 1/*1 genotype . as suggested by chen et al . , the pleasurable effects of acetaldehyde leading to higher positive expectancies in individuals with the aldh2 * 1/*2 compared with the aldh2 * 1/*1 genotype could be due to a development of tolerance to acetaldehyde . the present observation of the development of ad ( in aldh2 * 2/*2 individuals ) with low levels of alcohol consumption but with presumably high levels of acetaldehyde strongly implicates acetaldehyde in the causality of the dependence . the above conclusion might be weakened / challenged , however , by a comparison of our data on ad subjects with an analysis involving ethnicity , age and sex - matched controls ; this is currently ongoing in our laboratory . two clinical parameters -- namely , duration of ad and age at onset of ad -- correlated well with the protection - conferring property of the aldh2 glu487lys polymorphism . the duration of ad was found to be significantly longer ( p < 0.01 ) among individuals with the aldh2 * 1/*1 genotype , compared with aldh2 * 2/*2 subjects . the age at which patients developed ad , however , was significantly lower ( p < 0.01 ) in individuals with the aldh2 * 1/*1 genotype compared with aldh2 * 2/*2 subjects ( table 1 ) . alcohol - induced flushing is inherited as a dominant trait in asians . a substantial number of asians ( 50 - 80 per cent ) , compared with caucasians ( 3 - 12 per cent ) , exhibit flushing . acetaldehyde creates unpleasant adverse reactions by acting as a major deterrent to excessive alcohol drinking on the one hand , while giving the euphoric sensations that may reinforce alcohol drinking on the other . since people with the inactive aldh2 * 2 allele drink less often and consume less alcohol per occasion than those with the active aldh2 * 1 allele , our observation of a significantly lower age at onset and longer duration of ad among individuals with the aldh2 * 1/*1 genotype compared with those with the aldh2 * 2/*2 genotype seems to be in keeping with the available literature . significantly higher values of sgot and sgpt in individuals with the aldh2 * 1/*1 genotype , compared with the aldh2 * 2/*2 genotype ( table 1 ) , is suggestive of more liver damage due to higher alcohol consumption among aldh2 * 1/*1 subjects . our findings indicate that functional polymorphism of the gene coding for the aldh enzyme affects the propensity to develop ad . the most important finding of the study was the uniquely high frequency of the aldh2 * 2/*2 genotype ( among alcohol - dependent subjects ) being a risk - conferring factor for ad . further , based on the findings from this study , albeit based on a limited sample size , it could be hypothesised that the indian genotype is prone to slow metabolism of alcohol and high metabolism of acetaldehyde , and thereby prone to alcoholism . our findings should be viewed , however , in the perspective of the potential limitation posed by the absence of data from an ethnically , age- and sex - matched control population . further , these results not only warrant replication in larger sample sets , but also underscore the need for investigations on candidate gene polymorphisms from other biochemical pathways . the authors acknowledge the constructive suggestions from dr atul ambekar , national drug dependence treatment centre , all india institute of medical sciences , new delhi , india .

functional polymorphism in the genes encoding alcohol dehydrogenase ( adh ) 1b and aldehyde dehydrogenase ( aldh ) 2 are considered most important among several genetic determinants of alcohol dependence , a complex disorder . there is no report on the widely studied arg47his and glu487lys polymorphisms from indian alcoholdependent populations . in this paper , we report , for the first time , allelic and genotypic frequencies of arg47his and glu487lys single nucleotide polymorphisms ( snps ) in north indian alcohol - dependent subjects . a total of 174 alcohol - dependent males , recruited using dsm iv criteria ( american psychiatric association , 1994 ) , were genotyped using the polymerase chain reaction - restriction fragment length polymorphism method . the results obtained from genetic analysis were correlated with clinical parameters using student 's t - test or mann whitney 's u test . the highlight of the study findings was the uniquely high frequency of the aldh2 * 2/*2 genotype ( among alcohol - dependent subjects ) being a risk - conferring factor for alcohol dependence .

Introduction Materials and methods Subjects Genomic DNA extraction and genotyping Statistical analysis Results Discussion and conclusion Acknowledgments

alcohol dependence ( ad ; mendelian inheritance in man , ( mim ) % 1037800 ) , a chronic relapsing disorder , is a serious health concern globally . it is characterised by loss of sensitivity and development of tolerance to , and withdrawal symptoms and craving for alcohol . in addition , ad also leads to a plethora of disabling complications , such as hepatitis , hepatic cirrhosis , chronic pancreatitis , testicular atrophy and avascular necrosis of the hip joint . alcohol abuse disorders result from the interaction between an individual 's genetic and environmental susceptibility and repeated intake of alcohol over time . although the prevalence of ad in the asian population is low , a sizable number of people in india are suffering from alcohol use disorders . although india represents about one - sixth of the world 's population , there are very few data on genes / polymorphisms that confer susceptibility to ad in this population . genetic polymorphisms , particularly those of the alcohol metabolising enzymes alcohol dehydrogenase ( adh ) and aldehyde dehydrogenase ( aldh ) , have been largely implicated in the development of ad [ 16 - 18 ] . a highly suggestive piece of evidence for the linkage of three loci in the adh gene cluster on chromosome 4q was observed . on the basis of the genotype frequencies of adh1b and aldh2 polymorphisms , the risk for alcoholism in the japanese population was accurately estimated by higuchi et al . they concluded that the japanese population may be protected from alcoholism by inactive aldh2 and higher frequencies of atypical adh1b . functional polymorphisms in the genes encoding these enzymes influence the rate of synthesis and metabolism of acetaldehyde , the toxic metabolite of ethanol . the presence / absence of these polymorphisms in the population could modulate the probability of the development and relapse of ad , and confer protection / susceptibility to alcohol abuse and alcohol - related complications . the most widely studied functional polymorphisms in the ethanol metabolic pathway are the adh1b arg47his and aldh2 glu487lys polymorphisms . , in a case control analysis , found that individuals carrying one or two copies of the adh1b*2 allele and a single copy of aldh2 * 2 had the lowest risk ( odds ratios 0.04 - 0.05 ) for alcoholism , as compared with the adh1b*1/*1 and aldh2 * 1/*1 genotype . a considerable variation in the allele frequency of this polymorphism has been observed among different ethnic groups . due to the very low frequency of the adh1b*2 allele in the caucasian population reports on the adh1b*2 allele ( arg47his polymorphism ) frequency in the indian population are inconsistent . reported a 9.9 per cent adh1b*2 allele frequency in a heterogeneous sample from the indian population , whereas another study from india ( on the kachari population ) reported a 6.6 per cent frequency . recently , in a robust study involving 28 indian tribal populations , the polymorphism has been found to be monomorphic ( adh1b*1/*1 ) , with complete absence of the adh1b*2 allele . based on their findings , those authors argued that indians ( mainly those comprising the lower caste and working class ) could have developed tolerance to the adverse effects of alcohol , and thus the protection - conferring adh1b*2 allele has been selected out from the population . sensitivity to ethanol is highly associated with a functional polymorphism , glu487lys , and the 487lys ( aldh2 * 2 ) allele is responsible for a deficiency in aldh2 activity . high concentrations of acetaldehyde , due to the presence of the aldh2 * 2 allele , lead to adverse reactions to alcohol , which reduce the probability of heavy drinking vis - - vis ad and other alcohol - related problems . clinical and pharmacokinetic studies have indicated that individuals who show initial sensitivity to alcohol by virtue of their genetic make - up ( ie the presence ofthe aldh2 * 2 allele ) are discouraged from drinking excessive amounts of alcohol . found that aldh2 * 2/*2 subjects are at minimum risk for developing ad and alcohol - related organ damage , compared with those with the aldh2 * 1/*2 and aldh2 * 1/*1 genotypes . a significant variation in the allele frequency of the glu487lys polymorphism has been observed across different populations worldwide , with almost complete absence of the aldh2 * 2 allele in caucasians , africans and americans ( single nucleotide polymorphisms [ snp ] database ; national center for biotechnology information ) and a relatively higher frequency in east asians . studies on chinese and korean populations found that alcoholics are less likely to have the aldh2 * 2 allele than controls . pertinent to the east asian population , approximately 2 - 12 per cent of chinese and korean individuals are homozygous for the aldh2 * 2 allele ( aldh2 * 2/*2 genotype ) and appear to be almost completely protected . due to the paucity of indian data with regard to adh1b/ aldh2 gene polymorphisms , the present study aimed to characterise the arg47his and glu487lys polymorphisms in indian subjects with ad and to establish the genotype / phenotype correlation ( if any ) . in this study , 174 alcohol - dependent male subjects satisfying dsm 1v criteria derived from the diagnostic interview for genetic study ( digs ) , and aged between 18 and 60 years of age , from the national drug dependence treatment center at the all india institute of medical sciences , were recruited . the study was carried out in accordance with the declaration of helsinki ( 2000 ) of the world medical association . clinical details , including ethnicity , family history , age at first use of alcohol , quantity of alcohol consumed ( g / day ) , duration of alcohol use , duration of ad , age at onset of dependence , presence / absence of delirium and any other psychiatric or physical illness were assessed and recorded . genomic dna was extracted from peripheral blood leukocytes using the standard phenol - chloroform method . snp genotyping was done using the polymerase chain reaction ( pcr)-restriction fragment length polymorphism ( rflp ) approach . forward and reverse primer sequences used for the amplification of the adh1b arg47his snp were 5'-aatcttttctgaa tctgaacag-3 ' and 5'-gaaggggggtcacca ggttgc-3 ' , respectively ; and primer pairs for aldh2 glu487lys were 5'-caaattacagggt caagggct-3 ' ( forward ) and 5'-ccacactca cagttttctctt-3 ' ( reverse ) . 10 l of the amplified product was incubated at 37c for two hours with nmuci and mbo ii restriction enzymes ( re ) for adh1b and aldh2 , respectively . for the adh1b polymorphism , the presence of 95 base pair ( bp ) and 65 bp restriction fragments indicated the presence of wild - type arg and mutant his alleles , respectively . the presence of 134 bp and 123 bp fragments indicated the presence of wild - type glu and mutant lys alleles , respectively for the aldh2 snp . genotyping for approximately 10 per cent of the samples was repeated at random for quality control , and complete agreement was found . comparison of all clinical variables between subjects with different genotypic profiles for adh1b and aldh2 gene polymorphisms were carried out using the t - test or mann whitney 's u test , as appropriate ; p values < 0.05 were considered significant . in this study , 174 alcohol - dependent male subjects satisfying dsm 1v criteria derived from the diagnostic interview for genetic study ( digs ) , and aged between 18 and 60 years of age , from the national drug dependence treatment center at the all india institute of medical sciences , were recruited . the study was carried out in accordance with the declaration of helsinki ( 2000 ) of the world medical association . clinical details , including ethnicity , family history , age at first use of alcohol , quantity of alcohol consumed ( g / day ) , duration of alcohol use , duration of ad , age at onset of dependence , presence / absence of delirium and any other psychiatric or physical illness were assessed and recorded . genomic dna was extracted from peripheral blood leukocytes using the standard phenol - chloroform method . snp genotyping was done using the polymerase chain reaction ( pcr)-restriction fragment length polymorphism ( rflp ) approach . forward and reverse primer sequences used for the amplification of the adh1b arg47his snp were 5'-aatcttttctgaa tctgaacag-3 ' and 5'-gaaggggggtcacca ggttgc-3 ' , respectively ; and primer pairs for aldh2 glu487lys were 5'-caaattacagggt caagggct-3 ' ( forward ) and 5'-ccacactca cagttttctctt-3 ' ( reverse ) . 10 l of the amplified product was incubated at 37c for two hours with nmuci and mbo ii restriction enzymes ( re ) for adh1b and aldh2 , respectively . dna fragments in the re digested and undigested mixture were analysed on 10 per cent polyacrylamide gel electrophoresis ( page ) using one per cent tris - borate - edta ( tbe ) buffer and sized with the help of known dna markers . the presence of 134 bp and 123 bp fragments indicated the presence of wild - type glu and mutant lys alleles , respectively for the aldh2 snp . genotyping for approximately 10 per cent of the samples was repeated at random for quality control , and complete agreement was found . comparison of all clinical variables between subjects with different genotypic profiles for adh1b and aldh2 gene polymorphisms were carried out using the t - test or mann whitney 's u test , as appropriate ; p values < 0.05 were considered significant . for the aldh2 glu487lys snp , the genotypic frequencies were 0.73 ( 2 * 1/*1 ) , 0.16 ( 2 * 1/*2 ) and 0.11 ( 2 * 2/*2 ) , with a minor allele frequency ( aldh2 * 2 ) of 0.19 . the significance of association of various clinical parameters with aldh2 polymorphism is included in table 1 . about 80 per cent of subjects with the aldh2 * 1/*2 genotype , and 38 per cent with the aldh2 * 1/*1 genotype reported a history of severe alcohol withdrawal ( delirium ) during their alcohol drinking years . about 39 per cent of those with the aldh2 * 1/2 * 1 and 29 per cent of those with the aldh2 * 1/*2 genotype drank almost every day . subjects drinking excessive amounts ( > 60g / day ) , however , were more likely to have the aldh2 * 1/*1 or the aldh2 * 1/*2 genotype than the aldh2 * 2/*2 genotype . clinical details of the study population significant at p < 0.05 . the adh1b*2 and aldh2 * 2 alleles raise the levels of acetaldehyde by increasing the rate of synthesis and decreasing the rate of metabolism respectively , thus interacting additively but not synergistically . caucasians are largely monomorphic for the adh1b*1 allele , with a very low frequency of the aldh2 * 2 allele . since the adh1b*3 ( adh1b*369cys ) allele has been documented to be essentially specific to the african population , we did not genotype adh1b*3 in our study population . , the very low frequency of the adh1b*2 allele found in our population is surprising , since it is present at a substantial frequency in the neighbouring east asian population and confers protection against alcoholism . a very low frequency of adh1b*2 could indicate a selection pressure operating against the adh1b*2 allele in our population due to its inability / redundancy to protect the indian population from ad . commenting on the alcohol - consuming practice of the service / working and lower caste of indian society , reddy et al . suggested that , due to the heavy amount of alcohol consumption on a regular basis , most of these subjects might have developed a genetic adaptation to withstand drinking alcohol ( unlike most east asian populations with known sensitivity to alcohol consumption ) , thereby possibly requiring no protective mechanism and hence resulting in the absence of the adh1b*2 allele . could also support a very low frequency of the adh1b * 2 allele in the present study . a stronger influence of social setting and age than the effect of the adh1b*2 allele on alcohol drinking has also been found in a study in a jewish population , which supports the above - mentioned hypothesis of an environmental influence on genetic constitution with regard to the adh1b gene in the indian population . the aldh2 gene is located on chromosome 12q24 . the single bp difference ( g > a ; glu487lys ) in exon 12 causes the normal allele , aldh2 * 1 , to become a non - functional ( aldh2 * 2 ) allele , which codes for the inactive enzyme . the aldh2 * 2 allele , which is prevalent in asian populations but is extremely rare in non - asians , has the strongest protective association with ad . asian subjects homozygous for aldh2 * 2 have an almost zero risk of developing ad , whereas heterozygotes ( aldh2 * 1/2 * 2 ) are about one - third as likely to be alcoholic , compared with those without this allele . reported that the homozygous aldh2 * 2/*2 genotype is present at a very low frequency ( 0 - 0.3 per cent ) in alcoholic patients , compared with 3 - 12 per cent among non - alcoholic subjects . the heterozygous aldh2 * 1/*2 genotype has been found to be present in 6 - 17 per cent of alcohol - dependent subjects , compared with 30 - 45 per cent of non - alcoholic subjects . this difference in aldh2 * 2 allele frequency between alcoholic and non - alcoholic subjects has been postulated to be responsible for the phenomenon of adverse responses to alcohol consumption , such as facial flushing , nausea and tachycardia in individuals with the inactive form of the enzyme . in contrast to the reports from the neighbouring east asian population , the present study found comparatively higher allele ( aldh2 * 2 = 0.19 ) and genotypic ( aldh2 * 2/*2 = 0.11 , aldh2 * 1/*2 = 0.16 ) frequencies in alcohol - dependent individuals . a recent study from india is equally exciting , however , as it found the snp to be monomorphic ( aldh2 * 1/2 * 1 ) in healthy control subjects . in light of the findings of that recent report , as well as that of goedde et al . , the higher frequency of the inactive aldh2 allele observed in our indian alcohol - dependent subjects , compared with the control population , provides evidence for a direct role of acetaldehyde in the development of alcohol addiction ( despite its toxic actions ) . further , the amount of alcohol consumed by aldh2 * 2/*2 individuals is significantly lower than those with aldh2 * 1/*1 and aldh2 * 1/*2 genotypes ( table 1 ) . it should be noted that , although subjects with the aldh2 * 2/*2 genotype in this study are classified as alcohol dependent by dsm iv criteria , these individuals are only drinking three to five restaurant glasses per day ( of indian liquor , with 8 g alcohol / drink ) , which is not enough to produce ethanol intoxication , but rather acetaldehyde intoxication . when we compared our findings in alcohol - dependent subjects with those of an indian control population ( see bhaskar et al . , where the pleasurable effects of acetaldehyde led to higher positive expectancies in individuals with the aldh2 * 1/*2 compared with the aldh2 * 1/*1 genotype . , the pleasurable effects of acetaldehyde leading to higher positive expectancies in individuals with the aldh2 * 1/*2 compared with the aldh2 * 1/*1 genotype could be due to a development of tolerance to acetaldehyde . the present observation of the development of ad ( in aldh2 * 2/*2 individuals ) with low levels of alcohol consumption but with presumably high levels of acetaldehyde strongly implicates acetaldehyde in the causality of the dependence . two clinical parameters -- namely , duration of ad and age at onset of ad -- correlated well with the protection - conferring property of the aldh2 glu487lys polymorphism . the duration of ad was found to be significantly longer ( p < 0.01 ) among individuals with the aldh2 * 1/*1 genotype , compared with aldh2 * 2/*2 subjects . the age at which patients developed ad , however , was significantly lower ( p < 0.01 ) in individuals with the aldh2 * 1/*1 genotype compared with aldh2 * 2/*2 subjects ( table 1 ) . alcohol - induced flushing is inherited as a dominant trait in asians . a substantial number of asians ( 50 - 80 per cent ) , compared with caucasians ( 3 - 12 per cent ) , exhibit flushing . acetaldehyde creates unpleasant adverse reactions by acting as a major deterrent to excessive alcohol drinking on the one hand , while giving the euphoric sensations that may reinforce alcohol drinking on the other . since people with the inactive aldh2 * 2 allele drink less often and consume less alcohol per occasion than those with the active aldh2 * 1 allele , our observation of a significantly lower age at onset and longer duration of ad among individuals with the aldh2 * 1/*1 genotype compared with those with the aldh2 * 2/*2 genotype seems to be in keeping with the available literature . significantly higher values of sgot and sgpt in individuals with the aldh2 * 1/*1 genotype , compared with the aldh2 * 2/*2 genotype ( table 1 ) , is suggestive of more liver damage due to higher alcohol consumption among aldh2 * 1/*1 subjects . our findings indicate that functional polymorphism of the gene coding for the aldh enzyme affects the propensity to develop ad . the most important finding of the study was the uniquely high frequency of the aldh2 * 2/*2 genotype ( among alcohol - dependent subjects ) being a risk - conferring factor for ad . further , based on the findings from this study , albeit based on a limited sample size , it could be hypothesised that the indian genotype is prone to slow metabolism of alcohol and high metabolism of acetaldehyde , and thereby prone to alcoholism . our findings should be viewed , however , in the perspective of the potential limitation posed by the absence of data from an ethnically , age- and sex - matched control population . further , these results not only warrant replication in larger sample sets , but also underscore the need for investigations on candidate gene polymorphisms from other biochemical pathways .

hormone therapy ( ht ) has been the standard treatment option for postmenopausal symptom relief for decades . however , ht has been linked with increased breast cancer risk in a number of clinical trials including the women s health initiative ( whi ) . as a natural and perceived safe alternative to ht for postmenopausal system relief , however , rigorous interdisciplinary studies on the efficacy , potential toxicity , and health benefits of these botanicals continue to be in high demand . . the two major mechanisms of carcinogenesis are estrogen signaling ( hormonal pathway ) and metabolism of estrogens to reactive quinones ( chemical pathway , figure 1 ) . recently , a number of cohort studies analyzing the risk correlation between estrogen levels , estrogen metabolites , and breast cancer risk in postmenopausal women were conducted . the results indicated that higher estrogen levels were associated with increased risk of postmenopausal breast cancer , while enhanced estrogen 2-hydroxylation suggested a lower risk for breast cancer . various in vitro studies have supported this finding that estrogen 2-hydroxylation represents a detoxification pathway , whereas 4-hydroxylation is correlated with malignant transformation . p450 1a1 catalyzes the formation of 2-ohe1/e2 ( detoxification biomarker ) , which are clinically shown to be correlated with reduced breast cancer risk . p450 1b1 catalyzes the formation of 4-ohe1/e2 ( genotoxic biomarker ) , which is oxidized to the genotoxic estrogen-3,4-quinone ( 3,4-e1/e2-q ) and forms ros through redox cycling . the reactive quinone and ros contribute to estrogen carcinogenesis . chemopreventive botanicals are hypothesized to increase 2-hydroxylation and decrease 4-hydroxylation metabolism , as shown with green and red arrows . p450 1a1/1b1 are the major extra - hepatic p450 1 enzymes that metabolize estrogens into 2- or 4-hydroxylated forms , respectively ( figure 1 ) . the expression is mainly controlled by the upstream aryl hydrocarbon receptor ( ahr ) , which translocates into the nucleus upon activation and binds to the xenobiotic response element ( xre ) , initiating targeted gene transcription . in breast tissues , the 2- and 4-hydroxylated estrogen catechols can be further metabolized by catechol - o - methyl transferase ( comt ) to the more stable 2- and 4-methoxy ether metabolites , which can be used as biomarkers for 2- and 4-hydroxylation pathways . hops ( strobiles of humulus lupulus l. , cannabaceae ) have been traditionally used as a sleep aid and , more recently , by women for postmenopausal symptom relief . many biological activities of hops have been connected to a series of bioactive prenylated flavanones and chalcones such as 6-prenylnarigenin ( 6-pn ) , 8-prenylnarigenin ( 8-pn ) , isoxanthohumol ( ix ) , and xanthohumol ( xh ) ( figure 2 ) . the predominant prenylated chalcone , xh , has been shown to be an effective chemopreventive agent , inducing the detoxification enzyme nad(p)h : quinone oxidoreductase 1 ( nqo1 ) in both in vitro and in vivo studies . xh is metabolized to ix , desmethyl xanthohumol , 8-pn , and 6-pn as shown in figure 2 . 8-pn has been reported to be one of the most potent estrogen receptor alpha ( er ) phytoestrogens known to date , which is likely responsible for menopausal symptom relief . previously , we showed that a hop extract was able to reduce the potentially genotoxic estrogen 4-hydroxylation pathway and decrease estradiol ( e2 ) induced colony formation in human nontumorigenic breast epithelial mcf-10a cells . the uic / nih center for botanical dietary supplements research has further enriched a spent hop extract with respect to its estrogenic ( 8-pn ) and chemopreventive ( xh ) compounds ( figure 2 ) . the new standardized extract contained much higher levels of the marker compounds than the previous extract . the purpose of this study was to test the effect of this standardized hop extract , which has been used in human clinical trials , on estrogen metabolism . since a relatively low response was observed in mcf-10a cells , we also included the well characterized breast cancer mcf-7 cell line to confirm the bioactivities of the standardized hop extract as well as the effects of the four major prenylated marker compounds . the effects of this new hop extract and compounds on estrogen oxidative metabolism in the two breast cell lines were studied . their effects on p450 1a1/1b1 mrna expression and activity in mcf-10a and mcf-7 cells and inhibition of recombinant p450 1a1/1b1 activity were also measured . finally , xre activation was analyzed in both liver hepg2 cells and mcf-7 cells to confirm the mechanism of action . the results suggest that hops can selectively enhance p450 1a1 catalyzed estrogen 2-hydroxylation and potentially reduce breast cancer risk . all chemicals and reagents were purchased from sigma ( st . louis , mo ) or invitrogen ( carlsbad , ca ) unless otherwise stated . s enantiomers of 6-pn and 8-pn were purchased from sigma ( st . louis , mo ) . the ethanol extract of botanically authentic spent strobiles of humulus lupulus was obtained from hopsteiner ( mainburg , germany , and new york , ny ) and standardized to the prenylated polyphenol marker compounds 6-pn , 8-pn , ix , and xh as previously described ( figure 2 ) . briefly , standardization involved characterization by lc - uv , lc - ms / ms , and quantitative h nmr ( qhnmr ) . the same extract has been used in a phase i clinical trial in postmenopausal women . the concentrations of the four marker compounds in this extract were 1.2% 6-pn , 0.33% 8-pn , 0.99% ix , and 32% xh . mcf-10a and mcf-7 cells were obtained from american type culture collection ( manassas , va , usa ) . mcf-10a cells were cultured in 1:1 dulbecco s modified eagle medium and ham s f12 nutrient mixture ( dmem / f12 ) with 15 mm hepes and l - glutamine ( invitrogen ) , supplemented with 20 ng / ml epidermal growth factor , 100 ng / ml cholera toxin , 0.5 g / ml hydrocortisone , 10 g / ml insulin , 5% horse serum , and 1% penicillin mcf-7 cells were maintained in rpmi 1640 media supplemented with 10% fetal bovine serum , 1% glutamax , 1% ab / am , 1% nonessential amino acids , and 3 g / ml insulin . all experiments were done with cells under 15 passages in phenol - red free media supplemented with charcoal stripped serum and the same other ingredients . estradiol and all of the standard compounds of estrogen metabolites were obtained from steraloids inc . 4-methoxyestrone-1,4,16,16-d4 was obtained from cdn isotope ( pointe - claire , quebec ) and used as internal standard in the estrogen metabolites analysis . 2-methoxyestrone and 4-methoxyestrone were measured as indicators of the level of estrogen 2-hydroxylation and the 4-hydroxylation pathway as previously described with modifications . briefly , cells were estrogen starved for 24 h before being seeded into 6-well plates . cells were incubated with e2 ( 1 m ) in the presence and absence of hop extract ( 0.12.5 g / ml ) and 6-pn , 8-pn , ix , and xh ( 0.16 m ) for 2 days . cell media were then collected and spiked with 0.4 nm internal and 2 mm ascorbic acid . the organic layers were then combined and dried under a gentle flow of nitrogen . derivatization was performed with 100 l of 0.1 m nahco3 buffer ( ph 9.5 ) and 100 l of dansyl chloride in acetone ( 1 mg / ml ) at 60 c in a water bath with agitation for 10 min . samples were then cooled on ice , and 50 l of sample was analyzed by lc - ms / ms as described below . after derivation , all samples were analyzed by a positive ion electrospray tandem mass spectrometric method as previously described . briefly , lc - ms / ms was performed by using an agilent 1200 series nano flow lc system ( agilent technologies , aanta clara , ca ) coupled to an ab sciex triple quad 5500 system ( ab sciex , framingham , ma ) . the liquid chromatography separation was carried out with a 100 mm 3 mm i.d . waters beh c-18 column packed with 1.7 m particles ( waters , milford , ma ) and maintained at 40 c . multiple reactions monitoring transitions were selected as follows : 534.4171.2 for the detection of dansylated meoe1 and 538.4171.2 for dansylated meoe1-d4 . quantitation was performed using the analyst software ( applied biosystems , forster city , ca ) , and data were normalized to the e2 control treatment in each independent experiment . mcf-10a and mcf-7 cells at a density of 2.5 10 cells / ml were plated in 6-well plates and treated with dmso , hops , 6-pn , 8-pn , ix , or xh for 24 h. rna extraction , reverse transcription , and pcr were performed according to manufacturers protocols as previously described . the total rna was extracted according to qiagen s ( valencia , ca ) rneasy kit instructions . rna was reverse transcribed according to invitrogen s superscript iii first - strand synthesis system for rt - pcr . the resulting cdna ( 2 l ) was used for real - time pcr quantification using applied biosystems ( carlsbad , ca ) steponeplus real - time pcr system . taqman gene expression master mix and p450 1b1 primer with fam / mgb probe from applied biosystems were added to a 96-well reaction plate with cdna to perform real - time quantitative pcr ( one cycle of 50 c for 2 min , 95 c for 10 min , 40 cycles of 95 c for 15 s , and 60 c for 1 min ) . data were analyzed with the comparative ct ( ct ) method to determine fold difference in reference to the hprt1 endogenous control . erod assay measuring p450 1 enzyme activity was conducted both in cells and with recombinant p450 1a1 and 1b1 enzymes as previously described . briefly , 1 10 cells / well were plated and treated with hop extract or compounds for 2 days , cells were washed with pbs and incubated with 2.5 m 7-ethoxyresorufin and 1.5 mm salicylamide in pbs at 37 c . fluorescence was measured every minute with excitation at 530 nm and emission at 590 nm for 25 min with a biotek ( winooski , vt ) synergy h4 hybrid multi - mode microplate reader . for enzyme inhibition experiments , recombinant p450 1a1 and 1b1 protein with reductase p450 1a1 ( 0.15 pmole ) or 1b1 ( 0.8 pmole ) was preincubated with test compounds or vehicle for 5 min at 37 c in 200 l of 50 mm potassium phosphate buffer ( ph 7.4 ) with 1 mm nadph . the reaction was initiated by adding 7-ethoxyresorufin solution in potassium phosphate buffer to a final concentration of 2.5 m . fluorescence was measured every minute after 5 s of mixing for 25 min at 37 c . fluorescence with 7-ethoxyresorufin as substrate was linear for more than 15 min , and the reaction rate was determined from the slope of the linear regression curves plotted with data points measured in the first 15 min . mcf-7 cells were plated in 12-well plates overnight , and cells were transfected at 70% confluency with luciferase and renilla plasmids ( promega , madison , wi ) , xre pgl4.43 luciferase plasmid ( 1 g ) , and prl - tk ( 500 ng ) , using lipofectamine 2000 reagent ( invitrogen , grand island , ny ) for 6 h. after 6 h of transfection , cells were treated with hop extract / compounds with and without the presence of tcdd ( 10 nm ) for 24 h and lysed with passive lysis buffer . lysates were centrifuged and analyzed for luciferase activity according to promega s dual - luciferase reporter assay system protocol using the fluostar optima luminometer ( bmg labtechnologies , germany ) . the % of tcdd was obtained by setting tcdd s fold induction in the xre - luciferase assay as 100% . the fold induction of compounds was divided by the tcdd response and multiplied by 100 to obtain the % of tcdd response . the data were expressed as the mean sem of at least three independent experiments . significance was determined using one - way anova with dunnett s post - test , comparing treatment groups to the control ( * p < 0.05 ) . previously , we showed that a hop extract slightly decreased the estrogen 4-hydroxylation pathway and had no effect on the 2-hydroxylation pathway in mcf-10a cells in a six day experiment . in the current study , two day metabolism studies with the new clinical hop extract described in the material and methods showed that this hop extract stimulated estrogen 2-hydroxyaltion ( figure 3a ) . one problem with the mcf-10a cell line is its relatively low activities of p450 1a1/1b1 . in order to confirm the qualitative effects of hop extract and the bioactive marker compounds , additional experiments were done with the well characterized mcf-7 cells , which are known to be much more sensitive for p450 1a1/1b1 inductions . the data showed much higher overall induction of metabolism ( 1020-fold ) , and 2-meoe1 formation was preferred similar to the mcf-10a data ( figure 3b ) . although the preferential induction was observed in both cell lines , the differences in mcf-10a cells were more prominent than in mcf-7 cells with both hops and 6-pn treatment . 6-pn was the most potent compound tested , inducing 2-meoe1 50-fold compared to 40-fold for 4-meoe1 in mcf-7 cells and 3.5-fold for 2-meoe1 induction versus 2-fold for 4-meoe1 in mcf-10a cells ( figure 3c and d ) . in contrast to the mcf-10a cell experiments , 8-pn showed moderate induction of estrogen metabolism in mcf-7 cells , which could be due to the higher ahr mediated p450 1a1/1b1 induction in mcf-7 cells . overall , the results in mcf-7 cells are comparable with the mcf-10a data and suggested that hops and 6-pn preferentially induce estrogen 2-hydroxylation metabolism in breast cells . hop extract and 6-pn preferentially induced 2-hydroxylation metabolism in breast mcf-10a and mcf-7 cells . ( a ) mcf-10a cells and ( b ) mcf-7 cells were treated with e2 ( 1 m ) and the hop extract for 2 days , and media were collected and analyzed for 2-meoe1 and 4-meoe1 metabolite level by lc - ms / ms . ( c ) mcf-10a cells and ( d ) mcf-7 cells were treated with e2 ( 1 m ) and 6-pn , 8-pn , ix , xh ( 1 m ) , and tcdd ( 10 nm ) for 2 days , and media were analyzed for 2-meoe1 and 4-meoe1 metabolites . data were plotted as the means sem of three independent experiments and analyzed by one - way anova with dunnett s multiple comparison post - test to compare treatment groups with the control group , * p < 0.05 . p450 1a1/1b1 mrna levels were analyzed 24 h after treatment with hops and the bioactive compounds . in mcf-10a cells , hop extract significantly induced p450 1a1 mrna expression to 7-fold , with no significant effect on 1b1 ( figure 4a ) . in mcf-7 cells , the induction levels were significantly higher with 90- and 35-fold induction of p450 1a1 and 1b1 mrna expression ( figure 4b ) . regarding the hop compounds , the only compound that significantly increased p450 1a1 and 1b1 was 6-pn , with an increase to 16- and 2-fold , respectively , in mcf-10a cells ( figure 4c ) . in mcf-7 cells , qpcr analysis also showed that 6-pn preferentially increased p450 1a1 mrna levels to around 290-fold compared to the 25-fold induction of p450 1b1 ( figure 4d ) . 8-pn significantly induced p450 1a1 and 1b1 in mcf-7 cells ( figure 4d ) , yet not in mcf-10a cells ( figure 4c ) , and this induction in mcf-7 cells ( 90-fold and 20-fold , respectively ) was less than that of 6-pn . although these prenylated polyphenols share some common structural moieties , their bioactivities show remarkable differences . these data correlate with the results from estrogen oxidative metabolism , indicating that hops and 6-pn preferentially increased p450 1a1 mrna levels in breast cells . hop extract and 6-pn preferentially induced p450 1a1 mrna expression in mcf-10a and mcf-7 cells . ( a ) mcf-10a cells and ( b ) mcf-7 cells were treated with hop extract , and p450 1a1 and 1b1 mrna expression was analyzed after 24 h via qpcr . ( c ) mcf-10a and ( d ) mcf-7 cells were treated with 6-pn , 8-pn , ix , xh ( 1 m ) , and tcdd ( 10 nm ) for 24 h , and p450 1a1/1b1 mrna expression was analyzed via qpcr . results were plotted as the means sem of three independent experiments and analyzed by one - way anova with dunnett s multiple comparison post - test to compare treatment groups to the dmso control , * p < 0.05 . p450 1a1/1b1 activity was measured using the erod assay in both cell lines after 2 days of treatment with hop extract and bioactive compounds . in mcf-10a cells , in the presence of the hop extract , a significant dose responsive induction was observed to a maximum of 0.04 pmol / min / well resorufin formed ( figure 5a ) . in mcf-7 cells , the hop extract gave significantly higher erod activity compared to mcf-10a cells to a maximum of 0.25 pmol / min / well resorufin ( figure 5b ) . with the hop compounds in mcf-10a cells , only 6-pn increased p450 1a1/1b1 activity dose - dependently to 0.05 pmol / min / well resorufin ( figure 5c ) . xh moderately induced the p450 1 activity even though estrogen metabolism and p450 1a1/1b1 gene expression were not affected ; 8-pn and ix did not show significant effects . in mcf-7 cells , significant induction of p450 1a1/1b1 activity was observed with 6-pn and 8-pn ( 3 m ) to 0.8 and 0.2 pmol / min / well resorufin , while ix and xh did not have significant effects ( figure 5d ) . overall , the results from the erod activity assay were consistent with the results from estrogen metabolism and p450 1a1/1b1 mrna analysis . qualitatively , the data from these two cell lines were also comparable and indicated that hops and 6-pn strongly induced p450 1a1/1b1 activity in breast cells . p450 1a1/1b1 activity was analyzed in mcf-10a cells after 2-day treatment of ( a ) hop extract and ( c ) different doses of 6-pn , 8-pn , ix , and xh with the erod assay . p450 1a1/1b1 activity was analyzed in mcf-7 cells after 2-day treatment of ( b ) hop extract and ( d ) different doses of 6-pn , 8-pn , ix , and xh with the erod assay . results were represented as pmol / min / well resorufin formed and plotted as the means sem of three independent experiments and analyzed by one - way anova with dunnett s multiple comparison post - test to compare treatment groups to dmso control , * p < 0.05 . human hepatoma hepg2 cells and mcf-7 cells transfected with a xre - luciferase construct were used to measure the effect of bioactive compounds on ahr activation . the cells were incubated with the compounds for 24 h after transient transfection of the luciferase construct . of the four polyphenols , 6-pn and 8-pn significantly and dose - dependently increased xre - luciferase activity to around 6.5- and 10-fold of the control at 10 m , while xh and ix did not have significant effects ( figure 6a ) . in the presence of the ahr agonist tcdd ( 10 nm ) , a dose responsive decrease in xre luciferase activity was observed with 6-pn and 8-pn cotreatment ( figure 6b ) , which further suggested the interactions with ahr . in mcf-7 cells , 6-pn ( 5 m ) significantly induced the activity , while 8-pn ( 5 m ) had no effect , which correlated with the p450 1a1/1b1 activity and gene expression analysis . the results indicated some cell selectivity of 8-pn and also suggested 6-pn to be activating ahr in both breast and liver cell lines . finally , upon cotreatment of ahr antagonist ch223191 and 6-pn ( 1 m ) in mcf-7 cells , the erod activity was inhibited dose responsively ( figure 6d ) , which further confirmed 6-pn to be an ahr agonist . in summary , these results suggested that 6-pn acted as an ahr agonist in both breast and liver cells . hepg2 cells were incubated with ( a ) hop compounds alone and ( b ) 6-pn and 8-pn in the presence of tcdd ( 10 nm ) for 24 h before analysis of xre - luciferase reporter activity . ( c ) mcf-7 cells were incubated with 6-pn , 8-pn ( 5 m ) , and tcdd ( 10 nm ) for 24 h before analysis of xre - luciferase reporter activity . ( d ) p450 1a1/1b1 activity was measured via the erod assay in mcf-7 cells after cotreatment of 6-pn ( 1 m ) with ahr antagonist ch223191 ( 0.01 , 0.1 , 1 , and 10 m ) for 2 days . sem of three independent experiments and analyzed by one - way anova with dunnett s multiple comparison post - test to compare treatment groups to the control group , * p < 0.05 . various flavonoids have been previously reported to inhibit p450 1a1/1b1 activities . to study the inhibitory effects of hop compounds on p450 1a1/1b1 all four compounds acted as p450 1a1/1b1 inhibitors with ic50 values in the low micromolar range without selectivity for either p450 1a1 or 1b1 ( table 1 ) . however , the inhibitory activity of these compounds in cell culture experiments was considerably less than that with recombinant enzymes ( figures s2 and s3 ) . these data indicated the metabolism results were mainly the result of induction of p450 1a1/1b1 enzyme levels and that little direct inhibition of p450s should be observed at clinical concentrations of hop supplements . the values are expressed as the means sd from three independent dose responsive curves using recombinant p450 1a1 and p450 1b1 enzymes . estrogen exposure has long been linked with postmenopausal breast cancer risk , especially since the whi report in 2002 . estrogen carcinogenesis includes the hormonal mechanism involving classical er binding and estrogen signaling which promotes cell growth and the chemical mechanism where estrogens are converted to reactive quinones which modify dna leading to genotoxicity ( figure 1 ) . the estrogen 4-hydroxylation pathway is considered the genotoxic pathway by forming the electrophilic / redox active estrogen-3,4-quinone and reactive oxygen species ( ros ) which causes dna damage . contrary to estrogen 4-hydroxylation , estrogen 2-hydroxylation is a nongenotoxic pathway , and the metabolite , 2-methoxyestradiol , has been shown to have antiproliferative / anticancer activity . several recent clinical trials analyzing serum estrogen metabolite levels and the risk of postmenopausal breast cancer further support the 2-hydroxylation pathway as a marker for chemoprevention ; however , the data are inconclusive on the relationship between the estrogen 4-hydroxylation and breast cancer risk . p450 1a1 and 1b1 are the major enzymes in breast tissues that are responsible for the local estrogen 2- and 4-hydroxylation metabolism , respectively . these two enzymes share about 40% homology and are generally co - upregulated upon ahr activation . ahr is also responsible for the expression of several other phase i and phase ii enzymes , which are associated with the detoxification of environmental carcinogens as well as the potential activation of pro - carcinogens . p450 1b1 expression levels in tumors , as well as carcinogen - induced p450 1b1 levels in cancer cells , are higher than in normal tissues and cells . mcf-10a cells have been frequently used as a model to study estrogen chemical carcinogenesis due to the absence of er and they present a nontumorigenic phenotype . however , the ahr mediated p450 1a1/1b1 induction in mcf-10a cells is relatively low as reported previously and confirmed in the present study . data also suggest that the phase i and phase ii enzyme expression is variable in mcf-10a cells depending on the confluence level in cell culture . in addition , spontaneous expression of er might occur in mcf-10a cells after a certain number of passages . these potential problems and the variability observed in the current experiments prompted additional studies in the more robust mcf-7 cells . spink et al . showed that induction of p450 1a1/1b1 enzymes vary among different tumorigenic and nontumorigenic cell lines . they determined that mcf-7 cells had higher levels of tcdd - induced p450 1a1/1b1 mrna and almost 8-fold higher metabolic rates for tcdd - induced 4-meoe2 formation compared to those of mcf-10a cells . similarly , our results showed more than 15-fold increase in p450 1a1/1b1 gene induction with tcdd treatment in mcf-7 over mcf-10a cells ( figure 4c and d ) and about 10-fold higher metabolite formation with hop extract treatment in mcf-7 compared to that of mcf-10a cells ( figure 3a and b ) . since mcf-7 cells mirrored mcf-10a cells in the response to botanical treatment with higher induction levels , they serve as a better model to screen botanicals and compounds for the modulation of estrogen metabolism . in addition , the upstream p450 1a1/1b1 mrna induction trend among compounds ( 6-pn 8-pn and no effect with ix and xh ) corresponds with the metabolism and activity results in mcf-7 cells . however , due to the presence of er there is potential crosstalk between ahr and er signaling pathways , and ahr agonists have been reported to increase proteasomal degradation of er . the interesting relationship between er , ahr , and botanical modulation of estrogen metabolism will be the subject of future studies . botanicals have been previously shown to affect ahr activation , and the major compounds responsible were polyphenols . for example , resveratrol has been shown to induce xre activation to about 6-fold at 10 m in mcf-10a cells . in this study , hepg2 cells were used to compare the ahr activation by hop compounds because the xre - luciferase reporter activity in hepg2 cells were much higher compared to that in mcf-7 cells ( figure s7 ) . comparatively , in this study xre - luciferase activity was increased to 6.5-fold by 6-pn ( 10 m ) in hepg2 cells ( figure 6a ) . 8-pn displayed some cell selectivity with about 10-fold xre activation at 10 m in hepg2 cells but showed no effect in mcf-7 cells . preferential induction of p450 1a1 over p450 1b1 has been documented in the literature . quercetin ( 10 m ) and berberine ( > 5 m ) preferentially induced p450 1a1 over 1b1 in mcf-10f and mcf-7 cells , respectively . in contrast , benzo(a)pyrene ( 1 m ) preferentially increased p450 1b1 in human oral epithelial cells . however , the mechanisms of p450 1a1 and 1b1 preferential induction as well as in vivo effects need to be further studied . induction of xre activity and inhibition of tcdd induced xre activity further supported 6-pn to be an ahr agonist ( figure 6 ) . induction of p450 1 enzymes by natural flavonoids and flavonoid rich botanicals have been reported previously . naringenin , a flavanone with the same scaffold as the hop flavanones , does not induce p450 1a1/1b1 , while the effect of flavanones with a prenylated side chain has not been studied . results from this study indicated that prenylated naringenin derivatives exhibit a unique activity compared to that of the parent naringenin . in addition , the significant p450 1a1/1b1 induction by 6-pn compared to 8-pn may suggest that the positioning of the prenyl group is important for ahr activation . the effect of hop compounds on tcdd - induced xre - luciferase reporter activity was also measured . the results indicated that 6-pn and 8-pn had comparable effects in inhibiting tcdd induced xre - luciferase activity at micromolar levels ( figure 6b ) . resveratrol , as well as the scaffold parent compound naringenin , also demonstrated significant inhibitory effects on tcdd induced erod activity above 10 m in mcf-10a cells . similar studies looking at estrogen metabolism using a mcf-10f cell model observed decreased formation of 4-meoe1/e2 and dna adducts after cotreatment with resveratrol ( 25 m ) and tcdd ( 10 nm ) . in contrast , we previously showed that licochalcone a ( 10 m ) , a b - ring ( c-5 ) prenylated chalcone from licorice ( glycyrrhiza inflata ) , was an ahr antagonist and shut down estrogen oxidative metabolism in mcf-10a cells . it has also been shown that flavonoids generally exhibit more potent p450 1b1 inhibition over 1a1 . several ubiquitous , unsubstituted flavones and flavonols , such as quercetin , kaempferol , and apigenin , have been reported to be p450 1b1 inhibitors with ic50 values below 50 nm , while inhibiting p450 1a1 activity less potently . however , flavanones , like narigenin , are generally weak p450 1 inhibitors with ic50 values in the micromolar range , which might indicate the importance of the c - ring 23 double bond in p450 1 enzyme inhibition . several hydroxychalcones have been tested for the inhibition of p450 1a1 and 1b1 and showed ic50 values in the low micromolar range . our results as well as previous studies from henderson et al . showed that the prenylflavanones from hops are more potent inhibitors compared to the parent flavanone , naringenin . the hop compounds had similar inhibitory activities toward p450 1b1 , with ic50 values around 0.5 m ; 6-pn showed around 3-fold lower inhibitory activity to p450 1a1 ( ic50 0.6 m ) than 1b1 ( ic50 0.2 m ) ( table 1 ) . when compared to resveratrol , which inhibited p450 1a1 and 1b1 with ic50 values around 2 and 25 m , respectively the hop compounds have also been shown to inhibit p450 2c8 , 2c9 , and 19 with ic50 values in the low micromolar range . however , in contrast to the experiments with purified p450s , the hop compounds showed little to no inhibition in cells ( figure s3 ) , likely due to extensive metabolism . these data suggest that the effect on estrogen oxidative metabolism modulation was mainly contributed by the ahr agonist activity of 6-pn . the results from this study suggests that hop extracts should be standardized not only to 8-pn for estrogenic effects and to xh for chemopreventive properties but also to 6-pn for its potential modulation of estrogen metabolism . however , further studies are needed to test and confirm the activities in vivo . in conclusion , results from this study provided novel in vitro evidence that hops and its compound 6-pn preferentially induced the nontoxic estrogen 2-hydroxylation pathway in two different breast cell lines , which indicated a potentially protective role of hops to help reduce the risk of breast cancer through estrogen metabolism modulation . as hop dietary supplements are taken widely by women for postmenopausal symptom relief , it is important to expand our knowledge about the bioactivity and safety of 6-pn and related hop compounds . the clinical trial data have indicated long half - lives of these hop compounds . since the pharmacokinetic properties of the hop compounds would significantly influence their effect in vivo , the preferential 2-hydroxylation induction as well as modulation of p450 1a1/1b1 enzymes would need to be tested with in vivo models that will be studied in the future . the present data also confirm the importance of performing botanical standardization to several bioactive phyto - constituents simultaneously . accordingly , for hop extracts , suitable target markers are 8-pn ( estrogenic ) , xh ( chemopreventive ) , and , as shown in the present study , 6-pn ( ahr agonist ) as modulators of estrogen metabolism . assessing the levels of these compounds in standardized hop extracts will be beneficial for the health effects and enhance the safety of women consuming these herbal preparations . collectively , the present findings provide additional rationales for a meaningful chemical and biological standardization of safe and effective hop botanical supplements .

humulus lupulus l. ( hops ) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief . in addition to its efficacy for menopausal symptoms , hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer . in the present study , an enriched hop extract and the key bioactive compounds [ 6-prenylnarigenin ( 6-pn ) , 8-prenylnarigenin ( 8-pn ) , isoxanthohumol ( ix ) , and xanthohumol ( xh ) ] were tested for their effects on estrogen metabolism in breast cells ( mcf-10a and mcf-7 ) . the methoxyestrones ( 2-/4-meoe1 ) were analyzed as biomarkers for the nontoxic p450 1a1 catalyzed 2-hydroxylation and the genotoxic p450 1b1 catalyzed 4-hydroxylation pathways , respectively . the results indicated that the hop extract and 6-pn preferentially induced the 2-hydroxylation pathway in both cell lines . 8-pn only showed slight up - regulation of metabolism in mcf-7 cells , whereas ix and xh did not have significant effects in either cell line . to further explore the influence of hops and its bioactive marker compounds on p450 1a1/1b1 , mrna expression and ethoxyresorufin o - dealkylase ( erod ) activity were measured . the results correlated with the metabolism data and showed that hop extract and 6-pn preferentially enhanced p450 1a1 mrna expression and increased p450 1a1/1b1 activity . the aryl hydrocarbon receptor ( ahr ) activation by the isolated compounds was tested using xenobiotic response element ( xre ) luciferase construct transfected cells . 6-pn was found to be an ahr agonist that significantly induced xre activation and inhibited 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) induced xre activity . 6-pn mediated induction of erod activity was also inhibited by the ahr antagonist ch223191 . these data show that the hop extract and 6-pn preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through ahr mediated up - regulation of p450 1a1 , which further emphasizes the importance of standardization of botanical extracts to multiple chemical markers for both safety and desired bioactivity .

Introduction Materials and Methods Results Discussion

the results indicated that higher estrogen levels were associated with increased risk of postmenopausal breast cancer , while enhanced estrogen 2-hydroxylation suggested a lower risk for breast cancer . p450 1a1 catalyzes the formation of 2-ohe1/e2 ( detoxification biomarker ) , which are clinically shown to be correlated with reduced breast cancer risk . the expression is mainly controlled by the upstream aryl hydrocarbon receptor ( ahr ) , which translocates into the nucleus upon activation and binds to the xenobiotic response element ( xre ) , initiating targeted gene transcription . in breast tissues , the 2- and 4-hydroxylated estrogen catechols can be further metabolized by catechol - o - methyl transferase ( comt ) to the more stable 2- and 4-methoxy ether metabolites , which can be used as biomarkers for 2- and 4-hydroxylation pathways . hops ( strobiles of humulus lupulus l. , cannabaceae ) have been traditionally used as a sleep aid and , more recently , by women for postmenopausal symptom relief . many biological activities of hops have been connected to a series of bioactive prenylated flavanones and chalcones such as 6-prenylnarigenin ( 6-pn ) , 8-prenylnarigenin ( 8-pn ) , isoxanthohumol ( ix ) , and xanthohumol ( xh ) ( figure 2 ) . 8-pn has been reported to be one of the most potent estrogen receptor alpha ( er ) phytoestrogens known to date , which is likely responsible for menopausal symptom relief . the uic / nih center for botanical dietary supplements research has further enriched a spent hop extract with respect to its estrogenic ( 8-pn ) and chemopreventive ( xh ) compounds ( figure 2 ) . the purpose of this study was to test the effect of this standardized hop extract , which has been used in human clinical trials , on estrogen metabolism . since a relatively low response was observed in mcf-10a cells , we also included the well characterized breast cancer mcf-7 cell line to confirm the bioactivities of the standardized hop extract as well as the effects of the four major prenylated marker compounds . the effects of this new hop extract and compounds on estrogen oxidative metabolism in the two breast cell lines were studied . their effects on p450 1a1/1b1 mrna expression and activity in mcf-10a and mcf-7 cells and inhibition of recombinant p450 1a1/1b1 activity were also measured . the results suggest that hops can selectively enhance p450 1a1 catalyzed estrogen 2-hydroxylation and potentially reduce breast cancer risk . 2-methoxyestrone and 4-methoxyestrone were measured as indicators of the level of estrogen 2-hydroxylation and the 4-hydroxylation pathway as previously described with modifications . cells were incubated with e2 ( 1 m ) in the presence and absence of hop extract ( 0.12.5 g / ml ) and 6-pn , 8-pn , ix , and xh ( 0.16 m ) for 2 days . mcf-10a and mcf-7 cells at a density of 2.5 10 cells / ml were plated in 6-well plates and treated with dmso , hops , 6-pn , 8-pn , ix , or xh for 24 h. rna extraction , reverse transcription , and pcr were performed according to manufacturers protocols as previously described . mcf-7 cells were plated in 12-well plates overnight , and cells were transfected at 70% confluency with luciferase and renilla plasmids ( promega , madison , wi ) , xre pgl4.43 luciferase plasmid ( 1 g ) , and prl - tk ( 500 ng ) , using lipofectamine 2000 reagent ( invitrogen , grand island , ny ) for 6 h. after 6 h of transfection , cells were treated with hop extract / compounds with and without the presence of tcdd ( 10 nm ) for 24 h and lysed with passive lysis buffer . previously , we showed that a hop extract slightly decreased the estrogen 4-hydroxylation pathway and had no effect on the 2-hydroxylation pathway in mcf-10a cells in a six day experiment . in the current study , two day metabolism studies with the new clinical hop extract described in the material and methods showed that this hop extract stimulated estrogen 2-hydroxyaltion ( figure 3a ) . in order to confirm the qualitative effects of hop extract and the bioactive marker compounds , additional experiments were done with the well characterized mcf-7 cells , which are known to be much more sensitive for p450 1a1/1b1 inductions . the data showed much higher overall induction of metabolism ( 1020-fold ) , and 2-meoe1 formation was preferred similar to the mcf-10a data ( figure 3b ) . although the preferential induction was observed in both cell lines , the differences in mcf-10a cells were more prominent than in mcf-7 cells with both hops and 6-pn treatment . in contrast to the mcf-10a cell experiments , 8-pn showed moderate induction of estrogen metabolism in mcf-7 cells , which could be due to the higher ahr mediated p450 1a1/1b1 induction in mcf-7 cells . overall , the results in mcf-7 cells are comparable with the mcf-10a data and suggested that hops and 6-pn preferentially induce estrogen 2-hydroxylation metabolism in breast cells . hop extract and 6-pn preferentially induced 2-hydroxylation metabolism in breast mcf-10a and mcf-7 cells . ( a ) mcf-10a cells and ( b ) mcf-7 cells were treated with e2 ( 1 m ) and the hop extract for 2 days , and media were collected and analyzed for 2-meoe1 and 4-meoe1 metabolite level by lc - ms / ms . ( c ) mcf-10a cells and ( d ) mcf-7 cells were treated with e2 ( 1 m ) and 6-pn , 8-pn , ix , xh ( 1 m ) , and tcdd ( 10 nm ) for 2 days , and media were analyzed for 2-meoe1 and 4-meoe1 metabolites . p450 1a1/1b1 mrna levels were analyzed 24 h after treatment with hops and the bioactive compounds . in mcf-10a cells , hop extract significantly induced p450 1a1 mrna expression to 7-fold , with no significant effect on 1b1 ( figure 4a ) . in mcf-7 cells , the induction levels were significantly higher with 90- and 35-fold induction of p450 1a1 and 1b1 mrna expression ( figure 4b ) . regarding the hop compounds , the only compound that significantly increased p450 1a1 and 1b1 was 6-pn , with an increase to 16- and 2-fold , respectively , in mcf-10a cells ( figure 4c ) . in mcf-7 cells , qpcr analysis also showed that 6-pn preferentially increased p450 1a1 mrna levels to around 290-fold compared to the 25-fold induction of p450 1b1 ( figure 4d ) . 8-pn significantly induced p450 1a1 and 1b1 in mcf-7 cells ( figure 4d ) , yet not in mcf-10a cells ( figure 4c ) , and this induction in mcf-7 cells ( 90-fold and 20-fold , respectively ) was less than that of 6-pn . these data correlate with the results from estrogen oxidative metabolism , indicating that hops and 6-pn preferentially increased p450 1a1 mrna levels in breast cells . hop extract and 6-pn preferentially induced p450 1a1 mrna expression in mcf-10a and mcf-7 cells . ( a ) mcf-10a cells and ( b ) mcf-7 cells were treated with hop extract , and p450 1a1 and 1b1 mrna expression was analyzed after 24 h via qpcr . ( c ) mcf-10a and ( d ) mcf-7 cells were treated with 6-pn , 8-pn , ix , xh ( 1 m ) , and tcdd ( 10 nm ) for 24 h , and p450 1a1/1b1 mrna expression was analyzed via qpcr . p450 1a1/1b1 activity was measured using the erod assay in both cell lines after 2 days of treatment with hop extract and bioactive compounds . in mcf-7 cells , the hop extract gave significantly higher erod activity compared to mcf-10a cells to a maximum of 0.25 pmol / min / well resorufin ( figure 5b ) . with the hop compounds in mcf-10a cells , only 6-pn increased p450 1a1/1b1 activity dose - dependently to 0.05 pmol / min / well resorufin ( figure 5c ) . xh moderately induced the p450 1 activity even though estrogen metabolism and p450 1a1/1b1 gene expression were not affected ; 8-pn and ix did not show significant effects . in mcf-7 cells , significant induction of p450 1a1/1b1 activity was observed with 6-pn and 8-pn ( 3 m ) to 0.8 and 0.2 pmol / min / well resorufin , while ix and xh did not have significant effects ( figure 5d ) . overall , the results from the erod activity assay were consistent with the results from estrogen metabolism and p450 1a1/1b1 mrna analysis . qualitatively , the data from these two cell lines were also comparable and indicated that hops and 6-pn strongly induced p450 1a1/1b1 activity in breast cells . p450 1a1/1b1 activity was analyzed in mcf-10a cells after 2-day treatment of ( a ) hop extract and ( c ) different doses of 6-pn , 8-pn , ix , and xh with the erod assay . p450 1a1/1b1 activity was analyzed in mcf-7 cells after 2-day treatment of ( b ) hop extract and ( d ) different doses of 6-pn , 8-pn , ix , and xh with the erod assay . human hepatoma hepg2 cells and mcf-7 cells transfected with a xre - luciferase construct were used to measure the effect of bioactive compounds on ahr activation . of the four polyphenols , 6-pn and 8-pn significantly and dose - dependently increased xre - luciferase activity to around 6.5- and 10-fold of the control at 10 m , while xh and ix did not have significant effects ( figure 6a ) . in the presence of the ahr agonist tcdd ( 10 nm ) , a dose responsive decrease in xre luciferase activity was observed with 6-pn and 8-pn cotreatment ( figure 6b ) , which further suggested the interactions with ahr . in mcf-7 cells , 6-pn ( 5 m ) significantly induced the activity , while 8-pn ( 5 m ) had no effect , which correlated with the p450 1a1/1b1 activity and gene expression analysis . the results indicated some cell selectivity of 8-pn and also suggested 6-pn to be activating ahr in both breast and liver cell lines . finally , upon cotreatment of ahr antagonist ch223191 and 6-pn ( 1 m ) in mcf-7 cells , the erod activity was inhibited dose responsively ( figure 6d ) , which further confirmed 6-pn to be an ahr agonist . ( d ) p450 1a1/1b1 activity was measured via the erod assay in mcf-7 cells after cotreatment of 6-pn ( 1 m ) with ahr antagonist ch223191 ( 0.01 , 0.1 , 1 , and 10 m ) for 2 days . to study the inhibitory effects of hop compounds on p450 1a1/1b1 all four compounds acted as p450 1a1/1b1 inhibitors with ic50 values in the low micromolar range without selectivity for either p450 1a1 or 1b1 ( table 1 ) . these data indicated the metabolism results were mainly the result of induction of p450 1a1/1b1 enzyme levels and that little direct inhibition of p450s should be observed at clinical concentrations of hop supplements . several recent clinical trials analyzing serum estrogen metabolite levels and the risk of postmenopausal breast cancer further support the 2-hydroxylation pathway as a marker for chemoprevention ; however , the data are inconclusive on the relationship between the estrogen 4-hydroxylation and breast cancer risk . p450 1a1 and 1b1 are the major enzymes in breast tissues that are responsible for the local estrogen 2- and 4-hydroxylation metabolism , respectively . however , the ahr mediated p450 1a1/1b1 induction in mcf-10a cells is relatively low as reported previously and confirmed in the present study . showed that induction of p450 1a1/1b1 enzymes vary among different tumorigenic and nontumorigenic cell lines . since mcf-7 cells mirrored mcf-10a cells in the response to botanical treatment with higher induction levels , they serve as a better model to screen botanicals and compounds for the modulation of estrogen metabolism . in addition , the upstream p450 1a1/1b1 mrna induction trend among compounds ( 6-pn 8-pn and no effect with ix and xh ) corresponds with the metabolism and activity results in mcf-7 cells . in this study , hepg2 cells were used to compare the ahr activation by hop compounds because the xre - luciferase reporter activity in hepg2 cells were much higher compared to that in mcf-7 cells ( figure s7 ) . preferential induction of p450 1a1 over p450 1b1 has been documented in the literature . quercetin ( 10 m ) and berberine ( > 5 m ) preferentially induced p450 1a1 over 1b1 in mcf-10f and mcf-7 cells , respectively . induction of xre activity and inhibition of tcdd induced xre activity further supported 6-pn to be an ahr agonist ( figure 6 ) . naringenin , a flavanone with the same scaffold as the hop flavanones , does not induce p450 1a1/1b1 , while the effect of flavanones with a prenylated side chain has not been studied . the effect of hop compounds on tcdd - induced xre - luciferase reporter activity was also measured . the results indicated that 6-pn and 8-pn had comparable effects in inhibiting tcdd induced xre - luciferase activity at micromolar levels ( figure 6b ) . in contrast , we previously showed that licochalcone a ( 10 m ) , a b - ring ( c-5 ) prenylated chalcone from licorice ( glycyrrhiza inflata ) , was an ahr antagonist and shut down estrogen oxidative metabolism in mcf-10a cells . several ubiquitous , unsubstituted flavones and flavonols , such as quercetin , kaempferol , and apigenin , have been reported to be p450 1b1 inhibitors with ic50 values below 50 nm , while inhibiting p450 1a1 activity less potently . several hydroxychalcones have been tested for the inhibition of p450 1a1 and 1b1 and showed ic50 values in the low micromolar range . when compared to resveratrol , which inhibited p450 1a1 and 1b1 with ic50 values around 2 and 25 m , respectively the hop compounds have also been shown to inhibit p450 2c8 , 2c9 , and 19 with ic50 values in the low micromolar range . these data suggest that the effect on estrogen oxidative metabolism modulation was mainly contributed by the ahr agonist activity of 6-pn . in conclusion , results from this study provided novel in vitro evidence that hops and its compound 6-pn preferentially induced the nontoxic estrogen 2-hydroxylation pathway in two different breast cell lines , which indicated a potentially protective role of hops to help reduce the risk of breast cancer through estrogen metabolism modulation . since the pharmacokinetic properties of the hop compounds would significantly influence their effect in vivo , the preferential 2-hydroxylation induction as well as modulation of p450 1a1/1b1 enzymes would need to be tested with in vivo models that will be studied in the future . accordingly , for hop extracts , suitable target markers are 8-pn ( estrogenic ) , xh ( chemopreventive ) , and , as shown in the present study , 6-pn ( ahr agonist ) as modulators of estrogen metabolism .

approximately 273,000 individuals in the us are living with a spinal cord injury ( sci ) , and many are disabled ; 11.6% are employed 1 year postinjury and 35.2% are employed 20 years postinjury.1 pain is a frequent complication of sci that has been reported to develop in up to 96% of patients subsequent to their injury,2 and substantially adds to the patient burden by adversely impacting patients activities of daily living , quality of life , and social functioning.3,4 while post - sci pain can be categorized as nociceptive ( musculoskeletal , visceral , or other nociceptive ) , neuropathic ( at - level or below - level of injury ) , other pain , or unknown pain,5 neuropathic pain ( nep ) develops in about 50% of sci patients . this nep results from a lesion or disease of the somatosensory nervous system related to the injury6,7 and increases the complexity of sci management since nep is challenging to treat.8 recommendations for the pharmacologic management of sci - nep include antidepressants , antiepileptic drugs ( aeds ) , opioids , and intrathecal medications,911 and in the us , only pregabalin , an aed , has received approval by the us food and drug administration for the treatment of nep associated with sci.12 since complete pain reduction is seldom attained , the main goal of treatment is to reduce pain to a level considered acceptable by the patient . although studies have evaluated the treatment of pain following sci , recent data on the economic burden of sci , specifically for sci - associated nep , are limited . direct and indirect costs for hospitalization and rehabilitation in the us , including lifelong direct costs , have been measured following sci in some recent studies.1,1317 a 2007 study of us veteran health administration patients with a minimum duration of sci of 2 years showed average annual costs ranging from $ 17,561 to $ 28,334 , depending on level and completeness of sci.18 in the only study that estimated the direct medical costs among patients with sci - nep , overall annualized direct medical costs of $ 8,636 per patient were reported.19 that study , which recruited the population from community - based physician practices , stratified patients by self - reported pain severity and found that sci - nep costs increased with greater pain , with the highest costs among those with severe pain ( $ 11,666 ) . however , it is likely that not all sci survivors are covered by private health insurance at the time of injury . one option to fill this coverage gap is the us medicaid program , which provides financial assistance for medical and health - related services to more than 8 million disabled individuals.20 thus , the current retrospective , longitudinal study was undertaken to evaluate health care resource utilization ( hru ) and its associated costs , specifically among medicaid beneficiaries with nep secondary to sci . data for this analysis were derived from administrative medical and pharmacy claims in the truven health analytics marketscan multi - state medicaid database between january 1 , 2005 and june 30 , 2012 . this database includes complete longitudinal records of inpatient services , outpatient services , long - term care , nursing home , home health care , and prescription drug claims covered under medicaid programs in 12 geographically diverse states . all database records are de - identified and fully compliant with us patient confidentiality requirements , including the health insurance portability and accountability act ( hipaa ) of 1996 . the databases have been evaluated and certified by an independent third party to be in compliance with the conditions set forth in sections 164.514 ( a)(b)1ii of the hipaa privacy rule regarding the determination and documentation of statistically de - identified data . there was no interaction with any subjects , and the databases do not include any individually identifiable data ( eg , no names , addresses , social security or medical record numbers , or any other identifiers ) . from the available data , two cohorts were formed of patients who met the eligibility criteria for each cohort ; one cohort consisted of sci patients with associated nep ( sci - nep ) , and the second cohort consisted of sci patients without nep ( sci only ) . the sci - only cohort served as a control to help determine the contribution of nep to hru and costs during sci management . initial identification of patients with sci was based on an inpatient or outpatient medical claim for sci between january 1 , 2006 and june 30 , 2011 using icd-9-cm diagnosis codes of 344.0x ( quadriplegia and quadriparesis ) , 344.1x ( paraplegia ) , 344.6x ( cauda equina syndrome ) , 806.xx ( fracture of vertebral column with sci ) , or 952.xx ( sci without evidence of spinal bone injury ) in any position ; patients were required to be 18 years of age at the date of the first sci claim . a minimum of 6 months of continuous medical and pharmacy medicaid eligibility was required prior to the first sci claim with the sci diagnosis . from this population , the sci - nep cohort was identified by either a diagnosis of central nep ( icd-9-cm code 338.0x ) or a pharmacy claim for an nep - related aed or nep - related antidepressant drug within 12 months following first sci diagnosis . the use of nep - related medications as a proxy to identify nep was initiated because the central nep diagnostic code is rarely used in medical claims . the nep - related aeds were carbamazepine , gabapentin , lamotrigine , oxcarbazepine , phenytoin , pregabalin , topiramate , and valproic acid ; nep - related antidepressants included the serotonin - norepinephrine inhibitors ( snris ) duloxetine , venlafaxine , milnacipran , and desvenlafaxine and the tricyclic antidepressants ( tca ) amitriptyline , desipramine , doxepin , imipramine , and nortriptyline . medications for defining sci - nep were chosen based on a literature review and practitioner recommendations,6,811,21 and patients in this cohort could not have nep - related claims , including for nep - related aeds or antidepressants , during the 6 months before the first sci diagnosis . the date of the first nep diagnosis according to the above criteria was the sci - nep index date . all sci - nep patients were required to have continuous medicaid enrollment for 6 months preindex ( baseline ) and 12 months postindex . exclusion criteria included a diagnosis of any of the following conditions for which drugs prescribed for the treatment of sci - nep may also be used : epilepsy ( icd-9-cm codes 345.xx or 780.39 ) , amyotrophic lateral sclerosis ( icd-9-cm code 335.20 ) , multiple sclerosis ( icd-9-cm code 340.xx ) , diabetic peripheral neuropathy ( icd-9-cm codes 250.6x , 357.2x ) , or postherpetic neuralgia ( icd-9-cm code 053.1x ) . medical or pharmacy claims for medications used to treat multiple sclerosis ( intramuscular or subcutaneous interferon beta-1a , interferon beta-1b , glatiramer acetate , fingolimod ) or amyotrophic lateral sclerosis ( riluzole ) anytime during the study period were also cause for exclusion . patients included in the sci - only cohort were required to meet the same eligibility criteria as the sci - nep patients except without the nep diagnosis and to have no use of the nep - related medications ( aeds or antidepressants ) at any time during the study period . index dates for the sci - only cohort were assigned according to the date of their sci diagnosis and the number of days between their diagnosis and the index date for a randomly selected sci - nep patient . propensity score matching was conducted using a logistic regression model to predict the probability that a patient will develop nep.22 this model resulted in a propensity score based on preindex characteristics including age , sex , health plan type , index year , deyo - charlson comorbidity index ( dci ) score,23 number of three - digit icd-9-cm codes , number of unique drugs , specific type of sci diagnosis , presence of specific comorbid conditions , use of specific concomitant medications , and preindex all - cause health care expenditures . the purpose of matching the propensity score is to ensure similarity between the groups ( sci - nep and sci only ) with respect to observed covariates , so that observed differences in outcomes can be more confidently ascribed to the distinguishing factor ( nep ) ; therefore , the propensity score matching adjusts for the observed covariates in the model . matching from the sci pool was performed without replacement using an algorithm of one - to - one nearest neighbor matching.24 demographic and clinical characteristics were captured during the preindex baseline period for both cohorts . in addition to type of sci and specified comorbidities , clinical characteristics included the following comorbidity indexes : dci , number of unique three - digit icd-9-cm codes , and number of unique outpatient medications . clinical characteristics were also captured during the postindex period . to identify traumatic sci in the absence of a specific icd-9-cm code , the variable trauma - related sci was created as a proxy by searching the medical claims of each included patient for a diagnosis code of vertebral column fracture ( 806.xx ) or late effects of sci ( 907.2x ) at any time on or after the initial sci diagnosis.25 categories of hru and expenditures included inpatient admissions , emergency department ( ed ) visits , physician office visits , sci and pain - related procedures , and outpatient prescriptions . these resources were measured during the 12-month postindex period for sci - nep and sci - only patients . expenditures were determined using the gross covered payments on medical and pharmacy claims , ie , the amount eligible for payment to the provider before applying deductibles , copayments , or coordination of benefits . payments in all years were adjusted to 2012 us dollars using the medical care component of the bureau of labor statistics consumer price index.26 patient demographics , clinical characteristics , and 6-month preindex total health care expenditures were summarized descriptively . postindex use of concomitant medications , sci - related procedures , pain - related procedures , resource utilization , and costs were assessed using univariate and bivariate descriptive summaries . statistical significance , with a value of 0.05 specified a priori , between the sci - nep and sci - only cohorts was tested using chi - square or fisher s exact test for categorical variables and two - sample student s t - tests for quantitative variables . generalized linear models ( glms ) were used to further adjust the propensity score - matched results for effects of potential confounding or risk factors by relating health care resource and economic outcomes with patient demographic and clinical characteristics . in these models , binary outcomes ( ie , hospital admissions and ed visits ) were fitted with a glm using a binomial distribution and its canonical link , and count outcomes ( ie , number of hospital admissions or ed visits per patient ) were modeled using a poisson or negative binomial distribution ; when the number of events was zero for many individuals , the corresponding zero - inflated version was applied.27 cost data were modeled using a traditional glm , assuming an underlying gamma distribution and a log link , and an ordinary least squares model fit to the log transformed positive expenditures retransformed to predict costs , with bootstrapping for standard errors and confidence intervals.28 analyses were conducted using sas version 9.2 ( sas institute inc . , cary , nc , usa ) . data for this analysis were derived from administrative medical and pharmacy claims in the truven health analytics marketscan multi - state medicaid database between january 1 , 2005 and june 30 , 2012 . this database includes complete longitudinal records of inpatient services , outpatient services , long - term care , nursing home , home health care , and prescription drug claims covered under medicaid programs in 12 geographically diverse states . all database records are de - identified and fully compliant with us patient confidentiality requirements , including the health insurance portability and accountability act ( hipaa ) of 1996 . the databases have been evaluated and certified by an independent third party to be in compliance with the conditions set forth in sections 164.514 ( a)(b)1ii of the hipaa privacy rule regarding the determination and documentation of statistically de - identified data . there was no interaction with any subjects , and the databases do not include any individually identifiable data ( eg , no names , addresses , social security or medical record numbers , or any other identifiers ) . from the available data , two cohorts were formed of patients who met the eligibility criteria for each cohort ; one cohort consisted of sci patients with associated nep ( sci - nep ) , and the second cohort consisted of sci patients without nep ( sci only ) . the sci - only cohort served as a control to help determine the contribution of nep to hru and costs during sci management . initial identification of patients with sci was based on an inpatient or outpatient medical claim for sci between january 1 , 2006 and june 30 , 2011 using icd-9-cm diagnosis codes of 344.0x ( quadriplegia and quadriparesis ) , 344.1x ( paraplegia ) , 344.6x ( cauda equina syndrome ) , 806.xx ( fracture of vertebral column with sci ) , or 952.xx ( sci without evidence of spinal bone injury ) in any position ; patients were required to be 18 years of age at the date of the first sci claim . a minimum of 6 months of continuous medical and pharmacy medicaid eligibility was required prior to the first sci claim with the sci diagnosis . from this population , the sci - nep cohort was identified by either a diagnosis of central nep ( icd-9-cm code 338.0x ) or a pharmacy claim for an nep - related aed or nep - related antidepressant drug within 12 months following first sci diagnosis . the use of nep - related medications as a proxy to identify nep was initiated because the central nep diagnostic code is rarely used in medical claims . the nep - related aeds were carbamazepine , gabapentin , lamotrigine , oxcarbazepine , phenytoin , pregabalin , topiramate , and valproic acid ; nep - related antidepressants included the serotonin - norepinephrine inhibitors ( snris ) duloxetine , venlafaxine , milnacipran , and desvenlafaxine and the tricyclic antidepressants ( tca ) amitriptyline , desipramine , doxepin , imipramine , and nortriptyline . medications for defining sci - nep were chosen based on a literature review and practitioner recommendations,6,811,21 and patients in this cohort could not have nep - related claims , including for nep - related aeds or antidepressants , during the 6 months before the first sci diagnosis . the date of the first nep diagnosis according to the above criteria was the sci - nep index date . all sci - nep patients were required to have continuous medicaid enrollment for 6 months preindex ( baseline ) and 12 months postindex . exclusion criteria included a diagnosis of any of the following conditions for which drugs prescribed for the treatment of sci - nep may also be used : epilepsy ( icd-9-cm codes 345.xx or 780.39 ) , amyotrophic lateral sclerosis ( icd-9-cm code 335.20 ) , multiple sclerosis ( icd-9-cm code 340.xx ) , diabetic peripheral neuropathy ( icd-9-cm codes 250.6x , 357.2x ) , or postherpetic neuralgia ( icd-9-cm code 053.1x ) . medical or pharmacy claims for medications used to treat multiple sclerosis ( intramuscular or subcutaneous interferon beta-1a , interferon beta-1b , glatiramer acetate , fingolimod ) or amyotrophic lateral sclerosis ( riluzole ) anytime during the study period were also cause for exclusion . patients included in the sci - only cohort were required to meet the same eligibility criteria as the sci - nep patients except without the nep diagnosis and to have no use of the nep - related medications ( aeds or antidepressants ) at any time during the study period . index dates for the sci - only cohort were assigned according to the date of their sci diagnosis and the number of days between their diagnosis and the index date for a randomly selected sci - nep patient . propensity score matching was conducted using a logistic regression model to predict the probability that a patient will develop nep.22 this model resulted in a propensity score based on preindex characteristics including age , sex , health plan type , index year , deyo - charlson comorbidity index ( dci ) score,23 number of three - digit icd-9-cm codes , number of unique drugs , specific type of sci diagnosis , presence of specific comorbid conditions , use of specific concomitant medications , and preindex all - cause health care expenditures . the purpose of matching the propensity score is to ensure similarity between the groups ( sci - nep and sci only ) with respect to observed covariates , so that observed differences in outcomes can be more confidently ascribed to the distinguishing factor ( nep ) ; therefore , the propensity score matching adjusts for the observed covariates in the model . matching from the sci pool was performed without replacement using an algorithm of one - to - one nearest neighbor matching.24 in addition to type of sci and specified comorbidities , clinical characteristics included the following comorbidity indexes : dci , number of unique three - digit icd-9-cm codes , and number of unique outpatient medications . clinical characteristics were also captured during the postindex period . to identify traumatic sci in the absence of a specific icd-9-cm code , the variable trauma - related sci was created as a proxy by searching the medical claims of each included patient for a diagnosis code of vertebral column fracture ( 806.xx ) or late effects of sci ( 907.2x ) at any time on or after the initial sci diagnosis.25 categories of hru and expenditures included inpatient admissions , emergency department ( ed ) visits , physician office visits , sci and pain - related procedures , and outpatient prescriptions . these resources were measured during the 12-month postindex period for sci - nep and sci - only patients . expenditures were determined using the gross covered payments on medical and pharmacy claims , ie , the amount eligible for payment to the provider before applying deductibles , copayments , or coordination of benefits . payments in all years were adjusted to 2012 us dollars using the medical care component of the bureau of labor statistics consumer price index.26 patient demographics , clinical characteristics , and 6-month preindex total health care expenditures were summarized descriptively . postindex use of concomitant medications , sci - related procedures , pain - related procedures , resource utilization , and costs were assessed using univariate and bivariate descriptive summaries . statistical significance , with a value of 0.05 specified a priori , between the sci - nep and sci - only cohorts was tested using chi - square or fisher s exact test for categorical variables and two - sample student s t - tests for quantitative variables . generalized linear models ( glms ) were used to further adjust the propensity score - matched results for effects of potential confounding or risk factors by relating health care resource and economic outcomes with patient demographic and clinical characteristics . in these models , binary outcomes ( ie , hospital admissions and ed visits ) were fitted with a glm using a binomial distribution and its canonical link , and count outcomes ( ie , number of hospital admissions or ed visits per patient ) were modeled using a poisson or negative binomial distribution ; when the number of events was zero for many individuals , the corresponding zero - inflated version was applied.27 cost data were modeled using a traditional glm , assuming an underlying gamma distribution and a log link , and an ordinary least squares model fit to the log transformed positive expenditures retransformed to predict costs , with bootstrapping for standard errors and confidence intervals.28 analyses were conducted using sas version 9.2 ( sas institute inc . , cary , nc , usa ) . the sci - nep and sci - only cohorts each consisted of 546 patients who met all eligibility criteria ; incremental attrition is shown in table 1 . initiation of nep - related aeds ( n=351 ; 64.3% ) was the most common index event , followed by nep - related tcas ( n=115 ; 21.1% ) , nep - related snris ( n=76 ; 13.9% ) , and four patients ( 0.7% ) with central nep diagnosis . of the 351 aed index events , most were with gabapentin ( n=262 ; 74.6% ) , followed by pregabalin ( n=53 ; 15.1% ) and topiramate ( n=17 ; 4.8% ) . index events with tcas were primarily with amitriptyline ( 83 of 115 ; 72.2% ) while initiation of nep - related snris was primarily with duloxetine and venlafaxine , 57.9% and 34.2% of the snri index events , respectively . as shown in table 2 , the sci - nep cohort was primarily female ( 56.0% ) and white ( 58.6% ) , with an average age of 40.1 years ( standard deviation [ sd ] 13.8 ) , and the sci - only cohort was also primarily female ( 54.8% ) and white ( 58.6% ) , with an average age of 41.5 ( sd 16.0 ) years . both cohorts were similarly comprised of predominantly urban populations ( sci - nep 70.3% , sci only 74.4% ; p=0.137 ) , and small but significant differences were observed for the types of insurance plans ( table 2 ) . although the proportion of patients with each type of sci was similar between cohorts , trauma - related sci diagnosis codes were significantly more common in the sci - nep cohort ( 30.4% versus 22.3% ; p=0.003 ) ( table 3 ) . both cohorts were characterized by a substantial comorbidity burden at baseline , as indicated by the mean values for the comorbidity indexes and the prevalence of specific conditions . musculoskeletal pain was present in half of both cohorts and gastrointestinal conditions in approximately one - third . except for long - acting opioids , which were used by a significantly higher proportion of sci - nep patients during the preindex period ( 11.4% versus 7.7% ; p=0.039 ) , preindex medication utilization was not significantly different between the cohorts ( table 3 ) . however , spine decompression and stabilization procedures were significantly higher preindex in the sci - nep cohort relative to sci only , 12.8% versus 7.0% ( p=0.001 ) and 10.4% versus 6.2% ( p=0.012 ) , respectively . relative to the preindex period , there was greater use of all pain - related medication classes during the 12-month follow - up period in the sci - nep cohort , with short - acting opioids most frequently reported ( 86.8% ) , followed by anxiolytics ( 58.6% ) ; preindex use of these medications was 68.5% and 34.8% , respectively . also in contrast to the preindex period , medication utilization during follow - up was significantly higher in the sci - nep cohort relative to sci only for every medication category ( all p<0.01 ; figure 1 ) . the difference in proportion was greatest for muscle relaxants ( 58.6% versus 29.7% ) , short - acting opioids ( 86.8% versus 61.9% ) , and anxiolytics ( 54.0% versus 31.0% ) while the magnitude of the difference was highest for topical analgesics ( 9.2% versus 2.9% ) , long - acting analgesics ( 22.5% versus 8.6% ) , and sedative / hypnotics ( 34.1% versus 15.0% ) . in the sci - nep cohort , during the 12-month follow - up period , the most commonly used resource was outpatient medications , for which all patients had a claim , followed by physician office visits ( 93.4% ) , and while almost two - thirds ( 62.6% ) of this cohort had ed visits , only 34.6% had inpatient admissions ( table 4 ) . as also shown in table 4 , hru among the sci - nep patients was significantly higher relative to sci - only patients for the proportion with ed visits , physician office visits , sci- and pain - related procedures , and outpatient drug claims . among patients with outpatient drug claims , medication utilization was significantly higher for sci - nep , with 67.6 ( sd 54.2 ) claims per patient compared with 50.1 ( sd 51.5 ) claims per patient for sci only ( p<0.001 ) . there were no differences between the cohorts for inpatient admissions or number of admissions per patient admitted , but there was a trend toward significance for the longer length of hospital stay that was observed in the sci - nep cohort , 7.5 ( sd 28.7 ) days versus 4.7 ( sd 16.1 ) days ( p=0.050 ) . further adjustment of the covariates confirmed the lack of a difference between cohorts for hospitalizations , and further , found no difference in the rate of ed visits , even though the difference between the matched cohorts for the rate of ed visits was statistically significant . as shown in table 5 , the mean ( sd ) all - cause total expenditures for sci - nep patients of $ 37,333 ( $ 64,334 ) were higher than the $ 31,186 ( $ 52,252 ) for sci - only patients , but the difference was not significant ( p<0.083 ) ; median costs were $ 15,411 and $ 11,477 , respectively . however , significantly higher expenditures were observed for several hru categories including physician office visits , sci- and pain - related outpatient services , and outpatient medications . the highest mean expenditures were for inpatient admissions ( sci - nep , $ 18,122 [ $ 55,828 ] ; sci only , $ 6,575 [ $ 28,438 ] ; p=0.140 ) . sci- and pain - related outpatient visits and procedures averaged $ 3,543 ( $ 9,829 ) per sci - nep patient and $ 1,702 ( $ 5,703 ) per sci - only patient ( p=0.010 ) , and for outpatient prescriptions , the values were $ 4,433 ( $ 6,248 ) per sci - nep patient and $ 3,276 ( $ 6,357 ) per sci - only patient ( p=0.002 ) . outpatient prescriptions accounted for 11.9% of total expenditures for sci - nep and 10.5% for sci only . mean all - cause expenditures estimated using the ordinary least squares model to further adjust for covariates were $ 47,518 for sci - nep and $ 30,150 for sci only , resulting in a significant incremental economic burden of $ 17,369 ( 95% confidence interval $ 9,753 to $ 25,555 ; p<0.0001 ) for sci - nep . factors significantly associated with increased costs included preindex expenditures ( p=0.005 ) , quadriplegia ( p<0.001 ) , paraplegia ( p<0.001 ) , cauda equina syndrome ( p=0.032 ) , trauma - related sci ( p=0.026 ) , dci score ( p=0.004 ) , and number of preindex outpatient drugs ( p<0.001 ) . in contrast , factors associated with lower all - cause expenditures were health maintenance organization health plans ( p<0.001 ) , preindex nonsteroidal anti - inflammatory drug ( nsaid ) use ( p=0.036 ) , and spine decompression ( p=0.036 ) . in addressing the need for characterizing the economic burden of sci - nep , a previous burden of illness study estimated direct medical costs of $ 8,636 among patients with this condition.19 however , the estimated costs in that study , which was performed in the general population , were derived from survey questions specific to sci - nep . consequently , the overall economic burden may be substantially underestimated , at least for the medicaid population , relative to the all - cause expenditures of $ 47,518 reported here , and the incremental difference of $ 17,368 compared with sci patients without nep . these excess expenditures resulted from greater hru and associated costs among the sci - nep patients during the 12-month follow - up period , and were observed despite the matching at baseline for demographic and clinical characteristics . while higher hru and costs with sci - nep may not be surprising , this is the first study to describe such an economic burden in a medicaid population . sci generally results in an array of medical and psychological issues , including depression , anxiety , muscle spasms , gastrointestinal , genitourinary , and other visceral problems , as were found in the preindex comorbidities and concomitant medications of both the sci - nep and sci - only cohorts ( table 3).3,21,29,30 while these study populations were well matched for preindex comorbid burden , especially notable was the postindex medication burden , with significantly higher prescription medication utilization overall in the sci - nep cohort , particularly for medications used in pain management such as long- and short - acting opioids , anxiolytics , nsaids , and topical analgesics . berger et al31 had raised a concern in their 2004 study of painful neuropathic disorders that patients with high comorbid burdens typically receiving pain - related opioids and nsaids may not be receiving optimal treatment . the increased use of these medications by sci - nep patients , as well as the other medication classes such as muscle relaxants , sedatives , non - nep - related antidepressants and systemic steroids , provides evidence of the need for a more encompassing approach to treatment by recognizing the multifactorial nature of pain following sci . importantly , substance abuse was found preindex in more than one - quarter of each cohort , 28.2% of sci - nep and 26.7% of sci - only patients . these proportions are higher than the 7.6% reported among the 5.0 million patients in the entire marketscan multi - state medicaid database using claims from july 2007 through june 2012 . the particularly high rate of substance abuse across both sci cohorts supports the need in these patients for a multimodal approach that includes greater use of nonopioid pharmacotherapy . the adjusted economic burden of sci - nep during the year following nep diagnosis ( $ 47,518 ) was higher than that of sci - only patients ( $ 30,149 ) , representing a significant incremental burden of $ 17,369 . in both cohorts , the primary driver of total expenditures was inpatient admissions while the main driver of outpatient costs was outpatient prescriptions . these values also evince the generally high cost of sci , with and without nep , as they are both higher than the $ 15,749 per person that was reported as the average annual payment to the 8.8 million disabled medicaid recipients ( representing 19% of all medicaid beneficiaries ) according to the 2011 centers for medicare & medicaid services supplement , and higher than the mean cost of $ 21,450 ( range $ 17,561 to $ 28,334 depending on level and completeness of sci ) in a 2007 veterans health administration study of sci.18,20 a strength of this study is its use of propensity score matching , which resulted in cohorts with very few differences preindex . this matching increased the likelihood that the differences in hru and costs observed during follow - up are due to the onset of nep , the distinguishing difference between the cohorts at the index event . the results were further confirmed using statistical modeling to provide an additional level of robustness by accounting for covariates that may have had effects beyond their impact on the propensity score . nevertheless , results of this study should be interpreted within the context of its limitations . because absolute causality can not be determined , including any inferences on the effects of nep and comorbidities on resource utilization and expenditures , the observed relationships should be considered associative . moreover , the similarity between groups was based on observed covariates that were considered potential confounders or risk factors and that were incorporated into the models . however , it is also possible that there were covariates that were not observed and therefore not incorporated into the models . in terms of potential measurement misclassification , the use of claims is also associated with the potential for misclassifying , undercoding , or overcoding the diagnoses of interest . the use of aeds and antidepressants as proxies to identify nep represents another limitation , and despite excluding conditions for which these medications are approved for use , it is still possible that prescription of these medications after sci diagnosis may not have been related to nep . however , it is important to note that the need for such a proxy was due to a virtual lack of use of icd-9-cm code 338.0x , and use of these drugs during the pre - sci period was an exclusionary criterion to reduce the likelihood that they were prescribed for reasons other than nep . while excluding patients with depression would have reduced this likelihood , this was not done since it would have further risked biasing the study populations . additionally , the data did not specify whether nep was classified as at - level or below - level , which may be relevant from the clinical perspective since different treatment modalities may be used if the pain is at - level versus below - level.6,8 similarly , the nep itself may not have been related to sci . however , this study required both that the sci diagnosis precedes the first nep diagnosis , and that there was no evidence of nep within the 6-month period prior to sci . these requirements are supported by previous studies showing that of 64% of patients reporting pain at 6 months and 81% within 12 months , 60% reported nep.32,33 while this study required an sci diagnosis within 12 months prior to the first nep diagnosis , the original sci injury date as well as other diagnoses or procedures associated with sci may have occurred prior to our period of observation . prescription claims data can not be linked with specific diagnoses , and therefore our definition of nep - related medications may be over or underrepresented . finally , this analysis focused on a sample of us medicaid beneficiaries , the results of which may not be generalizable to the overall us medicaid population . this study suggests the magnitude of the economic burden associated with nep in patients with sci in a medicaid population , with estimated annual expenditures of $ 47,518 per patient during the year following nep onset . these expenditures result from use of a variety of health care resources in high proportions of patients , and the overall burden with regard to both hru and costs is significantly greater than among sci patients without nep . future research is warranted to explore differences between sci patients who develop nep and those who do not to garner further evidence for developing an effective multifactorial approach to treating patients with sci - associated nep .

backgroundthe study aimed to evaluate health care resource utilization ( hru ) and costs for neuropathic pain ( nep ) secondary to spinal cord injury ( sci ) among medicaid beneficiaries.methodsthe retrospective longitudinal cohort study used medicaid beneficiary claims with sci and evidence of nep ( sci - nep cohort ) matched with a cohort without nep ( sci - only cohort ) . patients had continuous medicaid eligibility 6 months pre- and 12 months postindex , defined by either a diagnosis of central nep ( icd-9-cm code 338.0x ) or a pharmacy claim for an nep - related antiepileptic or antidepressant drug within 12 months following first sci diagnosis . demographics , clinical characteristics , hru , and expenditures were compared between cohorts.resultspropensity score - matched cohorts each consisted of 546 patients . postindex percentages of patients with physician office visits , emergency department visits , sci- and pain - related procedures , and outpatient prescription utilization were all significantly higher for sci - nep ( p<0.001 ) . using regression models to account for covariates , adjusted mean expenditures were us$47,518 for sci - nep and us$30,150 for sci only , yielding incremental costs of us$17,369 ( 95% confidence interval us$9,753 to us$26,555 ) for sci - nep . factors significantly associated with increased cost included sci type , trauma - related sci , and comorbidity burden.conclusionsignificantly higher hru and total costs were incurred by medicaid patients with nep secondary to sci compared with matched sci - only patients .

Introduction Materials and methods Data source Subject selection and study period Outcome measures Statistical analysis Results Discussion Conclusion

approximately 273,000 individuals in the us are living with a spinal cord injury ( sci ) , and many are disabled ; 11.6% are employed 1 year postinjury and 35.2% are employed 20 years postinjury.1 pain is a frequent complication of sci that has been reported to develop in up to 96% of patients subsequent to their injury,2 and substantially adds to the patient burden by adversely impacting patients activities of daily living , quality of life , and social functioning.3,4 while post - sci pain can be categorized as nociceptive ( musculoskeletal , visceral , or other nociceptive ) , neuropathic ( at - level or below - level of injury ) , other pain , or unknown pain,5 neuropathic pain ( nep ) develops in about 50% of sci patients . this nep results from a lesion or disease of the somatosensory nervous system related to the injury6,7 and increases the complexity of sci management since nep is challenging to treat.8 recommendations for the pharmacologic management of sci - nep include antidepressants , antiepileptic drugs ( aeds ) , opioids , and intrathecal medications,911 and in the us , only pregabalin , an aed , has received approval by the us food and drug administration for the treatment of nep associated with sci.12 since complete pain reduction is seldom attained , the main goal of treatment is to reduce pain to a level considered acceptable by the patient . direct and indirect costs for hospitalization and rehabilitation in the us , including lifelong direct costs , have been measured following sci in some recent studies.1,1317 a 2007 study of us veteran health administration patients with a minimum duration of sci of 2 years showed average annual costs ranging from $ 17,561 to $ 28,334 , depending on level and completeness of sci.18 in the only study that estimated the direct medical costs among patients with sci - nep , overall annualized direct medical costs of $ 8,636 per patient were reported.19 that study , which recruited the population from community - based physician practices , stratified patients by self - reported pain severity and found that sci - nep costs increased with greater pain , with the highest costs among those with severe pain ( $ 11,666 ) . one option to fill this coverage gap is the us medicaid program , which provides financial assistance for medical and health - related services to more than 8 million disabled individuals.20 thus , the current retrospective , longitudinal study was undertaken to evaluate health care resource utilization ( hru ) and its associated costs , specifically among medicaid beneficiaries with nep secondary to sci . from the available data , two cohorts were formed of patients who met the eligibility criteria for each cohort ; one cohort consisted of sci patients with associated nep ( sci - nep ) , and the second cohort consisted of sci patients without nep ( sci only ) . the sci - only cohort served as a control to help determine the contribution of nep to hru and costs during sci management . initial identification of patients with sci was based on an inpatient or outpatient medical claim for sci between january 1 , 2006 and june 30 , 2011 using icd-9-cm diagnosis codes of 344.0x ( quadriplegia and quadriparesis ) , 344.1x ( paraplegia ) , 344.6x ( cauda equina syndrome ) , 806.xx ( fracture of vertebral column with sci ) , or 952.xx ( sci without evidence of spinal bone injury ) in any position ; patients were required to be 18 years of age at the date of the first sci claim . from this population , the sci - nep cohort was identified by either a diagnosis of central nep ( icd-9-cm code 338.0x ) or a pharmacy claim for an nep - related aed or nep - related antidepressant drug within 12 months following first sci diagnosis . medications for defining sci - nep were chosen based on a literature review and practitioner recommendations,6,811,21 and patients in this cohort could not have nep - related claims , including for nep - related aeds or antidepressants , during the 6 months before the first sci diagnosis . all sci - nep patients were required to have continuous medicaid enrollment for 6 months preindex ( baseline ) and 12 months postindex . exclusion criteria included a diagnosis of any of the following conditions for which drugs prescribed for the treatment of sci - nep may also be used : epilepsy ( icd-9-cm codes 345.xx or 780.39 ) , amyotrophic lateral sclerosis ( icd-9-cm code 335.20 ) , multiple sclerosis ( icd-9-cm code 340.xx ) , diabetic peripheral neuropathy ( icd-9-cm codes 250.6x , 357.2x ) , or postherpetic neuralgia ( icd-9-cm code 053.1x ) . patients included in the sci - only cohort were required to meet the same eligibility criteria as the sci - nep patients except without the nep diagnosis and to have no use of the nep - related medications ( aeds or antidepressants ) at any time during the study period . index dates for the sci - only cohort were assigned according to the date of their sci diagnosis and the number of days between their diagnosis and the index date for a randomly selected sci - nep patient . the purpose of matching the propensity score is to ensure similarity between the groups ( sci - nep and sci only ) with respect to observed covariates , so that observed differences in outcomes can be more confidently ascribed to the distinguishing factor ( nep ) ; therefore , the propensity score matching adjusts for the observed covariates in the model . to identify traumatic sci in the absence of a specific icd-9-cm code , the variable trauma - related sci was created as a proxy by searching the medical claims of each included patient for a diagnosis code of vertebral column fracture ( 806.xx ) or late effects of sci ( 907.2x ) at any time on or after the initial sci diagnosis.25 categories of hru and expenditures included inpatient admissions , emergency department ( ed ) visits , physician office visits , sci and pain - related procedures , and outpatient prescriptions . these resources were measured during the 12-month postindex period for sci - nep and sci - only patients . payments in all years were adjusted to 2012 us dollars using the medical care component of the bureau of labor statistics consumer price index.26 patient demographics , clinical characteristics , and 6-month preindex total health care expenditures were summarized descriptively . postindex use of concomitant medications , sci - related procedures , pain - related procedures , resource utilization , and costs were assessed using univariate and bivariate descriptive summaries . statistical significance , with a value of 0.05 specified a priori , between the sci - nep and sci - only cohorts was tested using chi - square or fisher s exact test for categorical variables and two - sample student s t - tests for quantitative variables . from the available data , two cohorts were formed of patients who met the eligibility criteria for each cohort ; one cohort consisted of sci patients with associated nep ( sci - nep ) , and the second cohort consisted of sci patients without nep ( sci only ) . the sci - only cohort served as a control to help determine the contribution of nep to hru and costs during sci management . initial identification of patients with sci was based on an inpatient or outpatient medical claim for sci between january 1 , 2006 and june 30 , 2011 using icd-9-cm diagnosis codes of 344.0x ( quadriplegia and quadriparesis ) , 344.1x ( paraplegia ) , 344.6x ( cauda equina syndrome ) , 806.xx ( fracture of vertebral column with sci ) , or 952.xx ( sci without evidence of spinal bone injury ) in any position ; patients were required to be 18 years of age at the date of the first sci claim . from this population , the sci - nep cohort was identified by either a diagnosis of central nep ( icd-9-cm code 338.0x ) or a pharmacy claim for an nep - related aed or nep - related antidepressant drug within 12 months following first sci diagnosis . medications for defining sci - nep were chosen based on a literature review and practitioner recommendations,6,811,21 and patients in this cohort could not have nep - related claims , including for nep - related aeds or antidepressants , during the 6 months before the first sci diagnosis . all sci - nep patients were required to have continuous medicaid enrollment for 6 months preindex ( baseline ) and 12 months postindex . exclusion criteria included a diagnosis of any of the following conditions for which drugs prescribed for the treatment of sci - nep may also be used : epilepsy ( icd-9-cm codes 345.xx or 780.39 ) , amyotrophic lateral sclerosis ( icd-9-cm code 335.20 ) , multiple sclerosis ( icd-9-cm code 340.xx ) , diabetic peripheral neuropathy ( icd-9-cm codes 250.6x , 357.2x ) , or postherpetic neuralgia ( icd-9-cm code 053.1x ) . patients included in the sci - only cohort were required to meet the same eligibility criteria as the sci - nep patients except without the nep diagnosis and to have no use of the nep - related medications ( aeds or antidepressants ) at any time during the study period . index dates for the sci - only cohort were assigned according to the date of their sci diagnosis and the number of days between their diagnosis and the index date for a randomly selected sci - nep patient . the purpose of matching the propensity score is to ensure similarity between the groups ( sci - nep and sci only ) with respect to observed covariates , so that observed differences in outcomes can be more confidently ascribed to the distinguishing factor ( nep ) ; therefore , the propensity score matching adjusts for the observed covariates in the model . to identify traumatic sci in the absence of a specific icd-9-cm code , the variable trauma - related sci was created as a proxy by searching the medical claims of each included patient for a diagnosis code of vertebral column fracture ( 806.xx ) or late effects of sci ( 907.2x ) at any time on or after the initial sci diagnosis.25 categories of hru and expenditures included inpatient admissions , emergency department ( ed ) visits , physician office visits , sci and pain - related procedures , and outpatient prescriptions . these resources were measured during the 12-month postindex period for sci - nep and sci - only patients . payments in all years were adjusted to 2012 us dollars using the medical care component of the bureau of labor statistics consumer price index.26 patient demographics , clinical characteristics , and 6-month preindex total health care expenditures were summarized descriptively . postindex use of concomitant medications , sci - related procedures , pain - related procedures , resource utilization , and costs were assessed using univariate and bivariate descriptive summaries . statistical significance , with a value of 0.05 specified a priori , between the sci - nep and sci - only cohorts was tested using chi - square or fisher s exact test for categorical variables and two - sample student s t - tests for quantitative variables . generalized linear models ( glms ) were used to further adjust the propensity score - matched results for effects of potential confounding or risk factors by relating health care resource and economic outcomes with patient demographic and clinical characteristics . the sci - nep and sci - only cohorts each consisted of 546 patients who met all eligibility criteria ; incremental attrition is shown in table 1 . initiation of nep - related aeds ( n=351 ; 64.3% ) was the most common index event , followed by nep - related tcas ( n=115 ; 21.1% ) , nep - related snris ( n=76 ; 13.9% ) , and four patients ( 0.7% ) with central nep diagnosis . as shown in table 2 , the sci - nep cohort was primarily female ( 56.0% ) and white ( 58.6% ) , with an average age of 40.1 years ( standard deviation [ sd ] 13.8 ) , and the sci - only cohort was also primarily female ( 54.8% ) and white ( 58.6% ) , with an average age of 41.5 ( sd 16.0 ) years . both cohorts were similarly comprised of predominantly urban populations ( sci - nep 70.3% , sci only 74.4% ; p=0.137 ) , and small but significant differences were observed for the types of insurance plans ( table 2 ) . although the proportion of patients with each type of sci was similar between cohorts , trauma - related sci diagnosis codes were significantly more common in the sci - nep cohort ( 30.4% versus 22.3% ; p=0.003 ) ( table 3 ) . however , spine decompression and stabilization procedures were significantly higher preindex in the sci - nep cohort relative to sci only , 12.8% versus 7.0% ( p=0.001 ) and 10.4% versus 6.2% ( p=0.012 ) , respectively . also in contrast to the preindex period , medication utilization during follow - up was significantly higher in the sci - nep cohort relative to sci only for every medication category ( all p<0.01 ; figure 1 ) . in the sci - nep cohort , during the 12-month follow - up period , the most commonly used resource was outpatient medications , for which all patients had a claim , followed by physician office visits ( 93.4% ) , and while almost two - thirds ( 62.6% ) of this cohort had ed visits , only 34.6% had inpatient admissions ( table 4 ) . as also shown in table 4 , hru among the sci - nep patients was significantly higher relative to sci - only patients for the proportion with ed visits , physician office visits , sci- and pain - related procedures , and outpatient drug claims . among patients with outpatient drug claims , medication utilization was significantly higher for sci - nep , with 67.6 ( sd 54.2 ) claims per patient compared with 50.1 ( sd 51.5 ) claims per patient for sci only ( p<0.001 ) . as shown in table 5 , the mean ( sd ) all - cause total expenditures for sci - nep patients of $ 37,333 ( $ 64,334 ) were higher than the $ 31,186 ( $ 52,252 ) for sci - only patients , but the difference was not significant ( p<0.083 ) ; median costs were $ 15,411 and $ 11,477 , respectively . however , significantly higher expenditures were observed for several hru categories including physician office visits , sci- and pain - related outpatient services , and outpatient medications . the highest mean expenditures were for inpatient admissions ( sci - nep , $ 18,122 [ $ 55,828 ] ; sci only , $ 6,575 [ $ 28,438 ] ; p=0.140 ) . sci- and pain - related outpatient visits and procedures averaged $ 3,543 ( $ 9,829 ) per sci - nep patient and $ 1,702 ( $ 5,703 ) per sci - only patient ( p=0.010 ) , and for outpatient prescriptions , the values were $ 4,433 ( $ 6,248 ) per sci - nep patient and $ 3,276 ( $ 6,357 ) per sci - only patient ( p=0.002 ) . mean all - cause expenditures estimated using the ordinary least squares model to further adjust for covariates were $ 47,518 for sci - nep and $ 30,150 for sci only , resulting in a significant incremental economic burden of $ 17,369 ( 95% confidence interval $ 9,753 to $ 25,555 ; p<0.0001 ) for sci - nep . factors significantly associated with increased costs included preindex expenditures ( p=0.005 ) , quadriplegia ( p<0.001 ) , paraplegia ( p<0.001 ) , cauda equina syndrome ( p=0.032 ) , trauma - related sci ( p=0.026 ) , dci score ( p=0.004 ) , and number of preindex outpatient drugs ( p<0.001 ) . in contrast , factors associated with lower all - cause expenditures were health maintenance organization health plans ( p<0.001 ) , preindex nonsteroidal anti - inflammatory drug ( nsaid ) use ( p=0.036 ) , and spine decompression ( p=0.036 ) . in addressing the need for characterizing the economic burden of sci - nep , a previous burden of illness study estimated direct medical costs of $ 8,636 among patients with this condition.19 however , the estimated costs in that study , which was performed in the general population , were derived from survey questions specific to sci - nep . these excess expenditures resulted from greater hru and associated costs among the sci - nep patients during the 12-month follow - up period , and were observed despite the matching at baseline for demographic and clinical characteristics . while higher hru and costs with sci - nep may not be surprising , this is the first study to describe such an economic burden in a medicaid population . sci generally results in an array of medical and psychological issues , including depression , anxiety , muscle spasms , gastrointestinal , genitourinary , and other visceral problems , as were found in the preindex comorbidities and concomitant medications of both the sci - nep and sci - only cohorts ( table 3).3,21,29,30 while these study populations were well matched for preindex comorbid burden , especially notable was the postindex medication burden , with significantly higher prescription medication utilization overall in the sci - nep cohort , particularly for medications used in pain management such as long- and short - acting opioids , anxiolytics , nsaids , and topical analgesics . the increased use of these medications by sci - nep patients , as well as the other medication classes such as muscle relaxants , sedatives , non - nep - related antidepressants and systemic steroids , provides evidence of the need for a more encompassing approach to treatment by recognizing the multifactorial nature of pain following sci . importantly , substance abuse was found preindex in more than one - quarter of each cohort , 28.2% of sci - nep and 26.7% of sci - only patients . these values also evince the generally high cost of sci , with and without nep , as they are both higher than the $ 15,749 per person that was reported as the average annual payment to the 8.8 million disabled medicaid recipients ( representing 19% of all medicaid beneficiaries ) according to the 2011 centers for medicare & medicaid services supplement , and higher than the mean cost of $ 21,450 ( range $ 17,561 to $ 28,334 depending on level and completeness of sci ) in a 2007 veterans health administration study of sci.18,20 a strength of this study is its use of propensity score matching , which resulted in cohorts with very few differences preindex . however , this study required both that the sci diagnosis precedes the first nep diagnosis , and that there was no evidence of nep within the 6-month period prior to sci . these requirements are supported by previous studies showing that of 64% of patients reporting pain at 6 months and 81% within 12 months , 60% reported nep.32,33 while this study required an sci diagnosis within 12 months prior to the first nep diagnosis , the original sci injury date as well as other diagnoses or procedures associated with sci may have occurred prior to our period of observation . these expenditures result from use of a variety of health care resources in high proportions of patients , and the overall burden with regard to both hru and costs is significantly greater than among sci patients without nep .

the estimated prevalence has been reported from 16.8% to 58%.15 frequent drooling may cause skin maceration and infection , body fluid loss , and recurrent pneumonia.6,7 at school and at home , children with salivary secretions may cause damage to books , teaching materials and furniture , and it even interferes with social relationships.6,8,9 van der burg et al8,10 reported that children with cp that drool are often avoided by other children , and familiar and unfamiliar adults ( including their parents ) . hockstein et al9 reported that drooling in children with cp could interfere with their education and increase their dependent level of care . such studies suggest that drooling might be associated with a reduced quality of life among children with cp . previous studies have shown that there are many factors that interfere with the health - related quality of life ( hrqol ) in children with cp including : motor , cognitive , language , and social impairment.1114 although the prevalence of drooling is high among children with cp , there are few articles regarding its relationship to the hrqol in these children . most prior studies8,10,15 have used modified questionnaires or qualitative methods to evaluate the relationship between drooling and hrqol . in this study , standardized measurement of the hrqol in children with cp , with and without drooling , was investigated . in addition , the relationship between drooling and hrqol was evaluated , as well as the factors that predict the variability of hrqol in these children . children with cp that attended one medical center hospital and two early intervention institutions at daytime for an early intervention and habilitation program in taiwan were enrolled . children with cp , aged 2 to 6 years , without drooling ( with a drooling ranking score of 2 according to the drooling rating scale developed by thomas - stonell and greenberg16 ) , were the control group . children with cp , aged 2 to 6 years , with drooling ( a drooling ranking score > 2 ) , were the study group . the exclusion criteria were : ( 1 ) children with cp combined with other problems such as congenital malformation or metabolic disorder , ( 2 ) children taking anticholinergic drugs over the past 2 months , and ( 3 ) children with an acute infection or other systemic disease . children with cp were enrolled consecutively from february 2011 to july 2011 . the study was conducted according to the criteria of the declaration of helsinki and the review board of the university hospital approved this study . forty - seven children were included in the study : 14 did not drool ( mean age : 43.2 13.8 months , diplegia 17% , quadriplegia 12.8% ) and 33 did drool ( mean age : 48.9 14.4 months , diplegia 10.6% , quadriplegia 59.6% ) . in the group without drooling , there were eight boys and six girls , and in the group with drooling , there were 22 boys and 11 girls ( table 1 ) . previous studies4,17 have reported that drooling in infancy usually resolves by 18 months of age . the children that drool beyond 4 years of age are considered abnormal and require further treatment . therefore , the participants were divided into groups ; those less than 4 years old ( > 2 years and < 4 years , as the age group where drooling was supposed to stop and conservative management of drooling was acceptable ) and those older than 4 years of age ( 4 years , as the age group where drooling was abnormal and required further treatment ) . the ranking of drooling was evaluated using the drooling rating scale as described by thomas - stonell and greenberg.16 the drooling rating scale was rated using two subscales : ( 1 ) drooling severity with a scale from 1 to 5 , where 1 = never drools , 2 = mild drooling causing wet lips only , 3 = moderate drooling causing wet lips and chin , 4 = severe drooling where clothing becomes damp , and 5 = profuse drooling causing clothing , hands , and the subjects in general to get wet ; ( 2 ) drooling frequency with a scale from 1 to 4 , where 1 = never drools , 2 = occasionally drools , 3 = frequently drools , and 4 = constantly drools . the drooling ranking score was determined by adding the two subscales together ( drooling severity + drooling frequency ) . the drooling ranking score was rated from 2 to 9 , with a ranking score of 2 representing no drooling and a ranking score of 9 as the most severe level of drooling . the parents or the children s primary caregivers rated the drooling , at least 1 hour before or after meals . the hrqol of these children was rated using the pediatric quality of life inventory version 4.0 ( pedsql 4.0 ) for toddlers ( ages 24 ) and young children ( ages 57 ) using the generic core scales , which combined four subscales that included : ( 1 ) physical functioning , ( 2 ) emotional functioning , ( 3 ) social functioning , and ( 4 ) school functioning . the scoring dimensions included : ( 1 ) a physical health summary score that represented a physical functioning scale score , and ( 2 ) a psychosocial health summary score that was the sum of the items over the number of items answered in the emotional , social , and school functioning scales . parents or the primary caregivers rated the pedsql 4.0 scores ( internal consistency reliability : total scale score cronbach s = 0.9 for parents / proxy ; physical health summary score cronbach s = 0.88 for parents / proxy ; psychosocial health summary score cronbach s = 0.86 for parents / proxy).18 the developmental status of the children was evaluated using the developmental screening test for 06-year - old children ( chinese version , content validity 0.890.93),19 which was also rated by the parents or primary caregivers . this test evaluated five developmental domains ( language , social - personality , gross motor , fine motor , and cognition ) ; the highest scores achieved for each domain were considered the developmental levels . the screening test was correlated with the bayley scales of infant development i ( criterion - related validity r = 0.229~0.566 ; p < 0.05).19 the mean differences in hrqol scores among the two groups were analyzed with the mann the correlation of the drooling ranking score , the developmental status , and the hrqol was analyzed with the pearson product moment correlation coefficient . statistical analyses were performed using spss software ( v 12.0 ; ibm , armonk , ny ) . children with cp that attended one medical center hospital and two early intervention institutions at daytime for an early intervention and habilitation program in taiwan were enrolled . children with cp , aged 2 to 6 years , without drooling ( with a drooling ranking score of 2 according to the drooling rating scale developed by thomas - stonell and greenberg16 ) , were the control group . children with cp , aged 2 to 6 years , with drooling ( a drooling ranking score > 2 ) , were the study group . the exclusion criteria were : ( 1 ) children with cp combined with other problems such as congenital malformation or metabolic disorder , ( 2 ) children taking anticholinergic drugs over the past 2 months , and ( 3 ) children with an acute infection or other systemic disease . children with cp were enrolled consecutively from february 2011 to july 2011 . the study was conducted according to the criteria of the declaration of helsinki and the review board of the university hospital approved this study . forty - seven children were included in the study : 14 did not drool ( mean age : 43.2 13.8 months , diplegia 17% , quadriplegia 12.8% ) and 33 did drool ( mean age : 48.9 14.4 months , diplegia 10.6% , quadriplegia 59.6% ) . in the group without drooling , there were eight boys and six girls , and in the group with drooling , there were 22 boys and 11 girls ( table 1 ) . previous studies4,17 have reported that drooling in infancy usually resolves by 18 months of age . the children that drool beyond 4 years of age are considered abnormal and require further treatment . therefore , the participants were divided into groups ; those less than 4 years old ( > 2 years and < 4 years , as the age group where drooling was supposed to stop and conservative management of drooling was acceptable ) and those older than 4 years of age ( 4 years , as the age group where drooling was abnormal and required further treatment ) . the ranking of drooling was evaluated using the drooling rating scale as described by thomas - stonell and greenberg.16 the drooling rating scale was rated using two subscales : ( 1 ) drooling severity with a scale from 1 to 5 , where 1 = never drools , 2 = mild drooling causing wet lips only , 3 = moderate drooling causing wet lips and chin , 4 = severe drooling where clothing becomes damp , and 5 = profuse drooling causing clothing , hands , and the subjects in general to get wet ; ( 2 ) drooling frequency with a scale from 1 to 4 , where 1 = never drools , 2 = occasionally drools , 3 = frequently drools , and 4 = constantly drools . the drooling ranking score was determined by adding the two subscales together ( drooling severity + drooling frequency ) . the drooling ranking score was rated from 2 to 9 , with a ranking score of 2 representing no drooling and a ranking score of 9 as the most severe level of drooling . the parents or the children s primary caregivers rated the drooling , at least 1 hour before or after meals . the hrqol of these children was rated using the pediatric quality of life inventory version 4.0 ( pedsql 4.0 ) for toddlers ( ages 24 ) and young children ( ages 57 ) using the generic core scales , which combined four subscales that included : ( 1 ) physical functioning , ( 2 ) emotional functioning , ( 3 ) social functioning , and ( 4 ) school functioning . the scoring dimensions included : ( 1 ) a physical health summary score that represented a physical functioning scale score , and ( 2 ) a psychosocial health summary score that was the sum of the items over the number of items answered in the emotional , social , and school functioning scales . parents or the primary caregivers rated the pedsql 4.0 scores ( internal consistency reliability : total scale score cronbach s = 0.9 for parents / proxy ; physical health summary score cronbach s = 0.88 for parents / proxy ; psychosocial health summary score cronbach s = 0.86 for parents / proxy).18 the developmental status of the children was evaluated using the developmental screening test for 06-year - old children ( chinese version , content validity 0.890.93),19 which was also rated by the parents or primary caregivers . this test evaluated five developmental domains ( language , social - personality , gross motor , fine motor , and cognition ) ; the highest scores achieved for each domain were considered the developmental levels . the screening test was correlated with the bayley scales of infant development i ( criterion - related validity r = 0.229~0.566 ; p < 0.05).19 the mean differences in hrqol scores among the two groups were analyzed with the mann the correlation of the drooling ranking score , the developmental status , and the hrqol was analyzed with the pearson product moment correlation coefficient . statistical analyses were performed using spss software ( v 12.0 ; ibm , armonk , ny ) . there were no significant correlations with sex and age of those that drooled compared to those that did not drool ( table 1 ) . however , the diagnosis of diplegia or quadriplegia was associated with a significant difference ; there were more children with quadriplegia that drooled and fewer with diplegia that drooled ( p < 0.01 , table 1 ) . table 1 also shows a comparison of the developmental status between those that drooled and those that did not drool . the language developmental status ( in months ) of the children that drooled was significantly lower compared to the children that did not drool ( 19.64 17.90 vs 42.36 13.45 ; p < 0.01 ) . the cognitive developmental status of the children that drooled was lower than in the children that did not drool ( 19.76 17.88 vs 43.36 17.76 ; p < 0.01 ) . there was no significant difference in the gross motor developmental status between the children that drooled and those that did not drool ( 12.88 12.54 vs 16.36 13.16 ; p > 0.05 ) . there were no significant differences in the hrqol ( physical health summary score and psychosocial health summary score ) by sex ( 18.96 17.43 vs 24.49 22.16 , t = 0.95 , p > 0.05 and 49.20 16.68 vs 43.51 18.31 , t = 1.09 , p > 0.05 , respectively ; table 2 ) and by age group ( 25.57 19.66 vs 16.90 28.25 , t = 1.57 , p > 0.05 and 52.13 16.43 vs 42.75 17.19 , t = 1.91 , p > 0.05 , respectively ) . the physical health summary scores of the quadriplegic group were significantly lower than those of the diplegic group ( 34.19 18.69 vs 15.90 17.11 , t = 3.20 , p < 0.01 ) ; however , there were no significant differences observed on the psychosocial health summary scores ( 53.48 10.70 vs 44.72 18.82 , t = 1.58 , p > 0.05 ) . for the children that drooled , both the physical health summary scores and the psychosocial health summary scores were significantly lower than the scores for the children that did not drool ( 31.97 22.22 vs 16.29 15.97 , t = 2.73 , p < 0.01 and 57.09 12.21 vs 42.92 17.57 , t = 2.74 , p < 0.01 , respectively ) . in order to determine which variables ( developmental status and drooling ranking scales ) could be enrolled in stepwise regression to predict the variability of hrqol , those predictor variables which were significant correlated with hrqol were enrolled in the stepwise regression study . the drooling ranking score was negatively correlated with the physical health summary score ( r = 0.355 ; p < 0.05 ) and psychosocial health summary score ( r = 0.381 ; p < 0.01 ) . the developmental status ( language , gross motor , and cognition ) of the children was positively correlated with both the physical health summary score ( r = 0.477 , 0.716 , and 0.503 , respectively ; p < 0.01 ) and the psychosocial health summary score ( r = 0.522 , 0.383 , and 0.516 , respectively , p < 0.01 ) . thus the drooling ranking score and the developmental status of language , gross motor , and cognition factors were included in the stepwise regression analysis because they were significantly correlated with hrqol . for variability of the physical health summary score , the developmental level of language and cognition was excluded after the stepwise regression ( due to p > 0.1 ) . the drooling ranking score and gross motor development predicted 56.6% of the variation of the physical health summary score ( r = 0.566 ; p < 0.01 ) . for the prediction of variability of the psychosocial health summary score , the drooling ranking score , gross motor development , and cognitive development scores were excluded after stepwise regression ( due to p > 0.1 ) . the language development level predicted 25.6% of the variation of the psychosocial health summary score ( r = 0.256 ; p < 0.01 ) . the frequency of drooling in the quadriplegic children was greater than in the diplegic children . this result is compatible with the findings of hegde and pani3 that showed that patients with quadriplegia had the most severe drooling , followed by children with diplegia and the least affected children had athetoid cp . but other reports showed opposite results and they found the drooling is more prevalent and intense in children with dyskinetic cp than in children with spastic cp.5,20 the main etiologies of drooling include : impaired postural control as well as oral motor and swallowing abnormalities.3,2022 the quadriplegic children with cp are more likely to develop drooling due to their more extensive brain dysfunction and poor oral motor and sensory function compared to the diplegic children with cp . in this paper , there was no dyskinetic - type cp enrolled for study so we can not compare the drooling problem with other cp subtypes . the results of this study showed that the language and cognitive development of the children that drooled was lower than in the children that did not drool ; however , the gross motor development showed no significant difference between these two groups ( table 1 ) . these findings are compatible with the report of senner et al23 that showed that children who drooled had more severe dysarthria and impaired nonverbal intelligence , but their gross motor status was not more impaired ; although impaired motor control was considered one of the contributing factors of drooling.21,22 children at different developmental stages may have different findings and explanations for their disease.24,25 younger children were less likely to be perceived as performing less well on the hrqol questions than older children.26 it is possible that finding no significant hrqol differences between age groups , in this study , was due to the younger ages of our study groups . previous studies12,14 also revealed that the hrqol ( both physical and psychosocial ) of quadriplegic children was lower than that of diplegic children . in this study , the physical health summary scores of quadriplegic children were lower than diplegic children ; however , the psychosocial health summary score was not . the possible explanations for this result include : ( 1 ) most of the subjects attended early intervention institutions or a hospital program , where they possibly had a more supported and structured environment and received less negative feedback from social interaction;10,14 ( 2 ) most of the subjects were young and cognitively impaired ; the impairment was known since birth and they received support from their caregivers to accomplish daily activities , and therefore had fewer experiences of negative psychosocial well - being;11,27 ( 3 ) the number of subjects was too small to show a statistically significant difference in psychosocial health scores . the results of this study showed that the physical health summary scores and psychosocial health summary scores were significantly lower in the children with cp that drooled than in the children with cp that did not drool . these results were compatible with previous studies showing that drooling may lead to health - related problems such as skin maceration , recurrent pneumonia , and malnutrition.7,28 although our result showed a significant level of correlation between drooling and psychosocial hrqol , but the correlation coefficient showed only a lower level of correlation ( r = 0.381 ; p < 0.01 ) . some studies have reported that drooling was associated with impaired social relationships of these children with adults and their peers ; however , few showed negative emotional reactions due to drooling.8,10,15 van der burg et al10 reported that children with cp who drooled showed few negative emotional reactions because they attended special education schools and the drooling problem seems to be acceptable and ignored in these places . although the language and cognitive developmental status was positively correlated with the physical health summary score , these two variables were excluded from the stepwise regression model because of the statistical result of p 0.1 . thus , the most important variables left in the stepwise regression model were gross motor development and rank of drooling , and they predicted 56.6% of the variation of the physical health summary score . in predicting the variation associated with the psychosocial health summary score , the level of drooling , gross motor development and cognitive development were excluded from the stepwise regression model ( p 0.1 ) ; the remaining language development predicted 25.6% of the variation of the psychosocial health summary score . dickinson et al11 showed that gross motor development correlated most with the physical wellness of children with cp ; intellectual disability and language impairment significantly interfered with psychosocial wellness . however , drooling was not investigated as a problem that interfered with the quality of life in this prior study . in our study , only the ranking of drooling / gross motor development and language development were considered as important factors associated with the physical health summary score and psychosocial health summary score . further investigation of these possible variables ( eg , family or institutional factors , gross motor function classification system of children with cp ) that might be correlated with the hrqol of children with cp requires further research . although cognitive development was excluded in the stepwise regression model , its importance can not be overlooked due to nearly the same correlation coefficient associated with the psychosocial health summary score as with the language development score . however , the excluded variables did not add much to predicting the variability of the psychosocial health summary score , when it was included . because the subjects in this study were too young or too cognitively impaired , a parent / proxy report was used to assess the drooling and hrqol of the enrolled children . a bias might have been introduced by parents that were bothered by their children s drooling and inclined to rate their children s hrqol lower . as in the study by davis et al,29 there was discordance between parent / proxy and child self - report because they responded to the hrqol questionnaire items differently . but in this study , the definitions of drooling severity and frequency were clearly defined for parents and primary caregivers . in addition , the standard hrqol questionnaires were used for the hrqol evaluation , and were not likely significantly affected by bias . although a self - reported hrqol is standard for the perceived hrqol , the parents are valuable proxies to assess their children s hrqol if the children are too young or too cognitively impaired to complete a self - reported hrqol.10,30 therefore , the findings of this study based on parent / proxy reports showed that children with cp that drooled had a lower hrqol are important . this was a cross - sectional study and focused only on 2- to 6-year - old children with diplegic and quadriplegic cp from a university hospital and early intervention institutions . because our subjects in this paper were young and recruited from localized areas in taiwan , and contained only two cp subtypes , the results can not be generalized to all ages and all groups of children with cp . this was a cross - sectional study and focused only on 2- to 6-year - old children with diplegic and quadriplegic cp from a university hospital and early intervention institutions . because our subjects in this paper were young and recruited from localized areas in taiwan , and contained only two cp subtypes , the results can not be generalized to all ages and all groups of children with cp . in conclusion , the standard assessment inventory was used to evaluate the correlation of drooling on the hrqol of children with cp . the more severe the drooling was ( without considering the type of cp ) , the lower the physical and psychosocial health quality of life was in the children with cp . the gross motor development level and ranking of drooling predicted the physical health score better , and the language development level predicted the psychosocial health score better . with regard to providing early intervention programs for children with cp , their developmental status should be assessed as well as their drooling problem , which has a negative correlation on their hrqol . the focus of this study was on young children with cp ; further evaluation is needed to determine similar correlations among older children with cp .

objectiveto investigate the association between drooling in children with cerebral palsy ( cp ) and their health - related quality of life ( hrqol ) , as well as the possible variables that predict their hrqol.methoda cross - sectional design was used for this study . children with cp , without other identified disease , aged 2 to 6 years , who drool ( n = 33 ) or did not drool ( n = 14 ) , were included . the dependent variables were the physical health summary scores and the psychosocial health summary scores of the pediatric quality of life inventory version 4.0 . the t test , pearson product moment correlation , mann whitney u test and stepwise regression analysis were used for statistical analysis.resultsthe physical health and psychosocial health summary scores of the children that drooled ( 16.29 15.97 and 42.92 17.57 , respectively ) were lower than for the children that did not drool ( 31.97 22.22 and 57.09 12.21 , respectively ; p < 0.01 ) . the drooling ranking score was negatively correlated with the physical health summary score ( r = 0.355 ; p < 0.05 ) and the psychosocial health summary score ( r = 0.381 ; p < 0.01 ) . the stepwise regression showed that gross motor development and the drooling ranking score predicted 56.6% of the variability of the physical health summary score ( r2 = 0.566 ; p < 0.01 ) . the language development score predicted 25.6% of the variability of the psychosocial health summary score ( r2 = 0.256 ; p < 0.01).conclusiondrooling was associated with a lower hrqol . prediction of the physical health summary score was more closely associated with gross motor development and the drooling ranking scores . prediction of the psychosocial health summary score was more closely associated with the language development of children with cp aged 2 to 6 years .

Introduction Materials and methods Participants Outcome measures Data analysis Results Discussion Study limitations Conclusion

hockstein et al9 reported that drooling in children with cp could interfere with their education and increase their dependent level of care . such studies suggest that drooling might be associated with a reduced quality of life among children with cp . previous studies have shown that there are many factors that interfere with the health - related quality of life ( hrqol ) in children with cp including : motor , cognitive , language , and social impairment.1114 although the prevalence of drooling is high among children with cp , there are few articles regarding its relationship to the hrqol in these children . in this study , standardized measurement of the hrqol in children with cp , with and without drooling , was investigated . in addition , the relationship between drooling and hrqol was evaluated , as well as the factors that predict the variability of hrqol in these children . children with cp , aged 2 to 6 years , without drooling ( with a drooling ranking score of 2 according to the drooling rating scale developed by thomas - stonell and greenberg16 ) , were the control group . children with cp , aged 2 to 6 years , with drooling ( a drooling ranking score > 2 ) , were the study group . the study was conducted according to the criteria of the declaration of helsinki and the review board of the university hospital approved this study . forty - seven children were included in the study : 14 did not drool ( mean age : 43.2 13.8 months , diplegia 17% , quadriplegia 12.8% ) and 33 did drool ( mean age : 48.9 14.4 months , diplegia 10.6% , quadriplegia 59.6% ) . therefore , the participants were divided into groups ; those less than 4 years old ( > 2 years and < 4 years , as the age group where drooling was supposed to stop and conservative management of drooling was acceptable ) and those older than 4 years of age ( 4 years , as the age group where drooling was abnormal and required further treatment ) . the ranking of drooling was evaluated using the drooling rating scale as described by thomas - stonell and greenberg.16 the drooling rating scale was rated using two subscales : ( 1 ) drooling severity with a scale from 1 to 5 , where 1 = never drools , 2 = mild drooling causing wet lips only , 3 = moderate drooling causing wet lips and chin , 4 = severe drooling where clothing becomes damp , and 5 = profuse drooling causing clothing , hands , and the subjects in general to get wet ; ( 2 ) drooling frequency with a scale from 1 to 4 , where 1 = never drools , 2 = occasionally drools , 3 = frequently drools , and 4 = constantly drools . the drooling ranking score was determined by adding the two subscales together ( drooling severity + drooling frequency ) . the drooling ranking score was rated from 2 to 9 , with a ranking score of 2 representing no drooling and a ranking score of 9 as the most severe level of drooling . the hrqol of these children was rated using the pediatric quality of life inventory version 4.0 ( pedsql 4.0 ) for toddlers ( ages 24 ) and young children ( ages 57 ) using the generic core scales , which combined four subscales that included : ( 1 ) physical functioning , ( 2 ) emotional functioning , ( 3 ) social functioning , and ( 4 ) school functioning . the scoring dimensions included : ( 1 ) a physical health summary score that represented a physical functioning scale score , and ( 2 ) a psychosocial health summary score that was the sum of the items over the number of items answered in the emotional , social , and school functioning scales . parents or the primary caregivers rated the pedsql 4.0 scores ( internal consistency reliability : total scale score cronbach s = 0.9 for parents / proxy ; physical health summary score cronbach s = 0.88 for parents / proxy ; psychosocial health summary score cronbach s = 0.86 for parents / proxy).18 the developmental status of the children was evaluated using the developmental screening test for 06-year - old children ( chinese version , content validity 0.890.93),19 which was also rated by the parents or primary caregivers . the screening test was correlated with the bayley scales of infant development i ( criterion - related validity r = 0.229~0.566 ; p < 0.05).19 the mean differences in hrqol scores among the two groups were analyzed with the mann the correlation of the drooling ranking score , the developmental status , and the hrqol was analyzed with the pearson product moment correlation coefficient . children with cp , aged 2 to 6 years , without drooling ( with a drooling ranking score of 2 according to the drooling rating scale developed by thomas - stonell and greenberg16 ) , were the control group . children with cp , aged 2 to 6 years , with drooling ( a drooling ranking score > 2 ) , were the study group . forty - seven children were included in the study : 14 did not drool ( mean age : 43.2 13.8 months , diplegia 17% , quadriplegia 12.8% ) and 33 did drool ( mean age : 48.9 14.4 months , diplegia 10.6% , quadriplegia 59.6% ) . therefore , the participants were divided into groups ; those less than 4 years old ( > 2 years and < 4 years , as the age group where drooling was supposed to stop and conservative management of drooling was acceptable ) and those older than 4 years of age ( 4 years , as the age group where drooling was abnormal and required further treatment ) . the drooling ranking score was determined by adding the two subscales together ( drooling severity + drooling frequency ) . the drooling ranking score was rated from 2 to 9 , with a ranking score of 2 representing no drooling and a ranking score of 9 as the most severe level of drooling . the hrqol of these children was rated using the pediatric quality of life inventory version 4.0 ( pedsql 4.0 ) for toddlers ( ages 24 ) and young children ( ages 57 ) using the generic core scales , which combined four subscales that included : ( 1 ) physical functioning , ( 2 ) emotional functioning , ( 3 ) social functioning , and ( 4 ) school functioning . the scoring dimensions included : ( 1 ) a physical health summary score that represented a physical functioning scale score , and ( 2 ) a psychosocial health summary score that was the sum of the items over the number of items answered in the emotional , social , and school functioning scales . parents or the primary caregivers rated the pedsql 4.0 scores ( internal consistency reliability : total scale score cronbach s = 0.9 for parents / proxy ; physical health summary score cronbach s = 0.88 for parents / proxy ; psychosocial health summary score cronbach s = 0.86 for parents / proxy).18 the developmental status of the children was evaluated using the developmental screening test for 06-year - old children ( chinese version , content validity 0.890.93),19 which was also rated by the parents or primary caregivers . the screening test was correlated with the bayley scales of infant development i ( criterion - related validity r = 0.229~0.566 ; p < 0.05).19 the mean differences in hrqol scores among the two groups were analyzed with the mann the correlation of the drooling ranking score , the developmental status , and the hrqol was analyzed with the pearson product moment correlation coefficient . there were no significant correlations with sex and age of those that drooled compared to those that did not drool ( table 1 ) . however , the diagnosis of diplegia or quadriplegia was associated with a significant difference ; there were more children with quadriplegia that drooled and fewer with diplegia that drooled ( p < 0.01 , table 1 ) . table 1 also shows a comparison of the developmental status between those that drooled and those that did not drool . the language developmental status ( in months ) of the children that drooled was significantly lower compared to the children that did not drool ( 19.64 17.90 vs 42.36 13.45 ; p < 0.01 ) . the cognitive developmental status of the children that drooled was lower than in the children that did not drool ( 19.76 17.88 vs 43.36 17.76 ; p < 0.01 ) . there was no significant difference in the gross motor developmental status between the children that drooled and those that did not drool ( 12.88 12.54 vs 16.36 13.16 ; p > 0.05 ) . there were no significant differences in the hrqol ( physical health summary score and psychosocial health summary score ) by sex ( 18.96 17.43 vs 24.49 22.16 , t = 0.95 , p > 0.05 and 49.20 16.68 vs 43.51 18.31 , t = 1.09 , p > 0.05 , respectively ; table 2 ) and by age group ( 25.57 19.66 vs 16.90 28.25 , t = 1.57 , p > 0.05 and 52.13 16.43 vs 42.75 17.19 , t = 1.91 , p > 0.05 , respectively ) . the physical health summary scores of the quadriplegic group were significantly lower than those of the diplegic group ( 34.19 18.69 vs 15.90 17.11 , t = 3.20 , p < 0.01 ) ; however , there were no significant differences observed on the psychosocial health summary scores ( 53.48 10.70 vs 44.72 18.82 , t = 1.58 , p > 0.05 ) . for the children that drooled , both the physical health summary scores and the psychosocial health summary scores were significantly lower than the scores for the children that did not drool ( 31.97 22.22 vs 16.29 15.97 , t = 2.73 , p < 0.01 and 57.09 12.21 vs 42.92 17.57 , t = 2.74 , p < 0.01 , respectively ) . in order to determine which variables ( developmental status and drooling ranking scales ) could be enrolled in stepwise regression to predict the variability of hrqol , those predictor variables which were significant correlated with hrqol were enrolled in the stepwise regression study . the drooling ranking score was negatively correlated with the physical health summary score ( r = 0.355 ; p < 0.05 ) and psychosocial health summary score ( r = 0.381 ; p < 0.01 ) . the developmental status ( language , gross motor , and cognition ) of the children was positively correlated with both the physical health summary score ( r = 0.477 , 0.716 , and 0.503 , respectively ; p < 0.01 ) and the psychosocial health summary score ( r = 0.522 , 0.383 , and 0.516 , respectively , p < 0.01 ) . thus the drooling ranking score and the developmental status of language , gross motor , and cognition factors were included in the stepwise regression analysis because they were significantly correlated with hrqol . for variability of the physical health summary score , the developmental level of language and cognition was excluded after the stepwise regression ( due to p > 0.1 ) . the drooling ranking score and gross motor development predicted 56.6% of the variation of the physical health summary score ( r = 0.566 ; p < 0.01 ) . for the prediction of variability of the psychosocial health summary score , the drooling ranking score , gross motor development , and cognitive development scores were excluded after stepwise regression ( due to p > 0.1 ) . the language development level predicted 25.6% of the variation of the psychosocial health summary score ( r = 0.256 ; p < 0.01 ) . this result is compatible with the findings of hegde and pani3 that showed that patients with quadriplegia had the most severe drooling , followed by children with diplegia and the least affected children had athetoid cp . but other reports showed opposite results and they found the drooling is more prevalent and intense in children with dyskinetic cp than in children with spastic cp.5,20 the main etiologies of drooling include : impaired postural control as well as oral motor and swallowing abnormalities.3,2022 the quadriplegic children with cp are more likely to develop drooling due to their more extensive brain dysfunction and poor oral motor and sensory function compared to the diplegic children with cp . the results of this study showed that the language and cognitive development of the children that drooled was lower than in the children that did not drool ; however , the gross motor development showed no significant difference between these two groups ( table 1 ) . these findings are compatible with the report of senner et al23 that showed that children who drooled had more severe dysarthria and impaired nonverbal intelligence , but their gross motor status was not more impaired ; although impaired motor control was considered one of the contributing factors of drooling.21,22 children at different developmental stages may have different findings and explanations for their disease.24,25 younger children were less likely to be perceived as performing less well on the hrqol questions than older children.26 it is possible that finding no significant hrqol differences between age groups , in this study , was due to the younger ages of our study groups . in this study , the physical health summary scores of quadriplegic children were lower than diplegic children ; however , the psychosocial health summary score was not . the possible explanations for this result include : ( 1 ) most of the subjects attended early intervention institutions or a hospital program , where they possibly had a more supported and structured environment and received less negative feedback from social interaction;10,14 ( 2 ) most of the subjects were young and cognitively impaired ; the impairment was known since birth and they received support from their caregivers to accomplish daily activities , and therefore had fewer experiences of negative psychosocial well - being;11,27 ( 3 ) the number of subjects was too small to show a statistically significant difference in psychosocial health scores . the results of this study showed that the physical health summary scores and psychosocial health summary scores were significantly lower in the children with cp that drooled than in the children with cp that did not drool . these results were compatible with previous studies showing that drooling may lead to health - related problems such as skin maceration , recurrent pneumonia , and malnutrition.7,28 although our result showed a significant level of correlation between drooling and psychosocial hrqol , but the correlation coefficient showed only a lower level of correlation ( r = 0.381 ; p < 0.01 ) . some studies have reported that drooling was associated with impaired social relationships of these children with adults and their peers ; however , few showed negative emotional reactions due to drooling.8,10,15 van der burg et al10 reported that children with cp who drooled showed few negative emotional reactions because they attended special education schools and the drooling problem seems to be acceptable and ignored in these places . although the language and cognitive developmental status was positively correlated with the physical health summary score , these two variables were excluded from the stepwise regression model because of the statistical result of p 0.1 . thus , the most important variables left in the stepwise regression model were gross motor development and rank of drooling , and they predicted 56.6% of the variation of the physical health summary score . in predicting the variation associated with the psychosocial health summary score , the level of drooling , gross motor development and cognitive development were excluded from the stepwise regression model ( p 0.1 ) ; the remaining language development predicted 25.6% of the variation of the psychosocial health summary score . dickinson et al11 showed that gross motor development correlated most with the physical wellness of children with cp ; intellectual disability and language impairment significantly interfered with psychosocial wellness . however , drooling was not investigated as a problem that interfered with the quality of life in this prior study . in our study , only the ranking of drooling / gross motor development and language development were considered as important factors associated with the physical health summary score and psychosocial health summary score . further investigation of these possible variables ( eg , family or institutional factors , gross motor function classification system of children with cp ) that might be correlated with the hrqol of children with cp requires further research . although cognitive development was excluded in the stepwise regression model , its importance can not be overlooked due to nearly the same correlation coefficient associated with the psychosocial health summary score as with the language development score . however , the excluded variables did not add much to predicting the variability of the psychosocial health summary score , when it was included . because the subjects in this study were too young or too cognitively impaired , a parent / proxy report was used to assess the drooling and hrqol of the enrolled children . in addition , the standard hrqol questionnaires were used for the hrqol evaluation , and were not likely significantly affected by bias . although a self - reported hrqol is standard for the perceived hrqol , the parents are valuable proxies to assess their children s hrqol if the children are too young or too cognitively impaired to complete a self - reported hrqol.10,30 therefore , the findings of this study based on parent / proxy reports showed that children with cp that drooled had a lower hrqol are important . this was a cross - sectional study and focused only on 2- to 6-year - old children with diplegic and quadriplegic cp from a university hospital and early intervention institutions . because our subjects in this paper were young and recruited from localized areas in taiwan , and contained only two cp subtypes , the results can not be generalized to all ages and all groups of children with cp . this was a cross - sectional study and focused only on 2- to 6-year - old children with diplegic and quadriplegic cp from a university hospital and early intervention institutions . because our subjects in this paper were young and recruited from localized areas in taiwan , and contained only two cp subtypes , the results can not be generalized to all ages and all groups of children with cp . in conclusion , the standard assessment inventory was used to evaluate the correlation of drooling on the hrqol of children with cp . the more severe the drooling was ( without considering the type of cp ) , the lower the physical and psychosocial health quality of life was in the children with cp . the gross motor development level and ranking of drooling predicted the physical health score better , and the language development level predicted the psychosocial health score better . with regard to providing early intervention programs for children with cp , their developmental status should be assessed as well as their drooling problem , which has a negative correlation on their hrqol . the focus of this study was on young children with cp ; further evaluation is needed to determine similar correlations among older children with cp .

chronic pain represents a major healthcare problem which seriously impairs the quality of sufferers working and social lives , and the most common single location is the lower back.1 chronic low back pain ( lbp ) is difficult to treat because of the range of causative mechanisms that may be involved ; this difficulty is compounded by the limited efficacy of current pharmacological agents , and exemplified by the inconsistency of existing treatment patterns . moreover , optimal pain management demands not only effective pain relief , but a precise balance of analgesia , tolerability , and beneficial effect on functionality / quality of life . in september 2009 , an international panel of clinical pain specialists met to discuss the challenges of chronic lbp , and to review the preclinical and clinical data relating to the new analgesic agent , tapentadol , which may offer a novel treatment option for chronic lbp . based on a literature search of pubmed before the meeting other presentations came from articles relating to the changes occurring in chronic lbp and the preclinical and clinical testing of tapentadol , as well as data on file from completed clinical trials . a 2006 survey of 46,394 adults in 15 european countries and israel found that 19% of respondents suffered from moderate to severe chronic pain ( 5 on a 10-point numeric rating scale [ nrs ] ) and nearly half received inadequate pain management.1 other surveys in norway2 and denmark3 have produced similar results . the economic implications for society are significant in terms of the utilization of healthcare resources.4 in the pan - european study , the most common location of chronic pain was the back , in 47% of sufferers ( 5% upper back , 18% lower back , 24% unspecified).1 this finding is consistent with other sources of prevalence data on low back pain , indicating an overall prevalence of 80.5 per 1000 population.5 it affects more women than men and the prevalence increases with age.5,6 in the us , lbp accounts for almost 30 million physician visits per year56% of which are to primary care physicians.7 thus lbp imposes a significant economic burden . in sweden alone , the cost of lbp in 2001 was estimated to be 1860 million,8 while in the us the overall cost of back pain is estimated to exceed us$15 billion per year.9 lbp is defined as pain located in the lumbosacral region of the spine , the most frequent location being the fourth and fifth lumbar segment . it may be classified according to its etiology ; mechanical or nonspecific lbp has no serious underlying pathology or nerve root compromise , in contrast to secondary lbp.10 it may also be classified according to its duration into 1 of 3 phases : acute , subacute ( intermittent ) , or chronic . definitions vary , but lbp is generally regarded as chronic if symptoms persist for more than 3 to 6 months.11 the transition to chronic lbp involves structural and neurophysiological changes , such as peripheral and central sensitization.12 at this point the patient s symptoms may be exacerbated by psychosocial factors such as irritability , emotional distress , social withdrawal , excessive consumption of alcohol or medication , and loss of income.13 the result may be persistent disability , depression , or anxiety . the impact on quality of life is considerable.14 early diagnosis is therefore important , to incorporate these aspects of the condition into the management of low back pain and increase the chance of recovery.15 a 2006 survey of 46,394 adults in 15 european countries and israel found that 19% of respondents suffered from moderate to severe chronic pain ( 5 on a 10-point numeric rating scale [ nrs ] ) and nearly half received inadequate pain management.1 other surveys in norway2 and denmark3 have produced similar results . the economic implications for society are significant in terms of the utilization of healthcare resources.4 in the pan - european study , the most common location of chronic pain was the back , in 47% of sufferers ( 5% upper back , 18% lower back , 24% unspecified).1 this finding is consistent with other sources of prevalence data on low back pain , indicating an overall prevalence of 80.5 per 1000 population.5 it affects more women than men and the prevalence increases with age.5,6 in the us , lbp accounts for almost 30 million physician visits per year56% of which are to primary care physicians.7 thus lbp imposes a significant economic burden . in sweden alone , the cost of lbp in 2001 was estimated to be 1860 million,8 while in the us the overall cost of back pain is estimated to exceed us$15 billion per year.9 lbp is defined as pain located in the lumbosacral region of the spine , the most frequent location being the fourth and fifth lumbar segment . it may be classified according to its etiology ; mechanical or nonspecific lbp has no serious underlying pathology or nerve root compromise , in contrast to secondary lbp.10 it may also be classified according to its duration into 1 of 3 phases : acute , subacute ( intermittent ) , or chronic . definitions vary , but lbp is generally regarded as chronic if symptoms persist for more than 3 to 6 months.11 the transition to chronic lbp involves structural and neurophysiological changes , such as peripheral and central sensitization.12 at this point the patient s symptoms may be exacerbated by psychosocial factors such as irritability , emotional distress , social withdrawal , excessive consumption of alcohol or medication , and loss of income.13 the result may be persistent disability , depression , or anxiety . the impact on quality of life is considerable.14 early diagnosis is therefore important , to incorporate these aspects of the condition into the management of low back pain and increase the chance of recovery.15 despite the substantial economic burden of chronic lbp , there is a lack of consensus on the best treatment.16 patients have very different levels of impairment , disability , and chronicity . cases of low impairment and disability may best be treated by simple evidence - based therapies exercises , cognitive - behavioral interventions , and medication but the multimechanistic nature of more severe chronic lbp means that no single intervention is likely to be effective.12,17 the complexity of treatment is further increased because chronic lbp may present with nociceptive pain , neuropathic pain , or both components.18,19 one survey using the painde - tect questionnaire found that 37% of chronic lbp patients suffered from neuropathic pain , while 35% had nociceptive pain and results for the remaining patients were unclear.20 a neuropathic component can complicate diagnosis for several reasons.21 firstly , the signs and symptoms can vary between patients and within individual patients over time . the clinical picture may be obscured by psychosocial factors , which need to be addressed . this is important because neuropathic pain often fails to respond to commonly prescribed analgesic therapy,21 treatment with single agent drugs being limited by incomplete efficacy and dose - limiting adverse effects.22 the major pharmacological agents currently used on - label to treat chronic lbp include nonopioids , such as paracetamol , nonsteroidal anti - inflammatory drugs ( nsaids ) , cyclooxygenase-2 ( cox-2 ) inhibitors , and opioids , while antidepressants , anticonvulsants , and local anesthetics may also be prescribed . for example , efficacy may be limited by an undetected neuropathic component , and poor tolerability may lead to an unfavorable efficacy / side effect ratio . opioid - related side effects , in particular , affect 73% to 90% of patients treated for longer than 3 months and are directly responsible for high withdrawal rates of 20% to 40%.23 treatment may also be compromised by drug drug interactions or the development of tolerance , demonstrating the urgent need for analgesics which are effective and safe in long - term use . these limitations can be addressed by combining agents with different mechanisms of action , to produce additive or synergistic analgesic effects and minimize drug - induced adverse events.24 this multimechanistic approach takes into account the diversity of underlying pain mechanisms and the different pharmacological principles required for their successful treatment it reduces the convenience of treatment , and thus compliance , and creates the potential for pharmacokinetic drug drug interactions.25 these may increase side effects or reduce analgesia , and the elderly , in particular , may be at increased risk.25 therefore the safety and efficacy of specific combinations must be empirically evaluated,22 but up to now clinical study data are inadequate . an alternative multimechanistic approach is to develop novel compounds which possess multiple mechanisms of action . tapentadol ( see table 1 ) has been developed to have broad analgesic activity by combining known analgesic principles ( -opioid receptor agonism and noradrenaline reuptake inhibition ) in a single molecule , and to offer a better balance between efficacy and tolerability than classical opioids . an immediate release formulation has recently been registered in the us to treat moderate to severe acute pain , and both immediate and prolonged formulations are currently under - going registration in europe for the treatment of severe acute and chronic pain . tapentadol s agonistic activity at the -opioid receptors of afferent pain fibres inhibits the release of excitatory neurotransmitters , and reduces the upward transmission of pain signals . via its -opioid action in the brain , tapentadol also influences the release of neurotransmitters by the descending pain pathways , producing a further inhibition of pain.26 like other systemic opioids , tapentadol acts at all these sites and its overall analgesic effect is enhanced by synergy between them.27 opioids can be effective in chronic neuropathic pain , but decreased potency requires higher doses in order to achieve sufficient analgesia.28,29 this characteristic of neuropathic pain , and the development of tolerance , both potentially increase the incidence of side effects . tapentadol addresses this problem by virtue of its second analgesic mechanism of action , ie , noradrenaline reuptake inhibition , particularly in chronic neuropathic pain . this property has been reported by studies in a number of animal models.30 noradrenaline reuptake inhibition exerts its antinociceptive effect via the descending pain pathways , where the increased synaptic noradrenaline binds to -2 receptors and thereby reduces pain signals to the brain.31 there is strong evidence of synergistic antinociception between this mechanism of action and -opioid agonism.3235 other analgesic agents also possess more than 1 mechanism of action ; for example , at first sight the established weak opioid tramadol appears similar to tapentadol , since it is a -opioid receptor agonist which also affects the monoaminergic system . however , there are important differences . both tapentadol s mechanisms of action reside in a single molecule that is metabolized via o - glucuronidation to an inactive metabolite.36 by contrast , tramadol is a racemic mixture of 2 enantiomers and produces an active metabolite . while the ( )-enantiomer and ( + ) -enantiomer of the parent compound produce noradrenaline reuptake inhibition and serotonin reuptake inhibition , respectively , weak -opioid receptor agonism resides mainly in the ( + ) -enantiomer of o - desmethyl - tramadol , the major active metabolite . thus , the relative contribution of the different mechanisms of action to the overall analgesic effect changes over time.36 as the parent molecule is metabolised , noradrenaline reuptake inhibition and serotonin reuptake inhibition diminish , and -opioid receptor agonism increases . this produces a complex time- and metabolism - dependent pattern of pharmacological activities.36 by contrast , tapentadol does not rely on metabolic activation to achieve full -opioid receptor agonism . also , tramadol is metabolized mainly by the cytochrome p450 system , which is polymorphic in humans , so that poor metabolizers do not experience satisfactory analgesia with standard doses.36 further differences favoring tapentadol over tramadol are that evidence suggests that analgesia is more readily obtained by noradrenaline reuptake inhibition than by serotonin reuptake inhibition.36,37 despite having an affinity to the -opioid receptor 50 times lower than that of morphine , in vivo testing in rats and mice found that intravenous tapentadol was only 2 to 3 times less potent than morphine in acute nociceptive pain.36,37 in 2 animal models of chronic neuropathic pain it was broadly equipotent to morphine , despite this much lower binding affinity , which is important in terms of opioid side effects.36 thus noradrenaline reuptake inhibition may make a greater contribution to the analgesic efficacy of tapentadol in chronic pain than in acute pain . this hypothesis is supported by a study which combined tapentadol with either the -opioid antagonist naloxone or the -2 antagonist yohimbine.30 in figure 1 , the dose - response curve shifted much further to the right when the -opioid action of tapentadol was antagonised in acute pain . in chronic pain , however , the reverse applied ; antagonizing noradrenaline reuptake inhibition had the greater effect.30 low affinity for the -opioid receptor and the presence of noradrenaline reuptake inhibition suggest that tapentadol might produce fewer opioid - related side effects than classical -opioid receptor agonists . the emetic potential of tapentadol and morphine has been compared in ferrets ; tapentadol produced fewer retches and vomits per animal , and the duration of these effects was shorter.37 in mice , tapentadol had a weaker inhibitory effect than morphine on gastrointestinal motility.37 when the development of tolerance was investigated in rats , complete tolerance was significantly delayed ( p < 0.0001 ) in animals receiving tapentadol ( 23 days ) compared with those receiving morphine ( 10 days).36 to summarise , tapentadol s -opioid action decreased ascending pain messages as well as increasing pain inhibition via the descending pathways . simultaneous blocking of the noradrenaline transporter further enhanced its analgesic effects via the increased activation of -2 receptors . thus there is a possible synergy between the mechanisms of action and also the sites of action . because its distinct pharmacological profile differentiates tapentadol from other centrally acting analgesics , it has been proposed that tapentadol should be classified by its 2 mechanisms of action , -opioid receptor agonism ( mor ) and noradrenaline reuptake inhibition ( nri ) , as a mor - nri compound.38 tapentadol has been subject to an extensive testing program ; to date , around 8000 patients have participated in phase iii clinical trials with either the immediate - release ( ir ) or prolonged - release ( pr ) formulation . chronic lbp was 1 major pain model used in the phase iii clinical program and has been included in the current phase iiib program , to gather further evidence on the use of tapentadol in this prevalent chronic pain condition . to evaluate the efficacy and safety of multiple doses of tapentadol pr , 981 patients with chronic lbp were recruited to a double - blind , randomized , active- and placebo - controlled , phase iii study.39 a 3-week titration phase allowed patients to achieve their optimal individual dose of tapentadol pr ( 100250 mg twice a day ) , oxycodone controlled release ( cr ; 2050 mg twice a day ) , or placebo . during the following 12-week maintenance phase , patients were not permitted rescue medication but were allowed to adjust their dosage , to reflect clinical practice . the primary endpoint was the mean change in pain intensity at week 12 or over the entire 12-week period , using the last observation carried forward ( locf ) imputation . demographic and baseline characteristics were consistent across treatment groups , the majority of subjects being women and below 65 years of age.39 severe pain ( nrs 6 ) was reported by 88.5% of subjects and 53.4% had prior opioid experience . as can be seen from figure 2 , both active treatment groups produced a comparable , statistically significant , reduction in pain intensity over the maintenance period ( both p < 0.001 vs placebo).39 reductions were similar in opioid - nave and opioid - experienced subjects . the results for tapentadol were supported by most secondary outcome parameters and also by more conservative imputations , such as worst observation carried forward ( wocf ) and baseline observation carried forward ( bocf).39 tapentadol also performed significantly better than placebo in all categories of the brief pain inventory , in overall sleep quality ratings and in the patients global impression of change ( pgic).39 tapentadol was well tolerated , the incidence of typical opioid adverse events being approximately half that of oxycodone.39 at dosages providing similar analgesic effects , tapentadol produced numerically lower levels of constipation ( 13.8% vs 26.8% ) , nausea ( 20.1% vs 34.5% ) , vomiting ( 9.1% vs 19.2% ) , dizziness ( 11.9% vs 17.1% ) and pruritis ( 7.2% vs 16.8%).39 cns effects tended to be milder in the tapentadol group than the oxycodone group.39 these tolerability findings are consistent with tapentadol having a -sparing effect , owing to the contribution of the nri component . figure 3 shows the percentage of patients in the different treatment groups who discontinued treatment over the study period . the main cause of discontinuation in the active treatment groups was treatment - emergent adverse events , predominantly gastrointestinal side effects . discontinuations for this reason were lower in the tapentadol group than the oxycodone group , the disparity being particularly marked during the titration phase.39 the efficacy of tapentadol in treating purely neuropathic pain has been investigated in 395 patients suffering from painful diabetic neuropathy.40 after a 3-week open - label titration phase , subjects who responded to tapentadol were assigned in a 1:1 ratio to receive either tapentadol pr or placebo for a 12-week , fixed - dose , double - blind phase . rescue medication was allowed during this phase , and the doses of tapentadol spanned the entire therapeutic range , from 100 mg twice a day to 250 mg twice a day . the primary endpoint was to show a statistically significant difference in pain intensity between tapentadol and placebo at week 12 , using an 11-point nrs and the locf imputation . this was achieved ( p < 0.001 ) ( see figure 4 ) and efficacy was confirmed by more conservative analyses of the primary endpoint , including bocf , wocf , and the proportions of patients showing 30% and 50% improvement.40 long - term safety has been evaluated in a phase iii , open - label , randomized , active - controlled study.41 a total of 1117 patients with chronic lbp or osteoarthritis were randomised ( 4:1 ) to receive tapentadol pr or oxycodone cr . a 1-week titration phase enabled subjects to determine their optimal dose of analgesic , within the range of 100 to 250 mg twice a day for tapentadol pr , or 20 to 50 mg twice a day for oxycodone cr . this was followed by a 51-week maintenance phase , during which patients were encouraged to stay on a stable dose , but could adjust it if necessary , and were allowed paracetamol rescue medication . the primary endpoint was to determine the safety of tapentadol over 1 year.41 the majority of subjects were women and under 65 years of age . around two - thirds suffered from chronic lbp . at baseline , 89.4% had severe pain and the median pain intensity was 8.0 on an 11-point nrs . the mean dose of tapentadol for those who completed the study remained unchanged over the 1-year period.41 figure 5b shows that the mean pain intensity also remained unchanged , indicating that tapentadol produced a consistent reduction in pain over the total study period . there were fewer gastrointestinal and cns adverse events in patients receiving tapentadol than in those receiving oxycodone.42 fewer patients in the tapentadol group withdrew from treatment because of adverse events ( figure 5a ) , and fewer patients in the tapentadol group discontinued treatment for any reason.42 in this study , therefore , the tolerability of tapentadol was superior to that of oxycodone over a period of 1 year . in these clinical trials , tapentadol provided reliable analgesia in chronic lbp , comparable to that of oxycodone , a representative traditional opioid . it was effective against both nociceptive and neuropathic pain . at equianalgesic doses , tapentadol produced a lower incidence of side effects than oxycodone , leading to fewer patients withdrawing from treatment . clinical studies in patients with acute and chronic osteoarthritis pain , and with acute postoperative pain , have produced similar results.41,43,44 further clinical trials are currently under way comparing tapentadol with other analgesics . tapentadol ( see table 1 ) has been developed to have broad analgesic activity by combining known analgesic principles ( -opioid receptor agonism and noradrenaline reuptake inhibition ) in a single molecule , and to offer a better balance between efficacy and tolerability than classical opioids . an immediate release formulation has recently been registered in the us to treat moderate to severe acute pain , and both immediate and prolonged formulations are currently under - going registration in europe for the treatment of severe acute and chronic pain . tapentadol s agonistic activity at the -opioid receptors of afferent pain fibres inhibits the release of excitatory neurotransmitters , and reduces the upward transmission of pain signals . via its -opioid action in the brain , tapentadol also influences the release of neurotransmitters by the descending pain pathways , producing a further inhibition of pain.26 like other systemic opioids , tapentadol acts at all these sites and its overall analgesic effect is enhanced by synergy between them.27 opioids can be effective in chronic neuropathic pain , but decreased potency requires higher doses in order to achieve sufficient analgesia.28,29 this characteristic of neuropathic pain , and the development of tolerance , both potentially increase the incidence of side effects . tapentadol addresses this problem by virtue of its second analgesic mechanism of action , ie , noradrenaline reuptake inhibition , particularly in chronic neuropathic pain . this property has been reported by studies in a number of animal models.30 noradrenaline reuptake inhibition exerts its antinociceptive effect via the descending pain pathways , where the increased synaptic noradrenaline binds to -2 receptors and thereby reduces pain signals to the brain.31 there is strong evidence of synergistic antinociception between this mechanism of action and -opioid agonism.3235 other analgesic agents also possess more than 1 mechanism of action ; for example , at first sight the established weak opioid tramadol appears similar to tapentadol , since it is a -opioid receptor agonist which also affects the monoaminergic system . however , there are important differences . both tapentadol s mechanisms of action reside in a single molecule that is metabolized via o - glucuronidation to an inactive metabolite.36 by contrast , tramadol is a racemic mixture of 2 enantiomers and produces an active metabolite . while the ( )-enantiomer and ( + ) -enantiomer of the parent compound produce noradrenaline reuptake inhibition and serotonin reuptake inhibition , respectively , weak -opioid receptor agonism resides mainly in the ( + ) -enantiomer of o - desmethyl - tramadol , the major active metabolite . thus , the relative contribution of the different mechanisms of action to the overall analgesic effect changes over time.36 as the parent molecule is metabolised , noradrenaline reuptake inhibition and serotonin reuptake inhibition diminish , and -opioid receptor agonism increases . this produces a complex time- and metabolism - dependent pattern of pharmacological activities.36 by contrast , tapentadol does not rely on metabolic activation to achieve full -opioid receptor agonism . also , tramadol is metabolized mainly by the cytochrome p450 system , which is polymorphic in humans , so that poor metabolizers do not experience satisfactory analgesia with standard doses.36 further differences favoring tapentadol over tramadol are that evidence suggests that analgesia is more readily obtained by noradrenaline reuptake inhibition than by serotonin reuptake inhibition.36,37 despite having an affinity to the -opioid receptor 50 times lower than that of morphine , in vivo testing in rats and mice found that intravenous tapentadol was only 2 to 3 times less potent than morphine in acute nociceptive pain.36,37 in 2 animal models of chronic neuropathic pain it was broadly equipotent to morphine , despite this much lower binding affinity , which is important in terms of opioid side effects.36 thus noradrenaline reuptake inhibition may make a greater contribution to the analgesic efficacy of tapentadol in chronic pain than in acute pain . this hypothesis is supported by a study which combined tapentadol with either the -opioid antagonist naloxone or the -2 antagonist yohimbine.30 in figure 1 , the dose - response curve shifted much further to the right when the -opioid action of tapentadol was antagonised in acute pain . in chronic pain , however , the reverse applied ; antagonizing noradrenaline reuptake inhibition had the greater effect.30 low affinity for the -opioid receptor and the presence of noradrenaline reuptake inhibition suggest that tapentadol might produce fewer opioid - related side effects than classical -opioid receptor agonists . the emetic potential of tapentadol and morphine has been compared in ferrets ; tapentadol produced fewer retches and vomits per animal , and the duration of these effects was shorter.37 in mice , tapentadol had a weaker inhibitory effect than morphine on gastrointestinal motility.37 when the development of tolerance was investigated in rats , complete tolerance was significantly delayed ( p < 0.0001 ) in animals receiving tapentadol ( 23 days ) compared with those receiving morphine ( 10 days).36 to summarise , tapentadol s -opioid action decreased ascending pain messages as well as increasing pain inhibition via the descending pathways . simultaneous blocking of the noradrenaline transporter further enhanced its analgesic effects via the increased activation of -2 receptors . thus there is a possible synergy between the mechanisms of action and also the sites of action . because its distinct pharmacological profile differentiates tapentadol from other centrally acting analgesics , it has been proposed that tapentadol should be classified by its 2 mechanisms of action , -opioid receptor agonism ( mor ) and noradrenaline reuptake inhibition ( nri ) , as a mor - nri compound.38 tapentadol has been subject to an extensive testing program ; to date , around 8000 patients have participated in phase iii clinical trials with either the immediate - release ( ir ) or prolonged - release ( pr ) formulation . chronic lbp was 1 major pain model used in the phase iii clinical program and has been included in the current phase iiib program , to gather further evidence on the use of tapentadol in this prevalent chronic pain condition . to evaluate the efficacy and safety of multiple doses of tapentadol pr , 981 patients with chronic lbp were recruited to a double - blind , randomized , active- and placebo - controlled , phase iii study.39 a 3-week titration phase allowed patients to achieve their optimal individual dose of tapentadol pr ( 100250 mg twice a day ) , oxycodone controlled release ( cr ; 2050 mg twice a day ) , or placebo . during the following 12-week maintenance phase , patients were not permitted rescue medication but were allowed to adjust their dosage , to reflect clinical practice . the primary endpoint was the mean change in pain intensity at week 12 or over the entire 12-week period , using the last observation carried forward ( locf ) imputation . demographic and baseline characteristics were consistent across treatment groups , the majority of subjects being women and below 65 years of age.39 severe pain ( nrs 6 ) was reported by 88.5% of subjects and 53.4% had prior opioid experience . as can be seen from figure 2 , both active treatment groups produced a comparable , statistically significant , reduction in pain intensity over the maintenance period ( both p < 0.001 vs placebo).39 reductions were similar in opioid - nave and opioid - experienced subjects . the results for tapentadol were supported by most secondary outcome parameters and also by more conservative imputations , such as worst observation carried forward ( wocf ) and baseline observation carried forward ( bocf).39 tapentadol also performed significantly better than placebo in all categories of the brief pain inventory , in overall sleep quality ratings and in the patients global impression of change ( pgic).39 tapentadol was well tolerated , the incidence of typical opioid adverse events being approximately half that of oxycodone.39 at dosages providing similar analgesic effects , tapentadol produced numerically lower levels of constipation ( 13.8% vs 26.8% ) , nausea ( 20.1% vs 34.5% ) , vomiting ( 9.1% vs 19.2% ) , dizziness ( 11.9% vs 17.1% ) and pruritis ( 7.2% vs 16.8%).39 cns effects tended to be milder in the tapentadol group than the oxycodone group.39 these tolerability findings are consistent with tapentadol having a -sparing effect , owing to the contribution of the nri component . figure 3 shows the percentage of patients in the different treatment groups who discontinued treatment over the study period . the main cause of discontinuation in the active treatment groups was treatment - emergent adverse events , predominantly gastrointestinal side effects . discontinuations for this reason were lower in the tapentadol group than the oxycodone group , the disparity being particularly marked during the titration phase.39 the efficacy of tapentadol in treating purely neuropathic pain has been investigated in 395 patients suffering from painful diabetic neuropathy.40 after a 3-week open - label titration phase , subjects who responded to tapentadol were assigned in a 1:1 ratio to receive either tapentadol pr or placebo for a 12-week , fixed - dose , double - blind phase . rescue medication was allowed during this phase , and the doses of tapentadol spanned the entire therapeutic range , from 100 mg twice a day to 250 mg twice a day . the primary endpoint was to show a statistically significant difference in pain intensity between tapentadol and placebo at week 12 , using an 11-point nrs and the locf imputation . this was achieved ( p < 0.001 ) ( see figure 4 ) and efficacy was confirmed by more conservative analyses of the primary endpoint , including bocf , wocf , and the proportions of patients showing 30% and 50% improvement.40 long - term safety has been evaluated in a phase iii , open - label , randomized , active - controlled study.41 a total of 1117 patients with chronic lbp or osteoarthritis were randomised ( 4:1 ) to receive tapentadol pr or oxycodone cr . a 1-week titration phase enabled subjects to determine their optimal dose of analgesic , within the range of 100 to 250 mg twice a day for tapentadol pr , or 20 to 50 mg twice a day for oxycodone cr . this was followed by a 51-week maintenance phase , during which patients were encouraged to stay on a stable dose , but could adjust it if necessary , and were allowed paracetamol rescue medication . the primary endpoint was to determine the safety of tapentadol over 1 year.41 the majority of subjects were women and under 65 years of age . around two - thirds suffered from chronic lbp . at baseline , 89.4% had severe pain and the median pain intensity was 8.0 on an 11-point nrs . the mean dose of tapentadol for those who completed the study remained unchanged over the 1-year period.41 figure 5b shows that the mean pain intensity also remained unchanged , indicating that tapentadol produced a consistent reduction in pain over the total study period . there were fewer gastrointestinal and cns adverse events in patients receiving tapentadol than in those receiving oxycodone.42 fewer patients in the tapentadol group withdrew from treatment because of adverse events ( figure 5a ) , and fewer patients in the tapentadol group discontinued treatment for any reason.42 in this study , therefore , the tolerability of tapentadol was superior to that of oxycodone over a period of 1 year . in these clinical trials , tapentadol provided reliable analgesia in chronic lbp , comparable to that of oxycodone , a representative traditional opioid . it was effective against both nociceptive and neuropathic pain . at equianalgesic doses , tapentadol produced a lower incidence of side effects than oxycodone , leading to fewer patients withdrawing from treatment . clinical studies in patients with acute and chronic osteoarthritis pain , and with acute postoperative pain , have produced similar results.41,43,44 further clinical trials are currently under way comparing tapentadol with other analgesics . the treatment of chronic lbp is challenging for various reasons , and requires a multidisciplinary approach to treatment , encompassing pharmacological , psychological , and physical therapies . the efficacy of classical opioids can be compromised by dose restrictions as a result of side effects or the development of tolerance . tapentadol combines -opioid receptor agonism and noradrenaline reuptake inhibition in a single molecule , with the object of achieving an opioid - sparing effect , that is , to produce fewer opioid - related side effects for a given level of analgesia . to date , preclinical and clinical data support the pharmacological classification of tapentadol as an mor - nri30,38 and suggest that its efficacy / tolerability ratio may be better than those of classical opioids.36,37,39,41 further research is required , however , particularly into efficacy and safety in patients with pure neuropathic pain and to provide specific data on lbp with a neuropathic component .

chronic pain affects approximately 1 in 5 people in europe , and around half of sufferers receive inadequate pain management . the most common location is the lower back . pharmacological treatment of this condition is challenging because of the range of causative mechanisms and the difficulty of balancing analgesic efficacy and tolerability . an international panel of clinical pain specialists met in september , 2009 , to discuss the treatment of chronic low back pain , and to review preclinical and clinical data relating to the new analgesic , tapentadol . a lack of consensus exists on the best treatment for low back pain . the range of regularly prescribed pharmacological agents extends from nonopioids ( paracetamol , nsaids , and cox-2 inhibitors ) to opioids , antidepressants and anticonvulsants . pain relief may be compromised , however , by an undetected neuropathic component or intolerable side effects . treatment is potentially life - long and effective analgesics are urgently needed , with demonstrable long - term safety . combining separate agents with different mechanisms of action could overcome the limitations of present pharmacological therapy , but clinical evidence for this approach is currently lacking . tapentadol combines -opioid agonism with noradrenaline reuptake inhibition in a single molecule . there is strong evidence of synergistic antinociception between these two mechanisms of action . in preclinical and clinical testing , tapentadol has shown efficacy against both nociceptive and neuropathic pain . preclinical data indicate that tapentadol s -opioid agonism makes a greater contribution to analgesia in acute pain , while noradrenaline reuptake inhibition makes a greater contribution in chronic neuropathic pain models . tapentadol also produces fewer adverse events than oxycodone at equianalgesic doses , and thus may have a -sparing effect. current evidence indicates that tapentadol s efficacy / tolerability ratio may be better than those of classical opioids . however , further research is needed to establish its role in pain management .

Introduction Prevalence of low back pain Complexity of treating low back pain Discussion The pharmacology of tapentadol Tapentadol: preclinical testing Tapentadol: clinical trials in chronic LBP Conclusion

chronic pain represents a major healthcare problem which seriously impairs the quality of sufferers working and social lives , and the most common single location is the lower back.1 chronic low back pain ( lbp ) is difficult to treat because of the range of causative mechanisms that may be involved ; this difficulty is compounded by the limited efficacy of current pharmacological agents , and exemplified by the inconsistency of existing treatment patterns . in september 2009 , an international panel of clinical pain specialists met to discuss the challenges of chronic lbp , and to review the preclinical and clinical data relating to the new analgesic agent , tapentadol , which may offer a novel treatment option for chronic lbp . based on a literature search of pubmed before the meeting other presentations came from articles relating to the changes occurring in chronic lbp and the preclinical and clinical testing of tapentadol , as well as data on file from completed clinical trials . the economic implications for society are significant in terms of the utilization of healthcare resources.4 in the pan - european study , the most common location of chronic pain was the back , in 47% of sufferers ( 5% upper back , 18% lower back , 24% unspecified).1 this finding is consistent with other sources of prevalence data on low back pain , indicating an overall prevalence of 80.5 per 1000 population.5 it affects more women than men and the prevalence increases with age.5,6 in the us , lbp accounts for almost 30 million physician visits per year56% of which are to primary care physicians.7 thus lbp imposes a significant economic burden . the impact on quality of life is considerable.14 early diagnosis is therefore important , to incorporate these aspects of the condition into the management of low back pain and increase the chance of recovery.15 a 2006 survey of 46,394 adults in 15 european countries and israel found that 19% of respondents suffered from moderate to severe chronic pain ( 5 on a 10-point numeric rating scale [ nrs ] ) and nearly half received inadequate pain management.1 other surveys in norway2 and denmark3 have produced similar results . the economic implications for society are significant in terms of the utilization of healthcare resources.4 in the pan - european study , the most common location of chronic pain was the back , in 47% of sufferers ( 5% upper back , 18% lower back , 24% unspecified).1 this finding is consistent with other sources of prevalence data on low back pain , indicating an overall prevalence of 80.5 per 1000 population.5 it affects more women than men and the prevalence increases with age.5,6 in the us , lbp accounts for almost 30 million physician visits per year56% of which are to primary care physicians.7 thus lbp imposes a significant economic burden . the impact on quality of life is considerable.14 early diagnosis is therefore important , to incorporate these aspects of the condition into the management of low back pain and increase the chance of recovery.15 despite the substantial economic burden of chronic lbp , there is a lack of consensus on the best treatment.16 patients have very different levels of impairment , disability , and chronicity . cases of low impairment and disability may best be treated by simple evidence - based therapies exercises , cognitive - behavioral interventions , and medication but the multimechanistic nature of more severe chronic lbp means that no single intervention is likely to be effective.12,17 the complexity of treatment is further increased because chronic lbp may present with nociceptive pain , neuropathic pain , or both components.18,19 one survey using the painde - tect questionnaire found that 37% of chronic lbp patients suffered from neuropathic pain , while 35% had nociceptive pain and results for the remaining patients were unclear.20 a neuropathic component can complicate diagnosis for several reasons.21 firstly , the signs and symptoms can vary between patients and within individual patients over time . this is important because neuropathic pain often fails to respond to commonly prescribed analgesic therapy,21 treatment with single agent drugs being limited by incomplete efficacy and dose - limiting adverse effects.22 the major pharmacological agents currently used on - label to treat chronic lbp include nonopioids , such as paracetamol , nonsteroidal anti - inflammatory drugs ( nsaids ) , cyclooxygenase-2 ( cox-2 ) inhibitors , and opioids , while antidepressants , anticonvulsants , and local anesthetics may also be prescribed . for example , efficacy may be limited by an undetected neuropathic component , and poor tolerability may lead to an unfavorable efficacy / side effect ratio . these limitations can be addressed by combining agents with different mechanisms of action , to produce additive or synergistic analgesic effects and minimize drug - induced adverse events.24 this multimechanistic approach takes into account the diversity of underlying pain mechanisms and the different pharmacological principles required for their successful treatment it reduces the convenience of treatment , and thus compliance , and creates the potential for pharmacokinetic drug drug interactions.25 these may increase side effects or reduce analgesia , and the elderly , in particular , may be at increased risk.25 therefore the safety and efficacy of specific combinations must be empirically evaluated,22 but up to now clinical study data are inadequate . tapentadol ( see table 1 ) has been developed to have broad analgesic activity by combining known analgesic principles ( -opioid receptor agonism and noradrenaline reuptake inhibition ) in a single molecule , and to offer a better balance between efficacy and tolerability than classical opioids . an immediate release formulation has recently been registered in the us to treat moderate to severe acute pain , and both immediate and prolonged formulations are currently under - going registration in europe for the treatment of severe acute and chronic pain . via its -opioid action in the brain , tapentadol also influences the release of neurotransmitters by the descending pain pathways , producing a further inhibition of pain.26 like other systemic opioids , tapentadol acts at all these sites and its overall analgesic effect is enhanced by synergy between them.27 opioids can be effective in chronic neuropathic pain , but decreased potency requires higher doses in order to achieve sufficient analgesia.28,29 this characteristic of neuropathic pain , and the development of tolerance , both potentially increase the incidence of side effects . tapentadol addresses this problem by virtue of its second analgesic mechanism of action , ie , noradrenaline reuptake inhibition , particularly in chronic neuropathic pain . this property has been reported by studies in a number of animal models.30 noradrenaline reuptake inhibition exerts its antinociceptive effect via the descending pain pathways , where the increased synaptic noradrenaline binds to -2 receptors and thereby reduces pain signals to the brain.31 there is strong evidence of synergistic antinociception between this mechanism of action and -opioid agonism.3235 other analgesic agents also possess more than 1 mechanism of action ; for example , at first sight the established weak opioid tramadol appears similar to tapentadol , since it is a -opioid receptor agonist which also affects the monoaminergic system . both tapentadol s mechanisms of action reside in a single molecule that is metabolized via o - glucuronidation to an inactive metabolite.36 by contrast , tramadol is a racemic mixture of 2 enantiomers and produces an active metabolite . thus , the relative contribution of the different mechanisms of action to the overall analgesic effect changes over time.36 as the parent molecule is metabolised , noradrenaline reuptake inhibition and serotonin reuptake inhibition diminish , and -opioid receptor agonism increases . also , tramadol is metabolized mainly by the cytochrome p450 system , which is polymorphic in humans , so that poor metabolizers do not experience satisfactory analgesia with standard doses.36 further differences favoring tapentadol over tramadol are that evidence suggests that analgesia is more readily obtained by noradrenaline reuptake inhibition than by serotonin reuptake inhibition.36,37 despite having an affinity to the -opioid receptor 50 times lower than that of morphine , in vivo testing in rats and mice found that intravenous tapentadol was only 2 to 3 times less potent than morphine in acute nociceptive pain.36,37 in 2 animal models of chronic neuropathic pain it was broadly equipotent to morphine , despite this much lower binding affinity , which is important in terms of opioid side effects.36 thus noradrenaline reuptake inhibition may make a greater contribution to the analgesic efficacy of tapentadol in chronic pain than in acute pain . in chronic pain , however , the reverse applied ; antagonizing noradrenaline reuptake inhibition had the greater effect.30 low affinity for the -opioid receptor and the presence of noradrenaline reuptake inhibition suggest that tapentadol might produce fewer opioid - related side effects than classical -opioid receptor agonists . the emetic potential of tapentadol and morphine has been compared in ferrets ; tapentadol produced fewer retches and vomits per animal , and the duration of these effects was shorter.37 in mice , tapentadol had a weaker inhibitory effect than morphine on gastrointestinal motility.37 when the development of tolerance was investigated in rats , complete tolerance was significantly delayed ( p < 0.0001 ) in animals receiving tapentadol ( 23 days ) compared with those receiving morphine ( 10 days).36 to summarise , tapentadol s -opioid action decreased ascending pain messages as well as increasing pain inhibition via the descending pathways . because its distinct pharmacological profile differentiates tapentadol from other centrally acting analgesics , it has been proposed that tapentadol should be classified by its 2 mechanisms of action , -opioid receptor agonism ( mor ) and noradrenaline reuptake inhibition ( nri ) , as a mor - nri compound.38 tapentadol has been subject to an extensive testing program ; to date , around 8000 patients have participated in phase iii clinical trials with either the immediate - release ( ir ) or prolonged - release ( pr ) formulation . the results for tapentadol were supported by most secondary outcome parameters and also by more conservative imputations , such as worst observation carried forward ( wocf ) and baseline observation carried forward ( bocf).39 tapentadol also performed significantly better than placebo in all categories of the brief pain inventory , in overall sleep quality ratings and in the patients global impression of change ( pgic).39 tapentadol was well tolerated , the incidence of typical opioid adverse events being approximately half that of oxycodone.39 at dosages providing similar analgesic effects , tapentadol produced numerically lower levels of constipation ( 13.8% vs 26.8% ) , nausea ( 20.1% vs 34.5% ) , vomiting ( 9.1% vs 19.2% ) , dizziness ( 11.9% vs 17.1% ) and pruritis ( 7.2% vs 16.8%).39 cns effects tended to be milder in the tapentadol group than the oxycodone group.39 these tolerability findings are consistent with tapentadol having a -sparing effect , owing to the contribution of the nri component . this was achieved ( p < 0.001 ) ( see figure 4 ) and efficacy was confirmed by more conservative analyses of the primary endpoint , including bocf , wocf , and the proportions of patients showing 30% and 50% improvement.40 long - term safety has been evaluated in a phase iii , open - label , randomized , active - controlled study.41 a total of 1117 patients with chronic lbp or osteoarthritis were randomised ( 4:1 ) to receive tapentadol pr or oxycodone cr . it was effective against both nociceptive and neuropathic pain . at equianalgesic doses , tapentadol produced a lower incidence of side effects than oxycodone , leading to fewer patients withdrawing from treatment . tapentadol ( see table 1 ) has been developed to have broad analgesic activity by combining known analgesic principles ( -opioid receptor agonism and noradrenaline reuptake inhibition ) in a single molecule , and to offer a better balance between efficacy and tolerability than classical opioids . an immediate release formulation has recently been registered in the us to treat moderate to severe acute pain , and both immediate and prolonged formulations are currently under - going registration in europe for the treatment of severe acute and chronic pain . via its -opioid action in the brain , tapentadol also influences the release of neurotransmitters by the descending pain pathways , producing a further inhibition of pain.26 like other systemic opioids , tapentadol acts at all these sites and its overall analgesic effect is enhanced by synergy between them.27 opioids can be effective in chronic neuropathic pain , but decreased potency requires higher doses in order to achieve sufficient analgesia.28,29 this characteristic of neuropathic pain , and the development of tolerance , both potentially increase the incidence of side effects . tapentadol addresses this problem by virtue of its second analgesic mechanism of action , ie , noradrenaline reuptake inhibition , particularly in chronic neuropathic pain . this property has been reported by studies in a number of animal models.30 noradrenaline reuptake inhibition exerts its antinociceptive effect via the descending pain pathways , where the increased synaptic noradrenaline binds to -2 receptors and thereby reduces pain signals to the brain.31 there is strong evidence of synergistic antinociception between this mechanism of action and -opioid agonism.3235 other analgesic agents also possess more than 1 mechanism of action ; for example , at first sight the established weak opioid tramadol appears similar to tapentadol , since it is a -opioid receptor agonist which also affects the monoaminergic system . both tapentadol s mechanisms of action reside in a single molecule that is metabolized via o - glucuronidation to an inactive metabolite.36 by contrast , tramadol is a racemic mixture of 2 enantiomers and produces an active metabolite . thus , the relative contribution of the different mechanisms of action to the overall analgesic effect changes over time.36 as the parent molecule is metabolised , noradrenaline reuptake inhibition and serotonin reuptake inhibition diminish , and -opioid receptor agonism increases . also , tramadol is metabolized mainly by the cytochrome p450 system , which is polymorphic in humans , so that poor metabolizers do not experience satisfactory analgesia with standard doses.36 further differences favoring tapentadol over tramadol are that evidence suggests that analgesia is more readily obtained by noradrenaline reuptake inhibition than by serotonin reuptake inhibition.36,37 despite having an affinity to the -opioid receptor 50 times lower than that of morphine , in vivo testing in rats and mice found that intravenous tapentadol was only 2 to 3 times less potent than morphine in acute nociceptive pain.36,37 in 2 animal models of chronic neuropathic pain it was broadly equipotent to morphine , despite this much lower binding affinity , which is important in terms of opioid side effects.36 thus noradrenaline reuptake inhibition may make a greater contribution to the analgesic efficacy of tapentadol in chronic pain than in acute pain . in chronic pain , however , the reverse applied ; antagonizing noradrenaline reuptake inhibition had the greater effect.30 low affinity for the -opioid receptor and the presence of noradrenaline reuptake inhibition suggest that tapentadol might produce fewer opioid - related side effects than classical -opioid receptor agonists . the emetic potential of tapentadol and morphine has been compared in ferrets ; tapentadol produced fewer retches and vomits per animal , and the duration of these effects was shorter.37 in mice , tapentadol had a weaker inhibitory effect than morphine on gastrointestinal motility.37 when the development of tolerance was investigated in rats , complete tolerance was significantly delayed ( p < 0.0001 ) in animals receiving tapentadol ( 23 days ) compared with those receiving morphine ( 10 days).36 to summarise , tapentadol s -opioid action decreased ascending pain messages as well as increasing pain inhibition via the descending pathways . because its distinct pharmacological profile differentiates tapentadol from other centrally acting analgesics , it has been proposed that tapentadol should be classified by its 2 mechanisms of action , -opioid receptor agonism ( mor ) and noradrenaline reuptake inhibition ( nri ) , as a mor - nri compound.38 tapentadol has been subject to an extensive testing program ; to date , around 8000 patients have participated in phase iii clinical trials with either the immediate - release ( ir ) or prolonged - release ( pr ) formulation . the results for tapentadol were supported by most secondary outcome parameters and also by more conservative imputations , such as worst observation carried forward ( wocf ) and baseline observation carried forward ( bocf).39 tapentadol also performed significantly better than placebo in all categories of the brief pain inventory , in overall sleep quality ratings and in the patients global impression of change ( pgic).39 tapentadol was well tolerated , the incidence of typical opioid adverse events being approximately half that of oxycodone.39 at dosages providing similar analgesic effects , tapentadol produced numerically lower levels of constipation ( 13.8% vs 26.8% ) , nausea ( 20.1% vs 34.5% ) , vomiting ( 9.1% vs 19.2% ) , dizziness ( 11.9% vs 17.1% ) and pruritis ( 7.2% vs 16.8%).39 cns effects tended to be milder in the tapentadol group than the oxycodone group.39 these tolerability findings are consistent with tapentadol having a -sparing effect , owing to the contribution of the nri component . this was achieved ( p < 0.001 ) ( see figure 4 ) and efficacy was confirmed by more conservative analyses of the primary endpoint , including bocf , wocf , and the proportions of patients showing 30% and 50% improvement.40 long - term safety has been evaluated in a phase iii , open - label , randomized , active - controlled study.41 a total of 1117 patients with chronic lbp or osteoarthritis were randomised ( 4:1 ) to receive tapentadol pr or oxycodone cr . it was effective against both nociceptive and neuropathic pain . at equianalgesic doses , tapentadol produced a lower incidence of side effects than oxycodone , leading to fewer patients withdrawing from treatment . the treatment of chronic lbp is challenging for various reasons , and requires a multidisciplinary approach to treatment , encompassing pharmacological , psychological , and physical therapies . tapentadol combines -opioid receptor agonism and noradrenaline reuptake inhibition in a single molecule , with the object of achieving an opioid - sparing effect , that is , to produce fewer opioid - related side effects for a given level of analgesia . to date , preclinical and clinical data support the pharmacological classification of tapentadol as an mor - nri30,38 and suggest that its efficacy / tolerability ratio may be better than those of classical opioids.36,37,39,41 further research is required , however , particularly into efficacy and safety in patients with pure neuropathic pain and to provide specific data on lbp with a neuropathic component .

abdominal pain is a common reason to seek medical care at the emergency department ( ed ) [ 1 , 2 ] . for about half of the patients , some sort of radiological examination is requested [ 35 ] . it is therefore important to evaluate different management strategies at the ed for improvement of diagnostic accuracy to optimize patient care and the use of health care resources . it is possible to perform ultrasound ( us ) examinations bedside and they do not have any known side effects , which makes them suitable for the use at the ed . bedside abdominal us performed at the ed , as well as computer tomography ( ct ) examination at an early stage , has been shown to increase diagnostic accuracy as well as diagnostic certainty when a patient presents with abdominal pain of unknown origin [ 711 ] . abdominal ultrasound is known to increase diagnostic accuracy for patients presenting with upper right abdominal pain [ 1214 ] . the results for diagnosing appendicitis with the help of us are still controversial and the diagnostic accuracy is operator dependent . several studies , though , have shown good results of us for diagnosis of appendicitis [ 1618 ] . an immediate ultrasound examination may not only increase diagnostic accuracy , but also provide additional information making it easier for the surgeon to determine the patient s need for operation at an earlier stage [ 4 , 13 ] . the aim of this study was to determine the effect of surgeon - performed us bedside at the ed , based on several patient characteristics , on diagnostic accuracy and further management of patients admitted to the ed for abdominal pain . the study was conducted between february 2004 and june 2005 at the ed of stockholm south general hospital , a public general hospital with a catchment area of about 600,000 inhabitants . nine surgeons with at least 2 years experience of surgery after completing internship participated in the study . the surgeons attended a 1-week course given by a specialist in ultrasound examination followed by 3 weeks of training in the radiological department in abdominal ultrasound , under the guidance of an ultrasound specialist . the surgeons were trained in detecting the following disease states : gallbladder stones , cholecystitis , wide bile ducts , hydronephrosis , abdominal aortic aneurysms , ovarial cysts , free abdominal fluid , pleura fluid collections , large abdominal masses , inflamed appendix , diverticulitis , intestinal obstruction , liver disease and large kidney stones . all patients , 18 years or older , admitted to the emergency ward for abdominal pain were eligible to participate in the study . exclusion criteria were pregnancy , previously diagnosed abdominal condition , acute conditions needing immediate care , inability to communicate with the investigator , drug or alcohol addiction and dementia . eight hundred patients were enrolled for the study . after inclusion , the patients were examined by the study surgeon . the study surgeon set a first preliminary diagnosis and then opened a sealed randomization envelope randomizing the patient to us or not . if randomized to the us group , the examination was performed with one out of two handheld , 2.55 mhz or 4.36 mhz , curved array transducers ( b k medical , denmark , hawk 2102 , transducers type 8665 and 8802 ) screening the entire abdomen . the two groups were subsequently managed according to clinical routine as decided by the study surgeon . the correct diagnosis was defined as the final diagnosis set by a senior surgeon 68 weeks after the patient had entered the study , based on information in the patient records . the senior surgeon was not aware of the preliminary diagnosis set by the surgeon at the ed . the final diagnosis was then compared with the preliminary diagnosis , with or without us examination . all information on the patients collected in the ed was entered by the study surgeon on a case report form . additional data about the patients who were admitted to the hospital for in - patient care were collected from the patient records and entered on a complementary case report form , designed for the admission period . we examined selected outcomes in different subgroups based on body mass index ( bmi ) , age , level of c - reactive protein ( crp ) , signs of peritonitis , symptoms predictable for appendicitis ( pain and tenderness in lower right abdomen ) , gallbladder disease ( pain and tenderness in upper right abdomen ) and first preliminary diagnosis of appendicitis , gallbladder disease or non - specific abdominal pain set at the ed before randomization . the outcomes analyzed were diagnostic accuracy , admission rate and amount of further examinations ordered at the ed [ us examinations and computer tomography ( ct ) scans from the radiological department or any other further examinations ] . in the bmi groups , we also examined level of difficulty and reliability of the us examination as assessed by the examining surgeon . for patients with signs of peritonitis chi - square test was used to compare groups regarding diagnostic accuracy , amount of requested complimentary examinations and hospital admission . if surgery was needed , we also compared the groups regarding when the decision on whether or not to perform surgery was taken . the results were regarded as significant if p was less than 0.05 , two - tailed . the sample size was calculated on the basis of the primary outcome of the study , diagnostic accuracy , presented in an earlier article . the patients received oral and written information from the study surgeon , and were included after informed consent . the study was approved by the institutional review board at karolinska institutet , stockholm , sweden . chi - square test was used to compare groups regarding diagnostic accuracy , amount of requested complimentary examinations and hospital admission . if surgery was needed , we also compared the groups regarding when the decision on whether or not to perform surgery was taken . the results were regarded as significant if p was less than 0.05 , two - tailed . the sample size was calculated on the basis of the primary outcome of the study , diagnostic accuracy , presented in an earlier article . the patients received oral and written information from the study surgeon , and were included after informed consent . the study was approved by the institutional review board at karolinska institutet , stockholm , sweden . among the 800 patients randomized in the study , one patient was missing due to loss of the study protocol and eight patients in each group did not fulfill the inclusion criteria . thus , 392 patients in the ultrasound group and 391 patients in the control group were eligible for statistical analysis ( fig . 1 ) . two patients in the ultrasound group and one patient in the control group switched groups.fig . 1study flow chart the baseline characteristics for the study subjects are shown in table 1.table 1baseline characteristics of patients with abdominal pain at the edcharacteristicsultrasound ( n = 392)non - ultrasound ( n = 391)meansdn%meansdn%age47204819height172917210weight73167316bmi ( body mass index)24.84.524.84.3gender male16040.817143.7 female23259.222056.3abdominal related comorbidity7619.47819.9comorbidity related to heart or diabetes6616.87418.9history of abdominal malignancy61.5123.1history of other malignancy112.8143.6other comorbidity13233.712331.5admission for abdominal pain within 1 year12432.013735.3referral for admission9224.412632.9duration of pain 08 h4414.84314.4 824 h9933.29732.4 > 24 h14749.315150.5 can not answer82.782.7affected general condition9023.37419.1tenderness33886.434789.2rigidity5113.14912.6palpable mass235.9297.5actual vas ( of pain)4.32.84.42.6maximal anamnestic vas ( of pain)7.62.67.61.8temperature37.00.837.00.7vas ( of pain ) = visual analogue scale ( scale 010 , 0 represents no pain at all , 10 represents unbearable pain ) baseline characteristics of patients with abdominal pain at the ed vas ( of pain ) = visual analogue scale ( scale 010 , 0 represents no pain at all , 10 represents unbearable pain ) table 2 summarizes the benefits of bedside us in the different evaluated subgroups . some sort of benefit was seen in all groups except among the patients with a preliminary first set diagnosis of appendicitis , where the intervention groups were equal regarding all outcomes.table 2benefits of us examinations in different subgroupsdiagnostic accuracyadmission frequencyrequested us at radiological departmentrequested ct at radiological departmentany other examination requestedbmi < 25xx 25xxxxcrp < 10xx 10xxxlower abdominal symptomsxupper abdominal symptomsxxxgallbladder diseasexxappendicitisnsapxxxperitonitisxxxage < 30xx 3059xxxx 60xx statistically significant benefits benefits of us examinations in different subgroups x statistically significant benefits in the age group of 3059 years , diagnostic accuracy was higher and requests for ct examinations were fewer among the patients examined with us . in all age groups , fewer complementary radiological us examinations were ordered in the us group as well as fewer further examinations except from the oldest group ( table 3).table 3results based on age groupsage < 30 ( n = 177)age 3059 ( n = 388)age 60 ( n = 218)us ( n = 87 ) [ % ( n)]non - us ( n = 90 ) [ % ( n)]p valueus ( n = 198 ) [ % ( n)]non - us ( n = 190 ) [ % ( n)]p valueus ( n = 107 ) [ % ( n)]non - us ( n = 111 ) [ % ( n)]p valuediagnostic accuracy65 ( 56)60 ( 52)0.46868 ( 130)58 ( 109)0.04258 ( 61)52 ( 55)0.405admission38 ( 33)47 ( 42)0.24040 ( 79)44 ( 84)0.39052 ( 56)63 ( 70)0.109ultrasound ordered3 ( 3)27 ( 24)<0.00110 ( 19)28 ( 53)<0.00111 ( 12)27 ( 30)0.003ct ordered2 ( 2)2 ( 2)0.9735 ( 9)12 ( 22)0.01119 ( 20)14 ( 15)0.297no other examination ordered59 ( 51)38 ( 34)0.00651 ( 101)29 ( 55)<0.00137 ( 39)27 ( 30)0.134partially missing data in maximum seven patients per group and analysis results based on age groups partially missing data in maximum seven patients per group and analysis in the group with bmi 25 or more , the diagnostic accuracy was higher and there were fewer requests for computer tomography examinations if the patient had been examined with us . the frequency of complementary us examinations at the radiological department or other further examinations was lower in the us group regardless of bmi ( table 4).table 4results based on body mass index ( bmi ) and c - reactive protein ( crp)bmi < 25 ( n = 436)bmi 25 ( n = 317)crp < 10 ( n = 403)crp 10 ( n = 367)us ( n = 223 ) [ % ( n)]non - us ( n = 213 ) [ % ( n)]p valueus ( n = 160 ) [ % ( n)]non - us ( n = 157 ) [ % ( n)]p valueus ( n = 219 ) [ % ( n)]non - us ( n = 184 ) [ % ( n)]p valueus ( n = 163 ) [ % ( n)]non - us ( n = 204 ) [ % ( n)]p valuediagnostic accuracy62 ( 135)58 ( 119)0.41667 ( 105)54 ( 85)0.02065 ( 142)62 ( 112)0.50663 ( 98)52 ( 103)0.047admission44 ( 97)48 ( 102)0.35843 ( 69)52 ( 82)0.10525 ( 55)34 ( 62)0.05968 ( 111)65 ( 132)0.495ultrasound ordered7 ( 15)25 ( 53)<0.00111 ( 18)29 ( 45)<0.0017 ( 16)26 ( 48)<0.00111 ( 18)28 ( 57)<0.001ct ordered8 ( 18)7 ( 15)0.7057 ( 11)14 ( 22)0.0414 ( 8)4 ( 8)0.71914 ( 22)14 ( 29)0.843no other examination ordered47 ( 104)33 ( 70)0.00454 ( 85)27 ( 42)<0.00151 ( 110)34 ( 62)0.00146 ( 75)28 ( 57)<0.001partially missing data in maximum nine patients per group and analysis results based on body mass index ( bmi ) and c - reactive protein ( crp ) partially missing data in maximum nine patients per group and analysis there was a significant difference in frequency of difficulty in performing us , when comparing patients with bmi 25 or more with bmi less than 25 ( 59 vs. 23% , p < 0.001 ) , whereas reliability was virtually the same between the groups ( 83 vs. 89% , p = 0.205 ) . diagnostic accuracy for the us group among the patients with elevated crp - level was higher , but not among patients with normal crp . admission frequency or request of ct examination did not differ between the intervention groups regardless of crp - level . the number of requested radiological us examinations or any other further examination was lower among the patients in the us group regardless of crp - level ( table 4 ) . a diagnosis of gallbladder concrement and/or cholecystitis set as the first emergency diagnosis before randomization was associated with a decreased number of further us examinations and further examinations in the us group ( table 5 ) . in the patients presenting with symptoms of gallbladder disease ( pain and tenderness in right upper abdomen ) , there was also a higher diagnostic accuracy as well as fewer requested radiological us examinations and further examinations , if examined with bedside us at the ed ( table 6).table 5results based on first set preliminary diagnosis at the edfirst emergency diagnosis set as gallbladder concrement and/or cholecystitis ( n = 61)appendicitis abscess as first emergency diagnosis ( n = 55)nsap as first emergency diagnosis ( n = 370)us ( n = 27 ) [ % ( n)]non - us n = 34 [ % ( n)]p valueus ( n = 31 ) [ % ( n)]non - us n = 24 [ % ( n)]p valueus ( n = 189 ) [ % ( n)]non - us ( n = 181 ) [ % ( n)]p valuediagnostic accuracy70 ( 19)62 ( 21)0.48264 ( 20)54 ( 13)0.43462 ( 118)55 ( 99)0.306admission52 ( 14)62 ( 21)0.43797 ( 30)100 ( 24)0.37531 ( 59)45 ( 81)0.007ultrasound ordered26 ( 7)82 ( 27)<0.0016 ( 2)21 ( 5)0.1125 ( 10)30 ( 55)0.001ct ordered0 ( 0)0 ( 0)13 ( 4)8 ( 2)0.59010 ( 19)10 ( 19)0.902no other examination ordered63 ( 17)18 ( 6)<0.00171 ( 22)46 ( 11)0.05948 ( 91)32 ( 57)0.001partially missing data in maximum three patients per group and analysistable 6results based on symptoms and signspain and tenderness in right upper abdomen ( n = 101)pain and tenderness in right lower abdomen ( n = 187)peritonitis ( n = 100)us n = 54 [ % ( n)]non - us ( n = 47 ) [ % ( n)]p valueus n = 91 [ % ( n)]non - us ( n = 96 ) [ % ( n)]p valueus ( n = 51 ) [ % ( n)]non - us ( n = 49 ) [ % ( n)]p valuediagnostic accuracy72 ( 38)52 ( 24)0.04559 ( 53)54 ( 51)0.47674 ( 37)54 ( 26)0.041admission50 ( 27)49 ( 23)0.91562 ( 56)58 ( 56)0.65590 ( 46)84 ( 41)0.332ultrasound ordered22 ( 12)74 ( 34)<0.0013 ( 3)22 ( 21)<0.00110 ( 5)29 ( 14)0.017ct ordered9 ( 5)6 ( 3)0.6158 ( 9)9 ( 8)0.91116 ( 8)24 ( 12)0.271no other examination ordered54 ( 29)17 ( 8)<0.00150 ( 45)45 ( 43)0.52353 ( 27)22 ( 11)0.002partially missing data in maximum one patient per group and analysisof these patients , 23 in us group and 26 in non - us group were admitted for surgery . 14 ( 60.9% ) in us group were admitted already at ed and 5 ( 19.2% ) in non - us group , p = 0.003 results based on first set preliminary diagnosis at the ed partially missing data in maximum three patients per group and analysis results based on symptoms and signs partially missing data in maximum one patient per group and analysis of these patients , 23 in us group and 26 in non - us group were admitted for surgery . 14 ( 60.9% ) in us group were admitted already at ed and 5 ( 19.2% ) in non - us group , p = 0.003 the only difference found among the patients with symptoms of appendicitis ( pain and tenderness in right lower abdomen ) was in the request for complementary us examinations at the radiological department with fewer requests in the intervention group ( table 6 ) . in 100 patients , the physical examination of the patient showed signs of peritonitis . among these patients , bedside us had an effect of higher diagnostic accuracy and fewer requests for radiological us examinations or any other further examination . as expected , the admission frequency was high in this group of patients , and did not differ between the comparison groups ( table 6 ) . from the patients in this critically ill group , 23 patients in the us group and 26 in the non - us group were admitted for surgery . of these patients requiring surgery , 14 ( 60.9% ) in the us group and 5 ( 19.2% ) in the non - us group were admitted for surgery with the decision taken while still at the ed ( p = 0.003 ) . the number of radiological us and further examinations was also lower in the group examined with bedside us ( table 5 ) . among the 800 patients randomized in the study , one patient was missing due to loss of the study protocol and eight patients in each group did not fulfill the inclusion criteria . thus , 392 patients in the ultrasound group and 391 patients in the control group were eligible for statistical analysis ( fig . 1 ) . two patients in the ultrasound group and one patient in the control group switched groups.fig . 1study flow chart the baseline characteristics for the study subjects are shown in table 1.table 1baseline characteristics of patients with abdominal pain at the edcharacteristicsultrasound ( n = 392)non - ultrasound ( n = 391)meansdn%meansdn%age47204819height172917210weight73167316bmi ( body mass index)24.84.524.84.3gender male16040.817143.7 female23259.222056.3abdominal related comorbidity7619.47819.9comorbidity related to heart or diabetes6616.87418.9history of abdominal malignancy61.5123.1history of other malignancy112.8143.6other comorbidity13233.712331.5admission for abdominal pain within 1 year12432.013735.3referral for admission9224.412632.9duration of pain 08 h4414.84314.4 824 h9933.29732.4 > 24 h14749.315150.5 can not answer82.782.7affected general condition9023.37419.1tenderness33886.434789.2rigidity5113.14912.6palpable mass235.9297.5actual vas ( of pain)4.32.84.42.6maximal anamnestic vas ( of pain)7.62.67.61.8temperature37.00.837.00.7vas ( of pain ) = visual analogue scale ( scale 010 , 0 represents no pain at all , 10 represents unbearable pain ) baseline characteristics of patients with abdominal pain at the ed vas ( of pain ) = visual analogue scale ( scale 010 , 0 represents no pain at all , 10 represents unbearable pain ) some sort of benefit was seen in all groups except among the patients with a preliminary first set diagnosis of appendicitis , where the intervention groups were equal regarding all outcomes.table 2benefits of us examinations in different subgroupsdiagnostic accuracyadmission frequencyrequested us at radiological departmentrequested ct at radiological departmentany other examination requestedbmi < 25xx 25xxxxcrp < 10xx 10xxxlower abdominal symptomsxupper abdominal symptomsxxxgallbladder diseasexxappendicitisnsapxxxperitonitisxxxage < 30xx 3059xxxx 60xx statistically significant benefits benefits of us examinations in different subgroups x statistically significant benefits in the age group of 3059 years , diagnostic accuracy was higher and requests for ct examinations were fewer among the patients examined with us . in all age groups , fewer complementary radiological us examinations were ordered in the us group as well as fewer further examinations except from the oldest group ( table 3).table 3results based on age groupsage < 30 ( n = 177)age 3059 ( n = 388)age 60 ( n = 218)us ( n = 87 ) [ % ( n)]non - us ( n = 90 ) [ % ( n)]p valueus ( n = 198 ) [ % ( n)]non - us ( n = 190 ) [ % ( n)]p valueus ( n = 107 ) [ % ( n)]non - us ( n = 111 ) [ % ( n)]p valuediagnostic accuracy65 ( 56)60 ( 52)0.46868 ( 130)58 ( 109)0.04258 ( 61)52 ( 55)0.405admission38 ( 33)47 ( 42)0.24040 ( 79)44 ( 84)0.39052 ( 56)63 ( 70)0.109ultrasound ordered3 ( 3)27 ( 24)<0.00110 ( 19)28 ( 53)<0.00111 ( 12)27 ( 30)0.003ct ordered2 ( 2)2 ( 2)0.9735 ( 9)12 ( 22)0.01119 ( 20)14 ( 15)0.297no other examination ordered59 ( 51)38 ( 34)0.00651 ( 101)29 ( 55)<0.00137 ( 39)27 ( 30)0.134partially missing data in maximum seven patients per group and analysis results based on age groups partially missing data in maximum seven patients per group and analysis in the group with bmi 25 or more , the diagnostic accuracy was higher and there were fewer requests for computer tomography examinations if the patient had been examined with us . the frequency of complementary us examinations at the radiological department or other further examinations was lower in the us group regardless of bmi ( table 4).table 4results based on body mass index ( bmi ) and c - reactive protein ( crp)bmi < 25 ( n = 436)bmi 25 ( n = 317)crp < 10 ( n = 403)crp 10 ( n = 367)us ( n = 223 ) [ % ( n)]non - us ( n = 213 ) [ % ( n)]p valueus ( n = 160 ) [ % ( n)]non - us ( n = 157 ) [ % ( n)]p valueus ( n = 219 ) [ % ( n)]non - us ( n = 184 ) [ % ( n)]p valueus ( n = 163 ) [ % ( n)]non - us ( n = 204 ) [ % ( n)]p valuediagnostic accuracy62 ( 135)58 ( 119)0.41667 ( 105)54 ( 85)0.02065 ( 142)62 ( 112)0.50663 ( 98)52 ( 103)0.047admission44 ( 97)48 ( 102)0.35843 ( 69)52 ( 82)0.10525 ( 55)34 ( 62)0.05968 ( 111)65 ( 132)0.495ultrasound ordered7 ( 15)25 ( 53)<0.00111 ( 18)29 ( 45)<0.0017 ( 16)26 ( 48)<0.00111 ( 18)28 ( 57)<0.001ct ordered8 ( 18)7 ( 15)0.7057 ( 11)14 ( 22)0.0414 ( 8)4 ( 8)0.71914 ( 22)14 ( 29)0.843no other examination ordered47 ( 104)33 ( 70)0.00454 ( 85)27 ( 42)<0.00151 ( 110)34 ( 62)0.00146 ( 75)28 ( 57)<0.001partially missing data in maximum nine patients per group and analysis results based on body mass index ( bmi ) and c - reactive protein ( crp ) partially missing data in maximum nine patients per group and analysis there was a significant difference in frequency of difficulty in performing us , when comparing patients with bmi 25 or more with bmi less than 25 ( 59 vs. 23% , p < 0.001 ) , whereas reliability was virtually the same between the groups ( 83 vs. 89% , p = 0.205 ) . diagnostic accuracy for the us group among the patients with elevated crp - level was higher , but not among patients with normal crp . admission frequency or request of ct examination did not differ between the intervention groups regardless of crp - level . the number of requested radiological us examinations or any other further examination was lower among the patients in the us group regardless of crp - level ( table 4 ) . a diagnosis of gallbladder concrement and/or cholecystitis set as the first emergency diagnosis before randomization was associated with a decreased number of further us examinations and further examinations in the us group ( table 5 ) . in the patients presenting with symptoms of gallbladder disease ( pain and tenderness in right upper abdomen ) , there was also a higher diagnostic accuracy as well as fewer requested radiological us examinations and further examinations , if examined with bedside us at the ed ( table 6).table 5results based on first set preliminary diagnosis at the edfirst emergency diagnosis set as gallbladder concrement and/or cholecystitis ( n = 61)appendicitis abscess as first emergency diagnosis ( n = 55)nsap as first emergency diagnosis ( n = 370)us ( n = 27 ) [ % ( n)]non - us n = 34 [ % ( n)]p valueus ( n = 31 ) [ % ( n)]non - us n = 24 [ % ( n)]p valueus ( n = 189 ) [ % ( n)]non - us ( n = 181 ) [ % ( n)]p valuediagnostic accuracy70 ( 19)62 ( 21)0.48264 ( 20)54 ( 13)0.43462 ( 118)55 ( 99)0.306admission52 ( 14)62 ( 21)0.43797 ( 30)100 ( 24)0.37531 ( 59)45 ( 81)0.007ultrasound ordered26 ( 7)82 ( 27)<0.0016 ( 2)21 ( 5)0.1125 ( 10)30 ( 55)0.001ct ordered0 ( 0)0 ( 0)13 ( 4)8 ( 2)0.59010 ( 19)10 ( 19)0.902no other examination ordered63 ( 17)18 ( 6)<0.00171 ( 22)46 ( 11)0.05948 ( 91)32 ( 57)0.001partially missing data in maximum three patients per group and analysistable 6results based on symptoms and signspain and tenderness in right upper abdomen ( n = 101)pain and tenderness in right lower abdomen ( n = 187)peritonitis ( n = 100)us n = 54 [ % ( n)]non - us ( n = 47 ) [ % ( n)]p valueus n = 91 [ % ( n)]non - us ( n = 96 ) [ % ( n)]p valueus ( n = 51 ) [ % ( n)]non - us ( n = 49 ) [ % ( n)]p valuediagnostic accuracy72 ( 38)52 ( 24)0.04559 ( 53)54 ( 51)0.47674 ( 37)54 ( 26)0.041admission50 ( 27)49 ( 23)0.91562 ( 56)58 ( 56)0.65590 ( 46)84 ( 41)0.332ultrasound ordered22 ( 12)74 ( 34)<0.0013 ( 3)22 ( 21)<0.00110 ( 5)29 ( 14)0.017ct ordered9 ( 5)6 ( 3)0.6158 ( 9)9 ( 8)0.91116 ( 8)24 ( 12)0.271no other examination ordered54 ( 29)17 ( 8)<0.00150 ( 45)45 ( 43)0.52353 ( 27)22 ( 11)0.002partially missing data in maximum one patient per group and analysisof these patients , 23 in us group and 26 in non - us group were admitted for surgery . 14 ( 60.9% ) in us group were admitted already at ed and 5 ( 19.2% ) in non - us group , p = 0.003 results based on first set preliminary diagnosis at the ed partially missing data in maximum three patients per group and analysis results based on symptoms and signs partially missing data in maximum one patient per group and analysis of these patients , 23 in us group and 26 in non - us group were admitted for surgery . 14 ( 60.9% ) in us group were admitted already at ed and 5 ( 19.2% ) in non - us group , p = 0.003 the only difference found among the patients with symptoms of appendicitis ( pain and tenderness in right lower abdomen ) was in the request for complementary us examinations at the radiological department with fewer requests in the intervention group ( table 6 ) . in 100 patients , the physical examination of the patient showed signs of peritonitis . among these patients , bedside us had an effect of higher diagnostic accuracy and fewer requests for radiological us examinations or any other further examination . as expected , the admission frequency was high in this group of patients , and did not differ between the comparison groups ( table 6 ) . from the patients in this critically ill group , 23 patients in the us group and 26 in the non - us group were admitted for surgery . of these patients requiring surgery , 14 ( 60.9% ) in the us group and 5 ( 19.2% ) in the non - us group were admitted for surgery with the decision taken while still at the ed ( p = 0.003 ) . the number of radiological us and further examinations was also lower in the group examined with bedside us ( table 5 ) . this study is based on a large randomized clinical trial from which we have proceeded with a thorough subgroup analysis . the overall results of the randomized clinical trial have previously been presented in two earlier papers [ 4 , 10 ] . our results show that surgeon - performed us is of higher value in overweight patients ( bmi 25 or more ) compared to patients with lower bmi . in this specific group , we did not only have an effect on diagnostic accuracy but also a decrease in requests for radiological us examinations as well as further examinations including ct scan . this is quite surprising since a high bmi is generally considered to hamper us examinations , a fact which is supported by a previous study showing a lower diagnostic accuracy in overweight patients ( bmi > 25 ) for diagnosing appendicitis . our results endorse the fact that us is more difficult to perform in overweight patients , but that it is still of great value . . this may give the additional bedside us examination a relatively high value for diagnosing and further management purpose . the slightly larger number of examinations ordered in the overweight group may also be because the surgeon feels insecure of the clinical examination performed . one should point out that although the surgeons in our study considered the us more difficult to perform in the bmi > 25 group , they considered the performed examination reliable to the same extent in both weight groups . this is supported by the fact that more ct scans were not ordered in the overweight us groups . the bedside us examination also gave a higher diagnostic accuracy in the group with elevated crp . appendicitis and cholecystitis , diagnoses that are normally connected with elevated crp , also had high diagnostic accuracy which might be an explanation for this finding . we consider the finding important , since an elevated crp generally indicates a more serious abdominal condition with need for immediate surgical treatment . another category of severely ill patients , with a high risk of needing immediate surgery , are those with signs of peritonitis . in this group , we likewise had a higher diagnostic accuracy with the help of us examination . even more important though , these patients were , to a higher rate , admitted to surgery while still at the ed . this may of course reduce the risk of complications due to doctor s delay . regarding age groups , the lowest diagnostic accuracy at the ed for both intervention groups we could though not show any increase in diagnostic accuracy in these older patients with the help of us . the only age group showing a significantly higher diagnostic accuracy with the help of us was the middle age group between 30 and 59 years . when grouping the patients according to symptoms suggesting appendicitis ( right lower abdominal pain ) and gallbladder disease ( right upper abdominal pain ) , there was only an increase in diagnostic accuracy for the patients with right upper abdominal pain . this is in line with earlier studies showing a high diagnostic accuracy with the help of us for this group of patients [ 2224 ] . the need for radiological examinations was lower among patients presenting with either right upper or lower abdominal pain , if examined with bedside us which shows that the study surgeon had confidence in the us performed bedside and did not require a confirmation of the us by another radiological examination . gallbladder disease and appendicitis are diagnoses in which it is earlier shown that us is of diagnostic value [ 1517 , 25 ] . however , we could not show any effect on diagnostic accuracy if one of these specific diagnoses was set at the ed as the first preliminary diagnosis . a reason for this might be that us does not contribute to the same extent to the diagnosis when the clinical and laboratory tests point to a specific diagnosis . in the group with localized pain and tenderness in the right upper abdomen , the relative effect of the us examination is probably higher , which gives us a small , but significant , higher diagnostic accuracy with the us examination . the importance of the us examination is , however , best illustrated by the fact that three times as many us examinations were ordered at the radiological department in the group not examined with bedside us . this was true both if gallbladder disease was set as first diagnosis as well as if there were symptoms of the disease ( table 5 ) . from this , we draw the conclusion that bedside us is indeed of great value in these patients , not to set the diagnosis , but to confirm it before surgery . acute nsap , generally defined as acute abdominal pain of under 7 days duration and for which there is no diagnosis after examination and baseline investigations , is a common cause for admission at the ed , including about half of the patients admitted at the ed for abdominal pain . the need for admission to a hospital ward for these patients was significantly reduced in our study when the patients were examined with us at the ed . a question that might be raised is the possible long - term side effects of the method . in an earlier study , we have nevertheless not found any differences in 2-year health care consumption or mortality between patients examined with bedside ultrasound or not at the ed when admitted for abdominal pain . it is reasonable to assume that a single ultrasound examination has very little impact on mortality and long - term health condition , but a great impact on the management of the actual condition , as shown in this study . the outcomes measured were primary and secondary outcomes for the study including the whole group of patients . this makes the statistical power less and the detected differences yield lower evidence when the results are generalized . the strengths of our study are that this is a randomized study including a large number of patients , and that we have achieved a nearly complete follow - up . all the more , the data were collected prospectively , and the large number of patients included makes the power in the comparisons in the subgroups acceptable . this study shows that surgeon - performed us at the ed for abdominal pain can be helpful in several ways for the majority of patients admitted to the ed for abdominal pain . the benefit is even more pronounced among patients that are overweight . for patients with peritonitis , taking into account other shown benefits and the lack of adverse effects , we find the method well worth consideration for implementation at the ed .

purposeto evaluate the effect of surgeon - performed ultrasound on acute abdomen in specific patient subgroups regarding the diagnostic accuracy and further management.methodseight hundred patients attending the emergency department at stockholm south general hospital , sweden , for abdominal pain , were randomized to either receive or not receive surgeon - performed ultrasound as a complement to routine management . patients were divided into subgroups based on patient characteristics , symptoms or first preliminary diagnosis set at the emergency department before randomization . outcomes measured were diagnostic accuracy , admission rate and requests for further examinations . timing of surgery was evaluated for patients with peritonitis.resultsincreased diagnostic accuracy was seen in patients with body mass index > 25 , elevated c - reactive protein , peritonitis , age 3059 years and/or upper abdominal pain . decreased need for further examinations and/or fewer admissions were seen in all groups except in patients with a preliminary diagnosis of appendicitis . among patients with non - specific abdominal pain , admission frequency was decreased with 14% when ultrasound was used ( p = 0.007 ) . among patients with peritonitis , requiring surgery , 61% in the ultrasound group were admitted for surgery directly from the emergency department compared to 19% in the control group.conclusionin different ways , surgeon - performed ultrasound is helpful for the majority of patients admitted to the emergency department for abdominal pain . taking into account other shown benefits and the lack of adverse effects , we find the method worth consideration for routine implementation .

Introduction Methods Statistical analysis Ethical considerations Results Participation and background data What benefits were shown in the subgroups? Age Body mass index C-reactive protein Gallbladder disease Appendicitis Peritonitis Non-specific abdominal pain Discussion Conclusion

the aim of this study was to determine the effect of surgeon - performed us bedside at the ed , based on several patient characteristics , on diagnostic accuracy and further management of patients admitted to the ed for abdominal pain . we examined selected outcomes in different subgroups based on body mass index ( bmi ) , age , level of c - reactive protein ( crp ) , signs of peritonitis , symptoms predictable for appendicitis ( pain and tenderness in lower right abdomen ) , gallbladder disease ( pain and tenderness in upper right abdomen ) and first preliminary diagnosis of appendicitis , gallbladder disease or non - specific abdominal pain set at the ed before randomization . some sort of benefit was seen in all groups except among the patients with a preliminary first set diagnosis of appendicitis , where the intervention groups were equal regarding all outcomes.table 2benefits of us examinations in different subgroupsdiagnostic accuracyadmission frequencyrequested us at radiological departmentrequested ct at radiological departmentany other examination requestedbmi < 25xx 25xxxxcrp < 10xx 10xxxlower abdominal symptomsxupper abdominal symptomsxxxgallbladder diseasexxappendicitisnsapxxxperitonitisxxxage < 30xx 3059xxxx 60xx statistically significant benefits benefits of us examinations in different subgroups x statistically significant benefits in the age group of 3059 years , diagnostic accuracy was higher and requests for ct examinations were fewer among the patients examined with us . in all age groups , fewer complementary radiological us examinations were ordered in the us group as well as fewer further examinations except from the oldest group ( table 3).table 3results based on age groupsage < 30 ( n = 177)age 3059 ( n = 388)age 60 ( n = 218)us ( n = 87 ) [ % ( n)]non - us ( n = 90 ) [ % ( n)]p valueus ( n = 198 ) [ % ( n)]non - us ( n = 190 ) [ % ( n)]p valueus ( n = 107 ) [ % ( n)]non - us ( n = 111 ) [ % ( n)]p valuediagnostic accuracy65 ( 56)60 ( 52)0.46868 ( 130)58 ( 109)0.04258 ( 61)52 ( 55)0.405admission38 ( 33)47 ( 42)0.24040 ( 79)44 ( 84)0.39052 ( 56)63 ( 70)0.109ultrasound ordered3 ( 3)27 ( 24)<0.00110 ( 19)28 ( 53)<0.00111 ( 12)27 ( 30)0.003ct ordered2 ( 2)2 ( 2)0.9735 ( 9)12 ( 22)0.01119 ( 20)14 ( 15)0.297no other examination ordered59 ( 51)38 ( 34)0.00651 ( 101)29 ( 55)<0.00137 ( 39)27 ( 30)0.134partially missing data in maximum seven patients per group and analysis results based on age groups partially missing data in maximum seven patients per group and analysis in the group with bmi 25 or more , the diagnostic accuracy was higher and there were fewer requests for computer tomography examinations if the patient had been examined with us . the frequency of complementary us examinations at the radiological department or other further examinations was lower in the us group regardless of bmi ( table 4).table 4results based on body mass index ( bmi ) and c - reactive protein ( crp)bmi < 25 ( n = 436)bmi 25 ( n = 317)crp < 10 ( n = 403)crp 10 ( n = 367)us ( n = 223 ) [ % ( n)]non - us ( n = 213 ) [ % ( n)]p valueus ( n = 160 ) [ % ( n)]non - us ( n = 157 ) [ % ( n)]p valueus ( n = 219 ) [ % ( n)]non - us ( n = 184 ) [ % ( n)]p valueus ( n = 163 ) [ % ( n)]non - us ( n = 204 ) [ % ( n)]p valuediagnostic accuracy62 ( 135)58 ( 119)0.41667 ( 105)54 ( 85)0.02065 ( 142)62 ( 112)0.50663 ( 98)52 ( 103)0.047admission44 ( 97)48 ( 102)0.35843 ( 69)52 ( 82)0.10525 ( 55)34 ( 62)0.05968 ( 111)65 ( 132)0.495ultrasound ordered7 ( 15)25 ( 53)<0.00111 ( 18)29 ( 45)<0.0017 ( 16)26 ( 48)<0.00111 ( 18)28 ( 57)<0.001ct ordered8 ( 18)7 ( 15)0.7057 ( 11)14 ( 22)0.0414 ( 8)4 ( 8)0.71914 ( 22)14 ( 29)0.843no other examination ordered47 ( 104)33 ( 70)0.00454 ( 85)27 ( 42)<0.00151 ( 110)34 ( 62)0.00146 ( 75)28 ( 57)<0.001partially missing data in maximum nine patients per group and analysis results based on body mass index ( bmi ) and c - reactive protein ( crp ) partially missing data in maximum nine patients per group and analysis there was a significant difference in frequency of difficulty in performing us , when comparing patients with bmi 25 or more with bmi less than 25 ( 59 vs. 23% , p < 0.001 ) , whereas reliability was virtually the same between the groups ( 83 vs. 89% , p = 0.205 ) . in the patients presenting with symptoms of gallbladder disease ( pain and tenderness in right upper abdomen ) , there was also a higher diagnostic accuracy as well as fewer requested radiological us examinations and further examinations , if examined with bedside us at the ed ( table 6).table 5results based on first set preliminary diagnosis at the edfirst emergency diagnosis set as gallbladder concrement and/or cholecystitis ( n = 61)appendicitis abscess as first emergency diagnosis ( n = 55)nsap as first emergency diagnosis ( n = 370)us ( n = 27 ) [ % ( n)]non - us n = 34 [ % ( n)]p valueus ( n = 31 ) [ % ( n)]non - us n = 24 [ % ( n)]p valueus ( n = 189 ) [ % ( n)]non - us ( n = 181 ) [ % ( n)]p valuediagnostic accuracy70 ( 19)62 ( 21)0.48264 ( 20)54 ( 13)0.43462 ( 118)55 ( 99)0.306admission52 ( 14)62 ( 21)0.43797 ( 30)100 ( 24)0.37531 ( 59)45 ( 81)0.007ultrasound ordered26 ( 7)82 ( 27)<0.0016 ( 2)21 ( 5)0.1125 ( 10)30 ( 55)0.001ct ordered0 ( 0)0 ( 0)13 ( 4)8 ( 2)0.59010 ( 19)10 ( 19)0.902no other examination ordered63 ( 17)18 ( 6)<0.00171 ( 22)46 ( 11)0.05948 ( 91)32 ( 57)0.001partially missing data in maximum three patients per group and analysistable 6results based on symptoms and signspain and tenderness in right upper abdomen ( n = 101)pain and tenderness in right lower abdomen ( n = 187)peritonitis ( n = 100)us n = 54 [ % ( n)]non - us ( n = 47 ) [ % ( n)]p valueus n = 91 [ % ( n)]non - us ( n = 96 ) [ % ( n)]p valueus ( n = 51 ) [ % ( n)]non - us ( n = 49 ) [ % ( n)]p valuediagnostic accuracy72 ( 38)52 ( 24)0.04559 ( 53)54 ( 51)0.47674 ( 37)54 ( 26)0.041admission50 ( 27)49 ( 23)0.91562 ( 56)58 ( 56)0.65590 ( 46)84 ( 41)0.332ultrasound ordered22 ( 12)74 ( 34)<0.0013 ( 3)22 ( 21)<0.00110 ( 5)29 ( 14)0.017ct ordered9 ( 5)6 ( 3)0.6158 ( 9)9 ( 8)0.91116 ( 8)24 ( 12)0.271no other examination ordered54 ( 29)17 ( 8)<0.00150 ( 45)45 ( 43)0.52353 ( 27)22 ( 11)0.002partially missing data in maximum one patient per group and analysisof these patients , 23 in us group and 26 in non - us group were admitted for surgery . 14 ( 60.9% ) in us group were admitted already at ed and 5 ( 19.2% ) in non - us group , p = 0.003 results based on first set preliminary diagnosis at the ed partially missing data in maximum three patients per group and analysis results based on symptoms and signs partially missing data in maximum one patient per group and analysis of these patients , 23 in us group and 26 in non - us group were admitted for surgery . of these patients requiring surgery , 14 ( 60.9% ) in the us group and 5 ( 19.2% ) in the non - us group were admitted for surgery with the decision taken while still at the ed ( p = 0.003 ) . 1study flow chart the baseline characteristics for the study subjects are shown in table 1.table 1baseline characteristics of patients with abdominal pain at the edcharacteristicsultrasound ( n = 392)non - ultrasound ( n = 391)meansdn%meansdn%age47204819height172917210weight73167316bmi ( body mass index)24.84.524.84.3gender male16040.817143.7 female23259.222056.3abdominal related comorbidity7619.47819.9comorbidity related to heart or diabetes6616.87418.9history of abdominal malignancy61.5123.1history of other malignancy112.8143.6other comorbidity13233.712331.5admission for abdominal pain within 1 year12432.013735.3referral for admission9224.412632.9duration of pain 08 h4414.84314.4 824 h9933.29732.4 > 24 h14749.315150.5 can not answer82.782.7affected general condition9023.37419.1tenderness33886.434789.2rigidity5113.14912.6palpable mass235.9297.5actual vas ( of pain)4.32.84.42.6maximal anamnestic vas ( of pain)7.62.67.61.8temperature37.00.837.00.7vas ( of pain ) = visual analogue scale ( scale 010 , 0 represents no pain at all , 10 represents unbearable pain ) baseline characteristics of patients with abdominal pain at the ed vas ( of pain ) = visual analogue scale ( scale 010 , 0 represents no pain at all , 10 represents unbearable pain ) some sort of benefit was seen in all groups except among the patients with a preliminary first set diagnosis of appendicitis , where the intervention groups were equal regarding all outcomes.table 2benefits of us examinations in different subgroupsdiagnostic accuracyadmission frequencyrequested us at radiological departmentrequested ct at radiological departmentany other examination requestedbmi < 25xx 25xxxxcrp < 10xx 10xxxlower abdominal symptomsxupper abdominal symptomsxxxgallbladder diseasexxappendicitisnsapxxxperitonitisxxxage < 30xx 3059xxxx 60xx statistically significant benefits benefits of us examinations in different subgroups x statistically significant benefits in the age group of 3059 years , diagnostic accuracy was higher and requests for ct examinations were fewer among the patients examined with us . the frequency of complementary us examinations at the radiological department or other further examinations was lower in the us group regardless of bmi ( table 4).table 4results based on body mass index ( bmi ) and c - reactive protein ( crp)bmi < 25 ( n = 436)bmi 25 ( n = 317)crp < 10 ( n = 403)crp 10 ( n = 367)us ( n = 223 ) [ % ( n)]non - us ( n = 213 ) [ % ( n)]p valueus ( n = 160 ) [ % ( n)]non - us ( n = 157 ) [ % ( n)]p valueus ( n = 219 ) [ % ( n)]non - us ( n = 184 ) [ % ( n)]p valueus ( n = 163 ) [ % ( n)]non - us ( n = 204 ) [ % ( n)]p valuediagnostic accuracy62 ( 135)58 ( 119)0.41667 ( 105)54 ( 85)0.02065 ( 142)62 ( 112)0.50663 ( 98)52 ( 103)0.047admission44 ( 97)48 ( 102)0.35843 ( 69)52 ( 82)0.10525 ( 55)34 ( 62)0.05968 ( 111)65 ( 132)0.495ultrasound ordered7 ( 15)25 ( 53)<0.00111 ( 18)29 ( 45)<0.0017 ( 16)26 ( 48)<0.00111 ( 18)28 ( 57)<0.001ct ordered8 ( 18)7 ( 15)0.7057 ( 11)14 ( 22)0.0414 ( 8)4 ( 8)0.71914 ( 22)14 ( 29)0.843no other examination ordered47 ( 104)33 ( 70)0.00454 ( 85)27 ( 42)<0.00151 ( 110)34 ( 62)0.00146 ( 75)28 ( 57)<0.001partially missing data in maximum nine patients per group and analysis results based on body mass index ( bmi ) and c - reactive protein ( crp ) partially missing data in maximum nine patients per group and analysis there was a significant difference in frequency of difficulty in performing us , when comparing patients with bmi 25 or more with bmi less than 25 ( 59 vs. 23% , p < 0.001 ) , whereas reliability was virtually the same between the groups ( 83 vs. 89% , p = 0.205 ) . in the patients presenting with symptoms of gallbladder disease ( pain and tenderness in right upper abdomen ) , there was also a higher diagnostic accuracy as well as fewer requested radiological us examinations and further examinations , if examined with bedside us at the ed ( table 6).table 5results based on first set preliminary diagnosis at the edfirst emergency diagnosis set as gallbladder concrement and/or cholecystitis ( n = 61)appendicitis abscess as first emergency diagnosis ( n = 55)nsap as first emergency diagnosis ( n = 370)us ( n = 27 ) [ % ( n)]non - us n = 34 [ % ( n)]p valueus ( n = 31 ) [ % ( n)]non - us n = 24 [ % ( n)]p valueus ( n = 189 ) [ % ( n)]non - us ( n = 181 ) [ % ( n)]p valuediagnostic accuracy70 ( 19)62 ( 21)0.48264 ( 20)54 ( 13)0.43462 ( 118)55 ( 99)0.306admission52 ( 14)62 ( 21)0.43797 ( 30)100 ( 24)0.37531 ( 59)45 ( 81)0.007ultrasound ordered26 ( 7)82 ( 27)<0.0016 ( 2)21 ( 5)0.1125 ( 10)30 ( 55)0.001ct ordered0 ( 0)0 ( 0)13 ( 4)8 ( 2)0.59010 ( 19)10 ( 19)0.902no other examination ordered63 ( 17)18 ( 6)<0.00171 ( 22)46 ( 11)0.05948 ( 91)32 ( 57)0.001partially missing data in maximum three patients per group and analysistable 6results based on symptoms and signspain and tenderness in right upper abdomen ( n = 101)pain and tenderness in right lower abdomen ( n = 187)peritonitis ( n = 100)us n = 54 [ % ( n)]non - us ( n = 47 ) [ % ( n)]p valueus n = 91 [ % ( n)]non - us ( n = 96 ) [ % ( n)]p valueus ( n = 51 ) [ % ( n)]non - us ( n = 49 ) [ % ( n)]p valuediagnostic accuracy72 ( 38)52 ( 24)0.04559 ( 53)54 ( 51)0.47674 ( 37)54 ( 26)0.041admission50 ( 27)49 ( 23)0.91562 ( 56)58 ( 56)0.65590 ( 46)84 ( 41)0.332ultrasound ordered22 ( 12)74 ( 34)<0.0013 ( 3)22 ( 21)<0.00110 ( 5)29 ( 14)0.017ct ordered9 ( 5)6 ( 3)0.6158 ( 9)9 ( 8)0.91116 ( 8)24 ( 12)0.271no other examination ordered54 ( 29)17 ( 8)<0.00150 ( 45)45 ( 43)0.52353 ( 27)22 ( 11)0.002partially missing data in maximum one patient per group and analysisof these patients , 23 in us group and 26 in non - us group were admitted for surgery . 14 ( 60.9% ) in us group were admitted already at ed and 5 ( 19.2% ) in non - us group , p = 0.003 results based on first set preliminary diagnosis at the ed partially missing data in maximum three patients per group and analysis results based on symptoms and signs partially missing data in maximum one patient per group and analysis of these patients , 23 in us group and 26 in non - us group were admitted for surgery . of these patients requiring surgery , 14 ( 60.9% ) in the us group and 5 ( 19.2% ) in the non - us group were admitted for surgery with the decision taken while still at the ed ( p = 0.003 ) . this study shows that surgeon - performed us at the ed for abdominal pain can be helpful in several ways for the majority of patients admitted to the ed for abdominal pain . for patients with peritonitis , taking into account other shown benefits and the lack of adverse effects , we find the method well worth consideration for implementation at the ed .

inuit communities are located in the nunavik region of northern qubec , including the ungava bay and hudson bay areas ( 11 ) . first nations communities , in contrast , are more numerous and dispersed throughout qubec . publicly funded universal health care covers obstetric services and is available to all quebecers , including indigenous populations , although health care delivery varies by community . in ungava bay , care is typically provided by physicians , whereas in hudson bay midwives predominate ( 11 ) . we extracted singleton infants from the qubec live birth file from 1981 to 2008 ( n=2,310,466 ) . we excluded 18,229 births missing gestational age ( 0.8% ) , and an additional 176 births missing maternal age ( 0.01% ) . the birth file is compiled from birth certificates and provides complete coverage of births of residents of qubec ( 12,13 ) . ptb categories were also examined according to severity of gestational age at birth : ( a ) extreme ptb ( 27 weeks ) ; ( b ) very ptb ( 2831 weeks ) ; and ( c ) moderate ptb ( 3236 weeks ) ( 14 ) . ultrasound estimates of gestational age are typically more accurate than estimates based on menstruation ( 1517 ) , however , ultrasound examinations may not have been fully implemented in the 1980s . indigenous populations were identified using 2 indicators : municipality of residence and self - reported parental language . in qubec , municipalities can be used to identify inuit- ( n=14 ) and first nations- ( n=45 ) inhabited areas ( also known as reserves or territories ) , defined as such by statistics canada for census purposes . all remaining municipalities not coded as indigenous were grouped in a separate category hereafter denoted rest of qubec . hence , 3 types of areas were available for analyses ( inuit - inhabited , first nations - inhabited , rest of qubec ) . language was expressed categorically ( inuit , first nations , french / english , other , unknown ) . first nations languages included all dialects of remaining indigenous populations of north america . according to the 2006 census , 89% of self - identified inuit and 46% of self - identified first nations reported an indigenous mother tongue in qubec ( 10 ) . maternal mother tongue was used to identify language , but language spoken at home and paternal mother tongue were also used to capture an additional 428 inuit and 2,040 first nations infants . language and type of area were analysed as a joint variable to identify inuit and first nations language births by area . categories for the joint language - by - area type indicator of indigenous status included ( a ) inuit language speakers in inuit - inhabited areas , ( b ) french / english speakers in inuit - inhabited areas , ( c ) first nations language speakers in first nations - inhabited areas , ( d ) french / english speakers in first nations - inhabited areas , ( e ) inuit language speakers in the rest of qubec , ( f ) first nations language speakers in the rest of qubec , ( g ) french / english speakers in the rest of qubec ( referent ) , ( h ) other . it is important to note that french / english speakers in inuit - inhabited areas represent individuals of inuit ethnicity who reported french / english language on birth certificates , or non - inuit migrants working temporarily in inuit - inhabited areas ( 11 ) . french / english speakers in first nations - inhabited areas most likely represent a mix of first nations and non - indigenous individuals . available covariates included maternal age ( < 20 , 2034 and35 years ) , parity ( 0 , 1 and2 previous deliveries ) , education ( no high school diploma , high school diploma , some post - secondary , some university or more , unknown ) and marital status ( legally married , not legally married ) . several studies have identified these variables as potential confounders of the relation between indigenous ethnicity and adverse birth outcomes ( 2,18 ) . due to the lack of variability in indigenous populations , immigration status ptb rates were computed according to indigenous status , maternal age , education , marital status , parity and birth year . fisher 's exact 95% confidence intervals ( cis ) , or wald 95% cis for large samples , were computed for proportions ( http://www.openepi.com/oe2.3/proportion/ proportion.htm ) . cox proportional hazard regression was employed to estimate hazard ratios ( hr ) and 95% cis of ptb for indigenous status in models that were unadjusted and adjusted for maternal age , education , marital status , parity and birth year . cox regression is increasingly used to examine perinatal outcomes such as ptb , because pregnancy is a dynamic process that evolves over time and results in a specific event , birth ( 19,20 ) . as the data were hierarchical with births nested in municipalities , clustering was accounted for with the robust sandwich estimator ( 21 ) . statistical package for social sciences ( spss , www.spss.com , version 17.0 for windows ) software was used for descriptive statistics , and sas software ( statistical analysis system , http://www.sas.com , version 9.2 ) for regression models . individual consent was thus not sought , and formal ethical approval was waived by the research ethics committee of the university of montral hospital centre . we performed a retrospective cohort study of the population of births in the province of qubec , canada , from 1981 to 2008 . according to the 2006 census , qubec had a population of over 7.4 million , of which approximately 0.1% was inuit and 0.9% first nations ( 10 ) . inuit communities are located in the nunavik region of northern qubec , including the ungava bay and hudson bay areas ( 11 ) . first nations communities , in contrast , are more numerous and dispersed throughout qubec . publicly funded universal health care covers obstetric services and is available to all quebecers , including indigenous populations , although health care delivery varies by community . in ungava bay , care is typically provided by physicians , whereas in hudson bay midwives predominate ( 11 ) . we extracted singleton infants from the qubec live birth file from 1981 to 2008 ( n=2,310,466 ) . we excluded 18,229 births missing gestational age ( 0.8% ) , and an additional 176 births missing maternal age ( 0.01% ) . the birth file is compiled from birth certificates and provides complete coverage of births of residents of qubec ( 12,13 ) . ptb categories were also examined according to severity of gestational age at birth : ( a ) extreme ptb ( 27 weeks ) ; ( b ) very ptb ( 2831 weeks ) ; and ( c ) moderate ptb ( 3236 weeks ) ( 14 ) . ultrasound estimates of gestational age are typically more accurate than estimates based on menstruation ( 1517 ) , however , ultrasound examinations may not have been fully implemented in the 1980s . indigenous populations were identified using 2 indicators : municipality of residence and self - reported parental language . in qubec , municipalities can be used to identify inuit- ( n=14 ) and first nations- ( n=45 ) inhabited areas ( also known as reserves or territories ) , defined as such by statistics canada for census purposes . all remaining municipalities not coded as indigenous were grouped in a separate category hereafter denoted rest of qubec . hence , 3 types of areas were available for analyses ( inuit - inhabited , first nations - inhabited , rest of qubec ) . language was expressed categorically ( inuit , first nations , french / english , other , unknown ) . first nations languages included all dialects of remaining indigenous populations of north america . according to the 2006 census , 89% of self - identified inuit and 46% of self - identified first nations reported an indigenous mother tongue in qubec ( 10 ) . maternal mother tongue was used to identify language , but language spoken at home and paternal mother tongue were also used to capture an additional 428 inuit and 2,040 first nations infants . language and type of area were analysed as a joint variable to identify inuit and first nations language births by area . categories for the joint language - by - area type indicator of indigenous status included ( a ) inuit language speakers in inuit - inhabited areas , ( b ) french / english speakers in inuit - inhabited areas , ( c ) first nations language speakers in first nations - inhabited areas , ( d ) french / english speakers in first nations - inhabited areas , ( e ) inuit language speakers in the rest of qubec , ( f ) first nations language speakers in the rest of qubec , ( g ) french / english speakers in the rest of qubec ( referent ) , ( h ) other . it is important to note that french / english speakers in inuit - inhabited areas represent individuals of inuit ethnicity who reported french / english language on birth certificates , or non - inuit migrants working temporarily in inuit - inhabited areas ( 11 ) . french / english speakers in first nations - inhabited areas most likely represent a mix of first nations and non - indigenous individuals . available covariates included maternal age ( < 20 , 2034 and35 years ) , parity ( 0 , 1 and2 previous deliveries ) , education ( no high school diploma , high school diploma , some post - secondary , some university or more , unknown ) and marital status ( legally married , not legally married ) . several studies have identified these variables as potential confounders of the relation between indigenous ethnicity and adverse birth outcomes ( 2,18 ) . due to the lack of variability in indigenous populations , immigration status ptb rates were computed according to indigenous status , maternal age , education , marital status , parity and birth year . fisher 's exact 95% confidence intervals ( cis ) , or wald 95% cis for large samples , were computed for proportions ( http://www.openepi.com/oe2.3/proportion/ proportion.htm ) . cox proportional hazard regression was employed to estimate hazard ratios ( hr ) and 95% cis of ptb for indigenous status in models that were unadjusted and adjusted for maternal age , education , marital status , parity and birth year . cox regression is increasingly used to examine perinatal outcomes such as ptb , because pregnancy is a dynamic process that evolves over time and results in a specific event , birth ( 19,20 ) . as the data were hierarchical with births nested in municipalities , clustering was accounted for with the robust sandwich estimator ( 21 ) . statistical package for social sciences ( spss , www.spss.com , version 17.0 for windows ) software was used for descriptive statistics , and sas software ( statistical analysis system , http://www.sas.com , version 9.2 ) for regression models . individual consent was thus not sought , and formal ethical approval was waived by the research ethics committee of the university of montral hospital centre . in inuit - inhabited areas , 4,851 infants ( 0.2% ) were born to inuit language speakers and 680 infants ( 0.03% ) to french / english speakers ( table i ) . in contrast , 11,678 infants ( 0.5% ) were born to first nations language speakers and 8,962 infants ( 0.4% ) to french / english speakers in first nations - inhabited areas . there were 513 births ( 0.02% ) to inuit language speakers and 3,836 births to first nations language speakers ( 0.2% ) in the rest of qubec . rates of preterm birth according to maternal characteristics , singleton live births , qubec , 19812008a ci , confidence interval . may not sum to total as unknown or other . the overall rate of ptb was 5.9% . compared with french / english speakers in the rest of qubec ( 5.8% ) , ptb rates were elevated for inuit and french / english speakers in inuit - inhabited areas ( 9.5 and 12.7% , respectively ) , and for inuit language speakers in the rest of qubec ( 12.7% ) . in contrast , rates for first nations ( 5.9% ) and french / english speakers ( 6.7% ) in first nations - inhabited areas and first nations language speakers in the rest of qubec ( 6.0% ) were only slightly higher compared with french / english speakers in the rest of qubec . similar patterns were observed when ptb was examined by severity according to gestational age ( table ii ) . compared with french / english speakers in the rest of qubec , inuit language speakers in the rest of qubec and french / english speakers in inuit - inhabited areas had the highest rates of moderate ( 10.3 and 11.0% vs. 5.1% , respectively ) and very ptb ( 1.95 and 1.32% vs. 0.44% , respectively ) . rates of extreme ptb were higher for inuit language speakers in inuit - inhabited areas ( 0.49% ) and the rest of qubec ( 0.39% ) , as well as for french / english speakers in first nations - inhabited areas ( 0.42% ) , compared with french / english speakers in the rest of qubec ( 0.27% ) . preterm birth rates according to severity by gestational age and indigenous status , singleton live births , qubec , 19812008a ci , confidence interval . may not sum to total as the other language category is not shown . whereas ptb rates increased over time for french / english language speakers in the rest of qubec , rates tended to decrease slightly for inuit language and french / english speakers in inuit - inhabited areas , and were generally stable for inuit language speakers in the rest of qubec ( table iii ) . the ptb rate increased for all other groups , especially first nations language speakers in first nations - inhabited areas and the rest of qubec . preterm birth rates according to birth year and indigenous status , singleton live births , qubec , 19812008a ci , confidence interval . relative to french / english speakers in the rest of qubec , the hazard of ptb was greatest for inuit language speakers in the rest of qubec ( adjusted hr 1.98 , 95% ci : 1.552.53 ) and french / english speakers in inuit - inhabited areas ( adjusted hr 1.97 , 95% ci : 1.592.43 , table iv ) . the hazard of ptb for inuit language speakers in inuit - inhabited areas was also elevated , but slightly weaker ( adjusted hr 1.28 , 95% ci : 1.171.41 ) . adjustment for maternal characteristics attenuated the associations , but did not change the direction of findings . this was not the case for first nations language speakers in first nations - inhabited areas and the rest of qubec , who upon adjustment had a lower hazard of ptb relative to french / english speakers in the rest of qubec ( hr < 0.9 ) . association between indigenous status and preterm birth , singleton live births , qubec , 19812008 hazard ratio ( hr ) and 95% confidence interval ( ci ) , adjusted for maternal age , education , marital status , parity and birth year . analyses stratified by birth year showed a persistently elevated hazard of ptb for inuit language speakers in the rest of qubec , and decreasing disparities for inuit language and french / english speakers in inuit - inhabited areas relative to french / english speakers in the rest of qubec ( table v ) . in contrast , hrs increased over calendar time for first nations language speakers . while the hazard was initially protective against ptb for first nations language speakers in the rest of qubec relative to french / english speakers in the rest of qubec ( hr 0.83 ) , the hazard was higher in the most recent period ( hr 1.32 ) . for first nations language speakers in first nations - inhabited areas , the initial protective association in 19811989 ( hr 0.71 ) disappeared by 20002008 ( hr 0.99 ) . association between indigenous status and preterm birth by birth year , singleton live births , qubec , 19812008 hazard ratio ( hr ) and 95% confidence interval ( ci ) , adjusted for type of area , maternal age , education , marital status , and parity . we observed higher rates of ptb in inuit - inhabited areas compared with french / english speakers in the rest of qubec . disparities for inuit groups persisted or decreased slightly over time relative to french / english speakers in the rest of qubec , but increased for first nations populations , although disparities generally remained greater for inuit groups . interestingly , inuit language speakers had a lower risk of ptb relative to french / english in inuit - inhabited areas , though their risk was nonetheless higher relative to french / english speakers in the rest of qubec . overall , the highest risks of ptb were observed for french / english speakers in inuit - inhabited areas , and inuit language speakers in the rest of qubec , relative to french / english speakers in the rest of qubec . these findings indicate a need for effective interventions to improve perinatal health in inuit - inhabited areas and for inuit language speakers in the rest of qubec . in general , our findings were consistent with previous reports of higher odds of ptb in inuit - inhabited areas compared with other areas ( 1,3,9 ) , as well as for inuit language compared with french / english speakers ( 8,22 ) . the lower rates of ptb for first nations language speakers in first nations - inhabited areas and the rest of qubec were consistent with those noted in other studies ( 1,2,8 ) . however , these studies grouped inuit individuals in the rest of qubec with the referent . previous studies also did not distinguish between the different linguistic groups living in inuit - inhabited areas . in fact , we found that in inuit - inhabited areas , inuit language speakers had a lower risk of ptb relative to french / english speakers . why this happens is unclear , but inuit people who report french / english as their language may be more acculturated to western society , and less protected by traditional lifestyle factors . behavioural risk factors for ptb such as smoking , substance use and poor nutritional habits ( 2326 ) , as well as psychosocial problems such as domestic violence , emotional stress , depression and anxiety ( 23,2729 ) may potentially be greater in acculturated inuit women with weaker ties to traditional culture and language ( 30 ) . it is important to note that a substantial proportion of french / english speakers in inuit - inhabited areas likely represent inuit individuals , but may also include some non - inuit individuals ( e.g. non - indigenous residents of qubec employed in inuit - inhabited areas ) the higher risk of ptb in all inuit categories may be related to environmental , cultural and lifestyle factors that potentially differ from french / english speakers in the rest of qubec ( 27,31 ) . there is , however , little evidence that midwife care is a risk factor for ptb for inuit women ( 11 ) . furthermore , midwife care is typically provided by inuit language speakers , whereas physician - led care is provided in french or english ( 11 ) , suggesting that cultural differences in communication or language barriers ( 27,31,33 ) are unlikely to explain the associations . indigenous mothers prefer to give birth in their community , and may be reticent to hospitalisation for treatment of preterm labour ( 27,34 ) . women with risk factors for preterm labour and high - risk pregnancies are nonetheless recommended for evacuation to larger facilities prior to term ( 35 ) , although the majority of transfers for preterm labour without ruptured membranes succeed in delivering at term ( 36 ) . thus , the potential role of obstetric interventions in driving ptb rates among the inuit is unclear . it is possible that rural isolation is related to the higher risks in inuit - inhabited areas , as challenges accessing medical care may be greater due to remoteness and limited resources ( 22,37 ) . ptb rates in remote rural indigenous populations of australia and the us are also high compared with non - indigenous populations , and it has been speculated that this may be due to differences in access to health care , socio - demographic , behavioural , cultural and environmental factors ( 4043 ) . results suggested that disparities between inuit populations and french / english speakers in the rest of qubec were stable over time , though there tended to be a decrease in inuit - inhabited areas . the decrease in inequalities , however , was most likely caused by a relative increase in ptb rates among french / english speakers in the rest of qubec , coupled with a slight improvement in rates in inuit - inhabited areas . wider use of ultrasound dating over time may explain some of the rate increase among french / english speakers in the rest of canada ( as menstrual dating tends to overestimate gestational age ) ( 16 ) . in inuit - inhabited areas , access to ultrasound is more limited and may have been adopted more gradually over time ( 36 ) , which suggests that ptb rates in the inuit are likely underestimated in all periods , and that disparities are likely greater in more recent periods . data on other potentially time - varying covariates such as obstetric interventions were , however , not available , which precludes a more detailed interpretation of time trends ( 44 ) . another important issue is that the initially protective association against ptb for first nations populations relative to french / english speakers in the rest of qubec has gradually disappeared over time , likely because ptb rates in first nations have caught up to the general population . nonetheless , disparities in ptb continue to be greater for inuit populations than for first nations . this study was limited by lack of data on several maternal risk factors such as smoking , substance use and obesity that could have influenced the observed associations ( 5,4547 ) . the extent to which our findings may be related to obesity , however , is unclear , because first nations populations also have high obesity rates ( 23 ) , but lower ptb rates . though we accounted for education , we may not have captured other aspects of socioeconomic status including income and occupation . misclassification of indigenous status through the use of language may have occurred and potentially attenuated the associations , as such individuals would have been grouped with french / english or other language categories . specifically , we could not capture inuit and first nations births in the rest of qubec among mothers who had reported french / english language , or mtis indigenous groups not identifiable through language or area indicators ( 0.4% of quebecers are mtis ) ( 10 ) . results for inuit language speakers in the rest of qubec and french / english speakers in inuit - inhabited areas should be interpreted with caution in light of small numbers . last , our findings may not generalise to other settings , especially places without universal health insurance . in summary , we found persistently elevated risks of ptb in inuit - inhabited areas and for inuit language speakers in the rest of qubec . the underlying pathways leading to these perinatal health disparities require further investigation to target prevention of ptb in inuit and northern populations . this study was limited by lack of data on several maternal risk factors such as smoking , substance use and obesity that could have influenced the observed associations ( 5,4547 ) . the extent to which our findings may be related to obesity , however , is unclear , because first nations populations also have high obesity rates ( 23 ) , but lower ptb rates . though we accounted for education , we may not have captured other aspects of socioeconomic status including income and occupation . misclassification of indigenous status through the use of language may have occurred and potentially attenuated the associations , as such individuals would have been grouped with french / english or other language categories . specifically , we could not capture inuit and first nations births in the rest of qubec among mothers who had reported french / english language , or mtis indigenous groups not identifiable through language or area indicators ( 0.4% of quebecers are mtis ) ( 10 ) . results for inuit language speakers in the rest of qubec and french / english speakers in inuit - inhabited areas should be interpreted with caution in light of small numbers . last , our findings may not generalise to other settings , especially places without universal health insurance . in summary , we found persistently elevated risks of ptb in inuit - inhabited areas and for inuit language speakers in the rest of qubec . the underlying pathways leading to these perinatal health disparities require further investigation to target prevention of ptb in inuit and northern populations . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

objectivesto evaluate preterm birth ( ptb ) for inuit and first nations vs. non - indigenous populations in the province of qubec , canada.study designretrospective cohort study.methodswe evaluated singleton live births for qubec residents , 19812008 ( n = 2,310,466 ) . municipality of residence ( inuit - inhabited , first nations - inhabited , rest of qubec ) and language ( inuit , first nations , french / english ) were used to identify inuit and first nations births . the outcome was ptb ( < 37 completed weeks ) . cox proportional hazards regression was employed to estimate hazard ratios ( hr ) and 95% confidence intervals ( ci ) of ptb , adjusting for maternal age , education , marital status , parity and birth year.resultsptb rates were higher for inuit language speakers in inuit - inhabited areas and the rest of qubec compared with french / english speakers in the rest of qubec , and disparities persisted over time . relative to french / english speakers in the rest of qubec , inuit language speakers in the rest of qubec had the highest risk of ptb ( hr 1.98 , 95% ci : 1.622.41 ) . the risk was also elevated for inuit language speakers in inuit - inhabited areas , though to a lesser extent ( hr 1.29 , 95% ci : 1.181.41 ) . in contrast , first nations language speakers in first nations - inhabited areas and the rest of qubec had similar or lower risks of ptb relative to french / english speakers in the rest of qubec.conclusionsinuit populations , especially those outside inuit - inhabited areas , have persistently elevated risks of ptb , indicating a need for strategies to prevent ptb in this population .

Materials and methods Study design and setting Data and variables Statistical analysis Results Discussion Limitations Conflict of interest and funding

inuit communities are located in the nunavik region of northern qubec , including the ungava bay and hudson bay areas ( 11 ) . first nations communities , in contrast , are more numerous and dispersed throughout qubec . publicly funded universal health care covers obstetric services and is available to all quebecers , including indigenous populations , although health care delivery varies by community . ptb categories were also examined according to severity of gestational age at birth : ( a ) extreme ptb ( 27 weeks ) ; ( b ) very ptb ( 2831 weeks ) ; and ( c ) moderate ptb ( 3236 weeks ) ( 14 ) . indigenous populations were identified using 2 indicators : municipality of residence and self - reported parental language . in qubec , municipalities can be used to identify inuit- ( n=14 ) and first nations- ( n=45 ) inhabited areas ( also known as reserves or territories ) , defined as such by statistics canada for census purposes . all remaining municipalities not coded as indigenous were grouped in a separate category hereafter denoted rest of qubec . hence , 3 types of areas were available for analyses ( inuit - inhabited , first nations - inhabited , rest of qubec ) . language was expressed categorically ( inuit , first nations , french / english , other , unknown ) . according to the 2006 census , 89% of self - identified inuit and 46% of self - identified first nations reported an indigenous mother tongue in qubec ( 10 ) . maternal mother tongue was used to identify language , but language spoken at home and paternal mother tongue were also used to capture an additional 428 inuit and 2,040 first nations infants . language and type of area were analysed as a joint variable to identify inuit and first nations language births by area . categories for the joint language - by - area type indicator of indigenous status included ( a ) inuit language speakers in inuit - inhabited areas , ( b ) french / english speakers in inuit - inhabited areas , ( c ) first nations language speakers in first nations - inhabited areas , ( d ) french / english speakers in first nations - inhabited areas , ( e ) inuit language speakers in the rest of qubec , ( f ) first nations language speakers in the rest of qubec , ( g ) french / english speakers in the rest of qubec ( referent ) , ( h ) other . it is important to note that french / english speakers in inuit - inhabited areas represent individuals of inuit ethnicity who reported french / english language on birth certificates , or non - inuit migrants working temporarily in inuit - inhabited areas ( 11 ) . french / english speakers in first nations - inhabited areas most likely represent a mix of first nations and non - indigenous individuals . available covariates included maternal age ( < 20 , 2034 and35 years ) , parity ( 0 , 1 and2 previous deliveries ) , education ( no high school diploma , high school diploma , some post - secondary , some university or more , unknown ) and marital status ( legally married , not legally married ) . due to the lack of variability in indigenous populations , immigration status ptb rates were computed according to indigenous status , maternal age , education , marital status , parity and birth year . fisher 's exact 95% confidence intervals ( cis ) , or wald 95% cis for large samples , were computed for proportions ( http://www.openepi.com/oe2.3/proportion/ proportion.htm ) . cox proportional hazard regression was employed to estimate hazard ratios ( hr ) and 95% cis of ptb for indigenous status in models that were unadjusted and adjusted for maternal age , education , marital status , parity and birth year . cox regression is increasingly used to examine perinatal outcomes such as ptb , because pregnancy is a dynamic process that evolves over time and results in a specific event , birth ( 19,20 ) . statistical package for social sciences ( spss , www.spss.com , version 17.0 for windows ) software was used for descriptive statistics , and sas software ( statistical analysis system , http://www.sas.com , version 9.2 ) for regression models . we performed a retrospective cohort study of the population of births in the province of qubec , canada , from 1981 to 2008 . according to the 2006 census , qubec had a population of over 7.4 million , of which approximately 0.1% was inuit and 0.9% first nations ( 10 ) . inuit communities are located in the nunavik region of northern qubec , including the ungava bay and hudson bay areas ( 11 ) . first nations communities , in contrast , are more numerous and dispersed throughout qubec . we excluded 18,229 births missing gestational age ( 0.8% ) , and an additional 176 births missing maternal age ( 0.01% ) . ptb categories were also examined according to severity of gestational age at birth : ( a ) extreme ptb ( 27 weeks ) ; ( b ) very ptb ( 2831 weeks ) ; and ( c ) moderate ptb ( 3236 weeks ) ( 14 ) . indigenous populations were identified using 2 indicators : municipality of residence and self - reported parental language . in qubec , municipalities can be used to identify inuit- ( n=14 ) and first nations- ( n=45 ) inhabited areas ( also known as reserves or territories ) , defined as such by statistics canada for census purposes . all remaining municipalities not coded as indigenous were grouped in a separate category hereafter denoted rest of qubec . hence , 3 types of areas were available for analyses ( inuit - inhabited , first nations - inhabited , rest of qubec ) . language was expressed categorically ( inuit , first nations , french / english , other , unknown ) . according to the 2006 census , 89% of self - identified inuit and 46% of self - identified first nations reported an indigenous mother tongue in qubec ( 10 ) . maternal mother tongue was used to identify language , but language spoken at home and paternal mother tongue were also used to capture an additional 428 inuit and 2,040 first nations infants . language and type of area were analysed as a joint variable to identify inuit and first nations language births by area . categories for the joint language - by - area type indicator of indigenous status included ( a ) inuit language speakers in inuit - inhabited areas , ( b ) french / english speakers in inuit - inhabited areas , ( c ) first nations language speakers in first nations - inhabited areas , ( d ) french / english speakers in first nations - inhabited areas , ( e ) inuit language speakers in the rest of qubec , ( f ) first nations language speakers in the rest of qubec , ( g ) french / english speakers in the rest of qubec ( referent ) , ( h ) other . it is important to note that french / english speakers in inuit - inhabited areas represent individuals of inuit ethnicity who reported french / english language on birth certificates , or non - inuit migrants working temporarily in inuit - inhabited areas ( 11 ) . french / english speakers in first nations - inhabited areas most likely represent a mix of first nations and non - indigenous individuals . available covariates included maternal age ( < 20 , 2034 and35 years ) , parity ( 0 , 1 and2 previous deliveries ) , education ( no high school diploma , high school diploma , some post - secondary , some university or more , unknown ) and marital status ( legally married , not legally married ) . due to the lack of variability in indigenous populations , immigration status ptb rates were computed according to indigenous status , maternal age , education , marital status , parity and birth year . fisher 's exact 95% confidence intervals ( cis ) , or wald 95% cis for large samples , were computed for proportions ( http://www.openepi.com/oe2.3/proportion/ proportion.htm ) . cox proportional hazard regression was employed to estimate hazard ratios ( hr ) and 95% cis of ptb for indigenous status in models that were unadjusted and adjusted for maternal age , education , marital status , parity and birth year . cox regression is increasingly used to examine perinatal outcomes such as ptb , because pregnancy is a dynamic process that evolves over time and results in a specific event , birth ( 19,20 ) . statistical package for social sciences ( spss , www.spss.com , version 17.0 for windows ) software was used for descriptive statistics , and sas software ( statistical analysis system , http://www.sas.com , version 9.2 ) for regression models . in inuit - inhabited areas , 4,851 infants ( 0.2% ) were born to inuit language speakers and 680 infants ( 0.03% ) to french / english speakers ( table i ) . in contrast , 11,678 infants ( 0.5% ) were born to first nations language speakers and 8,962 infants ( 0.4% ) to french / english speakers in first nations - inhabited areas . there were 513 births ( 0.02% ) to inuit language speakers and 3,836 births to first nations language speakers ( 0.2% ) in the rest of qubec . rates of preterm birth according to maternal characteristics , singleton live births , qubec , 19812008a ci , confidence interval . compared with french / english speakers in the rest of qubec ( 5.8% ) , ptb rates were elevated for inuit and french / english speakers in inuit - inhabited areas ( 9.5 and 12.7% , respectively ) , and for inuit language speakers in the rest of qubec ( 12.7% ) . in contrast , rates for first nations ( 5.9% ) and french / english speakers ( 6.7% ) in first nations - inhabited areas and first nations language speakers in the rest of qubec ( 6.0% ) were only slightly higher compared with french / english speakers in the rest of qubec . compared with french / english speakers in the rest of qubec , inuit language speakers in the rest of qubec and french / english speakers in inuit - inhabited areas had the highest rates of moderate ( 10.3 and 11.0% vs. 5.1% , respectively ) and very ptb ( 1.95 and 1.32% vs. 0.44% , respectively ) . rates of extreme ptb were higher for inuit language speakers in inuit - inhabited areas ( 0.49% ) and the rest of qubec ( 0.39% ) , as well as for french / english speakers in first nations - inhabited areas ( 0.42% ) , compared with french / english speakers in the rest of qubec ( 0.27% ) . preterm birth rates according to severity by gestational age and indigenous status , singleton live births , qubec , 19812008a ci , confidence interval . whereas ptb rates increased over time for french / english language speakers in the rest of qubec , rates tended to decrease slightly for inuit language and french / english speakers in inuit - inhabited areas , and were generally stable for inuit language speakers in the rest of qubec ( table iii ) . the ptb rate increased for all other groups , especially first nations language speakers in first nations - inhabited areas and the rest of qubec . preterm birth rates according to birth year and indigenous status , singleton live births , qubec , 19812008a ci , confidence interval . relative to french / english speakers in the rest of qubec , the hazard of ptb was greatest for inuit language speakers in the rest of qubec ( adjusted hr 1.98 , 95% ci : 1.552.53 ) and french / english speakers in inuit - inhabited areas ( adjusted hr 1.97 , 95% ci : 1.592.43 , table iv ) . the hazard of ptb for inuit language speakers in inuit - inhabited areas was also elevated , but slightly weaker ( adjusted hr 1.28 , 95% ci : 1.171.41 ) . this was not the case for first nations language speakers in first nations - inhabited areas and the rest of qubec , who upon adjustment had a lower hazard of ptb relative to french / english speakers in the rest of qubec ( hr < 0.9 ) . association between indigenous status and preterm birth , singleton live births , qubec , 19812008 hazard ratio ( hr ) and 95% confidence interval ( ci ) , adjusted for maternal age , education , marital status , parity and birth year . analyses stratified by birth year showed a persistently elevated hazard of ptb for inuit language speakers in the rest of qubec , and decreasing disparities for inuit language and french / english speakers in inuit - inhabited areas relative to french / english speakers in the rest of qubec ( table v ) . in contrast , hrs increased over calendar time for first nations language speakers . while the hazard was initially protective against ptb for first nations language speakers in the rest of qubec relative to french / english speakers in the rest of qubec ( hr 0.83 ) , the hazard was higher in the most recent period ( hr 1.32 ) . for first nations language speakers in first nations - inhabited areas , the initial protective association in 19811989 ( hr 0.71 ) disappeared by 20002008 ( hr 0.99 ) . association between indigenous status and preterm birth by birth year , singleton live births , qubec , 19812008 hazard ratio ( hr ) and 95% confidence interval ( ci ) , adjusted for type of area , maternal age , education , marital status , and parity . we observed higher rates of ptb in inuit - inhabited areas compared with french / english speakers in the rest of qubec . disparities for inuit groups persisted or decreased slightly over time relative to french / english speakers in the rest of qubec , but increased for first nations populations , although disparities generally remained greater for inuit groups . interestingly , inuit language speakers had a lower risk of ptb relative to french / english in inuit - inhabited areas , though their risk was nonetheless higher relative to french / english speakers in the rest of qubec . overall , the highest risks of ptb were observed for french / english speakers in inuit - inhabited areas , and inuit language speakers in the rest of qubec , relative to french / english speakers in the rest of qubec . these findings indicate a need for effective interventions to improve perinatal health in inuit - inhabited areas and for inuit language speakers in the rest of qubec . in general , our findings were consistent with previous reports of higher odds of ptb in inuit - inhabited areas compared with other areas ( 1,3,9 ) , as well as for inuit language compared with french / english speakers ( 8,22 ) . the lower rates of ptb for first nations language speakers in first nations - inhabited areas and the rest of qubec were consistent with those noted in other studies ( 1,2,8 ) . however , these studies grouped inuit individuals in the rest of qubec with the referent . previous studies also did not distinguish between the different linguistic groups living in inuit - inhabited areas . in fact , we found that in inuit - inhabited areas , inuit language speakers had a lower risk of ptb relative to french / english speakers . why this happens is unclear , but inuit people who report french / english as their language may be more acculturated to western society , and less protected by traditional lifestyle factors . it is important to note that a substantial proportion of french / english speakers in inuit - inhabited areas likely represent inuit individuals , but may also include some non - inuit individuals ( e.g. non - indigenous residents of qubec employed in inuit - inhabited areas ) the higher risk of ptb in all inuit categories may be related to environmental , cultural and lifestyle factors that potentially differ from french / english speakers in the rest of qubec ( 27,31 ) . furthermore , midwife care is typically provided by inuit language speakers , whereas physician - led care is provided in french or english ( 11 ) , suggesting that cultural differences in communication or language barriers ( 27,31,33 ) are unlikely to explain the associations . it is possible that rural isolation is related to the higher risks in inuit - inhabited areas , as challenges accessing medical care may be greater due to remoteness and limited resources ( 22,37 ) . ptb rates in remote rural indigenous populations of australia and the us are also high compared with non - indigenous populations , and it has been speculated that this may be due to differences in access to health care , socio - demographic , behavioural , cultural and environmental factors ( 4043 ) . results suggested that disparities between inuit populations and french / english speakers in the rest of qubec were stable over time , though there tended to be a decrease in inuit - inhabited areas . the decrease in inequalities , however , was most likely caused by a relative increase in ptb rates among french / english speakers in the rest of qubec , coupled with a slight improvement in rates in inuit - inhabited areas . wider use of ultrasound dating over time may explain some of the rate increase among french / english speakers in the rest of canada ( as menstrual dating tends to overestimate gestational age ) ( 16 ) . in inuit - inhabited areas , access to ultrasound is more limited and may have been adopted more gradually over time ( 36 ) , which suggests that ptb rates in the inuit are likely underestimated in all periods , and that disparities are likely greater in more recent periods . another important issue is that the initially protective association against ptb for first nations populations relative to french / english speakers in the rest of qubec has gradually disappeared over time , likely because ptb rates in first nations have caught up to the general population . misclassification of indigenous status through the use of language may have occurred and potentially attenuated the associations , as such individuals would have been grouped with french / english or other language categories . specifically , we could not capture inuit and first nations births in the rest of qubec among mothers who had reported french / english language , or mtis indigenous groups not identifiable through language or area indicators ( 0.4% of quebecers are mtis ) ( 10 ) . results for inuit language speakers in the rest of qubec and french / english speakers in inuit - inhabited areas should be interpreted with caution in light of small numbers . in summary , we found persistently elevated risks of ptb in inuit - inhabited areas and for inuit language speakers in the rest of qubec . the underlying pathways leading to these perinatal health disparities require further investigation to target prevention of ptb in inuit and northern populations . misclassification of indigenous status through the use of language may have occurred and potentially attenuated the associations , as such individuals would have been grouped with french / english or other language categories . specifically , we could not capture inuit and first nations births in the rest of qubec among mothers who had reported french / english language , or mtis indigenous groups not identifiable through language or area indicators ( 0.4% of quebecers are mtis ) ( 10 ) . results for inuit language speakers in the rest of qubec and french / english speakers in inuit - inhabited areas should be interpreted with caution in light of small numbers . in summary , we found persistently elevated risks of ptb in inuit - inhabited areas and for inuit language speakers in the rest of qubec . the underlying pathways leading to these perinatal health disparities require further investigation to target prevention of ptb in inuit and northern populations .

posterior vitreous detachment ( pvd ) is a common phenomenon frequently related with aging of ocular structures . the presence of persistent vitreomacular adhesions exerting tractional forces ( vitreomacular traction , vmt ) may be associated with the development of macular hole ( mh ) [ 2 , 3 ] . these alterations in the symptomatic phase may cause visual disturbances , including photopsia , metamorphopsia , blurred vision , and decreased visual acuity , which in addition of causing visual - related problems may affect negatively the patient 's health - related quality of life . the introduction of optical coherence tomography ( oct ) has allowed a more accurate visualization of the macular anatomy and better knowledge of the pathophysiology of the process , including measurement and assessment of mh characteristics [ 57 ] , facilitating treatment decision - making . the vitreous gel is responsible for the stabilization of the eyeball through collagen fibers ( mainly type ii collagen ) . collagen fibers are running in an anteroposterior direction through the vitreous center , convering in the anterior vitreous base , and inserting into the posterior vitreous cortex . spaces between the collagen fibrils are maintained by the protein opticin and the glycosaminoglycan chondroitin sulphate . spaces between the collagen fibrils are mostly filled with water ( 98% of the vitreous gel component ) and hyaluronic acid , which provides the gel - like consistency of the vitreous . the vitreoretinal interface is a complex anatomical structure composed by the union between the retina and the vitreous . densely packed collagen fibrils of the posterior vitreous cortex ( 100300 m in thickness ) lie over the macula and are superficially inserted into the internal limiting membrane ( ilm ) of the retina by means of adhesion molecules , such as laminin , fibronectin , and proteoglycans , which interact with opticin in the vitreous gel . adherences are more firmly attached to the retina at the vitreous base , optic disc , and fovea , as well as along the major retinal blood vessels . the vitreomacular junction has an annular shape , with a diameter of 3 - 4 mm . the set of events that occur as the eye ages are associated with a series of physiological changes in the vitreous gel , with progressive liquefaction ( at the age of 80 , around 50% of the vitreous gel has been liquefied ) and gradual destruction of the collagen - hyaluronic acid network . this occurs as a result of the development of fluid - filled pockets beginning in front of the macula , which over the time coalesce and enlarge , resulting in a weakened adhesion between the vitreous and the retina . this gradually predisposes to pvd , defined as separation of the posterior cortex from the ilm of the retina , which represents the final step of the normal vitreous aging process [ 11 , 12 ] . pvd is an insidious process that occurs over the course of months or years , being asymptomatic in many cases until complete separation of the vitreous from the macula and optic nerve , which is the final stage . however , the anterior attachment to the vitreous base is very strong and remains for a long time . acute symptoms of complete pvd include photopsia ( by vitreous traction on the peripheral retina ) and floaters by condensation of the vitreous collagen , glial tissue , or blood around the optic nerve . studies in healthy adults have shown that focal perifoveal pvd occurs in 50% of subjects aged between 30 and 39 , whereas complete pvd is found in 50% of subjects aged 70 years or older [ 13 , 14 ] . in addition to advanced age , pvd is more frequent in postmenopausal women by the effects of decreased estrogens on the connective tissue ( within the vitreous gel ) , as well as in the presence of myopia . the normal process of pvd due to vitreous aging may be complicated by the presence of vitreomacular adhesions between the cortex and the macular area , resulting from vitreous syneresis . these adherences may be focal or extensive , affecting the foveola only or a wide region of the macular area and the optic disc . simple vitreomacular adhesion ( vma ) is not associated with distortion of the macular architecture . however , these adherences may exert traction forces on the macula ( vmt ) , increasing secondarily during ocular saccades . this may cause retinal distortion and foveal detachment . on the other hand , continuous anteroposterior traction by vitreous contraction may cause alterations , such as cystoid macular edema . full - thickness mh is an anatomic defect in the fovea with interruption of all neural retinal layers . with the use of high - resolution oct , it has been shown that idiopathic mhs are initiated during perifoveal pvd as a consequence of the dynamic anteroposterior vmt process . this anteroposterior vmt may cause intraretinal cavitation with progression to dehiscence of the outer retinal layers and complete detachment of the cyst roof giving rise to a full - thickness defect . stages of the development of mh from focal vmt to complete aperture together with accompanying symptoms have been described by gass [ 18 , 19 ] . the introduction of enzymatic vitreolysis , which can result in the liberation of vmt , opens highly interesting new perspectives in this field . a few studies have been specifically addressed to the epidemiology of idiopathic vmt due to the overlapping of this condition with other ophthalmological diseases . a prevalence of isolated idiopathic vmt , without mh , has been estimated as approximately 22.5 cases per 100 000 of the general population , with an incidence of 0.6/100 000 persons - year . in different observational and intervention studies , the mean age of patients with vmt was around 6570 years ( range 4864 ) , with a predominance of females [ 4 , 15 ] . regarding the prevalence of mh , it has been reported around 0.1 to 0.8 in adults aged > 40 years , with an age - adjusted incidence of 7.8 cases per 100 000 of the general population per year . also , the risk of development of mh in the fellow eyes , without manifestations of pvd , has been estimated at around 712% after 5 years and 17% at 20 years . approximately two - thirds of patients with mh are women , and the disease is unilateral in 80% of cases . an increase in serum fibrinogen level has been reported as a risk factor for mh , whereas the use of estrogen replacement therapy in women decreases the risk . in subjects with myopia , now , nearly two decades since the introduction of oct , it is possible to assess and define the pathologic progression of disorders affecting vitreoretinal interface with a high level of accuracy and reproducibility . on the basis of oct - derived anatomic findings , a unified classification scheme for disease of the vitreomacular interface has been developed . with this purpose , a group of experts in diseases of the vitreoretinal interface ( international vitreomacular traction study group , ivts ) have proposed a classification system for diseases of the vitreomacular interface . this evidence - based classification is a clinically applicable system that is predictive of therapeutic outcomes and is useful for the execution and comparative analysis of clinical studies . vma represents a specific stage of partial vitreous detachment in the perifoveal area without retinal abnormalities . in previous classifications , vma is the equivalent of a stage 1 pvd [ 11 , 15 , 27 , 28 ] . vma is characterized by elevation of the cortical vitreous above the retinal surface , with the vitreous remaining attached within a 3 mm radius of the fovea ( as defined arbitrarily ) . the angle between the vitreous and the inner retinal surface is acute , and the retina displays no abnormalities in contour or morphological features of oct . vma is not accompanied by visual impairment and may be considered a normal finding in the natural course of pvd . also , vma may be subclassified by the size of the adhesion into focal ( 1500 m ) or broad ( > 1500 m ) . the cutoff of 1500 m corresponds to the area of increased vitreous adhesion to the fovea . vma usually resolves spontaneously as part of the normal process of pvd , although it may progress to vmt and , for this reason , periodic monitoring with oct is necessary . macular traction due to progression of pvd causes anatomic changes in contour of the foveal surface , intraretinal pseudocyst formation , and disappearance of foveolar depression , which typically results in reduced or distorted vision . the following anatomic criteria should be present at least in one oct image to classify an eye as having vmt : ( a ) evidence of perifoveal vitreous cortex detachment from the retinal surface , ( b ) attachment of the vitreous cortex to the macula within a 3 mm radius of the fovea , and ( c ) association of this attachment with distortion of the foveal surface , intraretinal structural changes , foveal detachment from the retinal pigment epithelium ( rpe ) , or a combination of these findings , without full - thickness interruption of all retinal layers . vmt can also be subclassified as focal or broad ( using the same cutoff of 1500 m ) depending on the width of the vitreous attachment . distortion of the foveal profile , formation of intraretinal cysts , intraretinal cavitation , subretinal fluid , and , even , rpe detachment can be observed . on the other hand , proliferation of residual of vitreous tissue provides the anatomic substrate to form an epiretinal membrane ( erm ) , which in turn may appear at any stage of vitreous separation . although spontaneous resolution of vmt may occur , traction on a large surface or the presence of erm is poor prognostic factor . in symptomatic patients , enzymatic vitreolysis or vitrectomy may be indicated . as stated above , full - thickness mh is an anatomic defect in the fovea featuring interruption of all neural retinal layers . the observation of the anatomic opening on several scans through the fovea is an unequivocal sign . according to the aperture size , mhs are considered small ( < 250 m ) , medium ( 250 to 400 m ) , and large ( diameter > 400 m ) . also , on the basis of oct findings , mh can be categorized according to the presence or absence of vmt . only patients with mh and concomitant vmt are candidates for pharmacologic vitreolysis . the correlations between mh stages commonly used in clinical practice and oct - based images proposed by the ivts group are shown in table 1 . primary mh results from vitreous traction on the fovea from anomalous pvd ( incomplete or inadequate separation of the vitreoretinal interface ) , whereas secondary mhs are caused by other pathologic conditions and do not have preexisting or concurrent vmt . secondary mhs have been reported in cases of blunt ocular trauma , lightning strike , high myopia [ 25 , 31 ] , macular schisis , macular telangiectasia type 2 , occlusion of the central retinal vein , diabetic macular edema , uveitis , and age - related macular degeneration . the availability of oct , particularly spectral domain oct ( sd - oct ) , has allowed a more accurate diagnosis and precise assessment of adhesion of the vitreous to the macula , differentiating vma from vmt . before the introduction of oct , only patients with advanced vma could have been diagnosed by biomicroscopy and , for this reason , the rates of spontaneous deterioration reported were high ( 64% ) . studies using sd - oct have shown that incomplete vitreous detachment with persistent vitreoretinal adhesions is more frequently observed than by clinical diagnosis . during the physiological process of pvd , the vitreous remains attached to the foveal region in the last stages ( figure 1 ) , so that , vma can be considered a normal stage in the natural history of pvd associated with vitreous aging [ 13 , 26 ] . only when symptoms are present or when foveal anatomic changes are observed , vma can be considered a pathological process . recently , john et al . investigated the spontaneous clinical course in 106 eyes of 81 patients identified as having vma by sd - oct and classified into three grades , with a mean follow - up of 18 months ( range 1 to 91 ) . the authors defined three grades to classify adherence : grade 1 ( 41% ) was incomplete cortical vitreous separation with attachment at the fovea , grade 2 ( 52% ) was the grade 1 findings and any intraretinal cysts , and grade 3 ( 7% ) was the grade 2 findings and the presence of subretinal fluid . by the last follow - up , spontaneous release of vma occurred in 32% of cases ( 34 eyes , in 30% , 30% , and 57% of grades 1 , 2 , and 3 , resp . ) . no changes were observed in 23 , 31 , and 2 eyes ( 52% of the total ) , and progression occurred in 7 , 8 , and 1 eye of grades 1 , 2 , and 3 , respectively ( 16% of the total ) . the authors conclude that the clinical course of patients with vma managed by initial observation was generally favourable in asymptomatic patients or with minimal symptoms of vmt . studying the retinal surface with sd - oct , it has been observed that pvd appears to begin in the perifoveal region , with a slow clinical course taking even years until complete separation of the vitreous from the papilla ( figure 2 ) . in most patients this process is asymptomatic but , in some cases , pvd may be complicated by macular pathology . in a oct study of eyes with macular edema secondary to vmt , published in 2012 , complete and spontaneous resolution of traction the clinical course of vma , particularly in asymptomatic patients , remains to be fully elucidated . systematic examination with sd - oct has been associated with an increase in diagnostic rates and has allowed assessing more accurately the course of this physiological process that may evolve into vmt , remain stable , or resolve spontaneously . therefore , in the presence of a vma syndrome , the first approach is to reexamine the patients using oct at a period of 3 months . even in cases of evolution to a vmt syndrome , observation still remains an option , given the possibility of spontaneous resolution of vmt . ocriplasmin is a truncated form of human plasmin that induces liquefaction of the vitreous and separation of the vitreous cortex from the retinal surface due to proteolytic activity against main components of the vitreomacular adhesion . the efficacy and safety of ocriplasmin have been evaluated in two pivotal , phase 3 clinical trials ( tg - mv-006 y tg - mv-007 ) carried out in the united states and europe . both studies were very similar except for the ratio of randomized assignments to ocriplasmin and placebo , which was 2 : 1 in the tg - mv-006 study and 3 : 1 in the tg - mv-007 . overall , 652 patients were randomized , 464 were assigned to treatment with a single intravitreal injection of ocriplasmin ( 125 g ) and 188 to a placebo intravitreal injection . the primary endpoint was the pharmacologic resolution of vma at day 28 , as determined by oct . secondary endpoints included the percentage of patients with complete pvd and nonsurgical closure of full - thickness mh at day 28 . eligible patients had symptomatic focal vma as seen on oct and a best - corrected visual acuity of 20/25 or less . exclusion criteria were high myopia ( more than 8 diopters or axial length > 26 mm ) , prior vitrectomy or prior laser photocoagulation of the macula , and other eye diseases that may affect visual acuity . patients with a mh > 400 m in diameter were also excluded . of note , the presence of an erm was not a criterion for exclusion . at day 28 , vma resolved in 26.5% of ocriplasmin - injected eyes and in 10.1% of placebo - injected eyes ( p < 0.001 ) . the between - group differences did not change substantially at 6 months ( 26.9% ocriplasmin versus 13.3% placebo , p = 0.001 ) . also , 72% of patients with resolution of vma showed the release during the first seven days . results of adhesion release were better in patients without erm ( 37.4% in the ocriplasmin group versus 14.3% in the placebo group , p < 0.001 ) . with regard to secondary variables ( day 28 ) , 13.4% of patients treated with ocriplasmin showed total pvd as compared to 3.7% of those treated with placebo ( p < 0.001 ) . also , nonsurgical closure of full - thickness mh was achieved in 40.6% of ocriplasmin - treated patients and in 10.6% of placebo - treated patients ( p < 0.001 ) . according to the investigator 's criteria , all patients could be treated with vitrectomy in the framework of the study if macular disease did not resolve . at 6 months , vitrectomy was performed in 17.7% of patients in the ocriplasmin group and in 26.6% of those in the placebo group ( p = 0.02 ) . at 6 months , there were statistically significant differences in favour of ocriplasmin in the gain of two or more lines ( 23.7% versus 11.2% , p < 0.001 ) or three or more lines ( 12.3% versus 6.4% , p = 0.02 ) . most adverse events were related to the development of pvd induced by ocriplasmin injection ( floaters and photopsia ) . there was a slightly higher incidence of retinal tears or detachments in the placebo group , which was attributed to the higher proportion of patients treated by means of vitrectomy in this group . the favourable results obtained in both clinical trials allowed approval of the use of intravitreal injection of ocriplasmin for the treatment of symptomatic vmt and mh by the food and drug administration ( fda ) in the united states , in november 2012 , and by the european medicines agency ( ema ) in may 2013 . outside the context of clinical trials , recent reports have provided data of the use of ocriplasmin in daily practice . in a retrospective study , 17 patients with symptomatic vmt were treated with a single intravitreal injection of ocriplasmin 0.125 mg . by day 28 , resolution of vmt was verified by sdoct in eight patients ( 47.1% ) , 7 of which ( 87.5% ) had already experienced release by day 7 . those who did not have traction release showed no statistically significant change in vma diameter . four of the five patients ( 80% ) with mh at baseline experienced resolution of their mh after injection . significant differences in visual acuity were not observed ( 20/49 at baseline and 20/46 at final follow - up ) . it should be noted that patients meeting the four positive predictor criteria ( younger than 65 years , no erm at baseline , traction < 1500 m , and phakic lens status ) showed a response rate of 75% ( three of four eyes ) . transient outer segment ellipsoid zone loss was documented in 7 cases ( 41.1% ) and subretinal fluid presence following injection was noted in 5 cases ( 29.4% ) . in another study of 19 patients with symptomatic vma treated with intravitreal ocriplasmin , resolution of vma results were significantly affected by lens status , with adhesion release in 53% of phakic patients , whereas no case of resolution of adhesions was observed in pseudophakic patients . also , closure of mhs after treatment was found in 3 of 6 patients ( 50% ) . visual acuity remains stable , with a slight tendency towards improvement in the majority of cases . only one patient showed an important loss of visual acuity ( from 20/70 to 20/200 ) due to progression of vmt to a full - thickness mh . the proportion of patients who had any ocular adverse event was 68.4% in the ocriplasmin group and 53.5% in the placebo group ( p < 0.001 ) . the most common complications included vitreous floaters ( ocriplasmin 16.8% versus placebo 7.5% , p = 0.002 ) , photopsia ( 11.8% versus 2.7% , p < 0.001 ) , blurred vision ( 8.6% versus 3.2% , p = 0.01 ) , and visual impairment ( 5.4% versus 1.6% , p = 0.02 ) . there were no differences between the groups in terms of severe ocular adverse events , including development of mh ( 5.2% versus 8.6% ) , retinal detachment ( 0% versus 1.6% ) , and reduced visual acuity ( 0.6% versus 0.5% ) . however , since the real - world use of the drug began , there have been some unfavourable reports of visual disturbances after ocriplasmin injection , including transient but profound visual decline , raising concerns regarding its safety . of 976 patients receiving ocriplasmin injection in clinical trials , 9 patients were reported to have experienced an acute decrease in vision , some to the hand motions level , within 24 hours of injection . in 8 of these 9 patients , in the clinical trials of ocriplasmin , dyschromatopsia , and electroretinographic ( erg ) changes occurred in a significantly greater number of eyes treated with ocriplasmin than in eyes receiving placebo [ 20 , 40 ] . freund et al . recently reported a single case report demonstrating changes seen in the outer photoreceptor segments by sd - oct . since the clinical trials used only time - domain oct with inferior resolution to sd - oct , it is possible that these cases may have been overlooked . in another study in which 17 patients were included , almost all the patients who responded to the treatment ( 7/8 ) had ellipsoid zone changes on the sd - oct ( figure 3 ) . these patients also had transient reduction of visual acuity and demonstrated subretinal fluid during the release process with almost the exact time course as the loss of the os ellipsoid zone . the loss of the os ellipsoid zone occurred after an average of 5 days after injection of ocriplasmin and the mean time of resolution on oct was 29.3 days . the occurrence and resolution of subretinal fluid occurred at an average of 4.8 days and 30 days after injection , respectively . however , in a retrospective review of 62 eyes with symptomatic vma treated with ocriplasmin , subretinal fluid appeared in 37% of cases , with persistence of fluid in 30% of cases after 5 months of follow - up . other studies have also shown resolution of the ellipsoid zone changes in most patients within weeks or months after ocriplasmin injection [ 43 , 44 ] . alteration of the ellipsoid zone on sd - oct and a significant decrease in erg amplitudes have been also reported in two patients with release of symptomatic vmt after ocriplasmin injection [ 45 , 46 ] . it is possible that this transient effect of the medication may be due to a diffuse enzymatic effect of the protease on the photoreceptors or the retinal pigment epithelium throughout the retina . the greater reduction in scotopic function compared with photopic function suggests that rod photoreceptors may be more susceptible than cone photoreceptors to the effects of ocriplasmin . if this transient affect occurs for both rods and cones , it may explain the dyschromatopsia , contrast sensitivity changes , dark adaptation issues , and erg changes reported in the ocriplasmin clinical trials . an ongoing phase 3b , 24-month randomized clinical trial which will evaluate erg and microperimetry in ocriplasmin - treated eyes compared to sham , will provide additional clarifications on the observed egr changes and dyschromatopsia events ( oasis study ; ntc01429441 ) already reported . surgery of idiopathic mh with ilm peeling is a very safe procedure , with good anatomic and functional results and scarce postoperative complications . data provided by clinical trials have shown that peeling of the ilm significantly increases mh closure rates and is also associated with significantly lower percentages of reoperation and reopening . therefore , ilm peeling is a cost - effective technique and the procedure of choice for all patients with idiopathic full - thickness mh susceptible to undergo surgical treatment [ 4853 ] . broad ilm peeling to the vascular arcades is recommended , so that tangential traction forces on the mh edges are removed facilitating approximation and closure . in cases of large mh ( > 400 m ) with increased risk of failure of primary surgery , alternative techniques have been proposed , such as the inverted ilm flap technique in which instead of completely removing the ilm , a remnant attached to the margins of the mh is left in place . this ilm remnant is then inverted upside down to cover the mh . with the use of this technique closure rates of 98% compared to 88% with the standard technique have been achieved . for refractory mh to the standard technique or for secondary mh after vitrectomy when peeling of the ilm has been already performed , an autologous transplantation of the ilm remnants introduced into the hole with subsequent gas tamponade contributes to the improvement of anatomic and visual outcomes . vital dyes have become effective and useful tools for identifying ocular tissues during vitrectomy , thereby facilitating ilm peeling and ensuring complete removal of this delicate membrane . the most frequently used vital dyes include triamcinolone acetonide suspension in balanced salt solution ( bss ) ( triesence ) , indocyanine green and infracyanine green , brilliant blue , and trypan blue with brilliant blue ( membrane blue - dual ) . triamcinolone suspension in bss is not a true dye but is very useful for the identification of vitreous remnants and the posterior hyaloid . deposition of crystals on the ilm surface helps the achievement of complete removal of the membrane , although it is less effective than vital dyes because triamcinolone does not increase the rigidity of ilm . indocyanine green and infracyanine green possess a great affinity for the matrix components of the ilm and produce intense staining of the ilm . besides the ability of indocyanine and infracyanine green to stain the ilm , they cause an increase in the biomechanical stiffness of the ilm , thereby facilitating its peeling . although in europe they are no longer used because of potential toxicity , they continue to be used in the united states [ 58 , 59 ] . brilliant blue has a remarkable affinity for the ilm and , although ilm staining is less intense than that achieved with indocyanine green , causes adequate staining of the ilm and may be used without fluid - air exchange . in europe the combination of trypan blue and brilliant blue allows staining of the erm , posterior hyaloid , and ilm simultaneously . this combination has a lower density than water and bss and circumvents the need for fluid - air exchange . there is controversy regarding posturing in mh surgery . although most authors recommend face - down posturing 90% of time for 10 days , different studies have reported successful hole closure in the absence of face - down positioning , given that isolation of the macula by gas tamponade maintaining the macula dried seems to be the most important factor for closure [ 6163 ] . in this respect , oct studies have shown that hole closure occurs during the first postoperative day independently of the types of gas tamponade and posturing , so that after vitrectomy with wide ilm peeling , gas tamponade would be sufficient ( preferably short - acting gases , such as sf6 ) at nonexpansible concentration , without the need of face - down posturing , avoiding the prone position during 3 to 5 days . this approach may be also indicated for phakic patients because it does not seem to increase the incidence of cataracts . combined phacovitrectomy or sequential vitrectomy and phacoemulsification are safe and effective for the treatment of mh , with equivalent anatomic and functional results . in most cases , idiopathic mh affects patients older than 50 years in which some degree of lens opacity is frequent . moreover , cataract develops in 75% to 95% of patients undergoing vitrectomy for mh within 3 years after surgery . for this reason , both cost and discomfort are lower with a single surgical procedure , and functional recovery is more rapid . combined phacovitrectomy may also decrease the risk of reopening after cataract extraction in the two - step surgical approach [ 66 , 67 ] . however , combined vitrectomy , phacoemulsification , and intraocular lens ( iol ) implantation may be associated with complications , including a high degree of postoperative anterior chamber inflammation and a higher risk of iol dislocation or papillary capture , generally as a result of excess gas tamponade and/or poor compliance to positioning . therefore , the decision of the combined versus the two - step procedure should be individualized according to the characteristics of each case and the patient 's and surgeon 's preferences . in the study of the moorfields macular hole ( mmhs ) group , an overall closure rate of 81% at 2 years was achieved in mhs stages 2 , 3 , and 4 as well as an improvement in visual acuity of 6/36 to 6/18 , which was clearly superior to results obtained in the observation group . in the vitrectomy for treatment of macular hole study ( vmhs ) , the rate of anatomic closure was 69% and the final visual acuity was higher in the operated than in nonoperated eyes ( 20/115 versus 20/166 ) . once peeling of the ilm has become popular , closure rates of 90% to 100% were reported [ 7175 ] . however , the use of indocyanine green was associated with potential toxicity in some cases and , for this reason , trypan blue and brilliant blue are in widespread use in some countries , with closure rates of 94% to 100% , without apparent severe side effects [ 7780 ] . despite its clear indication and safety in mh surgery , ilm peeling is a traumatic procedure that has acute effects on the underlying retinal nerve fiber layer . ilm peeling often results in temporary swelling of the arcuate nerve fiber layer ( sanfl ) which may be the earliest manifestation of dissociated nerve fiber layer ( donfl ) which occurs later in the postoperative period . however it is probably a transient feature that does not affect visual recovery . although peeling of the ilm has been widely adopted in mh surgery , the high percentages of hole closure obtained in the years prior to systematic ilm peeling add uncertainty about whether to use it in all cases . recently , spiteri cornish et al . [ 81 , 82 ] carried out a systematic review and meta - analysis to assess the success of hm surgery with ilm peeling compared with the nonpeeling technique . four randomized clinical trials comparing both techniques were identified [ 48 , 49 , 51 , 81 , 82 ] . there was no evidence of a difference in the primary outcome ( distance visual acuity at six months ) , nor in distance visual acuity at 12 months between randomized groups . overall , 66.2% achieved a visual acuity equal or greater than 69 letters on etdrs charts ( corresponding snellen visual acuity 20/40 ) and 77.9% gained more than three etdrs lines . improvement of visual acuity was higher in patients in which primary anatomic closure was achieved ( final visual acuity 72.8 7.6 letters and a mean improvement of 21.6 7.1 letters ) than in eyes in which further surgery was required ( 66.4 8.6 and 17.4 7.7 letters , resp . ) . however , visual improvement was obtained somewhat earlier in the ilm peeling group and , at 3 months , improvement was greater if ilm peeling was performed . in addition , the percentage of primary closure was higher in the ilm peeling as compared with no peeling ( 89.9% versus 50.3% , with an odds ratio ( or ) of 9.27 and 95% confidence interval [ ci ] of 4.9817.24 ) . when reoperations were excluded from the analysis , the ilm peeling group continued to have more favourable results ( or 3.99 , 95% ci 1.639.75 ) . also , in mh stage 2 , the efficacy rate was better for ilm peeling than no peeling ( 91.6% versus 61.3% , with an or of 6.19 ; 95% ci 1.6523.20 ) [ 48 , 49 , 83 ] . this higher success rate was not accompanied by an increase of perioperative complications , neither in the reports in which the ilm was stained with indocyanine green . in the meta - analysis , the rate of intraoperative complications was 19.32% for the ilm peeling group as compared with 21.1% for the nonpeeling group ( or 0.94 , 95% ci 0.471.87 ) . the most frequent intraoperative complications were small retinal hemorrhage ( 619% ) , retinal tears ( 5.432% ) , retinal detachment ( 26% ) , and choroidal hemorrhage ( 03% ) . according to these data , the authors conclude that ilm peeling offers more favourable cost - effectiveness compared with no peeling in mh surgery [ 81 , 82 ] . although anatomic closure in mh surgery is achieved in more than 90% of cases , sometimes it does not correlate well with improvement in visual acuity . multiple studies using oct have assessed hole configuration in an attempt to establish a correlation with postoperative visual acuity [ 8491 ] , emphasizing the importance of changes in the outer retina . defined a macular hole index ( mhi ) as a ratio of hole height to base diameter of hole , calculated from oct transverse images of the macular area , establishing that a mhi 0.5 was correlated with better postoperative visual acuity than mih < 0.5 . ruiz - moreno et al . described the diameter hole index ( dhi ) as a ratio between minimum hole diameter and base diameter , showing the minimum diameter was the best preoperative predictive prognostic factor . different studies have shown a direct correlation between integrity of the hyperreflective line as is / os junction of photoreceptors and postoperative improvement of visual acuity . in the study of kitaya et al . , postoperative vision 0.7 was correlated with good reconstitution of the is / os junction . however , sano et al . showed that a continuous is / os line was not a reliable prognostic factor in the early postoperative period given that abnormalities of the is / os line seen on sd - oct can be gradually repaired , with achievement of a continuous is / os line at 6 months . spaide and curcio assessed the correlation of the outer retina analyzed by means of sd - oct and histopathological findings , showing that the hyperreflective line identified as is / os junction of photoreceptors corresponded to the ellipsoid portion of the photoreceptor inner segment , containing mitochondria . wakabayashi et al . using sd - oct described that reconstitution of the external limiting membrane ( elm ) was more important to predict subsequent restoration of the foveal photoreceptor layer than the ellipsoid zone restoration . restoration of elm seems to be a necessary factor for reconstitution of the ellipsoid band , with subsequent migration of photoreceptors and complete closure of the full - thickness mh . ruiz - moreno et al . have analyzed 164 eyes with mh treated by vitrectomy and ilm peeling showing that restoration of the ellipsoid portion of the photoreceptor inner segment is an important prognostic factor for visual rehabilitation after mh surgery . reopening of the hole ( figure 4 ) is one of the best known complications after initially successful mh treatment with vitreous surgery [ 67 , 91101 ] . peeling of the ilm during primary mh surgery is one of the factors that has been mostly related to the incidence of reopening , varying between 0% and 8% in eyes with ilm peeling [ 67 , 9597 ] and between 2% and 16% in eyes with no peeling [ 67 , 9395 , 97 ] . the variable percentages reported in the studies are due in part to differences in the length of follow - up , with higher rates associated with prolonged follow - up periods . paques et al . reported a 9.5% incidence with a mean follow - up of 2 years , whereas scott et al . found a 12% incidence with a mean follow - up of 7 years . kumagai et al . analyzed the results of surgery in a series of 877 cases of mh , increasing the reopening percentage to 28.1% with no ilm peeling . the incidence of recurrence was 0.39% in eyes with peeling of the ilm , increasing to 7.2% with no peeling . besides no peeling , statistically significant risk factors for reopening were myopia of more than 6 diopters and intraoperative retinal tears . retinal tears treated with laser may be one of the factors that increase the development of erm , with subsequent tangential traction and reopening of the mh . no peeling of the ilm may be associated with a higher risk of erm formation [ 92 , 93 , 97 , 100 ] . yoshida and kishi observed the presence of erm in all cases of reopening of the hole . however , kumagai et al . did not report erm in none of the cases with hole reopening assessed by sd - oct . in relation to the incidence of reopening with bilateral mh , duker et al . reported bilateral reopening in 38% of cases , christmas et al . in 59% , scott et al . in 38% , and kumagai et al . in 14.9% . cataract surgery in the postoperative period of mh surgery has been involved in the reopening of mh . paques et al . observed that 73% of cases of hole reopening occurred after a secondary cataract surgery . bhatnagar et al . reported that in the presence of cystic macular edema after secondary cataract surgery , there was a sevenfold increase in the risk of reopened holes , and garca - arum et al and sheidow and gonder reported cystoid edema in combined surgical procedures and that the incidence of hole reopening did not increase in secondary cataracts . with regard to treatment of persisting mh , ilm peeling and erm removal should be performed in those cases in which these procedures were not performed at the initial macular surgery , together with long - acting gas tamponade ( c3f8 ) and strict face - down positioning during the first postoperative days . in these patients , when ilm peeling and removal of the erm have been performed in the first surgical procedure , the success of reoperation decreases . in a series of 30 patients reported by d'souza et al . with initial ilm peel who underwent repeat surgery involving vitrectomy , enlargement of ilm rhexis , and gas tamponade with c3f8 , more extensive ilm peeling causing tangential traction due to fibrosis of dissection margin may contribute to the anatomic closure . the use of growth factors , such as platelet - derived growth factors as a stimulus of glial progenitor cells , may be useful if the ilm has been adequately peeled ( figure 5 ) , as well as the use of heavy silicone oil in patients with positioning difficulties . based on the aforementioned data and as shown in the schematic representation in figure 6 , patients with vma can be observed without the need of any intervention . in cases of vmt if the patient is asymptomatic , a follow - up control at 3 months may be sufficient . during this interval , the patient should be advised to perform periodic self - examinations with the amsler grid or monocular reading tests . in case of symptoms , intensity and disability should be assessed . there is no consensus criterion regarding the degree of vision loss that should be considered significant and amenable to treatment . however , in the tg - mv-006 y tg - mv-007 clinical trials , patients with visual acuity equal or lower than 20/25 were eligible , so that this level of visual impairment can be already considered to be susceptible of treatment . also , other causes that may justify decreased visual acuity should be excluded . metamorphopsia clinically significant for the patient and visual loss progression are also key factors at the time of adopting a more interventional therapeutic attitude . despite these considerations , a period of observation may be an option for these patients , because spontaneous resolution is still possible . in case of deciding an active treatment , the presence of other associated macular diseases , such as erm , should be excluded . when traction is 1500 m , enzymatic vitreolysis with ocriplasmin is the treatment of choice . in the presence of > 1500 m traction or erm , surgical treatment with vitrectomy 400 m in size with mvt and in the absence of erm , enzymatic vitreolysis with ocriplasmin is again the most recommendable option . in cases of holes > 400 m , or in the absence of evident vmt , or in the presence of erm , vitrectomy is the first option . patients undergoing enzymatic vitreolysis with intravitreal injection of ocriplasmin should be evaluated at 7 and 30 days . most cases of vmt or mh resolve within the first week of treatment and also at this time the occurrence of potential treatment - related complications should be excluded . if resolution of vmt and/or hole closure had not occurred after a month of treatment , the likelihood of success is highly improbable and vitrectomy can be performed . patients with lamellar mh or pseudomacular holes in which traction is usually absent are also candidates for enzymatic vitreolysis . in cases of vmt associated with other retinal diseases , such as age - related macular degeneration , diabetic macular edema , or vitreomacular interface pathology in the myope , it is still too early to make a recommendation on the impact of enzymatic vitreolysis with ocriplasmin in the treatment of these conditions , and we should await for results of ongoing clinical trials on this topic . finally , in all cases , the final decision regarding treatment with enzymatic vitreolysis with ocriplasmin or vitrectomy should be consensuated with the patient . all cases in which the use of ocriplasmin is considered a first treatment option can be successfully treated by means of vitrectomy . also , it may be possible that patients who initially are not ideal candidates for enzymatic vitreolysis may have their pathologic condition solved by treatment with ocriplasmin . favourable prognostic factors for the choice of vitreolysis have been identified including young age and phakic status , but difficulties to maintain postoperative face - down posture or the waiting lists for vitrectomy are variables that should also be considered . enzymatic vitreolysis based on the intravitreal injection of ocriplasmin is a treatment option with proven efficacy and adequate safety profile in selected patients with vmt and mh . in cases of vmt , treatment with ocriplasmin is indicated when traction is 1500 m and in the absence of concurrent macular diseases ( erm ) . in the case of mh , the hole diameter should be 400 , traction has to be present , and erm should be absent . when resolution of the process after one month of the procedure is not achieved , vitrectomy with ilm peeling would be the surgical treatment of choice .

the paper presents a review of the sequence of events of posterior vitreous detachment ( pvd ) , vitreomacular adhesion ( vma ) , vitreomacular traction ( vmt ) , and macular hole ( mh ) from their pathophysiological aspects , clinical features , diagnostic implications , and current management strategies . a treatment algorithm to be used in clinical practice in patients with vma , vmt , and mh based on the presence of symptoms , visual acuity , associated epiretinal membrane , and width of the vitreous attachment is presented . observation , pharmacologic vitreolysis with ocriplasmin , and surgical treatment are positioned as treatment options in the different steps of the therapeutic algorithm , with clear indications of the paths to be followed according to the initial presenting manifestations and the patient 's clinical course .

1. Introduction 2. Diagnosis, Definition, and Classification of VMT and MH 3. Treatment Options 4. Practical Considerations: Therapeutic Algorithm 5. Conclusions

posterior vitreous detachment ( pvd ) is a common phenomenon frequently related with aging of ocular structures . the presence of persistent vitreomacular adhesions exerting tractional forces ( vitreomacular traction , vmt ) may be associated with the development of macular hole ( mh ) [ 2 , 3 ] . these alterations in the symptomatic phase may cause visual disturbances , including photopsia , metamorphopsia , blurred vision , and decreased visual acuity , which in addition of causing visual - related problems may affect negatively the patient 's health - related quality of life . collagen fibers are running in an anteroposterior direction through the vitreous center , convering in the anterior vitreous base , and inserting into the posterior vitreous cortex . spaces between the collagen fibrils are mostly filled with water ( 98% of the vitreous gel component ) and hyaluronic acid , which provides the gel - like consistency of the vitreous . the vitreoretinal interface is a complex anatomical structure composed by the union between the retina and the vitreous . densely packed collagen fibrils of the posterior vitreous cortex ( 100300 m in thickness ) lie over the macula and are superficially inserted into the internal limiting membrane ( ilm ) of the retina by means of adhesion molecules , such as laminin , fibronectin , and proteoglycans , which interact with opticin in the vitreous gel . adherences are more firmly attached to the retina at the vitreous base , optic disc , and fovea , as well as along the major retinal blood vessels . the set of events that occur as the eye ages are associated with a series of physiological changes in the vitreous gel , with progressive liquefaction ( at the age of 80 , around 50% of the vitreous gel has been liquefied ) and gradual destruction of the collagen - hyaluronic acid network . this occurs as a result of the development of fluid - filled pockets beginning in front of the macula , which over the time coalesce and enlarge , resulting in a weakened adhesion between the vitreous and the retina . pvd is an insidious process that occurs over the course of months or years , being asymptomatic in many cases until complete separation of the vitreous from the macula and optic nerve , which is the final stage . however , the anterior attachment to the vitreous base is very strong and remains for a long time . acute symptoms of complete pvd include photopsia ( by vitreous traction on the peripheral retina ) and floaters by condensation of the vitreous collagen , glial tissue , or blood around the optic nerve . in addition to advanced age , pvd is more frequent in postmenopausal women by the effects of decreased estrogens on the connective tissue ( within the vitreous gel ) , as well as in the presence of myopia . the normal process of pvd due to vitreous aging may be complicated by the presence of vitreomacular adhesions between the cortex and the macular area , resulting from vitreous syneresis . these adherences may be focal or extensive , affecting the foveola only or a wide region of the macular area and the optic disc . simple vitreomacular adhesion ( vma ) is not associated with distortion of the macular architecture . however , these adherences may exert traction forces on the macula ( vmt ) , increasing secondarily during ocular saccades . a prevalence of isolated idiopathic vmt , without mh , has been estimated as approximately 22.5 cases per 100 000 of the general population , with an incidence of 0.6/100 000 persons - year . in different observational and intervention studies , the mean age of patients with vmt was around 6570 years ( range 4864 ) , with a predominance of females [ 4 , 15 ] . approximately two - thirds of patients with mh are women , and the disease is unilateral in 80% of cases . on the basis of oct - derived anatomic findings , a unified classification scheme for disease of the vitreomacular interface has been developed . vma is characterized by elevation of the cortical vitreous above the retinal surface , with the vitreous remaining attached within a 3 mm radius of the fovea ( as defined arbitrarily ) . the angle between the vitreous and the inner retinal surface is acute , and the retina displays no abnormalities in contour or morphological features of oct . macular traction due to progression of pvd causes anatomic changes in contour of the foveal surface , intraretinal pseudocyst formation , and disappearance of foveolar depression , which typically results in reduced or distorted vision . the following anatomic criteria should be present at least in one oct image to classify an eye as having vmt : ( a ) evidence of perifoveal vitreous cortex detachment from the retinal surface , ( b ) attachment of the vitreous cortex to the macula within a 3 mm radius of the fovea , and ( c ) association of this attachment with distortion of the foveal surface , intraretinal structural changes , foveal detachment from the retinal pigment epithelium ( rpe ) , or a combination of these findings , without full - thickness interruption of all retinal layers . vmt can also be subclassified as focal or broad ( using the same cutoff of 1500 m ) depending on the width of the vitreous attachment . on the other hand , proliferation of residual of vitreous tissue provides the anatomic substrate to form an epiretinal membrane ( erm ) , which in turn may appear at any stage of vitreous separation . according to the aperture size , mhs are considered small ( < 250 m ) , medium ( 250 to 400 m ) , and large ( diameter > 400 m ) . also , on the basis of oct findings , mh can be categorized according to the presence or absence of vmt . only patients with mh and concomitant vmt are candidates for pharmacologic vitreolysis . the correlations between mh stages commonly used in clinical practice and oct - based images proposed by the ivts group are shown in table 1 . primary mh results from vitreous traction on the fovea from anomalous pvd ( incomplete or inadequate separation of the vitreoretinal interface ) , whereas secondary mhs are caused by other pathologic conditions and do not have preexisting or concurrent vmt . the availability of oct , particularly spectral domain oct ( sd - oct ) , has allowed a more accurate diagnosis and precise assessment of adhesion of the vitreous to the macula , differentiating vma from vmt . during the physiological process of pvd , the vitreous remains attached to the foveal region in the last stages ( figure 1 ) , so that , vma can be considered a normal stage in the natural history of pvd associated with vitreous aging [ 13 , 26 ] . the authors defined three grades to classify adherence : grade 1 ( 41% ) was incomplete cortical vitreous separation with attachment at the fovea , grade 2 ( 52% ) was the grade 1 findings and any intraretinal cysts , and grade 3 ( 7% ) was the grade 2 findings and the presence of subretinal fluid . no changes were observed in 23 , 31 , and 2 eyes ( 52% of the total ) , and progression occurred in 7 , 8 , and 1 eye of grades 1 , 2 , and 3 , respectively ( 16% of the total ) . the authors conclude that the clinical course of patients with vma managed by initial observation was generally favourable in asymptomatic patients or with minimal symptoms of vmt . studying the retinal surface with sd - oct , it has been observed that pvd appears to begin in the perifoveal region , with a slow clinical course taking even years until complete separation of the vitreous from the papilla ( figure 2 ) . in a oct study of eyes with macular edema secondary to vmt , published in 2012 , complete and spontaneous resolution of traction the clinical course of vma , particularly in asymptomatic patients , remains to be fully elucidated . therefore , in the presence of a vma syndrome , the first approach is to reexamine the patients using oct at a period of 3 months . ocriplasmin is a truncated form of human plasmin that induces liquefaction of the vitreous and separation of the vitreous cortex from the retinal surface due to proteolytic activity against main components of the vitreomacular adhesion . exclusion criteria were high myopia ( more than 8 diopters or axial length > 26 mm ) , prior vitrectomy or prior laser photocoagulation of the macula , and other eye diseases that may affect visual acuity . of note , the presence of an erm was not a criterion for exclusion . according to the investigator 's criteria , all patients could be treated with vitrectomy in the framework of the study if macular disease did not resolve . the favourable results obtained in both clinical trials allowed approval of the use of intravitreal injection of ocriplasmin for the treatment of symptomatic vmt and mh by the food and drug administration ( fda ) in the united states , in november 2012 , and by the european medicines agency ( ema ) in may 2013 . in another study of 19 patients with symptomatic vma treated with intravitreal ocriplasmin , resolution of vma results were significantly affected by lens status , with adhesion release in 53% of phakic patients , whereas no case of resolution of adhesions was observed in pseudophakic patients . visual acuity remains stable , with a slight tendency towards improvement in the majority of cases . the most common complications included vitreous floaters ( ocriplasmin 16.8% versus placebo 7.5% , p = 0.002 ) , photopsia ( 11.8% versus 2.7% , p < 0.001 ) , blurred vision ( 8.6% versus 3.2% , p = 0.01 ) , and visual impairment ( 5.4% versus 1.6% , p = 0.02 ) . there were no differences between the groups in terms of severe ocular adverse events , including development of mh ( 5.2% versus 8.6% ) , retinal detachment ( 0% versus 1.6% ) , and reduced visual acuity ( 0.6% versus 0.5% ) . in 8 of these 9 patients , in the clinical trials of ocriplasmin , dyschromatopsia , and electroretinographic ( erg ) changes occurred in a significantly greater number of eyes treated with ocriplasmin than in eyes receiving placebo [ 20 , 40 ] . however , in a retrospective review of 62 eyes with symptomatic vma treated with ocriplasmin , subretinal fluid appeared in 37% of cases , with persistence of fluid in 30% of cases after 5 months of follow - up . it is possible that this transient effect of the medication may be due to a diffuse enzymatic effect of the protease on the photoreceptors or the retinal pigment epithelium throughout the retina . if this transient affect occurs for both rods and cones , it may explain the dyschromatopsia , contrast sensitivity changes , dark adaptation issues , and erg changes reported in the ocriplasmin clinical trials . therefore , ilm peeling is a cost - effective technique and the procedure of choice for all patients with idiopathic full - thickness mh susceptible to undergo surgical treatment [ 4853 ] . broad ilm peeling to the vascular arcades is recommended , so that tangential traction forces on the mh edges are removed facilitating approximation and closure . in cases of large mh ( > 400 m ) with increased risk of failure of primary surgery , alternative techniques have been proposed , such as the inverted ilm flap technique in which instead of completely removing the ilm , a remnant attached to the margins of the mh is left in place . deposition of crystals on the ilm surface helps the achievement of complete removal of the membrane , although it is less effective than vital dyes because triamcinolone does not increase the rigidity of ilm . besides the ability of indocyanine and infracyanine green to stain the ilm , they cause an increase in the biomechanical stiffness of the ilm , thereby facilitating its peeling . although in europe they are no longer used because of potential toxicity , they continue to be used in the united states [ 58 , 59 ] . although most authors recommend face - down posturing 90% of time for 10 days , different studies have reported successful hole closure in the absence of face - down positioning , given that isolation of the macula by gas tamponade maintaining the macula dried seems to be the most important factor for closure [ 6163 ] . therefore , the decision of the combined versus the two - step procedure should be individualized according to the characteristics of each case and the patient 's and surgeon 's preferences . in the study of the moorfields macular hole ( mmhs ) group , an overall closure rate of 81% at 2 years was achieved in mhs stages 2 , 3 , and 4 as well as an improvement in visual acuity of 6/36 to 6/18 , which was clearly superior to results obtained in the observation group . in the vitrectomy for treatment of macular hole study ( vmhs ) , the rate of anatomic closure was 69% and the final visual acuity was higher in the operated than in nonoperated eyes ( 20/115 versus 20/166 ) . ilm peeling often results in temporary swelling of the arcuate nerve fiber layer ( sanfl ) which may be the earliest manifestation of dissociated nerve fiber layer ( donfl ) which occurs later in the postoperative period . although peeling of the ilm has been widely adopted in mh surgery , the high percentages of hole closure obtained in the years prior to systematic ilm peeling add uncertainty about whether to use it in all cases . there was no evidence of a difference in the primary outcome ( distance visual acuity at six months ) , nor in distance visual acuity at 12 months between randomized groups . improvement of visual acuity was higher in patients in which primary anatomic closure was achieved ( final visual acuity 72.8 7.6 letters and a mean improvement of 21.6 7.1 letters ) than in eyes in which further surgery was required ( 66.4 8.6 and 17.4 7.7 letters , resp . ) in addition , the percentage of primary closure was higher in the ilm peeling as compared with no peeling ( 89.9% versus 50.3% , with an odds ratio ( or ) of 9.27 and 95% confidence interval [ ci ] of 4.9817.24 ) . the most frequent intraoperative complications were small retinal hemorrhage ( 619% ) , retinal tears ( 5.432% ) , retinal detachment ( 26% ) , and choroidal hemorrhage ( 03% ) . multiple studies using oct have assessed hole configuration in an attempt to establish a correlation with postoperative visual acuity [ 8491 ] , emphasizing the importance of changes in the outer retina . defined a macular hole index ( mhi ) as a ratio of hole height to base diameter of hole , calculated from oct transverse images of the macular area , establishing that a mhi 0.5 was correlated with better postoperative visual acuity than mih < 0.5 . showed that a continuous is / os line was not a reliable prognostic factor in the early postoperative period given that abnormalities of the is / os line seen on sd - oct can be gradually repaired , with achievement of a continuous is / os line at 6 months . spaide and curcio assessed the correlation of the outer retina analyzed by means of sd - oct and histopathological findings , showing that the hyperreflective line identified as is / os junction of photoreceptors corresponded to the ellipsoid portion of the photoreceptor inner segment , containing mitochondria . restoration of elm seems to be a necessary factor for reconstitution of the ellipsoid band , with subsequent migration of photoreceptors and complete closure of the full - thickness mh . yoshida and kishi observed the presence of erm in all cases of reopening of the hole . reported that in the presence of cystic macular edema after secondary cataract surgery , there was a sevenfold increase in the risk of reopened holes , and garca - arum et al and sheidow and gonder reported cystoid edema in combined surgical procedures and that the incidence of hole reopening did not increase in secondary cataracts . the use of growth factors , such as platelet - derived growth factors as a stimulus of glial progenitor cells , may be useful if the ilm has been adequately peeled ( figure 5 ) , as well as the use of heavy silicone oil in patients with positioning difficulties . based on the aforementioned data and as shown in the schematic representation in figure 6 , patients with vma can be observed without the need of any intervention . however , in the tg - mv-006 y tg - mv-007 clinical trials , patients with visual acuity equal or lower than 20/25 were eligible , so that this level of visual impairment can be already considered to be susceptible of treatment . in case of deciding an active treatment , the presence of other associated macular diseases , such as erm , should be excluded . when traction is 1500 m , enzymatic vitreolysis with ocriplasmin is the treatment of choice . in the presence of > 1500 m traction or erm , surgical treatment with vitrectomy 400 m in size with mvt and in the absence of erm , enzymatic vitreolysis with ocriplasmin is again the most recommendable option . in cases of holes > 400 m , or in the absence of evident vmt , or in the presence of erm , vitrectomy is the first option . in cases of vmt associated with other retinal diseases , such as age - related macular degeneration , diabetic macular edema , or vitreomacular interface pathology in the myope , it is still too early to make a recommendation on the impact of enzymatic vitreolysis with ocriplasmin in the treatment of these conditions , and we should await for results of ongoing clinical trials on this topic . finally , in all cases , the final decision regarding treatment with enzymatic vitreolysis with ocriplasmin or vitrectomy should be consensuated with the patient . enzymatic vitreolysis based on the intravitreal injection of ocriplasmin is a treatment option with proven efficacy and adequate safety profile in selected patients with vmt and mh . in cases of vmt , treatment with ocriplasmin is indicated when traction is 1500 m and in the absence of concurrent macular diseases ( erm ) . in the case of mh , the hole diameter should be 400 , traction has to be present , and erm should be absent .

soil - transmitted helminths ( sth ) or geohelminths infect more than 2 billion people worldwide , especially in developing countries located in tropical and subtropical regions . the most prevalent species are ascaris lumbricoides ( roundworm ) , trichuris trichiura ( whipworm ) , and hookworms ( necator americanus and ancylostoma duodenale ) . children and women of childbearing age are among the high - risk groups for these parasitic diseases . the morbidity caused by sth infections is highly associated with the size of the worm population residing in the intestines ( also known as worm burden ) . infections of heavy intensity are more likely to cause impaired physical growth and cognitive development as well as micronutrient deficiencies , including iron - deficiency anaemia [ 1 , 2 ] . further , due to their chronic and insidious nature , even light intensity infections may compromise health outcomes . to reduce the morbidity caused by these parasitoses , the world health organization ( who ) recommends cyclical ( annual or biannual ) benzimidazole - based mass drug administration to groups at high - risk of infection , especially pre- and schoolchildren . the basic premise of this strategy is that even though this intervention may not decrease transmission and prevalence , it will in time reduce hosts ' worm burden with the ensuing reduction in health impact . in addition to health impact , sth infections affect the human host in more subtle manner : as extracellular metazoan parasites , helminths trigger type-2 immune response mediated by t - helper type 2 ( th2 ) cells . this response is typically characterized by expansion of mast cells , eosinophils , basophils , group 2 innate lymphoid cells ( ilc-2 ) , and alternatively activated macrophages ( aams ) , as well as increased production of th2 cytokines ( e.g. , il-4 , il-5 , il-9 , and il-13 ) and ige [ 35 ] . on the other hand , intracellular pathogens such as viruses , bacteria , protozoa , and fungi elicit a type-1 response , characterized by increased numbers of phagocytic neutrophils and macrophages , as well as cytotoxic cd8 t cells and th1 cells . antigen - presenting dendritic cells ( dcs ) secrete interleukin- ( il- ) 12 promoting differentiation of naive cd4 cells , first into null t - helper ( th0 ) and then into th1 . via production of interferon - gamma ( ifn- ) and il-2 to a lesser degree , th1 cells direct , maintain , and enhance the antimicrobial effect of type-1 response . both subsets of t - helper cells naturally , a healthy resolution of infections relies on a dynamic combination of the two [ 5 , 7 ] . chronic helminth infections such as the ones caused by sth exhibit a modified type-2 response , in which a superimposition of regulatory mechanisms exerted over the basic response pattern takes place . this regulation is mainly achieved by the expansion and induction of regulatory t cells ( treg ) . increased levels of anti - inflammatory cytokines il-10 and transforming growth factor beta ( tgf- ) are hallmarks of this response [ 4 , 8 , 9 ] . it has been suggested that , during chronic helminthiases , this anti - inflammatory network may play a key role in the lower prevalence of allergic disease observed in th2-skewed populations [ 7 , 1012 ] . conversely , helminth - induced il-10-mediated immunosuppression has raised concerns for populations in which sth infections coexist with other morbidities such as hiv / aids , malaria , and tuberculosis , as clearance for the latter depends on an active and timely type-1 response [ 8 , 1316 ] . in this context , successful deworming treatment has been proposed as a practical and inexpensive way to rebalance the immune response [ 17 , 18 ] . recent studies have observed that after administration of anthelminthic medication , eosinophil counts , il-10 levels [ 1921 ] , and ige concentrations show a significant decrease from pretreatment values . moreover , in hiv-1 infected individuals , deworming was shown to promote a significant decrease in plasma hiv rna levels [ 18 , 22 ] and cd8 t cells counts . also , a randomized control trial in kenya demonstrated a significant increase in cd4 t cell counts after deworming . other studies , however , found no evidence of this effect [ 18 , 19 ] . the principal aim of the present study was to characterize the immune profile in schoolchildren infected with geohelminths . the study was conducted in april 2014 and data were collected from schoolchildren living in linaca , a rural community of the municipality of tatumbla , department of francisco morazn in honduras . the study was approved by brock university 's bioscience research ethics board ( file number bu 12 - 262 , dated june 27 , 2013 ) as well as by the research ethics board of the master 's program in infectious and zoonotic diseases , school of microbiology , national autonomous university of honduras , unah ( file number cei - meiz 01 - 2013 , dated september 23 , 2013 ) . the school of linaca participates in the honduras national deworming program , which provides enrolled children with a 400 mg single - dose albendazole annually . at the time of the study , children who had not received deworming treatment from other sources in the past 3 months were included in the study . basic demographic and epidemiological data of participant children were obtained using a structured questionnaire during a 5-minute face - to - face interview conducted in spanish . body weight and height of participants were measured twice by different researchers and their average was used to calculate the following anthropometric indicators : ( a ) height - for - age z - score ( haz ) , ( b ) weight - for - age z - score ( waz ) , and ( c ) body - mass - index - for - age z - score ( baz ) . these indicators were used to ascertain children 's nutritional status as per international parameters : stunted growth ( chronic malnutrition ) , thinness , and underweight , respectively . a same - day samples were kept in portable coolers and examined early afternoon at the laboratory facilities of unah 's school of microbiology . the presence of intestinal parasites was assessed using both kato - katz and formol - ethyl acetate concentration methods . for the latter , microscopic examination of kato - katz smears was done between 30 and 60 min of preparation . helminth eggs were identified and counted and the number of eggs per gram ( epg ) was calculated . infection intensities were classified as light , moderate , or heavy , based on the epg calculations , according to who criteria . diagnostic accuracy was ensured by having 100% of negative and 10% of positive smears read again by a different researcher immediately after the first reading . formol - ethyl acetate was done one month after sample collection and sediments were observed with dry and immersion oil objective lenses in order to identify helminth and protozoa stages , respectively . blood samples obtained from the cubital vein were collected in 3 ml tubes of each k2edta and serum separator and clot activator ( bd vacutainer , nj , usa ) . the latter were centrifuged within 4 hours and serum was stored at 21c until immunological analysis . hematological automated analyses were performed within 4 hours of sample collection and were done with an abx pentra 120 ( horiba - abx - sas , montpellier , france ) . according to the parameters established in the most recent ( 2011 - 2012 ) demographic and health survey in honduras ( dhs - hn ) , anaemia was defined as hb concentration < 12 g / dl . eosinophilia was defined according to international standards as eosinophil count 500/l in peripheral blood . mild , moderate , or severe eosinophilia were defined if cell counts were 5001500/l , > 15005000/l , or > 5000/l , respectively . cytokine concentrations in serum samples were measured using a magpix magnetic beads platform ( luminex xmap , austin , tx , usa ) . multiplex panels containing magnetic beads covered with fluorescent dyed conjugated to a monoclonal antibody specific for each target molecule were used to quantify th1/th2 cytokines ( il-2 , il-6 , il-8 , il-12p70 , ifn- , gm - csf , tnf- , il-4 , il-5 , and il-13 ) and the regulatory cytokine il-10 . serum concentrations of total ige were also quantified using magnetic beads platform but with singleplex kits , bio - plex pro human ige isotyping ( bio - rad laboratories , inc . hyper - ige was defined as a serum concentration exceeding 100 iu / ml and was classified as mild ( > 100399 iu / ml ) , moderate ( > 399999 cytokine and ige tests were performed according to the manufacturer 's instructions ( bio - rad laboratories , inc . descriptive statistics were used to characterize the study population . point prevalence with 95% confidence intervals ( 95% ci ) was calculated for overall sth infections and for each parasite species , as well as for mixed sth infections ( i.e. , infected with two or more species ) . associations between nutritional indicators and parasite infections were explored using both univariate and multivariable logistic regression models . unadjusted and adjusted odds ratio ( or and adj . or ) with 95% ci were determined . due to the non - gaussian distribution of the immunological markers , nonparametric methods were used as follows . geometric means and kruskal - wallis test was used to assess differences among groups , followed by dunn 's test to investigate individual group differences . all statistical analyses were conducted using stata 13 ( statacorp lp , tx , usa ) and the level of significance was defined as p < 0.05 . table 1 summarizes the characteristics and parasitological findings in the study population . a total of 225 schoolchildren attending grades 1 to 6 ( age 613 ) participated in the study . most of the children lived in households with piped water ( 88% ) and flushing toilet and/or latrines ( 98% ) but 40% lived in households with earthen floor . qualitative kato - katz results were 100% correlated with those of formol - ethyl acetate . no cases of s. stercoralis or hookworm infections were found with either technique but one case of taenia solium was identified by the presence of gravid proglottids in the stool sample . among pathogenic protozoa , giardia intestinalis and entamoeba histolytica / dispar were present in 4% and 9% of the samples , respectively . prevalence for t. trichiura and a. lumbricoides was 22.2% ( 95% ci = 17.228.2 ) and 20.4% ( 95% ci = 15.626.2 ) , respectively . mixed sth infections represented 43% of all infections observed . while the majority ( 84% ) of trichuriasis cases were light intensity , about one - third ( 32.6% ) of ascariasis cases were moderate - to - heavy intensity ( table 1 ) . only 20% of moderate - to - heavy infections with both a. lumbricoides and t. trichiura occurred as single infections . in the multivariable analysis , almost three - quarters ( 71.7% ) of ascariasis cases occurred in boys and they had almost three times the odds of having a. lumbricoides infections compared to girls ( adj . or = 2.82 , 95% ci = 1.345.94 , and p = 0.006 ) . children living in households with earthen floor had twice the odds of helminthic infections ( adj . or = 2.29 , 95% ci = 1.154.55 , and p = 0.018 ) . conversely , children with access to piped water in their households showed 64% reduced odds of ascariasis ( adj . about 12% of the children were overweight , and this condition was significantly higher in boys than girls ( 73% versus 27% , p = 0.036 ) ; only one of these children was stunted . multivariable logistic models controlling for age and sex were constructed , and adjusted or were calculated . in this population , stunting was significantly associated with the age of participants as well as with trichuriasis . per every year of age , the odds of stunting increased in 34% ( adj . or = 1.34 , 95% ci = 1.031.73 , and p = 0.025 ) . similarly , children harbouring t. trichiura infections had almost four times the odds of being stunted ( adj . or = 3.93 , 95% ci = 1.0314.93 , and p = 0.045 ) when compared with children without sth infection . further , children harbouring moderate - to - heavy trichuriasis had an additional 70% increased odds of being stunted ( adj . or = 6.64 , 95% ci = 1.1937.09 , and p = 0.031 ) . figures 1 and 2 depict the variation observed in eosinophils count ( cells/l ) and serum levels of ige by sth infection and infection intensity . the geometric means and 95% ci of these parameters by sth infection the overall prevalence of eosinophilia was 31.7% ( 95% ci = 25.938.1 ) , and mild eosinophilia accounted for the vast majority ( 91.5% ) of cases . multivariable logistic models found no association between eosinophilia and sex or age of the studied children . significantly higher mean counts of eosinophils were found in children infected with t. trichiura alone or in those harbouring mixed infections compared to nonparasitized children . children harbouring mixed infections had 2.5-fold increased odds of having eosinophilia when compared to nonparasitized children ( adj . or = 2.59 , 95% ci = 1.135.90 , and p = 0.023 ) . similar increased odds were observed in children with single infection by t. trichiura , although this effect was only marginally significant ( adj . or = 2.48 , 95% ci = 0.986.26 , eosinophilia was positively correlated with intensity of infection by both a. lumbricoides and t. trichiura ( rs = 0.26 , p < 0.001 and rs = 0.33 , p < 0.001 , resp . ) . children with moderate - to - heavy ascariasis had five times the odds of presenting eosinophilia ( adj . or = 5.23 , 95% ci = 1.6916.12 , and p = 0.004 ) compared to their nonparasitized counterparts . likewise , moderate - to - heavy trichuriasis was associated with a 7-fold increased odds of eosinophilia ( adj . or = 7.19 , 95% ci = 1.4136.70 , and p = 0.018 ) . the overall prevalence of hyper - ige was 90.2% ( 95% ci = 85.593.5 ) . more than half of these cases ( 51.0% ) were severe , whereas mild and moderate hyper - ige accounted for 24.3% and 24.7% of the cases , respectively . no significant correlation was found between ige levels and sex or age of participant children . compared to those without sth infections , children with mixed sth infections had significantly higher mean levels of ige ( table 2 ) . there was a moderate positive correlation between ige serum levels and eosinophils count ( rs = 0.43 , p < 0.001 ) . similarly , ige levels were positively correlated with intensity of infection of both a. lumbricoides and t. trichiura . children with moderate - to - heavy ascariasis or trichuriasis had significantly higher mean values of ige compared to those uninfected or with only light infections ( p < 0.001 ) ( figure 2 ) . table 2 summarizes the geometric mean values and 95% ci of these cytokines by sth infection . in general , cytokine mean concentrations did not differ significantly when comparing the parasitized with the nonparasitized group . however , children with single infections by a. lumbricoides had significantly higher levels of il-10 and il-13 compared to children without infection , with single t. trichiura infections or with mixed infections ( p = 0.018 and p = 0.004 , resp . ) . spearman 's rank correlation coefficients showed a significant , although weak , negative correlation between il-10 levels and age ( rs = 0.23 , p < 0.001 ) . il-10 levels were not significantly correlated with infection intensity , whereas for il-13 a weak but significant positive correlation with a. lumbricoides infection intensity was found ( rs = 0.22 , p = 0.012 ) . significantly lower values of ifn- were found in children with single infections by t. trichiura ( p = 0.043 ) and these values showed a weak , negative correlation with t. trichiura infection intensity , although this correlation was not statistically significant ( rs = 0.17 , p = 0.069 ) ( figure 3 ) . on the other hand , ifn- values were significantly negatively associated with a. lumbricoides infection intensity ( p = 0.007 ) . children with moderate - to - heavy ascariasis had lower ifn- mean values ( 37.6 [ 15.790.0 ] pg / ml ) compared with those having no or light infections ( 131.5 [ 98.4175.8 ] pg / ml and 214.4 [ 99.0464.4 ] pg / ml , resp . ) ( figure 3 ) . th2/th1 ratios , as well as il-4/ifn- and il-10/ifn- , were also calculated ; significantly higher il-4/ifn- ratios were obtained in children with moderate - to - heavy infections by a. lumbricoides ( 0.020 [ 0.0100.042 ] ) , compared to those without ( 0.011 [ 0.080.014 ] ) or light infections ( 0.007 [ 0.0020.022 ] ) ( p = 0.046 and p = 0.021 , resp . ) a very similar pattern in il-4/ifn- ratio was found for t. trichiura infection intensity , although differences were only marginally significant ( p = 0.057 and p = 0.059 , resp . ) . finally , the potential effect of pathogenic protozoa in children 's immune response was assessed but this assessment was done only in children without sth infections . it was found that ifn- mean values were significantly higher in children harbouring pathogenic protozoa ( i.e. , g. intestinalis and/or e. histolytica / dispar ) compared to those infected with commensals only or to those with no protozoa at all ( 332.9 [ 146.7755.8 ] pg / ml , 93.7 [ 60.9144.2 ] pg / ml , and 145.6 [ 93.8225 ] pg / ml , resp . ) . il-10/ifn- ratios were significantly lower in children infected with pathogenic protozoa when compared to those uninfected or infected with commensals only ( 0.044 [ 0.0190.105 ] , 0.095 [ 0.0620.144 ] , and 0.124 [ 0.0720.214 ] , resp . ) ( figure 4 ) . no statistically significant associations could be established between protozoa infection and eosinophil counts , ige levels , or the remaining cytokines studied . the parasitological findings of this study demonstrate a moderately high ( 30% ) overall sth prevalence and specific prevalence for t. trichiura and a. lumbricoides of about 20% among schoolchildren living in linaca . school teachers reported that the school is reached by the national deworming program and thus receives a single - dose 400 mg albendazole tablet per child on a yearly basis . no records are kept at the school in terms of deworming tablets intake and no parasitological baseline data have been obtained during governmental surveys ( data reviewed in ) . to our knowledge , prior to the present study , two parasitological surveys had been undertaken in the study community . in 1985 , a study investigating causes of diarrhea in honduras included a small number of preschoolchildren and found prevalence of 48% and 30% for a. lumbricoides and compared to the sth prevalence found in the present study , it appears that there has been an important decrease in sth prevalence among linaca 's children . due to insufficient data on deworming , we did not attempt to identify if this strategy has served an important function in decreasing sth prevalence or at least worm burden . thus , whether or not deworming has contributed to sth prevalence reduction remains an open question . our findings , however , show that improved household conditions were strong protective factors for ascariasis . in this case , there might be a synergy between the built environment and anthelminthic medication , which may not be sufficient to protect against t. trichiura infection as one dose of alb treatment is largely ineffective to clear this parasite . even though a similar proportion of children were found with either excess weight ( 12% ) or chronic malnutrition ( stunted growth , ~10% ) , the latter similarly , despite the fact that trichuriasis prevalence was at a moderate level and that the majority of infections were light , a significant association was documented between stunting and both t. trichiura infection and infection intensity . this finding aligns with observations from other studies conducted in honduras , ethiopia , mexico , and brazil [ 33 , 34 ] . the concerning association between trichuriasis and chronic malnutrition , along with the fact that t. trichiura has been found consistently as the most prevalent geohelminth in honduras [ 27 , 35 , 36 ] , suggests that ( i ) a reexamination of the current deworming guidelines are necessary ; ( ii ) improvements in sanitary conditions and health education are indispensable components of sth control initiatives ; and ( iii ) it is important to monitor for potential benzimidazole resistance in this parasite . it has been shown that , in developing countries , eosinophilia is most commonly induced by tissue - invasive parasites , particularly helminths [ 25 , 38 ] . sth infections have been largely associated with eosinophilia , especially in early stages of infection , when larval migration occurs . the association between eosinophilia and sth in the present study is consistent with results from previous work conducted in honduras [ 39 , 40 ] and other countries such as brazil [ 41 , 42 ] , philippines , indonesia , and spain . whereas other causes of eosinophilia can not be ruled out , it is likely that , in this particular group of children , such eosinophilia was reactive ( i.e. , secondary to an external stimulus ) and most likely associated to sth infection . this inference is supported by data contained in linaca 's health centre morbidity report for 2014 , which shows that < 2% of children 's visits were due to asthma or allergic dermatitis ( linaca health care centre 2014 report , unpublished ) . moreover , a comprehensive socioeconomic and life conditions study conducted in the municipality of tatumbla , where linaca is located , contained no reference to allergic conditions being a health issue among inhabitants . rather , it revealed that childhood acute respiratory infections accounted for > 50% of visits to the health centre . the use of eosinophilia as a biomarker for helminthiases remains without consensus . as a predictor of current helminthic infections , some authors consider eosinophilia 's predictive value very limited , while others regard it as a suitable indicator of helminthiases in people from tropical and subtropical areas [ 26 , 42 , 44 , 46 ] . future studies should investigate eosinophilia at the individual level in order to better understand this dynamic relationship . our study revealed strikingly high prevalence of hyper - ige among the studied children ; it also highlighted a significant association between sth infections and increased total ige levels . such findings are consistent with observations in studies conducted in nigeria [ 47 , 48 ] , ecuador , venezuela , spain , and brazil [ 41 , 51 , 52 ] . helminth - induced ige is mostly characterized by a nonspecific polyclonal stimulation with only a small fraction of parasite - specific ige . however , it is the parasite - specific ige which plays an important role in helminth clearance as well as in preventing reinfection [ 48 , 50 , 5355 ] . in a study of venezuelan children , hagel and collaborators demonstrated that total ige levels were inversely correlated with parasite - specific ige levels . they also showed that reinfection with a. lumbricoides was significantly associated with high pretreatment total ige but low parasite - specific ige levels . another interesting finding in the present investigation was the significant association observed between high total ige levels and intensity of infection ( i.e. , worm burden ) . some studies have shown that specific ige levels reflect the parasite infection intensity ; therefore , higher specific antibody levels would be expected in lightly infected individuals when compared with those heavily infected . total ige levels in the studied children were significantly positively correlated with eosinophils counts ; this finding is not surprising due to the ability of ige to induce a cytotoxic response against helminthic parasites mediated by mast cells and eosinophils . it has been demonstrated that helminth infections strongly induce an immune response involving elevated th2 cytokines ( i.e. , il-4 , il-5 , il-9 , and il-13 ) , ige , iga , eosinophilia , and mucus secretion [ 5860 ] . helminth - caused tissue damage triggers the response by releasing danger - associated molecular patterns ( damps ) and cytokine alarmins , particularly il-25 and il-33 . these alarmins promote the activation of basophils and innate lymphoid cells ( ilc-2 ) to support a type-2 innate immune response needed to signal the type-2 adaptive immunity . in this context , large quantities of il-5 and il-13 are primarily produced by ilc-2 cells . il-5 promotes the eosinophils differentiation and activation which promote il-4 production , the main primer of cd4 th2 cells activation and expansion , with further release of il-4 , il-5 , and il-13 by this type of cells [ 3 , 6062 ] . in this environment , il-4 and il-13 induce aams , which mainly promote tissue repair and fibrosis by expression of different markers such as arginase-1 , ym1 , ym2 , and relm-. these aams also express regulatory il-10 and tgf- able to downregulate inflammatory response [ 35 , 9 , 6163 ] . instead , the majority of children showed either low or nondetectable values of this cytokine . this was an unexpected finding since il-5 is essential for eosinophilic differentiation , proliferation , and activation . since the participant children have probably experienced repeated sth infections since they were much younger , their immune response may have already changed to a modified type 2 response , where inflammatory th2 shifted to an attenuated phenotype characterized by the shutdown of effector cytokines such as il-5 , retention of il-4 , and reinforcement of anti - inflammatory cytokines such as il-10 and/or tgf- [ 4 , 9 ] . instead , as recently determined by fulkerson and colleagues , an alternative il-5-independent pathway for promoting eosinophilia might be in place . this pathway would contribute to persistent eosinophil differentiation and survival even after a complete il-5 withdrawal . finally , periodic deworming followed by reinfection along several years is likely disturbing the antiparasitic immune responses as described in the literature . we found that il-13 levels were significantly increased in children infected with a. lumbricoides and that these levels were positively correlated with worm burden . il-13 is a th2 pleiotropic cytokine with an important function in the intestinal epithelia , controlling the rate of transit of epithelial cells toward the outmost layer and promoting a harsh environment that might help in gut parasite expulsion ( i.e. , increased luminal fluids , mucus , from goblet cells , increased edema , and muscle contractility ) [ 7 , 60 , 61 ] . gallo and colleagues demonstrated that il-13 may also be produced by th1 and th17 cells , suggesting that this cytokine can have either a pro- or anti - inflammatory effect , depending on the environment . nevertheless , demonstrating an association between sth and il-13 overproduction has not been achieved by other research groups in india and brazil . some studies in cameroon [ 68 , 69 ] found high levels of il-13 associated with lower intensity of infection , but the present study did not establish such association . it can be produced by diverse type of cells such as macrophages , dendritic cells ( dc ) , b cells , and various subsets of cd4 and cd8 t cells . regardless of the type of infection , il-10 limits th1 and th2 effector responses by suppressing function of macrophages and dcs . the timing , site of its production , and strength may favor ( a ) simultaneous pathogen clearance and suppression of downstream pathologies ; ( b ) potential benefit to both the host and the pathogen ( limiting pathology and allowing persistent infection ) ; ( c ) severe tissue damage ; or ( d ) overwhelming infection [ 70 , 71 ] . in our study , statistically significant higher il-10 values were found in children with ascariasis , although they did not correlate with infection intensity of this parasite . similar findings have been frequently reported by other researchers [ 66 , 69 , 7274 ] , with some exceptions . this is congruent with previous studies conducted in a different community in honduras as well as with other studies in nigeria and cameroon [ 68 , 76 ] . although its efficacy is species - dependent , albendazole generally reduces the duration and/or intensity of helminth infections . in our study , ifn- levels it is well known that ifn- is downregulated by type-2 cytokines making our finding consistent with the literature . ifn- is the most important type-1 cytokine and is produced by a diverse type of cells from both innate and adaptive immune arms such as natural killer ( nk ) , cd4 th1 , cd8 tc , and eosinophils . it interacts with macrophages to activate direct antimicrobial and antitumor mechanisms as well as upregulating antigen processing and presentation pathways . the overall interpretation of the different cytokines , their levels , and relationships with helminthic infections is an interesting challenge . in moderate - to - heavy ascariasis this response was characterized by upregulation of il-13 and downregulation of ifn- , as well as by significantly higher il-4/ifn- ratio and ige levels . results from t. trichiura infections were not as well - defined as those from ascariasis . nonetheless and similarly to a. lumbricoides , infections of high intensity were associated with lower values of ifn- and higher il-4/ifn- ratios and ige levels . these findings confirm that a type-2 response against this parasite was also displayed in our study population . as mentioned earlier , a type-1 immune response is needed for the control and clearance of intestinal protozoa such as entamoeba histolytica , cryptosporidium spp . , and giardia intestinalis . although , particularly in the case of g. intestinalis , mixed th1-th2 responses have been reported , human and animal studies have shown that increased levels of ifn- play a crucial role in orchestrating the immune response and appear to be one of the main signatures in individuals with these parasitoses [ 7881 ] . this description is in agreement with our findings of significantly higher ( up to 3-fold ) levels of ifn- found in children harbouring g. intestinalis and/or e. histolytica / dispar without the immune influence of sth infections ( i.e. , negative for helminthic infections ) . the significantly lower il10/ifn- ratios observed in this group of children indicate a th1-biased response . additionally , in a 6-year longitudinal study in bolivia , blackwell and colleagues described an antagonist relationship between g. intestinalis and geohelminths where giardiasis was less likely to be present in helminth - infected individuals likewise , they also found that infection with helminths was less likely for individuals with giardiasis . our data , though cross - sectional , support these observations : none of the children infected with giardia intestinalis had ascariasis and only 10% of them had concomitant trichuriasis . since clearance and protective immunity against g. intestinalis require also th2 cytokines and antibodies , iga , igg , and ige [ 78 , 79 , 81 ] , a plausible scenario has been suggested : helminth - induced th2 response might provide cross - immunity against this protozoan . a proper interpretation of our findings must be done considering the limitations of the present study . firstly , its cross - sectional nature only allows establishing associations but no causality and , therefore , strong inferences can not be made . secondly , we did not obtain a complete medical history from participating children or an accurate deworming history . this is important as a variety of unmeasured confounding variables ( e.g. , bacterial and viral infections , autoimmune diseases , asthma , and other allergic diseases , as well as environmental and genetic factors ) may have contributed to skew the th1/th2 balance among research participants . thirdly , we measured circulating levels of cytokines , which may not be an accurate representation of local immune responses in the gut milieu . notwithstanding , the study has several strengths : an adequate sample size , determination of both helminthic and protozoal infections , and the use of highly sensitive and accurate methodology for biomarkers analysis . this is the first study providing a comprehensive immune profile in honduran children infected with geohelminths or pathogenic protozoa . our study shows that higher intensities of sth infections are associated with a polarized th2 response , characterized by reduced proinflammatory and increased regulatory cytokines , eosinophilia , and hyper - ige . the interpretation of the host 's immune response against helminth represents challenging undertaking due to the high complexity of multiple host - parasite interactions and potential unmeasured confounders . pivotal to characterizing and understanding the immune response is obtaining a meticulous clinical history from study participants , their deworming history , and very importantly the existence of multiple infections including pathogenic protozoa . further , the role of the intestinal microbiota and its interplay with the host and its parasites can no longer be ignored .

soil - transmitted helminth infections typically induce a type-2 immune response ( th2 ) , but no immunoepidemiological studies have been undertaken in honduras , an endemic country where the main control strategy is children 's annual deworming . we aimed to characterize the immune profile of honduran schoolchildren harbouring these parasitoses . demographic and epidemiological data were obtained through a survey ; nutritional status was assessed through anthropometry ; intestinal parasites were diagnosed by formol - ether and kato - katz ; and blood samples were collected to determine immunological markers including th1/th2 cytokines , ige , and eosinophil levels . a total of 225 children participated in the study , all of whom had received deworming during the national campaign five months prior to the study . trichuriasis and ascariasis prevalence were 22.2% and 20.4% , respectively . stunting was associated with both age and trichuriasis , whereas ascariasis was associated with sex and household conditions . helminth infections were strongly associated with eosinophilia and hyper - ige as well as with a th2-polarized response ( increased levels of il-13 , il-10 , and il4/ifn- ratios and decreased levels of ifn- ) . pathogenic protozoa infections were associated with a th1 response characterized by elevated levels of ifn- and decreased il10/ifn- ratios . even at low prevalence levels , sth infections affect children 's nutrition and play a polarizing role in their immune system .

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

soil - transmitted helminths ( sth ) or geohelminths infect more than 2 billion people worldwide , especially in developing countries located in tropical and subtropical regions . the most prevalent species are ascaris lumbricoides ( roundworm ) , trichuris trichiura ( whipworm ) , and hookworms ( necator americanus and ancylostoma duodenale ) . the morbidity caused by sth infections is highly associated with the size of the worm population residing in the intestines ( also known as worm burden ) . infections of heavy intensity are more likely to cause impaired physical growth and cognitive development as well as micronutrient deficiencies , including iron - deficiency anaemia [ 1 , 2 ] . in addition to health impact , sth infections affect the human host in more subtle manner : as extracellular metazoan parasites , helminths trigger type-2 immune response mediated by t - helper type 2 ( th2 ) cells . this response is typically characterized by expansion of mast cells , eosinophils , basophils , group 2 innate lymphoid cells ( ilc-2 ) , and alternatively activated macrophages ( aams ) , as well as increased production of th2 cytokines ( e.g. on the other hand , intracellular pathogens such as viruses , bacteria , protozoa , and fungi elicit a type-1 response , characterized by increased numbers of phagocytic neutrophils and macrophages , as well as cytotoxic cd8 t cells and th1 cells . via production of interferon - gamma ( ifn- ) and il-2 to a lesser degree , th1 cells direct , maintain , and enhance the antimicrobial effect of type-1 response . increased levels of anti - inflammatory cytokines il-10 and transforming growth factor beta ( tgf- ) are hallmarks of this response [ 4 , 8 , 9 ] . it has been suggested that , during chronic helminthiases , this anti - inflammatory network may play a key role in the lower prevalence of allergic disease observed in th2-skewed populations [ 7 , 1012 ] . conversely , helminth - induced il-10-mediated immunosuppression has raised concerns for populations in which sth infections coexist with other morbidities such as hiv / aids , malaria , and tuberculosis , as clearance for the latter depends on an active and timely type-1 response [ 8 , 1316 ] . in this context , successful deworming treatment has been proposed as a practical and inexpensive way to rebalance the immune response [ 17 , 18 ] . recent studies have observed that after administration of anthelminthic medication , eosinophil counts , il-10 levels [ 1921 ] , and ige concentrations show a significant decrease from pretreatment values . the principal aim of the present study was to characterize the immune profile in schoolchildren infected with geohelminths . the study was conducted in april 2014 and data were collected from schoolchildren living in linaca , a rural community of the municipality of tatumbla , department of francisco morazn in honduras . the study was approved by brock university 's bioscience research ethics board ( file number bu 12 - 262 , dated june 27 , 2013 ) as well as by the research ethics board of the master 's program in infectious and zoonotic diseases , school of microbiology , national autonomous university of honduras , unah ( file number cei - meiz 01 - 2013 , dated september 23 , 2013 ) . the school of linaca participates in the honduras national deworming program , which provides enrolled children with a 400 mg single - dose albendazole annually . at the time of the study , children who had not received deworming treatment from other sources in the past 3 months were included in the study . basic demographic and epidemiological data of participant children were obtained using a structured questionnaire during a 5-minute face - to - face interview conducted in spanish . body weight and height of participants were measured twice by different researchers and their average was used to calculate the following anthropometric indicators : ( a ) height - for - age z - score ( haz ) , ( b ) weight - for - age z - score ( waz ) , and ( c ) body - mass - index - for - age z - score ( baz ) . these indicators were used to ascertain children 's nutritional status as per international parameters : stunted growth ( chronic malnutrition ) , thinness , and underweight , respectively . the presence of intestinal parasites was assessed using both kato - katz and formol - ethyl acetate concentration methods . for the latter , microscopic examination of kato - katz smears was done between 30 and 60 min of preparation . formol - ethyl acetate was done one month after sample collection and sediments were observed with dry and immersion oil objective lenses in order to identify helminth and protozoa stages , respectively . blood samples obtained from the cubital vein were collected in 3 ml tubes of each k2edta and serum separator and clot activator ( bd vacutainer , nj , usa ) . according to the parameters established in the most recent ( 2011 - 2012 ) demographic and health survey in honduras ( dhs - hn ) , anaemia was defined as hb concentration < 12 g / dl . mild , moderate , or severe eosinophilia were defined if cell counts were 5001500/l , > 15005000/l , or > 5000/l , respectively . multiplex panels containing magnetic beads covered with fluorescent dyed conjugated to a monoclonal antibody specific for each target molecule were used to quantify th1/th2 cytokines ( il-2 , il-6 , il-8 , il-12p70 , ifn- , gm - csf , tnf- , il-4 , il-5 , and il-13 ) and the regulatory cytokine il-10 . hyper - ige was defined as a serum concentration exceeding 100 iu / ml and was classified as mild ( > 100399 iu / ml ) , moderate ( > 399999 cytokine and ige tests were performed according to the manufacturer 's instructions ( bio - rad laboratories , inc . descriptive statistics were used to characterize the study population . point prevalence with 95% confidence intervals ( 95% ci ) was calculated for overall sth infections and for each parasite species , as well as for mixed sth infections ( i.e. due to the non - gaussian distribution of the immunological markers , nonparametric methods were used as follows . table 1 summarizes the characteristics and parasitological findings in the study population . a total of 225 schoolchildren attending grades 1 to 6 ( age 613 ) participated in the study . qualitative kato - katz results were 100% correlated with those of formol - ethyl acetate . no cases of s. stercoralis or hookworm infections were found with either technique but one case of taenia solium was identified by the presence of gravid proglottids in the stool sample . among pathogenic protozoa , giardia intestinalis and entamoeba histolytica / dispar were present in 4% and 9% of the samples , respectively . prevalence for t. trichiura and a. lumbricoides was 22.2% ( 95% ci = 17.228.2 ) and 20.4% ( 95% ci = 15.626.2 ) , respectively . only 20% of moderate - to - heavy infections with both a. lumbricoides and t. trichiura occurred as single infections . multivariable logistic models controlling for age and sex were constructed , and adjusted or were calculated . in this population , stunting was significantly associated with the age of participants as well as with trichuriasis . or = 6.64 , 95% ci = 1.1937.09 , and p = 0.031 ) . the geometric means and 95% ci of these parameters by sth infection the overall prevalence of eosinophilia was 31.7% ( 95% ci = 25.938.1 ) , and mild eosinophilia accounted for the vast majority ( 91.5% ) of cases . or = 2.59 , 95% ci = 1.135.90 , and p = 0.023 ) . likewise , moderate - to - heavy trichuriasis was associated with a 7-fold increased odds of eosinophilia ( adj . or = 7.19 , 95% ci = 1.4136.70 , and p = 0.018 ) . the overall prevalence of hyper - ige was 90.2% ( 95% ci = 85.593.5 ) . more than half of these cases ( 51.0% ) were severe , whereas mild and moderate hyper - ige accounted for 24.3% and 24.7% of the cases , respectively . compared to those without sth infections , children with mixed sth infections had significantly higher mean levels of ige ( table 2 ) . however , children with single infections by a. lumbricoides had significantly higher levels of il-10 and il-13 compared to children without infection , with single t. trichiura infections or with mixed infections ( p = 0.018 and p = 0.004 , resp . ) il-10 levels were not significantly correlated with infection intensity , whereas for il-13 a weak but significant positive correlation with a. lumbricoides infection intensity was found ( rs = 0.22 , p = 0.012 ) . on the other hand , ifn- values were significantly negatively associated with a. lumbricoides infection intensity ( p = 0.007 ) . th2/th1 ratios , as well as il-4/ifn- and il-10/ifn- , were also calculated ; significantly higher il-4/ifn- ratios were obtained in children with moderate - to - heavy infections by a. lumbricoides ( 0.020 [ 0.0100.042 ] ) , compared to those without ( 0.011 [ 0.080.014 ] ) or light infections ( 0.007 [ 0.0020.022 ] ) ( p = 0.046 and p = 0.021 , resp . ) finally , the potential effect of pathogenic protozoa in children 's immune response was assessed but this assessment was done only in children without sth infections . it was found that ifn- mean values were significantly higher in children harbouring pathogenic protozoa ( i.e. il-10/ifn- ratios were significantly lower in children infected with pathogenic protozoa when compared to those uninfected or infected with commensals only ( 0.044 [ 0.0190.105 ] , 0.095 [ 0.0620.144 ] , and 0.124 [ 0.0720.214 ] , resp . ) no statistically significant associations could be established between protozoa infection and eosinophil counts , ige levels , or the remaining cytokines studied . no records are kept at the school in terms of deworming tablets intake and no parasitological baseline data have been obtained during governmental surveys ( data reviewed in ) . to our knowledge , prior to the present study , two parasitological surveys had been undertaken in the study community . in 1985 , a study investigating causes of diarrhea in honduras included a small number of preschoolchildren and found prevalence of 48% and 30% for a. lumbricoides and compared to the sth prevalence found in the present study , it appears that there has been an important decrease in sth prevalence among linaca 's children . even though a similar proportion of children were found with either excess weight ( 12% ) or chronic malnutrition ( stunted growth , ~10% ) , the latter similarly , despite the fact that trichuriasis prevalence was at a moderate level and that the majority of infections were light , a significant association was documented between stunting and both t. trichiura infection and infection intensity . this finding aligns with observations from other studies conducted in honduras , ethiopia , mexico , and brazil [ 33 , 34 ] . the concerning association between trichuriasis and chronic malnutrition , along with the fact that t. trichiura has been found consistently as the most prevalent geohelminth in honduras [ 27 , 35 , 36 ] , suggests that ( i ) a reexamination of the current deworming guidelines are necessary ; ( ii ) improvements in sanitary conditions and health education are indispensable components of sth control initiatives ; and ( iii ) it is important to monitor for potential benzimidazole resistance in this parasite . sth infections have been largely associated with eosinophilia , especially in early stages of infection , when larval migration occurs . the association between eosinophilia and sth in the present study is consistent with results from previous work conducted in honduras [ 39 , 40 ] and other countries such as brazil [ 41 , 42 ] , philippines , indonesia , and spain . moreover , a comprehensive socioeconomic and life conditions study conducted in the municipality of tatumbla , where linaca is located , contained no reference to allergic conditions being a health issue among inhabitants . rather , it revealed that childhood acute respiratory infections accounted for > 50% of visits to the health centre . our study revealed strikingly high prevalence of hyper - ige among the studied children ; it also highlighted a significant association between sth infections and increased total ige levels . such findings are consistent with observations in studies conducted in nigeria [ 47 , 48 ] , ecuador , venezuela , spain , and brazil [ 41 , 51 , 52 ] . helminth - induced ige is mostly characterized by a nonspecific polyclonal stimulation with only a small fraction of parasite - specific ige . however , it is the parasite - specific ige which plays an important role in helminth clearance as well as in preventing reinfection [ 48 , 50 , 5355 ] . they also showed that reinfection with a. lumbricoides was significantly associated with high pretreatment total ige but low parasite - specific ige levels . some studies have shown that specific ige levels reflect the parasite infection intensity ; therefore , higher specific antibody levels would be expected in lightly infected individuals when compared with those heavily infected . total ige levels in the studied children were significantly positively correlated with eosinophils counts ; this finding is not surprising due to the ability of ige to induce a cytotoxic response against helminthic parasites mediated by mast cells and eosinophils . it has been demonstrated that helminth infections strongly induce an immune response involving elevated th2 cytokines ( i.e. , il-4 , il-5 , il-9 , and il-13 ) , ige , iga , eosinophilia , and mucus secretion [ 5860 ] . these alarmins promote the activation of basophils and innate lymphoid cells ( ilc-2 ) to support a type-2 innate immune response needed to signal the type-2 adaptive immunity . il-5 promotes the eosinophils differentiation and activation which promote il-4 production , the main primer of cd4 th2 cells activation and expansion , with further release of il-4 , il-5 , and il-13 by this type of cells [ 3 , 6062 ] . since the participant children have probably experienced repeated sth infections since they were much younger , their immune response may have already changed to a modified type 2 response , where inflammatory th2 shifted to an attenuated phenotype characterized by the shutdown of effector cytokines such as il-5 , retention of il-4 , and reinforcement of anti - inflammatory cytokines such as il-10 and/or tgf- [ 4 , 9 ] . some studies in cameroon [ 68 , 69 ] found high levels of il-13 associated with lower intensity of infection , but the present study did not establish such association . it can be produced by diverse type of cells such as macrophages , dendritic cells ( dc ) , b cells , and various subsets of cd4 and cd8 t cells . this is congruent with previous studies conducted in a different community in honduras as well as with other studies in nigeria and cameroon [ 68 , 76 ] . ifn- is the most important type-1 cytokine and is produced by a diverse type of cells from both innate and adaptive immune arms such as natural killer ( nk ) , cd4 th1 , cd8 tc , and eosinophils . it interacts with macrophages to activate direct antimicrobial and antitumor mechanisms as well as upregulating antigen processing and presentation pathways . the overall interpretation of the different cytokines , their levels , and relationships with helminthic infections is an interesting challenge . in moderate - to - heavy ascariasis this response was characterized by upregulation of il-13 and downregulation of ifn- , as well as by significantly higher il-4/ifn- ratio and ige levels . results from t. trichiura infections were not as well - defined as those from ascariasis . nonetheless and similarly to a. lumbricoides , infections of high intensity were associated with lower values of ifn- and higher il-4/ifn- ratios and ige levels . although , particularly in the case of g. intestinalis , mixed th1-th2 responses have been reported , human and animal studies have shown that increased levels of ifn- play a crucial role in orchestrating the immune response and appear to be one of the main signatures in individuals with these parasitoses [ 7881 ] . this description is in agreement with our findings of significantly higher ( up to 3-fold ) levels of ifn- found in children harbouring g. intestinalis and/or e. histolytica / dispar without the immune influence of sth infections ( i.e. the significantly lower il10/ifn- ratios observed in this group of children indicate a th1-biased response . , bacterial and viral infections , autoimmune diseases , asthma , and other allergic diseases , as well as environmental and genetic factors ) may have contributed to skew the th1/th2 balance among research participants . thirdly , we measured circulating levels of cytokines , which may not be an accurate representation of local immune responses in the gut milieu . notwithstanding , the study has several strengths : an adequate sample size , determination of both helminthic and protozoal infections , and the use of highly sensitive and accurate methodology for biomarkers analysis . this is the first study providing a comprehensive immune profile in honduran children infected with geohelminths or pathogenic protozoa . our study shows that higher intensities of sth infections are associated with a polarized th2 response , characterized by reduced proinflammatory and increased regulatory cytokines , eosinophilia , and hyper - ige . the interpretation of the host 's immune response against helminth represents challenging undertaking due to the high complexity of multiple host - parasite interactions and potential unmeasured confounders . pivotal to characterizing and understanding the immune response is obtaining a meticulous clinical history from study participants , their deworming history , and very importantly the existence of multiple infections including pathogenic protozoa .

compensatory eye movements ( cem ) is a general term for a number of different reflexes that keep an image fixed on the retina during movements of the body and the head ( e.g. delgado - garcia , 2000 ) . as such , these eye movements have a specific and well - defined goal : to prevent movement of the visual image on the retina , often called retinal slip , during fixation . the circuitry of the cem system ( figure 1 ) is different from the circuitry for other eye movements such as saccades , although all the eye movement systems converge in the oculomotor nuclei of the brainstem ( buttner - ennever and buttner , 1992 ) . for horizontal eye movements these are the abducens nucleus ( ab ) and the nucleus prepositus hypoglossi ( nph ) . all cem - related input to these brainstem structures comes from the vestibular nuclei ( vn ) . the horizontal compensatory eye movement ( cem ) system . the optokinetic reflex ( okr ) uses visual input from the retina to stabilize the eye while the vestibulo - ocular reflex ( okr ) responds to vestibular information from the labyrinth . this figure emphasizes the distinction between sensory feedback ( black ) and motor signals ( red).blue and purple represent central stages of processing , and are added for comparison with other figures . cblm : cerebellar cortex ; aos / nrtp : accessory optic system and nucleus reticularis tegmentum pontis ; vn : vestibular nucleus ; nph : neuclus prepositus hypoglossi ; omn / ab : oculomotor nucleus and abducens . the optokinetic reflex ( okr ) is a closed loop system that directly responds to retinal slip , generating eye movements with the direction and magnitude of measured retinal slip . the aos , in turn projects to the vn and the cerebellum , through the nucleus reticularis tegmenti pontis ( nrtp ; gerrits et al . , 1984 ; langer et al . , 1985 ; glickstein et al . , 1994 ) . the okr has a response delay of about 80 ms ( e.g winkelman and frens , 2006 ) , mostly because of the inherent delay involved in visual processing ( graf et al . , 1988 ) . in keeping with this , the okr is only responsive to low velocity stimuli . for compensation of higher velocity stimuli , the cem system depends on the vestibulo - ocular reflex ( vor ) which uses vestibular input to estimate head movement and generate oppositely directed eye movements . the two systems are complementary : the vor compensates for higher frequencies while the okr compensates for the lower velocities ( collewijn , 1989 ) . both the vor and okr are adaptive , meaning that the mapping of stimulus to appropriate eye response can be tuned to match changing response properties of the eye and its supporting tissues ( usually collectively called the plant ) or changes in the sensitivity of the sensory organs ( blazquez et al . , 2004 ; boyden et al . , 2004 ; andreescu et al . , 2005 ; gittis and du lac , 2006 ) . changes in either plant response properties , sensory sensitivity , or environmental changes in the relationship of vision and vestibular input to movement will change the appropriate mapping from stimulus to response and thus the system must change the mapping so that retinal slip continues to be appropriately compensated . there is ample evidence that the flocculus , a small section of the cerebellar cortex is critical in this plasticity ( e.g. lisberger et al . , 1984 ) . the purkinje cells ( p - cells ) of the flocculus project only to the vn . there are sites of plasticity both at the level of the parallel fibre synapses to these p - cells , as well as at the p - cell / vn synapses ( raymond et al . in addition , the cerebellum has an important role in ongoing performance beyond its role in plasticity : the performance of the okr decreases dramatically after floccular lesions ( takemori and cohen , 1974 ; zee et al . , 1981 ) , while the vor is less affected ( waespe et al . , 1983 ; van neerven et al . , 1989 ) . there has been a long history of using models based on the principles of control theory to describe the control of eye movements generally and cem in particular . starting with the seminal work of david robinson ( for review robinson , 1981 ) , this tradition has generally posited a neural implementation of an inverse model that maps stimuli to command signals ( skavenski and robinson , 1973 ) . an inverse model , literally speaking , is a control process that inverts the plant ; that is , the plant converts control signals into motion , so an inverse model converts desired motion into the appropriate control signals ( jordan and rumelhart , 1992 ; figure 2a ) . ( a ) inverse and ( b ) forward models . the inverse model inverts this process , producing the motor commands that are appropriate for a given movement . a forward model mimics this process , estimating the movement that will be produced by the plant . the cerebellar floculus is thought by many to implement a form of inverse model ( kawato and gomi , 1992 , and see also lisberger , 2009 , for review of these ideas in relation to the smooth pursuit system ) . while this idea has many adherents , there are also alternative proposals . perhaps most famously , llins ( 1988 ) proposed that the cerebellum is involved in adjusting movement timing to facilitate coordination , rather than in generating compensatory movement commands . 2008 ) argued that synchrony and oscillatory activity in the inferior olive are compatible with a cerebellar timing mechanism driven by olivary harmonics . d'angelo and de zeeuw ( 2009 ) in contrast , focus on the temporal dynamics of the cerebellar granular layer . a somewhat more eclectic model that also focuses on timing is braitenberg 's model of the cerebellum as a system for generating sequences of movement in precise time relationship ( braitenberg et al . , 1997 ) . while each of these models can legitimately claim to explain important data , there is no doubt that the inverse model understanding of the cerebellum in cem is the most widely accepted . the controversy about timing models and adaptation models of the cerebellum has been going on for a long time ( miles and lisberger , 1981 ; ivry and keele , 1989 ; simpson et al . , 1996 ) . there are those who believe the two different approaches are mutually compatible ( mauk et al . , 2000 ) . it is not our intention , in any case , to take on this issue . the inverse model framework can be contrasted with a forward model ( wolpert and miall , 1996 ; todorov and jordan , 2002 ; see figure 2b ) which simulates the activity of the plant : it converts the current state and the control signals into a prediction of what the plant will actually do . the bottom line is : inverse models output motor commands and forward models output estimates of state . the focus on a neural inverse model of the oculomotor plant reflected a perspective that the central problem in oculomotor control is producing the appropriate motor commands once the goal is given . researchers in other motor systems notably arm movements followed in the footsteps of the pioneering work in oculomotor research and focused on the inverse model problem and the question of how appropriate motor commands are generated , given a particular desired movement . this approach was reinforced by the explanatory power of hypothesized desired trajectories ( flash and hogan , 1985 ; uno et al . , 1989 ) , and the apparent tendency of subjects to correct movements ( shadmehr and mussa - ivaldi , 1994 ; donchin et al . , 2003 ) . however , the possibility that a forward model also plays a role has been hypothesized for a long time ( e.g. wolpert and miall , 1996 ; kawato , 1999 ) . one recent radical proposal has been that the system does not work with either a desired trajectory or an inverse model ( todorov , 2004 ) . under this approach , the problem of predicting the results of motor commands is no less central than the problem of generating those motor commands in the first place . the reason such state prediction is so important is because it allows stable feedback control . feedback control is the use of the measured or predicted state of the system to generate ongoing motor commands . this form of control can be simpler and more flexible than open - loop control . sensory systems are both slow and noisy , this is inevitably a problem in physiological motor control . however , predictions of the state must be combined with actual sensory feedback in order for the control loop to remain robust in the face of unpredicted perturbations . thus , the framework ( which we will call the state - predicting feedback control , spfc , framework ) is built out of three essential building blocks ( figure 3 ; todorov , 2004 ; shadmehr and krakauer , 2008 ) . the forward model takes the current estimate of state and the motor commands and produces an initial prediction . the state estimator combines this prediction with actual sensory feedback to produce a better estimate of the current state . the feedback controller uses the current estimate of state in order to decide what motor commands to generate . it either replaces or incorporates the inverse model on which the tradition of robinson had focused . we propose that this framework is an appropriate description of cem control and that it can be mapped onto cem physiology in a manner that is consistent with experimental evidence . the spfc framework proposes that a feedback controller is optimized to produce motor commands that achieve task goals . in order to do this effectively , it uses an estimate of the current situation that is derived from a combination of feedback from the sensory system and forward model estimation that depends on efferent copy . how could such a computational scheme be implemented in the known anatomy and physiology of cem ? nevertheless , shadmehr and krakauer ( 2008 ) have recently proposed that neural structures involved in the control of arm movements can , in fact , be mapped onto the control structure described by these boxes . they suggest that motor cortex , in combination with the basal ganglia , implements a feedback controller implementing a control policy that maximizes successful performance . they support this using data from patients with parkinson 's disease ( mazzoni et al . , 2007 ) , and hemiparesis ( raghavan et al . , 2006 state estimation is hypothesized to occur in parietal cortex based on findings in patients with parietal lesions ( wolpert et al . , 1998 ) . finally , on the basis of the cerebellar role in in - flight adjustment of saccades ( quaia et al . , 2000 ) and anticipatory postural adjustments ( nowak et al . , 2007 ) , they claim the forward model is implemented in the cerebellum . since the cem system is located in brain stem nuclei and the cerebellum , and neither motor cortex nor parietal cortex is instrumental , our effort to ascribe computational functions to physiological correlates in the cem will necessarily produce different results . we will argue that , for cem , the most suitable mapping would be that the oculomotor nuclei and integrators ( robinson 's inverse plant ) combine to form a feedback controller . the cerebellar cortex ( and not the whole cerebellum ) generates a forward model , and the vn combine forward model output with current inputs to produce the state estimate . the feedback controller maps current estimate of state onto the appropriate motor command . in the language of control system experts , this could be approximated as a transformation ( 1)un = ln(x^n ) where un is a vector of length ku describing the motor commands at the n time step . in our case , this would be the command driving the ocular musculature ; each element of un represents the activation directed at a single muscle . x^n is a vector of length kx describing our current estimate of state ; the elements of x^n reflect variables like estimated eye position , eye velocity , and possibly include estimated head position and velocity and even desired eye position and velocity . of course , both the state and the motor vectors could , the point is that the feedback controller implements a function , l , that translates its input , an estimated state vector , x^ , into its output , the motor command vector , u. by definition , the output of a controller is motor command so whatever produces the motor commands must necessarily be implementing a controller . in our case , motor command is the activity that drives the muscles , and the motoneurons of the abducens nucleus are the output of the feedback controller , at least for horizontal motion . a subtler question regards whether any other related nuclei are also included . in cem , the controller must know the desired fixation point and it must receive an estimate of the current eye position . it calculates the vectorial difference between these two and generates motoneuron activity which will move the eye in the direction indicated by this vector . the brainstem circuit that traditionally constitutes the inverse plant meets these requirements ( buttner - ennever and buttner , 1992 ; glasauer , 2007 ) , even though there is debate on how the computation in the plant is achieved ( see below ) . the traditional view is that a displacement or velocity input is directly fed to the abducens output neurons that project to the eye muscles . in order to overcome the low - pass filter properties of the plant , an integrated version of this input the so - called oculomotor integrators are responsible for this indirect pathway ( e.g. mcfarland and fuchs , 1992 ; moschovakis , 1997 ) . recent work on the nph , the putative horizontal integrator , undermines this view since neurons in the nph are found to encode the whole motor command , u , rather than only the integrated part ( green et al . , 2007 ; ghasia et al . , 2008 ) , as shown in figure 4 . is that nph output serves feedback purposes . indeed , on the basis of the finding that nph feedback encodes propose that the feedback is updating a cerebellar forward model ( see figure 3 ) . nph neurons ( dashed , orange ) behave like the motor command , represented by the solid , red curve . the black lines show the activity of a hypothetical neuron that would encode eye position , with a constant gain and phase . nph and ab activity were taken from ( green et al . , 2007 ) . in both the ab and the nph , the cem circuit is shared with the other eye movement systems ( i.e. saccades and smooth pursuit ) . this fits the role of feedback controller , since the efference copy needed by the forward model should contain all oculomotor output in order to produce an optimal estimate of state ( see below ) . that is , we can use a forward model to generate an estimate about current state from our earlier estimate and our knowledge of system dynamics . we assume , for the purpose of simplicity , that the actual dynamics of the system can be described as linearly combining previous state and motor command : ( 2)xn+1=axn+bun+n x is the actual state , whose estimate is discussed above ( x^n ) . both x and x^n have the same size , but the latter is the brain 's estimate and the former is the actual quantity . a is a kx kx matrix , b is a kx ku matrix , and the forward model estimate would be generated from the previous estimate using a similar equation ( 3)x^fm , n+1=a^x^n+b^un where a^ and b^ represent the forward model 's estimates of system dynamics . notice that in this formulation , which is commonly used , the estimate of state used to calculate the forward model is not the same as the estimate produced by the forward model in the previous step . that is , we have x^fm , n+1 on the left side of the equation but x^n on the right hand side . what we mean by this is that we may improve the estimate generated by the forward model ( for instance , by incorporating information from sensory inputs ) before we use it in the forward model 's next step . the figure shows sensory input ( black line ) reaching the cerebellum in addition to the current estimates of state ( purple ) and efferent copy ( red ) . this is drawn to reflect the realities described in figure 1 , which shows that sensory input does reach the cerebellar cortex . this includes retinal input from aos ( which is routed through the inferior olive and climbing fibers ) and nrtp ( which comes through mossy fibers ) . part of the visual input , especially the part arriving through aos , may play a role in adaptation processes discussed below . on the other hand , sensory input that has a direct effect on cerebellar activity 3 . it may nevertheless be consistent with the cerebellum producing a predictive estimate of state based on all the available information . we assume that the forward model has no knowledge of the random fluctuations in the state represented by the noise term . however , we expect the forward model to be plastic . that is , if the state prediction of the forward model is consistently wrong the model should change . it has been amply demonstrated that the cerebellum receives efference copy from many motor systems . specifically , the cortical area responsible for cem , the flocculus , receives direct projections from the nph ( sato et al . , 1983 ; langer et al . , 1985 furthermore , it receives a strong input from the vn ( sato et al . , 1983 ; langer et al . , 1985 ; gerrits et al . 1993 ) , and we will argue later that this is the most likely candidate for a state estimator . the key issue in claiming that cerebellar cortex produces a forward model is to show that the output uses efference copy to generate an estimate of state . figure 5 shows that spike triggered averaging ( sta ) of the eye velocity reveals that the neural activity does not predict or follow the movement with a large latency . rather , the correlation peaks at a latency close to zero , or even slightly negative ( winkelman and frens , 2007 ) . because the activity does not precede the eye movement , it can not be causing it . similarly , because it does not follow the eye movement , it can not reflect purely sensory information . the flocculus thus processes efferent copy to produce an output that represents the current state faithfully , which is exactly what one expects from the forward model . ( a ) shows a simple spike triggered average of eye velocity in response to white noise optokinetic stimulation . the white noise stimulus was provided by a panaromic projector system and consisted of a hexagonal matrix of green patches that were rotated coherently around the animal according to a three dimensional gaussian white noise process filtered through a 20-hz low - pass filter . note that the curve of this neuron peaks slightly before 0 ms , i.e. the p - cell is active slightly after the actual movement . ( b ) summarizes the timing of the peak in 71 purkinje cells , showing activity that more or less coincides with the movement ( winkelman and frens , 2007 ) . the first source of information is the forward model , and the second source is sensory input . in our case , we can formalize the relationship between state and sensory input using the equation ( 4)yn = h(xn)+n yn is a vector of length ky whose components reflect all the different inputs from the head and eye . n is a noise term reflecting the fact that the activity in our sensory system is not a faithful representation of the state . the function h is meant to characterize the process of sensation . in engineering applications , these two sources of information about state the forward model and sensation or observation are often combined using a kalman filter ( 5)x^n+1=x^fm , n+1+kn[ynh^(x^n ) ] the kalman filter uses the forward model 's estimate of the next state , x^fm , n+1 , as a basis for the combined estimate . ynh^(x^n ) : the difference between the actual observation , yn , and the observation expected from our current estimate of state , h^(x^n ) . the matrix kn , of size kx ky , is called the kalman gain and it quantifies both the way different sensors are relevant to different aspects of state and the relative reliability of sensation and forward model estimation . in box 1 , we also explain how sensory delays lead to alternative formulations for state estimation . whatever the details of the calculation by which state is estimated , a number of essential points can be made regarding its physiological and behavioral correlates . first , sensory estimation is a combination of internal predictions and currently available sensory information . second , the way those two sources of information are combined should reflect their reliability : if sensory input is noisy , then the system should rely more on the forward model and vice versa . third , the input / output relations of the system give us insight into the specific calculation being performed . the kalman filter model is popular in engineering applications in part because it is possible , in certain circumstances , to calculate the optimal value for the kalman gain , kn , and , for this value , the estimate produced is as close as possible to the true value of the state . 5 describes a kalman filter only when the function h(x ) is linear and the value of the gain is set to the kalman gain . however , in the field of motor control the term is often used more loosely . the kalman filter updates the estimate of state produced by the forward model , x^fm using the discrepancy between our prediction of sensory feedback , h^(x^ ) , and the actual sensory feedback , y. this discrepancy is often called the sensory prediction error . one concern in using the kalman filter as a model of the activity of the vn is that there is no evidence that vn actually calculates anything like the sensory prediction error . in eq . 5 , two different estimates of state are used , x^fm , n+1 and x^n . a true kalman filter , uses only one of these estimates , x^fm , n+1 . that is : ( 6)x^n+1=x^fm , n+1+kn(yn+1hx^fm , n+1 ) this is because the true kalman filter does n't include sensory delay . in that case , the kalman filter can be rewritten as a weighted average : ( 7)x^n+1=(ikn h^)x^fm , n+1+knyn+1 with i signifying the identity matrix . this version of the equation calculates a weighted average of prediction ( x^fm , n+1 ) and sensation ( yn+1 ) and does not calculate a sensory prediction error ( yn+1hx^fm , n+1 ) . this means that a network that calculates a state estimate based on optimal mixing for forward model prediction and noisy sensory data does not need to calculate a sensory prediction error . while this may not make any difference computationally , it does make a difference in terms of our physiological predictions . if feedback is delayed by d time steps , then yndh^x^n would compare predictions about the current state with sensory information from a while ago ( we assume for this discussion that sensation h is linear ) . one class of solutions which includes the smith predictor ( wolpert and miall , 1996 ) is to compare the delayed sensation , ynd , to a delayed state estimate . in the brain , we do not have delay registers , but we can estimate the past state from the current one , or , for that matter from the output of the forward model , x^nd = r^x^fm , n+1 , where r^ performs backwards linear estimation of the state such as estimating previous position from current position and velocity . since d is substantial ( around 100 ms ) , the estimating backward using the current output of the state estimate , x^n , is relatively similar to using the current output of the forward model , x^fm , n+1 , since both are relatively similar compared to x^nd . this leads us to a modified kalman filter ( 8)x^n+1=x^fm , n+1+kn(yndh^r^x^fm , n+1 ) that can also be written as a weighted average ( 9)x^n+1=(ih^r^)x^fm , n+1+knynd we have simulated this process using a kalman filter tracking a particle driven by a sinusoidal force with a frequency of 2 hz , using a time step of 10 ms . filter receives the noisy sensory data with 0 delay ; the buffered filter receives the sensory data with a 100 ms delay , but keeps track of the last 10 estimates of state and updates them as the delayed sensory information arrives ; the linear estimator follows eq . it is clear from figure b1 that the linear estimator performs nearly as well as the buffered version in this case . the sum squared error of the buffered kalman filter is 12 times greater than a filter without delay while that of the linear estimation filter is 15 times greater . this shows that a reasonable state estimator can be developed that is based primarily on weighted averages of the forward prediction and sensation , even in cases of significant delay . we suggest that the vestibular nucleus has the characteristics necessary for generating such an estimate . its output should be a state estimate that reflects more recent sensory input than the forward model . if we accept that the flocculus generates a forward model , then the input requirements are met by the vn . all floccular output is directed to the vn , and sensory information about the head and eye converges here . as a matter of fact , the vn are quite inappropriately named . one key study that has looked into the exact properties of the output of the vn is stahl and simpson ( 1995 ) . they first receive input from the flocculus ( ftns ) while the rest , 80% of the neurons in the vn , do not ( non - ftns ) . the two groups of neurons have distinctly different behaviors , as seen in figure 6 . the firing of the non - ftns predicts ( with almost zero lead ) the firing of the neurons in the abducens nucleus . this , in combination with the fact that all non - ftns project to the abducens nucleus , suggests that the non - ftns might be a good candidate for the estimate of state that actually drives the feedback controller . the relationship of the ftns to sensory ( vestibular ) input , motor output ( abducens nucleus ) and actual eye movement is more complex . first , the ftns lead the non - ftns , suggesting that they are the first step in a two step computation , or perhaps an earlier step in a complex computation . roughly 60% do not project to the midbrain stahl and simpson ( 1995 ) . second , the relationship of ftn activity to actual eye movement is better in the dark than in the light , consistent with the idea that ftn activity reflects the predictions of a forward model which has a greater influence on the controller when sensory input is compromised . this suggestion is reinforced by the fact that the difference between light and dark nearly disappears when target velocity and acceleration are increased ( the target oscillates at a higher frequency ) because in these situations , the vestibular input is much more reliable than visual input , and so the importance of the forward model would not be different in the light and the dark . timing of activity of ftns in the vn , compared to non - ftns , abducens nucleus , vestibular efferents , and the actual eye movement . all phases are given with respect to a vestibular stimulus that was either given in the dark ( left panel ) , or in the light ( right panel ) . finally , the notion that these neurons carry the full 3d properties of the eye movement , while the actual motor command itself does not , suggests that the vn carries an estimate of state rather than a motor command ( ghasia et al . , 2008 ) . roy and cullen ( 2004 ) show that activity of vestibular neurons that normally reflects gaze shifts is suppressed during gaze shifts involving active head movements . this is consistent with the idea that the vestibular nucleus activity reflects the activity of a forward model incorporating efferent copy of commands to the neck muscles . cancellation of vestibular nucleus activity during active head movements suggests that vestibular nucleus activity reflects the position of the eyes in the head rather than the position of eyes in extrinsic space . this concerns the issue of adaptation . in many control systems , the plant , the environment and the sensory system for instance , in the case of eye movements , the physiological fluctuations in muscle strength change the effects of motor commands and putting on glasses ( which change visual magnification and have different characteristics in different parts of visual space ) or contact lenses ( which change the weight of the eye ) can change the way movements of the eye affect visual input . it is possible that the different forms of adaptation happen simultaneously : the forward model changes in response to sensory prediction error ; the sensory prediction optimally re - weights sensation and prediction ; the feedback controller adjusts the motor commands associated with the current state . thus , we must be clear , when we discuss physiological correlates , to specify where we think plasticity may be taking place , and which neurons carry the signals that drive the plasticity and in what coordinates these signals are represented . the mechanisms of plasticity of the cerebellar cortex have been well studied . the most widespread hypothesis is that climbing fibre ( cf ) projections ( that produce purkinje cell complex spikes ) encode errors that modify the pf - pc synapses through ltd ( ito , 1986 , 2006 ; simpson et al . , 1996 ) . there is evidence for other forms of plasticity as well ( hansel et al . , 2001 ; coesmans et al . , 2004 nonetheless , many researchers accept the role of the cf as a teacher signal . if we accept that cf activity carries some form of error signal that drives plasticity , we must face the question of what sort of error it really carries . until recently , on the other hand , such a signal is not optimal for modifying a forward model ( fm ) . adaptation in a forward model should reduce discrepancies between the estimated and the actual state ; it should adapt in response to an error that reflects such discrepancies . consequently the cf should report unexpected retinal slip rather than any retinal slip ( see figure 7 for an example ) . such signals have been found in the flocculus ( frens et al . , 2001 ; winkelman and frens , 2006 ) , as well as in the visual pathways projecting to the inferior olive ( ilg and hoffmann , 1991 , 1996 ) . complex spike ( cs ) modulation as a result of sinusoidal optokinetic stimulation . in ( a ) the behavior of the animal varied with the relative luminances of the moving and the static pattern . the frequency of the fitted sine wave equals the frequency of the stimulus ( 0.1 hz ) . note that the modulation in ( a ) and ( b ) is virtually identical , as are the cem made by the animal ( gain 0.60 and 0.58 , respectively ) . consequently the predicted slip ( caused by the eye movement over the static pattern ) is not reflected in the cs ( frens et al . , 2001 ) . plasticity in the vn ( pugh and raman , 2006 , 2009 ) , guided by the cerebellar projection may be the mechanism underlying the weighting required for the optimal state estimation proposed in box 1 . ( 2008 ) showed that early recovery of the vor from labyrinthectomy is cerebellar independent while later recovery is cerebellar dependent . of course , this argues strongly for non - cerebellar mechanisms of plasticity in the cem system . suggest that the early , non - cerebellar recovery reflects plasticity in the vestibular nucleus . our model suggests that this may result from a reweighting of the different inputs to the state estimator . indeed , our model makes a strong prediction : the early stage of recovery from vor will not depend on calculations related to the forward model while the later stage will have such a dependence . we were not able to find any studies of addressing the possibility of plasticity in the ab or nph . however , gaze stability is affected by vor adaptation , and one reasonable explanation for this would be adaptation of the gain of the oculomotor integrator ( tiliket et al . , 1994 ) . we propose that cem are generated by a spfc framework where specific functional roles can be ascribed to specific nuclei in the cem circuitry . the strength of the spfc framework has been demonstrated by many groups ( wolpert and miall , 1996 ; todorov and jordan , 2002 ; shadmehr and krakauer , 2008 ) . recently , it has also been applied to describe eye movements ( glasauer , 2007 ; ghasia et al . , 2008 ) . because the physiology and anatomy underlying cem is relatively well known , we are able to describe this mapping in more detail and with more precision than was possible in a similar attempt to describe the control of reaching movements ( shadmehr and krakauer , 2008 ) . the timing and nature of the signals that can be recorded in the flocculus , the vn , and the brainstem structures support our hypothesis . also , plasticity in the flocculus and in the vn and the purported olivary error signals can be understood in terms of this framework . our model can be contrasted with the classical approach , where the output of the cerebellum is an inverse model ( kawato and gomi , 1992 ) . the difference in the role played in the cerebellar output is , perhaps , the most salient difference between the two approaches , but there are other differences as well . for instance , the classical approach does not explain the separate function of the three different areas flocculus , vestibular nucleus , and brainstem motor nuclei that generate a cascading series of motor commands . in contrast , the spfc framework ascribes clear and distinct functions to each of these areas . however , making an experimental distinction between the output of a forward model and the output of an inverse model can be quite difficult . work by kawato 's group has shown that position , velocity and acceleration regress onto firing rate with a combined r of above 0.7 ( shidara et al . , 1993 ) . this has been widely regarded as evidence that the cerebellum implements an inverse model . however , in follow up work , the kawato group disavows this idea and claims that the floccular output can not represent the main part of the motor command to the eyes ( gomi et al . , 1998 ) . perhaps the two most convincing arguments in this respect come from our group and that of dora angelaki , as described above . both of these lines of reasoning argue in favor of the forward model interpretation . our model enjoys a family resemblance with previously presented schemes , notably the shadmehr and krakauer ( 2008 ) model of reaching movement control and the green et al . ( 2007 ) model for cem . however , there are also key differences between our model and the others . one key issue in this regard is whether the output of the vestibular nucleus describes the upcoming motor command or the current estimate of state . because cem do not obey listing 's law , the use of the representation of violation of listing 's law ( as was used to great effect in ghasia et al . , 2008 ) ( 2008 ) do suggest that many neurons in the vestibular nucleus represent state , and ( 2 ) if the vestibular nucleus is implementing an inverse model and the flocculus is implementing a forward model , it is unclear where state and prediction should be combined . our model is also different from the one used by shadmehr and krakauer to describe reaching movements ( shadmehr and krakauer , 2008 ) . shadmehr and krakauer suggest that the output of the deep cerebellar nuclei ( dcn ) reflects the output of a forward model ( x^fm ) . this is necessary in their scheme because they propose , based on evidence from errors in reaching movements , that the parietal cortex calculates an estimate of state , and thus they propose that the forward model output from the cerebellum should drive this estimate of state . one might say that the dcn is considered the output of the forward model because it more directly projects to the cortex , although the role of the ventrolateral thalamus which relays the dcn projection to cortex is not considered in their framework . in our system , the vn seem to be located appropriately to combine forward model prediction based on efferent feedback with delayed sensory information . thus , in our system , it is not the cerebellum but specifically the cerebellar cortex which generates a forward model prediction . this difference between our hypothesis and that of shadmehr and krakauer might arise for a number of reasons as both models are speculative . shadmehr and krakauer did not consider the cerebellar cortex and dcn separately or ascribe any role at all to the thalamus relay station . however , it is possible that the computation carried out by the cerebellum in the two systems is different and both models are correct . one important aspect of the shadmehr and krakauer analysis of the reaching movement system has to do with the role they ascribe to the basal ganglia in determining the mapping of estimated state to motor command . their framework explicitly uses the language of optimal feedback control , popularized in our field by todorov ( 2004 ) . in optimal feedback control , the controller produces a command which will lead to the best possible combination of task success and energy conservation . in different tasks or with different weight attached to energy conservation , the controller will map states onto motor commands differently . in the scheme put forward by shadmehr and krakauer , the role of the basal ganglia is to work with the motor cortex to learn to produce such optimal motor commands . there is no equivalent of the basal ganglia in the cem system , and it is quite possible that the cem does not implement an optimal controller : the cem system is a reflex system and the cost function may be very consistent relative to the costs associated with reaching movements in different tasks . another way in which our model differs from previous theories is that we explicitly reject the widespread hypothesis that state estimation is computed using a kalman filter ( see box 1 ) . rather , it seems that the vn calculation of current state reflects a process with two or more stages , where floccular target neurons perform a first stage of estimation and are then integrated into the broader calculation . the use of a forward model is useful when sensory signals are either noisy or have a large delay . the latter is specifically the case for the retinal slip signals that drive the visual component of cem ( the okr ) , which have a delay of 80 ms , whereas the vestibular afferents have a delay of only a few ms . this may explain why lesions of the flocculus primarily affect the okr , and only influence the plasticity of the vor , but not its performance ( waespe et al . , 1983 ; although the cem circuit is well studied , there are still many holes in our knowledge . for instance it is known that different vn neurons project to the brain stem and to the flocculus . perhaps the vn calculates two different state estimates or perhaps its projection forward to the brain stem motor nuclei includes partial calculation of the motor command . resolving this issue will need to wait until more data is available . also the finding that there are neurons at two levels of signal processing that strongly resemble the firing of the abducens ( the non - ftns in the vn , stahl and simpson , 1995 , and the cells in the nph green et al . , 2007 ) requires further experimentation , for instance during eye movements that are mechanically perturbed . an spfc framework can not successfully adapt to changes in the plant unless the feedback controller can adapt . since recent findings have obscured the functional difference between abducens nucleus and nph ( green et al . , 2007 ; ghasia et al . , 2008 ) , one tempting hypothesis is that nph serves as the adaptive component of the feedback controller . however , this is only speculation until some data on plasticity in the two nuclei becomes available . foveate species have smooth pursuit , which they can use to voluntarily reduce retinal slip . in the afoveate rabbit , for instance , in an experimental paradigm , where the visual environment rotates along with a vestibular stimulus , the vn modulate only at high frequencies , along with the actual eye movement ( stahl and simpson , 1995 ) . in the ( foveate ) primate , this correlation is less robust ( miles , 1974 ; waespe and henn , 1978 ) , since the smooth pursuit system can modify the eye movements . thus , the vn appear to represent an estimate of the eye state faithfully in the rabbit ( because cem are the only eye movements present ) , but this relation is harder to study in primates , since cem and sp are harder to distinguish . in sum , we believe that the spfc model for the cem accounts for the available data on the anatomy and physiology of the brain areas involved . it solves important conundrums , especially the timing of the activity of p - cells involved in cem . while the model remains speculative , it seems to us to be the most reasonable basis for continued exploration of the neural mechanisms involved in stabilizing the eye during fixation . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the compensatory eye movement ( cem ) system maintains a stable retinal image , integrating information from different sensory modalities to compensate for head movements . inspired by recent models of the physiology of limb movements , we suggest that cem can be modeled as a control system with three essential building blocks : a forward model that predicts the effects of motor commands ; a state estimator that integrates sensory feedback into this prediction ; and , a feedback controller that translates a state estimate into motor commands . we propose a specific mapping of nuclei within the cem system onto these control functions . specifically , we suggest that the flocculus is responsible for generating the forward model prediction and that the vestibular nuclei integrate sensory feedback to generate an estimate of current state . finally , the brainstem motor nuclei in the case of horizontal compensation this means the abducens nucleus and the nucleus prepositus hypoglossi implement a feedback controller , translating state into motor commands . while these efforts to understand the physiological control system as a feedback control system are in their infancy , there is the intriguing possibility that cem and targeted voluntary movements use the same cerebellar circuitry in fundamentally different ways .

Compensatory Eye Movements The State Predicting Feedback Controller The Feedback Controller The Forward Model The State Estimator Adaptation Discussion Conflict of Interest Statement

compensatory eye movements ( cem ) is a general term for a number of different reflexes that keep an image fixed on the retina during movements of the body and the head ( e.g. the circuitry of the cem system ( figure 1 ) is different from the circuitry for other eye movements such as saccades , although all the eye movement systems converge in the oculomotor nuclei of the brainstem ( buttner - ennever and buttner , 1992 ) . for horizontal eye movements these are the abducens nucleus ( ab ) and the nucleus prepositus hypoglossi ( nph ) . the horizontal compensatory eye movement ( cem ) system . for compensation of higher velocity stimuli , the cem system depends on the vestibulo - ocular reflex ( vor ) which uses vestibular input to estimate head movement and generate oppositely directed eye movements . both the vor and okr are adaptive , meaning that the mapping of stimulus to appropriate eye response can be tuned to match changing response properties of the eye and its supporting tissues ( usually collectively called the plant ) or changes in the sensitivity of the sensory organs ( blazquez et al . there is ample evidence that the flocculus , a small section of the cerebellar cortex is critical in this plasticity ( e.g. a forward model mimics this process , estimating the movement that will be produced by the plant . a somewhat more eclectic model that also focuses on timing is braitenberg 's model of the cerebellum as a system for generating sequences of movement in precise time relationship ( braitenberg et al . while each of these models can legitimately claim to explain important data , there is no doubt that the inverse model understanding of the cerebellum in cem is the most widely accepted . the inverse model framework can be contrasted with a forward model ( wolpert and miall , 1996 ; todorov and jordan , 2002 ; see figure 2b ) which simulates the activity of the plant : it converts the current state and the control signals into a prediction of what the plant will actually do . the focus on a neural inverse model of the oculomotor plant reflected a perspective that the central problem in oculomotor control is producing the appropriate motor commands once the goal is given . researchers in other motor systems notably arm movements followed in the footsteps of the pioneering work in oculomotor research and focused on the inverse model problem and the question of how appropriate motor commands are generated , given a particular desired movement . however , the possibility that a forward model also plays a role has been hypothesized for a long time ( e.g. under this approach , the problem of predicting the results of motor commands is no less central than the problem of generating those motor commands in the first place . feedback control is the use of the measured or predicted state of the system to generate ongoing motor commands . however , predictions of the state must be combined with actual sensory feedback in order for the control loop to remain robust in the face of unpredicted perturbations . thus , the framework ( which we will call the state - predicting feedback control , spfc , framework ) is built out of three essential building blocks ( figure 3 ; todorov , 2004 ; shadmehr and krakauer , 2008 ) . the forward model takes the current estimate of state and the motor commands and produces an initial prediction . the state estimator combines this prediction with actual sensory feedback to produce a better estimate of the current state . the feedback controller uses the current estimate of state in order to decide what motor commands to generate . we propose that this framework is an appropriate description of cem control and that it can be mapped onto cem physiology in a manner that is consistent with experimental evidence . the spfc framework proposes that a feedback controller is optimized to produce motor commands that achieve task goals . in order to do this effectively , it uses an estimate of the current situation that is derived from a combination of feedback from the sensory system and forward model estimation that depends on efferent copy . they suggest that motor cortex , in combination with the basal ganglia , implements a feedback controller implementing a control policy that maximizes successful performance . , 2007 ) , they claim the forward model is implemented in the cerebellum . since the cem system is located in brain stem nuclei and the cerebellum , and neither motor cortex nor parietal cortex is instrumental , our effort to ascribe computational functions to physiological correlates in the cem will necessarily produce different results . we will argue that , for cem , the most suitable mapping would be that the oculomotor nuclei and integrators ( robinson 's inverse plant ) combine to form a feedback controller . the cerebellar cortex ( and not the whole cerebellum ) generates a forward model , and the vn combine forward model output with current inputs to produce the state estimate . in the language of control system experts , this could be approximated as a transformation ( 1)un = ln(x^n ) where un is a vector of length ku describing the motor commands at the n time step . of course , both the state and the motor vectors could , the point is that the feedback controller implements a function , l , that translates its input , an estimated state vector , x^ , into its output , the motor command vector , u. by definition , the output of a controller is motor command so whatever produces the motor commands must necessarily be implementing a controller . in our case , motor command is the activity that drives the muscles , and the motoneurons of the abducens nucleus are the output of the feedback controller , at least for horizontal motion . in cem , the controller must know the desired fixation point and it must receive an estimate of the current eye position . the brainstem circuit that traditionally constitutes the inverse plant meets these requirements ( buttner - ennever and buttner , 1992 ; glasauer , 2007 ) , even though there is debate on how the computation in the plant is achieved ( see below ) . recent work on the nph , the putative horizontal integrator , undermines this view since neurons in the nph are found to encode the whole motor command , u , rather than only the integrated part ( green et al . indeed , on the basis of the finding that nph feedback encodes propose that the feedback is updating a cerebellar forward model ( see figure 3 ) . in both the ab and the nph , the cem circuit is shared with the other eye movement systems ( i.e. this fits the role of feedback controller , since the efference copy needed by the forward model should contain all oculomotor output in order to produce an optimal estimate of state ( see below ) . that is , we can use a forward model to generate an estimate about current state from our earlier estimate and our knowledge of system dynamics . we assume , for the purpose of simplicity , that the actual dynamics of the system can be described as linearly combining previous state and motor command : ( 2)xn+1=axn+bun+n x is the actual state , whose estimate is discussed above ( x^n ) . both x and x^n have the same size , but the latter is the brain 's estimate and the former is the actual quantity . a is a kx kx matrix , b is a kx ku matrix , and the forward model estimate would be generated from the previous estimate using a similar equation ( 3)x^fm , n+1=a^x^n+b^un where a^ and b^ represent the forward model 's estimates of system dynamics . notice that in this formulation , which is commonly used , the estimate of state used to calculate the forward model is not the same as the estimate produced by the forward model in the previous step . what we mean by this is that we may improve the estimate generated by the forward model ( for instance , by incorporating information from sensory inputs ) before we use it in the forward model 's next step . we assume that the forward model has no knowledge of the random fluctuations in the state represented by the noise term . however , we expect the forward model to be plastic . that is , if the state prediction of the forward model is consistently wrong the model should change . specifically , the cortical area responsible for cem , the flocculus , receives direct projections from the nph ( sato et al . 1993 ) , and we will argue later that this is the most likely candidate for a state estimator . the key issue in claiming that cerebellar cortex produces a forward model is to show that the output uses efference copy to generate an estimate of state . the flocculus thus processes efferent copy to produce an output that represents the current state faithfully , which is exactly what one expects from the forward model . the first source of information is the forward model , and the second source is sensory input . in engineering applications , these two sources of information about state the forward model and sensation or observation are often combined using a kalman filter ( 5)x^n+1=x^fm , n+1+kn[ynh^(x^n ) ] the kalman filter uses the forward model 's estimate of the next state , x^fm , n+1 , as a basis for the combined estimate . ynh^(x^n ) : the difference between the actual observation , yn , and the observation expected from our current estimate of state , h^(x^n ) . whatever the details of the calculation by which state is estimated , a number of essential points can be made regarding its physiological and behavioral correlates . second , the way those two sources of information are combined should reflect their reliability : if sensory input is noisy , then the system should rely more on the forward model and vice versa . the kalman filter model is popular in engineering applications in part because it is possible , in certain circumstances , to calculate the optimal value for the kalman gain , kn , and , for this value , the estimate produced is as close as possible to the true value of the state . the kalman filter updates the estimate of state produced by the forward model , x^fm using the discrepancy between our prediction of sensory feedback , h^(x^ ) , and the actual sensory feedback , y. this discrepancy is often called the sensory prediction error . one concern in using the kalman filter as a model of the activity of the vn is that there is no evidence that vn actually calculates anything like the sensory prediction error . in that case , the kalman filter can be rewritten as a weighted average : ( 7)x^n+1=(ikn h^)x^fm , n+1+knyn+1 with i signifying the identity matrix . this means that a network that calculates a state estimate based on optimal mixing for forward model prediction and noisy sensory data does not need to calculate a sensory prediction error . in the brain , we do not have delay registers , but we can estimate the past state from the current one , or , for that matter from the output of the forward model , x^nd = r^x^fm , n+1 , where r^ performs backwards linear estimation of the state such as estimating previous position from current position and velocity . since d is substantial ( around 100 ms ) , the estimating backward using the current output of the state estimate , x^n , is relatively similar to using the current output of the forward model , x^fm , n+1 , since both are relatively similar compared to x^nd . this shows that a reasonable state estimator can be developed that is based primarily on weighted averages of the forward prediction and sensation , even in cases of significant delay . we suggest that the vestibular nucleus has the characteristics necessary for generating such an estimate . its output should be a state estimate that reflects more recent sensory input than the forward model . if we accept that the flocculus generates a forward model , then the input requirements are met by the vn . they first receive input from the flocculus ( ftns ) while the rest , 80% of the neurons in the vn , do not ( non - ftns ) . the firing of the non - ftns predicts ( with almost zero lead ) the firing of the neurons in the abducens nucleus . this , in combination with the fact that all non - ftns project to the abducens nucleus , suggests that the non - ftns might be a good candidate for the estimate of state that actually drives the feedback controller . the relationship of the ftns to sensory ( vestibular ) input , motor output ( abducens nucleus ) and actual eye movement is more complex . second , the relationship of ftn activity to actual eye movement is better in the dark than in the light , consistent with the idea that ftn activity reflects the predictions of a forward model which has a greater influence on the controller when sensory input is compromised . this suggestion is reinforced by the fact that the difference between light and dark nearly disappears when target velocity and acceleration are increased ( the target oscillates at a higher frequency ) because in these situations , the vestibular input is much more reliable than visual input , and so the importance of the forward model would not be different in the light and the dark . timing of activity of ftns in the vn , compared to non - ftns , abducens nucleus , vestibular efferents , and the actual eye movement . finally , the notion that these neurons carry the full 3d properties of the eye movement , while the actual motor command itself does not , suggests that the vn carries an estimate of state rather than a motor command ( ghasia et al . this is consistent with the idea that the vestibular nucleus activity reflects the activity of a forward model incorporating efferent copy of commands to the neck muscles . cancellation of vestibular nucleus activity during active head movements suggests that vestibular nucleus activity reflects the position of the eyes in the head rather than the position of eyes in extrinsic space . in many control systems , the plant , the environment and the sensory system for instance , in the case of eye movements , the physiological fluctuations in muscle strength change the effects of motor commands and putting on glasses ( which change visual magnification and have different characteristics in different parts of visual space ) or contact lenses ( which change the weight of the eye ) can change the way movements of the eye affect visual input . it is possible that the different forms of adaptation happen simultaneously : the forward model changes in response to sensory prediction error ; the sensory prediction optimally re - weights sensation and prediction ; the feedback controller adjusts the motor commands associated with the current state . adaptation in a forward model should reduce discrepancies between the estimated and the actual state ; it should adapt in response to an error that reflects such discrepancies . in ( a ) the behavior of the animal varied with the relative luminances of the moving and the static pattern . of course , this argues strongly for non - cerebellar mechanisms of plasticity in the cem system . suggest that the early , non - cerebellar recovery reflects plasticity in the vestibular nucleus . we propose that cem are generated by a spfc framework where specific functional roles can be ascribed to specific nuclei in the cem circuitry . the timing and nature of the signals that can be recorded in the flocculus , the vn , and the brainstem structures support our hypothesis . also , plasticity in the flocculus and in the vn and the purported olivary error signals can be understood in terms of this framework . the difference in the role played in the cerebellar output is , perhaps , the most salient difference between the two approaches , but there are other differences as well . for instance , the classical approach does not explain the separate function of the three different areas flocculus , vestibular nucleus , and brainstem motor nuclei that generate a cascading series of motor commands . however , making an experimental distinction between the output of a forward model and the output of an inverse model can be quite difficult . however , in follow up work , the kawato group disavows this idea and claims that the floccular output can not represent the main part of the motor command to the eyes ( gomi et al . both of these lines of reasoning argue in favor of the forward model interpretation . one key issue in this regard is whether the output of the vestibular nucleus describes the upcoming motor command or the current estimate of state . because cem do not obey listing 's law , the use of the representation of violation of listing 's law ( as was used to great effect in ghasia et al . , 2008 ) ( 2008 ) do suggest that many neurons in the vestibular nucleus represent state , and ( 2 ) if the vestibular nucleus is implementing an inverse model and the flocculus is implementing a forward model , it is unclear where state and prediction should be combined . shadmehr and krakauer suggest that the output of the deep cerebellar nuclei ( dcn ) reflects the output of a forward model ( x^fm ) . this is necessary in their scheme because they propose , based on evidence from errors in reaching movements , that the parietal cortex calculates an estimate of state , and thus they propose that the forward model output from the cerebellum should drive this estimate of state . one might say that the dcn is considered the output of the forward model because it more directly projects to the cortex , although the role of the ventrolateral thalamus which relays the dcn projection to cortex is not considered in their framework . in our system , the vn seem to be located appropriately to combine forward model prediction based on efferent feedback with delayed sensory information . thus , in our system , it is not the cerebellum but specifically the cerebellar cortex which generates a forward model prediction . however , it is possible that the computation carried out by the cerebellum in the two systems is different and both models are correct . in the scheme put forward by shadmehr and krakauer , the role of the basal ganglia is to work with the motor cortex to learn to produce such optimal motor commands . there is no equivalent of the basal ganglia in the cem system , and it is quite possible that the cem does not implement an optimal controller : the cem system is a reflex system and the cost function may be very consistent relative to the costs associated with reaching movements in different tasks . rather , it seems that the vn calculation of current state reflects a process with two or more stages , where floccular target neurons perform a first stage of estimation and are then integrated into the broader calculation . also the finding that there are neurons at two levels of signal processing that strongly resemble the firing of the abducens ( the non - ftns in the vn , stahl and simpson , 1995 , and the cells in the nph green et al . in the afoveate rabbit , for instance , in an experimental paradigm , where the visual environment rotates along with a vestibular stimulus , the vn modulate only at high frequencies , along with the actual eye movement ( stahl and simpson , 1995 ) . thus , the vn appear to represent an estimate of the eye state faithfully in the rabbit ( because cem are the only eye movements present ) , but this relation is harder to study in primates , since cem and sp are harder to distinguish . in sum , we believe that the spfc model for the cem accounts for the available data on the anatomy and physiology of the brain areas involved . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the online version of this article ( doi:10.1007/s10953 - 015 - 0321 - 5 ) contains supplementary material , which is available to authorized users . the capture of sulfur dioxide ( so2 ) has drawn significant attention because so2 is one of the most harmful air pollutants , mainly originating from the combustion of fossil fuels . the most traditional and widely used technology for so2 capture from flue gases is limestone scrubbing . this process has certain disadvantages , including irreversibility of the reaction and a large amount of waste . recently , the absorption of so2 in ionic liquids ( ils ) was suggested as an alternative . the high absorption capacity and the good reversibility of the absorption process as well as unique and tunable properties of ils have caused a growing interest for the past decade [ 48 ] . so2 absorption by ils can occur in a physical or in a chemical way [ 4 , 5 , 8 ] from which only in the former case a full and simple recovery is possible . therefore , the appropriate media for the full or partial recovery of so2 depending on the purpose can be chosen . the physical absorption of so2 in ils is almost independent of the type of anion and cation , whereas in case of the chemical absorption the dependence on the type of il becomes more pronounced with the nature of the anion playing the crucial role [ 810 ] . such a principle difference in the behavior on the microscopic scale might be better understood when theoretical methods are used . static gas - phase calculations of interaction energies and the assigment of principle interaction types based on those values were investigated in several articles [ 1013 ] . also , molecular dynamics simulations employing empirical force fields were used for evaluating some physical properties of the systems [ 1417 ] . in order to understand the mechanism of so2 solvation in more detail , solute thus , the investigation of specific interactions between so2 and the il components , as well as understanding how the interactions influence the conditions in the il , can be provided from a valuable theoretical background which aids in the design of ils with desired properties . in this work , we have employed ab initio molecular dynamics ( aimd ) simulations to obtain insight into the structural and dynamic properties of the so2il systems . to do so , we have chosen 1-ethyl-3-methylimidazolium thiocyanate ( [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] ) as a model system . this il is one of the most promising candidate for large - scale application , possessing one of the highest capacities of so2 absorption , a rapid absorption rate , and excellent reversibility . moreover , the solubilities of other gases in this il are significantly lower than for so2 ( comparing the molar fraction of gases in [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] at 1 bar and 293 k : so2 is 75 % , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { nh}_3$$\end{document}nh316.3 % , co21.2 % ) , which is a requirement for separation processes . recently the structural properties of pure [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] [ 20 , 21 ] and its mixtures with another ionic liquid , as well as carbon dioxide ( co2 ) absorption in imidazolium and ethylammonium ionic liquids [ 2325 ] , have been studied from aimd , providing a solid background for the current investigation . thus , the knowledge gathered here provides a more thorough understanding of so2 solvation in ils . moreover , similarities and differences can be identified between so2 and co2 with respect to their solvation in ils . the mixture of [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] with so2 ( 32:1 molar ratio ) was studied from aimd . a system containing 32 ion pairs of [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] and one so2 molecule was simulated in a cubic box with a size of 2031.4 pm ( this corresponds to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho = 1.086\hbox { g}{\cdot}{\text{cm}}^{-3}$$\end{document}=1.086gcm-3 ) with periodic boundary conditions ( fig . 1 ) . for details on the preparation of the starting geometry for the system , see the supporting information.fig . 1representative snapshot of the simulation boxes : 1-ethyl-3-methylimidazolium thiocyanate in stick ( cations in blue , anions in red ) and so2 ( in green ) in ball - and - stick representation representative snapshot of the simulation boxes : 1-ethyl-3-methylimidazolium thiocyanate in stick ( cations in blue , anions in red ) and so2 ( in green ) in ball - and - stick representation the aimd simulations were carried out with the cp2k program package , using the quickstep module with the orbital transformation method for faster convergence . the electronic structure was calculated employing the density functional theory utilizing the blyp - d3 functional with the empirical dispersion correction ( d3 with zero dumping ) from grimme , since the dispersion - corrected exchange - correlation functional has provided reasonable results for ionic liquids [ 20 , 3032 ] . the molecularly optimized double-\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\zeta $ $ \end{document} basis set ( molopt - dzvp - sr - gth ) with corresponding goedecker the density smoothing for the electron density ( nn10_smooth ) and its derivative ( nn10 ) was used . the cutoff criterion for hoover chain thermostats [ 3739 ] with a time constant of 100 fs for individual atoms for a total of 5.0 ps and for the complete system in the main run . for the equilibration ( 5.0 ps ) the time step 0.5 fs was used . since this value provided a high energy drift ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2.8 \times 10^{-5}\hbox { a.u.}{\cdot}\hbox { fs}^{-1}$$\end{document}2.810 - 5a.u.fs-1 ) in the beginning of the main run ( first 22.3 ps ) , a shorter time step ( 0.25 fs ) was applied . this decreased the energy drift by one order to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$3.7 \times 10^{-6}\hbox { a.u.}{\cdot}\hbox { fs}^{-1}$$\end{document}3.710 - 6a.u.fs-1 ) , and we excluded the first 25.0 ps ( 22.3 with 0.5 and 2.7 ps with 0.25 fs time steps ) of the main run from further consideration . static quantum chemical calculations were performed from the density functional theory ( dft ) and wave function theory with the orca program ( version 3.0.0 ) . geometry optimization was performed on the b3lyp - d3(bj)/def2-tzvpp level , whereas the final energy calculations were done based on dft geometry applying the ccsd(t ) level of theory . extrapolation to the complete basis set limit was carried out according to a two - point extrapolation scheme separately for hartree the calculation of so2 gas frequencies was performed on blyp - d3(bj)/def2-tzvpp in order to be consistent with the exchange correlation functional , which was applied for bulk simulation . molecule representations were visualized using pymol , and all graphs were created using gnuplot 4.6 . the atom labeling used in the following discussion 2ball - and - stick representation of the cation , the anion , and the so2 molecule . n : blue ; c : orange ; o : red , h : white and s : yellow . ( please note that the cation s atoms are marked without primes , the anion with single primes , and the atoms of the so2 with double primes . ) ball - and - stick representation of the cation , the anion , and the so2 molecule . n : blue ; c : orange ; o : red , h : white and s : yellow . ( please note that the cation s atoms are marked without primes , the anion with single primes , and the atoms of the so2 with double primes . ) to gain insight into how so2 in low concentrations influences the structure of [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] , we compared corresponding radial distribution functions ( rdfs ) of the pure il to the system under study ( see supporting information figs . this is consistent with md simulations of systems with a higher concentration of so2 in ils [ 10 , 17 ] as well as with the results obtained previously for co2 in ethylammonium nitrate and in 1-ethyl-3-methylimidazolium acetate [ 23 , 24 ] . to reveal how so2 enters the il structure and to characterize the solvation shell of the solute molecule the latter analysis provides valuable information about the time development or the average of surface covering of a certain particle by other particles or groups of atoms . similar to co2 in 1-ethyl-3-methylimidazolium acetate [ 23 , 24 ] , so2 is surrounded only by one anion and five cations on average in the first solvation shell ( the numbers were defined based on the value of integral in the first minimum of the corresponding rdf between centers of mass of solute and ions , see fig . 3a it is apparent that a similar ratio of anions to cations is obtained from the average so2 surface covering by anions and cations ( 18 vs. 82 % ) . both results rdf as well as voronoi around so2.fig . 3 top time - development of the so2 surface covering by ions ( a , blue lines are cation atoms , and red lines are anion atoms ) and cation atoms ( b , black lines are alkyl hydrogen atoms , red lines are ring hydrogen atoms and blue lines are ring atoms ) . bottom representative so2 voronoi surfaces , color code for the surfaces is similar with those for graphs above . ( note grey color was used for the segments belonging to the anion for the bottom right voronoi surfaces ) top time - development of the so2 surface covering by ions ( a , blue lines are cation atoms , and red lines are anion atoms ) and cation atoms ( b , black lines are alkyl hydrogen atoms , red lines are ring hydrogen atoms and blue lines are ring atoms ) . bottom representative so2 voronoi surfaces , color code for the surfaces is similar with those for graphs above . ( note grey color was used for the segments belonging to the anion for the bottom right voronoi surfaces ) since a relatively large number of cations was detected in the first solvent shell of so2 , it is worth determining in detail which functional groups are important , and how this situation of so2 solvation compares to the co2 solvation by ils . to reveal the role of the specific interactions , especially the weak interactions , in the so2 solvation , we have separated the so2 surface coverage from the cations into three groups : ring hydrogen atoms ( h2 , h4 , h5 ) , heavy ring atoms ( n1 , c2 , n3 , c4 , c5 ) , and alkyl hydrogen atoms ( h6 , h7 , h8 ) . it is a reasonable first approximation that a larger coverage ( i.e. , closest neighbor ) corresponds to a more important role in the solute solvation . the ring hydrogen atoms possess a relatively strong interaction with the solute but also with the anion . the strong nature of the latter interaction was shown in several quantum chemical studies [ 46 , 47 ] . if there are contacts between the so2 and the cation ring other than via the ring hydrogen atoms , these are most likely weak solute\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document}system interactions . also the alkyl hydrogen atom contacts to the solute represents weak dispersion interaction [ 46 , 47 ] . interestingly , the alkyl hydrogen atoms ( 48 % ) and heavy ring atoms ( 15 % ) coverages , corresponding to the weak interactions , dominate over the coverage of the ring hydrogen atoms ( 19 % ) which correspond to strong interactions . moreover , these portions are comparable with those from the non - polar part of ethylammonium nitrate to co2 solvation . from these results it is apparent that weak interactions are important not only for the solvation of co2 [ 2325 ] but also for the solvation of so2 . to support this observation the increase of the cation s side chain results in the increase of the so2 solubility [ 5 , 10 , 48 ] which agrees with our findings . the rdfs , which reflect probabilities of finding two atoms at certain distances normalized by the density , of the cation s hydrogen atoms ( h2 , h4h5 , h6h8 ) with the oxygen atoms of the sulfur dioxide ( o ) and the anion tail atoms ( n and s ) , are presented in fig . 4 on a there is a noticeable similarity in the position of the first maximum for the cation - n and cation - o functions albeit with the difference that the o(so2 ) peaks are less pronounced than the n(\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]- ) peaks . remarkably , the interplay of co2 with a cation in ethylammonium nitrate or in 1-ethyl-3-methylimidazolium acetate [ 23 , 24 ] shows the opposite behavior , i.e. , there are no such peaks between the oxygen atoms of co2 and the acidic hydrogen atoms of the cation . thus , there is no contact of a co2 with the cation via the acidic hydrogen atoms . the co2 solvation rather takes the form that it competes with dispersion forces in the system such as anion cation side chain , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document} stacking , and side chain - side chain [ 46 , 47 ] interactions . these essential differences in the structure of solvated so2 and the solvated co2 might be one of the reasons for the significantly higher solubility of the former gas in ils.fig . the rdfs between hydrogen atoms and selected atoms form anion and so2 are presented on a c , whereas d contains the rdfs between s ( from so2 ) and selected atoms from cation and anion cation anion and ion the rdfs between hydrogen atoms and selected atoms form anion and so2 are presented on a c , whereas d contains the rdfs between s ( from so2 ) and selected atoms from cation and anion since not only the nature of the anion and the side chain of the cation are important for the so2 solvation but also the role of acidic hydrogen atoms , we have examined how the cation exchange in principle influences the so2 solubility . for example , the experimental findings on the cation exchange of 1-alkylpyridinium to 1-alkyl-3-methylimidazolium show a slight decrease in the so2 solubility in ils with chloride , bistriflimide , and tetrafluoroborate anions [ 10 , 11 ] . this changes significantly when ils with a thiocyanate anion are considered . when the 1-butylpyridinium cation is exchanged for the 1-ethyl-3-methylimidazolium cation , both with the thiocyanate anion , the so2 solubility increases from 2.6 to 3.0 mol per one mol il ( at 0.1 mpa and 293 k ) [ 10 , 12 ] . these contrasting experimental observations might be related to different so2 solvation mechanisms which can be explained by microscopic insight given for example by simulations , i.e. , in the 1-ethyl-3-methylimidazolium thiocyanate ionic liquid we have detected frequent conformations in which the ring hydrogen atoms of the cations are close to the oxygen atoms of the so2 and the sulfur atoms of the anions are close to the sulfur atom of the so2 . it is likely that this newly observed conformation , which we have termed linker conformation or linker effect , is responsible for the good incorporation of so2 in this particular ionic liquid by the formation of the following linked structure h2 , h4 , h5(cation)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}o(so2)s(so2)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}s(anion ) . possible linker bonds between ring hydrogen atoms of the 1-butylpyridinium cation and so2 should be weaker due to the lower acidity compared with those for the 1-ethyl-3-methylimidazolium cation . thus , the incorporation of so2 into the network of hydrogen bonds is limited in the case of ils consisting of 1-butylpyridinium cations . only one anion was found in the first solvation shell of so2 . to understand the nature of the intermolecular forces between the anion and the so2 , we have carried out static quantum chemical calculations regarding the formation of the anion the results from these calculations showed comparable interaction energies for possible adducts ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-68.6\ , \hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-68.6kjmol-1 for the thiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-65.7\,\hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-65.7kjmol-1 for the isothiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}{\cdot}\hbox { so}_2]^-$$\end{document}[scnso2]- ) . thus , it is likely that the formation of both complexes might occur during the simulation with almost equal probability . nevertheless , the rdfs for ss and ns distances indicate the formation of the thiocyanate adduct is dominant in the system under investigation as shown in fig . 4d . the time development of the distances between the s atom and the s or n atoms of all anions , as shown in fig . 5a and b , indicates that so2 interacts with one [ scn]\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^-$$\end{document}- over the majority of the simulation time , see in fig . 5a , black line . however , a temporary anion exchange ( red , blue and green lines in fig . the substitution of the s atom to the n atom coordination or the change from thiocyanate - so2 to isothiocyanate - so2 aducct occurs at around 12 , 14 , and 36 ps ( see fig . this transition in the anion coordination at so2 is in good agreement with the experimental results of the thiocyanate anion complex formation with so2 in dilute solutions of acetonitrile . thus , the absorption of so2 in the ionic liquid under consideration does not take place with a pure thiocyanate - so2 complex generation , rather an equilibrium mixture of thiocyanate and isothiocyanate adducts will form.fig . time development of distances between s atom of so2 and s ( a ) or n ( b ) atoms of all 32 anions . curves corresponding to anions that approach the so2 molecule closer than 350 pm during the simulation are colored black , red , blue and green . time development of distances between s atom of so2 and s ( a ) or n ( b ) atoms of all 32 anions . curves corresponding to anions that approach the so2 molecule closer than 350 pm during the simulation are colored black , red , blue and green . curves corresponding to other anions are colored grey to understand the structure of the dominant thiocyanate - so2 adduct in more detail , we have compared the geometrical parameters from aimd simulation in bulk and from the static quantum chemical calculation of the isolated adducts with data from crystal structures of the potassium 1,4,7,10,13,16-hexaoxacyclooctadecane thiocyanate - so2 adduct [ k(18-crown-6)][\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { ncs}{\cdot}\hbox { so}_2$$\end{document}ncsso2 ] and the tetramethylammonium thiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { nme}_4][\hbox { ncs}{\cdot}\hbox { so}_2]$$\end{document}[nme4][ncsso2 ] ( see fig . 6 and table 1 ) . both calculated structures agree well with the crystallographic data for the thiocyanate - so2 adducts , see table 1 . the differences in distances and angles are explained by the different surroundings for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- , particularly , the c-s-s-com dihedral angle could be smaller in absolute values due to the stabilization of the selected structure . considering the most probable distance between s and s atoms ( 283 pm ) , we also found good agreement with experimental values of s\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots$$\end{document}o distances in complexes of so2 with diethyl ester ( 287 pm ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { h}_2\hbox { o}$$\end{document}h2o ( 282 pm ) .fig . 6 ball - and - stick representation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- complex . for structural parameters see table 1 table 1structural parameters for the thiocyanate - so2 adductbulk gas [ k(18-crown-6)]\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot } \hbox { so}_2]^{\rm c}$$\end{document}[ncsso2]c \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { nme}_4][\hbox { ncs}{\cdot}\hbox { so}_2]^{\rm c}$$\end{document}[nme4][ncsso2]c ss ( pm)283271274301oso ( )116115119115css ( )96979990sscom ( )109112103107csscom ( ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pm $ $ \end{document}1421399174 the maximum of corresponding distribution function from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf{32}\mathbf{p}+\mathbf{so}_\mathbf{2}$$\end{document}32p+so2 trajectory ( see supporting information figs . s4s6 ) the structure was optimized in the gas - phase on b3lyp - d3/def2-tzvpp level of theory the distance and angles were evaluated from crystallographic structure from refs . and ball - and - stick representation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- complex . for structural parameters see table 1 structural parameters for the thiocyanate - so2 adduct the maximum of corresponding distribution function from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf{32}\mathbf{p}+\mathbf{so}_\mathbf{2}$$\end{document}32p+so2 trajectory ( see supporting information figs . s4s6 ) the structure was optimized in the gas - phase on b3lyp - d3/def2-tzvpp level of theory the distance and angles were evaluated from crystallographic structure from refs . and to clarify how the vibrational frequencies of the so2 and the thiocyanate anion change upon thiocyanate - so2 adduct formation , power spectra for the adduct have been calculated and compared with the gas - phase vibrational frequencies for so2 and the power spectra for the remaining anions ( fig . moreover , we summarize the information on the calculated and experimental data for unbound ( thiocyanate in bulk and so2 in gas - phase ) and bound states ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- ) of the so2 and the thiocyanate anion ( table 2 ) . it is apparent that the direction of relative shifts for the bands ( blue or red shift ) are in good agreement with the experimental results , whereas the absolute position of the maximum for the absorption band is not reproduced satisfactorily due to the deficiency of the blyp functional , as has been oberserved previously . interestingly , the experimental infrared spectra for so2 dissolved in thiocyanate ils [ 10 , 12 ] indicate that the absorption bands of dissolved so2 are almost at the same position as the fundamental frequencies of so2 in the gas - phase . care has to be taken in interpretation of the total spectra since some of the adduct bands might overlap with other bands or have low intensity compared to the rest of unbound so2.table 2vibrational frequencies in cm for so2 , thiocyanate and thiocyanate - so2 adductscalc.expt.so2 ( gas ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]-(bulk ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- so2 ( gas ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]-(bulk ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { nc}{\cdot}\hbox { so}_2]^-$$\end{document}[ncso2]- \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document}(cn)201920442052 2090 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu _ { as}(\hbox { so}_2)$$\end{document}as(so2 ) 127311711362 1152 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu _ { sym}(\hbox { so}_2)$$\end{document}sym(so2 ) 108610151151 1107 ( 1065 ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document}(cs)739722732 725 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( \hbox { so}_2)$$\end{document}(so2 ) 485484528 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta $ $ \end{document}(scn)472454 the data were taken for the cyanide - so2 adduct vibrational frequencies in cm for so2 , thiocyanate and thiocyanate - so2 adducts the data were taken for the cyanide - so2 adduct fig . 7power spectra 31 anions ( red ) and thiocyanate - so2 adduct ( green ) . vertical dashed black lines mark so2 frequencies from gas - phase calculation on the blyp - d3(bj)/def2-tzvpp level of theory power spectra 31 anions ( red ) and thiocyanate - so2 adduct ( green ) . vertical dashed black lines mark so2 frequencies from gas - phase calculation on the blyp - d3(bj)/def2-tzvpp level of theory to gain insight into how so2 in low concentrations influences the structure of [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] , we compared corresponding radial distribution functions ( rdfs ) of the pure il to the system under study ( see supporting information figs . this is consistent with md simulations of systems with a higher concentration of so2 in ils [ 10 , 17 ] as well as with the results obtained previously for co2 in ethylammonium nitrate and in 1-ethyl-3-methylimidazolium acetate [ 23 , 24 ] . to reveal how so2 enters the il structure and to characterize the solvation shell of the solute molecule the latter analysis provides valuable information about the time development or the average of surface covering of a certain particle by other particles or groups of atoms . similar to co2 in 1-ethyl-3-methylimidazolium acetate [ 23 , 24 ] , so2 is surrounded only by one anion and five cations on average in the first solvation shell ( the numbers were defined based on the value of integral in the first minimum of the corresponding rdf between centers of mass of solute and ions , see fig . 3a it is apparent that a similar ratio of anions to cations is obtained from the average so2 surface covering by anions and cations ( 18 vs. 82 % ) . both results rdf as well as voronoi around so2.fig . 3 top time - development of the so2 surface covering by ions ( a , blue lines are cation atoms , and red lines are anion atoms ) and cation atoms ( b , black lines are alkyl hydrogen atoms , red lines are ring hydrogen atoms and blue lines are ring atoms ) . bottom representative so2 voronoi surfaces , color code for the surfaces is similar with those for graphs above . ( note grey color was used for the segments belonging to the anion for the bottom right voronoi surfaces ) top time - development of the so2 surface covering by ions ( a , blue lines are cation atoms , and red lines are anion atoms ) and cation atoms ( b , black lines are alkyl hydrogen atoms , red lines are ring hydrogen atoms and blue lines are ring atoms ) . bottom representative so2 voronoi surfaces , color code for the surfaces is similar with those for graphs above . ( note grey color was used for the segments belonging to the anion for the bottom right voronoi surfaces ) since a relatively large number of cations was detected in the first solvent shell of so2 , it is worth determining in detail which functional groups are important , and how this situation of so2 solvation compares to the co2 solvation by ils . to reveal the role of the specific interactions , especially the weak interactions , in the so2 solvation , we have separated the so2 surface coverage from the cations into three groups : ring hydrogen atoms ( h2 , h4 , h5 ) , heavy ring atoms ( n1 , c2 , n3 , c4 , c5 ) , and alkyl hydrogen atoms ( h6 , h7 , h8 ) . it is a reasonable first approximation that a larger coverage ( i.e. , closest neighbor ) corresponds to a more important role in the solute solvation . the ring hydrogen atoms possess a relatively strong interaction with the solute but also with the anion . the strong nature of the latter interaction was shown in several quantum chemical studies [ 46 , 47 ] . if there are contacts between the so2 and the cation ring other than via the ring hydrogen atoms , these are most likely weak solute\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document}system interactions . also the alkyl hydrogen atom contacts to the solute represents weak dispersion interaction [ 46 , 47 ] . interestingly , the alkyl hydrogen atoms ( 48 % ) and heavy ring atoms ( 15 % ) coverages , corresponding to the weak interactions , dominate over the coverage of the ring hydrogen atoms ( 19 % ) which correspond to strong interactions . moreover , these portions are comparable with those from the non - polar part of ethylammonium nitrate to co2 solvation . from these results it is apparent that weak interactions are important not only for the solvation of co2 [ 2325 ] but also for the solvation of so2 . to support this observation the increase of the cation s side chain results in the increase of the so2 solubility [ 5 , 10 , 48 ] which agrees with our findings . the rdfs , which reflect probabilities of finding two atoms at certain distances normalized by the density , of the cation s hydrogen atoms ( h2 , h4h5 , h6h8 ) with the oxygen atoms of the sulfur dioxide ( o ) and the anion tail atoms ( n and s ) , are presented in fig . 4 on a there is a noticeable similarity in the position of the first maximum for the cation - n and cation - o functions albeit with the difference that the o(so2 ) peaks are less pronounced than the n(\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]- ) peaks . remarkably , the interplay of co2 with a cation in ethylammonium nitrate or in 1-ethyl-3-methylimidazolium acetate [ 23 , 24 ] shows the opposite behavior , i.e. , there are no such peaks between the oxygen atoms of co2 and the acidic hydrogen atoms of the cation . thus , there is no contact of a co2 with the cation via the acidic hydrogen atoms . the co2 solvation rather takes the form that it competes with dispersion forces in the system such as anion cation side chain , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document} stacking , and side chain - side chain [ 46 , 47 ] interactions . these essential differences in the structure of solvated so2 and the solvated co2 might be one of the reasons for the significantly higher solubility of the former gas in ils.fig . the rdfs between hydrogen atoms and selected atoms form anion and so2 are presented on a c , whereas d contains the rdfs between s ( from so2 ) and selected atoms from cation and anion cation anion and ion the rdfs between hydrogen atoms and selected atoms form anion and so2 are presented on a c , whereas d contains the rdfs between s ( from so2 ) and selected atoms from cation and anion since not only the nature of the anion and the side chain of the cation are important for the so2 solvation but also the role of acidic hydrogen atoms , we have examined how the cation exchange in principle influences the so2 solubility . for example , the experimental findings on the cation exchange of 1-alkylpyridinium to 1-alkyl-3-methylimidazolium show a slight decrease in the so2 solubility in ils with chloride , bistriflimide , and tetrafluoroborate anions [ 10 , 11 ] . this changes significantly when ils with a thiocyanate anion are considered . when the 1-butylpyridinium cation is exchanged for the 1-ethyl-3-methylimidazolium cation , both with the thiocyanate anion , the so2 solubility increases from 2.6 to 3.0 mol per one mol il ( at 0.1 mpa and 293 k ) [ 10 , 12 ] . these contrasting experimental observations might be related to different so2 solvation mechanisms which can be explained by microscopic insight given for example by simulations , i.e. , in the 1-ethyl-3-methylimidazolium thiocyanate ionic liquid we have detected frequent conformations in which the ring hydrogen atoms of the cations are close to the oxygen atoms of the so2 and the sulfur atoms of the anions are close to the sulfur atom of the so2 . it is likely that this newly observed conformation , which we have termed linker conformation or linker effect , is responsible for the good incorporation of so2 in this particular ionic liquid by the formation of the following linked structure h2 , h4 , h5(cation)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}o(so2)s(so2)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}s(anion ) . possible linker bonds between ring hydrogen atoms of the 1-butylpyridinium cation and so2 should be weaker due to the lower acidity compared with those for the 1-ethyl-3-methylimidazolium cation . thus , the incorporation of so2 into the network of hydrogen bonds is limited in the case of ils consisting of 1-butylpyridinium cations . only one anion was found in the first solvation shell of so2 . to understand the nature of the intermolecular forces between the anion and the so2 , we have carried out static quantum chemical calculations regarding the formation of the anion the results from these calculations showed comparable interaction energies for possible adducts ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-68.6\ , \hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-68.6kjmol-1 for the thiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-65.7\,\hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-65.7kjmol-1 for the isothiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}{\cdot}\hbox { so}_2]^-$$\end{document}[scnso2]- ) . thus , it is likely that the formation of both complexes might occur during the simulation with almost equal probability . nevertheless , the rdfs for ss and ns distances indicate the formation of the thiocyanate adduct is dominant in the system under investigation as shown in fig . 4d . the time development of the distances between the s atom and the s or n atoms of all anions , as shown in fig . 5a and b , indicates that so2 interacts with one [ scn]\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^-$$\end{document}- over the majority of the simulation time , see in fig . 5a , black line . however , a temporary anion exchange ( red , blue and green lines in fig . the substitution of the s atom to the n atom coordination or the change from thiocyanate - so2 to isothiocyanate - so2 aducct occurs at around 12 , 14 , and 36 ps ( see fig . this transition in the anion coordination at so2 is in good agreement with the experimental results of the thiocyanate anion complex formation with so2 in dilute solutions of acetonitrile . thus , the absorption of so2 in the ionic liquid under consideration does not take place with a pure thiocyanate - so2 complex generation , rather an equilibrium mixture of thiocyanate and isothiocyanate adducts will form.fig . time development of distances between s atom of so2 and s ( a ) or n ( b ) atoms of all 32 anions . curves corresponding to anions that approach the so2 molecule closer than 350 pm during the simulation are colored black , red , blue and green . time development of distances between s atom of so2 and s ( a ) or n ( b ) atoms of all 32 anions . curves corresponding to anions that approach the so2 molecule closer than 350 pm during the simulation are colored black , red , blue and green . curves corresponding to other anions are colored grey to understand the structure of the dominant thiocyanate - so2 adduct in more detail , we have compared the geometrical parameters from aimd simulation in bulk and from the static quantum chemical calculation of the isolated adducts with data from crystal structures of the potassium 1,4,7,10,13,16-hexaoxacyclooctadecane thiocyanate - so2 adduct [ k(18-crown-6)][\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { ncs}{\cdot}\hbox { so}_2$$\end{document}ncsso2 ] and the tetramethylammonium thiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { nme}_4][\hbox { ncs}{\cdot}\hbox { so}_2]$$\end{document}[nme4][ncsso2 ] ( see fig . 6 and table 1 ) . both calculated structures agree well with the crystallographic data for the thiocyanate - so2 adducts , see table 1 . the differences in distances and angles are explained by the different surroundings for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- , particularly , the c-s-s-com dihedral angle could be smaller in absolute values due to the stabilization of the selected structure . considering the most probable distance between s and s atoms ( 283 pm ) , we also found good agreement with experimental values of s\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots$$\end{document}o distances in complexes of so2 with diethyl ester ( 287 pm ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { h}_2\hbox { o}$$\end{document}h2o ( 282 pm ) .fig . 6 ball - and - stick representation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- complex . for structural parameters see table 1 table 1structural parameters for the thiocyanate - so2 adductbulk gas [ k(18-crown-6)]\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot } \hbox { so}_2]^{\rm c}$$\end{document}[ncsso2]c \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { nme}_4][\hbox { ncs}{\cdot}\hbox { so}_2]^{\rm c}$$\end{document}[nme4][ncsso2]c ss ( pm)283271274301oso ( )116115119115css ( )96979990sscom ( )109112103107csscom ( ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pm $ $ \end{document}1421399174 the maximum of corresponding distribution function from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf{32}\mathbf{p}+\mathbf{so}_\mathbf{2}$$\end{document}32p+so2 trajectory ( see supporting information figs . s4s6 ) the structure was optimized in the gas - phase on b3lyp - d3/def2-tzvpp level of theory the distance and angles were evaluated from crystallographic structure from refs . and ball - and - stick representation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- complex . for structural parameters see table 1 structural parameters for the thiocyanate - so2 adduct the maximum of corresponding distribution function from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf{32}\mathbf{p}+\mathbf{so}_\mathbf{2}$$\end{document}32p+so2 trajectory ( see supporting information figs . s4s6 ) the structure was optimized in the gas - phase on b3lyp - d3/def2-tzvpp level of theory the distance and angles were evaluated from crystallographic structure from refs . and to clarify how the vibrational frequencies of the so2 and the thiocyanate anion change upon thiocyanate - so2 adduct formation , power spectra for the adduct have been calculated and compared with the gas - phase vibrational frequencies for so2 and the power spectra for the remaining anions ( fig moreover , we summarize the information on the calculated and experimental data for unbound ( thiocyanate in bulk and so2 in gas - phase ) and bound states ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- ) of the so2 and the thiocyanate anion ( table 2 ) . it is apparent that the direction of relative shifts for the bands ( blue or red shift ) are in good agreement with the experimental results , whereas the absolute position of the maximum for the absorption band is not reproduced satisfactorily due to the deficiency of the blyp functional , as has been oberserved previously . interestingly , the experimental infrared spectra for so2 dissolved in thiocyanate ils [ 10 , 12 ] indicate that the absorption bands of dissolved so2 are almost at the same position as the fundamental frequencies of so2 in the gas - phase . care has to be taken in interpretation of the total spectra since some of the adduct bands might overlap with other bands or have low intensity compared to the rest of unbound so2.table 2vibrational frequencies in cm for so2 , thiocyanate and thiocyanate - so2 adductscalc.expt.so2 ( gas ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]-(bulk ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- so2 ( gas ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]-(bulk ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { nc}{\cdot}\hbox { so}_2]^-$$\end{document}[ncso2]- \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document}(cn)201920442052 2090 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu _ { as}(\hbox { so}_2)$$\end{document}as(so2 ) 127311711362 1152 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu _ { sym}(\hbox { so}_2)$$\end{document}sym(so2 ) 108610151151 1107 ( 1065 ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document}(cs)739722732 725 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( \hbox { so}_2)$$\end{document}(so2 ) 485484528 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta $ $ \end{document}(scn)472454 the data were taken for the cyanide - so2 adduct vibrational frequencies in cm for so2 , thiocyanate and thiocyanate - so2 adducts 7power spectra 31 anions ( red ) and thiocyanate - so2 adduct ( green ) . vertical dashed black lines mark so2 frequencies from gas - phase calculation on the blyp - d3(bj)/def2-tzvpp level of theory power spectra 31 anions ( red ) and thiocyanate - so2 adduct ( green ) . vertical dashed black lines mark so2 frequencies from gas - phase calculation on the blyp - d3(bj)/def2-tzvpp level of theory obtaining a picture , with microscopic resolution , of the solvation of small gas molecules ( like so2 ) is crucial to understanding the varying solubilities in different ionic liquids . in this article we have provided a detailed investigation of so2 solvation in the [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] ionic liquid from aimd . contacts between the so2 and groups that donate weak interactions , like the alkyl hydrogen atoms as well as the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document}-system of the cation , are numerous in the first solvent shell , whereas only one single thiocyanate anion is found in the first solvent shell forming an anion - so2 complex . the dynamics of the anion exchange at the so2 was investigated and the formation of different anion - so2 adducts were detected . the geometry of the most probable thiocyanate - so2 adduct of our bulk simulations resembles those from the static gas - phase calculations and from the available crystal structure , namely we find a pronounced sulfur sulfur bridge between the anion and the so2 which in a few instances is replaced by a n(anion)s(so2 ) isothiocynate - adduct . the qualitative and quantitative agreements between calculated and experimental frequencies , as well as potentially important bands for identification of the absorption of so2 , were detected . more interestingly , and in clear contrast to co2 , we observed that so2 is capable of forming a hydrogen bond with the acidic ring protons of the cation . thus instead of showing only the usual solvation pattern , we observed that the so2 molecule is incorporated into the ionic liquid network , a contact which we called a linker effect , i.e. , so2 can interact strongly with both the cation and the anion at the same time . undoubtly , this linker effect plays a crucial role in the high solubility of so2 in the [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] il and does not occur in the solvation of co2 . it was previously found that while co2 interacts with the anions of ils , it does not form hydrogen bonds by accepting the acidic protons of either the imidazolium or the ammonium cation . therefore , co2 it is not incorporated in the hydrogen bonding network of the ionic liquid . on the contrary , for the solvation of so2 in the [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] ionic liquid , a network of h(cation)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots \hbox { o}(\hbox { so}_2 ) { -}{\hbox { s}}(\hbox { so}_2)\cdots \hbox { s}$$\end{document}o(so2)-s(so2)s(anion ) , with so2 being a linker molecule , can be fabricated . this means , that given the right combination of cation and anion where the so2 is able to form hydrogen bonds with the cation and a sulfur sulfur bridge ( or a similar bond leading to a strong adduct ) with the anion , a good solubility of the so2 in the il should be observed . thus , the design of potential ionic liquids containing good hydrogen bond donor ability in the cation and sulfur atoms free to from sulfur sulfur bridges or similar strong adducts in the anion should not only lead to a specific absorption of this particular gas molecule , but also to the application of particular purposes of this gas - il mixture where a more extended hydrogen bond network is needed .

we have carried out an ab initio molecular dynamics study on the sulfur dioxide ( so2 ) solvation in 1-ethyl-3-methylimidazolium thiocyanate for which we have observed that both cations and anions play an essential role in the solvation of so2 . whereas , the anions tend to form a thiocyanate- and much less often an isothiocyanate - so2 adduct , the cations create a cage around so2 with those groups of atoms that donate weak interactions like the alkyl hydrogen atoms as well as the heavy atoms of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document}-system . despite these similarities between the solvation of so2 and co2 in ionic liquids , an essential difference was observed with respect to the acidic protons . whereas co2 avoids accepting hydrogen bonds form the acidic hydrogen atoms of the cations , so2 can from o(so2)h(cation ) hydrogen bonds and thus together with the strong anion - adduct it actively integrates in the hydrogen bond network of this particular ionic liquid . the fact that so2 acts in this way was termed a linker effect by us , because the so2 can be situated between cation and anion operating as a linker between them . the particular contacts are the h(cation)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}o(so2 ) hydrogen bond and a s(anion)s(so2 ) sulfur bridge . clearly , this observation provides a possible explanation for the question of why the so2 solubility in these ionic liquids is so high.electronic supplementary materialthe online version of this article ( doi:10.1007/s10953 - 015 - 0321 - 5 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Computational Details Results and Discussion CationAnion and CationSO AnionSO Conclusion Electronic supplementary material

the online version of this article ( doi:10.1007/s10953 - 015 - 0321 - 5 ) contains supplementary material , which is available to authorized users . it is likely that this newly observed conformation , which we have termed linker conformation or linker effect , is responsible for the good incorporation of so2 in this particular ionic liquid by the formation of the following linked structure h2 , h4 , h5(cation)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}o(so2)s(so2)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}s(anion ) . to understand the nature of the intermolecular forces between the anion and the so2 , we have carried out static quantum chemical calculations regarding the formation of the anion the results from these calculations showed comparable interaction energies for possible adducts ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-68.6\ , \hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-68.6kjmol-1 for the thiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-65.7\,\hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-65.7kjmol-1 for the isothiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}{\cdot}\hbox { so}_2]^-$$\end{document}[scnso2]- ) . care has to be taken in interpretation of the total spectra since some of the adduct bands might overlap with other bands or have low intensity compared to the rest of unbound so2.table 2vibrational frequencies in cm for so2 , thiocyanate and thiocyanate - so2 adductscalc.expt.so2 ( gas ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]-(bulk ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- so2 ( gas ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}]^-$$\end{document}[scn]-(bulk ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { nc}{\cdot}\hbox { so}_2]^-$$\end{document}[ncso2]- \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document}(cn)201920442052 2090 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu _ { as}(\hbox { so}_2)$$\end{document}as(so2 ) 127311711362 1152 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu _ { sym}(\hbox { so}_2)$$\end{document}sym(so2 ) 108610151151 1107 ( 1065 ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document}(cs)739722732 725 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( \hbox { so}_2)$$\end{document}(so2 ) 485484528 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta $ $ \end{document}(scn)472454 the data were taken for the cyanide - so2 adduct vibrational frequencies in cm for so2 , thiocyanate and thiocyanate - so2 adducts the data were taken for the cyanide - so2 adduct fig . it is likely that this newly observed conformation , which we have termed linker conformation or linker effect , is responsible for the good incorporation of so2 in this particular ionic liquid by the formation of the following linked structure h2 , h4 , h5(cation)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}o(so2)s(so2)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots $ $ \end{document}s(anion ) . to understand the nature of the intermolecular forces between the anion and the so2 , we have carried out static quantum chemical calculations regarding the formation of the anion the results from these calculations showed comparable interaction energies for possible adducts ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-68.6\ , \hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-68.6kjmol-1 for the thiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-65.7\,\hbox { kj}{\cdot}\hbox { mol}^{-1}$$\end{document}-65.7kjmol-1 for the isothiocyanate - so2 adduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { scn}{\cdot}\hbox { so}_2]^-$$\end{document}[scnso2]- ) . and to clarify how the vibrational frequencies of the so2 and the thiocyanate anion change upon thiocyanate - so2 adduct formation , power spectra for the adduct have been calculated and compared with the gas - phase vibrational frequencies for so2 and the power spectra for the remaining anions ( fig moreover , we summarize the information on the calculated and experimental data for unbound ( thiocyanate in bulk and so2 in gas - phase ) and bound states ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hbox { ncs}{\cdot}\hbox { so}_2]^-$$\end{document}[ncsso2]- ) of the so2 and the thiocyanate anion ( table 2 ) . in this article we have provided a detailed investigation of so2 solvation in the [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] ionic liquid from aimd . contacts between the so2 and groups that donate weak interactions , like the alkyl hydrogen atoms as well as the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document}-system of the cation , are numerous in the first solvent shell , whereas only one single thiocyanate anion is found in the first solvent shell forming an anion - so2 complex . undoubtly , this linker effect plays a crucial role in the high solubility of so2 in the [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] il and does not occur in the solvation of co2 . on the contrary , for the solvation of so2 in the [ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hbox { c}_2\hbox { c}_1\hbox { im}$$\end{document}c2c1im][scn ] ionic liquid , a network of h(cation)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cdots \hbox { o}(\hbox { so}_2 ) { -}{\hbox { s}}(\hbox { so}_2)\cdots \hbox { s}$$\end{document}o(so2)-s(so2)s(anion ) , with so2 being a linker molecule , can be fabricated .

we investigated the relationship between glycemic control and cardiovascular events using a nested case - control design . data were derived from the kaiser permanente southern california ( kpsc ) health plan , which contains information on patient demographics , diagnoses , prescriptions , laboratory results , and medical and hospital encounters . the kpsc membership includes 3.3 million individuals , representing 15% of the underlying population in southern california . membership is largely employer based ( 5% of the kpsc population is medicaid eligible and 11% is medicare eligible ) . the racial composition is as follows : 42.9% non - hispanic white ; 23.2% hispanic white ; 14.4% black ; 9.6% asian / pacific islander ; 0.2% american indian / alaskan ; 9.4% other ; and 0.3% two or more races . adult patients ( aged 18 years ) with type 2 diabetes were identified based on two recorded type 2 diabetes diagnoses between january 2002 and december 2007 and either an a1c > 7.5% or a prescription for oral hypoglycemic medication or insulin . patients with a diagnosis of polycystic ovarian syndrome , gestational diabetes , or serious illnesses including hiv / aids , cancer , sickle cell disease , cystic fibrosis , organ transplant , liver failure , or respiratory failure were excluded from the study . case subjects were defined using a primary composite end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes ( mi , stroke , heart failure , or arrhythmia ) between january 2005 and december 2007 . control subjects without the primary end point during the time window were eligible for matching . control subjects were assigned a pseudo event date equal to the index date of their matched case subject . we excluded patients without 3 years of continuous kpsc membership plus drug benefits prior to their index date , patients whose first type 2 diabetes diagnosis occurred after their index date , and patients with no recorded a1c in the observation window . case and control subjects were assigned to a1c categories based on their average a1c measured over the 3 years prior to their index date . sensitivity analyses were performed using their median a1c and most recent a1c prior to the index date . the study 's a1c categories are consistent with the accord study ( 6 , > 68 [ comparison group ] , and > 8% ) . a power analysis indicated that 672 case subjects , matched in a one - to - four ratio to control subjects , would be necessary to have 90% power to detect an odds ratio of 1.15 between a1c categories , adopting the two - sided 0.05 significance level ( 12 ) . we compared baseline characteristics between case and control subjects using the two - sided t test for continuous variables and the statistic for categorical variables . a conditional logistic regression model was fitted to estimate the odds ratio of the primary end point in patients with a1c 6 and > 8% , relative to patients with a1c between > 6 and 8% , adjusting for potential confounders . we used a stratified model for statistically significant interaction terms . in a post hoc analysis , we fitted a separate model for patients on antipsychotic medications . we adjusted for a number of laboratory , diagnostic , and prescription covariates in the multivariate analysis . a dichotomous variable was created to indicate if the patient 's a1c was measured at least six times during the 3-year preindex period . variability in preindex a1c values was defined as the difference between the minimum and maximum a1c , with an absolute difference of 1% considered as clinically meaningful ( 5,10 ) . cholesterol levels ( ldl and hdl ) measured in the year prior to index date also were included as covariates in the analyses . concurrent diabetes medications were categorized into six commonly prescribed regimens within kpsc : insulin monotherapy ; metformin monotherapy ; sulfonylurea monotherapy ; insulin plus oral medications ; other oral medications or combinations ; and no diabetes medication . medication adherence with diabetic drugs was measured using the proportion of days covered over the year prior to index ( 13 ) . dichotomous variables also were defined to reflect the use of statins , ace inhibitors , angiotensin receptor blockers ( arbs ) , other antihypertensives , antiplatelets , first- and second - generation antipsychotics , antiarrhythmics , tricyclic antidepressants , erythropoietin - stimulating agents ( esas ) , and -agonists . dichotomous variables were created to reflect cardiovascular events during the 3-year preindex period , including hospitalizations for mi , stroke , heart failure , or arrhythmia . diagnoses of retinopathy , nephropathy , chronic kidney disease , neuropathy , and prior amputations suggesting microvascular disease were included as covariates . finally , severe episodes of hypoglycemia requiring emergency - department services or hospitalizations were captured using dichotomous variables . data were derived from the kaiser permanente southern california ( kpsc ) health plan , which contains information on patient demographics , diagnoses , prescriptions , laboratory results , and medical and hospital encounters . the kpsc membership includes 3.3 million individuals , representing 15% of the underlying population in southern california . membership is largely employer based ( 5% of the kpsc population is medicaid eligible and 11% is medicare eligible ) . the racial composition is as follows : 42.9% non - hispanic white ; 23.2% hispanic white ; 14.4% black ; 9.6% asian / pacific islander ; 0.2% american indian / alaskan ; 9.4% other ; and 0.3% two or more races . adult patients ( aged 18 years ) with type 2 diabetes were identified based on two recorded type 2 diabetes diagnoses between january 2002 and december 2007 and either an a1c > 7.5% or a prescription for oral hypoglycemic medication or insulin . patients with a diagnosis of polycystic ovarian syndrome , gestational diabetes , or serious illnesses including hiv / aids , cancer , sickle cell disease , cystic fibrosis , organ transplant , liver failure , or respiratory failure were excluded from the study . case subjects were defined using a primary composite end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes ( mi , stroke , heart failure , or arrhythmia ) between january 2005 and december 2007 . control subjects without the primary end point during the time window were eligible for matching . control subjects were assigned a pseudo event date equal to the index date of their matched case subject . we excluded patients without 3 years of continuous kpsc membership plus drug benefits prior to their index date , patients whose first type 2 diabetes diagnosis occurred after their index date , and patients with no recorded a1c in the observation window . case and control subjects were assigned to a1c categories based on their average a1c measured over the 3 years prior to their index date . sensitivity analyses were performed using their median a1c and most recent a1c prior to the index date . the study 's a1c categories are consistent with the accord study ( 6 , > 68 [ comparison group ] , and > 8% ) . a power analysis indicated that 672 case subjects , matched in a one - to - four ratio to control subjects , would be necessary to have 90% power to detect an odds ratio of 1.15 between a1c categories , adopting the two - sided 0.05 significance level ( 12 ) . we compared baseline characteristics between case and control subjects using the two - sided t test for continuous variables and the statistic for categorical variables . a conditional logistic regression model was fitted to estimate the odds ratio of the primary end point in patients with a1c 6 and > 8% , relative to patients with a1c between > 6 and 8% , adjusting for potential confounders . we used a stratified model for statistically significant interaction terms . in a post hoc analysis , we fitted a separate model for patients on antipsychotic medications . we adjusted for a number of laboratory , diagnostic , and prescription covariates in the multivariate analysis . a dichotomous variable was created to indicate if the patient 's a1c was measured at least six times during the 3-year preindex period . variability in preindex a1c values was defined as the difference between the minimum and maximum a1c , with an absolute difference of 1% considered as clinically meaningful ( 5,10 ) . cholesterol levels ( ldl and hdl ) measured in the year prior to index date also were included as covariates in the analyses . concurrent diabetes medications were categorized into six commonly prescribed regimens within kpsc : insulin monotherapy ; metformin monotherapy ; sulfonylurea monotherapy ; insulin plus oral medications ; other oral medications or combinations ; and no diabetes medication . medication adherence with diabetic drugs was measured using the proportion of days covered over the year prior to index ( 13 ) . dichotomous variables also were defined to reflect the use of statins , ace inhibitors , angiotensin receptor blockers ( arbs ) , other antihypertensives , antiplatelets , first- and second - generation antipsychotics , antiarrhythmics , tricyclic antidepressants , erythropoietin - stimulating agents ( esas ) , and -agonists . dichotomous variables were created to reflect cardiovascular events during the 3-year preindex period , including hospitalizations for mi , stroke , heart failure , or arrhythmia . diagnoses of retinopathy , nephropathy , chronic kidney disease , neuropathy , and prior amputations suggesting microvascular disease were included as covariates . finally , severe episodes of hypoglycemia requiring emergency - department services or hospitalizations were captured using dichotomous variables . a pool of 254,118 type 2 diabetic patients was identified , from which a total of 16,589 case subjects met the end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes . after matching and applying the exclusion criteria , a total of 44,628 control subjects the mean age was 65.5 10.5 years , and 57% of the subjects were male . case subjects were twice as likely as control subjects to use insulin and were less likely to use statins , ace inhibitors , or arbs . approximately 90% of the population , of both case and control subjects , had a cardiovascular diagnosis . compared with control subjects , case subjects were four times more likely to have had a cardiovascular event in 3-year preindex period and approximately four times more likely to have had a severe episode of hypoglycemia . finally , the high percentage of -agonist use reflects the high prevalence of asthma and chronic obstructive pulmonary disease within the population of both case and control subjects . prior cardiovascular event = hospitalization for mi , stroke , heart failure , or arrhythmia in the 3-year preindex period . prior severe hypoglycemia = emergency - room visit or hospitalization with a primary diagnosis of hypoglycemia . in the unadjusted logistic model , patients with a 3-year average a1c of 6% were 18% more likely to experience a cardiovascular event than patients with an average a1c of > 68% ( odds ratio 1.18 [ 95% ci 1.111.25 ] ; p < 0.0001 ) . patients with an average a1c of > 8% were 31% more likely to have an event ( 1.31 [ 1.241.38 ] ; p < 0.0001 ) than the comparison group . patients with an average a1c of 6% were 20% more likely to experience a cardiovascular event , and patients with an average a1c of > 8% experienced a 16% increase in likelihood of a cardiovascular event compared with patients with an average a1c between > 6 and 8% , after adjusting for potential confounders . conditional logistic regression model of cardiovascular events * odds ratio estimated using multivariate analysis controlling for all covariates listed in table . prior cardiovascular event = hospitalization for mi , stroke , heart failure , arrhythmia in the 3-year preindex period . prior severe hypoglycemia = emergency - room visit or hospitalization with primary diagnosis of hypoglycemia . compared with the group with no diabetes medication use , patients using insulin ( alone or in combination ) experienced a 2.5-fold increase in the risk of a cardiovascular event , whereas patients treated with sulfonylurea monotherapy and other combinations of oral medications experienced an increased risk of 55% . adherence to diabetes medications conferred a significant protective effect , with each 10% increase in proportion of days covered being associated with a 44% decrease in the risk of a cardiovascular event . statins , ace inhibitors , and arbs were associated with a decrease in odds , whereas antipsychotics , esas and tricyclic antidepressants were associated with an increased risk of cardiovascular events . complications such as prior amputations , severe hypoglycemia , and history of previous cardiovascular events were significantly associated with cardiovascular events , as were indicators of microvascular disease ( including nephropathy , neuropathy , and retinopathy ) . high ldl ( > 100 mg / dl ) also was significantly associated with cardiovascular events , whereas high hdl ( > 40 mg / dl ) was protective . the stratified analysis evaluating the impact of a1c categories on cardiovascular outcomes within selected populations are presented in table 3 . for all subgroups , an average a1c of > 8% was associated with an increased risk of cardiovascular events ; however , for those with high ldl or those taking cardiovascular medications other than ace inhibitors and arbs , an a1c 6% was not significantly associated with event risk . conditional logistic regression model of cardiovascular events , stratified by subgroup ldl units are in mg / dl . other cardiovascular drugs include antiplatelets and antihypertensives other than ace inhibitors and arbs . * odds ratio for a1c category compared with a1c 68% ( reference ) . model controlled for number of a1c tests in the 3-year index period , a1c range , diabetes medications , proportion of days covered of diabetes medications , use of statins , ace inhibitors , arbs , antiarrhythmics , tricyclic antidepressants , esas , -agonists , diagnosis of hypertension , heart failure or peripheral vascular disease , retinopathy , nephropathy , neuropathy , prior cardiovascular events in 3-year preindex period , and severe hypoglycemia . model controlled for prior cardiovascular events , hdl level , use of -agonists , and esas . the final two groups in table 3 reflect that 2,539 patients taking antipsychotics had a different risk profile from those not taking antipsychotics . for patients taking antipsychotics , an a1c > 8% was associated with a threefold - increased odds of cardiovascular events , whereas a1c 6% was not significantly associated with cardiovascular events . for those not taking antipsychotics , the risk profile resembled that of the overall study , with both a1c categories showing statistically significant increases in cardiovascular risk relative to the > 68% group . defining glycemic categories based on the median a1c yielded similar results in which the risk of cardiovascular events in patients with median a1c 6% was 21% higher than for patients with a a1c between > 6 and 8% . patients with median a1c > 8% were at 10% increased risk of cardiovascular events . using the most recent a1c prior to index , patients with a1c 6% were 41% more likely to have an event than those near target , whereas those with a1c > 8% were at a 5% increased odds of a cardiovascular event . a pool of 254,118 type 2 diabetic patients was identified , from which a total of 16,589 case subjects met the end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes . after matching and applying the exclusion criteria , a total of 44,628 control subjects the mean age was 65.5 10.5 years , and 57% of the subjects were male . case subjects were twice as likely as control subjects to use insulin and were less likely to use statins , ace inhibitors , or arbs . approximately 90% of the population , of both case and control subjects , had a cardiovascular diagnosis . compared with control subjects , case subjects were four times more likely to have had a cardiovascular event in 3-year preindex period and approximately four times more likely to have had a severe episode of hypoglycemia . finally , the high percentage of -agonist use reflects the high prevalence of asthma and chronic obstructive pulmonary disease within the population of both case and control subjects . prior cardiovascular event = hospitalization for mi , stroke , heart failure , or arrhythmia in the 3-year preindex period . prior severe hypoglycemia = emergency - room visit or hospitalization with a primary diagnosis of hypoglycemia . in the unadjusted logistic model , patients with a 3-year average a1c of 6% were 18% more likely to experience a cardiovascular event than patients with an average a1c of > 68% ( odds ratio 1.18 [ 95% ci 1.111.25 ] ; p < 0.0001 ) . patients with an average a1c of > 8% were 31% more likely to have an event ( 1.31 [ 1.241.38 ] ; p < 0.0001 ) than the comparison group . patients with an average a1c of 6% were 20% more likely to experience a cardiovascular event , and patients with an average a1c of > 8% experienced a 16% increase in likelihood of a cardiovascular event compared with patients with an average a1c between > 6 and 8% , after adjusting for potential confounders . conditional logistic regression model of cardiovascular events * odds ratio estimated using multivariate analysis controlling for all covariates listed in table . prior cardiovascular event = hospitalization for mi , stroke , heart failure , arrhythmia in the 3-year preindex period . prior severe hypoglycemia = emergency - room visit or hospitalization with primary diagnosis of hypoglycemia . compared with the group with no diabetes medication use , patients using insulin ( alone or in combination ) experienced a 2.5-fold increase in the risk of a cardiovascular event , whereas patients treated with sulfonylurea monotherapy and other combinations of oral medications experienced an increased risk of 55% . adherence to diabetes medications conferred a significant protective effect , with each 10% increase in proportion of days covered being associated with a 44% decrease in the risk of a cardiovascular event . statins , ace inhibitors , and arbs were associated with a decrease in odds , whereas antipsychotics , esas and tricyclic antidepressants were associated with an increased risk of cardiovascular events . complications such as prior amputations , severe hypoglycemia , and history of previous cardiovascular events were significantly associated with cardiovascular events , as were indicators of microvascular disease ( including nephropathy , neuropathy , and retinopathy ) . high ldl ( > 100 mg / dl ) also was significantly associated with cardiovascular events , whereas high hdl ( > 40 mg / dl ) was protective . the stratified analysis evaluating the impact of a1c categories on cardiovascular outcomes within selected populations are presented in table 3 . for all subgroups , an average a1c of > 8% was associated with an increased risk of cardiovascular events ; however , for those with high ldl or those taking cardiovascular medications other than ace inhibitors and arbs , an a1c 6% was not significantly associated with event risk . conditional logistic regression model of cardiovascular events , stratified by subgroup ldl units are in mg / dl . other cardiovascular drugs include antiplatelets and antihypertensives other than ace inhibitors and arbs . * odds ratio for a1c category compared with a1c 68% ( reference ) . model controlled for number of a1c tests in the 3-year index period , a1c range , diabetes medications , proportion of days covered of diabetes medications , use of statins , ace inhibitors , arbs , antiarrhythmics , tricyclic antidepressants , esas , -agonists , diagnosis of hypertension , heart failure or peripheral vascular disease , retinopathy , nephropathy , neuropathy , prior cardiovascular events in 3-year preindex period , and severe hypoglycemia . model controlled for prior cardiovascular events , hdl level , use of -agonists , and esas . the final two groups in table 3 reflect that 2,539 patients taking antipsychotics had a different risk profile from those not taking antipsychotics . for patients taking antipsychotics , an a1c > 8% was associated with a threefold - increased odds of cardiovascular events , whereas a1c 6% was not significantly associated with cardiovascular events . for those not taking antipsychotics , the risk profile resembled that of the overall study , with both a1c categories showing statistically significant increases in cardiovascular risk relative to the > 68% group . our model was robust to changes in the definition of glycemic control . defining glycemic categories based on the median a1c yielded similar results in which the risk of cardiovascular events in patients with median a1c 6% was 21% higher than for patients with a a1c between > 6 and 8% . patients with median a1c > 8% were at 10% increased risk of cardiovascular events . using the most recent a1c prior to index , patients with a1c 6% were 41% more likely to have an event than those near target , whereas those with a1c > 8% were at a 5% increased odds of a cardiovascular event . in this study , patients with type 2 diabetes who achieved mean a1c levels of 6% or who failed to decrease their a1c to < 8% over a 3-year period were at increased risk for cardiovascular events compared with patients with mean a1c levels between > 6 and 8% . although treatment effects varied across subgroups with different risk profiles , these subgroup analyses are consistent with the core results . these results are consistent with the accord trial , which found a significant increase in all - cause death and cardiovascular death in the intensively treated arm ( 11 ) . our study also lends support to the results of a recent retrospective cohort study by currie et al . ( 14 ) , which found a u - shaped association between survival and a1c . in this study , patients with the lowest a1c levels ( median 6.4% ) were at a 52% increased risk of all - cause mortality relative to patients with a median a1c of 7.5% . in addition , patients in the highest a1c group ( median a1c 10.5% ) were at 79% increased relative risk . however , our results differ from that of the uk prospective diabetes study ( ukpds ) 10-year follow - up , which demonstrated that intensive treatment was associated with a 15% relative risk reduction in mi ( relative risk 0.85 [ 95% ci 0.740.97 ] ; p = 0.01 ) ( 5 ) . the characteristics of our patient population are comparable with the accord trial and the study by currie et al . ( 14 ) in terms of age and cardiovascular risk profile and less comparable with the ukpds . our study sample was composed of mostly elderly subjects ( mean age 65.5 years ) , whereas the ukpds recruited younger , newly diagnosed patients ( mean age 53 years ) , with < 8% of the population having a history of cardiovascular disease . by contrast , 90% of our study population had comorbid hypertension and 60% had microvascular disease . the accord trial also included patients with previous cardiovascular events or multiple cardiovascular risk factors . the study by currie et al . included patients with a history of medication escalation , and 63% of the population had a smoking history . these results suggest that in elderly patients with a high cardiovascular risk profile , intensive glycemic control should be initiated with caution . the medication regimens used in the different studies also warrant discussion . in our study , metformin monotherapy was not associated with excess cardiovascular risk , whereas other combinations of oral drugs , including sulfonylureas , exhibited an increased risk of events . similarly , in the ukpds , the relative risk reductions for death and mi were greater for patients receiving metformin - based regimens than for those on sulfonylurea - based regimens . in our study , insulin use alone or in combination with oral medications was associated with a > 2.5-fold increased risk of cardiovascular events . ( 15 ) , which identified a lower survival rate in insulin users compared with oral medication users . the mechanism for the excess risk associated with insulin and sulfonylurea use is not clearly understood ; however , it is possible that hypoglycemia plays a role . it has been proposed that hypoglycemia may precipitate cardiac arrhythmias through hypokalemia and sympatho - adrenal activation ( 15,16 ) . in addition , glucose variability has been implicated as a factor in oxidative stress and vascular inflammation ( 17 ) . taking antipsychotics also appeared to confer an increased risk of cardiovascular events , primarily driven by the use of first - generation antipsychotics , which are associated with cardiac rhythm disturbances and qtc prolongation ( 18 ) . to a lesser extent , second - generation antipsychotics , which are associated with weight gain and metabolic disturbances , also contributed to this effect ( 19 ) . as expected , our data show that patients taking antipsychotics differed significantly in all clinical characteristics from those not taking antipsychotics ; thus , it is likely that the two subgroups have different risk profiles . the secondary analysis revealed that intensive glycemic control in patients taking antipsychotics was associated with cardiovascular events to a lesser extent than in patients not taking antipsychotics . it is possible that any cardiovascular benefits of intensive glycemic control may take longer to become apparent , as shown in ukpds . however , even short - term cardiovascular risk associated with average a1c levels < 6% is a significant finding with clinical implications . second , the use of conditional logistic regression in our analysis resulted in odds ratios that may overestimate relative risk when the absolute rate of events is high ( 20 ) . although the magnitude of risk may be overestimated , the direction of risk associated with intensive glycemic control is valid . third , the nonrandomized design may subject the results to treatment selection bias ; however , the completeness of the kpsc database allowed us to control for multiple potential confounders , including laboratory results and hospitalization data . additionally , bmi data were not available for 66% of the study population and smoking status data generally are missing . finally , duration of diabetes was not available , which was an important determinant of risk associated with intensive treatment based on post hoc analyses of vadt ( 21 ) . our findings suggest that with respect to a1c control , aggressive lowering may not be appropriate for all type 2 diabetic patients . although the potential for selection bias precludes us from drawing causal conclusions about the relationship between mean a1c and cardiovascular risk , our findings , together with those of the accord trial and the study by currie et al . whereas uncontrolled hyperglycemia is an established risk factor for microvascular and macrovascular disease , intensive a1c control may not be the best approach for all type 2 diabetic patients . a given individual , aggressive treatment strategies should carefully weigh the benefits of preventing microvascular complications with the risk of precipitating cardiovascular events . further research is needed to identify the types of patients for whom intensive glycemic control would be most appropriate , as well as selection of appropriate medication regimens . ultimately , mitigating cardiovascular risk requires a multifactorial approach , glycemic control coupled with lipid lowering and blood pressure control , as well as lifestyle interventions ( 22 ) .

objectivetype 2 diabetes is associated with increased cardiovascular risk . the role of aggressive glycemic control in preventing cardiovascular events is unclear . a nested case - control study design was used to evaluate the association between average a1c and cardiovascular outcomes.research design and methodsadults with type 2 diabetes were identified among members of kaiser permanente southern california . type 2 diabetes was identified based on icd-9 diagnosis codes and either a1c > 7.5% or prescriptions for hypoglycemic agents . case subjects were defined based on nonfatal myocardial infarction , nonfatal stroke , or death attributed to cardiovascular events during a 3-year window . four type 2 diabetes control subjects were matched to each case subject based on age , sex , and index date for the corresponding case . a conditional logistic regression model was used to estimate the odds ratio of cardiovascular events and compare three patient groups based on average a1c measured in the preindex period ( 6 , > 68 , > 8%).resultsa total of 44,628 control subjects were matched to 11,157 case subjects . patients with an average a1c 6% were 20% more likely to experience a cardiovascular event than the group with an average a1c of > 68% ( p < 0.0001 ) . patients with an average a1c > 8% experienced a 16% increase in the likelihood of a cardiovascular event ( p < 0.0001 ) . we found evidence of statistical interaction with a1c category and ldl level ( p = 0.0002 ) , use of cardiovascular medications ( p = 0.02 ) , and use of antipsychotics ( p = 0.001).conclusionshigh - risk patients with type 2 diabetes who achieved mean a1c levels of 6% or failed to decrease their a1c to < 8% are at increased risk for cardiovascular events .

RESEARCH DESIGN AND METHODS Study sample Statistical analyses Covariates RESULTS Study population Primary analysis Sensitivity analysis CONCLUSIONS

we investigated the relationship between glycemic control and cardiovascular events using a nested case - control design . data were derived from the kaiser permanente southern california ( kpsc ) health plan , which contains information on patient demographics , diagnoses , prescriptions , laboratory results , and medical and hospital encounters . adult patients ( aged 18 years ) with type 2 diabetes were identified based on two recorded type 2 diabetes diagnoses between january 2002 and december 2007 and either an a1c > 7.5% or a prescription for oral hypoglycemic medication or insulin . patients with a diagnosis of polycystic ovarian syndrome , gestational diabetes , or serious illnesses including hiv / aids , cancer , sickle cell disease , cystic fibrosis , organ transplant , liver failure , or respiratory failure were excluded from the study . case subjects were defined using a primary composite end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes ( mi , stroke , heart failure , or arrhythmia ) between january 2005 and december 2007 . control subjects were assigned a pseudo event date equal to the index date of their matched case subject . we excluded patients without 3 years of continuous kpsc membership plus drug benefits prior to their index date , patients whose first type 2 diabetes diagnosis occurred after their index date , and patients with no recorded a1c in the observation window . case and control subjects were assigned to a1c categories based on their average a1c measured over the 3 years prior to their index date . the study 's a1c categories are consistent with the accord study ( 6 , > 68 [ comparison group ] , and > 8% ) . a power analysis indicated that 672 case subjects , matched in a one - to - four ratio to control subjects , would be necessary to have 90% power to detect an odds ratio of 1.15 between a1c categories , adopting the two - sided 0.05 significance level ( 12 ) . a conditional logistic regression model was fitted to estimate the odds ratio of the primary end point in patients with a1c 6 and > 8% , relative to patients with a1c between > 6 and 8% , adjusting for potential confounders . we adjusted for a number of laboratory , diagnostic , and prescription covariates in the multivariate analysis . cholesterol levels ( ldl and hdl ) measured in the year prior to index date also were included as covariates in the analyses . dichotomous variables also were defined to reflect the use of statins , ace inhibitors , angiotensin receptor blockers ( arbs ) , other antihypertensives , antiplatelets , first- and second - generation antipsychotics , antiarrhythmics , tricyclic antidepressants , erythropoietin - stimulating agents ( esas ) , and -agonists . dichotomous variables were created to reflect cardiovascular events during the 3-year preindex period , including hospitalizations for mi , stroke , heart failure , or arrhythmia . data were derived from the kaiser permanente southern california ( kpsc ) health plan , which contains information on patient demographics , diagnoses , prescriptions , laboratory results , and medical and hospital encounters . adult patients ( aged 18 years ) with type 2 diabetes were identified based on two recorded type 2 diabetes diagnoses between january 2002 and december 2007 and either an a1c > 7.5% or a prescription for oral hypoglycemic medication or insulin . patients with a diagnosis of polycystic ovarian syndrome , gestational diabetes , or serious illnesses including hiv / aids , cancer , sickle cell disease , cystic fibrosis , organ transplant , liver failure , or respiratory failure were excluded from the study . case subjects were defined using a primary composite end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes ( mi , stroke , heart failure , or arrhythmia ) between january 2005 and december 2007 . control subjects were assigned a pseudo event date equal to the index date of their matched case subject . we excluded patients without 3 years of continuous kpsc membership plus drug benefits prior to their index date , patients whose first type 2 diabetes diagnosis occurred after their index date , and patients with no recorded a1c in the observation window . case and control subjects were assigned to a1c categories based on their average a1c measured over the 3 years prior to their index date . sensitivity analyses were performed using their median a1c and most recent a1c prior to the index date . the study 's a1c categories are consistent with the accord study ( 6 , > 68 [ comparison group ] , and > 8% ) . a power analysis indicated that 672 case subjects , matched in a one - to - four ratio to control subjects , would be necessary to have 90% power to detect an odds ratio of 1.15 between a1c categories , adopting the two - sided 0.05 significance level ( 12 ) . a conditional logistic regression model was fitted to estimate the odds ratio of the primary end point in patients with a1c 6 and > 8% , relative to patients with a1c between > 6 and 8% , adjusting for potential confounders . we adjusted for a number of laboratory , diagnostic , and prescription covariates in the multivariate analysis . cholesterol levels ( ldl and hdl ) measured in the year prior to index date also were included as covariates in the analyses . dichotomous variables also were defined to reflect the use of statins , ace inhibitors , angiotensin receptor blockers ( arbs ) , other antihypertensives , antiplatelets , first- and second - generation antipsychotics , antiarrhythmics , tricyclic antidepressants , erythropoietin - stimulating agents ( esas ) , and -agonists . dichotomous variables were created to reflect cardiovascular events during the 3-year preindex period , including hospitalizations for mi , stroke , heart failure , or arrhythmia . a pool of 254,118 type 2 diabetic patients was identified , from which a total of 16,589 case subjects met the end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes . after matching and applying the exclusion criteria , a total of 44,628 control subjects the mean age was 65.5 10.5 years , and 57% of the subjects were male . case subjects were twice as likely as control subjects to use insulin and were less likely to use statins , ace inhibitors , or arbs . approximately 90% of the population , of both case and control subjects , had a cardiovascular diagnosis . compared with control subjects , case subjects were four times more likely to have had a cardiovascular event in 3-year preindex period and approximately four times more likely to have had a severe episode of hypoglycemia . prior cardiovascular event = hospitalization for mi , stroke , heart failure , or arrhythmia in the 3-year preindex period . in the unadjusted logistic model , patients with a 3-year average a1c of 6% were 18% more likely to experience a cardiovascular event than patients with an average a1c of > 68% ( odds ratio 1.18 [ 95% ci 1.111.25 ] ; p < 0.0001 ) . patients with an average a1c of > 8% were 31% more likely to have an event ( 1.31 [ 1.241.38 ] ; p < 0.0001 ) than the comparison group . patients with an average a1c of 6% were 20% more likely to experience a cardiovascular event , and patients with an average a1c of > 8% experienced a 16% increase in likelihood of a cardiovascular event compared with patients with an average a1c between > 6 and 8% , after adjusting for potential confounders . conditional logistic regression model of cardiovascular events * odds ratio estimated using multivariate analysis controlling for all covariates listed in table . prior cardiovascular event = hospitalization for mi , stroke , heart failure , arrhythmia in the 3-year preindex period . compared with the group with no diabetes medication use , patients using insulin ( alone or in combination ) experienced a 2.5-fold increase in the risk of a cardiovascular event , whereas patients treated with sulfonylurea monotherapy and other combinations of oral medications experienced an increased risk of 55% . adherence to diabetes medications conferred a significant protective effect , with each 10% increase in proportion of days covered being associated with a 44% decrease in the risk of a cardiovascular event . statins , ace inhibitors , and arbs were associated with a decrease in odds , whereas antipsychotics , esas and tricyclic antidepressants were associated with an increased risk of cardiovascular events . complications such as prior amputations , severe hypoglycemia , and history of previous cardiovascular events were significantly associated with cardiovascular events , as were indicators of microvascular disease ( including nephropathy , neuropathy , and retinopathy ) . high ldl ( > 100 mg / dl ) also was significantly associated with cardiovascular events , whereas high hdl ( > 40 mg / dl ) was protective . for all subgroups , an average a1c of > 8% was associated with an increased risk of cardiovascular events ; however , for those with high ldl or those taking cardiovascular medications other than ace inhibitors and arbs , an a1c 6% was not significantly associated with event risk . conditional logistic regression model of cardiovascular events , stratified by subgroup ldl units are in mg / dl . * odds ratio for a1c category compared with a1c 68% ( reference ) . model controlled for number of a1c tests in the 3-year index period , a1c range , diabetes medications , proportion of days covered of diabetes medications , use of statins , ace inhibitors , arbs , antiarrhythmics , tricyclic antidepressants , esas , -agonists , diagnosis of hypertension , heart failure or peripheral vascular disease , retinopathy , nephropathy , neuropathy , prior cardiovascular events in 3-year preindex period , and severe hypoglycemia . model controlled for prior cardiovascular events , hdl level , use of -agonists , and esas . for patients taking antipsychotics , an a1c > 8% was associated with a threefold - increased odds of cardiovascular events , whereas a1c 6% was not significantly associated with cardiovascular events . defining glycemic categories based on the median a1c yielded similar results in which the risk of cardiovascular events in patients with median a1c 6% was 21% higher than for patients with a a1c between > 6 and 8% . patients with median a1c > 8% were at 10% increased risk of cardiovascular events . using the most recent a1c prior to index , patients with a1c 6% were 41% more likely to have an event than those near target , whereas those with a1c > 8% were at a 5% increased odds of a cardiovascular event . a pool of 254,118 type 2 diabetic patients was identified , from which a total of 16,589 case subjects met the end point of nonfatal mi , nonfatal stroke , or death attributed to cardiovascular causes . after matching and applying the exclusion criteria , a total of 44,628 control subjects the mean age was 65.5 10.5 years , and 57% of the subjects were male . case subjects were twice as likely as control subjects to use insulin and were less likely to use statins , ace inhibitors , or arbs . approximately 90% of the population , of both case and control subjects , had a cardiovascular diagnosis . compared with control subjects , case subjects were four times more likely to have had a cardiovascular event in 3-year preindex period and approximately four times more likely to have had a severe episode of hypoglycemia . prior cardiovascular event = hospitalization for mi , stroke , heart failure , or arrhythmia in the 3-year preindex period . in the unadjusted logistic model , patients with a 3-year average a1c of 6% were 18% more likely to experience a cardiovascular event than patients with an average a1c of > 68% ( odds ratio 1.18 [ 95% ci 1.111.25 ] ; p < 0.0001 ) . patients with an average a1c of > 8% were 31% more likely to have an event ( 1.31 [ 1.241.38 ] ; p < 0.0001 ) than the comparison group . patients with an average a1c of 6% were 20% more likely to experience a cardiovascular event , and patients with an average a1c of > 8% experienced a 16% increase in likelihood of a cardiovascular event compared with patients with an average a1c between > 6 and 8% , after adjusting for potential confounders . conditional logistic regression model of cardiovascular events * odds ratio estimated using multivariate analysis controlling for all covariates listed in table . prior cardiovascular event = hospitalization for mi , stroke , heart failure , arrhythmia in the 3-year preindex period . compared with the group with no diabetes medication use , patients using insulin ( alone or in combination ) experienced a 2.5-fold increase in the risk of a cardiovascular event , whereas patients treated with sulfonylurea monotherapy and other combinations of oral medications experienced an increased risk of 55% . adherence to diabetes medications conferred a significant protective effect , with each 10% increase in proportion of days covered being associated with a 44% decrease in the risk of a cardiovascular event . statins , ace inhibitors , and arbs were associated with a decrease in odds , whereas antipsychotics , esas and tricyclic antidepressants were associated with an increased risk of cardiovascular events . complications such as prior amputations , severe hypoglycemia , and history of previous cardiovascular events were significantly associated with cardiovascular events , as were indicators of microvascular disease ( including nephropathy , neuropathy , and retinopathy ) . high ldl ( > 100 mg / dl ) also was significantly associated with cardiovascular events , whereas high hdl ( > 40 mg / dl ) was protective . for all subgroups , an average a1c of > 8% was associated with an increased risk of cardiovascular events ; however , for those with high ldl or those taking cardiovascular medications other than ace inhibitors and arbs , an a1c 6% was not significantly associated with event risk . conditional logistic regression model of cardiovascular events , stratified by subgroup ldl units are in mg / dl . * odds ratio for a1c category compared with a1c 68% ( reference ) . model controlled for number of a1c tests in the 3-year index period , a1c range , diabetes medications , proportion of days covered of diabetes medications , use of statins , ace inhibitors , arbs , antiarrhythmics , tricyclic antidepressants , esas , -agonists , diagnosis of hypertension , heart failure or peripheral vascular disease , retinopathy , nephropathy , neuropathy , prior cardiovascular events in 3-year preindex period , and severe hypoglycemia . model controlled for prior cardiovascular events , hdl level , use of -agonists , and esas . for patients taking antipsychotics , an a1c > 8% was associated with a threefold - increased odds of cardiovascular events , whereas a1c 6% was not significantly associated with cardiovascular events . for those not taking antipsychotics , the risk profile resembled that of the overall study , with both a1c categories showing statistically significant increases in cardiovascular risk relative to the > 68% group . our model was robust to changes in the definition of glycemic control . defining glycemic categories based on the median a1c yielded similar results in which the risk of cardiovascular events in patients with median a1c 6% was 21% higher than for patients with a a1c between > 6 and 8% . patients with median a1c > 8% were at 10% increased risk of cardiovascular events . using the most recent a1c prior to index , patients with a1c 6% were 41% more likely to have an event than those near target , whereas those with a1c > 8% were at a 5% increased odds of a cardiovascular event . in this study , patients with type 2 diabetes who achieved mean a1c levels of 6% or who failed to decrease their a1c to < 8% over a 3-year period were at increased risk for cardiovascular events compared with patients with mean a1c levels between > 6 and 8% . these results are consistent with the accord trial , which found a significant increase in all - cause death and cardiovascular death in the intensively treated arm ( 11 ) . ( 14 ) , which found a u - shaped association between survival and a1c . in this study , patients with the lowest a1c levels ( median 6.4% ) were at a 52% increased risk of all - cause mortality relative to patients with a median a1c of 7.5% . however , our results differ from that of the uk prospective diabetes study ( ukpds ) 10-year follow - up , which demonstrated that intensive treatment was associated with a 15% relative risk reduction in mi ( relative risk 0.85 [ 95% ci 0.740.97 ] ; p = 0.01 ) ( 5 ) . ( 14 ) in terms of age and cardiovascular risk profile and less comparable with the ukpds . our study sample was composed of mostly elderly subjects ( mean age 65.5 years ) , whereas the ukpds recruited younger , newly diagnosed patients ( mean age 53 years ) , with < 8% of the population having a history of cardiovascular disease . the accord trial also included patients with previous cardiovascular events or multiple cardiovascular risk factors . included patients with a history of medication escalation , and 63% of the population had a smoking history . these results suggest that in elderly patients with a high cardiovascular risk profile , intensive glycemic control should be initiated with caution . in our study , metformin monotherapy was not associated with excess cardiovascular risk , whereas other combinations of oral drugs , including sulfonylureas , exhibited an increased risk of events . in our study , insulin use alone or in combination with oral medications was associated with a > 2.5-fold increased risk of cardiovascular events . the mechanism for the excess risk associated with insulin and sulfonylurea use is not clearly understood ; however , it is possible that hypoglycemia plays a role . taking antipsychotics also appeared to confer an increased risk of cardiovascular events , primarily driven by the use of first - generation antipsychotics , which are associated with cardiac rhythm disturbances and qtc prolongation ( 18 ) . the secondary analysis revealed that intensive glycemic control in patients taking antipsychotics was associated with cardiovascular events to a lesser extent than in patients not taking antipsychotics . however , even short - term cardiovascular risk associated with average a1c levels < 6% is a significant finding with clinical implications . second , the use of conditional logistic regression in our analysis resulted in odds ratios that may overestimate relative risk when the absolute rate of events is high ( 20 ) . although the magnitude of risk may be overestimated , the direction of risk associated with intensive glycemic control is valid . finally , duration of diabetes was not available , which was an important determinant of risk associated with intensive treatment based on post hoc analyses of vadt ( 21 ) . although the potential for selection bias precludes us from drawing causal conclusions about the relationship between mean a1c and cardiovascular risk , our findings , together with those of the accord trial and the study by currie et al .

high - throughput techniques have produced vast amounts of sequence , expression and structure data . one of these techniques , dna microarray analysis , produces information on relative expression levels for thousands of genes simultaneously . in addition , large collections of microarray data contain information about concerted changes in transcript levels in these datasets beyond the original purpose of each dataset . microarray data have been collected in several general repositories , including arrayexpress ( 1 ) , gene expression omnibus ( geo ) ( 2 ) and the center for information biology gene expression database ( cibex ) ( 3 ) . however , it is generally difficult for experimental researchers who lack bioinformatics expertise to retrieve the information they seek . for the model plant arabidopsis thaliana , the arabidopsis information resource ( tair ) ( 4 ) and the nottingham arabidopsis stock centre arrays ( nascarrays ) ( 5 ) are species - specific repositories for microarray data . although it is easy to search the data for individual genes or samples , it is still difficult to retrieve gene - to - gene relationships or simply browse gene expression patterns . the comprehensive systems - biology database ( csb.db ) ( 6 ) , botany array resource ( bar ) ( 7 ) , arabidopsis co - expression tool ( act ) ( 8) and genevestigator ( 9 ) provide co - expressed gene relationships calculated from the array data stored in tair and/or the nascarrays . such gene relationship information is valuable for predicting gene function , because co - expressed genes are often involved in the same or related pathways . approaches for specifying experimental targets using such co - expressed relationships have already been reported ( 1013 ) . clique finder returns a group of co - expressed genes from the co - expressed gene network , although it does not show the network itself ( 8) . visualized expression patterns are provided in bar ( 7 ) , because a gene expression pattern is difficult to comprehend from lists of expression values . interactive cis - element prediction from co - expressed genes is also provided in bar ( 7 ) . it could enforce the estimation of regulatory scheme of the target genes deduced from the limited amount of experimentally determined cis elements , which are stored in cis - element databases ( 14,15 ) . although these databases are well equipped with such user - friendly tools , additional interfaces are still required . the following three points need to be addressed : ( i ) co - expressed gene lists are too long to allow visualization of complete co - expression patterns . ( ii ) although the color - coded heat maps provided by bar ( 7 ) are suitable for comparison of a set of genes , they do not represent quantitative expression changes well . ( iii ) the interactive cis - element prediction tool provided by bar ( 7 ) can extract cis elements from the user - selected genes , but the results are hard to exhaustively compare between different queries . to support the needs of non - bioinformatics experts , we constructed a database named a.thaliana trans - factor and cis - element prediction database ( atted - ii ) for retrieving gene - to - gene relationships similar to the other databases for co - expressed genes . ( i ) network representation of co - expressed gene relationships , which , in addition to the original lists of co - expressed genes , facilitates understanding of the structural basis of gene co - expression ( 16 ) . ( ii ) stored pre - calculated results for cis - element prediction are linked to every gene and every functional category , and are presented along with several characteristics of the cis elements . ( iii ) the gene expression patterns are graphically represented the display of bar graphs for individual genes makes it easy to see quantitative expression changes for many experimental conditions . atted - ii is based on the framework of the a.thaliana tissue - specific expression database ( atted ) , which was opened in 2003 as the repository for our original data for tissue - specific gene expression ( 17 ) . with the subsequent availability of public microarray data , we imported these data into the original atted and stored the calculated co - expression information and the predicted cis elements . atted - ii now contains co - expressed gene networks for 22 263 loci and for 1102 functional categories as well as predicted cis elements represented by 304 heptamers . all expression data for these contents are genechip data ( 25k ) released by tair ( 4 ) , update of which will be incorporated into atted - ii . in the following sections , the contents of atted - ii and its usefulness are described with examples for obtaining information on co - expressed genes ( figure 1 ) and predicted cis elements ( figure 2 ) . screenshots of atted - ii pages relevant to the search for wounding - responsive gene groups . target in tables in ( c ) or ( d ) indicates predicted subcellular localization . for example , c , c indicates both targetp and wolf psort predict that the gene product is localized in chloroplasts . yellow - colored agi codes in the table in ( c ) indicate genes in the co - expressed gene network . pink - colored agi codes in the table in ( d ) indicate query genes used to search for co - expressed genes . screenshots of atted - ii pages relevant to the search for a predicted regulatory scheme of at5g62490 . results of a statistical test for the presence of a cis element in the 50 co - expressed genes . atted - ii contains four types of pages ; locus page , co - expressed genes page , cis element page and functional category page . the locus page contains information for each of 31 128 loci , which covers all arabidopsis protein - coding loci defined by tair ( 4 ) . of these loci , 22 263 have gene expression patterns measured by genechip . for each locus , co - expressed genes are calculated and the 12 most strongly correlated genes are represented in the locus page as a network and a list . these co - expressed genes are supported by the following information provided in the same locus page : organized functional gene annotation from other databases , subcellular localization predicted by targetp ( 18 ) and wolf psort ( 19 ) , and graphs of the gene expression patterns . predicted cis elements in the 200 bp region upstream from the transcription start site ( tss ) are also shown to support co - expressed gene relationships ( figure 2c ) . the upstream sequence used in atted - ii is obtained from tair ( 4 ) , where for the loci without tss annotation the sequences from translational start site are provided . details about the predicted cis elements can be obtained in the cis element page.a co - expressed genes page is prepared for each of the 22 263 loci for which there are gene expression data . this page contains a longer list of co - expressed genes ( 300 genes ) than that found in locus page and presents statistical test results of the nearest 50 genes for functional categories and for cis - element appearances ( figure 2d ) . for extended analysis , whole lists including more than 300 co - expressed genes are downloadable from atted - ii as tab - delimited text files.the cis element page provides information for each predicted cis element . in this prediction , the extracted heptamers are matched with reported cis elements stored in the database of plant cis - acting regulatory dna elements ( place ) ( 14 ) ( figure 2e ) . the cis - element profiles for position and for experimental conditions are provided to reduce false positives and to consider biological functions ( figure 2e ) . also provided are results of a statistical test for functions of the genes having a predicted cis element and a list of transcription factors predicted to bind the cis element . list of transcription factors in atted - ii were downloaded from agris ( 20).a functional category page is constructed for each gene function category using the 3481 terms defined by go ( 21 ) , kegg ( 22 ) , aracyc ( 23 ) and kappa - view ( 24 ) . the 15 most significant co - expressed genes are selected from the genes annotated by each functional term , and co - expressed gene networks are drawn ( figure 1c ) . because two - thirds of the categories have no significantly co - expressed genes , the networks are drawn for 1102 categories in total . lists of all genes in the categories are described under the network ( figure 1c ) . the method for selecting the genes used to draw the networks is described in the help page in atted - ii . the locus page contains information for each of 31 128 loci , which covers all arabidopsis protein - coding loci defined by tair ( 4 ) . of these loci , 22 263 have gene expression patterns measured by genechip . for each locus , co - expressed genes are calculated and the 12 most strongly correlated genes are represented in the locus page as a network and a list . these co - expressed genes are supported by the following information provided in the same locus page : organized functional gene annotation from other databases , subcellular localization predicted by targetp ( 18 ) and wolf psort ( 19 ) , and graphs of the gene expression patterns . predicted cis elements in the 200 bp region upstream from the transcription start site ( tss ) are also shown to support co - expressed gene relationships ( figure 2c ) . the upstream sequence used in atted - ii is obtained from tair ( 4 ) , where for the loci without tss annotation the sequences from translational start site are provided . a co - expressed genes page is prepared for each of the 22 263 loci for which there are gene expression data . this page contains a longer list of co - expressed genes ( 300 genes ) than that found in locus page and presents statistical test results of the nearest 50 genes for functional categories and for cis - element appearances ( figure 2d ) . for extended analysis , whole lists including more than 300 co - expressed genes the cis element page provides information for each predicted cis element . in this prediction , the extracted heptamers are matched with reported cis elements stored in the database of plant cis - acting regulatory dna elements ( place ) ( 14 ) ( figure 2e ) . the cis - element profiles for position and for experimental conditions are provided to reduce false positives and to consider biological functions ( figure 2e ) . also provided are results of a statistical test for functions of the genes having a predicted cis element and a list of transcription factors predicted to bind the cis element . list of transcription factors in atted - ii were downloaded from agris ( 20 ) . a functional category page is constructed for each gene function category using the 3481 terms defined by go ( 21 ) , kegg ( 22 ) , aracyc ( 23 ) and kappa - view ( 24 ) . the 15 most significant co - expressed genes are selected from the genes annotated by each functional term , and co - expressed gene networks are drawn ( figure 1c ) . because two - thirds of the categories have no significantly co - expressed genes , the networks are drawn for 1102 categories in total . lists of all genes in the categories are described under the network ( figure 1c ) . the method for selecting the genes used to draw the networks is described in the help page in atted - ii . as an example , consider the study of plant defense systems for wounding and injury . as a non - motile organism , a plant can not move to a more favorable place but must bear and adapt to its environments using complex defence mechanisms . identification of the genes in a system is the first step for deeper understandings of the system . for gene identification , it is efficient to list and classify genes of co - functional candidates using co - expressed genes ( 1013 ) . for this purpose ( note that larger figures for this example are shown in the help page in atted - ii . ) . search wounding as a keyword using the search form to the right of the title logo in atted - ii ( figure 1a).as a result , a go category click the category id ( figure 1b).the functional category page for response to wounding provides co - expressed gene networks of 15 representative genes in this category ( figure 1c ) . the agi code , which is locus i d in arabidopsis ( 25 ) , a short description and the node i d are enclosed by the circle . the thickness of the lines connecting the circles corresponds to the relative strength of co - expression . the table under the network contains longer descriptions , where the genes in the networks are numbered by node ids and highlighted by yellow - colored agi codes . to consult each network , the information of the external links in each locus page is useful in addition to the information presented in atted - ii . in this example , publications for each locus provided by tair ( 4 ) were referred for the interpretation of the results obtained . each gene network clearly corresponds to a biological function in response to wounding as follows : network 1 contains five genes , four of which are enzymes for biosynthesis of jasmonic acid ( ja ) , which is the phytohormone that has a central signaling role in the wounding response . the other gene , atmyc2 , encodes a transcription factor that regulates ja - responsive genes ( 26 ) . gene expression profiles prepared in each locus page show that all of these genes are upregulated at an early stage of wounding ( data not shown in figure 1).network 2 contains five genes that are enzymes for biosynthesis of flavonoid , one of the major secondary metabolites functioning as a phytoalexin or protectant against photodamage.network 3 contains three genes that are enzymes for volatile signaling compounds for defence response . jmt and at3g11480 are enzymes used to produce methylated forms of ja and salicylic acid , respectively ( 27,28 ) . attps03 is an enzyme for terpene biosynthesis ( 29).network 4 contains two ja - responsive genes . cori3 is an enzyme , cystine cs lyase ( 30 ) , which is regulated by ja signaling . although lox2 had been thought to act in ja biosynthesis ( 31 ) , this gene is known to respond to wounding and ja stimulus at a late stage ( 32,33 ) and thus may be involved in ja synthesis at a late stage or function for other purpose in the wounding response . in this example , four groups of wounding - responsive genes are found . to make a complete list of genes related to the wounding response , next search for co - expressed genes from each group . to select genes to query , click on the checkboxes to the left of each appropriate entry for example , the group of genes for the ja biosynthetic pathway above and the click the button search co - expressed genes just above the table.the result of the co - expressed gene search is a list of the 300 most strongly co - expressed genes from the five query genes for ja biosynthesis ( figure 1d ) . this list contains putative functional and unknown genes in addition to many known ja biosynthesis genes . in the same way , these lists of genes are supported by other biological information or cis - element information and can be used to design experiments to identify gene functions . wounding as a keyword using the search form to the right of the title logo in atted - ii ( figure 1a ) . as a result , a go category the functional category page for response to wounding provides co - expressed gene networks of 15 representative genes in this category ( figure 1c ) . the agi code , which is locus i d in arabidopsis ( 25 ) , a short description and the node i d are enclosed by the circle . the thickness of the lines connecting the circles corresponds to the relative strength of co - expression . the table under the network contains longer descriptions , where the genes in the networks are numbered by node ids and highlighted by yellow - colored agi codes . to consult each network , the information of the external links in each locus page is useful in addition to the information presented in atted - ii . in this example , publications for each locus provided by tair ( 4 ) were referred for the interpretation of the results obtained . each gene network clearly corresponds to a biological function in response to wounding as follows : network 1 contains five genes , four of which are enzymes for biosynthesis of jasmonic acid ( ja ) , which is the phytohormone that has a central signaling role in the wounding response . the other gene , atmyc2 , encodes a transcription factor that regulates ja - responsive genes ( 26 ) . gene expression profiles prepared in each locus page show that all of these genes are upregulated at an early stage of wounding ( data not shown in figure 1).network 2 contains five genes that are enzymes for biosynthesis of flavonoid , one of the major secondary metabolites functioning as a phytoalexin or protectant against photodamage.network 3 contains three genes that are enzymes for volatile signaling compounds for defence response . jmt and at3g11480 are enzymes used to produce methylated forms of ja and salicylic acid , respectively ( 27,28 ) . attps03 is an enzyme for terpene biosynthesis ( 29).network 4 contains two ja - responsive genes . cori3 is an enzyme , cystine cs lyase ( 30 ) , which is regulated by ja signaling . although lox2 had been thought to act in ja biosynthesis ( 31 ) , this gene is known to respond to wounding and ja stimulus at a late stage ( 32,33 ) and thus may be involved in ja synthesis at a late stage or function for other purpose in the wounding response . in this example , four groups of wounding - responsive genes are found . to make a complete list of genes related to the wounding response , next search for co - expressed genes from each group . to select genes to query , click on the checkboxes to the left of each appropriate entry for example , the group of genes for the ja biosynthetic pathway above and the click the button search co - expressed genes just above the table . network 1 contains five genes , four of which are enzymes for biosynthesis of jasmonic acid ( ja ) , which is the phytohormone that has a central signaling role in the wounding response . the other gene , atmyc2 , encodes a transcription factor that regulates ja - responsive genes ( 26 ) . gene expression profiles prepared in each locus page show that all of these genes are upregulated at an early stage of wounding ( data not shown in figure 1 ) . network 2 contains five genes that are enzymes for biosynthesis of flavonoid , one of the major secondary metabolites functioning as a phytoalexin or protectant against photodamage . network 3 contains three genes that are enzymes for volatile signaling compounds for defence response . jmt and at3g11480 are enzymes used to produce methylated forms of ja and salicylic acid , respectively ( 27,28 ) . cori3 is an enzyme , cystine cs lyase ( 30 ) , which is regulated by ja signaling . although lox2 had been thought to act in ja biosynthesis ( 31 ) , this gene is known to respond to wounding and ja stimulus at a late stage ( 32,33 ) and thus may be involved in ja synthesis at a late stage or function for other purpose in the wounding response . in this example , four groups of wounding - responsive genes are found . to make a complete list of genes related to the wounding response , next search for co - expressed genes from each group . to select genes to query , click on the checkboxes to the left of each appropriate entry for example , the group of genes for the ja biosynthetic pathway above and the click the button the result of the co - expressed gene search is a list of the 300 most strongly co - expressed genes from the five query genes for ja biosynthesis ( figure 1d ) . this list contains putative functional and unknown genes in addition to many known ja biosynthesis genes . in the same way these lists of genes are supported by other biological information or cis - element information and can be used to design experiments to identify gene functions . information of predicted cis elements can provide support for proposed co - expressed gene relationships . when co - expressed genes share particular cis elements in their promoters , we can expect that these genes are co - regulated through these cis elements and that they function together . as an example in obtaining information on predicted cis elements , consider the regulation of an abscisic acid ( aba)-responsive protein at5g62490 . atted - ii indicates that this gene can be up - regulated at embryogenesis through an aba - responsive element ( abre ) ( figure 2 ) . ( note that larger figures for this example are shown in the help page in atted - ii . ) search at5g62490 as an agi code using the search form to the right of the title logo ( figure 2a).click the agi code in the resulting table to go to the locus page ( figure 2b).in the locus page for at5g62490 , cis elements are predicted at 6062 bp and 101103 bp upstream from the tss ( figure 2c ) . although six predicted heptamers are provided , they are grouped into two cis elements for position , gacacgtgt and ccacgtggc , sharing cacgtg core . the co - expressed gene list indicates that this gene is co - expressed with genes for late embryogenesis . graphs of at5g62490s expression pattern are also provided , showing that this gene is upregulated by osmotic and salt stresses as well as aba treatment ( data not shown in figure 2c ) . dramatic expression during embryogenesis is also shown , suggesting that this gene may function in embryogenesis . link to top 300 co - expressed gene list.in the co - expressed genes page for at5g62490 , appearance of acacgtg in the co - expressed genes is statistically abundant , suggesting that these genes , including at5g62490 itself , are commonly regulated through this cis element ( figure 2d ) . to obtain detailed information on this cis element , return to the locus page and click the link acacgtg to go to the cis element page.in the cis element page for acacgtg ( figure 2e ) , users can see that this predicted heptamer corresponds to the reported cis elements named abre and g - box . the abre is a central cis element of aba signal transduction in response to stress treatment ( 34 ) . this element prefers the position from 50 to 100 bp upstream of the tss , indicating that the abre - like element found in at5g62490 appears in an appropriate position ( 6062 bp upstream ) . the graph for cis - element specificity for various experimental conditions shows that genes containing this cis element in their promoters are usually up - regulated responding to aba as well as osmotic or salt stress ( data not shown in figure 2 ) . this pattern is consistent with the expression pattern of at5g62490 , supporting the possibility that this gene is regulated through this cis element . a list of transcription factor candidates that bind this heptamer is also provided on this page . this list includes known transcription factors to bind aba - responsive cis elements or g - box . in fact , abf3 , a well - known transcription factor to bind abre ( 35 ) , is found in the list , although its correlation value is not high for this sequence itself . when we focus on the regulation during embryogenesis , rd26 ( at4g27410 ) is a good candidate because this gene is highly expressed during embryogenesis and up - regulated by aba as well as osmotic or salt stress , as indicated by their expression patterns on their respective locus page . furthermore , rd26 functions in aba - dependent stress signaling ( 36 ) , and its homologous transcription factors recognize a cacg core ( 37 ) . g - box binding factor 3 ( gbf3 , at2g46270 ) is another candidate , since it binds g - box sequence for aba signaling ( 38 ) . the cis element pages for the second group of heptamers found in the locus page for at5g62490 contain almost identical information except for the position preference , which is at 100 bp upstream of the tss , indicating that the second cis element also appears at its most appropriate position ( 101103 bp ) . search at5g62490 as an agi code using the search form to the right of the title logo ( figure 2a ) . click the agi code in the resulting table to go to the locus page ( figure 2b ) . in the locus page for at5g62490 , cis elements are predicted at 6062 bp and 101103 bp upstream from the tss ( figure 2c ) . although six predicted heptamers are provided , they are grouped into two cis elements for position , gacacgtgt and ccacgtggc , sharing cacgtg core . the co - expressed gene list indicates that this gene is co - expressed with genes for late embryogenesis . graphs of at5g62490s expression pattern are also provided , showing that this gene is upregulated by osmotic and salt stresses as well as aba treatment ( data not shown in figure 2c ) . dramatic expression during embryogenesis is also shown , suggesting that this gene may function in embryogenesis . link to top 300 co - expressed gene list. in the co - expressed genes page for at5g62490 , appearance of acacgtg in the co - expressed genes is statistically abundant , suggesting that these genes , including at5g62490 itself , are commonly regulated through this cis element ( figure 2d ) . to obtain detailed information on this cis element , return to the locus page and click the link in the cis element page for acacgtg ( figure 2e ) , users can see that this predicted heptamer corresponds to the reported cis elements named abre and g - box . the abre is a central cis element of aba signal transduction in response to stress treatment ( 34 ) . this element prefers the position from 50 to 100 bp upstream of the tss , indicating that the abre - like element found in at5g62490 appears in an appropriate position ( 6062 bp upstream ) . the graph for cis - element specificity for various experimental conditions shows that genes containing this cis element in their promoters are usually up - regulated responding to aba as well as osmotic or salt stress ( data not shown in figure 2 ) . this pattern is consistent with the expression pattern of at5g62490 , supporting the possibility that this gene is regulated through this cis element . a list of transcription factor candidates that bind this heptamer is also provided on this page . this list includes known transcription factors to bind aba - responsive cis elements or g - box . in fact , abf3 , a well - known transcription factor to bind abre ( 35 ) , is found in the list , although its correlation value is not high for this sequence itself . when we focus on the regulation during embryogenesis , rd26 ( at4g27410 ) is a good candidate because this gene is highly expressed during embryogenesis and up - regulated by aba as well as osmotic or salt stress , as indicated by their expression patterns on their respective locus page . furthermore , rd26 functions in aba - dependent stress signaling ( 36 ) , and its homologous transcription factors recognize a cacg core ( 37 ) . g - box binding factor 3 ( gbf3 , at2g46270 ) is another candidate , since it binds g - box sequence for aba signaling ( 38 ) . the cis element pages for the second group of heptamers found in the locus page for at5g62490 contain almost identical information except for the position preference , which is at 100 bp upstream of the tss , indicating that the second cis element also appears at its most appropriate position ( 101103 bp ) . in summary , information in atted - ii suggested the following regulatory scheme for the test case of at5g62490 . the aba signal up - regulates at5g62490 together with co - expressed genes for late embryogenesis , potentially through two abres and the transcription factors rd26 and gbf3 . for example , using locus/(agi code of the gene of interest).html will bring up the locus page of the gene of interest . the profiles were constructed from 1388 genechip data downloaded from tair ( 4 ) , which were manually divided into 53 experimental groups and normalized by rma method ( 39 ) . to quantify the similarity of the gene expression profiles , pairwise pearson 's correlation coefficients were used ( 40 ) . the 12 most strongly correlated genes for each gene ( including the gene itself ) were used to generate the co - expressed gene networks . the 300 most strongly correlated genes for each gene were listed in the co - expressed genes page . the lists of all co - expressed genes are downloadable from atted - ii as tab - delimited text files . co - expressed gene networks were pictured by simply drawing a line between co - expressed genes . details of the method and description of the experiments used in atted - ii are available in the help pages of atted - ii . previous reports on comprehensive cis element prediction for arabidopsis have used reported cis elements to search the genome ( 20,41 ) or have extracted conserved motifs from the genome ( 42,43 ) . our approach using microarray data complements their approaches . to estimate gene regulatory schemes , cis elements in the region 200 bp upstream of the tss , which is downloaded from tair ( 4 ) , were predicted based on the method described previously ( 44 ) . briefly , oligomers were used as cis - element candidates , and correlations between gene expression changes and presence of the cis - element candidates in the gene promoters were estimated by calculation of pearson 's correlation coefficients . this value is named ceg ( correlation between gene expression and a defined gene group ) in atted - ii . we modified this method to distinguish the position of the heptamers from the tss to reduce false positives . moreover , estimation of expression changes using the extracted cis elements by a linear model ( 44 ) was best for cis elements within 200 bp upstream of the tss , indicating that most of the cis elements predicted out of this region are false positives ( data not shown ) . these results agreed with a previous report about aba - responsive elements ( 34 ) . thus we decided to use the region 200 bp upstream from the tss . as a result , 304 heptamers were extracted from all 16 384 ( = 4 ) possible heptamers and registered in atted - ii . the profiles were constructed from 1388 genechip data downloaded from tair ( 4 ) , which were manually divided into 53 experimental groups and normalized by rma method ( 39 ) . to quantify the similarity of the gene expression profiles , pairwise pearson 's correlation coefficients were used ( 40 ) . the 12 most strongly correlated genes for each gene ( including the gene itself ) were used to generate the co - expressed gene networks . the 300 most strongly correlated genes for each gene were listed in the co - expressed genes page . the lists of all co - expressed genes are downloadable from atted - ii as tab - delimited text files . co - expressed gene networks were pictured by simply drawing a line between co - expressed genes . details of the method and description of the experiments used in atted - ii are available in the help pages of atted - ii . previous reports on comprehensive cis element prediction for arabidopsis have used reported cis elements to search the genome ( 20,41 ) or have extracted conserved motifs from the genome ( 42,43 ) . our approach using microarray data complements their approaches . to estimate gene regulatory schemes , cis elements in the region 200 bp upstream of the tss , which is downloaded from tair ( 4 ) , were predicted based on the method described previously ( 44 ) . briefly , oligomers were used as cis - element candidates , and correlations between gene expression changes and presence of the cis - element candidates in the gene promoters were estimated by calculation of pearson 's correlation coefficients . this value is named ceg ( correlation between gene expression and a defined gene group ) in atted - ii . we modified this method to distinguish the position of the heptamers from the tss to reduce false positives . moreover , estimation of expression changes using the extracted cis elements by a linear model ( 44 ) was best for cis elements within 200 bp upstream of the tss , indicating that most of the cis elements predicted out of this region are false positives ( data not shown ) . these results agreed with a previous report about aba - responsive elements ( 34 ) . thus we decided to use the region 200 bp upstream from the tss . as a result , 304 heptamers were extracted from all 16 384 ( = 4 ) possible heptamers and registered in atted - ii .

publicly available database of co - expressed gene sets would be a valuable tool for a wide variety of experimental designs , including targeting of genes for functional identification or for regulatory investigation . here , we report the construction of an arabidopsis thaliana trans - factor and cis - element prediction database ( atted - ii ) that provides co - regulated gene relationships based on co - expressed genes deduced from microarray data and the predicted cis elements . atted - ii ( ) includes the following features : ( i ) lists and networks of co - expressed genes calculated from 58 publicly available experimental series , which are composed of 1388 genechip data in a.thaliana ; ( ii ) prediction of cis - regulatory elements in the 200 bp region upstream of the transcription start site to predict co - regulated genes amongst the co - expressed genes ; and ( iii ) visual representation of expression patterns for individual genes . atted - ii can thus help researchers to clarify the function and regulation of particular genes and gene networks .

INTRODUCTION DATABASE CONTENTS EXAMPLE 1: OBTAINING CO-EXPRESSED GENES FOR A PARTICULAR FUNCTION EXAMPLE 2: OBTAINING PREDICTED OTHER METHODS TO ACCESS PAGES IN ATTED-II DATA SOURCES AND CALCULATION Definition of co-expressed genes in ATTED-II Prediction of

microarray data have been collected in several general repositories , including arrayexpress ( 1 ) , gene expression omnibus ( geo ) ( 2 ) and the center for information biology gene expression database ( cibex ) ( 3 ) . for the model plant arabidopsis thaliana , the arabidopsis information resource ( tair ) ( 4 ) and the nottingham arabidopsis stock centre arrays ( nascarrays ) ( 5 ) are species - specific repositories for microarray data . although it is easy to search the data for individual genes or samples , it is still difficult to retrieve gene - to - gene relationships or simply browse gene expression patterns . the comprehensive systems - biology database ( csb.db ) ( 6 ) , botany array resource ( bar ) ( 7 ) , arabidopsis co - expression tool ( act ) ( 8) and genevestigator ( 9 ) provide co - expressed gene relationships calculated from the array data stored in tair and/or the nascarrays . such gene relationship information is valuable for predicting gene function , because co - expressed genes are often involved in the same or related pathways . approaches for specifying experimental targets using such co - expressed relationships have already been reported ( 1013 ) . clique finder returns a group of co - expressed genes from the co - expressed gene network , although it does not show the network itself ( 8) . interactive cis - element prediction from co - expressed genes is also provided in bar ( 7 ) . it could enforce the estimation of regulatory scheme of the target genes deduced from the limited amount of experimentally determined cis elements , which are stored in cis - element databases ( 14,15 ) . the following three points need to be addressed : ( i ) co - expressed gene lists are too long to allow visualization of complete co - expression patterns . ( ii ) although the color - coded heat maps provided by bar ( 7 ) are suitable for comparison of a set of genes , they do not represent quantitative expression changes well . ( iii ) the interactive cis - element prediction tool provided by bar ( 7 ) can extract cis elements from the user - selected genes , but the results are hard to exhaustively compare between different queries . to support the needs of non - bioinformatics experts , we constructed a database named a.thaliana trans - factor and cis - element prediction database ( atted - ii ) for retrieving gene - to - gene relationships similar to the other databases for co - expressed genes . ( i ) network representation of co - expressed gene relationships , which , in addition to the original lists of co - expressed genes , facilitates understanding of the structural basis of gene co - expression ( 16 ) . ( ii ) stored pre - calculated results for cis - element prediction are linked to every gene and every functional category , and are presented along with several characteristics of the cis elements . ( iii ) the gene expression patterns are graphically represented the display of bar graphs for individual genes makes it easy to see quantitative expression changes for many experimental conditions . atted - ii is based on the framework of the a.thaliana tissue - specific expression database ( atted ) , which was opened in 2003 as the repository for our original data for tissue - specific gene expression ( 17 ) . with the subsequent availability of public microarray data , we imported these data into the original atted and stored the calculated co - expression information and the predicted cis elements . atted - ii now contains co - expressed gene networks for 22 263 loci and for 1102 functional categories as well as predicted cis elements represented by 304 heptamers . all expression data for these contents are genechip data ( 25k ) released by tair ( 4 ) , update of which will be incorporated into atted - ii . in the following sections , the contents of atted - ii and its usefulness are described with examples for obtaining information on co - expressed genes ( figure 1 ) and predicted cis elements ( figure 2 ) . yellow - colored agi codes in the table in ( c ) indicate genes in the co - expressed gene network . pink - colored agi codes in the table in ( d ) indicate query genes used to search for co - expressed genes . screenshots of atted - ii pages relevant to the search for a predicted regulatory scheme of at5g62490 . results of a statistical test for the presence of a cis element in the 50 co - expressed genes . atted - ii contains four types of pages ; locus page , co - expressed genes page , cis element page and functional category page . for each locus , co - expressed genes are calculated and the 12 most strongly correlated genes are represented in the locus page as a network and a list . these co - expressed genes are supported by the following information provided in the same locus page : organized functional gene annotation from other databases , subcellular localization predicted by targetp ( 18 ) and wolf psort ( 19 ) , and graphs of the gene expression patterns . predicted cis elements in the 200 bp region upstream from the transcription start site ( tss ) are also shown to support co - expressed gene relationships ( figure 2c ) . the upstream sequence used in atted - ii is obtained from tair ( 4 ) , where for the loci without tss annotation the sequences from translational start site are provided . details about the predicted cis elements can be obtained in the cis element page.a co - expressed genes page is prepared for each of the 22 263 loci for which there are gene expression data . this page contains a longer list of co - expressed genes ( 300 genes ) than that found in locus page and presents statistical test results of the nearest 50 genes for functional categories and for cis - element appearances ( figure 2d ) . for extended analysis , whole lists including more than 300 co - expressed genes are downloadable from atted - ii as tab - delimited text files.the cis element page provides information for each predicted cis element . in this prediction , the extracted heptamers are matched with reported cis elements stored in the database of plant cis - acting regulatory dna elements ( place ) ( 14 ) ( figure 2e ) . list of transcription factors in atted - ii were downloaded from agris ( 20).a functional category page is constructed for each gene function category using the 3481 terms defined by go ( 21 ) , kegg ( 22 ) , aracyc ( 23 ) and kappa - view ( 24 ) . the 15 most significant co - expressed genes are selected from the genes annotated by each functional term , and co - expressed gene networks are drawn ( figure 1c ) . because two - thirds of the categories have no significantly co - expressed genes , the networks are drawn for 1102 categories in total . the method for selecting the genes used to draw the networks is described in the help page in atted - ii . for each locus , co - expressed genes are calculated and the 12 most strongly correlated genes are represented in the locus page as a network and a list . these co - expressed genes are supported by the following information provided in the same locus page : organized functional gene annotation from other databases , subcellular localization predicted by targetp ( 18 ) and wolf psort ( 19 ) , and graphs of the gene expression patterns . predicted cis elements in the 200 bp region upstream from the transcription start site ( tss ) are also shown to support co - expressed gene relationships ( figure 2c ) . the upstream sequence used in atted - ii is obtained from tair ( 4 ) , where for the loci without tss annotation the sequences from translational start site are provided . a co - expressed genes page is prepared for each of the 22 263 loci for which there are gene expression data . this page contains a longer list of co - expressed genes ( 300 genes ) than that found in locus page and presents statistical test results of the nearest 50 genes for functional categories and for cis - element appearances ( figure 2d ) . for extended analysis , whole lists including more than 300 co - expressed genes the cis element page provides information for each predicted cis element . in this prediction , the extracted heptamers are matched with reported cis elements stored in the database of plant cis - acting regulatory dna elements ( place ) ( 14 ) ( figure 2e ) . the 15 most significant co - expressed genes are selected from the genes annotated by each functional term , and co - expressed gene networks are drawn ( figure 1c ) . because two - thirds of the categories have no significantly co - expressed genes , the networks are drawn for 1102 categories in total . the method for selecting the genes used to draw the networks is described in the help page in atted - ii . for gene identification , it is efficient to list and classify genes of co - functional candidates using co - expressed genes ( 1013 ) . for this purpose ( note that larger figures for this example are shown in the help page in atted - ii . ) search wounding as a keyword using the search form to the right of the title logo in atted - ii ( figure 1a).as a result , a go category click the category id ( figure 1b).the functional category page for response to wounding provides co - expressed gene networks of 15 representative genes in this category ( figure 1c ) . the thickness of the lines connecting the circles corresponds to the relative strength of co - expression . to consult each network , the information of the external links in each locus page is useful in addition to the information presented in atted - ii . each gene network clearly corresponds to a biological function in response to wounding as follows : network 1 contains five genes , four of which are enzymes for biosynthesis of jasmonic acid ( ja ) , which is the phytohormone that has a central signaling role in the wounding response . to make a complete list of genes related to the wounding response , next search for co - expressed genes from each group . to select genes to query , click on the checkboxes to the left of each appropriate entry for example , the group of genes for the ja biosynthetic pathway above and the click the button search co - expressed genes just above the table.the result of the co - expressed gene search is a list of the 300 most strongly co - expressed genes from the five query genes for ja biosynthesis ( figure 1d ) . in the same way , these lists of genes are supported by other biological information or cis - element information and can be used to design experiments to identify gene functions . wounding as a keyword using the search form to the right of the title logo in atted - ii ( figure 1a ) . as a result , a go category the functional category page for response to wounding provides co - expressed gene networks of 15 representative genes in this category ( figure 1c ) . the agi code , which is locus i d in arabidopsis ( 25 ) , a short description and the node i d are enclosed by the circle . the thickness of the lines connecting the circles corresponds to the relative strength of co - expression . to consult each network , the information of the external links in each locus page is useful in addition to the information presented in atted - ii . each gene network clearly corresponds to a biological function in response to wounding as follows : network 1 contains five genes , four of which are enzymes for biosynthesis of jasmonic acid ( ja ) , which is the phytohormone that has a central signaling role in the wounding response . to make a complete list of genes related to the wounding response , next search for co - expressed genes from each group . to select genes to query , click on the checkboxes to the left of each appropriate entry for example , the group of genes for the ja biosynthetic pathway above and the click the button search co - expressed genes just above the table . network 1 contains five genes , four of which are enzymes for biosynthesis of jasmonic acid ( ja ) , which is the phytohormone that has a central signaling role in the wounding response . to make a complete list of genes related to the wounding response , next search for co - expressed genes from each group . to select genes to query , click on the checkboxes to the left of each appropriate entry for example , the group of genes for the ja biosynthetic pathway above and the click the button the result of the co - expressed gene search is a list of the 300 most strongly co - expressed genes from the five query genes for ja biosynthesis ( figure 1d ) . in the same way these lists of genes are supported by other biological information or cis - element information and can be used to design experiments to identify gene functions . information of predicted cis elements can provide support for proposed co - expressed gene relationships . when co - expressed genes share particular cis elements in their promoters , we can expect that these genes are co - regulated through these cis elements and that they function together . as an example in obtaining information on predicted cis elements , consider the regulation of an abscisic acid ( aba)-responsive protein at5g62490 . atted - ii indicates that this gene can be up - regulated at embryogenesis through an aba - responsive element ( abre ) ( figure 2 ) . ( note that larger figures for this example are shown in the help page in atted - ii . ) search at5g62490 as an agi code using the search form to the right of the title logo ( figure 2a).click the agi code in the resulting table to go to the locus page ( figure 2b).in the locus page for at5g62490 , cis elements are predicted at 6062 bp and 101103 bp upstream from the tss ( figure 2c ) . the co - expressed gene list indicates that this gene is co - expressed with genes for late embryogenesis . link to top 300 co - expressed gene list.in the co - expressed genes page for at5g62490 , appearance of acacgtg in the co - expressed genes is statistically abundant , suggesting that these genes , including at5g62490 itself , are commonly regulated through this cis element ( figure 2d ) . the graph for cis - element specificity for various experimental conditions shows that genes containing this cis element in their promoters are usually up - regulated responding to aba as well as osmotic or salt stress ( data not shown in figure 2 ) . the cis element pages for the second group of heptamers found in the locus page for at5g62490 contain almost identical information except for the position preference , which is at 100 bp upstream of the tss , indicating that the second cis element also appears at its most appropriate position ( 101103 bp ) . the co - expressed gene list indicates that this gene is co - expressed with genes for late embryogenesis . link to top 300 co - expressed gene list. in the co - expressed genes page for at5g62490 , appearance of acacgtg in the co - expressed genes is statistically abundant , suggesting that these genes , including at5g62490 itself , are commonly regulated through this cis element ( figure 2d ) . this element prefers the position from 50 to 100 bp upstream of the tss , indicating that the abre - like element found in at5g62490 appears in an appropriate position ( 6062 bp upstream ) . the graph for cis - element specificity for various experimental conditions shows that genes containing this cis element in their promoters are usually up - regulated responding to aba as well as osmotic or salt stress ( data not shown in figure 2 ) . when we focus on the regulation during embryogenesis , rd26 ( at4g27410 ) is a good candidate because this gene is highly expressed during embryogenesis and up - regulated by aba as well as osmotic or salt stress , as indicated by their expression patterns on their respective locus page . the cis element pages for the second group of heptamers found in the locus page for at5g62490 contain almost identical information except for the position preference , which is at 100 bp upstream of the tss , indicating that the second cis element also appears at its most appropriate position ( 101103 bp ) . in summary , information in atted - ii suggested the following regulatory scheme for the test case of at5g62490 . the aba signal up - regulates at5g62490 together with co - expressed genes for late embryogenesis , potentially through two abres and the transcription factors rd26 and gbf3 . the profiles were constructed from 1388 genechip data downloaded from tair ( 4 ) , which were manually divided into 53 experimental groups and normalized by rma method ( 39 ) . the 12 most strongly correlated genes for each gene ( including the gene itself ) were used to generate the co - expressed gene networks . the 300 most strongly correlated genes for each gene were listed in the co - expressed genes page . the lists of all co - expressed genes are downloadable from atted - ii as tab - delimited text files . co - expressed gene networks were pictured by simply drawing a line between co - expressed genes . details of the method and description of the experiments used in atted - ii are available in the help pages of atted - ii . to estimate gene regulatory schemes , cis elements in the region 200 bp upstream of the tss , which is downloaded from tair ( 4 ) , were predicted based on the method described previously ( 44 ) . briefly , oligomers were used as cis - element candidates , and correlations between gene expression changes and presence of the cis - element candidates in the gene promoters were estimated by calculation of pearson 's correlation coefficients . moreover , estimation of expression changes using the extracted cis elements by a linear model ( 44 ) was best for cis elements within 200 bp upstream of the tss , indicating that most of the cis elements predicted out of this region are false positives ( data not shown ) . as a result , 304 heptamers were extracted from all 16 384 ( = 4 ) possible heptamers and registered in atted - ii . the profiles were constructed from 1388 genechip data downloaded from tair ( 4 ) , which were manually divided into 53 experimental groups and normalized by rma method ( 39 ) . the 12 most strongly correlated genes for each gene ( including the gene itself ) were used to generate the co - expressed gene networks . the 300 most strongly correlated genes for each gene were listed in the co - expressed genes page . the lists of all co - expressed genes are downloadable from atted - ii as tab - delimited text files . co - expressed gene networks were pictured by simply drawing a line between co - expressed genes . details of the method and description of the experiments used in atted - ii are available in the help pages of atted - ii . previous reports on comprehensive cis element prediction for arabidopsis have used reported cis elements to search the genome ( 20,41 ) or have extracted conserved motifs from the genome ( 42,43 ) . to estimate gene regulatory schemes , cis elements in the region 200 bp upstream of the tss , which is downloaded from tair ( 4 ) , were predicted based on the method described previously ( 44 ) . briefly , oligomers were used as cis - element candidates , and correlations between gene expression changes and presence of the cis - element candidates in the gene promoters were estimated by calculation of pearson 's correlation coefficients . moreover , estimation of expression changes using the extracted cis elements by a linear model ( 44 ) was best for cis elements within 200 bp upstream of the tss , indicating that most of the cis elements predicted out of this region are false positives ( data not shown ) .

in greece , as elsewhere , both the scientific community and policy makers are interested in measuring the health status and health related quality of life of the population . the instruments used for this purpose are mostly health measures developed in other countries which have been translated into greek and applied into the greek context . the majority of the health related quality of life studies with greek samples is orientated towards measuring the impact of diseases on patients ' quality of life using either disease specific or generic instruments [ 1 - 8 ] . few studies have attempted to assess the health related quality of life of healthy individuals [ 9 - 12 ] . the sf-36 is a generic instrument which has been used to measure , assess and evaluate the quality of life of different greek populations [ 1 - 3 ] . this study used short form 36 ( sf-36 ) to assess the health of the personnel of hospitals members of the network of health promoting hospitals . the network of health promoting hospitals is a who initiative and in greece , the hellenic network of health promoting hospitals ( hnhph ) was established in 1998 . the greek version of sf-36 took its final form after two forward and backward translations and a pilot study conducted to assess its semantics and linguistic adaptation . the feasibility of the greek sf-36 was evaluated by assessing the response rates of each question both in the initial pilot study and the then conducted main study . the convergent validity and discriminant validity of the instrument were assessed by the multitrait - multimethod matrix . items and scales like physical functioning ( pf ) and general health ( gh ) correlated and converge with all five dimensions ( mobility , self care , usual activities , pain / discomfort , anxiety / depression ) of the eq-5d instrument and mental health ( mh ) scale with its anxiety / depression dimension . discrimination power was also found between dissimilar dimensions of the sf-36 and the eq-5d . therefore sf-36 considered being an acceptable and sensitive instrument for measuring health - related quality in greece . the main purpose of the present study was to assess the health status and health related quality of life of the personnel of the hellenic network of health promotion hospitals . additionally it aims to contribute to the validation of the sf-36 by contributing to the accumulation of different types of evidence showing that the instrument measures what is supposed and intended to measure . in the present study sf-36 was administered to a representative sample of the personnel of seven hospitals ( both public and private ) . these seven hospitals were the first members of the hellenic network of health promotion hospitals ( hnhph ) in the major area of athens . the survey took place in 19992000 . from a sampling population of 7,155 persons working at the seven hospitals , a two - stage proportional stratification was explored ; the first stage was based on a workplace criterion ( stratification according the hospital each one employee was working ) while the second stage was based on a professional criterion ( stratification according the occupational group each one employee belonged to ) . the study sample was drawn ( using alphabetical lists ) proportionately to the number of employees in each hospital and professional category ( stepwise technique ) . the final stratified random sample consisted of 395 employees who constitute about 5% of the experimental population . the questionnaire was self - administered and the participants were adults of working age of both sexes . in each hospital the questionnaires were distributed to the participants and then collected by the hospital representative to the hellenic network of health promotion hospitals . all participants were assigned to the following professional categories : administrative , auxiliary and technical , medical doctors and nurses . the technical category included all technical personnel ( mostly engineers but not blue collar workers ) and the auxiliary occupational category included all the manual hospital workers . different kind of hospitals participated in this study including a university hospital ( i ) , three hospitals belonging to the national health system of greece ( ii , v and vi ) , two private hospitals ( one profit and another non - profit)(iv and vii ) and one belonging to the national foundation of social insurance ( iii ) . the other instruments administered along with sf-36 referred to health behaviours ( e.g. smoking and nutritional habits ) and the interaction between work and health . all sf-36 items were coded , summed and transformed on a scale from 0 ( worst possible health state ) to 100 ( best possible health state ) . the researchers were aware of the existence of a second ( newer ) version of the sf-36 . but since , at the time the survey took place , this was still developing and has not been tested in greece and translated into greek , it was decided to use the first version of the instrument . the missing values were substituted according to the method the sf-36 developers have suggested in order to gain scores for missing values . the descriptive statistics for all eight scales of sf-36 analyzed by sex , age , profession and workplace ( hospital ) are provided . an important issue was , also , to assess the construct validity of the greek version of sf-36 ; to assess the extent to which the questionnaire supported pre - defined hypotheses and working assumptions . this was done by determining the extent to which scores variations on different dimensions of the questionnaire reflected the expected distribution of health status for the study population . in other words construct validity was assessed by examining the ability of the greek version of sf-36 to detect expected health differences and variations between the various subgroups of the study population . the expected health differences are : a ) men to report higher scores ( better health status ) than women ( according to who the expectation of lost healthy years at birth due to poor health for women in greece was 2.4 years more than the equivalent for men and the percentage of total life expectancy lost due to poor health was 2.4% more for women than it was for men ) b ) higher professional status employees to have higher scores ( better health status ) than their lower professional status colleagues , see for example and c ) older participants to report poorer physical health status than the young participants . regarding the significance of the observed differences between the various subgroups of the sample both parametric ( t - test and anova ) and non - parametric ( mann - whitney and kruskall - wallis ) tests were performed . the 95% confidence intervals for scores of all eight dimensions of sf-36 the level of significance was set at p = 0.05 and the bonferroni correction was used to control for the effect of multiple testing . in the present study sf-36 was administered to a representative sample of the personnel of seven hospitals ( both public and private ) . these seven hospitals were the first members of the hellenic network of health promotion hospitals ( hnhph ) in the major area of athens . the survey took place in 19992000 . from a sampling population of 7,155 persons working at the seven hospitals , a two - stage proportional stratification was explored ; the first stage was based on a workplace criterion ( stratification according the hospital each one employee was working ) while the second stage was based on a professional criterion ( stratification according the occupational group each one employee belonged to ) . the study sample was drawn ( using alphabetical lists ) proportionately to the number of employees in each hospital and professional category ( stepwise technique ) . the final stratified random sample consisted of 395 employees who constitute about 5% of the experimental population . the questionnaire was self - administered and the participants were adults of working age of both sexes . in each hospital the questionnaires were distributed to the participants and then collected by the hospital representative to the hellenic network of health promotion hospitals . all participants were assigned to the following professional categories : administrative , auxiliary and technical , medical doctors and nurses . the technical category included all technical personnel ( mostly engineers but not blue collar workers ) and the auxiliary occupational category included all the manual hospital workers . different kind of hospitals participated in this study including a university hospital ( i ) , three hospitals belonging to the national health system of greece ( ii , v and vi ) , two private hospitals ( one profit and another non - profit)(iv and vii ) and one belonging to the national foundation of social insurance ( iii ) . the sf-36 was administered first and prior to other instruments . the other instruments administered along with sf-36 referred to health behaviours ( e.g. smoking and nutritional habits ) and the interaction between work and health . all sf-36 items were coded , summed and transformed on a scale from 0 ( worst possible health state ) to 100 ( best possible health state ) . the researchers were aware of the existence of a second ( newer ) version of the sf-36 . but since , at the time the survey took place , this was still developing and has not been tested in greece and translated into greek , it was decided to use the first version of the instrument . the missing values were substituted according to the method the sf-36 developers have suggested in order to gain scores for missing values . the descriptive statistics for all eight scales of sf-36 analyzed by sex , age , profession and workplace ( hospital ) are provided . an important issue was , also , to assess the construct validity of the greek version of sf-36 ; to assess the extent to which the questionnaire supported pre - defined hypotheses and working assumptions . this was done by determining the extent to which scores variations on different dimensions of the questionnaire reflected the expected distribution of health status for the study population . in other words construct validity was assessed by examining the ability of the greek version of sf-36 to detect expected health differences and variations between the various subgroups of the study population . the expected health differences are : a ) men to report higher scores ( better health status ) than women ( according to who the expectation of lost healthy years at birth due to poor health for women in greece was 2.4 years more than the equivalent for men and the percentage of total life expectancy lost due to poor health was 2.4% more for women than it was for men ) b ) higher professional status employees to have higher scores ( better health status ) than their lower professional status colleagues , see for example and c ) older participants to report poorer physical health status than the young participants . regarding the significance of the observed differences between the various subgroups of the sample both parametric ( t - test and anova ) and non - parametric ( mann - whitney and kruskall - wallis ) tests were performed . the 95% confidence intervals for scores of all eight dimensions of sf-36 were calculated in order to assess the statistical precision of the estimates . the level of significance was set at p = 0.05 and the bonferroni correction was used to control for the effect of multiple testing . the age distribution of our sample seems to be consistent with that of the personnel of hnhph . the majority of the population was from 30 to 49 years old ( 72.2 ) and employees of public hospitals belonging to the national health service ( nhs ) of greece ( 65.1% ) . the breakdown of the sample by gender , age , occupation and workplace ( hospitals ) is shown in table 1 . the sociodemographic characteristics of the sample # university hospital + national health system hospital * * institution of social security hospital + + private hospitals the scores for the eight dimensions of sf-36 were calculated using algorithms and following the sf-36 developers ' instructions . as expected the three scales measuring well - being ( general health , vitality and mental health ) have lower mean scale scores and the scales measuring health - related limitation apart from the social functioning scale ( namely physical functioning , role physical , bodily pain and role emotional ) had higher mean scale scores . the mean scale score for social functioning was unexpectedly low and the possible reasons for this inconsistency are discussed further on . the distributions were markedly negatively skewed with the participants scoring mostly towards the positive end of the scales . this is , generally , indicative of a better state of health for the majority of our sample . the most negatively skewed scales were physical functioning , role physical , bodily pain and role emotional . all scales had 7 or more levels apart from the role emotional and role physical scales , which had 4 and 5 levels , respectively . especially as expected physical functioning , general health , vitality and mental health have 20 or more levels . this existence of 7 or more levels for the 6 scales gave reason for treating them as continuous variables . in a healthy population the number of participants scoring the lowest possible level ( 0 ) ( floor effect ) are expected to be very low while that of participants scoring the highest possible level ( 100 ) ( ceiling effect ) for the functional limitations scales are expected to be high . these expectations were confirmed for all scales in this study . the role emotional and role physical scales had the most profound floor and ceiling effects . on the role emotional 163 participants scored 100 ( 59.5% ) and 34 scored 0 ( 12.4% ) . while on the role physical the equivalent were 165 participants ( 58.9 ) ( ceiling effect ) and 28 ( 10% ) ( floor effect ) . descriptive statistics and features of scores distribution for sf-36 * percentage of participants with worst and best possible score , respectively table 3 provides data in the form of descriptive statistics broken down by sex , age , occupational status and hospitals . also it provides information regarding the statistical significance of the observed differences among the various subgroups of the study population on all sf-36 scales after controlling for the effect of multiple testing with bonferroni correction . using both parametric ( t - test and anova ) and non - parametric ( mann - whitney and kruskall - wallis ) tests it was found that all the sex differences , most of the occupation - related differences ( apart from these observed on the role emotional and the mental health scales ) and some of the age differences ( those observed on the social functioning , role emotional and mental health scales ) were statistically significant ( p<0.05 ) . the means and standard deviations on all sf-36 dimensions broken down by sex , age , occupation and workplace bold & italics characters indicate statistical significant scoring differences between the subgroups of the population ( after controlling for multiple testing with bonferroni correction ) . # university hospital + national health system hospital * * institution of social security hospital + + private hospitals regarding sex differences women reported poorer health status than men on all eight scales of the sf-36 . with respect to age , older participants ( > 50 years old ) reported , generally , better health than their younger counter partners . important scoring differences were found on all scales of sf-36 among the various professional categories . medical doctors and technical personnel ( mostly engineers ) reported far better health status than nurses and the auxiliary personnel . the health status of the administrative personnel was of an intermediate level between that of nurses and auxiliary personnel and that of medical doctors and technical personnel . some differences were found regarding the impact of the hospital where the participants work ( workplace ) on their health status but none of them was statistically significant ( p > 0.05 ) . regarding the validation of the instrument our results suggest that it has construct validity since it detects and shows health differences expected to exist between various subgroups of our sample ( the health differences between men and women and high and low occupational status employees ) . the only discrepancy between the expected and observed health differences appears between the age groups . the older participants reported the best scores on some physical health dimensions ( e.g. role physical and bodily pain ) and the younger participants reported the worst while the opposite was expected . this study constitutes the first attempt to assess the health status and health - related quality of life of such a healthy working population in greece and is one of the first applications of sf-36 to a healthy population in greece . sf-36 has been applied only once before to a healthy population in greece ( 3 ) the data of the present study reveal two major issues . the first issue is the health inequalities existing among employees of greek hospitals of the hellenic network of health promoting hospitals . the second is the considerable differences between the scores of a healthy greek population reported in this study and those ( national norms ) reported in studies from northern america and western europe . with respect to that second issue , a comparison between the results of the present study and those of other similar studies assessing the health related quality of life of health professionals ( after adjusting for age ) would has been more valid and methodologically sounder . unfortunately the existing literature on health - related quality of life of the health professionals is not rich . most studies found assessing the health status of health professionals have used other generic health instruments ( mostly general health questionnaire ghq ) and therefore a direct comparison between their findings and the results of the present study could not be made . only one study has been identified reporting scores on sf-36 scales for a sample of registered nurses in new zealand . the mean scores on six sf-36 scales reported in that study are much higher ( difference greater than five points ) than these reported by greek nurses in the present study . the only exceptions were the vitality and role emotional scales where the greek hospital nurses reported higher scores than their counterparts with only the difference on vitality scale to be greater than five points . the first major issue this study reveals is that of the notable health inequalities among the employees in greek hospitals . our results show that the employees included in this study are far from constituting a homogenous group of employees with similar health status and health related quality of life . the greatest inequalities observed were those referring to and reflecting differences in professional status , sex and age among the various subgroups of the study sample . specifically , the sex differences in the health status of the participants show similar patterns to those presented in other studies and constitute an indication of the construct validity of the greek version of sf-36 . men scored higher than women on all 8 dimensions of sf-36 and all these differences were statistically significant ( p < 0.05 ) . these findings are consistent with normative data from usa and canada ( where men scored higher than women on all 8 dimensions of sf-36 ) . but the sex differences observed in this study are much larger than those observed elsewhere . in this study the sex differences observed are greater than five points ( difference considered to be clinically and socially relevant ) on all 8 sf-36 dimensions . these sex differences to some extent seem to reflect the disadvantageous position of woman in a predominantly male societal structure . perhaps this is even more the case for greece which is a mediterranean country where women social position is traditionally worst than that of men . nevertheless it is possible the great magnitude of these sex differences observed in this study not only to be a reflection of women 's disadvantageous position in society but also a result of professional differences between men ( who were mostly medical doctors ) and women ( who were mostly nurses and auxiliary personnel ) . this perhaps could also be related to the contradiction , well known both for women in greece and in many other countries , between their higher than that of men life expectancy and ( at the same time ) higher morbidity rates . another interesting finding of this study is the important variability of the scores by age . the oldest group of our sample ( < 50 years old ) reported , generally , a better health status compared to any other age group of the sample whilst the younger participants ( 2029 and 3039 years old ) the worst ones . the statistical analysis showed that the only statistically significant scoring differences ( p < 0.05 ) were those observed on mental health , social functioning , role emotional dimensions whilst those ones observed on the vitality dimension was marginally non - significant ( p = 0.054 ) . older participants reported higher scores on all mental health dimensions ( mental health , social functioning , role emotional and vitality ) and this is consistent with findings of other studies showing that older people reported better mental health . but unexpectedly , older participants reported , as well , the highest score on the role physical and bodily pain scales , which practically means that they had a better than their younger counterpartners state of physical health . given that these unexpected age - related differences on physical health dimensions are not statistically significant and that the number of the older individuals participating in the study is too small ( n = 27 ) no safe conclusion could be drawn about them and the construct validity of the instrument . these unexpected health differences could be spurious and any relevant conclusion should be considered as preliminary . nevertheless if this is not the case , a reasonable explanation for these non - consistent physical health differences between the age groups could be that they are a result of the higher professional status of the older participants . the general health scale although not belonging to the core of the physical component , gives a more balanced account of physical health between the various age groups . on this scale the great differences between the young workers do not exist and all the age groups have reported similar score a pattern reported , also , in other studies . interesting findings are , also , those related to the professional status of the participants . it seems that our sample could be divided according to a professional status criterion , into three clearly distinct professional groups . one group with a good state of health consisting mainly of medical doctors and technical staff ( mostly engineers ) ; a second professional group consisting of administrative personnel whose scores were satisfactory but lower than those of the first group and a third one consisted of nurses and auxiliary staff which scored lower than the other two occupational groups . the medical doctors had the highest scores on the physical health dimensions while the technical personnel had its highest scores on mental health dimensions . these two professions ( constituting the healthiest professional group of the present study ) have reported quite similar results with differences meaningful greater than 5 points only on three dimensions of sf-36 ( bodily pain , vitality and role emotional ) . the administrative personnel hold constantly a position between the better offs ( medical doctors and technical personnel ) and the most disadvantage occupational groups ( nurses and auxiliary staff ) on , almost , all the sf-36 dimensions ( apart from the vitality and role emotional scales ) . their scores are consistent with their professional status in the working environment of the greek hospitals not as high as that of the medical doctors but higher and less health damaging than that of nurses and auxiliary personnel . nurses who are the second biggest professional group included in this study have scored low on all dimensions of sf-36 . their poor health status and health related quality of life primarily reflect the difficulties they face in their everyday life and most importantly reflects the difficulty of being a woman working a job with a low professional ( and consequently social ) status in a predominantly male , highly competitive environment . they work a highly stressful and demanding profession [ 30 - 32 ] which is not very well rewarded . a finding strengthening further this assumption about the stressfulness of the nursing profession in addition it should be taken into account that nurses of our sample were predominantly women and this might have an independent contribution to their higher than men morbidity rates . the auxiliary personnel seem to have by far the worst health status and poorer health related quality of life . they have reported the lowest scores on all sf-36 dimensions apart from that of mental health where nurses scored worst . the difference of 28.5 points observed on the bodily pain scale between the medical doctors ( 84.9 ) a profession of high social and professional status and the auxiliary personnel ( 56.4 ) who are unskilled manual workers of low social and professional status , is the greatest one observed in the present study . these findings are consistent with recent health research arguing about the impact of social gradient within the workplace and occupational hierarchy on health and well - being of people ( see for example ) and show that the greek version of sf-36 should be seen as an instrument having construct validity . regarding the workplace no safe conclusions can be drawn on differences among the seven hospitals included in this study . nevertheless , it seems that there are not any important health differences among the workers of our sample that could be attributed to workplace differences . the second major issue revealed is that of the observed differences between this study results and the national norms of other countries . although the population of this study does not represent the general greek population and its scores do not constitute the national norms of greece a comparison between the normative data of other countries and those of this study can produce useful preliminary though , since the sample of this study could be considerably different from the greek national norm conclusions regarding the differences between greece and other countries . especially since there are not any published normative data for greece and there is a lack of knowledge about the health related quality of life of the general population in greece . the participants in this study scored considerably lower on the 8 domains of the sf-36 than their counter partners in other studies from western europe and north america . table 4 shows a comparison between the swedish , canadian , uk , italian and us normative data and those of the present study . the greek scores are lower than the canadian , uk and swedish norms almost in every scale . in comparison to the us norms the greek scores were lower in role physical , general health , social functioning , role emotional and mental health . the italian norms are higher than those of the present study on the physical functioning , physical role , social functioning and role emotional scales and lower on the bodily pain , general health and vitality while for mental health scale both the italian norms and the results of this study are the same . however the magnitude of the differences between the italian norms and the greek results is quite small ( the only exemption is the difference observed on the social functioning scale 5.1 ) indicating a hidden influence of ( mediterranean ) culture on health outcomes as measured by sf-36 . comparison of the national norms of various countries with the scores of the present study * the scores of present study do not represent the greek national norms the present study has several limitations . the most important is that the comparison between the scores of this study and various national norms is a priori problematic since it is not age - adjusted and the study sample is not necessarily representative of the greek general population . but as already mentioned this comparison is only indicative and preliminary and is done only because the national greek norms have not been published . consequently any conclusion drawn from this comparison should be treated with caution . another limitation is the de facto small number of older participants which put into question the value of the observed health differences between the age groups of the sample . this study reveals the existence of considerable health inequalities among the employees in the greek hospitals . these health inequalities underscore the need for immediate interventions to tackle them and initiatives to support women , young and low professional status workers in the greek hospitals . moreover the findings of this study constitute an indication of the construct validity of the greek version of sf-36 . yt conceived of the study , coordinated both the original study and paper drafting and corrected the final draft . yy participated in drafting the validity and reliability section of the manuscript and commented on the final draft .

backgroundthe main aim of the study was to assess the health status and health related quality of life of the personnel of the hellenic network of health promotion hospitals . the instrument used was sf-36 . an additional aim was to contribute to the validation of the sf-36.methodsthe study instrument was administered to 347 randomly selected employees from seven hospitals within major athens area . completed questionnaire were obtained by 292 employees . the statistical significance of the observed differences was tested with parametric ( t - test and anova ) and non - parametric tests ( mann - whitney and kruskall - wallis ) . also , since the greek national norms have not been published yet , the mean scores on all eight sf-36 dimensions of this study were compared with the u.s and several european national norms just to assess the extent to what there are significant differences between a greek healthy population and the general populations of several other countries.resultsmedical doctors and technical personnel ( mostly engineers ) reported better health status than nurses and administrative and auxiliary personnel ; women reported poorer health status than men on all eight sf-36 dimensions ; younger employees reported poorer health status than their older counterpartners . moreover the mean scores on all sf-36 dimensions reported by the participants on this study were considerably lower than the u.s and many european national norms . also the study results constitute an indication of the sf-36 construct validity.conclusionthe findings of this study show that there are major and intense health inequalities among the employees in greek hospitals .

Background Methods Sample Instrument Statistical Analysis Results Discussion Conclusion Authors' contributions

in greece , as elsewhere , both the scientific community and policy makers are interested in measuring the health status and health related quality of life of the population . the instruments used for this purpose are mostly health measures developed in other countries which have been translated into greek and applied into the greek context . the majority of the health related quality of life studies with greek samples is orientated towards measuring the impact of diseases on patients ' quality of life using either disease specific or generic instruments [ 1 - 8 ] . few studies have attempted to assess the health related quality of life of healthy individuals [ 9 - 12 ] . the sf-36 is a generic instrument which has been used to measure , assess and evaluate the quality of life of different greek populations [ 1 - 3 ] . this study used short form 36 ( sf-36 ) to assess the health of the personnel of hospitals members of the network of health promoting hospitals . the network of health promoting hospitals is a who initiative and in greece , the hellenic network of health promoting hospitals ( hnhph ) was established in 1998 . the greek version of sf-36 took its final form after two forward and backward translations and a pilot study conducted to assess its semantics and linguistic adaptation . the feasibility of the greek sf-36 was evaluated by assessing the response rates of each question both in the initial pilot study and the then conducted main study . the convergent validity and discriminant validity of the instrument were assessed by the multitrait - multimethod matrix . items and scales like physical functioning ( pf ) and general health ( gh ) correlated and converge with all five dimensions ( mobility , self care , usual activities , pain / discomfort , anxiety / depression ) of the eq-5d instrument and mental health ( mh ) scale with its anxiety / depression dimension . discrimination power was also found between dissimilar dimensions of the sf-36 and the eq-5d . the main purpose of the present study was to assess the health status and health related quality of life of the personnel of the hellenic network of health promotion hospitals . additionally it aims to contribute to the validation of the sf-36 by contributing to the accumulation of different types of evidence showing that the instrument measures what is supposed and intended to measure . in the present study sf-36 was administered to a representative sample of the personnel of seven hospitals ( both public and private ) . these seven hospitals were the first members of the hellenic network of health promotion hospitals ( hnhph ) in the major area of athens . the study sample was drawn ( using alphabetical lists ) proportionately to the number of employees in each hospital and professional category ( stepwise technique ) . in each hospital the questionnaires were distributed to the participants and then collected by the hospital representative to the hellenic network of health promotion hospitals . all participants were assigned to the following professional categories : administrative , auxiliary and technical , medical doctors and nurses . the technical category included all technical personnel ( mostly engineers but not blue collar workers ) and the auxiliary occupational category included all the manual hospital workers . different kind of hospitals participated in this study including a university hospital ( i ) , three hospitals belonging to the national health system of greece ( ii , v and vi ) , two private hospitals ( one profit and another non - profit)(iv and vii ) and one belonging to the national foundation of social insurance ( iii ) . smoking and nutritional habits ) and the interaction between work and health . but since , at the time the survey took place , this was still developing and has not been tested in greece and translated into greek , it was decided to use the first version of the instrument . an important issue was , also , to assess the construct validity of the greek version of sf-36 ; to assess the extent to which the questionnaire supported pre - defined hypotheses and working assumptions . this was done by determining the extent to which scores variations on different dimensions of the questionnaire reflected the expected distribution of health status for the study population . in other words construct validity was assessed by examining the ability of the greek version of sf-36 to detect expected health differences and variations between the various subgroups of the study population . the expected health differences are : a ) men to report higher scores ( better health status ) than women ( according to who the expectation of lost healthy years at birth due to poor health for women in greece was 2.4 years more than the equivalent for men and the percentage of total life expectancy lost due to poor health was 2.4% more for women than it was for men ) b ) higher professional status employees to have higher scores ( better health status ) than their lower professional status colleagues , see for example and c ) older participants to report poorer physical health status than the young participants . regarding the significance of the observed differences between the various subgroups of the sample both parametric ( t - test and anova ) and non - parametric ( mann - whitney and kruskall - wallis ) tests were performed . the 95% confidence intervals for scores of all eight dimensions of sf-36 the level of significance was set at p = 0.05 and the bonferroni correction was used to control for the effect of multiple testing . in the present study sf-36 was administered to a representative sample of the personnel of seven hospitals ( both public and private ) . these seven hospitals were the first members of the hellenic network of health promotion hospitals ( hnhph ) in the major area of athens . the study sample was drawn ( using alphabetical lists ) proportionately to the number of employees in each hospital and professional category ( stepwise technique ) . in each hospital the questionnaires were distributed to the participants and then collected by the hospital representative to the hellenic network of health promotion hospitals . all participants were assigned to the following professional categories : administrative , auxiliary and technical , medical doctors and nurses . the technical category included all technical personnel ( mostly engineers but not blue collar workers ) and the auxiliary occupational category included all the manual hospital workers . different kind of hospitals participated in this study including a university hospital ( i ) , three hospitals belonging to the national health system of greece ( ii , v and vi ) , two private hospitals ( one profit and another non - profit)(iv and vii ) and one belonging to the national foundation of social insurance ( iii ) . smoking and nutritional habits ) and the interaction between work and health . the researchers were aware of the existence of a second ( newer ) version of the sf-36 . but since , at the time the survey took place , this was still developing and has not been tested in greece and translated into greek , it was decided to use the first version of the instrument . the missing values were substituted according to the method the sf-36 developers have suggested in order to gain scores for missing values . an important issue was , also , to assess the construct validity of the greek version of sf-36 ; to assess the extent to which the questionnaire supported pre - defined hypotheses and working assumptions . this was done by determining the extent to which scores variations on different dimensions of the questionnaire reflected the expected distribution of health status for the study population . in other words construct validity was assessed by examining the ability of the greek version of sf-36 to detect expected health differences and variations between the various subgroups of the study population . the expected health differences are : a ) men to report higher scores ( better health status ) than women ( according to who the expectation of lost healthy years at birth due to poor health for women in greece was 2.4 years more than the equivalent for men and the percentage of total life expectancy lost due to poor health was 2.4% more for women than it was for men ) b ) higher professional status employees to have higher scores ( better health status ) than their lower professional status colleagues , see for example and c ) older participants to report poorer physical health status than the young participants . regarding the significance of the observed differences between the various subgroups of the sample both parametric ( t - test and anova ) and non - parametric ( mann - whitney and kruskall - wallis ) tests were performed . the 95% confidence intervals for scores of all eight dimensions of sf-36 were calculated in order to assess the statistical precision of the estimates . the age distribution of our sample seems to be consistent with that of the personnel of hnhph . the majority of the population was from 30 to 49 years old ( 72.2 ) and employees of public hospitals belonging to the national health service ( nhs ) of greece ( 65.1% ) . the sociodemographic characteristics of the sample # university hospital + national health system hospital * * institution of social security hospital + + private hospitals the scores for the eight dimensions of sf-36 were calculated using algorithms and following the sf-36 developers ' instructions . the distributions were markedly negatively skewed with the participants scoring mostly towards the positive end of the scales . also it provides information regarding the statistical significance of the observed differences among the various subgroups of the study population on all sf-36 scales after controlling for the effect of multiple testing with bonferroni correction . using both parametric ( t - test and anova ) and non - parametric ( mann - whitney and kruskall - wallis ) tests it was found that all the sex differences , most of the occupation - related differences ( apart from these observed on the role emotional and the mental health scales ) and some of the age differences ( those observed on the social functioning , role emotional and mental health scales ) were statistically significant ( p<0.05 ) . the means and standard deviations on all sf-36 dimensions broken down by sex , age , occupation and workplace bold & italics characters indicate statistical significant scoring differences between the subgroups of the population ( after controlling for multiple testing with bonferroni correction ) . # university hospital + national health system hospital * * institution of social security hospital + + private hospitals regarding sex differences women reported poorer health status than men on all eight scales of the sf-36 . with respect to age , older participants ( > 50 years old ) reported , generally , better health than their younger counter partners . medical doctors and technical personnel ( mostly engineers ) reported far better health status than nurses and the auxiliary personnel . the health status of the administrative personnel was of an intermediate level between that of nurses and auxiliary personnel and that of medical doctors and technical personnel . some differences were found regarding the impact of the hospital where the participants work ( workplace ) on their health status but none of them was statistically significant ( p > 0.05 ) . regarding the validation of the instrument our results suggest that it has construct validity since it detects and shows health differences expected to exist between various subgroups of our sample ( the health differences between men and women and high and low occupational status employees ) . this study constitutes the first attempt to assess the health status and health - related quality of life of such a healthy working population in greece and is one of the first applications of sf-36 to a healthy population in greece . the first issue is the health inequalities existing among employees of greek hospitals of the hellenic network of health promoting hospitals . the second is the considerable differences between the scores of a healthy greek population reported in this study and those ( national norms ) reported in studies from northern america and western europe . with respect to that second issue , a comparison between the results of the present study and those of other similar studies assessing the health related quality of life of health professionals ( after adjusting for age ) would has been more valid and methodologically sounder . unfortunately the existing literature on health - related quality of life of the health professionals is not rich . most studies found assessing the health status of health professionals have used other generic health instruments ( mostly general health questionnaire ghq ) and therefore a direct comparison between their findings and the results of the present study could not be made . the mean scores on six sf-36 scales reported in that study are much higher ( difference greater than five points ) than these reported by greek nurses in the present study . the only exceptions were the vitality and role emotional scales where the greek hospital nurses reported higher scores than their counterparts with only the difference on vitality scale to be greater than five points . the first major issue this study reveals is that of the notable health inequalities among the employees in greek hospitals . our results show that the employees included in this study are far from constituting a homogenous group of employees with similar health status and health related quality of life . the greatest inequalities observed were those referring to and reflecting differences in professional status , sex and age among the various subgroups of the study sample . specifically , the sex differences in the health status of the participants show similar patterns to those presented in other studies and constitute an indication of the construct validity of the greek version of sf-36 . in this study the sex differences observed are greater than five points ( difference considered to be clinically and socially relevant ) on all 8 sf-36 dimensions . nevertheless it is possible the great magnitude of these sex differences observed in this study not only to be a reflection of women 's disadvantageous position in society but also a result of professional differences between men ( who were mostly medical doctors ) and women ( who were mostly nurses and auxiliary personnel ) . another interesting finding of this study is the important variability of the scores by age . the oldest group of our sample ( < 50 years old ) reported , generally , a better health status compared to any other age group of the sample whilst the younger participants ( 2029 and 3039 years old ) the worst ones . older participants reported higher scores on all mental health dimensions ( mental health , social functioning , role emotional and vitality ) and this is consistent with findings of other studies showing that older people reported better mental health . given that these unexpected age - related differences on physical health dimensions are not statistically significant and that the number of the older individuals participating in the study is too small ( n = 27 ) no safe conclusion could be drawn about them and the construct validity of the instrument . nevertheless if this is not the case , a reasonable explanation for these non - consistent physical health differences between the age groups could be that they are a result of the higher professional status of the older participants . the general health scale although not belonging to the core of the physical component , gives a more balanced account of physical health between the various age groups . on this scale the great differences between the young workers do not exist and all the age groups have reported similar score a pattern reported , also , in other studies . interesting findings are , also , those related to the professional status of the participants . one group with a good state of health consisting mainly of medical doctors and technical staff ( mostly engineers ) ; a second professional group consisting of administrative personnel whose scores were satisfactory but lower than those of the first group and a third one consisted of nurses and auxiliary staff which scored lower than the other two occupational groups . these two professions ( constituting the healthiest professional group of the present study ) have reported quite similar results with differences meaningful greater than 5 points only on three dimensions of sf-36 ( bodily pain , vitality and role emotional ) . the administrative personnel hold constantly a position between the better offs ( medical doctors and technical personnel ) and the most disadvantage occupational groups ( nurses and auxiliary staff ) on , almost , all the sf-36 dimensions ( apart from the vitality and role emotional scales ) . their scores are consistent with their professional status in the working environment of the greek hospitals not as high as that of the medical doctors but higher and less health damaging than that of nurses and auxiliary personnel . nurses who are the second biggest professional group included in this study have scored low on all dimensions of sf-36 . their poor health status and health related quality of life primarily reflect the difficulties they face in their everyday life and most importantly reflects the difficulty of being a woman working a job with a low professional ( and consequently social ) status in a predominantly male , highly competitive environment . a finding strengthening further this assumption about the stressfulness of the nursing profession in addition it should be taken into account that nurses of our sample were predominantly women and this might have an independent contribution to their higher than men morbidity rates . the auxiliary personnel seem to have by far the worst health status and poorer health related quality of life . they have reported the lowest scores on all sf-36 dimensions apart from that of mental health where nurses scored worst . the difference of 28.5 points observed on the bodily pain scale between the medical doctors ( 84.9 ) a profession of high social and professional status and the auxiliary personnel ( 56.4 ) who are unskilled manual workers of low social and professional status , is the greatest one observed in the present study . these findings are consistent with recent health research arguing about the impact of social gradient within the workplace and occupational hierarchy on health and well - being of people ( see for example ) and show that the greek version of sf-36 should be seen as an instrument having construct validity . regarding the workplace no safe conclusions can be drawn on differences among the seven hospitals included in this study . nevertheless , it seems that there are not any important health differences among the workers of our sample that could be attributed to workplace differences . the second major issue revealed is that of the observed differences between this study results and the national norms of other countries . although the population of this study does not represent the general greek population and its scores do not constitute the national norms of greece a comparison between the normative data of other countries and those of this study can produce useful preliminary though , since the sample of this study could be considerably different from the greek national norm conclusions regarding the differences between greece and other countries . especially since there are not any published normative data for greece and there is a lack of knowledge about the health related quality of life of the general population in greece . the participants in this study scored considerably lower on the 8 domains of the sf-36 than their counter partners in other studies from western europe and north america . the greek scores are lower than the canadian , uk and swedish norms almost in every scale . the italian norms are higher than those of the present study on the physical functioning , physical role , social functioning and role emotional scales and lower on the bodily pain , general health and vitality while for mental health scale both the italian norms and the results of this study are the same . however the magnitude of the differences between the italian norms and the greek results is quite small ( the only exemption is the difference observed on the social functioning scale 5.1 ) indicating a hidden influence of ( mediterranean ) culture on health outcomes as measured by sf-36 . comparison of the national norms of various countries with the scores of the present study * the scores of present study do not represent the greek national norms the present study has several limitations . the most important is that the comparison between the scores of this study and various national norms is a priori problematic since it is not age - adjusted and the study sample is not necessarily representative of the greek general population . but as already mentioned this comparison is only indicative and preliminary and is done only because the national greek norms have not been published . another limitation is the de facto small number of older participants which put into question the value of the observed health differences between the age groups of the sample . this study reveals the existence of considerable health inequalities among the employees in the greek hospitals . these health inequalities underscore the need for immediate interventions to tackle them and initiatives to support women , young and low professional status workers in the greek hospitals . moreover the findings of this study constitute an indication of the construct validity of the greek version of sf-36 .

numerous studies have shown associations of particulate matter air pollution characterized as particles smaller than 10 m ( pm10 ) or 2.5 m ( pm2.5 ) and adverse health effects . much less is known about health effects of particles smaller than 0.1 m , also known as ultrafine particles ( ufp ) , which may be more toxic because of their potential to penetrate deeper into the lungs , their higher biological reactivity per surface area , and their potential uptake in the bloodstream . ufp contributes only a small fraction to particle mass and thus ufp is not well reflected by pm10 or pm2.5 measurements . the lack of data on health effects of long - term ufp exposure is related to a lack of routine monitoring and models describing the large spatial variation of ufp . therefore , there is a need for models that provide long - term ufp exposure estimates at a fine spatial scale . land use regression ( lur ) models are a common approach in epidemiology to assess air pollution exposure at a fine spatial scale , using predictor variables from geographic information systems ( gis ) . lur models for pm2.5 and no2 are typically built on data from ( bi)weekly measurements at 2080 monitoring sites per study area . however , because of high costs and labor - intensive operation of ufp monitors , this approach is not attractive for ufp . recent studies developed ufp lur models based on short - term monitoring or mobile monitoring campaigns conducted while driving . previously published short - term and mobile ufp models substantially differed in model structure ( gis predictors included in the model ) and model performance ( percentage explained variability ( r ) ) . due to differences in area size , number of monitoring sites , duration , and frequency of monitoring , monitoring equipment , gis predictor variables , and model development procedures , it is unclear whether the difference in model structure and performance is due to inherent differences between study areas or due to these methodological issues . a recent study showed that models based on short - term and mobile monitoring in the same study area resulted in comparable model structures and highly correlated predictions at external sites . most studies develop a single best model , which is applied for exposure assessment in epidemiological studies . due to correlations between predictor variables , it is likely that alternative models can be developed which explain variability almost equally well . gulliver developed and interpreted four no2 models in the framework of 4-fold hold - out validation ( hv ) . wang applied model predictions of 40 models from a cross - validation method to predict subject s exposure to no2 in an epidemiological study . very few studies have developed multiple models for short - term monitoring designs ( hankey , 2015 ) . little is known about the robustness of model predictions at external sites by applying multiple models developed on one monitoring data set . using external sites is important as for short - term and mobile monitoring , the monitoring sites used for model development may differ systematically from the often residential addresses to which the model are applied , for example , in distance to roads . we performed a harmonized short - term monitoring campaign contemporaneously in six european study areas . we developed ten lur models per area based on 90% subsets of the sites , following a common modeling approach . our aims were to develop lur models for predicting spatial patterns in ufp for six european study areas ; to assess the agreement in lur model structure and performance within and between study areas . a further aim was to evaluate the performance of a model using the ufp concentration data from six study areas combined . important new contributions of this paper include ( a ) the evaluation of the robustness of model predictions at external residential sites , not included in model development in all six areas ; ( b ) validation of the models with ufp monitoring data with longer monitoring duration at residential external sites in two of the areas ; ( c ) an evaluation of the potential to develop a model for a large geographic area and comparison with performance of local models . in basel ( switzerland ) , heraklion ( greece ) , amsterdam , maastricht , and utrecht ( the netherlands , three cities collectively referred to as the netherlands ) , norwich ( united kingdom ) , sabadell ( spain ) , and turin ( italy ) , monitoring sites were selected based on criteria applied before in the escape and music studies , and evaluated by a team of experts from all centers ( supporting information ( si ) 1 ) . in each area , 160 sites were selected ( 240 in the netherlands because multiple cities were studied ) . for large spatial contrast in traffic intensities and land use , seven types of monitoring site were defined : traffic , urban background , urban green , water , highway , industry , and regional background , as applied before . measurements were made as close as possible to home faades , but not on private property . traffic sites were monitored close to home faades along a major road with > 10 000 vehicles / day , not on curbsides . urban background sites were close to home faades > 100 m away from a major road . urban green sites were at the edge of a park , water sites adjacent to a canal or a river , highway sites were within 100 m from a highway , industry sites were in a mixed industrial - residential zone , and regional background sites were outside the study city . traffic sites represented approximately 40% of the total sites in all areas . in all areas , a harmonized short - term monitoring campaign was conducted contemporaneously between january 2014 and february 2015 , measuring each monitoring site three times in different seasons ( summer , winter and spring / autumn ) . measurements were taken on monday - friday , and site types were visited in random order . at each visit , ufp concentrations were measured for 30 min following a prescribed protocol , and a gps coordinate was taken . to avoid rush hour influences and increase comparability between monitoring sites , measurements were taken between 9.00 am and 4.00 pm . during the entire measurement campaign , reference site ufp measurements were conducted in each area to allow temporal adjustment of local data . the reference site was an urban background location in the study area ( si 1 ) . in the large study area of the netherlands , the reference site was in one of the areas ( utrecht ) , 40 km from amsterdam and 140 km from maastricht . ufp was monitored in all study areas using a cpc 3007 ( tsi inc . , shoreview , mn ) , operating at a flow of 100 ml / min measuring particles ranging from 101000 nm at 1 s intervals . the cpc 3007 does not specifically measure ufp , but ufp typically dominates particle number . the reference sites in the netherlands and in heraklion were also equipped with a cpc , operating at identical settings , whereas other areas used a minidisc ( testo ag , lenzkirch , germany ) , because of the limited number of cpcs available . the minidisc operated at a flow of 1000 ml / min measuring particles from 10300 nm at 1 s intervals . we colocated the two instruments used in each study area regularly to check comparability . in the netherlands , norwich and sabadell , the mean ratio of the two instruments was close to unity ( si 2 ) . in turin , the cpc used at the short - term sites gave 27% lower readings than the minidisc used at the reference site . in heraklion , the monitoring site cpc gave 41% higher ufp readings than the reference site cpc with large variation . we did not correct for these differences , as the reference site measurements is used only to correct for temporal variation . qa / qc included zero checks before and after measurements and regular colocation of all ufp monitors per study area at the local reference site for at least 3 h per exercise . all site and reference measurements were averaged over the corresponding period , after removing measurements with error codes of the instrument ( e.g. , deviating flow ) . extreme reference site 30 min measurements , defined as more than four interquartile ranges ( iqrs ) lower or higher than the 25th or 75th percentile , were flagged and individually inspected , as they might indicate local sources near the reference site ( e.g. , diesel - powered grass mower near the dutch site ) not reflective of concentration patterns in the wider area . we identified 15 , 30 min reference observations as indicative of local sources ( 3 in the netherlands , 10 in norwich , 2 in sabadell ) , 0.5% of all reference site observations . in turin , reference site measurements were missing for 65% of the 480 , 30 min measurement periods due to misinterpretation of the protocol . a regression model using routine nox , hour of the day , barometric pressure and relative humidity , fit on the valid 35% of the data ( r 62% ) , was applied to impute the missing 30 min reference site observations ( si 3 ) . in norwich 17% of the reference site data was missing due to operational problems . a regression model built on routine and meteorological data ( r 50% ) was used to impute these missing observations ( si 3 ) . in the other areas , no predictive model could be developed ( percentage missing < 10% in netherlands , basel , and sabadell and 18% in heraklion ) . to improve assessment of spatial contrasts between sites , the ufp concentration at the local reference site was used to adjust monitored ufp levels for temporal variability in three steps , following procedures of previous studies . first , the mean reference ufp concentration of the corresponding interval was subtracted from the annual mean concentration at the reference site . third , the adjusted average ufp concentration was calculated as the average of three adjusted samples from one site . application of the ratio method ( accounting for differences between two instruments ) resulted in unrealistic averages due to large individual ratios ( up to 8) on days with low ufp concentrations at the reference site . gps coordinates from three site visits were averaged and manually corrected for optimal accuracy in position relative to roads on detailed road maps . predictor variables were generated locally for each of these sites in a gis , using coordinates and digital data sets on traffic , heavy traffic , population density , land use and restaurant density . predictors and buffer sizes were similar to these used in the escape and music studies , supplemented with airport land use and restaurant data because of studies documenting increased outdoor ufp concentrations related to emissions from airports and restaurants , and the inclusion of restaurants in a previous ufp model . traffic and heavy traffic predictors were collected at buffer radii of 50 , 100 , 300 , 500 , and 1000 m from the best available road network data ( si 4 ) . population and land use predictors at radii of 100 , 300 , 500 , 1000 , and 5000 m ( land use defined as airport only radii of 1000 and 5000 m ) were collected from population density data from the european environmental agency and corine land use data sets ( coordination of information on the environment ) . number of restaurants was collected at radii of 100 , 300 , 500 , 1000 , and 5000 m using the open street map application turbo overpass . heavy traffic data from basel , heraklion , sabadell and turin were not available in a gis . restaurant data do not cover all restaurants in the city as inclusion in the database is not free ( si 4 ) . restaurant data were not used for heraklion , since the number of amenities was underreported and did not reflect realistic distributions across neighborhoods . we used external sites to test the robustness of predictions of the 10 lur models . residential addresses of 3148 subjects per study area participating in the exposomics study were used for all areas except heraklion . in heraklion , 50 randomly selected addresses were used . gis predictors for subject s home addresses were collected to test robustness of model predictions . additionally , in basel and the netherlands 24 h average outdoor ufp concentrations were monitored at the home faade with minidiscs in three seasons . study period and study area were harmonized between the short - term monitoring campaign and residential outdoor measurements . the temporally adjusted average ufp concentration was used for external model validation when at least two valid 24 h observations were available . lur models were developed centrally by applying procedures equivalent to procedures applied in the escape and music studies . briefly , temporal - variation adjusted 30 min average ufp concentration per site was used as dependent variable in a linear regression model , using gis predictors as explanatory variables . predictors that were not available for all areas or present in less than 50% of the areas were not used in the combined area model . predictors were selected using a supervised stepwise selection procedure , selecting the variable with the largest adjusted r to the model if the direction of effect was as defined a priori and did not change the direction of effect of previously included variables . this process was continued until no more variables provided a gain in adjusted r. variables included were checked for p - values ( removed when p - value > 0.10 ) , collinearity ( removed when variance inflation factor > 3 ) , and influential observations ( if cook s d > 1 the model was further examined ) . in each area , 10 models were developed to evaluate robustness of model structure and model predictions at the external sites , following the 10-fold cross - validation approach . first , monitoring sites were stratified by site type ( traffic vs nontraffic ) and subsequently randomly distributed in 10 groups . next , each time 90% ( 9 groups ) of the sites was used for model development and 10% ( 1 group ) for validation . the model r and root mean square error ( rmse ) were obtained from each individual model , the hv r and rmse were obtained by predicting ufp levels in each validation set and regressing these against measured values over all pooled random draws . in basel and the netherlands , an additional validation was obtained by testing modeled against measured 24 h outdoor ufp concentrations at the external sites . we calculated bias , defined as the average of modeled minus measured ufp . for model structure comparison , we classified predictors in nearby traffic ( traffic predictors , radius 100 m ) , distant traffic ( traffic predictors , radius > 100 m ) , population , industry , port , airport , restaurants , and green space . predictions from the 10 models at external sites in a specific area were compared with scatterplots and correlation coefficients . predictions were performed after truncation of predictors such that they were within the range in the model development data ( truncation applied on one site in basel , two sites in heraklion , two sites in the netherlands , three sites in norwich ) . external sites are residential addresses of participants in the personal exposure monitoring survey . ten models on combining data from all areas were developed following the procedure described before , additionally stratifying sites by study area prior to stratification by site type . to account for systematic differences in background concentration between study areas , we specified random intercepts using a linear mixed - effect model after the supervised stepwise model development procedure . we further evaluated random slopes to account for differences in emissions due to , for example , composition of the vehicle fleet across areas . leave one area out validation ( loaov ) was applied to explore applicability of combined lur models in areas without measurements . all short - term sites from one area were excluded and one model was developed for all other areas . a random intercept per area was introduced and the loaov r and rmse were obtained by evaluating modeled and measured ufp levels in the excluded area . for basel and the netherlands loaov models were also compared with measurements at the external sites . gis predictors were generated locally in arcgis ( esri , redlands , ca ) ( land use , population , and traffic predictors ) and in the overpass turbo and qgis applications ( restaurant predictors ) . local data cleaning and calculations per center were performed using the statistical package available ( sas , stata , r ) , final checks and model building were performed using the statistical package r 3.2.2 . for lur model development , 160 monitoring sites per city in basel , heraklion , sabadell and turin , 161 sites in norwich and 242 sites in the netherlands were monitored ( total 1043 sites ) . adjusted average ufp observation were included for lur modeling when based on at least two 30 min site observations , corrected for corresponding reference measurements , leading to loss of 1 site in basel , 2 sites in the netherlands and 10 sites in heraklion , an overall loss of 13 sites ( 1.2% ) . there was large variability in adjusted average ufp concentrations among sites in all study areas ( figure 2 ) . concentrations were highest at the traffic sites and industrial sites in turin and sabadell . the average within site standard deviation after temporal adjustment was 6985 particles / cm , 51% of the overall mean across study areas . the netherlands is comprised out of the cities of amsterdam , utrecht , and maastricht . model r differed between areas , ranging from on average 28% in sabadell to 48% in the netherlands . model r and rmse of the ten models within areas were very similar ( table 1 ) . within an area , the 10 models typically contained one to three predictor categories ( e.g. , nearby traffic ) in all 10 models ( figure 3 ) . other predictor variables were included in a selection of models , such as port -included in 6 of 10 models in the netherlands- or industry which was included in 6 of 10 models in turin . the exact predictor variables ( e.g. , traffic intensity nearest road ) and coefficients differed more among the 10 models ( si 5 ) . between study areas more difference in model structures was seen ( figure 3 , si 5 ) . nearby traffic was included in all models , population was included in 46 of 60 models ( not at all in sabadell ) , industry was included in 41 of 60 models ( not at all in basel ) , and restaurant data were included in all local models in basel and sabadell , but not in any model of the other study areas . selection of predictor categories in the 10 models per study area . explained variability ( r ) , root mean square error ( rmse ) , and bias ( difference between modeled and measured ufp ) from the 10 models at model development , holdout validation ( hv , based on pooled analysis ) , and at application on external sites . model robustness expressed in mean and sd of predicted ufp / cm and intraclass correlation coefficient ( icc ) of 10 models at application on external sites . hv r decreased by 720% compared to model r and rmse increased by about 10% ( table 1 ) . the models predicted ufp variability at external sites with longer duration monitoring substantially better ( r in basel 53% and in the netherlands 50% ) at the external sites , there was virtually no bias for the netherlands and a modest 20% systematic overestimation at the basel sites . consistent with the modest differences in local model structure , ufp predictions among models per area were highly correlated ( figure 4 , table1 ) . predictions in individual models showed high similarity in basel , the netherlands , sabadell , and turin ( icc 0.960.98 ) and more variation in norwich and especially heraklion ( figure 4 and si 6 ) . because of the high consistency of models , we also developed models based on 100% of the sites ( si 7 ) . in each area , models were very similar to the 10 models per area . ; predicted ufp concentrations of each model plotted against each other for basel ( a , highly similar ) and heraklion ( b , more variation ) in the lower panel , together with the pearson correlation coefficient in the upper panel . red lines represent the best fit lines ; * * * = p - value < 0.001 . a random slope per area did not improve prediction and was not included ( si 5 and 8) . models built on short - term sites from all areas resulted in a model r of 34% with low sd ( table 2 ) . every model consisted of predictors representing nearby traffic , distant traffic , population and industry ( si 5 ) . modeled concentrations of the 10 models on external sites hv r over all areas was close to the model r. hv r and rmse of the combined model assessed per area were similar to hv r and rmse of local models ( table 3 ) . validation r at external sites in both basel and the netherlands was higher than hv r , comparable to performance in local models . explained variability ( r ) , root mean square error ( rmse ) , and bias ( difference between modeled and measured ufp ) from the 10 models at model development , holdout validation ( hv , based on pooled analysis ) , and at application on external sites . model robustness expressed in mean and sd of predicted ufp / cm and intraclass correlation coefficient ( icc ) of 10 models at application on external sites . based on values prior to introduction of random intercept leave one area out validation ( loaov ) models are based on combined model with one complete area excluded in model development , on which the model is subsequently tested . holdout validation ( hv ) r , root mean square error ( rmse ) , and bias ( difference between modeled and measured ufp ) of local and combined area models repeated from tables 1 and 2 . local and combined area external r , rmse and bias are based on average predicted ufp concentration of the 10 models . we further tested the combined model by dropping complete areas from the model development ( si 9 ) . the loaov r was close to the hv r of local and combined models . when applying loaov models on external sites , it performed equally well as local and combined area models in basel , where in the netherlands r decreased and rmse increased ( table 3 ) . systematic overestimation ( heraklion , turin ) and underestimation ( sabadell , switzerland ) up to about 30% of the overall mean were found for combined models excluding complete areas . at external sites overestimation of about 2% ( netherlands ) and 20% ( basel ) were found . measurements at the external sites were 24 h averages , including night - time with typically lower concentrations . lur models for ufp were developed in six european areas based on harmonized short - term monitoring campaigns and a common modeling approach . the 10 models developed within each area were generally robust in model structure and in prediction at external sites . validation at external sites with repeated 24 h monitoring in two of the six areas showed substantially higher rs ( 5053% ) . a combined area model explained ufp variability at external sites from two areas equally well as local models . predictor categories selected in the 10 models per area had high agreement , resulting in highly correlated model predictions at external sites . exact predictors in final models could differ , but due to correlation of predictors within a predictor category , modeled ufp concentrations were highly correlated . variables like traffic intensity and heavy traffic intensity on the nearest road , variables of two adjacent buffer categories ( e.g. , 300 and 500 m ) as well as population and address density , were correlated as observed before . predicted ufp levels from local models were very consistent in four of the areas , with slightly higher variability in heraklion and norwich . in these two areas more moderate correlations were found with 2 of the 10 models which included the predictor traffic intensity divided by distance . in heraklion , one of the models with lower correlation had a lower coefficient for traffic on the nearest road ( the main predictor in the heraklion models ) compared to the other nine models . this likely contributed to the more modest correlation with other models . despite harmonized monitoring and modeling approaches , differences in model r , rmse and structure were found between the six areas , which were much larger than differences between the 10 models within an area . models from all areas included nearby traffic often traffic intensity at the nearest street- , consistent with the major influence of motorized traffic emissions on urban ufp concentrations . nearby traffic variables predicted a substantial contrast in ufp , of typically 40006000 particles / cm for a difference between the 10th and 90th percentile of the predictor . the relatively high number of traffic predictors offered is another potential explanation , however the inclusion of many more near compared to distant traffic predictors argues in favor of the source interpretation . population density was included in all 10 models in four of the six areas , 6 out of 10 models in heraklion , and in none of the models in sabadell . this is possibly due to the lower population variability in this moderate sized town . industry , port , airport and restaurants were included in models of only one or a few areas . port in a 5 km buffer was only represented in heraklion and the netherlands , not located within this radius in the other areas . airport was not selected in the netherlands , probably because few sites were located within a 5 km radius of an airport . the inclusion of these nontraffic sources is consistent with studies documenting that ufp emissions are related to multiple combustion sources . we do not have a clear explanation of the difference in model r between the six study areas . differences in model r could be due to the characteristics of the study area such as size and complexity , but also to differences in the variability of gis predictor variables . different performance of our temporal adjustment may have contributed to variability in model r as well . in norwich and especially turin , imputation of measurements at the reference site was used to avoid missing values . the current local model rs , ranging from 28% to 48% , and predictors used in these models are comparable to those reported of spatial lur models in previous short - term monitoring work . in girona province , spain , a model with only traffic predictors captured 36% of ufp variability at 644 sites measured for a single 15 min period . for vancouver , canada , a single measurement at 80 locations resulted in model rs from 29 to 53% including traffic population , port and restaurant predictors . in amsterdam and rotterdam , the netherlands , 37% of ufp variability at 160 sites was explained with traffic , population and port predictors . our spatial models can be applied for assessing long - term average exposures . we did not develop spatiotemporal models , further including temporal predictors such as temperature , to allow temporally more refined estimates . a low hv r , however , does not imply that models do not provide valid predictions , as argued previously . current ufp models predicted repeated 24 h measurements from basel and the netherlands substantially better than the hv r suggested . for both areas a moderately high r of around 50% was found , compared to hv r of 18 and 35% in basel and the netherlands . we previously documented higher external validation r related to longer averaging times at the external validation sites relative to the model development sites in two studies . our spatial predictors are constant in time and therefore can not explain remaining temporal variation in short - term measurements . repeated 24 h measurements likely reflect long - term average ufp concentrations better than short - term monitoring , because these observations are less affected by temporal exposure variation . model r and hv r from short - term monitoring may not be the metric that should be leading in assessing model performance . based on these metrics models from the netherlands ( r = 48% ) were better than basel models ( r = 30% ) , but at external sites models performed equally well . this suggests that testing on external sites with longer - term monitoring is a better tool to assess performance . long - term ufp concentration data are however not routinely available and thus require a dedicated monitoring effort , as illustrated in a recent swiss study where external validation from routine monitoring was available for four sites for ufp and 80100 sites for pm10 and no2 . model and hv r were lower in our and most other short - term and mobile lur models for ufp compared to lur models developed for pollutants such as no2 and pm2.5 . the large spatial variation of ufp may be more difficult to model , but the use of short - term averages for ufp compared to much longer average times for no2 and pm2.5 likely explains part of the difference in model r. in a swiss study , based upon 2 week monitoring periods , model r was similar for ufp and pm2.5 absorbance and higher than for pm2.5 and no2 . combined model r was 34% with very high consistency across the 10 models , almost similar pooled hv r , and identical predictor categories represented . within the different areas combined models performed almost similar to local models in hv r and rmse . the relatively modest differences in ufp concentrations across study areas and the dominance of traffic as the major predictor may have contributed to the possibility to develop combined models that were only slightly less predictive than the local models . while ufp concentrations were somewhat higher in sabadell and turin , the difference with the other areas was lower than previously reported for pollutants such as pm2.5 , no2 and black carbon . the rationale for developing combined area models is especially that combined area models may be applied in areas without monitoring more readily than single area models . models for large geographical areas for other pollutants are increasingly developed and our study suggests that this approach is feasible for ufp as well . increased model validity related to using more sites is another rationale . problems with developing combined models include availability and comparability of predictor data and assumptions of the same effect of a specific predictor ( e.g. , traffic nearest road ) on concentrations . for example different traffic compositions may result in different associations to traffic related predictors per area , but this was not observed in the current study ( si 8) . if a predictor variable ( source ) is present in a few areas only , it is difficult to distinguish the influence of this source from other systematic differences between areas . in the current study , this potentially contributed to the lower model r compared to local models from heraklion , the netherlands , or turin in the current study , since ufp variability can no longer be explained by port or restaurant . lur models with short - term sites from one area excluded explained ufp variability in basel equally well as local and combined models , where loaov r remained at 53% . in the netherlands loaov r dropped by 10% and the netherlands study area was the only individual study area that covered a large geographical area with both large cities and smaller towns . the loaov model in contrast to the local model did not include 5000 m population and address density , accounting for these urbanistic related differences . these results suggest that transferability of models to independent areas is more difficult , but this could only be tested in two areas . the use of local sites in the development of lur models seems to be beneficial for model fit at independent sites , as shown for the netherlands . we suggest to apply all the 10 models we developed to assess long - term ufp exposure in epidemiological studies and to perform 10 epidemiological analyses . this will allow assessment of the consistency of epidemiological associations obtained with these 10 different models , improving assessment of uncertainty of effect estimates beyond standard errors . the number of models could be extended , using , for example , monte carlo approaches . applying multiple models will likely provide consistent associations for models with high agreement , but more variation for areas with lower iccs ( norwich and heraklion ) . alternatively , exposure could be the average of 10 models ( for example by bayesian model averaging ) applied at cohort addresses . exposure estimates will in both cases depend less on specific selected gis variables compared to using a single best model based on model r. this is particularly of interest for variables for which it is unclear whether they are causally related to ufp or are proxies for other variables . an example is the variable port in the netherlands , which was selected in six of ten models . port has been a predictor in previous ufp lur models , but in the current study could also represent other differences between the city of amsterdam ( with port ) and the other two dutch cities without ports . the inclusion of port in 6 of 10 models may reflect the uncertainty of the importance of this variable . the lack of inclusion of port in some models was not due to too few sites with a nonzero value : 77 of the sites had a nonzero value . for epidemiological studies within the study areas covered by monitoring , we suggest to primarily use the local models . although our study did not show large differences in performance compared to the combined model , the inclusion of more specific predictors in the local model favors its use . the combined model could be applied as a further test of consistency of epidemiological findings . as our study areas did not cover very large metropolitan areas ( london , paris ) , northern or central and eastern europe , rural areas , nor altitude differences we therefore advise to apply the combined model in urban areas similar to the monitored areas . a combined model is furthermore more useful in multicity studies than in single city studies , particularly if between - city contrasts in exposure are exploited .

long - term ultrafine particle ( ufp ) exposure estimates at a fine spatial scale are needed for epidemiological studies . land use regression ( lur ) models were developed and evaluated for six european areas based on repeated 30 min monitoring following standardized protocols . in each area ; basel ( switzerland ) , heraklion ( greece ) , amsterdam , maastricht , and utrecht ( the netherlands ) , norwich ( united kingdom ) , sabadell ( spain ) , and turin ( italy ) , 160240 sites were monitored to develop lur models by supervised stepwise selection of gis predictors . for each area and all areas combined , 10 models were developed in stratified random selections of 90% of sites . ufp prediction robustness was evaluated with the intraclass correlation coefficient ( icc ) at 3150 external sites per area . models from basel and the netherlands were validated against repeated 24 h outdoor measurements . structure and model r2 of local models were similar within , but varied between areas ( e.g. , 3843% turin ; 2531% sabadell ) . robustness of predictions within areas was high ( icc 0.730.98 ) . external validation r2 was 53% in basel and 50% in the netherlands . combined area models were robust ( icc 0.931.00 ) and explained ufp variation almost equally well as local models . in conclusion , robust ufp lur models could be developed on short - term monitoring , explaining around 50% of spatial variance in longer - term measurements .

Introduction Materials and Methods Results Discussion

much less is known about health effects of particles smaller than 0.1 m , also known as ultrafine particles ( ufp ) , which may be more toxic because of their potential to penetrate deeper into the lungs , their higher biological reactivity per surface area , and their potential uptake in the bloodstream . therefore , there is a need for models that provide long - term ufp exposure estimates at a fine spatial scale . land use regression ( lur ) models are a common approach in epidemiology to assess air pollution exposure at a fine spatial scale , using predictor variables from geographic information systems ( gis ) . recent studies developed ufp lur models based on short - term monitoring or mobile monitoring campaigns conducted while driving . previously published short - term and mobile ufp models substantially differed in model structure ( gis predictors included in the model ) and model performance ( percentage explained variability ( r ) ) . due to differences in area size , number of monitoring sites , duration , and frequency of monitoring , monitoring equipment , gis predictor variables , and model development procedures , it is unclear whether the difference in model structure and performance is due to inherent differences between study areas or due to these methodological issues . a recent study showed that models based on short - term and mobile monitoring in the same study area resulted in comparable model structures and highly correlated predictions at external sites . due to correlations between predictor variables , it is likely that alternative models can be developed which explain variability almost equally well . gulliver developed and interpreted four no2 models in the framework of 4-fold hold - out validation ( hv ) . very few studies have developed multiple models for short - term monitoring designs ( hankey , 2015 ) . little is known about the robustness of model predictions at external sites by applying multiple models developed on one monitoring data set . using external sites is important as for short - term and mobile monitoring , the monitoring sites used for model development may differ systematically from the often residential addresses to which the model are applied , for example , in distance to roads . we performed a harmonized short - term monitoring campaign contemporaneously in six european study areas . we developed ten lur models per area based on 90% subsets of the sites , following a common modeling approach . our aims were to develop lur models for predicting spatial patterns in ufp for six european study areas ; to assess the agreement in lur model structure and performance within and between study areas . important new contributions of this paper include ( a ) the evaluation of the robustness of model predictions at external residential sites , not included in model development in all six areas ; ( b ) validation of the models with ufp monitoring data with longer monitoring duration at residential external sites in two of the areas ; ( c ) an evaluation of the potential to develop a model for a large geographic area and comparison with performance of local models . in basel ( switzerland ) , heraklion ( greece ) , amsterdam , maastricht , and utrecht ( the netherlands , three cities collectively referred to as the netherlands ) , norwich ( united kingdom ) , sabadell ( spain ) , and turin ( italy ) , monitoring sites were selected based on criteria applied before in the escape and music studies , and evaluated by a team of experts from all centers ( supporting information ( si ) 1 ) . in each area , 160 sites were selected ( 240 in the netherlands because multiple cities were studied ) . in all areas , a harmonized short - term monitoring campaign was conducted contemporaneously between january 2014 and february 2015 , measuring each monitoring site three times in different seasons ( summer , winter and spring / autumn ) . during the entire measurement campaign , reference site ufp measurements were conducted in each area to allow temporal adjustment of local data . in the large study area of the netherlands , the reference site was in one of the areas ( utrecht ) , 40 km from amsterdam and 140 km from maastricht . the reference sites in the netherlands and in heraklion were also equipped with a cpc , operating at identical settings , whereas other areas used a minidisc ( testo ag , lenzkirch , germany ) , because of the limited number of cpcs available . in the netherlands , norwich and sabadell , the mean ratio of the two instruments was close to unity ( si 2 ) . all site and reference measurements were averaged over the corresponding period , after removing measurements with error codes of the instrument ( e.g. extreme reference site 30 min measurements , defined as more than four interquartile ranges ( iqrs ) lower or higher than the 25th or 75th percentile , were flagged and individually inspected , as they might indicate local sources near the reference site ( e.g. we identified 15 , 30 min reference observations as indicative of local sources ( 3 in the netherlands , 10 in norwich , 2 in sabadell ) , 0.5% of all reference site observations . in the other areas , no predictive model could be developed ( percentage missing < 10% in netherlands , basel , and sabadell and 18% in heraklion ) . predictors and buffer sizes were similar to these used in the escape and music studies , supplemented with airport land use and restaurant data because of studies documenting increased outdoor ufp concentrations related to emissions from airports and restaurants , and the inclusion of restaurants in a previous ufp model . heavy traffic data from basel , heraklion , sabadell and turin were not available in a gis . we used external sites to test the robustness of predictions of the 10 lur models . gis predictors for subject s home addresses were collected to test robustness of model predictions . additionally , in basel and the netherlands 24 h average outdoor ufp concentrations were monitored at the home faade with minidiscs in three seasons . study period and study area were harmonized between the short - term monitoring campaign and residential outdoor measurements . lur models were developed centrally by applying procedures equivalent to procedures applied in the escape and music studies . predictors that were not available for all areas or present in less than 50% of the areas were not used in the combined area model . predictors were selected using a supervised stepwise selection procedure , selecting the variable with the largest adjusted r to the model if the direction of effect was as defined a priori and did not change the direction of effect of previously included variables . in each area , 10 models were developed to evaluate robustness of model structure and model predictions at the external sites , following the 10-fold cross - validation approach . first , monitoring sites were stratified by site type ( traffic vs nontraffic ) and subsequently randomly distributed in 10 groups . in basel and the netherlands , an additional validation was obtained by testing modeled against measured 24 h outdoor ufp concentrations at the external sites . predictions were performed after truncation of predictors such that they were within the range in the model development data ( truncation applied on one site in basel , two sites in heraklion , two sites in the netherlands , three sites in norwich ) . leave one area out validation ( loaov ) was applied to explore applicability of combined lur models in areas without measurements . a random intercept per area was introduced and the loaov r and rmse were obtained by evaluating modeled and measured ufp levels in the excluded area . for basel and the netherlands loaov models were also compared with measurements at the external sites . gis predictors were generated locally in arcgis ( esri , redlands , ca ) ( land use , population , and traffic predictors ) and in the overpass turbo and qgis applications ( restaurant predictors ) . for lur model development , 160 monitoring sites per city in basel , heraklion , sabadell and turin , 161 sites in norwich and 242 sites in the netherlands were monitored ( total 1043 sites ) . adjusted average ufp observation were included for lur modeling when based on at least two 30 min site observations , corrected for corresponding reference measurements , leading to loss of 1 site in basel , 2 sites in the netherlands and 10 sites in heraklion , an overall loss of 13 sites ( 1.2% ) . the netherlands is comprised out of the cities of amsterdam , utrecht , and maastricht . model r differed between areas , ranging from on average 28% in sabadell to 48% in the netherlands . within an area , the 10 models typically contained one to three predictor categories ( e.g. other predictor variables were included in a selection of models , such as port -included in 6 of 10 models in the netherlands- or industry which was included in 6 of 10 models in turin . nearby traffic was included in all models , population was included in 46 of 60 models ( not at all in sabadell ) , industry was included in 41 of 60 models ( not at all in basel ) , and restaurant data were included in all local models in basel and sabadell , but not in any model of the other study areas . selection of predictor categories in the 10 models per study area . explained variability ( r ) , root mean square error ( rmse ) , and bias ( difference between modeled and measured ufp ) from the 10 models at model development , holdout validation ( hv , based on pooled analysis ) , and at application on external sites . model robustness expressed in mean and sd of predicted ufp / cm and intraclass correlation coefficient ( icc ) of 10 models at application on external sites . the models predicted ufp variability at external sites with longer duration monitoring substantially better ( r in basel 53% and in the netherlands 50% ) at the external sites , there was virtually no bias for the netherlands and a modest 20% systematic overestimation at the basel sites . predictions in individual models showed high similarity in basel , the netherlands , sabadell , and turin ( icc 0.960.98 ) and more variation in norwich and especially heraklion ( figure 4 and si 6 ) . in each area , models were very similar to the 10 models per area . ; predicted ufp concentrations of each model plotted against each other for basel ( a , highly similar ) and heraklion ( b , more variation ) in the lower panel , together with the pearson correlation coefficient in the upper panel . models built on short - term sites from all areas resulted in a model r of 34% with low sd ( table 2 ) . modeled concentrations of the 10 models on external sites hv r over all areas was close to the model r. hv r and rmse of the combined model assessed per area were similar to hv r and rmse of local models ( table 3 ) . validation r at external sites in both basel and the netherlands was higher than hv r , comparable to performance in local models . explained variability ( r ) , root mean square error ( rmse ) , and bias ( difference between modeled and measured ufp ) from the 10 models at model development , holdout validation ( hv , based on pooled analysis ) , and at application on external sites . model robustness expressed in mean and sd of predicted ufp / cm and intraclass correlation coefficient ( icc ) of 10 models at application on external sites . holdout validation ( hv ) r , root mean square error ( rmse ) , and bias ( difference between modeled and measured ufp ) of local and combined area models repeated from tables 1 and 2 . local and combined area external r , rmse and bias are based on average predicted ufp concentration of the 10 models . when applying loaov models on external sites , it performed equally well as local and combined area models in basel , where in the netherlands r decreased and rmse increased ( table 3 ) . at external sites overestimation of about 2% ( netherlands ) and 20% ( basel ) were found . measurements at the external sites were 24 h averages , including night - time with typically lower concentrations . lur models for ufp were developed in six european areas based on harmonized short - term monitoring campaigns and a common modeling approach . the 10 models developed within each area were generally robust in model structure and in prediction at external sites . validation at external sites with repeated 24 h monitoring in two of the six areas showed substantially higher rs ( 5053% ) . a combined area model explained ufp variability at external sites from two areas equally well as local models . predictor categories selected in the 10 models per area had high agreement , resulting in highly correlated model predictions at external sites . exact predictors in final models could differ , but due to correlation of predictors within a predictor category , modeled ufp concentrations were highly correlated . models from all areas included nearby traffic often traffic intensity at the nearest street- , consistent with the major influence of motorized traffic emissions on urban ufp concentrations . population density was included in all 10 models in four of the six areas , 6 out of 10 models in heraklion , and in none of the models in sabadell . port in a 5 km buffer was only represented in heraklion and the netherlands , not located within this radius in the other areas . airport was not selected in the netherlands , probably because few sites were located within a 5 km radius of an airport . differences in model r could be due to the characteristics of the study area such as size and complexity , but also to differences in the variability of gis predictor variables . the current local model rs , ranging from 28% to 48% , and predictors used in these models are comparable to those reported of spatial lur models in previous short - term monitoring work . current ufp models predicted repeated 24 h measurements from basel and the netherlands substantially better than the hv r suggested . for both areas a moderately high r of around 50% was found , compared to hv r of 18 and 35% in basel and the netherlands . our spatial predictors are constant in time and therefore can not explain remaining temporal variation in short - term measurements . repeated 24 h measurements likely reflect long - term average ufp concentrations better than short - term monitoring , because these observations are less affected by temporal exposure variation . model r and hv r from short - term monitoring may not be the metric that should be leading in assessing model performance . based on these metrics models from the netherlands ( r = 48% ) were better than basel models ( r = 30% ) , but at external sites models performed equally well . this suggests that testing on external sites with longer - term monitoring is a better tool to assess performance . long - term ufp concentration data are however not routinely available and thus require a dedicated monitoring effort , as illustrated in a recent swiss study where external validation from routine monitoring was available for four sites for ufp and 80100 sites for pm10 and no2 . model and hv r were lower in our and most other short - term and mobile lur models for ufp compared to lur models developed for pollutants such as no2 and pm2.5 . the large spatial variation of ufp may be more difficult to model , but the use of short - term averages for ufp compared to much longer average times for no2 and pm2.5 likely explains part of the difference in model r. in a swiss study , based upon 2 week monitoring periods , model r was similar for ufp and pm2.5 absorbance and higher than for pm2.5 and no2 . within the different areas combined models performed almost similar to local models in hv r and rmse . the relatively modest differences in ufp concentrations across study areas and the dominance of traffic as the major predictor may have contributed to the possibility to develop combined models that were only slightly less predictive than the local models . while ufp concentrations were somewhat higher in sabadell and turin , the difference with the other areas was lower than previously reported for pollutants such as pm2.5 , no2 and black carbon . for example different traffic compositions may result in different associations to traffic related predictors per area , but this was not observed in the current study ( si 8) . in the current study , this potentially contributed to the lower model r compared to local models from heraklion , the netherlands , or turin in the current study , since ufp variability can no longer be explained by port or restaurant . lur models with short - term sites from one area excluded explained ufp variability in basel equally well as local and combined models , where loaov r remained at 53% . in the netherlands loaov r dropped by 10% and the netherlands study area was the only individual study area that covered a large geographical area with both large cities and smaller towns . the use of local sites in the development of lur models seems to be beneficial for model fit at independent sites , as shown for the netherlands . we suggest to apply all the 10 models we developed to assess long - term ufp exposure in epidemiological studies and to perform 10 epidemiological analyses . port has been a predictor in previous ufp lur models , but in the current study could also represent other differences between the city of amsterdam ( with port ) and the other two dutch cities without ports . for epidemiological studies within the study areas covered by monitoring , we suggest to primarily use the local models .

many critically ill patients admitted to the intensive care unit ( icu ) enter a state of negative energy balance during the first 34 days following admission [ 1 , 2 ] . this energy deficit often progresses during their icu stay and may result in malnutrition and adverse outcomes . multiple factors contribute to energy deficit , including increased metabolism [ 3 , 4 ] , delays in the initiation of feeding , and inadequate caloric provision . indeed , a number of studies have demonstrated that target caloric intake is achieved in only 5075% of icu patients and that as many as 25% of patients receive only 1,0001,500 kcal / day . prolonged negative energy balance within the icu is associated with serious complications [ 1 , 2 ] . in two separate studies , progressive negative energy balance was strongly correlated with increased numbers of infectious complications , particularly sepsis [ 1 , 2 ] . in addition , cumulative total energy deficit has been correlated with increased length of mechanical ventilation , length of icu stay , total number of complications , and duration of antibiotic use . delayed initiation of feeding and/or negative energy balance in critical care patients may also be associated with higher icu and in - hospital mortality rates [ 5 , 6 ] , while early initiation of feeding results in improved caloric intake . this article aims to review well - known papers that evaluate potential biologic effects of lipids when provided as a parenteral energy source and to provide future perspectives on the use of parenteral lipid emulsions ( les ) in critically ill patients . given the association between negative energy balance / malnutrition and both morbidity and mortality , ensuring that critically ill patients receive adequate caloric and nutrient intake should be a high priority for intensive care clinicians . current guidelines recommend that all icu patients who tolerate enteral nutrition ( en ) should receive en ( approximately 2530 kcal / kg per day ) if they are not expected to be on a full oral diet within 3 days . however , within the icu setting en may not be feasible or can not be established at rates that provide adequate nutrition for a number of reasons . for example , en may be frequently interrupted because of diagnostic investigations , surgery , diarrhea , vomiting , mechanical problems ( e.g. , tube displacement ) or patient transfers . en may be contraindicated in patients with anatomic gastrointestinal disorders , severe diarrhea , and reduced intestinal blood flow . parenteral nutrition ( pn ) is therefore recommended under certain circumstances ( table 1 ) .table 1potential indications for parenteral nutrition in intensive care patients [ 810]parenteral nutrition as a supplement to enteral nutritionparenteral nutrition aloneenteral nutrition is insufficient to meet target caloric intakeintolerance to enteral nutritionsuppression of gastrointestinal activity ( e.g. , immediately following injury or surgery)major gut failure ( e.g. , extensive intestinal resection)conditions preventing adequate nutrient absorption ( e.g. , inflammatory bowel disease , gastric outlet obstruction , intractable vomiting , severe diarrhea , paralytic ileus ) potential indications for parenteral nutrition in intensive care patients [ 810 ] despite these recommendations , pn is often underused . although pn was once a popular means of administering nutrients , utilization has decreased in recent years because of concerns regarding metabolic complications associated with overfeeding [ 12 , 13 ] and an increased risk of septic complications . it has been suggested that en is required in order to maintain gut function and is less likely to cause bacterial translocation than pn , although other perspectives exist [ 12 , 14 ] . however , available literature suggests that supplementation of en with pn enhances caloric intake and nutritional status [ 1 , 15 ] and that pn is as safe as en in critically ill patients [ 16 , 17 ] . the systemic inflammatory response is associated with metabolic stress in critically ill patients , including an overall increase in metabolic rate , production of reactive species , insulin resistance , and alterations in substrate utilization that result in hyperglycemia ( partly because of increased gluconeogenesis ) , lipolysis , and increased proteolysis relative to protein synthesis , potentially resulting in negative nitrogen balance [ 3 , 4 , 11 , 18 , 19 ] . the nutrition provided to patients within the icu setting should therefore aim to blunt this catabolic state and enhance anabolic activity during recovery while avoiding overfeeding . administration of dextrose must often be balanced with infusion of insulin to maintain euglycemia and minimize increases in carbon dioxide ( co2 ) production . lipids are generally administered to provide 30% of total calories , but dosage reduction should be considered when triglyceride concentrations are > 400 mg / dl . nutritional requirements vary depending upon the level of metabolic stress ; therefore , energy requirements , as well as blood glucose and electrolyte concentrations , should be monitored on a daily basis , and the composition of artificial nutrition adjusted as required . early pn formulations consisted primarily of high concentrations of glucose and amino acids in order to provide adequate calories and were often associated with a number of complications . prolonged use of these formulations was associated with essential fatty acid ( efa ) deficiency because they did not provide linoleic acid ( la ; 18:2-6 ) or -linolenic acid ( ala ; 18:3-3 ) , which are not synthesized by the body and must be obtained from the diet [ 2022 ] . furthermore , the high dextrose loads provided by early pn solutions were associated with a variety of other complications , such as excessive co2 production , metabolic stress ( increased concentrations of cortisol , epinephrine , and glucagon ) , fever , and hepatic steatosis . as critical illnesses can be associated with impaired glucose tolerance , overzealous infusion of hypertonic dextrose solutions was often also associated with hyperglycemia , which , if undetected and untreated , can progress to hyperosmolar nonketotic coma . hyperglycemia can also be associated with an increased incidence of complications in critically ill patients , such as severe infections , multiple organ failure , and increased mortality rates . lipids provide a more energy - dense source of calories ( approximately 9 kcal / g ) than either amino acids ( 4 kcal / g ) or dextrose monohydrate ( 3.4 kcal / g ) . therefore , the fluid volume of pn required to achieve adequate caloric intake can be substantially reduced . the reduced fluid volume and increase in osmolarity of formulations incorporating les permit the safe administration of pn via the peripheral and central routes . formulations with osmolarities 900 mosm / l can be administered peripherally , while highly osmolar formulations can be administered via central veins , which may be of importance in critically ill patients requiring fluid restriction [ 10 , 25 ] . currently available parenteral les also contain sufficient la and ala to prevent efa deficiency . perhaps most importantly , the use of les in pn is associated with a reduction in the metabolic complications related to excessive hypertonic glucose infusion because the dextrose load is correspondingly reduced . results of a study involving critically ill patients with gastric carcinoma , sepsis , colitis or pancreatitis demonstrated that replacement of one - third of the total calories contained in a conventional glucose - amino acid pn formulation with a le maintained or increased patients body weight . plasma glucose concentrations were maintained or reduced , and no cases of hyperglycemia , hyperosmolar nonketotic coma or hypertriglyceridemia were observed . another study found that icu patients receiving parenteral fluids unintentionally received 150600 kcal / day dextrose as a constituent of various fluids and drugs . therefore , the administration of pn containing les may help to prevent hyperglycemia and its complications . it should be noted that , despite the potential benefits of parenteral lipids , the use of les may be limited in some patients . triglycerides and other components of les form artificial chylomicrons , which are hydrolyzed in the body into free fatty acids ( fa ) and small remnant particles taken up by the liver [ 22 , 27 , 28 ] ; the presence of excess phospholipids can also form liposomes , which interfere with lipid metabolism and may result in hypercholesterolemia [ 21 , 22 , 28 ] . when parenteral lipids are given in excess of the liver s ability to process them , hyperlipidemia and hepatic steatosis may occur [ 21 , 22 , 28 ] . parenteral les also commonly contain phytosterols , which may be present in the circulation in quantities large enough to induce cholestasis . although hepatic impairment is most common among patients on long - term pn [ 28 , 29 ] , critically ill patients are also at increased risk because plasma levels of fa increase with metabolic stress [ 27 , 30 ] . therefore , parenteral les should be used with caution in septic patients [ 27 , 28 , 30 ] and patients with other conditions known to impair hepatic clearance of fa [ 28 , 30 ] . in addition to the duration and dose of parenteral les and the patient s level of metabolic stress , the oil source of the le may also affect the relative risk of developing abnormal liver function [ 21 , 22 , 28 , 31 ] . although les contain numerous biologically active compounds , triacylglycerols providing fa are their primary component . fatty acids are classified according to their structure , in terms of their hydrocarbon chain length ( short , medium or long ) , degree of saturation ( number of double bonds ) , and location of double bonds ( counted from the methyl carbon of the hydrocarbon chain ) ( table 2 ) [ 21 , 22 , 32 ] . fatty acids play key roles in determining the structural integrity and fluidity of cell membranes and can give rise to several important bioactive mediators [ 21 , 22 , 33 ] . they can also regulate the expression of a variety of genes and modulate cell signaling pathways , such as those involved in apoptosis , inflammation , and cell - mediated immune responses . for example , longer - chain fa , such as arachidonic acid ( aa ; 20:4-6 ) , eicosapentaenoic acid ( epa ; 20:5-3 ) , and docosahexaenoic acid ( dha ; 22:6-3 ) , are involved in the generation of pro- and anti - inflammatory lipid mediators .table 2fatty acid nomenclature and key dietary sources [ 22 , 32]common namechemical namechemical structure [ length of hydrocarbon chain ( c atoms ) : number of double bonds and position of first double bond]dietary sourcescapricdecanoic10:0coconut oillauricdodecanoic12:0coconut oilmyristictetradecanoic14:0milkpalmitichexadecanoic16:0milk , eggs , animal fats , meat , cocoa butter , palm oil , fish and fish oilspalmitoleic9-hexadecenoic16:1-7fish and fish oilsstearicoctadecanoic18:0milk , eggs , animal fats , meat , cocoa butteroleic9-octadecenoic18:1-9milk , eggs , animal fats , meat , cocoa butter , olive oillinoleic9,12-octadecadienoic18:2-6seeds , seed oils , eggs , animal fats , meatarachidonic5,8,11,14-eicosatetraenoic20:4-6meat , egg lipids , algal oils-linolenic9,12,15-octadecatrienoic18:3-3seeds , seed oils , green leaves , nutseicosapentaenoic5,8,11,14,17-eicosapentaenoic20:5-3fish and fish oilsdocosapentaenoic7,10,13,16,19-docosapentaenoic22:5-3fish and fish oilsdocosahexaenoic4,7,10,13,16,19-docosahexaenoic22:6-3fish and fish oils , algal oils fatty acid nomenclature and key dietary sources [ 22 , 32 ] the majority of early les , which were first included in pn during the 1960s , were derived from soybean oil , which contains a high concentration of both la and ala [ 21 , 22 , 33 ] . these early les were demonstrated to efficiently deliver nonglucose energy , thereby reducing the adverse effects associated with intake of high dextrose concentrations . furthermore , they provided efa and the fat - soluble vitamins e and k . however , studies published during the 1970s and 1980s found that soybean - oil- and cottonseed - oil - based les were associated with a number of adverse immunological effects , such as reduced migration and phagocytosis of granulocytes , which resulted in increased rates of infections , including sepsis [ 3437 ] . the cottonseed - oil - based le ( lipomul ) had such adverse clinical effects ( e.g. , hemolytic anemia ) that it has since been removed from the market . in an effort to address concerns associated with much of this research focused on minimizing complications to which critically ill patients are particularly susceptible , including oxidative stress , alterations in cell - mediated immunity , inflammation , and thrombosis . although these are important issues to consider , well - controlled clinical data in critically ill patients are limited , and discrepancies may be observed between studies because of the heterogeneity of the study designs , patient populations , and specific les used . reactive oxygen species ( ros ) can react with and damage cell membranes , lipids , proteins , and dna through oxidation . under normal circumstances , ros may be produced in increased amounts by cells such as neutrophils and macrophages as part of a natural immune response . levels of ros are balanced by the neutralizing activity of antioxidant molecules and enzymes , thereby preventing excessive damage to the host [ 38 , 39 ] . oxidative stress occurs when an imbalance develops because of high ros levels and/or low antioxidant levels . during critical illness , increased ros and inflammatory mediator production occurs against a background of compromised antioxidant activity , which is partly due to preexisting nutritional deficiencies and/or suboptimal provision of artificial nutrition . depletion of the antioxidants selenium and zinc has been observed in trauma and burn patients , while surgery is associated with reductions in vitamins a , c , and e. this state of imbalance can cause tissue damage and may play an important role in the development of sepsis and multiple organ failure , among other complications ( table 3 ) [ 38 , 39].table 3diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage septic shockacute respiratory distress syndromesystemic inflammatory response syndromedisseminated intravascular coagulationmultiple organ dysfunctionburnscardiovascular diseasediabetes mellitustraumareperfusion injurycancer diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage a key consequence of oxidative stress is lipid peroxidation , where ros react with the double bond of unsaturated lipids , producing unstable lipid peroxides that may cause cell death [ 38 , 39 ] . the high number of double bonds present in -6 and -3 polyunsaturated fatty acids ( pufa ) provide targets for lipid peroxidation , and these fa may therefore be associated with an increased risk of oxidative stress . for this reason , the antioxidant -tocopherol ( vitamin e ) is sometimes added to pufa - rich les in clinical practice . another approach to minimize oxidative stress is the partial replacement of pufa - rich oils with alternative fa sources , such as oils rich in medium - chain triglycerides ( mct ; derived from coconut oil containing medium - chain fa such as capric acid ) , which are more resistant to oxidative damage . in one study of adults requiring pn , a 1:1 mixture of mct ( derived from coconut oil ) and soybean oil ( mct / soybean oil le ) supplemented with -tocopherol demonstrated a reduced propensity to lipid peroxidation when compared with a conventional soybean - oil - based le ( table 4 ) . however , a separate study comparing a soybean - oil - based le with a mct / soybean oil le supplemented with -tocopherol in patients undergoing abdominal surgery found no difference in lipid peroxidation between the two les .table 4key clinical studies evaluating biological and clinical effects of lipid emulsionsclinical studypopulationdesigntreatment groupskey findingslipid peroxidation / antioxidant activity gobel et al . premature infants ( 2837 weeks gestation ; n = 45)prospective , randomized , double - blind trialintralipid vs. clinoleic for 7 days , starting 72 h after birthurinary malondialdehyde excretion at day 7 : no difference between groups ( data not provided ) goulet et al . children with gastrointestinal disorders ( at home ; n = 18)prospective , randomized trialintralipid vs. clinoleic for 2 monthsldl peroxidation index at day 60 : 55.1 ( clinoleic ) vs. 63.3 mol / l ( intralipid ; ns)ldl + vldl peroxidation index at day 60 : 83.7 ( clinoleic ) vs. 104.6 mol / l ( intralipid ; p = 0.0027 ) linseisen et al . adults undergoing abdominal surgery ( n = 33)prospective , randomized , double - blind trialintralipid vs. lipoplus for 5 daystotal plasma cholesterol oxidation products concentration at day 6 : 45.5 ( lipoplus ) vs. 40.9 mol / l ( intralipid ; ns)total antioxidant capacity at day 6 : 153 ( lipoplus ) vs. 129 mol / l ( intralipid ; ns ) manuel - y - keenoy et al . adults requiring total pn ( n = 24)prospective , randomized , double - blind trialintralipid vs. lipofundin mct + -tocopherol ( 200 mg / dl ) for 11 daysserum concentrations of -tocopherol at day 11 : significantly higher for lipofundin mct vs. intralipid ( p = 0.006)malondialdehyde / mg ldl- and vldl - cholesterol at day 11 : significantly reduced for lipofundin mct vs. intralipid ( p = 0.022)immunosuppression / infection rates battistella et al . adult trauma patients ( n = 60)prospective , randomized , double - blind trialintralipid vs. pn containing no lipids for 10 daysmean duration of mechanical ventilation : 27 ( intralipid ) vs. 15 days ( no lipids ; p = 0.01)mean icu stay : 29 ( intralipid ) vs. 18 days ( no lipids ; p = 0.02)mean hospital stay : 39 ( intralipid ) vs. 27 days ( no lipids ; p = 0.03)total number of infectious complications : 72 ( intralipid ) vs. 39 ( no lipids ; p < 0.05)number of patients with pneumonia : 22 ( intralipid ) vs. 13 ( no lipids ; p = 0.05)number of patients with sepsis : 13 ( intralipid ) vs. 5 ( no lipids ; p = 0.04 ) weiss et al . adults undergoing abdominal surgery ( n = 24)prospective , randomized triallipoven + omegaven vs. lipoven for 5 daysmean icu stay : 4.1 ( lipoven + omegaven ) vs. 9.1 days ( lipoven ; ns)mean hospital stay : 17.8 ( lipoven + omegaven ) vs. 23.5 days ( lipoven ; ns)number of infectious complications : 5 in each group grau et al . adults undergoing laparotomy ( n = 72)prospective , randomized , double - blind trialintralipid vs. lipofundin - mctincidence of intra - abdominal abscesses : 32% ( intralipid ) vs. 8% ( lipofundin - mct ; p < 0.05)incidence of in - hospital mortality : 36% ( intralipid ) vs. 15% ( lipofundin - mct ; ns ) garcia - de - lorenzo et al . adults with severe burns ( n = 22)prospective , randomized , double - blind trialclinoleic vs. lipofundin - mct for 6 daysmean icu stay : 32.9 ( clinoleic ) vs. 41.8 days ( lipofundin - mct ; ns)mean hospital stay : 57.0 ( clinoleic ) vs. 64.9 days ( lipofundin - mct ; ns)incidence of infections : 6 patients in each groupincidence of in - icu mortality : 36% ( clinoleic ) vs. 27% ( lipofundin - mct ) huschak et al . adult multiple trauma patients ( n = 33)prospective , randomized , open - label studyclinoleic ( lipid - to - glucose ratio = 3:1 ) vs. lipofundin - mct ( lipid - to - glucose ratio = 1:3 ) for 6 daysmean duration of mechanical ventilation : 13 ( clinoleic / low glucose ) vs. 24 days ( lipofundin - mct / high glucose ; p = 0.01)mean icu stay : 18 ( clinoleic / low glucose ) vs. 25 days ( lipofundin - mct / high glucose ; p = 0.04)mean hospital stay : 80 ( clinoleic / low glucose ) vs. 85 days ( lipofundin - mct / high glucose ; ns)mean sepsis score significantly lower for clinoleic / low glucose vs. lipofundin - mct / high glucose on days 514 ( p adults undergoing abdominal surgery ( n = 256)prospective , randomized studyintralipid vs. lipoplus for 5 daysmean icu stay : 6.3 ( intralipid ) vs. 4.1 days ( lipoplus ; ns)mean hospital stay : 21.9 ( intralipid ) vs. 17.2 days ( lipoplus ; p = 0.0061)incidence of pyrexia : 24.0% ( intralipid ) vs. 17.3% ( lipoplus ; ns)incidence of catheter - related sepsis : 3.9% ( intralipid ) vs. 3.1% ( lipoplus ; ns)incidence of pneumonia : 3.9% ( intralipid ) vs. 0.8% ( lipoplus ; ns)incidence of mortality : 1.6% ( intralipid ) vs. 4.7% ( lipoplus ; ns ) mateu - de antonio et al . adults requiring pn ( n = 42)retrospective , observational studyintralipid vs. clinoleicmean icu stay : 31.3 ( intralipid ) vs. 25.2 days ( clinoleic ; ns)mean hospital stay : 46.5 ( intralipid ) vs 45.4 days ( clinoleic ; ns)incidence of sepsis : 50% ( intralipid ) vs. 43.5% ( clinoleic ; ns)incidence of mortality : 44% ( intralipid ) vs. 47.8% ( clinoleic ; ns ) heller et al . adults requiring total pn ( n = 661)prospective , open - label studyintralipid + omegaven for 3 daysmean icu stay : significantly decreased at omegaven doses > 0.05 g / kg per day ( p < 0.001)mean hospital stay : significantly decreased at omegaven doses > 0.05 g / kg per day ( p < 0.001)requirement for antibiotics : significantly higher at omegaven doses < 0.05 vs. 0.150.2 g / kg per day ( p < 0.001)survival rate : significantly lower at omegaven doses < 0.05 vs. 0.10.2 g / kg per day ( p < 0.001)inflammation gogos et al . severely ill adults ( n = 20)prospective , randomized studylipofundin - s vs. lipofundin - mct for 30 dayscirculating tnf- concentrations : no differences between groupsendotoxin - induced tnf- release at day 30 vs. baseline : 185.4 vs. 95.3 pg / ml ( lipofundin - s ; p < 0.01 ) ; 90.7 vs. 103.8 pg / ml ( lipofundin - mct ; ns ) koller et al . adults undergoing abdominal surgery ( n = 30)prospective , randomized , double - blind studyintralipid vs. lipoplus for 5 daysday 6 : significant increase in ltb5 release vs. baseline with lipoplus but not intralipid ( p = 0.0104)no difference between groups for ltc5 releaseday 6 ltb5/ltb4 ratio : significant increase vs. baseline with lipoplus but not intralipid ( p < 0.02 ) wichmann et al . adults undergoing abdominal surgery ( n = 256)prospective , randomized studyintralipid vs. lipoplus for 5 daysday 6 : significant increase in ltb5 release vs. day 1 in both groups ( p < 0.01)day 6 ltb5/ltb4 ratio : significant increase vs. day 1 with lipoplus ( p = 0.002 ) but not intralipid mayer et al . adults with sepsis ( n = 21)prospective , randomized studylipoven vs. omegaven for 5 daysendotoxin - induced tnf- , il-1 , il-6 , il-8 release ( days 1 - 18 ) : significant increase from baseline with lipoven ; significant decrease from baseline with omegaven mateu - de antonio et al . adults requiring pn ( n = 42)retrospective , observational studyintralipid vs. clinoleicmean concentrations of c - reactive protein at end of pn : 8.4 ( intralipid ) vs. 11.0 mg / l ( clinoleic ; ns)thrombosis porta et al . adult critically ill patients ( n = 23)prospective , randomized , open - label studyintralipid vs. lipofundin - mct for 7 daysno significant changes in platelet aggregation in either treatment group roulet et al . adults undergoing total esophagectomy ( n = 19)prospective , randomized studylipoven vs. lipoven + omegaven for 7 daysmean bleeding time : 3.1 ( lipoven ) vs. 3.3 min ( lipoven + omegaven ; ns)mean maximal collagen - induced platelet aggregation : 91% ( lipoven ) vs. 87% ( lipoven + omegaven ; ns)mean maximal adenosine diphosphate - induced platelet aggregation : 78% ( lipoven ) vs. 77% ( lipoven + omegaven ; ns)all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise statedldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin key clinical studies evaluating biological and clinical effects of lipid emulsions all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise stated ldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin metabolism of mct differs from that of long - chain triglycerides ( lct ) . unlike longer - chain fa , mct require little carnitine for mitochondrial entry , and it has been suggested that their more rapid breakdown may impart an increased production of ketones in critically ill patients . however , this is thought to be a transient phenomenon that is reversible upon discontinuation of mct infusion and rarely causes clinical problems . however , formulations containing mct should not be used in patients who develop ketosis or acidosis in the icu setting . it has been suggested that monounsaturated fa ( mufa ; often derived from olive oil , which also provides antioxidants ) with only one double bond , such as oleic acid ( oa ; 18:1-9 ) , may be less susceptible to lipid peroxidation than -6 and -3 pufa with several double bonds . in vitro studies indicated that cells treated with oa or olive oil were associated with less mitochondrial ros production than cells treated with certain pufa ( e.g. , dha ) or a soybean - oil - based le [ 45 , 46 ] . in a preclinical rodent study , lipid peroxidation was lower among mice administered oa or olive oil by gavage than among mice administered pufa ( i.e. , la or dha ) or fish oil . in children requiring pn , a le containing 80% olive oil led to lower concentrations of certain lipid peroxides than a soybean - oil - based le ( table 4 ) . in a separate study involving preterm infants , peroxidation markers were similar between an olive - oil - rich le supplemented with -tocopherol and a conventional soybean - oil - based le , whereas vitamin e status was enhanced with olive - oil - rich le . fatty acids have been shown to modulate the immune system in a number of ways , influencing cell signaling , gene expression , and apoptosis . in particular , -6 pufa have been associated with reduced migration and phagocytic activity of neutrophils and macrophages , decreased lymphocyte reactivity to microbial antigens , and inhibition of antibody - dependent cellular cytotoxicity [ 34 , 35 , 5052 ] . the consequences of these immunosuppressive actions have been demonstrated within the icu setting . when compared with pn containing no lipids administered to trauma patients , pn containing a soybean - oil - based le was associated with higher rates of infection and significantly longer durations of mechanical ventilation , longer icu stays , and longer hospital stays ( table 4 ) . in patients with septic shock , those receiving a soybean - oil - based le showed increased leukocyte counts and reduced neutrophil cytotoxic activity , while those receiving a fish - oil - based le experienced opposite effects . however , excessive ( hypercaloric ) feeding may also have contributed to immunosuppression observed in some older studies . among surgical patients receiving pn , a soybean - oil - based le supplemented with fish oil was associated with a shorter icu and overall hospital stay when compared with a le that did not contain fish oil ; however , the rates of infection were similar between groups ( table 4 ) . when a soybean - oil - based le was supplemented with fish oil , higher doses were associated with a reduction in the length of icu and overall hospital stay , significantly reduced antibiotic requirement , and significantly increased survival . in comparison with soybean oil , mct are generally described as immune neutral ; however , effects upon certain components of the immune system have been described . in two ex vivo studies , mct and mct / soybean oil les increased monocyte activation and neutrophil adhesion and degranulation [ 57 , 58 ] . in contrast , another ex vivo study reported that mct and mct / soybean oil les were both associated with impaired neutrophil killing of candida albicans . an in vitro study showed that a mct / soybean oil le inhibited the proliferation of t - lymphocytes but not lymphokine - activated killer cells . there are few clinical data evaluating the effect of mct on immune function . in a study involving healthy volunteers , administration of an mct / soybean oil le a second study of healthy subjects found that , in contrast to intermittent infusion of soybean - oil - based le , infusion of pn containing mct / soybean oil le did not impair the clearance of 99tc - sulfur colloid by the reticuloendothelial system . in pediatric surgical patients receiving pn , an mct / soybean oil le was associated with a significantly increased lymphocyte count when compared with a soybean - oil - based le . another study compared an mct / soybean oil le with a soybean - oil - based le in severely undernourished patients undergoing laparotomy . in that study , the incidence of intra - abdominal abscesses was significantly lower in the mct / soybean oil group than the soybean oil group , but there were no significant differences between the groups for other infections ( table 4 ) . lipid emulsions containing high concentrations of olive oil may have less impact on the host immune response than soybean - oil - based or mct / soybean oil les , with little effect on lymphocytes , natural killer cells , and neutrophils . in an in vitro study , the percentage of lymphocytes undergoing apoptosis or necrosis was higher following incubation with 200 m la ( 77% ) than with 200 m oa ( 23% ) ; both were higher than the control group ( 3% ) . another study showed that lymphocyte activation was dose - dependently inhibited by soybean - oil - based les but not an olive - oil - rich le . in contrast to a mct / soybean oil le , an olive - oil - rich le had little effect on neutrophil activation , phagocytosis , generation of ros or chemotaxis . the immunosupportive effects of an olive - oil - rich emulsion may be reflected clinically by the low incidence of infectious complications reported in severely burned patients receiving pn in the icu : in a study evaluating this patient population , the incidences of sepsis and multiple organ failure and the duration of mechanical ventilation , icu stay , and hospital stay were comparable for patients receiving olive - oil - rich and mct / soybean oil les ( table 4 ) . administration of soybean - oil - based les is associated with high blood levels of the -6 pufa la and its metabolite aa , the further metabolism of which may produce proinflammatory eicosanoids [ e.g. , prostaglandins , thromboxanes , and leukotrienes ( lt ) ] that can regulate additional inflammatory mediators [ e.g. , tumor necrosis factor ( tnf)- ] . this hypothesis is supported by a study reporting that malnourished , severely ill patients experienced significantly increased total production of tnf- when receiving pn with a soybean - oil - based le but not an mct / soybean oil le . although there has been much focus on the proinflammatory effects of aa - derived eicosanoids , lipoxins derived from aa have potent inflammation - resolving effects . conversely , the long - chain -3 pufas epa and dha found in fish oils are thought to possess anti - inflammatory properties because they are readily incorporated into cell membranes and thereby impact aa metabolism [ 18 , 32 ] , because the metabolism of epa is associated with production of less biologically potent eicosanoids than those produced from aa [ 18 , 32 ] , and because epa and dha are precursors of resolvins with powerful inflammation - resolving properties [ 18 , 69 ] . indeed , when soybean oil was partially replaced with fish oil ( 3:1 ratio ) in surgical patients receiving pn , lt synthesis was shifted from the proinflammatory ltb4 ( produced from aa ) to the less potent ltb5 ( produced from epa ) ( table 4 ) [ 70 , 71 ] . this change was associated with significantly reduced concentrations of the inflammatory mediators interleukin ( il)-6 and tnf- . in patients with sepsis , concentrations of il-1 , il-6 , il-8 , and tnf- released from mononuclear leukocytes were significantly increased following administration of a soybean - oil - based le compared with a decrease of approximately 30% following administration of a fish - oil - based le . lipid emulsions rich in olive oil have not been well studied with regard to their effect on inflammation ; however , a few studies have demonstrated that oa has relatively few effects on the production of inflammatory mediators . in a preclinical study , lipopolysaccharide - induced production of il-1 , il-6 , il-8 , and tnf- by neutrophils was not altered by an olive - oil - rich le , compared with significant reductions in il-1 with soybean - oil - based and mct / soybean oil les . in another study , infectious complication rates , mortality rates , and length of icu stay were similar among critically ill patients receiving pn containing soybean - oil - based and olive - oil - rich les . thrombosis is a common and serious complication for many critically ill , surgical , and trauma patients , as these states may be associated with changes in the availability of clotting factors and alterations in the fibrinolytic pathway , resulting in intravascular coagulation . however , the effects of les on coagulation have not been extensively assessed . in one study , platelet aggregation was inhibited immediately following intravenous infusion of a fish - oil - based le to healthy volunteers , but had returned to normal at 24 h . in surgical patients receiving pn , the latency to collagen - induced platelet aggregation and time to maximal platelet aggregation were significantly longer for a soybean - oil - based le than for a fish - oil - enriched le ; however , adenosine - diphosphate - induced platelet aggregation and bleeding time were unchanged . another study found no change in platelet aggregation for critically ill patients receiving either soybean - oil - based or mct / soybean oil les . the effects of parenteral olive - oil - rich les on thrombosis have not been systematically evaluated . however , in one study of patients undergoing hemodialysis , the need to change hemofilters due to blood coagulation was significantly reduced among patients receiving pn containing an olive - oil - rich le compared with a soybean - oil - based le . although les contain numerous biologically active compounds , triacylglycerols providing fa are their primary component . fatty acids are classified according to their structure , in terms of their hydrocarbon chain length ( short , medium or long ) , degree of saturation ( number of double bonds ) , and location of double bonds ( counted from the methyl carbon of the hydrocarbon chain ) ( table 2 ) [ 21 , 22 , 32 ] . fatty acids play key roles in determining the structural integrity and fluidity of cell membranes and can give rise to several important bioactive mediators [ 21 , 22 , 33 ] . they can also regulate the expression of a variety of genes and modulate cell signaling pathways , such as those involved in apoptosis , inflammation , and cell - mediated immune responses . for example , longer - chain fa , such as arachidonic acid ( aa ; 20:4-6 ) , eicosapentaenoic acid ( epa ; 20:5-3 ) , and docosahexaenoic acid ( dha ; 22:6-3 ) , are involved in the generation of pro- and anti - inflammatory lipid mediators .table 2fatty acid nomenclature and key dietary sources [ 22 , 32]common namechemical namechemical structure [ length of hydrocarbon chain ( c atoms ) : number of double bonds and position of first double bond]dietary sourcescapricdecanoic10:0coconut oillauricdodecanoic12:0coconut oilmyristictetradecanoic14:0milkpalmitichexadecanoic16:0milk , eggs , animal fats , meat , cocoa butter , palm oil , fish and fish oilspalmitoleic9-hexadecenoic16:1-7fish and fish oilsstearicoctadecanoic18:0milk , eggs , animal fats , meat , cocoa butteroleic9-octadecenoic18:1-9milk , eggs , animal fats , meat , cocoa butter , olive oillinoleic9,12-octadecadienoic18:2-6seeds , seed oils , eggs , animal fats , meatarachidonic5,8,11,14-eicosatetraenoic20:4-6meat , egg lipids , algal oils-linolenic9,12,15-octadecatrienoic18:3-3seeds , seed oils , green leaves , nutseicosapentaenoic5,8,11,14,17-eicosapentaenoic20:5-3fish and fish oilsdocosapentaenoic7,10,13,16,19-docosapentaenoic22:5-3fish and fish oilsdocosahexaenoic4,7,10,13,16,19-docosahexaenoic22:6-3fish and fish oils , algal oils fatty acid nomenclature and key dietary sources [ 22 , 32 ] the majority of early les , which were first included in pn during the 1960s , were derived from soybean oil , which contains a high concentration of both la and ala [ 21 , 22 , 33 ] . these early les were demonstrated to efficiently deliver nonglucose energy , thereby reducing the adverse effects associated with intake of high dextrose concentrations . furthermore , they provided efa and the fat - soluble vitamins e and k . however , studies published during the 1970s and 1980s found that soybean - oil- and cottonseed - oil - based les were associated with a number of adverse immunological effects , such as reduced migration and phagocytosis of granulocytes , which resulted in increased rates of infections , including sepsis [ 3437 ] . the cottonseed - oil - based le ( lipomul ) had such adverse clinical effects ( e.g. , hemolytic anemia ) that it has since been removed from the market . in an effort to address concerns associated with the soybean - oil - based les , alternative sources of fa were investigated . much of this research focused on minimizing complications to which critically ill patients are particularly susceptible , including oxidative stress , alterations in cell - mediated immunity , inflammation , and thrombosis . although these are important issues to consider , well - controlled clinical data in critically ill patients are limited , and discrepancies may be observed between studies because of the heterogeneity of the study designs , patient populations , and specific les used . reactive oxygen species ( ros ) can react with and damage cell membranes , lipids , proteins , and dna through oxidation . under normal circumstances , ros may be produced in increased amounts by cells such as neutrophils and macrophages as part of a natural immune response . levels of ros are balanced by the neutralizing activity of antioxidant molecules and enzymes , thereby preventing excessive damage to the host [ 38 , 39 ] . oxidative stress occurs when an imbalance develops because of high ros levels and/or low antioxidant levels . during critical illness , increased ros and inflammatory mediator production occurs against a background of compromised antioxidant activity , which is partly due to preexisting nutritional deficiencies and/or suboptimal provision of artificial nutrition . depletion of the antioxidants selenium and zinc has been observed in trauma and burn patients , while surgery is associated with reductions in vitamins a , c , and e. this state of imbalance can cause tissue damage and may play an important role in the development of sepsis and multiple organ failure , among other complications ( table 3 ) [ 38 , 39].table 3diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage septic shockacute respiratory distress syndromesystemic inflammatory response syndromedisseminated intravascular coagulationmultiple organ dysfunctionburnscardiovascular diseasediabetes mellitustraumareperfusion injurycancer diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage a key consequence of oxidative stress is lipid peroxidation , where ros react with the double bond of unsaturated lipids , producing unstable lipid peroxides that may cause cell death [ 38 , 39 ] . the high number of double bonds present in -6 and -3 polyunsaturated fatty acids ( pufa ) provide targets for lipid peroxidation , and these fa may therefore be associated with an increased risk of oxidative stress . for this reason , the antioxidant -tocopherol ( vitamin e ) is sometimes added to pufa - rich les in clinical practice . another approach to minimize oxidative stress is the partial replacement of pufa - rich oils with alternative fa sources , such as oils rich in medium - chain triglycerides ( mct ; derived from coconut oil containing medium - chain fa such as capric acid ) , which are more resistant to oxidative damage . in one study of adults requiring pn , a 1:1 mixture of mct ( derived from coconut oil ) and soybean oil ( mct / soybean oil le ) supplemented with -tocopherol demonstrated a reduced propensity to lipid peroxidation when compared with a conventional soybean - oil - based le ( table 4 ) . however , a separate study comparing a soybean - oil - based le with a mct / soybean oil le supplemented with -tocopherol in patients undergoing abdominal surgery found no difference in lipid peroxidation between the two les .table 4key clinical studies evaluating biological and clinical effects of lipid emulsionsclinical studypopulationdesigntreatment groupskey findingslipid peroxidation / antioxidant activity gobel et al . premature infants ( 2837 weeks gestation ; n = 45)prospective , randomized , double - blind trialintralipid vs. clinoleic for 7 days , starting 72 h after birthurinary malondialdehyde excretion at day 7 : no difference between groups ( data not provided ) goulet et al . children with gastrointestinal disorders ( at home ; n = 18)prospective , randomized trialintralipid vs. clinoleic for 2 monthsldl peroxidation index at day 60 : 55.1 ( clinoleic ) vs. 63.3 mol / l ( intralipid ; ns)ldl + vldl peroxidation index at day 60 : 83.7 ( clinoleic ) vs. 104.6 mol / l ( intralipid ; p = 0.0027 ) linseisen et al . adults undergoing abdominal surgery ( n = 33)prospective , randomized , double - blind trialintralipid vs. lipoplus for 5 daystotal plasma cholesterol oxidation products concentration at day 6 : 45.5 ( lipoplus ) vs. 40.9 mol / l ( intralipid ; ns)total antioxidant capacity at day 6 : 153 ( lipoplus ) vs. 129 mol / l ( intralipid ; ns ) manuel - y - keenoy et al . adults requiring total pn ( n = 24)prospective , randomized , double - blind trialintralipid vs. lipofundin mct + -tocopherol ( 200 mg / dl ) for 11 daysserum concentrations of -tocopherol at day 11 : significantly higher for lipofundin mct vs. intralipid ( p = 0.006)malondialdehyde / mg ldl- and vldl - cholesterol at day 11 : significantly reduced for lipofundin mct vs. intralipid ( p = 0.022)immunosuppression / infection rates battistella et al . adult trauma patients ( n = 60)prospective , randomized , double - blind trialintralipid vs. pn containing no lipids for 10 daysmean duration of mechanical ventilation : 27 ( intralipid ) vs. 15 days ( no lipids ; p = 0.01)mean icu stay : 29 ( intralipid ) vs. 18 days ( no lipids ; p = 0.02)mean hospital stay : 39 ( intralipid ) vs. 27 days ( no lipids ; p = 0.03)total number of infectious complications : 72 ( intralipid ) vs. 39 ( no lipids ; p < 0.05)number of patients with pneumonia : 22 ( intralipid ) vs. 13 ( no lipids ; p = 0.05)number of patients with sepsis : 13 ( intralipid ) vs. 5 ( no lipids ; p = 0.04 ) weiss et al . adults undergoing abdominal surgery ( n = 24)prospective , randomized triallipoven + omegaven vs. lipoven for 5 daysmean icu stay : 4.1 ( lipoven + omegaven ) vs. 9.1 days ( lipoven ; ns)mean hospital stay : 17.8 ( lipoven + omegaven ) vs. 23.5 days ( lipoven ; ns)number of infectious complications : 5 in each group grau et al . adults undergoing laparotomy ( n = 72)prospective , randomized , double - blind trialintralipid vs. lipofundin - mctincidence of intra - abdominal abscesses : 32% ( intralipid ) vs. 8% ( lipofundin - mct ; p < 0.05)incidence of in - hospital mortality : 36% ( intralipid ) vs. 15% ( lipofundin - mct ; ns ) garcia - de - lorenzo et al . adults with severe burns ( n = 22)prospective , randomized , double - blind trialclinoleic vs. lipofundin - mct for 6 daysmean icu stay : 32.9 ( clinoleic ) vs. 41.8 days ( lipofundin - mct ; ns)mean hospital stay : 57.0 ( clinoleic ) vs. 64.9 days ( lipofundin - mct ; ns)incidence of infections : 6 patients in each groupincidence of in - icu mortality : 36% ( clinoleic ) vs. 27% ( lipofundin - mct ) huschak et al . adult multiple trauma patients ( n = 33)prospective , randomized , open - label studyclinoleic ( lipid - to - glucose ratio = 3:1 ) vs. lipofundin - mct ( lipid - to - glucose ratio = 1:3 ) for 6 daysmean duration of mechanical ventilation : 13 ( clinoleic / low glucose ) vs. 24 days ( lipofundin - mct / high glucose ; p = 0.01)mean icu stay : 18 ( clinoleic / low glucose ) vs. 25 days ( lipofundin - mct / high glucose ; p = 0.04)mean hospital stay : 80 ( clinoleic / low glucose ) vs. 85 days ( lipofundin - mct / high glucose ; ns)mean sepsis score significantly lower for clinoleic / low glucose vs. lipofundin - mct / high glucose on days 514 ( p adults undergoing abdominal surgery ( n = 256)prospective , randomized studyintralipid vs. lipoplus for 5 daysmean icu stay : 6.3 ( intralipid ) vs. 4.1 days ( lipoplus ; ns)mean hospital stay : 21.9 ( intralipid ) vs. 17.2 days ( lipoplus ; p = 0.0061)incidence of pyrexia : 24.0% ( intralipid ) vs. 17.3% ( lipoplus ; ns)incidence of catheter - related sepsis : 3.9% ( intralipid ) vs. 3.1% ( lipoplus ; ns)incidence of pneumonia : 3.9% ( intralipid ) vs. 0.8% ( lipoplus ; ns)incidence of mortality : 1.6% ( intralipid ) vs. 4.7% ( lipoplus ; ns ) mateu - de antonio et al . adults requiring pn ( n = 42)retrospective , observational studyintralipid vs. clinoleicmean icu stay : 31.3 ( intralipid ) vs. 25.2 days ( clinoleic ; ns)mean hospital stay : 46.5 ( intralipid ) vs 45.4 days ( clinoleic ; ns)incidence of sepsis : 50% ( intralipid ) vs. 43.5% ( clinoleic ; ns)incidence of mortality : 44% ( intralipid ) vs. 47.8% ( clinoleic ; ns ) heller et al . adults requiring total pn ( n = 661)prospective , open - label studyintralipid + omegaven for 3 daysmean icu stay : significantly decreased at omegaven doses > 0.05 g / kg per day ( p < 0.001)mean hospital stay : significantly decreased at omegaven doses > 0.05 g / kg per day ( p < 0.001)requirement for antibiotics : significantly higher at omegaven doses < 0.05 vs. 0.150.2 g / kg per day ( p < 0.001)survival rate : significantly lower at omegaven doses < 0.05 vs. 0.10.2 g / kg per day ( p < 0.001)inflammation gogos et al . severely ill adults ( n = 20)prospective , randomized studylipofundin - s vs. lipofundin - mct for 30 dayscirculating tnf- concentrations : no differences between groupsendotoxin - induced tnf- release at day 30 vs. baseline : 185.4 vs. 95.3 pg / ml ( lipofundin - s ; p < 0.01 ) ; 90.7 vs. 103.8 pg / ml ( lipofundin - mct ; ns ) koller et al . adults undergoing abdominal surgery ( n = 30)prospective , randomized , double - blind studyintralipid vs. lipoplus for 5 daysday 6 : significant increase in ltb5 release vs. baseline with lipoplus but not intralipid ( p = 0.0104)no difference between groups for ltc5 releaseday 6 ltb5/ltb4 ratio : significant increase vs. baseline with lipoplus but not intralipid ( p < 0.02 ) wichmann et al . adults undergoing abdominal surgery ( n = 256)prospective , randomized studyintralipid vs. lipoplus for 5 daysday 6 : significant increase in ltb5 release vs. day 1 in both groups ( p < 0.01)day 6 ltb5/ltb4 ratio : significant increase vs. day 1 with lipoplus ( p = 0.002 ) but not intralipid mayer et al . adults with sepsis ( n = 21)prospective , randomized studylipoven vs. omegaven for 5 daysendotoxin - induced tnf- , il-1 , il-6 , il-8 release ( days 1 - 18 ) : significant increase from baseline with lipoven ; significant decrease from baseline with omegaven mateu - de antonio et al . adults requiring pn ( n = 42)retrospective , observational studyintralipid vs. clinoleicmean concentrations of c - reactive protein at end of pn : 8.4 ( intralipid ) vs. 11.0 mg / l ( clinoleic ; ns)thrombosis porta et al . adult critically ill patients ( n = 23)prospective , randomized , open - label studyintralipid vs. lipofundin - mct for 7 daysno significant changes in platelet aggregation in either treatment group roulet et al . adults undergoing total esophagectomy ( n = 19)prospective , randomized studylipoven vs. lipoven + omegaven for 7 daysmean bleeding time : 3.1 ( lipoven ) vs. 3.3 min ( lipoven + omegaven ; ns)mean maximal collagen - induced platelet aggregation : 91% ( lipoven ) vs. 87% ( lipoven + omegaven ; ns)mean maximal adenosine diphosphate - induced platelet aggregation : 78% ( lipoven ) vs. 77% ( lipoven + omegaven ; ns)all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise statedldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin key clinical studies evaluating biological and clinical effects of lipid emulsions all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise stated ldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin metabolism of mct differs from that of long - chain triglycerides ( lct ) . unlike longer - chain fa , mct require little carnitine for mitochondrial entry , and it has been suggested that their more rapid breakdown may impart an increased production of ketones in critically ill patients . however , this is thought to be a transient phenomenon that is reversible upon discontinuation of mct infusion and rarely causes clinical problems . however , formulations containing mct should not be used in patients who develop ketosis or acidosis in the icu setting . it has been suggested that monounsaturated fa ( mufa ; often derived from olive oil , which also provides antioxidants ) with only one double bond , such as oleic acid ( oa ; 18:1-9 ) , may be less susceptible to lipid peroxidation than -6 and -3 pufa with several double bonds . in vitro studies indicated that cells treated with oa or olive oil were associated with less mitochondrial ros production than cells treated with certain pufa ( e.g. , dha ) or a soybean - oil - based le [ 45 , 46 ] . in a preclinical rodent study , lipid peroxidation was lower among mice administered oa or olive oil by gavage than among mice administered pufa ( i.e. , la or dha ) or fish oil . in children requiring pn , a le containing 80% olive oil led to lower concentrations of certain lipid peroxides than a soybean - oil - based le ( table 4 ) . in a separate study involving preterm infants , peroxidation markers were similar between an olive - oil - rich le supplemented with -tocopherol and a conventional soybean - oil - based le , whereas vitamin e status was enhanced with olive - oil - rich le . fatty acids have been shown to modulate the immune system in a number of ways , influencing cell signaling , gene expression , and apoptosis . in particular , -6 pufa have been associated with reduced migration and phagocytic activity of neutrophils and macrophages , decreased lymphocyte reactivity to microbial antigens , and inhibition of antibody - dependent cellular cytotoxicity [ 34 , 35 , 5052 ] . the consequences of these immunosuppressive actions have been demonstrated within the icu setting . when compared with pn containing no lipids administered to trauma patients , pn containing a soybean - oil - based le was associated with higher rates of infection and significantly longer durations of mechanical ventilation , longer icu stays , and longer hospital stays ( table 4 ) . in patients with septic shock , those receiving a soybean - oil - based le showed increased leukocyte counts and reduced neutrophil cytotoxic activity , while those receiving a fish - oil - based le experienced opposite effects . however , excessive ( hypercaloric ) feeding may also have contributed to immunosuppression observed in some older studies . among surgical patients receiving pn , a soybean - oil - based le supplemented with fish oil was associated with a shorter icu and overall hospital stay when compared with a le that did not contain fish oil ; however , the rates of infection were similar between groups ( table 4 ) . when a soybean - oil - based le was supplemented with fish oil , higher doses were associated with a reduction in the length of icu and overall hospital stay , significantly reduced antibiotic requirement , and significantly increased survival . in comparison with soybean oil , mct are generally described as immune neutral ; however , effects upon certain components of the immune system have been described . in two ex vivo studies , mct and mct / soybean oil les increased monocyte activation and neutrophil adhesion and degranulation [ 57 , 58 ] . in contrast , another ex vivo study reported that mct and mct / soybean oil les were both associated with impaired neutrophil killing of candida albicans . an in vitro study showed that a mct / soybean oil le inhibited the proliferation of t - lymphocytes but not lymphokine - activated killer cells . there are few clinical data evaluating the effect of mct on immune function . in a study involving healthy volunteers , administration of an mct / soybean oil le a second study of healthy subjects found that , in contrast to intermittent infusion of soybean - oil - based le , infusion of pn containing mct / soybean oil le did not impair the clearance of 99tc - sulfur colloid by the reticuloendothelial system . in pediatric surgical patients receiving pn , an mct / soybean oil le was associated with a significantly increased lymphocyte count when compared with a soybean - oil - based le . another study compared an mct / soybean oil le with a soybean - oil - based le in severely undernourished patients undergoing laparotomy . in that study , the incidence of intra - abdominal abscesses was significantly lower in the mct / soybean oil group than the soybean oil group , but there were no significant differences between the groups for other infections ( table 4 ) . lipid emulsions containing high concentrations of olive oil may have less impact on the host immune response than soybean - oil - based or mct / soybean oil les , with little effect on lymphocytes , natural killer cells , and neutrophils . in an in vitro study , the percentage of lymphocytes undergoing apoptosis or necrosis was higher following incubation with 200 m la ( 77% ) than with 200 m oa ( 23% ) ; both were higher than the control group ( 3% ) . another study showed that lymphocyte activation was dose - dependently inhibited by soybean - oil - based les but not an olive - oil - rich le . in contrast to a mct / soybean oil le , an olive - oil - rich le had little effect on neutrophil activation , phagocytosis , generation of ros or chemotaxis . the immunosupportive effects of an olive - oil - rich emulsion may be reflected clinically by the low incidence of infectious complications reported in severely burned patients receiving pn in the icu : in a study evaluating this patient population , the incidences of sepsis and multiple organ failure and the duration of mechanical ventilation , icu stay , and hospital stay were comparable for patients receiving olive - oil - rich and mct / soybean oil les ( table 4 ) . administration of soybean - oil - based les is associated with high blood levels of the -6 pufa la and its metabolite aa , the further metabolism of which may produce proinflammatory eicosanoids [ e.g. , prostaglandins , thromboxanes , and leukotrienes ( lt ) ] that can regulate additional inflammatory mediators [ e.g. , tumor necrosis factor ( tnf)- ] . this hypothesis is supported by a study reporting that malnourished , severely ill patients experienced significantly increased total production of tnf- when receiving pn with a soybean - oil - based le but not an mct / soybean oil le . although there has been much focus on the proinflammatory effects of aa - derived eicosanoids , lipoxins derived from aa have potent inflammation - resolving effects . conversely , the long - chain -3 pufas epa and dha found in fish oils are thought to possess anti - inflammatory properties because they are readily incorporated into cell membranes and thereby impact aa metabolism [ 18 , 32 ] , because the metabolism of epa is associated with production of less biologically potent eicosanoids than those produced from aa [ 18 , 32 ] , and because epa and dha are precursors of resolvins with powerful inflammation - resolving properties [ 18 , 69 ] . indeed , when soybean oil was partially replaced with fish oil ( 3:1 ratio ) in surgical patients receiving pn , lt synthesis was shifted from the proinflammatory ltb4 ( produced from aa ) to the less potent ltb5 ( produced from epa ) ( table 4 ) [ 70 , 71 ] . this change was associated with significantly reduced concentrations of the inflammatory mediators interleukin ( il)-6 and tnf- . in patients with sepsis , concentrations of il-1 , il-6 , il-8 , and tnf- released from mononuclear leukocytes were significantly increased following administration of a soybean - oil - based le compared with a decrease of approximately 30% following administration of a fish - oil - based le . lipid emulsions rich in olive oil have not been well studied with regard to their effect on inflammation ; however , a few studies have demonstrated that oa has relatively few effects on the production of inflammatory mediators . in a preclinical study , lipopolysaccharide - induced production of il-1 , il-6 , il-8 , and tnf- by neutrophils was not altered by an olive - oil - rich le , compared with significant reductions in il-1 with soybean - oil - based and mct / soybean oil les . in another study , infectious complication rates , mortality rates , and length of icu stay were similar among critically ill patients receiving pn containing soybean - oil - based and olive - oil - rich les . thrombosis is a common and serious complication for many critically ill , surgical , and trauma patients , as these states may be associated with changes in the availability of clotting factors and alterations in the fibrinolytic pathway , resulting in intravascular coagulation . in one study , platelet aggregation was inhibited immediately following intravenous infusion of a fish - oil - based le to healthy volunteers , but had returned to normal at 24 h . in surgical patients receiving pn , the latency to collagen - induced platelet aggregation and time to maximal platelet aggregation were significantly longer for a soybean - oil - based le than for a fish - oil - enriched le ; however , adenosine - diphosphate - induced platelet aggregation and bleeding time were unchanged . another study found no change in platelet aggregation for critically ill patients receiving either soybean - oil - based or mct / soybean oil les . the effects of parenteral olive - oil - rich les on thrombosis have not been systematically evaluated . however , in one study of patients undergoing hemodialysis , the need to change hemofilters due to blood coagulation was significantly reduced among patients receiving pn containing an olive - oil - rich le compared with a soybean - oil - based le . reactive oxygen species ( ros ) can react with and damage cell membranes , lipids , proteins , and dna through oxidation . under normal circumstances , ros may be produced in increased amounts by cells such as neutrophils and macrophages as part of a natural immune response . levels of ros are balanced by the neutralizing activity of antioxidant molecules and enzymes , thereby preventing excessive damage to the host [ 38 , 39 ] . oxidative stress occurs when an imbalance develops because of high ros levels and/or low antioxidant levels . during critical illness , increased ros and inflammatory mediator production occurs against a background of compromised antioxidant activity , which is partly due to preexisting nutritional deficiencies and/or suboptimal provision of artificial nutrition . depletion of the antioxidants selenium and zinc has been observed in trauma and burn patients , while surgery is associated with reductions in vitamins a , c , and e. this state of imbalance can cause tissue damage and may play an important role in the development of sepsis and multiple organ failure , among other complications ( table 3 ) [ 38 , 39].table 3diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage septic shockacute respiratory distress syndromesystemic inflammatory response syndromedisseminated intravascular coagulationmultiple organ dysfunctionburnscardiovascular diseasediabetes mellitustraumareperfusion injurycancer diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage a key consequence of oxidative stress is lipid peroxidation , where ros react with the double bond of unsaturated lipids , producing unstable lipid peroxides that may cause cell death [ 38 , 39 ] . the high number of double bonds present in -6 and -3 polyunsaturated fatty acids ( pufa ) provide targets for lipid peroxidation , and these fa may therefore be associated with an increased risk of oxidative stress . for this reason , the antioxidant -tocopherol ( vitamin e ) is sometimes added to pufa - rich les in clinical practice . another approach to minimize oxidative stress is the partial replacement of pufa - rich oils with alternative fa sources , such as oils rich in medium - chain triglycerides ( mct ; derived from coconut oil containing medium - chain fa such as capric acid ) , which are more resistant to oxidative damage . in one study of adults requiring pn , a 1:1 mixture of mct ( derived from coconut oil ) and soybean oil ( mct / soybean oil le ) supplemented with -tocopherol demonstrated a reduced propensity to lipid peroxidation when compared with a conventional soybean - oil - based le ( table 4 ) . however , a separate study comparing a soybean - oil - based le with a mct / soybean oil le supplemented with -tocopherol in patients undergoing abdominal surgery found no difference in lipid peroxidation between the two les .table 4key clinical studies evaluating biological and clinical effects of lipid emulsionsclinical studypopulationdesigntreatment groupskey findingslipid peroxidation / antioxidant activity gobel et al . premature infants ( 2837 weeks gestation ; n = 45)prospective , randomized , double - blind trialintralipid vs. clinoleic for 7 days , starting 72 h after birthurinary malondialdehyde excretion at day 7 : no difference between groups ( data not provided ) goulet et al . children with gastrointestinal disorders ( at home ; n = 18)prospective , randomized trialintralipid vs. clinoleic for 2 monthsldl peroxidation index at day 60 : 55.1 ( clinoleic ) vs. 63.3 mol / l ( intralipid ; ns)ldl + vldl peroxidation index at day 60 : 83.7 ( clinoleic ) vs. 104.6 mol / l ( intralipid ; p = 0.0027 ) linseisen et al . adults undergoing abdominal surgery ( n = 33)prospective , randomized , double - blind trialintralipid vs. lipoplus for 5 daystotal plasma cholesterol oxidation products concentration at day 6 : 45.5 ( lipoplus ) vs. 40.9 mol / l ( intralipid ; ns)total antioxidant capacity at day 6 : 153 ( lipoplus ) vs. 129 mol / l ( intralipid ; ns ) manuel - y - keenoy et al . adults requiring total pn ( n = 24)prospective , randomized , double - blind trialintralipid vs. lipofundin mct + -tocopherol ( 200 mg / dl ) for 11 daysserum concentrations of -tocopherol at day 11 : significantly higher for lipofundin mct vs. intralipid ( p = 0.006)malondialdehyde / mg ldl- and vldl - cholesterol at day 11 : significantly reduced for lipofundin mct vs. intralipid ( p = 0.022)immunosuppression / infection rates battistella et al . adult trauma patients ( n = 60)prospective , randomized , double - blind trialintralipid vs. pn containing no lipids for 10 daysmean duration of mechanical ventilation : 27 ( intralipid ) vs. 15 days ( no lipids ; p = 0.01)mean icu stay : 29 ( intralipid ) vs. 18 days ( no lipids ; p = 0.02)mean hospital stay : 39 ( intralipid ) vs. 27 days ( no lipids ; p = 0.03)total number of infectious complications : 72 ( intralipid ) vs. 39 ( no lipids ; p < 0.05)number of patients with pneumonia : 22 ( intralipid ) vs. 13 ( no lipids ; p = 0.05)number of patients with sepsis : 13 ( intralipid ) vs. 5 ( no lipids ; p = 0.04 ) weiss et al . adults undergoing abdominal surgery ( n = 24)prospective , randomized triallipoven + omegaven vs. lipoven for 5 daysmean icu stay : 4.1 ( lipoven + omegaven ) vs. 9.1 days ( lipoven ; ns)mean hospital stay : 17.8 ( lipoven + omegaven ) vs. 23.5 days ( lipoven ; ns)number of infectious complications : 5 in each group grau et al . adults undergoing laparotomy ( n = 72)prospective , randomized , double - blind trialintralipid vs. lipofundin - mctincidence of intra - abdominal abscesses : 32% ( intralipid ) vs. 8% ( lipofundin - mct ; p < 0.05)incidence of in - hospital mortality : 36% ( intralipid ) vs. 15% ( lipofundin - mct ; ns ) garcia - de - lorenzo et al . adults with severe burns ( n = 22)prospective , randomized , double - blind trialclinoleic vs. lipofundin - mct for 6 daysmean icu stay : 32.9 ( clinoleic ) vs. 41.8 days ( lipofundin - mct ; ns)mean hospital stay : 57.0 ( clinoleic ) vs. 64.9 days ( lipofundin - mct ; ns)incidence of infections : 6 patients in each groupincidence of in - icu mortality : 36% ( clinoleic ) vs. 27% ( lipofundin - mct ) huschak et al . adult multiple trauma patients ( n = 33)prospective , randomized , open - label studyclinoleic ( lipid - to - glucose ratio = 3:1 ) vs. lipofundin - mct ( lipid - to - glucose ratio = 1:3 ) for 6 daysmean duration of mechanical ventilation : 13 ( clinoleic / low glucose ) vs. 24 days ( lipofundin - mct / high glucose ; p = 0.01)mean icu stay : 18 ( clinoleic / low glucose ) vs. 25 days ( lipofundin - mct / high glucose ; p = 0.04)mean hospital stay : 80 ( clinoleic / low glucose ) vs. 85 days ( lipofundin - mct / high glucose ; ns)mean sepsis score significantly lower for clinoleic / low glucose vs. lipofundin - mct / high glucose on days 514 ( p < 0.05 ) wichmann et al . adults undergoing abdominal surgery ( n = 256)prospective , randomized studyintralipid vs. lipoplus for 5 daysmean icu stay : 6.3 ( intralipid ) vs. 4.1 days ( lipoplus ; ns)mean hospital stay : 21.9 ( intralipid ) vs. 17.2 days ( lipoplus ; p = 0.0061)incidence of pyrexia : 24.0% ( intralipid ) vs. 17.3% ( lipoplus ; ns)incidence of catheter - related sepsis : 3.9% ( intralipid ) vs. 3.1% ( lipoplus ; ns)incidence of pneumonia : 3.9% ( intralipid ) vs. 0.8% ( lipoplus ; ns)incidence of mortality : 1.6% ( intralipid ) vs. 4.7% ( lipoplus ; ns ) mateu - de antonio et al . adults requiring pn ( n = 42)retrospective , observational studyintralipid vs. clinoleicmean icu stay : 31.3 ( intralipid ) vs. 25.2 days ( clinoleic ; ns)mean hospital stay : 46.5 ( intralipid ) vs 45.4 days ( clinoleic ; ns)incidence of sepsis : 50% ( intralipid ) vs. 43.5% ( clinoleic ; ns)incidence of mortality : 44% ( intralipid ) vs. 47.8% ( clinoleic ; ns ) heller et al . adults requiring total pn ( n = 661)prospective , open - label studyintralipid + omegaven for 3 daysmean icu stay : significantly decreased at omegaven doses > 0.05 g / kg per day ( p < 0.001)mean hospital stay : significantly decreased at omegaven doses > 0.05 g / kg per day ( p < 0.001)requirement for antibiotics : significantly higher at omegaven doses < 0.05 vs. 0.150.2 g / kg per day ( p < 0.001)survival rate : significantly lower at omegaven doses < 0.05 vs. 0.10.2 g / kg per day ( p < 0.001)inflammation gogos et al . severely ill adults ( n = 20)prospective , randomized studylipofundin - s vs. lipofundin - mct for 30 dayscirculating tnf- concentrations : no differences between groupsendotoxin - induced tnf- release at day 30 vs. baseline : 185.4 vs. 95.3 pg / ml ( lipofundin - s ; p < 0.01 ) ; 90.7 vs. 103.8 pg / ml ( lipofundin - mct ; ns ) koller et al . adults undergoing abdominal surgery ( n = 30)prospective , randomized , double - blind studyintralipid vs. lipoplus for 5 daysday 6 : significant increase in ltb5 release vs. baseline with lipoplus but not intralipid ( p = 0.0104)no difference between groups for ltc5 releaseday 6 ltb5/ltb4 ratio : significant increase vs. baseline with lipoplus but not intralipid ( p < 0.02 ) wichmann et al . adults undergoing abdominal surgery ( n = 256)prospective , randomized studyintralipid vs. lipoplus for 5 daysday 6 : significant increase in ltb5 release vs. day 1 in both groups ( p < 0.01)day 6 ltb5/ltb4 ratio : significant increase vs. day 1 with lipoplus ( p = 0.002 ) but not intralipid mayer et al . adults with sepsis ( n = 21)prospective , randomized studylipoven vs. omegaven for 5 daysendotoxin - induced tnf- , il-1 , il-6 , il-8 release ( days 1 - 18 ) : significant increase from baseline with lipoven ; significant decrease from baseline with omegaven mateu - de antonio et al . adults requiring pn ( n = 42)retrospective , observational studyintralipid vs. clinoleicmean concentrations of c - reactive protein at end of pn : 8.4 ( intralipid ) vs. 11.0 mg / l ( clinoleic ; ns)thrombosis porta et al . adult critically ill patients ( n = 23)prospective , randomized , open - label studyintralipid vs. lipofundin - mct for 7 daysno significant changes in platelet aggregation in either treatment group roulet et al . adults undergoing total esophagectomy ( n = 19)prospective , randomized studylipoven vs. lipoven + omegaven for 7 daysmean bleeding time : 3.1 ( lipoven ) vs. 3.3 min ( lipoven + omegaven ; ns)mean maximal collagen - induced platelet aggregation : 91% ( lipoven ) vs. 87% ( lipoven + omegaven ; ns)mean maximal adenosine diphosphate - induced platelet aggregation : 78% ( lipoven ) vs. 77% ( lipoven + omegaven ; ns)all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise statedldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin key clinical studies evaluating biological and clinical effects of lipid emulsions all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise stated ldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin metabolism of mct differs from that of long - chain triglycerides ( lct ) . unlike longer - chain fa , mct require little carnitine for mitochondrial entry , and it has been suggested that their more rapid breakdown may impart an increased production of ketones in critically ill patients . however , this is thought to be a transient phenomenon that is reversible upon discontinuation of mct infusion and rarely causes clinical problems . however , formulations containing mct should not be used in patients who develop ketosis or acidosis in the icu setting . it has been suggested that monounsaturated fa ( mufa ; often derived from olive oil , which also provides antioxidants ) with only one double bond , such as oleic acid ( oa ; 18:1-9 ) , may be less susceptible to lipid peroxidation than -6 and -3 pufa with several double bonds . in vitro studies indicated that cells treated with oa or olive oil were associated with less mitochondrial ros production than cells treated with certain pufa ( e.g. , dha ) or a soybean - oil - based le [ 45 , 46 ] . in a preclinical rodent study , lipid peroxidation was lower among mice administered oa or olive oil by gavage than among mice administered pufa ( i.e. , la or dha ) or fish oil . in children requiring pn , a le containing 80% olive oil led to lower concentrations of certain lipid peroxides than a soybean - oil - based le ( table 4 ) . in a separate study involving preterm infants , peroxidation markers were similar between an olive - oil - rich le supplemented with -tocopherol and a conventional soybean - oil - based le , whereas vitamin e status was enhanced with olive - oil - rich le . fatty acids have been shown to modulate the immune system in a number of ways , influencing cell signaling , gene expression , and apoptosis . in particular , -6 pufa have been associated with reduced migration and phagocytic activity of neutrophils and macrophages , decreased lymphocyte reactivity to microbial antigens , and inhibition of antibody - dependent cellular cytotoxicity [ 34 , 35 , 5052 ] . the consequences of these immunosuppressive actions have been demonstrated within the icu setting . when compared with pn containing no lipids administered to trauma patients , pn containing a soybean - oil - based le was associated with higher rates of infection and significantly longer durations of mechanical ventilation , longer icu stays , and longer hospital stays ( table 4 ) . in patients with septic shock , those receiving a soybean - oil - based le showed increased leukocyte counts and reduced neutrophil cytotoxic activity , while those receiving a fish - oil - based le experienced opposite effects . however , excessive ( hypercaloric ) feeding may also have contributed to immunosuppression observed in some older studies . among surgical patients receiving pn , a soybean - oil - based le supplemented with fish oil was associated with a shorter icu and overall hospital stay when compared with a le that did not contain fish oil ; however , the rates of infection were similar between groups ( table 4 ) . when a soybean - oil - based le was supplemented with fish oil , higher doses were associated with a reduction in the length of icu and overall hospital stay , significantly reduced antibiotic requirement , and significantly increased survival . in comparison with soybean oil , mct are generally described as immune neutral ; however , effects upon certain components of the immune system have been described . in two ex vivo studies , mct and mct / soybean oil les increased monocyte activation and neutrophil adhesion and degranulation [ 57 , 58 ] . in contrast , another ex vivo study reported that mct and mct / soybean oil les were both associated with impaired neutrophil killing of candida albicans . an in vitro study showed that a mct / soybean oil le inhibited the proliferation of t - lymphocytes but not lymphokine - activated killer cells . there are few clinical data evaluating the effect of mct on immune function . in a study involving healthy volunteers , administration of an mct / soybean oil le a second study of healthy subjects found that , in contrast to intermittent infusion of soybean - oil - based le , infusion of pn containing mct / soybean oil le did not impair the clearance of 99tc - sulfur colloid by the reticuloendothelial system . in pediatric surgical patients receiving pn , an mct / soybean oil le was associated with a significantly increased lymphocyte count when compared with a soybean - oil - based le . another study compared an mct / soybean oil le with a soybean - oil - based le in severely undernourished patients undergoing laparotomy . in that study , the incidence of intra - abdominal abscesses was significantly lower in the mct / soybean oil group than the soybean oil group , but there were no significant differences between the groups for other infections ( table 4 ) . lipid emulsions containing high concentrations of olive oil may have less impact on the host immune response than soybean - oil - based or mct / soybean oil les , with little effect on lymphocytes , natural killer cells , and neutrophils . in an in vitro study , the percentage of lymphocytes undergoing apoptosis or necrosis was higher following incubation with 200 m la ( 77% ) than with 200 m oa ( 23% ) ; both were higher than the control group ( 3% ) . another study showed that lymphocyte activation was dose - dependently inhibited by soybean - oil - based les but not an olive - oil - rich le . in contrast to a mct / soybean oil le , an olive - oil - rich le had little effect on neutrophil activation , phagocytosis , generation of ros or chemotaxis . the immunosupportive effects of an olive - oil - rich emulsion may be reflected clinically by the low incidence of infectious complications reported in severely burned patients receiving pn in the icu : in a study evaluating this patient population , the incidences of sepsis and multiple organ failure and the duration of mechanical ventilation , icu stay , and hospital stay were comparable for patients receiving olive - oil - rich and mct / soybean oil les ( table 4 ) . administration of soybean - oil - based les is associated with high blood levels of the -6 pufa la and its metabolite aa , the further metabolism of which may produce proinflammatory eicosanoids [ e.g. , prostaglandins , thromboxanes , and leukotrienes ( lt ) ] that can regulate additional inflammatory mediators [ e.g. , tumor necrosis factor ( tnf)- ] . this hypothesis is supported by a study reporting that malnourished , severely ill patients experienced significantly increased total production of tnf- when receiving pn with a soybean - oil - based le but not an mct / soybean oil le . although there has been much focus on the proinflammatory effects of aa - derived eicosanoids , lipoxins derived from aa have potent inflammation - resolving effects . conversely , the long - chain -3 pufas epa and dha found in fish oils are thought to possess anti - inflammatory properties because they are readily incorporated into cell membranes and thereby impact aa metabolism [ 18 , 32 ] , because the metabolism of epa is associated with production of less biologically potent eicosanoids than those produced from aa [ 18 , 32 ] , and because epa and dha are precursors of resolvins with powerful inflammation - resolving properties [ 18 , 69 ] . indeed , when soybean oil was partially replaced with fish oil ( 3:1 ratio ) in surgical patients receiving pn , lt synthesis was shifted from the proinflammatory ltb4 ( produced from aa ) to the less potent ltb5 ( produced from epa ) ( table 4 ) [ 70 , 71 ] . this change was associated with significantly reduced concentrations of the inflammatory mediators interleukin ( il)-6 and tnf- . in patients with sepsis , concentrations of il-1 , il-6 , il-8 , and tnf- released from mononuclear leukocytes were significantly increased following administration of a soybean - oil - based le compared with a decrease of approximately 30% following administration of a fish - oil - based le . lipid emulsions rich in olive oil have not been well studied with regard to their effect on inflammation ; however , a few studies have demonstrated that oa has relatively few effects on the production of inflammatory mediators . in a preclinical study , lipopolysaccharide - induced production of il-1 , il-6 , il-8 , and tnf- by neutrophils was not altered by an olive - oil - rich le , compared with significant reductions in il-1 with soybean - oil - based and mct / soybean oil les . in another study , infectious complication rates , mortality rates , and length of icu stay were similar among critically ill patients receiving pn containing soybean - oil - based and olive - oil - rich les . thrombosis is a common and serious complication for many critically ill , surgical , and trauma patients , as these states may be associated with changes in the availability of clotting factors and alterations in the fibrinolytic pathway , resulting in intravascular coagulation . however , the effects of les on coagulation have not been extensively assessed . in one study , platelet aggregation was inhibited immediately following intravenous infusion of a fish - oil - based le to healthy volunteers , but had returned to normal at 24 h . in surgical patients receiving pn , the latency to collagen - induced platelet aggregation and time to maximal platelet aggregation were significantly longer for a soybean - oil - based le than for a fish - oil - enriched le ; however , adenosine - diphosphate - induced platelet aggregation and bleeding time were unchanged . another study found no change in platelet aggregation for critically ill patients receiving either soybean - oil - based or mct / soybean oil les . the effects of parenteral olive - oil - rich les on thrombosis have not been systematically evaluated . however , in one study of patients undergoing hemodialysis , the need to change hemofilters due to blood coagulation was significantly reduced among patients receiving pn containing an olive - oil - rich le compared with a soybean - oil - based le . a summary of the key characteristics of available parenteral les is presented in table 5 ; in some countries ( i.e. , the usa ) only soybean - oil - based les are available . it has been suggested that the ratio of la to ala is important because of competition between these fa for a number of enzymes , thereby potentially influencing the production of eicosanoid and eicosanoid - like inflammatory mediators . however , a number of studies have suggested that the absolute concentrations of certain pufa are more important than their ratio in determining their biologic effects [ 80 , 81 ] . in addition , the fa content of les can vary depending upon the specific source of oil ( e.g. , epa and dha within different fish oils ) .table 5key characteristics of widely available parenteral lipid emulsions [ 22 , 43 , 73 , 89]intralipidlipovenlipofundin - mctstructolipidomegavenlipoplusclinoleicsmoflipidmanufacturerfresenius - kabi , germanyfresenius - kabi , germanyb . braun , germanybaxter , francefresenius - kabi , germanyoil source ( % by weight)soybeansoybeancoconut ( 50% ) , soybean ( 50%)coconut ( 36% ) , soybean ( 64%)fish ( 100%)coconut ( 50% ) , soybean ( 40% ) , fish ( 10%)olive ( 80% ) , soybean ( 20%)coconut ( 30% ) , soybean ( 30% ) , olive ( 25% ) , fish ( 15%)typical fa composition ( % of total fa ) caproic0.5tracetrace caprylic28.5263010 capric201019.511 lauric1tracetrace myristic50.5trace1 palmitic11127.571261210 stearic45234.52.523.5 palmitoleic90.51.51.5 oleic242411141586231 linoleic535329354.524.51920 -linolenic884.541.83.52.52 arachidonic2 eicosapentaenoic203.53 docosapentaenoic23trace docosahexaenoic122.52 -tocopherol ( mol / l)871325021650556275500fa fatty acids key characteristics of widely available parenteral lipid emulsions [ 22 , 43 , 73 , 89 ] the stability of les , with respect to phase separation and presence of large globules , is also an important issue in clinical settings in which the final concentrations of lipid components and emulsifiers are of key importance . all les eventually become unstable when diluted above a certain threshold within the pn formulation . current regulations in the usa require that globules > 5 m do not exceed 0.05% ( weight / volume ) of the emulsion . in general , mct / soybean oil and olive - oil - rich les have demonstrated greater stability when compared with soybean - oil- and safflower - oil - based les , although stability may vary between manufacturers [ 82 , 83 ] . the stability of mct / soybean oil les may be due to the inclusion of shorter - chain lipids , which exhibit a lower free energy when dispersed in water , resulting in greater miscibility between phases and reduced physicochemical stress on emulsifying agents compared with les containing predominantly longer - chain triglycerides . olive - oil - rich les contain sodium oleate , which acts as an additional emulsifying agent and thereby augments stability . in addition , the stability of pn formulations that contain les may be affected by interactions between fa and other commonly administered compounds , such as carnitine , heparin , and some vitamins [ 8486 ] . lipid emulsions demonstrate different biologic effects depending upon their specific fa content , which may translate into beneficial effects for selected patients ( table 6 ) . the biologic effects associated with les are likely to benefit a majority of patients receiving parenteral les , including those on long - term total pn , but may have the greatest importance for patients under metabolic stress . therefore , physicians need to consider several issues when selecting an le as part of a pn regimen.table 6potential therapeutic applications of lipid emulsionscell function and proliferation provide sufficient fatty acids improve metabolism and limit / reverse energy deficitoxidative stress limit the contribution of lipid peroxidation to oxidative stress maintain or increase antioxidant concentrationsintrinsic immune function support the immune system and limit immunosuppression reduce the incidence of infectious complicationsinflammation prevent / regulate hyperinflammation , especially important for patients with pre - existing inflammation ( e.g. , surgery , sepsis , chronic inflammatory diseases ) potential therapeutic applications of lipid emulsions all currently available les provide sufficient -6 and -3 efa , with the exception of 100% fish oil le , which should generally only be used as a pharmacological agent or as a supplement to other les [ 21 , 22 ] . although there are conflicting views regarding the comparative utility of different le formulations in critically ill patients , there is growing consensus that les based entirely on soybean oil should be avoided in favor of emulsions in which the la and ala content is partially replaced by mct , olive oil providing mufa or fish oil providing epa and dha . this is particularly true for patients with highly proinflammatory states , such as surgical , trauma , burn , and septic patients [ 8 , 87 ] . in addition , the clinical use of les should not exacerbate oxidative stress in critically ill patients . unfortunately , evidence on the differential effects of les in critically ill patients remains limited . furthermore , inconsistencies of findings between studies have suggested that the biologic effects of fa may vary with the medical condition and level of metabolic stress ; therefore , further clinical trials exploring the effects of fa in different subpopulations of critically ill patients are highly desirable . a summary of the key characteristics of available parenteral les is presented in table 5 ; in some countries ( i.e. , the usa ) only soybean - oil - based les are available . it has been suggested that the ratio of la to ala is important because of competition between these fa for a number of enzymes , thereby potentially influencing the production of eicosanoid and eicosanoid - like inflammatory mediators . however , a number of studies have suggested that the absolute concentrations of certain pufa are more important than their ratio in determining their biologic effects [ 80 , 81 ] . in addition , the fa content of les can vary depending upon the specific source of oil ( e.g. , epa and dha within different fish oils ) .table 5key characteristics of widely available parenteral lipid emulsions [ 22 , 43 , 73 , 89]intralipidlipovenlipofundin - mctstructolipidomegavenlipoplusclinoleicsmoflipidmanufacturerfresenius - kabi , germanyfresenius - kabi , germanyb . braun , germanybaxter , francefresenius - kabi , germanyoil source ( % by weight)soybeansoybeancoconut ( 50% ) , soybean ( 50%)coconut ( 36% ) , soybean ( 64%)fish ( 100%)coconut ( 50% ) , soybean ( 40% ) , fish ( 10%)olive ( 80% ) , soybean ( 20%)coconut ( 30% ) , soybean ( 30% ) , olive ( 25% ) , fish ( 15%)typical fa composition ( % of total fa ) caproic0.5tracetrace caprylic28.5263010 capric201019.511 lauric1tracetrace myristic50.5trace1 palmitic11127.571261210 stearic45234.52.523.5 palmitoleic90.51.51.5 oleic242411141586231 linoleic535329354.524.51920 -linolenic884.541.83.52.52 arachidonic2 eicosapentaenoic203.53 docosapentaenoic23trace docosahexaenoic122.52 -tocopherol ( mol / l)871325021650556275500fa fatty acids key characteristics of widely available parenteral lipid emulsions [ 22 , 43 , 73 , 89 ] the stability of les , with respect to phase separation and presence of large globules , is also an important issue in clinical settings in which the final concentrations of lipid components and emulsifiers are of key importance . all les eventually become unstable when diluted above a certain threshold within the pn formulation . current regulations in the usa require that globules > 5 m do not exceed 0.05% ( weight / volume ) of the emulsion . in general , mct / soybean oil and olive - oil - rich les have demonstrated greater stability when compared with soybean - oil- and safflower - oil - based les , although stability may vary between manufacturers [ 82 , 83 ] . the stability of mct / soybean oil les may be due to the inclusion of shorter - chain lipids , which exhibit a lower free energy when dispersed in water , resulting in greater miscibility between phases and reduced physicochemical stress on emulsifying agents compared with les containing predominantly longer - chain triglycerides . olive - oil - rich les contain sodium oleate , which acts as an additional emulsifying agent and thereby augments stability . in addition , the stability of pn formulations that contain les may be affected by interactions between fa and other commonly administered compounds , such as carnitine , heparin , and some vitamins [ 8486 ] . lipid emulsions demonstrate different biologic effects depending upon their specific fa content , which may translate into beneficial effects for selected patients ( table 6 ) . the biologic effects associated with les are likely to benefit a majority of patients receiving parenteral les , including those on long - term total pn , but may have the greatest importance for patients under metabolic stress . therefore , physicians need to consider several issues when selecting an le as part of a pn regimen.table 6potential therapeutic applications of lipid emulsionscell function and proliferation provide sufficient fatty acids improve metabolism and limit / reverse energy deficitoxidative stress limit the contribution of lipid peroxidation to oxidative stress maintain or increase antioxidant concentrationsintrinsic immune function support the immune system and limit immunosuppression reduce the incidence of infectious complicationsinflammation prevent / regulate hyperinflammation , especially important for patients with pre - existing inflammation ( e.g. , surgery , sepsis , chronic inflammatory diseases ) potential therapeutic applications of lipid emulsions all currently available les provide sufficient -6 and -3 efa , with the exception of 100% fish oil le , which should generally only be used as a pharmacological agent or as a supplement to other les [ 21 , 22 ] . although there are conflicting views regarding the comparative utility of different le formulations in critically ill patients , there is growing consensus that les based entirely on soybean oil should be avoided in favor of emulsions in which the la and ala content is partially replaced by mct , olive oil providing mufa or fish oil providing epa and dha . this is particularly true for patients with highly proinflammatory states , such as surgical , trauma , burn , and septic patients [ 8 , 87 ] . in addition , the clinical use of les should not exacerbate oxidative stress in critically ill patients . unfortunately , evidence on the differential effects of les in critically ill patients remains limited . furthermore , inconsistencies of findings between studies have suggested that the biologic effects of fa may vary with the medical condition and level of metabolic stress ; therefore , further clinical trials exploring the effects of fa in different subpopulations of critically ill patients are highly desirable . energy deficit is a major problem among icu patients and is associated with an increased incidence of complications , length of stay , and mortality . pn , either alone or in combination with en , can improve caloric delivery to critically ill patients , preventing or correcting energy deficits and improving outcomes . lipids are an important source of calories in artificial nutrition , and they have demonstrated a wide range of biologic activities that may benefit a variety of patients receiving pn , as well as those receiving en with or without pn supplementation . parenteral lipid emulsions derived from soybean oil are the most extensively evaluated formulations in preclinical and clinical studies and have demonstrated efficacy and safety in delivering vital nutrition to critically ill patients . newer les that utilize partial substitution of soybean oil with mct , olive oil or fish oil either alone or in combination have demonstrated potential benefits in terms of reduced impacts on oxidative stress and differential effects on cell - mediated immunity and inflammation . however , few published studies have evaluated the biologic effects of newer parenteral les , and data assessing the clinical benefits of these newer formulations are limited and sometimes inconsistent because of the heterogeneity of the study designs and patient populations . ongoing research to further characterize and compare the biologic properties of lipids given parenterally , as well as enterally , will be an important resource for physicians , especially those managing critically ill patients , who are often under metabolic stress . , the prescription of les should be based upon the limited clinical data available , the range of available les , cost implications , and an understanding of the potential biologic effects of their components , bearing in mind the situation and therapeutic goals of the individual patient .

backgroundenergy deficit is a common and serious problem in intensive care units and is associated with increased rates of complications , length of stay , and mortality . parenteral nutrition ( pn ) , either alone or in combination with enteral nutrition , can improve nutrient delivery to critically ill patients . lipids provide a key source of calories within pn formulations , preventing or correcting energy deficits and improving outcomes.discussionin this article , we review the role of parenteral lipid emulsions ( les ) in the management of critically ill patients and highlight important biologic activities associated with lipids . soybean - oil - based les with high contents of polyunsaturated fatty acids ( pufa ) were the first widely used formulations in the intensive care setting . however , they may be associated with increased rates of infection and lipid peroxidation , which can exacerbate oxidative stress . more recently developed parenteral les employ partial substitution of soybean oil with oils providing medium - chain triglycerides , -9 monounsaturated fatty acids or -3 pufa . many of these les have demonstrated reduced effects on oxidative stress , immune responses , and inflammation . however , the effects of these les on clinical outcomes have not been extensively evaluated.conclusionsongoing research using adequately designed and well - controlled studies that characterize the biologic properties of les should assist clinicians in selecting les within the critical care setting . prescription of pn containing les should be based on available clinical data , while considering the individual patient s physiologic profile and therapeutic requirements .

Introduction Role of parenteral nutrition in the intensive care setting Individualizing nutrition in critically ill patients The role of lipid emulsions in parenteral nutrition Evolution of parenteral lipid emulsions Fatty acids Early parenteral lipid emulsions Minimization of risks associated with parenteral lipids in critical care patients Oxidative stress Cell-mediated immunity Inflammation Thrombosis Choosing a parenteral lipid emulsion for the critically ill patient Components of commercially available lipid emulsions Potential therapeutic roles for lipid emulsions Conclusions

this article aims to review well - known papers that evaluate potential biologic effects of lipids when provided as a parenteral energy source and to provide future perspectives on the use of parenteral lipid emulsions ( les ) in critically ill patients . however , studies published during the 1970s and 1980s found that soybean - oil- and cottonseed - oil - based les were associated with a number of adverse immunological effects , such as reduced migration and phagocytosis of granulocytes , which resulted in increased rates of infections , including sepsis [ 3437 ] . depletion of the antioxidants selenium and zinc has been observed in trauma and burn patients , while surgery is associated with reductions in vitamins a , c , and e. this state of imbalance can cause tissue damage and may play an important role in the development of sepsis and multiple organ failure , among other complications ( table 3 ) [ 38 , 39].table 3diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage septic shockacute respiratory distress syndromesystemic inflammatory response syndromedisseminated intravascular coagulationmultiple organ dysfunctionburnscardiovascular diseasediabetes mellitustraumareperfusion injurycancer diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage a key consequence of oxidative stress is lipid peroxidation , where ros react with the double bond of unsaturated lipids , producing unstable lipid peroxides that may cause cell death [ 38 , 39 ] . the high number of double bonds present in -6 and -3 polyunsaturated fatty acids ( pufa ) provide targets for lipid peroxidation , and these fa may therefore be associated with an increased risk of oxidative stress . however , a separate study comparing a soybean - oil - based le with a mct / soybean oil le supplemented with -tocopherol in patients undergoing abdominal surgery found no difference in lipid peroxidation between the two les .table 4key clinical studies evaluating biological and clinical effects of lipid emulsionsclinical studypopulationdesigntreatment groupskey findingslipid peroxidation / antioxidant activity gobel et al . adults undergoing total esophagectomy ( n = 19)prospective , randomized studylipoven vs. lipoven + omegaven for 7 daysmean bleeding time : 3.1 ( lipoven ) vs. 3.3 min ( lipoven + omegaven ; ns)mean maximal collagen - induced platelet aggregation : 91% ( lipoven ) vs. 87% ( lipoven + omegaven ; ns)mean maximal adenosine diphosphate - induced platelet aggregation : 78% ( lipoven ) vs. 77% ( lipoven + omegaven ; ns)all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise statedldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin key clinical studies evaluating biological and clinical effects of lipid emulsions all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise stated ldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin metabolism of mct differs from that of long - chain triglycerides ( lct ) . when compared with pn containing no lipids administered to trauma patients , pn containing a soybean - oil - based le was associated with higher rates of infection and significantly longer durations of mechanical ventilation , longer icu stays , and longer hospital stays ( table 4 ) . among surgical patients receiving pn , a soybean - oil - based le supplemented with fish oil was associated with a shorter icu and overall hospital stay when compared with a le that did not contain fish oil ; however , the rates of infection were similar between groups ( table 4 ) . when a soybean - oil - based le was supplemented with fish oil , higher doses were associated with a reduction in the length of icu and overall hospital stay , significantly reduced antibiotic requirement , and significantly increased survival . in a study involving healthy volunteers , administration of an mct / soybean oil le a second study of healthy subjects found that , in contrast to intermittent infusion of soybean - oil - based le , infusion of pn containing mct / soybean oil le did not impair the clearance of 99tc - sulfur colloid by the reticuloendothelial system . lipid emulsions containing high concentrations of olive oil may have less impact on the host immune response than soybean - oil - based or mct / soybean oil les , with little effect on lymphocytes , natural killer cells , and neutrophils . the immunosupportive effects of an olive - oil - rich emulsion may be reflected clinically by the low incidence of infectious complications reported in severely burned patients receiving pn in the icu : in a study evaluating this patient population , the incidences of sepsis and multiple organ failure and the duration of mechanical ventilation , icu stay , and hospital stay were comparable for patients receiving olive - oil - rich and mct / soybean oil les ( table 4 ) . administration of soybean - oil - based les is associated with high blood levels of the -6 pufa la and its metabolite aa , the further metabolism of which may produce proinflammatory eicosanoids [ e.g. in another study , infectious complication rates , mortality rates , and length of icu stay were similar among critically ill patients receiving pn containing soybean - oil - based and olive - oil - rich les . thrombosis is a common and serious complication for many critically ill , surgical , and trauma patients , as these states may be associated with changes in the availability of clotting factors and alterations in the fibrinolytic pathway , resulting in intravascular coagulation . however , the effects of les on coagulation have not been extensively assessed . the effects of parenteral olive - oil - rich les on thrombosis have not been systematically evaluated . however , studies published during the 1970s and 1980s found that soybean - oil- and cottonseed - oil - based les were associated with a number of adverse immunological effects , such as reduced migration and phagocytosis of granulocytes , which resulted in increased rates of infections , including sepsis [ 3437 ] . depletion of the antioxidants selenium and zinc has been observed in trauma and burn patients , while surgery is associated with reductions in vitamins a , c , and e. this state of imbalance can cause tissue damage and may play an important role in the development of sepsis and multiple organ failure , among other complications ( table 3 ) [ 38 , 39].table 3diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage septic shockacute respiratory distress syndromesystemic inflammatory response syndromedisseminated intravascular coagulationmultiple organ dysfunctionburnscardiovascular diseasediabetes mellitustraumareperfusion injurycancer diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage a key consequence of oxidative stress is lipid peroxidation , where ros react with the double bond of unsaturated lipids , producing unstable lipid peroxides that may cause cell death [ 38 , 39 ] . the high number of double bonds present in -6 and -3 polyunsaturated fatty acids ( pufa ) provide targets for lipid peroxidation , and these fa may therefore be associated with an increased risk of oxidative stress . however , a separate study comparing a soybean - oil - based le with a mct / soybean oil le supplemented with -tocopherol in patients undergoing abdominal surgery found no difference in lipid peroxidation between the two les .table 4key clinical studies evaluating biological and clinical effects of lipid emulsionsclinical studypopulationdesigntreatment groupskey findingslipid peroxidation / antioxidant activity gobel et al . adults undergoing total esophagectomy ( n = 19)prospective , randomized studylipoven vs. lipoven + omegaven for 7 daysmean bleeding time : 3.1 ( lipoven ) vs. 3.3 min ( lipoven + omegaven ; ns)mean maximal collagen - induced platelet aggregation : 91% ( lipoven ) vs. 87% ( lipoven + omegaven ; ns)mean maximal adenosine diphosphate - induced platelet aggregation : 78% ( lipoven ) vs. 77% ( lipoven + omegaven ; ns)all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise statedldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin key clinical studies evaluating biological and clinical effects of lipid emulsions all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise stated ldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin metabolism of mct differs from that of long - chain triglycerides ( lct ) . when compared with pn containing no lipids administered to trauma patients , pn containing a soybean - oil - based le was associated with higher rates of infection and significantly longer durations of mechanical ventilation , longer icu stays , and longer hospital stays ( table 4 ) . among surgical patients receiving pn , a soybean - oil - based le supplemented with fish oil was associated with a shorter icu and overall hospital stay when compared with a le that did not contain fish oil ; however , the rates of infection were similar between groups ( table 4 ) . when a soybean - oil - based le was supplemented with fish oil , higher doses were associated with a reduction in the length of icu and overall hospital stay , significantly reduced antibiotic requirement , and significantly increased survival . in a study involving healthy volunteers , administration of an mct / soybean oil le a second study of healthy subjects found that , in contrast to intermittent infusion of soybean - oil - based le , infusion of pn containing mct / soybean oil le did not impair the clearance of 99tc - sulfur colloid by the reticuloendothelial system . the immunosupportive effects of an olive - oil - rich emulsion may be reflected clinically by the low incidence of infectious complications reported in severely burned patients receiving pn in the icu : in a study evaluating this patient population , the incidences of sepsis and multiple organ failure and the duration of mechanical ventilation , icu stay , and hospital stay were comparable for patients receiving olive - oil - rich and mct / soybean oil les ( table 4 ) . administration of soybean - oil - based les is associated with high blood levels of the -6 pufa la and its metabolite aa , the further metabolism of which may produce proinflammatory eicosanoids [ e.g. in another study , infectious complication rates , mortality rates , and length of icu stay were similar among critically ill patients receiving pn containing soybean - oil - based and olive - oil - rich les . thrombosis is a common and serious complication for many critically ill , surgical , and trauma patients , as these states may be associated with changes in the availability of clotting factors and alterations in the fibrinolytic pathway , resulting in intravascular coagulation . the effects of parenteral olive - oil - rich les on thrombosis have not been systematically evaluated . depletion of the antioxidants selenium and zinc has been observed in trauma and burn patients , while surgery is associated with reductions in vitamins a , c , and e. this state of imbalance can cause tissue damage and may play an important role in the development of sepsis and multiple organ failure , among other complications ( table 3 ) [ 38 , 39].table 3diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage septic shockacute respiratory distress syndromesystemic inflammatory response syndromedisseminated intravascular coagulationmultiple organ dysfunctionburnscardiovascular diseasediabetes mellitustraumareperfusion injurycancer diseases and intensive care unit states associated with reactive oxygen species - mediated tissue damage a key consequence of oxidative stress is lipid peroxidation , where ros react with the double bond of unsaturated lipids , producing unstable lipid peroxides that may cause cell death [ 38 , 39 ] . the high number of double bonds present in -6 and -3 polyunsaturated fatty acids ( pufa ) provide targets for lipid peroxidation , and these fa may therefore be associated with an increased risk of oxidative stress . however , a separate study comparing a soybean - oil - based le with a mct / soybean oil le supplemented with -tocopherol in patients undergoing abdominal surgery found no difference in lipid peroxidation between the two les .table 4key clinical studies evaluating biological and clinical effects of lipid emulsionsclinical studypopulationdesigntreatment groupskey findingslipid peroxidation / antioxidant activity gobel et al . adults undergoing total esophagectomy ( n = 19)prospective , randomized studylipoven vs. lipoven + omegaven for 7 daysmean bleeding time : 3.1 ( lipoven ) vs. 3.3 min ( lipoven + omegaven ; ns)mean maximal collagen - induced platelet aggregation : 91% ( lipoven ) vs. 87% ( lipoven + omegaven ; ns)mean maximal adenosine diphosphate - induced platelet aggregation : 78% ( lipoven ) vs. 77% ( lipoven + omegaven ; ns)all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise statedldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin key clinical studies evaluating biological and clinical effects of lipid emulsions all lipid emulsions administered as part of total parenteral nutrition within the icu setting , unless otherwise stated ldl low - density lipoprotein , ns not significant , vldl very low - density lipoprotein , pn parenteral nutrition , icu intensive care unit , mct medium - chain triglycerides , tnf tumor necrosis factor , lt leukotriene , il interleukin metabolism of mct differs from that of long - chain triglycerides ( lct ) . when compared with pn containing no lipids administered to trauma patients , pn containing a soybean - oil - based le was associated with higher rates of infection and significantly longer durations of mechanical ventilation , longer icu stays , and longer hospital stays ( table 4 ) . among surgical patients receiving pn , a soybean - oil - based le supplemented with fish oil was associated with a shorter icu and overall hospital stay when compared with a le that did not contain fish oil ; however , the rates of infection were similar between groups ( table 4 ) . when a soybean - oil - based le was supplemented with fish oil , higher doses were associated with a reduction in the length of icu and overall hospital stay , significantly reduced antibiotic requirement , and significantly increased survival . in a study involving healthy volunteers , administration of an mct / soybean oil le a second study of healthy subjects found that , in contrast to intermittent infusion of soybean - oil - based le , infusion of pn containing mct / soybean oil le did not impair the clearance of 99tc - sulfur colloid by the reticuloendothelial system . the immunosupportive effects of an olive - oil - rich emulsion may be reflected clinically by the low incidence of infectious complications reported in severely burned patients receiving pn in the icu : in a study evaluating this patient population , the incidences of sepsis and multiple organ failure and the duration of mechanical ventilation , icu stay , and hospital stay were comparable for patients receiving olive - oil - rich and mct / soybean oil les ( table 4 ) . administration of soybean - oil - based les is associated with high blood levels of the -6 pufa la and its metabolite aa , the further metabolism of which may produce proinflammatory eicosanoids [ e.g. in another study , infectious complication rates , mortality rates , and length of icu stay were similar among critically ill patients receiving pn containing soybean - oil - based and olive - oil - rich les . thrombosis is a common and serious complication for many critically ill , surgical , and trauma patients , as these states may be associated with changes in the availability of clotting factors and alterations in the fibrinolytic pathway , resulting in intravascular coagulation . however , the effects of les on coagulation have not been extensively assessed . the effects of parenteral olive - oil - rich les on thrombosis have not been systematically evaluated . in general , mct / soybean oil and olive - oil - rich les have demonstrated greater stability when compared with soybean - oil- and safflower - oil - based les , although stability may vary between manufacturers [ 82 , 83 ] . in general , mct / soybean oil and olive - oil - rich les have demonstrated greater stability when compared with soybean - oil- and safflower - oil - based les , although stability may vary between manufacturers [ 82 , 83 ] . energy deficit is a major problem among icu patients and is associated with an increased incidence of complications , length of stay , and mortality . pn , either alone or in combination with en , can improve caloric delivery to critically ill patients , preventing or correcting energy deficits and improving outcomes . parenteral lipid emulsions derived from soybean oil are the most extensively evaluated formulations in preclinical and clinical studies and have demonstrated efficacy and safety in delivering vital nutrition to critically ill patients . newer les that utilize partial substitution of soybean oil with mct , olive oil or fish oil either alone or in combination have demonstrated potential benefits in terms of reduced impacts on oxidative stress and differential effects on cell - mediated immunity and inflammation . , the prescription of les should be based upon the limited clinical data available , the range of available les , cost implications , and an understanding of the potential biologic effects of their components , bearing in mind the situation and therapeutic goals of the individual patient .

pertussis , an acute infectious illness of the respiratory tract remains endemic in developed nations despite high vaccination coverage [ 7 , 8 , 16 ] . while the early use of whole - cell vaccine was highly effective in reducing the incidence of reported pertussis in the united states in the 1970s , there has been a resurgence of reported pertussis over the last 15 years [ 8 , 16 , 31 ] . worldwide , there are an estimated 50 million cases occurring annually ( 90% of which are in developing countries ) , and there are as many as 400,0000 pertussis - related deaths [ 16 , 31 ] . it is general consensus , moreover , that the reported incidence of pertussis is considerably lower than its actual incidence [ 8 , 31 ] . though in the prevaccine era pertussis was regarded as a childhood disease affecting primarily young children , pertussis epidemiology in the postvaccine era is different [ 8 , 17 ] . infants are the most vulnerable group with the highest rates of complications and mortality , yet adolescents and adults now comprise a significant percentage of cases and a conduit of infection for the infants [ 8 , 13 , 17 ] . pcr , culture , and serology are the mainstay of the laboratory diagnosis of pertussis , with various factors affecting the sensitivity and specificity of each modality [ 8 , 24 , 36 ] . however , in recent years , pcr has become an increasingly more popular tool and has significantly contributed to the increasing laboratory diagnosis of pertussis [ 8 , 27 , 36 ] . advances have also been made with regard to prevention and disease control , with experts from 17 countries recently establishing the global pertussis initiative ( gpi ) with the aim of analyzing the status of pertussis and enhancing existing immunization strategies [ 8 , 15 ] . before the introduction of the whole - cell pertussis vaccine in the1940s , there were approximately 200,000 cases reported annually in the us . immunizations reduced disease rates and in 1976 pertussis incidence reached a nadir of 1,010 reported cases [ 13 , 37 ] . however , since that time , there has been a substantial increase in the number of cases reported [ 8 , 13 , 31 ] . indeed , over the past 15 years , there has been a marked increase in the incidence of pertussis with reported disease in the us reaching a rate of 8.9 per 100,000 in 2004 with nearly 19,000 provisional reported cases [ 7 , 17 ] . it is also well established that , despite high vaccination coverage for primary immunization in infants and children , pertussis continues to be a global concern with increased incidence in many countries including argentina , australia , canada , italy , japan , the netherlands , switzerland and the us . it is also widely noted that in recent years there is a general shift in the age distribution of pertussis , with adults and adolescents an underrecognized but significant source of infection for neonates and infants [ 8 , 13 , 1517 ] . data from the euvac - net project , a network for the epidemiologic surveillance and control of communicable diseases in the european community , demonstrate that between 1998 and 2002 , the rate of disease incidence remained stable at a high rate among children less than 1 year old . nevertheless , these data indicate that the incidence rate among adults doubled in 5 years . similarly , centers for disease control and prevention ( cdc ) surveillance data from 19902003 demonstrate that the reported incidence of pertussis among adolescents has substantially increased with a nearly ten - fold rise . moreover , when comparing pertussis disease rates in 19901993 , recent cdc data from 2004 reveal a nearly 19-fold increase in the number of cases in persons aged 1019 years and a 16-fold increase in persons over 20 years . several factors have been proposed as underlying the increasing incidence of pertussis disease including waning immunity with subsequent atypical disease manifestations , increasing awareness by public health personnel with subsequent enhanced surveillance and improved laboratory diagnostics [ 8 , 17 , 31 ] . waning of both vaccine - induced immunity and infection - acquired immunity is widely cited as an important reason for recent epidemiologic trends [ 7 , 8 , 36 , 37 ] . while the assessment of the duration of immunity afforded after either natural infection or vaccination is complex , individuals are clearly susceptible to initial infection / reinfection after vaccination or previous pertussis illness , respectively . studies vary in their estimation of protection against disease with protective immunity after natural infection waning 720 years after illness , and duration of immunity after vaccination waning at approximately 412 years in children . yet , regardless of the precise interval , when individuals do contract pertussis after the waning of their immunity , their disease manifestations are frequently atypical [ 8 , 17 , 27 ] . as such such underdiagnosis poses a potentially serious public - health concern in that those untreated persons with protracted cough continue to unknowingly transmit the disease to others . finally , it has been proposed that the increased incidence rates may also be a function of enhanced surveillance as well as improved and more sensitive diagnostic lab techniques ( e.g. , pcr ) , in that such techniques allow for the diagnosis of cases that would probably have been missed in the past [ 8 , 17 , 27 , 35 ] . nevertheless , it is important to note , that the current estimates are likely to be , if anything , an underrepresentation of the true incidence of disease [ 8 , 31 ] . first , the clinical diagnosis of pertussis is complicated by underconsulting , particularly among adolescents and adults . second , with prolonged cough often being their only clinical feature , by the time these adolescents and adults finally do seek medical attention , it is often too late to culture or detect the organism by pcr , thus potentially resulting in a missed diagnosis [ 8 , 17 , 31 , 35 ] . moreover , the wide heterogeneity in disease expression , modification of disease by immunization , mixed infection , inconsistent definition , and insensitive nonstandardized , poorly performed , or lack of available laboratory tests , further complicate physician diagnosis . while the classic or typical pertussis may be easily recognized , it is seen less often since general immunization began . instead , atypical pertussis , usually characterized by the absence of whoop and often a somewhat shorter duration of cough , is more common than classical pertussis among adolescents and adults [ 8 , 17 ] . and finally , it should be noted that , immunized young children that are pcr positive for b pertussis can be asymptomatic [ 29 , 31 ] . regardless of whether an individual displays classical pertussis signs and symptoms or a more protracted , atypical cough , pertussis may not be suspected because of the misconception among many physicians that pertussis is a childhood disease [ 8 , 17 ] . co - occurrence of other infections like influenza a or b , adenovirus , and rsv may also complicate the clinical diagnosis . and , finally , even when diagnosed , pertussis is often underreported . indeed , sutter and cochi report that in the us , only an estimated 11.6% of pertussis cases were actually reported [ 17 , 30 ] . thus multiple institutional , clinical , and laboratory factors diminish the true assessment of pertussis incidence , and the current data clearly are an underestimation of the true burden of disease . because accurate diagnosis of pertussis can not be made by clinical signs and symptoms alone , there is a need for improved laboratory diagnosis of pertussis . while several laboratory techniques exist for the identification of b. pertussis , namely , culture , serology and pcr , overall , several practical factors may adversely affect the sensitivity of its laboratory diagnosis . delayed specimen collection , poor specimen collection techniques , specimen transport problems , and lab media contamination are but a few of the practical constraints often influencing the outcomes of the laboratory diagnosis of pertussis . moreover , previous exposure to the organism , patient s age , stage of disease , previous antibiotic administration , and immunization are other factors that may have a substantial impact on the sensitivity of the tests . finally , limited access to diagnostic or laboratory methods , in both developed and developing countries , undoubtedly affects b. pertussis laboratory confirmation [ 8 , 24 ] . culture b. pertussis is a fastidious gram - negative cocobacillus , and its isolation from nasopharyngeal secretions remains the gold standard for diagnosis . culture requires collection of a posterior nasopharyngeal specimen with a dacron or calcium alginate swab . to increase the yield of positive cultures , specimens should be immediately plated onto selective regan lowe agar or bordet gengou medium , selective media that are seldom readily available in physician 's offices because of their cost and short shelf - life [ 17 , 24 ] . the main reasons for failure of bacterial growth in culture , from correctly collected and transported specimens , stem from bacterial and fungal contamination and the lack of fresh media . generally , 710 days are required to grow , isolate , and identify the organism , an obvious limitation of the culture method.the timing of obtaining specimens for culture is also of paramount importance and greatly affects its yield . the proportion of patients testing positive for pertussis by culture is highest when the initial specimens are obtained early in the course of illness , i.e. , during the early catarrhal phase of the illness when the organism is present in the nasopharynx in sufficient quantity . however , adults and adolescents often present late in the course of their illness , thereby greatly reducing the likelihood of culturing the organism [ 17 , 24 ] . studies also demonstrate that proportions of positive cultures decline in patients who have been previously immunized and undoubtedly in those in whom antibiotics have been started . thus , given the limited window of opportunity for positive culture , it is important to stress that a negative culture does not exclude pertussis . finally , it is important to emphasize , that despite its low yield , culture should be attempted , as the bacterial isolates are needed for genotypic and phenotypic analysis . pcr the use of pcr for the diagnosis of pertussis is rapidly evolving as it provides a sensitive , rapid means for laboratory diagnosis in circumstances in which the probability of a positive culture is low [ 8 , 17 , 32 , 35 ] . notably , the cdc and world health organization ( who ) now include a positive pcr in their lab definition of pertussis . while the sensitivity of pcr also decreases somewhat with the duration of cough and among previously immunized individuals , it is nevertheless a significantly more robust tool for diagnosis for those in the later stages of the disease or for those who have already received antibiotics [ 17 , 35 ] . specifically , in their 2005 consensus paper , the european research programme for improved pertussis strain characterization and surveillance ( eupertstrain ) state that the real - time pcr is more sensitive than culture for the detection of b. pertussis , especially after the first 34 weeks of coughing and after antibiotic therapy has been initiated [ 16 , 27 ] . in a prospective study in which nasopharyngeal samples were obtained simultaneously for both pcr and culture , the identification of b. pertussis infections was nearly four - fold higher with pcr [ 8 , 28 ] . finally , pcr is an invaluable tool for the diagnosis of pertussis among young infants since the yield of culture is low and serology is problematic in this age group [ 1 , 17].as with culture , important factors for the successful application of pcr in the diagnosis of infection by bordetella species include proper sample collection and preparation . for example , a dacron swab with a fine flexible wire shaft , and not calcium alginate , is the recommended swab . after obtaining the nasopharyngeal sample , the swab should be shaken vigorously in saline solution , the swab discarded and the vial sealed for further processing . appropriate primer selection , amplification conditions , and controls are also essential for effective pcr testing . primers have been derived from four chromosomal regions and common primers employed in pcr detection systems include is481 , is1001 ptp1 , and ptp2 [ 8 , 24 ] . inherent with the high sensitivity , false positive results are a well - recognized problem associated with the pcr diagnosis of pertussis and other respiratory illnesses . while at the present time , pcr is not routinely available and its methods need more standardization , optimization , and quality control , in the future , an internationally accepted standardized kit might be available , which would facilitate the expanded use of pcr for pertussis diagnosis [ 16 , 35 ] . serology natural infection with b. pertussis is followed by an increase in serum levels of iga , igm , and igg antibodies to specific pertussis antigen whereas the primary immunization of children induces mainly igm and igg antibodies . during the past 15 years , elisas have constituted the mainstay of serologic diagnosis using specific b. pertussis protein as antigens , and the serologic diagnosis of pertussis is suspected with increases in iga or igg antibody titers to pertussis toxin ( pt ) , filamentous hemmaglutinin ( fha ) , pertactin , fimbriae or sonicated whole organisms in two serum samples collected 24 weeks apart . notably , these antibody responses to fha are not specific to b. pertussis , but also occur following other bordetella species ; moreover , these antibodies may be cross - reacting epitopes to other bacteria including h. influenzae and m. pneumoniae . thus , the greatest sensitivity and specificity for the serological diagnosis of b. pertussis infection is by elisa measurement of igg and iga antibodies to pt demonstrating at least a two - fold rise in titer between acute- and convalescent - phase sera .still , the main problem in the serologic diagnosis of b. pertussis by elisa is the frequent delay in obtaining the acute - phase specimen . in individuals with re - infections , there is a rapid increase in titer such that if a delayed acute - phase sample is obtained , the titer is likely to have already peaked , thereby hampering the detection of the significant titer increase between the acute- and convalescent - phase serum samples . notably , for those individuals not recently immunized , a single - serum sample elisa may circumvent the problem , as ill patients will have significantly higher elisa titers than the geometric mean titers ( gmt ) of healthy controls [ 2325 ] . with this in mind , although a rise in pt iga is more suggestive of a recent antibody response , it is less consistent than a pt igg rise ; hence , in adolescents and adults , a single high value of igg or iga antibodies to pt suggests pertussis infection [ 17 , 24 , 35 ] . indeed , de melker et al . demonstrated that an igg concentration to pt of at least 100 units / ml in a single serum sample was diagnostic of either a recent or active pertussis infection .the serological diagnosis of pertussis among infants also has notable limitations . some culture - positive patients , particularly infants younger than 3 months , do not develop measurable antibodies , a finding that calls into question the utility of even obtaining a serum specimen for serology in young infants .in summary , despite the shortcomings of serology , a single - sample serology test can be a useful tool , particularly among older patients presenting late in the course of their illness when culture and pcr testing are negative . use of antibiotics in the treatment and prevention of pertussis antimicrobial agents administered early in the course of disease , i.e. , during the catarrhal stage , may ameliorate the disease ; although , after the cough is established antibiotics do not have a discernable effect on the course of the illness , but rather are recommended to limit the spread of organisms to other individuals .erythromycin , clarithromycin or azithromycin are now considered first - line agents for treatment ( and prophylaxis ) of pertussis in individuals 6 months of age or older ( table 1 ) . the antibiotic choice for infants younger than 6 months of age , however , requires special attention . the fda has not yet approved azithromycin or clarithromycin for use in infants younger than 6 months ; however , the 2006 aap endorsed red book lists azithromycin as the preferred macrolide for this age group because of the risk of idiopathic hypertrophic pyloric stenosis associated with erythromycin . notably however , there was a recent report of infantile hypertrophic pyloric stenosis among two young infants treated with azithromycin for pertussis . table 1recommended antimicrobial therapy and postexposure prophylaxis for pertussis in infants , children , adolescents , and adults agerecommended drugsalternativeazithromycinerythromycinclarithromycintmp - smx<1 mo10 mg / kg per day as a single dose for 5 days4050 mg / kg per day in 4 divided doses for 14 daysnot recommendedcontraindicated at < 2 mo of age15 mosee abovesee above15 mg / kg per day in 2 divided doses for 7 days2 mo of age : tmp , 8 mg / kg per day ; smx , 40 mg / kg per day in 2 doses for 14 days6 mo and children10 mg / kg as a single dose on day 1 ( maximum 500 mg ) ; then 5 mg / kg per day as a single dose on days 25 ( maximum 250 mg / day)see above ( maximum 2 g / day)see above ( maximum 1 g / day)see aboveadolescents and adults500 mg as a single dose on day 1 , then 250 mg as a single dose on days 252 g / day in 4 divided doses for 14 days1 g / day in 2 divided doses for 7 daystmp , 300 mg / day ; smx , 1600 mg / day in 2 divided doses for 14 daysused with permission of the american academy of pediatrics . red book : 2006 report of the committee on infectious diseases book , american academy of pediatrics , 2006tmp trimethoprim , smx sulfamethoxazolepreferred macrolide for this age because of risk of idiopathic hypertrophic pyloric stenosis associated with erythromycin recommended antimicrobial therapy and postexposure prophylaxis for pertussis in infants , children , adolescents , and adults used with permission of the american academy of pediatrics . red book : 2006 report of the committee on infectious diseases book , american academy of pediatrics , 2006 tmp trimethoprim , smx sulfamethoxazole preferred macrolide for this age because of risk of idiopathic hypertrophic pyloric stenosis associated with erythromycin postexposure prophylaxis the american academy of pediatrics 2006 red book recommends that chemoprophylaxis be administered to all household contacts and other close contacts , regardless of age and immunization status . the rationale behind this recommendation is that administration of chemoprophylaxis to asymptomatic contacts within 21 days of onset of cough in the index patient can limit secondary transmission . other countries like the united kingdom limit the use of prophylaxis for the protection of only those with the greatest risk from pertussis , namely , young infants [ 11 , 33 ] . notably , an evidence - based review of literature on the use of erythromycin in preventing secondary transmission of pertussis to close contacts concluded that in countries where effective pertussis vaccines are in use , chemoprophyalxis should be limited to those most susceptible to the complications of pertussis ( i.e. , unimmunized or partially immunized infants ) and to those individuals who come in close contact with the latter [ 11 , 12 ] . regardless of the policy , the agents , dose , and duration of prophylaxis are the same as for treatment of pertussis . pertussis vaccines licensed for use in infants , children , and adults vary across countries . these vaccines differ both in terms of their active ingredients and in terms of the other diseases for which coverage is provided ( e.g. , polio , diptheria ) . for example , repevax ( sanofi pasteur ) contains diptheria , tetanus , pertussis ( acellular , component ) as well as inactivated polio , whereas adacel contains only tetanus toxoid , reduced diphtheria toxoid , and acellular pertussis . , many potential immunization strategies have been proposed to improve pertussis control ( table 2 ) . universal immunization of adolescents and adults , selective perinatal immunization of women who recently gave birth , and close contacts of newborns , are but a few of the strategies that were discussed by the global pertussis initiative ( gpi ) , which first convened in 2001 . the second gpi convened in 2005 and reiterated several intervention strategies to address the ongoing severe pertussis disease among neonates and infants . table 2immunization strategies assessed by gpi participants ( see , table 1 , pg . universal adult immunizationreduce morbidity in adultsreduce transmission to young infantsdevelop herd immunityreduce morbidity in older children2 . selective immunization of new mothers , family , and close contacts of newbornsreduce transmission to infantsreduce morbidity in adults , particularly young adults3 . preschool booster at 4 years of agereduce morbidity in 4- to 6-year oldsreduce transmission to infantsdevelop herd immunity7 . reinforce and/or improve the current infant and toddler immunization strategyreduce morbidity and mortality in infants , toddlers , and childrenreduce overall circulation of pertussisused with permission from lippincott williams & wilkinsentries represent the consensus of opinion of the gpi participants immunization strategies assessed by gpi participants ( see , table 1 , pg . s70 ) used with permission from lippincott williams & wilkins entries represent the consensus of opinion of the gpi participants immunization of adolescents as previously noted , the incidence of pertussis among adolescents is increasing and these individuals then serve as a reservoir of infection to unvaccinated or incompletely vaccinated infants . two tdap vaccines ( boostrix and adacel ) are licensed for use in the us . recently , the cdcs advisory committee on immunization practices ( acip ) has recommended routine tdap for adolescents from 1118 years [ 3 , 13 , 16 ] . several other countries including canada , austria , australia , france , and germany have also introduced the universal immunization of adolescents . in germany , for example , the current immunization schedule recommends dtap at 2 , 3 , 4 , and 1114 months , and dtap at 56 years and at 917 years . for a complete overview of the pertussis vaccination in other european countries please access the euvac.net website . future studies will be needed to evaluate the duration of protection afforded and the potential need for an adult booster . immunization of adults the adult pertussis trial ( apert ) , a study sponsored by the united states national institute of health ( nih ) , has recently demonstrated the efficacy of acellular pertussis vaccines in preventing pertussis disease in adults ( and adolescents ) . [ 16 , 20 , 21 , 34 ] . to date , only adacel is licensed for use in adults , and the recommended adult immunization schedule in the us ( october 2006september 2007 ) now recommends that tdap replace a single dose of td for adults < 65 years who have not previously received a dose of tdap ( either in the primary series , booster or for wound management ) . given the increased public awareness of adolescent and adult pertussis , in conjunction with perhaps an increased awareness about vaccines in general ( e.g. , hpv and influenza ) , the general public may be more receptive to a universal adult vaccination against pertussis . the expected benefits of such programs would be to build up herd immunity and reduce disease . alternatively , the selective vaccination of only those adults at highest risk of transmitting b. pertussis to vulnerable infants is likely to decrease both the incidence and the impact of pertussis on young infants . regardless of the approach used , successful adult vaccination programs must include education and public awareness . cocoon strategy the vaccination of household members , including parents and siblings of newborn infants , has been recently coined the cocoon strategy . recent studies have demonstrated that parents are frequently the source of pertussis infection to their infants [ 2 , 13 , 16 , 19 , 22 ] . while implementation of this strategy is expected to lead to only modest reductions in typical adult cases , there is a strong indirect effect on infants and young children . in countries where universal immunization of adults is not yet feasible , many experts consider such targeted immunization as presently , the cocoon strategy is recommended in several european countries , including australia , france , germany , and austria . maternal vaccination although there is efficient placental transfer of pertussis antibodies , low maternal levels and rapid decay in newborns render the infants vulnerable to life - threatening pertussis [ 16 , 18 ] . maternal immunization during pregnancy might afford some degree of protection to mother and infant during a vulnerable period , and the use of tdap during pregnancy is currently under consideration . neonatal vaccination given the resurgence of reported pertussis in infant populations noted in multiple countries , and the high morbidity and mortality in this age group , newborn pertussis immunization is a potentially attractive strategy [ 5 , 16 , 19 ] . it is still unclear , however , if such a strategy will induce sufficient and timely immunity for this targeted immunization . despite the increasing awareness of b. pertussis , it continues to affect millions of people worldwide . while classical pertussis was once regarded as a child s disease , today , pertussis poses a serious threat to infants , and greatly affects adolescents adults , now functioning as reservoirs of infection . while advances in molecular biology have undoubtedly increased the capacity to diagnose pertussis , work is still needed to standardize laboratory techniques . the increased awareness of the pertussis problem among experts and the lay public will hopefully pave the way for the implementation of various vaccine strategies to enhance its control .

despite high vaccination coverage , over the last fifteen years there has been a worldwide resurgence of b. pertussis infection . while classical pertussis in the prevaccine era was primarily a childhood disease , today with widespread vaccination , there has been a shift in the incidence of disease to adolescents and adults . centers of disease control and prevention ( cdc ) data from 2004 reveal a nearly 19-fold increase in the number of cases in individuals 1019 years and a 16-fold increase in persons over 20 years . indeed adolescent and adults play a significant role in the transmission of pertussis to neonates and infants who are vulnerable to substantial morbidity and mortality from pertussis infection . several explanations have been proposed to explain the increasing incidence of disease , with waning immunity after natural infection or immunization being widely cited as a significant factor . improving molecular biology diagnostic techniques , namely pcr assays , also accounts for the increasing laboratory diagnosis of pertussis . expanding vaccination strategies including universal immunization of adolescents , targeted immunization of adults , and in particular , healthcare workers , childcare providers and parents of newborns , will likely improve pertussis control . with pertussis continuing to pose a serious threat to infants , and greatly affecting adolescents and adults , there remains a need to : ( a ) increase the awareness of physicians as to the growing pertussis problem , ( b ) standardize diagnostic techniques , and ( c ) implement various new vaccine strategies to enhance its control .

Introduction Epidemiology of pertussis Laboratory diagnosis of pertussis Prevention of pertussis: vaccination strategies Conclusion

pertussis , an acute infectious illness of the respiratory tract remains endemic in developed nations despite high vaccination coverage [ 7 , 8 , 16 ] . while the early use of whole - cell vaccine was highly effective in reducing the incidence of reported pertussis in the united states in the 1970s , there has been a resurgence of reported pertussis over the last 15 years [ 8 , 16 , 31 ] . worldwide , there are an estimated 50 million cases occurring annually ( 90% of which are in developing countries ) , and there are as many as 400,0000 pertussis - related deaths [ 16 , 31 ] . it is general consensus , moreover , that the reported incidence of pertussis is considerably lower than its actual incidence [ 8 , 31 ] . though in the prevaccine era pertussis was regarded as a childhood disease affecting primarily young children , pertussis epidemiology in the postvaccine era is different [ 8 , 17 ] . infants are the most vulnerable group with the highest rates of complications and mortality , yet adolescents and adults now comprise a significant percentage of cases and a conduit of infection for the infants [ 8 , 13 , 17 ] . pcr , culture , and serology are the mainstay of the laboratory diagnosis of pertussis , with various factors affecting the sensitivity and specificity of each modality [ 8 , 24 , 36 ] . however , in recent years , pcr has become an increasingly more popular tool and has significantly contributed to the increasing laboratory diagnosis of pertussis [ 8 , 27 , 36 ] . advances have also been made with regard to prevention and disease control , with experts from 17 countries recently establishing the global pertussis initiative ( gpi ) with the aim of analyzing the status of pertussis and enhancing existing immunization strategies [ 8 , 15 ] . before the introduction of the whole - cell pertussis vaccine in the1940s , there were approximately 200,000 cases reported annually in the us . however , since that time , there has been a substantial increase in the number of cases reported [ 8 , 13 , 31 ] . indeed , over the past 15 years , there has been a marked increase in the incidence of pertussis with reported disease in the us reaching a rate of 8.9 per 100,000 in 2004 with nearly 19,000 provisional reported cases [ 7 , 17 ] . it is also well established that , despite high vaccination coverage for primary immunization in infants and children , pertussis continues to be a global concern with increased incidence in many countries including argentina , australia , canada , italy , japan , the netherlands , switzerland and the us . it is also widely noted that in recent years there is a general shift in the age distribution of pertussis , with adults and adolescents an underrecognized but significant source of infection for neonates and infants [ 8 , 13 , 1517 ] . data from the euvac - net project , a network for the epidemiologic surveillance and control of communicable diseases in the european community , demonstrate that between 1998 and 2002 , the rate of disease incidence remained stable at a high rate among children less than 1 year old . similarly , centers for disease control and prevention ( cdc ) surveillance data from 19902003 demonstrate that the reported incidence of pertussis among adolescents has substantially increased with a nearly ten - fold rise . moreover , when comparing pertussis disease rates in 19901993 , recent cdc data from 2004 reveal a nearly 19-fold increase in the number of cases in persons aged 1019 years and a 16-fold increase in persons over 20 years . several factors have been proposed as underlying the increasing incidence of pertussis disease including waning immunity with subsequent atypical disease manifestations , increasing awareness by public health personnel with subsequent enhanced surveillance and improved laboratory diagnostics [ 8 , 17 , 31 ] . waning of both vaccine - induced immunity and infection - acquired immunity is widely cited as an important reason for recent epidemiologic trends [ 7 , 8 , 36 , 37 ] . while the assessment of the duration of immunity afforded after either natural infection or vaccination is complex , individuals are clearly susceptible to initial infection / reinfection after vaccination or previous pertussis illness , respectively . studies vary in their estimation of protection against disease with protective immunity after natural infection waning 720 years after illness , and duration of immunity after vaccination waning at approximately 412 years in children . finally , it has been proposed that the increased incidence rates may also be a function of enhanced surveillance as well as improved and more sensitive diagnostic lab techniques ( e.g. , pcr ) , in that such techniques allow for the diagnosis of cases that would probably have been missed in the past [ 8 , 17 , 27 , 35 ] . nevertheless , it is important to note , that the current estimates are likely to be , if anything , an underrepresentation of the true incidence of disease [ 8 , 31 ] . first , the clinical diagnosis of pertussis is complicated by underconsulting , particularly among adolescents and adults . second , with prolonged cough often being their only clinical feature , by the time these adolescents and adults finally do seek medical attention , it is often too late to culture or detect the organism by pcr , thus potentially resulting in a missed diagnosis [ 8 , 17 , 31 , 35 ] . moreover , the wide heterogeneity in disease expression , modification of disease by immunization , mixed infection , inconsistent definition , and insensitive nonstandardized , poorly performed , or lack of available laboratory tests , further complicate physician diagnosis . instead , atypical pertussis , usually characterized by the absence of whoop and often a somewhat shorter duration of cough , is more common than classical pertussis among adolescents and adults [ 8 , 17 ] . and finally , it should be noted that , immunized young children that are pcr positive for b pertussis can be asymptomatic [ 29 , 31 ] . regardless of whether an individual displays classical pertussis signs and symptoms or a more protracted , atypical cough , pertussis may not be suspected because of the misconception among many physicians that pertussis is a childhood disease [ 8 , 17 ] . indeed , sutter and cochi report that in the us , only an estimated 11.6% of pertussis cases were actually reported [ 17 , 30 ] . thus multiple institutional , clinical , and laboratory factors diminish the true assessment of pertussis incidence , and the current data clearly are an underestimation of the true burden of disease . because accurate diagnosis of pertussis can not be made by clinical signs and symptoms alone , there is a need for improved laboratory diagnosis of pertussis . while several laboratory techniques exist for the identification of b. pertussis , namely , culture , serology and pcr , overall , several practical factors may adversely affect the sensitivity of its laboratory diagnosis . delayed specimen collection , poor specimen collection techniques , specimen transport problems , and lab media contamination are but a few of the practical constraints often influencing the outcomes of the laboratory diagnosis of pertussis . moreover , previous exposure to the organism , patient s age , stage of disease , previous antibiotic administration , and immunization are other factors that may have a substantial impact on the sensitivity of the tests . culture b. pertussis is a fastidious gram - negative cocobacillus , and its isolation from nasopharyngeal secretions remains the gold standard for diagnosis . to increase the yield of positive cultures , specimens should be immediately plated onto selective regan lowe agar or bordet gengou medium , selective media that are seldom readily available in physician 's offices because of their cost and short shelf - life [ 17 , 24 ] . generally , 710 days are required to grow , isolate , and identify the organism , an obvious limitation of the culture method.the timing of obtaining specimens for culture is also of paramount importance and greatly affects its yield . the proportion of patients testing positive for pertussis by culture is highest when the initial specimens are obtained early in the course of illness , i.e. studies also demonstrate that proportions of positive cultures decline in patients who have been previously immunized and undoubtedly in those in whom antibiotics have been started . pcr the use of pcr for the diagnosis of pertussis is rapidly evolving as it provides a sensitive , rapid means for laboratory diagnosis in circumstances in which the probability of a positive culture is low [ 8 , 17 , 32 , 35 ] . specifically , in their 2005 consensus paper , the european research programme for improved pertussis strain characterization and surveillance ( eupertstrain ) state that the real - time pcr is more sensitive than culture for the detection of b. pertussis , especially after the first 34 weeks of coughing and after antibiotic therapy has been initiated [ 16 , 27 ] . in a prospective study in which nasopharyngeal samples were obtained simultaneously for both pcr and culture , the identification of b. pertussis infections was nearly four - fold higher with pcr [ 8 , 28 ] . finally , pcr is an invaluable tool for the diagnosis of pertussis among young infants since the yield of culture is low and serology is problematic in this age group [ 1 , 17].as with culture , important factors for the successful application of pcr in the diagnosis of infection by bordetella species include proper sample collection and preparation . primers have been derived from four chromosomal regions and common primers employed in pcr detection systems include is481 , is1001 ptp1 , and ptp2 [ 8 , 24 ] . inherent with the high sensitivity , false positive results are a well - recognized problem associated with the pcr diagnosis of pertussis and other respiratory illnesses . while at the present time , pcr is not routinely available and its methods need more standardization , optimization , and quality control , in the future , an internationally accepted standardized kit might be available , which would facilitate the expanded use of pcr for pertussis diagnosis [ 16 , 35 ] . serology natural infection with b. pertussis is followed by an increase in serum levels of iga , igm , and igg antibodies to specific pertussis antigen whereas the primary immunization of children induces mainly igm and igg antibodies . during the past 15 years , elisas have constituted the mainstay of serologic diagnosis using specific b. pertussis protein as antigens , and the serologic diagnosis of pertussis is suspected with increases in iga or igg antibody titers to pertussis toxin ( pt ) , filamentous hemmaglutinin ( fha ) , pertactin , fimbriae or sonicated whole organisms in two serum samples collected 24 weeks apart . thus , the greatest sensitivity and specificity for the serological diagnosis of b. pertussis infection is by elisa measurement of igg and iga antibodies to pt demonstrating at least a two - fold rise in titer between acute- and convalescent - phase sera .still , the main problem in the serologic diagnosis of b. pertussis by elisa is the frequent delay in obtaining the acute - phase specimen . in individuals with re - infections , there is a rapid increase in titer such that if a delayed acute - phase sample is obtained , the titer is likely to have already peaked , thereby hampering the detection of the significant titer increase between the acute- and convalescent - phase serum samples . with this in mind , although a rise in pt iga is more suggestive of a recent antibody response , it is less consistent than a pt igg rise ; hence , in adolescents and adults , a single high value of igg or iga antibodies to pt suggests pertussis infection [ 17 , 24 , 35 ] . demonstrated that an igg concentration to pt of at least 100 units / ml in a single serum sample was diagnostic of either a recent or active pertussis infection .the serological diagnosis of pertussis among infants also has notable limitations . use of antibiotics in the treatment and prevention of pertussis antimicrobial agents administered early in the course of disease , i.e. , during the catarrhal stage , may ameliorate the disease ; although , after the cough is established antibiotics do not have a discernable effect on the course of the illness , but rather are recommended to limit the spread of organisms to other individuals .erythromycin , clarithromycin or azithromycin are now considered first - line agents for treatment ( and prophylaxis ) of pertussis in individuals 6 months of age or older ( table 1 ) . table 1recommended antimicrobial therapy and postexposure prophylaxis for pertussis in infants , children , adolescents , and adults agerecommended drugsalternativeazithromycinerythromycinclarithromycintmp - smx<1 mo10 mg / kg per day as a single dose for 5 days4050 mg / kg per day in 4 divided doses for 14 daysnot recommendedcontraindicated at < 2 mo of age15 mosee abovesee above15 mg / kg per day in 2 divided doses for 7 days2 mo of age : tmp , 8 mg / kg per day ; smx , 40 mg / kg per day in 2 doses for 14 days6 mo and children10 mg / kg as a single dose on day 1 ( maximum 500 mg ) ; then 5 mg / kg per day as a single dose on days 25 ( maximum 250 mg / day)see above ( maximum 2 g / day)see above ( maximum 1 g / day)see aboveadolescents and adults500 mg as a single dose on day 1 , then 250 mg as a single dose on days 252 g / day in 4 divided doses for 14 days1 g / day in 2 divided doses for 7 daystmp , 300 mg / day ; smx , 1600 mg / day in 2 divided doses for 14 daysused with permission of the american academy of pediatrics . red book : 2006 report of the committee on infectious diseases book , american academy of pediatrics , 2006tmp trimethoprim , smx sulfamethoxazolepreferred macrolide for this age because of risk of idiopathic hypertrophic pyloric stenosis associated with erythromycin recommended antimicrobial therapy and postexposure prophylaxis for pertussis in infants , children , adolescents , and adults used with permission of the american academy of pediatrics . other countries like the united kingdom limit the use of prophylaxis for the protection of only those with the greatest risk from pertussis , namely , young infants [ 11 , 33 ] . notably , an evidence - based review of literature on the use of erythromycin in preventing secondary transmission of pertussis to close contacts concluded that in countries where effective pertussis vaccines are in use , chemoprophyalxis should be limited to those most susceptible to the complications of pertussis ( i.e. regardless of the policy , the agents , dose , and duration of prophylaxis are the same as for treatment of pertussis . pertussis vaccines licensed for use in infants , children , and adults vary across countries . , many potential immunization strategies have been proposed to improve pertussis control ( table 2 ) . universal immunization of adolescents and adults , selective perinatal immunization of women who recently gave birth , and close contacts of newborns , are but a few of the strategies that were discussed by the global pertussis initiative ( gpi ) , which first convened in 2001 . the second gpi convened in 2005 and reiterated several intervention strategies to address the ongoing severe pertussis disease among neonates and infants . selective immunization of new mothers , family , and close contacts of newbornsreduce transmission to infantsreduce morbidity in adults , particularly young adults3 . reinforce and/or improve the current infant and toddler immunization strategyreduce morbidity and mortality in infants , toddlers , and childrenreduce overall circulation of pertussisused with permission from lippincott williams & wilkinsentries represent the consensus of opinion of the gpi participants immunization strategies assessed by gpi participants ( see , table 1 , pg . s70 ) used with permission from lippincott williams & wilkins entries represent the consensus of opinion of the gpi participants immunization of adolescents as previously noted , the incidence of pertussis among adolescents is increasing and these individuals then serve as a reservoir of infection to unvaccinated or incompletely vaccinated infants . two tdap vaccines ( boostrix and adacel ) are licensed for use in the us . several other countries including canada , austria , australia , france , and germany have also introduced the universal immunization of adolescents . in germany , for example , the current immunization schedule recommends dtap at 2 , 3 , 4 , and 1114 months , and dtap at 56 years and at 917 years . immunization of adults the adult pertussis trial ( apert ) , a study sponsored by the united states national institute of health ( nih ) , has recently demonstrated the efficacy of acellular pertussis vaccines in preventing pertussis disease in adults ( and adolescents ) . to date , only adacel is licensed for use in adults , and the recommended adult immunization schedule in the us ( october 2006september 2007 ) now recommends that tdap replace a single dose of td for adults < 65 years who have not previously received a dose of tdap ( either in the primary series , booster or for wound management ) . given the increased public awareness of adolescent and adult pertussis , in conjunction with perhaps an increased awareness about vaccines in general ( e.g. alternatively , the selective vaccination of only those adults at highest risk of transmitting b. pertussis to vulnerable infants is likely to decrease both the incidence and the impact of pertussis on young infants . cocoon strategy the vaccination of household members , including parents and siblings of newborn infants , has been recently coined the cocoon strategy . recent studies have demonstrated that parents are frequently the source of pertussis infection to their infants [ 2 , 13 , 16 , 19 , 22 ] . in countries where universal immunization of adults is not yet feasible , many experts consider such targeted immunization as presently , the cocoon strategy is recommended in several european countries , including australia , france , germany , and austria . maternal vaccination although there is efficient placental transfer of pertussis antibodies , low maternal levels and rapid decay in newborns render the infants vulnerable to life - threatening pertussis [ 16 , 18 ] . neonatal vaccination given the resurgence of reported pertussis in infant populations noted in multiple countries , and the high morbidity and mortality in this age group , newborn pertussis immunization is a potentially attractive strategy [ 5 , 16 , 19 ] . despite the increasing awareness of b. pertussis , it continues to affect millions of people worldwide . while classical pertussis was once regarded as a child s disease , today , pertussis poses a serious threat to infants , and greatly affects adolescents adults , now functioning as reservoirs of infection . while advances in molecular biology have undoubtedly increased the capacity to diagnose pertussis , work is still needed to standardize laboratory techniques . the increased awareness of the pertussis problem among experts and the lay public will hopefully pave the way for the implementation of various vaccine strategies to enhance its control .

telomerase is the enzyme responsible for the maintenance of telomeres , which cap and protect the ends of chromosomes . telomeres are made of tandem copies of a simple dna repeat , ( ttaggg)n , which the enzyme telomerase synthesizes . in cells that lack telomerase , telomeres shorten with each round of cell division and this attrition eventually limits cellular lifespan . after a finite number of cell divisions , cells without telomerase undergo a growth arrest termed replicative senescence . during cancer development , this limit poses an obstacle that tumour cells must overcome to become malignant . in the great majority of cancers , overcoming replicative senescence is achieved initially by inactivating important cell cycle checkpoints and eventually by up - regulation of telomerase expression once telomeres have become critically short . this reactivation of telomerase stabilizes the telomeres and extends cellular lifespan . to escape senescence and gain cellular immortality , the great majority of cancers up - regulate telomerase . as a consequence , telomerase is detected in more than 85% of all cases of cancer [ 3 , 4 ] , making the enzyme an attractive target for cancer therapeutics . in cancer cells , telomerase inhibition has been shown to lead to telomere shortening and , after sufficient cell divisions , this attrition can cause an uncapping of telomeres and induction of apoptosis [ 5 , 6 ] . in this review , we discuss the biological and biochemical properties of telomerase , its use as a diagnostic and prognostic marker for cancers and its value as a target for cancer therapeutics . telomeres protect the ends of chromosomes from degradation , interchromosomal fusions and other forms of inadequate recombination [ 7 , 8 ] . a second vital function of telomeres is to hide the ends of chromosomes from dna damage - sensing mechanisms , which would otherwise detect the ends as double - stranded dna breaks ( ds - dna breaks ) . telomeric dna repeats serve as anchor for the recruitment of dna - binding factors telomeric repeat factor ( trf)1 , trf2 and protection of telomeres 1 ( pot1 ) in combination with other telomere associated proteins , which together form a protective capping complex originally termed telosome and more recently shelterin [ 8 , 9 ] . although most of the telomere is made of duplex telomeric dna , all telomeres end with a g - rich single - stranded 3-overhang of 50 to 300 bases . evidence suggests that this extension is sequestered into a large looping structure , termed a t - loop . formation of this structure involves the looping of the telomere and the insertion of its 3-telomeric overhang into upstream duplex telomeric dna [ 8 , 10 ] . it has been proposed that t - loops are especially well adapted to shield the ends of chromosomes from dna damage - sensing mechanisms and other activities . however , for these protective features to be in place , telomeres may have to be of a minimum required size . yet , because they lack telomerase , most normal human cells will shorten their telomeres with cell divisions until the shortest telomere has become uncapped ( and perhaps unlooped ) , giving rise to a persistent dna damage signal [ 2 , 11 , 12 ] . telomeres shorten because of problems associated with the replication of linear dna molecules , the so - called end replication problems. when replication forks reach a telomere , problems are experienced at the levels of both the leading and lagging strand synthesis . on the lagging strand , internal priming by the last okazaki fragment and removal of the rna primer creates a gap of unreplicated dna [ 1 , 2 ] . on the leading strand , post - replication excision by the apollo nuclease is needed to produce a single - stranded 3-telomeric overhang that can serve as substrate for pot1 binding and t - loop formation [ 14 , 15 ] . the net result is a loss of telomeric dna repeats each time cells divide [ 1 , 2 ] . as cells divide and telomeric dna repeats are lost , telomeres become dysfunctional , lose their protective features and become uncapped ( i.e. recognized as ds - dna breaks by the dna damage sensing and dna repair machinery ) . the response of human cells to uncapped telomeres varies depending on whether these cells have functional dna damage and cell cycle checkpoints ( fig . , an uncapped telomere will be recognized as a ds - dna break by dna damage sensing mechanisms , the activation of which will induce atm kinase activity , phosphorylation of p53 and up - regulation of cell cycle inhibitor p21[11 , 12 , 16 , 17 ] . a late response involving the up - regulation of p16 and activation of downstream retinoblastoma susceptibility protein ( rb ) can also be observed [ 1618 ] . the net result is an irreversible cell cycle arrest and establishment of the senescent state . in cancer cells lacking functional checkpoints , these dna damage signals will be ignored and the cell will continue to divide and shorten its telomeres . eventually , the uncapped telomeres can serve as substrate for non - homologous end joining ( nhej ) and through this process , can become fused to other dysfunctional telomeres [ 7 , 8 , 19 ] . at anaphase , the dicentric chromatids generated by these fusions will fail to segregate properly and , as the cells continue to divide , there will be recurrent cycles of anaphase - bridge , breakage and fusion . invariably , such cycles lead to a loss of genomic integrity and to a state of crisis characterized by p53-independent apoptosis [ 6 , 20 ] . during cancer development , the induction of senescence or crisis poses an obstacle that tumour cells must overcome to become malignant . in the great majority of cancers , overcoming these limits to cellular lifespan is achieved by means of telomerase expression . as cells divide and telomeric dna repeats are lost , telomeres lose their protective shelterins ( grey spheres ) , become unfolded ( loss of the t - loop ) and are recognized as ds - dna breaks . in cells with intact checkpoints , the uncapped telomere leads to the activation of the atm kinase , phosphorylation of p53 and up - regulation of the p21 gene ( atm / p53/p21 cascade ) . once induced , p21 inhibits pcna and cyclin - dependent kinases cdk2 and cdk4 , thereby blocking the cell cycle . a late response involving the up - regulation of the p16 gene is also observed . once induced , p16 blocks cdk4 , the activity of which is required for inactivation of rb , a powerful inhibitor of the cell cycle ( p16/cdk4/rb cascade ) . acting in concert , these signalling pathways cause the cells to enter an irreversible state of growth arrest ( senescence ) . in cancer cells that lack components of these pathways ( most commonly p53 , p16 or rb ) , the dna damage signals emanating from uncapped telomeres will be ignored and the cells will bypass senescence and with division , progressive telomere erosion will continue . when many telomeres become uncapped , the very short telomeres will serve as substrate for nhej and through this process , will become associated and/or fused to other dysfunctional telomeres . at anaphase , the dicentric chromatids produced by these fusions will fail to segregate properly ( anaphase bridges ) , will break ( breakages ) and will again be involved in fusion events with other dysfunctional chromosomes ( fusions ) . as cells continue to divide , these recurrent cycles of anaphase bridges , breakages and fusions will lead to a state of crisis characterized by p53-independent apoptosis ( crisis ) . only a very rare human cell can bypass crisis and when a cell accomplishes this , a mechanism to maintain telomeres must be engaged . telomerase compensates for the effects of the end replication problems by the synthesis of new telomeric dna repeats . in human beings , the telomerase complex has an estimated mass of over 1000 kd and may function as a homodimer . the enzyme telomerase is composed of several subunits , two of which are essential for its activity : the protein htert ( human telomerase reverse transcriptase ) and the small nuclear rna htr ( human telomerase rna ) [ 2225 ] . the latter contains a short sequence ( 5-cuaacccuaa-3 ) that serves as a template for the synthesis of telomeric dna repeats and htert provides catalytic activity ( fig . the template hybridizes and aligns itself with the overhang , after which htert elongates the overhang while copying the template into dna . each step adds a 6-base telomeric dna repeat to the overhang , thereby elongating the telomere . the enzyme is generally but not always processive and is capable of adding many more repeats to the same telomere before falling off its substrate . to detect and measure the activity of telomerase , the telomeric repeat amplification protocol ( trap ) is most commonly used . in the trap assay , products of the telomerase reaction are quantified following pcr amplification [ 26 , 27 ] . the assay is very sensitive and incorporates an internal standard ( itas ) with which to normalize the signals for variations in pcr efficiency . telomerase activity is calculated as the ratio of the intensity of the telomeric products over that of the itas . using the trap assay , the activity of the enzyme can be measured in a wide variety of samples , from very small tissue biopsies , bodily fluids to cell pellets . the main pathway that cells use to bypass crisis is to activate the ribonucleoprotein telomerase complex . when telomerase binds to a telomere , its rna template region ( shaded ) hybridizes ( asterisk ) to the end of the 3-telomeric overhang . next , the htert subunit acts as a reverse transcriptase and copies the rna template into dna ( dotted arrow ) . grn163l ( imetelstat ) is a n3-p5 thio - phosphoramidate oligonucleotide complementary to sequence of the rna template ( sequence shown ) and which has been modified to carry a palmitate group at its 5-end . when grn163l hybridizes ( asterisk ) to the rna template , the active site is blocked and the enzyme is inhibited . at the early stages of human development , telomerase activity is ubiquitously present throughout the embryo [ 29 , 30 ] . at these stages , telomerase is needed to compensate for the massive numbers of cell divisions needed to complete embryogenesis . at the later stages , htert expression is repressed and telomerase activity becomes undetectable in most tissues , the exact timing of repression varying depending on the tissue . at birth , the activity is absent from most human cells with the exception of dividing male germ cell lineages and of rare proliferative cells of the blood , skin and gastrointestinal tract [ 29 , 30 ] . in tissues that divide rapidly , tissue homeostasis is maintained by the continuous proliferation and differentiation of adult stem cells . to support the proliferation of these stem cells throughout a lifetime , it is believed that some telomerase activity is needed to slow down but not completely compensate for the rate of telomere loss . the mechanism for partial telomere maintenance in proliferative stem cells is not well understood but in embryonic stem cells , telomerase completely compensates for telomere loss at each cell division . indeed , much remains to be learned about the regulation of telomerase in normal stem cells . a notable exception to the almost universal absence of telomerase in human tissues is cancer . a survey of the published literature established telomerase as one of the best markers of cancer cells . although most normal tissues lack the activity , telomerase is detected in more than 85% of human cancers , irrespective of the tumour type [ 24 ] . in most cases , the activity is localized to the tumour and the activity is absent from the surrounding normal tissues . human cancer cells commonly possess mechanisms that compensate for telomere attrition and provide them with cellular immortality , a hallmark of cancer . in the vast majority of cancers , this mechanism is the up - regulation of telomerase activity . malignancies that develop from telomerase - negative tissues are almost always positive for the activity . even in cancer cells originating from telomerase - positive precursors , this ability to maintain telomeres is a fundamental property of cancer cells , which normal somatic human cells do not possess . this lack of telomere attrition provides cancer cells with cellular immortality , a capacity for unlimited number of cell divisions . this conversion to cellular immortality is needed to allow the many stages of carcinogenesis to proceed unimpeded by telomere attrition , giving the cancer cells the ability to invade without being halted by the induction of senescence or crisis [ 2 , 16 ] . in some cases , however , telomerase is not detected in a tumour due to the engagement of an alternative telomere lengthening mechanism or because the tumour is self - limiting [ 33 , 34 ] . in stage ivs ( 4s ) neuroblastoma , for example , telomeres are extremely short and there is no detectable telomerase activity . amazingly , these tumours almost always regress spontaneously . this indicates that telomerase is not essential for cancer development as these children with neuroblastoma are clearly born with an advanced cancer . however , and perhaps more importantly , these results indicate that to have a sustained tumour that can progress , a mechanism to maintain telomeres must be engaged . because of examples such as neuroblastoma where telomerase negative tumours regress , and because most cancer cells are immortal due to telomerase reactivation , targeting telomerase is an attractive strategy for cancer therapy . in the following sections , we will discuss three approaches , each using an entirely different strategy to target telomerase - expressing cancer cells . the first of these approaches consists of blocking the activity of the enzyme to limit the lifespan of telomerase - expressing cancer cells . inhibiting telomerase in telomerase - expressing cancer cells leads to telomere shortening and when sufficient telomere erosion has taken place , one of two anti - proliferative barriers will be observed : either senescence or crisis [ 2 , 35 ] . the two barriers are different and whether a telomerase - inhibited cancer cell experiences one or the other is dictated by the functionality of its dna damage response and cell cycle checkpoints ( fig . cells that possess functional checkpoints , such as primary human cells , undergo senescence as soon as the shortest telomere becomes uncapped [ 2 , 35 ] . crisis , which represents the preferred outcome , is a form of p53-independent apoptosis that generally leads to the death of the cancer cell [ 5 , 6 ] . because they lack functional checkpoints , most often because of mutations in the p53 and rb pathways , cancer cells will typically ignore these signals and will continue to shorten their telomeres until crisis is induced [ 2 , 35 , 36 ] . because sufficient telomere shortening must occur before senescence or crisis are induced , a delay should be expected before the effects of telomerase inhibition are observed [ 35 , 37 ] . this delay may preclude the use of these inhibitors as a primary line of treatment , but to block the incidence of recurrence following conventional therapy , these inhibitors are expected to be especially well suited ( fig . , residual cancer cells may survive and after multiple rounds of cell divisions , these cells can give rise to a new tumour . in the presence of a telomerase inhibitor , these residual cells would be predicted to shorten their telomeres as they divide , causing some to experience crisis before detectable tumour formation . for these reasons , telomerase inhibitors are now being tested in combination with conventional cancer therapy and in maintenance clinical trials aimed at reducing recurrence after surgery , chemotherapy or radiation therapy . combining standard chemotherapy with telomerase inhibition . standard therapies have been optimized to kill bulk tumour cells , not necessarily the cancer stem cells which tend to express multidrug resistance . after standard chemotherapy , the residual cancer cells that survive may have become enriched in cancer stem cells . after multiple rounds of cell divisions , these cancer stem cells will give rise to a new tumour ( arrow pointing up ) and the heterogeneity of the original tumour is re - established . if the patient is treated with a telomerase inhibitor following chemotherapy ( arrow pointing down ) , these residual cancer stem cells would be predicted to lose telomeric dna repeats as they divide , forcing them to enter crisis after a limited number of cell divisions . the hope is that using telomerase inhibitors in a cancer maintenance setting may lead to more durable responses . for inhibiting telomerase , the template region of the human telomerase rna ( htr ) presents an accessible target for oligonucleotide - based inhibitors [ 3841 ] . oligonucleotides that can hybridize to the template region of htr , as shown previously , can be used to inhibit telomerase ( fig . n3-p5thio - phosphoramidate oligonucleotides complementary to the htr template have been found to be especially good telomerase inhibitors . these compounds are water soluble , acid stable , resistant to nucleases and demonstrate high thermal stability of duplexes formed with their complementary rna strands [ 4246 ] . one compound , grn163 ( geron corporation , menlo park , ca , usa ) caused telomerase inhibition and subsequent telomere shortening in a number of cancer cell lines [ 4550 ] . as with most anionic oligonucleotides , repeated transfection of grn163 with cationic lipophilic carriers was required for efficient intracellular uptake , because naked oligonucleotides are poorly internalized . a second - generation of grn163 analogues , modified by lipidation , was made to facilitate cellular uptake . one such compound , grn163l ( formally known as imetelstat ) , is a grn163 oligonucleotide modified to carry a 5-terminal palmitoyl ( c16 ) moiety conjugated to the n3-p5 thio - phosphoramidate backbone ( 5-palmitate - tagggttagacaa - nh2 - 3 ) . grn163l is lipid soluble and does not rely on any membrane transporter for cellular uptake . when compared to grn163 , grn163l displayed greatly improved uptake and telomerase inhibition [ 46 , 48 , 49 ] . the effects of grn163l have been investigated in cancer cells lines and in mice bearing human tumour xenografts . in a wide variety of cancer cell lines , grn163l inhibited telomerase with ic50 in the nanomolar range . in cancer cells chronically exposed to grn163l , telomeres shorten with cell divisions and after a delay , a state of crisis is induced with evidence of chromosomal fusions , anaphase bridges and widespread apoptosis . this inhibition of cellular growth by grn163l has been reported in cancer cell lines of diverse origins , including multiple myeloma and tumours of the bladder , breast , liver , lung and stomach [ 45 , 48 , 5154 ] . in mice bearing human tumour xenografts , the maximum tolerated dose was 1000 mg / kg and telomerase inhibition could readily be detected with just 5 mg / kg . even when administered as a single agent , grn163l could block the growth of implanted tumour cells . in mice carrying liver cancer xenografts , grn163l alone inhibited tumour growth , in addition to sensitizing the tumour cells to conventional chemotherapy . in a xenograft model of lung cancer metastasis , administration of a single dose of grn163l was sufficient to prevent the growth of lung metastases . first , most primary cancers have very short telomeres , which could include some that may already be critically short . however , in some instances what is observed in vitro and in vivo does not concur . thus , it is possible that grn163l may also be exerting telomerase - independent effects on tumour growth through mechanisms that remains poorly characterized . however , on the basis of these and other pre - clinical studies , grn163l has entered clinical trials in patients afflicted with different forms of cancer . phase i trials of grn163l have just been completed in patients with chronic lymphocytic leukaemia , multiple myeloma and solid tumours ( e.g. breast cancer and non - small cell lung cancer ( nsclc ) . from these trials , administered by intravenous infusions , grn163l was found to have good safety profiles and excellent pharmacokinetics and biodistribution properties . at doses of 7.5 mg / kg and above , the level of exposure to grn163l was higher than the exposure that was required in xenograft models to inhibit telomerase and tumour growth ( dr . mark j. ratain , aacr - nci - eortc 2009 international conference , boston , ma , usa ) . dose escalation proceeded to 11.7 mg / kg but due to haematological toxicities , this dose was considered to exceed the maximum tolerated dose . based on these and other considerations , 9.4 mg / kg was chosen as the recommended dose for phase ii clinical trials . in combination trials , the only notable toxicities observed at this dose were reversible anaemia , thrombocytopenia and neutropenia , and it may be that some of these toxicities were due to concurrent use of standard chemotherapy . starting at 9.4 mg / kg , phase ii trials are now being initiated for nsclc to evaluate grn163l in a maintenance setting , with grn163l given on days 1 and 8 of a 21 day cycle . it is well established that the standard first line induction therapy ( debulking ) for patients with advanced nsclc produces partial responses in up to 35% of patients . thus , 14 weeks after completing four to six cycles of induction chemotherapy , patients will be treated with imetelstat ( grn163l ) , bevacizumab ( avastin , an angiogenesis inhibitor ) or observation ( e.g. no maintenance therapy ) . combinations of imetelstat and bevacizumab may be permitted in some individuals as well as cross - over from one to the other maintenance therapy . approximately 100 patients will be enrolled in these studies with the main end - point being objective and progression - free survival . in addition to standard biomarkers , this trial will collect other tissues ( e.g. peripheral blood mononuclear cells , circulating tumour cells , dna / rna and serum ) to assay for a variety of other predictive markers . there will be approximately 25 cancer sites involved in the united states , canada and germany ( geron corporation ; personal communications , 2010 ) . in other phase ii trials , grn163l will also be tested in combination with paclitaxel in patients with advanced breast cancer with the primary end - point being progression free survival . finally , grn163l will be tested as a single therapeutic agent for multiple myeloma , with one of the end - points being reduction in myeloma progenitor cells and essential thrombocythemia , where haematological and molecular responses will be assessed . mounting evidence suggests that , in many cases , recurrences are the products of residual cancer cells that possess properties of stem cells . therefore , an important question will be whether grn163l can target these cancer stem cells efficaciously . according to a consensus definition , a tumour cell is a cancer stem cell if it can self - renew , initiate the formation of new tumours , and regenerate all of the heterogeneous lineages of cancer cell types that comprise a tumour . rare cells that fit this definition have been identified in a growing number of diseases , from haematological malignancies to brain , breast and pancreatic cancer [ 5759 ] . conventional therapies have been optimized to kill bulk tumour cells , not necessarily the cancer stem cells which tend to express multidrug resistance pumps [ 59 , 60 ] . when these cancer stem cells divide in the presence of a telomerase inhibitor , their telomeres should shorten and this attrition would be expected to compromise the ability of these cells to self - renew ( fig . these predictions have been verified in the context of multiple myeloma as well as prostate , brain , breast and pancreatic cancer [ 6164 ] . hence , cancer stem cells isolated from multiple myeloma specimens and cell lines showed reduced proliferation with 100-fold fewer colonies after 5 weeks of exposure to grn163l . likewise , telomerase was inhibited and telomeres shortened in cancer stem cells after treatment of prostate cancer cells with grn163l . in addition , the abundance of cancer stem cells was reduced following grn163l treatment of prostate , pancreatic and breast cancer cell lines [ 62 , 64 ] . finally , a key characteristic of cancer stem cells isolated from brain and breast cancer is their ability to give rise to free - floating spheres , respectively called neurospheres and mammospheres . treatment of breast cancer cell lines or primary brain tumour cells with grn163l reduced the number of mammospheres and neurospheres that cancer stem cells could form [ 62 , 63 ] . taken together , these studies demonstrate broad anti - cancer stem cell activity of grn163l , thereby confirming the rationale for using this drug to block disease recurrence after conventional cancer therapy . inhibiting telomerase in telomerase - expressing cancer cells leads to telomere shortening and when sufficient telomere erosion has taken place , one of two anti - proliferative barriers will be observed : either senescence or crisis [ 2 , 35 ] . the two barriers are different and whether a telomerase - inhibited cancer cell experiences one or the other is dictated by the functionality of its dna damage response and cell cycle checkpoints ( fig . cells that possess functional checkpoints , such as primary human cells , undergo senescence as soon as the shortest telomere becomes uncapped [ 2 , 35 ] . crisis , which represents the preferred outcome , is a form of p53-independent apoptosis that generally leads to the death of the cancer cell [ 5 , 6 ] . because they lack functional checkpoints , most often because of mutations in the p53 and rb pathways , cancer cells will typically ignore these signals and will continue to shorten their telomeres until crisis is induced [ 2 , 35 , 36 ] . because sufficient telomere shortening must occur before senescence or crisis are induced , a delay should be expected before the effects of telomerase inhibition are observed [ 35 , 37 ] . this delay may preclude the use of these inhibitors as a primary line of treatment , but to block the incidence of recurrence following conventional therapy , these inhibitors are expected to be especially well suited ( fig . 3 ) . after conventional therapy , residual cancer cells may survive and after multiple rounds of cell divisions , these cells can give rise to a new tumour . in the presence of a telomerase inhibitor , these residual cells would be predicted to shorten their telomeres as they divide , causing some to experience crisis before detectable tumour formation . for these reasons , telomerase inhibitors are now being tested in combination with conventional cancer therapy and in maintenance clinical trials aimed at reducing recurrence after surgery , chemotherapy or radiation therapy . combining standard chemotherapy with telomerase inhibition . standard therapies have been optimized to kill bulk tumour cells , not necessarily the cancer stem cells which tend to express multidrug resistance . after standard chemotherapy , the residual cancer cells that survive may have become enriched in cancer stem cells . after multiple rounds of cell divisions , these cancer stem cells will give rise to a new tumour ( arrow pointing up ) and the heterogeneity of the original tumour is re - established . if the patient is treated with a telomerase inhibitor following chemotherapy ( arrow pointing down ) , these residual cancer stem cells would be predicted to lose telomeric dna repeats as they divide , forcing them to enter crisis after a limited number of cell divisions . the hope is that using telomerase inhibitors in a cancer maintenance setting may lead to more durable responses . for inhibiting telomerase , the template region of the human telomerase rna ( htr ) presents an accessible target for oligonucleotide - based inhibitors [ 3841 ] . oligonucleotides that can hybridize to the template region of htr , as shown previously , can be used to inhibit telomerase ( fig . n3-p5thio - phosphoramidate oligonucleotides complementary to the htr template have been found to be especially good telomerase inhibitors . these compounds are water soluble , acid stable , resistant to nucleases and demonstrate high thermal stability of duplexes formed with their complementary rna strands [ 4246 ] . one compound , grn163 ( geron corporation , menlo park , ca , usa ) caused telomerase inhibition and subsequent telomere shortening in a number of cancer cell lines [ 4550 ] . as with most anionic oligonucleotides , repeated transfection of grn163 with cationic lipophilic carriers was required for efficient intracellular uptake , because naked oligonucleotides are poorly internalized . a second - generation of grn163 analogues , modified by lipidation , was made to facilitate cellular uptake . one such compound , grn163l ( formally known as imetelstat ) , is a grn163 oligonucleotide modified to carry a 5-terminal palmitoyl ( c16 ) moiety conjugated to the n3-p5 thio - phosphoramidate backbone ( 5-palmitate - tagggttagacaa - nh2 - 3 ) . grn163l is lipid soluble and does not rely on any membrane transporter for cellular uptake . when compared to grn163 , grn163l displayed greatly improved uptake and telomerase inhibition [ 46 , 48 , 49 ] . the effects of grn163l have been investigated in cancer cells lines and in mice bearing human tumour xenografts . in a wide variety of cancer cell lines , grn163l inhibited telomerase with ic50 in the nanomolar range . in cancer cells chronically exposed to grn163l , telomeres shorten with cell divisions and after a delay , a state of crisis is induced with evidence of chromosomal fusions , anaphase bridges and widespread apoptosis . this inhibition of cellular growth by grn163l has been reported in cancer cell lines of diverse origins , including multiple myeloma and tumours of the bladder , breast , liver , lung and stomach [ 45 , 48 , 5154 ] . in mice bearing human tumour xenografts , the maximum tolerated dose was 1000 mg / kg and telomerase inhibition could readily be detected with just 5 mg / kg . even when administered as a single agent , grn163l could block the growth of implanted tumour cells . in mice carrying liver cancer xenografts , grn163l alone inhibited tumour growth , in addition to sensitizing the tumour cells to conventional chemotherapy . in a xenograft model of lung cancer metastasis , administration of a single dose of grn163l was sufficient to prevent the growth of lung metastases . first , most primary cancers have very short telomeres , which could include some that may already be critically short . however , in some instances what is observed in vitro and in vivo does not concur . thus , it is possible that grn163l may also be exerting telomerase - independent effects on tumour growth through mechanisms that remains poorly characterized . however , on the basis of these and other pre - clinical studies , grn163l has entered clinical trials in patients afflicted with different forms of cancer . phase i trials of grn163l have just been completed in patients with chronic lymphocytic leukaemia , multiple myeloma and solid tumours ( e.g. breast cancer and non - small cell lung cancer ( nsclc ) . from these trials , the bioavailability , pharmacokinetics , pharmacodynamics and tolerability over multiple cycles have been determined . administered by intravenous infusions , grn163l was found to have good safety profiles and excellent pharmacokinetics and biodistribution properties . at doses of 7.5 mg / kg and above , the level of exposure to grn163l was higher than the exposure that was required in xenograft models to inhibit telomerase and tumour growth ( dr . mark j. ratain , aacr - nci - eortc 2009 international conference , boston , ma , usa ) . dose escalation proceeded to 11.7 mg / kg but due to haematological toxicities , this dose was considered to exceed the maximum tolerated dose . based on these and other considerations , 9.4 mg / kg was chosen as the recommended dose for phase ii clinical trials . in combination trials , the only notable toxicities observed at this dose were reversible anaemia , thrombocytopenia and neutropenia , and it may be that some of these toxicities were due to concurrent use of standard chemotherapy . starting at 9.4 mg / kg , phase ii trials are now being initiated for nsclc to evaluate grn163l in a maintenance setting , with grn163l given on days 1 and 8 of a 21 day cycle . it is well established that the standard first line induction therapy ( debulking ) for patients with advanced nsclc produces partial responses in up to 35% of patients . thus , 14 weeks after completing four to six cycles of induction chemotherapy , patients will be treated with imetelstat ( grn163l ) , bevacizumab ( avastin , an angiogenesis inhibitor ) or observation ( e.g. no maintenance therapy ) . combinations of imetelstat and bevacizumab may be permitted in some individuals as well as cross - over from one to the other maintenance therapy . approximately 100 patients will be enrolled in these studies with the main end - point being objective and progression - free survival . in addition to standard biomarkers , this trial will collect other tissues ( e.g. peripheral blood mononuclear cells , circulating tumour cells , dna / rna and serum ) to assay for a variety of other predictive markers . there will be approximately 25 cancer sites involved in the united states , canada and germany ( geron corporation ; personal communications , 2010 ) . in other phase ii trials , grn163l will also be tested in combination with paclitaxel in patients with advanced breast cancer with the primary end - point being progression free survival . finally , grn163l will be tested as a single therapeutic agent for multiple myeloma , with one of the end - points being reduction in myeloma progenitor cells and essential thrombocythemia , where haematological and molecular responses will be assessed . mounting evidence suggests that , in many cases , recurrences are the products of residual cancer cells that possess properties of stem cells . therefore , an important question will be whether grn163l can target these cancer stem cells efficaciously . according to a consensus definition , a tumour cell is a cancer stem cell if it can self - renew , initiate the formation of new tumours , and regenerate all of the heterogeneous lineages of cancer cell types that comprise a tumour . rare cells that fit this definition have been identified in a growing number of diseases , from haematological malignancies to brain , breast and pancreatic cancer [ 5759 ] . conventional therapies have been optimized to kill bulk tumour cells , not necessarily the cancer stem cells which tend to express multidrug resistance pumps [ 59 , 60 ] . when these cancer stem cells divide in the presence of a telomerase inhibitor , their telomeres should shorten and this attrition would be expected to compromise the ability of these cells to self - renew ( fig . these predictions have been verified in the context of multiple myeloma as well as prostate , brain , breast and pancreatic cancer [ 6164 ] . hence , cancer stem cells isolated from multiple myeloma specimens and cell lines showed reduced proliferation with 100-fold fewer colonies after 5 weeks of exposure to grn163l . likewise , telomerase was inhibited and telomeres shortened in cancer stem cells after treatment of prostate cancer cells with grn163l . in addition , the abundance of cancer stem cells was reduced following grn163l treatment of prostate , pancreatic and breast cancer cell lines [ 62 , 64 ] . finally , a key characteristic of cancer stem cells isolated from brain and breast cancer is their ability to give rise to free - floating spheres , respectively called neurospheres and mammospheres . treatment of breast cancer cell lines or primary brain tumour cells with grn163l reduced the number of mammospheres and neurospheres that cancer stem cells could form [ 62 , 63 ] . taken together , these studies demonstrate broad anti - cancer stem cell activity of grn163l , thereby confirming the rationale for using this drug to block disease recurrence after conventional cancer therapy . a different approach to target telomerase - expressing cancer cells is to use a vaccine to stimulate the patient s own immune system to attack tumour cells if they display htert peptides at their surface through major histocompatibility complex ( mhc ) presentation . peptides displayed by mhc presentation are generated from within the cells by the limited proteolysis of cellular proteins , capture of the resulting peptides by mhc class i molecules , their transport to the plasma membrane and presentation at the surface by mhc class i molecules . in telomerase - targeted immunotherapy , immune cells are exposed to a vaccine that consists of antigen - presenting cells that were either exposed to high levels of an immunogenic htert peptide or modified to overexpress an immunogenic fragment of htert . once administered to a patient , the antigen - presenting cells elicit the activation and expansion of telomerase - specific cytotoxic t lymphocytes , which then can recognize and kill the telomerase - expressing tumour cells . basic studies in human and murine systems have reported evidence of cytotoxic t cells that recognize dominant epitopes from htert and can kill tumour cells displaying these peptides [ 66 , 67 ] . htert - based vaccines have also been reported to generate cytotoxic t cells against modified , low - affinity cryptic htert epitopes and these t cells protected animals against tumour challenges . in phase i clinical trials , tumour vaccines against htert have resulted in the induction of t - cell immune responses with minimal toxicities [ 6870 ] . a frequent problem that limits the broad applicability of cancer vaccines is the heterogeneous expression of the tumour antigen within a tumour . however , in the case of telomerase , the antigen is both universally expressed in cancers and critical for maintenance of the malignant phenotype . already , several htert - based vaccines have been developed but the most advanced vaccines are the gv1001 ( gemvax ) and grnvac1 ( geron vaccine ) . gv1001 is a 16-mer peptide from the active site of human tert ( aa 611626 ) [ 68 , 69 ] . developed by kael - gemvax ( gwangju , south korea ) as an injectable formulation , the vaccinating gv1001 peptide is eventually trimmed and processed for presentation by mhc class i and class ii molecules , allowing it to elicit the activity of telomerase - specific cytotoxic ( cd8 ) and helper ( cd4 ) t cells . in phase i / ii clinical trials for nsclc , hepatocellular carcinoma and non - resectable pancreatic carcinoma , gv1001 has been well tolerated and shown to induce htert - specific t - cell responses [ 6870 ] . gv1001 is currently in a randomized phase iii clinical trial in patients with locally advanced or metastatic pancreatic cancer ( clinicaltrials.gov identifier : nct00425360 ) . patients will receive doublet therapy with gemcitabine and capecitabine either with or without gv1001 vaccination , with overall survival as the main end - point . the trial is lead by the national cancer research institute in england and will enrol over 1100 pancreatic cancer patients across england . grnvac1 is a preparation of antigen - presenting cells transduced with mrnas encoding a near full - length htert protein . in this case , the antigen - presenting cells are autologous dendritic cells isolated from the patients own tissues . dendritic cells are a professional antigen - presenting cell whose main function is to process and display antigens . in these cells , the htert fragment should be processed into a multitude of epitopes to elicit a polyclonal t - cell response , which could potentially recognize any htert peptide displayed by the patient s tumour . currently , grnvac1 has completed phase ii trials in patients with acute myelogenous leukaemia and metastatic prostate cancer ( clinicaltrials.gov identifier : nct00510133 and nct01153113 , respectively ) . so far , results show that grnvac1 is safe and well tolerated , with htert - specific immune responses detected in a large fraction of patients . relapse - free survival in some patients with aml at high risk of relapse is encouraging . results from these and other similar trials will determine if htert has potential has a target for broad - spectrum cancer immunotherapy . the htert promoter is very tightly regulated and the promoter is generally inactive in cells that lack telomerase . hence , a third approach to target telomerase - expressing cancer cells is to use the htert promoter to drive the expression of a suicide gene and/or control the replication of a lytic virus . in the first strategy , the htert promoter drives the expression of a pro - apoptotic protein , such as trail ( tumour necrosis factor - related apoptosis - inducing ligand ) or prodrug - activating enzyme [ 7378 ] . viruses carrying the suicide gene are injected in a tumour and telomerase - expressing cancer cells are then killed by the administration of a prodrug , which the activating enzyme converts to a toxin . a second strategy , currently in clinical trials , is to use the htert promoter as a switch that controls the replication of a lytic virus . viral proteins e1a and e1b are required for replication of the adenovirus , and if the e1 gene is redesigned to be controlled by the htert promoter , the modified virus can only replicate in cells expressing telomerase [ 8083 ] . one such virus , telomelysin ( obp-301 ) , is currently being used in phase i clinical trials for various types of tumours . in pre - clinical studies , this and other similarly engineered adenovirus showed great promises for the selective and efficacious targeting of telomerase - expressing cells . in vitro , the modified viruses could replicate in a wide variety of human cancer cell lines , effectively inducing cytolysis [ 8083 ] . in normal human cells lacking telomerase , replication and cytotoxicity were greatly attenuated . in mice implanted with human lung , prostate or liver cancer cells , intratumoral injection of the virus effectively retarded tumour growth and extended the survival of mice , including mice with large tumour burden [ 8083 ] . when injected into large xenografts , the virus could reduce the size of the tumours with formation of a massive ulceration at the site of injection . viruses were not detected in tissues outside of the injected tumours , except for the presence of circulating viruses in the blood as well as spreading of the virus to other distant tumour sites . viral particles released after cytolysis can infect other nearby cells or spread to the rest of the body via blood or lymphatic flow , potentially allowing the virus to reach other tumour sites , including distant metastases . based on these and other studies , a phase i clinical trial was conducted in patients with advanced solid tumours to assess the clinical safety of the obp-301 virus , also known as telomelysin . sixteen patients with a variety of solid tumours were enrolled and given a single intratumoral injection of the virus , after which safety , response and pharmacodynamics were evaluated . one patient had a partial response at the injected malignant lesion and seven patients demonstrated stable disease 56 days after treatment . these studies demonstrated the feasibility of targeting telomerase - expressing cancer cells with oncolytic viruses . second - generation viruses that combine the two strategies by using the htert promoter to control both viral replication and the expression of a suicide gene have also been designed [ 85 , 86 ] . studies of these other second - generation viruses should determine if these oncolytic viruses have potential in the clinics for broad - spectrum cancer therapy . the maintenance of telomeres by the cellular ribonucleoprotein enzyme telomerase is of well - documented importance for cancer [ 2 , 8790 ] . telomerase , a cellular reverse transcriptase that adds dna to the ends of chromosomes , is reactivated or up - regulated in the vast majority of advanced malignancies , and is thus an almost universal target for the treatment of human cancers . most human tumours not only express telomerase but also have very short telomeres , whereas telomerase activity is absent or at lower levels in normal tissues which also have longer telomeres . this relationship between activation of telomerase activity and short telomeres in human malignancies makes the inhibition of telomerase a valuable target for cancer therapeutics . importantly , the mode of action of telomerase inhibitors predicts minimal side effects on normal stem cells that can express telomerase . in this review , we summarized the role of telomeres and telomerase in cancer and reviewed the current status of ongoing telomerase clinical trials . but many questions still remain , including : what are the key safety concerns , such as the effects of telomerase inhibitors on normal stem cells ? is telomerase activity regulated by the same mechanisms in cancer stem cells as it is in normal stem cells ? how can telomerase inhibitors affect cancer stem cells if they are more quiescent than the more differentiated bulk tumour cells ? will human cancers become resistant to telomerase inhibitors ? clearly more basic research will be required as initial results from clinical trials emerge . in summary , the multitude of studies on human telomeres and telomerase highlight the complex interplay between signals emanating from dysfunctional telomeres and tumour suppressor pathways that regulate stem cell biology , aging and cancer . a more complete understanding of these relationships should lead to more effective clinical trials . the authors confirm that there are no conflicts with the exception that they have conducted collaborative research with geron corporation , menlo park , ca , usa .

abstractthe role of telomeres and telomerase as a target for cancer therapeutics is an area of continuing interest . this review is intended to provide an update on the field , pointing to areas in which our knowledge remains deficient and exploring the details of the most promising areas being advanced into clinical trials . topics that will be covered include the role of dysfunctional telomeres in cellular aging and how replicative senescence provides an initial barrier to the emergence of immortalized cells , a hallmark of cancer . as an important translational theme , this review will consider possibilities for selectively targeting telomeres and telomerase to enhance cancer therapy . the role of telomerase as an immunotherapy , as a gene therapy approach using telomerase promoter driven oncolytic viruses and as a small oligonucleotide targeted therapy ( imetelstat ) will be discussed .

Targeting telomerase-expressing cancer cells Human telomeres act as a mitotic clock Telomerase expression and cellular lifespan Telomerase inhibition to block tumour growth Telomerase-targeted immunotherapy Targeting telomerase-expressing cells with oncolytic viruses Concluding remarks Conflict of interest

telomerase is the enzyme responsible for the maintenance of telomeres , which cap and protect the ends of chromosomes . in the great majority of cancers , overcoming replicative senescence is achieved initially by inactivating important cell cycle checkpoints and eventually by up - regulation of telomerase expression once telomeres have become critically short . this reactivation of telomerase stabilizes the telomeres and extends cellular lifespan . as a consequence , telomerase is detected in more than 85% of all cases of cancer [ 3 , 4 ] , making the enzyme an attractive target for cancer therapeutics . in cancer cells , telomerase inhibition has been shown to lead to telomere shortening and , after sufficient cell divisions , this attrition can cause an uncapping of telomeres and induction of apoptosis [ 5 , 6 ] . in this review , we discuss the biological and biochemical properties of telomerase , its use as a diagnostic and prognostic marker for cancers and its value as a target for cancer therapeutics . telomeric dna repeats serve as anchor for the recruitment of dna - binding factors telomeric repeat factor ( trf)1 , trf2 and protection of telomeres 1 ( pot1 ) in combination with other telomere associated proteins , which together form a protective capping complex originally termed telosome and more recently shelterin [ 8 , 9 ] . although most of the telomere is made of duplex telomeric dna , all telomeres end with a g - rich single - stranded 3-overhang of 50 to 300 bases . on the lagging strand , internal priming by the last okazaki fragment and removal of the rna primer creates a gap of unreplicated dna [ 1 , 2 ] . on the leading strand , post - replication excision by the apollo nuclease is needed to produce a single - stranded 3-telomeric overhang that can serve as substrate for pot1 binding and t - loop formation [ 14 , 15 ] . , an uncapped telomere will be recognized as a ds - dna break by dna damage sensing mechanisms , the activation of which will induce atm kinase activity , phosphorylation of p53 and up - regulation of cell cycle inhibitor p21[11 , 12 , 16 , 17 ] . the net result is an irreversible cell cycle arrest and establishment of the senescent state . eventually , the uncapped telomeres can serve as substrate for non - homologous end joining ( nhej ) and through this process , can become fused to other dysfunctional telomeres [ 7 , 8 , 19 ] . at anaphase , the dicentric chromatids generated by these fusions will fail to segregate properly and , as the cells continue to divide , there will be recurrent cycles of anaphase - bridge , breakage and fusion . in the great majority of cancers , overcoming these limits to cellular lifespan is achieved by means of telomerase expression . as cells divide and telomeric dna repeats are lost , telomeres lose their protective shelterins ( grey spheres ) , become unfolded ( loss of the t - loop ) and are recognized as ds - dna breaks . in cells with intact checkpoints , the uncapped telomere leads to the activation of the atm kinase , phosphorylation of p53 and up - regulation of the p21 gene ( atm / p53/p21 cascade ) . once induced , p16 blocks cdk4 , the activity of which is required for inactivation of rb , a powerful inhibitor of the cell cycle ( p16/cdk4/rb cascade ) . in cancer cells that lack components of these pathways ( most commonly p53 , p16 or rb ) , the dna damage signals emanating from uncapped telomeres will be ignored and the cells will bypass senescence and with division , progressive telomere erosion will continue . when many telomeres become uncapped , the very short telomeres will serve as substrate for nhej and through this process , will become associated and/or fused to other dysfunctional telomeres . telomerase compensates for the effects of the end replication problems by the synthesis of new telomeric dna repeats . in human beings , the telomerase complex has an estimated mass of over 1000 kd and may function as a homodimer . the enzyme is generally but not always processive and is capable of adding many more repeats to the same telomere before falling off its substrate . in the trap assay , products of the telomerase reaction are quantified following pcr amplification [ 26 , 27 ] . telomerase activity is calculated as the ratio of the intensity of the telomeric products over that of the itas . when telomerase binds to a telomere , its rna template region ( shaded ) hybridizes ( asterisk ) to the end of the 3-telomeric overhang . next , the htert subunit acts as a reverse transcriptase and copies the rna template into dna ( dotted arrow ) . grn163l ( imetelstat ) is a n3-p5 thio - phosphoramidate oligonucleotide complementary to sequence of the rna template ( sequence shown ) and which has been modified to carry a palmitate group at its 5-end . at the later stages , htert expression is repressed and telomerase activity becomes undetectable in most tissues , the exact timing of repression varying depending on the tissue . a notable exception to the almost universal absence of telomerase in human tissues is cancer . a survey of the published literature established telomerase as one of the best markers of cancer cells . in most cases , the activity is localized to the tumour and the activity is absent from the surrounding normal tissues . human cancer cells commonly possess mechanisms that compensate for telomere attrition and provide them with cellular immortality , a hallmark of cancer . in the vast majority of cancers , this mechanism is the up - regulation of telomerase activity . even in cancer cells originating from telomerase - positive precursors , this ability to maintain telomeres is a fundamental property of cancer cells , which normal somatic human cells do not possess . this lack of telomere attrition provides cancer cells with cellular immortality , a capacity for unlimited number of cell divisions . this indicates that telomerase is not essential for cancer development as these children with neuroblastoma are clearly born with an advanced cancer . because of examples such as neuroblastoma where telomerase negative tumours regress , and because most cancer cells are immortal due to telomerase reactivation , targeting telomerase is an attractive strategy for cancer therapy . the first of these approaches consists of blocking the activity of the enzyme to limit the lifespan of telomerase - expressing cancer cells . crisis , which represents the preferred outcome , is a form of p53-independent apoptosis that generally leads to the death of the cancer cell [ 5 , 6 ] . because sufficient telomere shortening must occur before senescence or crisis are induced , a delay should be expected before the effects of telomerase inhibition are observed [ 35 , 37 ] . for these reasons , telomerase inhibitors are now being tested in combination with conventional cancer therapy and in maintenance clinical trials aimed at reducing recurrence after surgery , chemotherapy or radiation therapy . standard therapies have been optimized to kill bulk tumour cells , not necessarily the cancer stem cells which tend to express multidrug resistance . the hope is that using telomerase inhibitors in a cancer maintenance setting may lead to more durable responses . for inhibiting telomerase , the template region of the human telomerase rna ( htr ) presents an accessible target for oligonucleotide - based inhibitors [ 3841 ] . oligonucleotides that can hybridize to the template region of htr , as shown previously , can be used to inhibit telomerase ( fig . n3-p5thio - phosphoramidate oligonucleotides complementary to the htr template have been found to be especially good telomerase inhibitors . one compound , grn163 ( geron corporation , menlo park , ca , usa ) caused telomerase inhibition and subsequent telomere shortening in a number of cancer cell lines [ 4550 ] . one such compound , grn163l ( formally known as imetelstat ) , is a grn163 oligonucleotide modified to carry a 5-terminal palmitoyl ( c16 ) moiety conjugated to the n3-p5 thio - phosphoramidate backbone ( 5-palmitate - tagggttagacaa - nh2 - 3 ) . in cancer cells chronically exposed to grn163l , telomeres shorten with cell divisions and after a delay , a state of crisis is induced with evidence of chromosomal fusions , anaphase bridges and widespread apoptosis . however , on the basis of these and other pre - clinical studies , grn163l has entered clinical trials in patients afflicted with different forms of cancer . dose escalation proceeded to 11.7 mg / kg but due to haematological toxicities , this dose was considered to exceed the maximum tolerated dose . combinations of imetelstat and bevacizumab may be permitted in some individuals as well as cross - over from one to the other maintenance therapy . in addition to standard biomarkers , this trial will collect other tissues ( e.g. finally , grn163l will be tested as a single therapeutic agent for multiple myeloma , with one of the end - points being reduction in myeloma progenitor cells and essential thrombocythemia , where haematological and molecular responses will be assessed . therefore , an important question will be whether grn163l can target these cancer stem cells efficaciously . according to a consensus definition , a tumour cell is a cancer stem cell if it can self - renew , initiate the formation of new tumours , and regenerate all of the heterogeneous lineages of cancer cell types that comprise a tumour . in addition , the abundance of cancer stem cells was reduced following grn163l treatment of prostate , pancreatic and breast cancer cell lines [ 62 , 64 ] . finally , a key characteristic of cancer stem cells isolated from brain and breast cancer is their ability to give rise to free - floating spheres , respectively called neurospheres and mammospheres . cells that possess functional checkpoints , such as primary human cells , undergo senescence as soon as the shortest telomere becomes uncapped [ 2 , 35 ] . crisis , which represents the preferred outcome , is a form of p53-independent apoptosis that generally leads to the death of the cancer cell [ 5 , 6 ] . because sufficient telomere shortening must occur before senescence or crisis are induced , a delay should be expected before the effects of telomerase inhibition are observed [ 35 , 37 ] . for these reasons , telomerase inhibitors are now being tested in combination with conventional cancer therapy and in maintenance clinical trials aimed at reducing recurrence after surgery , chemotherapy or radiation therapy . after multiple rounds of cell divisions , these cancer stem cells will give rise to a new tumour ( arrow pointing up ) and the heterogeneity of the original tumour is re - established . the hope is that using telomerase inhibitors in a cancer maintenance setting may lead to more durable responses . for inhibiting telomerase , the template region of the human telomerase rna ( htr ) presents an accessible target for oligonucleotide - based inhibitors [ 3841 ] . oligonucleotides that can hybridize to the template region of htr , as shown previously , can be used to inhibit telomerase ( fig . one such compound , grn163l ( formally known as imetelstat ) , is a grn163 oligonucleotide modified to carry a 5-terminal palmitoyl ( c16 ) moiety conjugated to the n3-p5 thio - phosphoramidate backbone ( 5-palmitate - tagggttagacaa - nh2 - 3 ) . when compared to grn163 , grn163l displayed greatly improved uptake and telomerase inhibition [ 46 , 48 , 49 ] . in cancer cells chronically exposed to grn163l , telomeres shorten with cell divisions and after a delay , a state of crisis is induced with evidence of chromosomal fusions , anaphase bridges and widespread apoptosis . this inhibition of cellular growth by grn163l has been reported in cancer cell lines of diverse origins , including multiple myeloma and tumours of the bladder , breast , liver , lung and stomach [ 45 , 48 , 5154 ] . even when administered as a single agent , grn163l could block the growth of implanted tumour cells . however , on the basis of these and other pre - clinical studies , grn163l has entered clinical trials in patients afflicted with different forms of cancer . thus , 14 weeks after completing four to six cycles of induction chemotherapy , patients will be treated with imetelstat ( grn163l ) , bevacizumab ( avastin , an angiogenesis inhibitor ) or observation ( e.g. approximately 100 patients will be enrolled in these studies with the main end - point being objective and progression - free survival . in addition to standard biomarkers , this trial will collect other tissues ( e.g. peripheral blood mononuclear cells , circulating tumour cells , dna / rna and serum ) to assay for a variety of other predictive markers . finally , grn163l will be tested as a single therapeutic agent for multiple myeloma , with one of the end - points being reduction in myeloma progenitor cells and essential thrombocythemia , where haematological and molecular responses will be assessed . therefore , an important question will be whether grn163l can target these cancer stem cells efficaciously . according to a consensus definition , a tumour cell is a cancer stem cell if it can self - renew , initiate the formation of new tumours , and regenerate all of the heterogeneous lineages of cancer cell types that comprise a tumour . in addition , the abundance of cancer stem cells was reduced following grn163l treatment of prostate , pancreatic and breast cancer cell lines [ 62 , 64 ] . finally , a key characteristic of cancer stem cells isolated from brain and breast cancer is their ability to give rise to free - floating spheres , respectively called neurospheres and mammospheres . peptides displayed by mhc presentation are generated from within the cells by the limited proteolysis of cellular proteins , capture of the resulting peptides by mhc class i molecules , their transport to the plasma membrane and presentation at the surface by mhc class i molecules . in telomerase - targeted immunotherapy , immune cells are exposed to a vaccine that consists of antigen - presenting cells that were either exposed to high levels of an immunogenic htert peptide or modified to overexpress an immunogenic fragment of htert . once administered to a patient , the antigen - presenting cells elicit the activation and expansion of telomerase - specific cytotoxic t lymphocytes , which then can recognize and kill the telomerase - expressing tumour cells . in phase i clinical trials , tumour vaccines against htert have resulted in the induction of t - cell immune responses with minimal toxicities [ 6870 ] . a frequent problem that limits the broad applicability of cancer vaccines is the heterogeneous expression of the tumour antigen within a tumour . however , in the case of telomerase , the antigen is both universally expressed in cancers and critical for maintenance of the malignant phenotype . developed by kael - gemvax ( gwangju , south korea ) as an injectable formulation , the vaccinating gv1001 peptide is eventually trimmed and processed for presentation by mhc class i and class ii molecules , allowing it to elicit the activity of telomerase - specific cytotoxic ( cd8 ) and helper ( cd4 ) t cells . in phase i / ii clinical trials for nsclc , hepatocellular carcinoma and non - resectable pancreatic carcinoma , gv1001 has been well tolerated and shown to induce htert - specific t - cell responses [ 6870 ] . in these cells , the htert fragment should be processed into a multitude of epitopes to elicit a polyclonal t - cell response , which could potentially recognize any htert peptide displayed by the patient s tumour . results from these and other similar trials will determine if htert has potential has a target for broad - spectrum cancer immunotherapy . hence , a third approach to target telomerase - expressing cancer cells is to use the htert promoter to drive the expression of a suicide gene and/or control the replication of a lytic virus . viruses carrying the suicide gene are injected in a tumour and telomerase - expressing cancer cells are then killed by the administration of a prodrug , which the activating enzyme converts to a toxin . a second strategy , currently in clinical trials , is to use the htert promoter as a switch that controls the replication of a lytic virus . viral proteins e1a and e1b are required for replication of the adenovirus , and if the e1 gene is redesigned to be controlled by the htert promoter , the modified virus can only replicate in cells expressing telomerase [ 8083 ] . in pre - clinical studies , this and other similarly engineered adenovirus showed great promises for the selective and efficacious targeting of telomerase - expressing cells . in mice implanted with human lung , prostate or liver cancer cells , intratumoral injection of the virus effectively retarded tumour growth and extended the survival of mice , including mice with large tumour burden [ 8083 ] . viral particles released after cytolysis can infect other nearby cells or spread to the rest of the body via blood or lymphatic flow , potentially allowing the virus to reach other tumour sites , including distant metastases . based on these and other studies , a phase i clinical trial was conducted in patients with advanced solid tumours to assess the clinical safety of the obp-301 virus , also known as telomelysin . these studies demonstrated the feasibility of targeting telomerase - expressing cancer cells with oncolytic viruses . studies of these other second - generation viruses should determine if these oncolytic viruses have potential in the clinics for broad - spectrum cancer therapy . the maintenance of telomeres by the cellular ribonucleoprotein enzyme telomerase is of well - documented importance for cancer [ 2 , 8790 ] . telomerase , a cellular reverse transcriptase that adds dna to the ends of chromosomes , is reactivated or up - regulated in the vast majority of advanced malignancies , and is thus an almost universal target for the treatment of human cancers . this relationship between activation of telomerase activity and short telomeres in human malignancies makes the inhibition of telomerase a valuable target for cancer therapeutics . in this review , we summarized the role of telomeres and telomerase in cancer and reviewed the current status of ongoing telomerase clinical trials . but many questions still remain , including : what are the key safety concerns , such as the effects of telomerase inhibitors on normal stem cells ? clearly more basic research will be required as initial results from clinical trials emerge . in summary , the multitude of studies on human telomeres and telomerase highlight the complex interplay between signals emanating from dysfunctional telomeres and tumour suppressor pathways that regulate stem cell biology , aging and cancer .

all reactions utilizing air- and moisture - sensitive reagents were performed in dried glassware under an atmosphere of dry nitrogen . dry solvents ( thf , toluene and dcm ) were obtained from a braun mb - sps-800 . for thin - layer chromatography ( tlc ) analysis , merck precoated plates ( silica gel 60 f254 , art 5715 , 0.25 mm ) were used . column chromatography was performed on fluka silica gel ( 60 , 220440 mesh ) . h nmr , c nmr , and p nmr spectra were measured on bruker 400 or 500 mhz spectrometers . deuterated solvents ( methanol - d4 and chlorofrom - d ) were obtained from sigma - aldrich and used as supplied . the following compounds were synthesized according to literature procedures : 1 , d10-diethyl phosphite , 4-pyridyl diphenylphosphine l1 , 4-pyridyl diphenylphosphine oxide l2 , 4-pyridylmethyl diethylphosphonate l8 , 3-pyridylmethyl diethylphosphonate l9 . detailed synthetic procedures and characterization data can be found in the supporting information . p - h2 was produced by cooling h2 gas over fe2o3 at 30 k. samples involved the analysis of 5 mm concentrations of 1 and 5 or 6 equiv of substrate ( l1-l12 ) in methanol - d4 ( 0.6 or 3.0 ml ) solution that was located in either a 5 or 8 mm nmr tube fitted with a j. young s tap . the resulting solutions were degassed prior to the introduction of p - h2 at a pressure of 3 bar . samples were then shaken for 10 s in a specified fringe field of either a 9.4 t bruker avance ( iii ) nmr spectrometer or a 7 t bruker biospec preclinical mri scanner . they were then rapidly transported into the main magnetic field of the instrument for subsequent probing by nmr or mri methods . historically , n - heteroaromatic molecules have been shown to be excellent receptors for polarization transfer to h and c nuclei under sabre . we therefore synthesized a phosphine ( l1 ) , a phosphine oxide ( l2 ) , a phosphine sulfide ( l3 ) and a phosphonate ester ( l4 ) which incorporated a 4-substituted pyridine motif , as detailed in the supporting information ( figure 1 ) . we selected the 4-substitution pattern for this study in order to minimize any steric interactions between the hyperpolarization target l and the imes ligand in the catalyst 2 . we expected this restriction to promote activity in the widest range of analytes possible . a series of samples were then prepared in methanol - d4 that contained l1-l4 , at a concentration of 5 mm , and the catalyst precursor 1 at a 17% catalyst loading . these samples were probed after sabre had taken place at 298 k on a 400 mhz nmr spectrometer by the application of a simple 90 rf - pulse . we will discuss the effects of sabre on the pyridyl h polarization , then the p polarization , and finally the polarization received by the other substituent groups . when this 90 pulse was applied to the h nuclei of these samples , only l3 proved to exhibit weak pyridyl proton polarization . for example , the two h nmr signals of l4 , that were derived from the pyridine functionality , showed a 1499-fold total signal enhancement when the initial polarization transfer step was undertaken in a 45 g field . this high h ptc was reflected in the fact that the corresponding p signal exhibited a similarly impressive 545-fold signal enhancement . for p , ptc was maximized through transfer at 0.5 g rather than the 45 g field observed for its h nuclei . h and p signal intensity gains for analytes l1-l4 under sabre conditions using 1 as the catalyst . a series of control experiments were then undertaken on triphenylphosphine , triphenylphosphine oxide , and phenyldiethylphosphonate in order to explore the role that the pyridine substituent might play in the hyperpolarization transfer process . these experiments revealed that no polarization transfer took place into the h or p nuclei of these materials under the same sabre conditions as used to hyperpolarize l14 described above . based on this information it is clear that the pyridine motif is necessary for successful ptc under these conditions . this is achieved by the formation of an active sabre catalyst as typified in scheme 1 . we note that this situation differs from that reported when the related [ ircl(h)2(pph3)3 ] complex was used to successfully polarize triphenylphosphine at temperatures between 333 and 353 k by koptyug et al . in this case however , when 1 reacts with an excess of pph3 a series of new and rapidly relaxing hydride ligand signals are observed at 7.33 , 7.98 , and 8.59 which are indicative of the formation of [ ir(h)2(h2)(imes)(pph3)2]cl . the formation of such species precludes the observation of sabre in these samples because of their role in the rapid quenching of p - h2 ( see the supporting information ) . as a consequence of the formation of 2a - l4 , when the corresponding h nmr spectra are examined under sabre conditions polarized hydride resonances appear as a singlet in the region around 22.15 for the pair of chemically equivalent hydride ligands ( see the supporting information ) . characteristic signals for l4 , as a ligand in 2a - l4 , are also seen to be polarized in the associated p nmr spectra . we illustrate selected regions of these hyperpolarized h and p nmr spectra after ptc at 0.5 g , in figures 2 and 3 respectively to detail some of these effects . the hyperpolarized h nmr trace shown in figure 2 confirms that both of the two pairs of equivalent ring protons of bound l4 receive magnetization , while those of the axial group remain unaffected . this is manifested in the detection of emission signals for the free analyte at 8.76 and for the equatorial ligand at 8.50 . the magnetic state that produces this emission effect is single spin order in nature and associated with an enhanced but inverted zeeman population . in contrast , the corresponding pair of 3,5-ring protons in l4 produce antiphase peaks that are separated by 13.5 hz . this splitting corresponds to the value of jph in l4 and requires that a heteronuclear two - spin order proton - phosphorus term is created through sabre . application of a 90 rf - pulse to the h nuclei of this term creates the visible antiphase signal that is seen ; the creation of homonuclear proton terms of this type is a well - established characteristic of phip . for l4 , sabre enhanced h nmr spectrum ( lower ) and corresponding thermal reference spectrum ( upper , x64 vertical expansion ) of 2a - l4 and l4 ( = equatorial ligand signal , = axial ligand signal of 2a - l4 ) . the hyperpolarized p nmr spectrum of the l4 sample , collected as a single transient , also showed two antiphase peaks , at 14.44 and 13.73 for free and equatorially bound l4 respectively ( figure 3 ) . the signal for free l4 showed a 545-fold increase in size after ptc at 0.5 g relative to the corresponding thermally polarized trace . the separation between the point of maximum displacement and minimum displacement for the signal due to l4 in this nmr trace is 27.0 hz ( smaller jph couplings are hidden within the peak envelope ) and twice that of the separation shown in the h nmr spectrum . this difference confirms the creation of a proton - phosphorus two spin order term , which leads to a triplet when excited wherein the central feature has zero intensity ( 1:0 : + 1 ) . sabre enhanced p nmr spectrum ( lower ) and thermal reference spectrum ( upper , x64 vertical expansion ) of 2a - l4 and l4 ( = equatorial ligand , = axial ligand of 2a - l4 ) . interestingly , when 2a - l4 is probed by exsy methods both the axial and equatorial l4 ligands are observed to undergo exchange with the free ligand pool present in bulk solution . the exchange profile seen for axial - l4 suggests that there is some in - cage recombination of this ligand , a process which is not observed for equatorial - l4 ( see the supporting information ) . the axial ligand loss rate is 0.645 0.010 s at 283 k and slightly more rapid than equatorial ligand loss rate which is 0.522 0.007 s. the observed hyperpolarization seen in the equatorial - l4h and p signals is therefore a consequence of sabre prior to ligand loss . we note that pyridylphosphonates find widespread use within a biological setting as lysophosphatidic acid receptor antagonists , nucleotide analogues , and enzyme inhibitors . importantly , related compounds have been shown to exhibit low toxicity in rat studies ( ld50 of 3.2 g / kg ( oral ) ) . as a consequence of these facts and the observation that l4 delivers high levels of visible p hyperpolarization this was achieved by the synthesis of the phosphonate derivatives l5-l12 which are shown in table 1 , alongside the levels of h and p hyperpolarization that results from their analysis under sabre . for p , 5 mm concentrations of 1 with 5 equiv of l7 and 6 of equivalents of l5-l12 were employed at ( a ) 45 g and ( b ) 0.5 g. in order to examine the effect that the regiochemistry of substitution played on the polarization level we replaced the 4- substitution pattern exhibited by l1-l4 with 2- and 3- substitutions in the form of substrates l5 and l6 . polarization transfer into 2-pyridylphosphonate l5 proved to be unsuccessful , and this is due to the formation of a complex that yields a very broad hydride resonance at 31.3 which does not show phip . upon cooling the solution to 233 k this hydride ligand signal separates into two resonances at 30.2 and 32.1 which confirms that this complex is a dihydride . the corresponding hydride ligand signals yield an average t1 value of 0.56 s at 253 k and are therefore classically bonded and terminal in character . nonetheless , the presence of this complex in solution provides a pathway to rapidly destroy the p - h2 present , presumably via the formation of a dihydrogen - dihydride complex , with the result that there is very little observable polarization transfer into l5 . the measured h signal enhancement at 45 g for l6 was 2866-fold , and the corresponding p signal enhancement was 336-fold . clearly , the change to a 3-substitution pattern has improved the level of h ptc relative to 4-substitution but surprisingly decreased the efficiency for p transfer . the synthesis of the bis - phosphonate l7 was therefore undertaken , as this material contains two chemically equivalent p nuclei and one fewer pyridyl proton than the monosubstituted derivatives . we speculated that better p - ptc might result as a consequence of this change on the basis that each p - h2 molecule contains a specific amount of latent polarization , and consequently when shared with fewer acceptor sites a net gain in the level of observable transfer might result . the corresponding h and p signal enhancements for l7 were 2271- and 741-fold respectively when 6 equiv of the analyte was employed , and therefore this hypothesis is confirmed . however , one further and somewhat unexpected observation was made , the enhanced steric bulk of l7 results in a change in the active complex such that 2b - l7 results wherein one coordination site is occupied by chloride ( see the supporting information ) . the two hydride signals for this complex appear at 23.74 and 24.06 . because of this change in catalyst form we found that reducing the number of equivalents of l7 to 5 led to an improvement in ptc , giving h and p signal enhancements of 3689- and 860-fold after transfer at 45 and 0.5 g respectively . in order to further test the efficiency of polarization transfer to p in these systems we added a series of spacers between the pyridyl carbon and the phosphorus center . in l8 and l9 this corresponds to the introduction of a ch2 group , while for l10 it is nme and for l11 it is o. these analytes were sabre active , but while the pyridyl ring protons remained strongly enhanced the spacer acted to substantially reduce the levels of p polarization . this change again results in a reduction in the number of proton environments able to accept magnetization . while successful , the levels of both h and p signal enhancement were again lower than those achieved for the pyridylphosphonates . we now address the potential of these substrates to act as mri agents for which in - phase p signals are desirable . as we have indicated , all of the described p signals that were created through ptc in a 0.5 g appear in antiphase . while this effect can be refocused to produce an in - phase signal , we proposed that changing the polarization transfer field ( ptf ) might achieve a similar result . this would be achieved by changing the relative proportions of single spin ( in - phase ) and two spin ( antiphase ) components and result in a predominantly in - phase signal being obtained . a series of experiments were therefore performed in which the ptf was increased using a variable field polarizer and the resulting p signal monitored . significant in - phase contributions were observed after transfer at 45 g without any significant reduction in the overall p signal enhancement ( 216-fold vs 336-fold for l6 and 703-fold vs 860-fold for l7 at 45 and 0.5 g respectively ) . while ptfs above 45 g did result in an increase in the relative single spin contribution , a sharp decline in the overall signal enhancement was noted . for l6 the relative proportions of the single spin and two spin terms are 0.08:1 and 0.23:1 when the ptf is 0.5 and 45 g respectively . in contrast , when l7 is examined , the relative proportions are 0.06:1 at 0.5 g and 0.88:1 at 45 g respectively . we conclude from this that l7 is potentially the better mri probe for these model studies . this is because the signal for l4 proved to remain predominately antiphase at both 0.5 g ( 0.15:1 ) and 45 g ( 0.17:1 ) , and it is therefore less suitable for such measurements . sabre hyperpolarized p nmr traces for l4 [ a ] , l6 [ b ] , and l7 [ c ] that result after polarization transfer catalysis in a 45 or 0.5 g field . further examination of the hyperpolarized h nmr spectra revealed that the signal enhancements described so far also extend into the ethyl groups of the phosphonate . typically these h signal enhancements were 50-fold per ethyl group and reflect a relayed h - p - h transfer process . it was therefore reasoned that deuteration of the phosphonate ethyl esters would increase the level of retained p polarization as transfer to h is less efficient due to the large frequency difference that exists between them . therefore , we synthesized d10-l4 , d10-l6 , and d20-l7 and analyzed them under analogous conditions to their protio - counterparts ( figure 5 ) . the levels of hyperpolarization seen in the aromatic proton signals of d10-l4 , d10-l6 , and d20-l7 proved to remain comparable to those of their protio analogues , but the p signals were found to dramatically increase in intensity . structure of substrates d10-l4 , d10-l6 , and d20-l7 and their signal intensity gains , alongside those of their protio counterparts l4 , l6 , and l7 , under sabre after ptc at the specified ptf . substrate d20-l7 yielded a p signal gain of 3588 ( 2.3% polarization ) after ptc at 0.5 g. a 2251-fold signal enhancement ( 1.4% polarization ) is , however , achieved after ptc at 45 g with significant in - phase character being visible in the detected signal . we note this is an order of magnitude larger than previously reported for p enhancements using this technique and can be achieved without the need for elevated temperatures during the polarization transfer step . in a further development , we completed magnetization transfer by inserting the sample into a -magnetic shield , which attenuates the earth s field by a factor of 3000-fold , prior to moving it into the 9.4 t magnet for examination . while this created in - phase p magnetization , it would therefore appear that simply changing the polarization transfer field to a readily accessible value between 0.5 and 130 g reflects the optimal route to achieve in - phase signal . we also note that complexes of type 1 are known to catalyze hydrogen deuterium exchange , and , as such , there is the potential to detect 1-proton - phip in such analytes . hence , we prepared a sample of d20-l7 and 1 in methanol - d4 under 3-bar of h2 and followed it by h nmr for a period of 10 days . at this point , if the p enhancements that were seen here were due to a 1-proton - phip , then the enhancement levels should remain comparable to those observed at day 1 . this suggests that when such samples are examined they should be prepared shortly before their use . [ a]-[c ] thermally polarized p images of l6 , l7 , and d20-l7 , respectively collected over 2048 averages . the repetition time was 20 s. [ d]-[f ] hyperpolarized images of l6 , l7 , and d20-l7 , respectively collected in a single shot after ptc at 45 g. ( te / fov / slice thickness were 3.2 ms/4 4 cm/5 mm respectively ) . [ g ] 2048-average thermal image and [ h ] 1 scan hyperpolarized p image of d20-l7 collected on a 7 t biospec 70/30 preclinical scanner . ( te / fov / slice thickness were 12 ms/3 3 cm/10 mm respectively ) . samples employed 5 mm concentrations of 1 in methanol - d4 ( 3 ml ) with l6 ( 6 equiv ) , l7 or d20-l7 ( 5 equiv ) and 3 bar of p - h2 . snr values were calculated by dividing the mean signal intensity value in the region with signal by the standard deviation of the residual signal seen in an analogously sized noise region . we have thus far exemplified the ability to hyperpolarize p , and we next evaluated the spin lattice relaxation times ( t1 ) of the analytes l4 , d10-l4 , l6 , d10-l6 , l7 , and d20-l7 . the t1 relaxation values of the zeeman based p polarization are 8.3 , 7.6 , 9.2 , 9.3 , 5.3 , and 6.0 s respectively in methanol - d4 . for d20-l7 they increase to 6.5 s in ethanol - d6 and decrease to 3.6 s in d2o . these t1 values , although short , do offer the opportunity to obtain images using the sabre technique . with a number of these substrates displaying favorable results we were hopeful that it would be possible to collect mri with improved spatial resolution and short acquisition times . a series of hyperpolarized samples were therefore interrogated in a 9.4 t vertical bore scanner using the rapid acquisition with relaxation enhancement ( rare ) pulse sequence . we employed an echo train length of 32 and a matrix size 32 32 with zero filling to 128 128 . this resulted in a total single scan acquisition time of 500 ms . for comparison purposes , thermal images of l6 , l7 , and d20-l7 were recorded with 2048 averages , as shown in figure 6 . under hyperpolarization conditions , the largest gains in signal - to - noise ratio ( snr ) were observed for d20-l7 ( 94.4 ) and reflected a > 8-fold improvement on that of the thermal control ( 11.4 ) . similar images were also recorded on a 7 t biospec 70/30 preclinical scanner using d20-l7 as the imaging agent ( figures 6 g and 6h ) . a > 6-fold improvement in snr was achieved in the single scan hyperpolarized image ( snr = 27.7 ) when compared to the corresponding 2048-average thermal average ( snr = 4.5 ) . we note that insertion of the sample tube into the horizontal scanner caused more residual motion of the sample than was apparent in the analogous measurements on the vertical bore scanner . in addition , the time taken to transfer the sample to the magnet is ca . 5 s longer for the horizontal scanner , and the magnetic fields experienced by the sample during transfer differ . a combination of these effects could account for the reduction in observed snr that is observed in the hyperpolarized images that were recorded on the biospec . we have demonstrated that the h and p nuclei of a series of pyridyl substituted phosphonate esters , a phosphine , and a phosphine oxide can be efficiently hyperpolarized using sabre . substitution at the 3-position delivered the greatest efficiency in polarization transfer catalysis to h and p , compared to substitution at the 2- and 4- positions . in the ligands that showed high performance , the phosphorus center was connected directly to the pyridyl ring such that a strong jph coupling of 13.5 hz exists between protons in the pyridyl ring and the p nucleus . these were shown to receive polarization via a relay mechanism which first polarized the pyridyl protons , then the p nucleus , and finally the ethyl protons . the net gain for the ethyl protons was of the order of 50-fold per ethyl group in comparison to the 23 thousand level shown by the aromatic protons . the effect of leakage of the p magnetization to the h nuclei of these ethyl groups was shown to reduce the level of retained p polarization . when the h and p polarization levels are compared between the protio forms of l4 and l6 and their deuterated ethyl counterparts d10-l4 and d10-l6 , it could be seen that the best levels of h polarization result for three - substituted d10-l6 . furthermore , the p polarization levels are broadly similar for these two substrates at 1% . however , this raw observation masks the fact that for symmetrical d10-l4 antiphase signal dominates at all monitored polarization transfer fields . in addition , the t1 values for d10-l4 are much less than those for four - substituted d10-l6 in the presence of 2 . the result of the small t1 of d10-l4 is that it must actually be better p polarized under sabre than d10-l6 ; we observe the magnetization ca . 3 s after polarization transfer has ceased and relaxation has reduced the hyperpolarized signal amplitude . these observations confirm that 3-substituted pyridines are more suitable candidates for the future rational design of mri contrast agents that exploit sabre . deuterium incorporation into the ethyl groups has also been shown to increase the level of p signal enhancement . the symmetric bis-3 - 5-substituted derivative l7 was also synthesized , which proved to deliver strong p polarization . we have shown that this is a consequence of more efficient transfer into the three remaining pyridyl protons and that this synthetic strategy would enable the most atom efficient route to delivering such a pyridyl substituted agent . as a consequence , for d20-l7 , the corresponding p signal gain exceeds 3500-fold ( 2.3% polarization ) which is an order of magnitude larger than that obtained using other approaches . we also prepared systems where a c , n , or o based spacer was introduced to separate the h and p functionalities . while the h nuclei of the pyridyl groups in these analytes still retained high levels of hyperpolarization , the level of transfer into the p center was reduced by an order of magnitude . we concluded that the addition of a spacer was therefore undesirable . in these studies , the process of sabre was found to result in the creation of two types of p hyperpolarization . these are single - spin zeeman magnetization and longitudinal two - spin heteronuclear h p magnetization . when the corresponding terms were probed by a radio frequency pulse to p they yield in - phase and antiphase signals , respectively . as an in - phase signal is desirable when investigating these hyperpolarized substrates using traditional mri sequences we demonstrated that if the magnetic field experienced by the sample at the point of polarization transfer catalysis is 45 g , optimal in - phase p signal enhancement results under sabre in these systems . interrogation of the resulting samples on a 9.4 t vertical bore scanner proved to allow images to be collected in a single scan at a 5 mm concentration . the snr of a typical image was 94.4 and far exceeds that of a similar 12 h measurement under normal conditions which employed 2048 averages . although the t1 values for these p signals proved to be around 6 s , we have demonstrated that we can obtain good images on phantoms . we are working toward creating further phosphorus containing molecules that have longer t1 values which might ultimately be used for in vivo applications . we expect that the methods of levitt and warren will be particularly relevant here .

traditional 31p nmr or mri measurements suffer from low sensitivity relative to 1h detection and consequently require longer scan times . we show here that hyperpolarization of 31p nuclei through reversible interactions with parahydrogen can deliver substantial signal enhancements in a range of regioisomeric phosphonate esters containing a heteroaromatic motif which were synthesized in order to identify the optimum molecular scaffold for polarization transfer . a 3588-fold 31p signal enhancement ( 2.34% polarization ) was returned for a partially deuterated pyridyl substituted phosphonate ester . this hyperpolarization level is sufficient to allow single scan 31p mr images of a phantom to be recorded at a 9.4 t observation field in seconds that have signal - to - noise ratios of up to 94.4 when the analyte concentration is 10 mm . in contrast , a 12 h 2048 scan measurement under standard conditions yields a signal - to - noise ratio of just 11.4 . 31p - hyperpolarized images are also reported from a 7 t preclinical scanner .

Materials and Methods Results and Discussion Conclusions

dry solvents ( thf , toluene and dcm ) were obtained from a braun mb - sps-800 . deuterated solvents ( methanol - d4 and chlorofrom - d ) were obtained from sigma - aldrich and used as supplied . the following compounds were synthesized according to literature procedures : 1 , d10-diethyl phosphite , 4-pyridyl diphenylphosphine l1 , 4-pyridyl diphenylphosphine oxide l2 , 4-pyridylmethyl diethylphosphonate l8 , 3-pyridylmethyl diethylphosphonate l9 . the resulting solutions were degassed prior to the introduction of p - h2 at a pressure of 3 bar . samples were then shaken for 10 s in a specified fringe field of either a 9.4 t bruker avance ( iii ) nmr spectrometer or a 7 t bruker biospec preclinical mri scanner . they were then rapidly transported into the main magnetic field of the instrument for subsequent probing by nmr or mri methods . historically , n - heteroaromatic molecules have been shown to be excellent receptors for polarization transfer to h and c nuclei under sabre . we therefore synthesized a phosphine ( l1 ) , a phosphine oxide ( l2 ) , a phosphine sulfide ( l3 ) and a phosphonate ester ( l4 ) which incorporated a 4-substituted pyridine motif , as detailed in the supporting information ( figure 1 ) . we selected the 4-substitution pattern for this study in order to minimize any steric interactions between the hyperpolarization target l and the imes ligand in the catalyst 2 . we expected this restriction to promote activity in the widest range of analytes possible . a series of samples were then prepared in methanol - d4 that contained l1-l4 , at a concentration of 5 mm , and the catalyst precursor 1 at a 17% catalyst loading . these samples were probed after sabre had taken place at 298 k on a 400 mhz nmr spectrometer by the application of a simple 90 rf - pulse . for example , the two h nmr signals of l4 , that were derived from the pyridine functionality , showed a 1499-fold total signal enhancement when the initial polarization transfer step was undertaken in a 45 g field . this high h ptc was reflected in the fact that the corresponding p signal exhibited a similarly impressive 545-fold signal enhancement . h and p signal intensity gains for analytes l1-l4 under sabre conditions using 1 as the catalyst . a series of control experiments were then undertaken on triphenylphosphine , triphenylphosphine oxide , and phenyldiethylphosphonate in order to explore the role that the pyridine substituent might play in the hyperpolarization transfer process . these experiments revealed that no polarization transfer took place into the h or p nuclei of these materials under the same sabre conditions as used to hyperpolarize l14 described above . we note that this situation differs from that reported when the related [ ircl(h)2(pph3)3 ] complex was used to successfully polarize triphenylphosphine at temperatures between 333 and 353 k by koptyug et al . in this case however , when 1 reacts with an excess of pph3 a series of new and rapidly relaxing hydride ligand signals are observed at 7.33 , 7.98 , and 8.59 which are indicative of the formation of [ ir(h)2(h2)(imes)(pph3)2]cl . the formation of such species precludes the observation of sabre in these samples because of their role in the rapid quenching of p - h2 ( see the supporting information ) . as a consequence of the formation of 2a - l4 , when the corresponding h nmr spectra are examined under sabre conditions polarized hydride resonances appear as a singlet in the region around 22.15 for the pair of chemically equivalent hydride ligands ( see the supporting information ) . characteristic signals for l4 , as a ligand in 2a - l4 , are also seen to be polarized in the associated p nmr spectra . we illustrate selected regions of these hyperpolarized h and p nmr spectra after ptc at 0.5 g , in figures 2 and 3 respectively to detail some of these effects . in contrast , the corresponding pair of 3,5-ring protons in l4 produce antiphase peaks that are separated by 13.5 hz . this splitting corresponds to the value of jph in l4 and requires that a heteronuclear two - spin order proton - phosphorus term is created through sabre . application of a 90 rf - pulse to the h nuclei of this term creates the visible antiphase signal that is seen ; the creation of homonuclear proton terms of this type is a well - established characteristic of phip . for l4 , sabre enhanced h nmr spectrum ( lower ) and corresponding thermal reference spectrum ( upper , x64 vertical expansion ) of 2a - l4 and l4 ( = equatorial ligand signal , = axial ligand signal of 2a - l4 ) . the hyperpolarized p nmr spectrum of the l4 sample , collected as a single transient , also showed two antiphase peaks , at 14.44 and 13.73 for free and equatorially bound l4 respectively ( figure 3 ) . the signal for free l4 showed a 545-fold increase in size after ptc at 0.5 g relative to the corresponding thermally polarized trace . this difference confirms the creation of a proton - phosphorus two spin order term , which leads to a triplet when excited wherein the central feature has zero intensity ( 1:0 : + 1 ) . the exchange profile seen for axial - l4 suggests that there is some in - cage recombination of this ligand , a process which is not observed for equatorial - l4 ( see the supporting information ) . for p , 5 mm concentrations of 1 with 5 equiv of l7 and 6 of equivalents of l5-l12 were employed at ( a ) 45 g and ( b ) 0.5 g. in order to examine the effect that the regiochemistry of substitution played on the polarization level we replaced the 4- substitution pattern exhibited by l1-l4 with 2- and 3- substitutions in the form of substrates l5 and l6 . polarization transfer into 2-pyridylphosphonate l5 proved to be unsuccessful , and this is due to the formation of a complex that yields a very broad hydride resonance at 31.3 which does not show phip . nonetheless , the presence of this complex in solution provides a pathway to rapidly destroy the p - h2 present , presumably via the formation of a dihydrogen - dihydride complex , with the result that there is very little observable polarization transfer into l5 . the measured h signal enhancement at 45 g for l6 was 2866-fold , and the corresponding p signal enhancement was 336-fold . clearly , the change to a 3-substitution pattern has improved the level of h ptc relative to 4-substitution but surprisingly decreased the efficiency for p transfer . the synthesis of the bis - phosphonate l7 was therefore undertaken , as this material contains two chemically equivalent p nuclei and one fewer pyridyl proton than the monosubstituted derivatives . we speculated that better p - ptc might result as a consequence of this change on the basis that each p - h2 molecule contains a specific amount of latent polarization , and consequently when shared with fewer acceptor sites a net gain in the level of observable transfer might result . the corresponding h and p signal enhancements for l7 were 2271- and 741-fold respectively when 6 equiv of the analyte was employed , and therefore this hypothesis is confirmed . however , one further and somewhat unexpected observation was made , the enhanced steric bulk of l7 results in a change in the active complex such that 2b - l7 results wherein one coordination site is occupied by chloride ( see the supporting information ) . the two hydride signals for this complex appear at 23.74 and 24.06 . because of this change in catalyst form we found that reducing the number of equivalents of l7 to 5 led to an improvement in ptc , giving h and p signal enhancements of 3689- and 860-fold after transfer at 45 and 0.5 g respectively . in order to further test the efficiency of polarization transfer to p in these systems we added a series of spacers between the pyridyl carbon and the phosphorus center . in l8 and l9 this corresponds to the introduction of a ch2 group , while for l10 it is nme and for l11 it is o. these analytes were sabre active , but while the pyridyl ring protons remained strongly enhanced the spacer acted to substantially reduce the levels of p polarization . this change again results in a reduction in the number of proton environments able to accept magnetization . while successful , the levels of both h and p signal enhancement were again lower than those achieved for the pyridylphosphonates . as we have indicated , all of the described p signals that were created through ptc in a 0.5 g appear in antiphase . while this effect can be refocused to produce an in - phase signal , we proposed that changing the polarization transfer field ( ptf ) might achieve a similar result . this would be achieved by changing the relative proportions of single spin ( in - phase ) and two spin ( antiphase ) components and result in a predominantly in - phase signal being obtained . significant in - phase contributions were observed after transfer at 45 g without any significant reduction in the overall p signal enhancement ( 216-fold vs 336-fold for l6 and 703-fold vs 860-fold for l7 at 45 and 0.5 g respectively ) . while ptfs above 45 g did result in an increase in the relative single spin contribution , a sharp decline in the overall signal enhancement was noted . for l6 the relative proportions of the single spin and two spin terms are 0.08:1 and 0.23:1 when the ptf is 0.5 and 45 g respectively . in contrast , when l7 is examined , the relative proportions are 0.06:1 at 0.5 g and 0.88:1 at 45 g respectively . sabre hyperpolarized p nmr traces for l4 [ a ] , l6 [ b ] , and l7 [ c ] that result after polarization transfer catalysis in a 45 or 0.5 g field . further examination of the hyperpolarized h nmr spectra revealed that the signal enhancements described so far also extend into the ethyl groups of the phosphonate . typically these h signal enhancements were 50-fold per ethyl group and reflect a relayed h - p - h transfer process . substrate d20-l7 yielded a p signal gain of 3588 ( 2.3% polarization ) after ptc at 0.5 g. a 2251-fold signal enhancement ( 1.4% polarization ) is , however , achieved after ptc at 45 g with significant in - phase character being visible in the detected signal . we note this is an order of magnitude larger than previously reported for p enhancements using this technique and can be achieved without the need for elevated temperatures during the polarization transfer step . in a further development , we completed magnetization transfer by inserting the sample into a -magnetic shield , which attenuates the earth s field by a factor of 3000-fold , prior to moving it into the 9.4 t magnet for examination . while this created in - phase p magnetization , it would therefore appear that simply changing the polarization transfer field to a readily accessible value between 0.5 and 130 g reflects the optimal route to achieve in - phase signal . hence , we prepared a sample of d20-l7 and 1 in methanol - d4 under 3-bar of h2 and followed it by h nmr for a period of 10 days . [ a]-[c ] thermally polarized p images of l6 , l7 , and d20-l7 , respectively collected over 2048 averages . the repetition time was 20 s. [ d]-[f ] hyperpolarized images of l6 , l7 , and d20-l7 , respectively collected in a single shot after ptc at 45 g. ( te / fov / slice thickness were 3.2 ms/4 4 cm/5 mm respectively ) . [ g ] 2048-average thermal image and [ h ] 1 scan hyperpolarized p image of d20-l7 collected on a 7 t biospec 70/30 preclinical scanner . for d20-l7 they increase to 6.5 s in ethanol - d6 and decrease to 3.6 s in d2o . with a number of these substrates displaying favorable results we were hopeful that it would be possible to collect mri with improved spatial resolution and short acquisition times . a series of hyperpolarized samples were therefore interrogated in a 9.4 t vertical bore scanner using the rapid acquisition with relaxation enhancement ( rare ) pulse sequence . we employed an echo train length of 32 and a matrix size 32 32 with zero filling to 128 128 . this resulted in a total single scan acquisition time of 500 ms . for comparison purposes , thermal images of l6 , l7 , and d20-l7 were recorded with 2048 averages , as shown in figure 6 . under hyperpolarization conditions , the largest gains in signal - to - noise ratio ( snr ) were observed for d20-l7 ( 94.4 ) and reflected a > 8-fold improvement on that of the thermal control ( 11.4 ) . similar images were also recorded on a 7 t biospec 70/30 preclinical scanner using d20-l7 as the imaging agent ( figures 6 g and 6h ) . a > 6-fold improvement in snr was achieved in the single scan hyperpolarized image ( snr = 27.7 ) when compared to the corresponding 2048-average thermal average ( snr = 4.5 ) . a combination of these effects could account for the reduction in observed snr that is observed in the hyperpolarized images that were recorded on the biospec . we have demonstrated that the h and p nuclei of a series of pyridyl substituted phosphonate esters , a phosphine , and a phosphine oxide can be efficiently hyperpolarized using sabre . substitution at the 3-position delivered the greatest efficiency in polarization transfer catalysis to h and p , compared to substitution at the 2- and 4- positions . in the ligands that showed high performance , the phosphorus center was connected directly to the pyridyl ring such that a strong jph coupling of 13.5 hz exists between protons in the pyridyl ring and the p nucleus . when the h and p polarization levels are compared between the protio forms of l4 and l6 and their deuterated ethyl counterparts d10-l4 and d10-l6 , it could be seen that the best levels of h polarization result for three - substituted d10-l6 . however , this raw observation masks the fact that for symmetrical d10-l4 antiphase signal dominates at all monitored polarization transfer fields . 3 s after polarization transfer has ceased and relaxation has reduced the hyperpolarized signal amplitude . deuterium incorporation into the ethyl groups has also been shown to increase the level of p signal enhancement . the symmetric bis-3 - 5-substituted derivative l7 was also synthesized , which proved to deliver strong p polarization . we have shown that this is a consequence of more efficient transfer into the three remaining pyridyl protons and that this synthetic strategy would enable the most atom efficient route to delivering such a pyridyl substituted agent . as a consequence , for d20-l7 , the corresponding p signal gain exceeds 3500-fold ( 2.3% polarization ) which is an order of magnitude larger than that obtained using other approaches . while the h nuclei of the pyridyl groups in these analytes still retained high levels of hyperpolarization , the level of transfer into the p center was reduced by an order of magnitude . we concluded that the addition of a spacer was therefore undesirable . in these studies , the process of sabre was found to result in the creation of two types of p hyperpolarization . when the corresponding terms were probed by a radio frequency pulse to p they yield in - phase and antiphase signals , respectively . as an in - phase signal is desirable when investigating these hyperpolarized substrates using traditional mri sequences we demonstrated that if the magnetic field experienced by the sample at the point of polarization transfer catalysis is 45 g , optimal in - phase p signal enhancement results under sabre in these systems . interrogation of the resulting samples on a 9.4 t vertical bore scanner proved to allow images to be collected in a single scan at a 5 mm concentration . the snr of a typical image was 94.4 and far exceeds that of a similar 12 h measurement under normal conditions which employed 2048 averages . although the t1 values for these p signals proved to be around 6 s , we have demonstrated that we can obtain good images on phantoms . we are working toward creating further phosphorus containing molecules that have longer t1 values which might ultimately be used for in vivo applications .

in august and september 2005 , 29 g. biloba food supplements were sampled in the netherlands . claims for a positive influence on memory and/or concentration or improvement of blood circulation were most frequently mentioned . only four products did n't have any claims on the label ( see table 2 ) . analytical data and claims on products investigated note : na , not analyzed ( method not suitable ) ; & , not present . to determine the degree to which the content of the substances typical for g. biloba were analog to what was declared on the label , the main components of g. biloba , flavone glycosides ( also known as flavonoids ) , and terpene lactones were analyzed . flavone glycosides were analyzed by using high - pressure liquid chromatography ( hplc ) according to the method of mesbah et al . ( 15 ) , but with an extra hydrolysis step added to the pretreatment step . without this hydrolysis step , terpene lactones were analyzed using hplc with refractive index - detection ( ri ) , also based on the method of mesbah et al . ( 15 ) , but with ri - detection instead of the uv - detection , because uv - detection leads to interfering peaks . besluit geneesmiddelen wet ( 18 ) states that the amount of the active substance present in a pharmaceutical product has to be 90110% of the declared amount . to prove the presence of a 24% standardized g. biloba extract in the products , the amount of bilobalide compared to the total amount of terpene lactones was calculated . a ratio of 3959% was used as a reference , corresponding to the ratio used in the european scientific cooperative on phytotherapy ( escop ) monograph ( 19 ) . three commonly encountered claimed health effects for g. biloba were selected : improved blood circulation , effects on symptoms of old age , and improved memory . examples of such claims used are : helps for cold hands and feet ; improves memory ; protects against symptoms of old age ; natural memory booster. per health effect a literature search was conducted in publicly available , objective scientific data : reference and text books and monographs , e.g. escop and who ( 11 , 19);scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . reference and text books and monographs , e.g. escop and who ( 11 , 19 ) ; scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . an overview of the available literature per health effect can be found in table 3 , including not only studies in healthy volunteers ( n=24 ) , but also some important patient studies ( n=11 ) . the passclaim criteria were applied to assess if the health claims can be supported by the totality of the data and weighing of the evidence ( 8) . the criteria are that the product needs to be characterized and administered in amounts consistent with its intended consumption . studies should include representative human study groups , have appropriate controls , an adequate study duration , characterized background diets , and monitor compliance of the study group . the target variable should change in a statistically significant way and the change should be biologically meaningful for the target group . dependent on the quality of the data , per health claim a conclusion was drawn as to the substantiation of the claim with reference to the strengths and weaknesses therein . meta - analyses and systematic reviews gci scale by physician cognition , change from baseline mood and emotional function raven 's progressive matrices visual - auditory learning stroop color word test odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) weschler memory scale - revised : immediate and delayed paragraph recall delayed matching - to - sample test long - term episodic memory frontal lobe function two tests selected from cantab sustained attention digit symbol substitution test speed of comprehension test symbol digit modalities test rey auditory verbal learning test cognometer battery of tests ( simple rt and working memory ) digit span backwards ( p<0.05 ) working memory speed ( p<0.05 ) rey auditory verbal learning test , delay list ( p<0.01 ) these sign changes indicate significant egb761 related improvements specifically in memory processes . sternberg memory scanning test vocabulary and digit span subtests wais - r wechsler adult intelligence scale - revised reading span test prose recall test cognitive abilities testing woodcock - johnson psych - educational battery - revised self - ordered pointing odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) information on toxicity of g. biloba was obtained by searching literature databases ( medline , pubmed , toxline ) , international documentation ( commission e monograph , escop monograph , hager 's handbuch , european medicines agency emea information on animal drugs , cbg college ter beoordeling van geneesmiddelen [ dutch medicines evaluation board ] information on drugs , who , herbalgram information , national toxicology program ntp ) , and the internet . in the commission e monograph , escop monograph , and hager 's handbuch , toxicity of the standardized extract egb 761 is described . the toxicity evaluation in the current evaluation is based on those reports as well as recent ntp studies and recent literature , predominantly case reports , and reports on interaction with frequently / commonly used medication . in addition to g. biloba and the gb - ste , data on gas were considered for evaluation . because of their toxic properties , the presence of gas is limited in standardized g. biloba preparations to maximum 5 ppm ( 11 ) . however , in non - standardized g. biloba preparations , gas can be present at higher levels . in august and september 2005 , 29 g. biloba food supplements were sampled in the netherlands . claims for a positive influence on memory and/or concentration or improvement of blood circulation were most frequently mentioned . only four products did n't have any claims on the label ( see table 2 ) . analytical data and claims on products investigated note : na , not analyzed ( method not suitable ) ; & , not present . to determine the degree to which the content of the substances typical for g. biloba were analog to what was declared on the label , the main components of g. biloba , flavone glycosides ( also known as flavonoids ) , and terpene lactones were analyzed . flavone glycosides were analyzed by using high - pressure liquid chromatography ( hplc ) according to the method of mesbah et al . ( 15 ) , but with an extra hydrolysis step added to the pretreatment step . without this hydrolysis step , terpene lactones were analyzed using hplc with refractive index - detection ( ri ) , also based on the method of mesbah et al . ( 15 ) , but with ri - detection instead of the uv - detection , because uv - detection leads to interfering peaks . besluit geneesmiddelen wet ( 18 ) states that the amount of the active substance present in a pharmaceutical product has to be 90110% of the declared amount . to prove the presence of a 24% standardized g. biloba extract in the products , the amount of bilobalide compared to the total amount of terpene lactones was calculated . a ratio of 3959% was used as a reference , corresponding to the ratio used in the european scientific cooperative on phytotherapy ( escop ) monograph ( 19 ) . three commonly encountered claimed health effects for g. biloba were selected : improved blood circulation , effects on symptoms of old age , and improved memory . examples of such claims used are : helps for cold hands and feet ; improves memory ; protects against symptoms of old age ; natural memory booster. per health effect a literature search was conducted in publicly available , objective scientific data : reference and text books and monographs , e.g. escop and who ( 11 , 19);scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . reference and text books and monographs , e.g. escop and who ( 11 , 19 ) ; scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . an overview of the available literature per health effect can be found in table 3 , including not only studies in healthy volunteers ( n=24 ) , but also some important patient studies ( n=11 ) . the passclaim criteria were applied to assess if the health claims can be supported by the totality of the data and weighing of the evidence ( 8) . the criteria are that the product needs to be characterized and administered in amounts consistent with its intended consumption . studies should include representative human study groups , have appropriate controls , an adequate study duration , characterized background diets , and monitor compliance of the study group . the target variable should change in a statistically significant way and the change should be biologically meaningful for the target group . dependent on the quality of the data , per health claim a conclusion was drawn as to the substantiation of the claim with reference to the strengths and weaknesses therein . meta - analyses and systematic reviews gci scale by physician cognition , change from baseline mood and emotional function raven 's progressive matrices visual - auditory learning stroop color word test odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) weschler memory scale - revised : immediate and delayed paragraph recall delayed matching - to - sample test long - term episodic memory frontal lobe function two tests selected from cantab sustained attention digit symbol substitution test speed of comprehension test symbol digit modalities test rey auditory verbal learning test cognometer battery of tests ( simple rt and working memory ) digit span backwards ( p<0.05 ) working memory speed ( p<0.05 ) rey auditory verbal learning test , delay list ( p<0.01 ) these sign changes indicate significant egb761 related improvements specifically in memory processes . sternberg memory scanning test vocabulary and digit span subtests wais - r wechsler adult intelligence scale - revised reading span test prose recall test cognitive abilities testing woodcock - johnson psych - educational battery - revised self - ordered pointing odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) information on toxicity of g. biloba was obtained by searching literature databases ( medline , pubmed , toxline ) , international documentation ( commission e monograph , escop monograph , hager 's handbuch , european medicines agency emea information on animal drugs , cbg college ter beoordeling van geneesmiddelen [ dutch medicines evaluation board ] information on drugs , who , herbalgram information , national toxicology program ntp ) , and the internet . in the commission e monograph , escop monograph , and hager 's handbuch , toxicity of the standardized extract egb 761 is described . the toxicity evaluation in the current evaluation is based on those reports as well as recent ntp studies and recent literature , predominantly case reports , and reports on interaction with frequently / commonly used medication . in addition to g. biloba and the gb - ste , data on gas were considered for evaluation . because of their toxic properties , the presence of gas is limited in standardized g. biloba preparations to maximum 5 ppm ( 11 ) . however , in non - standardized g. biloba preparations , gas can be present at higher levels . for 16 products , both the detailed content and the recommended dose were declared on the label ( see table 2 ) . for 13 products , the declaration was unclear , e.g. the recommended dose was stated , but the amount of flavonoids and terpene lactones in this dose was not stated in the declaration . only two of the 29 products met the pharmaceutical guideline ( the amount present in the product is 90110% of the declared amount ) for terpene lactones . for flavonoids , only four products met the criteria of a ratio of 3959% ( the amount of bilobalide compared to the total amount of terpene lactones ) . based on the daily dose and the concentration of terpene lactones and flavonoids found in the products , a dose range of substances typical for g. biloba was calculated ; 16 products did not meet the dose range for terpene lactones and flavonoids for a 24% standardized extract . in summary , when the components of herbal supplements were evaluated according to the guideline for pharmaceuticals , that require the presence of 90110% of the declared amount , one out of 29 g. biloba products meets the guideline.1 the amount of terpene lactones and flavonoids found vary between 27358% for terpene lactones and between 86418% for flavonoids . the ( combined ) passclaim criteria were checked per health effect , for the selected studies ( see table 3 ) . the final step of the assessment , passclaim criterion 6 , weighing of the evidence , is neither worked out in detail in passclaim ( 8) , nor in the efsa guidance document ( 7 ) . in practice , this is done now by efsa on a case - by - case basis ( 2 ) . in our assessment we have used the who criteria ( 20 ) , which categorizes data into convincing evidence ( consistent results from adequately powered , randomized placebo - controlled trials of the claimed substance in human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim ) , probable evidence ( consistent results from small randomized placebo - controlled trials of the claimed substance or active ingredients contained therein in sufficient numbers of human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim and/or human epidemiological studies ) , possible ( or supporting ) evidence ( consistent results from animal or in vitro studies of the claimed substance or active ingredients contained therein and assessing endpoints relevant to the claim ) and insufficient evidence ( inconsistent results and/or studies which do not consider the substance which is the subject of the claim and/or the target population and/or endpoints relevant to the claim ) . a detailed description and comments on individual meta - analyses , systematic reviews , and individual studies are given in table 3 . treatment of the disease claudication intermittens is one of the best - known uses of g. biloba . there is a registered medicine on the market containing g. biloba , tavonin , which is used for patients that do n't respond ( enough ) to walking exercise ( 16 ) . in patients with claudicatio intermittens , a positive effect at a dose of 120 mg / day was seen in two meta - analyses ( 21 , 22 ) . however , a recent systematic review that included 739 patients from 14 trials ( with different dosage and duration of treatment ) did n't find a significant effect on walking distance in people with claudication intermittens ( 23 ) . such clinical studies , however , may not be considered pertinent to a claim for the general population . it is an inaccurate expression that is often used to describe a collection of symptoms associated with dementia ( 11 ) or the effects of reduced cerebral blood flow in the elderly ( 24 ) . for the purpose of this evaluation , therefore , cerebrovascular insufficiency has been considered a disease . a systematic review by kleijnen and knipschild including patients with mild to moderate cerebrovascular insufficiency showed that treatment with 112160 mg / day g. biloba for 46 weeks was effective for cerebrovascular insufficiency ( 24 ) . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . santos et al . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . another study by the same group also described a decreased blood viscosity ( measured using wells - brookfield cone / plate viscometer dv - i ) in 25 adult men taking 80 mg gb - ste a day ( 26 ) . a randomized double blind placebo - controlled trial with 240 mg / day egb761 for 3 weeks in 27 older subjects found a vasoregulation role of g. biloba ( 27 ) . unfortunately , the studies described above included different blood flow parameters , therefore , comparison of the studies is complicated . we concluded there was insufficient evidence that gb - ste use results in improved blood circulation in healthy subjects . to enable scientific evaluation , a claim must include in the wording some reference to the target population and the health effect . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . if submitted through article 13.5 , the dossier could be returned to the applicants who in some cases would be afforded the opportunity to re - word the claim to enable evaluation subsequent to re - submission . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . most studies of g. biloba and aging have considered cognitive endpoints in subjects with mild impairment ( 13 ) or alzheimer and dementia patients ( 2831 ) with inconsistent results for a beneficial effect of g. biloba . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . a large trial including 262 healthy subjects aged 60 years and over , observed improved performance on validated memory tests with gb - ste ( 180 mg / day , 6 weeks ) ( 34 ) . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . the observed effect , however , was weak and disappeared when other variables were controlled . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . a double blind controlled study showed no effect of gb - ste 120 mg / day on the memory of healthy young males ( n=104 ) ( 41 ) . in conclusion , there is insufficient evidence to substantiate the claim that g. biloba can improve memory in healthy subjects . in the open literature there are no ( animal ) data on acute toxicity , carcinogenicity , reproduction toxicity , teratogenicity , neurotoxicity or immunotoxicity , but information on these toxicological endpoints are available in the commission e monograph , escop monograph , the emea report , and a recent ntp study ( 10 , 19 , 42 ) . however , this should be interpreted with care as g. biloba is an herbal preparation consisting of many components , which may show interactions . indeed , most reports on kinetics focus on known ingredients of g. biloba such as ginkgolides a and b and bilobalide . for gb - ste , low acute toxicity has been reported for rodents with ld50 values ranging from 1,100 ( intravenous , rats and mice ) to 7,725 mg / kg body weight ( bw ) ( oral , mice ) . a 13-week gavage study in mice and rats with gb - ste ( egb761 dose levels 0 , 65.5 [ rats only ] , 125 , 250 , 500 , 1,000 ( rats and mice ) , and 2,000 ( mice only ) mg / kg bw per day ) showed an increase in liver weight at all dose levels tested , and a dose - related increase in hepatocyte hypertrophy in male and female mice at and above 250 mg / kg bw and in male rats at all dose levels . in oral studies with rats and dogs during 6 months , a dose- and time - related mild temporary vasodilatation in cranial blood vessels was observed in dogs dosed at and above 100 mg gb - ste / kg bw from 35 days on ( 19 ) . although gb - ste was reported not mutagenic in ( among other tests ) a ames test by commission e 1994 and escop 2003 , recently ntp ( 43 ) reported a positive ames test . an in vivo micronucleus assay was negative for male mice , whereas for female mice the results were equivocal . escop ( 19 ) reported no carcinogenic effects in a 2-year study in rats at dose levels of 4 , 20 , and 100 mg / kg bw . g / kg bw day ) and rabbits ( up to 0.9 g / kg bw day ) did not show embryotoxic , teratogenic or reproduction toxicity effects ( 19 ) . gb - ste also contains gas , to which immunotoxic , cytotoxic , mutagenic , and carcinogenic properties are ascribed ( 4448 ) . as a consequence , a maximum level for the presence of gas in the gb - ste was set at 5 mg / kg ( 11 ) . however , as not all g. biloba preparations included in the present study contain gb - ste , maximum levels of 5 mg / kg are not guaranteed , which may pose a health risk ( 49 ) . indeed , chiu et al . ( 50 ) report higher levels ( 16733 times ) than 5 ppm in 13 of 14 g. biloba preparations . reportedly , g. biloba is generally well tolerated by human ( 19 , 51 ) , albeit that some mild side effects have been reported [ gastrointestinal complaints , headache , allergic skin reactions , nausea , dizziness , restlessness , heart palpitation , and weakness ( 16 , 22 , 51 , 52 ) ] . in addition to these mild effects , in several case studies side effects have been reported related to blood platelets , hemorrhage , and blood coagulation , which is in agreement with the paf - inhibiting properties of g. biloba ( 5361 ) . in these case reports , reference is made to a ginkgo extract or a ginkgo containing preparation, without further specifications of the g. biloba preparation used . the duration and dose of intake of g. biloba prior to the reported paf - related side effects ranges from 2 weeks to 2 years , respectively , 80160 mg / day , well within the recommended dose range . as for case studies on effects on bleeding , available data on interactions with drugs are also mainly associated with drugs involved in the treatment of blood coagulation and bleeding ( 53 , 6264 ) . the escop monograph ( 19 ) reports that there is no clear evidence for gb - ste on blood coagulation ( alone or in combination with medicines ) . however , various case reports of interactions with drugs have been reported ( 16 , 53 , 54 , 6571 ) . interactions of herbal preparations with drugs are particularly relevant for drugs with a narrow therapeutic margin , it can result in more or stronger side effects or effect on the bioavailability of the drug ( 72 , 73 ) . although a direct causal relationship between g. biloba intake and observed effects in case studies is lacking ( 74 ) , the absence of other risk factors , the paf - inhibiting activity of g. biloba , and the disappearance of effects upon cessation of intake suggest such causality ( 5961 ) . the who has indicated to be careful with the use of g. biloba in combination with drugs that affect blood coagulation or platelet aggregation and to stop g. biloba use before a diagnostic treatment or an operation ( 49 , 75 ) . summarizing , the potential safety issues associated with g. biloba are severalfold . first , in most literature reports on g. biloba , the preparation used is not specified . animal toxicity studies typically were performed with gb - ste , whereas in case studies no specification on the g. biloba preparation is given . second , because of the immunotoxic , cytotoxic , carcinogenic , and genotoxic potential of gas , the maximum level of gas in gb - ste is limited to 5 ppm . however , if a preparation of g. biloba does not contain the gb - ste , a higher level of gas can not be excluded . finally , g. biloba has paf - antagonist activity , which may result in effects on platelet aggregation and blood coagulation . indeed , there are various case studies reporting hemorrhages with intake of g. biloba preparations alone or in combination with drugs affecting platelet aggregation and blood coagulation . moreover , the dose levels of case reports are within the range of recommended dose levels . nevertheless , exact dose levels are unknown . as a result of abovementioned available data , there was insufficient evidence to substantiate the three selected health claims for g. biloba , when applying the passclaim criteria . first , a clear definition of what sort of study sample constitutes a healthy target population is lacking . asp and bryngelsson ( 3 ) concluded that passclaim is useful for applicants for health claims and for the agencies that evaluate the evidence . since herbal food supplements are intended for healthy persons , health claims for herbal food supplements have to be proven in studies performed with healthy persons who are representative of the target population . however , distinction between healthy , complaint and illness is not always clear ( definition of a healthy person is not clear ) . for this analysis we excluded patient studies , which left only a small number of available studies per health effect . secondly , it is unclear to which degree a herbal supplement of an applicant should be identical to the product used in the human studies . should they be exactly the same , or should they contain the same standardized extract ? this study showed that most studies are performed reportedly using standardized g. biloba extract , but also that nearly every product found in the dutch shops was not in conformity with the label declaration . when a claim can be substantiated using those studies , the product that sets the claim should resemble the standardized extract . thirdly , the passclaim criteria 4 and 5 require consideration of specific markers , or endpoints , depending on the type of claim made . finally , the procedure for weighing the evidence was not described in detail in the passclaim project . in contrast to the usa , where qualified health claims are allowed ( 77 ) , the eu regulation 1924/2006 was not designed for supporting qualified health claims . however , qualification of the evidence according to the who criteria has proved useful for scientific purposes . the value of such criteria in consumer communication has not yet been established . indeed , in the usa experience , consumers do not make the anticipated distinction between substantiated and not substantiated ( = qualified ) health claims ( 2 , 7780 ) . hence the need for interdisciplinary groups of expert scientists to monitor and evaluate such claims . firstly , there was no case with product - specific information , delivered by a manufacturer , to evaluate . it is possible that information is missing , e.g. information from negative or null trials or unpublished proprietary or confidential studies . in future , efsa evaluations will endeavor to include all available information , including confidential data , for the substantiation of article 14 and 13.5 claims . secondly , regarding the g. biloba products that were included in this study , the sampling of these products was random from dutch shops . that the search was not exhaustive leaves the possibility that there were products available for purchase which were not analyzed and which contained at least the minimum amount of g. biloba extract . the finding that the standardized 24% of g. biloba extract is often not incorporated into the food supplements , nevertheless , raises doubts as to the efficacy of such products . this analysis has also implied that it is not possible to establish a safe level for intake of g. biloba . review of the science provided insufficient evidence with which to substantiate the three claims for g. biloba . in conclusion , the claim g. biloba and improvement in the symptoms of old age could not be evaluated . a cause and effect relationship between g. biloba and improved circulation or memory has not been established . for 16 products , both the detailed content and the recommended dose were declared on the label ( see table 2 ) . for 13 products , the declaration was unclear , e.g. the recommended dose was stated , but the amount of flavonoids and terpene lactones in this dose was not stated in the declaration . only two of the 29 products met the pharmaceutical guideline ( the amount present in the product is 90110% of the declared amount ) for terpene lactones . for flavonoids , only four products met the criteria of a ratio of 3959% ( the amount of bilobalide compared to the total amount of terpene lactones ) . based on the daily dose and the concentration of terpene lactones and flavonoids found in the products , a dose range of substances typical for g. biloba was calculated ; 16 products did not meet the dose range for terpene lactones and flavonoids for a 24% standardized extract . in summary , when the components of herbal supplements were evaluated according to the guideline for pharmaceuticals , that require the presence of 90110% of the declared amount , one out of 29 g. biloba products meets the guideline.1 the amount of terpene lactones and flavonoids found vary between 27358% for terpene lactones and between 86418% for flavonoids . the ( combined ) passclaim criteria were checked per health effect , for the selected studies ( see table 3 ) . the final step of the assessment , passclaim criterion 6 , weighing of the evidence , is neither worked out in detail in passclaim ( 8) , nor in the efsa guidance document ( 7 ) . in practice , this is done now by efsa on a case - by - case basis ( 2 ) . in our assessment we have used the who criteria ( 20 ) , which categorizes data into convincing evidence ( consistent results from adequately powered , randomized placebo - controlled trials of the claimed substance in human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim ) , probable evidence ( consistent results from small randomized placebo - controlled trials of the claimed substance or active ingredients contained therein in sufficient numbers of human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim and/or human epidemiological studies ) , possible ( or supporting ) evidence ( consistent results from animal or in vitro studies of the claimed substance or active ingredients contained therein and assessing endpoints relevant to the claim ) and insufficient evidence ( inconsistent results and/or studies which do not consider the substance which is the subject of the claim and/or the target population and/or endpoints relevant to the claim ) . a detailed description and comments on individual meta - analyses , systematic reviews , and individual studies are given in table 3 . treatment of the disease claudication intermittens is one of the best - known uses of g. biloba . there is a registered medicine on the market containing g. biloba , tavonin , which is used for patients that do n't respond ( enough ) to walking exercise ( 16 ) . in patients with claudicatio intermittens , a positive effect at a dose of 120 mg / day was seen in two meta - analyses ( 21 , 22 ) . however , a recent systematic review that included 739 patients from 14 trials ( with different dosage and duration of treatment ) did n't find a significant effect on walking distance in people with claudication intermittens ( 23 ) . such clinical studies , however , may not be considered pertinent to a claim for the general population . it is an inaccurate expression that is often used to describe a collection of symptoms associated with dementia ( 11 ) or the effects of reduced cerebral blood flow in the elderly ( 24 ) . for the purpose of this evaluation , therefore , cerebrovascular insufficiency has been considered a disease . a systematic review by kleijnen and knipschild including patients with mild to moderate cerebrovascular insufficiency showed that treatment with 112160 mg / day g. biloba for 46 weeks was effective for cerebrovascular insufficiency ( 24 ) . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . santos et al . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . another study by the same group also described a decreased blood viscosity ( measured using wells - brookfield cone / plate viscometer dv - i ) in 25 adult men taking 80 mg gb - ste a day ( 26 ) . a randomized double blind placebo - controlled trial with 240 mg / day egb761 for 3 weeks in 27 older subjects found a vasoregulation role of g. biloba ( 27 ) . unfortunately , the studies described above included different blood flow parameters , therefore , comparison of the studies is complicated . we concluded there was insufficient evidence that gb - ste use results in improved blood circulation in healthy subjects . to enable scientific evaluation , a claim must include in the wording some reference to the target population and the health effect . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . if submitted through article 13.5 , the dossier could be returned to the applicants who in some cases would be afforded the opportunity to re - word the claim to enable evaluation subsequent to re - submission . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . most studies of g. biloba and aging have considered cognitive endpoints in subjects with mild impairment ( 13 ) or alzheimer and dementia patients ( 2831 ) with inconsistent results for a beneficial effect of g. biloba . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . a large trial including 262 healthy subjects aged 60 years and over , observed improved performance on validated memory tests with gb - ste ( 180 mg / day , 6 weeks ) ( 34 ) . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . the observed effect , however , was weak and disappeared when other variables were controlled . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . a double blind controlled study showed no effect of gb - ste 120 mg / day on the memory of healthy young males ( n=104 ) ( 41 ) . in conclusion , there is insufficient evidence to substantiate the claim that g. biloba can improve memory in healthy subjects . treatment of the disease claudication intermittens is one of the best - known uses of g. biloba . there is a registered medicine on the market containing g. biloba , tavonin , which is used for patients that do n't respond ( enough ) to walking exercise ( 16 ) . in patients with claudicatio intermittens , a positive effect at a dose of 120 mg / day was seen in two meta - analyses ( 21 , 22 ) . however , a recent systematic review that included 739 patients from 14 trials ( with different dosage and duration of treatment ) did n't find a significant effect on walking distance in people with claudication intermittens ( 23 ) . such clinical studies , however , may not be considered pertinent to a claim for the general population . it is an inaccurate expression that is often used to describe a collection of symptoms associated with dementia ( 11 ) or the effects of reduced cerebral blood flow in the elderly ( 24 ) . for the purpose of this evaluation , therefore , cerebrovascular insufficiency has been considered a disease . a systematic review by kleijnen and knipschild including patients with mild to moderate cerebrovascular insufficiency showed that treatment with 112160 mg / day g. biloba for 46 weeks was effective for cerebrovascular insufficiency ( 24 ) . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . santos et al . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . another study by the same group also described a decreased blood viscosity ( measured using wells - brookfield cone / plate viscometer dv - i ) in 25 adult men taking 80 mg gb - ste a day ( 26 ) . a randomized double blind placebo - controlled trial with 240 mg / day egb761 for 3 weeks in 27 older subjects found a vasoregulation role of g. biloba ( 27 ) . unfortunately , the studies described above included different blood flow parameters , therefore , comparison of the studies is complicated . we concluded there was insufficient evidence that gb - ste use results in improved blood circulation in healthy subjects . to enable scientific evaluation , a claim must include in the wording some reference to the target population and the health effect . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . if submitted through article 13.5 , the dossier could be returned to the applicants who in some cases would be afforded the opportunity to re - word the claim to enable evaluation subsequent to re - submission . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . most studies of g. biloba and aging have considered cognitive endpoints in subjects with mild impairment ( 13 ) or alzheimer and dementia patients ( 2831 ) with inconsistent results for a beneficial effect of g. biloba . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . a large trial including 262 healthy subjects aged 60 years and over , observed improved performance on validated memory tests with gb - ste ( 180 mg / day , 6 weeks ) ( 34 ) . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . the observed effect , however , was weak and disappeared when other variables were controlled . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . a double blind controlled study showed no effect of gb - ste 120 mg / day on the memory of healthy young males ( n=104 ) ( 41 ) . in conclusion , there is insufficient evidence to substantiate the claim that g. biloba can improve memory in healthy subjects . in the open literature there are no ( animal ) data on acute toxicity , carcinogenicity , reproduction toxicity , teratogenicity , neurotoxicity or immunotoxicity , but information on these toxicological endpoints are available in the commission e monograph , escop monograph , the emea report , and a recent ntp study ( 10 , 19 , 42 ) . however , this should be interpreted with care as g. biloba is an herbal preparation consisting of many components , which may show interactions . indeed , most reports on kinetics focus on known ingredients of g. biloba such as ginkgolides a and b and bilobalide . for gb - ste , low acute toxicity has been reported for rodents with ld50 values ranging from 1,100 ( intravenous , rats and mice ) to 7,725 mg / kg body weight ( bw ) ( oral , mice ) . a 13-week gavage study in mice and rats with gb - ste ( egb761 dose levels 0 , 65.5 [ rats only ] , 125 , 250 , 500 , 1,000 ( rats and mice ) , and 2,000 ( mice only ) mg / kg bw per day ) showed an increase in liver weight at all dose levels tested , and a dose - related increase in hepatocyte hypertrophy in male and female mice at and above 250 mg / kg bw and in male rats at all dose levels . in oral studies with rats and dogs during 6 months , a dose- and time - related mild temporary vasodilatation in cranial blood vessels was observed in dogs dosed at and above 100 mg gb - ste / kg bw from 35 days on ( 19 ) . although gb - ste was reported not mutagenic in ( among other tests ) a ames test by commission e 1994 and escop 2003 , recently ntp ( 43 ) reported a positive ames test . an in vivo micronucleus assay was negative for male mice , whereas for female mice the results were equivocal . escop ( 19 ) reported no carcinogenic effects in a 2-year study in rats at dose levels of 4 , 20 , and 100 mg / kg bw . g / kg bw day ) and rabbits ( up to 0.9 g / kg bw day ) did not show embryotoxic , teratogenic or reproduction toxicity effects ( 19 ) . gb - ste also contains gas , to which immunotoxic , cytotoxic , mutagenic , and carcinogenic properties are ascribed ( 4448 ) . as a consequence , a maximum level for the presence of gas in the gb - ste was set at 5 mg / kg ( 11 ) . however , as not all g. biloba preparations included in the present study contain gb - ste , maximum levels of 5 mg / kg are not guaranteed , which may pose a health risk ( 49 ) . ( 50 ) report higher levels ( 16733 times ) than 5 ppm in 13 of 14 g. biloba preparations . reportedly , g. biloba is generally well tolerated by human ( 19 , 51 ) , albeit that some mild side effects have been reported [ gastrointestinal complaints , headache , allergic skin reactions , nausea , dizziness , restlessness , heart palpitation , and weakness ( 16 , 22 , 51 , 52 ) ] . in addition to these mild effects , in several case studies side effects have been reported related to blood platelets , hemorrhage , and blood coagulation , which is in agreement with the paf - inhibiting properties of g. biloba ( 5361 ) . in these case reports , reference is made to a ginkgo extract or a ginkgo containing preparation, without further specifications of the g. biloba preparation used . the duration and dose of intake of g. biloba prior to the reported paf - related side effects ranges from 2 weeks to 2 years , respectively , 80160 mg / day , well within the data on interactions with drugs are also mainly associated with drugs involved in the treatment of blood coagulation and bleeding ( 53 , 6264 ) . the escop monograph ( 19 ) reports that there is no clear evidence for gb - ste on blood coagulation ( alone or in combination with medicines ) . however , various case reports of interactions with drugs have been reported ( 16 , 53 , 54 , 6571 ) . interactions of herbal preparations with drugs are particularly relevant for drugs with a narrow therapeutic margin , it can result in more or stronger side effects or effect on the bioavailability of the drug ( 72 , 73 ) . although a direct causal relationship between g. biloba intake and observed effects in case studies is lacking ( 74 ) , the absence of other risk factors , the paf - inhibiting activity of g. biloba , and the disappearance of effects upon cessation of intake suggest such causality ( 5961 ) . the who has indicated to be careful with the use of g. biloba in combination with drugs that affect blood coagulation or platelet aggregation and to stop g. biloba use before a diagnostic treatment or an operation ( 49 , 75 ) . summarizing , the potential safety issues associated with g. biloba are severalfold . first , in most literature reports on g. biloba , the preparation used is not specified . animal toxicity studies typically were performed with gb - ste , whereas in case studies no specification on the g. biloba preparation is given . second , because of the immunotoxic , cytotoxic , carcinogenic , and genotoxic potential of gas , the maximum level of gas in gb - ste is limited to 5 ppm . however , if a preparation of g. biloba does not contain the gb - ste , a higher level of gas can not be excluded . finally , g. biloba has paf - antagonist activity , which may result in effects on platelet aggregation and blood coagulation . indeed , there are various case studies reporting hemorrhages with intake of g. biloba preparations alone or in combination with drugs affecting platelet aggregation and blood coagulation . moreover , the dose levels of case reports are within the range of recommended dose levels . nevertheless , exact dose levels are unknown . as a result of abovementioned available data , there was insufficient evidence to substantiate the three selected health claims for g. biloba , when applying the passclaim criteria first , a clear definition of what sort of study sample constitutes a healthy target population is lacking . asp and bryngelsson ( 3 ) concluded that passclaim is useful for applicants for health claims and for the agencies that evaluate the evidence . since herbal food supplements are intended for healthy persons , health claims for herbal food supplements have to be proven in studies performed with healthy persons who are representative of the target population . however , distinction between healthy , complaint and illness is not always clear ( definition of a healthy person is not clear ) . for this analysis we excluded patient studies , which left only a small number of available studies per health effect . secondly , it is unclear to which degree a herbal supplement of an applicant should be identical to the product used in the human studies . should they be exactly the same , or should they contain the same standardized extract ? this study showed that most studies are performed reportedly using standardized g. biloba extract , but also that nearly every product found in the dutch shops was not in conformity with the label declaration . when a claim can be substantiated using those studies , the product that sets the claim should resemble the standardized extract . thirdly , the passclaim criteria 4 and 5 require consideration of specific markers , or endpoints , depending on the type of claim made . finally , the procedure for weighing the evidence was not described in detail in the passclaim project . in contrast to the usa , where qualified health claims are allowed ( 77 ) , the eu regulation 1924/2006 was not designed for supporting qualified health claims . however , qualification of the evidence according to the who criteria has proved useful for scientific purposes . the value of such criteria in consumer communication has not yet been established . indeed , in the usa experience , consumers do not make the anticipated distinction between substantiated and not substantiated ( = qualified ) health claims ( 2 , 7780 ) . hence the need for interdisciplinary groups of expert scientists to monitor and evaluate such claims . firstly , there was no case with product - specific information , delivered by a manufacturer , to evaluate . it is possible that information is missing , e.g. information from negative or null trials or unpublished proprietary or confidential studies . in future , efsa evaluations will endeavor to include all available information , including confidential data , for the substantiation of article 14 and 13.5 claims . secondly , regarding the g. biloba products that were included in this study , the sampling of these products was random from dutch shops . that the search was not exhaustive leaves the possibility that there were products available for purchase which were not analyzed and which contained at least the minimum amount of g. biloba extract . the finding that the standardized 24% of g. biloba extract is often not incorporated into the food supplements , nevertheless , raises doubts as to the efficacy of such products . this analysis has also implied that it is not possible to establish a safe level for intake of g. biloba . review of the science provided insufficient evidence with which to substantiate the three claims for g. biloba . in conclusion , the claim g. biloba and improvement in the symptoms of old age could not be evaluated . a cause and effect relationship between g. biloba and improved circulation or memory has not been established . the main conclusions in this study were : three selected health claims for g. biloba could not be substantiated.neither safety nor efficacy can be guaranteed at the recommended daily dose.the content of g. biloba containing products often did not conform to what was declared on the label.a multidisciplinary approach is needed to get insight in the relevant issues for health claim evaluation.moreover , the passclaim criteria need to be developed in more detail when used for general application on health claim substantiation for herbal supplements and there is also a need for clarification of the study conditions that are required for claim substantiation , especially with respect to patient studies . a multidisciplinary approach is recommended to assess health claims : analyses of substances typical for g. biloba in the products and toxicological risk assessment are needed for a full assessment . this provides a good insight into the several aspects that are significant for the evaluation of health claims for herbal substances . the content of g. biloba containing products often did not conform to what was declared on the label . a multidisciplinary approach is needed to get insight in the relevant issues for health claim evaluation . the authors have not received any funding or benefits from industry to conduct this study .

backgroundeuropean regulation 1924/2006 states that all health claims made on foods need to be substantiated scientifically.objectiveto apply the passclaim criteria for the scientific substantiation of health claims on foods to herbal supplements containing ginkgo biloba . evaluation of three selected claimed health effects for g. biloba ( improvement of blood circulation , improvement of symptoms of old age , and improvement of memory ) was achieved through review of publicly available scientific data . a total of 35 human intervention studies were evaluated . commercially available products claimed to contain mainly g. biloba ( n=29 ) were randomly sampled in the netherlands and analyzed for their content on ginkgo extract . also , a toxicological risk assessment was performed.resultsthe three selected health claims investigated could not be substantiated . this was mainly because of a lack of data from studies in healthy volunteers . in most studies results performed with a 24% standardized g. biloba extract were described . however , our chemical analysis showed that 25 of the 29 sampled products did not contain the required minimum 24% standardized extract . moreover , in most preparations the content of substances typical for g. biloba did not conform to what was declared on the label . since toxicity data for g. biloba are very limited , a safety limit could not be established.conclusionsevidence is lacking for three health claims of herbal products with g. biloba . neither safety nor efficacy can be guaranteed at the recommended daily dose . the multidisciplinary approach described in this paper provides good insight into issues that are relevant for the evaluation of health claims for herbal food supplements .

Methods Product analysis Health claim substantiation Toxicity Results and discussion Product analysis Health claim substantiation Health effect 1: improved blood circulation Health effect 2: improvement of symptoms of old age Health effect 3: improved memory Toxicity Human studies Applicability of the PASSCLAIM criteria Limitations of the study Conclusions and recommendations Conflict of interest and funding

in august and september 2005 , 29 g. biloba food supplements were sampled in the netherlands . claims for a positive influence on memory and/or concentration or improvement of blood circulation were most frequently mentioned . only four products did n't have any claims on the label ( see table 2 ) . to determine the degree to which the content of the substances typical for g. biloba were analog to what was declared on the label , the main components of g. biloba , flavone glycosides ( also known as flavonoids ) , and terpene lactones were analyzed . to prove the presence of a 24% standardized g. biloba extract in the products , the amount of bilobalide compared to the total amount of terpene lactones was calculated . three commonly encountered claimed health effects for g. biloba were selected : improved blood circulation , effects on symptoms of old age , and improved memory . examples of such claims used are : helps for cold hands and feet ; improves memory ; protects against symptoms of old age ; natural memory booster. search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . an overview of the available literature per health effect can be found in table 3 , including not only studies in healthy volunteers ( n=24 ) , but also some important patient studies ( n=11 ) . the passclaim criteria were applied to assess if the health claims can be supported by the totality of the data and weighing of the evidence ( 8) . dependent on the quality of the data , per health claim a conclusion was drawn as to the substantiation of the claim with reference to the strengths and weaknesses therein . in the commission e monograph , escop monograph , and hager 's handbuch , toxicity of the standardized extract egb 761 is described . because of their toxic properties , the presence of gas is limited in standardized g. biloba preparations to maximum 5 ppm ( 11 ) . however , in non - standardized g. biloba preparations , gas can be present at higher levels . in august and september 2005 , 29 g. biloba food supplements were sampled in the netherlands . claims for a positive influence on memory and/or concentration or improvement of blood circulation were most frequently mentioned . only four products did n't have any claims on the label ( see table 2 ) . to determine the degree to which the content of the substances typical for g. biloba were analog to what was declared on the label , the main components of g. biloba , flavone glycosides ( also known as flavonoids ) , and terpene lactones were analyzed . besluit geneesmiddelen wet ( 18 ) states that the amount of the active substance present in a pharmaceutical product has to be 90110% of the declared amount . to prove the presence of a 24% standardized g. biloba extract in the products , the amount of bilobalide compared to the total amount of terpene lactones was calculated . three commonly encountered claimed health effects for g. biloba were selected : improved blood circulation , effects on symptoms of old age , and improved memory . examples of such claims used are : helps for cold hands and feet ; improves memory ; protects against symptoms of old age ; natural memory booster. search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . an overview of the available literature per health effect can be found in table 3 , including not only studies in healthy volunteers ( n=24 ) , but also some important patient studies ( n=11 ) . the passclaim criteria were applied to assess if the health claims can be supported by the totality of the data and weighing of the evidence ( 8) . in the commission e monograph , escop monograph , and hager 's handbuch , toxicity of the standardized extract egb 761 is described . because of their toxic properties , the presence of gas is limited in standardized g. biloba preparations to maximum 5 ppm ( 11 ) . however , in non - standardized g. biloba preparations , gas can be present at higher levels . for 16 products , both the detailed content and the recommended dose were declared on the label ( see table 2 ) . the recommended dose was stated , but the amount of flavonoids and terpene lactones in this dose was not stated in the declaration . only two of the 29 products met the pharmaceutical guideline ( the amount present in the product is 90110% of the declared amount ) for terpene lactones . based on the daily dose and the concentration of terpene lactones and flavonoids found in the products , a dose range of substances typical for g. biloba was calculated ; 16 products did not meet the dose range for terpene lactones and flavonoids for a 24% standardized extract . in summary , when the components of herbal supplements were evaluated according to the guideline for pharmaceuticals , that require the presence of 90110% of the declared amount , one out of 29 g. biloba products meets the guideline.1 the amount of terpene lactones and flavonoids found vary between 27358% for terpene lactones and between 86418% for flavonoids . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . however , as not all g. biloba preparations included in the present study contain gb - ste , maximum levels of 5 mg / kg are not guaranteed , which may pose a health risk ( 49 ) . in addition to these mild effects , in several case studies side effects have been reported related to blood platelets , hemorrhage , and blood coagulation , which is in agreement with the paf - inhibiting properties of g. biloba ( 5361 ) . in these case reports , reference is made to a ginkgo extract or a ginkgo containing preparation, without further specifications of the g. biloba preparation used . interactions of herbal preparations with drugs are particularly relevant for drugs with a narrow therapeutic margin , it can result in more or stronger side effects or effect on the bioavailability of the drug ( 72 , 73 ) . although a direct causal relationship between g. biloba intake and observed effects in case studies is lacking ( 74 ) , the absence of other risk factors , the paf - inhibiting activity of g. biloba , and the disappearance of effects upon cessation of intake suggest such causality ( 5961 ) . summarizing , the potential safety issues associated with g. biloba are severalfold . first , in most literature reports on g. biloba , the preparation used is not specified . second , because of the immunotoxic , cytotoxic , carcinogenic , and genotoxic potential of gas , the maximum level of gas in gb - ste is limited to 5 ppm . however , if a preparation of g. biloba does not contain the gb - ste , a higher level of gas can not be excluded . as a result of abovementioned available data , there was insufficient evidence to substantiate the three selected health claims for g. biloba , when applying the passclaim criteria . since herbal food supplements are intended for healthy persons , health claims for herbal food supplements have to be proven in studies performed with healthy persons who are representative of the target population . this study showed that most studies are performed reportedly using standardized g. biloba extract , but also that nearly every product found in the dutch shops was not in conformity with the label declaration . thirdly , the passclaim criteria 4 and 5 require consideration of specific markers , or endpoints , depending on the type of claim made . finally , the procedure for weighing the evidence was not described in detail in the passclaim project . indeed , in the usa experience , consumers do not make the anticipated distinction between substantiated and not substantiated ( = qualified ) health claims ( 2 , 7780 ) . the finding that the standardized 24% of g. biloba extract is often not incorporated into the food supplements , nevertheless , raises doubts as to the efficacy of such products . review of the science provided insufficient evidence with which to substantiate the three claims for g. biloba . in conclusion , the claim g. biloba and improvement in the symptoms of old age could not be evaluated . for 16 products , both the detailed content and the recommended dose were declared on the label ( see table 2 ) . the recommended dose was stated , but the amount of flavonoids and terpene lactones in this dose was not stated in the declaration . based on the daily dose and the concentration of terpene lactones and flavonoids found in the products , a dose range of substances typical for g. biloba was calculated ; 16 products did not meet the dose range for terpene lactones and flavonoids for a 24% standardized extract . in summary , when the components of herbal supplements were evaluated according to the guideline for pharmaceuticals , that require the presence of 90110% of the declared amount , one out of 29 g. biloba products meets the guideline.1 the amount of terpene lactones and flavonoids found vary between 27358% for terpene lactones and between 86418% for flavonoids . such clinical studies , however , may not be considered pertinent to a claim for the general population . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . in addition to these mild effects , in several case studies side effects have been reported related to blood platelets , hemorrhage , and blood coagulation , which is in agreement with the paf - inhibiting properties of g. biloba ( 5361 ) . in these case reports , reference is made to a ginkgo extract or a ginkgo containing preparation, without further specifications of the g. biloba preparation used . the duration and dose of intake of g. biloba prior to the reported paf - related side effects ranges from 2 weeks to 2 years , respectively , 80160 mg / day , well within the data on interactions with drugs are also mainly associated with drugs involved in the treatment of blood coagulation and bleeding ( 53 , 6264 ) . interactions of herbal preparations with drugs are particularly relevant for drugs with a narrow therapeutic margin , it can result in more or stronger side effects or effect on the bioavailability of the drug ( 72 , 73 ) . although a direct causal relationship between g. biloba intake and observed effects in case studies is lacking ( 74 ) , the absence of other risk factors , the paf - inhibiting activity of g. biloba , and the disappearance of effects upon cessation of intake suggest such causality ( 5961 ) . summarizing , the potential safety issues associated with g. biloba are severalfold . animal toxicity studies typically were performed with gb - ste , whereas in case studies no specification on the g. biloba preparation is given . second , because of the immunotoxic , cytotoxic , carcinogenic , and genotoxic potential of gas , the maximum level of gas in gb - ste is limited to 5 ppm . however , if a preparation of g. biloba does not contain the gb - ste , a higher level of gas can not be excluded . as a result of abovementioned available data , there was insufficient evidence to substantiate the three selected health claims for g. biloba , when applying the passclaim criteria first , a clear definition of what sort of study sample constitutes a healthy target population is lacking . since herbal food supplements are intended for healthy persons , health claims for herbal food supplements have to be proven in studies performed with healthy persons who are representative of the target population . this study showed that most studies are performed reportedly using standardized g. biloba extract , but also that nearly every product found in the dutch shops was not in conformity with the label declaration . when a claim can be substantiated using those studies , the product that sets the claim should resemble the standardized extract . finally , the procedure for weighing the evidence was not described in detail in the passclaim project . review of the science provided insufficient evidence with which to substantiate the three claims for g. biloba . in conclusion , the claim g. biloba and improvement in the symptoms of old age could not be evaluated . the main conclusions in this study were : three selected health claims for g. biloba could not be substantiated.neither safety nor efficacy can be guaranteed at the recommended daily dose.the content of g. biloba containing products often did not conform to what was declared on the label.a multidisciplinary approach is needed to get insight in the relevant issues for health claim evaluation.moreover , the passclaim criteria need to be developed in more detail when used for general application on health claim substantiation for herbal supplements and there is also a need for clarification of the study conditions that are required for claim substantiation , especially with respect to patient studies . a multidisciplinary approach is recommended to assess health claims : analyses of substances typical for g. biloba in the products and toxicological risk assessment are needed for a full assessment . this provides a good insight into the several aspects that are significant for the evaluation of health claims for herbal substances . the content of g. biloba containing products often did not conform to what was declared on the label .

the measurement of drug levels is a vital part of any comprehensive assessment of antimalarial drug efficacy . defining the pharmacokinetic properties of the antimalarial drugs has provided a basis for optimising dosing regimens and has explained why intrinsically efficacious antimalarial drugs do not always work . dihydroartemisinin - piperaquine ( dp ) is one of the leading fixed - dose artemisinin - based combination treatments ( acts ) , and has recently been recommended as a first - line treatment for uncomplicated falciparum malaria by the world health organisation . dihydroartemisinin ( dha ) is a rapidly eliminated artemisinin derivative and the active metabolite of artesunate , artemether and artemotil . it has an elimination half - life of approximately 3080 min [ 11 , 31 , 32 ] . piperaquine is a bisquinoline compound , structurally related to chloroquine but efficacious against chloroquine - resistant parasites . although the drug is more than 40 years old and was used extensively in china between 1978 and 1992 , estimates of the pharmacokinetic parameters of piperaquine have been published only in the last 5 years . terminal elimination half - life ( t ) estimates produced by different laboratories have varied between 11 and > 60 days [ 13 , 14 , 22 , 2830 ] with studies longer sampling duration and employing more sensitive assays giving longer estimates . the oral bioavailability of piperaquine has been shown to be increased by co - administration with fat , which could account for some of the observed inter - individual variation in pharmacokinetic profiles , although a more recent evaluation of piperaquine pharmacokinetics in vietnamese volunteers found moderately wide variability in total drug exposure ( auc , area under the plasma concentration time curve ) which was not related to concomitant food intake . studies to date have used plasma drug assays , although results of whole blood assays at a single time point have also been reported . in two separate clinical trials in laos and myanmar , piperaquine levels were measured from single samples taken on day 7 after treatment , as measurements at this time point provide a useful surrogate of in - vivo drug exposure . venous plasma levels were measured in the study in laos and whole blood levels ( taken onto filter paper ) were analysed in the myanmar study . the mean [ 95% confidence limit ( 95% ci ) ] day 7 plasma piperaquine concentration in the laos study was 31.8 ( 28.834.8 ) ng / ml ( n = 104 ) , whereas in the myanmar study , the mean capillary whole blood value was 72 ng / ml [ median ( interquartile range ) 62 ( 5082 ) ng / ml ] ( n = 156 ) . thus , concentrations assessed from capillary whole blood were approximately double those measured in plasma at the same time point . the requirements for multiple venepunctures and a cold chain to transport samples to an analytical laboratory are major impediments in conducting community - based studies of antimalarial drugs that incorporate the measurement of drug concentrations . assays have been adapted to the field with the development of sensitive methods capable of measuring concentrations in very small volume capillary blood samples , often taken onto filter paper , and by the use of sparse sampling schedules coupled with non - linear mixed - effects modelling to generate pharmacokinetic estimates and investigate sources of variation . the principal aim of this study was to compare piperaquine concentrations in samples of venous plasma and venous whole blood with those in capillary whole blood taken simultaneously and to determine the relationship between them . this was part of a detailed pharmacokinetic study nested into two larger randomised trials of the efficacy and safety of dp which have been reported previously [ 1 , 2 , 30 ] . it took place in clinics of the shoklo malaria research unit along the thai - myanmar border . karen or burmese patients , aged > 2 years , with symptomatic uncomplicated falciparum infection , randomised to treatment with dp and to the nested pharmacokinetic study , were eligible for inclusion . the study was explained to patients in their own language , and written consent was obtained ( thumbprint if patients were unable to read or write ) . the total dose was 7 mg / kg body weight of dha and 55 mg / kg piperaquine ( artekin holley pharmaceutical co , guangzhou , china ) split into four doses at 0 , 8 , 24 h and 48 h , respectively , or three doses at 0 , 24 and 48 h , respectively . a maximum of four sets of post - treatment samples was collected from each patient . these were selected randomly from the following time windows after the first dose of dp : 04 , 812 , 2448 , 4852 h ( three - dose arm ) or 48 , 1224 , 2848 , 5272 h ( four - dose arm ) , and 7 , 14 , 21 , 28 , 35 , 42 , 49 , 56 or 63 days ( both groups ) . from the 4.5-ml blood sample taken by venesection , 1.5 ml was transferred to a cryo tube , and the remainder was transferred into a sodium heparin tube ; this sample was centrifuged for 10 min at 1500 g and the plasma transferred by pipette to a cryo tube before freezing in liquid nitrogen . capillary samples were taken at the same time from a fingerprick into heparinised capillary tubes . the blood was then transferred to a cryo tube to be frozen immediately in liquid nitrogen . samples were transferred to the main laboratory in batches where they were stored at 80c . after all samples were collected , they were transferred on dry ice to the pharmacology laboratory in the faculty of tropical medicine , mahidol university , bangkok . piperaquine concentrations were measured using a high - throughput assay utilising solid phase extraction ( spe ) and liquid chromatography with ultra - violet detection [ 1517 ] . the lower limit of quantification ( lloq ) was higher for the blood assays than for the plasma assay , mainly because of differences in sample volumes but also because the recovery of piperaquine from blood is slightly lower than that from plasma . the plasma assay could accommodate 1 ml while the blood assay only accommodated 500 l . a larger volume of blood resulted in an unacceptably high back - pressure in the spe step . the plasma assay used a 1-ml sample , the blood assay used 0.5 ml and the capillary blood assay used 0.1 ml . the sample volumes and the impact on assay sensitivity are summarised in table 1 . table 1assay sensitivity and quality control resultsparametersvenous plasma ( n = 486 samples)venous blood ( n = 186 samples)capillary blood ( n = 178 samples)control concentration ( ng / ml)20.010061359.6400200059.64002000mean value19.698.063656.4409.3197959.3410.52113sd1.34.1266.537.01104.914.1127rsd ( % ) 6.74.14.111.69.05.58.33.46.0sample volume ( l)1000500100lloq ( ng / ml)2.51537.33sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantificationthere were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing assay sensitivity and quality control results sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantification there were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing the relationships between piperaquine concentrations in the venous plasma and venous blood , venous plasma and capillary blood and capillary blood and venous blood were investigated using regression modelling . since all concentrations were log - normally distributed , they were modelled after a logarithmic transformation . the relationship between the concentrations was modelled as a power function , and the optimal fractional polynomial function was found using the 10 ( statacorp , college station , tx ) . the final model was a random intercept model to account for multiple measurements per subject and was adjusted for time since the first dose or log - parasitaemia , if either improved the model significantly , as assessed by the wald test . for example , the relationship between venous plasma and venous whole blood concentrations was estimated using the following model structure : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ log{\left ( { c\,venous\,blood } \right)_{ij } } = \beta + fp\left ( { log{{\left ( { c\,venous\,plasma } \right)}_{ij } } } \right ) + { \beta_1}{x_{1ij } } + { \beta_2}{x_{2ij } } + { v_i } + { e_{ij } } $ $ \end{document}where fp(x ) is a fractional polynomial 1 x + ... + m x ; p1< ... <pm are integer or fractional powers ; x1 and x2 are other covariates , such as time from the first dose or log of parasitaemia on the day of sampling , vi n(0,b ) is a subject - specific residual and eij n(0, ) is a usual residual . parasitaemia at the time of sampling was estimated from the fitted log - linear decline in parasite densities assuming a first - order elimination process . for fitting purposes parasitaemia was assumed to be equal to half of the detection limit for the first negative slide . the utility of the models was assessed by examining the relative difference between the predicted and observed ( measured ) concentrations on the original scale ; for example : ( predicted venous plasma concentration measured venous plasma concentration)/measured venous plasma concentration 100% . approval for the study was granted by the faculty of tropical medicine ethical committee , mahidol university , bangkok , thailand and the oxford tropical ethics committee ( oxtrec ) , uk . this was part of a detailed pharmacokinetic study nested into two larger randomised trials of the efficacy and safety of dp which have been reported previously [ 1 , 2 , 30 ] . it took place in clinics of the shoklo malaria research unit along the thai - myanmar border . karen or burmese patients , aged > 2 years , with symptomatic uncomplicated falciparum infection , randomised to treatment with dp and to the nested pharmacokinetic study , were eligible for inclusion . the study was explained to patients in their own language , and written consent was obtained ( thumbprint if patients were unable to read or write ) . the total dose was 7 mg / kg body weight of dha and 55 mg / kg piperaquine ( artekin holley pharmaceutical co , guangzhou , china ) split into four doses at 0 , 8 , 24 h and 48 h , respectively , or three doses at 0 , 24 and 48 h , respectively . a maximum of four sets of post - treatment samples was collected from each patient . these were selected randomly from the following time windows after the first dose of dp : 04 , 812 , 2448 , 4852 h ( three - dose arm ) or 48 , 1224 , 2848 , 5272 h ( four - dose arm ) , and 7 , 14 , 21 , 28 , 35 , 42 , 49 , 56 or 63 days ( both groups ) . from the 4.5-ml blood sample taken by venesection , 1.5 ml was transferred to a cryo tube , and the remainder was transferred into a sodium heparin tube ; this sample was centrifuged for 10 min at 1500 g and the plasma transferred by pipette to a cryo tube before freezing in liquid nitrogen . capillary samples were taken at the same time from a fingerprick into heparinised capillary tubes . the blood was then transferred to a cryo tube to be frozen immediately in liquid nitrogen . samples were transferred to the main laboratory in batches where they were stored at 80c . after all samples were collected , they were transferred on dry ice to the pharmacology laboratory in the faculty of tropical medicine , mahidol university , bangkok . piperaquine concentrations were measured using a high - throughput assay utilising solid phase extraction ( spe ) and liquid chromatography with ultra - violet detection [ 1517 ] . the lower limit of quantification ( lloq ) was higher for the blood assays than for the plasma assay , mainly because of differences in sample volumes but also because the recovery of piperaquine from blood is slightly lower than that from plasma . the plasma assay could accommodate 1 ml while the blood assay only accommodated 500 l . a larger volume of blood resulted in an unacceptably high back - pressure in the spe step . the plasma assay used a 1-ml sample , the blood assay used 0.5 ml and the capillary blood assay used 0.1 ml . the sample volumes and the impact on assay sensitivity are summarised in table 1 . table 1assay sensitivity and quality control resultsparametersvenous plasma ( n = 486 samples)venous blood ( n = 186 samples)capillary blood ( n = 178 samples)control concentration ( ng / ml)20.010061359.6400200059.64002000mean value19.698.063656.4409.3197959.3410.52113sd1.34.1266.537.01104.914.1127rsd ( % ) 6.74.14.111.69.05.58.33.46.0sample volume ( l)1000500100lloq ( ng / ml)2.51537.33sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantificationthere were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing assay sensitivity and quality control results sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantification there were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing the relationships between piperaquine concentrations in the venous plasma and venous blood , venous plasma and capillary blood and capillary blood and venous blood were investigated using regression modelling . since all concentrations were log - normally distributed , they were modelled after a logarithmic transformation . the relationship between the concentrations was modelled as a power function , and the optimal fractional polynomial function was found using the the final model was a random intercept model to account for multiple measurements per subject and was adjusted for time since the first dose or log - parasitaemia , if either improved the model significantly , as assessed by the wald test . for example , the relationship between venous plasma and venous whole blood concentrations was estimated using the following model structure : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ log{\left ( { c\,venous\,blood } \right)_{ij } } = \beta + fp\left ( { log{{\left ( { c\,venous\,plasma } \right)}_{ij } } } \right ) + { \beta_1}{x_{1ij } } + { \beta_2}{x_{2ij } } + { v_i } + { e_{ij } } $ $ \end{document}where fp(x ) is a fractional polynomial 1 x + ... + m x ; p1< ... <pm are integer or fractional powers ; x1 and x2 are other covariates , such as time from the first dose or log of parasitaemia on the day of sampling , vi n(0,b ) is a subject - specific residual and eij n(0, ) is a usual residual . parasitaemia at the time of sampling was estimated from the fitted log - linear decline in parasite densities assuming a first - order elimination process . for fitting purposes parasitaemia was assumed to be equal to half of the detection limit for the first negative slide . the utility of the models was assessed by examining the relative difference between the predicted and observed ( measured ) concentrations on the original scale ; for example : ( predicted venous plasma concentration measured venous plasma concentration)/measured venous plasma concentration 100% . approval for the study was granted by the faculty of tropical medicine ethical committee , mahidol university , bangkok , thailand and the oxford tropical ethics committee ( oxtrec ) , uk . a median of three ( range 14 ) sets of samples were collected from the same patient . piperaquine was detectable in all of the plasma samples but undetectable in three venous blood samples and 20 capillary blood samples . the median piperaquine concentrations were 136.1 ( range 0907.4 ) ng / ml in capillary samples , 83.9 ( 0852.5 ) ng / ml in venous samples and 36.3 ( 2.8345.1 ) ng / ml in venous plasma samples . the lloq of the assays , coefficients of variation and the quality control results are shown in table 1 . table 2characteristics of 97 patients with uncomplicated falciparum malaria treated with dihydroartemisinin - piperaquine ( dp ) in the pharmacokinetic studypatient characteristicsvaluesmale , n ( % ) 58 ( 60)age ( years)25 ( 355)weight ( kg)48 ( 1274)haematocrit on enrolment ( % ) 39 ( 2052)three - dose dp treatment , n ( % ) 47 ( 49)parasitaemia/l , geometric mean10,887 ( 83223,872)piperaquine total dose , mg / kg53.3 ( 4074)the results are expressed as the median value with the range given in parenthesis unless stated otherwise characteristics of 97 patients with uncomplicated falciparum malaria treated with dihydroartemisinin - piperaquine ( dp ) in the pharmacokinetic study the results are expressed as the median value with the range given in parenthesis unless stated otherwise there were three pairs of samples for which piperaquine was below the limit of detection in venous blood but detectable in plasma taken at the same time ( plasma concentrations 4.3 , 4.8 and 40.0 ng / ml , respectively ) . for the other paired concentrations , venous blood concentrations were nearly always higher than venous plasma concentrations with a median ( 90% range ) ratio of venous blood concentration to venous plasma concentration of 2.15 ( 0.915.26 ) . this ratio was strongly negatively correlated with plasma concentration ( spearman s = 0.578 , p < 0.001 ) and parasitaemia on the day of sampling ( spearman s = 0.417 , p < 0.001 and positively correlated with the interval between dosing and sampling ( spearman s = 0.550 , p < 0.001 ) . the best model to describe the relationship between venous blood log - concentration and venous plasma log - concentration was linear and included log - parasitaemia at the time of sampling ( p < 0.001 ) ( table 3 ) . the fit was considerably worse at low plasma concentrations , particularly if the time and parasite covariates were not included ( fig . 1b ) shows a small decrease in the difference between observed and predicted log concentrations [ by 0.109 ( 95% ci 0.0330.184 per 1 u ) ] , which corresponds to much larger differences on the original scale . table 3summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole bloodparameterstransformationcoefficient95% confidence intervalp valuelog(venous blood concentration ) = f(venous plasma concentration ) plasma concentrationlog(x)0.7540.6970.8110.000 parasitaemialog10(x + 1)0.1250.167 to 0.0820.001 constant ( )1.7521.5381.9660.000 b0.2100.1380.3190.005 0.3580.3120.410 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2560.1090.468log(venous plasma concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9740.8641.084<0.001 constant ( )1.0721.637 to 0.506<0.001 b0.2940.1920.4490.002 0.4560.3910.530 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2930.1250.525log(venous blood concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9200.9030.938<0.001 parasitaemialog10(x + 1)0.1120.145 to 0.078<0.001 constant ( )0 b0.2520.1890.338<0.001 0.2640.2280.306 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.4770.3060.653b , between subject variation ; , residual variationlikelihood ratio test of b = 0fig . altman plot for measured and predicted venous blood concentration summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole blood b , between subject variation ; , residual variation likelihood ratio test of b = 0 assessment of the relationship between venous blood and venous plasma concentrations of piperaquine . model was adjusted for parasitaemia at the time of sampling . a predicted versus measured venous blood concentrations . natural log scale . altman plot for measured and predicted venous blood concentration in 20 pairs of samples piperaquine was undetectable in capillary blood but detectable in plasma taken at the same time [ median plasma concentrations 4.8 ( 90% range 2.940.0 ) ng / ml ] . median capillary blood levels at which piperaquine was detectable in both venous plasma and capillary blood samples were 3.5-fold ( 90% range 1.77.4 ) higher than those measured in plasma . this ratio was not correlated with plasma concentration , the time of sampling or parasitaemia on the day of sampling . the best model to describe the relationship between log plasma concentration and log capillary concentration was linear without covariates ( table 3 ) . predicted ( from piperaquine capillary concentrations ) versus measured piperaquine venous plasma concentrations are shown in fig . 2b ) shows a decrease in the difference between observed and predicted log concentrations [ by 0.218 ( 90% range 0.1130.323 ) per 1 u ] , which corresponds to much larger differences on the original scale . altman plot for measured and predicted venous plasma concentration assessment of the relationship between venous plasma and capillary blood concentrations of piperaquine . altman plot for measured and predicted venous plasma concentration piperaquine was undetectable in 20 capillary blood samples ; of these , piperaquine was also undetectable in three corresponding samples of venous blood . of the remaining 17 samples , the median venous concentration ( 90% range ) was 20.55 ( 12.3268.65 ) ng / ml . on average , capillary drug levels were 1.66-fold higher ( 90% range 0.923.03 ) than the levels measured in corresponding venous blood samples . this ratio was strongly negatively correlated with the interval between dosing and sampling ( spearman s = 0.45 , p < 0.001 ) and positively correlated with parasitaemia on the day of sampling ( spearman s = 0.45 , p < 0.001 ) . the best model to describe the relationship between venous log - concentration and capillary log - concentration was linear and included log - parasitaemia at the time of sampling ( p < 0.001 ) ( table 3 ) . 3 ) shows a small decrease in the difference between observed and predicted log concentrations [ by 0.128 ( 90% range 0.0520.204 ) per 1 u ] which would be 0.175 ( 90% range 0.920.258 ) if parasitaemia was not included in the model . differences on the original scale are larger , but the majority ( 89% , 125/141 ) of measures remain within 50% . overall , 58% ( 82/141 ) of predicted venous blood concentrations ( from capillary concentrations ) were within 20% of the measured venous concentrations , while 70% ( 99/141 ) were within 30% . the discrepancies were higher for low concentrations , with only 41% ( 7/17 ) of predicted venous blood concentrations remaining within the 20 or 30% range for venous concentrations < 30 ng / ml . for concentrations > 30 ng / ml , these proportions were 60% ( 75/124 ) and 74% ( 92/124 ) , respectively . these values remained the same if the cut - off was taken as 40 or 50 ng / ml . nearly all ( 93% , or 96/103 ) capillary blood concentrations taken between 24 h after the first dose and before or on day 21 were > 30 ng / ml . from day 28 onwards , the majority ( 77% ) of concentrations were < 30 ng / ml . after day 3 , when parasitaemia had cleared , the relationship between venous piperaquine concentrations and capillary concentrations became simpler : venous concentration = ( capillary concentration ) . among samples taken after day 3 and before day 28 , 70% ( 42/60 ) of the predicted venous blood concentrations were within 30% of the measured venous blood concentrations , and 62% ( 37/60 ) of predictions were within 20% ; consequently , the observed versus expected agreement was good in this time interval . altman plot for measured and predicted venous blood concentration assessment of the relationship between venous blood and capillary blood concentrations of piperaquine . model was adjusted for parasitaemia at the time of sampling . a predicted versus measured venous blood concentrations . altman plot for measured and predicted venous blood concentration figure 4 compares the predictive utilities of all models . 4distribution of the relative difference ( % ) between measured and predicted values of piperaquine for : a venous blood concentration predicted from venous plasma concentration , adjusted for parasitaemia , b venous blood concentration predicted from venous plasma concentration , unadjusted for parasitaemia , c venous plasma concentration predicted from capillary blood concentration , d venous blood concentration predicted from capillary blood concentration , adjusted for parasitaemia , e venous blood concentration predicted from capillary blood concentration , unadjusted for parasitaemia . boundaries of the boxes 25th and 75th centiles , line in the box median distribution of the relative difference ( % ) between measured and predicted values of piperaquine for : a venous blood concentration predicted from venous plasma concentration , adjusted for parasitaemia , b venous blood concentration predicted from venous plasma concentration , unadjusted for parasitaemia , c venous plasma concentration predicted from capillary blood concentration , d venous blood concentration predicted from capillary blood concentration , adjusted for parasitaemia , e venous blood concentration predicted from capillary blood concentration , unadjusted for parasitaemia . there were three pairs of samples for which piperaquine was below the limit of detection in venous blood but detectable in plasma taken at the same time ( plasma concentrations 4.3 , 4.8 and 40.0 ng / ml , respectively ) . for the other paired concentrations , venous blood concentrations were nearly always higher than venous plasma concentrations with a median ( 90% range ) ratio of venous blood concentration to venous plasma concentration of 2.15 ( 0.915.26 ) . this ratio was strongly negatively correlated with plasma concentration ( spearman s = 0.578 , p < 0.001 ) and parasitaemia on the day of sampling ( spearman s = 0.417 , p < 0.001 and positively correlated with the interval between dosing and sampling ( spearman s = 0.550 , p < 0.001 ) . the best model to describe the relationship between venous blood log - concentration and venous plasma log - concentration was linear and included log - parasitaemia at the time of sampling ( p < 0.001 ) ( table 3 ) . the fit was considerably worse at low plasma concentrations , particularly if the time and parasite covariates were not included ( fig . 1b ) shows a small decrease in the difference between observed and predicted log concentrations [ by 0.109 ( 95% ci 0.0330.184 per 1 u ) ] , which corresponds to much larger differences on the original scale . table 3summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole bloodparameterstransformationcoefficient95% confidence intervalp valuelog(venous blood concentration ) = f(venous plasma concentration ) plasma concentrationlog(x)0.7540.6970.8110.000 parasitaemialog10(x + 1)0.1250.167 to 0.0820.001 constant ( )1.7521.5381.9660.000 b0.2100.1380.3190.005 0.3580.3120.410 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2560.1090.468log(venous plasma concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9740.8641.084<0.001 constant ( )1.0721.637 to 0.506<0.001 b0.2940.1920.4490.002 0.4560.3910.530 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2930.1250.525log(venous blood concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9200.9030.938<0.001 parasitaemialog10(x + 1)0.1120.145 to 0.078<0.001 constant ( )0 b0.2520.1890.338<0.001 0.2640.2280.306 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.4770.3060.653b , between subject variation ; , residual variationlikelihood ratio test of b = 0fig . altman plot for measured and predicted venous blood concentration summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole blood b , between subject variation ; , residual variation likelihood ratio test of b = 0 assessment of the relationship between venous blood and venous plasma concentrations of piperaquine . in 20 pairs of samples piperaquine was undetectable in capillary blood but detectable in plasma taken at the same time [ median plasma concentrations 4.8 ( 90% range 2.940.0 ) ng / ml ] . median capillary blood levels at which piperaquine was detectable in both venous plasma and capillary blood samples were 3.5-fold ( 90% range 1.77.4 ) higher than those measured in plasma . this ratio was not correlated with plasma concentration , the time of sampling or parasitaemia on the day of sampling . the best model to describe the relationship between log plasma concentration and log capillary concentration was linear without covariates ( table 3 ) . predicted ( from piperaquine capillary concentrations ) versus measured piperaquine venous plasma concentrations are shown in fig . 2b ) shows a decrease in the difference between observed and predicted log concentrations [ by 0.218 ( 90% range 0.1130.323 ) per 1 u ] , which corresponds to much larger differences on the original scale . altman plot for measured and predicted venous plasma concentration assessment of the relationship between venous plasma and capillary blood concentrations of piperaquine . piperaquine was undetectable in 20 capillary blood samples ; of these , piperaquine was also undetectable in three corresponding samples of venous blood . of the remaining 17 samples , the median venous concentration ( 90% range ) was 20.55 ( 12.3268.65 ) ng / ml . on average , capillary drug levels were 1.66-fold higher ( 90% range 0.923.03 ) than the levels measured in corresponding venous blood samples . this ratio was strongly negatively correlated with the interval between dosing and sampling ( spearman s = 0.45 , p < 0.001 ) and positively correlated with parasitaemia on the day of sampling ( spearman s = 0.45 , p < 0.001 ) . the best model to describe the relationship between venous log - concentration and capillary log - concentration was linear and included log - parasitaemia at the time of sampling ( p < 0.001 ) ( table 3 ) . 3 ) shows a small decrease in the difference between observed and predicted log concentrations [ by 0.128 ( 90% range 0.0520.204 ) per 1 u ] which would be 0.175 ( 90% range 0.920.258 ) if parasitaemia was not included in the model . differences on the original scale are larger , but the majority ( 89% , 125/141 ) of measures remain within 50% . overall , 58% ( 82/141 ) of predicted venous blood concentrations ( from capillary concentrations ) were within 20% of the measured venous concentrations , while 70% ( 99/141 ) were within 30% . the discrepancies were higher for low concentrations , with only 41% ( 7/17 ) of predicted venous blood concentrations remaining within the 20 or 30% range for venous concentrations < 30 ng / ml . for concentrations > 30 ng / ml , these proportions were 60% ( 75/124 ) and 74% ( 92/124 ) , respectively . these values remained the same if the cut - off was taken as 40 or 50 ng / ml . nearly all ( 93% , or 96/103 ) capillary blood concentrations taken between 24 h after the first dose and before or on day 21 were > 30 ng / ml . from day 28 onwards , the majority ( 77% ) of concentrations were < 30 ng / ml . after day 3 , when parasitaemia had cleared , the relationship between venous piperaquine concentrations and capillary concentrations became simpler : venous concentration = ( capillary concentration ) . among samples taken after day 3 and before day 28 , 70% ( 42/60 ) of the predicted venous blood concentrations were within 30% of the measured venous blood concentrations , and 62% ( 37/60 ) of predictions were within 20% ; consequently , the observed versus expected agreement was good in this time interval . altman plot for measured and predicted venous blood concentration assessment of the relationship between venous blood and capillary blood concentrations of piperaquine . altman plot for measured and predicted venous blood concentration figure 4 compares the predictive utilities of all models . 4distribution of the relative difference ( % ) between measured and predicted values of piperaquine for : a venous blood concentration predicted from venous plasma concentration , adjusted for parasitaemia , b venous blood concentration predicted from venous plasma concentration , unadjusted for parasitaemia , c venous plasma concentration predicted from capillary blood concentration , d venous blood concentration predicted from capillary blood concentration , adjusted for parasitaemia , e venous blood concentration predicted from capillary blood concentration , unadjusted for parasitaemia . boundaries of the boxes 25th and 75th centiles , line in the box median distribution of the relative difference ( % ) between measured and predicted values of piperaquine for : a venous blood concentration predicted from venous plasma concentration , adjusted for parasitaemia , b venous blood concentration predicted from venous plasma concentration , unadjusted for parasitaemia , c venous plasma concentration predicted from capillary blood concentration , d venous blood concentration predicted from capillary blood concentration , adjusted for parasitaemia , e venous blood concentration predicted from capillary blood concentration , unadjusted for parasitaemia . many antimalarial drugs are eliminated slowly from the body , and the measurement of drug concentrations in small volumes of whole blood taken from a finger prick sample is more convenient and more acceptable in young children . this allows community - based assessments , even in very remote settings , providing essential information on drug efficacy , adherence and toxicity . the new act dihydroartemisinin - piperaquine is likely to have a major role in antimalarial treatment , so the development of field - based tools for its assessment is important . the results of this study show that measurements of piperaquine in venous plasma , venous or capillary blood are not readily interchangeable across the whole range of concentrations encountered in patients with current dosing . piperaquine concentrations were highest in capillary whole blood , followed in decreasing concentrations by venous whole blood and venous plasma . the higher concentrations of piperaquine detected in whole blood compared to plasma suggests that piperaquine concentrates within blood cells . these results are consistent with previous findings that capillary blood concentrations were approximately double those measured in plasma at day 7 [ 18 , 25 ] . studies of chloroquine pharmacokinetics have reported concentrations in erythrocytes that are usually two to fivefold higher than those in plasma . this underlies the differences between plasma and serum concentrations and contributes to the variability in plasma concentrations . the duration and force of centrifugation has been shown to influence the concentrations of chloroquine and its metabolite desethylchloroquine in plasma , with concentrations of both drugs decreasing as the centrifugal force increases until relatively stable levels are reached at 500 g ( due to variable removal of white cells and platelets ) . the measurement of whole blood concentrations avoids this potential confounder and is now the generally accepted method for measuring chloroquine in therapeutic assessments . in the seminal study of gustafasson et al . of 11 healthy volunteers in whom chloroquine concentrations were measured in plasma and erythrocytes for 38 weeks after dosage , the disposition of chloroquine in erythrocytes paralleled that in plasma . there is a consistent increase in platelet count ( often doubling ) and haematocrit ( circa 10% ) in patients recovering from acute uncomplicated malaria . neutrophil counts also tend to increase . in acute malaria , patients may be dehydrated and haemoconcentrated when they present , and there are reduced concentrations of albumin ( which binds acidic drugs ) and marked increases in the concentrations of the acute phase protein 1-acid glycoprotein ( binds basic drugs ) . in addition , capillary blood samples are affected by factors affecting peripheral perfusion and tissue fluid concentrations as they contain a variable admixture of interstitial fluid . in cases of acute malaria , patients are usually febrile and vasodilated , whereas following recovery there may be peripheral vasoconstriction , leading to greater tissue fluid admixture in capillary samples . previous studies in patients and volunteers without malaria have revealed that the haematocrit and leucocyte levels tend to be higher in capillary blood samples than in venous blood samples , while platelet numbers tend to be similar or lower [ 57,27 , 34 ] . haematocrit measurements have also been observed to be more variable on repeated capillary sampling than on repeated venous sampling . for certain drugs , such as neuroleptics , the red cell concentrations have been reported to correlate better with therapeutic effects or dose than plasma concentrations [ 4 , 8 , 26 ] . hinderling has argued that for drugs with a kb / p ( blood to plasma concentration ratio ) > 2.0 , measuring concentrations in whole blood or erythrocytes rather than in plasma lowers the lloq . the increased sensitivity permits the kinetics of the drug to be followed up for at least one additional half - life . in our study , the lloq was higher in the blood assays than the plasma assay , mainly because of differences in sample volumes but also because the recovery of piperaquine from blood is slightly lower than that from plasma . the plasma assay used a 1-ml sample to obtain a lloq of 2.5 ng / ml , while the capillary blood assay used a 0.1-ml sample to obtain a lloq of 37 ng / ml . the model for the relationship between piperaquine concentrations measured in venous and capillary blood adjusted for parasitaemia showed the least amount of variability between observed and predicted concentrations , but this may be attributable to the large proportion of undetectable results at the lower end of the concentration range for the capillary measurements . the relationship between piperaquine concentrations in plasma , venous blood and capillary bloods is variable and not predictable at low concentrations . further studies are recommended to characterise the compartmentalisation in blood cells , to compare estimates of piperaquine pharmacokinetic parameters in plasma and whole blood and to provide information on the optimum samples for field studies in terms of drug measurement . a simple approach for most field studies is to take a single measurement of drug concentration on day 7 . at this point , parasitaemia has almost invariably cleared , and a good approximation of the venous concentration is ( capillary concentration ) . thus , despite certain limitations , the capillary blood piperaquine measurement could prove to be useful in field assessment studies of therapeutic efficacy and adherence .

purposedihydroartemisinin - piperaquine ( dp ) is a fixed - dose artemisinin - based combination treatment . field pharmacokinetic studies would be simplified and facilitated by being able to use small volume capillary assays rather than venous blood . the aim of this study was to describe the relationship between piperaquine concentrations measured in capillary blood , venous blood and venous plasma.methodssamples of plasma , whole blood obtained by venesection and capillary blood were taken simultaneously from patients with uncomplicated plasmodium falciparum malaria treated with dp between 0 and 9 weeks after treatment . piperaquine concentrations in venous and capillary samples were measured using solid phase extraction and analysis by liquid chromatography with ultraviolet detection.resultsa total of 161 sets of the three measures were obtained from 54 patients . piperaquine concentrations in the venous blood samples were approximately twofold higher and those in the capillary blood samples were threefold higher than the corresponding venous plasma concentrations . capillary blood piperaquine concentrations were approximately 1.7-fold higher than venous blood concentrations , and this difference also increased with time.conclusiondifferences in whole blood and plasma levels of piperaquine suggest compartmentalisation of the drug within blood cells , as also occurs with the structurally related quinoline chloroquine . the relationship between piperaquine concentrations in the venous plasma , venous blood and capillary blood is variable and unpredictable at low concentrations . however , within the range of concentrations usually present in patients between 3 and 21 days after treatment with currently recommended doses , the relationship between capillary and venous whole blood is predictable ; consequently , capillary blood sampling can be used in field assessments .

Background Study design and methods Study site and population DP dosing regimen Sampling Statistical analysis Ethical approval Results Venous blood versus venous plasma Venous plasma versus capillary blood Capillary blood versus venous blood Discussion

dihydroartemisinin - piperaquine ( dp ) is one of the leading fixed - dose artemisinin - based combination treatments ( acts ) , and has recently been recommended as a first - line treatment for uncomplicated falciparum malaria by the world health organisation . the principal aim of this study was to compare piperaquine concentrations in samples of venous plasma and venous whole blood with those in capillary whole blood taken simultaneously and to determine the relationship between them . table 1assay sensitivity and quality control resultsparametersvenous plasma ( n = 486 samples)venous blood ( n = 186 samples)capillary blood ( n = 178 samples)control concentration ( ng / ml)20.010061359.6400200059.64002000mean value19.698.063656.4409.3197959.3410.52113sd1.34.1266.537.01104.914.1127rsd ( % ) 6.74.14.111.69.05.58.33.46.0sample volume ( l)1000500100lloq ( ng / ml)2.51537.33sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantificationthere were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing assay sensitivity and quality control results sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantification there were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing the relationships between piperaquine concentrations in the venous plasma and venous blood , venous plasma and capillary blood and capillary blood and venous blood were investigated using regression modelling . for example , the relationship between venous plasma and venous whole blood concentrations was estimated using the following model structure : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ log{\left ( { c\,venous\,blood } \right)_{ij } } = \beta + fp\left ( { log{{\left ( { c\,venous\,plasma } \right)}_{ij } } } \right ) + { \beta_1}{x_{1ij } } + { \beta_2}{x_{2ij } } + { v_i } + { e_{ij } } $ $ \end{document}where fp(x ) is a fractional polynomial 1 x + ... + m x ; p1< ... <pm are integer or fractional powers ; x1 and x2 are other covariates , such as time from the first dose or log of parasitaemia on the day of sampling , vi n(0,b ) is a subject - specific residual and eij n(0, ) is a usual residual . table 1assay sensitivity and quality control resultsparametersvenous plasma ( n = 486 samples)venous blood ( n = 186 samples)capillary blood ( n = 178 samples)control concentration ( ng / ml)20.010061359.6400200059.64002000mean value19.698.063656.4409.3197959.3410.52113sd1.34.1266.537.01104.914.1127rsd ( % ) 6.74.14.111.69.05.58.33.46.0sample volume ( l)1000500100lloq ( ng / ml)2.51537.33sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantificationthere were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing assay sensitivity and quality control results sd , standard deviation ; rsd , relative standard deviation ; lloq , lower limit of quantification there were more plasma samples than venous blood and capillary blood samples because not all patients in the larger pharmacokinetic study gave all types of blood for testing the relationships between piperaquine concentrations in the venous plasma and venous blood , venous plasma and capillary blood and capillary blood and venous blood were investigated using regression modelling . for example , the relationship between venous plasma and venous whole blood concentrations was estimated using the following model structure : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ log{\left ( { c\,venous\,blood } \right)_{ij } } = \beta + fp\left ( { log{{\left ( { c\,venous\,plasma } \right)}_{ij } } } \right ) + { \beta_1}{x_{1ij } } + { \beta_2}{x_{2ij } } + { v_i } + { e_{ij } } $ $ \end{document}where fp(x ) is a fractional polynomial 1 x + ... + m x ; p1< ... <pm are integer or fractional powers ; x1 and x2 are other covariates , such as time from the first dose or log of parasitaemia on the day of sampling , vi n(0,b ) is a subject - specific residual and eij n(0, ) is a usual residual . table 2characteristics of 97 patients with uncomplicated falciparum malaria treated with dihydroartemisinin - piperaquine ( dp ) in the pharmacokinetic studypatient characteristicsvaluesmale , n ( % ) 58 ( 60)age ( years)25 ( 355)weight ( kg)48 ( 1274)haematocrit on enrolment ( % ) 39 ( 2052)three - dose dp treatment , n ( % ) 47 ( 49)parasitaemia/l , geometric mean10,887 ( 83223,872)piperaquine total dose , mg / kg53.3 ( 4074)the results are expressed as the median value with the range given in parenthesis unless stated otherwise characteristics of 97 patients with uncomplicated falciparum malaria treated with dihydroartemisinin - piperaquine ( dp ) in the pharmacokinetic study the results are expressed as the median value with the range given in parenthesis unless stated otherwise there were three pairs of samples for which piperaquine was below the limit of detection in venous blood but detectable in plasma taken at the same time ( plasma concentrations 4.3 , 4.8 and 40.0 ng / ml , respectively ) . for the other paired concentrations , venous blood concentrations were nearly always higher than venous plasma concentrations with a median ( 90% range ) ratio of venous blood concentration to venous plasma concentration of 2.15 ( 0.915.26 ) . table 3summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole bloodparameterstransformationcoefficient95% confidence intervalp valuelog(venous blood concentration ) = f(venous plasma concentration ) plasma concentrationlog(x)0.7540.6970.8110.000 parasitaemialog10(x + 1)0.1250.167 to 0.0820.001 constant ( )1.7521.5381.9660.000 b0.2100.1380.3190.005 0.3580.3120.410 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2560.1090.468log(venous plasma concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9740.8641.084<0.001 constant ( )1.0721.637 to 0.506<0.001 b0.2940.1920.4490.002 0.4560.3910.530 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2930.1250.525log(venous blood concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9200.9030.938<0.001 parasitaemialog10(x + 1)0.1120.145 to 0.078<0.001 constant ( )0 b0.2520.1890.338<0.001 0.2640.2280.306 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.4770.3060.653b , between subject variation ; , residual variationlikelihood ratio test of b = 0fig . altman plot for measured and predicted venous blood concentration summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole blood b , between subject variation ; , residual variation likelihood ratio test of b = 0 assessment of the relationship between venous blood and venous plasma concentrations of piperaquine . among samples taken after day 3 and before day 28 , 70% ( 42/60 ) of the predicted venous blood concentrations were within 30% of the measured venous blood concentrations , and 62% ( 37/60 ) of predictions were within 20% ; consequently , the observed versus expected agreement was good in this time interval . for the other paired concentrations , venous blood concentrations were nearly always higher than venous plasma concentrations with a median ( 90% range ) ratio of venous blood concentration to venous plasma concentration of 2.15 ( 0.915.26 ) . table 3summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole bloodparameterstransformationcoefficient95% confidence intervalp valuelog(venous blood concentration ) = f(venous plasma concentration ) plasma concentrationlog(x)0.7540.6970.8110.000 parasitaemialog10(x + 1)0.1250.167 to 0.0820.001 constant ( )1.7521.5381.9660.000 b0.2100.1380.3190.005 0.3580.3120.410 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2560.1090.468log(venous plasma concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9740.8641.084<0.001 constant ( )1.0721.637 to 0.506<0.001 b0.2940.1920.4490.002 0.4560.3910.530 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.2930.1250.525log(venous blood concentration ) = f(capillary blood concentration ) capillary concentrationlog(x)0.9200.9030.938<0.001 parasitaemialog10(x + 1)0.1120.145 to 0.078<0.001 constant ( )0 b0.2520.1890.338<0.001 0.2640.2280.306 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho = \sigma_b^2/\left ( { \sigma_b^2 + { \sigma^2 } } \right ) $ $ \end{document}0.4770.3060.653b , between subject variation ; , residual variationlikelihood ratio test of b = 0fig . altman plot for measured and predicted venous blood concentration summary of the models used to describe the relationship between the concentrations of piperaquine in venous plasma and those in capillary and venous whole blood b , between subject variation ; , residual variation likelihood ratio test of b = 0 assessment of the relationship between venous blood and venous plasma concentrations of piperaquine . among samples taken after day 3 and before day 28 , 70% ( 42/60 ) of the predicted venous blood concentrations were within 30% of the measured venous blood concentrations , and 62% ( 37/60 ) of predictions were within 20% ; consequently , the observed versus expected agreement was good in this time interval . altman plot for measured and predicted venous blood concentration assessment of the relationship between venous blood and capillary blood concentrations of piperaquine . the results of this study show that measurements of piperaquine in venous plasma , venous or capillary blood are not readily interchangeable across the whole range of concentrations encountered in patients with current dosing . piperaquine concentrations were highest in capillary whole blood , followed in decreasing concentrations by venous whole blood and venous plasma . the model for the relationship between piperaquine concentrations measured in venous and capillary blood adjusted for parasitaemia showed the least amount of variability between observed and predicted concentrations , but this may be attributable to the large proportion of undetectable results at the lower end of the concentration range for the capillary measurements . the relationship between piperaquine concentrations in plasma , venous blood and capillary bloods is variable and not predictable at low concentrations .

this phase ii trial was a prospective randomized double - blind placebo - controlled parallel - group study conducted between april 2007 and may 2008 at 118 sites in the u.s . ( n = 106 ) , mexico ( n = 9 ) , chile ( n = 2 ) , and the dominican republic ( n = 1 ) ( online appendix 1 , available at http://care.diabetesjournals.org/cgi/content/full/dc09-0366/dc1 ) . men and women of non - childbearing potential ( age 1875 years ) were eligible for inclusion if diagnosed with type 2 diabetes 3 months before screening . subjects were drug nave ( diet and exercise ) or treated with metformin monotherapy and stable for > 3 months before prescreening ( 1 week prior to screening visit ) . only subjects treated with metformin monotherapy were eligible for the exenatide arm ( consistent with labeling ) . additional inclusion criteria included bmi 20 and 40 kg / m and a1c at screening 7 and 10% . exclusion criteria included any oral antidiabetic monotherapy ( except metformin ) 3 months prior to screening or insulin < 1 month prior to screening and not used for > 7 days ; pancreatitis within 5 years ; significant cardiovascular , cerebrovascular , renal , or hepatobiliary diseases ; fasting serum triglycerides 800 mg / dl ( 9 mmol / l ) at screening ; or hematological profiles considered to be clinically significant . lipid - lowering medications must have been maintained at the same dose for 3 months prior to enrollment , and no prescription or over - the - counter weight - loss drugs were permitted . the study protocol was approved by an institutional review board and conducted in accordance with the declaration of helsinki . written informed consent was obtained from all subjects at prescreening . a data safety monitoring committee of independent experts assessed safety data on an ongoing basis . subjects were randomized into 1 of 10 treatment arms : double - blind placebo ( matched to albiglutide arms ) ; albiglutide weekly ( 4 , 15 , or 30 mg ) , every 2 weeks ( biweekly ; 15 , 30 , or 50 mg ) , or monthly ( 50 or 100 mg ) ; or open - label exenatide ( 5 g twice daily for 4 weeks followed by 12 weeks of 10 g twice daily ) . albiglutide ( formulated in 10 mmol / l sodium phosphate , 4.2% trehalose , 2.8% mannitol , 0.01% polysorbate-80 ) and placebo ( same formulation without active principle ) were administered in the physician 's office over the course of 16 weeks . subjects receiving 4 mg albiglutide were given 1.0 ml ( 4 mg / ml solution ) . subjects receiving 15 , 30 , or 50 mg albiglutide were given 0.32 , 0.65 , or 1.0 ml ( 50 mg / ml solution ) . subjects receiving 100 mg albiglutide were given two 1.0-ml injections ( 50 mg / ml solution , > 1 inch apart ) . subjects receiving exenatide initiated treatment in the physician 's office and subsequently self - administered according to the package insert . after 16 weeks , subjects entered an 11-week washout phase to assess safety and immunogenicity . a1c and fasting plasma glucose ( fpg ) measurements were performed at screening , baseline , and weeks 2 ( fpg only ) , 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . fasting fructosamine , c - peptide , glucagon , insulin , and lipids were measured at baseline and weeks 5 , 8 , 12 , and 16 . -cell function was calculated using homeostasis model assessment ( 24 ) . adverse event assessments and safety analyses were conducted throughout the study . immunogenicity samples were taken at baseline and weeks 1 , 4 , 8 , 12 , and 16 and were screened for anti - albiglutide antibodies via elisa ( 20 ) . plasma samples were collected to characterize the pharmacokinetics of albiglutide , quantified by elisa ( 20 ) at baseline and at weeks 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . population pharmacokinetics analysis was performed using a nonlinear mixed - effect modeling approach with nonmem software ( icon development solutions , ellicott city , md ) . a1c , fpg , and albiglutide concentrations were obtained at weeks 17 , 18 , 20 , 23 , and 27 ; fasting fructosamine , c - peptide , glucagon , insulin , and lipid profiles were obtained at weeks 20 and 27 ; and immunogenicity assessments were examined at weeks 20 , 23 , and 27 . the primary objective was to evaluate the dose response of albiglutide for safety and efficacy . with 30 subjects planned in each arm , a two - sided 95% ci for each group mean response had a half - width of 0.36% on the a1c scale , assuming a standard deviation of 1.00% . the primary efficacy end point was change from baseline a1c at week 16 versus placebo across different doses within each schedule ( weekly , biweekly , and monthly ) . the primary analysis was an ancova model with main effects for group and prior metformin therapy , adjusting for baseline a1c . comparisons were made on the intent - to - treat population , defined as all randomly assigned subjects with at least one postbaseline assessment of the primary end point , using last observation carried forward . the safety population included all randomized subjects who received at least one dose of any medication . an interim analysis was conducted at 8 weeks for administrative purposes by an independent statistical analysis group ; blinding was retained for study investigators and study personnel with daily operational responsibility . no formal interim inferential hypothesis testing was conducted ; the study conduct was unaltered based on the results of the interim analysis . subjects were randomized into 1 of 10 treatment arms : double - blind placebo ( matched to albiglutide arms ) ; albiglutide weekly ( 4 , 15 , or 30 mg ) , every 2 weeks ( biweekly ; 15 , 30 , or 50 mg ) , or monthly ( 50 or 100 mg ) ; or open - label exenatide ( 5 g twice daily for 4 weeks followed by 12 weeks of 10 g twice daily ) . albiglutide ( formulated in 10 mmol / l sodium phosphate , 4.2% trehalose , 2.8% mannitol , 0.01% polysorbate-80 ) and placebo ( same formulation without active principle ) were administered in the physician 's office over the course of 16 weeks . subjects receiving 4 mg albiglutide were given 1.0 ml ( 4 mg / ml solution ) . subjects receiving 15 , 30 , or 50 mg albiglutide were given 0.32 , 0.65 , or 1.0 ml ( 50 mg / ml solution ) . subjects receiving 100 mg albiglutide were given two 1.0-ml injections ( 50 mg / ml solution , > 1 inch apart ) . subjects receiving exenatide initiated treatment in the physician 's office and subsequently self - administered according to the package insert . after 16 weeks a1c and fasting plasma glucose ( fpg ) measurements were performed at screening , baseline , and weeks 2 ( fpg only ) , 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . fasting fructosamine , c - peptide , glucagon , insulin , and lipids were measured at baseline and weeks 5 , 8 , 12 , and 16 . -cell function was calculated using homeostasis model assessment ( 24 ) . adverse event assessments and safety analyses were conducted throughout the study . immunogenicity samples were taken at baseline and weeks 1 , 4 , 8 , 12 , and 16 and were screened for anti - albiglutide antibodies via elisa ( 20 ) . plasma samples were collected to characterize the pharmacokinetics of albiglutide , quantified by elisa ( 20 ) at baseline and at weeks 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . population pharmacokinetics analysis was performed using a nonlinear mixed - effect modeling approach with nonmem software ( icon development solutions , ellicott city , md ) . a1c , fpg , and albiglutide concentrations were obtained at weeks 17 , 18 , 20 , 23 , and 27 ; fasting fructosamine , c - peptide , glucagon , insulin , and lipid profiles were obtained at weeks 20 and 27 ; and immunogenicity assessments were examined at weeks 20 , 23 , and 27 . a1c and fasting plasma glucose ( fpg ) measurements were performed at screening , baseline , and weeks 2 ( fpg only ) , 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . fasting fructosamine , c - peptide , glucagon , insulin , and lipids were measured at baseline and weeks 5 , 8 , 12 , and 16 . -cell function was calculated using homeostasis model assessment ( 24 ) . adverse event assessments and safety analyses were conducted throughout the study . immunogenicity samples were taken at baseline and weeks 1 , 4 , 8 , 12 , and 16 and were screened for anti - albiglutide antibodies via elisa ( 20 ) . plasma samples were collected to characterize the pharmacokinetics of albiglutide , quantified by elisa ( 20 ) at baseline and at weeks 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . population pharmacokinetics analysis was performed using a nonlinear mixed - effect modeling approach with nonmem software ( icon development solutions , ellicott city , md ) . a1c , fpg , and albiglutide concentrations were obtained at weeks 17 , 18 , 20 , 23 , and 27 ; fasting fructosamine , c - peptide , glucagon , insulin , and lipid profiles were obtained at weeks 20 and 27 ; and immunogenicity assessments were examined at weeks 20 , 23 , and 27 . the primary objective was to evaluate the dose response of albiglutide for safety and efficacy . with 30 subjects planned in each arm , a two - sided 95% ci for each group mean response had a half - width of 0.36% on the a1c scale , assuming a standard deviation of 1.00% . the primary efficacy end point was change from baseline a1c at week 16 versus placebo across different doses within each schedule ( weekly , biweekly , and monthly ) . the primary analysis was an ancova model with main effects for group and prior metformin therapy , adjusting for baseline a1c . comparisons were made on the intent - to - treat population , defined as all randomly assigned subjects with at least one postbaseline assessment of the primary end point , using last observation carried forward . the safety population included all randomized subjects who received at least one dose of any medication . an interim analysis was conducted at 8 weeks for administrative purposes by an independent statistical analysis group ; blinding was retained for study investigators and study personnel with daily operational responsibility . no formal interim inferential hypothesis testing was conducted ; the study conduct was unaltered based on the results of the interim analysis . of 361 subjects randomized , 356 received treatment and were included in the safety analysis , 345 were included in the efficacy analysis , and 255 completed the 16-week trial . withdrawal rates were similar across groups ( online appendix 2 ) ; the most frequent reason for withdrawal was adverse events . adverse events occurring in at least one subject leading to withdrawal included hyperglycemia ( 011.8% , n = 24 , mostly in placebo and lower - dose groups ) , gi events ( 011.4% , n = 10 , across groups ) , injection site events ( 09.7% , n = 11 , mostly in higher - dose groups ) , and hypertriglyceridemia ( 05.7% , n = 2 of 35 receiving 50 mg albiglutide biweekly , deemed unrelated to study drug ; 0% in other groups ) . other reasons for withdrawal included loss to follow - up , protocol violations , and voluntary withdrawal . a similar proportion of subjects receiving placebo or albiglutide were drug nae ( 25.734.4% ) or receiving metformin monotherapy . the groups were similar in terms of race / ethnicity ( 43.871.0% caucasian ; 87.1 and 12.9% of subjects were from u.s . and latin american clinics , respectively ) , and the rates of dyslipidemia , hypertension , and coronary artery disease were 50.080.0 , 47.167.6 , and 015.2% , respectively . after 16 weeks , albiglutide significantly reduced a1c in a generally dose - dependent manner within each dose schedule ( table 1 ) ( fig . mean a1c reductions from baseline in subjects receiving the highest dose in each treatment schedule were 0.87 , 0.79 , and 0.87% for 30 mg weekly , 50 mg biweekly , and 100 mg monthly , respectively , versus placebo ( 0.17% ) or exenatide ( 0.54% ) . a1c reductions ( based on ancova model ) for the highest doses compared with placebo were statistically significant : 30 mg weekly , 0.62% ( 95% ci 1.03 to 0.22 ) , p < 0.003 ; 50 mg biweekly , 0.57% ( 0.96 to 0.19 ) , p < 0.003 ; and 100 mg monthly , 0.60% ( 0.99 to 0.22 ) , p < 0.002 . numerically greater reductions in a1c were observed in subjects with baseline a1c 8.5% . change from baseline in glycemic parameters at 16 weeks data are means sd for the intent - to - treat population , last observation carried forward . the proportion of subjects achieving the ada target a1c ( < 7.0% ) at week 16 increased with increasing doses of albiglutide within each schedule ; similar proportions of subjects achieved target a1c at the highest albiglutide dose among the three schedules . more subjects receiving albiglutide 30 mg weekly ( 52% ) , 50 mg biweekly ( 53% ) , and 100 mg monthly ( 48% ) achieved a1c < 7.0% compared with subjects receiving placebo ( 20% ) and exenatide ( 35% ) ( fig . the time course of albiglutide - induced changes in fpg demonstrated that each schedule elicited a dose - dependent fpg reduction over 16 weeks , with no changes in fpg observed with placebo . rapid reductions in fpg were observed , with similar fpg reductions at the 16-week end point for each of the highest doses ( fig . statistically significant reductions were seen for fpg compared with placebo at week 16 ( 1.38 mmol / l [ p < 0.02 ] , 1.16 mmol / l [ p < 0.03 ] , and 1.17 mmol / l [ p < 0.03 ] for 30 mg weekly , 50 mg biweekly , and 100 mg monthly albiglutide doses , respectively ) . the 4- and the greatest fluctuations in fpg over time were observed in subjects receiving albiglutide 100 mg monthly ( fig . exenatide was associated with a relatively consistent fpg profile over time that was numerically less than fpg reductions seen with the highest doses of albiglutide ( fig . small improvements in -cell function ( assessed by homeostasis model assessment of -cell function ) were noted in subjects receiving albiglutide ( online appendix 4 ) . a consistent trend in weight reduction was noted with average weight loss ranging from 1.1 to 1.7 kg in subjects receiving the highest albiglutide dose in each schedule . these reductions were numerically greater than those for subjects receiving placebo ( 0.7 kg ) but less than those for subjects receiving exenatide ( 2.4 kg ) . albiglutide and exenatide tended to reduce mean systolic and diastolic blood pressure but did not significantly change the plasma lipoprotein profile ( online appendix 4 ) . the percentage of subjects reporting at least one adverse event was similar across groups ( 6785% ) . the most frequently reported adverse events included nausea ( 11.854.3% ) , vomiting ( 041.2% ) , headache ( 5.923.5% ) , dizziness ( 5.714.3% ) , nasopharyngitis ( 5.711.4% ) , back pain ( 014.3% ) , influenza ( 09.7% ) , upper respiratory tract infections ( 015.2% ) , and local skin reactions ( 2.928.6% ) ( online appendix 5 ) . the proportion of subjects who experienced nausea and/or vomiting was lower with administration of 30 mg albiglutide compared with the proportion of subjects receiving higher doses . in the 30-mg weekly arm , 29.0% of subjects experienced nausea and/or vomiting , compared with 54.3% in the 50-mg biweekly group and 55.9% in the 100-mg monthly group . the percentage of exenatide subjects who experienced nausea and/or vomiting also was higher ( 45.7% ) than in the 30-mg weekly albiglutide group . 2 ) of nausea and/or vomiting revealed that the proportion of subjects experiencing these adverse events each week was low in the 30-mg weekly arm ( < 10% ) and declined over the course of the study , with no reports of nausea or vomiting after 8 weeks ( fig . although the proportion of nausea and/or vomiting for the 50-mg biweekly dose was greater than for the 30-mg weekly dose , it also declined over the study period ( fig . subjects receiving the 100-mg monthly dose also experienced higher rates of nausea and/or vomiting , with peak incidence occurring following each monthly dose administration . the overall rate was higher for 100-mg monthly than for the other albiglutide groups ( fig . 2c ) . the incidence of nausea and/or vomiting with exenatide reached 20% by week 2 , increased at week 5 to a peak incidence of 29% ( due to label - based titration ) , and also declined over the study period ( fig . time course of nausea and vomiting as adverse events . the percentage of subjects experiencing vomiting with or without nausea ( ) or nausea ( ) adverse events during each week of the 16-week trial for albiglutide 30 mg weekly ( a ) , albiglutide 50 mg biweekly ( b ) , albiglutide 100 mg monthly ( c ) , placebo ( d ) , and exenatide ( e ) . other adverse events were less common than gi - related events and were similar across groups with no dose - dependent trends . documented hypoglycemia was not increased with albiglutide ( 03.1% ) relative to placebo ( 3.9% ) and exenatide ( 2.9% ) . cardiac - related adverse events ( eight subjects ; six assessed as severe ) were distributed across groups , with no dose - dependent trends ( online appendix 5 ) . eight subjects ( 2.5% ) had confirmed positive anti - albiglutide antibodies at least once in the placebo and albiglutide arms after baseline measurement . however , two subjects tested positive prior to albiglutide treatment , and one subject who tested positive received placebo . the remaining five albiglutide - positive subjects were detected in the weekly and biweekly arms . the appearance of anti - albiglutide antibodies was largely transient : one subject remained positive at week 27 . antibodies were non - neutralizing and low titer and showed cross - reactivity with glp-1 in four of five subjects . there was no obvious association between the presence of anti - albiglutide antibodies and either efficacy or safety . injection site local skin reactions , most of which were small , were observed in the study and were more common in the albiglutide groups ( 2.928.6% ) compared with placebo ( 5.9% ) and exenatide ( 2.9% ) . injection site reactions tended to occur once per person in subjects receiving 30 mg albiglutide and approximately twice per person in subjects receiving higher albiglutide doses . skin reactions were not associated with positive ige antibodies or neutralizing antibodies and did not worsen upon repeated dosing or exhibit dose dependency . steady - state albiglutide levels were reached within 4 to 5 weeks of the first dose ( online appendix 6 ) . greater peak / trough fluctuations in circulating albiglutide concentrations were observed with less frequent administration of higher doses . of 361 subjects randomized , 356 received treatment and were included in the safety analysis , 345 were included in the efficacy analysis , and 255 completed the 16-week trial . withdrawal rates were similar across groups ( online appendix 2 ) ; the most frequent reason for withdrawal was adverse events . adverse events occurring in at least one subject leading to withdrawal included hyperglycemia ( 011.8% , n = 24 , mostly in placebo and lower - dose groups ) , gi events ( 011.4% , n = 10 , across groups ) , injection site events ( 09.7% , n = 11 , mostly in higher - dose groups ) , and hypertriglyceridemia ( 05.7% , n = 2 of 35 receiving 50 mg albiglutide biweekly , deemed unrelated to study drug ; 0% in other groups ) . other reasons for withdrawal included loss to follow - up , protocol violations , and voluntary withdrawal . a similar proportion of subjects receiving placebo or albiglutide were drug nae ( 25.734.4% ) or receiving metformin monotherapy . the groups were similar in terms of race / ethnicity ( 43.871.0% caucasian ; 87.1 and 12.9% of subjects were from u.s . and latin american clinics , respectively ) , and the rates of dyslipidemia , hypertension , and coronary artery disease were 50.080.0 , 47.167.6 , and 015.2% , respectively . after 16 weeks , albiglutide significantly reduced a1c in a generally dose - dependent manner within each dose schedule ( table 1 ) ( fig . 1a ) . mean a1c reductions from baseline in subjects receiving the highest dose in each treatment schedule were 0.87 , 0.79 , and 0.87% for 30 mg weekly , 50 mg biweekly , and 100 mg monthly , respectively , versus placebo ( 0.17% ) or exenatide ( 0.54% ) . a1c reductions ( based on ancova model ) for the highest doses compared with placebo were statistically significant : 30 mg weekly , 0.62% ( 95% ci 1.03 to 0.22 ) , p < 0.003 ; 50 mg biweekly , 0.57% ( 0.96 to 0.19 ) , p < 0.003 ; and 100 mg monthly , 0.60% ( 0.99 to 0.22 ) , p < 0.002 . numerically greater reductions in a1c were observed in subjects with baseline a1c 8.5% . change from baseline in glycemic parameters at 16 weeks data are means sd for the intent - to - treat population , last observation carried forward . the proportion of subjects achieving the ada target a1c ( < 7.0% ) at week 16 increased with increasing doses of albiglutide within each schedule ; similar proportions of subjects achieved target a1c at the highest albiglutide dose among the three schedules . more subjects receiving albiglutide 30 mg weekly ( 52% ) , 50 mg biweekly ( 53% ) , and 100 mg monthly ( 48% ) achieved a1c < 7.0% compared with subjects receiving placebo ( 20% ) and exenatide ( 35% ) ( fig . the time course of albiglutide - induced changes in fpg demonstrated that each schedule elicited a dose - dependent fpg reduction over 16 weeks , with no changes in fpg observed with placebo . rapid reductions in fpg were observed , with similar fpg reductions at the 16-week end point for each of the highest doses ( fig . statistically significant reductions were seen for fpg compared with placebo at week 16 ( 1.38 mmol / l [ p < 0.02 ] , 1.16 mmol / l [ p < 0.03 ] , and 1.17 mmol / l [ p < 0.03 ] for 30 mg weekly , 50 mg biweekly , and 100 mg monthly albiglutide doses , respectively ) . the 4- and 15-mg weekly dose regimens of albiglutide reduced fpg but were less effective . the greatest fluctuations in fpg over time were observed in subjects receiving albiglutide 100 mg monthly ( fig . exenatide was associated with a relatively consistent fpg profile over time that was numerically less than fpg reductions seen with the highest doses of albiglutide ( fig . small improvements in -cell function ( assessed by homeostasis model assessment of -cell function ) were noted in subjects receiving albiglutide ( online appendix 4 ) . there was no significant difference in weight reduction among groups . a consistent trend in weight reduction was noted with average weight loss ranging from 1.1 to 1.7 kg in subjects receiving the highest albiglutide dose in each schedule . these reductions were numerically greater than those for subjects receiving placebo ( 0.7 kg ) but less than those for subjects receiving exenatide ( 2.4 kg ) . albiglutide and exenatide tended to reduce mean systolic and diastolic blood pressure but did not significantly change the plasma lipoprotein profile ( online appendix 4 ) . the percentage of subjects reporting at least one adverse event was similar across groups ( 6785% ) . the most frequently reported adverse events included nausea ( 11.854.3% ) , vomiting ( 041.2% ) , headache ( 5.923.5% ) , dizziness ( 5.714.3% ) , nasopharyngitis ( 5.711.4% ) , back pain ( 014.3% ) , influenza ( 09.7% ) , upper respiratory tract infections ( 015.2% ) , and local skin reactions ( 2.928.6% ) ( online appendix 5 ) . the proportion of subjects who experienced nausea and/or vomiting was lower with administration of 30 mg albiglutide compared with the proportion of subjects receiving higher doses . in the 30-mg weekly arm , 29.0% of subjects experienced nausea and/or vomiting , compared with 54.3% in the 50-mg biweekly group and 55.9% in the 100-mg monthly group . the percentage of exenatide subjects who experienced nausea and/or vomiting also was higher ( 45.7% ) than in the 30-mg weekly albiglutide group . 2 ) of nausea and/or vomiting revealed that the proportion of subjects experiencing these adverse events each week was low in the 30-mg weekly arm ( < 10% ) and declined over the course of the study , with no reports of nausea or vomiting after 8 weeks ( fig . although the proportion of nausea and/or vomiting for the 50-mg biweekly dose was greater than for the 30-mg weekly dose , it also declined over the study period ( fig . subjects receiving the 100-mg monthly dose also experienced higher rates of nausea and/or vomiting , with peak incidence occurring following each monthly dose administration . the overall rate was higher for 100-mg monthly than for the other albiglutide groups ( fig . 2c ) . the incidence of nausea and/or vomiting with exenatide reached 20% by week 2 , increased at week 5 to a peak incidence of 29% ( due to label - based titration ) , and also declined over the study period ( fig . the percentage of subjects experiencing vomiting with or without nausea ( ) or nausea ( ) adverse events during each week of the 16-week trial for albiglutide 30 mg weekly ( a ) , albiglutide 50 mg biweekly ( b ) , albiglutide 100 mg monthly ( c ) , placebo ( d ) , and exenatide ( e ) . other adverse events were less common than gi - related events and were similar across groups with no dose - dependent trends . documented hypoglycemia was not increased with albiglutide ( 03.1% ) relative to placebo ( 3.9% ) and exenatide ( 2.9% ) . cardiac - related adverse events ( eight subjects ; six assessed as severe ) were distributed across groups , with no dose - dependent trends ( online appendix 5 ) . eight subjects ( 2.5% ) had confirmed positive anti - albiglutide antibodies at least once in the placebo and albiglutide arms after baseline measurement . however , two subjects tested positive prior to albiglutide treatment , and one subject who tested positive received placebo . the remaining five albiglutide - positive subjects were detected in the weekly and biweekly arms . the appearance of anti - albiglutide antibodies was largely transient : one subject remained positive at week 27 . antibodies were non - neutralizing and low titer and showed cross - reactivity with glp-1 in four of five subjects . there was no obvious association between the presence of anti - albiglutide antibodies and either efficacy or safety . injection site local skin reactions , most of which were small , were observed in the study and were more common in the albiglutide groups ( 2.928.6% ) compared with placebo ( 5.9% ) and exenatide ( 2.9% ) . injection site reactions tended to occur once per person in subjects receiving 30 mg albiglutide and approximately twice per person in subjects receiving higher albiglutide doses . skin reactions were not associated with positive ige antibodies or neutralizing antibodies and did not worsen upon repeated dosing or exhibit dose dependency . steady - state albiglutide levels were reached within 4 to 5 weeks of the first dose ( online appendix 6 ) . greater peak / trough fluctuations in circulating albiglutide concentrations were observed with less frequent administration of higher doses . in this study , the dose- and time - dependent effects of albiglutide , a long - acting glp-1 receptor agonist , was evaluated to identify potential dose regimens for future studies . within each dose schedule , albiglutide was associated with generally dose - dependent a1c reductions that were significantly different from placebo . maximum doses in each schedule ( albiglutide 30 mg weekly , 50 mg biweekly , and 100 mg monthly ) elicited similar responses in a1c , providing meaningful reductions within the range of 0.80.9% from a mean baseline a1c of 8.0% . albiglutide also significantly reduced fpg at week 16 compared with placebo , with reductions observed at the time of the first assessment ( 2 weeks postdose ) . in a previous study , fpg reductions variability in glycemic response appeared to be related to circulating concentrations of albiglutide . with a plasma half - life of 5 days and at doses sufficient to achieve consistent therapeutic response ( i.e. , 30 mg ) , weekly dosing provided consistent fpg reduction . greater fpg fluctuations were observed following biweekly or monthly dosing , despite similar a1c reductions . albiglutide 30-mg weekly dosing elicited steady and consistent improvements in fpg reductions , with a more favorable nausea / vomiting profile than exenatide . when dosed biweekly , 50 mg albiglutide also improved glycemic indexes but with higher gi adverse event rates , possibly related to the higher initial dose . in all dosing schedules however , when dosed monthly , albiglutide did not produce stable fpg reductions between dosing and was associated with higher gi event rates . the increase in fpg fluctuation and gi events in the biweekly and monthly regimens is likely due to fluctuations in plasma albiglutide concentrations resulting from less frequent dosing . an escalating - dose titration for the biweekly regimen might have resulted in a lower frequency of gi events . this approach may be tested in future studies to explore the possibility of using biweekly dosing as a maintenance option in patients who respond well and tolerate the initial weekly regimen . mechanistically , the reasons for differences in the tolerability of albiglutide and exenatide are unknown but may be due to differences in pharmacokinetics , including gradual absorption ( tmax is 3 days vs. 2.1 h for albiglutide and exenatide , respectively ) and a long plasma half - life of 5 days resulting in a steady state achieved after 45 doses for 30 mg albiglutide weekly . the slow accumulation of albiglutide may ameliorate gi intolerability often observed with exenatide , and albiglutide 's relative impermeance to the central nervous system may result in a more benign profile with respect to nausea and vomiting than exenatide ( 21 ) . weight loss was similar across albiglutide arms and numerically less than with exenatide , which had a higher baseline bmi . exploratory post hoc analyses indicated that there was no obvious correlation between reduction in a1c and weight loss for all albiglutide doses ( data not shown ) . however , larger and longer - term studies will determine the true effect on weight and cardiometabolic parameters . immunogenicity was closely monitored owing to the possible appearance of neutralizing antibodies or the development of immediate hypersensitivity reactions . in this study , anti - albiglutide antibodies however , the observation that positive titers of anti - albiglutide antibodies were detected in three subjects at baseline suggests that the immunogenicity rate may be overestimated . exenatide , which has 53% homology to human glp-1 ( 14 ) , is associated with antibody development following administration with twice - daily ( 1518 ) and weekly ( 25 ) formulations ( > 40% ) . antibody formation may attenuate efficacy , especially among patients developing high levels of anti - exenatide antibodies ( 25 ) . first , the number of subjects in each arm is relatively small compared with phase iii studies . second , relative to the number of subjects , the dropout rate was high owing to adverse events , loss to follow - up , and voluntary withdrawals . third , the duration of active treatment was 16 weeks , so the magnitude or durability of response can not be fully appreciated . fourth , no escalating doses were tested for biweekly and monthly dosing , which may have attenuated gi adverse events and fpg fluctuations . finally , because exenatide was administered open label , formal statistical comparisons were not conducted between exenatide and albiglutide . in conclusion , albiglutide improved glucose control in a dose - dependent manner when given weekly and biweekly . higher monthly doses of albiglutide were efficacious , but their use was constrained by the higher frequency of gi - related adverse events . weekly albiglutide significantly improved glycemic control with an acceptable safety and tolerability profile and modest weight loss without increasing the risk of hypoglycemia or immunological response in subjects with type 2 diabetes . future studies may elucidate whether titration dosing or biweekly scheduling could be options for patients who respond to and tolerate the initial weekly regimen .

objectiveto evaluate the efficacy , safety , and tolerability of incremental doses of albiglutide , a long - acting glucagon - like peptide-1 receptor agonist , administered with three dosing schedules in patients with type 2 diabetes inadequately controlled with diet and exercise or metformin monotherapy.research design and methodsin this randomized multicenter double - blind parallel - group study , 356 type 2 diabetic subjects with similar mean baseline characteristics ( age 54 years , diabetes duration 4.9 years , bmi 32.1 kg / m2 , a1c 8.0% ) received subcutaneous placebo or albiglutide ( weekly [ 4 , 15 , or 30 mg ] , biweekly [ 15 , 30 , or 50 mg ] , or monthly [ 50 or 100 mg ] ) or exenatide twice daily as an open - label active reference ( per labeling in metformin subjects only ) over 16 weeks followed by an 11-week washout period . the main outcome measure was change from baseline a1c of albiglutide groups versus placebo at week 16.resultsdose-dependent reductions in a1c were observed within all albiglutide schedules . mean a1c was similarly reduced from baseline by albiglutide 30 mg weekly , 50 mg biweekly ( every 2 weeks ) , and 100 mg monthly ( 0.87 , 0.79 , and 0.87% , respectively ) versus placebo ( 0.17% , p < 0.004 ) and exenatide ( 0.54% ) . weight loss ( 1.1 to 1.7 kg ) was observed with these three albiglutide doses with no significant between - group effects . the incidence of gastrointestinal adverse events in subjects receiving albiglutide 30 mg weekly was less than that observed for the highest biweekly and monthly doses of albiglutide or exenatide.conclusionsweekly albiglutide administration significantly improved glycemic control and elicited weight loss in type 2 diabetic patients , with a favorable safety and tolerability profile .

RESEARCH DESIGN AND METHODS Randomization Assessments On therapy. Eleven-week washout. Statistical analysis RESULTS Subject disposition and baseline characteristics Efficacy Safety and tolerability Pharmacokinetics CONCLUSIONS Supplementary Material

this phase ii trial was a prospective randomized double - blind placebo - controlled parallel - group study conducted between april 2007 and may 2008 at 118 sites in the u.s . men and women of non - childbearing potential ( age 1875 years ) were eligible for inclusion if diagnosed with type 2 diabetes 3 months before screening . subjects were drug nave ( diet and exercise ) or treated with metformin monotherapy and stable for > 3 months before prescreening ( 1 week prior to screening visit ) . subjects were randomized into 1 of 10 treatment arms : double - blind placebo ( matched to albiglutide arms ) ; albiglutide weekly ( 4 , 15 , or 30 mg ) , every 2 weeks ( biweekly ; 15 , 30 , or 50 mg ) , or monthly ( 50 or 100 mg ) ; or open - label exenatide ( 5 g twice daily for 4 weeks followed by 12 weeks of 10 g twice daily ) . albiglutide ( formulated in 10 mmol / l sodium phosphate , 4.2% trehalose , 2.8% mannitol , 0.01% polysorbate-80 ) and placebo ( same formulation without active principle ) were administered in the physician 's office over the course of 16 weeks . subjects receiving 15 , 30 , or 50 mg albiglutide were given 0.32 , 0.65 , or 1.0 ml ( 50 mg / ml solution ) . after 16 weeks , subjects entered an 11-week washout phase to assess safety and immunogenicity . a1c and fasting plasma glucose ( fpg ) measurements were performed at screening , baseline , and weeks 2 ( fpg only ) , 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . plasma samples were collected to characterize the pharmacokinetics of albiglutide , quantified by elisa ( 20 ) at baseline and at weeks 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . the primary efficacy end point was change from baseline a1c at week 16 versus placebo across different doses within each schedule ( weekly , biweekly , and monthly ) . subjects were randomized into 1 of 10 treatment arms : double - blind placebo ( matched to albiglutide arms ) ; albiglutide weekly ( 4 , 15 , or 30 mg ) , every 2 weeks ( biweekly ; 15 , 30 , or 50 mg ) , or monthly ( 50 or 100 mg ) ; or open - label exenatide ( 5 g twice daily for 4 weeks followed by 12 weeks of 10 g twice daily ) . albiglutide ( formulated in 10 mmol / l sodium phosphate , 4.2% trehalose , 2.8% mannitol , 0.01% polysorbate-80 ) and placebo ( same formulation without active principle ) were administered in the physician 's office over the course of 16 weeks . subjects receiving 15 , 30 , or 50 mg albiglutide were given 0.32 , 0.65 , or 1.0 ml ( 50 mg / ml solution ) . after 16 weeks a1c and fasting plasma glucose ( fpg ) measurements were performed at screening , baseline , and weeks 2 ( fpg only ) , 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . plasma samples were collected to characterize the pharmacokinetics of albiglutide , quantified by elisa ( 20 ) at baseline and at weeks 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . a1c and fasting plasma glucose ( fpg ) measurements were performed at screening , baseline , and weeks 2 ( fpg only ) , 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . plasma samples were collected to characterize the pharmacokinetics of albiglutide , quantified by elisa ( 20 ) at baseline and at weeks 4 , 5 , 7 , 8 , 9 , 12 , 15 , and 16 . the primary objective was to evaluate the dose response of albiglutide for safety and efficacy . the primary efficacy end point was change from baseline a1c at week 16 versus placebo across different doses within each schedule ( weekly , biweekly , and monthly ) . of 361 subjects randomized , 356 received treatment and were included in the safety analysis , 345 were included in the efficacy analysis , and 255 completed the 16-week trial . adverse events occurring in at least one subject leading to withdrawal included hyperglycemia ( 011.8% , n = 24 , mostly in placebo and lower - dose groups ) , gi events ( 011.4% , n = 10 , across groups ) , injection site events ( 09.7% , n = 11 , mostly in higher - dose groups ) , and hypertriglyceridemia ( 05.7% , n = 2 of 35 receiving 50 mg albiglutide biweekly , deemed unrelated to study drug ; 0% in other groups ) . a similar proportion of subjects receiving placebo or albiglutide were drug nae ( 25.734.4% ) or receiving metformin monotherapy . and latin american clinics , respectively ) , and the rates of dyslipidemia , hypertension , and coronary artery disease were 50.080.0 , 47.167.6 , and 015.2% , respectively . mean a1c reductions from baseline in subjects receiving the highest dose in each treatment schedule were 0.87 , 0.79 , and 0.87% for 30 mg weekly , 50 mg biweekly , and 100 mg monthly , respectively , versus placebo ( 0.17% ) or exenatide ( 0.54% ) . a1c reductions ( based on ancova model ) for the highest doses compared with placebo were statistically significant : 30 mg weekly , 0.62% ( 95% ci 1.03 to 0.22 ) , p < 0.003 ; 50 mg biweekly , 0.57% ( 0.96 to 0.19 ) , p < 0.003 ; and 100 mg monthly , 0.60% ( 0.99 to 0.22 ) , p < 0.002 . numerically greater reductions in a1c were observed in subjects with baseline a1c 8.5% . change from baseline in glycemic parameters at 16 weeks data are means sd for the intent - to - treat population , last observation carried forward . the proportion of subjects achieving the ada target a1c ( < 7.0% ) at week 16 increased with increasing doses of albiglutide within each schedule ; similar proportions of subjects achieved target a1c at the highest albiglutide dose among the three schedules . more subjects receiving albiglutide 30 mg weekly ( 52% ) , 50 mg biweekly ( 53% ) , and 100 mg monthly ( 48% ) achieved a1c < 7.0% compared with subjects receiving placebo ( 20% ) and exenatide ( 35% ) ( fig . the time course of albiglutide - induced changes in fpg demonstrated that each schedule elicited a dose - dependent fpg reduction over 16 weeks , with no changes in fpg observed with placebo . rapid reductions in fpg were observed , with similar fpg reductions at the 16-week end point for each of the highest doses ( fig . statistically significant reductions were seen for fpg compared with placebo at week 16 ( 1.38 mmol / l [ p < 0.02 ] , 1.16 mmol / l [ p < 0.03 ] , and 1.17 mmol / l [ p < 0.03 ] for 30 mg weekly , 50 mg biweekly , and 100 mg monthly albiglutide doses , respectively ) . the 4- and the greatest fluctuations in fpg over time were observed in subjects receiving albiglutide 100 mg monthly ( fig . exenatide was associated with a relatively consistent fpg profile over time that was numerically less than fpg reductions seen with the highest doses of albiglutide ( fig . small improvements in -cell function ( assessed by homeostasis model assessment of -cell function ) were noted in subjects receiving albiglutide ( online appendix 4 ) . a consistent trend in weight reduction was noted with average weight loss ranging from 1.1 to 1.7 kg in subjects receiving the highest albiglutide dose in each schedule . these reductions were numerically greater than those for subjects receiving placebo ( 0.7 kg ) but less than those for subjects receiving exenatide ( 2.4 kg ) . the most frequently reported adverse events included nausea ( 11.854.3% ) , vomiting ( 041.2% ) , headache ( 5.923.5% ) , dizziness ( 5.714.3% ) , nasopharyngitis ( 5.711.4% ) , back pain ( 014.3% ) , influenza ( 09.7% ) , upper respiratory tract infections ( 015.2% ) , and local skin reactions ( 2.928.6% ) ( online appendix 5 ) . 2 ) of nausea and/or vomiting revealed that the proportion of subjects experiencing these adverse events each week was low in the 30-mg weekly arm ( < 10% ) and declined over the course of the study , with no reports of nausea or vomiting after 8 weeks ( fig . the incidence of nausea and/or vomiting with exenatide reached 20% by week 2 , increased at week 5 to a peak incidence of 29% ( due to label - based titration ) , and also declined over the study period ( fig . the percentage of subjects experiencing vomiting with or without nausea ( ) or nausea ( ) adverse events during each week of the 16-week trial for albiglutide 30 mg weekly ( a ) , albiglutide 50 mg biweekly ( b ) , albiglutide 100 mg monthly ( c ) , placebo ( d ) , and exenatide ( e ) . documented hypoglycemia was not increased with albiglutide ( 03.1% ) relative to placebo ( 3.9% ) and exenatide ( 2.9% ) . cardiac - related adverse events ( eight subjects ; six assessed as severe ) were distributed across groups , with no dose - dependent trends ( online appendix 5 ) . injection site local skin reactions , most of which were small , were observed in the study and were more common in the albiglutide groups ( 2.928.6% ) compared with placebo ( 5.9% ) and exenatide ( 2.9% ) . injection site reactions tended to occur once per person in subjects receiving 30 mg albiglutide and approximately twice per person in subjects receiving higher albiglutide doses . of 361 subjects randomized , 356 received treatment and were included in the safety analysis , 345 were included in the efficacy analysis , and 255 completed the 16-week trial . adverse events occurring in at least one subject leading to withdrawal included hyperglycemia ( 011.8% , n = 24 , mostly in placebo and lower - dose groups ) , gi events ( 011.4% , n = 10 , across groups ) , injection site events ( 09.7% , n = 11 , mostly in higher - dose groups ) , and hypertriglyceridemia ( 05.7% , n = 2 of 35 receiving 50 mg albiglutide biweekly , deemed unrelated to study drug ; 0% in other groups ) . a similar proportion of subjects receiving placebo or albiglutide were drug nae ( 25.734.4% ) or receiving metformin monotherapy . and latin american clinics , respectively ) , and the rates of dyslipidemia , hypertension , and coronary artery disease were 50.080.0 , 47.167.6 , and 015.2% , respectively . mean a1c reductions from baseline in subjects receiving the highest dose in each treatment schedule were 0.87 , 0.79 , and 0.87% for 30 mg weekly , 50 mg biweekly , and 100 mg monthly , respectively , versus placebo ( 0.17% ) or exenatide ( 0.54% ) . a1c reductions ( based on ancova model ) for the highest doses compared with placebo were statistically significant : 30 mg weekly , 0.62% ( 95% ci 1.03 to 0.22 ) , p < 0.003 ; 50 mg biweekly , 0.57% ( 0.96 to 0.19 ) , p < 0.003 ; and 100 mg monthly , 0.60% ( 0.99 to 0.22 ) , p < 0.002 . numerically greater reductions in a1c were observed in subjects with baseline a1c 8.5% . change from baseline in glycemic parameters at 16 weeks data are means sd for the intent - to - treat population , last observation carried forward . the proportion of subjects achieving the ada target a1c ( < 7.0% ) at week 16 increased with increasing doses of albiglutide within each schedule ; similar proportions of subjects achieved target a1c at the highest albiglutide dose among the three schedules . more subjects receiving albiglutide 30 mg weekly ( 52% ) , 50 mg biweekly ( 53% ) , and 100 mg monthly ( 48% ) achieved a1c < 7.0% compared with subjects receiving placebo ( 20% ) and exenatide ( 35% ) ( fig . the time course of albiglutide - induced changes in fpg demonstrated that each schedule elicited a dose - dependent fpg reduction over 16 weeks , with no changes in fpg observed with placebo . rapid reductions in fpg were observed , with similar fpg reductions at the 16-week end point for each of the highest doses ( fig . statistically significant reductions were seen for fpg compared with placebo at week 16 ( 1.38 mmol / l [ p < 0.02 ] , 1.16 mmol / l [ p < 0.03 ] , and 1.17 mmol / l [ p < 0.03 ] for 30 mg weekly , 50 mg biweekly , and 100 mg monthly albiglutide doses , respectively ) . the greatest fluctuations in fpg over time were observed in subjects receiving albiglutide 100 mg monthly ( fig . exenatide was associated with a relatively consistent fpg profile over time that was numerically less than fpg reductions seen with the highest doses of albiglutide ( fig . small improvements in -cell function ( assessed by homeostasis model assessment of -cell function ) were noted in subjects receiving albiglutide ( online appendix 4 ) . a consistent trend in weight reduction was noted with average weight loss ranging from 1.1 to 1.7 kg in subjects receiving the highest albiglutide dose in each schedule . these reductions were numerically greater than those for subjects receiving placebo ( 0.7 kg ) but less than those for subjects receiving exenatide ( 2.4 kg ) . the most frequently reported adverse events included nausea ( 11.854.3% ) , vomiting ( 041.2% ) , headache ( 5.923.5% ) , dizziness ( 5.714.3% ) , nasopharyngitis ( 5.711.4% ) , back pain ( 014.3% ) , influenza ( 09.7% ) , upper respiratory tract infections ( 015.2% ) , and local skin reactions ( 2.928.6% ) ( online appendix 5 ) . 2 ) of nausea and/or vomiting revealed that the proportion of subjects experiencing these adverse events each week was low in the 30-mg weekly arm ( < 10% ) and declined over the course of the study , with no reports of nausea or vomiting after 8 weeks ( fig . the incidence of nausea and/or vomiting with exenatide reached 20% by week 2 , increased at week 5 to a peak incidence of 29% ( due to label - based titration ) , and also declined over the study period ( fig . the percentage of subjects experiencing vomiting with or without nausea ( ) or nausea ( ) adverse events during each week of the 16-week trial for albiglutide 30 mg weekly ( a ) , albiglutide 50 mg biweekly ( b ) , albiglutide 100 mg monthly ( c ) , placebo ( d ) , and exenatide ( e ) . documented hypoglycemia was not increased with albiglutide ( 03.1% ) relative to placebo ( 3.9% ) and exenatide ( 2.9% ) . injection site local skin reactions , most of which were small , were observed in the study and were more common in the albiglutide groups ( 2.928.6% ) compared with placebo ( 5.9% ) and exenatide ( 2.9% ) . injection site reactions tended to occur once per person in subjects receiving 30 mg albiglutide and approximately twice per person in subjects receiving higher albiglutide doses . in this study , the dose- and time - dependent effects of albiglutide , a long - acting glp-1 receptor agonist , was evaluated to identify potential dose regimens for future studies . maximum doses in each schedule ( albiglutide 30 mg weekly , 50 mg biweekly , and 100 mg monthly ) elicited similar responses in a1c , providing meaningful reductions within the range of 0.80.9% from a mean baseline a1c of 8.0% . albiglutide also significantly reduced fpg at week 16 compared with placebo , with reductions observed at the time of the first assessment ( 2 weeks postdose ) . , 30 mg ) , weekly dosing provided consistent fpg reduction . when dosed biweekly , 50 mg albiglutide also improved glycemic indexes but with higher gi adverse event rates , possibly related to the higher initial dose . the increase in fpg fluctuation and gi events in the biweekly and monthly regimens is likely due to fluctuations in plasma albiglutide concentrations resulting from less frequent dosing . mechanistically , the reasons for differences in the tolerability of albiglutide and exenatide are unknown but may be due to differences in pharmacokinetics , including gradual absorption ( tmax is 3 days vs. 2.1 h for albiglutide and exenatide , respectively ) and a long plasma half - life of 5 days resulting in a steady state achieved after 45 doses for 30 mg albiglutide weekly . the slow accumulation of albiglutide may ameliorate gi intolerability often observed with exenatide , and albiglutide 's relative impermeance to the central nervous system may result in a more benign profile with respect to nausea and vomiting than exenatide ( 21 ) . exploratory post hoc analyses indicated that there was no obvious correlation between reduction in a1c and weight loss for all albiglutide doses ( data not shown ) . fourth , no escalating doses were tested for biweekly and monthly dosing , which may have attenuated gi adverse events and fpg fluctuations . higher monthly doses of albiglutide were efficacious , but their use was constrained by the higher frequency of gi - related adverse events . weekly albiglutide significantly improved glycemic control with an acceptable safety and tolerability profile and modest weight loss without increasing the risk of hypoglycemia or immunological response in subjects with type 2 diabetes .

diabetic retinopathy is one of the leading preventable causes of visual impairment in the uk . treatment can prevent vision loss , but requires early detection and careful monitoring to be most effective . the prevalence of diabetic retinopathy at diagnosis of type 2 diabetes is a useful indirect measure of how well a healthcare system is performing with respect to diabetes detection ; where type 2 diabetes is present for a long time prior to diagnosis prevalence rates of diabetic retinopathy at diagnosis will be high . prevalence at diagnosis also indicates to what extent there is an urgency to perform retinal screening after diagnosis . finally , understanding the characteristics of those patients with type 2 diabetes who have diabetic retinopathy at diagnosis is of practical use for targeting of screening . the aim of this study was to examine the prevalence and determinants of diabetic retinopathy among people with newly diagnosed type 2 diabetes in scotland ( population 5.1 million ) . we also assess the coverage , uptake and rapidity of retinal screening delivery in this population . the data used were from an anonymised extract of the scottish care information diabetes collaboration ( sci - dc ) , a clinical database that holds data on people diagnosed with diabetes in scotland . the sci - dc database was rolled out across scotland from 2000 and the estimated coverage of the total diabetic population is around 99% . sci - dc captures key diabetes - related data items , such as bmi , hba1c , lipids and bp , from all hospitals and 1,100 general practices in scotland . sci - dc data were linked to death records held by the national records of scotland using probabilistic linkage . the national roll out of the scottish diabetic retinopathy screening ( drs ) programme to improve the availability of high - quality retinal screening in scotland began in 2006 , attaining nationwide coverage by january 2007 . all eligible patients ( aged 12 years and over ) registered on the sci - dc are invited to participate in this programme . all new registrants are automatically entered as new patients on the drs database , which triggers the appointment process . those who are already attending eye clinics for diabetic eye disease , those declining screening and those who are too unfit or frail for screening are suspended from the programme , with their status reviewed at least every 3 years . the retinal examination involves a single - field digital photograph , with mydriasis if required , with centralised grading or , when photographic images are ungradable , slit - lamp examination . slit - lamp examination gradings were not available for all health boards for the whole period of the study , but were included for analysis when available . the use of a single central - field digital photograph for the detection of sight - threatening retinopathy has been validated [ 68 ] . the programme includes quality - control protocols to ensure the quality of the photographs and the grading . the subsequent action taken is determined by the most severe finding in the worst eye . visual acuity is often unaffected during the early stages of diabetic retinopathy , but may deteriorate as the severity of the retinopathy and maculopathy worsens with proliferative retinopathy ( r4 ) and referable maculopathy ( m2 ) , both of which are sight - threatening conditions . previous analyses from the pilot phase of the drs programme estimate the prevalence of diabetic retinopathy at 20% and the referral rate for eye disease at 3% . the most recent data for the years 20092010 indicate a stable referral rate of 3.5% .table 1grading scheme of the scottish diabetic retinopathy screening collaborationgradeexplanation / descriptionretinopathy r0no diabetic retinopathy r1 ( mild)bdr mildat least one dot haemorrhage or microaneurysm with or without hard exudates r2 ( moderate)bdr moderatefour or more blot haemorrhages ( i.e. airlie house standard photograph 2a ) in one hemi - field only ( inferior and superior hemi - fields delineated by a line passing through the centre of the fovea and optic disc ) r3 ( severe)bdr severeany of the following features : four or more blot haemorrhages ( i.e. airlie house standard photograph 2a ) in both inferior and superior hemi - fields venous beading ( airlie house standard photograph 6a ) irma ( airlie house standard photograph 8a ) r4 ( proliferative)pdrany of the following features new vessels vitreous haemorrhagemaculopathy m1 ( observable)lesions within a radius of > 1 but < 2 disc diameters of the centre of the foveaany hard exudates m2 ( referable)lesions within a radius of < 1 disc diameter of the centre of the foveaany blot haemorrhagesany hard exudatesadapted from scottish diabetic retinopathy grading scheme bdr , background diabetic retinopathy ; irma , intraretinal microvascular abnormalities ; pdr , proliferative diabetic retinopathy grading scheme of the scottish diabetic retinopathy screening collaboration adapted from scottish diabetic retinopathy grading scheme bdr , background diabetic retinopathy ; irma , intraretinal microvascular abnormalities ; pdr , proliferative diabetic retinopathy for this analysis , the retinopathy / maculopathy grade for an individual was defined as the grade of the worst eye . we extracted data on all those registered on sci - dc with type 2 diabetes diagnosed between 1 january 2005 and 31 may 2008 ( the most recently available capture of new patients ) . drs data for this cohort were available up to the end of 2010 , as were data for other covariates including sex , age , bmi , hba1c , bp , total cholesterol and socioeconomic status ( as assessed by the scottish index of multiple deprivation [ simd ] , a measure indexed by residence ) . for covariates , the measurement used was the one made at the time nearest to the diagnosis of type 2 diabetes . when no measure was available within 180 days of diagnosis , the data were considered to be missing ; the exception was bmi , for which data were considered missing when no observation was available within 365 days of diagnosis . for individuals diagnosed prior to the launch of the drs programme , the time to screening was calculated as the time from diagnosis to the first sci - dc entry representing retinal examination , regardless of the source ; for those diagnosed after the launch of the drs programme , the time to screening was calculated as the time to first drs screening . the primary date of type 2 diabetes diagnosis was based on the date entered into the sci - dc by clinicians ; if multiple dates were entered we took the earliest date . this date was checked against multiple data sources including prescription and hospital discharge records for any prior evidence of type 2 diabetes . we excluded 2,406 individuals ( 4.4% of potentially eligible individuals ) where there was significant discrepancy over the date of diagnosis ( i.e. a difference in date of diagnosis of > 120 days ) . where there was a discrepancy of < 120 days we selected the earliest date for our analyses . diabetes type was determined according to type recorded in sci - dc with the addition of an algorithm to identify individuals at high risk of being mislabelled based on age of diagnosis and early use of insulin . approval was obtained from the scotland a research ethics committee , the caldicott ( data privacy ) guardian for the 14 scottish health boards and the isd privacy advisory committee . we used t tests to compare continuous variables and tests to compare dichotomous variables among people screened within 1 year of diagnosis vs those screened later , and people who had successful screening vs those with ungradable photographic images . logistic regression was used to examine independent associations of variables with the prevalence of retinopathy at screening . cox proportional hazards models were used to examine independent associations of variables with time from diagnosis to first retinal screening . to control for observed clustering of times to first screening , associated with the year of diagnosis ( i.e. the strong relationship between time to screening and year of diagnosis ) , we fitted a multivariate mixed - effects cox proportional hazards model , with year of diagnosis taken as a random effect , and age , sex and any of the variables that were significantly associated in univariate analysis entered as fixed effects . we used t tests to compare continuous variables and tests to compare dichotomous variables among people screened within 1 year of diagnosis vs those screened later , and people who had successful screening vs those with ungradable photographic images . logistic regression was used to examine independent associations of variables with the prevalence of retinopathy at screening . cox proportional hazards models were used to examine independent associations of variables with time from diagnosis to first retinal screening . to control for observed clustering of times to first screening , associated with the year of diagnosis ( i.e. the strong relationship between time to screening and year of diagnosis ) , we fitted a multivariate mixed - effects cox proportional hazards model , with year of diagnosis taken as a random effect , and age , sex and any of the variables that were significantly associated in univariate analysis entered as fixed effects . there were 51,526 people with newly diagnosed type 2 diabetes eligible for the study . over half were male ( n = 28,576 [ 55% ] ) and the mean age at diagnosis was 61.8 years ( sd 12.8 years ) . as of 31 december 2010 , 47,090 ( 91.4% ) people had attended a retinal screening examination , with 25,322 ( 53.8% of the screened population ) screened within 1 year of diagnosis . a total of 4,436 ( 8.6% ) had not attended a drs screening ( table 2 ) . the leading reason for not being screened related to ill health , with 2,143 ( 4.2% ) dying prior to the end of 2010 . ( among those who died before screening , the median time from diabetes diagnosis to death was 375 days , interquartile range [ iqr ] 169657 days . ) suspension from the programme because of eye clinic attendance affected only 0.8% of the population . the proportion of unscreened individuals declined over time : of all individuals diagnosed in 2005 , 1,917 ( 12.1% ) had not been screened as of 31 december 2010 , while for subsequent years those numbers fell to 1,283 ( 10.1% ) in 2006 , 941 ( 8.2% ) in 2007 and 238 ( 5.4% ) in 2008.table 2screening the newly diagnosed type 2 populationretinopathy screening statusnumber ( % ) of newly diagnosed patients with type 2 diabetes ( n = 51,526)died before screening2,143 ( 4.1)already under the care of eye clinic / retinal screening outside the drs system399 ( 0.8)unscreened for other reasons ( including choice not to enter screening programme , poor health or no longer resident in scotland)1,894 ( 3.7)total not screened before end 20104,436 ( 8.6%)ungradable images with no slit - lamp examination data3,567 ( 6.9)at least one graded screening result available43,523 ( 84.5)total screened before end 201047,090 ( 91.4 ) screening the newly diagnosed type 2 population complete covariate data for the period around diagnosis of type 2 diabetes were available for the majority of individuals with a record of screening ( n = 40,194 , 85.4% ) . the proportions of missing data by variable were : 8.5% for hba1c ( n = 4,006 ) ; 6.3% for total cholesterol ( n = 2,832 ) ; 5.2% for bp ( n = 2,470 ) ; and 0.6% for simd ( n = 293 ) . of the 47,090 who were screened , the median time to first retinal screening was 315 days ( iqr 111607 days ) . however , time to first screening was strongly related to year of diagnosis ( fig . 1 ) ; individuals diagnosed in 2005 had a median time to any documented screening of almost 18 months ( median = 540 days , iqr 258747 ) falling to <3 months ( median = 83 days , iqr 51135 ) in 2008 . in a mixed - effects multivariate cox proportional hazards model , with year of diagnosis treated as a random effect and the other variables as fixed effects , male sex ( hr 1.03 , 95% ci 1.01 , 1.05 ) , older age ( hr 1.01 , 95% ci 1.00 , 1.02 per 10 years of age ) , systolic bp 135 mmhg ( hr 1.07 , 95% ci 1.04 , 1.10 ) , diastolic bp 80 mmhg ( hr 1.03 , 95% ci 1.01 , 1.04 ) , total cholesterol 4.5 mmol / l ( hr 1.05 , 95% ci 1.03 , 1.07 ) and lower socioeconomic status ( hr 1.02 , 95% ci 1.00 , 1.04 ) were all statistically significantly associated with longer time to screening , while obesity was associated with a shorter time to screening ( hr 0.97 , 95% ci 0.95 , 0.99 ) within a multivariate model ( p < 0.05 ) . 1median time to retinal screening from diagnosis of type 2 diabetes in days , by year of diabetes diagnosis . error bars indicate the 25th to 75th percentiles ; dotted line indicates 1 year median time to retinal screening from diagnosis of type 2 diabetes in days , by year of diabetes diagnosis . error bars indicate the 25th to 75th percentiles ; dotted line indicates 1 year the prevalence of any diabetic retinopathy at first screening was 19.3% and that of referable diabetic retinopathy was 1.9% ( table 3 ) , with only 0.7% having r3 or r4 grade retinopathy.table 3prevalence of retinopathy at first screening for all people successfully screenedfindingfrequency , n ( % ) ( n = 43,523)no eye disease35,114 ( 80.7 ) r0 and no maculopathy ( m0)35,114 ( 80.7)non - referable eye disease7,568 ( 17.4 ) r1 and no maculopathy ( m0)7,341 ( 16.9 ) r2 and no maculopathy ( m0)39 ( 0.1 ) r0 or r1 or r2 with non - referable maculopathy ( m1)188 ( 0.4)referable eye disease841 ( 1.9 ) r0 or r1 or r2 with referable maculopathy ( m2)523 ( 1.2 ) r3 any maculopathy190 ( 0.4 ) r4 any maculopathy128 ( 0.3 ) prevalence of retinopathy at first screening for all people successfully screened the prevalence of diabetic retinopathy varied by time to screening ; for individuals screened within 1 year of diagnosis ( n = 25,322 ) the prevalence of any diabetic retinopathy was 18.3% and 1.6% for referable diabetic retinopathy vs 20.5% and 2.3% , respectively , for people screened more than a year after diagnosis ( p < 0.0001 for both comparisons ) . the prevalence was highest for those first screened > 2 years after diagnosis ( n = 7,512 ) who had a prevalence of any diabetic retinopathy of 20.7% and of referable diabetic retinopathy of 2.7% . individuals screened within 3 months of diagnosis ( n = 9,354 ) had a prevalence of any diabetic retinopathy of 18.5% and referable diabetic retinopathy of 1.4% . details of the diagnosis for the eye disease causing follow - up with the eye clinics were not available for 0.8% of this population . if we assume all these people ( n = 399 [ see table 2 ] ) are attending an eye clinic because of diabetic retinopathy , then the upper limit of any diabetic retinopathy for the population is 20.0% and 2.6% for referable diabetic retinopathy . when the drs programme does not obtain satisfactory photographs , slit - lamp examinations are undertaken . however , results from these examinations have not routinely been entered into the central database until recently . within the current analyses 578 individuals had results available from slit - lamp examinations and are categorised according to the retinopathy status found by slit - lamp examination . overall , 7.6% of those screened during the study period had an ungradable image and no slit - lamp examination result available . individuals with ungradable images were older ( mean age 72 years ) , had higher systolic bp ( 140.7 mmhg ) , lower diastolic bp ( mean 77.8 mmhg ) , lower total cholesterol ( mean 4.99 mmol / l ) , lower hba1c ( 7.9% [ 63 mmol / mol ] ) , and lower bmi ( mean 30.1 kg / m ) when compared with those who had successful screening ( p < 0.001 for all differences using the t test ) . of the 3,567 people with ungradable images at their first screening , 2,198 ( 61.6% ) had no diabetic retinopathy at their next screening , with 356 ( 10.0% ) having evidence of diabetic retinopathy , while the remaining 1,013 ( 28.4% ) had persistently ungradable images . if we assume that this subsequent finding of referable diabetic retinopathy had been present at the first examination and that persistently ungraded eyes all represent diabetic retinopathy then the overall rate of diabetic retinopathy at first screening in the study would increase from 19.3% to 19.9% . in univariate logistic regression models the following variables were associated with the presence of retinopathy at first screening : male sex , lower bmi , higher hba1c , longer time to first retinopathy screening , lower socioeconomic status and higher systolic and diastolic bp . there was no association with age at diagnosis or total cholesterol ( data not shown ) . in a logistic regression model that included all the variables associated with retinopathy in the univariate analyses , together with age , the factors independently associated with retinopathy were male sex , lower bmi , higher hba1c , higher systolic bp and longer time to first retinopathy screening ( table 4 ) . when those not screened because of eye clinic attendance were included in the above model as having retinopathy these risk factor relationships did not change appreciably ( data not shown).table 4characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy statusall ( n = 47,090)no diabetic retinopathy ( n = 35,114)diabetic retinopathy ( n = 8,409)or for diabetic retinopathy vs no diabetic retinopathy ( 95% ci)p valuemale sex26,341 ( 55.9%)19,654 ( 56%)5,103 ( 60.7%)1.19 ( 1.14 , 1.25)<0.001age ( years)61.3 12.460.4 12.060.6 12.11.02 ( 0.99 , 1.04)0.163bmi ( kg / m)32.0 6.432.2 6.431.7 6.40.87 ( 0.82 , 0.93)<0.001hba1c ( % ) 8.1 2.18.0 2.18.4 2.21.07 ( 1.06 , 1.08)0.001hba1c ( mmol / mol)65.0 23.163.9 23.168.3 24.21.06 ( 1.05 , 1.08)<0.001systolic bp ( mmhg)139.9 86.8139.5 99.6141.1 24.1diastolic bp ( mmhg)80.9 1280.9 12.281.8 11.41.01 ( 0.98 , 1.03)0.572higher socioeconomic status21,308 ( 45.2%)15,993 ( 45.5%)3,704 ( 44.0%)0.96 ( 0.91 , 1.01)0.122median time to screening ( days)315 ( 111607)305 ( 109601)353 ( 116625)1.12 ( 1.071.17)<0.001data are mean sd , median with iqr or frequency with percentageors and p values were computed by multiple logistic regression with a model that included all variablesors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy status data are mean sd , median with iqr or frequency with percentage ors and p values were computed by multiple logistic regression with a model that included all variables ors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year of the 47,090 who were screened , the median time to first retinal screening was 315 days ( iqr 111607 days ) . however , time to first screening was strongly related to year of diagnosis ( fig . 1 ) ; individuals diagnosed in 2005 had a median time to any documented screening of almost 18 months ( median = 540 days , iqr 258747 ) falling to <3 months ( median = 83 days , iqr 51135 ) in 2008 . in a mixed - effects multivariate cox proportional hazards model , with year of diagnosis treated as a random effect and the other variables as fixed effects , male sex ( hr 1.03 , 95% ci 1.01 , 1.05 ) , older age ( hr 1.01 , 95% ci 1.00 , 1.02 per 10 years of age ) , systolic bp 135 mmhg ( hr 1.07 , 95% ci 1.04 , 1.10 ) , diastolic bp 80 mmhg ( hr 1.03 , 95% ci 1.01 , 1.04 ) , total cholesterol 4.5 mmol / l ( hr 1.05 , 95% ci 1.03 , 1.07 ) and lower socioeconomic status ( hr 1.02 , 95% ci 1.00 , 1.04 ) were all statistically significantly associated with longer time to screening , while obesity was associated with a shorter time to screening ( hr 0.97 , 95% ci 0.95 , 0.99 ) within a multivariate model ( p < 0.05 ) . 1median time to retinal screening from diagnosis of type 2 diabetes in days , by year of diabetes diagnosis . error bars indicate the 25th to 75th percentiles ; dotted line indicates 1 year median time to retinal screening from diagnosis of type 2 diabetes in days , by year of diabetes diagnosis . the prevalence of any diabetic retinopathy at first screening was 19.3% and that of referable diabetic retinopathy was 1.9% ( table 3 ) , with only 0.7% having r3 or r4 grade retinopathy.table 3prevalence of retinopathy at first screening for all people successfully screenedfindingfrequency , n ( % ) ( n = 43,523)no eye disease35,114 ( 80.7 ) r0 and no maculopathy ( m0)35,114 ( 80.7)non - referable eye disease7,568 ( 17.4 ) r1 and no maculopathy ( m0)7,341 ( 16.9 ) r2 and no maculopathy ( m0)39 ( 0.1 ) r0 or r1 or r2 with non - referable maculopathy ( m1)188 ( 0.4)referable eye disease841 ( 1.9 ) r0 or r1 or r2 with referable maculopathy ( m2)523 ( 1.2 ) r3 any maculopathy190 ( 0.4 ) r4 any maculopathy128 ( 0.3 ) prevalence of retinopathy at first screening for all people successfully screened the prevalence of diabetic retinopathy varied by time to screening ; for individuals screened within 1 year of diagnosis ( n = 25,322 ) the prevalence of any diabetic retinopathy was 18.3% and 1.6% for referable diabetic retinopathy vs 20.5% and 2.3% , respectively , for people screened more than a year after diagnosis ( p < 0.0001 for both comparisons ) . the prevalence was highest for those first screened > 2 years after diagnosis ( n = 7,512 ) who had a prevalence of any diabetic retinopathy of 20.7% and of referable diabetic retinopathy of 2.7% . individuals screened within 3 months of diagnosis ( n = 9,354 ) had a prevalence of any diabetic retinopathy of 18.5% and referable diabetic retinopathy of 1.4% . details of the diagnosis for the eye disease causing follow - up with the eye clinics were not available for 0.8% of this population . if we assume all these people ( n = 399 [ see table 2 ] ) are attending an eye clinic because of diabetic retinopathy , then the upper limit of any diabetic retinopathy for the population is 20.0% and 2.6% for referable diabetic retinopathy . when the drs programme does not obtain satisfactory photographs , slit - lamp examinations are undertaken . however , results from these examinations have not routinely been entered into the central database until recently . within the current analyses 578 individuals had results available from slit - lamp examinations and are categorised according to the retinopathy status found by slit - lamp examination . overall , 7.6% of those screened during the study period had an ungradable image and no slit - lamp examination result available . individuals with ungradable images were older ( mean age 72 years ) , had higher systolic bp ( 140.7 mmhg ) , lower diastolic bp ( mean 77.8 mmhg ) , lower total cholesterol ( mean 4.99 mmol / l ) , lower hba1c ( 7.9% [ 63 mmol / mol ] ) , and lower bmi ( mean 30.1 kg / m ) when compared with those who had successful screening ( p < 0.001 for all differences using the t test ) . of the 3,567 people with ungradable images at their first screening , 2,198 ( 61.6% ) had no diabetic retinopathy at their next screening , with 356 ( 10.0% ) having evidence of diabetic retinopathy , while the remaining 1,013 ( 28.4% ) had persistently ungradable images . if we assume that this subsequent finding of referable diabetic retinopathy had been present at the first examination and that persistently ungraded eyes all represent diabetic retinopathy then the overall rate of diabetic retinopathy at first screening in the study would increase from 19.3% to 19.9% . in univariate logistic regression models the following variables were associated with the presence of retinopathy at first screening : male sex , lower bmi , higher hba1c , longer time to first retinopathy screening , lower socioeconomic status and higher systolic and diastolic bp . there was no association with age at diagnosis or total cholesterol ( data not shown ) . in a logistic regression model that included all the variables associated with retinopathy in the univariate analyses , together with age , the factors independently associated with retinopathy were male sex , lower bmi , higher hba1c , higher systolic bp and longer time to first retinopathy screening ( table 4 ) . when those not screened because of eye clinic attendance were included in the above model as having retinopathy these risk factor relationships did not change appreciably ( data not shown).table 4characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy statusall ( n = 47,090)no diabetic retinopathy ( n = 35,114)diabetic retinopathy ( n = 8,409)or for diabetic retinopathy vs no diabetic retinopathy ( 95% ci)p valuemale sex26,341 ( 55.9%)19,654 ( 56%)5,103 ( 60.7%)1.19 ( 1.14 , 1.25)<0.001age ( years)61.3 12.460.4 12.060.6 12.11.02 ( 0.99 , 1.04)0.163bmi ( kg / m)32.0 6.432.2 6.431.7 6.40.87 ( 0.82 , 0.93)<0.001hba1c ( % ) 8.1 2.18.0 2.18.4 2.21.07 ( 1.06 , 1.08)0.001hba1c ( mmol / mol)65.0 23.163.9 23.168.3 24.21.06 ( 1.05 , 1.08)<0.001systolic bp ( mmhg)139.9 86.8139.5 99.6141.1 24.1diastolic bp ( mmhg)80.9 1280.9 12.281.8 11.41.01 ( 0.98 , 1.03)0.572higher socioeconomic status21,308 ( 45.2%)15,993 ( 45.5%)3,704 ( 44.0%)0.96 ( 0.91 , 1.01)0.122median time to screening ( days)315 ( 111607)305 ( 109601)353 ( 116625)1.12 ( 1.071.17)<0.001data are mean sd , median with iqr or frequency with percentageors and p values were computed by multiple logistic regression with a model that included all variablesors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy status data are mean sd , median with iqr or frequency with percentage ors and p values were computed by multiple logistic regression with a model that included all variables ors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year diabetic retinopathy remains a major complication of type 2 diabetes and requires early detection for best treatment . the drs programme had screened 91.4% of all people in scotland newly diagnosed with type 2 diabetes by 31 december 2010 . the median time from diabetes diagnosis to retinopathy screening declined throughout the study period , with participants diagnosed in 2008 having a median wait to screening of 83 days . the prevalence of any diabetic retinopathy among people with newly diagnosed type 2 diabetes was 19.3% , which is almost half the prevalence of any diabetic retinopathy , 3539% , reported by the ukpds . the major strengths of the current study are its use of national - level data , which include the results of retinal photography screening for diabetic retinopathy and covariate data from the time of type 2 diabetes diagnosis . the drs is the only form of diabetic retinopathy screening recognised for primary care payments in scotland , so it is the dominant method of diabetic retinopathy screening . scotland also has a means for linking an individual s medical data from a variety of sources via a unique medical identifier , which allows the incorporation of data from many sources . the richness of the data sources allowed us to use a variety of data to determine diabetes type . this meant we were able to exclude individuals from the study who showed strong evidence for having type 1 diabetes even if originally classified as having type 2 diabetes . similarly , we could interrogate a number of data sources to ensure that the individuals included in the study had a consistent date of diagnosis . the drs programme is aimed at detecting sight - threatening diabetic retinopathy and relies on a single - field photograph per eye , as has been validated as a means for identifying sight - threatening diabetic retinopathy [ 68 ] . this approach is less sensitive than the seven - fields - per - eye approach used in the wisconsin epidemiologic study of diabetic retinopathy and will miss mild disease , such as peripheral microaneurysms . we also lack data for the presence of diabetic retinopathy among the small proportion of individuals in scotland who obtain their screening outside the drs programme . this is primarily via ophthalmology clinics and , as < 1% of the population attend such screenings , even if we assumed all of these individuals had diabetic retinopathy it would not make a major difference to our prevalence estimate ( 20.0% vs 19.3% ) . in the ukpds , which recruited patients with new - onset type 2 diabetes aged 2565 between 1983 and 1991 , the prevalence of any diabetic retinopathy was 35% in women and 39% in men . our data are not directly comparable as the ukpds used four fields and so was more likely to detect early grades of peripheral diabetic retinopathy than our study . however , much has changed since the ukpds , including the diagnostic criteria for diabetes as well as health policy in the uk . there is now a greater emphasis on screening for type 2 diabetes . unlike the nhs in england and wales , the nhs in scotland has not adopted systematic screening for type 2 diabetes ; however , risk profiling for cardiovascular disease , including testing for type 2 diabetes , has been encouraged . identifying obese patients who are at high risk for type 2 diabetes is also included in the quality and outcomes framework , a series of standards for primary care practices that provides additional funds on the basis of meeting specific targets . the lower prevalence of diabetic retinopathy at diabetes diagnosis reported in the current study suggests that these system - wide changes have reduced delays in diabetes diagnosis . our findings are in line with reports from recent population studies in which the prevalence of diabetic retinopathy ranged from 6% to 23% [ 3 , 14 , 1821 ] . the lowest estimates ( 6.2% in australia and 10.2% in the usa ) come from studies that undertook simultaneous diabetes diagnosis and retinal screening . diabetic retinopathy also occurs in non - diabetic populations [ 22 , 23 ] , with estimates ranging from 5.2% in the pima indians to 8% in the general us population . in australia the prevalence of diabetic retinopathy was 5.8% for those with normal glucose tolerance and 6.7% in people with impaired glucose tolerance or impaired fasting glucose . this suggests that even with moves to minimise delay in diagnosis of diabetes through diabetes screening programmes we would not anticipate achieving a prevalence of diabetic retinopathy at the time of diagnosis of less than 5% . it also raises the question of whether factors other than dysglycaemia may be relevant to the development of diabetic retinopathy in some individuals . the importance of glycaemia , blood pressure and diabetes duration as risk factors for diabetic retinopathy is already well established [ 13 , 14 , 2527 ] . results concerning the relationship between bmi and risk for diabetic retinopathy are inconsistent , with both positive [ 26 , 28 ] and negative associations [ 2931 ] reported . we have not reported the associations with smoking or triacylglycerols because there were insufficient data for individuals in the study at the time of diagnosis . of the risk factors for delays in screening found in the current study only high systolic bp was also associated with the presence of diabetic retinopathy at first screening and none of the factors measured had a clinically significant impact on delays in screening . while delays in screening are a concern , the knowledge that the prevalence of diabetic retinopathy and sight - threatening diabetic retinopathy is low even for those screened after 24 months is reassuring . the usa has now started diagnosing diabetes based on the presence of an elevated hba1c . it is unknown how hba1c criteria will impact on time to diagnosis in the population and the net effect could be earlier diagnosis because of greater ease in carrying out the test ( i.e. no requirement for fasting or glucose challenge ) , or later diagnosis as hba1c diagnosis detects fewer individuals than the standard glucose tolerance tests . our data suggest that a delay in diagnosis of up to 2 years would have a minimal impact on the prevalence of sight - threatening diabetic retinopathy . the nationwide drs programme has successfully screened over 90% of individuals with newly diagnosed type 2 diabetes in scotland , with the majority being screened within 12 months of diagnosis . delays in screening have become less common over time , indicating improvements in the system as the drs programme attained full national coverage , with a current median time to screening of <3 months . when diabetic retinopathy screening was within 3 months of diabetes diagnosis , the prevalence of any diabetic retinopathy was 18.5% and 1.4% for referable diabetic retinopathy . while these prevalences are much lower than those reported in the past they remain higher than estimates from population screening , suggesting that there is still room for earlier diagnosis of type 2 diabetes in this population . however , even among individuals not screened until after a year of diagnosis the prevalence of referable eye disease remains very low . this work was supported by the wellcome trust through the scottish health informatics programme ( ship ) grant ( ref wt086113 ) . ship is a collaboration between the universities of aberdeen , dundee , edinburgh , glasgow and st andrews and the information services division of nhs scotland . all authors made substantial contributions to the conception and design , acquisition of data , or analysis and interpretation of data as well as to the drafting or revising of the manuscript . in detail , hcl contributed to the design of the study and interpretation of the data . hmc contributed to the conception and design of the study , interpretation of the data and revision of the manuscript . dc , jao , gpl , mb , jd , nl and sp made substantial contributions to the design of the study , acquisition and interpretation of the data and revised the manuscript . rsl , jam , adm , ns and shw contributed to the interpretation of the data and made revisions to the manuscript . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

aims / hypothesisthe aim of this study was to examine the prevalence of and risk factors for diabetic retinopathy in people with newly diagnosed type 2 diabetes mellitus , using scottish national data.methodswe identified individuals diagnosed with type 2 diabetes mellitus in scotland between january 2005 and may 2008 using data from the national diabetes database . we calculated the prevalence of retinopathy and ors for risk factors associated with retinopathy at first screening.resultsof the 51,526 people with newly diagnosed type 2 diabetes mellitus identified , 91.4% had been screened by 31 december 2010 . the median time to first screening was 315 days ( interquartile range [ iqr ] 111607 days ) , but by 2008 the median was 83 days ( iqr 51135 days ) . the prevalence at first screening of any retinopathy was 19.3% , and for referable retinopathy it was 1.9% . for individuals screened after a year the prevalence of any retinopathy was 20.5% and referable retinopathy was 2.3% . any retinopathy at screening was associated with male sex ( or 1.19 , 95% ci 1.14 , 1.25 ) , hba1c ( or 1.07 , 95% ci 1.06 , 1.08 per 1% [ 11 mmol / mol ] increase ) , systolic bp ( or 1.06 , 95% ci 1.05 , 1.08 per 10 mmhg increase ) , time to screening ( or for screening > 1 year post diagnosis = 1.12 , 95% ci 1.07 , 1.17 ) and obesity ( or 0.87 , 95% ci 0.82 , 0.93 ) in multivariate analysis.conclusions/interpretationthe prevalence of retinopathy at first screening is lower than in previous uk studies , consistent with earlier diagnosis of diabetes . most newly diagnosed type 2 diabetic patients in scotland are screened within an acceptable interval and the prevalence of referable disease is low , even in those with delayed screening .

Introduction Methods Statistical analysis Results Time to screening Prevalence of retinopathy and maculopathy Ungradable images (R6) Risk factors associated with early diabetic retinopathy Discussion Conclusions Funding Duality of interest Contribution statement Open Access

the aim of this study was to examine the prevalence and determinants of diabetic retinopathy among people with newly diagnosed type 2 diabetes in scotland ( population 5.1 million ) . there were 51,526 people with newly diagnosed type 2 diabetes eligible for the study . ( among those who died before screening , the median time from diabetes diagnosis to death was 375 days , interquartile range [ iqr ] 169657 days . ) the proportion of unscreened individuals declined over time : of all individuals diagnosed in 2005 , 1,917 ( 12.1% ) had not been screened as of 31 december 2010 , while for subsequent years those numbers fell to 1,283 ( 10.1% ) in 2006 , 941 ( 8.2% ) in 2007 and 238 ( 5.4% ) in 2008.table 2screening the newly diagnosed type 2 populationretinopathy screening statusnumber ( % ) of newly diagnosed patients with type 2 diabetes ( n = 51,526)died before screening2,143 ( 4.1)already under the care of eye clinic / retinal screening outside the drs system399 ( 0.8)unscreened for other reasons ( including choice not to enter screening programme , poor health or no longer resident in scotland)1,894 ( 3.7)total not screened before end 20104,436 ( 8.6%)ungradable images with no slit - lamp examination data3,567 ( 6.9)at least one graded screening result available43,523 ( 84.5)total screened before end 201047,090 ( 91.4 ) screening the newly diagnosed type 2 population complete covariate data for the period around diagnosis of type 2 diabetes were available for the majority of individuals with a record of screening ( n = 40,194 , 85.4% ) . of the 47,090 who were screened , the median time to first retinal screening was 315 days ( iqr 111607 days ) . in a mixed - effects multivariate cox proportional hazards model , with year of diagnosis treated as a random effect and the other variables as fixed effects , male sex ( hr 1.03 , 95% ci 1.01 , 1.05 ) , older age ( hr 1.01 , 95% ci 1.00 , 1.02 per 10 years of age ) , systolic bp 135 mmhg ( hr 1.07 , 95% ci 1.04 , 1.10 ) , diastolic bp 80 mmhg ( hr 1.03 , 95% ci 1.01 , 1.04 ) , total cholesterol 4.5 mmol / l ( hr 1.05 , 95% ci 1.03 , 1.07 ) and lower socioeconomic status ( hr 1.02 , 95% ci 1.00 , 1.04 ) were all statistically significantly associated with longer time to screening , while obesity was associated with a shorter time to screening ( hr 0.97 , 95% ci 0.95 , 0.99 ) within a multivariate model ( p < 0.05 ) . error bars indicate the 25th to 75th percentiles ; dotted line indicates 1 year median time to retinal screening from diagnosis of type 2 diabetes in days , by year of diabetes diagnosis . error bars indicate the 25th to 75th percentiles ; dotted line indicates 1 year the prevalence of any diabetic retinopathy at first screening was 19.3% and that of referable diabetic retinopathy was 1.9% ( table 3 ) , with only 0.7% having r3 or r4 grade retinopathy.table 3prevalence of retinopathy at first screening for all people successfully screenedfindingfrequency , n ( % ) ( n = 43,523)no eye disease35,114 ( 80.7 ) r0 and no maculopathy ( m0)35,114 ( 80.7)non - referable eye disease7,568 ( 17.4 ) r1 and no maculopathy ( m0)7,341 ( 16.9 ) r2 and no maculopathy ( m0)39 ( 0.1 ) r0 or r1 or r2 with non - referable maculopathy ( m1)188 ( 0.4)referable eye disease841 ( 1.9 ) r0 or r1 or r2 with referable maculopathy ( m2)523 ( 1.2 ) r3 any maculopathy190 ( 0.4 ) r4 any maculopathy128 ( 0.3 ) prevalence of retinopathy at first screening for all people successfully screened the prevalence of diabetic retinopathy varied by time to screening ; for individuals screened within 1 year of diagnosis ( n = 25,322 ) the prevalence of any diabetic retinopathy was 18.3% and 1.6% for referable diabetic retinopathy vs 20.5% and 2.3% , respectively , for people screened more than a year after diagnosis ( p < 0.0001 for both comparisons ) . individuals screened within 3 months of diagnosis ( n = 9,354 ) had a prevalence of any diabetic retinopathy of 18.5% and referable diabetic retinopathy of 1.4% . individuals with ungradable images were older ( mean age 72 years ) , had higher systolic bp ( 140.7 mmhg ) , lower diastolic bp ( mean 77.8 mmhg ) , lower total cholesterol ( mean 4.99 mmol / l ) , lower hba1c ( 7.9% [ 63 mmol / mol ] ) , and lower bmi ( mean 30.1 kg / m ) when compared with those who had successful screening ( p < 0.001 for all differences using the t test ) . if we assume that this subsequent finding of referable diabetic retinopathy had been present at the first examination and that persistently ungraded eyes all represent diabetic retinopathy then the overall rate of diabetic retinopathy at first screening in the study would increase from 19.3% to 19.9% . in univariate logistic regression models the following variables were associated with the presence of retinopathy at first screening : male sex , lower bmi , higher hba1c , longer time to first retinopathy screening , lower socioeconomic status and higher systolic and diastolic bp . in a logistic regression model that included all the variables associated with retinopathy in the univariate analyses , together with age , the factors independently associated with retinopathy were male sex , lower bmi , higher hba1c , higher systolic bp and longer time to first retinopathy screening ( table 4 ) . when those not screened because of eye clinic attendance were included in the above model as having retinopathy these risk factor relationships did not change appreciably ( data not shown).table 4characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy statusall ( n = 47,090)no diabetic retinopathy ( n = 35,114)diabetic retinopathy ( n = 8,409)or for diabetic retinopathy vs no diabetic retinopathy ( 95% ci)p valuemale sex26,341 ( 55.9%)19,654 ( 56%)5,103 ( 60.7%)1.19 ( 1.14 , 1.25)<0.001age ( years)61.3 12.460.4 12.060.6 12.11.02 ( 0.99 , 1.04)0.163bmi ( kg / m)32.0 6.432.2 6.431.7 6.40.87 ( 0.82 , 0.93)<0.001hba1c ( % ) 8.1 2.18.0 2.18.4 2.21.07 ( 1.06 , 1.08)0.001hba1c ( mmol / mol)65.0 23.163.9 23.168.3 24.21.06 ( 1.05 , 1.08)<0.001systolic bp ( mmhg)139.9 86.8139.5 99.6141.1 24.1diastolic bp ( mmhg)80.9 1280.9 12.281.8 11.41.01 ( 0.98 , 1.03)0.572higher socioeconomic status21,308 ( 45.2%)15,993 ( 45.5%)3,704 ( 44.0%)0.96 ( 0.91 , 1.01)0.122median time to screening ( days)315 ( 111607)305 ( 109601)353 ( 116625)1.12 ( 1.071.17)<0.001data are mean sd , median with iqr or frequency with percentageors and p values were computed by multiple logistic regression with a model that included all variablesors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy status data are mean sd , median with iqr or frequency with percentage ors and p values were computed by multiple logistic regression with a model that included all variables ors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year of the 47,090 who were screened , the median time to first retinal screening was 315 days ( iqr 111607 days ) . 1 ) ; individuals diagnosed in 2005 had a median time to any documented screening of almost 18 months ( median = 540 days , iqr 258747 ) falling to <3 months ( median = 83 days , iqr 51135 ) in 2008 . in a mixed - effects multivariate cox proportional hazards model , with year of diagnosis treated as a random effect and the other variables as fixed effects , male sex ( hr 1.03 , 95% ci 1.01 , 1.05 ) , older age ( hr 1.01 , 95% ci 1.00 , 1.02 per 10 years of age ) , systolic bp 135 mmhg ( hr 1.07 , 95% ci 1.04 , 1.10 ) , diastolic bp 80 mmhg ( hr 1.03 , 95% ci 1.01 , 1.04 ) , total cholesterol 4.5 mmol / l ( hr 1.05 , 95% ci 1.03 , 1.07 ) and lower socioeconomic status ( hr 1.02 , 95% ci 1.00 , 1.04 ) were all statistically significantly associated with longer time to screening , while obesity was associated with a shorter time to screening ( hr 0.97 , 95% ci 0.95 , 0.99 ) within a multivariate model ( p < 0.05 ) . the prevalence of any diabetic retinopathy at first screening was 19.3% and that of referable diabetic retinopathy was 1.9% ( table 3 ) , with only 0.7% having r3 or r4 grade retinopathy.table 3prevalence of retinopathy at first screening for all people successfully screenedfindingfrequency , n ( % ) ( n = 43,523)no eye disease35,114 ( 80.7 ) r0 and no maculopathy ( m0)35,114 ( 80.7)non - referable eye disease7,568 ( 17.4 ) r1 and no maculopathy ( m0)7,341 ( 16.9 ) r2 and no maculopathy ( m0)39 ( 0.1 ) r0 or r1 or r2 with non - referable maculopathy ( m1)188 ( 0.4)referable eye disease841 ( 1.9 ) r0 or r1 or r2 with referable maculopathy ( m2)523 ( 1.2 ) r3 any maculopathy190 ( 0.4 ) r4 any maculopathy128 ( 0.3 ) prevalence of retinopathy at first screening for all people successfully screened the prevalence of diabetic retinopathy varied by time to screening ; for individuals screened within 1 year of diagnosis ( n = 25,322 ) the prevalence of any diabetic retinopathy was 18.3% and 1.6% for referable diabetic retinopathy vs 20.5% and 2.3% , respectively , for people screened more than a year after diagnosis ( p < 0.0001 for both comparisons ) . individuals with ungradable images were older ( mean age 72 years ) , had higher systolic bp ( 140.7 mmhg ) , lower diastolic bp ( mean 77.8 mmhg ) , lower total cholesterol ( mean 4.99 mmol / l ) , lower hba1c ( 7.9% [ 63 mmol / mol ] ) , and lower bmi ( mean 30.1 kg / m ) when compared with those who had successful screening ( p < 0.001 for all differences using the t test ) . if we assume that this subsequent finding of referable diabetic retinopathy had been present at the first examination and that persistently ungraded eyes all represent diabetic retinopathy then the overall rate of diabetic retinopathy at first screening in the study would increase from 19.3% to 19.9% . in univariate logistic regression models the following variables were associated with the presence of retinopathy at first screening : male sex , lower bmi , higher hba1c , longer time to first retinopathy screening , lower socioeconomic status and higher systolic and diastolic bp . in a logistic regression model that included all the variables associated with retinopathy in the univariate analyses , together with age , the factors independently associated with retinopathy were male sex , lower bmi , higher hba1c , higher systolic bp and longer time to first retinopathy screening ( table 4 ) . when those not screened because of eye clinic attendance were included in the above model as having retinopathy these risk factor relationships did not change appreciably ( data not shown).table 4characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy statusall ( n = 47,090)no diabetic retinopathy ( n = 35,114)diabetic retinopathy ( n = 8,409)or for diabetic retinopathy vs no diabetic retinopathy ( 95% ci)p valuemale sex26,341 ( 55.9%)19,654 ( 56%)5,103 ( 60.7%)1.19 ( 1.14 , 1.25)<0.001age ( years)61.3 12.460.4 12.060.6 12.11.02 ( 0.99 , 1.04)0.163bmi ( kg / m)32.0 6.432.2 6.431.7 6.40.87 ( 0.82 , 0.93)<0.001hba1c ( % ) 8.1 2.18.0 2.18.4 2.21.07 ( 1.06 , 1.08)0.001hba1c ( mmol / mol)65.0 23.163.9 23.168.3 24.21.06 ( 1.05 , 1.08)<0.001systolic bp ( mmhg)139.9 86.8139.5 99.6141.1 24.1diastolic bp ( mmhg)80.9 1280.9 12.281.8 11.41.01 ( 0.98 , 1.03)0.572higher socioeconomic status21,308 ( 45.2%)15,993 ( 45.5%)3,704 ( 44.0%)0.96 ( 0.91 , 1.01)0.122median time to screening ( days)315 ( 111607)305 ( 109601)353 ( 116625)1.12 ( 1.071.17)<0.001data are mean sd , median with iqr or frequency with percentageors and p values were computed by multiple logistic regression with a model that included all variablesors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year characteristics near to diagnosis of diabetes mellitus by subsequent retinopathy status data are mean sd , median with iqr or frequency with percentage ors and p values were computed by multiple logistic regression with a model that included all variables ors for continuous variables are per ten units except for : hba1c , which is given per 1% unit ( 11 mmol / mol ) ; bmi , which is presented for obese vs non - obese ; and time to screening , which is presented for screened after 1 year vs screened within 1 year diabetic retinopathy remains a major complication of type 2 diabetes and requires early detection for best treatment . the drs programme had screened 91.4% of all people in scotland newly diagnosed with type 2 diabetes by 31 december 2010 . the prevalence of any diabetic retinopathy among people with newly diagnosed type 2 diabetes was 19.3% , which is almost half the prevalence of any diabetic retinopathy , 3539% , reported by the ukpds . the major strengths of the current study are its use of national - level data , which include the results of retinal photography screening for diabetic retinopathy and covariate data from the time of type 2 diabetes diagnosis . of the risk factors for delays in screening found in the current study only high systolic bp was also associated with the presence of diabetic retinopathy at first screening and none of the factors measured had a clinically significant impact on delays in screening . when diabetic retinopathy screening was within 3 months of diabetes diagnosis , the prevalence of any diabetic retinopathy was 18.5% and 1.4% for referable diabetic retinopathy .

a sample of 30 hiv - infected individuals from a integrated counselling and testing centre ( ictc ) in vadodara , gujarat , india were selected to participate in individual face - to - face in - depth interviews after pilot testing . hiv - infected patients were eligible to participate in the study if they were : ( 1 ) english - or gujarati - speaking ; ( 2 ) 18 years or older ; and ( 3 ) diagnosed with hiv for at least 3 months . all interviews were conducted in a private area by trained , bilingual staff after taking written informed consent . each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide . two note - takers were present at each interview to document the dialogue verbatim and take notes , including non - verbal communication . two focus groups were convened , one with seven medical doctors and the other with eight non - medical providers . hiv service providers were eligible to participate if they were : ( 1 ) gujarati - speaking ; ( 2 ) 18 years or over ; and ( 3 ) had worked with hiv - positive patients for more than 1 year as either a medical doctor , a non - governmental organization staff member , integrated counselling and testing counselor , or peer educator for hiv - positive persons . each of the focus groups lasted about 2 hours and was audio - recorded with prior permission of the participants . the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation . the entire research protocol was reviewed and approved by institutional review board ( irb ) of both the university of north carolina at chapel hill , usa and the medical college , baroda , india . the in - depth interviews and focus group guides were developed by research teams in india and the u.s . in addition , a community advisory board ( cab ) , comprising experts from the field of social work , medical providers , and hiv consultants was convened to discuss and refine the content of the qualitative instruments . the in - depth interview guide was designed to assess topics pertaining to hiv disclosure processes , perceived barriers and facilitators to disclosure , and hiv - associated stigma and discrimination related to disclosure among hiv - positive individuals . the following topics were explored with the hiv service providers : ( 1 ) secondary prevention services provided to plwha ; ( 2 ) the advice they gave to plwha regarding whom to disclose to and why ; and ( 3 ) the consequences that they perceived their clients faced after disclosure . extensive notes of in - depth interviews were recorded in gujarati , and translated into english . the focus group discussions were transcribed verbatim in gujarati from the audio - recordings and then the transcripts were translated into english . in the early phase of coding , we based initial themes on topics covered in the interview / focus group discussion ( fgd ) guides . in the later phases , in - depth interview and fgd transcripts were read several times and content analyzed , respectively using the technique of open coding to discover conceptual patterns , or themes , in the text . research staff in india and u.s . coded the 8 of the 30 interview transcripts independently and then met to discuss the codes and both the fgds , and refined the code definitions . once all of the interviews were coded , a reliability exercise was conducted for the in - depth interviews . based on the reliability exercise , over 90% of the codes matched with the codebook developed by the study staff . in all , two codebooks were generated for the interview and fgds respectively . all interviews and fgds were coded using maxqda software , 2007 ( berlin , germany ) . once the interview and fgd codebooks were generated , a triangulation technique was used to analyze which themes from the hiv - positive individuals and from hiv service providers respective perspectives supported as well as which contradicted one another . a sample of 30 hiv - infected individuals from a integrated counselling and testing centre ( ictc ) in vadodara , gujarat , india were selected to participate in individual face - to - face in - depth interviews after pilot testing . hiv - infected patients were eligible to participate in the study if they were : ( 1 ) english - or gujarati - speaking ; ( 2 ) 18 years or older ; and ( 3 ) diagnosed with hiv for at least 3 months . all interviews were conducted in a private area by trained , bilingual staff after taking written informed consent . each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide . two note - takers were present at each interview to document the dialogue verbatim and take notes , including non - verbal communication . two focus groups were convened , one with seven medical doctors and the other with eight non - medical providers . hiv service providers were eligible to participate if they were : ( 1 ) gujarati - speaking ; ( 2 ) 18 years or over ; and ( 3 ) had worked with hiv - positive patients for more than 1 year as either a medical doctor , a non - governmental organization staff member , integrated counselling and testing counselor , or peer educator for hiv - positive persons . each of the focus groups lasted about 2 hours and was audio - recorded with prior permission of the participants . the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation . the entire research protocol was reviewed and approved by institutional review board ( irb ) of both the university of north carolina at chapel hill , usa and the medical college , baroda , india . a sample of 30 hiv - infected individuals from a integrated counselling and testing centre ( ictc ) in vadodara , gujarat , india were selected to participate in individual face - to - face in - depth interviews after pilot testing . hiv - infected patients were eligible to participate in the study if they were : ( 1 ) english - or gujarati - speaking ; ( 2 ) 18 years or older ; and ( 3 ) diagnosed with hiv for at least 3 months . all interviews were conducted in a private area by trained , bilingual staff after taking written informed consent . each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide . two note - takers were present at each interview to document the dialogue verbatim and take notes , including non - verbal communication . two focus groups were convened , one with seven medical doctors and the other with eight non - medical providers . hiv service providers were eligible to participate if they were : ( 1 ) gujarati - speaking ; ( 2 ) 18 years or over ; and ( 3 ) had worked with hiv - positive patients for more than 1 year as either a medical doctor , a non - governmental organization staff member , integrated counselling and testing counselor , or peer educator for hiv - positive persons . each of the focus groups lasted about 2 hours and was audio - recorded with prior permission of the participants . the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation . the entire research protocol was reviewed and approved by institutional review board ( irb ) of both the university of north carolina at chapel hill , usa and the medical college , baroda , india . the in - depth interviews and focus group guides were developed by research teams in india and the u.s . in addition , a community advisory board ( cab ) , comprising experts from the field of social work , medical providers , and hiv consultants was convened to discuss and refine the content of the qualitative instruments . the in - depth interview guide was designed to assess topics pertaining to hiv disclosure processes , perceived barriers and facilitators to disclosure , and hiv - associated stigma and discrimination related to disclosure among hiv - positive individuals . the following topics were explored with the hiv service providers : ( 1 ) secondary prevention services provided to plwha ; ( 2 ) the advice they gave to plwha regarding whom to disclose to and why ; and ( 3 ) the consequences that they perceived their clients faced after disclosure . extensive notes of in - depth interviews were recorded in gujarati , and translated into english . the focus group discussions were transcribed verbatim in gujarati from the audio - recordings and then the transcripts were translated into english . in the early phase of coding , we based initial themes on topics covered in the interview / focus group discussion ( fgd ) guides . in the later phases , in - depth interview and fgd transcripts were read several times and content analyzed , respectively using the technique of open coding to discover conceptual patterns , or themes , in the text . research staff in india and u.s . coded the 8 of the 30 interview transcripts independently and then met to discuss the codes and both the fgds , and refined the code definitions . once all of the interviews were coded , a reliability exercise was conducted for the in - depth interviews . based on the reliability exercise , over 90% of the codes matched with the codebook developed by the study staff . in all , two codebooks were generated for the interview and fgds respectively . all interviews and fgds were coded using maxqda software , 2007 ( berlin , germany ) . once the interview and fgd codebooks were generated , a triangulation technique was used to analyze which themes from the hiv - positive individuals and from hiv service providers respective perspectives supported as well as which contradicted one another . there were 30 hiv - infected patients , equal number of men and women , who participated in the study . their demographic details have been represented in table 1 . among the total patients , majority ( both men and women ) were from the younger age groups of 20 - 30 years and 31 - 40 years as compared to the older age groups of 41 - 50 years and 51 - 60 years . about three - fourth of the patients were married , a couple of female patients were widowed , a couple of patients ( equal number of men and women ) were divorced , few patients ( more women than men ) were separated from their spouse and a couple of patients ( equal number of men and women ) were unmarried at the time of the study . demographic details of plwha attending ssg hospital , vadodara the highest level of education among the patients was up to graduation which was found among a couple of female patients . in case of the male patients , less than half of them were educated up to 10 standard and about one - third were educated up to 12 standard . there were equal number of men and women among one - fifth of the patients who were illiterate . as regards employment , more than half of the patients ( more men than women ) were employed at the time of the study . among the patients selected for the study from vadodara district , less than half were from urban areas , one - third were from urban slums one - fifth were from rural areas and a couple of patients were from tribal areas . stigma associated with hiv was described as a barrier to disclosure because participants perceived hiv - positive status to have a negative impact on plwha and their children . the potential for hiv stigma to limit children 's marriage prospects was particularly worrisome and cited as a reason not to disclose one 's serostatus to people other than a spouse . a widowed female participant aged 30 years said , it would have adverse effects on my son ; the school might create problems for him . people may break relations and behave badly with me and my family as they consider hiv as a bad disease . in addition , a 33-year - old widow and a mother of three children , anxious about their future said , i fear that if i disclose my hiv positive status to everyone , it might affect my daughters marriage prospects . participants perceived that many of their friends and family held misconceptions about hiv which would lead to increased stigma and mistreatment after disclosure . a married female aged 28 years said , it would create a lot of problems for us , even if there is a slight indication in the society . people believe that one is infected by hiv only because of illicit relations . a separated female participant aged 42 years narrated , i had not disclosed my positive status to relatives and neighbors , but my husband revealed my positive status in his family and neighborhood . now , all my in - laws have cut off their relations with me and my son and neighbors discriminate against us . a female medical service provider stated plwha should not disclose their hiv status to the community due to hiv - related discrimination . the desire to protect others from the pain of knowing about their illness motivated some participants not to disclose . this concern seemed particularly salient in decisions about disclosing to parents , particularly if the parents were old or in poor health , or because they feared that the reality would be too painful and agonizing for their parents . i have not revealed my hiv positive status to my mother as i know that she would not be able to bear the reality . hiv service providers pointed out that in some cases , hiv - positive male participants withheld disclosure to their prospective spouse for fear of rejection , in turn passing infection on to her after marriage . a remarried male participant , aged 44 years , who had not disclosed his serostatus to his wife , discussed his dilemma saying , i have never practiced sex without using condoms saying i do nt want children . but my wife ( who is unaware of my serostatus ) fights a lot with me as she wants children . there were instances where family members disclosed participants hiv - positive status to others without their consent or will or in an , upset and disappointed by his ex - wife 's disclosure of his hiv status said , she told her brother , who in turn told her parents and then my father came to know . in half of the participants , physical incapacity and health problems were cited as reasons why their hiv - positive status was disclosed involuntarily to family members . a married male participant aged 44 years who disclosed his hiv - positive status due to his ill health , stated i have only told my mother as i was feeling weak and i could not even walk . medical providers stated that it was important for hiv - positive patients to disclose to their families , to protect caregivers from the potential risk of hiv infection . a married hiv - infected female participant aged 32 years , described how her and my husband 's condition did not improve at all , so the doctor asked to get the hiv test done . my report also came positive . while involuntary disclosure was a common thread among the participants , some discussed experiences of voluntary disclosure . voluntary disclosure occurred when participants felt morally obligated to divulge their serostatus to a spouse so s / he could get tested for hiv . a divorced male participant aged 30 years , who voluntarily disclosed his hiv - positive status to his wife stated , after reading the report , i thought this should not be kept a secret . the next day i took my wife and 11-year - old daughter for the test . another reason participants gave for voluntarily disclosing their hiv - positive status was to obtain emotional and physical support from family . an example is this explanation from a married female participant aged 26 years , who gave her reason for the decision to disclose her and her husband 's hiv - positive status to their parents , we told this to our parents only as they care for us . there are many consequences of disclosure [ figure 1 ] , but a couple of salient themes are presented below . a severe consequence of disclosure was family disruption leading to separation and divorce . a female participant aged 28 years , my mother - in law told me not to keep the child because i had hiv , the child could get it from me . but the doctors told us to take medicines and assured us with a 99% guarantee that the child would be hiv - negative and asked us to decide whether we want to keep the child or not . a divorced female participant aged 28 years expressed her grief , the doctor told me to get my cd4 count tested . so the doctor told me to abort the child as my cd4 count was low . consequences of disclosure some married female participants living in joint families , talked about being blamed by their mothers - in - law for either placing their son at risk for hiv or for being the source of their son 's hiv infection . my mother - in - law held me responsible for her son 's hiv infection . other women became aware of their serostatus only after being remarried and getting tested for hiv when they became pregnant . a divorced female participant aged 28 years , said with a lot of emotion , my first husband died due to hiv . my in - laws never told me about his hiv positive status even after his death . during my second marriage , i came to know about my being hiv positive when i was pregnant again . i advised him that whenever you get married , you either marry a positive person like yourself or inform the person about your positive status beforehand . after a few months , that same person came for getting his wife 's hiv test done . his wife was found positive ; he had not informed his wife about his hiv - positive status before marriage . although majority of patients described the barriers of disclosure , some hiv service providers and patients described the benefits . a female non - medical service provider explained , we try to explain to the patients that if they disclose their status to their families , they would take better care of them . if you tell any member of your family , who is close to you , then it would be better for you . medical service providers believed that hiv - positive patients would benefit from treatment by telling their providers about their seropositive status when going for any kind of treatment . this would also allow doctors to screen for tuberculosis , one of the most common opportunistic infections among plwha in india . a married female participant said , those persons in the neighborhood to whom i have disclosed my status , are good . they say that this disease does not spread by talking to an infected person or touching , so there is no harm in even eating food prepared by me . there were 30 hiv - infected patients , equal number of men and women , who participated in the study . their demographic details have been represented in table 1 . among the total patients , majority ( both men and women ) were from the younger age groups of 20 - 30 years and 31 - 40 years as compared to the older age groups of 41 - 50 years and 51 - 60 years . about three - fourth of the patients were married , a couple of female patients were widowed , a couple of patients ( equal number of men and women ) were divorced , few patients ( more women than men ) were separated from their spouse and a couple of patients ( equal number of men and women ) were unmarried at the time of the study . demographic details of plwha attending ssg hospital , vadodara the highest level of education among the patients was up to graduation which was found among a couple of female patients . in case of the male patients , less than half of them were educated up to 10 standard and about one - third were educated up to 12 standard . there were equal number of men and women among one - fifth of the patients who were illiterate . as regards employment , more than half of the patients ( more men than women ) were employed at the time of the study . among the patients selected for the study from vadodara district , less than half were from urban areas , one - third were from urban slums one - fifth were from rural areas and a couple of patients were from tribal areas . stigma associated with hiv was described as a barrier to disclosure because participants perceived hiv - positive status to have a negative impact on plwha and their children . the potential for hiv stigma to limit children 's marriage prospects was particularly worrisome and cited as a reason not to disclose one 's serostatus to people other than a spouse . a widowed female participant aged 30 years said , it would have adverse effects on my son ; the school might create problems for him . people may break relations and behave badly with me and my family as they consider hiv as a bad disease . in addition , a 33-year - old widow and a mother of three children , anxious about their future said , i fear that if i disclose my hiv positive status to everyone , it might affect my daughters marriage prospects . participants perceived that many of their friends and family held misconceptions about hiv which would lead to increased stigma and mistreatment after disclosure . a married female aged 28 years said , it would create a lot of problems for us , even if there is a slight indication in the society . people believe that one is infected by hiv only because of illicit relations . a separated female participant aged 42 years narrated , i had not disclosed my positive status to relatives and neighbors , but my husband revealed my positive status in his family and neighborhood . now , all my in - laws have cut off their relations with me and my son and neighbors discriminate against us . a female medical service provider stated plwha should not disclose their hiv status to the community due to hiv - related discrimination . the desire to protect others from the pain of knowing about their illness motivated some participants not to disclose . this concern seemed particularly salient in decisions about disclosing to parents , particularly if the parents were old or in poor health , or because they feared that the reality would be too painful and agonizing for their parents . i have not revealed my hiv positive status to my mother as i know that she would not be able to bear the reality . hiv service providers pointed out that in some cases , hiv - positive male participants withheld disclosure to their prospective spouse for fear of rejection , in turn passing infection on to her after marriage . a remarried male participant , aged 44 years , who had not disclosed his serostatus to his wife , discussed his dilemma saying , i have never practiced sex without using condoms saying i do nt want children . but my wife ( who is unaware of my serostatus ) fights a lot with me as she wants children . there were instances where family members disclosed participants hiv - positive status to others without their consent or will or in an , upset and disappointed by his ex - wife 's disclosure of his hiv status said , she told her brother , who in turn told her parents and then my father came to know . in half of the participants , physical incapacity and health problems were cited as reasons why their hiv - positive status was disclosed involuntarily to family members . a married male participant aged 44 years who disclosed his hiv - positive status due to his ill health , stated i have only told my mother as i was feeling weak and i could not even walk . medical providers stated that it was important for hiv - positive patients to disclose to their families , to protect caregivers from the potential risk of hiv infection . a married hiv - infected female participant aged 32 years , described how her and my husband 's condition did not improve at all , so the doctor asked to get the hiv test done . my report also came positive . while involuntary disclosure was a common thread among the participants , some discussed experiences of voluntary disclosure . voluntary disclosure occurred when participants felt morally obligated to divulge their serostatus to a spouse so s / he could get tested for hiv . a divorced male participant aged 30 years , who voluntarily disclosed his hiv - positive status to his wife stated , the next day i took my wife and 11-year - old daughter for the test . another reason participants gave for voluntarily disclosing their hiv - positive status was to obtain emotional and physical support from family . an example is this explanation from a married female participant aged 26 years , who gave her reason for the decision to disclose her and her husband 's hiv - positive status to their parents , we told this to our parents only as they care for us . there are many consequences of disclosure [ figure 1 ] , but a couple of salient themes are presented below . a severe consequence of disclosure was family disruption leading to separation and divorce . disclosure also impacted reproductive decision - making . a 21-year - old pregnant female participant , talked about her dilemma , my mother - in law told me not to keep the child because i had hiv , the child could get it from me . but the doctors told us to take medicines and assured us with a 99% guarantee that the child would be hiv - negative and asked us to decide whether we want to keep the child or not . a divorced female participant aged 28 years expressed her grief , the doctor told me to get my cd4 count tested . so the doctor told me to abort the child as my cd4 count was low . consequences of disclosure some married female participants living in joint families , talked about being blamed by their mothers - in - law for either placing their son at risk for hiv or for being the source of their son 's hiv infection . a married female participant , aged 35 years , said , my mother - in - law held me responsible for her son 's hiv infection . other women became aware of their serostatus only after being remarried and getting tested for hiv when they became pregnant . a divorced female participant aged 28 years , said with a lot of emotion , my first husband died due to hiv . my in - laws never told me about his hiv positive status even after his death . during my second marriage , i came to know about my being hiv positive when i was pregnant again . i advised him that whenever you get married , you either marry a positive person like yourself or inform the person about your positive status beforehand . after a few months , that same person came for getting his wife 's hiv test done . his wife was found positive ; he had not informed his wife about his hiv - positive status before marriage . although majority of patients described the barriers of disclosure , some hiv service providers and patients described the benefits . a female non - medical service provider explained , we try to explain to the patients that if they disclose their status to their families , they would take better care of them . if you tell any member of your family , who is close to you , then it would be better for you . medical service providers believed that hiv - positive patients would benefit from treatment by telling their providers about their seropositive status when going for any kind of treatment . this would also allow doctors to screen for tuberculosis , one of the most common opportunistic infections among plwha in india . a married female participant said , those persons in the neighborhood to whom i have disclosed my status , are good . they say that this disease does not spread by talking to an infected person or touching , so there is no harm in even eating food prepared by me . negative reactions influence the behavior of plwha and can undermine hiv disclosure . in india , hiv is perceived by some to be associated with an immoral lifestyle , which may contribute to hiv - related discrimination and stigmatization . such perceptions can negatively affect families , communities , workplaces , schools , and health - care settings . findings from this study revealed several factors which influenced the decision to disclose one 's positive hiv status , including perceived hiv - associated stigma , fear of discrimination , and family breakdown . fear of social stigma prevented hiv - positive participants from disclosing their status immediately to their partners and families which contributed to social isolation . this study also revealed that most of the hiv - positive wives disclosed their status to their husbands , whereas only a few of the hiv - positive husbands revealed their status to their wives . in a cross - sectional study conducted in kolkata , all of the female patients ( 100% ) had disclosed their serostatus to their sexual partners , as compared to 65% of male patients . lack of disclosure and resulting secrecy on the husband 's part contributed to conflict in the relationship . in one case , the husband insisted on condom use to protect his wife , but his wife resisted condom use because of her desire for children , which resulted in intimate partner violence ( ipv ) . the wife 's decision to have children may have been different had she known about her husband 's hiv status , which may have prevented ipv altogether . hiv service providers have an important role , as they may help foster hiv disclosure . in this study , many non - medical service providers explained the benefits of disclosure to their clients including spousal and family support . however , some findings of the present study also suggest discriminatory practices by hiv service providers which undermine the delivery of hiv prevention services including facilitating disclosure . in one qualitative study conducted in southern india , hiv - positive women felt discriminated against by their own health - care providers . one study reported that negative attitudes and discriminatory behaviors by providers towards plwha indicated a need for ethics training . additional and ongoing training in these areas are critical in creating environments supportive of hiv disclosure . our findings also showed that involuntary disclosure was more common than voluntary disclosure among hiv - infected participants . voluntary disclosure occurred when participants felt morally obliged to divulge their serostatus to their spouse or due to obtain emotional support from their family . a similar pattern regarding disclosure emerged from discussions with health service providers except that they felt that patients also disclosed their hiv - positive status to close friends . in western countries , disclosure to friends is more common as they may be more supportive than family members . however , in the present study , fear of discrimination by friends , peers , and colleagues at their workplace led to non - disclosure of hiv - positive status by participants . this study indicated some benefits of disclosure to family including : spousal support , care from other family members , protecting the seronegative spouse from hiv , and avoiding unintended pregnancy . however , although there are several benefits to disclosure , including the prompt hiv testing of sexual partners , it may not be enough given that hiv - related stigma can prevent hiv testing and treatment , which reinforces the notion that that the person who has the illness can be identified as the marked other via testing . addressing hiv - related stigma as a way to increase hiv disclosure remains a critical area for intervention . given the cultural context in india , gender - specific approaches to enhance hiv disclosure need to be considered . there has been a suggestion in previous studies about individualized approaches to post - test counseling , including enhanced support services for hiv - positive women and public education to de - stigmatize hiv - disease , particularly because hiv - positive women are more often at the receiving end of discrimination and hiv - associated stigma when compared to men . in addition , hiv - positive women are looked upon with suspicion and their moral character is questioned , especially when they are tested earlier than their spouse . our study showed similar findings with respect to hiv - positive test results having a disproportionate negative impact on women . couples - based counseling models which focus on both the seropositive and seronegative partner may also be critical in preventing hiv transmission . hiv prevention counseling in integrated counselling and treatment centre must address issues of fear of disclosure , potential negative consequences of disclosure , including stigma and discrimination when disclosing hiv - positive serostatus . training local counselors and other hiv service providers in the identified barriers and facilitators of serostatus disclosure is recommended .

background : human immunodeficiency virus ( hiv ) disclosure offers important benefits to people living with hiv / aids . however , fear of discrimination , blame , and disruption of family relationships can make disclosure a difficult decision . barriers to hiv disclosure are influenced by the particular culture within which the individuals live . although many studies have assessed such barriers in the u.s . , very few studies have explored the factors that facilitate or prevent hiv disclosure in india . understanding these factors is critical to the refinement , development , and implementation of a counseling intervention to facilitate disclosure.materials and methods : to explore these factors , we conducted 30 in - depth interviews in the local language with hiv- positive individuals from the integrated counselling and testing centre in gujarat , india , assessing the experiences , perceived barriers , and facilitators to disclosure . to triangulate the findings , we conducted two focus group discussions with hiv medical and non - medical service providers , respectively.results:perceived hiv - associated stigma , fear of discrimination , and fear of family breakdown acted as barriers to hiv disclosure . most people living with hiv / aids came to know of their hiv status due to poor physical health , spousal hiv - positive status , or a positive hiv test during pregnancy . some wives only learned of their husbands hiv positive status after their husbands died . the focus group participants confirmed similar findings . disclosure had serious implications for individuals living with hiv , such as divorce , maltreatment , ostracism , and decisions regarding child bearing.interpretation and conclusion : the identified barriers and facilitators in the present study can be used to augment training of hiv service providers working in voluntary counseling and testing centers in india .

MATERIALS AND METHODS Participant selection and recruitment In-depth interviews with PLWHA Focus groups with HIV service providers Study tools and training Data analysis RESULTS Demographic profile of HIV-infected patients Fear of stigmatization Reasons for nondisclosure Involuntary vs. voluntary disclosure Consequences of disclosure Benefits of disclosure DISCUSSION CONCLUSIONS

a sample of 30 hiv - infected individuals from a integrated counselling and testing centre ( ictc ) in vadodara , gujarat , india were selected to participate in individual face - to - face in - depth interviews after pilot testing . hiv - infected patients were eligible to participate in the study if they were : ( 1 ) english - or gujarati - speaking ; ( 2 ) 18 years or older ; and ( 3 ) diagnosed with hiv for at least 3 months . each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide . two focus groups were convened , one with seven medical doctors and the other with eight non - medical providers . hiv service providers were eligible to participate if they were : ( 1 ) gujarati - speaking ; ( 2 ) 18 years or over ; and ( 3 ) had worked with hiv - positive patients for more than 1 year as either a medical doctor , a non - governmental organization staff member , integrated counselling and testing counselor , or peer educator for hiv - positive persons . each of the focus groups lasted about 2 hours and was audio - recorded with prior permission of the participants . the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation . the entire research protocol was reviewed and approved by institutional review board ( irb ) of both the university of north carolina at chapel hill , usa and the medical college , baroda , india . the in - depth interviews and focus group guides were developed by research teams in india and the u.s . in addition , a community advisory board ( cab ) , comprising experts from the field of social work , medical providers , and hiv consultants was convened to discuss and refine the content of the qualitative instruments . the in - depth interview guide was designed to assess topics pertaining to hiv disclosure processes , perceived barriers and facilitators to disclosure , and hiv - associated stigma and discrimination related to disclosure among hiv - positive individuals . the following topics were explored with the hiv service providers : ( 1 ) secondary prevention services provided to plwha ; ( 2 ) the advice they gave to plwha regarding whom to disclose to and why ; and ( 3 ) the consequences that they perceived their clients faced after disclosure . extensive notes of in - depth interviews were recorded in gujarati , and translated into english . the focus group discussions were transcribed verbatim in gujarati from the audio - recordings and then the transcripts were translated into english . in the early phase of coding , we based initial themes on topics covered in the interview / focus group discussion ( fgd ) guides . in the later phases , in - depth interview and fgd transcripts were read several times and content analyzed , respectively using the technique of open coding to discover conceptual patterns , or themes , in the text . research staff in india and u.s . once all of the interviews were coded , a reliability exercise was conducted for the in - depth interviews . once the interview and fgd codebooks were generated , a triangulation technique was used to analyze which themes from the hiv - positive individuals and from hiv service providers respective perspectives supported as well as which contradicted one another . a sample of 30 hiv - infected individuals from a integrated counselling and testing centre ( ictc ) in vadodara , gujarat , india were selected to participate in individual face - to - face in - depth interviews after pilot testing . hiv - infected patients were eligible to participate in the study if they were : ( 1 ) english - or gujarati - speaking ; ( 2 ) 18 years or older ; and ( 3 ) diagnosed with hiv for at least 3 months . each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide . two focus groups were convened , one with seven medical doctors and the other with eight non - medical providers . hiv service providers were eligible to participate if they were : ( 1 ) gujarati - speaking ; ( 2 ) 18 years or over ; and ( 3 ) had worked with hiv - positive patients for more than 1 year as either a medical doctor , a non - governmental organization staff member , integrated counselling and testing counselor , or peer educator for hiv - positive persons . the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation . a sample of 30 hiv - infected individuals from a integrated counselling and testing centre ( ictc ) in vadodara , gujarat , india were selected to participate in individual face - to - face in - depth interviews after pilot testing . hiv - infected patients were eligible to participate in the study if they were : ( 1 ) english - or gujarati - speaking ; ( 2 ) 18 years or older ; and ( 3 ) diagnosed with hiv for at least 3 months . each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide . two focus groups were convened , one with seven medical doctors and the other with eight non - medical providers . hiv service providers were eligible to participate if they were : ( 1 ) gujarati - speaking ; ( 2 ) 18 years or over ; and ( 3 ) had worked with hiv - positive patients for more than 1 year as either a medical doctor , a non - governmental organization staff member , integrated counselling and testing counselor , or peer educator for hiv - positive persons . the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation . the in - depth interviews and focus group guides were developed by research teams in india and the u.s . in addition , a community advisory board ( cab ) , comprising experts from the field of social work , medical providers , and hiv consultants was convened to discuss and refine the content of the qualitative instruments . the in - depth interview guide was designed to assess topics pertaining to hiv disclosure processes , perceived barriers and facilitators to disclosure , and hiv - associated stigma and discrimination related to disclosure among hiv - positive individuals . the following topics were explored with the hiv service providers : ( 1 ) secondary prevention services provided to plwha ; ( 2 ) the advice they gave to plwha regarding whom to disclose to and why ; and ( 3 ) the consequences that they perceived their clients faced after disclosure . extensive notes of in - depth interviews were recorded in gujarati , and translated into english . the focus group discussions were transcribed verbatim in gujarati from the audio - recordings and then the transcripts were translated into english . in the early phase of coding , we based initial themes on topics covered in the interview / focus group discussion ( fgd ) guides . in the later phases , in - depth interview and fgd transcripts were read several times and content analyzed , respectively using the technique of open coding to discover conceptual patterns , or themes , in the text . research staff in india and u.s . once all of the interviews were coded , a reliability exercise was conducted for the in - depth interviews . once the interview and fgd codebooks were generated , a triangulation technique was used to analyze which themes from the hiv - positive individuals and from hiv service providers respective perspectives supported as well as which contradicted one another . there were 30 hiv - infected patients , equal number of men and women , who participated in the study . among the total patients , majority ( both men and women ) were from the younger age groups of 20 - 30 years and 31 - 40 years as compared to the older age groups of 41 - 50 years and 51 - 60 years . stigma associated with hiv was described as a barrier to disclosure because participants perceived hiv - positive status to have a negative impact on plwha and their children . in addition , a 33-year - old widow and a mother of three children , anxious about their future said , i fear that if i disclose my hiv positive status to everyone , it might affect my daughters marriage prospects . a married female aged 28 years said , it would create a lot of problems for us , even if there is a slight indication in the society . a female medical service provider stated plwha should not disclose their hiv status to the community due to hiv - related discrimination . this concern seemed particularly salient in decisions about disclosing to parents , particularly if the parents were old or in poor health , or because they feared that the reality would be too painful and agonizing for their parents . i have not revealed my hiv positive status to my mother as i know that she would not be able to bear the reality . hiv service providers pointed out that in some cases , hiv - positive male participants withheld disclosure to their prospective spouse for fear of rejection , in turn passing infection on to her after marriage . there were instances where family members disclosed participants hiv - positive status to others without their consent or will or in an , upset and disappointed by his ex - wife 's disclosure of his hiv status said , she told her brother , who in turn told her parents and then my father came to know . in half of the participants , physical incapacity and health problems were cited as reasons why their hiv - positive status was disclosed involuntarily to family members . a married male participant aged 44 years who disclosed his hiv - positive status due to his ill health , stated i have only told my mother as i was feeling weak and i could not even walk . medical providers stated that it was important for hiv - positive patients to disclose to their families , to protect caregivers from the potential risk of hiv infection . a married hiv - infected female participant aged 32 years , described how her and my husband 's condition did not improve at all , so the doctor asked to get the hiv test done . a divorced male participant aged 30 years , who voluntarily disclosed his hiv - positive status to his wife stated , after reading the report , i thought this should not be kept a secret . another reason participants gave for voluntarily disclosing their hiv - positive status was to obtain emotional and physical support from family . an example is this explanation from a married female participant aged 26 years , who gave her reason for the decision to disclose her and her husband 's hiv - positive status to their parents , we told this to our parents only as they care for us . consequences of disclosure some married female participants living in joint families , talked about being blamed by their mothers - in - law for either placing their son at risk for hiv or for being the source of their son 's hiv infection . my mother - in - law held me responsible for her son 's hiv infection . a divorced female participant aged 28 years , said with a lot of emotion , my first husband died due to hiv . my in - laws never told me about his hiv positive status even after his death . during my second marriage , i came to know about my being hiv positive when i was pregnant again . i advised him that whenever you get married , you either marry a positive person like yourself or inform the person about your positive status beforehand . his wife was found positive ; he had not informed his wife about his hiv - positive status before marriage . although majority of patients described the barriers of disclosure , some hiv service providers and patients described the benefits . a female non - medical service provider explained , we try to explain to the patients that if they disclose their status to their families , they would take better care of them . medical service providers believed that hiv - positive patients would benefit from treatment by telling their providers about their seropositive status when going for any kind of treatment . a married female participant said , those persons in the neighborhood to whom i have disclosed my status , are good . there were 30 hiv - infected patients , equal number of men and women , who participated in the study . among the total patients , majority ( both men and women ) were from the younger age groups of 20 - 30 years and 31 - 40 years as compared to the older age groups of 41 - 50 years and 51 - 60 years . stigma associated with hiv was described as a barrier to disclosure because participants perceived hiv - positive status to have a negative impact on plwha and their children . in addition , a 33-year - old widow and a mother of three children , anxious about their future said , i fear that if i disclose my hiv positive status to everyone , it might affect my daughters marriage prospects . now , all my in - laws have cut off their relations with me and my son and neighbors discriminate against us . a female medical service provider stated plwha should not disclose their hiv status to the community due to hiv - related discrimination . this concern seemed particularly salient in decisions about disclosing to parents , particularly if the parents were old or in poor health , or because they feared that the reality would be too painful and agonizing for their parents . i have not revealed my hiv positive status to my mother as i know that she would not be able to bear the reality . hiv service providers pointed out that in some cases , hiv - positive male participants withheld disclosure to their prospective spouse for fear of rejection , in turn passing infection on to her after marriage . there were instances where family members disclosed participants hiv - positive status to others without their consent or will or in an , upset and disappointed by his ex - wife 's disclosure of his hiv status said , she told her brother , who in turn told her parents and then my father came to know . in half of the participants , physical incapacity and health problems were cited as reasons why their hiv - positive status was disclosed involuntarily to family members . a married male participant aged 44 years who disclosed his hiv - positive status due to his ill health , stated i have only told my mother as i was feeling weak and i could not even walk . medical providers stated that it was important for hiv - positive patients to disclose to their families , to protect caregivers from the potential risk of hiv infection . a married hiv - infected female participant aged 32 years , described how her and my husband 's condition did not improve at all , so the doctor asked to get the hiv test done . a divorced male participant aged 30 years , who voluntarily disclosed his hiv - positive status to his wife stated , the next day i took my wife and 11-year - old daughter for the test . another reason participants gave for voluntarily disclosing their hiv - positive status was to obtain emotional and physical support from family . an example is this explanation from a married female participant aged 26 years , who gave her reason for the decision to disclose her and her husband 's hiv - positive status to their parents , we told this to our parents only as they care for us . a 21-year - old pregnant female participant , talked about her dilemma , my mother - in law told me not to keep the child because i had hiv , the child could get it from me . consequences of disclosure some married female participants living in joint families , talked about being blamed by their mothers - in - law for either placing their son at risk for hiv or for being the source of their son 's hiv infection . other women became aware of their serostatus only after being remarried and getting tested for hiv when they became pregnant . a divorced female participant aged 28 years , said with a lot of emotion , my first husband died due to hiv . my in - laws never told me about his hiv positive status even after his death . during my second marriage , i came to know about my being hiv positive when i was pregnant again . i advised him that whenever you get married , you either marry a positive person like yourself or inform the person about your positive status beforehand . after a few months , that same person came for getting his wife 's hiv test done . his wife was found positive ; he had not informed his wife about his hiv - positive status before marriage . although majority of patients described the barriers of disclosure , some hiv service providers and patients described the benefits . a female non - medical service provider explained , we try to explain to the patients that if they disclose their status to their families , they would take better care of them . medical service providers believed that hiv - positive patients would benefit from treatment by telling their providers about their seropositive status when going for any kind of treatment . this would also allow doctors to screen for tuberculosis , one of the most common opportunistic infections among plwha in india . a married female participant said , those persons in the neighborhood to whom i have disclosed my status , are good . in india , hiv is perceived by some to be associated with an immoral lifestyle , which may contribute to hiv - related discrimination and stigmatization . findings from this study revealed several factors which influenced the decision to disclose one 's positive hiv status , including perceived hiv - associated stigma , fear of discrimination , and family breakdown . fear of social stigma prevented hiv - positive participants from disclosing their status immediately to their partners and families which contributed to social isolation . this study also revealed that most of the hiv - positive wives disclosed their status to their husbands , whereas only a few of the hiv - positive husbands revealed their status to their wives . the wife 's decision to have children may have been different had she known about her husband 's hiv status , which may have prevented ipv altogether . hiv service providers have an important role , as they may help foster hiv disclosure . in this study , many non - medical service providers explained the benefits of disclosure to their clients including spousal and family support . however , some findings of the present study also suggest discriminatory practices by hiv service providers which undermine the delivery of hiv prevention services including facilitating disclosure . in one qualitative study conducted in southern india , hiv - positive women felt discriminated against by their own health - care providers . additional and ongoing training in these areas are critical in creating environments supportive of hiv disclosure . a similar pattern regarding disclosure emerged from discussions with health service providers except that they felt that patients also disclosed their hiv - positive status to close friends . however , in the present study , fear of discrimination by friends , peers , and colleagues at their workplace led to non - disclosure of hiv - positive status by participants . this study indicated some benefits of disclosure to family including : spousal support , care from other family members , protecting the seronegative spouse from hiv , and avoiding unintended pregnancy . however , although there are several benefits to disclosure , including the prompt hiv testing of sexual partners , it may not be enough given that hiv - related stigma can prevent hiv testing and treatment , which reinforces the notion that that the person who has the illness can be identified as the marked other via testing . addressing hiv - related stigma as a way to increase hiv disclosure remains a critical area for intervention . given the cultural context in india , gender - specific approaches to enhance hiv disclosure need to be considered . there has been a suggestion in previous studies about individualized approaches to post - test counseling , including enhanced support services for hiv - positive women and public education to de - stigmatize hiv - disease , particularly because hiv - positive women are more often at the receiving end of discrimination and hiv - associated stigma when compared to men . in addition , hiv - positive women are looked upon with suspicion and their moral character is questioned , especially when they are tested earlier than their spouse . our study showed similar findings with respect to hiv - positive test results having a disproportionate negative impact on women . hiv prevention counseling in integrated counselling and treatment centre must address issues of fear of disclosure , potential negative consequences of disclosure , including stigma and discrimination when disclosing hiv - positive serostatus . training local counselors and other hiv service providers in the identified barriers and facilitators of serostatus disclosure is recommended .

in this article , we study trends in family - formation behavior since 1960 in the countries that used to be called the eastern bloc . in this connection , the account of the second demographic transition ( sdt ) is very attractive , both as a generalized summarizing description and because of its underlying theory of value change in the direction of increasing tolerance in family matters and of women s increasing autonomy ( lesthaeghe and van de kaa 1986 ; lesthaeghe and surkyn 2002 ; for a recent , independent assessment , see sobotka 2008 ) . the sdt account consists of a narrative of changes in demographic behavior and of an explanation for those changes . the changes on which the narrative focuses are a decline in marriage formation , an increase in non - marital cohabitation , a general decrease in fertility ( particularly at higher birth orders ) but an increase in non - marital childbearing , an increase in union disruption , and a postponement of marriage and childbearing . briefly stated , the explanation given is that these developments are caused by ideational changes regarding family life and childbearing , i.e. , changes in norms , values , beliefs , and attitudes , sometimes operating in tandem with political , economic , and social changes . there is ample evidence of most of the demographic developments in the sdt narrative all over europe , particularly concerning fertility trends ; see for instance frejka et al . there is also already quite some literature on recent changes in union - formation behavior in central and eastern europe and on their interpretation ( carlson and klinger 1987 ; lesthaeghe and surkyn 2002 ; aassve et al . 2004 ; spder 2004 and 2005 , zakharov 2005 , gerber and berman 2005 ; koytcheva 2006 , thornton and philipov 2007 ; kostova 2007 , muresan ( 2007a , b ) , and bradatan and kulcsar 2008).1 too little attention has been given so far to the finer structure of the trends in union - formation risks in the region , however , which is surprising , given that ideational change must be a force behind the growth in non - marital cohabitation and therefore a prime indicator of the very explanation given for the sdt . in this article , we focus therefore on non - marital cohabitation as a competitor to conventional marriage . our account is for bulgaria , romania , russia , and hungary , for we are fortunate in having early access to the data from the first round of the gender and generations surveys ( ggs ) in the first three of these countries and to their close counterpart for hungary.2 all the surveys have used a random sample of women and men of all relevant ages . in this present study , we use the data for women only ; for sample sizes ( in terms of years of exposure ) see table 1.table 1person - years of exposure since 1960total person - years of exposureyear of data collectionas childless , not pregnantas childless , pregnantat parity 1russia35,1613733,8652004bulgaria40,0573601,9892004romania33,9312901,4162005hungary49,7474559512001notethe first wave of the hungarian ggs ( originally called turning points of the life - course ) was conducted in november 2001 through january 2002 , but we do not use data collected in 2002 in our studysource our own calculations based on ggs data person - years of exposure since 1960 notethe first wave of the hungarian ggs ( originally called turning points of the life - course ) was conducted in november 2001 through january 2002 , but we do not use data collected in 2002 in our study source our own calculations based on ggs data we started this investigation in a descriptive mood and without any strong preconceived ideas or hypotheses about union - entry trends , but with a series of open questions . we were curious to see to what extent the fall of communism around 1990 might have given a particular impetus to developments in union formation across the four countries , and what commonalities we could find in the patterns of such developments . the single - country background and previous literature has been described succinctly for russia and bulgaria by philipov and jasilioniene ( 2007),3 for romania by muresan ( 2007a , b),4 and for hungary by spder ( 2005 ) . the three former authors have provided extensive survival tables for russia , bulgaria , and romania in the spirit of andersson and philipov ( 2002 ) , who gave such tables for hungary and fifteen other european countries for an earlier period . following carlson and klinger ( 1987 ) ; spder ( 2004 , 2005 ) maintained that post - divorce non - marital cohabitation has old roots in hungary and that consensual first unions gained considerable ground in that country well before the regime change . we focus on first unions and find similar patterns also for russia , romania , and bulgaria . table 2 contains some highlights for the three ggs countries for which period survival tables are available,5 and we see that there was considerable cohabitation already around the late 1980s and that in bulgaria and russia , it had outflanked direct marriage at least by the early 21st century . according to this table , romania seems to be in a different category , where marriage had held up much better than in bulgaria and russia . statistics like those of table 2 have been derived from straightforward occurrence / exposure rates , with no standardization nor any other attempt at hedging against compositional effects ; so , we started out wondering to what extent the considerable differences between the countries would hold up to closer scrutiny.table 2entry into marital and non - marital unions as competing events in bulgaria , romania , and russia . womenperiodbulgariarussiaromaniaever entered into cohabitation marriageever entered into cohabitation marriageever entered into cohabitation marriage19851989543734622076199019946032465019992003631462333556source philipov and jasilioniene ( 2007 ) , table a8source muresan ( 2007b ) , tables 5.5 and 5.61980198919962005 entry into marital and non - marital unions as competing events in bulgaria , romania , and russia . women source philipov and jasilioniene ( 2007 ) , table a8 source muresan ( 2007b ) , tables 5.5 and 5.6 in demography , one of the ways to handle compositional effects is by using standardization , and we have applied this method in the form of an unusual variant of intensity regression where entry into marriage and into a non - marital union are studied jointly as competing risks in a manner that permits direct comparison between the two types of union formation in each of the four countries.6 this procedure has been described most fully by hoem and kostova ( 2008 ) , to whom we refer for mathematical aspects of the approach we use . ( we give some further discussion of such items in an appendix to this article . ) this article can be regarded as a further elaboration of the bulgarian data and an extension to the three other countries for which we also have data . based on data in a monthly format for the years since 1960 , we have used proportional - hazards event - history analysis with a piecewise constant baseline intensity to reflect the impact of a woman s age , formally using the type of union formed as a fixed covariate in addition to the other fixed and time - varying covariates available to us ( the determinants ) . among the determinants we have included a time - varying covariate that we call pregnancy - and - parity status . it provides a differentiation between ( i ) non - pregnant childless women ( ii ) pregnant childless women,7 and ( iii ) mothers , i.e. , women at parities 1 and above . the first of these three groups overwhelmingly dominates the exposures to the risk of first - union formation ( table 1 ) and we report most of our results for this group alone . since our focus is on the changing trends in union formation , we display the interaction between calendar time and union type in our descriptions below , and let the other available covariates appear as control variables . these are most importantly ( self - reported ) ethnicity , but also a number of covariates that are intended to reflect other aspects of the respondent s background , namely whether she grew up in an urban or rural region , whether she lived with both parents at age 15 , her number of siblings , her own educational attainment , and the educational attainments of her mother and father.8 these are standard covariates readily available in our data , except the respondent s own educational attainment . we would have used it more extensively if we had had enough information to make it a genuine time - varying covariate , but the data only contain the attainment made by the time of data collection plus the time at which the respondent had reached this level of education ( according to her own report ) , so we have had to impute a non - fixed covariate using a method developed by hoem and kreyenfeld ( 2006 ) . since this is not the real thing , we do not report the outcome here , nor do we report the risk patterns for our other control variables , mainly in order not to detract attention from our main focus on union - entry trends , but also because they do not contain any really notable surprises , particularly since bradatan and kulcsar ( 2008 ) went this way before us . among the findings that we do report is a strong drop in the marriage - formation risk in all the four countries and a counterpart increase in the risk of entry into non - marital unions , though surprisingly in bulgaria ( and possibly hungary ) this increase turned into a drop at the beginning of the 2000s.9 as one of our referees has pointed out , this may just be a sign of accelerated postponement of entry into a first union , which would be another typical trait of the sdt . to give a feeling for the size order of the relative union - formation risks in our four data sets in the twilight years of state communism , we attach table 3 , where for each country we display the ( two - way ) empirical interactions between the type of union formation ( marital and non - marital ) on the one hand and pregnancy - and - parity status on the other . the estimates have been produced by an intensity regression where age and calendar time appear formally as ( time - varying ) control variables not involved in any interactions , so the figures represent a kind of average over active childbearing ages and over the forty - odd years since 1960.table 3relative risk of first - union formation by parity - and - pregnancy status , for each type of union . 2004childless , not pregnantchildless , pregnantparity 1 ( mother)russia 19602004cohabitation0.502.340.48marriage ( direct)18.400.42bulgaria 19602004cohabitation1.3111.690.64marriage ( direct)117.070.47romania 19602005cohabitation0.241.700.16marriage ( direct)18.470.73hungary 19602001cohabitation0.321.360.50marriage ( direct)117.691.25note standardized with respect to age , ethnicity , calendar period , character of region where respondent grew up ( urban / rural ) , whether respondent lived with both parents at age 15 , number of siblings , own educational attainment , and mother s and father s educational attainments . some of these covariates have not been available for hungary . for romania the parents educational attainments were not included because of data quality problemssource our own calculations based on ggs data relative risk of first - union formation by parity - and - pregnancy status , for each type of union . our selected countries , 1960-ca . 2004 note standardized with respect to age , ethnicity , calendar period , character of region where respondent grew up ( urban / rural ) , whether respondent lived with both parents at age 15 , number of siblings , own educational attainment , and mother s and father s educational attainments . the parents educational attainments were not included because of data quality problems source our own calculations based on ggs data the general pattern is that as long as a woman was childless and not pregnant , the risk of entry into a non - marital union most often was low by comparison to the risk of marriage formation . note that our method allows for a direct comparison of the union - formation risks across the two types of unions in each country . ) not surprisingly , the union - formation intensities increased strongly if the woman became pregnant , and the increase was particularly strong for marriage formation . if she did not form a union before she had her ( first ) child , then the entry intensities largely went back to the size order they had before she became pregnant , or even to something smaller . it is as if the arrival of the first child is some kind of watershed , after which the woman was less attractive as a partner , or alternatively that the remaining women were less attracted by partnership . only in hungary , mothers still ran a ( somewhat ) higher risk of entry into a union , especially a marital union , than before they became pregnant . to get closer to the changing dynamics of union formation , we report the trends in ( standardized ) entry rates since 1960 in fig . 1 , computed separately for each of the four countries . these are relative risks of entry into cohabitation and into marriage for childless non - pregnant women10 in a two - way interaction between calendar period and decrement type , standardized for the control variables listed above . the basis of comparison is the country - specific risk of entry into a marital union for childless non - pregnant women in 19601964.11,12fig . non - pregnant childless women in russia , romania , bulgaria , and hungary , since 1960 . source our own calculations based on ggs data trends in the rates of union formation , by type of union . non - pregnant childless women in russia , romania , bulgaria , and hungary , since 1960 . source our own calculations based on ggs data the following patterns strike the eye : in bulgaria and hungary , marriage risks have decreased over time ever since the early 1980s ( roughly ) ; in russia they have decreased strongly since half a decade later , and in romania since another half a decade later again . in all the countries , the risks of entry into non - marital unions have increased ever since the 1960s , much as one would expect from descriptions of the sdt.13 taken together , these manifestations started well before the fall of communism , particularly for entry into consensual unions . developments of this nature have been noted earlier by gerber and berman ( 2005 ) and by spder ( 2004 , 2005 ) . as we just said , the marriage risk has fallen since the early 1980s , but the entry risk for cohabitation stabilized during the 1980s and 1990s . if anything , it dropped after the turn of the century . this looks like a deviation from ( standard ) patterns in the sdt , though one should note that the cohabitational entry risks continued to increase relative to the marriage risks throughout the whole period of our observation.14 , 15 romania is another exception from the general trend in the risks of entry into cohabitation , relative to that of marriage formation . even if the process of first union formation largely follows the trends observed in the other three countries , marriage was the dominating type of first union throughout the entire period of observation . if we add an interaction between age attained and decrement ( union type ) in the intensity regression that produces the standardized risk trends mentioned above , we get age profiles for the two entry risks as an extra bonus ( fig . 2 ) . we had expected entry into cohabitation to be shifted toward younger ages than the age profile for marriage formation , much as in the diagram for bulgaria , but the diagrams for russia , hungary and romania show how incorrect such a preconception could be.fig . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1960-ca . 2004 . source our own calculations based on ggs data age profiles of entry risks of union formation , by type of union . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1960-ca . 2004 . the findings presented in section 3 provide a neat and compact description of entry trends in the four countries , based on a standardization technique of a type that is ubiquitous in demography.16 standardization is known to summarize risk trends and differentials well under wide conditions , and to be robust against mild deviations from those conditions . one of the conditions that we have not addressed above is the assumption of a stable age profile in the risks , i.e. , we have behaved as if each of the two piecewise constant baseline hazards ( one for each decrement ) were the same for all calendar periods in the analysis . this may have simplified matters unduly ; after all , many authors document to their satisfaction that there has been a delay in union formation , so marriage and perhaps entry into cohabitation occur progressively later in life as calendar time increases . one question is , therefore , how robust the results above are against what may be a misspecification . to check on this question , we have estimated the hazard parameters once more , but now with a three - way interaction between age , period , and decrement.17 the outcome is given in fig . 3 , where to avoid needless complication , we have temporarily used five - year age groups and have concentrated on the years between 1980 and the survey date.18 for each country , we have plotted the age profiles of the rates of union formation for each period k , and we get the following graphical patterns , which can serve as a simple optical goodness - of - fit test of our basic specification.fig . 3age profiles of entry risks of union formation , by type of union and period . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1980-ca . source our own calculations based on ggs data age profiles of entry risks of union formation , by type of union and period . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1980-ca . source our own calculations based on ggs data for hungary the entry risk for marriage formation has indeed shifted steadily toward higher ages ; for entry into cohabitation they seem to have shifted somewhat in the opposite direction . for romania we also see a bit of a shift toward later ages in the risk of entry into marriage , while in bulgaria we can see a similar shift in the risk of entry into non - marital cohabitation . with some good will , one can even discern some tendency for the profile to shift a little toward younger ages in the risk diagrams for russia . all in all , perhaps there is only a mild deviation from the requirement of a stable age profile in bulgaria , russia , and romania . by way of conclusion , to get a realistic representation it looks as if we may be able to make do with our original intensity specification for russia , romania , and bulgaria , but not necessarily for hungary . for the latter country , we have therefore tried the specification with a three - way interaction between age , period , and decrement once more , but now with our finer age specification and with periods back to 1960 . the result is that for each age group we can essentially draw a diagram like that of the corresponding panel in fig . 1 ( details available from the first author ) . in our view , therefore , the whole story of the entry trends in hungary since the 1960s is adequately represented in fig . 1 , in any case . except for details , we draw the same conclusion concerning the intensity age profiles in fig . 2 . as we mentioned toward the end of section 3 , we have found that lately the risk of entry into cohabitation has dropped somewhat in bulgaria . to see whether this means that bulgarian women have given up on the sdt , at least as far as union formation is concerned , it pays to introduce an additional dimension , namely , the conversion of non - marital unions into marriages . 4a , which is similar to a corresponding figure presented by hoem and kostova ( 2008 , fig . 4 ) , but which is now constructed in a way that covers the whole period and the entire population of this study . in fig . 5 the same data are seen from a different angle , but it tells the same story , namely , that the sdt remains in progress in bulgaria . here is some further background information.fig . 4relative rates of conversion of cohabitation into marriage , by time since entry into cohabitation for each calendar period , women in bulgaria and hungary , 1960-ca . rates relative to a conversion during the first 6 months in the period 19601969 . 5relative rates of conversion of cohabitation into marriage , by calendar period for each duration since entry into cohabitation , bulgarian women , 19602004 . rates relative to a conversion during the first 6 months in the period 19601969 . source our own calculations based on ggs data relative rates of conversion of cohabitation into marriage , by time since entry into cohabitation for each calendar period , women in bulgaria and hungary , 1960-ca . rates relative to a conversion during the first 6 months in the period 19601969 . source our own calculations based on ggs data relative rates of conversion of cohabitation into marriage , by calendar period for each duration since entry into cohabitation , bulgarian women , 19602004 . rates relative to a conversion during the first 6 months in the period 19601969 . source our own calculations based on ggs data consensual unions seem to have been entrenched in bulgaria for a long time . ( note how high the bulgarian curve for entry into cohabitation is in fig . 1 . ) anecdotal evidence suggests that there may have been a long - standing pattern where couples who are engaged to be married , move in with one set of their parents and then marry only subsequently , when this fits the family economy and other practical circumstances ( observation by kostova 2007 ) . [ this fits well to the quick conversions of consensual to marital unions noted by koytcheva ( 2006 , sect . 7.1.1 ) based on bulgarian data sets different from the ggs . ] in our data , this would be recorded as an entry into a consensual union and a later conversion of the union into a marriage . 4a and 5 show that after the fall of communism , the conversion activity was scaled down considerably . a consensual union became a much more durable arrangement , fully in agreement with what a description of the sdt would predict . figure 4b extends this painlessly to hungary , for which , as we remember , we have found a similar drop in the two years right after the turn of the century ( fig . 1 ) . extensions to the data for romania and russia largely show the same pattern for conversion risks ( not documented here ) . the union - formation trends that we have revealed in this descriptive study of the four countries in central and eastern europe turn out to have several features in common . marriage formation has dropped in all the four countries since the fall of communism , and sometimes earlier . consensual unions have gained ground all the time until the end of the twentieth century , and only in bulgaria and hungary does popular interest in consensual - union formation seem to have been reduced somewhat thereafter . in all the four countries , the wind has gone out of the sails of conversions of consensual unions into marriages ; so non - marital unions have progressively stayed consensual longer . despite all commonalities , it is evident that the sdt , of which we have found some traces , is not a unitary movement that reached all the countries in central and eastern europe roughly at the same time and had the same features throughout , no more than it was in western europe . if anything , such a transition did not start simultaneously in all of the four countries , and above all , it began well before the fall of communism and before the societal transition to a market economy got underway around 1990 . if we take the distinct drop in marriage formation as a main marker of the start of the sdt as we study it in this article , then a rough estimate would be that it started in hungary and bulgaria after the early 1980s and in russia and romania half - a - decade and a full decade later , respectively . such differences should fit with the economic and social developments in the countries , but establishing such a correspondence is a matter of future research . in particular , the special trends in bulgaria ( and possibly hungary ) need further investigation , most likely by bringing in further dimensions in the analysis . we doubt whether it will be enough to continue to study standardized trends in decrement - specific union formation . in any case , our empirical findings have put similar observations made by lesthaeghe and surkyn ( 2002 ) ; gerber and berman ( 2005 ) ; zakharov ( 2005 ) , and spder ( 2004 , 2005 ) on a firmer empirical ground than before . as a final reflection on our findings , we want to underline that interpretations should be made with some prudence , for it is possible that the perception of what constitutes a consensual union has varied across countries and has changed over time , and also that reporting inaccuracy may have exaggerated the early growth of entry risks for consensual unions . in brief , the reporting accuracy depends on the respondents ability to recall and willingness to reveal cohabitational episodes . it is possible that cohabitational episodes that occurred long ago may have been forgotten or suppressed more often than more recent episodes,19 and if this is the case , cohabitational behavior at the beginning of our period of observation may have been more extensive than what we can report . if so , then the value change central to the sdt explanation may have been smaller than what meets the eye . for a discussion of the mathematical structure of our transition intensities , we note that the quantities plotted in figs . 1 , 2 are maximum - likelihood estimates of parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{kh } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{ih } $ $ \end{document } of multiplicative intensity functions that in its most general four - factor representation has the form1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijkh } = a_{ih } b_{jh } c_{kh } $ $ \end{document}where i is the age group , j is the background group , k is the calendar period , and h is the decrement.20 as we have noted , in the present application , the latter stands for the type of union formation , i.e. , for entry into a marital or non - marital union , represented by h = 1 and h = 2 , respectively , say . if we let a stand for the age factor , b for the combination of all the background factors that we mentioned as covariates in section 2 , c for the calendar period , and d for the decrement,21 then the above specification of the union - formation intensity can be written symbolically as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu = \,ad + bd + cd\ , , $ $ \end{document } where a double letter like ad or cd indicates that we include an interaction between the two factors involved ( a and d , say , represented in ( 1 ) by a double subscript on the a parameter ) and a plus sign indicates that an interaction has not been included . , 2 work as a fair representation of our data , is that for each type h of union entry , the age effect\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \ { a_{ih } \ } $ $ \end{document}is the same in all periodsk and for all levels j on the background factor , as is indicated here by the lack of subscripts k and j on the a parameter in ( 1 ) . in brief , there is a requirement of ( i ) stability and ( ii ) uniformity in the age effects for our standardization to work without problem . ( similar requirements must be satisfied for factor b. ) as we indicated in section 4 , we are of the opinion that the requirements on factor a cause no essential problem for our empirical analysis . we now turn to the issue of the interpretation of the intensity parameters as relative risks . if we had been willing to analyze each decrement separately , then we would be dealing with two individual intensities \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk1 } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk2 } $ $ \end{document } , and the very multiplicative specification of each of them , as in ( 1 ) , would make sure that the parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{jh } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{kh } $ $ \end{document } could be interpreted as relative risks , in the usual manner for single - decrement intensities ( hoem 1976 ) . ( for the baseline factor a , we operate with absolute risks , per 1,000 person - months for instance , and the issue of relative risks does not concern the a parameters . ) to secure parameter identification , we would impose side conditions of the type \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{{k_{0 } 1 } } = 1 $ $ \end{document}and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{{k_{0 } 2 } } = 1 $ $ \end{document } for a suitable period \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k_{0 } $ $ \end{document } , and we would use similar side conditions for the b parameters . when we want to analyze \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk1 } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk2 } $ $ \end{document}jointly for the purpose of seeing how one of them develops over time ( i.e. , as a function of k ) relative to the other , things turn out to be a bit more complicated . first , we drop one of the side conditions on each parameter set , and only require that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{{k_{0 } 1 } } = 1 $ $ \end{document } for the c parameters , say . for the period factor c , we are faced with two types of relative risks , as follows : ( i ) for any given type h of union formation , the intensity of union entry for a factor combination ( i , j , k ) , relative to the combination ( i , j , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k_{0 } $ $ \end{document } ) , is2.h\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijkh } /\mu_{{ijk_{0 } h } } \ , = \frac{{a_{ih } b_{jh } c_{kh } } } { { a_{ih } b_{jh } c_{{k_{0 } h } } } } \ , = \,\frac{{c_{kh } } } { { c_{{k_{0 } h } } } } . $ $ \end{document}thus in particular for h = 1,2.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk1 } /\mu_{{ijk_{0 } 1 } } \ , = \,c_{k1 } \ , , $ $ \end{document}(because of the side condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{{k_{0 } 1 } } = 1 $ $ \end{document } ) and we see that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{k1 } $ $ \end{document } is a relative risk in its own right . furthermore,2.2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk2 } /\mu_{{ijk_{0 } 2 } } \ , = \,c_{k2 } /c_{{k_{0 } 2 } } \ , , $ $ \end{document}which shows that up to a divisor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{{k_{0 } 2 } } $ $ \end{document } , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{k2 } $ $ \end{document } are relative risks also . all in all , we have established that the items \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{kh } $ $ \end{document } can essentially be interpreted as relative risks along each curve for every country in fig . 1 . thus ( 2.1 ) and ( 2.2 ) show that the form of the trend curves remains independent of the specification of the age and background parameters ( a and b ) . ( ii ) it remains to compare corresponding points on the two curves for each country , i.e. , to compare the curve point for the coordinate ( k , 1 ) with the curve point for ( k , 2 ) for each period k. note that as has essentially been shown before by hoem and kostova ( 2008 , end of appendix),3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk2 } /\mu_{ijk1 } \ , = \,\frac{{a_{i2 } b_{j2 } c_{k2 } } } { { a_{i1 } b_{j1 } c_{k1 } } } \ , = \,s_{ij } \frac{{c_{k2 } } } { { c_{k1 } } } \,{\text{with}}\,s_{ij } = \,\,\frac{{a_{i2 } b_{j2 } } } { { a_{i1 } b_{j1 } } } . $ $ \end{document}with the model specification \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu \ , = \,a + b + cd $ $ \end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{i2 } = a_{i1 } $ $ \end{document } for all i and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{j2 } = b_{j1 } $ $ \end{document } for all j , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_{ij } \equiv 1 $ $ \end{document}. we have used this model specification to produce all the curves in fig . 1 , and see that we can , therefore , compare directly the trend and level of the risk of entry into cohabitation with the corresponding risk of marriage formation for each country . our computer program will provide estimates for the a , b , and c parameters even if we do not have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_{ij } \equiv 1 $ $ \end{document } , and the estimates may have some interest in their own right , but we can no longer automatically interpret the c estimates as relative risks and they may easily deviate considerably from those produced by the specification \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu \ , = \,a + b + cd $ $ \end{document } , except in special cases . for instance , the specification \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu \ , = \,ad + b + cd $ $ \end{document } , produces separate age profiles for the two decrements , for it means that we have let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijkh } = a_{ih } b_{j } c_{kh } $ $ \end{document } , with two separate age profiles \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \ { a_{i1 } \ } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \ { a_{i2 } \ } $ $ \end{document } , which we have plotted for each country in fig . 2 . we see that for each country , the age profiles of the two entry risks largely coincide except in details ( i.e. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{i1 } \approx a_{i2 } $ $ \end{document } for all i ) , i.e. , the near - equality of the age profiles need not be such a terrible approximation , though for the details it manifestly is a tall order . so long as we are willing to accept approximations liberally , as one generally does when one practices standardization , the c parameters can therefore still be interpreted as relative risks , because according to ( 3 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { c_{k2 } } \mathord{\left/ { \vphantom { { c_{k2 } } { c_{k1 } } } } \right . \kern-\nulldelimiterspace } { c_{k1 } } } $ $ \end{document } largely represents the relative risk\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijk2 } /\mu_{ijk1 } ( remember that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{j1 } = b_{j2 } $ $ \end{document } for all j with the given intensity specification , so \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{j1 } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{j2 } $ $ \end{document } cancel in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_{ij } $ $ \end{document}. ) this also shows up in risk - trend diagrams that are much like those in fig . 1 except for minor details ( not displayed here ) . we run into trouble if we try to extend these ideas to the specification \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu = ad + bd + cd $ $ \end{document}. ( when b represents several background factors , each of them is interacted separately with the decrement factor . ) for bulgaria and romania , the corresponding c plots are much like those in fig . 1 , but for russia and hungary the inclusion of the interactions between the decrement and all the background factors produces c plots that really fail to represent properly the trends in union - formation risks . allowing the background factors to influence the two competing risks differentially ( as is the purpose of letting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijkh } = a_{ih } b_{jh } c_{kh } $ $ \end{document } with genuinely h - dependent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{jh } $ $ \end{document } ) results in a loss of control over the interpretation of the intensity parameters . we do not know how to interpret our parameters if they can not be taken as relative risks . if a differential impact of the background factors is important , one should probably abstain from a joint analysis of the competing risks and , instead study them separately until one understands better the implications of our formula ( 3 ) .

using data from the first round of the national gender and generations surveys of russia , romania , and bulgaria , and from a similar survey of hungary , which were all collected in recent years , we study rates of entry into marital and non - marital unions . we have used elements from the narrative of the second demographic transition ( sdt ) as a vehicle to give our analysis of the data from the four countries some coherence , and find what can be traces of the sdt in these countries . the details vary by country ; in particular , latter - day developments in union formation patterns did not start at the same time in all the countries , but in our assessment it began everywhere before communism fell , that is , before the societal transition to a market economy got underway in 1990 .

Introduction Method and Covariates Trends Over the Years Since 1960 Shifting Age Profiles Conversion of Non-Marital into Marital Unions Summary and Reflections A1. Stable and Uniform Age Profiles A2. The Interpretation of Period Coefficients of Our Study as Relative Risks

in this connection , the account of the second demographic transition ( sdt ) is very attractive , both as a generalized summarizing description and because of its underlying theory of value change in the direction of increasing tolerance in family matters and of women s increasing autonomy ( lesthaeghe and van de kaa 1986 ; lesthaeghe and surkyn 2002 ; for a recent , independent assessment , see sobotka 2008 ) . the changes on which the narrative focuses are a decline in marriage formation , an increase in non - marital cohabitation , a general decrease in fertility ( particularly at higher birth orders ) but an increase in non - marital childbearing , an increase in union disruption , and a postponement of marriage and childbearing . there is ample evidence of most of the demographic developments in the sdt narrative all over europe , particularly concerning fertility trends ; see for instance frejka et al . 2004 ; spder 2004 and 2005 , zakharov 2005 , gerber and berman 2005 ; koytcheva 2006 , thornton and philipov 2007 ; kostova 2007 , muresan ( 2007a , b ) , and bradatan and kulcsar 2008).1 too little attention has been given so far to the finer structure of the trends in union - formation risks in the region , however , which is surprising , given that ideational change must be a force behind the growth in non - marital cohabitation and therefore a prime indicator of the very explanation given for the sdt . in this article , we focus therefore on non - marital cohabitation as a competitor to conventional marriage . our account is for bulgaria , romania , russia , and hungary , for we are fortunate in having early access to the data from the first round of the gender and generations surveys ( ggs ) in the first three of these countries and to their close counterpart for hungary.2 all the surveys have used a random sample of women and men of all relevant ages . in this present study , we use the data for women only ; for sample sizes ( in terms of years of exposure ) see table 1.table 1person - years of exposure since 1960total person - years of exposureyear of data collectionas childless , not pregnantas childless , pregnantat parity 1russia35,1613733,8652004bulgaria40,0573601,9892004romania33,9312901,4162005hungary49,7474559512001notethe first wave of the hungarian ggs ( originally called turning points of the life - course ) was conducted in november 2001 through january 2002 , but we do not use data collected in 2002 in our studysource our own calculations based on ggs data person - years of exposure since 1960 notethe first wave of the hungarian ggs ( originally called turning points of the life - course ) was conducted in november 2001 through january 2002 , but we do not use data collected in 2002 in our study source our own calculations based on ggs data we started this investigation in a descriptive mood and without any strong preconceived ideas or hypotheses about union - entry trends , but with a series of open questions . we were curious to see to what extent the fall of communism around 1990 might have given a particular impetus to developments in union formation across the four countries , and what commonalities we could find in the patterns of such developments . the three former authors have provided extensive survival tables for russia , bulgaria , and romania in the spirit of andersson and philipov ( 2002 ) , who gave such tables for hungary and fifteen other european countries for an earlier period . following carlson and klinger ( 1987 ) ; spder ( 2004 , 2005 ) maintained that post - divorce non - marital cohabitation has old roots in hungary and that consensual first unions gained considerable ground in that country well before the regime change . we focus on first unions and find similar patterns also for russia , romania , and bulgaria . statistics like those of table 2 have been derived from straightforward occurrence / exposure rates , with no standardization nor any other attempt at hedging against compositional effects ; so , we started out wondering to what extent the considerable differences between the countries would hold up to closer scrutiny.table 2entry into marital and non - marital unions as competing events in bulgaria , romania , and russia . womenperiodbulgariarussiaromaniaever entered into cohabitation marriageever entered into cohabitation marriageever entered into cohabitation marriage19851989543734622076199019946032465019992003631462333556source philipov and jasilioniene ( 2007 ) , table a8source muresan ( 2007b ) , tables 5.5 and 5.61980198919962005 entry into marital and non - marital unions as competing events in bulgaria , romania , and russia . women source philipov and jasilioniene ( 2007 ) , table a8 source muresan ( 2007b ) , tables 5.5 and 5.6 in demography , one of the ways to handle compositional effects is by using standardization , and we have applied this method in the form of an unusual variant of intensity regression where entry into marriage and into a non - marital union are studied jointly as competing risks in a manner that permits direct comparison between the two types of union formation in each of the four countries.6 this procedure has been described most fully by hoem and kostova ( 2008 ) , to whom we refer for mathematical aspects of the approach we use . this article can be regarded as a further elaboration of the bulgarian data and an extension to the three other countries for which we also have data . based on data in a monthly format for the years since 1960 , we have used proportional - hazards event - history analysis with a piecewise constant baseline intensity to reflect the impact of a woman s age , formally using the type of union formed as a fixed covariate in addition to the other fixed and time - varying covariates available to us ( the determinants ) . since our focus is on the changing trends in union formation , we display the interaction between calendar time and union type in our descriptions below , and let the other available covariates appear as control variables . these are most importantly ( self - reported ) ethnicity , but also a number of covariates that are intended to reflect other aspects of the respondent s background , namely whether she grew up in an urban or rural region , whether she lived with both parents at age 15 , her number of siblings , her own educational attainment , and the educational attainments of her mother and father.8 these are standard covariates readily available in our data , except the respondent s own educational attainment . we would have used it more extensively if we had had enough information to make it a genuine time - varying covariate , but the data only contain the attainment made by the time of data collection plus the time at which the respondent had reached this level of education ( according to her own report ) , so we have had to impute a non - fixed covariate using a method developed by hoem and kreyenfeld ( 2006 ) . among the findings that we do report is a strong drop in the marriage - formation risk in all the four countries and a counterpart increase in the risk of entry into non - marital unions , though surprisingly in bulgaria ( and possibly hungary ) this increase turned into a drop at the beginning of the 2000s.9 as one of our referees has pointed out , this may just be a sign of accelerated postponement of entry into a first union , which would be another typical trait of the sdt . to give a feeling for the size order of the relative union - formation risks in our four data sets in the twilight years of state communism , we attach table 3 , where for each country we display the ( two - way ) empirical interactions between the type of union formation ( marital and non - marital ) on the one hand and pregnancy - and - parity status on the other . the parents educational attainments were not included because of data quality problems source our own calculations based on ggs data the general pattern is that as long as a woman was childless and not pregnant , the risk of entry into a non - marital union most often was low by comparison to the risk of marriage formation . it is as if the arrival of the first child is some kind of watershed , after which the woman was less attractive as a partner , or alternatively that the remaining women were less attracted by partnership . only in hungary , mothers still ran a ( somewhat ) higher risk of entry into a union , especially a marital union , than before they became pregnant . 1 , computed separately for each of the four countries . non - pregnant childless women in russia , romania , bulgaria , and hungary , since 1960 . non - pregnant childless women in russia , romania , bulgaria , and hungary , since 1960 . in all the countries , the risks of entry into non - marital unions have increased ever since the 1960s , much as one would expect from descriptions of the sdt.13 taken together , these manifestations started well before the fall of communism , particularly for entry into consensual unions . this looks like a deviation from ( standard ) patterns in the sdt , though one should note that the cohabitational entry risks continued to increase relative to the marriage risks throughout the whole period of our observation.14 , 15 romania is another exception from the general trend in the risks of entry into cohabitation , relative to that of marriage formation . we had expected entry into cohabitation to be shifted toward younger ages than the age profile for marriage formation , much as in the diagram for bulgaria , but the diagrams for russia , hungary and romania show how incorrect such a preconception could be.fig . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1960-ca . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1960-ca . the findings presented in section 3 provide a neat and compact description of entry trends in the four countries , based on a standardization technique of a type that is ubiquitous in demography.16 standardization is known to summarize risk trends and differentials well under wide conditions , and to be robust against mild deviations from those conditions . , we have behaved as if each of the two piecewise constant baseline hazards ( one for each decrement ) were the same for all calendar periods in the analysis . this may have simplified matters unduly ; after all , many authors document to their satisfaction that there has been a delay in union formation , so marriage and perhaps entry into cohabitation occur progressively later in life as calendar time increases . to check on this question , we have estimated the hazard parameters once more , but now with a three - way interaction between age , period , and decrement.17 the outcome is given in fig . 3 , where to avoid needless complication , we have temporarily used five - year age groups and have concentrated on the years between 1980 and the survey date.18 for each country , we have plotted the age profiles of the rates of union formation for each period k , and we get the following graphical patterns , which can serve as a simple optical goodness - of - fit test of our basic specification.fig . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1980-ca . non - pregnant childless women in russia , romania , bulgaria , and hungary , 1980-ca . for romania we also see a bit of a shift toward later ages in the risk of entry into marriage , while in bulgaria we can see a similar shift in the risk of entry into non - marital cohabitation . all in all , perhaps there is only a mild deviation from the requirement of a stable age profile in bulgaria , russia , and romania . by way of conclusion , to get a realistic representation it looks as if we may be able to make do with our original intensity specification for russia , romania , and bulgaria , but not necessarily for hungary . for the latter country , we have therefore tried the specification with a three - way interaction between age , period , and decrement once more , but now with our finer age specification and with periods back to 1960 . as we mentioned toward the end of section 3 , we have found that lately the risk of entry into cohabitation has dropped somewhat in bulgaria . to see whether this means that bulgarian women have given up on the sdt , at least as far as union formation is concerned , it pays to introduce an additional dimension , namely , the conversion of non - marital unions into marriages . 5 the same data are seen from a different angle , but it tells the same story , namely , that the sdt remains in progress in bulgaria . 4relative rates of conversion of cohabitation into marriage , by time since entry into cohabitation for each calendar period , women in bulgaria and hungary , 1960-ca . in our data , this would be recorded as an entry into a consensual union and a later conversion of the union into a marriage . figure 4b extends this painlessly to hungary , for which , as we remember , we have found a similar drop in the two years right after the turn of the century ( fig . the union - formation trends that we have revealed in this descriptive study of the four countries in central and eastern europe turn out to have several features in common . marriage formation has dropped in all the four countries since the fall of communism , and sometimes earlier . consensual unions have gained ground all the time until the end of the twentieth century , and only in bulgaria and hungary does popular interest in consensual - union formation seem to have been reduced somewhat thereafter . in all the four countries , the wind has gone out of the sails of conversions of consensual unions into marriages ; so non - marital unions have progressively stayed consensual longer . despite all commonalities , it is evident that the sdt , of which we have found some traces , is not a unitary movement that reached all the countries in central and eastern europe roughly at the same time and had the same features throughout , no more than it was in western europe . if anything , such a transition did not start simultaneously in all of the four countries , and above all , it began well before the fall of communism and before the societal transition to a market economy got underway around 1990 . if we take the distinct drop in marriage formation as a main marker of the start of the sdt as we study it in this article , then a rough estimate would be that it started in hungary and bulgaria after the early 1980s and in russia and romania half - a - decade and a full decade later , respectively . such differences should fit with the economic and social developments in the countries , but establishing such a correspondence is a matter of future research . as a final reflection on our findings , we want to underline that interpretations should be made with some prudence , for it is possible that the perception of what constitutes a consensual union has varied across countries and has changed over time , and also that reporting inaccuracy may have exaggerated the early growth of entry risks for consensual unions . 1 , 2 are maximum - likelihood estimates of parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{kh } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{ih } $ $ \end{document } of multiplicative intensity functions that in its most general four - factor representation has the form1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{ijkh } = a_{ih } b_{jh } c_{kh } $ $ \end{document}where i is the age group , j is the background group , k is the calendar period , and h is the decrement.20 as we have noted , in the present application , the latter stands for the type of union formation , i.e. if we let a stand for the age factor , b for the combination of all the background factors that we mentioned as covariates in section 2 , c for the calendar period , and d for the decrement,21 then the above specification of the union - formation intensity can be written symbolically as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu = \,ad + bd + cd\ , , $ $ \end{document } where a double letter like ad or cd indicates that we include an interaction between the two factors involved ( a and d , say , represented in ( 1 ) by a double subscript on the a parameter ) and a plus sign indicates that an interaction has not been included . , 2 work as a fair representation of our data , is that for each type h of union entry , the age effect\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \ { a_{ih } \ } $ $ \end{document}is the same in all periodsk and for all levels j on the background factor , as is indicated here by the lack of subscripts k and j on the a parameter in ( 1 ) . 1 , and see that we can , therefore , compare directly the trend and level of the risk of entry into cohabitation with the corresponding risk of marriage formation for each country . 1 , but for russia and hungary the inclusion of the interactions between the decrement and all the background factors produces c plots that really fail to represent properly the trends in union - formation risks . if a differential impact of the background factors is important , one should probably abstain from a joint analysis of the competing risks and , instead study them separately until one understands better the implications of our formula ( 3 ) .

disease mapping contains a set of statistical methods , which leads to generate accurate maps based on estimations of incidence , prevalence , and mortality of diseases . the oldest example of disease mapping is the address of cholera victims based on distance from water resources , which was presented in 1854 by john snow . due to importance of geographic distribution of disease rates in determination of risk factors , the most important aims of disease mapping contains describing spatial variation in prevalence of diseases in order to formulate hypothesis about causes of diseases , specification of high risk areas which needed increased notice and interference , generating an accurate map of disease risk in area for the purpose of better resource allocation , and risk estimation as well as producing disease atlase . there are different models and methods to develop maps of diseases including simple statistic illustration , informal methods , basic models , bayesian models , multilevel models , etc . bayesian approach to disease mapping consists of prior information about variation in disease rates , in addition to observed events in each area . it also could consider spatial pattern of disease in which closer geographic areas have more similar disease rates . bayesian models in disease mapping comprise variously wide range with every one of them has its own formulation , characteristics , advantages , and disadvantages . several studies were conducted about comparison of these models . however , except a few and limited studies , disease mapping models were not compared , and their general condition was not investigated . clayton and kaldor made comparisons about a limited set of models ( empirical bayesian and full bayesian models ) . they concluded that in spite of some differences among relative risk estimations in all methods , the order of relative risk is similar in the whole mapping area . other efforts have been carried out to find the sensitivity analysis of full bayesian models that are confined to a limited range of bayesian models . regarding the importance of empirical and full bayesian models , this paper describes and compares three bayesian models in study of relative risk estimation of suicide in counties of ilam province during 18 months from march of 2007 till october of 2008 . it can begin from safe and preventable behavior and end up to sever and unpreventable ones . according to the world health organization report , the worldwide number of suicide attempts leading to death was one million people annually , and the number of suicide commitment was 20 - 30 times more than this . it is also estimated that around 1,530,000 million people will die due to suicide , and 10 - 20 times more than this figure will commit suicide by the year2020 . it means that , averagely , one death due to suicide per 20 seconds and one suicide commitment per one to two second will happen by the year 2020 . lithuania and russia have the first ranks of suicide in the world . in these countries 46 and 41 suicidesper 100,000 ( one hundred thousand ) people have been committed suicide in these countries respectively . suicide as a social anomaly has a high prevalence in iran too . according to reports from welfare organization of iran , international statistics indicates that suicide rate in iran was 9 per 100,000 people , which contributed 1% of total number of deaths . recently , among all provinces of iran , ilam province has shown one of the highest suicide rates . this province located in the west of iran contains eight counties , and its population has been 545,787 people in year 2006 . there are some differences among its counties according to weather , cultural , and social conditions . in addition , some of the counties near the border of iraq have more post - war problems of 8-year iraq - iran war for a long time . therefore , because of importance of suicide as a complex psychological phenomenon , influenced by individual and environmental factors , investigation and comparison of suicide rate in all counties of ilam province seems necessary and worthwhile to study . concerning the importance of suicide and its high rate in ilam province and existence of only some descriptive reports about suicide incidence , it is essential to investigate geographical variation of suicide incidence rates among counties of ilam province using advanced statistical models . in this applied ecological study , suicide data of ilam province collected by ilam university of medical science during 18 months from march of 2007 till october of 2008 were analyzed . relative risk of a disease is calculated by use of proportion of having a disease in a group with exposure to a risk factor to a group without this risk factor . in this study , we consider relative risk as proportion of the cases committed suicide to expected numbers . data analysis was carried out using gamma- poisson , log - normal , and bym bayesian models in winbugs software . estimates from each model were compared with one another as well as with standardized mortality ratio as a classic non - bayesian model . the results of bayesian models were compared by use of deviance information criterion ( dic ) . first of all , regarding the importance of these methods , specific properties , and some differences among them , it is essential to explain briefly theoretical principles , advantages , disadvantages , and characterization of each model . the first step to evaluate a disease incidence within an area is to estimate the expected rate of incidence in that area . the proportion of observed cases to expected one is called standardized mortality ratio ( smr ) . in studies related to diseases incidence , we use the proportion of observed to expected disease incidence , which is called standardized incidence ratio ( sir ) . this ratio is an estimation of relative risk within each area . in order to utilize this method , the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , , oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation . , it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii . therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . to overcome these difficulties , hierarchical bayesian models play an important role in modeling the complexity of data structure in spatial epidemiology and can overcome smrs shortcomings . in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) . this model assumes that the number of deaths in areas follow a poisson distribution with mean eii . the prior distribution for relative risks is also considered gamma ( a , b ) . to obtain posterior distribution , gamma prior distribution for relative risks is combined with poisson likelihood . so , relative risk have posterior distribution gamma(a + oi , b + ei ) with mean which . thus , posterior mean for area i is weighted average of smr for area i and overall relative risk that weights are conversely related to smr variance . if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean . 2 . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method . although gamma prior distribution for relative risk is proper from mathematical point of view , it could be limiting because of difficulty with entering covariates . log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas . this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components : : trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al . is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) : trend component that is overall level of relative risk . ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al . is : if i and j were adjacent , wij = 1 , otherwise wij = 0 . vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters . the first step to evaluate a disease incidence within an area is to estimate the expected rate of incidence in that area . the proportion of observed cases to expected one is called standardized mortality ratio ( smr ) . in studies related to diseases incidence , we use the proportion of observed to expected disease incidence , which is called standardized incidence ratio ( sir ) . this ratio is an estimation of relative risk within each area . in order to utilize this method , the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , , oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation , it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii . therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . to overcome these difficulties , hierarchical bayesian models play an important role in modeling the complexity of data structure in spatial epidemiology and can overcome smrs shortcomings . in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) . this model assumes that the number of deaths in areas follow a poisson distribution with mean eii . the prior distribution for relative risks is also considered gamma ( a , b ) . to obtain posterior distribution , gamma prior distribution for relative risks is combined with poisson likelihood . so , relative risk have posterior distribution gamma(a + oi , b + ei ) with mean which . thus , posterior mean for area i is weighted average of smr for area i and overall relative risk that weights are conversely related to smr variance . if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean . the advantages of this method are : 1 . this model estimates full posterior , which allows hypothesis testing and calculates confidence intervals . 2 . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method . although gamma prior distribution for relative risk is proper from mathematical point of view , it could be limiting because of difficulty with entering covariates . log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas . this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components : : trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al . is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) : trend component that is overall level of relative risk . ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al . is : if i and j were adjacent , wij = 1 , otherwise wij = 0 . vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters . the first step to evaluate a disease incidence within an area is to estimate the expected rate of incidence in that area . the proportion of observed cases to expected one is called standardized mortality ratio ( smr ) . in studies related to diseases incidence , we use the proportion of observed to expected disease incidence , which is called standardized incidence ratio ( sir ) . this ratio is an estimation of relative risk within each area . in order to utilize this method , the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , , oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation , it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii . therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . to overcome these difficulties , hierarchical bayesian models play an important role in modeling the complexity of data structure in spatial epidemiology and can overcome smrs shortcomings . in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) . this model assumes that the number of deaths in areas follow a poisson distribution with mean eii . the prior distribution for relative risks is also considered gamma ( a , b ) . to obtain posterior distribution , gamma prior distribution for relative risks is combined with poisson likelihood . so , relative risk have posterior distribution gamma(a + oi , b + ei ) with mean which . thus , posterior mean for area i is weighted average of smr for area i and overall relative risk that weights are conversely related to smr variance . if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean . the advantages of this method are : 1 . this model estimates full posterior , which allows hypothesis testing and calculates confidence intervals . 2 . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method . although gamma prior distribution for relative risk is proper from mathematical point of view , it could be limiting because of difficulty with entering covariates . log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas . this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components : : trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al . is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) is introduced that is an uncorrelated overdispersion parameter . the prior distribution of this parameter is : vi ~ n ( 0 , v ) : trend component that is overall level of relative risk . ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . the conditional autoregressive model proposed by besag et al . is : if i and j were adjacent , wij = 1 , otherwise wij = 0 . vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters . information about ilam counties population , suicide numbers based on 2006 national census conducted by statistical center of iran and registered suicide cases in each county during 18-month study period from march 2007 till october 2008 is shown in table 1 . population of counties based on national census in year 2006 , the number of suicide cases , and its proportion to each county population , from march 2007 till october 2008 the expected number of suicide cases in each county is calculated by multiplying the provincial crude incidence rate by the county population or its estimation ( third column multiplied by fifth column in table 1 ) . expected number of suicide from march 2007 till october 2008 for estimating relative risk of suicide , at first , estimation based on standardized incidence ratio as classical non - bayesian model was calculated . according to this estimation , the relative risks in counties from highest to lowest ones are dare - shahr ( 2.311 ) , ilam ( 1.158 ) , dehloran ( 1.074 ) , eyvan ( 0.839 ) , abdanan ( 0.677 ) , mehran ( 0.471 ) , shirvan - cherdavel ( 0.302 ) , and malekshahi ( 0.293 ) . then , in order to apply and compare bayesian models in estimating relative risk of suicide in counties of ilam province , dates were fitted to gamma- poisson ( gp ) , log - normal ( ln ) , and full bayesian ( bym ) models by use of winbugs software . estimation of relative risk for suicide in each county was calculated and shown in tables 35 along with standard deviation and confidence interval . using these results , in addition to compare counties relative risks , we can compare three mentioned models . according to estimations made by fitting these models , relative risk estimations of this county using gamma- poisson , log - normal , and bym model were 2.243 , 2.275 , and 2.279 , respectively . estimation relative risk of suicide in ilam counties using gamma - poisson model estimation relative risk of suicide in ilam counties using log - normal model estimation relative risk of suicide in ilam counties using bym model estimation of relative risk for this county based on three mentioned models were 0.321 , 0.321 , and 0.319 respectively that indicates the lower relative risk than expected and the average rate in this province . regarding the confidence intervals resulted from the three models , dare - shahr and ilam counties shown to be significantly higher risk rather than average province ( relative risk more than 1 ) and shirvan - cherdavel , malekshahi , and mehran were found to be lower risk rather than the average amount in the province ( relative risk lower than 1 ) . moreover , the three models were compared by use of dic criterion [ table 6 ] . this criterion is obtained by summing two other criteria , which represents goodness of fit and model complexity respectively . although the estimations obtained from gamma - poisson , log - normal , and bym model represented some differences of standardized incidence ratios according to either amounts and counties incidence ranks , they showed approximately similar estimations for suicide relative risk . these results are in accordance with clayton and kaldor 's findings in comparing bayesian models . they showed that in spite of some differences among relative risk estimations , the ranks for each area are same in all three models . in all three models , the highest standard deviation belonged to dare - shahr county , which has the most number of suicides , and the lowest one was seen in shirvan - cherdavel county with the least ones . one justification for similarities between results of the models could be related to the low number of study areas and registered incidence cases . if the counts in lower levels such as districts or villages are available , their comparisons will be more valuable than recent results . according to the estimations resulted from fitting the models , the ranks of ilam province counties from highest to lowest relative risk of suicide are , in turn : dare - shahr , ilam , dehloran , eyvan , abdanan , mehran , malekshahi , and shirvan - cherdavel . this result is accordance with the only study of geographical variation of suicide incidence in ilam province counties during 1994 - 2001 , which showed that dare - shahr is the highest risk county . . it could be due to different data registry systems , underestimation in various data resources , and the differences in study periods . with regard to the importance of suicide incidence and its remarkable mortality ratio in ilam province , and also for the reason of having various suicide registry systems such as ilam university of medical science , welfare organization , forensic medicine , and the police , it is recommended to conduct more comprehensive survey covering the maximum recorded cases , repeat the study in longer period and in more valuable level of counties like districts or villages . additionally , it 's better to add time term to the study in order to investigate temporal variation along with spatial variation .

introduction : disease mapping includes a set of statistical techniques that provides maps based on estimates of diseases rates . bayesian ones are the most important models in this field . they consider prior information on changes in the disease rates in overall map and spatial pattern of the disease . these include a broad range of models with their own formulation , characteristics , strengths , and weaknesses . in the present study , we explain and compare three important and widely - used bayesian models in the study of evaluating relative risk of suicide in ilam province.materials and methods : in this applied - ecological research , suicide incidence in ilam province in 2008 and 2009 was analyzed by use of gamma - poisson , log - normal , and bym bayesian models . models were fitted to data using winbugs software.results:fitting the three models showed that darehshahr and shirvan - chrdavol had the highest and the lowest relative risk of suicide , respectively ( relative risks based on gamma - poisson , log - normal , and bym models were 2.243 , 2.275 , and 2.279 for dareshahr and 0.321 , 0.321 , and 0.319 for shirvan - chrdavol , respectively).conclusion : despite some differences in estimates , the ranks of relative risks in counties in all three models are the same . the counties based on the relative risks of suicide from the most to the least are : darehshahr , ilam , dehloran , eyvan , abdanan , mehran , malekshahi , and shirvan - chrdavol .

INTRODUCTION MATERIALS AND METHODS Diseases mapping models Standardized mortality ratio using maximum likelihood estimation Gamma-poisson model Log-normal model BYM model RESULTS CONCLUSION

disease mapping contains a set of statistical methods , which leads to generate accurate maps based on estimations of incidence , prevalence , and mortality of diseases . the oldest example of disease mapping is the address of cholera victims based on distance from water resources , which was presented in 1854 by john snow . due to importance of geographic distribution of disease rates in determination of risk factors , the most important aims of disease mapping contains describing spatial variation in prevalence of diseases in order to formulate hypothesis about causes of diseases , specification of high risk areas which needed increased notice and interference , generating an accurate map of disease risk in area for the purpose of better resource allocation , and risk estimation as well as producing disease atlase . there are different models and methods to develop maps of diseases including simple statistic illustration , informal methods , basic models , bayesian models , multilevel models , etc . bayesian approach to disease mapping consists of prior information about variation in disease rates , in addition to observed events in each area . it also could consider spatial pattern of disease in which closer geographic areas have more similar disease rates . bayesian models in disease mapping comprise variously wide range with every one of them has its own formulation , characteristics , advantages , and disadvantages . however , except a few and limited studies , disease mapping models were not compared , and their general condition was not investigated . clayton and kaldor made comparisons about a limited set of models ( empirical bayesian and full bayesian models ) . they concluded that in spite of some differences among relative risk estimations in all methods , the order of relative risk is similar in the whole mapping area . other efforts have been carried out to find the sensitivity analysis of full bayesian models that are confined to a limited range of bayesian models . regarding the importance of empirical and full bayesian models , this paper describes and compares three bayesian models in study of relative risk estimation of suicide in counties of ilam province during 18 months from march of 2007 till october of 2008 . according to the world health organization report , the worldwide number of suicide attempts leading to death was one million people annually , and the number of suicide commitment was 20 - 30 times more than this . it is also estimated that around 1,530,000 million people will die due to suicide , and 10 - 20 times more than this figure will commit suicide by the year2020 . lithuania and russia have the first ranks of suicide in the world . recently , among all provinces of iran , ilam province has shown one of the highest suicide rates . this province located in the west of iran contains eight counties , and its population has been 545,787 people in year 2006 . there are some differences among its counties according to weather , cultural , and social conditions . in addition , some of the counties near the border of iraq have more post - war problems of 8-year iraq - iran war for a long time . therefore , because of importance of suicide as a complex psychological phenomenon , influenced by individual and environmental factors , investigation and comparison of suicide rate in all counties of ilam province seems necessary and worthwhile to study . concerning the importance of suicide and its high rate in ilam province and existence of only some descriptive reports about suicide incidence , it is essential to investigate geographical variation of suicide incidence rates among counties of ilam province using advanced statistical models . in this applied ecological study , suicide data of ilam province collected by ilam university of medical science during 18 months from march of 2007 till october of 2008 were analyzed . relative risk of a disease is calculated by use of proportion of having a disease in a group with exposure to a risk factor to a group without this risk factor . in this study , we consider relative risk as proportion of the cases committed suicide to expected numbers . data analysis was carried out using gamma- poisson , log - normal , and bym bayesian models in winbugs software . the results of bayesian models were compared by use of deviance information criterion ( dic ) . first of all , regarding the importance of these methods , specific properties , and some differences among them , it is essential to explain briefly theoretical principles , advantages , disadvantages , and characterization of each model . this ratio is an estimation of relative risk within each area . in order to utilize this method , the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , , oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation . therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . to overcome these difficulties , hierarchical bayesian models play an important role in modeling the complexity of data structure in spatial epidemiology and can overcome smrs shortcomings . in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) . thus , posterior mean for area i is weighted average of smr for area i and overall relative risk that weights are conversely related to smr variance . if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method . log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas . this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components : : trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) : trend component that is overall level of relative risk . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters . this ratio is an estimation of relative risk within each area . in order to utilize this method , the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , , oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation , it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii . therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) . the prior distribution for relative risks is also considered gamma ( a , b ) . to obtain posterior distribution , gamma prior distribution for relative risks is combined with poisson likelihood . if the observed and expected counts are high , the estimator tends to smr , but when they are low , it tends to prior mean . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method . log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas . this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components : : trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) : trend component that is overall level of relative risk . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters . the first step to evaluate a disease incidence within an area is to estimate the expected rate of incidence in that area . in studies related to diseases incidence , we use the proportion of observed to expected disease incidence , which is called standardized incidence ratio ( sir ) . this ratio is an estimation of relative risk within each area . in order to utilize this method , the map should be divided into n non - overlapped adjacent areas ( i = 1 , 2 , , oi and ei are the number of observed and expected events in i area , respectively . during the study , ei was assumed constant and known and calculated by multiplying the overall crude incidence rate by the area population or its estimation , it is assumed that the number of events between areas are independent and follow poisson distribution with mean eii . therefore , the likelihood for oi is calculated by : also , logarithm of likelihood function is : so , the estimation of maximum likelihood is : although use of smr and sir is so common , there is some substantial shortcoming related to these estimations . in addition to observed numbers , bayesian approach in disease mapping contains prior information about geographical variation of disease rates in whole map . it can be also considered disease spatial correlation ( the tendency of nearer areas to the same rates of disease ) . the advantages of this method are : 1 . even if the real distribution of relative risks is gamma , this model can estimate mean and variance using maximum likelihood method , which is more valuable than moment method . although gamma prior distribution for relative risk is proper from mathematical point of view , it could be limiting because of difficulty with entering covariates . log - normal model is more flexible than gamma- poisson model for relative risks of disease : in this model , vi is included in order to consider spatial correlation and similarity of adjacent areas . this model was introduced by clayton and kaldor and extended by besag , york , and mollie , and its formulation is described as : log(i ) = + ui + vi in this model , relative risk parameter is splitted into three components : : trend component that is overall level of relative risk.ui : ( ( spatial overdispersion ) spatial correlated heterogeneity ) : it is logical that the close areas have similar relative risks . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . for this component , spatial correlation structure is used where estimates for relative risk in each area are dependent on adjacent areas . is : that , and if i and j were adjacent , wij = 1 , otherwise wij = 0.vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . the prior distribution of this parameter is : vi ~ n ( 0 , v ) : trend component that is overall level of relative risk . to take this similarities into account , the random variable ui which is uncorrelated with other { uj } sis included in the model . vi : ( non - spatial overdispersion ( spatial uncorrelated heterogeneity ) ) : by the formulation of the model for spatially correlated heterogeneities , the variance is dependent on the number of neighbors and independence could nt be defined well . in order to justify this problem , another component ( vi ) the prior distribution of this parameter is : vi ~ n ( 0 , v ) both u and v parameters control variability of u and v. to analysis of full bayesian , the prior distributions should be determined for these parameters . information about ilam counties population , suicide numbers based on 2006 national census conducted by statistical center of iran and registered suicide cases in each county during 18-month study period from march 2007 till october 2008 is shown in table 1 . population of counties based on national census in year 2006 , the number of suicide cases , and its proportion to each county population , from march 2007 till october 2008 the expected number of suicide cases in each county is calculated by multiplying the provincial crude incidence rate by the county population or its estimation ( third column multiplied by fifth column in table 1 ) . expected number of suicide from march 2007 till october 2008 for estimating relative risk of suicide , at first , estimation based on standardized incidence ratio as classical non - bayesian model was calculated . according to this estimation , the relative risks in counties from highest to lowest ones are dare - shahr ( 2.311 ) , ilam ( 1.158 ) , dehloran ( 1.074 ) , eyvan ( 0.839 ) , abdanan ( 0.677 ) , mehran ( 0.471 ) , shirvan - cherdavel ( 0.302 ) , and malekshahi ( 0.293 ) . then , in order to apply and compare bayesian models in estimating relative risk of suicide in counties of ilam province , dates were fitted to gamma- poisson ( gp ) , log - normal ( ln ) , and full bayesian ( bym ) models by use of winbugs software . estimation of relative risk for suicide in each county was calculated and shown in tables 35 along with standard deviation and confidence interval . using these results , in addition to compare counties relative risks , we can compare three mentioned models . according to estimations made by fitting these models , relative risk estimations of this county using gamma- poisson , log - normal , and bym model were 2.243 , 2.275 , and 2.279 , respectively . estimation relative risk of suicide in ilam counties using gamma - poisson model estimation relative risk of suicide in ilam counties using log - normal model estimation relative risk of suicide in ilam counties using bym model estimation of relative risk for this county based on three mentioned models were 0.321 , 0.321 , and 0.319 respectively that indicates the lower relative risk than expected and the average rate in this province . regarding the confidence intervals resulted from the three models , dare - shahr and ilam counties shown to be significantly higher risk rather than average province ( relative risk more than 1 ) and shirvan - cherdavel , malekshahi , and mehran were found to be lower risk rather than the average amount in the province ( relative risk lower than 1 ) . moreover , the three models were compared by use of dic criterion [ table 6 ] . although the estimations obtained from gamma - poisson , log - normal , and bym model represented some differences of standardized incidence ratios according to either amounts and counties incidence ranks , they showed approximately similar estimations for suicide relative risk . these results are in accordance with clayton and kaldor 's findings in comparing bayesian models . they showed that in spite of some differences among relative risk estimations , the ranks for each area are same in all three models . in all three models , the highest standard deviation belonged to dare - shahr county , which has the most number of suicides , and the lowest one was seen in shirvan - cherdavel county with the least ones . one justification for similarities between results of the models could be related to the low number of study areas and registered incidence cases . according to the estimations resulted from fitting the models , the ranks of ilam province counties from highest to lowest relative risk of suicide are , in turn : dare - shahr , ilam , dehloran , eyvan , abdanan , mehran , malekshahi , and shirvan - cherdavel . this result is accordance with the only study of geographical variation of suicide incidence in ilam province counties during 1994 - 2001 , which showed that dare - shahr is the highest risk county . it could be due to different data registry systems , underestimation in various data resources , and the differences in study periods . with regard to the importance of suicide incidence and its remarkable mortality ratio in ilam province , and also for the reason of having various suicide registry systems such as ilam university of medical science , welfare organization , forensic medicine , and the police , it is recommended to conduct more comprehensive survey covering the maximum recorded cases , repeat the study in longer period and in more valuable level of counties like districts or villages . additionally , it 's better to add time term to the study in order to investigate temporal variation along with spatial variation .

cannabinoid cb1 receptors and relevant endocannabinoids are widely distributed throughout the brain in mediating affective , cognitive , and motor behaviors [ 1 , 2 ] . much of the current neurochemical evidence suggests that their functional roles are mainly to modulate presynaptic neurotransmission impinging on postsynaptic neurons [ 36 ] . midbrain raphe serotonergic neurons are known as an important substrate of cannabinoids , which have been demonstrated operating in a plethora of neurological responses [ 69 ] . thus , the explicit understanding of cannabinoid modulations on midbrain raphe serotonergic neuronal activity has been for many years one of the central issues in the cannabinoid research . the dorsal ( drn ) raphe nuclei ( mrn ) are two principal sources of serotonergic projections to the forebrain . the drn serotonergic projections are likely associated with cognitive and affective activity [ 10 , 11 ] while mrn serotonergic neurons are more clinging to motor behavior [ 12 , 13 ] . some of the forebrain neurons , particularly gabaergic and glutamatergic afferents , in turn innervate raphe nuclei forming feedback circuits that control extreme fluctuations of 5-ht availability . the convergence of evidence suggests that gabaergic and glutamatergic afferents put different weights on controlling serotonergic activity in two raphe nuclei . gabaergic afferents have a strong and tonic inhibitory influence on serotonergic neurons in the drn but little to the mrn [ 1517 ] . however , less is known about the fundamental mechanism underlying neural circuit used by cannabinoids for 5-ht release in the forebrain . the primary goal of the present study was to determine how 5-ht efflux in somatodendritic and axon terminal regions is altered differently in response to administrations of cannabinoids . this study was carried out in the drn and one of serotonergic ascending projection sites , namely , nucleus accumbens ( nacc ) . the second goal was to test the hypothesis that there are direct and indirect effects of cannabinoids on 5-ht efflux in the nacc . specifically , the direct effect is simply to inhibit axonal terminal activity via the activation of cb1 receptors , resulting in reduction in 5-ht efflux . in contrast , the indirect effect is to disinhibit postsynaptic 5-ht neurons at the drn involving in gabaergic neurotransmission , resulting in an increase in 5-ht efflux . our results clearly support a conclusion that when cannabinoids are administered systemically in freely behaving animals , the actual 5-ht efflux is determined by the net effect of two opposite actions , resulting in only a small increase in 5-ht efflux in the nacc . adult male sprague - dawley rats purchased from charles river laboratories ( raleigh , nc , usa ) were pair housed with food and water available ad libitum in a temperature- and humidity - controlled facility and were maintained on a 12 h light / dark cycle ( lights on at 08:00 am ) . all animal use procedures were in strict accordance with the national institutes of health guide for the care and use of laboratory animals , and the study was specifically approved by the institutional animal care & use committee at florida atlantic university ( permit number / id : a10 - 05 ) . rats weighing from 300 to 350 g were anesthetized with a combination of xylazine ( 4 mg / kg i.p . ) and ketamine ( 80 mg / kg i.p . ) , and then guide cannulae ( 21-gauge stainless steel tubing ) were preimplanted as described previously in detail using standard techniques for stereotaxic surgery . the coordinates for guide cannulae in the drn were ap 1.2 relative to interaural zero , ml 4.0 , and dv 1.0 below the skull surface at a 32 angle lateral to midline ; and in the nacc , ap 10.7 , ml 1.4 , and dv 1.0 below the skull surface . the evening before the experiments , rats were briefly anesthetized with isoflurane , and aseptic dialysis probes were inserted through the guide cannulae . the target coordinates for the tip of the probe were as follows : in the drn , ap 1.2 mm relative to interaural zero , ml 0.6 mm relative to midline , and dv 5.56.4 mm below the skull surface ; in the nacc , ap 10.7 mm , ml 1.4 mm , and dv 6.08.5 mm . rats were then placed in a test chamber and attached to a fluid swivel that allowed animals to move freely . dialysis probes were perfused overnight with artificial cerebrospinal fluid ( acsf ) containing 140 mm nacl , 3.0 mm kcl , 1.5 mm cacl2 , 1.0 mm mgcl2 , 0.25 mm nah2po4 , and 1.0 mm na2hpo4 . samples were collected at 30-min intervals and analyzed by high - performance liquid chromatography with electrochemical detection ( hplc - ec ; eicom htec-500 ) in conjugation with an autoinjector ( cma 200 ) . in the 5-ht assay , mobile phase ( 0.1 m phosphate buffer at ph 6.0 , 500 mg / l 1-decanesulfonic acid , 50 mg / l edta , and 1.0% methanol ) was pumped at a rate of 0.50 ml / min . in the gaba assay , samples were prederivatized with o - phthaldialdehyde and 2-methy-2-propanethiol for 10 min at room temperature and then analyzed on a c18 column ( 100 3.2 mm , basi , usa ) . the mobile phase consisted of 40% acetonitrile , 0.1 m sodium acetate buffer , and 50 mg the flow rate of gaba separation was set at 0.7 ml / min . to investigate the neural circuit involving in cb1 receptors , drugs were examined by administering to rats through two distinct routes : systemic administration and local infusion . for the systemic administration , win 55,212 - 2 , cp 55940 , am251 , and sr 147778 being dissolved in saline containing 10% tween-80 and 20% dimethyl sulfoxide ( dmso ) were injected intraperitoneally at a volume of 1 ml / kg in body weight . win 55,212 - 2 mesylate and am251 were purchased from tocris cookson ( ellisville , mo , usa ) . control animals were administered with the vehicle that was used to dissolve the drugs . for the local infusion , drugs dissolved in the infusion medium were delivered directly into the drn or nacc . except for experiments in figure 3 , win 55,212 - 2 was usually dissolved in the artificial cerebrospinal fluid ( acsf ) containing 1.5% ethanol and 1.5% cremophor . cremophor and ethanol were purchased from sigma . in figure 3 , win 55,212 - 2 was dissolved in the acsf containing 1 m citalopram in addition to 1.5% ethanol and 1.5% cremophor . note that the purpose of this set of experiments was to test whether cannabinoids could directly inhibit 5-ht efflux released from dendrites in the drn or from axon terminals in the nacc . however , 5-ht basal release was already low in the regular microdialysis samples at which an inhibitory response to drugs is not easily detected by measuring reduction in 5-ht efflux . thus , citalopram , a selective serotonin reuptake inhibitor ( ssri ) , was employed to elevate the baseline so that reduction in 5-ht efflux , if any , could be reliably measured with the microdialysis approach . previous studies have demonstrated that citalopram added into the infusion medium at the concentration of 1 m would not alter the quality of the drug response . lastly , bicuculline at the concentration of 100 m was dissolved in the acsf before infusing into the drn . bicuculline was purchased as the methobromide - h2o salt from tocris cookson ( ellisville , mo , usa ) . in the control animals , the drn or nacc was locally infused with the vehicle ( isotonic ) that was used to dissolve the drugs . to reduce intersubject variability in group analysis , the data presented in figures are expressed as mean ( s.e.m . ) percentage of changes from averaged baseline measurements . mean baseline 5-ht levels were calculated as the average of four successive samples before drug administration . statistical analysis was performed by the two - way ( drug treatment time ) factorial anova . if significant interactions of drug treatment time course were found , further statistical analysis was carried out using the one - way anova followed by post hoc scheffe test in determining the significance of the respective time points . the first set of experiments was to determine whether systemic injection of win 55,212 - 2 ( win55 hereafter ) had an effect on 5-ht efflux in the brain . the doses used in this study were based on behavioral investigations at which the drug was effective to produce conditioning place preference [ 22 , 23 ] . as shown in figure 1(a ) , win55 at 2.5 and 5 mg / kg ( i.p . ) failed to elicit a significant effect on extracellular 5-ht in the drn . the conclusion was supported by the two - way anova test revealing that there was no significant effect of treatment doses ( fdose ( 2,21 ) = 1.598 , p = 0.2258 ) , time ( ftime ( 5,105 ) = 1.324 , p = 0.2599 ) , or interaction of time doses ( fint ( 10,105 ) = 0.54 , p = 0.8583 ) . in contrast , the same injection produced a dose- and time - dependent increase in the nacc ( figure 1(b ) ; fdose ( 2,26 ) = 11.854 , p = 0.0002 ; ftime ( 5,130 ) = 5.766 , p = 0.0002 ) . the post hoc scheffe test showed that the significant effect was apparent 30 min after injection . cp55940 ( cp55 hereafter ) , which is known for its high affinity to bind cannabinoid receptors , was used to reexamine the nacc response to cannabinoid injection . the dose used in this study was 0.25 mg / kg and 0.5 mg / kg . cp55 at 0.5 mg / kg produced over 50% increases in the nacc ( figure 1(c ) ) but not in the drn ( data not shown ) . the anova test revealed a significant effect of drug doses ( fdose ( 2,24 ) = 7.185 , p = 0.0036 ) and time ( ftime ( 5,120 ) = 6.766 , p = 0.0001 ) . the post hoc scheffe test showed that the significant increase occurred between 0.5 and 1.5 h after the injection . next , we tested whether the win55-induced increases in 5-ht efflux in the nacc could be attributable to the activation of cb1 receptors . sr 147778 is a newly synthesized chemical that has a highly potent antagonistic activity at cb1 receptors [ 25 , 26 ] . as shown in figure 2(a ) , sr 147778 ( 5 mg / kg , i.p . ) alone had no effect on 5-ht in the nacc . injection of sr 147778 30 min before win55 ( 5 mg / kg i.p . ) blocked the win55-evoked increases in 5-ht efflux in the nacc ( ftreatment ( 1,9 ) = 9.457 , p = 0.0132 ) . am251 pretreatment 30 min before win55 significantly blocked the win55-elicited increases in 5-ht in the nacc ( figure 2(b ) ; ftreatment ( 1,11 ) = 31.804 , p = 0.0002 ) . it has been suggested that endogenous opioidergic system may be one of the substrates responsible for the effect of cb1 receptor agonists in the cns [ 2729 ] . since opioids also cause regional selective effects on 5-ht efflux , we tested whether endogenous opioids are involved in the win55-induced increases in 5-ht in the nacc . if this was the case , the opioid receptor antagonist naloxone should be able to antagonize the effect . as shown in figure 2(c ) , naloxone alone ( 10 mg / kg , s.c . ) had no effect on 5-ht efflux . naloxone pretreatment failed to block the win55-induced increases in 5-ht efflux ( ftreatment ( 1,14 ) = 1.579 , p = 0.2295 ) , suggesting that endogenous opioids did not mediate the effect of cannabinoids on 5-ht efflux . we suggest that the existence of a neural circuit is responsible for regional selective effects of cannabinoids on 5-ht efflux in the cns . to test this hypothesis , cannabinoids were directly applied to the drn or nacc through microdialysis probes while 5-ht efflux was determined using the same probes . in this set of experiment , 1 m citalopram was included into the infusion medium to elevate the basal levels of 5-ht efflux . note that citalopram at such a low concentration would not alter the response of serotonergic neurons to drug administration . as shown in figure 3(a ) , win55 at 100 , 300 , and 1000 m infusing into the drn produced a robust increase in 5-ht efflux in a concentration - dependent manner ( fdose ( 3,19 ) = 5.273 , p = 0.0081 ) . in contrast , win55 infusion directly into the nacc caused a concentration - dependent reduction in 5-ht efflux in the nacc ( figure 3(b ) ; fdose ( 3,20 ) = 5.219 , p = 0.008 ) . thus , win55 produced two opposite effects on 5-ht efflux in the nacc , depending on the route of drug administrations . we suggest that the systemic win55-induced increases may be due to disinhibition of serotonergic neurons in the drn . if this was the case , we expected that local infusion of win55 to the drn would also elicit an increase in 5-ht in the nacc , similar to the systemic injection . to test this , we employed a dual - probe microdialysis technique . as shown in figure 4(a ) , win55 infusion into the drn caused a concentration - dependent increase in the nacc ( fdose ( 2,18 ) = 4.256 , p = 0.0307 ) . based on evidence that the direct effect of cannabinoids is inhibitory [ 31 , 32 ] , we further suggest that the win55-induced increases may be due to inhibition of gabaergic neurotransmission on the serotonergic neurons in the drn . if this was the case , bicuculline , a gabaa receptor antagonist , locally infusing into the drn would block the inhibitory impact of gabaergic neurotransmission and thereby disrupt the effect of win55 on the increased 5-ht efflux . as shown in figure 4(b ) , bicuculline pretreatment in the drn abolished the systemic win55 ( 5 mg / kg , i.p)-elicited increases in 5-ht efflux in the nacc ( ftreatment ( 1,14 ) = 3.153 , p = 0.0975 ) . lastly , to confirm the relationship between gabaergic presynaptic responses and cannabinoid administration , we measured changes in gaba efflux in the drn . as shown in figure 5 , there was a significant reduction in gaba efflux in the drn following injection of win55 and cp55 ( ftreatment ( 2,13 ) = 43.843 , p < 0.0001 ) . by measuring changes in 5-ht and gaba efflux in the drn and/or nacc , this present study demonstrated that there exists a neural circuit in the drn through which cannabinoids regulate serotonergic neuronal activity in the nacc . our data provide evidence demonstrating that cannabinoids can exert both indirect and direct effects on serotonergic neurons ; the direct effect of cannabinoids is inhibitory while the indirect effect via presynaptic circuit is excitatory . despite having opposite effects , we showed that systemic administration of exogenous cannabinoids such as win55 and cp55 has a modest excitatory net effect on 5-ht efflux in the nacc of drug - nave animals . this is consistent with the reports that the neurophysiologic function of cannabinoids is considered to be a neuromodulator that usually exerts their effect on both local principal neurons and all types of afferents [ 6 , 32 , 33 ] , resulting in a site - specific response . in the drn , gabaergic and glutamatergic components are two major afferents that synapse onto the serotonergic neurons , controlling 5-ht availability for the forebrain [ 14 , 35 ] . cb1 receptors are found to be widely expressed in the raphe afferents in addition to serotonergic cells . thus , cannabinoids can exert their effects on all regulatory afferents and principal neurons that form a neural circuit including gabaergic , glutamatergic , and serotonergic components . as such , the change in 5-ht efflux in response to systemic cannabinoids can not be exclusively a result of the direct action on serotonergic neurons but a complicated integration of inhibitory modulation of both inhibitory and excitatory neurotransmissions impinging on postsynaptic serotonergic neurons . thus , the effect of cannabinoids on 5-ht release would be a compromised result of excitatory and inhibitory influences , which may explain the findings that there were only modest changes in 5-ht efflux observed in the present study . the present findings that cannabinoids exert both direct and indirect effects on serotonergic neurons are well consistent with the previous reports that cannabinoids can either reduce [ 36 , 37 ] , elevate [ 38 , 39 ] , or bidirectionally alter serotonergic activity . theoretically , the inhibition of glutamatergic afferents would cause a reduction in 5-ht efflux while the inhibition of gabaergic afferents would result in an increase in 5-ht efflux . therefore , their net effects on serotonergic neurons are likely determined by their relative potency of tonic activity . physiologically , gabaergic afferents exert spontaneous inhibitory tone on serotonergic neurons while glutamatergic afferents almost have no measurable role in terms of spontaneous action . note that spontaneous action of glutamatergic afferents is apparent only at a specific behavioral state such as responding to auditory stimulation . therefore , it is conceivable that modulation of gabaergic afferents would be more prominent on 5-ht efflux than the glutamatergic influence . in general , presynaptic modulation of gabaergic inputs has been suggested to be the key mechanism responsible for several illicit drugs such as opioids , alcohol , and gamma hydroxybutyrate [ 18 , 41 , 42 ] . in the present study , we suggest that cannabinoids may use the same neural mechanism for their central action , which inhibits gabaergic neurotransmissions to the drn , thus resulting in disinhibition of serotonergic neurons ( disinhibition theory ) . this may explain our neurochemical data that 5-ht efflux is elevated in the drn - nacc pathway following the systemic administration of exogenous cannabinoids . we demonstrate that changes in 5-ht efflux are dependent not only on doses of cannabinoid injection but also on brain areas examined . specifically , systemic injection produced approximately a 50100% increase from baseline 5-ht in the nacc . in contrast , there was an insignificant effect in the drn following the same injection . the mechanism for the area - dependent efflux is not clearly understood but likely ascribed to regional circuits in the controlling of postsynaptic 5-ht efflux ( see discussion further below ) and also due to possible difference in the release of 5-ht in the two regions ( see a review by ) . the release of 5-ht from the serotonergic neurons is believed to take place at axon terminals where 5-ht - containing vesicles can be found . in the drn , there are numerous recurrent axon collaterals projected from other raphe nuclei , by which serotonergic neurons can be negatively regulated . recent development of in vivo neurochemistry has suggested that dendrites are also equipped with 5-ht - containing vesicles , and many neuroactive chemicals can influence a dendritic release [ 45 , 46 ] . this suggests that 5-ht efflux measured by microdialysis in the drn is derived from many sources involved in distinct mechanisms including dendritic release , recurrent axon collateral release , and axon terminal release . although it is difficult to determine the amounts of each release contributing to the 5-ht efflux in the drn , it is conceivable that dendritic release may be an important component in the extracellular measurement . much of available evidence suggests that the properties of dendritic release may be different from that at axons in the forebrain . for instance , the spontaneous 5-ht release is relatively higher in the drn compared with that from axon terminals [ 17 , 47 ] . it is known that the receptor subtype expression is different between the somatodendritic sites and axon terminals of serotonergic neurons [ 15 , 48 ] . altogether , drugs that cause changes in 5-ht efflux at somatodendritic sites of the drn would not necessarily produce the same response in forebrain areas that contain exclusively the axon terminals . we found that the neurochemical response to cannabinoids is depending on the route of drug administration . unlike systemic injection , local application of cannabinoids to the nacc failed to produce the increase in 5-ht . instead it should be kept in mind that citalopram at the concentration of 1 m was added in the infusion medium into the nacc . after the addition of citalopram , the basal level of 5-ht efflux before drug administration was elevated to ~4 pg / sample , almost 10 times greater than the physiological level . since citalopram effectively blocks reuptake of extracellular 5-ht released from local circuitry , a small reduction in 5-ht efflux can be , therefore , sensitively determined . this pharmacological approach has been widely employed in the in vivo 5-ht research using microdialysis techniques . furthermore , it has been demonstrated that a local application of citalopram at such a low concentration did not alter responsivity of serotonergic neurons to drug administration when the data were expressed as percentage changes relative to baseline . for comparison , citalopram was also infused into the drn for investigating the effects of cannabinoids on 5-ht efflux in the drn . as a result , win55 caused a concentration - dependent increase in the drn but a decrease in the nacc . this further supports that neural circuits that control 5-ht release in response to cannabinoids are different in two regions , regardless the addition of citalopram in the infusion medium . previous in vitro studies demonstrated that cannabinoids decrease 5-ht release from axon terminals [ 3 , 37 ] . this effect can be ascribed to a direct inhibitory activation of cb1 receptors located on the axon terminals . alternatively , excitatory presynaptic influence from glutamatergic afferents may be inhibited by cannabinoid application , as suggested in one study . however , there is no evidence implicating that 5-ht axon terminal in this region is tonically controlled by glutamatergic afferents . thus , reduction in 5-ht efflux can be considered to be a direct inhibitory effect of cannabinoids on serotonergic axon terminals as suggested in the previous studies [ 23 , 37 ] . this is consistent with the observation that cb1 receptors are expressed in the axon terminals that might directly respond to cannabinoids . in the present study , we found that the local infusion of win55 into the drn produced an increase in 5-ht in both the drn and nacc . these observations are in agreement with findings that excitatory stimulation of the drn serotonergic cells results in an increase in extracellular 5-ht in the nacc [ 49 , 50 ] . our data suggest that a cb1 receptor - mediated neural circuit that directly controls 5-ht release and transmission through the drn - nacc pathway would be activated by cannabinoids in the drn . the effect was apparent in response to 300 m and 1000 m , but not 100 m win55 . in this regard , relatively high concentrations of win55 were used during the local infusion for two reasons . first , the microdialysis membrane served as a barrier to the region of the brain . it has been demonstrated with a 1-mm microdialysis probe that only ~7% of infused substances can cross the barrier as examined in an in vitro study . second , the viscosity of the organic solvent in which win55 was dissolved was relatively high , which might have dampened the diffusion of the drug across the probe membrane . thus , it was necessary to utilize high concentrations in the infusion medium that drugs could effectively follow concentration gradients diffusing into local brain tissue . finally , we presented evidence that gabaergic neurotransmission in the drn is the major neural target for cannabinoid regulation , which is responsible for changes in 5-ht efflux of the drn - nacc pathway . this view is strengthened by the observation that gabaergic neurons have a strong tonic inhibition on serotonergic neurons in the drn [ 17 , 49 ] . thus , although the action of cannabinoids in the circuit appears to have multiple sites including gabaergic and glutamatergic and serotonergic neurons [ 6 , 39 , 51 ] , the net consequence would be that serotonergic neurons are disinhibited resulting in an increase in 5-ht efflux in the regions receiving drn projections . this hypothesis was experimentally approved in this study by testing with bicuculline , a gabaa receptor antagonist , in which blocking gaba activation in the drn abolished the increase in 5-ht in the nacc ( figure 4 ) . previous studies have shown that cannabinoids could reduce gaba efflux or gaba - mediated spontaneously inhibitory postsynaptic currents in many brain regions including but not limited to frontal cortex , pallidus , substantia nigra , and hippocampus [ 5255 ] . in the present study , we measured changes in gaba efflux in the drn , demonstrating that the reduction in gaba efflux was in correlation with the increase in 5-ht efflux , further supporting the role of gabaergic neurotransmission in the drn - nacc serotonergic pathway . collectively , systemic administration of cannabinoids may exert two opposite effects on serotonergic neurotransmission , resulting in a small increase in 5-ht efflux in the nacc .

in vivo microdialysis was used in this study to reveal the role of cannabinoids in regulating serotonin ( 5-ht ) efflux in the nucleus accumbens ( nacc ) and dorsal raphe nucleus ( drn ) . the cannabinoid cb1 receptor agonists win55212 - 2 and cp55940 systematically administered to rats caused significant increases in 5-ht efflux in the nacc but failed to have an effect in the drn . to reveal mechanisms underlying regionally selective responses , we tested the hypothesis that cannabinoids have both direct and indirect effects on 5-ht efflux , depending on the location of cb1 receptors in the neural circuit between drn and nacc . we showed that the direct effect of cannabinoids caused a reduction in 5-ht efflux whereas the indirect effect resulted in an increase . furthermore , the indirect effect was blocked by the gabaa receptor antagonist bicuculline in the drn , suggesting that the action is likely due to a presynaptic inhibition on gabaergic activity that exerts a tonic influence on neuronal circuits regulating 5-ht efflux . involvement of gabaergic neurons was confirmed by measuring changes in gaba efflux . taken together , our study suggests that cannabinoids may have direct and indirect effects on the 5-ht regulatory circuits , resulting in regionally selective changes of 5-ht efflux in the brain .

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

cannabinoid cb1 receptors and relevant endocannabinoids are widely distributed throughout the brain in mediating affective , cognitive , and motor behaviors [ 1 , 2 ] . thus , the explicit understanding of cannabinoid modulations on midbrain raphe serotonergic neuronal activity has been for many years one of the central issues in the cannabinoid research . the dorsal ( drn ) raphe nuclei ( mrn ) are two principal sources of serotonergic projections to the forebrain . gabaergic afferents have a strong and tonic inhibitory influence on serotonergic neurons in the drn but little to the mrn [ 1517 ] . however , less is known about the fundamental mechanism underlying neural circuit used by cannabinoids for 5-ht release in the forebrain . the primary goal of the present study was to determine how 5-ht efflux in somatodendritic and axon terminal regions is altered differently in response to administrations of cannabinoids . this study was carried out in the drn and one of serotonergic ascending projection sites , namely , nucleus accumbens ( nacc ) . the second goal was to test the hypothesis that there are direct and indirect effects of cannabinoids on 5-ht efflux in the nacc . specifically , the direct effect is simply to inhibit axonal terminal activity via the activation of cb1 receptors , resulting in reduction in 5-ht efflux . in contrast , the indirect effect is to disinhibit postsynaptic 5-ht neurons at the drn involving in gabaergic neurotransmission , resulting in an increase in 5-ht efflux . our results clearly support a conclusion that when cannabinoids are administered systemically in freely behaving animals , the actual 5-ht efflux is determined by the net effect of two opposite actions , resulting in only a small increase in 5-ht efflux in the nacc . the coordinates for guide cannulae in the drn were ap 1.2 relative to interaural zero , ml 4.0 , and dv 1.0 below the skull surface at a 32 angle lateral to midline ; and in the nacc , ap 10.7 , ml 1.4 , and dv 1.0 below the skull surface . the target coordinates for the tip of the probe were as follows : in the drn , ap 1.2 mm relative to interaural zero , ml 0.6 mm relative to midline , and dv 5.56.4 mm below the skull surface ; in the nacc , ap 10.7 mm , ml 1.4 mm , and dv 6.08.5 mm . in the 5-ht assay , mobile phase ( 0.1 m phosphate buffer at ph 6.0 , 500 mg / l 1-decanesulfonic acid , 50 mg / l edta , and 1.0% methanol ) was pumped at a rate of 0.50 ml / min . to investigate the neural circuit involving in cb1 receptors , drugs were examined by administering to rats through two distinct routes : systemic administration and local infusion . for the local infusion , drugs dissolved in the infusion medium were delivered directly into the drn or nacc . except for experiments in figure 3 , win 55,212 - 2 was usually dissolved in the artificial cerebrospinal fluid ( acsf ) containing 1.5% ethanol and 1.5% cremophor . in figure 3 , win 55,212 - 2 was dissolved in the acsf containing 1 m citalopram in addition to 1.5% ethanol and 1.5% cremophor . note that the purpose of this set of experiments was to test whether cannabinoids could directly inhibit 5-ht efflux released from dendrites in the drn or from axon terminals in the nacc . however , 5-ht basal release was already low in the regular microdialysis samples at which an inhibitory response to drugs is not easily detected by measuring reduction in 5-ht efflux . thus , citalopram , a selective serotonin reuptake inhibitor ( ssri ) , was employed to elevate the baseline so that reduction in 5-ht efflux , if any , could be reliably measured with the microdialysis approach . lastly , bicuculline at the concentration of 100 m was dissolved in the acsf before infusing into the drn . in the control animals , the drn or nacc was locally infused with the vehicle ( isotonic ) that was used to dissolve the drugs . the first set of experiments was to determine whether systemic injection of win 55,212 - 2 ( win55 hereafter ) had an effect on 5-ht efflux in the brain . the doses used in this study were based on behavioral investigations at which the drug was effective to produce conditioning place preference [ 22 , 23 ] . failed to elicit a significant effect on extracellular 5-ht in the drn . the conclusion was supported by the two - way anova test revealing that there was no significant effect of treatment doses ( fdose ( 2,21 ) = 1.598 , p = 0.2258 ) , time ( ftime ( 5,105 ) = 1.324 , p = 0.2599 ) , or interaction of time doses ( fint ( 10,105 ) = 0.54 , p = 0.8583 ) . in contrast , the same injection produced a dose- and time - dependent increase in the nacc ( figure 1(b ) ; fdose ( 2,26 ) = 11.854 , p = 0.0002 ; ftime ( 5,130 ) = 5.766 , p = 0.0002 ) . the post hoc scheffe test showed that the significant effect was apparent 30 min after injection . cp55940 ( cp55 hereafter ) , which is known for its high affinity to bind cannabinoid receptors , was used to reexamine the nacc response to cannabinoid injection . the dose used in this study was 0.25 mg / kg and 0.5 mg / kg . cp55 at 0.5 mg / kg produced over 50% increases in the nacc ( figure 1(c ) ) but not in the drn ( data not shown ) . the anova test revealed a significant effect of drug doses ( fdose ( 2,24 ) = 7.185 , p = 0.0036 ) and time ( ftime ( 5,120 ) = 6.766 , p = 0.0001 ) . the post hoc scheffe test showed that the significant increase occurred between 0.5 and 1.5 h after the injection . next , we tested whether the win55-induced increases in 5-ht efflux in the nacc could be attributable to the activation of cb1 receptors . alone had no effect on 5-ht in the nacc . blocked the win55-evoked increases in 5-ht efflux in the nacc ( ftreatment ( 1,9 ) = 9.457 , p = 0.0132 ) . am251 pretreatment 30 min before win55 significantly blocked the win55-elicited increases in 5-ht in the nacc ( figure 2(b ) ; ftreatment ( 1,11 ) = 31.804 , p = 0.0002 ) . it has been suggested that endogenous opioidergic system may be one of the substrates responsible for the effect of cb1 receptor agonists in the cns [ 2729 ] . since opioids also cause regional selective effects on 5-ht efflux , we tested whether endogenous opioids are involved in the win55-induced increases in 5-ht in the nacc . if this was the case , the opioid receptor antagonist naloxone should be able to antagonize the effect . had no effect on 5-ht efflux . naloxone pretreatment failed to block the win55-induced increases in 5-ht efflux ( ftreatment ( 1,14 ) = 1.579 , p = 0.2295 ) , suggesting that endogenous opioids did not mediate the effect of cannabinoids on 5-ht efflux . we suggest that the existence of a neural circuit is responsible for regional selective effects of cannabinoids on 5-ht efflux in the cns . to test this hypothesis , cannabinoids were directly applied to the drn or nacc through microdialysis probes while 5-ht efflux was determined using the same probes . in this set of experiment , 1 m citalopram was included into the infusion medium to elevate the basal levels of 5-ht efflux . as shown in figure 3(a ) , win55 at 100 , 300 , and 1000 m infusing into the drn produced a robust increase in 5-ht efflux in a concentration - dependent manner ( fdose ( 3,19 ) = 5.273 , p = 0.0081 ) . in contrast , win55 infusion directly into the nacc caused a concentration - dependent reduction in 5-ht efflux in the nacc ( figure 3(b ) ; fdose ( 3,20 ) = 5.219 , p = 0.008 ) . thus , win55 produced two opposite effects on 5-ht efflux in the nacc , depending on the route of drug administrations . we suggest that the systemic win55-induced increases may be due to disinhibition of serotonergic neurons in the drn . if this was the case , we expected that local infusion of win55 to the drn would also elicit an increase in 5-ht in the nacc , similar to the systemic injection . as shown in figure 4(a ) , win55 infusion into the drn caused a concentration - dependent increase in the nacc ( fdose ( 2,18 ) = 4.256 , p = 0.0307 ) . based on evidence that the direct effect of cannabinoids is inhibitory [ 31 , 32 ] , we further suggest that the win55-induced increases may be due to inhibition of gabaergic neurotransmission on the serotonergic neurons in the drn . if this was the case , bicuculline , a gabaa receptor antagonist , locally infusing into the drn would block the inhibitory impact of gabaergic neurotransmission and thereby disrupt the effect of win55 on the increased 5-ht efflux . as shown in figure 4(b ) , bicuculline pretreatment in the drn abolished the systemic win55 ( 5 mg / kg , i.p)-elicited increases in 5-ht efflux in the nacc ( ftreatment ( 1,14 ) = 3.153 , p = 0.0975 ) . lastly , to confirm the relationship between gabaergic presynaptic responses and cannabinoid administration , we measured changes in gaba efflux in the drn . as shown in figure 5 , there was a significant reduction in gaba efflux in the drn following injection of win55 and cp55 ( ftreatment ( 2,13 ) = 43.843 , p < 0.0001 ) . by measuring changes in 5-ht and gaba efflux in the drn and/or nacc , this present study demonstrated that there exists a neural circuit in the drn through which cannabinoids regulate serotonergic neuronal activity in the nacc . our data provide evidence demonstrating that cannabinoids can exert both indirect and direct effects on serotonergic neurons ; the direct effect of cannabinoids is inhibitory while the indirect effect via presynaptic circuit is excitatory . despite having opposite effects , we showed that systemic administration of exogenous cannabinoids such as win55 and cp55 has a modest excitatory net effect on 5-ht efflux in the nacc of drug - nave animals . this is consistent with the reports that the neurophysiologic function of cannabinoids is considered to be a neuromodulator that usually exerts their effect on both local principal neurons and all types of afferents [ 6 , 32 , 33 ] , resulting in a site - specific response . in the drn , gabaergic and glutamatergic components are two major afferents that synapse onto the serotonergic neurons , controlling 5-ht availability for the forebrain [ 14 , 35 ] . cb1 receptors are found to be widely expressed in the raphe afferents in addition to serotonergic cells . thus , cannabinoids can exert their effects on all regulatory afferents and principal neurons that form a neural circuit including gabaergic , glutamatergic , and serotonergic components . as such , the change in 5-ht efflux in response to systemic cannabinoids can not be exclusively a result of the direct action on serotonergic neurons but a complicated integration of inhibitory modulation of both inhibitory and excitatory neurotransmissions impinging on postsynaptic serotonergic neurons . thus , the effect of cannabinoids on 5-ht release would be a compromised result of excitatory and inhibitory influences , which may explain the findings that there were only modest changes in 5-ht efflux observed in the present study . the present findings that cannabinoids exert both direct and indirect effects on serotonergic neurons are well consistent with the previous reports that cannabinoids can either reduce [ 36 , 37 ] , elevate [ 38 , 39 ] , or bidirectionally alter serotonergic activity . theoretically , the inhibition of glutamatergic afferents would cause a reduction in 5-ht efflux while the inhibition of gabaergic afferents would result in an increase in 5-ht efflux . therefore , it is conceivable that modulation of gabaergic afferents would be more prominent on 5-ht efflux than the glutamatergic influence . in the present study , we suggest that cannabinoids may use the same neural mechanism for their central action , which inhibits gabaergic neurotransmissions to the drn , thus resulting in disinhibition of serotonergic neurons ( disinhibition theory ) . this may explain our neurochemical data that 5-ht efflux is elevated in the drn - nacc pathway following the systemic administration of exogenous cannabinoids . we demonstrate that changes in 5-ht efflux are dependent not only on doses of cannabinoid injection but also on brain areas examined . specifically , systemic injection produced approximately a 50100% increase from baseline 5-ht in the nacc . in contrast , there was an insignificant effect in the drn following the same injection . the mechanism for the area - dependent efflux is not clearly understood but likely ascribed to regional circuits in the controlling of postsynaptic 5-ht efflux ( see discussion further below ) and also due to possible difference in the release of 5-ht in the two regions ( see a review by ) . in the drn , there are numerous recurrent axon collaterals projected from other raphe nuclei , by which serotonergic neurons can be negatively regulated . this suggests that 5-ht efflux measured by microdialysis in the drn is derived from many sources involved in distinct mechanisms including dendritic release , recurrent axon collateral release , and axon terminal release . although it is difficult to determine the amounts of each release contributing to the 5-ht efflux in the drn , it is conceivable that dendritic release may be an important component in the extracellular measurement . much of available evidence suggests that the properties of dendritic release may be different from that at axons in the forebrain . for instance , the spontaneous 5-ht release is relatively higher in the drn compared with that from axon terminals [ 17 , 47 ] . altogether , drugs that cause changes in 5-ht efflux at somatodendritic sites of the drn would not necessarily produce the same response in forebrain areas that contain exclusively the axon terminals . we found that the neurochemical response to cannabinoids is depending on the route of drug administration . unlike systemic injection , local application of cannabinoids to the nacc failed to produce the increase in 5-ht . instead it should be kept in mind that citalopram at the concentration of 1 m was added in the infusion medium into the nacc . after the addition of citalopram , the basal level of 5-ht efflux before drug administration was elevated to ~4 pg / sample , almost 10 times greater than the physiological level . since citalopram effectively blocks reuptake of extracellular 5-ht released from local circuitry , a small reduction in 5-ht efflux can be , therefore , sensitively determined . this pharmacological approach has been widely employed in the in vivo 5-ht research using microdialysis techniques . for comparison , citalopram was also infused into the drn for investigating the effects of cannabinoids on 5-ht efflux in the drn . as a result , win55 caused a concentration - dependent increase in the drn but a decrease in the nacc . this effect can be ascribed to a direct inhibitory activation of cb1 receptors located on the axon terminals . however , there is no evidence implicating that 5-ht axon terminal in this region is tonically controlled by glutamatergic afferents . thus , reduction in 5-ht efflux can be considered to be a direct inhibitory effect of cannabinoids on serotonergic axon terminals as suggested in the previous studies [ 23 , 37 ] . this is consistent with the observation that cb1 receptors are expressed in the axon terminals that might directly respond to cannabinoids . in the present study , we found that the local infusion of win55 into the drn produced an increase in 5-ht in both the drn and nacc . these observations are in agreement with findings that excitatory stimulation of the drn serotonergic cells results in an increase in extracellular 5-ht in the nacc [ 49 , 50 ] . our data suggest that a cb1 receptor - mediated neural circuit that directly controls 5-ht release and transmission through the drn - nacc pathway would be activated by cannabinoids in the drn . first , the microdialysis membrane served as a barrier to the region of the brain . finally , we presented evidence that gabaergic neurotransmission in the drn is the major neural target for cannabinoid regulation , which is responsible for changes in 5-ht efflux of the drn - nacc pathway . this view is strengthened by the observation that gabaergic neurons have a strong tonic inhibition on serotonergic neurons in the drn [ 17 , 49 ] . thus , although the action of cannabinoids in the circuit appears to have multiple sites including gabaergic and glutamatergic and serotonergic neurons [ 6 , 39 , 51 ] , the net consequence would be that serotonergic neurons are disinhibited resulting in an increase in 5-ht efflux in the regions receiving drn projections . this hypothesis was experimentally approved in this study by testing with bicuculline , a gabaa receptor antagonist , in which blocking gaba activation in the drn abolished the increase in 5-ht in the nacc ( figure 4 ) . previous studies have shown that cannabinoids could reduce gaba efflux or gaba - mediated spontaneously inhibitory postsynaptic currents in many brain regions including but not limited to frontal cortex , pallidus , substantia nigra , and hippocampus [ 5255 ] . in the present study , we measured changes in gaba efflux in the drn , demonstrating that the reduction in gaba efflux was in correlation with the increase in 5-ht efflux , further supporting the role of gabaergic neurotransmission in the drn - nacc serotonergic pathway . collectively , systemic administration of cannabinoids may exert two opposite effects on serotonergic neurotransmission , resulting in a small increase in 5-ht efflux in the nacc .

bladder cancer ( bc ) is a heterogeneous disease ; therefore , pathologically similar tumors may behave differently . in approximately 70% of all cases , patients present with nonmuscle invasive bladder cancer ( nmibc ) , whereas the remaining 30% present with muscle invasive bladder cancer ( mibc ) . the standard treatment for nmibc is transurethral resection ( tur ) complemented by intravesical immunotherapy or chemotherapy to prevent recurrence and progression . numerous factors are likely involved in disease outcome , and many patients with nmibc experience disease recurrence and progression after primary treatment . furthermore , the potential of tumors to recur and progress to mibc is highly unpredictable . thus , more sensitive and noninvasive tumor markers that can detect and predict tumor recurrence , progression , and metastasis are required . research efforts worldwide have focused on identifying clinically useful tumor markers or potentially valuable therapeutic targets to improve current diagnostic and management strategies for patients with bc . recent advances in our understanding of epigenetic modifications , including dna methylation , histone modifications , and micrornas , have provided new opportunities for detecting , treating , and preventing cancer . the utility of dna methylation as a biomarker has attracted increasing attention in recent years because aberrant dna methylation is a major characteristic of bc and plays a crucial role in tumor initiation and progression . here , we review the current knowledge base and epigenetic issues involved in the detection and prediction of bc . while genetics refers to the study of information inherited on the basis of gene sequences , epigenetics is the study of reversible changes in gene function that can be inherited , or of other cell phenotypes that occur without any change in dna sequence . dna methylation occurs throughout the genome and involves the addition of a methyl group to the cytosine ring of the cpg dinucleotide . the methylation pattern is established during development and is normally maintained throughout the life of an individual . thus , dna methylation is a key regulator of gene transcription and genomic stability , and inappropriately altered dna methylation patterns are frequently detected as epigenetic changes in human cancers . mechanisms that generally regulate normal dna methylation patterns are impaired during tumorigenesis ; therefore , many cancers show global hypomethylation , which is accompanied by regional hypermethylation in some promoter sequences . aberrant methylation of tumor suppressor genes is the most well - categorized epigenetic change in human neoplasias . aberrant promoter methylation has been described for several genes in various malignant diseases , and each tumor type may have its own distinct pattern of methylation . because some epigenetic events occur early in the disease process , molecular diagnosis may facilitate detection before symptomatic or overt radiographic manifestations appear . much progress has been made in the field of bc epigenetics research ; examples are the biological characterization of methylation alterations and a move towards translational applications , including the development of potential new biomarkers for bc . because promoter hypermethylation is common in bc , potential dna methylation markers for bc have been identified in serum , bladder washes , urine samples , and cancer tissues . furthermore , methylation of these genes may facilitate cancer detection and/or correlate with a poor prognosis . thus , aberrant dna methylation events may serve as biological markers for early detection , effective treatment , and accurate prognosis of bc . previous studies of dna methylation - based biomarkers in bc focused on genes that are often methylated in other cancers . in 2001 , the promoter methylation profiles of ten different cancer - related genes from 98 bladder tumors were examined to evaluate their relationship with clinicopathological features and the aggressiveness of the disease . among these genes , four ( rassf1a , apc , cdh1 , and cdh13 ) showed high rates of methylation ( 35% , 35% , 36% , and 29% , respectively ) and these showed a significant correlation with various parameters associated with poor prognosis , such as tumor grade , growth pattern , muscle invasion , tumor stage , and ploidy status . a technique called methylation score ( m score ) analysis , which is based on a combination of methylation markers , was developed to increase the sensitivity / specificity of bc detection . the methylation status of six wnt - antagonist genes ( sfrp-1 , sfrp-2 , sfrp-4 , sfrp-5 , wif-1 , and dkk-3 ) was examined in bc tissues and corresponding normal bladder mucosa . the m score had a sensitivity of 77.2% and a specificity of 66.7% for bc detection , and yielded better results than analyses based on single genes . in addition , the m score was able to distinguish between superficial and invasive bladder tumors , with a sensitivity of 72.2% and a specificity of 61.1% , respectively , making it a useful staging biomarker . the evolution of classic single - gene dna methylation detection assays into genome - wide microarray based technologies , coupled with the development of cutting - edge bioinformatics approaches , has provided an unprecedented opportunity to investigate the role of aberrant dna methylation in the genesis and progression of bc . several high - throughput screening methods have been developed to simultaneously analyze the methylation status of hundreds of preselected genes using universal bead arrays . these methods have led to the discovery of methylation signatures that distinguish normal tissue from cancer tissue . wolff et al . used the goldengate methylation assay ( illumina , san diego , ca , usa ) , which comprises 1,370 cpg sites , to study methylation patterns in 49 samples from patients with nmibc , 38 from those with mibc ( with matched normal - appearing urothelium ) , and 12 samples of urothelia from age - matched cancer - free controls with no history of urothelial cancer . they found distinct patterns of hypomethylation in nmibc and widespread hypermethylation in mibc , confirming that the two pathways differ epigenetically as well as genetically . relative to control samples from urothelial cancer - free patients , invasive tumors had 526 hypermethylated loci ( 38% ) and noninvasive tumors had 132 hypermethylated loci ( 10% ) , of which 117 ( 89% ) overlapped with those found in the invasive tumors . normal - appearing urothelia samples taken from sites located at least 5 cm away from the invasive tumor had 169 hypermethylated loci ( 12% ) , of which 142 ( 89% ) were the same as those found in the invasive tumor . the authors concluded that these patterns were indicative of an epigenetic ' field defect ' , i.e. , methylation was already present in normal - looking cells before the onset of tumorigenesis . this finding suggests that methylation precedes tumorigenesis , which may have implications for the surveillance of patients by urine testing because methylation will presumably persist in the normal urothelium after tumor resection . currently , cancer recurrence or progression in bc patients is monitored by periodic cystoscopy and urine cytology , the frequency of which varies according to the risk factors associated with the disease . although cystoscopic examination is the gold standard for bc diagnosis , it is costly , involves substantial patient discomfort , and has variable sensitivity . moreover , the sensitivity of cytological analysis is low , particularly for low - grade transitional cell carcinomas , and its accuracy depends on the pathologist 's experience . frequent recurrence of bc after tur and its subsequent progression are problems for both patients and urologists . the challenge for the clinician is to develop reasonable surveillance protocols that facilitate cost - effective and noninvasive monitoring . to date , molecular biology and genetic studies have identified several potential markers in serum , bladder washes , urinary specimens , and cancer tissues . however , the limitations of currently available markers have increased interest in identifying other molecular parameters that provide a more accurate prognosis for bc patients . of particular interest is the epigenetic silencing of tumor suppressor genes cancer detection via abnormal dna methylation is quite powerful due to the inherent stability of dna compared with that of rna or proteins . because promoter hypermethylation is a frequent occurrence in bc , detecting cancer - specific hypermethylation events in various biological fluids ( urine / blood ) and tissues would be feasible as these biological materials are easily accessible . were the first to demonstrate the feasibility of diagnosing bc by detecting methylated dna in voided urine . they examined the methylation status of 7 genes ( rar , dapk , e - cadherin , p16 , p15 , gstp1 , and mgmt ) in 22 voided urine samples from bc patients and 17 from age- and sex - matched controls . a panel comprising some of these markers ( dapk , rar , e - cadherin , and p16 ) achieved a sensitivity of 91% and a specificity of 76% for detecting bc ; by comparison , cytology achieved a sensitivity and specificity of 46% and 100% , respectively . examination of matched tumor and urine samples identified no false positive urine samples when the tumor was negative for methylation , indicating that these markers were specific . the feasibility of detecting dna methylation or hypermethylation in voided urine , and its potential role as a tumor marker for bc , have since been examined in several studies ( table 1 ) . friedrich et al . examined dna methylation of apoptosis - associated genes in urine sediments . dapk methylation was detected in 22% of samples ( 8 of 37 ) , tert methylation in 51% ( 18 of 37 ) , and bcl2 methylation in 65% ( 24 of 37 ) . combined methylation analyses ( i.e. , dapk , bcl2 , and tert ) yielded both high sensitivity ( 78% ) and specificity ( 100% ) for detecting bc . similarly , hoque et al . examined the potential of detecting dna hypermethylation in voided urine and its promising role as a tumor marker for bc . they used a quantitative real - time pcr assay to examine promoter hypermethylation in dna present in urine sediments obtained from 175 bc patients and 94 age - matched control subjects . nine genes were examined : apc , p14 , cdh1 , gstp1 , mgmt , p14arf , rarb2 , rassf1a , and timp3 . combined methylation analysis based on four genes ( p14arf , p14 , mgmt , and gstp1 ) yielded 69% sensitivity and 100% specificity . more recently , renard et al . reported that methylated twist1 and nid2 genes were promising urine markers for bc based on a well - designed study approach to selecting and validating candidate genes . bc cell lines and bc - related patient samples were used to select the best candidate markers , which were then validated in methylation - specific polymerase chain reaction assays using 496 urine samples collected from three urology clinical sites . they identified two genes , twist1 and nid2 , that were frequently methylated in urine samples collected from bc patients , including those with early - stage and low - grade disease . the sensitivity of this 2-gene panel ( 90% ) was significantly better than that of cytology ( 48% ) , with comparable specificity ( 93% and 96% , respectively ) . the positive predictive value ( ppv ) and negative predictive value of the 2-gene panel was 86% and 95% , respectively . the clinical feasibility of twist1 and nid2 as urinary biomarkers for detecting bc were recently evaluated ; unfortunately , the different studies yielded different values for the sensitivity and specificity of twist1 and nid2 for detecting bc . examined the methylation patterns of twist1 and nid2 genes in urine samples from 24 bc patients and 15 controls . methylation of twist1 and nid2 was detected in 87.5% and 95.8% , respectively , of samples . the sensitivity of twist1 and nid2 gene methylation ( 87.5% and 95.8% , respectively ) for cancer detection was similar to that reported by renard et al . , and higher than that of urine cytology ( 62.5% ) . abern et al . examined urine samples from 111 bc patients in an attempt to externally validate a urine - based methylation assay that combined twist1 and nid2 . when samples were examined in accordance with the assay described by renard et al . , the sensitivity and specificity were 79% and 63% , respectively ; however , when optimized for the 111 samples examined , the sensitivity and specificity were 75% and 71% , respectively . re - examined the diagnostic utility of the twist1/nid2 gene methylation assay by using it to externally validate 209 urine samples obtained from bc patients . reinert et al . evaluated the clinical utility of methylation markers ( selected from genome - voided microarrays ) in urine samples from 119 bcs and 59 controls . they found that a 4-marker panel ( znf154 , hoxa9 , pou4f2 , and eomes ) achieved a sensitivity of 84% and a specificity of 96% for detecting bc . a validation study based on dna obtained from 184 bc patients and 35 controls showed that a panel of 6 methylation markers ( eomes , hoxa9 , pou4f2 , twist1 , vim , and znf154 ) had a sensitivity of 82%89% and a specificity of 94%100% for detecting bc . in addition , these methylation markers predicted recurrence within a 12 month follow - up period with a sensitivity of 8894% and a specificity of 43%67% . recently , su et al . reported changes in the levels of urinary methylation markers in 368 urine samples serially collected from 90 nmibc patients . they showed that a panel of 3 markers ( sox1 , irak3 , and l1-met ) discriminated between patients with and without recurrence ( with a sensitivity and specificity of 86%/89% and 80%/97% in the test and validation sets , respectively ) . this panel provided better resolution than either cytology or cystoscopy for the detection of early recurrence . in summary , modern techniques for examining dna methylation permit the sensitive and quantitative detection of methylated genes , with impressive results . however , methylation markers for bc diagnosis are still not as well - established as u.s . independent validation experiments often achieve lower sensitivity and/or specificity values because the cutoff values are only fitted to data obtained in the initial experiment . nevertheless , it is evident that methylation markers are more sensitive than cytology , and that some markers show specificity comparable with that of cytology . only a highly selective panel of methylation markers will increase the sensitivity and specificity of urine analysis in the clinic . in addition , future studies should use standardized assays and cutoff values to compare dna methylation markers with established markers in a large - scale well - designed prospective cohort . several studies show a positive association between the hypermethylation status of genes and a poor prognosis for bc patients ; indeed , some of these genes were identified as independent predictive factors of bc prognosis ( table 2 ) . they determined the methylation status of ten genes in 98 bc specimens and calculated the methylation index ( mi ) . of these ten genes , the methylation status of two ( cdh1 and fhit ) , and the mi , were significantly correlated with poor survival . . demonstrated that an increased rate of dapk methylation in bc specimens was significantly associated with a reduced time to recurrence . after adjusting for stage and grade , likewise , a study examining the methylation status of laminin-5-encoding genes showed that lamc2 methylation was strongly associated with poor survival . catto et al . analyzed hypermethylation at 11 cpg islands in a large cohort of urothelial carcinomas . compared with unmethylated tumors , methylation at these sites was significantly associated with advanced grade and stage . friedrich et al . examined the methylation status of 20 cancer - associated genes in 105 consecutive primary nmibc patients . among these genes , the methylation status of six ( socs-1 , stat-1 , bcl-2 , dapk , timp-3 , and cdh1 ) was associated with tumor recurrence . christoph et al . reported that apaf-1 and igfbp-3 methylation was an independent prognostic marker for recurrence in nmibc . a genome - wide study by kandimalla et al . revealed that the methylation status of tbx2 , tbx3 , gata2 , and zic4 was associated with cancer progression in both test and validation sets of pta tumors . however , multivariate analysis identified only tbx3 and gata2 methylation as an independent predictor of progression when compared with other clinicopathological variables . a study of 181 bc patients identified the methylation of hoxa9 , isl1 , and aldh1a3 as an independent predictor of disease recurrence and progression . sacristan et al . classified paraffin - embedded samples from 251 primary nmibc patients into subgroups ( pta low - grade [ lg ] , n=79 ; pt1lg , n=81 ; and pt1 high - grade [ hg ] , n=91 ) according to the methylation status of 25 tumor suppressor genes , and examined whether this could be used to predict the outcome . they found that methylation of rarb , cd44 , pax5a , gstp1 , igsf4 ( cadm1 ) , pycard , cdh13 , tp53 , and gata5 distinguished pta from pt1 tumors , whereas rarb , cd44 , gstp1 , igsf4 , chfr , pycard , tp53 , stk11 , and gata5 distinguished lg from hg tumors . multivariate analyses indicated that methylation of pax5a , wt1 , and brca1 was an independent predictor of recurrence in ptalg , that methylation of pax6 , atm , chfr , and rb1 was an independent predictor of recurrence in pt1lg disease , and that methylation of pycard was an independent predictor of recurrence in pt1hg disease . a significant association between hypermethylation of genes and poor survival has been reported for bc . multivariate analysis revealed that the overall degree of methylation was more significantly associated with subsequent progression and death than tumor stage . furthermore , epigenetic predictive models developed using artificial intelligence techniques identified the presence and timing of tumor progression with 97% specificity and 75% sensitivity . a study of 101 bc samples ( 56 nmibc and 45 mibc ) showed that methylation of sox9 was significantly associated with poor overall survival . kim et al . examined the association between runx3 inactivation and bc over a 50-month median follow - up period . multivariate cox regression analyses revealed that runx3 hypermethylation was the only strong predictor of bc progression , and that the methylation status of runx3 was significantly associated with cancer - specific survival . cebrian et al . examined the methylation status of kiss1 in 804 paraffin - embedded bc specimens . kiss1 methylation was associated with increasing stage , tumor grade , and poor disease - specific survival . a study of 133 bc patients found that methylation of cdh 13 was signif icantly associated with tumor recurrence and a poor prognosis . in addition , multivariate analysis indicated that cdh 13 was independently associated with poor outcome and the relative risk of death . examined the methylation status of 18 genes in paraffin - embedded primary bladder tumors ( n=61 ) and identified prognostic indicators of recurrence ( sfrp5 and h2afx ) , progression ( cacna1 g ) , and disease - specific survival ( sfrp5 ) . a recent study of pcdh17 promoter methylation in bc revealed an association between a significant reduction in survival and an independent predictor of overall survival . although data are sparse , several studies identified a significant association between methylation status and predicted responses to bacillus calmette - gurin ( bcg ) in high - risk bc patients . examined myopodin methylation in 170 t1g3 bc specimens , including a subset of 108 patients who underwent bcg treatment . univariate and multivariate analyses revealed that myopodin methylation was associated with an increased rate of recurrence and progression , and with shorter disease - specific overall survival . in the subset of patients treated with bcg , myopodin methylation was also associated with increased recurrence and progression , and with shorter disease - specific survival , with ppvs of 38.3% , 25.9% , and 14.8% , respectively . examined the methylation status of 25 tumor suppressor genes and its utility for predicting bcg responses in 91 patients with t1g3 high - risk bcs . multivariate analysis identified a combination of msh6 and thbs1 as the best predictor of progression . similarly , another study examined the utility of pmf-1 methylation for predicting the clinical outcome of 108 t1hg nmibc patients receiving treatment with bcg . the methylation status of these genes may serve to distinguish high - risk patients that respond to bcg from those who may require more aggressive therapeutic approaches . in summary , dna methylation is significantly associated with advanced stage , high rates of tumor progression , poor responses to bcg therapy , and increased mortality . however , numerous factors are involved in progression and survival , and future studies should perform multivariate analyses on large numbers of patients ; also , long - term follow - up is needed to confirm that the methylation status is independent of other variables . moreover , understanding the epigenetic changes that occur during the early steps of cancer progression may improve molecular strategies aimed at cancer prevention and/or early intervention . live , apoptotic , and necrotic tumor cells shed into circulation may be a source of measurable epigenetic biomarkers . relatively few studies have reported the identification of blood - based bc methylation markers ( table 3 ) . were the first to describe the presence of methylated dna in plasma samples from 27 bc patients . of these , p14arf and p16ink4a promoter methylation was detected in 87% and 40% , respectively , of samples . hypermethylation of p14arf in plasma was significantly associated with multicentric foci , larger tumors , and relapse . valenzuela et al . examined the methylation status of p16ink4a in serum samples from 86 bc patients and 49 controls , and showed sensitivity , specificity , and ppv for the detection of bc of 0.226 , 0.95 , and 0.98 , respectively . another study showed that the frequency of p16ink4a and dapk methylation in serum was 45% and 64.3% , respectively . studied the methylation status of apc , dapk , gstp1 , ptgs2 , tig1 , and reprimo in the serum of 45 bc patients and 45 controls . hypermethylation at apc , gstp1 , or tig1 distinguished bc from controls with 80% sensitivity and 93% specificity . hypermethylation correlated significantly with prognostically unfavorable clinicopathological parameters , and apc hypermethylation was significantly associated with cancer - specific mortality . recently , hauser et al . analyzed the dna hypermethylation patterns of apc , gstp1 , p14 , p16 , rarb , rassf1a , and timp3 in a prospective , multicenter cohort ( n=227 ) . they found that both the methylation level at each gene site and the number of methylated genes was higher in bc patients than in healthy individuals ; however , levels between bc patients and patients with nonmalignant disease were similar . the sensitivity and specificity of methylated genes for discriminating bc patients from healthy individuals were 62% and 89% , respectively . the authors concluded that dna methylation status has limited value as a biomarker in patients with noninvasive bc . several serum methylation markers ( cdh13 , pcdh10 , and pcdh17 ) are prognostic indicators for bc . although the frequency of cdh13 ( 30% , n=127 ) , pcdh10 ( 50% , n=117 ) and pcdh17 ( 52% , n=151 ) is low , none have been detected in control samples . moreover , the methylation pattern of cdh13 , pcdh10 , and pcdh17 is significantly associated with aggressive tumor characteristics ( tumor size , stage , and grade ) , and pcdh10 and pcdh17 are independent predictors of cancer - specific survival . in summary , hypermethylated genes can be detected in the blood , but the rate of methylation is relatively low and widely variable . few data are available regarding the prognostic value of blood - based hypermethylation markers in bc . thus , the clinical relevance of blood - based hypermethylation markers may remain limited ; however , future studies should shed light on its clinical value . it is clear that much has been discovered about the molecular events that underlie promoter methylation and its role in bc detection , recurrence , progression , and survival . however , the majority of studies have simply identified potential markers ; what is now needed is for these markers to be translated to the clinic . rigorous multicenter prospective validation studies involving large cohorts in a large clinical setting should be performed along with robust statistical analyses . cross talk between different molecular pathways and tumor heterogeneity mean that a single methylation marker would be of limited value for predicting disease status and outcome . nevertheless , our understanding of the epigenetic events that lead to urothelial tumorigenesis and prognosis is improving , and should allow clinicians to identify key epigenetic changes that can be targeted for detecting and predicting disease . methylation markers in bc will be valuable tools for stratifying heterogeneous bc patient populations into risk groups , which can then be used to guide clinical decision - making ( e.g. , observation versus adjuvant therapy ) . aberrant patterns of epigenetic modification could be crucial parameters for bc diagnosis , prognosis , and therapy .

urothelial carcinomas of the urinary bladder have diverse biological and functional characteristics , and numerous factors are likely to be involved in recurrence , progression , and patient survival . while several molecular markers used to evaluate the development and prognosis of bladder cancer have been studied , they are of limited value ; therefore , new molecular parameters useful for predicting the prognosis of bladder cancer patients ( particularly patients at high risk of progression and recurrence ) are required . recent progress in the understanding of epigenetic modification and gene silencing has provided new opportunities for the detection , treatment , and prevention of cancer . methylation is an important molecular mechanism in bladder cancer and may have utility as a prognostic and/or diagnostic marker . this review discusses the epigenetic issues involved in the detection and prediction of bladder cancer .

INTRODUCTION EPIGENETIC STUDIES IN BLADDER CANCER DETECTING METHYLATION MARKERS IN URINE SAMPLES PROGNOSTIC VALUE OF METHYLATION MARKERS IN TISSUES METHYLATION MARKERS IN THE BLOOD CONCLUSIONS

bladder cancer ( bc ) is a heterogeneous disease ; therefore , pathologically similar tumors may behave differently . in approximately 70% of all cases , patients present with nonmuscle invasive bladder cancer ( nmibc ) , whereas the remaining 30% present with muscle invasive bladder cancer ( mibc ) . numerous factors are likely involved in disease outcome , and many patients with nmibc experience disease recurrence and progression after primary treatment . thus , more sensitive and noninvasive tumor markers that can detect and predict tumor recurrence , progression , and metastasis are required . research efforts worldwide have focused on identifying clinically useful tumor markers or potentially valuable therapeutic targets to improve current diagnostic and management strategies for patients with bc . recent advances in our understanding of epigenetic modifications , including dna methylation , histone modifications , and micrornas , have provided new opportunities for detecting , treating , and preventing cancer . the utility of dna methylation as a biomarker has attracted increasing attention in recent years because aberrant dna methylation is a major characteristic of bc and plays a crucial role in tumor initiation and progression . here , we review the current knowledge base and epigenetic issues involved in the detection and prediction of bc . dna methylation occurs throughout the genome and involves the addition of a methyl group to the cytosine ring of the cpg dinucleotide . the methylation pattern is established during development and is normally maintained throughout the life of an individual . thus , dna methylation is a key regulator of gene transcription and genomic stability , and inappropriately altered dna methylation patterns are frequently detected as epigenetic changes in human cancers . mechanisms that generally regulate normal dna methylation patterns are impaired during tumorigenesis ; therefore , many cancers show global hypomethylation , which is accompanied by regional hypermethylation in some promoter sequences . aberrant promoter methylation has been described for several genes in various malignant diseases , and each tumor type may have its own distinct pattern of methylation . because some epigenetic events occur early in the disease process , molecular diagnosis may facilitate detection before symptomatic or overt radiographic manifestations appear . much progress has been made in the field of bc epigenetics research ; examples are the biological characterization of methylation alterations and a move towards translational applications , including the development of potential new biomarkers for bc . because promoter hypermethylation is common in bc , potential dna methylation markers for bc have been identified in serum , bladder washes , urine samples , and cancer tissues . furthermore , methylation of these genes may facilitate cancer detection and/or correlate with a poor prognosis . thus , aberrant dna methylation events may serve as biological markers for early detection , effective treatment , and accurate prognosis of bc . previous studies of dna methylation - based biomarkers in bc focused on genes that are often methylated in other cancers . in 2001 , the promoter methylation profiles of ten different cancer - related genes from 98 bladder tumors were examined to evaluate their relationship with clinicopathological features and the aggressiveness of the disease . among these genes , four ( rassf1a , apc , cdh1 , and cdh13 ) showed high rates of methylation ( 35% , 35% , 36% , and 29% , respectively ) and these showed a significant correlation with various parameters associated with poor prognosis , such as tumor grade , growth pattern , muscle invasion , tumor stage , and ploidy status . the methylation status of six wnt - antagonist genes ( sfrp-1 , sfrp-2 , sfrp-4 , sfrp-5 , wif-1 , and dkk-3 ) was examined in bc tissues and corresponding normal bladder mucosa . the m score had a sensitivity of 77.2% and a specificity of 66.7% for bc detection , and yielded better results than analyses based on single genes . in addition , the m score was able to distinguish between superficial and invasive bladder tumors , with a sensitivity of 72.2% and a specificity of 61.1% , respectively , making it a useful staging biomarker . the evolution of classic single - gene dna methylation detection assays into genome - wide microarray based technologies , coupled with the development of cutting - edge bioinformatics approaches , has provided an unprecedented opportunity to investigate the role of aberrant dna methylation in the genesis and progression of bc . several high - throughput screening methods have been developed to simultaneously analyze the methylation status of hundreds of preselected genes using universal bead arrays . used the goldengate methylation assay ( illumina , san diego , ca , usa ) , which comprises 1,370 cpg sites , to study methylation patterns in 49 samples from patients with nmibc , 38 from those with mibc ( with matched normal - appearing urothelium ) , and 12 samples of urothelia from age - matched cancer - free controls with no history of urothelial cancer . relative to control samples from urothelial cancer - free patients , invasive tumors had 526 hypermethylated loci ( 38% ) and noninvasive tumors had 132 hypermethylated loci ( 10% ) , of which 117 ( 89% ) overlapped with those found in the invasive tumors . normal - appearing urothelia samples taken from sites located at least 5 cm away from the invasive tumor had 169 hypermethylated loci ( 12% ) , of which 142 ( 89% ) were the same as those found in the invasive tumor . the authors concluded that these patterns were indicative of an epigenetic ' field defect ' , i.e. this finding suggests that methylation precedes tumorigenesis , which may have implications for the surveillance of patients by urine testing because methylation will presumably persist in the normal urothelium after tumor resection . currently , cancer recurrence or progression in bc patients is monitored by periodic cystoscopy and urine cytology , the frequency of which varies according to the risk factors associated with the disease . although cystoscopic examination is the gold standard for bc diagnosis , it is costly , involves substantial patient discomfort , and has variable sensitivity . moreover , the sensitivity of cytological analysis is low , particularly for low - grade transitional cell carcinomas , and its accuracy depends on the pathologist 's experience . the challenge for the clinician is to develop reasonable surveillance protocols that facilitate cost - effective and noninvasive monitoring . to date , molecular biology and genetic studies have identified several potential markers in serum , bladder washes , urinary specimens , and cancer tissues . however , the limitations of currently available markers have increased interest in identifying other molecular parameters that provide a more accurate prognosis for bc patients . of particular interest is the epigenetic silencing of tumor suppressor genes cancer detection via abnormal dna methylation is quite powerful due to the inherent stability of dna compared with that of rna or proteins . they examined the methylation status of 7 genes ( rar , dapk , e - cadherin , p16 , p15 , gstp1 , and mgmt ) in 22 voided urine samples from bc patients and 17 from age- and sex - matched controls . a panel comprising some of these markers ( dapk , rar , e - cadherin , and p16 ) achieved a sensitivity of 91% and a specificity of 76% for detecting bc ; by comparison , cytology achieved a sensitivity and specificity of 46% and 100% , respectively . the feasibility of detecting dna methylation or hypermethylation in voided urine , and its potential role as a tumor marker for bc , have since been examined in several studies ( table 1 ) . dapk methylation was detected in 22% of samples ( 8 of 37 ) , tert methylation in 51% ( 18 of 37 ) , and bcl2 methylation in 65% ( 24 of 37 ) . combined methylation analyses ( i.e. , dapk , bcl2 , and tert ) yielded both high sensitivity ( 78% ) and specificity ( 100% ) for detecting bc . examined the potential of detecting dna hypermethylation in voided urine and its promising role as a tumor marker for bc . nine genes were examined : apc , p14 , cdh1 , gstp1 , mgmt , p14arf , rarb2 , rassf1a , and timp3 . combined methylation analysis based on four genes ( p14arf , p14 , mgmt , and gstp1 ) yielded 69% sensitivity and 100% specificity . reported that methylated twist1 and nid2 genes were promising urine markers for bc based on a well - designed study approach to selecting and validating candidate genes . bc cell lines and bc - related patient samples were used to select the best candidate markers , which were then validated in methylation - specific polymerase chain reaction assays using 496 urine samples collected from three urology clinical sites . the positive predictive value ( ppv ) and negative predictive value of the 2-gene panel was 86% and 95% , respectively . the clinical feasibility of twist1 and nid2 as urinary biomarkers for detecting bc were recently evaluated ; unfortunately , the different studies yielded different values for the sensitivity and specificity of twist1 and nid2 for detecting bc . , and higher than that of urine cytology ( 62.5% ) . , the sensitivity and specificity were 79% and 63% , respectively ; however , when optimized for the 111 samples examined , the sensitivity and specificity were 75% and 71% , respectively . re - examined the diagnostic utility of the twist1/nid2 gene methylation assay by using it to externally validate 209 urine samples obtained from bc patients . reinert et al . they found that a 4-marker panel ( znf154 , hoxa9 , pou4f2 , and eomes ) achieved a sensitivity of 84% and a specificity of 96% for detecting bc . a validation study based on dna obtained from 184 bc patients and 35 controls showed that a panel of 6 methylation markers ( eomes , hoxa9 , pou4f2 , twist1 , vim , and znf154 ) had a sensitivity of 82%89% and a specificity of 94%100% for detecting bc . recently , su et al . reported changes in the levels of urinary methylation markers in 368 urine samples serially collected from 90 nmibc patients . they showed that a panel of 3 markers ( sox1 , irak3 , and l1-met ) discriminated between patients with and without recurrence ( with a sensitivity and specificity of 86%/89% and 80%/97% in the test and validation sets , respectively ) . this panel provided better resolution than either cytology or cystoscopy for the detection of early recurrence . independent validation experiments often achieve lower sensitivity and/or specificity values because the cutoff values are only fitted to data obtained in the initial experiment . nevertheless , it is evident that methylation markers are more sensitive than cytology , and that some markers show specificity comparable with that of cytology . only a highly selective panel of methylation markers will increase the sensitivity and specificity of urine analysis in the clinic . they determined the methylation status of ten genes in 98 bc specimens and calculated the methylation index ( mi ) . of these ten genes , the methylation status of two ( cdh1 and fhit ) , and the mi , were significantly correlated with poor survival . . analyzed hypermethylation at 11 cpg islands in a large cohort of urothelial carcinomas . compared with unmethylated tumors , methylation at these sites was significantly associated with advanced grade and stage . among these genes , the methylation status of six ( socs-1 , stat-1 , bcl-2 , dapk , timp-3 , and cdh1 ) was associated with tumor recurrence . christoph et al . a genome - wide study by kandimalla et al . revealed that the methylation status of tbx2 , tbx3 , gata2 , and zic4 was associated with cancer progression in both test and validation sets of pta tumors . however , multivariate analysis identified only tbx3 and gata2 methylation as an independent predictor of progression when compared with other clinicopathological variables . a study of 181 bc patients identified the methylation of hoxa9 , isl1 , and aldh1a3 as an independent predictor of disease recurrence and progression . classified paraffin - embedded samples from 251 primary nmibc patients into subgroups ( pta low - grade [ lg ] , n=79 ; pt1lg , n=81 ; and pt1 high - grade [ hg ] , n=91 ) according to the methylation status of 25 tumor suppressor genes , and examined whether this could be used to predict the outcome . they found that methylation of rarb , cd44 , pax5a , gstp1 , igsf4 ( cadm1 ) , pycard , cdh13 , tp53 , and gata5 distinguished pta from pt1 tumors , whereas rarb , cd44 , gstp1 , igsf4 , chfr , pycard , tp53 , stk11 , and gata5 distinguished lg from hg tumors . multivariate analyses indicated that methylation of pax5a , wt1 , and brca1 was an independent predictor of recurrence in ptalg , that methylation of pax6 , atm , chfr , and rb1 was an independent predictor of recurrence in pt1lg disease , and that methylation of pycard was an independent predictor of recurrence in pt1hg disease . multivariate analysis revealed that the overall degree of methylation was more significantly associated with subsequent progression and death than tumor stage . kim et al . multivariate cox regression analyses revealed that runx3 hypermethylation was the only strong predictor of bc progression , and that the methylation status of runx3 was significantly associated with cancer - specific survival . examined the methylation status of kiss1 in 804 paraffin - embedded bc specimens . kiss1 methylation was associated with increasing stage , tumor grade , and poor disease - specific survival . in addition , multivariate analysis indicated that cdh 13 was independently associated with poor outcome and the relative risk of death . examined the methylation status of 18 genes in paraffin - embedded primary bladder tumors ( n=61 ) and identified prognostic indicators of recurrence ( sfrp5 and h2afx ) , progression ( cacna1 g ) , and disease - specific survival ( sfrp5 ) . although data are sparse , several studies identified a significant association between methylation status and predicted responses to bacillus calmette - gurin ( bcg ) in high - risk bc patients . examined myopodin methylation in 170 t1g3 bc specimens , including a subset of 108 patients who underwent bcg treatment . univariate and multivariate analyses revealed that myopodin methylation was associated with an increased rate of recurrence and progression , and with shorter disease - specific overall survival . in the subset of patients treated with bcg , myopodin methylation was also associated with increased recurrence and progression , and with shorter disease - specific survival , with ppvs of 38.3% , 25.9% , and 14.8% , respectively . examined the methylation status of 25 tumor suppressor genes and its utility for predicting bcg responses in 91 patients with t1g3 high - risk bcs . multivariate analysis identified a combination of msh6 and thbs1 as the best predictor of progression . similarly , another study examined the utility of pmf-1 methylation for predicting the clinical outcome of 108 t1hg nmibc patients receiving treatment with bcg . in summary , dna methylation is significantly associated with advanced stage , high rates of tumor progression , poor responses to bcg therapy , and increased mortality . however , numerous factors are involved in progression and survival , and future studies should perform multivariate analyses on large numbers of patients ; also , long - term follow - up is needed to confirm that the methylation status is independent of other variables . moreover , understanding the epigenetic changes that occur during the early steps of cancer progression may improve molecular strategies aimed at cancer prevention and/or early intervention . live , apoptotic , and necrotic tumor cells shed into circulation may be a source of measurable epigenetic biomarkers . were the first to describe the presence of methylated dna in plasma samples from 27 bc patients . hypermethylation of p14arf in plasma was significantly associated with multicentric foci , larger tumors , and relapse . examined the methylation status of p16ink4a in serum samples from 86 bc patients and 49 controls , and showed sensitivity , specificity , and ppv for the detection of bc of 0.226 , 0.95 , and 0.98 , respectively . studied the methylation status of apc , dapk , gstp1 , ptgs2 , tig1 , and reprimo in the serum of 45 bc patients and 45 controls . hypermethylation correlated significantly with prognostically unfavorable clinicopathological parameters , and apc hypermethylation was significantly associated with cancer - specific mortality . analyzed the dna hypermethylation patterns of apc , gstp1 , p14 , p16 , rarb , rassf1a , and timp3 in a prospective , multicenter cohort ( n=227 ) . the sensitivity and specificity of methylated genes for discriminating bc patients from healthy individuals were 62% and 89% , respectively . the authors concluded that dna methylation status has limited value as a biomarker in patients with noninvasive bc . several serum methylation markers ( cdh13 , pcdh10 , and pcdh17 ) are prognostic indicators for bc . although the frequency of cdh13 ( 30% , n=127 ) , pcdh10 ( 50% , n=117 ) and pcdh17 ( 52% , n=151 ) is low , none have been detected in control samples . moreover , the methylation pattern of cdh13 , pcdh10 , and pcdh17 is significantly associated with aggressive tumor characteristics ( tumor size , stage , and grade ) , and pcdh10 and pcdh17 are independent predictors of cancer - specific survival . in summary , hypermethylated genes can be detected in the blood , but the rate of methylation is relatively low and widely variable . thus , the clinical relevance of blood - based hypermethylation markers may remain limited ; however , future studies should shed light on its clinical value . it is clear that much has been discovered about the molecular events that underlie promoter methylation and its role in bc detection , recurrence , progression , and survival . however , the majority of studies have simply identified potential markers ; what is now needed is for these markers to be translated to the clinic . cross talk between different molecular pathways and tumor heterogeneity mean that a single methylation marker would be of limited value for predicting disease status and outcome . nevertheless , our understanding of the epigenetic events that lead to urothelial tumorigenesis and prognosis is improving , and should allow clinicians to identify key epigenetic changes that can be targeted for detecting and predicting disease . methylation markers in bc will be valuable tools for stratifying heterogeneous bc patient populations into risk groups , which can then be used to guide clinical decision - making ( e.g. aberrant patterns of epigenetic modification could be crucial parameters for bc diagnosis , prognosis , and therapy .

while our armamentarium used to treat squamous cell cancers of the head and neck ( hnscc ) has greatly expanded with the addition of chemoradiation , intensity modulated radiation therapy ( imrt ) , and biological agents , the progression free survival rates have increased only slightly while the overall survival ( os ) rates have stagnated . cancers afflicting the oral cavity ( occ ) are especially prone to locoregional failure even after aggressive surgery followed by radiation and/or chemotherapy . aside from platinum - based chemotherapies , there has been little development of new systemic agents equipped to eradicate microscopic occ disease . for example , a major randomized trial investigating the use of an epidermal growth factor receptor ( egfr ) antagonist concurrent with radiotherapy in patients with hnscc did not include those with occ . this lack of targeted therapies , coupled with the aggressive nature of occ , highlights the need to identify genes that drive this malignancy in order to identify new targets . in contrast to occ , oropharyngeal cancers ( opc ) behave more favourably and also frequently demonstrate a distinct gene expression signature . in general , cure rates using standard therapeutic regimens are 10% higher for opc than for occ . this difference may partially be explained by the impact of the human papillomavirus ( hpv ) , which has been responsible for an opc epidemic . those with hpv - positive opc have a 5-year os of 70 - 90% , while those with hpv - negative opc have a 5-year os of less than 50% . furthermore , hpv - negative hnsccs often have mutations in p53 , a major tumour suppressor protein controlling genomic integrity , and a correspondingly worse prognosis . given the more favourable outcomes of patients with hpv - positive opc , there is a push to de - escalate their treatment . in addition , the unique molecular signature of hpv may enable better ways to target this disease specifically . therefore , in contrast to occs , understanding the distinct mutational landscapes in opcs may enable us to identify new molecular targets and , therefore , to de - escalate the toxicities associated with the current , non - targeted cytotoxic chemotherapies . much of our previous knowledge regarding the molecular characteristics of hnscc was derived from expression microarrays or other assays quantifying gene expression [ 11 - 15 ] . with half of the studied cancers derived from the oral cavity or oropharynx , these studies demonstrated at least four unique expression patterns in hnscc , including : an egfr , a mesenchymal , an epithelial , and an anti - oxidant expression pattern . most tumours with the egfr expression pattern recurred within 2 years , while the majority with the epithelial or anti - oxidant pattern never recurred . furthermore , these expression signatures could differentiate between hpv - positive and hpv - negative cancers , as well as prognosticate responses to therapy . nevertheless , these microarray expression patterns could only implicate a large set of genes involved in hnscc and have difficulty pinpointing the exact genes driving this disease . in addition , others have used cytogenetic approaches to identify structural changes in chromosomes [ 16 - 20 ] . these studies support the model whereby hnscc carcinogenesis begins through two distinct pathways : one caused by chemical carcinogens and the other by hpv oncogenes . later , these pathways share common chromosomal alterations during progression to invasive cancer . however , because these chromosomal changes are only detected on a megabase - pair level , the changes affecting the exact genes that drive the development of hnsccs remain largely unknown . therefore , much of our knowledge of genes that drive hnscc remains limited to p53 mutations , hpv oncogenes , and the egfr pathway . there may exist additional undiscovered driving mutations that may one day serve as new targets for novel therapies . the goal of this article is to review recent trends in identifying hnscc " driver " mutations , especially those occurring in occs and opcs . we define " driver " mutations as mutations in genes that confer a selective advantage to a clone enabling it to better survive or proliferate . this contrasts with " passenger " mutations that have little if any advantageous effect . we will first discuss the use of next generation sequencing ( ngs ) to catalogue point mutations prevalent in hnscc . next , we will review how some of these mutant genes have already been validated in genetically engineered mouse models ( gemm ) . finally , we will discuss how gemms may complement ngs by testing novel mutations identified by ngs as well as identify pathways observed in ngs analysis . thus , this review will examine recent trends in the identification and validation of novel targets , which may revolutionize our understanding of hnscc biology and usher in innovative treatment strategies . in the present article , the authors discuss ways to identify genetically engineered mouse models that supported the recent identification of mutant genes which likely acted as " driver " mutations in hnsccs . we searched relevant articles on pubmed ( www.ncbi.nlm.nih.gov ) regarding next generation sequencing and genetically engineered mouse models for head and neck cancer from 1990 to present . first , to identify mutant genes identified by ngs in hnscc samples we performed two searches . we queried ( 1 ) " genome sequencing " head and neck cancer and ( 2 ) " exome sequencing " head and neck cancer which returned 10 and 4 results , respectively . we selected two articles that specifically described ngs ( specifically whole exome sequencing ) in hnscc . from these articles , we compiled a list of commonly mutated genes and searched whether each gene has been described in an autochthonous head and neck cancer model using the query terms " transgenic mice head and neck cancer " . the specific genes searched and the resulting citations are : tp53 , 55 citations ; tp63 , 7 citations ; syne1,2 , 0 citations ; notch , 4 citations ; hpv , 21 citations ; pi3kca , 0 citations , pten , 11 citations ; ras , 53 citations ; prb , 13 citations ; fbxw7 , 0 citations , ripk4 , 0 citations ; dicer , 0 citations . in order to identify additional gemms for oral cavity / oropharynx cancer , we also searched all citations using the terms " transgenic mice head and neck cancer " that resulted in 392 total articles . we reviewed all article abstracts and selected articles describing gemms targeting genes subsequently identified in ngs of hnsccs . we also selected articles describing gemms of genes not identified by ngs to discuss pathways important in cancer but possibly missed by this approach . given the breadth of genomic analysis in hnsccs , we regret any omissions of gemms for oral cavity / oropharynx cancer . next generation sequencing ( ngs ) for oral cavity and oropharynx cancers ( occs and opcs ) conventional or " low throughput " dna sequencing provides one sequence read per dna sample . with this technique , the dna sample requires a homogeneous dna template to decipher a maximum sequence of approximately 1000 base - pairs ( bp ) long . given that the human genome contains 3.5 billion bp encoding 10 to 30,000 genes , it is not surprising that a single genome once required approximately 13 years and three billion dollars to sequence . ngs can complete the same task in days , at a cost of approaching a few thousand dollars . its efficiency has revolutionized the sequencing of entire genomes or , more commonly , " exomes " ( genomic libraries limited to a cell 's expressed sequences ) . this has allowed investigators to catalogue mutations in over twenty malignancies , including brain , breast , and prostate cancers ( the cancer genome atlas : http://cancergenome.nih.gov/ ) . ngs is essentially multiple " low throughput " dna sequencing reactions run in parallel in a single sample . since the genomic or exomic sequences can be very long , the dna or cdna is cut or sheared by mechanical means to generate many smaller fragments . each individual dna molecule is then amplified in order to enhance subsequent dna sequencing detection . these amplified dna clones are then sequenced in a massively - parralled fashion to generate multiple short dna sequences or " reads " . these shorter reads are then aligned using computer software in order to determine the longer genomic or exomic sequence . in regards to hnscc , the cancer genome atlas contains 312 separately sequenced hnscc genomes at the writing of this article . furthermore , two recent publications by agrawal et al . and stransky et al . have also detailed common mutations in 106 hnscc samples . here , the authors performed whole exome sequencing that can identify coding as well as splice - site mutations . this approach is limited because cancer causing mutations can occur in non - protein coding regions that affect the regulation of gene expression , and may cause altered expression of a normal gene in a tissue / cell type or at a temporal point in time that is different than in normal cells . of note , in the study by stransky et al . , 50 of 77 samples were of occ or opc . both publications compared sequences from tumour specimens and those of autologous tissue controls , and samples were micro - dissected to minimize contamination by adjacent stroma . the authors called mutations in tumours if the genetic changes were detected in the tumour but not the autologous control tissues . subsequently , many of these mutations were further validated in separate cohorts or by additional sequencing and mass spectrometric genotyping analysis . interestingly , the average number of mutations differed by almost ten - fold ( 19 vs. 130 ) with each group reporting a variety of mutations per tumour . this variation in mutations may reflect technical differences , such as differences in sample size ( 32 samples vs. 74 samples ) , as well as the degree of sequence coverage for each study ( 44 to 77-fold vs. 150-fold ) which can affect sequence accuracy . analyzed tumours harvested prior to chemotherapy or radiation , which can select for additional mutational events and also account for the lower average number of mutations in their study . finally , these differences may reflect differences in the accumulation of mutations due to cancer progression , additional dna damage or different stages of cancer . ngs has generated unexpected insights . while the finding that tumours arising from smokers had more mutations than those of non - smokers was expected , two tumours from non - smokers had the highest number of mutations in one study , suggesting genomic instability in hnscc may not be entirely tobacco dependent . certain germline genetic conditions , such as fanconi anemia , can affect dna repair pathways and predispose individuals to hnscc . therefore , these instances of increased mutations and genomic instability may be due to various mendelian cancer syndromes in addition to carcinogen exposure . consistent with epidemiologic studies suggestive of biological differences based on hpv status , hpv positive tumours contained approximately half the number of mutations as hpv negative tumours , independent of smoking status . in addition , tp53 mutations were inversely correlated with hpv positivity and found in up to 78% of hpv - negative tumours . has shown an inverse relationship between p53 mutations and hpv positivity in hnscc . compared to 25% of hpv - positive tumours , furthermore , only hpv - negative tumours had mutations that disrupted p53 function suggesting that most p53 mutations resulting in a functional significance were exclusive of hpv . therefore , ngs will continue to identify potential genes that are advantageous for hnsccs and further elucidate those already known such as hpv oncogenes or mutations in p53 . overall , genes previously implicated in hnscc and confirmed by whole - exome sequencing include tp53 , cdkn2a , hras , pten , pi3kca and rb . in multiple studies , in addition , ngs has identified new mutations in genes that regulate epithelial differentiation in up to 30% of tumours . inactivating point mutations in notch1 are particularly noteworthy ; in one study , point mutations affecting this gene occurred in 11% of the hnscc tumours and focal deletions were seen in two additional tumours . importantly , the identification of notch genes and others may represent the first new targets implicated in the genesis , as well as treatment , of hnscc . while advancements in sequencing may further pinpoint the structural changes causing head and neck cancer , these techniques , like previous technologies , fail to separate those changes that drive hnscc and those passenger mutations that provide no selection advantage . validation of driver mutations requires additional in vivo and in vitro models to confirm and to understand their importance in the biology of this disease . validation of next generation sequencing ( ngs ) with existing genetically engineered mouse models ( gemms ) using information gleaned from ngs , we may better understand the physiological significance and molecular mechanisms of several candidate genes driving the development of hnsccs . previous mouse models of hnscc relied mainly on chemical carcinogens such as coal tar , cigarette smoke , 9,10-dimethyl-1,2-benzanthracene ( dmba ) , and 4-nitroquinoline 1-oxide ( 4nqo ) . over the last 20 years , gemm have been developed to study how changes in the structure or expression of specific genes impact hnscc development in vivo . these mice have been further engineered to express these altered genes in a tissue - specific and temporal manner . below , we will describe some of these previously known mutations identified in ngs that also cause hnsccs in gemms . these observations indicate that gemms can be used to test whether novel mutations identified by ngs " drive " hnsccs . tp53 is one of the most frequently mutated genes in human cancers , including occ . loss of tp53 function may be nearly universal event in the development of hnscc by mutation , deletion , amplification of mdm2 , deletion of cdkn2a or expression of hpv oncogenes . in addition to p53 deletion , the more common way of inactivating p53 in occ is by mutation , which induces both dominant negative , as well as lesser understood gain - of - function mutations . with gain - of - function mutations , the tumour suppressive activities of p53 are inhibited , while other potentially progrowth functions of p53 are maintained . in fact , these gain - of - function mutations in p53 predict for worse outcome in hnsccs . the loss of p53 has been shown to be an initiating event in mouse models , where its deletion predisposed de novo tumour formation and greatly sensitized mice to chemical carcinogens . however , the majority of mice with p53 deletion or mutations in all tissues died rapidly due to lymphoma or other cancers before the impact of their p53 defects caused development of hnscc or squamous cell carcinomas ( scc ) at other cutaneous sites . in murine models where p53 defects successfully led to hnscc development , mice with mutations or loss of p53 were treated with carcinogens or bred to mice with additional genetic abnormalities . hnsccs developed in mice which possessed p53 defects in the germline or when p53 was conditionally disrupted in the basal epithelial layer of the oral cavity and skin . to conditionally delete p53 , mice expressing cre recombinase expressed under the keratin 5 or keratin 14 promoter which is active in the basal keratinocyte layer of the epithelium were bred to knock - in mice possessing floxed p53 alleles . since cre recombinase was expressing in the basal keratinocyte layer , the mice possessed disrupted p53 pathways in the epithelium of their skin and upper aerodigestive tract . transgenic mice expressing dominant negative p53 or mice with p53 haploinsufficiency in the germline experienced accelerated hnsccs after 4-nqo treatment compared to wild type mice . in addition , mice that lost p53 expression or had p53 gain - of - function mutations in the basal keratinocytes of the oral cavity developed invasive hnscc when tumours also expressed a mutant kras gene . these results confirmed the clinical observations where loss or mutation in p53 was an important event in at least 50% of hnsccs . loss of tp53 and hpv - positivity appear to be exclusive events . in one study using whole - exome sequencing , tp53 mutations were not identified in any of the hpv associated tumours but were found in 78% of the hpv - negative tumours . as such , investigation of hpv oncogenes will likely provide future insights into a distinct subset of tumours and will be described below . human papillomavirus ( hpv ) hpv - associated cancers likely arise due to the expression of the viral oncoproteins e6 and e7 . e7 binds to and inhibits the retinoblastoma protein ( rb ) enabling cells to progress through the cell cycle and to divide . hpv relies on e6 to bind p53 and to degrade it , enabling hpv infected cells to escape this safeguard . mice expressing high risk hpv16 oncogenes , e6 and e7 , from the promoter of the bovine keratin 6 gene develop focal epithelial hyperplasia on the tongue by 27 weeks of age . however , no tumours develop in these mice suggesting e6 and e7 alone were insufficient to drive tumourigenesis . furthermore , epithelial cells derived from hpv16 transgenic or from hpv18 immortalized cells can not form independent colonies in soft agar or tumours in syngeneic or immuno - compromised mice unless they are extensively cultivated in vitro . other available hpv transgenic mice that target expression via the a crystallin and keratin 14 promoter have a low incidence of epithelial malignancies that develop after 15 months in only 5 - 10% of mice . however , tumour development in the oral cavity has not been noted . taken together , these studies indicate oncogenes e6 and e7 from high - risk hpv can immortalize epithelial cells but additional genetic events are required for transformation . while e6 and e7 alone are not sufficient to drive tumour formation , mice that co - express mutant ras or those exposed to chemical carcinogens are highly susceptible to the development of tumours of the oral cavity . schreiber et al . demonstrated strong synergy between the mutant hras and hpv16 e6/e7 . in this model , mice expressing hras driven by the zeta - globin promoter , were crossed with transgenic mice that express hpv16-e6/e7 in epithelial tissues using a keratin 14 promoter driven . double transgenic mice developed dysplastic squamous papillomas of the transitional epithelium that involved the mouth , eye and ear beginning around 3 months of age . furthermore , k14-hpv - e6/e7 mice treated with 4nqo , a chemical carcinogen , developed oral scc . importantly , these e6/e7 driven tumours resembled the molecular characteristics of human hpv - positive occ , including overexpression of p16 , a surrogate for hpv infection . in addition , minichromosome maintenance protein 7 ( mcm7 ) was overexpressed in this model of hnscc , verifying a previous study on human cervical cancer . although e7 may play a more prominent role than e6 with regard to long term carcinogenesis , the development of hnsccs in mice likely required a synergy between e6 and e7 . it is believed that e7 may be the predominant initiating oncogene whereas e6 is thought to play a more important role in the progression to malignancy . in addition , e7 likely targeted multiple rb family members to cause hnscc as deletion of both p107 and rb recapitulates many features of hpv-16 e7 mice after 4nqo treatment . therefore , the development of hpv - positive hnsccs require both the inhibition of p53 pathways and rb family members by hpv e6 and e7 respectively as well as additional mutagenic events . to study the contribution of other genes to the development of hpv - associated cancers , several reports have studied mice that express hpv oncogenes and that harbour additional defects in other cellular genes . compared to the general population , fanconi anemia ( fa ) patients who reach 50 years of age are more likely to develop a solid tumour where the majority of these tumours are squamous cell cancers ( sccs ) involving the head and neck . in a study by kutler et al . , 84% of sccs in fa patients , of which the majority had hnsccs , tested positive for hpv and none of these sccs had p53 mutations . by contrast , van zeeberg et al . did not detect hpv signatures in hnsccs but did demonstrate that two - thirds of anogenital cancers contained hpv dna . although the aetiology of hnsccs in fa patients remains a hotly debated topic , it is likely that hpv plays an important role in this process . this relationship was demonstrated by park et al . who showed that mice expressing hpv16-e7 and deficient in the fa gene fancd2 developed sccs of the tongue and oesophagus at a higher frequency than that observed in control mice . here , the hpv oncogenes were driven by a k14 promoter and expressed in the basal epithelium of the oral cavity and skin . therefore , hpv oncogenes may cooperate with other cellular genes to cause hnscc and other cancers . although mutations in ras are present in only 4 - 5% of hnscc , alterations of in ras signalling occurs frequently in cancer . this often includes amplification of chromosome 7p11 , the locus for egfr and a downstream mediator of ras . in addition , promoter methylation of rassf1a , a negative regulator or ras , is frequently observed in occ . parallel to these observations , mice expressing a g12d mutation in kras developed benign squamous papillomas of the oral mucosa , tongue and palate by 16 - 24 weeks . although highly proliferative , these papillomas never progressed to malignancy suggesting a role for kras in the initiation , but not progression to scc . in another study , mice expressing mutant kras in the basal epithelium developed papillomas exclusively located within the oral mucosa . this indicates that ras requires other factors to increase genomic instability and that this ultimately can lead to the development of frank malignancy . several groups have used these models to study novel therapies for hnsccs . for example , rapamycin prevented tumour progression of benign or malignant tumours in mice possessing mutant k - ras , with or without loss of p53 , respectively . this preclinical model parallels recent finding using this small molecule inhibitor in hnscc patients . similarly , samuel et al . showed that deletion of rac1 prevented oral papilloma development in mutant kras mice , providing another possible therapeutic target for mutant kras mice . studies such as these with mutant kras mice demonstrate that genetically engineered mice can be used to identify novel targets and therapeutic regimens for hnsccs . using genetically engineered mouse models ( gemms ) to test for " driver " mutations identified by next generation sequencing ( ngs ) the significant amount of next ngs data provides a starting point to develop novel in vivo models for hnscc in order to better understand the biology and treatment of this disease . as mentioned above , ngs identified inactivating mutations in the notch gene family in 22% of the samples . originally described in drosophila , notch family members are transmembrane proteins that regulate cell - cell communication and differentiation . the predicted effect of this mutation is a truncation resulting in a loss - of - function mutant . additional mutations occurred in the extracellular ligand binding domain and splice junctions that were also likely inactivating in nature . these mutations are similar to those recently described for myeloid leukaemia but contrast sharply with notch activating mutations observed in other lymphocytic leukaemia 's and lymphomas . these results suggest that notch mutations may be context dependent whereby notch inhibition may promote some cancers while inhibiting others . non - chemically induced transgenic mice models of oral cavity cancers ( occ ) ngs = next generation sequencing . along these lines , a clinical trial using a notch inhibitor was stopped due to an unanticipated consequence of increased cutaneous cancers . similarly , mice with a tissue specific deletion of notch1 in the skin resulted in corneal hyperplasia and skin tumours as early as 8 months post inactivation . this group suggested that loss of notch1 drove skin cancers by elevating -catenin possibly resulting in de - differentiation of epithelial cells . as no respective gemms exist for notch driven occ , the precise role of the notch gene family remains unclear in hnscc and may be context - dependent . using ngs approaches , stransky et al . confirmed previous observations that cyclin d1 the cyclin d family promotes cellular proliferation by enabling cells to enter the s phase of the cell cycle in order to synthesize dna and prepare for cell division . amplifications or overexpression of cyclin d1 frequently occurs in scc leading to dysregulation of the cell cycle . in transgenic mice , expression of cyclin d1 was directed to the oral - oesophageal squamous epithelium using part of the epstein - barr virus ed - l2 promoter ( l2-cd1 ) . expression of cyclin d1 caused hyperplasia of the basal and suprabasal epithelia of the tongue , oesophagus and forestomach . these mice were treated with 20 to 50 ppm of 4nqo for 8 weeks and then observed for an additional 16 weeks . half of the l2-cd1 mice treated with 50 ppm of 4nqo , exhibited scc of the tongue and oesophagus by 16 weeks . furthermore , when mice both overexpressed ccnd1 and were haploinsufficient for p53 , invasive scc occurred by 12 months of age . cancers were evident in the buccal mucosa ( 12% ) , tongue ( 25% ) and upper and lower oesophagus ( 11 - 12% ) with 25% containing metastasis to lymph nodes . finally , these ngs approaches showed deletion or inactivating mutations in the phosphatase and tensin homolog ( pten ) gene . pten functions as a tumour suppressor by regulating akt which promotes cell survival and metabolism . mice expressing a myrisolated , and hence , constitutively active , akt ( myrakt ) under control of a bovine keratin 5 promoter , developed dysplastic lesions in the palate , cheeks and lips addin en.cite addin en.cite.data . when the epithelial cells expressed myrakt and also lost p53 expression , mice developed malignant tumours in the oral cavity , palate , tongue and lips with local metastasis to regional lymph nodes . therefore , as shown with notch1 , ccnd1 , and pten , mouse models complimented and were able to confirm candidate genes that drive hnscc as initially identified by ngs . however , one notable gene important in hnsccs but not covered with ngs involves the transforming growth factor ( tgf- ) signalling pathway . tgf- regulates cellular proliferation and differentiation as well as angiogenesis and immune suppression . this pathway is often mutated in cancer cells so that these cells become resistant to the anti - proliferative effects of tgf- but still benefit from its pro - angiogenic and immunosuppressive functions . although mutations in tgf- signalling were not found by whole - genome sequencing , tumours of the head and neck have frequent loss of chromosome 18q , which contains the smad2 , smad3 , smad4 and tbr2 genes . in addition , tgf- is well known to cause differentiation of epithelial cells and whole - exome sequencing identified up to 30% mutations in genes that play a role in terminal differentiation . therefore , future studies still require vigilance to examine candidate genes not identified by massive sequencing efforts or other high - throughput technologies . to this end , several mouse models have shown that loss of the tgf- signalling pathway in cancer cells resulted in hnsccs . after chemical carcinogen treatment , mice possessing deletion of tgfbr1 in their epithelium developed sccs of the oral cavity . these mice also developed regional and distant metastasis within one year after treatment . furthermore , tumours exhibited enhanced proliferation , reduced apoptosis and the tumour stroma appeared highly inflamed with high levels of tgf-. furthermore , the tgf- signalling pathway may also cooperate with the akt pathway to cause hnsccs ; mice that had lost both tgfbr1 and the akt inhibitor pten in their epithelia developed oral sccs within ten weeks . in addition , mice that lost other proteins involved in the tgf- signalling pathway such as tgfbr2 or the downstream effector molecules smad4 also developed hnsccs mainly affecting the oral cavity and regional lymphatics . consistent with previous studies , tumours and stroma from mutant smad4 mice had high levels of tgfb1 and inflammation . thus , these mouse models may provide additional insight into genes mediating hnsccs that were not observed using ngs and other powerful high throughput techniques . as with other high throughput technologies such as expression microarrays and comparative genomic hybridization , recent advances in ngs can identify both new candidates and novel structural information regarding genes that drive hnsccs . in addition to genes known to be involved in hnscc such as hpv oncoproteins e6 and e7 , p53 and ras , these studies have also identified novel mutations in genes such at notch1 and pten , among others . all of these genes have been show to accelerate the development of sccs in genetically engineered mice . furthermore , ngs along with other works have identified mutations in several novel pathways . for example , 22% of tumours contained mutations in spectrin repeat containing , nuclear envelope ( syne1 ) which may regulate cytoskeletal regulation . in addition , these studies reported that 3% of cancers had mutations in the endoribonucelase dicer , an important player in mirna genesis . still , it remains unclear which candidates actually promotes scc development as well as the mechanism by which this occurs . gemms provide a novel platform to better understand and validate these novel mutations that have been identified by sequencing hnsccs genomes . it has been shown that these gemms develop sccs when mice possess mutated genes known to be involved in hnsccs and continued study will allow the discovery and validation of novel " driver " mutations important in hnsccs . understanding how these novel mutations promote malignant transformation may enable us to target hnsccs more rationally . furthermore , these models will provide an in vivo platform to study the effectiveness of different strategies utilizing cytotoxic chemotherapy as well as other small molecule inhibitors . one caution with this approach centers on the extent to which regional differences in mutations contribute to tumour heterogeneity and possibly response to therapy . for example , gerlinger et al . showed that more that 60% of all somatic mutations differed among tumour regions . despite such heterogeneity , many subclonal populations exhibited convergent tumour evolution with distinct mutations affecting similar pathways . furthermore , epigenetic alterations also occur in hnsccs and may promote tumour growth . these changes may be missed in ngs and may be difficult to study in gemms . finally , mutations in mitochondrial dna are associated increased hnscc aggressiveness . such mutations can be studied with gemms but may be missed with ngs as these events turn off gene expression without causing mutations and lead to further tumour heterogeneity . therefore , coupling ngs with gemms will also be essential to understand which mutations and pathways drive hnsccs . thus , coupling ngs approaches with gemms will provide important platforms to investigate the best ways to target individual candidate genes and , more generally , those pathways essential to hnscc . mts is a recipient of the burroughs wellcome fund career award for medical scientists award .

abstractobjectivescancer is likely caused by alterations in gene structure or expression . recently , next generation sequencing has documented mutations in 106 head and neck squamous cell cancer genomes , suggesting several new candidate genes . however , it remains difficult to determine which mutations directly contributed to cancer . here , summarize the animal models which have already validated and may test cancer causing mutations identified by next generation sequencing approaches.material and methodswe reviewed the existing literature on genetically engineered mouse models and next generation sequencing ( ngs ) , as it relates to animal models of squamous cell cancers of the head and neck ( hnscc ) in pubmed.resultsnsg has identified an average of 19 to 130 distinct mutations per hnscc specimen . while many mutations likely had biological significance , it remains unclear which mutations were essential to , or " drive , " carcinogenesis . in contrast , " passenger " mutations also exist that provide no selection advantage . the genes identified by ngs included p53 , ras , human papillomavirus oncogenes , as well as novel genes such as notch1 , dicer and syne1,2 . animal models of hnscc have already validated some of these common gene mutations identified by ngs.conclusions the advent of next generation sequencing will provide new leads to the genetic changes occurring in squamous cell cancers of the head and neck . animal models will enable us to validate these new leads in order to better elucidate the biology of squamous cell cancers of the head and neck .

INTRODUCTION MATERIAL AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

while our armamentarium used to treat squamous cell cancers of the head and neck ( hnscc ) has greatly expanded with the addition of chemoradiation , intensity modulated radiation therapy ( imrt ) , and biological agents , the progression free survival rates have increased only slightly while the overall survival ( os ) rates have stagnated . this lack of targeted therapies , coupled with the aggressive nature of occ , highlights the need to identify genes that drive this malignancy in order to identify new targets . this difference may partially be explained by the impact of the human papillomavirus ( hpv ) , which has been responsible for an opc epidemic . furthermore , hpv - negative hnsccs often have mutations in p53 , a major tumour suppressor protein controlling genomic integrity , and a correspondingly worse prognosis . therefore , in contrast to occs , understanding the distinct mutational landscapes in opcs may enable us to identify new molecular targets and , therefore , to de - escalate the toxicities associated with the current , non - targeted cytotoxic chemotherapies . furthermore , these expression signatures could differentiate between hpv - positive and hpv - negative cancers , as well as prognosticate responses to therapy . these studies support the model whereby hnscc carcinogenesis begins through two distinct pathways : one caused by chemical carcinogens and the other by hpv oncogenes . therefore , much of our knowledge of genes that drive hnscc remains limited to p53 mutations , hpv oncogenes , and the egfr pathway . the goal of this article is to review recent trends in identifying hnscc " driver " mutations , especially those occurring in occs and opcs . we define " driver " mutations as mutations in genes that confer a selective advantage to a clone enabling it to better survive or proliferate . this contrasts with " passenger " mutations that have little if any advantageous effect . we will first discuss the use of next generation sequencing ( ngs ) to catalogue point mutations prevalent in hnscc . next , we will review how some of these mutant genes have already been validated in genetically engineered mouse models ( gemm ) . finally , we will discuss how gemms may complement ngs by testing novel mutations identified by ngs as well as identify pathways observed in ngs analysis . in the present article , the authors discuss ways to identify genetically engineered mouse models that supported the recent identification of mutant genes which likely acted as " driver " mutations in hnsccs . we searched relevant articles on pubmed ( www.ncbi.nlm.nih.gov ) regarding next generation sequencing and genetically engineered mouse models for head and neck cancer from 1990 to present . first , to identify mutant genes identified by ngs in hnscc samples we performed two searches . we queried ( 1 ) " genome sequencing " head and neck cancer and ( 2 ) " exome sequencing " head and neck cancer which returned 10 and 4 results , respectively . we selected two articles that specifically described ngs ( specifically whole exome sequencing ) in hnscc . from these articles , we compiled a list of commonly mutated genes and searched whether each gene has been described in an autochthonous head and neck cancer model using the query terms " transgenic mice head and neck cancer " . in order to identify additional gemms for oral cavity / oropharynx cancer , we also searched all citations using the terms " transgenic mice head and neck cancer " that resulted in 392 total articles . we also selected articles describing gemms of genes not identified by ngs to discuss pathways important in cancer but possibly missed by this approach . next generation sequencing ( ngs ) for oral cavity and oropharynx cancers ( occs and opcs ) conventional or " low throughput " dna sequencing provides one sequence read per dna sample . its efficiency has revolutionized the sequencing of entire genomes or , more commonly , " exomes " ( genomic libraries limited to a cell 's expressed sequences ) . each individual dna molecule is then amplified in order to enhance subsequent dna sequencing detection . these shorter reads are then aligned using computer software in order to determine the longer genomic or exomic sequence . have also detailed common mutations in 106 hnscc samples . here , the authors performed whole exome sequencing that can identify coding as well as splice - site mutations . this approach is limited because cancer causing mutations can occur in non - protein coding regions that affect the regulation of gene expression , and may cause altered expression of a normal gene in a tissue / cell type or at a temporal point in time that is different than in normal cells . the authors called mutations in tumours if the genetic changes were detected in the tumour but not the autologous control tissues . subsequently , many of these mutations were further validated in separate cohorts or by additional sequencing and mass spectrometric genotyping analysis . this variation in mutations may reflect technical differences , such as differences in sample size ( 32 samples vs. 74 samples ) , as well as the degree of sequence coverage for each study ( 44 to 77-fold vs. 150-fold ) which can affect sequence accuracy . analyzed tumours harvested prior to chemotherapy or radiation , which can select for additional mutational events and also account for the lower average number of mutations in their study . while the finding that tumours arising from smokers had more mutations than those of non - smokers was expected , two tumours from non - smokers had the highest number of mutations in one study , suggesting genomic instability in hnscc may not be entirely tobacco dependent . in addition , tp53 mutations were inversely correlated with hpv positivity and found in up to 78% of hpv - negative tumours . therefore , ngs will continue to identify potential genes that are advantageous for hnsccs and further elucidate those already known such as hpv oncogenes or mutations in p53 . in multiple studies , in addition , ngs has identified new mutations in genes that regulate epithelial differentiation in up to 30% of tumours . inactivating point mutations in notch1 are particularly noteworthy ; in one study , point mutations affecting this gene occurred in 11% of the hnscc tumours and focal deletions were seen in two additional tumours . importantly , the identification of notch genes and others may represent the first new targets implicated in the genesis , as well as treatment , of hnscc . while advancements in sequencing may further pinpoint the structural changes causing head and neck cancer , these techniques , like previous technologies , fail to separate those changes that drive hnscc and those passenger mutations that provide no selection advantage . validation of driver mutations requires additional in vivo and in vitro models to confirm and to understand their importance in the biology of this disease . validation of next generation sequencing ( ngs ) with existing genetically engineered mouse models ( gemms ) using information gleaned from ngs , we may better understand the physiological significance and molecular mechanisms of several candidate genes driving the development of hnsccs . previous mouse models of hnscc relied mainly on chemical carcinogens such as coal tar , cigarette smoke , 9,10-dimethyl-1,2-benzanthracene ( dmba ) , and 4-nitroquinoline 1-oxide ( 4nqo ) . over the last 20 years , gemm have been developed to study how changes in the structure or expression of specific genes impact hnscc development in vivo . below , we will describe some of these previously known mutations identified in ngs that also cause hnsccs in gemms . these observations indicate that gemms can be used to test whether novel mutations identified by ngs " drive " hnsccs . loss of tp53 function may be nearly universal event in the development of hnscc by mutation , deletion , amplification of mdm2 , deletion of cdkn2a or expression of hpv oncogenes . in addition to p53 deletion , the more common way of inactivating p53 in occ is by mutation , which induces both dominant negative , as well as lesser understood gain - of - function mutations . in fact , these gain - of - function mutations in p53 predict for worse outcome in hnsccs . the loss of p53 has been shown to be an initiating event in mouse models , where its deletion predisposed de novo tumour formation and greatly sensitized mice to chemical carcinogens . however , the majority of mice with p53 deletion or mutations in all tissues died rapidly due to lymphoma or other cancers before the impact of their p53 defects caused development of hnscc or squamous cell carcinomas ( scc ) at other cutaneous sites . to conditionally delete p53 , mice expressing cre recombinase expressed under the keratin 5 or keratin 14 promoter which is active in the basal keratinocyte layer of the epithelium were bred to knock - in mice possessing floxed p53 alleles . in addition , mice that lost p53 expression or had p53 gain - of - function mutations in the basal keratinocytes of the oral cavity developed invasive hnscc when tumours also expressed a mutant kras gene . in one study using whole - exome sequencing , tp53 mutations were not identified in any of the hpv associated tumours but were found in 78% of the hpv - negative tumours . human papillomavirus ( hpv ) hpv - associated cancers likely arise due to the expression of the viral oncoproteins e6 and e7 . mice expressing high risk hpv16 oncogenes , e6 and e7 , from the promoter of the bovine keratin 6 gene develop focal epithelial hyperplasia on the tongue by 27 weeks of age . however , no tumours develop in these mice suggesting e6 and e7 alone were insufficient to drive tumourigenesis . while e6 and e7 alone are not sufficient to drive tumour formation , mice that co - express mutant ras or those exposed to chemical carcinogens are highly susceptible to the development of tumours of the oral cavity . double transgenic mice developed dysplastic squamous papillomas of the transitional epithelium that involved the mouth , eye and ear beginning around 3 months of age . therefore , the development of hpv - positive hnsccs require both the inhibition of p53 pathways and rb family members by hpv e6 and e7 respectively as well as additional mutagenic events . compared to the general population , fanconi anemia ( fa ) patients who reach 50 years of age are more likely to develop a solid tumour where the majority of these tumours are squamous cell cancers ( sccs ) involving the head and neck . although the aetiology of hnsccs in fa patients remains a hotly debated topic , it is likely that hpv plays an important role in this process . who showed that mice expressing hpv16-e7 and deficient in the fa gene fancd2 developed sccs of the tongue and oesophagus at a higher frequency than that observed in control mice . here , the hpv oncogenes were driven by a k14 promoter and expressed in the basal epithelium of the oral cavity and skin . although mutations in ras are present in only 4 - 5% of hnscc , alterations of in ras signalling occurs frequently in cancer . parallel to these observations , mice expressing a g12d mutation in kras developed benign squamous papillomas of the oral mucosa , tongue and palate by 16 - 24 weeks . this indicates that ras requires other factors to increase genomic instability and that this ultimately can lead to the development of frank malignancy . for example , rapamycin prevented tumour progression of benign or malignant tumours in mice possessing mutant k - ras , with or without loss of p53 , respectively . studies such as these with mutant kras mice demonstrate that genetically engineered mice can be used to identify novel targets and therapeutic regimens for hnsccs . using genetically engineered mouse models ( gemms ) to test for " driver " mutations identified by next generation sequencing ( ngs ) the significant amount of next ngs data provides a starting point to develop novel in vivo models for hnscc in order to better understand the biology and treatment of this disease . as mentioned above , ngs identified inactivating mutations in the notch gene family in 22% of the samples . non - chemically induced transgenic mice models of oral cavity cancers ( occ ) ngs = next generation sequencing . as no respective gemms exist for notch driven occ , the precise role of the notch gene family remains unclear in hnscc and may be context - dependent . confirmed previous observations that cyclin d1 the cyclin d family promotes cellular proliferation by enabling cells to enter the s phase of the cell cycle in order to synthesize dna and prepare for cell division . amplifications or overexpression of cyclin d1 frequently occurs in scc leading to dysregulation of the cell cycle . in transgenic mice , expression of cyclin d1 was directed to the oral - oesophageal squamous epithelium using part of the epstein - barr virus ed - l2 promoter ( l2-cd1 ) . expression of cyclin d1 caused hyperplasia of the basal and suprabasal epithelia of the tongue , oesophagus and forestomach . half of the l2-cd1 mice treated with 50 ppm of 4nqo , exhibited scc of the tongue and oesophagus by 16 weeks . furthermore , when mice both overexpressed ccnd1 and were haploinsufficient for p53 , invasive scc occurred by 12 months of age . finally , these ngs approaches showed deletion or inactivating mutations in the phosphatase and tensin homolog ( pten ) gene . therefore , as shown with notch1 , ccnd1 , and pten , mouse models complimented and were able to confirm candidate genes that drive hnscc as initially identified by ngs . however , one notable gene important in hnsccs but not covered with ngs involves the transforming growth factor ( tgf- ) signalling pathway . tgf- regulates cellular proliferation and differentiation as well as angiogenesis and immune suppression . although mutations in tgf- signalling were not found by whole - genome sequencing , tumours of the head and neck have frequent loss of chromosome 18q , which contains the smad2 , smad3 , smad4 and tbr2 genes . in addition , tgf- is well known to cause differentiation of epithelial cells and whole - exome sequencing identified up to 30% mutations in genes that play a role in terminal differentiation . therefore , future studies still require vigilance to examine candidate genes not identified by massive sequencing efforts or other high - throughput technologies . to this end , several mouse models have shown that loss of the tgf- signalling pathway in cancer cells resulted in hnsccs . after chemical carcinogen treatment , mice possessing deletion of tgfbr1 in their epithelium developed sccs of the oral cavity . in addition , mice that lost other proteins involved in the tgf- signalling pathway such as tgfbr2 or the downstream effector molecules smad4 also developed hnsccs mainly affecting the oral cavity and regional lymphatics . thus , these mouse models may provide additional insight into genes mediating hnsccs that were not observed using ngs and other powerful high throughput techniques . as with other high throughput technologies such as expression microarrays and comparative genomic hybridization , recent advances in ngs can identify both new candidates and novel structural information regarding genes that drive hnsccs . in addition to genes known to be involved in hnscc such as hpv oncoproteins e6 and e7 , p53 and ras , these studies have also identified novel mutations in genes such at notch1 and pten , among others . all of these genes have been show to accelerate the development of sccs in genetically engineered mice . furthermore , ngs along with other works have identified mutations in several novel pathways . for example , 22% of tumours contained mutations in spectrin repeat containing , nuclear envelope ( syne1 ) which may regulate cytoskeletal regulation . still , it remains unclear which candidates actually promotes scc development as well as the mechanism by which this occurs . gemms provide a novel platform to better understand and validate these novel mutations that have been identified by sequencing hnsccs genomes . it has been shown that these gemms develop sccs when mice possess mutated genes known to be involved in hnsccs and continued study will allow the discovery and validation of novel " driver " mutations important in hnsccs . understanding how these novel mutations promote malignant transformation may enable us to target hnsccs more rationally . furthermore , these models will provide an in vivo platform to study the effectiveness of different strategies utilizing cytotoxic chemotherapy as well as other small molecule inhibitors . despite such heterogeneity , many subclonal populations exhibited convergent tumour evolution with distinct mutations affecting similar pathways . furthermore , epigenetic alterations also occur in hnsccs and may promote tumour growth . these changes may be missed in ngs and may be difficult to study in gemms . finally , mutations in mitochondrial dna are associated increased hnscc aggressiveness . such mutations can be studied with gemms but may be missed with ngs as these events turn off gene expression without causing mutations and lead to further tumour heterogeneity . therefore , coupling ngs with gemms will also be essential to understand which mutations and pathways drive hnsccs . thus , coupling ngs approaches with gemms will provide important platforms to investigate the best ways to target individual candidate genes and , more generally , those pathways essential to hnscc . mts is a recipient of the burroughs wellcome fund career award for medical scientists award .

hydrophilic soft contact lenses were invented in 1961 by czechoslovakian chemist professor otto wichterle and first commercially introduced in the united states by bausch & lomb in 1971 . in the more than three decades since their introduction , hydrophilic soft contact lenses have proven a comparatively safe and effective form of vision correction . the majority of complications associated with hydrophilic lenses are minor and self - limiting , however contact lens related microbial keratitis ( cl - mk ) stands out as a potentially sight threatening and sometimes life altering event . the relative risk of infectious keratitis has been extensively investigated ; however , the underling risk factors much less so . length of continuous lens wear , particularly overnight wear , has been identified as a key factor in lens - related microbial keratitis . several studies suggest that relative risk increases from approximately 4 in 10,000 for daily wear to 20 in 10,000 for extended wear ( poggio et al 1989 ; schein et al 1989 ; cheng et al 1999 ) . other factors have been recognized but generally are assumed to play contributory or relatively minor roles in the genesis of cl - mk ( weissman and mondino 2002 ) . they include hypoxia , non - compliance with lens care regimens , blepharitis , diabetes mellitus , epithelial trauma , steroid use , tobacco use , and therapeutic lens use . the role contact lens materials play in the genesis of cl - mk has been explored , albeit indirectly . post - approval surveillance and other studies regarding silicone hydrogel - based contact lenses have shown relatively little difference in the incidence of presumed microbial keratitis compared to conventional hydrogel lenses ( schein et al 2005 ) . the roles of potentially important contributory factors such as lens care products , their constituents , lens designs , and interactions among these elements have been largely ignored both clinically and scientifically . based on current knowledge the overwhelming majority of cl - mk cases have been attributed to infection by gram - negative bacteria , primarily pseudomonas and serratia spp ( cheng et al 1999 ; schaefera et al 2001 ; robertson et al 2007 ) . pseudomonas is one of the few pathogens known to be capable of penetrating an intact corneal epithelium ( fleiszig et al 1998 ) . cl - mk has been associated with other pathogens including acanthamoeba spp . and , rarely fungi . the incidence of reported fungal keratitis has been low compared to bacterial keratitis with the exception of occurrences in tropical climates and in surface or immune compromised individuals , especially those wearing extended wear or therapeutic bandage contact lenses . in south florida , which has historically been the epicenter of fungal keratitis in the us , a review of records from january 1982 and january 1992 found trauma to be the most common risk factor ( 44% ) ( rosa et al 1994 ) . five cases were linked to extended wear contact lenses ( 4% ) and one to a therapeutic bandage contact lens ( < 1% ) . in a retrospective review of culture - positive fungal keratitis at the singapore national eye center and singapore general hospital from january 1991 to december 1995 , twenty - nine cases of culture - positive fungal keratitis were reported . more than half of the patients had a history of ocular trauma while a quarter had prior topical corticosteroid therapy . reports describing a dramatic rise in the incidence of contact lens - related fungal keratitis later found to be associated with bausch and lomb renu with moistureloc multipurpose solution began in 2005 and continued into 2006 . on march 8 , 2006 , the united states centers for disease control ( cdc ) received a report of three patients with contact lens - associated fusarium keratitis from david s. chu , md , a new jersey ophthalmologist and cornea specialist ( us cdc 2006a ) . the outbreak had come to the united states and was soon to become a global mini - epidemic . this paper documents the fusarium outbreak with particular focus on the time line , epidemiology , mechanisms underlying disinfectant failure and the relevant pathophysiology of fungal infection in contact lens wearers . although renu with moistureloc s proclivity for fungal growth was first discovered accidentally in january of 2005 ( epstein 2007 ) its clinical impact remained unrecognized for more than a year . the first signs of an outbreak were discovered in hong kong and then in singapore . ophthalmologists at the hong kong hospital authority initially voiced concern in july 2005 during a periodic review of emerging cases regarding an unusually high yield of fusarium from patients clinical specimens sent for culture ( hong kong hospital authority 2006 ) . as a result , hong kong s health department issued an alert in august 2005 based on reports of four patients in public hospitals with cl - mk in july and august . these cases were reported to bausch & lomb and apparently were investigated ; however , a link between these emerging cases and b&l lens care products was not found . several months later investigators in singapore uncovered the relationship . on january 27 , 2006 , donald tan , md , the deputy director of singapore s national eye center ( snec ) sent a letter to the singapore ministry of health ( moh ) describing thirteen fusarium cases in contact lens wearers all of whom used bausch & lomb s renu with moistureloc multipurpose solution . as a result , the singapore ministry of health issued a public alert regarding the increasing incidence of contact lens related fungal corneal infections on february 17 , 2006 . the alert detailed nineteen patients that had been treated at snec for culture positive fungal keratitis due to fusarium since may 2005 and an additional three cases reported by changi general hospital . as the numbers of infected patients in singapore mounted , the moh advised that as a precautionary measure for contact lens users to discontinue using bausch and lomb s renu multipurpose contact lens solution for the time being on february 20 , 2006 ( update 2 ) . on february 21 ( update 3 ) , the singapore moh announced that bausch & lomb was voluntarily suspending sales of renu multipurpose solutions . they further announced in view of the potentially serious adverse visual consequences of fungal corneal infection , the ministry of health strongly advises all contact lens users as a precautionary measure to discontinue the use of b & l renu multipurpose contact lens solution for the time being , until the causes behind this recent increase in infections can be more clearly ascertained . b&l will advise consumers on what to do with existing stocks of their product . on april 12 , 2006 in an update on contact lens related fungal infections , the singapore moh reported the following : a comprehensive case - control study ( comparing contact lens users with infection and contact lens users without corneal infection was undertaken in feb mar 2006 to investigate risk factors for the spike in fungal corneal infection . the study found a strong association between corneal infection and the use of renu solution . this association remained strong even after taking into consideration sociodemographic , lens , hygiene and environmental factors . the findings are also consistent with recent observations made in the us and hong kong . david chu , the city of new york department of health and mental hygiene issued 2006 alert # 6 on march 14 , 2006 , warning both public and professional communities about the association of fusarium keratitis and soft contact lens wear . after meeting with representatives of bausch & lomb , on march 30 , 2006 officers of the american optometric association ( aoa ) and the aoa contact lens & cornea section met on march 31 , 2006 . an urgent response to the fusarium outbreak was approved and on april 3 , 2006 , the aoa issued a press release and a clinical advisory regarding the fusarium outbreak in the united states . optometrists in the united states were advised to be vigilant and consider a fungal etiology when encountering cl - mk . the press quickly disseminated this information and on april 5 , 2006 , good morning america , a national morning television program televised a segment on the fusarium outbreak . on march 31 , 2006 , bausch & lomb issued their first press release discussing the fusarium outbreak and their management of the situation . they described interaction with the us cdc and collaboration of the johns hopkins wilmer eye institute and the cdc to implement a surveillance program at leading corneal treatment centers in the us . top experts in corneal infectious disease as well as microbiologists specializing in fungi were retained to collect and culture clinical isolates of fusarium from patient samples and identify the genotypes of retained specimens in an attempt to determine if they represented uncommon variants . bausch & lomb also announced their collaboration with health authorities in singapore and hong kong in completing the aforementioned case control study . the remainder of the press release focused on poor patient compliance as a root cause of the outbreak and the results of extensive testing that confirmed that renu with moistureloc was sterile and effective . bausch & lomb again reminded contact lens wearers of the importance of proper lens care in an april 7 , 2006 press release . on april 10 , 2006 , the us centers for disease control and prevention ( cdc ) and us food and drug administration ( fda ) issued public health alerts about the rise in reports in the us of cl - related fusarium keratitis . of particular note was the geographic distribution , with many of the cases occurring in areas where fungal keratitis was rarely if ever clinically encountered . twenty - eight of the thirty case patients wore soft contact lenses , and twenty - six of those twenty - eight used different types of bausch & lomb renu brand contact lens solution in the month prior to the onset of infection . eight patients required corneal transplantation ( us cdc 2006a ) . in a terse response to the initial cdc report bausch and lomb announced on april 10 , 2006 , that it was temporarily suspending u.s . shipments of renu with moistureloc lens care solutions produced in its greenville , sc , manufacturing facility . this limited response to the worsening crisis was not well received by the press or the financial community with several critical articles and commentaries quickly appearing in the media . subsequently , in a press release issued april 13 , 2006 , bausch & lomb requested that retailers remove us - manufactured renu with moistureloc from shelves and recommended that consumers switch to another solution pending further investigation . the company also placed ads in usa today and regional newspapers on april 14 and 16 with bausch & lomb chairman and ceo ron zarrella explaining the situation and providing guidance on alternatives . in a may 5 , 2006 update , the us cdc reported 102 confirmed cases , twelve possible cases and eighty - one cases still under investigation from thirty - one us states and territories ( us cdc 2006b ) . of the fifty eight confirmed cases for which cdc had complete data : 56 wore contact lenses32 reported using any b&l renu with moistureloc15 reported using any b&l renu multiplus7 reported using any unspecified b&l renu3 reported using any amo product3 reported using any alcon product 56 wore contact lenses 32 reported using any b&l renu with moistureloc 15 reported using any b&l renu multiplus 7 reported using any unspecified b&l renu 3 reported using any amo product 3 reported using any alcon product some cases reported using more than one type of solution , therefore the solution categories are not mutually exclusive . in a response to the cdc , dated may 3 , 2006 ( which was initially released on may 2 ) , bausch & lomb offered the following : it would be expected that the distribution of lens care products associated with these cases would be roughly proportional to the products relative market share . in the small sample of cases cdc has analyzed to date , the 27-percent representation of renu multiplus solution is well below its approximate 40-percent market share . the 57-percent share of cases preliminarily reported for the company s moistureloc formula is significantly and disproportionately higher than its u.s . this disproportionate representation of the moistureloc formula in the cdc case reports is the reason bausch & lomb voluntarily withdrew moistureloc from the market while the investigation to determine the cause of these unusual infections continues . it should be noted that no explanation was offered for the disproportionately low incidence of cases associated with other products , particularly alcon laboratory s optifree products that were linked to only three cases ( 5% ) despite having a market share comparable to renu multiplus , which was associated with 27% of cases . a may 12 , 2006 , cdc press release detailed reports of 122 confirmed cases , fifteen possible cases and sixty cases still under investigation from thirty - three u.s . seventy - five reports included insufficient evidence to classify them as cases or carry other non - fusarium diagnoses . states or territories with at least one confirmed or possible case included : arkansas , arizona , california , connecticut , florida , georgia , iowa , illinois , kansas , kentucky , louisiana , massachusetts , maryland , michigan , missouri , north carolina , north dakota , new jersey , new york , ohio , oklahoma , pennsylvania , puerto rico , tennessee , texas and vermont . states where cases were currently under investigation included : indiana , minnesota , mississippi , nevada , oregon , rhode island , and virginia ( us cdc 2006c ) . with the evidence continuing to mount linking renu with moistureloc to the fungal keratitis outbreak , bausch & lomb , in a press conference and subsequent press release dated may 15 , 2006 , announced a global voluntary recall of moistureloc . ron zarrella , b&l ceo was quoted , after an extensive investigation involving thousands of tests , millions of dollars and collaboration with government agencies , health authorities and independent experts , there is no evidence of product contamination , tampering , counterfeiting or sterility failure . that leads us to conclude that some aspect of the moistureloc formula may be increasing the relative risk of fusarium infection in unusual circumstances . in a final update , fusarium keratitis united states , 20052006 published in the may 26 , 2006 mmwr , the cdc reported 130 confirmed cases and announced its final determination ( us cdc 2006d ) . among 125 contact lens wearers , 118 were able to identify which contact lens solution(s ) they had used during the month before onset of infection . seventy - five ( 64% ) reported using bausch & lomb s renu with moistureloc alone , fourteen ( 12% ) reported using moistureloc in combination with another product , eight ( 7% ) reported using an unspecified bausch and lomb solution , and twenty - one ( 18% ) reported using only products other than moistureloc , from various manufacturers . the results of the cdc case - control investigation indicated increased risk for fusarium keratitis associated with use of bausch & lomb s renu with moistureloc . the cdc data were subsequently updated and published in the journal of the american medical association . as of june 30 , 2006 , 164 confirmed cases were identified in thirty - three states and one us territory . fusarium was not recovered from the factory , warehouse , solution filtrate , or unopened solution bottles ; production of implicated lots was not clustered in time . among thirty - nine isolates tested , at least ten different fusarium species were identified , comprising nineteen unique multilocus genotypes ( chang et al 2006 ) . in singapore from march 1 , 2005 , to may 31 , 2006 , sixty - six patients ( sixty - eight affected eyes ) were diagnosed with contact lens related fusarium keratitis ; the estimated annual national incidence was 2.35 cases per 10,000 contact lens wearers with more than 98% wearing soft , disposable lenses . sixty - two patients ( 93.9% ) reported using renu solution with forty - two patients ( 63.6% ) specifically reporting use of renu with moistureloc . most patients ( 81.8% ) reported poor contact lens hygiene practices ( khor et al 2006 ) . results of a subsequent case - control study evaluating risk factors for contact lens - related fusarium keratitis in contact lens users in singapore from march 2005 through may 2006 has been published . it included 61 patients with fusarium keratitis and 188 population - based and 179 hospital - based control subjects . patients with fusarium keratitis were found to have been more likely to use bausch & lomb renu contact lens solutions than were either population - based or hospital - based control subjects . after controlling for age , sex , contact lens hygiene , and other factors , the risk associated with renu with moistureloc was 5 times higher compared to the still significant risk of using renu multiplus . the authors concluded that use of renu contact lens solutions significantly increased the risk of contact lens - related fusarium keratitis in singapore and called for further investigations into the role of renu multiplus in the development of fusarium keratitis in other populations ( saw et al 2007 ) . in the united states , several groups reported on the outbreak . bascom palmer eye institute in miami , florida , reported treating ten cases of soft contact lens - associated keratomycosis between 1969 and 1992 . three cases of fungal keratitis associated with contact lenses were seen between 1969 and 1977 , two between 1977 and 1982 and five between 1982 and 1992 . in contrast , between january 2004 and april 2006 , thirty - four cases at bascom palmer were attributed to fusarium infection ( alfonso , cantu - dibildox , et al 2006 ) . fungal pathogens in cl - mk increased from 26.7% ( eight of thirty ) to 50% ( eighteen of thirty - six ) of isolates in 2005 . fusarium species ( 66 of 122 , 54.1% ) were the most frequent fungal pathogens ( alfonso , miller , et al 2006 ) . fungal keratitis is rare in non - tropical or sub - tropical areas . despite this well recognized climate - associated pattern , fusarium cl - mk was reported in states including pennsylvania , ohio , and california even during the cold winter months ( bernal et al 2006 ; cohen 2006 ; jeng et al 2006 ) . a variety of possible etiologies were proposed and disseminated to professional groups and the press by bausch & lomb soon after the fusarium outbreak was identified in the us . during a march 30 , 2006 briefing on the status of the fusarium investigation presented to the aoa and aoa contact lens & cornea section leadership by b&l director of medical marketing gary orsborn , ms , od , the following possible etiologies were suggested : poor patient hygiene and non - compliance with manufacturer or practitioner recommendationscontamination of the manufacturing plant or some aspect of the manufacturing processcontamination of bottles after leaving the factory either during transport or storagethe recent asian tsunami and hurricane katrina causing shifts in the ecosystem resulting in new , more virulent strains of fusarium and possibly other fungisignificant diversity of genotypes found in clinical isolatesfusarium contamination of non - preserved fluoroquinolones drops used to treat cl - mk resulting in super - infection poor patient hygiene and non - compliance with manufacturer or practitioner recommendations contamination of the manufacturing plant or some aspect of the manufacturing process contamination of bottles after leaving the factory either during transport or storage the recent asian tsunami and hurricane katrina causing shifts in the ecosystem resulting in new , more virulent strains of fusarium and possibly other fungi significant diversity of genotypes found in clinical isolates fusarium contamination of non - preserved fluoroquinolones drops used to treat cl - mk resulting in super - infection of these , poor patient compliance was highlighted as the most likely etiologic factor . initial data from the singapore eye research institute found that more than 80% of infected patients reported poor hygienic practices . however , comparison to historical data suggests that patient non - compliance did not worsen significantly during the fusarium outbreak , certainly not sufficiently to explain such a large scale occurrence . claydon and efron reported contact lens non - compliance in the use of recommended care and maintenance regimens in 40 to 91% of contact lens patients with many who were confused or ignorant about their behavior ( claydon and efron 1994 ) . non - compliance as a primary factor was made even more unlikely by genetic analysis of clinical isolates , which revealed a diverse array of fusarium solani genotypes . the broad genetic diversity and the fact that a preponderance of phylogenetic species are viewed as commonly found and associated with sinks and drains suggest the source of inoculation in these cases of keratitis was most likely from strains resident in the water systems of the patients homes ( levy et al 2006 ) . simple logic would suggest that non - hygienic patients who failed to wash their hands prior to handling lenses would be less likely to encounter and transfer a fungal inoculum to the lens , case or eye than a compliant patient exposed to sinks and drains during hand washing . extensive investigation by both the us fda and b&l found no point source of contamination . no contamination was found in any bottles , and all tested product was found stable and effective . this additional information confirmed that the outbreak was due to disinfection failure occurring during product use by patients . the issue of climatic change was considered by several people including bausch and lomb s ceo , ronald zarrella . he was quoted in a washington times article published april 13 , 2006 there s been theories all the way from , has the tsunami ... hurricanes and the effect of environmental factors created mold levels that are unprecedented ? more recently , iyer , and coworkers from the university of florida at gainesville published a retrospective review of fungal keratitis at their institution from 1999 through june 2006 ( iyer et al 2006 ) . their results suggest that , at least at their institution , contact lenses represent a major risk factor for fungal keratitis ; furthermore , the incidence of contact lens related fungal keratitis was increasing even before the fusarium outbreak in 2005 and 2006 . fungal contamination of non - bak preserved antibiotic bottles has been anecdotally described in scientific posters and the ophthalmic press , but the clinical relevance has never been established ( mack 2006 ; ashford 2006 ) . other studies suggest that microbial contamination of the contents of an antibiotic bottle is unlikely . cultures of bottles used by individual patients and in a multidose office or hospital setting have demonstrated no clinically significant contamination ( mason et al 2005 ) . additional evidence that fluoroquinolones did not foster the fusarium outbreak is the reported efficacy of commercial non - preserved moxifloxacin 0.5% ( vigamox , alcon labs , ft . worth , tx ) against culture positive fusarium cases ( munir et al 2006 ) . while climatic change may be influencing shifts in the prevalence and severity of mycotic disease , other factors , specifically solution formulation issues and inadequate testing , are more likely key contributors to this epidemic ( us fda 2006 ) . in 1997 , the us food and drug administration adopted an updated standard for contact lens care product disinfection . a 172-page guidance document containing specific direction for still current subsequently , the international organization for standardization ( iso ) developed and promulgated a newer standard , which was designed to harmonize numerous differing country - specific standards ( rosenthal , sutton , et al 2002 ) . the iso 14729 procedure was introduced in 2001 ( iso 2001 ) . stand alone test which evaluates the disinfecting solution s innate antimicrobial activity . upon passing this test , the stand alone test assumes that in conjunction with rubbing and rinsing steps , the disinfectant will be capable of passing a more stringent regimen criteria . the regimen test evaluates the antimicrobial efficacy of the entire regimen as described in the package insert ( e.g. , rubbing , rinsing , and disinfecting ) . the us fda had come under harsh criticism during the fusarium outbreak for maintaining insufficiently robust standards and failing to revise them despite significant advances in contact lens materials and lens care products . evidence had been accumulating for several years regarding the interaction between disinfecting solutions and lens materials . loss of disinfecting efficacy after lenses were soaked in biguanide disinfecting products for various periods of time has been demonstrated in several studies ( rosenthal , henry , et al 2002 ; dannelley et al 2004 ) . current fda ( iso ) stand - alone testing protocols are in - vitro tests and do not incorporate contact lenses . postulating a mechanism that explains how a multipurpose contact lens disinfection solution could become a key element in a world wide outbreak of fungal keratitis is a daunting task . all modern contact lens solutions , including renu with moistureloc must pass relatively stringent testing that challenges effectiveness against a variety of potential pathogens including fusarium solani . in january 2005 , newly introduced bausch and lomb renu with moistureloc was subjected to a small pilot study exploring its compatibility with silicone hydrogel and conventional hydrogel lens materials ( epstein 2007 ) . new unworn lenses were placed directly in conventional soft lens flat - pack cases containing b&l renu with moisture - loc . when the lenses were checked approximately a week later , several were covered with what appeared to be fungus . united states , fungal contamination of contact lenses is rare and usually is observed as small isolated focal fungal colonies invading the matrix of the lens . consultation with several knowledgeable colleagues suggested that this was of no clinical significance and the lenses were placed in storage and forgotten about . the experiment was repeated and several generations of lenses with significant fungal growth were produced using the original solution as an inoculum ( epstein 2007 ) . previous research has shown how biocides can absorb into contact lens materials resulting in the solution losing disinfectant efficacy ( iso 2001 ; rosenthal , sutton , et al 2002 ) . recently , the contact lens uptake characteristics and fungicidal activity of alexidine , a small molecular weight monomeric biguanide in renu with moistureloc , has been compared to polyhexamethylene biguanide ( phmb ) in bausch & lomb renu multiplus and amo complete moistureplus and polyquad ( polyquaternium-1 ) and aldox ( myristamidopropyl dimethylamine ) used in opti - free replenish ( rosenthal et al 2006 ) . approximately 30% to 60% of the phmb and alexidine were depleted from the solution within six hours . diminished antimicrobial activity corresponded to decreases in disinfectant concentration throughout the course of the evaluation . the polyquaternium-1 based solution maintained biocide concentration and consequently retained nearly 100% of its fungicidal activity . of note , the diminishing alexidine concentration of renu with moistureloc was reflected in reduced antimicrobial efficacy . renu with moistureloc showed an average 37% less fungicidal activity and an average of 54% less alexidine after a 6-hour soak period and showed an average 84% less activity and an average 88% less alexidine biocide present in the 7-day old used solution . renu multiplus showed an average 71% less fungicidal activity with an average 19% less phmb after six hours and 94% less fungicidal activity with an average drop of 50% biocide after seven - days . loss of disinfection efficacy during the disinfection process appears the most likely initiator of sustained fusarium contamination of the solution , lens and case . as biocide is lost through contact lens material absorption , disinfectant efficacy is reduced and the kill curve rapidly diminishes . fusarium solani is a relatively hearty organism and may survive longer than most other pathogens that may be inadvertently inoculated by the patient during lens handling . despite the activity of alexidine against fusarium , clearly some organisms survive the initial high concentration of the biocide and persist while disinfectant levels fall to levels that are more hospitable to the organism . fusarium strains have shown several properties that aid in their survival and may account for their selection ( zhang et al 2006 ) . this is likely exacerbated by non - compliant reuse of the solution with topping off since the lens has been shown to absorb disinfectant ( rosenthal et al 2006 ) . however , the mere survival of fusarium organisms does not account for the level of infection observed . however , advances in lens care design as well as competitive forces prompted bausch & lomb and other companies to formulate products with additives that boost moisture retention to improve patient comfort . the moistureloc formula contains polyquaternium-10 , an organic compound commonly used in hair - care products . its cellulose content serves as a nutritive media that may facilitate fungal growth especially as disinfectant levels fall to sub - lethal levels . this accounts at least in part for the observed pattern of fungal growth on lenses . the persistence of fusarium during the decaying disinfection cycle supports the concept that renu with moistureloc may function as a selective culture media . research performed by b&l has shown how the accumulated polymer in moistureloc can serve to isolate and protect fungus . polymer coating the lens surface , as it was designed to do , would serve to shield fungal organisms from high levels of disinfectant likely present at the surface of the lens . as disinfectant levels decline , the growth curve would overtake and exceed the kill curve of the disinfectant . this interaction has never been observed with contact lens care products as most predicate products lacked nutritive properties and disinfectant levels were assumed to remain static ; however , these principals derive from current pharmacokinetic - pharmacodynamic models of in vivo antibiotic function ( mueller et al 2004 ) . furthermore , they offer one of the few logical explanations for the intensity and geographic diversity of this epidemic . humans have been exposed to microbial pathogens since the origin of the species . as a result , a variety of effective defenses to bacterial , viral , fungal protozoan , and parasitic infection have evolved . this is especially true of the ocular surface , which is habitually in direct contact with the environment . defenses against microbial infection include : rapid epithelial healing , tight junctions , and brisk cell turn over . these defenses are so effective that only five bacteria are capable of invading an intact cornea : neisseria gonorrhoeae , corynebacterium diphtheriae , listeria , haemophilus aegyptius , and invasive strains of pseudomonas aeruginosa ( fleiszig et all 1998 ) . penetration of these innate natural host barriers is therefore a key prerequisite for microbial infection . although host defenses are complex and interwoven , intact epithelium serves as a primary defense against infection . the importance of the epithelium in protection against infection is obvious when the barrier is breached , as in wounds and burns , where infection is an obvious major cause of mortality and morbidity ( janeway et al 2001 ) . , fungal keratitis is relatively rare , especially outside of tropical and subtropical areas . because fungi do not typically invade intact cornea , trauma , usually caused by plants or vegetable matter is considered the most common cause of fungal keratitis ( liesegang and forster 1980 ) . ocular and systemic disease and prior corticosteroid treatment are additional risk factors ( thomas 2003 ) . in developing an animal model of fusarium keratitis , forster and rebell found it necessary to scarify the cornea and pretreat with subconjunctival corticosteroids to obtain sustained culture - positive ulcers in high percentage of eyes ( forster and rebell 1975 ) . a review of the clinical and microbiology records of the new york eye and infirmary identified sixty - one cases of fungal keratitis in fifty - seven patients between january 1 , 1987 and june 1 , 2003 , a sixteen - year period . of 5083 positive corneal cultures , sixty - one eyes in fifty - seven patients were positive for fungus representing 1.2% of cases . human immunodeficiency virus ( hiv ) seropositivity , chronic ocular surface disease , and trauma were the most commonly associated risk factors ( ritterband et al 2006 ) . contact lens associated fungal keratitis is rarely encountered , even in subtropical climates . in a 10 year review of records from january 1982 to january 1992 at south florida s bascom palmer eye institute only 4% of fungal keratitis cases were associated with contact lens wear ( rosa et al 1994 ) . fungal infection in cosmetic contact lens wearers is rare , again representing only 4% of overall fungal keratitis cases in another large series ( wilhelmus et al 1988 ) . other estimates are as low as 1% of all cases of cl - mk ( rosenthal et al 2006 ) . fluorescein staining ( staining ) of the ocular surface reveals epithelial trauma and barrier disruption . staining is typically observed after instilling sodium fluorescein solution or an alternative vital dye and examining the eye with a biomicroscope using special lighting and , in some cases , filtering techniques . low grade corneal staining has been reported to occur spontaneously in successful contact lens wearers and non - contact lens wearers ( schwallie et al 1997 ; jalbert et al 1999 ) . corneal staining may be asymptomatic even in severe cases . as a result , poor correlation exists between observed staining and patient symptoms ( jones et al 2002 ; garofalo et al 2005 ; epstein 2006 ) . regardless of cause or extent , staining represents a breakdown of normally protective barriers and logically increases the risk of infection and inflammation ( carnt et al 2007 ) . over the past few years , numerous reports describing corneal staining in contact lens wearers using different combinations of multipurpose solutions ( mps ) and contact lens materials have been published , especially with the biguanide disinfectants such as bausch and lomb renu multiplus ( epstein 2002 ; jones et al 2002 ; pritchard et al 2003 ; lebow and schachet 2003 ; andrasko et al 2006 ) . bausch and lomb renu with moistureloc has also been associated with significant corneal staining in several investigations ( townsend et al 2005 ; lebow and schachet 2006 ) . the research of andrasko and associates has been of particular value in understanding contact lens material and mps product interactions . this series of well - constructed double masked ( ongoing ) studies has already evaluated more than fifty different combinations of contact lenses and lens care products . this data is charted on a color - coded grid for ease of interpretation ( http://www.staininggrid.com figure 1 ) . biguanide - based mps showed higher levels of corneal disruption when paired with silicone hydrogel lenses . in particular , the combination of b&l 's purevision ( balafilcon a ) lens and renu multiplus ( phmb ) mps produced the highest levels of average corneal staining area this finding is consistent with prior reports ( epstein 2002 ; jones et al 2002 ; lebow and schachet 2003 ) . b&l renu with moistureloc in combination with the conventional hydrogel vistakon acuvue 2 had relatively high levels of corneal staining 25% . although the cdc did not specifically report on the relation of lens type and infection rate , personal communication with clinicians at bascom palmer eye institute in miami , fl and the singapore national eye institute in singapore suggested the majority of contact lens - related fusarium infections were in patients wearing acuvue 2 or similar hydrophilic materials . the risk of developing microbial keratitis depends upon several factors including : the extent of epithelial barrier disruption , the magnitude of the infectious inoculum ( introduced or present ) within the ocular environment , the virulence of the organism and finally , the effectiveness of the host inflammatory response ( lawin - brussel et al 1993 ) . the larger the surface area of epithelial compromise , the greater the probability that an organism ( or multiple organisms ) will gain entry into damaged areas of epithelium or corneal stroma and cause infection . likewise , the larger the number of potentially infectious organisms present , the greater the probability of infection . increased virulence of a pathogen will increase the chances of infection while effective humeral defenses will decrease the odds of infection . the presence of a contact lens may play a role in infection by interfering with the normal ocular immune response . since the cornea is avascular , polymorphonuclear leukocytes ( pmn ) must migrate from the conjunctival circulation to the site of infection . this acute inflammatory response was blocked by a contact lens in a rabbit model of pseudomonas keratitis ( lawin - brussel et al 1995 ) . in that regard , the presence of a contact lens may have been a contributory factor in the renu with moistureloc outbreak . nearly 200 cases of fusarium keratitis were reported to the us cdc , sixty - six cases were reported in singapore and others occurred in malaysia , china , india and several european countries . the full extent of the outbreak may never be known . some question if bausch and lomb waited too long before issuing a global recall of renu with moistureloc , worsening an already bad situation . despite the convincing evidence of a link between the fusarium outbreak and renu with moistureloc established in singapore and mounting evidence in the us , b&l initially only stopped production and distribution , not sales or use of the product . several days later , a limited recall was initiated in the us and patients and professionals were advised to discontinue using moistureloc . angela j. panzarella , b&l vice president for global vision care , was quoted : i wish we had made the decision the first day not to sell or use it . although many of the facts of this outbreak will ultimately become clearer with time and additional investigation , there remains a great opportunity to learn from this experience . in analyzing the specific factors that contributed to widespread fusarium infection , comparison to normal trends of microbial keratitis cl - mk has been estimated to range from approximately 4 in 10,000 for daily wear to 20 in 10,000 for extended - wear ( schein et al 1989 ; cheng et al 1999 ) . at the height of the fusarium epidemic in singapore the estimated annual national incidence was 2.35 cases per 10,000 wearers ( khor et al 2006 ) . considering that fungal keratitis normally manifests as 1 in every 5 cases of microbial keratitis in singapore and contact lens related keratitis is generally a small fraction of that ( approximately 6% ) , it is obvious that the numbers reported in singapore are many times greater than expected ( wong et al 1997 ; houang et al 2001 ) . it is likely that analysis of annualized infection incidence during the fusarium outbreak would yield similar numbers for the us and other areas ( alfonso , cantu - dibildox et al 2006 ) . while some of the core elements of the infectious outbreak such as biocide uptake , loss of disinfection efficacy , and some of the effects of the polymeric additives have already been investigated and reported , some critically important pieces of this puzzle remain unconfirmed . five key elements of fusarium infection associated with renu with moistureloc are depicted in figure 2 . corneal staining likely plays an important role in the genesis of infection and deserves further investigation . of sixty - six patients infected in singapore , sixty - two reported using b&l renu solutions . of these , 63.6% reported using renu with moistureloc and the remaining 30.3% appear to have been using b&l renu multiplus . in the cdc data , even though this was below the expected usage rate based on market share , it still represents a disproportionate number compared to other brands . these data when viewed in the context of the high levels of corneal staining reported with biguanide based mps like renu multiplus , the recent data of rosenthal and coworkers regarding loss of disinfectant efficacy with renu multiplus and moistureloc and the report of iyer et al regarding the ongoing increase in fungal cl - mk paints a disturbing picture ; one that may be only the tip of the iceberg ( dannelley et al 2004 ; iyer et al 2006 ; mack 2006 ) . 60 million individuals throughout the world wear contact lenses . while the risk of infection associated with contact lenses is generally perceived as small , perhaps a more appropriate perspective is that infection rates may in reality be needlessly high , especially if we can use this experience to identify specific risk factors and learn to modify them . during the fusarium outbreak , many patients experienced intense pain , lost their sight or even their eyes . for these patients and for the millions of other contact lens wearers , it is critical that a thorough and comprehensive investigation be conducted . it is hoped that this paper will serve as a starting point and as a result contact lenses will continue to evolve into an ever safer and more effective form of vision correction .

the 20052006 outbreak of fusarium keratitis associated with soft hydrophilic contact lens wear was both unprecedented and unexpected . more than 250 cases have been reported worldwide that have primarily been associated with bausch & lomb renu with moistureloc and , more recently , with bausch and lomb renu multiplus multipurpose contact lens disinfecting solutions . this article documents the outbreak , presenting the time line and the historical and scientific basis for its occurrence . underlying causes are explored including likely mechanisms of contamination and subsequent corneal infection . thorough exploration of this unique occurrence affords the opportunity to examine contact lens and lens care actions and interactions and to develop greater understanding of possible associated risks and ways to minimize them .

Introduction Origins of the outbreak: Time line Origins of the problem Current disinfectant standards for contact lens care products Etiology of the fusarium outbreak The relation between corneal staining (barrier disruption) and infection Conclusion

hydrophilic soft contact lenses were invented in 1961 by czechoslovakian chemist professor otto wichterle and first commercially introduced in the united states by bausch & lomb in 1971 . in the more than three decades since their introduction , hydrophilic soft contact lenses have proven a comparatively safe and effective form of vision correction . the majority of complications associated with hydrophilic lenses are minor and self - limiting , however contact lens related microbial keratitis ( cl - mk ) stands out as a potentially sight threatening and sometimes life altering event . other factors have been recognized but generally are assumed to play contributory or relatively minor roles in the genesis of cl - mk ( weissman and mondino 2002 ) . they include hypoxia , non - compliance with lens care regimens , blepharitis , diabetes mellitus , epithelial trauma , steroid use , tobacco use , and therapeutic lens use . the roles of potentially important contributory factors such as lens care products , their constituents , lens designs , and interactions among these elements have been largely ignored both clinically and scientifically . based on current knowledge the overwhelming majority of cl - mk cases have been attributed to infection by gram - negative bacteria , primarily pseudomonas and serratia spp ( cheng et al 1999 ; schaefera et al 2001 ; robertson et al 2007 ) . cl - mk has been associated with other pathogens including acanthamoeba spp . more than half of the patients had a history of ocular trauma while a quarter had prior topical corticosteroid therapy . reports describing a dramatic rise in the incidence of contact lens - related fungal keratitis later found to be associated with bausch and lomb renu with moistureloc multipurpose solution began in 2005 and continued into 2006 . on march 8 , 2006 , the united states centers for disease control ( cdc ) received a report of three patients with contact lens - associated fusarium keratitis from david s. chu , md , a new jersey ophthalmologist and cornea specialist ( us cdc 2006a ) . this paper documents the fusarium outbreak with particular focus on the time line , epidemiology , mechanisms underlying disinfectant failure and the relevant pathophysiology of fungal infection in contact lens wearers . although renu with moistureloc s proclivity for fungal growth was first discovered accidentally in january of 2005 ( epstein 2007 ) its clinical impact remained unrecognized for more than a year . these cases were reported to bausch & lomb and apparently were investigated ; however , a link between these emerging cases and b&l lens care products was not found . on january 27 , 2006 , donald tan , md , the deputy director of singapore s national eye center ( snec ) sent a letter to the singapore ministry of health ( moh ) describing thirteen fusarium cases in contact lens wearers all of whom used bausch & lomb s renu with moistureloc multipurpose solution . as the numbers of infected patients in singapore mounted , the moh advised that as a precautionary measure for contact lens users to discontinue using bausch and lomb s renu multipurpose contact lens solution for the time being on february 20 , 2006 ( update 2 ) . on february 21 ( update 3 ) , the singapore moh announced that bausch & lomb was voluntarily suspending sales of renu multipurpose solutions . they further announced in view of the potentially serious adverse visual consequences of fungal corneal infection , the ministry of health strongly advises all contact lens users as a precautionary measure to discontinue the use of b & l renu multipurpose contact lens solution for the time being , until the causes behind this recent increase in infections can be more clearly ascertained . on april 12 , 2006 in an update on contact lens related fungal infections , the singapore moh reported the following : a comprehensive case - control study ( comparing contact lens users with infection and contact lens users without corneal infection was undertaken in feb mar 2006 to investigate risk factors for the spike in fungal corneal infection . the study found a strong association between corneal infection and the use of renu solution . david chu , the city of new york department of health and mental hygiene issued 2006 alert # 6 on march 14 , 2006 , warning both public and professional communities about the association of fusarium keratitis and soft contact lens wear . after meeting with representatives of bausch & lomb , on march 30 , 2006 officers of the american optometric association ( aoa ) and the aoa contact lens & cornea section met on march 31 , 2006 . on march 31 , 2006 , bausch & lomb issued their first press release discussing the fusarium outbreak and their management of the situation . bausch & lomb also announced their collaboration with health authorities in singapore and hong kong in completing the aforementioned case control study . the remainder of the press release focused on poor patient compliance as a root cause of the outbreak and the results of extensive testing that confirmed that renu with moistureloc was sterile and effective . bausch & lomb again reminded contact lens wearers of the importance of proper lens care in an april 7 , 2006 press release . twenty - eight of the thirty case patients wore soft contact lenses , and twenty - six of those twenty - eight used different types of bausch & lomb renu brand contact lens solution in the month prior to the onset of infection . in a terse response to the initial cdc report bausch and lomb announced on april 10 , 2006 , that it was temporarily suspending u.s . shipments of renu with moistureloc lens care solutions produced in its greenville , sc , manufacturing facility . subsequently , in a press release issued april 13 , 2006 , bausch & lomb requested that retailers remove us - manufactured renu with moistureloc from shelves and recommended that consumers switch to another solution pending further investigation . the company also placed ads in usa today and regional newspapers on april 14 and 16 with bausch & lomb chairman and ceo ron zarrella explaining the situation and providing guidance on alternatives . of the fifty eight confirmed cases for which cdc had complete data : 56 wore contact lenses32 reported using any b&l renu with moistureloc15 reported using any b&l renu multiplus7 reported using any unspecified b&l renu3 reported using any amo product3 reported using any alcon product 56 wore contact lenses 32 reported using any b&l renu with moistureloc 15 reported using any b&l renu multiplus 7 reported using any unspecified b&l renu 3 reported using any amo product 3 reported using any alcon product some cases reported using more than one type of solution , therefore the solution categories are not mutually exclusive . in a response to the cdc , dated may 3 , 2006 ( which was initially released on may 2 ) , bausch & lomb offered the following : it would be expected that the distribution of lens care products associated with these cases would be roughly proportional to the products relative market share . this disproportionate representation of the moistureloc formula in the cdc case reports is the reason bausch & lomb voluntarily withdrew moistureloc from the market while the investigation to determine the cause of these unusual infections continues . it should be noted that no explanation was offered for the disproportionately low incidence of cases associated with other products , particularly alcon laboratory s optifree products that were linked to only three cases ( 5% ) despite having a market share comparable to renu multiplus , which was associated with 27% of cases . with the evidence continuing to mount linking renu with moistureloc to the fungal keratitis outbreak , bausch & lomb , in a press conference and subsequent press release dated may 15 , 2006 , announced a global voluntary recall of moistureloc . seventy - five ( 64% ) reported using bausch & lomb s renu with moistureloc alone , fourteen ( 12% ) reported using moistureloc in combination with another product , eight ( 7% ) reported using an unspecified bausch and lomb solution , and twenty - one ( 18% ) reported using only products other than moistureloc , from various manufacturers . the results of the cdc case - control investigation indicated increased risk for fusarium keratitis associated with use of bausch & lomb s renu with moistureloc . in singapore from march 1 , 2005 , to may 31 , 2006 , sixty - six patients ( sixty - eight affected eyes ) were diagnosed with contact lens related fusarium keratitis ; the estimated annual national incidence was 2.35 cases per 10,000 contact lens wearers with more than 98% wearing soft , disposable lenses . sixty - two patients ( 93.9% ) reported using renu solution with forty - two patients ( 63.6% ) specifically reporting use of renu with moistureloc . results of a subsequent case - control study evaluating risk factors for contact lens - related fusarium keratitis in contact lens users in singapore from march 2005 through may 2006 has been published . patients with fusarium keratitis were found to have been more likely to use bausch & lomb renu contact lens solutions than were either population - based or hospital - based control subjects . after controlling for age , sex , contact lens hygiene , and other factors , the risk associated with renu with moistureloc was 5 times higher compared to the still significant risk of using renu multiplus . the authors concluded that use of renu contact lens solutions significantly increased the risk of contact lens - related fusarium keratitis in singapore and called for further investigations into the role of renu multiplus in the development of fusarium keratitis in other populations ( saw et al 2007 ) . three cases of fungal keratitis associated with contact lenses were seen between 1969 and 1977 , two between 1977 and 1982 and five between 1982 and 1992 . a variety of possible etiologies were proposed and disseminated to professional groups and the press by bausch & lomb soon after the fusarium outbreak was identified in the us . during a march 30 , 2006 briefing on the status of the fusarium investigation presented to the aoa and aoa contact lens & cornea section leadership by b&l director of medical marketing gary orsborn , ms , od , the following possible etiologies were suggested : poor patient hygiene and non - compliance with manufacturer or practitioner recommendationscontamination of the manufacturing plant or some aspect of the manufacturing processcontamination of bottles after leaving the factory either during transport or storagethe recent asian tsunami and hurricane katrina causing shifts in the ecosystem resulting in new , more virulent strains of fusarium and possibly other fungisignificant diversity of genotypes found in clinical isolatesfusarium contamination of non - preserved fluoroquinolones drops used to treat cl - mk resulting in super - infection poor patient hygiene and non - compliance with manufacturer or practitioner recommendations contamination of the manufacturing plant or some aspect of the manufacturing process contamination of bottles after leaving the factory either during transport or storage the recent asian tsunami and hurricane katrina causing shifts in the ecosystem resulting in new , more virulent strains of fusarium and possibly other fungi significant diversity of genotypes found in clinical isolates fusarium contamination of non - preserved fluoroquinolones drops used to treat cl - mk resulting in super - infection of these , poor patient compliance was highlighted as the most likely etiologic factor . however , comparison to historical data suggests that patient non - compliance did not worsen significantly during the fusarium outbreak , certainly not sufficiently to explain such a large scale occurrence . the broad genetic diversity and the fact that a preponderance of phylogenetic species are viewed as commonly found and associated with sinks and drains suggest the source of inoculation in these cases of keratitis was most likely from strains resident in the water systems of the patients homes ( levy et al 2006 ) . the issue of climatic change was considered by several people including bausch and lomb s ceo , ronald zarrella . more recently , iyer , and coworkers from the university of florida at gainesville published a retrospective review of fungal keratitis at their institution from 1999 through june 2006 ( iyer et al 2006 ) . in 1997 , the us food and drug administration adopted an updated standard for contact lens care product disinfection . the us fda had come under harsh criticism during the fusarium outbreak for maintaining insufficiently robust standards and failing to revise them despite significant advances in contact lens materials and lens care products . evidence had been accumulating for several years regarding the interaction between disinfecting solutions and lens materials . postulating a mechanism that explains how a multipurpose contact lens disinfection solution could become a key element in a world wide outbreak of fungal keratitis is a daunting task . all modern contact lens solutions , including renu with moistureloc must pass relatively stringent testing that challenges effectiveness against a variety of potential pathogens including fusarium solani . in january 2005 , newly introduced bausch and lomb renu with moistureloc was subjected to a small pilot study exploring its compatibility with silicone hydrogel and conventional hydrogel lens materials ( epstein 2007 ) . recently , the contact lens uptake characteristics and fungicidal activity of alexidine , a small molecular weight monomeric biguanide in renu with moistureloc , has been compared to polyhexamethylene biguanide ( phmb ) in bausch & lomb renu multiplus and amo complete moistureplus and polyquad ( polyquaternium-1 ) and aldox ( myristamidopropyl dimethylamine ) used in opti - free replenish ( rosenthal et al 2006 ) . of note , the diminishing alexidine concentration of renu with moistureloc was reflected in reduced antimicrobial efficacy . renu with moistureloc showed an average 37% less fungicidal activity and an average of 54% less alexidine after a 6-hour soak period and showed an average 84% less activity and an average 88% less alexidine biocide present in the 7-day old used solution . as biocide is lost through contact lens material absorption , disinfectant efficacy is reduced and the kill curve rapidly diminishes . however , the mere survival of fusarium organisms does not account for the level of infection observed . however , advances in lens care design as well as competitive forces prompted bausch & lomb and other companies to formulate products with additives that boost moisture retention to improve patient comfort . the persistence of fusarium during the decaying disinfection cycle supports the concept that renu with moistureloc may function as a selective culture media . this interaction has never been observed with contact lens care products as most predicate products lacked nutritive properties and disinfectant levels were assumed to remain static ; however , these principals derive from current pharmacokinetic - pharmacodynamic models of in vivo antibiotic function ( mueller et al 2004 ) . furthermore , they offer one of the few logical explanations for the intensity and geographic diversity of this epidemic . in developing an animal model of fusarium keratitis , forster and rebell found it necessary to scarify the cornea and pretreat with subconjunctival corticosteroids to obtain sustained culture - positive ulcers in high percentage of eyes ( forster and rebell 1975 ) . contact lens associated fungal keratitis is rarely encountered , even in subtropical climates . in a 10 year review of records from january 1982 to january 1992 at south florida s bascom palmer eye institute only 4% of fungal keratitis cases were associated with contact lens wear ( rosa et al 1994 ) . staining is typically observed after instilling sodium fluorescein solution or an alternative vital dye and examining the eye with a biomicroscope using special lighting and , in some cases , filtering techniques . low grade corneal staining has been reported to occur spontaneously in successful contact lens wearers and non - contact lens wearers ( schwallie et al 1997 ; jalbert et al 1999 ) . over the past few years , numerous reports describing corneal staining in contact lens wearers using different combinations of multipurpose solutions ( mps ) and contact lens materials have been published , especially with the biguanide disinfectants such as bausch and lomb renu multiplus ( epstein 2002 ; jones et al 2002 ; pritchard et al 2003 ; lebow and schachet 2003 ; andrasko et al 2006 ) . bausch and lomb renu with moistureloc has also been associated with significant corneal staining in several investigations ( townsend et al 2005 ; lebow and schachet 2006 ) . this series of well - constructed double masked ( ongoing ) studies has already evaluated more than fifty different combinations of contact lenses and lens care products . in particular , the combination of b&l 's purevision ( balafilcon a ) lens and renu multiplus ( phmb ) mps produced the highest levels of average corneal staining area this finding is consistent with prior reports ( epstein 2002 ; jones et al 2002 ; lebow and schachet 2003 ) . b&l renu with moistureloc in combination with the conventional hydrogel vistakon acuvue 2 had relatively high levels of corneal staining 25% . although the cdc did not specifically report on the relation of lens type and infection rate , personal communication with clinicians at bascom palmer eye institute in miami , fl and the singapore national eye institute in singapore suggested the majority of contact lens - related fusarium infections were in patients wearing acuvue 2 or similar hydrophilic materials . this acute inflammatory response was blocked by a contact lens in a rabbit model of pseudomonas keratitis ( lawin - brussel et al 1995 ) . in that regard , the presence of a contact lens may have been a contributory factor in the renu with moistureloc outbreak . nearly 200 cases of fusarium keratitis were reported to the us cdc , sixty - six cases were reported in singapore and others occurred in malaysia , china , india and several european countries . the full extent of the outbreak may never be known . some question if bausch and lomb waited too long before issuing a global recall of renu with moistureloc , worsening an already bad situation . despite the convincing evidence of a link between the fusarium outbreak and renu with moistureloc established in singapore and mounting evidence in the us , b&l initially only stopped production and distribution , not sales or use of the product . although many of the facts of this outbreak will ultimately become clearer with time and additional investigation , there remains a great opportunity to learn from this experience . five key elements of fusarium infection associated with renu with moistureloc are depicted in figure 2 . of these , 63.6% reported using renu with moistureloc and the remaining 30.3% appear to have been using b&l renu multiplus . these data when viewed in the context of the high levels of corneal staining reported with biguanide based mps like renu multiplus , the recent data of rosenthal and coworkers regarding loss of disinfectant efficacy with renu multiplus and moistureloc and the report of iyer et al regarding the ongoing increase in fungal cl - mk paints a disturbing picture ; one that may be only the tip of the iceberg ( dannelley et al 2004 ; iyer et al 2006 ; mack 2006 ) .

currently , the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use ( ich ) recommends sponsors submitting new drug applications to evaluate the drug s effects on cardiac repolarization by conducting a clinical thorough qt ( tqt ) study . this recommendation is set to investigate possible drug - induced prolongation of the qt interval and to prevent associated potentially fatal pro - arrhythmias , such as torsades de pointes . this growing concern for cardiac safety is because some drugs , which were not originally developed to treat cardiovascular diseases , were found to cause arrhythmias and were withdrawn from the market . since its publication in 2005 , ich guideline e14 has gained a substantial amount of interest , and the guideline s proposal to examine tqt is currently followed worldwide . although ich guideline e14 does not specify the use of moxifloxacin as a positive control , it has been the most widely and most commonly used positive control in tqt studies . the effects of moxifloxacin on qt interval have been well documented and compared with ibutilide , an intravenous formulation that is the only other positive control that has been used in published tqt studies , moxifloxacin is orally administered and is therefore a better choice for use in blinded studies . when ich guideline e14 was introduced in 2005 , the initial assumption was that ethnicity would not significantly affect the results of tqt studies . since then , clinical data challenging this assumption have been accumulating . unfortunately , two limitations have arisen to date : limited data evaluating inter - ethnic differences in baseline , drug - free qt intervals exist and evidence from tqt studies has been collected mostly from caucasian subjects or subjects that do not adequately represent ethnic differences . a known debate concerning which qt interval correction method qt intervals are influenced by the individual s heart rate and should be corrected ( heart rate - corrected qt ; qtc ) for investigational purposes . formulae that reflect individual heart rate include bazett s formula , fridericia s formula , and a correction using the individual qt / rr regression model . there was previously no consensus regarding which method to use in tqt studies , but as the data accumulated , it is now encouraged that newer correction formulae such as individual correction should be used . in addition , tqt studies may use either the time - matched baseline method or the pre - dose baseline method . ich guideline e14 recommends the use of the time - matched method for parallel studies and the use of the pre - dose method for crossover studies ; however , few studies have addressed the differences between the two baseline measurement methods . comparing the two methods may provide some insight into whether using different baseline measurement methods significantly affects the results of tqt studies . at present , no comparable published data collected from korean subjects exist that can be used to evaluate an investigational product s effects on qt interval during the drug development phase . furthermore , the effects of moxifloxacin 400 or 800 mg ( supratherapeutic dose ) on qt prolongation have not been fully assessed in healthy korean subjects , nor has the known diurnal variation been evaluated in this population . hence , an investigation is required to evaluate whether the usual positive control dose for tqt studies , moxifloxacin 400 mg , is adequate for korean subjects and to determine whether moxifloxacin can be used as a positive control in koreans , as outlined by ich guideline e14 . therefore , the aims of the present study were to evaluate qtc prolongation in healthy korean male subjects ( both at therapeutic and supratherapeutic doses of moxifloxacin ) , to assess the use of moxifloxacin as an adequate positive control , to compare qt interval correction methods , and to compare baseline measurement methods in korean subjects . healthy korean male subjects , aged 2040 years with body weight over 50 kg and within 20 % of ideal body weight ( calculated as : ( height in cm 100 ) 0.9 ) , were recruited to participate in this study and written informed consent was obtained prior to participation . eligibility was assessed based on medical history , physical examination , and laboratory test results ( hematology , routine clinical chemistry , urinalysis , and serology , including hepatitis b and c , and hiv ) at screening . twelve - lead electrocardiography ( ecg ) was taken at screening and subjects with ecg findings , such as qtc interval using fridericia s formula ( qtcf ) over 450 ms , pr interval above 200 ms or below 110 ms , intraventricular conduction delay with qrs over 120 ms , second- or third - degree atrioventricular block , pathologic q waves ( defined as q - wave over 40 ms or depth over 0.5 mv ) , ventricular preexcitation , and left or right bundle branch block , were excluded from the study . subjects with the following criteria were also excluded : family history of long qt syndrome , any torsades de pointes risk factors , such as sudden death , cardiac failure , hypokalemia , and arrhythmia , and history of hypersensitivity to drugs , including quinolone antibiotics . enrolled subjects were asked not to drink alcohol or caffeinated beverages and not to smoke from 24 h prior to hospitalization until the end of the study . this study was designed as a multi - center , randomized , open - label , placebo - controlled , three - way crossover trial . 1).fig . 1study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) on day 1 , baseline 12-lead ecgs were measured after 10 min of supine position using either mac5000 or mac5500 ( ge healthcare , milwaukee , wi , usa ; set at 25 mm / s ) at the following time points : 0 , 1 , 2 , 3 , 4 , 6 , 8 , 12 , 16 , and 24 h. ecgs were recorded once for every time point . on day 2 , subjects received one of the three treatments in each period according to their sequence group : placebo ( water ) , moxifloxacin 400 mg ( avelox tablets , bayer korea ltd . , seoul , korea ) , or moxifloxacin 800 mg . ecgs were then recorded at the corresponding time points in the same manner as on the baseline day . blood sampling for the pharmacokinetic ( pk ) analyses was conducted at the same time points as the ecg recordings for subjects who took moxifloxacin . when the procedures were to be processed at the same time , the ecg was taken first , after which point , the vital signs were measured and the pk sampling was conducted to minimize the influence of the other procedures on the ecg results . the plasma was immediately separated by centrifugation at 2,093g for 10 min at 4 c and was stored at 70 c until further analysis . a washout period of 7 days was selected on the basis of the terminal half - life and the effects of moxifloxacin on the qt interval . to minimize variability among the three study centers , each center used the same bottled water ( volvic , group danone s.a . , paris , france ) for drug administration and the same meal plans . to minimize variability between the ecg recording periods , the exact placement of landmarks ( e.g. , clavicle , nipples , and sternal notch ) and precordial electrodes were marked on a transparent plastic film for each subject during period i , and this film was used throughout the study for each individual subject . the exact places of precordial electrodes did not change over the whole course of the study . mary s hospital , seoul national university hospital , and seoul national university bundang hospital . all of the procedures were performed in accordance with the recommendations of the declaration of helsinki regarding biomedical research involving human subjects and the korean good clinical practice guidelines . qt intervals were measured automatically using the muse cv information system ( ge medical systems , milwaukee , wi , usa ) and the representative median value from 12 leads was taken . for all other values , including heart rate , pr interval , rr interval , and qrs interval , automatically calculated values from the mac5000 or mac5500 were used . the baseline - corrected difference in qtc ( qtc ) and the placebo - adjusted difference in qtc ( qtc ) were calculated using either bazett s formula ( qtcb = qt / rr ) , fridericia s formula ( qtcf = qt / rr ) , or an individual qt / rr linear regression model ( qtci ) . this was performed by first correcting the qt interval , then calculating qtc and qtc as follows : qtc = qt ( day 2 ) qt ( baseline ) and qtc = qtc ( treatment ) qtc ( placebo ) . : first , the qt interval vs. rr interval data obtained from each subject were plotted and fitted to a linear mixed model using the equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log qt_{ij } = b_{i } + \alpha_{i } \log rr_{ij } + e_{ij } , $ $ \end{document}logqtij = bi+ilogrrij+eij , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e^{{b_{i } } } $ $ \end{document}ebi is the subject - specific qt in seconds when the rr interval was 1 s , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \alpha_{i } $ $ \end{document}i is the slope of the log - transformed rr vs. qt relationship , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{ij } $ $ \end{document}eij is an error term . the subscripts i and j refer to the individual ( i ) and the measurement time ( j ) . this linear model was manipulated to yield a correction of the equation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{qtci } } = qt/(rr)^{{\alpha_{i } } } $ $ \end{document}qtci = qt/(rr)i . this correction from the placebo phase was applied to the data obtained during each subject s treatment phases . a repeated - measures analysis of variance taking the baseline qtc ( 1d ) as the covariate , and period , sequence , study site , dosing amount , and time as fixed effects , was used for the statistical analyses . the slopes and intercepts were estimated using qtc calculated by bazett s formula , fridericia s formula , and the individual linear regression method . in the present study , the time - matched baseline measurement was used in all qt interval calculations . however , to assess the differences between baseline measurement methods , an arbitrary pre - dose baseline at 0 h was chosen and qtc and qtc were calculated from it . the plasma concentrations of moxifloxacin were determined using api 3200 lc / ms / ms system ( applied biosystems , foster city , ca , usa ) . a volume of 200 l of plasma was deproteinized with 200 l of 10 % trichloroacetic acid containing the internal standard ( moxifloxacin - d4 , 5 g / ml ) . fifty microliters of the supernatant was diluted with 450 l of distilled water and 5 l of the dilution was injected onto a hypersil gold c18 column ( 50 3.0 mm , 5 m ) at a flow rate of 0.4 ml / min under isocratic conditions with 35 % methanol containing 0.1 % formic acid . analytes were detected using multiple - reaction monitoring in the electrospray positive - ionization mode of ms . the mass transitions were m / z 402.1 384.0 for moxifloxacin and m / z 406.2 388.2 for the internal standard . the lower limit of quantification was 100 ng / ml . the intra- and inter - day precisions ( relative standard deviation ) were below 3.94 % and the accuracy range was 97.73106.6 % . the following pk parameters were assessed using a non - compartmental method with phoenix winnonlin ( pharsight , mountain view , ca , usa ) : maximum observed drug concentration ( cmax ) , time to reach cmax following drug administration ( tmax ) , area under the plasma concentration - time curve ( auc ) from 0 h to the last measurable concentration ( auclast ) , auc from 0 h extrapolated to infinite time ( aucinf ) , terminal elimination half - life ( t1/2 ) , apparent clearance ( cl / f ) , and apparent volume of distribution ( vd / f ) . cmax and tmax were determined by direct inspection of individual pk data , whereas auclast and aucinf were calculated using the linear up / log - down method . the safety of subjects was assessed via vital sign measurements , physical examinations , adverse events , clinical laboratory tests , and 12-lead ecg . subjects were asked open - ended questions about their well - being , and adverse events were recorded and assessed based on their number of occurrences , the number of subjects who experienced adverse events , and their severity , seriousness , and causal relationship to moxifloxacin . healthy korean male subjects , aged 2040 years with body weight over 50 kg and within 20 % of ideal body weight ( calculated as : ( height in cm 100 ) 0.9 ) , were recruited to participate in this study and written informed consent was obtained prior to participation . eligibility was assessed based on medical history , physical examination , and laboratory test results ( hematology , routine clinical chemistry , urinalysis , and serology , including hepatitis b and c , and hiv ) at screening . twelve - lead electrocardiography ( ecg ) was taken at screening and subjects with ecg findings , such as qtc interval using fridericia s formula ( qtcf ) over 450 ms , pr interval above 200 ms or below 110 ms , intraventricular conduction delay with qrs over 120 ms , second- or third - degree atrioventricular block , pathologic q waves ( defined as q - wave over 40 ms or depth over 0.5 mv ) , ventricular preexcitation , and left or right bundle branch block , were excluded from the study . subjects with the following criteria were also excluded : family history of long qt syndrome , any torsades de pointes risk factors , such as sudden death , cardiac failure , hypokalemia , and arrhythmia , and history of hypersensitivity to drugs , including quinolone antibiotics . enrolled subjects were asked not to drink alcohol or caffeinated beverages and not to smoke from 24 h prior to hospitalization until the end of the study . this study was designed as a multi - center , randomized , open - label , placebo - controlled , three - way crossover trial . 1).fig . 1study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) on day 1 , baseline 12-lead ecgs were measured after 10 min of supine position using either mac5000 or mac5500 ( ge healthcare , milwaukee , wi , usa ; set at 25 mm / s ) at the following time points : 0 , 1 , 2 , 3 , 4 , 6 , 8 , 12 , 16 , and 24 h. ecgs were recorded once for every time point . on day 2 , subjects received one of the three treatments in each period according to their sequence group : placebo ( water ) , moxifloxacin 400 mg ( avelox tablets , bayer korea ltd . , seoul , korea ) , or moxifloxacin 800 mg . ecgs were then recorded at the corresponding time points in the same manner as on the baseline day . blood sampling for the pharmacokinetic ( pk ) analyses was conducted at the same time points as the ecg recordings for subjects who took moxifloxacin . when the procedures were to be processed at the same time , the ecg was taken first , after which point , the vital signs were measured and the pk sampling was conducted to minimize the influence of the other procedures on the ecg results . the plasma was immediately separated by centrifugation at 2,093g for 10 min at 4 c and was stored at 70 c until further analysis . a washout period of 7 days was selected on the basis of the terminal half - life and the effects of moxifloxacin on the qt interval . to minimize variability among the three study centers , each center used the same bottled water ( volvic , group danone s.a . , paris , france ) for drug administration and the same meal plans . to minimize variability between the ecg recording periods , the exact placement of landmarks ( e.g. , clavicle , nipples , and sternal notch ) and precordial electrodes were marked on a transparent plastic film for each subject during period i , and this film was used throughout the study for each individual subject . the exact places of precordial electrodes did not change over the whole course of the study . mary s hospital , seoul national university hospital , and seoul national university bundang hospital . all of the procedures were performed in accordance with the recommendations of the declaration of helsinki regarding biomedical research involving human subjects and the korean good clinical practice guidelines . qt intervals were measured automatically using the muse cv information system ( ge medical systems , milwaukee , wi , usa ) and the representative median value from 12 leads was taken . for all other values , including heart rate , pr interval , rr interval , and qrs interval , automatically calculated values from the mac5000 or mac5500 were used . the baseline - corrected difference in qtc ( qtc ) and the placebo - adjusted difference in qtc ( qtc ) were calculated using either bazett s formula ( qtcb = qt / rr ) , fridericia s formula ( qtcf = qt / rr ) , or an individual qt / rr linear regression model ( qtci ) . this was performed by first correcting the qt interval , then calculating qtc and qtc as follows : qtc = qt ( day 2 ) qt ( baseline ) and qtc = qtc ( treatment ) qtc ( placebo ) . first , the qt interval vs. rr interval data obtained from each subject were plotted and fitted to a linear mixed model using the equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log qt_{ij } = b_{i } + \alpha_{i } \log rr_{ij } + e_{ij } , $ $ \end{document}logqtij = bi+ilogrrij+eij , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e^{{b_{i } } } $ $ \end{document}ebi is the subject - specific qt in seconds when the rr interval was 1 s , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \alpha_{i } $ $ \end{document}i is the slope of the log - transformed rr vs. qt relationship , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{ij } $ $ \end{document}eij is an error term . the subscripts i and j refer to the individual ( i ) and the measurement time ( j ) . this linear model was manipulated to yield a correction of the equation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{qtci } } = qt/(rr)^{{\alpha_{i } } } $ $ \end{document}qtci = qt/(rr)i . this correction from the placebo phase was applied to the data obtained during each subject s treatment phases . a repeated - measures analysis of variance taking the baseline qtc ( 1d ) as the covariate , and period , sequence , study site , dosing amount , and time as fixed effects , was used for the statistical analyses . the slopes and intercepts were estimated using qtc calculated by bazett s formula , fridericia s formula , and the individual linear regression method . in the present study , the time - matched baseline measurement was used in all qt interval calculations . however , to assess the differences between baseline measurement methods , an arbitrary pre - dose baseline at 0 h was chosen and qtc and qtc were calculated from it . the plasma concentrations of moxifloxacin were determined using api 3200 lc / ms / ms system ( applied biosystems , foster city , ca , usa ) . a volume of 200 l of plasma was deproteinized with 200 l of 10 % trichloroacetic acid containing the internal standard ( moxifloxacin - d4 , 5 g / ml ) . fifty microliters of the supernatant was diluted with 450 l of distilled water and 5 l of the dilution was injected onto a hypersil gold c18 column ( 50 3.0 mm , 5 m ) at a flow rate of 0.4 ml / min under isocratic conditions with 35 % methanol containing 0.1 % formic acid . analytes were detected using multiple - reaction monitoring in the electrospray positive - ionization mode of ms . the mass transitions were m / z 402.1 384.0 for moxifloxacin and m / z 406.2 388.2 for the internal standard . the lower limit of quantification was 100 ng / ml . the intra- and inter - day precisions ( relative standard deviation ) were below 3.94 % and the accuracy range was 97.73106.6 % . the following pk parameters were assessed using a non - compartmental method with phoenix winnonlin ( pharsight , mountain view , ca , usa ) : maximum observed drug concentration ( cmax ) , time to reach cmax following drug administration ( tmax ) , area under the plasma concentration - time curve ( auc ) from 0 h to the last measurable concentration ( auclast ) , auc from 0 h extrapolated to infinite time ( aucinf ) , terminal elimination half - life ( t1/2 ) , apparent clearance ( cl / f ) , and apparent volume of distribution ( vd / f ) . cmax and tmax were determined by direct inspection of individual pk data , whereas auclast and aucinf were calculated using the linear up / log - down method . the safety of subjects was assessed via vital sign measurements , physical examinations , adverse events , clinical laboratory tests , and 12-lead ecg . subjects were asked open - ended questions about their well - being , and adverse events were recorded and assessed based on their number of occurrences , the number of subjects who experienced adverse events , and their severity , seriousness , and causal relationship to moxifloxacin . five subjects withdrew consent prior to the completion of the study and 33 subjects completed the study . the means standard deviation of subject demographic parameters were as follows : age 26.4 4.8 years , height 174.5 5.0 cm , weight 68.3 6.3 kg , and baseline qtcf 398.3 16.1 ms . there were no statistically significant differences in demographic characteristics ( age , height , weight , and baseline qtcf interval ) among the sequence groups and study centers ( data not shown ) . there were definite increases in qtc after moxifloxacin dosing ( fig . 2 ) . qtci at each measurement time is shown , with means and two - sided 90 % confidence intervals ( cis ) that did not include zero at any of the measurement times ( table 1 ) . the majority of the largest time - matched qtc occurred approximately 4 h after dosing ( table 2 ) . depending on the correction method used to calculate qtc , moxifloxacin 400 mg prolonged the qt interval from 12 ms ( qtci ) to 16 ms ( qtcb ) and moxifloxacin 800 mg prolonged qtc from 21 ms ( qtci ) to 29 ms ( qtcb).fig . 2baseline- and placebo - corrected qt ( qtc)-time profiles using : a bazett s formula , b fridericia s formula , and c the individually corrected method . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg)table 1baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms)time ( h)treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgmean90 % lower90 % uppermean90 % lower90 % upper110.236.6613.7915.1311.5718.7027.744.1811.3016.7313.1720.30310.997.4314.5520.4616.9024.02411.668.1015.2220.9617.4024.5369.906.3413.4617.6414.0821.2084.631.078.1916.9313.3720.49127.323.7610.8813.409.8316.96165.982.419.5410.887.3214.44248.825.2512.3815.4911.9319.05table 2largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci)]treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgtime ( h)mean [ 90 % ci]time ( h)mean [ 90 % ci]qtcb415.95 [ 10.81 , 21.09]3 28.83 [ 23.69 , 33.97]qtcf412.31 [ 8.38 , 16.24]4 23.14 [ 19.21 , 27.07]qtci411.66 [ 8.10 , 15.22]4 20.96 [ 17.40 , 24.53 ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model baseline- and placebo - corrected qt ( qtc)-time profiles using : a bazett s formula , b fridericia s formula , and c the individually corrected method . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg ) baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms ) largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci ) ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model an increase in plasma moxifloxacin concentration was weakly associated with qtc prolongation ( fig . the slopes of the regression lines using each correction method differed slightly ( qtcb : 0.0067 , qtcf : 0.00535 , and qtci : 0.0047 ) , while the correlation coefficients were similar for each correction method ( qtcb : 0.4344 , qtcf : 0.4346 , and qtci : 0.4220 ) . there was a statistically significant difference between the time - matched and pre - dose baseline measurement methods ( fig . 4 , p < 0.001 ) , but the time courses of the qtc profiles were similar between the two baseline correction methods ( p = 0.853 ) . qtci regression showed rate - correction coefficient ( ) values of 0.305 0.044 ( mean and standard deviation ) , with a minimum value of 0.207 and a maximum value of 0.413 ( data not shown ) , which is comparable to the value of qtcf ( 0.333).fig . 3plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . the equations for each regression line were as follows : a qtcb = 0.901 + 0.00670 moxifloxacin concentration , b qtcf = 1.39 + 0.00535 moxifloxacin concentration , and c qtci = 2.36 + 0.00470 moxifloxacin concentration ( open circle 400 mg , solid circle 800 mg)fig . 4comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . the equations for each regression line were as follows : a qtcb = 0.901 + 0.00670 moxifloxacin concentration , b qtcf = 1.39 + 0.00535 moxifloxacin concentration , and c qtci = 2.36 + 0.00470 moxifloxacin concentration ( open circle 400 mg , solid circle 800 mg ) comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group differences among study centers , sequence groups , periods , and treatment - time interaction did not influence the variation in qtc prolongation ( data not shown ) . qtc prolongation was affected by the different treatments , ( i.e. , moxifloxacin 400 or 800 mg ) and by time ( both p < 0.0001 ) . dose - dependent pk profiles were observed in the moxifloxacin concentration - time profiles ( fig . the median value for tmax was slightly greater in the moxifloxacin 800-mg group than in the moxifloxacin 400-mg group . certain parameters , such as t1/2 , cl / f , and vd / f did not significantly differ between the treatment groups , while other parameters , such as cmax and auclast , tended to increase two - fold as the dose doubled ( data not shown).fig . 5plasma concentration - time profiles after a single oral administration of moxifloxacin plasma concentration - time profiles after a single oral administration of moxifloxacin a total of 14 subjects reported 11 adverse events , which included chest discomfort , chill , diarrhea , dizziness , dry mouth , epistaxis , fever , nausea , paresthesia , pruritis , and rhinorrhea . among these , chest discomfort , diarrhea , and nausea were assessed to be either possibly or probably related to moxifloxacin . no serious adverse events were reported and all of the reported adverse events disappeared spontaneously . five subjects withdrew consent prior to the completion of the study and 33 subjects completed the study . the means standard deviation of subject demographic parameters were as follows : age 26.4 4.8 years , height 174.5 5.0 cm , weight 68.3 6.3 kg , and baseline qtcf 398.3 16.1 ms . there were no statistically significant differences in demographic characteristics ( age , height , weight , and baseline qtcf interval ) among the sequence groups and study centers ( data not shown ) . there were definite increases in qtc after moxifloxacin dosing ( fig . 2 ) . qtci at each measurement time is shown , with means and two - sided 90 % confidence intervals ( cis ) that did not include zero at any of the measurement times ( table 1 ) . the majority of the largest time - matched qtc occurred approximately 4 h after dosing ( table 2 ) . depending on the correction method used to calculate qtc , moxifloxacin 400 mg prolonged the qt interval from 12 ms ( qtci ) to 16 ms ( qtcb ) and moxifloxacin 800 mg prolonged qtc from 21 ms ( qtci ) to 29 ms ( qtcb).fig . 2baseline- and placebo - corrected qt ( qtc)-time profiles using : a bazett s formula , b fridericia s formula , and c the individually corrected method . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg)table 1baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms)time ( h)treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgmean90 % lower90 % uppermean90 % lower90 % upper110.236.6613.7915.1311.5718.7027.744.1811.3016.7313.1720.30310.997.4314.5520.4616.9024.02411.668.1015.2220.9617.4024.5369.906.3413.4617.6414.0821.2084.631.078.1916.9313.3720.49127.323.7610.8813.409.8316.96165.982.419.5410.887.3214.44248.825.2512.3815.4911.9319.05table 2largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci)]treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgtime ( h)mean [ 90 % ci]time ( h)mean [ 90 % ci]qtcb415.95 [ 10.81 , 21.09]3 28.83 [ 23.69 , 33.97]qtcf412.31 [ 8.38 , 16.24]4 23.14 [ 19.21 , 27.07]qtci411.66 [ 8.10 , 15.22]4 20.96 [ 17.40 , 24.53 ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model baseline- and placebo - corrected qt ( qtc)-time profiles using : a bazett s formula , b fridericia s formula , and c the individually corrected method . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg ) baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms ) largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci ) ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model an increase in plasma moxifloxacin concentration was weakly associated with qtc prolongation ( fig . the slopes of the regression lines using each correction method differed slightly ( qtcb : 0.0067 , qtcf : 0.00535 , and qtci : 0.0047 ) , while the correlation coefficients were similar for each correction method ( qtcb : 0.4344 , qtcf : 0.4346 , and qtci : 0.4220 ) . there was a statistically significant difference between the time - matched and pre - dose baseline measurement methods ( fig . 4 , p < 0.001 ) , but the time courses of the qtc profiles were similar between the two baseline correction methods ( p = 0.853 ) . qtci regression showed rate - correction coefficient ( ) values of 0.305 0.044 ( mean and standard deviation ) , with a minimum value of 0.207 and a maximum value of 0.413 ( data not shown ) , which is comparable to the value of qtcf ( 0.333).fig . 3plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . the equations for each regression line were as follows : a qtcb = 0.901 + 0.00670 moxifloxacin concentration , b qtcf = 1.39 + 0.00535 moxifloxacin concentration , and c qtci = 2.36 + 0.00470 moxifloxacin concentration ( open circle 400 mg , solid circle 800 mg)fig . 4comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . the equations for each regression line were as follows : a qtcb = 0.901 + 0.00670 moxifloxacin concentration , b qtcf = 1.39 + 0.00535 moxifloxacin concentration , and c qtci = 2.36 + 0.00470 moxifloxacin concentration ( open circle 400 mg , solid circle 800 mg ) comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group differences among study centers , sequence groups , periods , and treatment - time interaction did not influence the variation in qtc prolongation ( data not shown ) . qtc prolongation was affected by the different treatments , ( i.e. , moxifloxacin 400 or 800 mg ) and by time ( both p < 0.0001 ) . dose - dependent pk profiles were observed in the moxifloxacin concentration - time profiles ( fig . 5 ) . the median value for tmax was slightly greater in the moxifloxacin 800-mg group than in the moxifloxacin 400-mg group . certain parameters , such as t1/2 , cl / f , and vd / f did not significantly differ between the treatment groups , while other parameters , such as cmax and auclast , tended to increase two - fold as the dose doubled ( data not shown).fig . 5plasma concentration - time profiles after a single oral administration of moxifloxacin plasma concentration - time profiles after a single oral administration of moxifloxacin a total of 14 subjects reported 11 adverse events , which included chest discomfort , chill , diarrhea , dizziness , dry mouth , epistaxis , fever , nausea , paresthesia , pruritis , and rhinorrhea . among these , chest discomfort , diarrhea , and nausea were assessed to be either possibly or probably related to moxifloxacin . no serious adverse events were reported and all of the reported adverse events disappeared spontaneously . our study found a definite prolongation of the qtc interval after moxifloxacin dosing [ 11.66 ms in the moxifloxacin 400-mg group and 20.96 ms in the moxifloxacin 800-mg group ( qtci values ) ] . the mean differences and 90 % cis of qtci did not include zero at any of the measurement time points . a positive relationship between qt interval prolongation and moxifloxacin concentration ( r = 0.422 in qtci ) was also observed . the tmax of moxifloxacin 400 and 800 mg occurred 1 and 3 h after dosing , respectively , whereas the largest time - matched qtc was measured approximately 4 h after dosing . moxifloxacin 400 mg is known to cause a mean increase in the qtc interval of between 10 and 14 ms 24 h after a single oral dose [ 4 , 8 ] , which was consistent with the results of this study . in addition , a supratherapeutic dose of moxifloxacin ( 800 mg ) resulted in a nearly 2-fold increase in the qtc interval from baseline compared with the 400-mg dose , which was greater than the previous report by demolis et al . . although demolis et al . only used qtcb and qtcf values in their study , they found no relationship between the dose of moxifloxacin and qt interval lengthening , but found a positive relationship between qt interval prolongation and moxifloxacin concentration [ r = 0.35 , moxifloxacin concentration ( g / l ) vs. qt interval ( ms ) ; r = 0.72 , moxifloxacin concentration ( g / l ) vs. qt interval ( % ) ] . . differs from our study in that they performed submaximal exercise testing to allow for variation in rr intervals , which may explain the differences between the correlation coefficients ( moxifloxacin concentration vs. qt or qtc interval ) in the two studies . their findings with supratherapeutic doses of moxifloxacin differed from those of our study , in which an increase in the moxifloxacin dose almost doubled qtc . because there were no noticeable differences in pk parameters between the two studies , there is a possibility that korean subjects may show different susceptibility to supratherapeutic doses of moxifloxacin than caucasian subjects . nonetheless , our findings suggest that moxifloxacin induces a detectable effect of greater than 5 ms on qtc prolongation , which confirms the adequacy of the use of moxifloxacin as a positive control in korean tqt studies , explained by answer 1 in the ich e14 questions - and - answers document . data reported by florian et al . showed the sufficiency of linear concentration-qtcf model in describing the effect of moxifloxacin on qt interval . pooled data from 20 tqt studies were analyzed , and a mean slope of 3.1 ms per g / ml was estimated . this estimated slope is smaller when compared with the present study s slope ( 0.00535 ms per g / l for qtcf ) . although caucasians were more than 80 % of the dataset in florian et al . , it is unlikely that this difference is because of ethnicity . there seems to be a wide inter - individual variability in moxifloxacin - induced qt response , as the range of the slope varied greatly from 1.6 to 4.8 ms per g / ml even when the percentages of ethnic backgrounds were similar between studies . therefore , the difference in mean slopes of concentration-qtc models is likely because of individual variability . a study that recruited healthy japanese subjects , which reported the largest qtcf change from baseline as 11.6 ms ( 90 % ci 9.114.1 ) in a non - fasting state and 14.4 ms ( 90 % ci 11.916.8 ) in a fasting state , found no statistically significant differences between caucasian and japanese subjects in qtc interval prolongation . the value obtained in the fasting state was similar to the largest qtcf found in our study , but because direct comparison is not possible , this does not imply ethnic differences between japanese and korean subjects . it is worth noting , however , that there was a study ( clinicaltrials.gov identifier nct01876316 ) that compared moxifloxacin - induced qt prolongation in japanese and korean subjects , and this study has concluded there was no significant difference between the two ethnicities ( unpublished data ) . one study that used a quinidine intravenous infusion to evaluate differences in qt interval prolongation between caucasian and korean subjects reported that korean subjects were less sensitive to quinidine - induced qt prolongation than caucasian subjects . although this study contributed valuable korean qt prolongation study data , a difference exists : this study did not use moxifloxacin , a drug that is commonly used as a positive control in tqt studies . previously identified differences based on qt interval correction methods were observed : namely , the tendency of bazett s formula to extend to extreme values . this tendency was more evident in the moxifloxacin 800-mg group , where the largest time - matched qtcb was calculated to be 28.83 ms ( 90 % ci 23.6933.97 ) . therefore , a correction method using either fridericia s formula or individual correction may be a better choice for tqt studies in korean subjects , where individual correction would most likely be the best choice as noted previously . we also investigated different baseline measurement methods and found a statistically significant difference between two baseline measurement methods ; namely , a trend was observed in which the qtc from the time - matched baseline was measured to be lower than that from the pre - dose baseline . this finding may be because the time - matched baseline measurement corrects for diurnal variation . one limitation to our study is the fact we took only one pre - dose recording , while the usual pre - dose baseline measurement is conducted by taking the median qtc value from three pre - dose ecg recordings . therefore , an exact one - on - one comparison of the time - matched and pre - dose baseline methods was not appropriate . ich guideline e14 recommends that parallel studies use the time - matched baseline method and that crossover studies use the pre - dose baseline method . in contrast to the recommendations , our study was a crossover study that used the time - matched baseline method ; however , despite the identified limitations of our study , we think that the time - matched baseline measurement can also be used in crossover studies because of its merits in diurnal variation correction . a study by yan et al . suggested that parallel studies using time - matched baseline correction could show higher variation in qtcf and result in smaller correlation , probably because of a time lag between baseline measurement and dosing . yan et al . have also found slightly lower values for qtcf in crossover designs that used pre - dose baseline correction . because our study is unique in that we have set up a crossover study with time - matched baseline method , it is quite difficult to compare whether one baseline correction method is preferable in place of another . at present we speculated that by confirming the qt interval prolongation effects of moxifloxacin we could obtain comparable pilot data that could be used in qt interval prolongation studies in drug development targeting the korean population . by doing so , an effective safety monitoring plan can be established and guidelines for accurate ecg data collection and analysis may be presented . bazett s formula is not recommended for calculating the range of qt interval prolongation corrected by individual rr , but fridericia s formula and the individual correction method may be used interchangeably . the current study aimed to provide comparable pilot qt interval prolongation data in korean subjects that could be used in scientific and regulatory fields and was not necessarily focused on detecting inter - ethnic differences . however , because other studies have suggested that possible differences exist between ethnic groups , further studies are needed to evaluate and incorporate possible interethnic differences . in addition , because this study only included male subjects , gender differences were not evaluated . in summary , moxifloxacin 400 mg causes moderate qt interval prolongation and is an adequate positive control in korean tqt studies . our results indicate that caution should be exercised when a supratherapeutic dose of moxifloxacin is used in korean subjects . furthermore , the findings of the present study may be employed in drug development studies targeting the korean population and may also be applied to further research attempting to evaluate the cardiac safety of a drug in korean subjects .

background and objectivemoxifloxacin 400 mg is a widely used positive control in thorough qt ( tqt ) studies , but its qt - prolonging effects in korean subjects have not been studied . the present study was conducted to collect pilot data in korean subjects after moxifloxacin administration to evaluate the adequacy of moxifloxacin as a positive control.methodsthirty-eight , healthy , korean , male subjects were recruited for pharmacokinetic ( pk ) blood sampling and electrocardiography ( ecg ) recordings at three different study sites . on day 1 , a baseline 12-lead ecg was recorded , and on day 2 , ecg recordings were conducted after placebo , or moxifloxacin 400- or 800-mg administration . baseline - corrected , placebo - adjusted , corrected qt ( qtc ) values were calculated . blood samples were collected after moxifloxacin administration and pk parameters were assessed.resultsa total of 33 subjects completed the study . the largest time - matched qtc occurred approximately 4 h after dosing , with qtci ( qt interval corrected by individual qt - rr regression model ) values of 11.66 ms ( moxifloxacin 400 mg ) and 20.96 ms ( 800 mg ) . the mean and 90 % confidence intervals of qtci did not include zero at any of the measurement time points . there was a positive correlation between plasma moxifloxacin concentration and qtci ( r = 0.422 ) . dose - proportional pk profiles were observed.conclusionmoxifloxacin 400 mg is an adequate positive control in korean tqt studies . our results indicate that moxifloxacin 400 mg can be used to evaluate the cardiac safety of a drug in korean subjects .

Introduction Methods Subjects Study Design Pharmacodynamic Analyses Moxifloxacin Plasma Concentration Determinations Pharmacokinetic Analyses Safety Assessments Results Subject Demographics Pharmacodynamic Analyses Pharmacokinetic Analyses Safety Assessments Discussion Conclusion

currently , the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use ( ich ) recommends sponsors submitting new drug applications to evaluate the drug s effects on cardiac repolarization by conducting a clinical thorough qt ( tqt ) study . although ich guideline e14 does not specify the use of moxifloxacin as a positive control , it has been the most widely and most commonly used positive control in tqt studies . the effects of moxifloxacin on qt interval have been well documented and compared with ibutilide , an intravenous formulation that is the only other positive control that has been used in published tqt studies , moxifloxacin is orally administered and is therefore a better choice for use in blinded studies . at present , no comparable published data collected from korean subjects exist that can be used to evaluate an investigational product s effects on qt interval during the drug development phase . furthermore , the effects of moxifloxacin 400 or 800 mg ( supratherapeutic dose ) on qt prolongation have not been fully assessed in healthy korean subjects , nor has the known diurnal variation been evaluated in this population . hence , an investigation is required to evaluate whether the usual positive control dose for tqt studies , moxifloxacin 400 mg , is adequate for korean subjects and to determine whether moxifloxacin can be used as a positive control in koreans , as outlined by ich guideline e14 . therefore , the aims of the present study were to evaluate qtc prolongation in healthy korean male subjects ( both at therapeutic and supratherapeutic doses of moxifloxacin ) , to assess the use of moxifloxacin as an adequate positive control , to compare qt interval correction methods , and to compare baseline measurement methods in korean subjects . 1study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) on day 1 , baseline 12-lead ecgs were measured after 10 min of supine position using either mac5000 or mac5500 ( ge healthcare , milwaukee , wi , usa ; set at 25 mm / s ) at the following time points : 0 , 1 , 2 , 3 , 4 , 6 , 8 , 12 , 16 , and 24 h. ecgs were recorded once for every time point . on day 2 , subjects received one of the three treatments in each period according to their sequence group : placebo ( water ) , moxifloxacin 400 mg ( avelox tablets , bayer korea ltd . blood sampling for the pharmacokinetic ( pk ) analyses was conducted at the same time points as the ecg recordings for subjects who took moxifloxacin . the baseline - corrected difference in qtc ( qtc ) and the placebo - adjusted difference in qtc ( qtc ) were calculated using either bazett s formula ( qtcb = qt / rr ) , fridericia s formula ( qtcf = qt / rr ) , or an individual qt / rr linear regression model ( qtci ) . in the present study , the time - matched baseline measurement was used in all qt interval calculations . 1study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) study design pharmacokinetic sampling ( black shaded line ) ; 12-lead electrocardiogram ( grey shaded line ) on day 1 , baseline 12-lead ecgs were measured after 10 min of supine position using either mac5000 or mac5500 ( ge healthcare , milwaukee , wi , usa ; set at 25 mm / s ) at the following time points : 0 , 1 , 2 , 3 , 4 , 6 , 8 , 12 , 16 , and 24 h. ecgs were recorded once for every time point . on day 2 , subjects received one of the three treatments in each period according to their sequence group : placebo ( water ) , moxifloxacin 400 mg ( avelox tablets , bayer korea ltd . blood sampling for the pharmacokinetic ( pk ) analyses was conducted at the same time points as the ecg recordings for subjects who took moxifloxacin . the baseline - corrected difference in qtc ( qtc ) and the placebo - adjusted difference in qtc ( qtc ) were calculated using either bazett s formula ( qtcb = qt / rr ) , fridericia s formula ( qtcf = qt / rr ) , or an individual qt / rr linear regression model ( qtci ) . five subjects withdrew consent prior to the completion of the study and 33 subjects completed the study . qtci at each measurement time is shown , with means and two - sided 90 % confidence intervals ( cis ) that did not include zero at any of the measurement times ( table 1 ) . the majority of the largest time - matched qtc occurred approximately 4 h after dosing ( table 2 ) . depending on the correction method used to calculate qtc , moxifloxacin 400 mg prolonged the qt interval from 12 ms ( qtci ) to 16 ms ( qtcb ) and moxifloxacin 800 mg prolonged qtc from 21 ms ( qtci ) to 29 ms ( qtcb).fig . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg)table 1baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms)time ( h)treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgmean90 % lower90 % uppermean90 % lower90 % upper110.236.6613.7915.1311.5718.7027.744.1811.3016.7313.1720.30310.997.4314.5520.4616.9024.02411.668.1015.2220.9617.4024.5369.906.3413.4617.6414.0821.2084.631.078.1916.9313.3720.49127.323.7610.8813.409.8316.96165.982.419.5410.887.3214.44248.825.2512.3815.4911.9319.05table 2largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci)]treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgtime ( h)mean [ 90 % ci]time ( h)mean [ 90 % ci]qtcb415.95 [ 10.81 , 21.09]3 28.83 [ 23.69 , 33.97]qtcf412.31 [ 8.38 , 16.24]4 23.14 [ 19.21 , 27.07]qtci411.66 [ 8.10 , 15.22]4 20.96 [ 17.40 , 24.53 ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model baseline- and placebo - corrected qt ( qtc)-time profiles using : a bazett s formula , b fridericia s formula , and c the individually corrected method . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg ) baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms ) largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci ) ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model an increase in plasma moxifloxacin concentration was weakly associated with qtc prolongation ( fig . 3plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . 4comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . the equations for each regression line were as follows : a qtcb = 0.901 + 0.00670 moxifloxacin concentration , b qtcf = 1.39 + 0.00535 moxifloxacin concentration , and c qtci = 2.36 + 0.00470 moxifloxacin concentration ( open circle 400 mg , solid circle 800 mg ) comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group differences among study centers , sequence groups , periods , and treatment - time interaction did not influence the variation in qtc prolongation ( data not shown ) . dose - dependent pk profiles were observed in the moxifloxacin concentration - time profiles ( fig . qtci at each measurement time is shown , with means and two - sided 90 % confidence intervals ( cis ) that did not include zero at any of the measurement times ( table 1 ) . the majority of the largest time - matched qtc occurred approximately 4 h after dosing ( table 2 ) . depending on the correction method used to calculate qtc , moxifloxacin 400 mg prolonged the qt interval from 12 ms ( qtci ) to 16 ms ( qtcb ) and moxifloxacin 800 mg prolonged qtc from 21 ms ( qtci ) to 29 ms ( qtcb).fig . 2baseline- and placebo - corrected qt ( qtc)-time profiles using : a bazett s formula , b fridericia s formula , and c the individually corrected method . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg)table 1baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms)time ( h)treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgmean90 % lower90 % uppermean90 % lower90 % upper110.236.6613.7915.1311.5718.7027.744.1811.3016.7313.1720.30310.997.4314.5520.4616.9024.02411.668.1015.2220.9617.4024.5369.906.3413.4617.6414.0821.2084.631.078.1916.9313.3720.49127.323.7610.8813.409.8316.96165.982.419.5410.887.3214.44248.825.2512.3815.4911.9319.05table 2largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci)]treatmentmoxifloxacin 400 mgmoxifloxacin 800 mgtime ( h)mean [ 90 % ci]time ( h)mean [ 90 % ci]qtcb415.95 [ 10.81 , 21.09]3 28.83 [ 23.69 , 33.97]qtcf412.31 [ 8.38 , 16.24]4 23.14 [ 19.21 , 27.07]qtci411.66 [ 8.10 , 15.22]4 20.96 [ 17.40 , 24.53 ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model baseline- and placebo - corrected qt ( qtc)-time profiles using : a bazett s formula , b fridericia s formula , and c the individually corrected method . the data are presented as the arithmetic means standard deviation ( solid circle 400 mg , open circle 800 mg ) baseline- and placebo - adjusted qtci ( qt interval corrected by individual qt - rr regression ) mean differences and 90 % confidence intervals by time point ( ms ) largest time - matched qtc ( baseline- and placebo - adjusted corrected qt ) by treatment . least - squares mean difference adjusted by placebo [ 90 % confidence intervals ( ci ) ] qtcb corrected qt using bazett s formula , qtcf corrected qt using fridericia s formula , qtci corrected qt using individual qt / rr regression model an increase in plasma moxifloxacin concentration was weakly associated with qtc prolongation ( fig . 3plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . 4comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group plasma concentrations of moxifloxacin vs. corrected qt ( qtc ) scatter plot and regression lines using : a bazett s formula , b fridericia s formula , and c the individual correction method . the equations for each regression line were as follows : a qtcb = 0.901 + 0.00670 moxifloxacin concentration , b qtcf = 1.39 + 0.00535 moxifloxacin concentration , and c qtci = 2.36 + 0.00470 moxifloxacin concentration ( open circle 400 mg , solid circle 800 mg ) comparison of pre - dose baseline - corrected ( solid circle ) and time - matched ( open circle ) qtci ( mean differences with 90 % confidence intervals ) in a the moxifloxacin 400-mg group and b the moxifloxacin 800-mg group differences among study centers , sequence groups , periods , and treatment - time interaction did not influence the variation in qtc prolongation ( data not shown ) . our study found a definite prolongation of the qtc interval after moxifloxacin dosing [ 11.66 ms in the moxifloxacin 400-mg group and 20.96 ms in the moxifloxacin 800-mg group ( qtci values ) ] . the mean differences and 90 % cis of qtci did not include zero at any of the measurement time points . a positive relationship between qt interval prolongation and moxifloxacin concentration ( r = 0.422 in qtci ) was also observed . the tmax of moxifloxacin 400 and 800 mg occurred 1 and 3 h after dosing , respectively , whereas the largest time - matched qtc was measured approximately 4 h after dosing . only used qtcb and qtcf values in their study , they found no relationship between the dose of moxifloxacin and qt interval lengthening , but found a positive relationship between qt interval prolongation and moxifloxacin concentration [ r = 0.35 , moxifloxacin concentration ( g / l ) vs. qt interval ( ms ) ; r = 0.72 , moxifloxacin concentration ( g / l ) vs. qt interval ( % ) ] . because there were no noticeable differences in pk parameters between the two studies , there is a possibility that korean subjects may show different susceptibility to supratherapeutic doses of moxifloxacin than caucasian subjects . nonetheless , our findings suggest that moxifloxacin induces a detectable effect of greater than 5 ms on qtc prolongation , which confirms the adequacy of the use of moxifloxacin as a positive control in korean tqt studies , explained by answer 1 in the ich e14 questions - and - answers document . although this study contributed valuable korean qt prolongation study data , a difference exists : this study did not use moxifloxacin , a drug that is commonly used as a positive control in tqt studies . this tendency was more evident in the moxifloxacin 800-mg group , where the largest time - matched qtcb was calculated to be 28.83 ms ( 90 % ci 23.6933.97 ) . the current study aimed to provide comparable pilot qt interval prolongation data in korean subjects that could be used in scientific and regulatory fields and was not necessarily focused on detecting inter - ethnic differences . in summary , moxifloxacin 400 mg causes moderate qt interval prolongation and is an adequate positive control in korean tqt studies . our results indicate that caution should be exercised when a supratherapeutic dose of moxifloxacin is used in korean subjects . furthermore , the findings of the present study may be employed in drug development studies targeting the korean population and may also be applied to further research attempting to evaluate the cardiac safety of a drug in korean subjects .

prostacyclin ( pgi2 ) is a member of the prostaglandin family of bioactive lipids , and is a derivative of the 20-carbon , omega-6 fatty acid , arachidonic acid ( aa or 5,8,11,14-eicosatetraenoic acid ) . both cyclooxygenase enzymes ( cox-1 and cox-2 ) convert aa into the prostaglandin precursor pgh2 , which is subsequently synthesized into prostacyclin ( pgi2 ) via prostacyclin synthase ( pgis ; figure 1 ) . however , the majority of pgi2 produced in vivo , particularly within the systemic and pulmonary vasculature ( moncada et al . , 1977 ; catella - lawson et al . , 1999 ; mcadam et al . , 1999 ) , and other regions like the synovium ( brodie et al . , 1980 ; crofford et al . , 1994 ) , appears to be derived from cox-2 . pgi2 is unstable at physiological ph and , thus , has a very short half - life in vivo ( < 2 min ) , rapidly forming the inactive hydration product 6-keto - prostaglandin f1 ( 6-keto - pgf1 ; lewis and dollery , 1983 ; smyth and fitzgerald , 2002 ) . the actions of pgi2 are mediated through a seven - transmembrane - spanning g - protein coupled receptor ( gpcr ) , referred to as the ip receptor ( international union of pharmacology nomenclature ) . the ip receptor is a class a rhodopsin - like gpcr that couples predominately to the gs subunit of the heterotrimeric g - protein and mediates intracellular signaling via adenylyl cyclase ( ac ) activation and cyclic amp ( camp ) production ( boie et al . , animal studies have also shown that pgi2 may also signal through alternate gq- and gi - related pathways ( lawler et al . , 2001 ) , as well as nuclear receptor - mediated pathways , such as the peroxisome proliferator activated receptor gamma ( ppar ) pathway ( lim and dey , 2002 ) . stitham et al . ( 2003 ) have elucidated the putative binding pocket for the human ip receptor , which has been reported to also accommodate type e prostanoids ( i.e. , pge1 and pge2 ) in addition to its native ligand pgi2 and its analogs ( boie et al . , 1994 ; nakagawa et al . , 1994 ) . the physiological effects of pgi2 are vast with much remaining to be uncovered . within the vasculature , pgi2 serves as a potent vasodilator and is the major inhibitory prostanoid in platelet aggregation ( smyth et al . , 2009 ) , and has also been shown to inhibit vascular smooth muscle cell ( vsmc ) proliferation and de - differentiation ( fetalvero et al . , 2006 , 2007 ) . within the lungs , pgi2 reduces pulmonary blood pressure as well as bronchial hyper - responsiveness ( idzko et al . , 2007 ) . within the kidneys , pgi2 serves to regulate renal blood flow and glomerular filtration rate , as well as mediates the release of renin ( komhoff et al . , 1998 ) . in the nervous system , pgi2 has been shown to elicit nociceptive pain response ( murata et al . , 1997 ) . prostanoid biosynthesis pathway . the enzyme phospholipase a2 ( pla2 ) hydrolyzes arachidonic acid ( aa ) from the phospholipids of the extracellular membrane . arachidonic acid is modified by the cyclooxygenase ( cox ) enzymes ( cox-1 and cox-2 ) to form the intermediate precursor prostaglandin g2 ( pgg2 ) via the addition of two oxygen ( o2 ) molecules . prostaglandin h2 ( pgh2 ) is subsequently formed by the actions of peroxidase enzyme , which releases a single oxygen ( o2 ) molecule . as shown , all prostanoids are derived from the parent compound pgh2 and are formed via their respective synthase enzymes , namely prostaglandin i2 synthase ( pgis ) , prostaglandin e2 synthase ( pges-1 ) , prostaglandin d2 synthase ( pgds ) , prostaglandin f2 synthase ( pges-2 ) , and thromboxane a2 synthase ( tbxas-1 ) . as described , prostacyclin ( pgi2 ) is best known for its regulatory role within the cardiovascular system , where it promotes vsmc relaxation ( vasodilatation ) and inhibits platelet aggregation ( anti - thrombotic ) . the seminal work by vane ( 1971 ) demonstrating the inhibition of prostaglandin biosynthesis as the mechanism of action for aspirin ( acetylsalicylic acid ) and other aspirin - like drugs first highlighted the importance of the prostaglandin family of molecules , and set the stage for the development of many pharmacologic agents , such as traditional , non - selective non - steroidal anti - inflammatory drugs ( tnsaids ) and the newer selective cox-2 inhibitors . further work by davies et al . ( 1984 ) pinpointed particular prostaglandins , principally prostaglandin e2 ( pge2 ) and prostacyclin ( pgi2 ) , in the mediation of vascular permeability associated with the hyperemia and edema seen with acute inflammation . murata et al . ( 1997 ) demonstrated the involvement of prostacyclin ( pgi2)-mediated inflammatory swelling in vivo , using prostacyclin receptor deficient ( ip/ ) mice . in these critical experiments , it was shown that mice lacking the prostacyclin receptor had a reduced inflammatory response , as measured by percent change in vascular permeability using a carrageenan - induced paw - edema model ( murata et al . , 1997 ) . limb edema was decreased by approximately 50% in ip - deficient mice , similar to levels seen in mice pre - treated with the non - steroidal anti - inflammatory agent indomethacin . moreover , a significant reduction in lung exudate volume , using a carrageenan - induced pleurisy model , was also observed ( although data not shown ) for ip - deficient mice as well ( murata et al . , 1997 ) . in contrast , a study by takahashi et al . ( 2002 ) demonstrated the ip - deficient mice showed higher skin and airway immune responses ( relating to increased capillary permeability in these tissues ) in antigen - sensitized inflammation , suggesting a protective role for pgi2 in allergic inflammation . these studies ( among others ) have highlighted prostacyclin as a major endogenous mediator of inflammation both pro - inflammatory and anti - inflammatory , depending upon the tissue and pathological model being studied ( figure 2 ) . prostacyclin ( pgi2 ) serves as a protective , anti - inflammatory mediator in the processes of atherosclerosis and pulmonary vascular diseases , such as pulmonary arterial hypertension ( pah ) and idiopathic pulmonary fibrosis ( ipf ) . conversely , in rheumatological conditions , such as rheumatoid arthritis ( ra ) and osteoarthritis ( oa ) , pgi2 acts as a propagatory , pro - inflammatory molecule . while the majority of focus has centered around the role of prostaglandin e2 ( pge2 ) in rheumatoid arthritis ( ra ) , some studies have shown that the predominate prostaglandin detected within the synovial fluid of patients with ra is in fact prostacyclin ( pgi2 ; brodie et al . , 1980 ) . moreover , using both collagen - induced arthritis and collagen - antibody - induced arthritis models , honda et al . ( 2006 ) showed that prostacyclin receptor knockout ( ip/ ) mice exhibited significantly reduced clinical and histological arthritic scores versus control mice , in both arthritis models , placing further emphasis on receptor - mediated prostacyclin activity in the pathogenesis of ra . using a k / bxn serum - transfer arthritis model , chen et al . ( 2008 ) administered serum from arthritic k / bxn mice to induce an igg - mediated autoantibody - induced inflammatory arthritis to recipient mice lacking either prostaglandin e synthase-1 ( mpges-1/ ) or the prostacyclin receptor ( ip/ ) , in order to determine the relative importance of pge2 and pgi2 , respectively . findings revealed that mice deficient in prostaglandin e synthase-1 mpges-1 ( and therefore unable to produce pge2 ) , developed arthritis in normal fashion , whereas mice lacking the receptor for pgi2 demonstrated a significant decrease ( 31% versus wild - type ) in clinical arthritis . furthermore , using cox-1 and cox-2 knockout animals , it was shown that mice the lacking cox-1 isoform were resistant to the development of arthritic disease , while those lacking the cox-2 isoform remained vulnerable ( wang et al . , 2008 ) . these results convey two important points : ( 1 ) a substantial proportion of the prostanoid contribution to joint inflammation ( at least in the k / bxn serum - transfer arthritis model ) can be accounted for by pgi2 and its interaction with the ip receptor , and ( 2 ) selective cox-1 inhibition through genetic knockout prevented the development of disease , suggesting that cox-1-derived pgi2 is the major inflammatory mediator within this arthritis model ( wang et al . , 2008 ) . more importantly , such studies lend weight to the involvement of prostacyclin in chronic inflammatory disease processes , as well as being an acute mediator , and also call into question the paradigm regarding cox-1- and cox-2-derived prostaglandin functions in vivo ( i.e. , regulatory housekeeping versus inflammatory induction ) . interestingly , from the perspective of clinical therapies , there does not seem to be a difference in efficacy according to cox selectivity . ( 2008 ) showed , meta - analysis from a systematic review of 145 randomized controlled trials , examining the clinical effectiveness of a variety of cox-2 inhibitors ( including etodolac , meloxicam , celecoxib , rofecoxib , etoricoxib , valdecoxib , and lumiracoxib ) , showed similar efficacy compared to non - selective nsaids ( including naproxen , diclofenac , ibuprofen , loxoprofen , nabumetone , piroxicam , indomethacin , tenoxicam , and nimesulide ) in the symptomatic relief of both ra and osteoarthritis ( oa ) , but with superior gastrointestinal tolerability and protection against complicated upper gastrointestinal events ( e.g. , ulcers , bleeding , perforations ) the majority of evidence coming from oa populations . however , the amount of evidence for this gastro - protective effect varied considerably across individual drugs . moreover , an increased risk of myocardial infarction ( mi ) was also observed among those drugs with greater volume of evidence in terms of exposure in patient - years ( chen et al . , 2008 ) , presumably as a by - product of the discriminating suppression of cox-2-derived pgi2 , which has been shown to be cardioprotective ( murata et al . , 1997 ; cheng et al . , 2002 ; egan et al . , 2004 ) . along these same lines , as chronic inflammation has been linked to enhanced development of atherogenesis ( libby et al . , 2002 ) , individuals with ra may already be at increased risk for cardiovascular disease . in fact , a recent comparative study involving disease - duration - matched ra and diabetes mellitus ( dm ) patients found that ra was a substantial and independent cardiovascular risk factor , with similar severity to dm , with significantly worsened preclinical atherosclerotic markers , increased intima - to - media thickness , as well as lower flow - mediated dilatation ( measure of endothelial function ; stamatelopoulos et al . , 2009 ) . thus , the combination of chronic ra - mediated inflammation , perpetuating an increased atherosclerotic burden , along with cox inhibition therapy either selective ( cox-2 inhibitor ) or non - selective ( nsaids ) would undoubtedly increase cardiovascular risk drastically , given what we now know about pgi2 . thus , pgi2 is emerging as an important intermediary in inflammatory conditions such as rheumatoid and oa , with the dualistic purpose of pro - inflammatory mediator on the one hand involved in disease pathophysiology and cardioprotective factor on the other both of which are central in the consideration of pharmacologic therapies and adverse effects . while pulmonary fibrosis and pulmonary hypertension are distinct pathophysiological entities , they do share some commonalities , some of which involve pgi2 . pulmonary arterial hypertension ( pah ) is in fact a heterogeneous group of diseases sharing similarities in pathophysiological mechanisms , clinical presentation , and therapeutic approaches ( simonneau et al . , 2009 ) . the pathogenesis of pah is complex and incompletely understood , with both genetic and environmental factors contributing to altered vascular structure and function ( badesch et al . , 2007 ) . the main vascular changes in pah are vasoconstriction , vsmc proliferation , endothelial loss or dysfunction , and thrombosis ( farber and loscalzo , 2004 ) , which implicates a disruption of vascular hemostasis and its principle mediators , particularly pgi2 and txa2 , among others . this is evidenced by findings in patients with pah , whereby both the production of prostacyclin synthase ( pgis ) within small - to - medium pulmonary arteries , as well as urinary metabolites ( 6-keto - prostacyclin f2 ) of prostacyclin , were shown to be decreased , while levels of thromboxane metabolites ( thromboxane b2 ) were increased ( christman et al . interestingly , this imbalance of pgi2 and txa2 within the pulmonary vasculature , leading to increased mean pulmonary artery pressure , mimics that of the cardio - systemic vasculature system . in fact , many of the pathophysiological mechanisms identified in pah overlap with those involved in atherogenesis , including vascular smooth muscle and endothelial cell dysfunction , enhanced platelet activity and thrombosis , inflammation , and cellular chemotaxis ( essop , 2010 ) . owing to its potent vasodilatory , anti - thrombotic , and anti - proliferative effects , pgi2 has secured a central role in the treatment of pah . continuous intravenous epoprostenol ( synthetic pgi2 ) is the best - studied advanced therapy for pah and remains a first - line agent , particularly for those with severe disease ( who functional class iv ) , as it has been shown to improve overall symptoms , exercise capacity , and hemodynamic status in controlled clinical trials ( barst et al . , 2009 ) , as well as confer a survival benefit in both idiopathic and heritable forms of pah ( ipah and hpah ; barst et al . , 2009 ) . there are limitations to treatment with epoprostenol based upon its pharmacology ( plasma half - life = 35 min ) and long - term use requires a permanent central venous catheter and a portable infusion pump . analogs of pgi2 have also been used clinically , and are administered by a variety of routes , including intravenously ( e.g. , treprostinil and iloprost ) , subcutaneously ( e.g. , treprostinil ) , inhalation ( e.g. , iloprost ) , and orally ( e.g. , beraprost ) . these medications are generally more stable with longer half - lives , but have variable safety and efficacy equivalencies compared to epoprostenol , and clinical trials with these ( and other ) alternative agents are limited with respect to severe disease classification ( barst et al . , 1996 ) . other vasoactive therapies for pah include the dual endothelin receptor ( eta and etb ) antagonist ( e.g. , bosentan ) and phosphodiesterase type-5 ( pde-5 ) inhibitors , which have been proven effective , alone or in combination therapy , in milder forms of pah ( who functional class ii and iii ; rubin et al . , 2002 ; sitbon et al . , 2003 ; galie et al . , 2005 ; again , the central role of pgi2 , as both an inflammatory and hemodynamic mediator , puts it at the forefront in understanding the pathophysiology and pharmacological treatment of pulmonary vascular diseases , particularly pah . interestingly , pgi2 seems to have a similar safeguarding effect in the chronic inflammatory condition of idiopathic pulmonary fibrosis ( ipf ) , as recent studies have shown that cox-2-derived pgi2 serves a protective role against bleomycin - induced pulmonary fibrosis a major animal model for ipf that mimics the progressive fibrosis and interstitial inflammation of sub - pleural lung tissue in humans . ( 2006 ) , ip - deficient mice were more susceptible to bleomycin - induced pulmonary fibrosis , demonstrating increased collagen deposition and cellularity after bleomycin administration compared with the wild - type mice . these observations correlated with increases in quantitative measurements of histological lung scores and hydroxyproline levels within the lung parenchyma . similar results were found using cox-2-deficient mice , but were not supported using knockouts for either the ep2 or ep4 receptors , which bind cox-2-derived prostaglandin e2 ( pge2 ; lovgren et al . , 2006 ) . such findings put direct focus on the loss of cox-2-derived prostacyclin as a protective factor . while cox-2-derived prostacyclin is now well known for its protective effects within the cardiovascular system , such results provide compelling evidence for pgi2-mediated protection against fibrotic pathologies as well . a more recent animal study by zhu et al . ( 2010 ) also confirms these findings , pharmacologically , and identifies pgi2 as a potential new therapeutic agent for pulmonary fibrotic disease . using intra - peritoneal injections of iloprost , a stable pgi2 analog , it was demonstrated that a single dose of iloprost ( 200 g / kg ; prior to bleomycin injection ) could preclude pulmonary inflammation and fibrosis in mice ( zhu et al . , 2010 ) . pre - treatment with iloprost seemed to significantly reduce both inflammatory infiltration and collagen deposition with the pulmonary interstitium , as well as improve lung mechanics ( reduced static compliance and elevated tissue elastance ; zhu et al . however , the specific inflammatory cell subtype being suppressed could not be delineated in this current study . iloprost pre - treatment decreased production of pro - inflammatory and fibrotic cytokines , such as tnf - alpha , il-6 , and tgf - beta-1 , and increased release of anti - fibrotic mediators , including ifn - gamma and chemokine cxcl10/ip-10 , as measured by mrna expression levels or elisa . moreover , the cumulative mortality in iloprost - treated mice was 10% at day 21 versus 60% in the non - iloprost - treated cohort ( zhu et al . , 2010 ) . in human studies , inhaled iloprost has been proven efficacious in the treatment of various forms of pulmonary hypertension , including pulmonary hypertension secondary to pulmonary fibrosis ( olschewski et al . , 1999 ) . in fact , it has been suggested that the majority of vascular resistance in fibrotic lung disease is due to persistent vasoconstriction ( olschewski et al . , 1999 ) , which may explain the effectiveness of pgi2 analog therapy . certain studies also suggest a long - term clinical benefit from continued therapy with inhaled iloprost , which was well tolerated and required no substantial dose increase over a 2-year trial ( olschewski et al . , 2010 ) . in the united states , its use has been approved for pah new york heart association ( nyha ) functional class iii and iv ; for individuals with marked limitations or inability to carry on physical activity ( gomberg - maitland and olschewski , 2008 ) . thus , pgi2 seems to be an important effector in both these fibro - proliferative disorders of the lung , playing both a protective role against disease development , as well as a therapeutic role in symptom management . atherosclerosis is now known as an inflammatory disease , with the same complex cellular interactions involving monocytes , macrophages , lymphocytes , extracellular matrix ( ecm ) components , and connective tissue cells as seen in other chronic inflammatory and fibro - proliferative diseases ( e.g. , ra , pulmonary fibrosis , glomerulosclerosis ; ross , 1999 ) . the critical role of pgi2 in atherosclerosis is quickly emerging , with evidence spanning from molecular and cell biology to human clinical trials . mounting data has demonstrated the protective effect of prostacyclin activity against the development of atherothrombotic cardiovascular disease through the inhibition of various cellular processes , including platelet activation , leukocyte adhesion , as well as vsmc modulation . as such , pgi2 analogs ( e.g. , iloprost ) are able to down - regulate lymphocyte adhesion to endothelial cells , which suggests an ability to block the early events in atherosclerosis ( della bella et al . , 2001 ) . furthermore , interactions within the realm of lipid metabolism has lent further support toward the atheroprotective properties of pgi2 ( thiemermann , 1991 ) . specifically , hdl has been shown to induce cox-2 expression and pgi2 production in both endothelial and vsmcs ( pomerantz et al . , 1985 ; vinals et al . , 1997 , 1999 ) while , conversely , pgi2 has been shown to induce cholesterol ester hydrolase activity , which catalyzes the first step in the removal of cholesterol from foam cells , critical components in atherogenesis ( hajjar and weksler , 1983 ; weksler et al . , 1983 ; hajjar et al . , the effects of pgi2 on vsmcs are becoming an important target for understanding both the pathophysiology of atherothrombosis and the atheroprotective effects of prostacyclin . in mature blood vessels , vsmcs are quiescent and exhibit a differentiated , contractile phenotype . however , in response to vascular injury , these cells have been shown to re - enter the cell cycle , proliferate , migrate toward attractants , down - regulate expression of contractile proteins , and up - regulate synthesis of proteins , particularly ecm ( campbell et al . , 1988 ) . , 1995 ; zucker et al . , 1998 ) and anti - migratory ( blindt et al . , 2002 ) effects on smooth muscle cells . in advanced atherosclerotic lesions ( as well as restenotic lesions ) , expression levels of smooth - muscle - specific differentiation markers are markedly reduced ( wilcox , 1992 ; o'brien et al . , 1993 ) , and ( 2006 ) have shown , treatment with the stable pgi2 analog , iloprost , induces vsmc differentiation via a camp - pka - mediated signaling pathway . in similar fashion , kasza et al . ( 2009 ) went on to further show that , in addition to increased contractile protein expression and contractile morphology , iloprost - treated vsmcs up - regulate cox-2 expression , mediated not only by camp - pka , but also novel pathways involving erk-1/2 activation and akt-1 inhibition . in turn , the up - regulated cox-2 expression lead to subsequent pgi2 release ( i.e. , prostacyclin - induced prostacyclin release ) , which was shown to have a paracrine , positive - feedback effect on neighboring vsmcs ( not exposed to iloprost ) , inducing similar cellular responses ( kasza et al . , 2009 ) . thus , there appears to be a clear link between the major atheroprotective effects of prostacyclin and vsmc modulation . as such , the phenotypic change in vsmcs toward a proliferative and de - differentiated state , which is a hallmark occurrence in the progression of atherosclerosis and restenosis , necessitates a clear understanding of this regulatory process and is an extremely important area of research . mouse models have provided valuable insight into the role of prostacyclin in cardiovascular homeostasis and pathogenesis . ip - deficient ( ip/ ) mice display increased propensities toward thrombosis ( murata et al . , 1997 ) , intimal hyperplasia and restenosis ( cheng et al . , 2002 ) , and reperfusion injury ( xiao et al . , 2001 ) . moreover , in both atherogenic apolipoprotein e ( apo - e)- and low - density lipoprotein receptor ( ldl - r)-deficient backgrounds , mice lacking the ip receptor demonstrated greater atherosclerotic burden with higher platelet reactivity and leukocyte adhesion to endothelial cells ( egan et al . moreover , egan et al . ( 2004 ) went on to further demonstrate that , in female pre - menopausal ldl - r knockout mice , the atheroprotective effect of estrogen is significantly reduced in the absence of the ip receptor ( i.e. , double ldl - r and ip knockout ) . moreover , as long - term estrogen exposure was shown to increase cox-2 expression , as well as the formation of the pgi2 metabolite 6-keto - pgf1 in mouse aortic smooth muscle cells ( masmcs ) , this study suggests a significant contribution of estrogen - mediated , cox-2-derived pgi2 in the protection against atherogenesis ( egan et al . , 2004 ) . such in vivo findings highlight the important functional presence of prostacyclin activity in the maintenance of cardiovascular homeostasis and , in turn , implicate receptor - ligand ( hip - pgi2 ) dysfunction in the acceleration of atherogenesis and the subsequent development of related disorders , including stroke , mi , and hypertension . in humans , the importance of pgi2 in atherogenesis has also been shown . ( 2008 ) demonstrated that patients harboring a dysfunctional human prostacyclin receptor variant ( r212c ) exhibited an enhanced atherothrombotic phenotype , with a higher incidence of triple - vessel coronary artery disease ( cad ) and greater number of clinical cardiovascular events ( including mi , stroke , ptca , cabg , pvd , and unstable angina ) in high - risk patients versus age- and risk - factor - matched normal - allele patients . ( 2008 ) correlated the r212c prostacyclin receptor polymorphism , as well as two other synonymous variants , with intimal hyperplasia and progressive deep venous thrombosis , respectively . 2010 ) demonstrated that co - expression of the r212c variant , together with wild - type hip , resulted in dominant - negative inhibition of signaling through receptor homo - dimerization , with enhanced wild - type ip localization to the endoplasmic reticulum . interestingly , a similar effect was observed with co - expression of the r212c variant and the wild - type thromboxane receptor ( tp ) , which revealed r212c hetero - dimerization , with subsequent inhibition of tp receptor activity ( ibrahim et al . , 2010 ) . since this time , stitham , arehart , and colleagues have identified other functionally deficient prostacyclin receptor polymorphisms ( i.e. , r215c and l104r ) associated with increased cad ( ibrahim et al . , 2010 ) . while such mutations are rare , it is notable that single , heterozygous , point mutations within the prostacyclin receptor gene ( ptgir ) are associated with clinically significant disease changes . similarly , mutations and polymorphisms in the prostacyclin synthase ( pgis ) gene have been associated with essential hypertension , mi , and cerebral infarction ( iwai et al . , 1999 ; nakayama , 2005 ) . the sum of these results correspond not only with the aforementioned ip knockout mice studies , but also parallel findings from the world - wide withdrawal of the selective cox-2 inhibitors ( e.g. , rofecoxib and valdecoxib ) whose discriminating suppression of cox-2-derived prostacyclin ( pgi2 ) resulted in increased risk of cardiovascular events ( e.g. , mi and thrombotic stroke ) , particularly in predisposed patients ( fitzgerald , 2004 ; white et al . , 2004 ; bresalier et al . , 2005 ) these latter findings relating adverse cardiovascular events to unmatched suppression of cox-2-derived pgi2 have been widely discussed and demonstrate that a disrupted balance between pgi2 and txa2 favoring unopposed cox-1-derived txa2 production is responsible , at least in fair part , for pro - thrombotic and perhaps pro - atherogenic effects ( mcadam et al . , 1999 ; vane , 2002 ) . in a recent opinion article , rovati et al . ( 2010 ) proposed that concomitant tp receptor antagonism , along with selective cox-2 inhibition ( dual coxib - tp antagonists ) , may abrogate such adverse cardiovascular events ( caused by the imbalance between pgi2 and txa2 ) and improve the safety profile of selective cox-2 inhibitors . other groups have proposed this concept as well , but clinical trials have yet to be pursued . as another approach , capone et al . ( 2010 ) suggest assessment of cox-2 activity in whole blood ex vivo , perhaps in combination with biomarkers such as biochemical ( urinary levels of 6-keto - pgf1 ) and genetic ( ip receptor and other prostacyclin - related polymorphisms ) as potential surrogate endpoints to assess for prostacyclin synthesis in vivo as a predictor of cardiovascular risk . as the role of pgi2 is becoming more defined in inflammatory - related diseases , the development of novel agonists and antagonists for the ip receptor is at the forefront of research . as previously stated , the role of pgi2 in arthritic diseases ( ra and oa ) is one of a pro - inflammatory mediator , and the use of wide - ranging inhibitors of prostaglandin synthesis ( nsaids and selective cox-2 inhibitors ) has remained a mainstay of therapy . however , targeted antagonism of pgi2 activity has proven effective in reducing pain and inflammation in preclinical trials . using a mono - iodoacetate ( mia)-induced rodent model of chronic oa , pulichino et al . ( 2006 ) have shown that a novel arylamide compound ( keitz et al . , 2004 ) with specific ip receptor antagonism significantly reduced joint discomfort in a dose - dependent manner , and with similar efficacy to diclofenac as well as an mf - tricyclic cox-2 inhibitor . furthermore , in a collagen - antibody - induced model of ra , the same ip antagonist reduced mean scores for all arthritic parameters by 93% ( auc of the clinical scores ) when given in prophylactic mode 1 day prior to collagen antibody injection , in ip+/ mice ( pulichino et al . , 2006 ) . comparably , a 91% reduction in arthritic scores was observed for ip/ mice and a 98% reduction for cox-2-treated mice . interestingly , treatment in therapeutic mode ( 6 days post - injection ) had no effect on clinical scores ( pulichino et al . , 2006 ) . however , as discussed earlier , targeted pgi2 antagonism has the potential for increased risk of adverse cardiovascular effects , which may be even more pronounced than with broader - spectrum nsaids or selective cox-2 inhibitor therapies . for the treatment of pulmonary vascular disease such as pah and ipf , the vasoactive pgi2 analog formulations ( e.g. , epoprostenol , treprostinil , and iloprost ) are all in clinical use , but are reserved for advanced therapy for persistent disease ( barst et al . , 2009 ) . moreover , these agents have limitations , including short half - lives , parenteral ( non - oral ) routes of administration , and heterogeneous therapeutic response . thus , the quest for novel therapies for these diseases is ongoing . in preclinical studies of both pah and ipf , a novel , non - prostanoid prostacyclin receptor agonist ( ono-1301 , toray industries ) that also has potent inhibitory activity against thromboxane synthase ( tbxas1 ) murakami et al . ( 2006 ) have demonstrated attenuation of bleomycin - induced pulmonary fibrosis , as well as improved survival in mice , using ono-1301 . ( 2005 ) demonstrated similar protective results in a monocrotaline - induced pah model , whereby rats treated with ono-1301 showed improved pulmonary hemodynamics and survival , along with a reduction of vascular remodeling ( media hypertrophy ) and plasma txa2 metabolites . moreover , the pharmacokinetic profile ( half - life approximately 5.6 h ) of ono-1301 appears to be better than some of the pgi2 analogs in current therapeutic use ( antoniu , 2006 ) and , thus , if the same clinical efficacy can be proved in humans , ono-1301 could be a new therapy for pah and ipf . in lieu of this , several new orally available , non - prostanoid , selective ip receptor agonists have reached clinical trial status for pah , including apd811 ( arena pharmaceuticals ) , which is in phase i and ns-304 , a.k.a . , selexipag ( actelion pharmaceuticals ; kuwano et al . , 2007 , 2008 ) , which is currently in phase iii . with the imbalance between pgi2 and txa2 playing an important role in atherogenesis and atherothrombosis , the development of dual - acting compounds seems like a promising direction for the development of novel therapeutic agents . in a similar fashion to the aforementioned dual coxib - tp antagonists , both miyamoto and yamada have demonstrated the potential for tra-418 ( toray industries ) , a novel compound with both pgi2 ( ip receptor ) agonistic and txa2 ( tp receptor ) antagonistic activities ( miyamoto et al . in vitro studies showed that tra-418 inhibited platelet aggregation through impedance of both glycoprotein iib / iiia ( gpiib / iiia ) activation and p - selectin expression , which are key markers of platelet activation ( miyamoto et al . , 2003 ) . these results suggest that this compound may be useful as both an anti - thrombotic and anti - atherogenic agent . similarly , ohno et al . ( 2005 ) have developed a dual - acting benzofuran compound ( toray industries ) that possesses similar pgi2 agonism and txa2 antagonism , which maintains potent anti - platelet activity with minimal effect on blood vessel dilation . thus , while the current pool of pgi2-centered compounds is primarily investigational , and clinical safety and efficacy in human disease still largely unproven , their development emphasizes the centrality of pgi2 activity in various inflammatory - mediated pathologies , and provides exciting new directions to steer drug development . the culmination of data presented in this review reinforces the notion that bunting et al . ( 1983 ) put forth almost 30 years ago that a dynamic balance between the prostaglandins prostacyclin ( pgi2 ) and thromboxane a2 ( txa2 ; in addition to many other mediators ) is crucial in maintaining cardiovascular homeostasis , and has critical pathophysiological and therapeutic implications . however , we are just now realizing the potential breadth and scope of this seminal proposition with pgi2 playing a central role . our current knowledge of pgi2 , as both a physiological pathophysiological mediator and therapeutic agent , in a host of inflammatory - related diseases , is growing rapidly . as demonstrated , pgi2 has been shown to play protective roles in atherogenesis relating to cad , mi , stroke , and other cardiovascular abnormalities . it has also been shown to be involved in certain fibro - proliferative and pulmonary vascular diseases , such as ipf and pah , where it serves as both a protective factor and first - line pharmacotherapy . furthermore , in the setting of ra , pgi2 seems to play a pro - inflammatory role and , as evidence increases , perhaps may one day be considered a therapeutic target in ra and related disorders ( figure 2 ) . further study of this important prostaglandin , in both the realms of basic science and clinical medicine , is needed and will undoubtedly yield new insights into inflammatory disorders and pharmacological treatments . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

prostacyclin ( pgi2 ) is a member of the prostaglandin family of bioactive lipids . its best - characterized role is in the cardiovascular system , where it is released by vascular endothelial cells , serving as a potent vasodilator and inhibitor of platelet aggregation . in recent years , prostacyclin ( pgi2 ) has also been shown to promote differentiation and inhibit proliferation in vascular smooth muscle cells . in addition to these well - described homeostatic roles within the cardiovascular system , prostacyclin ( pgi2 ) also plays an important role as an inflammatory mediator . in this review , we focus on the contribution of prostacyclin ( pgi2 ) as both a pathophysiological mediator and therapeutic agent in three major inflammatory - mediated disease processes , namely rheumatoid arthritis , where it promotes disease progression ( pro - inflammatory ) , along with pulmonary vascular disease and atherosclerosis , where it inhibits disease progression ( anti - inflammatory ) . the emerging role of prostacyclin ( pgi2 ) in this context provides new opportunities for understanding the complex molecular basis for inflammatory - related diseases , and insights into the development of current and future anti - inflammatory treatments .

Pharmacology of Prostacyclin and Its Receptor Prostacyclin as an Inflammatory Mediator Role of Prostacyclin in Arthritis Role of Prostacyclin in Pulmonary Fibrosis and Pulmonary Hypertension Role of Prostacyclin in Atherosclerosis Novel Prostacyclin-Related Therapies in Inflammation Conclusion Conflict of Interest Statement

prostacyclin ( pgi2 ) is a member of the prostaglandin family of bioactive lipids , and is a derivative of the 20-carbon , omega-6 fatty acid , arachidonic acid ( aa or 5,8,11,14-eicosatetraenoic acid ) . both cyclooxygenase enzymes ( cox-1 and cox-2 ) convert aa into the prostaglandin precursor pgh2 , which is subsequently synthesized into prostacyclin ( pgi2 ) via prostacyclin synthase ( pgis ; figure 1 ) . , 1999 ) , and other regions like the synovium ( brodie et al . , 2001 ) , as well as nuclear receptor - mediated pathways , such as the peroxisome proliferator activated receptor gamma ( ppar ) pathway ( lim and dey , 2002 ) . , pge1 and pge2 ) in addition to its native ligand pgi2 and its analogs ( boie et al . within the vasculature , pgi2 serves as a potent vasodilator and is the major inhibitory prostanoid in platelet aggregation ( smyth et al . , 2009 ) , and has also been shown to inhibit vascular smooth muscle cell ( vsmc ) proliferation and de - differentiation ( fetalvero et al . in the nervous system , pgi2 has been shown to elicit nociceptive pain response ( murata et al . as shown , all prostanoids are derived from the parent compound pgh2 and are formed via their respective synthase enzymes , namely prostaglandin i2 synthase ( pgis ) , prostaglandin e2 synthase ( pges-1 ) , prostaglandin d2 synthase ( pgds ) , prostaglandin f2 synthase ( pges-2 ) , and thromboxane a2 synthase ( tbxas-1 ) . as described , prostacyclin ( pgi2 ) is best known for its regulatory role within the cardiovascular system , where it promotes vsmc relaxation ( vasodilatation ) and inhibits platelet aggregation ( anti - thrombotic ) . the seminal work by vane ( 1971 ) demonstrating the inhibition of prostaglandin biosynthesis as the mechanism of action for aspirin ( acetylsalicylic acid ) and other aspirin - like drugs first highlighted the importance of the prostaglandin family of molecules , and set the stage for the development of many pharmacologic agents , such as traditional , non - selective non - steroidal anti - inflammatory drugs ( tnsaids ) and the newer selective cox-2 inhibitors . ( 1984 ) pinpointed particular prostaglandins , principally prostaglandin e2 ( pge2 ) and prostacyclin ( pgi2 ) , in the mediation of vascular permeability associated with the hyperemia and edema seen with acute inflammation . ( 1997 ) demonstrated the involvement of prostacyclin ( pgi2)-mediated inflammatory swelling in vivo , using prostacyclin receptor deficient ( ip/ ) mice . these studies ( among others ) have highlighted prostacyclin as a major endogenous mediator of inflammation both pro - inflammatory and anti - inflammatory , depending upon the tissue and pathological model being studied ( figure 2 ) . prostacyclin ( pgi2 ) serves as a protective , anti - inflammatory mediator in the processes of atherosclerosis and pulmonary vascular diseases , such as pulmonary arterial hypertension ( pah ) and idiopathic pulmonary fibrosis ( ipf ) . conversely , in rheumatological conditions , such as rheumatoid arthritis ( ra ) and osteoarthritis ( oa ) , pgi2 acts as a propagatory , pro - inflammatory molecule . while the majority of focus has centered around the role of prostaglandin e2 ( pge2 ) in rheumatoid arthritis ( ra ) , some studies have shown that the predominate prostaglandin detected within the synovial fluid of patients with ra is in fact prostacyclin ( pgi2 ; brodie et al . ( 2008 ) administered serum from arthritic k / bxn mice to induce an igg - mediated autoantibody - induced inflammatory arthritis to recipient mice lacking either prostaglandin e synthase-1 ( mpges-1/ ) or the prostacyclin receptor ( ip/ ) , in order to determine the relative importance of pge2 and pgi2 , respectively . findings revealed that mice deficient in prostaglandin e synthase-1 mpges-1 ( and therefore unable to produce pge2 ) , developed arthritis in normal fashion , whereas mice lacking the receptor for pgi2 demonstrated a significant decrease ( 31% versus wild - type ) in clinical arthritis . these results convey two important points : ( 1 ) a substantial proportion of the prostanoid contribution to joint inflammation ( at least in the k / bxn serum - transfer arthritis model ) can be accounted for by pgi2 and its interaction with the ip receptor , and ( 2 ) selective cox-1 inhibition through genetic knockout prevented the development of disease , suggesting that cox-1-derived pgi2 is the major inflammatory mediator within this arthritis model ( wang et al . more importantly , such studies lend weight to the involvement of prostacyclin in chronic inflammatory disease processes , as well as being an acute mediator , and also call into question the paradigm regarding cox-1- and cox-2-derived prostaglandin functions in vivo ( i.e. ( 2008 ) showed , meta - analysis from a systematic review of 145 randomized controlled trials , examining the clinical effectiveness of a variety of cox-2 inhibitors ( including etodolac , meloxicam , celecoxib , rofecoxib , etoricoxib , valdecoxib , and lumiracoxib ) , showed similar efficacy compared to non - selective nsaids ( including naproxen , diclofenac , ibuprofen , loxoprofen , nabumetone , piroxicam , indomethacin , tenoxicam , and nimesulide ) in the symptomatic relief of both ra and osteoarthritis ( oa ) , but with superior gastrointestinal tolerability and protection against complicated upper gastrointestinal events ( e.g. , 2008 ) , presumably as a by - product of the discriminating suppression of cox-2-derived pgi2 , which has been shown to be cardioprotective ( murata et al . thus , the combination of chronic ra - mediated inflammation , perpetuating an increased atherosclerotic burden , along with cox inhibition therapy either selective ( cox-2 inhibitor ) or non - selective ( nsaids ) would undoubtedly increase cardiovascular risk drastically , given what we now know about pgi2 . thus , pgi2 is emerging as an important intermediary in inflammatory conditions such as rheumatoid and oa , with the dualistic purpose of pro - inflammatory mediator on the one hand involved in disease pathophysiology and cardioprotective factor on the other both of which are central in the consideration of pharmacologic therapies and adverse effects . pulmonary arterial hypertension ( pah ) is in fact a heterogeneous group of diseases sharing similarities in pathophysiological mechanisms , clinical presentation , and therapeutic approaches ( simonneau et al . the main vascular changes in pah are vasoconstriction , vsmc proliferation , endothelial loss or dysfunction , and thrombosis ( farber and loscalzo , 2004 ) , which implicates a disruption of vascular hemostasis and its principle mediators , particularly pgi2 and txa2 , among others . this is evidenced by findings in patients with pah , whereby both the production of prostacyclin synthase ( pgis ) within small - to - medium pulmonary arteries , as well as urinary metabolites ( 6-keto - prostacyclin f2 ) of prostacyclin , were shown to be decreased , while levels of thromboxane metabolites ( thromboxane b2 ) were increased ( christman et al . interestingly , this imbalance of pgi2 and txa2 within the pulmonary vasculature , leading to increased mean pulmonary artery pressure , mimics that of the cardio - systemic vasculature system . in fact , many of the pathophysiological mechanisms identified in pah overlap with those involved in atherogenesis , including vascular smooth muscle and endothelial cell dysfunction , enhanced platelet activity and thrombosis , inflammation , and cellular chemotaxis ( essop , 2010 ) . owing to its potent vasodilatory , anti - thrombotic , and anti - proliferative effects , pgi2 has secured a central role in the treatment of pah . continuous intravenous epoprostenol ( synthetic pgi2 ) is the best - studied advanced therapy for pah and remains a first - line agent , particularly for those with severe disease ( who functional class iv ) , as it has been shown to improve overall symptoms , exercise capacity , and hemodynamic status in controlled clinical trials ( barst et al . analogs of pgi2 have also been used clinically , and are administered by a variety of routes , including intravenously ( e.g. , iloprost ) , and orally ( e.g. , 2005 ; again , the central role of pgi2 , as both an inflammatory and hemodynamic mediator , puts it at the forefront in understanding the pathophysiology and pharmacological treatment of pulmonary vascular diseases , particularly pah . interestingly , pgi2 seems to have a similar safeguarding effect in the chronic inflammatory condition of idiopathic pulmonary fibrosis ( ipf ) , as recent studies have shown that cox-2-derived pgi2 serves a protective role against bleomycin - induced pulmonary fibrosis a major animal model for ipf that mimics the progressive fibrosis and interstitial inflammation of sub - pleural lung tissue in humans . such findings put direct focus on the loss of cox-2-derived prostacyclin as a protective factor . while cox-2-derived prostacyclin is now well known for its protective effects within the cardiovascular system , such results provide compelling evidence for pgi2-mediated protection against fibrotic pathologies as well . ( 2010 ) also confirms these findings , pharmacologically , and identifies pgi2 as a potential new therapeutic agent for pulmonary fibrotic disease . iloprost pre - treatment decreased production of pro - inflammatory and fibrotic cytokines , such as tnf - alpha , il-6 , and tgf - beta-1 , and increased release of anti - fibrotic mediators , including ifn - gamma and chemokine cxcl10/ip-10 , as measured by mrna expression levels or elisa . thus , pgi2 seems to be an important effector in both these fibro - proliferative disorders of the lung , playing both a protective role against disease development , as well as a therapeutic role in symptom management . atherosclerosis is now known as an inflammatory disease , with the same complex cellular interactions involving monocytes , macrophages , lymphocytes , extracellular matrix ( ecm ) components , and connective tissue cells as seen in other chronic inflammatory and fibro - proliferative diseases ( e.g. mounting data has demonstrated the protective effect of prostacyclin activity against the development of atherothrombotic cardiovascular disease through the inhibition of various cellular processes , including platelet activation , leukocyte adhesion , as well as vsmc modulation . specifically , hdl has been shown to induce cox-2 expression and pgi2 production in both endothelial and vsmcs ( pomerantz et al . , 1997 , 1999 ) while , conversely , pgi2 has been shown to induce cholesterol ester hydrolase activity , which catalyzes the first step in the removal of cholesterol from foam cells , critical components in atherogenesis ( hajjar and weksler , 1983 ; weksler et al . , the effects of pgi2 on vsmcs are becoming an important target for understanding both the pathophysiology of atherothrombosis and the atheroprotective effects of prostacyclin . however , in response to vascular injury , these cells have been shown to re - enter the cell cycle , proliferate , migrate toward attractants , down - regulate expression of contractile proteins , and up - regulate synthesis of proteins , particularly ecm ( campbell et al . , 2002 ) effects on smooth muscle cells . , 1993 ) , and ( 2006 ) have shown , treatment with the stable pgi2 analog , iloprost , induces vsmc differentiation via a camp - pka - mediated signaling pathway . , prostacyclin - induced prostacyclin release ) , which was shown to have a paracrine , positive - feedback effect on neighboring vsmcs ( not exposed to iloprost ) , inducing similar cellular responses ( kasza et al . as such , the phenotypic change in vsmcs toward a proliferative and de - differentiated state , which is a hallmark occurrence in the progression of atherosclerosis and restenosis , necessitates a clear understanding of this regulatory process and is an extremely important area of research . mouse models have provided valuable insight into the role of prostacyclin in cardiovascular homeostasis and pathogenesis . , 2002 ) , and reperfusion injury ( xiao et al . ( 2004 ) went on to further demonstrate that , in female pre - menopausal ldl - r knockout mice , the atheroprotective effect of estrogen is significantly reduced in the absence of the ip receptor ( i.e. moreover , as long - term estrogen exposure was shown to increase cox-2 expression , as well as the formation of the pgi2 metabolite 6-keto - pgf1 in mouse aortic smooth muscle cells ( masmcs ) , this study suggests a significant contribution of estrogen - mediated , cox-2-derived pgi2 in the protection against atherogenesis ( egan et al . such in vivo findings highlight the important functional presence of prostacyclin activity in the maintenance of cardiovascular homeostasis and , in turn , implicate receptor - ligand ( hip - pgi2 ) dysfunction in the acceleration of atherogenesis and the subsequent development of related disorders , including stroke , mi , and hypertension . in humans , the importance of pgi2 in atherogenesis has also been shown . ( 2008 ) demonstrated that patients harboring a dysfunctional human prostacyclin receptor variant ( r212c ) exhibited an enhanced atherothrombotic phenotype , with a higher incidence of triple - vessel coronary artery disease ( cad ) and greater number of clinical cardiovascular events ( including mi , stroke , ptca , cabg , pvd , and unstable angina ) in high - risk patients versus age- and risk - factor - matched normal - allele patients . interestingly , a similar effect was observed with co - expression of the r212c variant and the wild - type thromboxane receptor ( tp ) , which revealed r212c hetero - dimerization , with subsequent inhibition of tp receptor activity ( ibrahim et al . while such mutations are rare , it is notable that single , heterozygous , point mutations within the prostacyclin receptor gene ( ptgir ) are associated with clinically significant disease changes . similarly , mutations and polymorphisms in the prostacyclin synthase ( pgis ) gene have been associated with essential hypertension , mi , and cerebral infarction ( iwai et al . , rofecoxib and valdecoxib ) whose discriminating suppression of cox-2-derived prostacyclin ( pgi2 ) resulted in increased risk of cardiovascular events ( e.g. ( 2010 ) proposed that concomitant tp receptor antagonism , along with selective cox-2 inhibition ( dual coxib - tp antagonists ) , may abrogate such adverse cardiovascular events ( caused by the imbalance between pgi2 and txa2 ) and improve the safety profile of selective cox-2 inhibitors . ( 2010 ) suggest assessment of cox-2 activity in whole blood ex vivo , perhaps in combination with biomarkers such as biochemical ( urinary levels of 6-keto - pgf1 ) and genetic ( ip receptor and other prostacyclin - related polymorphisms ) as potential surrogate endpoints to assess for prostacyclin synthesis in vivo as a predictor of cardiovascular risk . as the role of pgi2 is becoming more defined in inflammatory - related diseases , the development of novel agonists and antagonists for the ip receptor is at the forefront of research . as previously stated , the role of pgi2 in arthritic diseases ( ra and oa ) is one of a pro - inflammatory mediator , and the use of wide - ranging inhibitors of prostaglandin synthesis ( nsaids and selective cox-2 inhibitors ) has remained a mainstay of therapy . , 2004 ) with specific ip receptor antagonism significantly reduced joint discomfort in a dose - dependent manner , and with similar efficacy to diclofenac as well as an mf - tricyclic cox-2 inhibitor . for the treatment of pulmonary vascular disease such as pah and ipf , the vasoactive pgi2 analog formulations ( e.g. ( 2005 ) demonstrated similar protective results in a monocrotaline - induced pah model , whereby rats treated with ono-1301 showed improved pulmonary hemodynamics and survival , along with a reduction of vascular remodeling ( media hypertrophy ) and plasma txa2 metabolites . with the imbalance between pgi2 and txa2 playing an important role in atherogenesis and atherothrombosis , the development of dual - acting compounds seems like a promising direction for the development of novel therapeutic agents . in vitro studies showed that tra-418 inhibited platelet aggregation through impedance of both glycoprotein iib / iiia ( gpiib / iiia ) activation and p - selectin expression , which are key markers of platelet activation ( miyamoto et al . thus , while the current pool of pgi2-centered compounds is primarily investigational , and clinical safety and efficacy in human disease still largely unproven , their development emphasizes the centrality of pgi2 activity in various inflammatory - mediated pathologies , and provides exciting new directions to steer drug development . the culmination of data presented in this review reinforces the notion that bunting et al . ( 1983 ) put forth almost 30 years ago that a dynamic balance between the prostaglandins prostacyclin ( pgi2 ) and thromboxane a2 ( txa2 ; in addition to many other mediators ) is crucial in maintaining cardiovascular homeostasis , and has critical pathophysiological and therapeutic implications . our current knowledge of pgi2 , as both a physiological pathophysiological mediator and therapeutic agent , in a host of inflammatory - related diseases , is growing rapidly . as demonstrated , pgi2 has been shown to play protective roles in atherogenesis relating to cad , mi , stroke , and other cardiovascular abnormalities . it has also been shown to be involved in certain fibro - proliferative and pulmonary vascular diseases , such as ipf and pah , where it serves as both a protective factor and first - line pharmacotherapy . furthermore , in the setting of ra , pgi2 seems to play a pro - inflammatory role and , as evidence increases , perhaps may one day be considered a therapeutic target in ra and related disorders ( figure 2 ) .

to evaluate the generalizability of daa trials , we examined the eligibility criteria of trial protocols . we performed a review of all published phase 3 trials that evaluated second - generation daas in individuals with hiv hcv coinfection as of november 2015 by searching pubmed and clinical trial registries ( clinicaltrials.gov ) . the daas that were identified included simeprevir , sofosbuvir , ledipasvir , grazoprevir / elbasvir , ombitasvir , paritaprevir / ritonavir / dasabuvir , faldaprevir , and daclatasvir . we further restricted trials to those where trial protocols were available . the following trials that met our inclusion criteria were analyzed : nct01479868 ; the photon-1 trial ( nct01667731 ) ; the turquoise - i trial ( nct01939197 ) ; the ion-4 trial ( nct02073656 ) ; and the ally-2 trial ( nct02032888 ) that evaluated simeprevir ; sofosbuvir ; ombitasvir , paritaprevir / ritonavir / dasabuvir ( 3d ) ; sofosbuvir / ledipasvir ; and daclatasvir / sofosbuvir , respectively [ 1719 , 22 , 23 ] . specific eligibility criteria used to assess the generalizability of each trial are listed in table 1 . supplementary table 1 summarizes permitted combination antiretroviral therapy ( cart ) regimens by each trial . we then used the canadian coinfection cohort ( ccc ) as a representative population to evaluate the percentage of current cohort participants that would be eligible to participate in these trials . hcv coinfected patients from 18 canadian centers that provide care to hiv - infected persons . briefly , participating centers include large urban tertiary care and community - based hospitals , private clinics , and street outreach programs in the attempt to capture a representative population in care . after obtaining informed consent , sociodemographic , behavioral , and medical data were prospectively collected via self - administered questionnaires / chart review and blood sampled every 6 months . research involving this cohort was approved by all of the institutional ethics boards of the participating centers . of the 1423 cohort participants , we excluded those who died ( n = 184 ) , withdrew from the study ( n = 107 ) , and were lost to follow - up ( defined as not completing a questionnaire within 18 months of the database closure ; n = 258 ) . of the 874 remaining participants , 615 ( 70% ) had evidence of chronic hcv infection ( hcv rna positive , based on each center 's standard of care ) . we further subdivided the cohort into those who had a documented hcv genotype that reflected the trial populations . a total of 410 coinfected individuals were infected with genotype 1 , 26 with genotype 2 , 94 with genotype 3 , and 11 with genotype 4 . the ion-4 trial evaluated those with genotypes 1 or 4 , and the ally-2 study was open to coinfected patients with genotypes 1 , 2 , 3 , 4 , 5 , or 6 . the diverse demographic , clinical , and risk profiles of active ccc participants overall and subdivided by trial target populations according to eligible genotypes are presented in table 2 . eighty percent of cohort participants had a history of injection drug use ( idu ) and 31% had been using injection drugs at their last cohort visit . poverty and history of incarceration were very common . despite these factors , 87% of the cohort received cart and the majority maintained hiv viral suppression with high cd4 cell counts . the cart regimens that were used were diverse ; an equal proportion received tenofovir and abacavir - based backbones and the majority use boosted protease inhibitors or efavirenz drugs with potential for drug drug interactions with some daas . the median duration of hcv infection was more than 20 years ; 15% had evidence of advanced fibrosis based on an aspartate - to - platelet ratio index ( apri ) score of > 1.5 , and 13% had a diagnosis of cirrhosis ( clinically verified ) . table 2.characteristics of the canadian coinfection cohort participants at last visit and according to specific trial target populationscharacteristictotal active patients ( n = 874)simeprevir and turquoise - i genotype 1 ( n = 410)photon-1 genotype 1 , 2 , or 3(n = 530)gt1 = 410gt2 = 26gt3 = 94)ion-4 genotype 1 or 4(n = 421)gt1 = 410gt4 = 11ally-2 genotype 1 , 2 , 3 , 4 , 5 , 6(n = 541)gt1 = 410gt2 = 26gt3 = 94gt4 = 11age , median ( iqr ) , y49 ( 43 , 55)47 ( 42 , 52)49 ( 43 , 55)49 ( 44 , 54)49 ( 43 , 55)female , no . ( % ) 244 ( 28)102 ( 25)147 ( 28)108 ( 26)153 ( 28)aboriginal , no . ( % ) 171 ( 20)81 ( 20)113 ( 21)81 ( 19)113 ( 21)gross annual income < $ 18 000 can , no . ( % ) 634 ( 73)311 ( 76)403 ( 76)317 ( 75)412 ( 76)history of incarceration , no . ( % ) 489 ( 56)234 ( 57)308 ( 58)236 ( 56)310 ( 57)current psychiatric diagnosis , no . ( % ) 80 ( 9)55 ( 13)65 ( 12)55 ( 13)65 ( 12)currently living in shelter or homeless , no . ( % ) 73 ( 8)43 ( 10)47 ( 9)43 ( 10)47 ( 9)history of idu , no . ( % ) ( % ) 259 ( 30)130 ( 32)68 ( 32)130 ( 31)168 ( 31)current alcohol use , no . ( % ) 497 ( 57)213 ( 52)278 ( 52)220 ( 53)285 ( 53)current alcohol abuse , no . ( % ) 132 ( 15)61 ( 15)81 ( 15)62 ( 15)82 ( 15)time since hiv diagnosis , median ( iqr ) , ( y)15.8 ( 9.6 , 21.4)15.8 ( 8.7 , 21.5)15.7 ( 8.5 , 21.2)15.7 ( 8.8 , 21.5)15.7 ( 8.5 , 21.3)undetectable hiv rna , no . ( % ) 680 ( 78)292 ( 71)388 ( 73)301 ( 72)397 ( 73)cd4 t - cell count , median ( iqr ) , ( cells / mm)500 ( 332 , 690)490 ( 300 , 674)480 ( 298 , 670)490 ( 300 , 680)480 ( 300 , 675)currently cart naive , no . ( % ) 23 ( 3)13 ( 3)17 ( 3)13 ( 3)17 ( 3)on cart , no . ( % ) 752 ( 86)356 ( 87)455 ( 86)366 ( 87)465 ( 86)nrti backbone , no . ( % ) tenofovir / emtricitabine318 ( 36)147 ( 36)191 ( 36)153 ( 36)197 ( 36 ) abacavir / lamivudine , % 317 ( 36)142 ( 35)186 ( 35)146 ( 35)190 ( 35)nnrti based , no . ( % ) efavirenz127 ( 15)54 ( 14)67 ( 13)57 ( 14)70 ( 13 ) nevirapine20 ( 2)11 ( 3)11 ( 2)12 ( 3)12 ( 2 ) rilpivirine22 ( 3)19 ( 5)23 ( 5)19 ( 5)23 ( 3 ) etravirine36 ( 4)18 ( 4)25 ( 5)18 ( 4)25 ( 5)protease inhibitors / ritonavir , no . ( % ) atazanavir164 ( 19)75 ( 18)100 ( 19)76 ( 18)101 ( 19 ) lopinavir76 ( 9)30 ( 7)42 ( 8)32 ( 8)44 ( 8) darunavir159 ( 18)79 ( 19)103 ( 19)81 ( 20)105 ( 19)integrase inhibitors raltegravir190 ( 22)90 ( 22)117 ( 22)93 ( 22)120 ( 22 ) dolutegravir27 ( 3)17 ( 4)17 ( 3)17 ( 4)17 ( 3 ) elvitegravir43 ( 5)19 ( 5)23 ( 4)19 ( 5)23 ( 4)duration of hcv infection , median ( iqr ) , y21.7 ( 13.7 , 30.0)21.4 ( 13.0 , 29.1)21.3 ( 13.2 , 29.3)21.0 ( 13.0 , 29.0)21.0 ( 13.0 , 29.0)prior hcv treatment experience , no . ( % ) 334 ( 38)113 ( 28)148 ( 28)119 ( 28)152 ( 28)current ast to platelet ratio index ( apri ) > 1.5 , no . ( % ) 130 ( 15)78 ( 19)109 ( 21)81 ( 19)112 ( 21)history of cirrhosis ( clinical diagnosis ) , no . ( % ) 115 ( 13)64 ( 16)78 ( 15)66 ( 16)80 ( 15)history of end stage liver disease diagnosis , no . ( % ) 129 ( 15)74 ( 18)89 ( 17)76 ( 18)91 ( 17)active patients ( n = 874 ) includes all active cohort participants . undetectable hiv rna ( rna < 50 copies / ml ) trials restricted participation to specific genotypes ; therefore , the cohort is subdivided into those genotypes . the ion-4 trial evaluated those with genotypes 1 or 4 , and the ally-2 study was open to coinfected patients with genotypes 1 , 2 , 3 , 4 , 5 , or 6.abbreviations : ast , aspartate aminotransferase ; cart , combined antiretroviral therapy ; gt , genotype ; hcv , hepatitis c virus ; hiv , human immunodeficiency virus ; idu , injection drug use ; iqr , interquartile range ; nnrti , nonnucleoside reverse - transcriptase inhibitor ; nrti , nucleoside reverse - transcriptase inhibitor . current idu is defined as use of any injection drugs within 6 months of last cohort visit ( self reported ) . current alcohol abuse is defined as drinking more than 2 units of alcohol on a typical day within 6 months of last cohort visit ( self reported ) . includes ascites , bleeding esophageal varices , portal hypertension , hepatocellular carcinoma , spontaneous bacterial peritonitis . characteristics of the canadian coinfection cohort participants at last visit and according to specific trial target populations active patients ( n = 874 ) includes all active cohort participants . undetectable hiv rna ( rna < 50 copies / ml ) . trials restricted participation to specific genotypes ; therefore , the cohort is subdivided into those genotypes . the ion-4 trial evaluated those with genotypes 1 or 4 , and the ally-2 study was open to coinfected patients with genotypes 1 , 2 , 3 , 4 , 5 , or 6 . abbreviations : ast , aspartate aminotransferase ; cart , combined antiretroviral therapy ; gt , genotype ; hcv , hepatitis c virus ; hiv , human immunodeficiency virus ; idu , injection drug use ; iqr , interquartile range ; nnrti , nonnucleoside reverse - transcriptase inhibitor ; nrti , nucleoside reverse - transcriptase inhibitor . single person poverty is considered an annual income of < $ 18 421/yr can . current idu is defined as use of any injection drugs within 6 months of last cohort visit ( self reported ) . current alcohol abuse is defined as drinking more than 2 units of alcohol on a typical day within 6 months of last cohort visit ( includes ascites , bleeding esophageal varices , portal hypertension , hepatocellular carcinoma , spontaneous bacterial peritonitis . after applying all trial eligibility criteria to the ccc participants , only 5.9% ( 24/410 ) would have been eligible to be screened for the simeprevir trial , 9.8% ( 52/530 ) for the photon-1 trial , 6.3% ( 26/410 ) for the turquoise - i trial , and 8.1% ( 34/421 ) for the ion-4 trial . the ally-2 trial stood out as being far more inclusive , with 43% ( 233/541 ) of the cohort eligible for screening . the most common reasons for noneligibility in all trials except the ally-2 trial were restriction to specific antiretroviral therapies that resulted in the exclusion of 63%79% of the cohort , followed by active drug use ( excluding marijuana ) , which excluded 53%55% of the cohort . figure 1 illustrates that even if antiretroviral eligibility criteria were not considered ( eg , assuming patients could be safely switched to other regimens compatible with daas under study ) , 74%77% of the cohort would still have been excluded , primarily due to active drug use for 4 of the 5 trials . among all trials , as many as 1 in 6 participants would have been excluded because of either detectable hiv rna ( 15%18% ) and/or not meeting minimal cd4 count requirements ( 3%19% ) . criteria related to safety concerns , specifically clinical cutoffs for anemia and renal and liver function resulted in relatively few exclusions . despite the enhanced ease and tolerability of all oral interferon - free daas , eligibility into these trials was just as exclusive as the trial with pegylated - interferon and ribavirin , with the notable exception of the ally-2 trial . table 1.selection of exclusion criteria each exclusivecriteriatrial - specificexclusion criteriasimeprevir trial ( gt1)n = 410no . ( % ) photon-1 trial ( gt 1 , 2 , or 3)n = 530no . ( % ) ally-2 trial ( gt 1 , 2 , 3 , or 4)n = 541no . ( % ) combined antiretroviral therapy regimenssupplementary table 1291 ( 71)336 ( 63)301 ( 73)334 ( 79)44 ( 8)active illicit drug use ( excluding marijuana)supplementary table 2221 ( 54)294 ( 55)221 ( 54)223 ( 53)nacd4 t - cell count ( cells / mm)<300<200<10077 ( 19)57 ( 11)39 ( 10)12 ( 3)47 ( 9)human immunodeficiency virus rna ( copies / ml)>50>4070 ( 17)82 ( 15)73 ( 18)71 ( 17)80 ( 15)active psychiatric disordernananana65 ( 12)neutrophils ( cells / mm)<1.5<1.2<0.7535 ( 9)na10 ( 2)na2(<1)albumin ( g / dl)<3.3<3.0<2.853 ( 11)25 ( 4)12 ( 3)19 ( 5)22 ( 4)hemoglobin ( g / dl)<110 ( female ) or < 120 ( male)<10044 ( 11)47 ( 9)35 ( 9)36 ( 9)9 ( 2)platelets ( cells / mm)<90,000<60,000<50,00033 ( 8)na8 ( 2)7 ( 2)8 ( 2)decompensated liver disease30 ( 7)38 ( 7)30 ( 7)31 ( 7)39 ( 7)aids illness14 ( 3)16 ( 3)14 ( 3)14 ( 3)21 ( 4)hypertension ( mmhg)systolic blood pressure 160 or diastolic blood pressure 100nananana14 ( 3)coinfection with hepatitis bhbsag positive13 ( 3)17 ( 3)13 ( 3)14 ( 3)18 ( 3)serum creatinine ( mg / dl ) or cockcroft - gault equation ( ml / min)<1.5<60 ml / min<50 ml / min9 ( 2)56 ( 11)40 ( 10)43 ( 10)41(8)age ( y)<18<18 and > 705 ( 1)3 ( < 1)5 ( 1)2 ( < 1)3(<1)body mass index ( kg / m)<1818 and>3818 and>35na22 ( 4)20 ( 5)20 ( 5)40 ( 7)bilirubin ( mg / dl)>3 > 23 ( < 1)3 ( < 1)3 ( < 1)3 ( < 1)3 ( < 1)international normalized ratio>1.54 ( < 1)4 ( < 1)4 ( < 1)5 ( < 1)naalpha - fetoprotein ( ng / ml)<50<1006 ( 1)na4(<1)na4(<1)aspartate aminotransferase ( u / l)<10 uln<7 uln3 ( < 1)5 ( < 1)5 ( 1)3 ( < 1)naalanine aminotransferase ( u / l)<10 uln<7 uln1 ( < 1)1 ( < 1)7 ( 2)1 ( < 1)1 ( < 1)abbreviations : na , not applicable ; uln , upper limit of normal . the n ( % ) of the cohort population excluded based on each of the individual criteria . photon-1 trial ( sofosbuvir ) allowed : tenofovir / emtricitabine with atazanavir / ritonavir , darunavir / ritonavir , efavirenz , raltegravir , or rilpivirine . turquoise - i trial ( ombitasvir , paritaprevir / ritonavir / dasabuvir ) allowed : tenofovir / emtricitabine with atazanavir / ritonavir , darunavir / ritonavir , raltegravir . ion-4 trial ( ledipasvir / sofosbuvir ) allowed : tenofovir / emtricitabine with efavirenz , raltegravir , or ripilvirine . active severe psychiatric disorders include , but are not limited to , schizophrenia , psychosis , bipolar disorder , posttraumatic stress disorder , mania , and similar . decompensated liver disease includes , but is not limited to , radiologic evidence of a history or presence of ascites , bleeding varices , or hepatic encephalopathy . figure 1.green figures represent the number of canadian coinfection cohort participants who would be eligible to be screened in nct01479868 ( trial evaluating simeprevir ) ; photon-1 : nct01667731 ( trial evaluating sofosbuvir ) ; turquoise - i : nct01939197 ( trial evaluating ombitasvir , paritaprevir / ritonavir / dasabuvir [ 3d ] ) ; ion-4 : nct02073656 ( trial evaluating ledipasvir / sofosbuvir ) ; and ally-2 : nct02032888 ( trial evaluating daclatasvir / sofosbuvir ) . selection of exclusion criteria each exclusive abbreviations : na , not applicable ; uln , upper limit of normal . the n ( % ) of the cohort population excluded based on each of the individual criteria . photon-1 trial ( sofosbuvir ) allowed : tenofovir / emtricitabine with atazanavir / ritonavir , darunavir / ritonavir , efavirenz , raltegravir , or rilpivirine . turquoise - i trial ( ombitasvir , paritaprevir / ritonavir / dasabuvir ) allowed : tenofovir / emtricitabine with atazanavir / ritonavir , darunavir / ritonavir , raltegravir . ion-4 trial ( ledipasvir / sofosbuvir ) allowed : tenofovir / emtricitabine with efavirenz , raltegravir , or ripilvirine . ally-2 trial ( daclatasvir / sofosbuvir ) only excluded unboosted protease inhibitors and cobicistat . active severe psychiatric disorders include , but are not limited to , schizophrenia , psychosis , bipolar disorder , posttraumatic stress disorder , mania , and similar . decompensated liver disease includes , but is not limited to , radiologic evidence of a history or presence of ascites , bleeding varices , or hepatic encephalopathy . green figures represent the number of canadian coinfection cohort participants who would be eligible to be screened in nct01479868 ( trial evaluating simeprevir ) ; photon-1 : nct01667731 ( trial evaluating sofosbuvir ) ; turquoise - i : nct01939197 ( trial evaluating ombitasvir , paritaprevir / ritonavir / dasabuvir [ 3d ] ) ; ion-4 : nct02073656 ( trial evaluating ledipasvir / sofosbuvir ) ; and ally-2 : nct02032888 ( trial evaluating daclatasvir / sofosbuvir ) . when clinical trials are internally valid , they are considered the gold standard for estimating treatment effects . trial results are used to support licensure , inform health authorities in conducting cost - effectiveness analyses , and guide clinical decision making . however , to make these inferences to the wider population , trials also need to be externally valid . here , we illustrate that the majority of hiv hcv - coinfected patients in clinical care would not be included in recent clinical trials that evaluated hcv therapy . therefore , daa trial results may have limited generalizability . in the last 5 years we have witnessed svr rates previously unimaginable , especially in hard - to - treat coinfected patients . however , it is important to evaluate how trial efficacy translates to real - world effectiveness , which is driven by factors such as adherence , loss to follow - up , and comorbidities . real - world data on the effectiveness of daas in hcv - monoinfected populations have been , on average , 5%15% lower than what was reported in phase 3 trials [ 26 , 27 ] . it is unclear what proportion of these lower svr rates are explained by patients not adhering to their medications or being lost to follow - up as opposed to poorer efficacy . in one real - world analysis of interferon - free therapies in 151 hcv - monoinfected patients , the authors reported svr rates of 88% ; 7% relapsed and 4% were lost to post - treatment follow - up and could not be assessed for svr . with the widespread use of daas by increasingly marginalized populations , higher failure rates than those seen in clinical trials could translate to hundreds of thousands of treatment failures with limited future treatment options . fundamentally , trial participants are different ; they include highly motivated people who may receive compensation and extensive support from trial staff , including for adherence . such extensive programs are not feasible in most real - world healthcare settings , although they might serve as an effective model of care . additionally , we observed that trial populations were , on average , healthier than the cohort population . this was evident by comparing baseline cd4 cell counts of trial participants to those in the ccc . regardless of minimum cutoffs , trial participants had higher cd4 cell counts ( between 31 and 139 cells / ml higher ) than the average ccc participant [ 1719 , 22 , 23 ] . while restriction into clinical trials for the purposes of protecting the safety of participants is legitimate , we found that the majority of exclusionary criteria were not related to safety but appear to be aimed at maximizing treatment response rates . in particular , exclusion of active drug users may have been overly conservative as studies have shown they can achieve comparable svrs as those not injecting drugs in well - supported settings . reinfection and interactions between illicit drugs and daas , however , remain a concern for the active drug - using population . however , this should not prevent the inclusion of this important subgroup of individuals , especially when the eradication of hcv in developed countries is contingent on expanding treatment to active drug users . on the contrary , more data on the effectiveness of daas in this population are urgently needed in order to support scaling - up treatment strategies . the eligibility criteria for the ally-2 study appeared to be far more inclusive with respect to permitting enrollment of stable people who use drugs , illustrating that it is possible to conduct studies that are more reflective of the target population . despite these broader criteria , it will be important for future studies to report on the number of active drug users enrolled . finally , given the prohibitive cost of treatments , restricting trials to ideal populations may also have profound effects on policy decisions , as evidenced by the state - level medicaid restrictions of sofosbuvir where the majority of us states require abstinence from drugs and alcohol despite international guidelines stating the opposite . for coinfected patients , hcv treatment is further complicated by potential drug drug interactions between cart and daas . while some drug drug interactions are well documented and exclusion of individuals taking these medications is justifiable , even if it were feasible to switch hiv regimens , the majority of the ccc participants would remain ineligible primarily due to active drug use and hiv viral load / cd4 cell count cutoffs or advanced liver disease . even though the ally-2 trial permitted the majority of cart regimens and stable drug users , 57% of the cohort would still have been excluded from participating in this trial . this is particularly alarming given that the ccc comprises individuals who are able to access care and maintain cart successfully . the ccc is open to all hiv - positive patients with evidence of hcv infection followed at participating sites without restriction and is estimated to include 23% of the total coinfected population in care in canada . since participants have access to universal healthcare , insurance does not restrict those who can attend clinics . although other socioeconomic determinants may affect access to care , this does not appear to be the case as cohort participants did have very high rates of substance abuse and poverty . representativeness of this cohort can likely be extended to individuals with health insurance in the united states and in certain european countries where the prevalence of active illicit drug use is similar to that in canada . additional factors such as overall willingness and motivation to participate in clinical trials were not assessed and may further reduce the proportion of coinfected trial participants . other clinical criteria such as evidence of malignancies or other significant illnesses , electrocardiographic abnormalities , clinical cutoffs for hcv rna , and glycosylated hemoglobin involve data that have not been routinely collected as part of the ccc and therefore they were not assessed . moreover , historically hcv trial protocols in coinfection have restricted participation into clinical trials based on the presence of hiv resistance . this was only an exclusionary criterion for the turquoise - i trial ( past virologic failure to more than 1 hiv-1 art regimen and specifically darunavir resistance ) . additionally , documentation on previous hcv treatment failures and clinical definitions of what constituted cirrhotic vs noncirrhotic patients could also have further excluded trial participation . taken together , populations from the photon-2 trial , which evaluated sofosbuvir ( nct01783678 ) , and the c - edge co - infection trial , which evaluated grazoprevir / elbasvir ( nct02105662 ) , were not included in this analysis because trial protocols were not published . based solely on the limited eligibility criteria available from published papers and publically from clinical trial registries , we would estimate that only 12.6% of cohort participants would have been eligible to be screened for the photon-2 trial and 10.2% for the c - edge trial . similar to the other trials , the most exclusive eligibility criterion was restriction to specific antiretroviral regimens , excluding 63% of the ccc from photon-2 and 80% from the c - edge trials [ 11 , 32 ] . thus , for the coinfected population , drug drug interactions will remain a limiting factor for those who can not be safely switched to alternative regimens . daas are the most expensive antivirals ever to be developed on a per pill basis , costing between $ 54 000 and $ 122 000 per treatment course in canada . clinical trials have demonstrated very high efficacy in people who do not reflect target populations and in ideal trial settings . despite breakthroughs in hcv treatments , many psychosocial disadvantages still require intervention in order to increase treatment uptake and obtain successful outcomes . unless mandated to do so , the pharmaceutical industry has little incentive to evaluate daas in representative populations . even when restriction is more inclusive , there is still no guarantee of enrolling representative populations . therefore , observational study designs that estimate unbiased treatment effects in the coinfected population will be essential to determine how effective these therapies will be in the real world . this work illustrates the need to evaluate the external validity of all marketed pharmaceuticals in order to determine whether trial populations represent target populations . if generalizability is found to be limited , then targeted phase 4 studies need to be considered . the advent of daas , especially interferon - free regimens , has given hope that the burden of liver disease can be reduced among hiv hcv - coinfected individuals and that hcv can ultimately be eliminated . it remains to be seen how effective these therapies will be for the average patient who urgently requires them .

trial results are used to support licensure , inform cost - effectiveness analyses , and guide clinical decision making . we found the majority of coinfected patients were not included in clinical trials of direct - acting antivirals , raising concerns about the generalizability of these trial results .

METHODS RESULTS DISCUSSION Supplementary Material

to evaluate the generalizability of daa trials , we examined the eligibility criteria of trial protocols . we performed a review of all published phase 3 trials that evaluated second - generation daas in individuals with hiv hcv coinfection as of november 2015 by searching pubmed and clinical trial registries ( clinicaltrials.gov ) . the daas that were identified included simeprevir , sofosbuvir , ledipasvir , grazoprevir / elbasvir , ombitasvir , paritaprevir / ritonavir / dasabuvir , faldaprevir , and daclatasvir . we further restricted trials to those where trial protocols were available . specific eligibility criteria used to assess the generalizability of each trial are listed in table 1 . supplementary table 1 summarizes permitted combination antiretroviral therapy ( cart ) regimens by each trial . hcv coinfected patients from 18 canadian centers that provide care to hiv - infected persons . briefly , participating centers include large urban tertiary care and community - based hospitals , private clinics , and street outreach programs in the attempt to capture a representative population in care . after obtaining informed consent , sociodemographic , behavioral , and medical data were prospectively collected via self - administered questionnaires / chart review and blood sampled every 6 months . research involving this cohort was approved by all of the institutional ethics boards of the participating centers . of the 1423 cohort participants , we excluded those who died ( n = 184 ) , withdrew from the study ( n = 107 ) , and were lost to follow - up ( defined as not completing a questionnaire within 18 months of the database closure ; n = 258 ) . we further subdivided the cohort into those who had a documented hcv genotype that reflected the trial populations . a total of 410 coinfected individuals were infected with genotype 1 , 26 with genotype 2 , 94 with genotype 3 , and 11 with genotype 4 . the ion-4 trial evaluated those with genotypes 1 or 4 , and the ally-2 study was open to coinfected patients with genotypes 1 , 2 , 3 , 4 , 5 , or 6 . the diverse demographic , clinical , and risk profiles of active ccc participants overall and subdivided by trial target populations according to eligible genotypes are presented in table 2 . poverty and history of incarceration were very common . despite these factors , 87% of the cohort received cart and the majority maintained hiv viral suppression with high cd4 cell counts . the cart regimens that were used were diverse ; an equal proportion received tenofovir and abacavir - based backbones and the majority use boosted protease inhibitors or efavirenz drugs with potential for drug drug interactions with some daas . the median duration of hcv infection was more than 20 years ; 15% had evidence of advanced fibrosis based on an aspartate - to - platelet ratio index ( apri ) score of > 1.5 , and 13% had a diagnosis of cirrhosis ( clinically verified ) . table 2.characteristics of the canadian coinfection cohort participants at last visit and according to specific trial target populationscharacteristictotal active patients ( n = 874)simeprevir and turquoise - i genotype 1 ( n = 410)photon-1 genotype 1 , 2 , or 3(n = 530)gt1 = 410gt2 = 26gt3 = 94)ion-4 genotype 1 or 4(n = 421)gt1 = 410gt4 = 11ally-2 genotype 1 , 2 , 3 , 4 , 5 , 6(n = 541)gt1 = 410gt2 = 26gt3 = 94gt4 = 11age , median ( iqr ) , y49 ( 43 , 55)47 ( 42 , 52)49 ( 43 , 55)49 ( 44 , 54)49 ( 43 , 55)female , no . ( % ) 634 ( 73)311 ( 76)403 ( 76)317 ( 75)412 ( 76)history of incarceration , no . ( % ) 489 ( 56)234 ( 57)308 ( 58)236 ( 56)310 ( 57)current psychiatric diagnosis , no . ( % ) 132 ( 15)61 ( 15)81 ( 15)62 ( 15)82 ( 15)time since hiv diagnosis , median ( iqr ) , ( y)15.8 ( 9.6 , 21.4)15.8 ( 8.7 , 21.5)15.7 ( 8.5 , 21.2)15.7 ( 8.8 , 21.5)15.7 ( 8.5 , 21.3)undetectable hiv rna , no . ( % ) 680 ( 78)292 ( 71)388 ( 73)301 ( 72)397 ( 73)cd4 t - cell count , median ( iqr ) , ( cells / mm)500 ( 332 , 690)490 ( 300 , 674)480 ( 298 , 670)490 ( 300 , 680)480 ( 300 , 675)currently cart naive , no . ( % ) 23 ( 3)13 ( 3)17 ( 3)13 ( 3)17 ( 3)on cart , no . ( % ) 752 ( 86)356 ( 87)455 ( 86)366 ( 87)465 ( 86)nrti backbone , no . ( % ) atazanavir164 ( 19)75 ( 18)100 ( 19)76 ( 18)101 ( 19 ) lopinavir76 ( 9)30 ( 7)42 ( 8)32 ( 8)44 ( 8) darunavir159 ( 18)79 ( 19)103 ( 19)81 ( 20)105 ( 19)integrase inhibitors raltegravir190 ( 22)90 ( 22)117 ( 22)93 ( 22)120 ( 22 ) dolutegravir27 ( 3)17 ( 4)17 ( 3)17 ( 4)17 ( 3 ) elvitegravir43 ( 5)19 ( 5)23 ( 4)19 ( 5)23 ( 4)duration of hcv infection , median ( iqr ) , y21.7 ( 13.7 , 30.0)21.4 ( 13.0 , 29.1)21.3 ( 13.2 , 29.3)21.0 ( 13.0 , 29.0)21.0 ( 13.0 , 29.0)prior hcv treatment experience , no . ( % ) 130 ( 15)78 ( 19)109 ( 21)81 ( 19)112 ( 21)history of cirrhosis ( clinical diagnosis ) , no . ( % ) 115 ( 13)64 ( 16)78 ( 15)66 ( 16)80 ( 15)history of end stage liver disease diagnosis , no . ( % ) 129 ( 15)74 ( 18)89 ( 17)76 ( 18)91 ( 17)active patients ( n = 874 ) includes all active cohort participants . the ion-4 trial evaluated those with genotypes 1 or 4 , and the ally-2 study was open to coinfected patients with genotypes 1 , 2 , 3 , 4 , 5 , or 6.abbreviations : ast , aspartate aminotransferase ; cart , combined antiretroviral therapy ; gt , genotype ; hcv , hepatitis c virus ; hiv , human immunodeficiency virus ; idu , injection drug use ; iqr , interquartile range ; nnrti , nonnucleoside reverse - transcriptase inhibitor ; nrti , nucleoside reverse - transcriptase inhibitor . current idu is defined as use of any injection drugs within 6 months of last cohort visit ( self reported ) . current alcohol abuse is defined as drinking more than 2 units of alcohol on a typical day within 6 months of last cohort visit ( self reported ) . trials restricted participation to specific genotypes ; therefore , the cohort is subdivided into those genotypes . the ion-4 trial evaluated those with genotypes 1 or 4 , and the ally-2 study was open to coinfected patients with genotypes 1 , 2 , 3 , 4 , 5 , or 6 . current alcohol abuse is defined as drinking more than 2 units of alcohol on a typical day within 6 months of last cohort visit ( includes ascites , bleeding esophageal varices , portal hypertension , hepatocellular carcinoma , spontaneous bacterial peritonitis . after applying all trial eligibility criteria to the ccc participants , only 5.9% ( 24/410 ) would have been eligible to be screened for the simeprevir trial , 9.8% ( 52/530 ) for the photon-1 trial , 6.3% ( 26/410 ) for the turquoise - i trial , and 8.1% ( 34/421 ) for the ion-4 trial . the most common reasons for noneligibility in all trials except the ally-2 trial were restriction to specific antiretroviral therapies that resulted in the exclusion of 63%79% of the cohort , followed by active drug use ( excluding marijuana ) , which excluded 53%55% of the cohort . figure 1 illustrates that even if antiretroviral eligibility criteria were not considered ( eg , assuming patients could be safely switched to other regimens compatible with daas under study ) , 74%77% of the cohort would still have been excluded , primarily due to active drug use for 4 of the 5 trials . despite the enhanced ease and tolerability of all oral interferon - free daas , eligibility into these trials was just as exclusive as the trial with pegylated - interferon and ribavirin , with the notable exception of the ally-2 trial . ( % ) combined antiretroviral therapy regimenssupplementary table 1291 ( 71)336 ( 63)301 ( 73)334 ( 79)44 ( 8)active illicit drug use ( excluding marijuana)supplementary table 2221 ( 54)294 ( 55)221 ( 54)223 ( 53)nacd4 t - cell count ( cells / mm)<300<200<10077 ( 19)57 ( 11)39 ( 10)12 ( 3)47 ( 9)human immunodeficiency virus rna ( copies / ml)>50>4070 ( 17)82 ( 15)73 ( 18)71 ( 17)80 ( 15)active psychiatric disordernananana65 ( 12)neutrophils ( cells / mm)<1.5<1.2<0.7535 ( 9)na10 ( 2)na2(<1)albumin ( g / dl)<3.3<3.0<2.853 ( 11)25 ( 4)12 ( 3)19 ( 5)22 ( 4)hemoglobin ( g / dl)<110 ( female ) or < 120 ( male)<10044 ( 11)47 ( 9)35 ( 9)36 ( 9)9 ( 2)platelets ( cells / mm)<90,000<60,000<50,00033 ( 8)na8 ( 2)7 ( 2)8 ( 2)decompensated liver disease30 ( 7)38 ( 7)30 ( 7)31 ( 7)39 ( 7)aids illness14 ( 3)16 ( 3)14 ( 3)14 ( 3)21 ( 4)hypertension ( mmhg)systolic blood pressure 160 or diastolic blood pressure 100nananana14 ( 3)coinfection with hepatitis bhbsag positive13 ( 3)17 ( 3)13 ( 3)14 ( 3)18 ( 3)serum creatinine ( mg / dl ) or cockcroft - gault equation ( ml / min)<1.5<60 ml / min<50 ml / min9 ( 2)56 ( 11)40 ( 10)43 ( 10)41(8)age ( y)<18<18 and > 705 ( 1)3 ( < 1)5 ( 1)2 ( < 1)3(<1)body mass index ( kg / m)<1818 and>3818 and>35na22 ( 4)20 ( 5)20 ( 5)40 ( 7)bilirubin ( mg / dl)>3 > 23 ( < 1)3 ( < 1)3 ( < 1)3 ( < 1)3 ( < 1)international normalized ratio>1.54 ( < 1)4 ( < 1)4 ( < 1)5 ( < 1)naalpha - fetoprotein ( ng / ml)<50<1006 ( 1)na4(<1)na4(<1)aspartate aminotransferase ( u / l)<10 uln<7 uln3 ( < 1)5 ( < 1)5 ( 1)3 ( < 1)naalanine aminotransferase ( u / l)<10 uln<7 uln1 ( < 1)1 ( < 1)7 ( 2)1 ( < 1)1 ( < 1)abbreviations : na , not applicable ; uln , upper limit of normal . turquoise - i trial ( ombitasvir , paritaprevir / ritonavir / dasabuvir ) allowed : tenofovir / emtricitabine with atazanavir / ritonavir , darunavir / ritonavir , raltegravir . ion-4 trial ( ledipasvir / sofosbuvir ) allowed : tenofovir / emtricitabine with efavirenz , raltegravir , or ripilvirine . active severe psychiatric disorders include , but are not limited to , schizophrenia , psychosis , bipolar disorder , posttraumatic stress disorder , mania , and similar . figure 1.green figures represent the number of canadian coinfection cohort participants who would be eligible to be screened in nct01479868 ( trial evaluating simeprevir ) ; photon-1 : nct01667731 ( trial evaluating sofosbuvir ) ; turquoise - i : nct01939197 ( trial evaluating ombitasvir , paritaprevir / ritonavir / dasabuvir [ 3d ] ) ; ion-4 : nct02073656 ( trial evaluating ledipasvir / sofosbuvir ) ; and ally-2 : nct02032888 ( trial evaluating daclatasvir / sofosbuvir ) . active severe psychiatric disorders include , but are not limited to , schizophrenia , psychosis , bipolar disorder , posttraumatic stress disorder , mania , and similar . when clinical trials are internally valid , they are considered the gold standard for estimating treatment effects . trial results are used to support licensure , inform health authorities in conducting cost - effectiveness analyses , and guide clinical decision making . here , we illustrate that the majority of hiv hcv - coinfected patients in clinical care would not be included in recent clinical trials that evaluated hcv therapy . therefore , daa trial results may have limited generalizability . in the last 5 years we have witnessed svr rates previously unimaginable , especially in hard - to - treat coinfected patients . however , it is important to evaluate how trial efficacy translates to real - world effectiveness , which is driven by factors such as adherence , loss to follow - up , and comorbidities . it is unclear what proportion of these lower svr rates are explained by patients not adhering to their medications or being lost to follow - up as opposed to poorer efficacy . in one real - world analysis of interferon - free therapies in 151 hcv - monoinfected patients , the authors reported svr rates of 88% ; 7% relapsed and 4% were lost to post - treatment follow - up and could not be assessed for svr . with the widespread use of daas by increasingly marginalized populations , higher failure rates than those seen in clinical trials could translate to hundreds of thousands of treatment failures with limited future treatment options . regardless of minimum cutoffs , trial participants had higher cd4 cell counts ( between 31 and 139 cells / ml higher ) than the average ccc participant [ 1719 , 22 , 23 ] . while restriction into clinical trials for the purposes of protecting the safety of participants is legitimate , we found that the majority of exclusionary criteria were not related to safety but appear to be aimed at maximizing treatment response rates . on the contrary , more data on the effectiveness of daas in this population are urgently needed in order to support scaling - up treatment strategies . despite these broader criteria , it will be important for future studies to report on the number of active drug users enrolled . finally , given the prohibitive cost of treatments , restricting trials to ideal populations may also have profound effects on policy decisions , as evidenced by the state - level medicaid restrictions of sofosbuvir where the majority of us states require abstinence from drugs and alcohol despite international guidelines stating the opposite . for coinfected patients , hcv treatment is further complicated by potential drug drug interactions between cart and daas . while some drug drug interactions are well documented and exclusion of individuals taking these medications is justifiable , even if it were feasible to switch hiv regimens , the majority of the ccc participants would remain ineligible primarily due to active drug use and hiv viral load / cd4 cell count cutoffs or advanced liver disease . even though the ally-2 trial permitted the majority of cart regimens and stable drug users , 57% of the cohort would still have been excluded from participating in this trial . since participants have access to universal healthcare , insurance does not restrict those who can attend clinics . representativeness of this cohort can likely be extended to individuals with health insurance in the united states and in certain european countries where the prevalence of active illicit drug use is similar to that in canada . additional factors such as overall willingness and motivation to participate in clinical trials were not assessed and may further reduce the proportion of coinfected trial participants . other clinical criteria such as evidence of malignancies or other significant illnesses , electrocardiographic abnormalities , clinical cutoffs for hcv rna , and glycosylated hemoglobin involve data that have not been routinely collected as part of the ccc and therefore they were not assessed . moreover , historically hcv trial protocols in coinfection have restricted participation into clinical trials based on the presence of hiv resistance . taken together , populations from the photon-2 trial , which evaluated sofosbuvir ( nct01783678 ) , and the c - edge co - infection trial , which evaluated grazoprevir / elbasvir ( nct02105662 ) , were not included in this analysis because trial protocols were not published . based solely on the limited eligibility criteria available from published papers and publically from clinical trial registries , we would estimate that only 12.6% of cohort participants would have been eligible to be screened for the photon-2 trial and 10.2% for the c - edge trial . daas are the most expensive antivirals ever to be developed on a per pill basis , costing between $ 54 000 and $ 122 000 per treatment course in canada . clinical trials have demonstrated very high efficacy in people who do not reflect target populations and in ideal trial settings . this work illustrates the need to evaluate the external validity of all marketed pharmaceuticals in order to determine whether trial populations represent target populations . if generalizability is found to be limited , then targeted phase 4 studies need to be considered .

adult male long - evans rats ( harlan , indianapolis , in ) , glp-1r mice , and their wild - type c57bl/6j littermates were housed individually in plastic rodent cages and maintained on a 12-h light / dark cycle with ad libitum access to water and pelleted rodent diet ( harlan teklad ) . rats and mice were outfitted with cannulas ( plastics one , roanoake , va ) aimed at the third cerebral ventricle , and correct cannula placement was verified as previously described ( 25,26 ) . all procedures were approved by the university of cincinnati institutional animal care and use committee . glp-1 and ex4 were obtained from bio nebraska ( lincoln , ne ) and american peptide ( sunnyvale , ca ) , respectively . the glp-1r antagonists his1 , glu8 ex4 ( dhex ) , and ex ( 9 - 39 ) ( ex9 ) were obtained from baylor college of medicine protein synthesis core ( houston , tx ) and tocris ( ellisville , mo ) , respectively . all peptides were dissolved in saline and administered either intracerebroventricularly in a volume of 1.0 l or intraperitoneally in a volume of 1.0 ml / kg . rats and mice were fed ad libitum at all times except for the mornings of study days . during this time , food was removed from the animals ' cages and weighed 4 h before lights off , and animals were assigned to weight - matched groups . pretreatment ( saline or glp-1r antagonist ) injections commenced 1 h before lights off , and treatment ( saline or glp-1r agonist ) injections commenced 30 min before lights off . injection order was counterbalanced across all experimental groups to evenly distribute subtle variations in timing of injections . food was returned to the animals ' cages at lights off , and food intake and body weight were measured at selected time points . first , dose - response curves for anorexia induced by intracerebroventricular glp-1 ( 0 , 0.3 , 1.0 , 3.0 , and 10.0 g ) and ex4 ( 0 , 0.01 , 0.03 , 0.1 , and 0.3 g ) were established . based on these results , time courses of anorexia induced by intracerebroventricular glp-1 ( 3.0 nmol , 10.0 g ) and ex4 ( 0.03 nmol , 0.1 g ) were compared , and conditioned taste aversion ( cta ) to the same doses of intracerebroventricular glp-1 and ex4 was assessed as previously described ( 27 ) . to assess the ability of central glp-1r antagonism to block anorexia induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline , dhex ( 10.0 g , ) , or ex9 ( 100.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . to assess the ability of peripheral glp-1r antagonism to block anorexia induced by peripheral ex4 , rats were pretreated with intraperitoneal saline or dhex ( 1.0 mg / kg ) and then treated with intraperitoneal saline or ex4 ( 10.0 g / kg ) . finally , to assess the ability of central ex4 to reduce food intake in glp-1r mice , mice were treated with intracerebroventricular saline or ex4 ( 1.0 g ) . to assess the ability of central glp-1r antagonism to block neuronal activation induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline or dhex ( 10.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . two hours later , rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 0.1 mol / l pbs followed by 4.0% paraformaldehyde / pbs . brains were postfixed at 4c for 24 h in 4.0% paraformaldehyde / pbs and stored at 4c in 30.0% sucrose / pbs . serial coronal forebrain sections and longitudinal hindbrain sections were collected at 35 m using a freezing microtome and stored at 20c in cryoprotectant . after washing with pbs , sections were incubated in 1.0% hydrogen peroxide / pbs for 10 min , followed by 1.0% sodium borohydride / pbs for 30 min . sections were blocked for 1 h in 0.1% bsa/0.4% triton - x-100/pbs and incubated overnight at room temperature in blocking solution containing rabbit anti c - fos diluted at 1:5,000 ( sc-52 ; santa cruz biotechnology , santa cruz , ca ) . the next morning , sections were washed and incubated at room temperature for 1 h in blocking solution containing biotinylated goat anti - rabbit igg diluted at 1:200 ( ba-1000 ; vector laboratories , burlingame , ca ) followed by 1 h in abc solution diluted 1:800 in pbs ( pk6100 ; vector laboratories ) and 10 min in dab - nickel solution . finally , sections were washed with 0.1 mol / l phosphate buffer , mounted on gelatin - coated slides , and cover slipped . for quantification of c - fos immunoreactivity in the central nucleus of the amygdala ( cea ) , paraventricular nucleus of the hypothalamus ( pvn ) , and nucleus of the solitary tract ( nts ) , digital images of sections were acquired using a digital camera attached to a zeiss microscope ( zeiss , thornwood , ny ) . for each brain , two sections per area were analyzed , and special care was taken to compare only sections within the same plane along the rostro - caudal ( cea and pvn ) or dorso - ventral ( nts ) axis . c - fos immunoreactivity was quantified as optical density using the national institutes of health program scion image . ins-1 cells were seeded in 35-mm six - well plates at a density of 2 10 cell / well in 1.5 ml of media consisting of rpmi-1640 supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) , 1.0 mmol / l sodium pyruvate , 2.0 mmol / l l - glutamine , 50.0 mol / l -mercaptoethanol , and 0.5 mg / ml gentamicin sulfate and grown in a 37c incubator in an atmosphere of 5% co2 and 95% air and 100% humidity for 3 days until nearly confluent . on day 4 , cells were washed with pbs and replaced with fresh media . on day 5 , cells were preincubated for 2 h in 2.0 ml of buffer consisting of krebs - ringer bicarbonate buffer ( krb ) supplemented with 0.1% bsa and 30 mg / dl glucose and then washed twice with 2.0 ml of the same buffer solution . cells were then incubated for 1 h in 1.0 ml of krb supplemented with 0.1% bsa , 200 mg / dl glucose , and 1.0 nmol / l glp-1 , 0.01 nmol / l ex4 , or 1.0 nmol / l ex4 with or without 100 nmol / l dhex . finally , incubation buffer was harvested , centrifuged , decanted , and stored at 20c for immunoreactive insulin ( iri ) assay , and cells were washed once with 1.0 ml of preincubation buffer and then extracted with 1.0 ml of acid ethanol for 2 h at 20c , after which acid ethanol was diluted 1:200 with tris assay buffer for iri assay in cell layer . data were analyzed using one- or two - way anova or two - way repeated - measures anova . glp-1 and ex4 were obtained from bio nebraska ( lincoln , ne ) and american peptide ( sunnyvale , ca ) , respectively . the glp-1r antagonists his1 , glu8 ex4 ( dhex ) , and ex ( 9 - 39 ) ( ex9 ) were obtained from baylor college of medicine protein synthesis core ( houston , tx ) and tocris ( ellisville , mo ) , respectively . all peptides were dissolved in saline and administered either intracerebroventricularly in a volume of 1.0 l or intraperitoneally in a volume of 1.0 ml / kg . rats and mice were fed ad libitum at all times except for the mornings of study days . during this time , food was removed from the animals ' cages and weighed 4 h before lights off , and animals were assigned to weight - matched groups . pretreatment ( saline or glp-1r antagonist ) injections commenced 1 h before lights off , and treatment ( saline or glp-1r agonist ) injections commenced 30 min before lights off . injection order was counterbalanced across all experimental groups to evenly distribute subtle variations in timing of injections . food was returned to the animals ' cages at lights off , and food intake and body weight were measured at selected time points . first , dose - response curves for anorexia induced by intracerebroventricular glp-1 ( 0 , 0.3 , 1.0 , 3.0 , and 10.0 g ) and ex4 ( 0 , 0.01 , 0.03 , 0.1 , and 0.3 g ) were established . based on these results , time courses of anorexia induced by intracerebroventricular glp-1 ( 3.0 nmol , 10.0 g ) and ex4 ( 0.03 nmol , 0.1 g ) were compared , and conditioned taste aversion ( cta ) to the same doses of intracerebroventricular glp-1 and ex4 was assessed as previously described ( 27 ) . to assess the ability of central glp-1r antagonism to block anorexia induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline , dhex ( 10.0 g , ) , or ex9 ( 100.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . to assess the ability of peripheral glp-1r antagonism to block anorexia induced by peripheral ex4 , rats were pretreated with intraperitoneal saline or dhex ( 1.0 mg / kg ) and then treated with intraperitoneal saline or ex4 ( 10.0 g / kg ) . finally , to assess the ability of central ex4 to reduce food intake in glp-1r mice , mice were treated with intracerebroventricular saline or ex4 ( 1.0 g ) . to assess the ability of central glp-1r antagonism to block neuronal activation induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline or dhex ( 10.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . two hours later , rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 0.1 mol / l pbs followed by 4.0% paraformaldehyde / pbs . brains were postfixed at 4c for 24 h in 4.0% paraformaldehyde / pbs and stored at 4c in 30.0% sucrose / pbs . serial coronal forebrain sections and longitudinal hindbrain sections were collected at 35 m using a freezing microtome and stored at 20c in cryoprotectant . after washing with pbs , sections were incubated in 1.0% hydrogen peroxide / pbs for 10 min , followed by 1.0% sodium borohydride / pbs for 30 min . sections were blocked for 1 h in 0.1% bsa/0.4% triton - x-100/pbs and incubated overnight at room temperature in blocking solution containing rabbit anti c - fos diluted at 1:5,000 ( sc-52 ; santa cruz biotechnology , santa cruz , ca ) . the next morning , sections were washed and incubated at room temperature for 1 h in blocking solution containing biotinylated goat anti - rabbit igg diluted at 1:200 ( ba-1000 ; vector laboratories , burlingame , ca ) followed by 1 h in abc solution diluted 1:800 in pbs ( pk6100 ; vector laboratories ) and 10 min in dab - nickel solution . finally , sections were washed with 0.1 mol / l phosphate buffer , mounted on gelatin - coated slides , and cover slipped . for quantification of c - fos immunoreactivity in the central nucleus of the amygdala ( cea ) , paraventricular nucleus of the hypothalamus ( pvn ) , and nucleus of the solitary tract ( nts ) , digital images of sections were acquired using a digital camera attached to a zeiss microscope ( zeiss , thornwood , ny ) . for each brain , two sections per area were analyzed , and special care was taken to compare only sections within the same plane along the rostro - caudal ( cea and pvn ) or dorso - ventral ( nts ) axis . c - fos immunoreactivity was quantified as optical density using the national institutes of health program scion image . ins-1 cells were seeded in 35-mm six - well plates at a density of 2 10 cell / well in 1.5 ml of media consisting of rpmi-1640 supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) , 1.0 mmol / l sodium pyruvate , 2.0 mmol / l l - glutamine , 50.0 mol / l -mercaptoethanol , and 0.5 mg / ml gentamicin sulfate and grown in a 37c incubator in an atmosphere of 5% co2 and 95% air and 100% humidity for 3 days until nearly confluent . on day 4 , cells were washed with pbs and replaced with fresh media . on day 5 , cells were preincubated for 2 h in 2.0 ml of buffer consisting of krebs - ringer bicarbonate buffer ( krb ) supplemented with 0.1% bsa and 30 mg / dl glucose and then washed twice with 2.0 ml of the same buffer solution . cells were then incubated for 1 h in 1.0 ml of krb supplemented with 0.1% bsa , 200 mg / dl glucose , and 1.0 nmol / l glp-1 , 0.01 nmol / l ex4 , or 1.0 nmol / l ex4 with or without 100 nmol / l dhex . finally , incubation buffer was harvested , centrifuged , decanted , and stored at 20c for immunoreactive insulin ( iri ) assay , and cells were washed once with 1.0 ml of preincubation buffer and then extracted with 1.0 ml of acid ethanol for 2 h at 20c , after which acid ethanol was diluted 1:200 with tris assay buffer for iri assay in cell layer . data were analyzed using one- or two - way anova or two - way repeated - measures anova . consistent with previous reports , intracerebroventricular glp-1 and ex4 elicited potent , dose - dependent reductions in 4-h food intake ( fig . 1a and b ; p < 0.05 , one - way anova with tukey 's post hoc test ) . however , ex4 significantly reduced food intake at doses much lower than those of glp-1 . specifically , 10.0 g of glp-1 and 0.1 g of ex4 produced comparable degrees of anorexia , reducing food intake to 56 and 45% of control values , respectively . these data indicate that , when administered into the third ventricle , ex4 is roughly 100-fold more potent than glp-1 at reducing food intake . a and b : dose - response curves for intracerebroventricular glp-1 ( a ) and ex4 ( b ) . c : time course of anorectic effects of intracerebroventricular glp-1 ( 3.0 nmol ) and ex4 ( 0.03 nmol ) over 24 h. , saline ; , glp-1 ( 3.0 nmol / l ) ; , ex4 ( 0.03 nmol / l ) . d : preference ratios for 0.1% saccharin versus total fluid intake during the 4-h two - bottle access to saccharin and water . saccharin was previously paired with intracerebroventricular saline , intracerebroventricular glp-1 ( 3.0 nmol ) , intracerebroventricular ex4 ( 0.03 nmol ) , or intraperitoneal licl ( 0.15 mol / l administered at 2.0% body wt ) . whereas 3.0 nmol ( 10.0 g ) of glp-1 and 0.03 nmol ( 0.1 g ) of ex4 both actively suppressed food intake up to 4 h , only ex4 elicited persistent anorexia that remained detectable throughout the 24 h of observation ( p < 0.05 , two - way repeated - measures anova with tukey 's post hoc test ) . furthermore , these doses of glp-1 and ex4 both led to the formation of a cta ( fig . 1d ; p < 0.05 , one - way anova with tukey 's post hoc test ) . interestingly , there was a strong trend toward a significantly lower preference ratio of ex4-treated rats versus glp-1treated rats ( p = 0.052 ) , suggesting that the aversive effects of ex4 were more pronounced than those of glp-1 . although previous studies have reported an inability to block certain effects of ex4 with glp-1r antagonists , these studies did not necessarily account for the significantly greater potency of ex4 over glp-1 . therefore , we sought to compare the ability of glp-1r antagonists to block anorexia and neuronal activation induced by doses of intracerebroventricular glp-1 and ex4 that produce effects of comparable magnitude . pretreatment with either 10.0 g of dhex or 100.0 g of ex9 caused near - complete blockade of anorexia induced by 10.0 g of glp-1 ( fig . 2a and c ; p < 0.05 by two - way anova with tukey 's post hoc test ) . however , whereas 0.1 g of ex4 and 10.0 g of glp-1 elicited comparable degrees of anorexia , the doses of dhex and ex9 that nearly abolished glp-1induced anorexia failed to block the anorectic effect of ex4 ( fig . 2b and d ) , although a nonsignificant trend was observed with dhex ( p = 0.148 ) . a and b : rats were pretreated with intracerebroventricular dhex ( 10 g ) followed by intracerebroventricular glp-1 ( 10 g ) ( a ) or ex4 ( 0.1 g ) ( b ) . c and d : rats were pretreated with intracerebroventricular ex9 ( 100 g ) followed by intracerebroventricular glp-1 ( 10 g ) ( c ) or ex4 ( 0.1 g ) ( d ) . cumulative 4-h food intake is shown . * p < 0.05 vs. sal / sal . # p < 0.05 vs. sal / glp-1 . to determine whether neuronal activation in response to glp-1 and ex4 was also differentially sensitive to glp-1r antagonism , the effect of intracerebroventricular dhex to block c - fos immunoreactivity induced by intracerebroventricular glp-1 and ex4 was compared . at the same doses as used above , glp-1 and ex4 both induced c - fos immunoreactivity in identical brain regions , including the cea , pvn , and the nts ( fig . 3a c ; p < 0.05 by two - way anova with tukey 's post hoc test ) . the magnitude of c - fos immunoreactivity induced by glp-1 and ex4 was similar in the pvn and nts , whereas glp-1 induced slightly more c - fos immunoreactivity than ex4 in the cea . in the cea , dhex significantly blocked c - fos immunoreactivity induced by glp-1 ( p < 0.05 ) ; however , in the pvn and the nts , this difference failed to reach statistical significance . nonetheless , for all three regions , the amount of c - fos immunoreactivity in brains treated with dhex and ex4 was significantly greater than that of brains treated with saline , dhex alone , or dhex and glp-1 ( p < 0.05 ) . these results , combined with the food intake data , suggest that cns actions of ex4 are relatively insensitive to competitive glp-1r antagonism . quantification of c - fos positive nuclei in the pvn ( a ) , nts ( b ) , and cea ( c ) of rats that were treated with intracerebroventricular saline or dhex ( 10 g ) followed by intracerebroventricular saline , glp-1 ( 10 g ) , or ex4 ( 0.1 g ) and killed 2 h later . * p < 0.05 vs. sal / sal . # p < 0.05 vs. sal / glp-1 . because dhex , a validated but lesser used glp-1r antagonist ( 28,30,31 ) , failed to block anorexia and neuronal activation induced by intracerebroventricular ex4 , we sought to determine whether dhex is an effective antagonist of ex4 in vitro by assessing its ability to block insulin secretion induced by ex4 in the rat pancreatic islet cell line ins-1 . as expected , 1.0 nmol / l glp-1 significantly augmented insulin secretion above that of glucose alone , and this effect was completely blocked by coincubation with 100 nmol / l dhex ( fig . 4 ; p < 0.05 by two - way anova with tukey 's post hoc test ) . however , in contrast to our in vivo data , 0.01 nmol / l ex4 failed to augment insulin secretion , whereas 1.0 nmol / l ex4 had an effect that was comparable to 1.0 nmol / l glp-1 . moreover , this effect was completely blocked by coincubation with 100 nmol / l dhex ( p < 0.05 ) . nmol / l ) on insulin secretion induced by glp-1 ( 1.0 nmol / l ) and ex4 ( 0.01 and 1.0 nmol / l ) in the presence of glucose ( 200 mg% ) . # p < 0.05 vs. glucose + glp-1 ( 1.0 nmol / l ) or glucose + ex4 ( 1.0 nmol / l ) . , saline ; , 100 nmol / l dhex . to determine whether the insensitivity of ex4 to glp-1r antagonism was specific to cns administration , we assessed the ability of dhex to block anorexia induced by intraperitoneal ex4 . as expected , 10 g / kg of intraperitoneal ex4 5 ; p < 0.05 by two - way anova with tukey 's post hoc test ) . dhex , the same 100-fold excess of antagonist that failed to block anorexia induced by intracerebroventricular ex4 , significantly attenuated this effect ( p < 0.05 ) . effect of intraperitoneal dhex ( 1.0 mg / kg ) on anorexia induced by intraperitoneal ex4 ( 10.0 g / kg ) . data are represented as means se . * p < 0.05 vs. sal / sal . the insensitivity of cns ex4 effects to glp-1r antagonism raises the possibility that ex4 may act in part via a glp-1r independent mechanism . to determine whether the glp-1r is required for the central anorectic effect of ex4 , intracerebroventricular ex4 was administered to wild - type and glp-1r mice . in wild - type mice , 1.0 g of intracerebroventricular ex4 elicited profound anorexia such that daily food intake and body weight were significantly reduced for up to 48 and 72 h , respectively ( fig . 6a and b ; p < 0.05 by two - way repeated - measures anova with tukey 's post hoc test ) . conversely , this same high dose of intracerebroventricular ex4 had no effect on food intake or body weight in glp-1r mice ( fig . a and b : wild - type mice received intracerebroventricular saline ( ) or ex4 ( 1.0 g ) ( ) . food intake ( a ) and body weight change ( b ) were measured over 96 h. c and d : glp-1r mice received intracerebroventricular saline or ex4 ( 1.0 g ) . food intake ( c ) and body weight change ( d ) were measured over 24 h. data are represented as means se . consistent with previous reports , intracerebroventricular glp-1 and ex4 elicited potent , dose - dependent reductions in 4-h food intake ( fig . 1a and b ; p < 0.05 , one - way anova with tukey 's post hoc test ) . however , ex4 significantly reduced food intake at doses much lower than those of glp-1 . specifically , 10.0 g of glp-1 and 0.1 g of ex4 produced comparable degrees of anorexia , reducing food intake to 56 and 45% of control values , respectively . these data indicate that , when administered into the third ventricle , ex4 is roughly 100-fold more potent than glp-1 at reducing food intake . a and b : dose - response curves for intracerebroventricular glp-1 ( a ) and ex4 ( b ) . c : time course of anorectic effects of intracerebroventricular glp-1 ( 3.0 nmol ) and ex4 ( 0.03 nmol ) over 24 h. , saline ; , glp-1 ( 3.0 nmol / l ) ; , ex4 ( 0.03 nmol / l ) . d : preference ratios for 0.1% saccharin versus total fluid intake during the 4-h two - bottle access to saccharin and water . saccharin was previously paired with intracerebroventricular saline , intracerebroventricular glp-1 ( 3.0 nmol ) , intracerebroventricular ex4 ( 0.03 nmol ) , or intraperitoneal licl ( 0.15 mol / l administered at 2.0% body wt ) . whereas 3.0 nmol ( 10.0 g ) of glp-1 and 0.03 nmol ( 0.1 g ) of ex4 both actively suppressed food intake up to 4 h , only ex4 elicited persistent anorexia that remained detectable throughout the 24 h of observation ( p < 0.05 , two - way repeated - measures anova with tukey 's post hoc test ) . furthermore , these doses of glp-1 and ex4 both led to the formation of a cta ( fig . 1d ; p < 0.05 , one - way anova with tukey 's post hoc test ) . interestingly , there was a strong trend toward a significantly lower preference ratio of ex4-treated rats versus glp-1treated rats ( p = 0.052 ) , suggesting that the aversive effects of ex4 were more pronounced than those of glp-1 . although previous studies have reported an inability to block certain effects of ex4 with glp-1r antagonists , these studies did not necessarily account for the significantly greater potency of ex4 over glp-1 . therefore , we sought to compare the ability of glp-1r antagonists to block anorexia and neuronal activation induced by doses of intracerebroventricular glp-1 and ex4 that produce effects of comparable magnitude . pretreatment with either 10.0 g of dhex or 100.0 g of ex9 caused near - complete blockade of anorexia induced by 10.0 g of glp-1 ( fig . 2a and c ; p < 0.05 by two - way anova with tukey 's post hoc test ) . however , whereas 0.1 g of ex4 and 10.0 g of glp-1 elicited comparable degrees of anorexia , the doses of dhex and ex9 that nearly abolished glp-1induced anorexia failed to block the anorectic effect of ex4 ( fig . 2b and d ) , although a nonsignificant trend was observed with dhex ( p = 0.148 ) . a and b : rats were pretreated with intracerebroventricular dhex ( 10 g ) followed by intracerebroventricular glp-1 ( 10 g ) ( a ) or ex4 ( 0.1 g ) ( b ) . c and d : rats were pretreated with intracerebroventricular ex9 ( 100 g ) followed by intracerebroventricular glp-1 ( 10 g ) ( c ) or ex4 ( 0.1 g ) ( d ) . cumulative 4-h food intake is shown . * p < 0.05 vs. sal / sal . # p < 0.05 vs. sal / glp-1 . to determine whether neuronal activation in response to glp-1 and ex4 was also differentially sensitive to glp-1r antagonism , the effect of intracerebroventricular dhex to block c - fos immunoreactivity induced by intracerebroventricular glp-1 and ex4 was compared . at the same doses as used above , glp-1 and ex4 both induced c - fos immunoreactivity in identical brain regions , including the cea , pvn , and the nts ( fig c ; p < 0.05 by two - way anova with tukey 's post hoc test ) . the magnitude of c - fos immunoreactivity induced by glp-1 and ex4 was similar in the pvn and nts , whereas glp-1 induced slightly more c - fos immunoreactivity than ex4 in the cea . in the cea , dhex significantly blocked c - fos immunoreactivity induced by glp-1 ( p < 0.05 ) ; however , in the pvn and the nts , this difference failed to reach statistical significance . nonetheless , for all three regions , the amount of c - fos immunoreactivity in brains treated with dhex and ex4 was significantly greater than that of brains treated with saline , dhex alone , or dhex and glp-1 ( p < 0.05 ) . these results , combined with the food intake data , suggest that cns actions of ex4 are relatively insensitive to competitive glp-1r antagonism . quantification of c - fos positive nuclei in the pvn ( a ) , nts ( b ) , and cea ( c ) of rats that were treated with intracerebroventricular saline or dhex ( 10 g ) followed by intracerebroventricular saline , glp-1 ( 10 g ) , or ex4 ( 0.1 g ) and killed 2 h later . * p < 0.05 vs. sal / sal . # p < 0.05 vs. sal / glp-1 . because dhex , a validated but lesser used glp-1r antagonist ( 28,30,31 ) , failed to block anorexia and neuronal activation induced by intracerebroventricular ex4 , we sought to determine whether dhex is an effective antagonist of ex4 in vitro by assessing its ability to block insulin secretion induced by ex4 in the rat pancreatic islet cell line ins-1 . as expected , 1.0 nmol / l glp-1 significantly augmented insulin secretion above that of glucose alone , and this effect was completely blocked by coincubation with 100 nmol / l dhex ( fig . 4 ; p < 0.05 by two - way anova with tukey 's post hoc test ) . however , in contrast to our in vivo data , 0.01 nmol / l ex4 failed to augment insulin secretion , whereas 1.0 nmol / l ex4 had an effect that was comparable to 1.0 nmol / l glp-1 . moreover , this effect was completely blocked by coincubation with 100 nmol / l dhex ( p < 0.05 ) . effect of dhex ( 100 nmol / l ) on insulin secretion induced by glp-1 ( 1.0 nmol / l ) and ex4 ( 0.01 and 1.0 nmol / l ) in the presence of glucose ( 200 mg% ) . * p < 0.05 vs. glucose . # p < 0.05 vs. glucose + glp-1 ( 1.0 nmol / l ) or glucose + ex4 ( 1.0 to determine whether the insensitivity of ex4 to glp-1r antagonism was specific to cns administration , we assessed the ability of dhex to block anorexia induced by intraperitoneal ex4 . as expected , 10 g / kg of intraperitoneal ex4 significantly reduced food intake at 4 h ( fig . 5 ; p < 0.05 by two - way anova with tukey 's post hoc test ) . surprisingly , pretreatment with 1.0 mg / kg i.p . dhex , the same 100-fold excess of antagonist that failed to block anorexia induced by intracerebroventricular ex4 , significantly attenuated this effect ( p < 0.05 ) . effect of intraperitoneal dhex ( 1.0 mg / kg ) on anorexia induced by intraperitoneal ex4 ( 10.0 g / kg ) * p < 0.05 vs. sal / sal . # p < 0.05 vs. sal / ex4 . the insensitivity of cns ex4 effects to glp-1r antagonism raises the possibility that ex4 may act in part via a glp-1r independent mechanism . to determine whether the glp-1r is required for the central anorectic effect of ex4 , intracerebroventricular ex4 was administered to wild - type and glp-1r mice . in wild - type mice , 1.0 g of intracerebroventricular ex4 elicited profound anorexia such that daily food intake and body weight were significantly reduced for up to 48 and 72 h , respectively ( fig . 6a and b ; p < 0.05 by two - way repeated - measures anova with tukey 's post hoc test ) . conversely , this same high dose of intracerebroventricular ex4 had no effect on food intake or body weight in glp-1r mice ( fig . a and b : wild - type mice received intracerebroventricular saline ( ) or ex4 ( 1.0 g ) ( ) . food intake ( a ) and body weight change ( b ) were measured over 96 h. c and d : glp-1r mice received intracerebroventricular saline or ex4 ( 1.0 g ) . food intake ( c ) and body weight change ( d ) were measured over 24 h. data are represented as means se . * because ex4 ( 14 ) , but not the dpp-4 inhibitor sitagliptin ( 16 ) , produces weight loss in patients , it is critical that we better understand the unique anorectic properties of ex4 . to this end , we report key distinctions between the central anorectic effects of ex4 and native glp-1 . not only do our data confirm that central glp-1 and ex4 differ significantly in potency and duration of action , but they also reveal novel differences between the two peptides regarding sensitivity to glp-1r antagonism . ex4 , when administered into the cns , reduces food intake in a manner distinct from that of glp-1 . consistent with previous reports ( 32,33 ) , central ex4 reduced 4-h food intake at doses 30- to 100-fold lower than those required by glp-1 to cause equivalent anorexia . importantly , this difference in potency at 4 h can not simply be explained by differences in duration of action , as both 3.0 nmol of glp-1 and 0.03 nmol of ex4 reduced food intake to a comparable extent from 0 to 2 h and 2 to 4 h. however , in contrast to glp-1 , ex4 dynamically reduced food intake over 24 h of observation , indicating that even at significantly lower doses , central ex4 exhibits a significantly longer duration of action . consistent with our food intake data , 0.1 g of central ex4 produced an almost identical degree of neuronal activation as 10.0 g of glp-1 in the pvn and the nts but interestingly not in the cea . it is possible that because 100-fold less ex4 than glp-1 was administered , less peptide diffused through the neuropil to the cea , which , unlike the pvn and the nts , does not abut the ventricular system . despite producing less neuronal activation in the cea , an area important for the formation of glp-1mediated cta ( 34 ) , central ex4 produced a lower preference ratio for saccharin than glp-1 , suggesting that ex4 induced a greater visceral illness response . although a proportional relationship between glp-1r mediated neuronal activity and behavioral responses has yet to be established , these data are significant because they suggest that enhanced visceral illness or aversive learning may in part underlie the potent anorectic effect of central ex4 . the above data and those of others support a role for ex4 as a highly potent , long - acting cns glp-1r agonist , yet the mechanism for this unique pharmacological profile remains unknown . however , in vitro and ex vivo comparisons of binding affinity have yielded equivocal results ( 3538 ) , and it remains unclear whether glp-1 and ex4 remain bound to cns glp-1r for different periods of time . however , data from baggio et al . ( 39 ) revealed no difference in the ability of glp-1 and ex4 to desensitize the glp-1r in vitro . moreover , because glp-1 and ex4 were administered as boluses , this hypothesis fails to adequately explain the present results . finally , it is possible that differential clearance and/or degradation of glp-1 versus ex4 account for their distinct pharmacological profiles within the cns . perhaps the most striking difference between central glp-1 and ex4 revealed by our data are their sensitivity to glp-1r antagonism . whereas dhex almost completely blocked anorexia and neuronal activation induced by glp-1 , it failed to significantly block that induced by an equipotent dose of ex4 . this phenomenon is not specific to dhex , as ex9 also failed to block anorexia induced by central ex4 . however , dhex is an effective antagonist of ex4 in vitro , as it completely blocked the enhancement of glucose - stimulated insulin secretion induced by ex4 in ins-1 cells . in addition , dhex is an effective antagonist of ex4 in vivo , as intraperitoneal dhex completely blocked anorexia induced by intraperitoneal ex4 . taken together , these data indicate that compared with glp-1 , ex4 is relatively insensitive to glp-1r antagonism . moreover , this phenomenon seems to be specific for central effects but not peripheral effects , many of which have been reported to be blocked by ex9 ( 4042 ) . certainly , previous studies ( 2224 ) have reported an inability to block ex4 effects with glp-1r antagonists . however , they may not have adequately accounted for the increased potency of ex4 versus glp-1 . here , we closely controlled for this difference and found that pretreatment with glp-1r antagonists significantly blocked anorexia induced by central glp-1 but not an equipotent and , importantly , 100-fold lower dose of ex4 . moreover , this phenomenon is not secondary to differences in agonist duration of action , as it was observed at early time points when both glp-1 and ex4 dynamically reduced food intake . nor is it secondary to the antagonist duration of action , as timing of pretreatment and treatment injections was consistent across all experiments , and similar trends were observed with both c - fos and food intake ( data not shown ) at 2 h. while intriguing , these data are difficult to reconcile with our other experiments . specifically , we found no differences between glp-1 and ex4 in either potency or sensitivity to dhex in vitro , although this discrepancy might easily be explained by obvious differences between animal models and immortalized cell lines . more difficult to explain , however , is the comparison to our peripheral ex4 food intake study , in which the same 100-fold excess of dhex , this time administered intraperitoneally , completely blocked intraperitoneal ex4-induced anorexia . consequently , it is possible that fundamental differences exist between central and peripheral glp-1r , which may occur at the level of posttranslational processing , protein protein interactions , or coupling to second - messenger systems . perhaps the most obvious explanation for the discrepancies between central glp-1 and ex4 is that the latter acts in part independently of the glp-1r . however , consistent with previous reports ( 18 ) , central ex4 had no effect on either food intake or body weight in glp-1r mice , suggesting that the glp-1r is required for these effects . although the lack of ex4 effects in glp-1r mice provides a strong basis to rule out glp-1r independence , there is some evidence for both functional ( 43 ) and structural ( 44 ) differences between the glp-1 systems of mice and rats . finally , other studies reporting an inability to block ex4 effects with glp-1r antagonists have all been conducted in rats ( 2224 ) . although none of these observations provides definitive evidence for glp-1r independent effects of ex4 , they do raise the possibility that ex4 may interact with the glp-1r in a species - dependent manner . although difficult to reconcile with the above data , our findings regarding central ex4 and glp-1r antagonists are consistent with several reports of in vivo effects of ex4 that are insensitive to glp-1r antagonists ( 2224 ) . recently , it was reported that central ex4 decreases ghrelin secretion in fasted rats ( 24 ) . not only was this effect insensitive to ex9 blockade , it was also elicited by ex9 alone , consistent with several in vitro reports of independent ex9 effects ( 4550 ) . whereas these data , like ours , fail to prove glp-1r independence of ex4 , they are nonetheless significant in that they provide potential mechanistic insight into the unique anorectic properties of central ex4 , particularly its duration of action . for instance , ongoing glp-1r signaling by ex4 may prevent circulating ghrelin levels from rising in response to ex4-mediated reductions in food intake , leading to an attenuation or delay in the subsequent drive to eat and thus a prolonged duration of anorexia . however , because our experiments used ad libitum fed rats , whose circulating ghrelin levels should be low , and because ex4 is more efficacious in fed versus fasted rats ( 51 ) , it seems unlikely that ex4 's effects on ghrelin secretion underlie either its increased potency acutely or its insensitivity to glp-1r antagonists in the present studies . because studies have generally found no effect of ex4 in glp-1r mice ( 1721 ) , it seems reasonable to cite strictly pharmacological differences when explaining discrepancies between in vivo effects of glp-1 and ex4 . however , in many ways , the existing data fail to adequately support this hypothesis . for instance , some in vitro studies have found ex4 to have greater potency and affinity for the glp-1r than native glp-1 ( 35 ) , but these differences , at least in potency , are significantly smaller than those reported here . regarding antagonist sensitivity , one potential explanation for our findings is that ex4 is more able to displace antagonists from the glp-1r . however , studies have generally reported little to no difference in the ability of glp-1 versus ex4 to displace radio - labeled ex9 ( 5255 ) . taken together , our data , combined with the existing literature , provide conclusive evidence for distinct pharmacological profiles of glp-1 and ex4 , yet further studies are needed to understand whether pharmacological differences alone are sufficient to explain the unique in vivo effects of ex4 . in conclusion , our data indicate that the central , but not peripheral , anorectic effect of ex4 is insensitive to glp-1r antagonism , yet glp-1r is required for this effect . these data suggest that there are important differences between the in vivo pharmacological properties of glp-1 and ex4 within the cns . moreover , they underscore the need for a greater understanding of how these ligands interact with cns glp-1r , particularly in light of recent data revealing novel roles of cns glp-1r activity in the regulation of peripheral glucose homeostasis and cardiovascular function ( 56,57 ) . such an understanding is critical if we are to maximize the therapeutic benefit of ex4 and other glp-1based therapies .

objectiveglucagon - like peptide ( glp)-1 is a regulatory peptide synthesized in the gut and the brain that plays an important role in the regulation of food intake . both glp-1 and exendin ( ex)-4 , a long - acting glp-1 receptor ( glp-1r ) agonist , reduce food intake when administered intracerebroventricularly , whereas ex4 is much more potent at suppressing food intake when given peripherally . it has generally been hypothesized that this difference is due to the relative pharmacokinetic profiles of glp-1 and ex4 , but it is possible that the two peptides control feeding via distinct mechanisms.research design and methodsin this study , the anorectic effects of intracerebroventricular glp-1 and ex4 , and the sensitivity of these effects to glp-1r antagonism , were compared in rats . in addition , the glp-1r dependence of the anorectic effect of intracerebroventricular ex4 was assessed in glp-1r/ mice.resultsintracerebroventricular ex4 was 100-fold more potent than glp-1 at reducing food intake , and this effect was insensitive to glp-1r antagonism . however , glp-1r antagonists completely blocked the anorectic effect of intraperitoneal ex4 . despite the insensitivity of intracerebroventricular ex4 to glp-1r antagonism , intracerebroventricular ex4 failed to reduce food intake in glp-1r/ mice.conclusionsthese data suggest that although glp-1rs are required for the actions of ex4 , there appear to be key differences in how glp-1 and ex4 interact with central nervous system glp-1r and in how ex4 interacts with glp-1r in the brain versus the periphery . a better understanding of these unique differences may lead to expansion and/or improvement of glp-1based therapies for type 2 diabetes and obesity .

RESEARCH DESIGN AND METHODS Peptides. Food intake studies. c-Fos immunohistochemistry. Tissue culture studies. Statistical analysis. RESULTS Comparison of intracerebroventricular GLP-1 and Ex4-induced anorexia. Sensitivity of intracerebroventricular GLP-1 and Ex4 to GLP-1r antagonism. Potency of Ex4 and sensitivity to GLP-1r antagonism in vitro. Sensitivity of intraperitoneal Ex4 to GLP-1r antagonism. Effect of intracerebroventricular Ex4 in wild-type and GLP-1r DISCUSSION

based on these results , time courses of anorexia induced by intracerebroventricular glp-1 ( 3.0 nmol , 10.0 g ) and ex4 ( 0.03 nmol , 0.1 g ) were compared , and conditioned taste aversion ( cta ) to the same doses of intracerebroventricular glp-1 and ex4 was assessed as previously described ( 27 ) . to assess the ability of central glp-1r antagonism to block anorexia induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline , dhex ( 10.0 g , ) , or ex9 ( 100.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . finally , to assess the ability of central ex4 to reduce food intake in glp-1r mice , mice were treated with intracerebroventricular saline or ex4 ( 1.0 g ) . to assess the ability of central glp-1r antagonism to block neuronal activation induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline or dhex ( 10.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . based on these results , time courses of anorexia induced by intracerebroventricular glp-1 ( 3.0 nmol , 10.0 g ) and ex4 ( 0.03 nmol , 0.1 g ) were compared , and conditioned taste aversion ( cta ) to the same doses of intracerebroventricular glp-1 and ex4 was assessed as previously described ( 27 ) . to assess the ability of central glp-1r antagonism to block anorexia induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline , dhex ( 10.0 g , ) , or ex9 ( 100.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . finally , to assess the ability of central ex4 to reduce food intake in glp-1r mice , mice were treated with intracerebroventricular saline or ex4 ( 1.0 g ) . to assess the ability of central glp-1r antagonism to block neuronal activation induced by central glp-1 and ex4 , rats were pretreated with intracerebroventricular saline or dhex ( 10.0 g ) and then treated with intracerebroventricular saline , glp-1 ( 10.0 g ) , or ex4 ( 0.1 g ) . consistent with previous reports , intracerebroventricular glp-1 and ex4 elicited potent , dose - dependent reductions in 4-h food intake ( fig . specifically , 10.0 g of glp-1 and 0.1 g of ex4 produced comparable degrees of anorexia , reducing food intake to 56 and 45% of control values , respectively . these data indicate that , when administered into the third ventricle , ex4 is roughly 100-fold more potent than glp-1 at reducing food intake . c : time course of anorectic effects of intracerebroventricular glp-1 ( 3.0 nmol ) and ex4 ( 0.03 nmol ) over 24 h. , saline ; , glp-1 ( 3.0 nmol / l ) ; , ex4 ( 0.03 nmol / l ) . whereas 3.0 nmol ( 10.0 g ) of glp-1 and 0.03 nmol ( 0.1 g ) of ex4 both actively suppressed food intake up to 4 h , only ex4 elicited persistent anorexia that remained detectable throughout the 24 h of observation ( p < 0.05 , two - way repeated - measures anova with tukey 's post hoc test ) . furthermore , these doses of glp-1 and ex4 both led to the formation of a cta ( fig . interestingly , there was a strong trend toward a significantly lower preference ratio of ex4-treated rats versus glp-1treated rats ( p = 0.052 ) , suggesting that the aversive effects of ex4 were more pronounced than those of glp-1 . although previous studies have reported an inability to block certain effects of ex4 with glp-1r antagonists , these studies did not necessarily account for the significantly greater potency of ex4 over glp-1 . therefore , we sought to compare the ability of glp-1r antagonists to block anorexia and neuronal activation induced by doses of intracerebroventricular glp-1 and ex4 that produce effects of comparable magnitude . however , whereas 0.1 g of ex4 and 10.0 g of glp-1 elicited comparable degrees of anorexia , the doses of dhex and ex9 that nearly abolished glp-1induced anorexia failed to block the anorectic effect of ex4 ( fig . to determine whether neuronal activation in response to glp-1 and ex4 was also differentially sensitive to glp-1r antagonism , the effect of intracerebroventricular dhex to block c - fos immunoreactivity induced by intracerebroventricular glp-1 and ex4 was compared . the magnitude of c - fos immunoreactivity induced by glp-1 and ex4 was similar in the pvn and nts , whereas glp-1 induced slightly more c - fos immunoreactivity than ex4 in the cea . in the cea , dhex significantly blocked c - fos immunoreactivity induced by glp-1 ( p < 0.05 ) ; however , in the pvn and the nts , this difference failed to reach statistical significance . these results , combined with the food intake data , suggest that cns actions of ex4 are relatively insensitive to competitive glp-1r antagonism . because dhex , a validated but lesser used glp-1r antagonist ( 28,30,31 ) , failed to block anorexia and neuronal activation induced by intracerebroventricular ex4 , we sought to determine whether dhex is an effective antagonist of ex4 in vitro by assessing its ability to block insulin secretion induced by ex4 in the rat pancreatic islet cell line ins-1 . as expected , 1.0 nmol / l glp-1 significantly augmented insulin secretion above that of glucose alone , and this effect was completely blocked by coincubation with 100 nmol / l dhex ( fig . however , in contrast to our in vivo data , 0.01 nmol / l ex4 failed to augment insulin secretion , whereas 1.0 nmol / l ex4 had an effect that was comparable to 1.0 nmol / l glp-1 . to determine whether the insensitivity of ex4 to glp-1r antagonism was specific to cns administration , we assessed the ability of dhex to block anorexia induced by intraperitoneal ex4 . dhex , the same 100-fold excess of antagonist that failed to block anorexia induced by intracerebroventricular ex4 , significantly attenuated this effect ( p < 0.05 ) . the insensitivity of cns ex4 effects to glp-1r antagonism raises the possibility that ex4 may act in part via a glp-1r independent mechanism . to determine whether the glp-1r is required for the central anorectic effect of ex4 , intracerebroventricular ex4 was administered to wild - type and glp-1r mice . consistent with previous reports , intracerebroventricular glp-1 and ex4 elicited potent , dose - dependent reductions in 4-h food intake ( fig . specifically , 10.0 g of glp-1 and 0.1 g of ex4 produced comparable degrees of anorexia , reducing food intake to 56 and 45% of control values , respectively . these data indicate that , when administered into the third ventricle , ex4 is roughly 100-fold more potent than glp-1 at reducing food intake . c : time course of anorectic effects of intracerebroventricular glp-1 ( 3.0 nmol ) and ex4 ( 0.03 nmol ) over 24 h. , saline ; , glp-1 ( 3.0 nmol / l ) ; , ex4 ( 0.03 nmol / l ) . whereas 3.0 nmol ( 10.0 g ) of glp-1 and 0.03 nmol ( 0.1 g ) of ex4 both actively suppressed food intake up to 4 h , only ex4 elicited persistent anorexia that remained detectable throughout the 24 h of observation ( p < 0.05 , two - way repeated - measures anova with tukey 's post hoc test ) . furthermore , these doses of glp-1 and ex4 both led to the formation of a cta ( fig . interestingly , there was a strong trend toward a significantly lower preference ratio of ex4-treated rats versus glp-1treated rats ( p = 0.052 ) , suggesting that the aversive effects of ex4 were more pronounced than those of glp-1 . although previous studies have reported an inability to block certain effects of ex4 with glp-1r antagonists , these studies did not necessarily account for the significantly greater potency of ex4 over glp-1 . therefore , we sought to compare the ability of glp-1r antagonists to block anorexia and neuronal activation induced by doses of intracerebroventricular glp-1 and ex4 that produce effects of comparable magnitude . however , whereas 0.1 g of ex4 and 10.0 g of glp-1 elicited comparable degrees of anorexia , the doses of dhex and ex9 that nearly abolished glp-1induced anorexia failed to block the anorectic effect of ex4 ( fig . to determine whether neuronal activation in response to glp-1 and ex4 was also differentially sensitive to glp-1r antagonism , the effect of intracerebroventricular dhex to block c - fos immunoreactivity induced by intracerebroventricular glp-1 and ex4 was compared . at the same doses as used above , glp-1 and ex4 both induced c - fos immunoreactivity in identical brain regions , including the cea , pvn , and the nts ( fig c ; p < 0.05 by two - way anova with tukey 's post hoc test ) . the magnitude of c - fos immunoreactivity induced by glp-1 and ex4 was similar in the pvn and nts , whereas glp-1 induced slightly more c - fos immunoreactivity than ex4 in the cea . in the cea , dhex significantly blocked c - fos immunoreactivity induced by glp-1 ( p < 0.05 ) ; however , in the pvn and the nts , this difference failed to reach statistical significance . these results , combined with the food intake data , suggest that cns actions of ex4 are relatively insensitive to competitive glp-1r antagonism . because dhex , a validated but lesser used glp-1r antagonist ( 28,30,31 ) , failed to block anorexia and neuronal activation induced by intracerebroventricular ex4 , we sought to determine whether dhex is an effective antagonist of ex4 in vitro by assessing its ability to block insulin secretion induced by ex4 in the rat pancreatic islet cell line ins-1 . as expected , 1.0 nmol / l glp-1 significantly augmented insulin secretion above that of glucose alone , and this effect was completely blocked by coincubation with 100 nmol / l dhex ( fig . however , in contrast to our in vivo data , 0.01 nmol / l ex4 failed to augment insulin secretion , whereas 1.0 nmol / l ex4 had an effect that was comparable to 1.0 nmol / l glp-1 . # p < 0.05 vs. glucose + glp-1 ( 1.0 nmol / l ) or glucose + ex4 ( 1.0 to determine whether the insensitivity of ex4 to glp-1r antagonism was specific to cns administration , we assessed the ability of dhex to block anorexia induced by intraperitoneal ex4 . dhex , the same 100-fold excess of antagonist that failed to block anorexia induced by intracerebroventricular ex4 , significantly attenuated this effect ( p < 0.05 ) . the insensitivity of cns ex4 effects to glp-1r antagonism raises the possibility that ex4 may act in part via a glp-1r independent mechanism . to determine whether the glp-1r is required for the central anorectic effect of ex4 , intracerebroventricular ex4 was administered to wild - type and glp-1r mice . not only do our data confirm that central glp-1 and ex4 differ significantly in potency and duration of action , but they also reveal novel differences between the two peptides regarding sensitivity to glp-1r antagonism . ex4 , when administered into the cns , reduces food intake in a manner distinct from that of glp-1 . importantly , this difference in potency at 4 h can not simply be explained by differences in duration of action , as both 3.0 nmol of glp-1 and 0.03 nmol of ex4 reduced food intake to a comparable extent from 0 to 2 h and 2 to 4 h. however , in contrast to glp-1 , ex4 dynamically reduced food intake over 24 h of observation , indicating that even at significantly lower doses , central ex4 exhibits a significantly longer duration of action . consistent with our food intake data , 0.1 g of central ex4 produced an almost identical degree of neuronal activation as 10.0 g of glp-1 in the pvn and the nts but interestingly not in the cea . it is possible that because 100-fold less ex4 than glp-1 was administered , less peptide diffused through the neuropil to the cea , which , unlike the pvn and the nts , does not abut the ventricular system . although a proportional relationship between glp-1r mediated neuronal activity and behavioral responses has yet to be established , these data are significant because they suggest that enhanced visceral illness or aversive learning may in part underlie the potent anorectic effect of central ex4 . however , in vitro and ex vivo comparisons of binding affinity have yielded equivocal results ( 3538 ) , and it remains unclear whether glp-1 and ex4 remain bound to cns glp-1r for different periods of time . ( 39 ) revealed no difference in the ability of glp-1 and ex4 to desensitize the glp-1r in vitro . finally , it is possible that differential clearance and/or degradation of glp-1 versus ex4 account for their distinct pharmacological profiles within the cns . however , dhex is an effective antagonist of ex4 in vitro , as it completely blocked the enhancement of glucose - stimulated insulin secretion induced by ex4 in ins-1 cells . in addition , dhex is an effective antagonist of ex4 in vivo , as intraperitoneal dhex completely blocked anorexia induced by intraperitoneal ex4 . taken together , these data indicate that compared with glp-1 , ex4 is relatively insensitive to glp-1r antagonism . here , we closely controlled for this difference and found that pretreatment with glp-1r antagonists significantly blocked anorexia induced by central glp-1 but not an equipotent and , importantly , 100-fold lower dose of ex4 . moreover , this phenomenon is not secondary to differences in agonist duration of action , as it was observed at early time points when both glp-1 and ex4 dynamically reduced food intake . more difficult to explain , however , is the comparison to our peripheral ex4 food intake study , in which the same 100-fold excess of dhex , this time administered intraperitoneally , completely blocked intraperitoneal ex4-induced anorexia . perhaps the most obvious explanation for the discrepancies between central glp-1 and ex4 is that the latter acts in part independently of the glp-1r . however , consistent with previous reports ( 18 ) , central ex4 had no effect on either food intake or body weight in glp-1r mice , suggesting that the glp-1r is required for these effects . although none of these observations provides definitive evidence for glp-1r independent effects of ex4 , they do raise the possibility that ex4 may interact with the glp-1r in a species - dependent manner . although difficult to reconcile with the above data , our findings regarding central ex4 and glp-1r antagonists are consistent with several reports of in vivo effects of ex4 that are insensitive to glp-1r antagonists ( 2224 ) . however , because our experiments used ad libitum fed rats , whose circulating ghrelin levels should be low , and because ex4 is more efficacious in fed versus fasted rats ( 51 ) , it seems unlikely that ex4 's effects on ghrelin secretion underlie either its increased potency acutely or its insensitivity to glp-1r antagonists in the present studies . because studies have generally found no effect of ex4 in glp-1r mice ( 1721 ) , it seems reasonable to cite strictly pharmacological differences when explaining discrepancies between in vivo effects of glp-1 and ex4 . for instance , some in vitro studies have found ex4 to have greater potency and affinity for the glp-1r than native glp-1 ( 35 ) , but these differences , at least in potency , are significantly smaller than those reported here . however , studies have generally reported little to no difference in the ability of glp-1 versus ex4 to displace radio - labeled ex9 ( 5255 ) . taken together , our data , combined with the existing literature , provide conclusive evidence for distinct pharmacological profiles of glp-1 and ex4 , yet further studies are needed to understand whether pharmacological differences alone are sufficient to explain the unique in vivo effects of ex4 . in conclusion , our data indicate that the central , but not peripheral , anorectic effect of ex4 is insensitive to glp-1r antagonism , yet glp-1r is required for this effect . these data suggest that there are important differences between the in vivo pharmacological properties of glp-1 and ex4 within the cns . moreover , they underscore the need for a greater understanding of how these ligands interact with cns glp-1r , particularly in light of recent data revealing novel roles of cns glp-1r activity in the regulation of peripheral glucose homeostasis and cardiovascular function ( 56,57 ) .

visual attention is the selection of visual information for purposes such as in - depth processing , perception , or action control . because we have to select information at all times , understanding attention is a key to an understanding of almost any form of cognition . to date , however , the mechanisms by which attention operates are not fully understood . one persistent debate in this area concerns the role of inhibition of irrelevant stimuli as one form of top - down control over attention . whereas some researchers believe that inhibition of attention is a response to initial capture of attention and , thus , follows preceding attentional capture by an irrelevant stimulus , other researchers believe that active inhibition of attentional capture by an irrelevant stimulus is possible right from the start of such a stimulus . to start with the first proposition , many researchers argued that salient objects capture attention in a bottom - up way ( cf . [ 3 , 4 ] ) . according to the salience model of attention , any visual stimulus that stands out among its surroundings by a strong feature contrast in color , orientation , or luminance may capture attention in an exogenous stimulus - driven way , regardless of the current goal of the observer ( cf . [ 5 , 6 ] ) . in line with this prediction , an irrelevant color singleton distractor that is a stimulus with a color different from its surrounding stimuli , such as one green circle among several red circles , interferes with finding a shape - defined target stimulus ( i.e. , the one rectangle among several circles ) ( cf . ) . this is the case although attending to the specific color of the singleton is neither necessary nor helpful for finding the target . such findings have been attributed to the bottom - up capture of attention by an irrelevant singleton . as a consequence , attention is thought to first be distracted away from the relevant target and only later be redirected towards the target . this is possible after a deliberate inhibition of the irrelevant stimulus , allowing attention to disengage from the distractor . findings by kim and cave are in general agreement with this late - inhibition or disengagement hypothesis . these authors used a probe stimulus as a second target in a combined search and probe reaction task . critically , the probe was shown after the search display with the probe either at the position of the search display 's shape - defined target or at the position of the search display 's color - singleton distractor . with an interval of 60 ms between search display and probe , kim and cave observed ( nonsignificantly ) faster responses to probes at color - distractor positions than to probes at shape - target positions . however , with a cue - target onset asynchrony ( ctoa ) of 150 ms between the color - singleton distractor ( cue ) and the probe ( target ) , responses to probes at the position of the color - singleton distractor were significantly delayed relative to responses to probes presented at the location of the shape target . like theeuwes et al . , kim and cave took their results as an indication of bottom - up capture by the color - singleton distractor with a short ctoa , giving way to disengagement and even active inhibition with a long ctoa . the core notion of the disengagement hypothesis , that is , the idea of active inhibition following initial allocation of attention towards a stimulus , is also at the heart of another well - known phenomenon called inhibition of return ( ior ) . ior denotes the finding that attracting visual attention toward one position in space by a cue delays a second attention shift to the same position at a later point in time [ 810 ] . ior is observed with long ctoas and corresponds to longer reaction times where cue and target are presented at the same position ( sp ) compared to cue and target at different positions ( dp ) . thus , the idea of stimuli initially triggering attentional capture and later inhibition is a very dominant notion found throughout the attention literature . however , recent findings by mcdonald et al . and by ansorge et al . are potentially in disagreement with this late - inhibition or disengagement hypothesis for irrelevant stimuli . ansorge et al . asked their participants to saccade to one out of four positions , varying randomly from trial to trial ( see figure 1 for an illustration of a similar stimulus and task sequence ) . prior to the saccades , participants were presented with a relevant or an irrelevant color singleton cue . participants only had to attend to the relevant cue because this cue indicated the position of a discrimination target later in the trial . in contrast , the participants were asked to ignore the irrelevant cue : when an irrelevant cue was presented , no target discrimination was required so that it was safe to ignore this cue . in addition , ignorance of the irrelevant cue was encouraged : because cue and saccade target positions were uncorrelated ( cf . ) and because saccades require a prior attention shift to the target position [ 13 , 14 ] , the participants could fully concentrate on the saccade task and ignore the irrelevant cues completely . in contrast , the participants were forced to shift their attention to the relevant cue for the encoding of its position for the discrimination task and at the potential cost of a suboptimal preparation of their saccades . all of these cues were nonpredictive of the saccade target position , and relevant and irrelevant cues had different fixed colors , so that the participants knew exactly which color they had to attend to ( e.g. , red ) and which color they could ignore ( e.g. , green ) . under these conditions , ansorge et al . studied the time course of selective attentional capture and/or inhibition by looking at the development of the saccadic latencies across the latency distribution , from quick to slow saccades . with a long ctoa , and with relevant cues , ior followed initial capture : initial capture among the fast responses was reflected in quicker saccades to a saccade target at the same position ( sp ) as the cue compared to slower saccades to a saccade target at a different position ( dp ) than the cue . with relevant cues , this pattern reversed into ior among the slower responses . in contrast , with a long ctoa and irrelevant cues , inhibition in the form of slower saccades to sp than dp targets was found right from the beginning and without preceding capture effects . these findings point to a form of proactive inhibition of irrelevant cues , completely preventing attentional capture by the irrelevant cues , rather than late disengagement . in fact , the only reliable capture effect for irrelevant cues showed up in a condition with a different procedure and no subsequent inhibition ( experiment 4 ) . thus , no conclusion could be drawn about transitions from capture to disengagement . a recent study by mcdonald et al . equally found evidence for proactive inhibition of irrelevant stimuli without a trace of preceding attention capture . to discern between capture and inhibition , mcdonald and colleagues used two lateralized components of the event - related potential ( erp ) : the n2pc ( cf . [ 15 , 16 ] ) or posterior contralateral negativity ( pcn ) and the pd [ 18 , 19 ] . the n2pc has been widely used to investigate both stimulus - driven capture and top - down contingent capture [ 2125 ] . importantly , when mcdonald et al . tested for initial capture of attention by irrelevant stimuli , all they found was a pd , that is , evidence for proactive inhibition of the irrelevant stimuli . this was found after splitting the erps into fast and slow responses : the quickest target responses of the participants indicated proactive inhibition of attentional capture by an irrelevant distractor . even so , it is not entirely clear whether the findings reflected only early inhibition or whether some capture of the irrelevant singletons occurred before it was suppressed . regarding the findings of ansorge et al . , these authors used saccadic latencies after relatively long ctoas ( > 200 ms ) . this method is relatively insensitive to the early attentional effects , so that preceding attention capture even by irrelevant cues might have gone unnoticed ( see their condition with a ctoa of 200 ms ) . regarding the findings of mcdonald et al . , it is possible that their observations reflected a mixture of weaker capture effects of the irrelevant distractors in some of the trials and of stronger inhibition of distraction in other trials . as a result , a net inhibitory pd effect could have masked evidence for early capture in the form of an n2pc in the study of mcdonald et al . at least in the slower responses , there was also clear evidence for this possibility : on slow response trials ( ) there was neither an early distractor ( ) nor a late target n2pc ( ) . the absence of either n2pc suggests that the target and distractor n2pc wave cancelled each other out ( ) thus , to test once more whether capture by irrelevant cues could precede subsequent inhibition , we combined the methods of ansorge et al . and of mcdonald et al . , using two different measures for early and late effects based on the very same trials . late inhibition was assessed through the presence of ior in saccadic reaction times after a sufficiently long ctoa of 1 s. this procedure allows for the registration of an early capture effect by all singletons , without masking by a concomitant n2pc by the targets . in this situation , in addition , we conducted a median - split of the erps on the basis of whether a fast or a slow saccade was given that allowed us to test whether the fastest responses were associated with a pd component , similar to mcdonald et al . . the aim of the current experiment was to investigate the connection between attention capture by relevant and irrelevant stimuli and ( subsequent ) inhibition . we examined within the same trials ( 1 ) the amount of initial capture of attention by an irrelevant cue and a relevant cue in the form of the n2pc and ( 2 ) the amount of inhibition in the form of an early pd and late saccadic inhibition of return . in detail , in the first display of each trial , we used one of two color - singleton cues : the first cue was relevant in half of the trials and it was irrelevant in the other half of the trials . a relevant first cue had a fixed color ( e.g. , it was green ) , known to the participant . the participant had to look for the relevant first cue and importantly , also covertly , attend to its location because it indicated the position of a subsequent discrimination target . we call this cue relevant to make clear that its color serves the same purpose as a searched - for feature of a target in a standard color - search task . in contrast , the irrelevant first cue had a different color ( e.g. , it was blue if the relevant cue was green ) , also known to the participants . therefore , the participants could have ignored this cue completely and were not required to shift spatial attention to its location . we call this cue irrelevant because its color served the same purpose as the color of an irrelevant distractor in a visual search display . that is , the color of the irrelevant first cue indicated with 100% certainty that this stimulus could be safely ignored . to test ior after the first cue , our participants had to encode the position of a second singleton cue in a second display for a saccade in a subsequent third display ( see figure 1 ) . importantly , positions of the first cue and of the second saccade cue were uncorrelated . because the participants have to allocate their attention to the position of the saccade target ( cf . [ 13 , 14 , 28 , 29 ] ) , they had to disengage their attention away from any first cue and to redirect it towards the position of the second cue in anticipation of the saccade target . also , the ctoa was 1 s long allowing for both inhibition ( or disengagement ) of attention and saccadic inhibition ( of return ) . we therefore expected saccadic inhibition with respect to the position of the first or covert cue ( [ 11 , 30 , 31 ] ; see also ) . the question is whether with irrelevant first cues a capture effect in the form of an n2pc precedes this inhibition effect or whether inhibition is observed from the start , in the form of a pd . also , in the relevant condition , an n2pc to the first cue was to be expected because an attention shift to this first cue was required to encode its position . twelve volunteers participated but one was excluded because her saccade latencies were more than three standard deviations slower than that of the other participants . the remaining participants ( with a mean age of 25 years and a male / female ratio of 6 : 5 ) reported normal or corrected - to - normal vision . written and informed consent was obtained from each participant before the experiment . visual stimuli were presented on a 19-inch crt color monitor ( sony multiscan g400 ) , with a screen resolution of 1,024 768 pixels and a refresh rate of 100 hz . the participants sat at a distance of 57 cm from the screen in a quiet , dimly lit room , with their head resting on a chin rest to ensure a constant viewing distance and a straight - ahead gaze direction . the first and second displays were presented for 50 ms and the last display for 1 s. all displays were separated by an interstimulus interval of 450 ms , such that the onset asynchrony between two displays was 500 ms . a gray central fixation cross was presented on a black background ( < 1 cd / m ) , visible throughout each trial . the first display consisted of six equidistant placeholders , each in the shape of the digital letter 8 ( with a size of 1.7 1 and with stroke strength of .3 ) . a placeholder was located per each of the positions at 0 , 60 , 120 , 180 , 240 , and 300 from the vertical meridian that is , the shape-8s were presented equally spaced on the circumference of a virtual circle centered on the screen , with an eccentricity of 7. five placeholders were presented in gray ( cielab color coordinates : 6.9 , 16.8 ) , and one was presented in a different color , either in green ( cielab : 30.2 , 24.9 ) or blue ( cielab : 46.9 , 89 ) . it was always shown at one of the four lateral positions but never presented above or below the fixation . the shape-8s were replaced by three letters e and three digits 3 , in digital notation . five of these shapes were presented in gray and one was presented in red ( cielab : 47.6 , 41.1 ) . this red stimulus was the second or saccade cue and it could also only appear at one of the four lateral positions . the red singleton was called a saccade cue because this second cue served as the cue for the saccade target in the subsequent display . also , in this display , one figure served as a discrimination target if it had been cued by a relevant first cue ( blue or green cue ) in the preceding display , with relevant first cue color fixed across trials and balanced across participants . positions of the discrimination target and second ( or red ) cue were uncorrelated across trials . consequently , in 25% of the trials the discrimination target and second or saccade cue were at the same position ( sp condition ) , and in 75% of the trials they were at different positions ( dp condition ) . this display consisted solely of six empty circles surrounding the stimulus positions as used in the preceding displays . the saccade display was presented for 1 s. the color of the first singleton cue in the first screen indicated whether the discrimination task in the second screen had to be performed on a given trial . for instance , a first green singleton was linked to the discrimination task while a first blue singleton could be ignored , or vice versa . in the discrimination task , participants had to encode and remember the shape of the digit presented in the second screen at the position of the relevant first singleton cue . this was necessary for the report of this figure at the end of the trials . as soon as the third display , the saccade display , appeared the saccade had to be executed . after the saccade was executed , in a relevant - cue trial , participants typed the identity of the discrimination target letter ( i.e. , whether the letter e or the digit 3 was presented ) by pressing the marked buttons # f and # j labeled left and right on a standard keyboard directly in front of the participants . if no discrimination was necessary ( i.e. , after irrelevant cues ) , this part of the trial was skipped . participants started the next trial in a self - pace manner , by pressing the space bar . participants were informed that the color singleton cues could only appear at the four lateral positions on the screen and that the position of the second or saccade singleton cue was independent of the position of the first singleton cue . blocks consisted of 64 trials and feedback was given about whether the target discrimination was correct and about whether the saccade was registered during the third screen . altogether ten blocks of trials were conducted , of which the first was training and not analyzed . each factor combination of the variables discrimination target ( e or 3 ) , first cue position ( above / left , above / right , below / left , and below / right ) , first cue color ( blue , green ) , and second cue 's position ( above / left , above / right , below / left , and below / right ) was equally likely and presented in a pseudorandom order within each block . saccades were recorded with an eyelink 1000 desktop mount system ( sr research , mississauga , on , canada ) with a 35 mm lens and eyelink software version 4.52 , sampling at 1,000 hz . a 9-point calibration was used to adjust the eye - tracker before the experiment and in advance of every single block . saccadic reaction time ( saccadic rt ) was calculated as the time between ( 1 ) the onset of the third display ( with the saccade - target stimulus circle ) and ( 2 ) the time of a local velocity minimum that immediately preceded the point in time at which eye velocity exceeded 80/s . a saccade counted as correct if it landed in an area of 1.5 around the center of the saccade target . saccade landing position was calculated as the x - y coordinates of the eye - tracker signal at the time at which eye velocity returned to a presaccadic baseline level . also , if the eyes started to move earlier than 100 ms after the saccade target , a trial was discarded . dc - eeg was recorded from 23 scalp electrodes mounted in an elastic cap at standard positions of the extended 10/20 system at sites fpz , f7 , f3 , fz , f4 , f8 , fc5 , fc6 , t7 , c3 , cz , c4 , t8 , cp5 , cp6 , p7 , p3 , pz , p4 , p8 , o1 , o2 , and oz . the continuous eeg was sampled at a rate of 1,000 hz with a digital low - pass filter of 50 hz . all scalp electrodes were online referenced to a noncephalic sternovertebral site , above the seventh vertebra and the right manilum sternum . the vertical eog ( electrodes below and above the left eye ) and the horizontal eog ( electrodes at the outer canthi ) were recorded bipolarly , so as to delete trials with eye movements during the critical eeg recording interval . trials with saccades earlier than 100 ms after the saccade target ( detected with the eye - tracker ) or muscular artifacts ( exceeding 80 v at any electrode ) , as well as trials in which the target was not correctly discriminated , were excluded from analysis . erps were calculated for 400 ms after the first cue 's onset relative to a 50 msec precue baseline . n2pc amplitudes in response to the first color cue were calculated separately for left and right and relevant and irrelevant cue , collapsed across all saccade target positions as mean erp amplitudes at locations p3/4 in the 160270 ms interval after cue onset . a switch box was implemented behind the parallel port of the master to send one unique synchronization trigger every 500 ms ( one for the onset of the first display , one for the second display , and one for the third display in each trial ) in parallel , separately to the two slaves , eye - tracker and eeg recorder . in total , 17.5% of all trials were excluded . trials with saccades faster than 100 ms and slower than 1 s after the saccade target accounted for 8.1% , trials with saccades towards the wrong target or with muscular artifacts for another 6.4% , and trials with a false identification of the discrimination target for 3% . to take the dynamics of the saccadic response into account , saccadic rts were sorted and grouped into five percentiles from fast to slow ( cf . ) . this was done to test our hypotheses about ior with differently fast responses because the amount of capture and of ior does vary over time and an effect that is absent in the average of all responses can well be present when looking at only the faster or only the slower responses ( e.g. , [ 11 , 34 ] ) . as can be seen in figure 2 , from fast responses on the left to slow responses on the right this was reflected in faster saccadic rts under dp conditions ( broken lines ) as compared to sp conditions ( solid lines ) , more so with the irrelevant cues ( red lines ) than with the relevant cues ( blue lines ) . a repeated - measures anova with the variables position ( same versus different position of first or covert cue and saccade cue / target ) , cue type ( relevant first cue or irrelevant first cue ) , and percentile ( 1st to 5th ) revealed inhibition at the location of the first or covert cue only among the slowest responses in the form of slower saccadic latencies in sp than dp conditions . this was reflected in a significant interaction of position and percentile , f(4,40 ) = 2.79 , p < .05 . from the 1st to the 5th quintile , saccadic inhibition ( saccadic rt in sp conditions minus saccadic rt in dp conditions ) was 0 ms , 1 ms , 4 ms , 3 ms , and 32 ms ( 1st to 4th quintile , all ts < 1 ; 5th quintile , t(9 ) = 2.29 , p < .05 ) . in addition , we found faster saccadic rts in trials with a relevant than an irrelevant cue in the first display ( 241 ms versus 273 ms ) , resulting in a marginally significant main effect for cue type , ( 1,10 ) = 4.72 , p = .055 . there was also a trivial main effect of percentile ( increasing saccadic rts with percentile ) , ( 4,10 ) = 94.56 , p < .01 . further , there was a numerically stronger inhibitory effect on saccades after the irrelevant cue ( 10 ms ) than after the relevant cue ( 2 ms ) , as would be expected based on an active inhibition explanation . however , the two - way interaction of relevance and position was not significant , ( 1,10 ) = .55 , p = .48 , as was the three - way interaction , f < 1 . in sum , saccadic inhibition was selectively present in the slowest saccades and it was largely independent of the type of cue that was used in the first display . figure 3 shows erps time - locked to the first cue 's onset at lateral posterior electrodes p3 and p4 contra- and ipsilateral to the first cue , separately for cues with a relevant color ( panel a ) , cues with an irrelevant color ( panel b ) , and difference waves ( i.e. , contra- minus ipsilateral activity for relevant and irrelevant cues , panel c ) . the differences are depicted together with topographical erp - difference maps for the time window of the n2pc ( 160 ms to 270 ms ) . all erps are relative to a baseline from 50 ms before the first cue to the onset of the first cue . as can be seen , there was an n2pc in the relevant and in the irrelevant cueing conditions . also , by looking at figure 3 , it seems as if the n2pc started later and was weaker in the irrelevant than in the relevant cueing condition . these observations were confirmed in a repeated - measures anova with the variables cue type ( relevant or irrelevant cue ) , laterality ( electrode ipsi- or contralateral to the first cue ) , and hemisphere ( right or left hemisphere ) . the analysis revealed a significant main effect for laterality , ( 1,10 ) = 7.3 , p < .05 , and a significant interaction of laterality and cue type , f(1,10 ) = 10.14 , p < .01 . however , if the cue was relevant , the n2pc was stronger ( contra- minus ipsilateral activity : 0.76 v ) and started earlier than if the cue was irrelevant ( .30 v ) , as was shown in section 2.2.3 . we were concerned that the choice of the electrode locations of the n2pc might have been unfortunate . therefore , we repeated our major analysis of the n2pc in a repeated - measures anova with the additional variable site ( p3/p4 , p7/p8 , and o1/o2 ) and the variables cue type ( relevant or irrelevant cue ) and laterality ( electrode ipsi- or contralateral to the first cue ) as before . besides replicating the main effect of laterality , f(1,10 ) = 6.86 , p < .05 , and an interaction of laterality and cue type , f(1,10 ) = 5.56 , p < .01 , there were no significant main effects , all fs < 1.40 and all ps > .28 , and no significant interactions including the three - way interaction of site , cue type , and laterality , all fs < 2.10 all ps > .15 . in addition , the anova was also repeated with the erps pooled across p3 , p7 , and o1 ( for the left side ) and across p4 , p8 , and o2 ( for the right side ) . this anova also confirmed a laterality effect , ( 1,10 ) = 6.86 , p < .05 , and an interaction of laterality and cue type , f(1,10 ) = 5.56 , p < .01 , and no main effect of cue type , f < 1 . to demonstrate the earlier onset of the n2pc with relevant cues than with irrelevant cues , we split the n2pc window into an early phase ( 160 ms to 215 ms after the cue onset ) and into a late phase ( 215 ms to 270 ms after the cue onset ; cf . ) . in the early window , an anova revealed a significant two - way interaction of laterality and cue type , ( 1,10 ) = 30.17 , p < .01 . post - hoc t - tests revealed that the contra - to - ipsilateral negativity difference ( .78 v ) was only significant in the relevant condition , ( 10 ) = 4.33 , p < .01 , but not in the irrelevant condition ( .02 v ) , t(10 ) = .08 , p = .93 . a similar anova of the late time window only led to a main effect of laterality , ( 1 , 10 ) = 8.10 , p < .05 . the contra - to - ipsilateral negativity difference was about similar in relevant ( .77 v ) and irrelevant ( .66 v ) cueing conditions . there was neither a main effect of cue type , nor of hemisphere , nor any interaction between the variables , all other fs < 2.10 all ps > .18 . recently , mcdonald and colleagues showed that irrelevant distractors elicited a pd among the fastest responses . we therefore also repeated our anova of the activity at p3 and p4 , with only the fastest 50% of the saccades and the two within - participant variables cue type ( relevant or irrelevant cue ) , and laterality ( electrode ipsi- or contralateral to the first cue ) . again , activity was more negative at contra- than ipsilateral electrodes , ( 1 , 10 ) = 11.16 , p < .05 . this time , however , the interaction was far from significant , f < 1 . in contrast to the findings of mcdonald et al . , a more prominent n2pc rather than a pd was observed with the irrelevant singleton cues during the fastest responses . in the present study , we tested whether irrelevant cues were proactively inhibited or whether they captured attention before being inhibited . in line with the latter possibility , relevant , and importantly also irrelevant , cues elicited an n2pc and both stimuli led to inhibition of saccades 1 s after the cues . this was reflected in slower saccadic rts to targets in sp than dp conditions . in other words , we found the typical ior effect , an observation in line with the late inhibition or disengagement hypothesis of theeuwes et al . . this finding is also in agreement with prior findings of ansorge et al . with relevant cues . in their study , these authors found a capture effect of the relevant cues when a ctoa of 200 ms was used . ansorge et al . also observed that ior started earlier with an irrelevant cue than with a relevant cue . this particular finding could not be observed in the present study . in the present study , among the slowest responses , ior with irrelevant cues this latter finding is thus also not so well in line with theeuwes et al . 's disengagement theory , according to which one would have expected stronger disengagement or ior after irrelevant than after relevant cues . according to disengagement theory , only the stronger disengagement of attention that follows irrelevant cues accounts for seemingly stronger capture effects by relevant than irrelevant cues . clearly , this prediction of the disengagement theory was not confirmed . in contrast , our results suggested a mixture of early capture differences with more capture by relevant than irrelevant cues and a later disengagement effect that was numerically stronger with irrelevant than relevant cues , as two sources contributing to stronger capture effects by relevant than irrelevant cues . concerning stronger capture by relevant than irrelevant cues , this was reflected in the n2pc . when we looked at the n2pc as an index of the initial capture of attention , we found a larger overall n2pc . this reflected on average an earlier start of the n2pc elicited by the relevant cue . these findings are in line with prior findings showing an earlier or temporally less variable capture effect and often even a selective capture effect for top - down matching than nonmatching cues [ 12 , 21 , 23 , 35 , 36 ] . this difference in capture for top - down matching as compared to nonmatching cues is typically assumed to reflect either of two processes : selective top - down tuning to sets of features so that initial capture is restricted to the cues matching the set [ 12 , 37 ] or less inhibition of attention captured by the top - down matching cue [ 1 , 38 ] . with the current procedure , we can not decide which of these interpretations holds true , that is , whether the temporally more variable or trailing onset of the n2pc by the irrelevant cues reflected less initial capture by these cues or a combination of initial capture by the irrelevant cues and proactive inhibition of the irrelevant cues . with respect to the latter , however , we did not find any evidence for strong early proactive inhibition of the irrelevant cues in the form of a pd . the trailing of the n2pc for irrelevant cues might be a tentative hint for some proactive inhibition . without any proactive influence , one would expect similar onset times of the n2pc for relevant and irrelevant stimuli ( although the initially smaller n2pc for irrelevant stimuli may camouflage its early onset ) . in particular , prior studies found proactive inhibition in the form of a pd when only looking at the fastest responses . in contrast to this finding , early or proactive inhibition was not associated with the fastest responses in the present study . this was evident when we sorted the erps as to whether they were recorded in a trial with a quick or slow saccade : among the fast saccades , the n2pcs of irrelevant and relevant cues became even more similar . this means that in the present study , more proactive inhibition would have counteracted the irrelevant cue 's n2pc onset in the trials with the slower saccades . which factors might account for the differences between the present study and the previous study by mcdonald et al . ? to reconcile the different findings , results from kiss and colleagues might be of interest . these authors presented target and distractor simultaneously ( similar to ) and found proactive inhibition of the irrelevant distractor in the form of a pd when the display was shown for 200 ms but an n2pc plus subsequent inhibition ( again in the form of a pd but occurring at a later point in time ) when the display was presented until a response was given . this might indicate that the irrelevant distractor elicits an n2pc and captures attention when the participants have time for their attention to shift to the target so that the distractor - elicited capture is not masked by a concomitant target - elicited n2pc . this might also explain why we found an n2pc of the irrelevant cues whereas most contingent - capture studies did not find any evidence for capture by irrelevant singleton distractors ( e.g. , [ 21 , 39 ] ) . with respect to the finding of an n2pc to the irrelevant cue in the present study and its absence in prior studies , a few other procedural differences might also play a role . first of all , the relevant cue was 100% valid ( 100% sp ) ; that is , it predicted the discrimination target position with certainty . although there was no discrimination target in the irrelevant target position it is possible that a bit of the general informative value of the relevant cues spilled over to the irrelevant cues . in other words , participants might have inadvertently attended to the irrelevant cue on at least some trials , for example , because they were not paying close enough attention to the color of the first cue . in support of this possibility , it would have been possible to find the relevant cues by the so - called singleton search strategy [ 40 , 41 ] . in fact , the use of two different relevant colors one ( e.g. , blue ) for the first display 's relevant cue and another one ( red ) for the saccade cue in the second display might have encouraged our participants to use a singleton search strategy rather than a feature search strategy . a few findings seem to indicate that the use of a top - down set containing two relevant colors leads to the erroneous capture of attention by an irrelevant color - singleton distractor in at least some trials ( cf . in addition , participants might have actively searched for even the irrelevant cues because these cues informed the participants that they would not have to discriminate between the different target orientations and keep the cue 's position in mind . the relatively long ctoa might have encouraged this strategy further because it would have allowed sufficient time to first willingly attend to each cue relevant and irrelevant and then to return attention to a neutral position after the irrelevant cue and before the onset of the target . even though this particularity of our procedure might explain why we did find an n2pc for both relevant and irrelevant cues , it is important to note that we were still able to ascertain two things : first , recording eeg we were able to demonstrate capture where behavioral measures only indicated inhibition . thus , although one might argue that the difference in the way participants processed relevant and irrelevant cues in our study was only small , our eeg measure was definitely sensitive to it . in sum , we might not have ended the debate over early proactive inhibition for complete prevention of capture once and for all with our study . however , we provide one more piece in the puzzle and another demonstration of the usefulness of combining eeg with behavioral measures to obtain a more complete picture of the processes engaged through a given paradigm . a further point that needs , it would be strange if different degrees of initial capture by relevant versus irrelevant cues ultimately lead to relatively similar degrees of ior by these stimuli . however , researchers had argued from very early on that capture and certain forms of inhibition could be partly independent processes . today , it is clear that nonattentional factors like motor inhibition and sensory habituation can also contribute to inhibition [ 8 , 49 , 50 ] . therefore , it is in principle possible to find similar degrees of late inhibition after different degrees of capture [ 5153 ] or even more inhibition following less capture by an irrelevant stimulus . along similar lines , prinzmetal et al . reported that attention capture and ior are differentially modulated by , on the one hand , the number of potential target locations and , on the other , the presence of distractor stimuli in the target display . dissociations of attention capture and ior are also in line with neurophysiological observations suggesting that the two effects arise at different stages of processing and may therefore be modulated differentially ( e.g. , ) . in more functional terms , prinzmetal et al . recently suggested that attention capture may best be described by a serial search mechanism , reminiscent of the attentional spotlight that ( at least for top - down matching cues ) is first allocated to the cued location and has to be redirected on invalid ( dp ) trials . ior , however , may better be accounted for by a decision process in a competitive accumulator model in which the decision to respond to a particular location previously visited by attention is systematically delayed ( see also ) . in conclusion , the two mechanisms proposed for attention capture and ior are very distinct , supporting the possibility for dissociations . previous studies such as gibson and amelio failed to find any evidence for ior with color singletons , a result that was ascribed to the special role of abrupt onsets for the occurrence of ior . here , we show that relevant and even irrelevant color singletons lead to ior when an eye movement instead of a manual response is used and when the saccadic rt distribution is taken into account . in line with this interpretation , godijn and theeuwes and more recently priess et al . and ansorge et al in conclusion , in line with the late inhibition or disengagement theory , we have shown that the irrelevant and the relevant distractor first both captured attention ( reflected in their n2pcs ) before they were actively inhibited ( reflected in saccadic ior ) . this lack of proactive inhibition was also found if only the fastest responses were analyzed . however , we found little indication that ior was stronger after irrelevant than relevant cues . therefore , it is not likely that disengagement was the only responsible process . early inhibition ( among the slower responses ) or contingent capture must have also contributed to the n2pc differences between relevant and irrelevant cues .

attentional capture is usually stronger for task - relevant than irrelevant stimuli , whereas irrelevant stimuli can trigger equal or even stronger amounts of inhibition than relevant stimuli . capture and inhibition , however , are typically assessed in separate trials , leaving it open whether or not inhibition of irrelevant stimuli is a consequence of preceding attentional capture by the same stimuli or whether inhibition is the only response to these stimuli . here , we tested the relationship between capture and inhibition in a setup allowing for estimates of the capture and inhibition based on the very same trials . we recorded saccadic inhibition after relevant and irrelevant stimuli . at the same time , we recorded the n2pc , an event - related potential , reflecting initial capture of attention . we found attentional capture not only for , relevant but importantly also for irrelevant stimuli , although the n2pc was stronger for relevant than irrelevant stimuli . in addition , inhibition of saccades was the same for relevant and irrelevant stimuli . we conclude with a discussion of the mechanisms that are responsible for these effects .

1. Introduction 2. Experiment 3. Discussion 4. Conclusion

to date , however , the mechanisms by which attention operates are not fully understood . one persistent debate in this area concerns the role of inhibition of irrelevant stimuli as one form of top - down control over attention . whereas some researchers believe that inhibition of attention is a response to initial capture of attention and , thus , follows preceding attentional capture by an irrelevant stimulus , other researchers believe that active inhibition of attentional capture by an irrelevant stimulus is possible right from the start of such a stimulus . according to the salience model of attention , any visual stimulus that stands out among its surroundings by a strong feature contrast in color , orientation , or luminance may capture attention in an exogenous stimulus - driven way , regardless of the current goal of the observer ( cf . in line with this prediction , an irrelevant color singleton distractor that is a stimulus with a color different from its surrounding stimuli , such as one green circle among several red circles , interferes with finding a shape - defined target stimulus ( i.e. this is the case although attending to the specific color of the singleton is neither necessary nor helpful for finding the target . such findings have been attributed to the bottom - up capture of attention by an irrelevant singleton . this is possible after a deliberate inhibition of the irrelevant stimulus , allowing attention to disengage from the distractor . critically , the probe was shown after the search display with the probe either at the position of the search display 's shape - defined target or at the position of the search display 's color - singleton distractor . however , with a cue - target onset asynchrony ( ctoa ) of 150 ms between the color - singleton distractor ( cue ) and the probe ( target ) , responses to probes at the position of the color - singleton distractor were significantly delayed relative to responses to probes presented at the location of the shape target . , kim and cave took their results as an indication of bottom - up capture by the color - singleton distractor with a short ctoa , giving way to disengagement and even active inhibition with a long ctoa . the core notion of the disengagement hypothesis , that is , the idea of active inhibition following initial allocation of attention towards a stimulus , is also at the heart of another well - known phenomenon called inhibition of return ( ior ) . ior is observed with long ctoas and corresponds to longer reaction times where cue and target are presented at the same position ( sp ) compared to cue and target at different positions ( dp ) . thus , the idea of stimuli initially triggering attentional capture and later inhibition is a very dominant notion found throughout the attention literature . are potentially in disagreement with this late - inhibition or disengagement hypothesis for irrelevant stimuli . in addition , ignorance of the irrelevant cue was encouraged : because cue and saccade target positions were uncorrelated ( cf . ) all of these cues were nonpredictive of the saccade target position , and relevant and irrelevant cues had different fixed colors , so that the participants knew exactly which color they had to attend to ( e.g. studied the time course of selective attentional capture and/or inhibition by looking at the development of the saccadic latencies across the latency distribution , from quick to slow saccades . with a long ctoa , and with relevant cues , ior followed initial capture : initial capture among the fast responses was reflected in quicker saccades to a saccade target at the same position ( sp ) as the cue compared to slower saccades to a saccade target at a different position ( dp ) than the cue . in contrast , with a long ctoa and irrelevant cues , inhibition in the form of slower saccades to sp than dp targets was found right from the beginning and without preceding capture effects . these findings point to a form of proactive inhibition of irrelevant cues , completely preventing attentional capture by the irrelevant cues , rather than late disengagement . in fact , the only reliable capture effect for irrelevant cues showed up in a condition with a different procedure and no subsequent inhibition ( experiment 4 ) . equally found evidence for proactive inhibition of irrelevant stimuli without a trace of preceding attention capture . to discern between capture and inhibition , mcdonald and colleagues used two lateralized components of the event - related potential ( erp ) : the n2pc ( cf . the n2pc has been widely used to investigate both stimulus - driven capture and top - down contingent capture [ 2125 ] . tested for initial capture of attention by irrelevant stimuli , all they found was a pd , that is , evidence for proactive inhibition of the irrelevant stimuli . this was found after splitting the erps into fast and slow responses : the quickest target responses of the participants indicated proactive inhibition of attentional capture by an irrelevant distractor . even so , it is not entirely clear whether the findings reflected only early inhibition or whether some capture of the irrelevant singletons occurred before it was suppressed . , it is possible that their observations reflected a mixture of weaker capture effects of the irrelevant distractors in some of the trials and of stronger inhibition of distraction in other trials . the absence of either n2pc suggests that the target and distractor n2pc wave cancelled each other out ( ) thus , to test once more whether capture by irrelevant cues could precede subsequent inhibition , we combined the methods of ansorge et al . , using two different measures for early and late effects based on the very same trials . in this situation , in addition , we conducted a median - split of the erps on the basis of whether a fast or a slow saccade was given that allowed us to test whether the fastest responses were associated with a pd component , similar to mcdonald et al . the aim of the current experiment was to investigate the connection between attention capture by relevant and irrelevant stimuli and ( subsequent ) inhibition . we examined within the same trials ( 1 ) the amount of initial capture of attention by an irrelevant cue and a relevant cue in the form of the n2pc and ( 2 ) the amount of inhibition in the form of an early pd and late saccadic inhibition of return . in detail , in the first display of each trial , we used one of two color - singleton cues : the first cue was relevant in half of the trials and it was irrelevant in the other half of the trials . we call this cue relevant to make clear that its color serves the same purpose as a searched - for feature of a target in a standard color - search task . we call this cue irrelevant because its color served the same purpose as the color of an irrelevant distractor in a visual search display . also , the ctoa was 1 s long allowing for both inhibition ( or disengagement ) of attention and saccadic inhibition ( of return ) . we therefore expected saccadic inhibition with respect to the position of the first or covert cue ( [ 11 , 30 , 31 ] ; see also ) . the question is whether with irrelevant first cues a capture effect in the form of an n2pc precedes this inhibition effect or whether inhibition is observed from the start , in the form of a pd . the first display consisted of six equidistant placeholders , each in the shape of the digital letter 8 ( with a size of 1.7 1 and with stroke strength of .3 ) . a placeholder was located per each of the positions at 0 , 60 , 120 , 180 , 240 , and 300 from the vertical meridian that is , the shape-8s were presented equally spaced on the circumference of a virtual circle centered on the screen , with an eccentricity of 7. five placeholders were presented in gray ( cielab color coordinates : 6.9 , 16.8 ) , and one was presented in a different color , either in green ( cielab : 30.2 , 24.9 ) or blue ( cielab : 46.9 , 89 ) . this red stimulus was the second or saccade cue and it could also only appear at one of the four lateral positions . consequently , in 25% of the trials the discrimination target and second or saccade cue were at the same position ( sp condition ) , and in 75% of the trials they were at different positions ( dp condition ) . the saccade display was presented for 1 s. the color of the first singleton cue in the first screen indicated whether the discrimination task in the second screen had to be performed on a given trial . in the discrimination task , participants had to encode and remember the shape of the digit presented in the second screen at the position of the relevant first singleton cue . this was necessary for the report of this figure at the end of the trials . after the saccade was executed , in a relevant - cue trial , participants typed the identity of the discrimination target letter ( i.e. , whether the letter e or the digit 3 was presented ) by pressing the marked buttons # f and # j labeled left and right on a standard keyboard directly in front of the participants . participants were informed that the color singleton cues could only appear at the four lateral positions on the screen and that the position of the second or saccade singleton cue was independent of the position of the first singleton cue . each factor combination of the variables discrimination target ( e or 3 ) , first cue position ( above / left , above / right , below / left , and below / right ) , first cue color ( blue , green ) , and second cue 's position ( above / left , above / right , below / left , and below / right ) was equally likely and presented in a pseudorandom order within each block . saccade landing position was calculated as the x - y coordinates of the eye - tracker signal at the time at which eye velocity returned to a presaccadic baseline level . n2pc amplitudes in response to the first color cue were calculated separately for left and right and relevant and irrelevant cue , collapsed across all saccade target positions as mean erp amplitudes at locations p3/4 in the 160270 ms interval after cue onset . trials with saccades faster than 100 ms and slower than 1 s after the saccade target accounted for 8.1% , trials with saccades towards the wrong target or with muscular artifacts for another 6.4% , and trials with a false identification of the discrimination target for 3% . a repeated - measures anova with the variables position ( same versus different position of first or covert cue and saccade cue / target ) , cue type ( relevant first cue or irrelevant first cue ) , and percentile ( 1st to 5th ) revealed inhibition at the location of the first or covert cue only among the slowest responses in the form of slower saccadic latencies in sp than dp conditions . in addition , we found faster saccadic rts in trials with a relevant than an irrelevant cue in the first display ( 241 ms versus 273 ms ) , resulting in a marginally significant main effect for cue type , ( 1,10 ) = 4.72 , p = .055 . however , the two - way interaction of relevance and position was not significant , ( 1,10 ) = .55 , p = .48 , as was the three - way interaction , f < 1 . in sum , saccadic inhibition was selectively present in the slowest saccades and it was largely independent of the type of cue that was used in the first display . , contra- minus ipsilateral activity for relevant and irrelevant cues , panel c ) . the differences are depicted together with topographical erp - difference maps for the time window of the n2pc ( 160 ms to 270 ms ) . as can be seen , there was an n2pc in the relevant and in the irrelevant cueing conditions . however , if the cue was relevant , the n2pc was stronger ( contra- minus ipsilateral activity : 0.76 v ) and started earlier than if the cue was irrelevant ( .30 v ) , as was shown in section 2.2.3 . we were concerned that the choice of the electrode locations of the n2pc might have been unfortunate . therefore , we repeated our major analysis of the n2pc in a repeated - measures anova with the additional variable site ( p3/p4 , p7/p8 , and o1/o2 ) and the variables cue type ( relevant or irrelevant cue ) and laterality ( electrode ipsi- or contralateral to the first cue ) as before . in addition , the anova was also repeated with the erps pooled across p3 , p7 , and o1 ( for the left side ) and across p4 , p8 , and o2 ( for the right side ) . to demonstrate the earlier onset of the n2pc with relevant cues than with irrelevant cues , we split the n2pc window into an early phase ( 160 ms to 215 ms after the cue onset ) and into a late phase ( 215 ms to 270 ms after the cue onset ; cf . ) we therefore also repeated our anova of the activity at p3 and p4 , with only the fastest 50% of the saccades and the two within - participant variables cue type ( relevant or irrelevant cue ) , and laterality ( electrode ipsi- or contralateral to the first cue ) . this time , however , the interaction was far from significant , f < 1 . in the present study , we tested whether irrelevant cues were proactively inhibited or whether they captured attention before being inhibited . in line with the latter possibility , relevant , and importantly also irrelevant , cues elicited an n2pc and both stimuli led to inhibition of saccades 1 s after the cues . in other words , we found the typical ior effect , an observation in line with the late inhibition or disengagement hypothesis of theeuwes et al . in their study , these authors found a capture effect of the relevant cues when a ctoa of 200 ms was used . according to disengagement theory , only the stronger disengagement of attention that follows irrelevant cues accounts for seemingly stronger capture effects by relevant than irrelevant cues . in contrast , our results suggested a mixture of early capture differences with more capture by relevant than irrelevant cues and a later disengagement effect that was numerically stronger with irrelevant than relevant cues , as two sources contributing to stronger capture effects by relevant than irrelevant cues . concerning stronger capture by relevant than irrelevant cues , this was reflected in the n2pc . when we looked at the n2pc as an index of the initial capture of attention , we found a larger overall n2pc . this reflected on average an earlier start of the n2pc elicited by the relevant cue . this difference in capture for top - down matching as compared to nonmatching cues is typically assumed to reflect either of two processes : selective top - down tuning to sets of features so that initial capture is restricted to the cues matching the set [ 12 , 37 ] or less inhibition of attention captured by the top - down matching cue [ 1 , 38 ] . with the current procedure , we can not decide which of these interpretations holds true , that is , whether the temporally more variable or trailing onset of the n2pc by the irrelevant cues reflected less initial capture by these cues or a combination of initial capture by the irrelevant cues and proactive inhibition of the irrelevant cues . with respect to the latter , however , we did not find any evidence for strong early proactive inhibition of the irrelevant cues in the form of a pd . the trailing of the n2pc for irrelevant cues might be a tentative hint for some proactive inhibition . without any proactive influence , one would expect similar onset times of the n2pc for relevant and irrelevant stimuli ( although the initially smaller n2pc for irrelevant stimuli may camouflage its early onset ) . in particular , prior studies found proactive inhibition in the form of a pd when only looking at the fastest responses . this was evident when we sorted the erps as to whether they were recorded in a trial with a quick or slow saccade : among the fast saccades , the n2pcs of irrelevant and relevant cues became even more similar . these authors presented target and distractor simultaneously ( similar to ) and found proactive inhibition of the irrelevant distractor in the form of a pd when the display was shown for 200 ms but an n2pc plus subsequent inhibition ( again in the form of a pd but occurring at a later point in time ) when the display was presented until a response was given . this might also explain why we found an n2pc of the irrelevant cues whereas most contingent - capture studies did not find any evidence for capture by irrelevant singleton distractors ( e.g. in other words , participants might have inadvertently attended to the irrelevant cue on at least some trials , for example , because they were not paying close enough attention to the color of the first cue . a few findings seem to indicate that the use of a top - down set containing two relevant colors leads to the erroneous capture of attention by an irrelevant color - singleton distractor in at least some trials ( cf . in addition , participants might have actively searched for even the irrelevant cues because these cues informed the participants that they would not have to discriminate between the different target orientations and keep the cue 's position in mind . the relatively long ctoa might have encouraged this strategy further because it would have allowed sufficient time to first willingly attend to each cue relevant and irrelevant and then to return attention to a neutral position after the irrelevant cue and before the onset of the target . even though this particularity of our procedure might explain why we did find an n2pc for both relevant and irrelevant cues , it is important to note that we were still able to ascertain two things : first , recording eeg we were able to demonstrate capture where behavioral measures only indicated inhibition . thus , although one might argue that the difference in the way participants processed relevant and irrelevant cues in our study was only small , our eeg measure was definitely sensitive to it . however , we provide one more piece in the puzzle and another demonstration of the usefulness of combining eeg with behavioral measures to obtain a more complete picture of the processes engaged through a given paradigm . a further point that needs , it would be strange if different degrees of initial capture by relevant versus irrelevant cues ultimately lead to relatively similar degrees of ior by these stimuli . however , researchers had argued from very early on that capture and certain forms of inhibition could be partly independent processes . therefore , it is in principle possible to find similar degrees of late inhibition after different degrees of capture [ 5153 ] or even more inhibition following less capture by an irrelevant stimulus . reported that attention capture and ior are differentially modulated by , on the one hand , the number of potential target locations and , on the other , the presence of distractor stimuli in the target display . dissociations of attention capture and ior are also in line with neurophysiological observations suggesting that the two effects arise at different stages of processing and may therefore be modulated differentially ( e.g. ior , however , may better be accounted for by a decision process in a competitive accumulator model in which the decision to respond to a particular location previously visited by attention is systematically delayed ( see also ) . here , we show that relevant and even irrelevant color singletons lead to ior when an eye movement instead of a manual response is used and when the saccadic rt distribution is taken into account . however , we found little indication that ior was stronger after irrelevant than relevant cues . therefore , it is not likely that disengagement was the only responsible process . early inhibition ( among the slower responses ) or contingent capture must have also contributed to the n2pc differences between relevant and irrelevant cues .

the poor survival statistics of epithelial ovarian cancer ( eoc ) are mentioned by way of introduction in almost all review literature pertaining to the disease . unfortunately , in the past forty years there have been only small improvements in overall ovarian cancer survival rates . specific challenges to the treatment of eoc include the problems of late detection , metastasis within the peritoneal cavity , drug resistance , and cancer recurrence even after initial response to treatment . up to 90% of eocs do not have an identified genetic component , and the development of specific and sensitive screening tools has proven elusive . a metabolic approach to the targeted treatment of eoc has the potential to address many of the issues that make this the most deadly gynecologic cancer . in recent years , it has been noticed that the influence of lifestyle , in particular the high - fat western diet , is associated with the multisite development of cancers . the state of chronic positive energy balance is linked to a cluster of conditions including impaired glucose regulation and insulin resistance , collectively called the metabolic syndrome . hyperglycemia is a distinguishing feature of over - nutrition and it is believed to be an independent risk factor for cancer development . to provide an idea of the clinical importance of hyperglycemia , it is estimated that the incidence of type two diabetes mellitus ( t2 dm ) , a common consequence of the syndrome , will double in many regions in the next fifteen years . however , the burden of t2 dm , where as many as one third of individuals are undiagnosed , almost certainly underestimates the true incidence of abnormal glucose homeostasis in the population . given the emerging association between hyperglycemia and cancer , it is conceivable that there will be an increase in the incidence of eoc in the near future . we hypothesize that hyperglycemia provides a nutrient - rich , growth signal - rich environment for epithelial ovarian cancer cells , where tumour formation and growth is encouraged by free radical - induced dna damage . we address possible cellular mechanisms by which a hyperglycemic environment may increase the rate of development of ovarian tumours , and discuss the implications for metabolically targeted eoc treatments . while significant associations have been reported between elevated glucose [ 4 , 5 ] , glycemic load , t2 dm [ 2 , 7 ] , and a number of cancers , there is little information to support the influence of preexisting hyperglycemia on eoc . however , much of the literature relating cancer and glucose abnormalities comes from clinical or epidemiological studies that were not originally designed to evaluate the effects of hyperglycemia on cancer development . this is a particular limitation when looking at eoc because of its relatively low population incidence . in addition , many of the studies used diabetic status or a single glucose measurement as a proxy for classifying glucose abnormalities , likely underestimating the true hyperglycemic population . the changing profile of insulin status over the course of t2 dm probably further obscured any associations and there was poor consideration of confounding variables such as insulin , obesity , medication , and time since diagnosis . the design of these population studies presumed that hyperglycemia was a direct and sufficient cause of ovarian cancer , when it may in fact be more important in the growth promotion of previously transformed cells . in this way , end - point analyses such as case - control or retrospective cohort studies a more useful consideration may be that of time to tumour development in patients with hyperglycemia . for example , in women already diagnosed with ovarian cancer , high glucose appears to be a poor prognostic factor . a further complication of these studies is that both hyperglycemia and eoc are notoriously quiet diseases in their early stages . this makes it very difficult from a population health standpoint to infer an association , or suggest causality , as the underlying pathologies of both diseases begin and may interact well before diagnosis . although population - based studies have not been supportive for a role of preexisting hyperglycemia in the development of ovarian cancer , recent basic science still suggests that eoc may be subject to the influence of high blood sugar . the rate of glucose uptake , which increases with increasing extracellular glucose , has been linked with tumour aggressiveness . eoc cells are also sensitive to complete glucose deprivation than nontransformed ovarian epithelial cells ; thus , they may also be very responsive to hyperglycemia . the impact of hyperinsulinemia on cancer has received much more research attention than the impact of hyperglycemia , although the two conditions are very closely related . insulin is mitogenic via its signaling through the insulin receptor and the insulin - like growth factor ( igf ) pathways and direct anabolic signaling which is mediated by changes in the insulin receptor ( ir ) population . expression of the ir is elevated in eoc , suggesting a tumour - promoting role in this cancer . however , we contend that the specific impact of hyperglycemia on eoc is also an important area of research as abnormalities in glucose metabolism typically underlie hyperinsulinemia . thus , although insulin has direct , isolated actions on tumour growth , changes in glucose metabolism predispose changes in insulin signaling . in addition , it is becoming clear that there are insulin - independent mechanisms of glucose action on cancer risk , particularly through energy - sensing pathways and glucotoxic damage . almost 80 years ago , dr . warburg observed that , compared to normal cells , cancer cells show a preference for glycolysis and lactate production over oxidative phosphorylation . because glycolysis is 18 times less efficient at producing atp , this glycolytic switch suggests that cancer cells have an inherently high need for glucose . furthermore , tumours are very active metabolically and require copious amounts of cellular fuel to meet growth demands . aerobic glycolysis has been successfully exploited in eoc diagnostics in which tumour visualization occurs through the detection of the differential uptake of glucose in cancer cells compared to normal cells . the use of fdg - pet ( 18-fluoro-2-deoxyglucose positron emission tomography ) demonstrates the association between tumour growth and energy availability . warburg 's initial observation was bolstered by evidence that tumours could induce host hypoglycemia in a tumour mass - dependent fashion [ 18 , 19 ] . in many tumour - bearing animals , there appeared to be host compensation for hypoglycemia at the level of the liver , with increased gluconeogenesis and glycogen mobilization . local hypoglycemia in the area around the tumour was particularly pronounced [ 18 , 20 ] . it was found that while tumours had the capacity to take up larger volumes of glucose in mildly hyperglycemic environments they were poor at compensating for low blood glucose by increasing glucose uptake [ 18 , 20 ] . an important role for the vasculature was identified in hyperglycemic conditions , as tumours were able to increase glucose uptake by increasing glucose transfer across the capillary walls . following these metabolic observations , a number of groups looked at the growth characteristics of tumours in hyperglycemic environments . it was reported widely that profound hypoinsulinemia usually caused by chemical destruction of pancreatic -cells consistently caused a decrease in tumour growth [ 19 , 21 , 22 ] . however , in diabetic animals , combined treatment of both antitumour and antihyperglycemia therapies gave the best tumour - reductive outcome . although they demonstrated a negative effect of hyperglycemia on tumour development , these early studies have a number of limitations . the alloxan used to induce diabetes was toxic and administered systemically , and so may have had effects outside the target endocrine cells within the pancreas . also , the studies that showed a decrease in tumour mass in the diabetic animals did not report the changes with respect to total animal mass , which is generally smaller in the diabetic animals . the studies also seem to make the assumption that all glucose taken up is immediately metabolized . however , it was noted independently by several groups that glucose uptake was too high to be fully explained by the amount of tumour growth [ 20 , 23 ] . these results suggest the possibility that cancer cells may be able to store fuel in times of high abundance . nigam et al . concluded that low glycogen was due to defective glycogen synthesis and reported low activities of key glyconeogenic enzymes phosphoglucomutase and glycogen synthetase as compared to normal tissues . the low tumour glycogen was also linked to abnormally high rates of glycogen breakdown by phosphorylase . a recent article looking at glycogen levels in human colorectal cancer , however , reported that tumour cells actually had higher glycogen content than normal tissue . the authors noted that there was less glycogen in poorly differentiated tumours compared to well - differentiated tumours , suggesting that low glycogen may be an indicator of a poor prognosis . they also found a very clear negative correlation between glycogen level and proliferation index . it seems likely that , given the high rate of fuel usage in a tumour , at normoglycemic levels , there would be little need for storage as most would be used immediately . this brings up an intriguing question : could hyperfueled conditions favour a storage phenotype in cancer cells ? this might explain the low growth rates of tumours in type one diabetic conditions . glycogen synthase kinase 3 ( gsk3 ) phosphorylates and inactivates glycogen synthase , preventing the formation of glycogen . high levels of gsk3 have been implicated in the progression of a number of cancers , including ovarian cancer . gsk3 affects tumour growth through many different mechanisms , including nf-b and wnt signaling activation . although it was not discussed in the literature reviewed here , gsk overexpression may be linked with glycogen storage and proliferation index . in summary , despite a number of investigations , carbohydrate metabolism by tumours we consider the possible effects of glucose on eoc development to be either permissive or contributing . permissive effects are those that alter the energy status of cells , allowing tumour cells greater access to fuel . contributing effects are those that directly damage protein or dna in some cancer - promoting way . persistent elevations in blood sugar occur once hypersecretion of insulin is no longer able to compensate for combined insulin resistance and high glucose levels . the failure of insulin to facilitate glucose entry into cells is evaluated on a continuum , meaning that patients may have significant pathological changes while being in a in fact , by time of diagnosis of t2 dm , hyperglycemia has already caused vascular complications in at least 20% of patients [ 3 , 27 ] . however , poor glycemic control is not solely due to impaired insulin signaling , as glucose has the ability to regulate its own clearance by mass action . glucose self - regulation is impaired in people with hyperglycemia , leading to a state of glucose resistance . chronic hyperglycemia downregulates enzymes responsible for glucose metabolism , including those of the energy - sensing amp - activated protein kinase ( ampk ) pathway . this results in fewer glucose transporters translocating to the cell surface , further impeding the cell 's ability to take up fuel . thus , the effects of glucose join insulin resistance in maintaining and exacerbating hyperglycemia . it is postulated that where there is energy available tumour cells will have a suitable soil to grow . the biological plausibility of this excess energy hypothesis has been supported by a number of in vitro studies : yamamoto et al . found that increasing glucose concentration in the culture media of mcf-7 breast cancer cells increased proliferation , mediated by an upregulation of cdk2 and cyclin d1 . in a line of choriocarcinoma cells , sustained hyperglycemia was found to stimulate the cell 's glucose transport system , increasing glucose uptake rates . in contrast , most nontransformed cells downregulate glucose transport in the presence of hyperglycemia . studies in human breast cancer xenografts also suggest that the amount of glucose metabolism is not determined by metabolic demand , but rather by substrate availability . together , these findings support the idea that the fuel availability in hyperglycemia may be permissive for cancer growth . in hyperglycemia - induced insulin resistance , the correlation between cancer risk and t2 dm suggests that where normal cells fail metabolically cancer cells excel . it is possible that in hyperglycemia cancer cells are inherently better at responding to the effects of insulin compared to insulin - resistant normal cells . in their 2004 paper , gatenby and gillies argue that mutations affecting substrate use can not be early events in carcinogenesis because they would offer no advantage when there are no constraints on fuel availability , which typically arise in a larger tumour mass . while this is true in a normal cellular environment , in hyperglycemia better access to the abundance of extracellular glucose , therefore , confers a selective growth advantage and could be an early marker of tumourigenic potential . if conditions such as dysglycemia and diabetes prove to be involved in eoc initiation as well as promotion , then we propose that the selective pressures of the energy status may be an early event in the formation of eoc tumours . cells that are best able to survive high glycemic conditions necessarily have a key characteristic of cancer cells , essentially obtaining self - sufficiency in growth signals . thus , cancers that arise in a hyperglycemic environment may represent an unregulated adaptive survival response . although there is currently no directly supportive data for this hypothesis , possible mechanisms for this relationship are described in the following sections . the consequences of chronic exposure to high glucose tend to be detrimental to cellular function and affect the physiology of the normal ovary . in fact , most long - term diabetic complications ( retinopathy , neuropathy , and nephropathy ) are consequences of hyperglycemia and can not be reversed despite glucose normalization . however , this damage might also provide a mutational advantage to some cells by altering cellular proteins or dna . cancer development is often thought of in terms of a series of hits . the conditions of the tumour microenvironment , many of them determined by an altered metabolic profile , have been shown to contribute to the genetic instability of cancer cells , providing the necessary hits for a more aggressive tumour . acidity , hypoxia , and formation of reactive oxygen species may all be enhanced in tumours in a hyperglycemic environment . in tumour cells , high glucose flux through the glycolytic pathway produces large quantities of lactate , resulting in tumour tissue with ph 0.5 units lower than normal tissue . cancerous cells adapt to this acidification , exhibiting maximal growth at the relatively low ph of about 6.8 . tumours also have a capacity , similar to working skeletal muscle , to share lactate between hypoxic and nonhypoxic cells , so it is not extruded as a waste product . despite these survival adaptations , tumour acidity has been shown to impair dna repair mechanisms and to upregulate angiogenic molecules such as vascular endothelial growth factor ( vegf ) and il-8 in order to enhance lactate clearance [ 43 , 44 ] . experimental evidence demonstrates that the acidic environment is supportive of tumourigenesis , increasing resistance to chemotherapy , mutation rate , and invasion capability . the acid - mediated tumour invasion hypothesis postulates that h ions from the tumour microenvironment diffuse down their concentration gradient into the surrounding normal tissue . because the normal cells can not survive the increase in acidity , the border of malignant tissue is progressively pushed forward . in fact , mathematical modeling has shown that tumour acid production alone can explain patterns of tumour growth . the effects of acidity are particularly important in a hyperglycemic environment because increased glucose flux through tumour cells has been shown to create a large increase in lactate production [ 33 , 49 ] . the characteristic microvascular damage caused by hyperglycemia may lead to periods of hypoxia , possibly through a nitric - oxide - mediated mechanism . the bioavailability of the vasodilator is decreased in diabetes as it is scavenged by superoxide radicals to form the highly reactive onoo molecule . transient hypoxia is thought to be one of the strongest pressures for cells to undergo transformation and is a central hypothesis explaining the glycolytic switch [ 13 , 53 ] . hypoxic conditions also increase the activity of hypoxia - inducible factor ( hif-1 ) and vegf , which are strongly associated with both tumour angiogenesis and eoc tumour aggressiveness [ 54 , 55 ] . levels of oxidative stress reflect the ability to balance production and elimination of highly reactive free radicals , which include the family of reactive oxygen species ( ros ) . oxidative stress is known to be higher in diabetic patients than in healthy individuals , and it is often cited as a unifying theory to explain tissue damage by hyperglycemia . because ros can also create dna damage through a number of mechanisms , it has similarly been proposed that carcinogenesis in general is caused by oxidative stress . this stress in ovarian epithelial cells specifically is thought to be a potential initiator of tumourigenesis . hyperglycemia also causes increased flux of glucose through the aldose - reductase ( polyol ) pathway , which has been postulated to increase sensitivity to oxidative stress by reducing regeneration of the antioxidant glutathione . while epidemiological studies evaluating antioxidant use in diabetes [ 52 , 61 ] and ovarian cancer have not been conclusive , preliminary results suggest that this therapeutic avenue is worth further exploration . a recent study of flavonoids with antioxidant effects found that they inhibited cell growth and vegf expression in ovarian cancer cells . much of the tissue damage and cellular dysfunction associated with hyperglycemia has been attributed to advanced glycation end products ( ages ) created by the nonenzymatic glycation of proteins . while age accumulation is a normal part of aging , it occurs at an accelerated rate in diabetes where progressive modifications can lead to irreversible cross - linking , impairing the actions of other molecules [ 64 , 65 ] . receptors for age ( rage ) mediate many more severe actions and potentiate the cellular response . rages are upregulated by presence of age ligands , and age - rage binding protects the ligands , allowing them to persist in the environment . age - rage interaction has been shown to stimulate tumour cell growth or invasiveness in pancreatic cancer , melanoma , and glioma , while blocking the rage inhibits tumour formation and metastasis [ 68 , 69 ] . the ovarian surface epithelium may be particularly susceptible to the effects of glycation damage because not only the tissue is well vascularized , but it is also in constant contact with peritoneal fluid , whose glucose content is reflective of blood glucose levels . mechanistically , age - rage signaling has been linked to induction of an inflammatory response in the vasculature , as well as an increase in matrix metalloproteinases ( mmps)-2 and -9 , and may , therefore , play a role in determining tumour invasiveness . because age - rage signaling seems to be part of the chronic rather than acute response , its contributions to the development of tumour formation are quite plausible . glucose reactivity in hyperglycemia can also lead to glucose autoxidation , generating hydroxide radicals , and contributing to the burden of oxidative stress . also , apart from rage signaling , glucose moieties on proteins can donate electrons to form hydrogen peroxide , directly activating nf-b [ 73 , 74 ] and contributing to an inflammatory response . there is evidence that changes to local tissue can enhance the possibility of tumour spread , possibly implicating glucose - induced damage to the peritoneal cavity as a permissive factor for ovarian tumour metastasis . glucose is a large , hydrophilic molecule that can not diffuse through the lipid bilayer of cells on its own , and thus requires specific transporter proteins . glucose enters cells by facilitated diffusion mainly through glucose transporters ( gluts ) , and the activation of glut genes is one of the earliest events in oncogenesis . because gluts have a role in glucose sensing and respond to extracellular glucose concentrations , these transporters may be very important in a hyperglycemic environment . glut1 in particular is highly expressed in ovarian cancer , where tumour status ( benign , borderline , or malignant ) is correlated with the level of glut1 expression . almost all invasive epithelial carcinomas are positive for glut1 , independent of stage , grade , or histological subtype [ 79 , 80 ] . antibodies to glut1 decrease proliferation , induce apoptosis in nonsmall cell lung cancer and breast cancer cell lines , and appear to synergize with a number of chemotherapeutics to enhance their apoptotic effects . very recently , another class of transporters , sodium / glucose cotransporters ( sglts ) , was shown to be associated with the epidermal growth factor receptor ( egfr ) in cancer cells . the authors of the study proposed that sglts may enhance tumourigenesis by making cells independent of the glucose concentration gradient , allowing them to take up fuel in any situation . this hypothesis is in line with the proposal made here that permissive effects of glucose are cancer causing : removing restrictions on fuel availability seems to enhance tumourigenesis . the egfr is particularly important in ovarian cancer ; it is normally expressed on ovarian surface epithelium and is often overexpressed in eoc . in both rats and humans , hyperglycemia has been shown to be a major cause of the systemic inflammatory response [ 99 , 100 ] . both oxidative stress and age - rage signaling are also implicated in promoting systemic inflammation in hyperglycemic environments . inflammation is thought to be associated with cancer development mechanistically because of rapid cell division , dna excision and repair , oxidative stress , and high concentrations of cytokines and prostaglandins ; all of which are promoters of mutagenesis . moreover , inflammation has been proposed as a unifying hypothesis for the development of eoc . the high concentrations of circulating growth - promoting and inflammatory cytokines as a result of hyperglycemia may mean that factors , which normally in an autocrine or paracrine fashion are instead coming from the systemic environment and exerting an endocrine effect , potentiate tumour growth . in support of this , animal knockout studies have shown that mmp production by the host may be more important in carcinogenesis than mmp production by tumour cells themselves . cytokines can affect eoc tumour growth by acting as growth factors , increasing angiogenesis , or an immunomodulatory pathway whereby they prevent cellular recognition and destruction of the tumour . a number of cytokines that are increased as part of systemic inflammation in diabetes also have tumour promoting effects in ovarian cancer . il-1 and tnf- are thought to increase production of il-6 , which promotes cell attachment and migration and also blocks apoptosis induced by cytotoxic agents . in addition , although tgf- normally inhibits epithelial cell proliferation , repeated exposure to high levels may attenuate the response of cancerous epithelial cells . the inflammatory hypothesis lends itself to testing with a variety of antiinflammatory drugs and indeed early studies show promise . a study evaluating human ovarian tumours in nude mice concluded that cyclooxygenase inhibitors limited tumour growth , in part through an antiangiogenic mechanism . epidemiologically , patients with chronic aspirin , nsaid , or acetaminophen use have been shown to have a reduced risk of eoc . however , as with antioxidant trials , these observational studies are still preliminary . recently , the inflammation associated with postovulatory follicle repair has received attention as a possible contributor to eoc promotion . the incessant ovulation hypothesis purports that the repeated damage and repair cycles associated with ovulation enhance the possibility for mutagenesis . incessant ovulation also increases the likelihood that inclusion cysts will form , trapping epithelial cells in the hormone - rich environment of the ovarian stroma [ 1 , 111 ] . if these trapped cells are inappropriately maintained , they are more likely to transform [ 111113 ] . wound healing in hyperglycemia is characteristically slow and almost certainly influenced by the effects of inflammation and damage from glycation . lowered nitric oxide bioavailability in combination with the tissue damage caused by hyperglycemia may be partly responsible . in one study age - rage blockade decreased expression of inflammatory cytokines and mmps resulting in normalization of wound closure in a genetic mouse model of diabetes . taken together , the mutagenic risk and the risk of entrapment in inclusion cysts from repeated ovulations , combined with impaired wound healing , might mean a greater risk for ovarian cancer development in a hyperglycemic environment . this idea provides a possible mechanism by which hyperglycemia may initiate cancer , in addition to playing a role in promotion of eoc from an unrelated transforming event . folkman , solid tumours must recruit new blood vessels in order to grow beyond 1 - 2 mm in size . most of the tumour vascularization occurs through angiogenesis , which is the development of new blood vessels from preexisting vasculature . the angiogenic process is regulated by a balance between pro- and anti - angiogenic factors and in ovarian cancer there is a concomitant overexpression of proangiogenic factors and an inhibition of anti - angiogenic molecules . there are numerous reports concluding that elevated glucose levels contribute to increased angiogenic processes . granulosa cell tumours of the ovary have been shown to have increased expression of members of both the glycolytic and angiogenic pathways . glucose directly increases expression of the potent proangiogenic factor vegf , which is thought to be the mechanism involved in the vascular complications associated with diabetes ( reviewed in ) . in a similar fashion to tumour cells , endothelial cells that comprise the tumour vasculature also increase their utilization of glucose . glucose transporter expression is increased in the hypoxic environment associated with most solid tumours , and glucose increases survival of both tumour epithelial and endothelial cells . because increased tumour vascularity is correlated with increased metastatic potential and tumour progression [ 121 , 122 ] unfortunately , inflammation may be self - promoting as increased tumour perfusion can act to further exacerbate the immune response . in addition to the direct effects of glucose , the effects of inflammation are likely mediated by vegf . inflammatory mediators upregulate vegf and vegf receptors , which are correlated with the clinical outcomes of ovarian cancer patients . for example , nf-b can promote angiogenesis by activating vegf and il-8 and may be central to inflammation - induced tumour growth and progression . the possible impact of hyperglycemia - related inflammation on cancer suggests that anti - angiogenic molecules such as thrombospondin-1 may be of great benefit in treating diabetic tumours . the relationship between angiogenesis , inflammation , and carcinogenesis is illustrated by the fact that a number of anti - angiogenic drugs that are promising in the treatment of cancer are also effective against chronic inflammatory diseases . because of the multitude of protumour effects of glucose , it is intuitive that glucose deprivation may be a potent antitumour treatment approach . from the literature , it is apparent that glucose is an important energy substrate , survival factor , and proangiogenic molecule . there are a number of antihyperglycemic treatments currently available for reducing serum blood glucose and these drugs may effectively inhibit glucose availability to the tumour . although the effects of antihyperglycemic drugs are well documented in diabetes , their effects in cancer are relatively unknown . preliminary reports show that these drugs may have multi - modal effects in slowing tumour growth . in an approach similar to that using anti - angiogenic drugs , the class of antihyperglycemic drugs such as metformin and rosiglitazone may reduce glucose availability to the tumour and essentially starve the tumour of nutrients . these drugs have also been shown to have direct effects on metabolic and signaling pathways that may be independent of glucose . metformin is in the biguanide class of antidiabetic drugs and decreases circulating glucose levels by suppressing hepatic production of glucose . metformin , by reducing insulin and glucose levels , reduced the size and increased latency of mammary adenocarcinomas in her-2/neu transgenic mice , demonstrating a potent antitumour effect . in vitro , metformin significantly inhibits the growth of epithelial ovarian cancer cells and may potentiate the effects of the common chemotherapy drug cisplatin . metformin may preferentially increase peripheral glucose uptake in skeletal muscle , as administration increases ampk activity in skeletal muscle and stimulates translocation of muscle glut-4 . this favoured packaging of glucose into skeletal muscle cells would decrease serum glucose levels and availability to the tumour cells resulting in nutrient depletion . stimulation of ampk by metformin also contributes to the reduced hepatocyte production of glucose . in fact , ampk activation is associated with an inhibition of tumourigenesis through apoptosis induction , decreased cell proliferation and may be a communal molecule utilized by metformin as well as a number of anti - tumour drugs that have been shown to have effects in eoc . c93 , resveratrol [ 13 , 136 ] , 2-deoxy - d - glucose , and aicar are targeted therapies that are effective in the treatment of ovarian cancer . interestingly , these molecules also cause the stimulation of ampk , indicating a common pathway intersection with metformin . although not yet investigated , there is a possibility that metformin may have a synergistic interaction with these molecules , in addition to its glucose deprivation effects . rosiglitazone is another antidiabetic agent in the thiazolidinedione class of drugs designed to reduce the hyperglycemia associated with this disease . rosiglitazone activates the peroxisome proliferator activated receptors ( ppar ) in target tissues , increasing insulin sensitivity and decreasing serum levels of glucose . as with metformin , rosiglitazone also stimulates increased expression of glut-4 causing glucose uptake in skeletal muscle . one of the mechanisms by which rosiglitazone may have a significant antitumour effect is through the inhibition of angiogenesis . rosiglitazone has been shown to inhibit vegf - induced angiogenesis and is suggested as a treatment option for vascular disorders associated with diabetes such as diabetic retinopathy , macular degeneration , and so forth . as vegf expression is significantly elevated in eoc and is responsible for some of the ovarian tumour vascularization ( reviewed in ) , rosiglitazone may have a bimodal anti - tumour effect by decreasing glucose availability and also by reducing tumour angiogenesis . simply by decreasing tumour vascularity an emerging view of cancer relies on an initiation - promotion paradigm that suggests a fundamental role of the tumour environment on cancer development . new data suggests that hyperglycemia may be a contributing factor to the onset and progression of eoc through a number of complex mechanisms ( summarized in figure 1 ) . we propose that hyperglycemia has important effects on both the progression and somatic evolution of epithelial ovarian cancer . altered glucose homeostasis is common in cancer patients , so antihyperglycemic therapies are applicable to even those who have normal blood sugar . although there are a number of cellular mechanisms through which hyperglycemia may effect the promotion or initiation of ovarian cancer , there is almost no in vivo experimental data exploring the link between hyperglycemia and eoc . further research in this area not only has applications in the development of cancer therapeutics , but also will provide new insights into eoc pathogenesis , early detection , and possible prevention .

epithelial ovarian cancer ( eoc ) is the most lethal gynecologic cancer and also one of the most poorly understood . other health issues that are affecting women with increasing frequency are obesity and diabetes , which are associated with dysglycemia and increased blood glucose . the warburg effect describes the ability of fast - growing cancer cells to preferentially metabolize glucose via anaerobic glycolysis rather than oxidative phosphorylation . recent epidemiological studies have suggested a role for hyperglycemia in the pathogenesis of a number of cancers . if hyperglycemia contributes to tumour growth and progression , then it is intuitive that antihyperglycemic drugs may also have an important antitumour role . preliminary reports suggest that these drugs not only reduce available plasma glucose , but also have direct effects on cancer cell viability through modification of molecular energy - sensing pathways . this review investigates the effect that hyperglycemia may have on eoc and the potential of antihyperglycemic drugs as therapeutic adjuncts .

1. Introduction 2. Hyperglycemia and EOC: Epidemiological Evidence 3. Hyperinsulinemia versus Hyperglycemia 4. Hyperglycemia 5. Inflammation and EOC 6. Glucose, Angiogenesis, and Tumour Formation 7. Antidiabetic Drugs as Targeted EOC Therapy 8. Summary and Conclusions

the poor survival statistics of epithelial ovarian cancer ( eoc ) are mentioned by way of introduction in almost all review literature pertaining to the disease . unfortunately , in the past forty years there have been only small improvements in overall ovarian cancer survival rates . up to 90% of eocs do not have an identified genetic component , and the development of specific and sensitive screening tools has proven elusive . a metabolic approach to the targeted treatment of eoc has the potential to address many of the issues that make this the most deadly gynecologic cancer . in recent years , it has been noticed that the influence of lifestyle , in particular the high - fat western diet , is associated with the multisite development of cancers . hyperglycemia is a distinguishing feature of over - nutrition and it is believed to be an independent risk factor for cancer development . to provide an idea of the clinical importance of hyperglycemia , it is estimated that the incidence of type two diabetes mellitus ( t2 dm ) , a common consequence of the syndrome , will double in many regions in the next fifteen years . however , the burden of t2 dm , where as many as one third of individuals are undiagnosed , almost certainly underestimates the true incidence of abnormal glucose homeostasis in the population . given the emerging association between hyperglycemia and cancer , it is conceivable that there will be an increase in the incidence of eoc in the near future . we hypothesize that hyperglycemia provides a nutrient - rich , growth signal - rich environment for epithelial ovarian cancer cells , where tumour formation and growth is encouraged by free radical - induced dna damage . while significant associations have been reported between elevated glucose [ 4 , 5 ] , glycemic load , t2 dm [ 2 , 7 ] , and a number of cancers , there is little information to support the influence of preexisting hyperglycemia on eoc . however , much of the literature relating cancer and glucose abnormalities comes from clinical or epidemiological studies that were not originally designed to evaluate the effects of hyperglycemia on cancer development . the design of these population studies presumed that hyperglycemia was a direct and sufficient cause of ovarian cancer , when it may in fact be more important in the growth promotion of previously transformed cells . for example , in women already diagnosed with ovarian cancer , high glucose appears to be a poor prognostic factor . although population - based studies have not been supportive for a role of preexisting hyperglycemia in the development of ovarian cancer , recent basic science still suggests that eoc may be subject to the influence of high blood sugar . the rate of glucose uptake , which increases with increasing extracellular glucose , has been linked with tumour aggressiveness . the impact of hyperinsulinemia on cancer has received much more research attention than the impact of hyperglycemia , although the two conditions are very closely related . insulin is mitogenic via its signaling through the insulin receptor and the insulin - like growth factor ( igf ) pathways and direct anabolic signaling which is mediated by changes in the insulin receptor ( ir ) population . however , we contend that the specific impact of hyperglycemia on eoc is also an important area of research as abnormalities in glucose metabolism typically underlie hyperinsulinemia . thus , although insulin has direct , isolated actions on tumour growth , changes in glucose metabolism predispose changes in insulin signaling . in addition , it is becoming clear that there are insulin - independent mechanisms of glucose action on cancer risk , particularly through energy - sensing pathways and glucotoxic damage . warburg observed that , compared to normal cells , cancer cells show a preference for glycolysis and lactate production over oxidative phosphorylation . because glycolysis is 18 times less efficient at producing atp , this glycolytic switch suggests that cancer cells have an inherently high need for glucose . aerobic glycolysis has been successfully exploited in eoc diagnostics in which tumour visualization occurs through the detection of the differential uptake of glucose in cancer cells compared to normal cells . the use of fdg - pet ( 18-fluoro-2-deoxyglucose positron emission tomography ) demonstrates the association between tumour growth and energy availability . an important role for the vasculature was identified in hyperglycemic conditions , as tumours were able to increase glucose uptake by increasing glucose transfer across the capillary walls . following these metabolic observations , a number of groups looked at the growth characteristics of tumours in hyperglycemic environments . although they demonstrated a negative effect of hyperglycemia on tumour development , these early studies have a number of limitations . also , the studies that showed a decrease in tumour mass in the diabetic animals did not report the changes with respect to total animal mass , which is generally smaller in the diabetic animals . however , it was noted independently by several groups that glucose uptake was too high to be fully explained by the amount of tumour growth [ 20 , 23 ] . the authors noted that there was less glycogen in poorly differentiated tumours compared to well - differentiated tumours , suggesting that low glycogen may be an indicator of a poor prognosis . this brings up an intriguing question : could hyperfueled conditions favour a storage phenotype in cancer cells ? high levels of gsk3 have been implicated in the progression of a number of cancers , including ovarian cancer . in summary , despite a number of investigations , carbohydrate metabolism by tumours we consider the possible effects of glucose on eoc development to be either permissive or contributing . the failure of insulin to facilitate glucose entry into cells is evaluated on a continuum , meaning that patients may have significant pathological changes while being in a in fact , by time of diagnosis of t2 dm , hyperglycemia has already caused vascular complications in at least 20% of patients [ 3 , 27 ] . chronic hyperglycemia downregulates enzymes responsible for glucose metabolism , including those of the energy - sensing amp - activated protein kinase ( ampk ) pathway . the biological plausibility of this excess energy hypothesis has been supported by a number of in vitro studies : yamamoto et al . found that increasing glucose concentration in the culture media of mcf-7 breast cancer cells increased proliferation , mediated by an upregulation of cdk2 and cyclin d1 . studies in human breast cancer xenografts also suggest that the amount of glucose metabolism is not determined by metabolic demand , but rather by substrate availability . together , these findings support the idea that the fuel availability in hyperglycemia may be permissive for cancer growth . it is possible that in hyperglycemia cancer cells are inherently better at responding to the effects of insulin compared to insulin - resistant normal cells . in their 2004 paper , gatenby and gillies argue that mutations affecting substrate use can not be early events in carcinogenesis because they would offer no advantage when there are no constraints on fuel availability , which typically arise in a larger tumour mass . while this is true in a normal cellular environment , in hyperglycemia better access to the abundance of extracellular glucose , therefore , confers a selective growth advantage and could be an early marker of tumourigenic potential . if conditions such as dysglycemia and diabetes prove to be involved in eoc initiation as well as promotion , then we propose that the selective pressures of the energy status may be an early event in the formation of eoc tumours . cells that are best able to survive high glycemic conditions necessarily have a key characteristic of cancer cells , essentially obtaining self - sufficiency in growth signals . although there is currently no directly supportive data for this hypothesis , possible mechanisms for this relationship are described in the following sections . the conditions of the tumour microenvironment , many of them determined by an altered metabolic profile , have been shown to contribute to the genetic instability of cancer cells , providing the necessary hits for a more aggressive tumour . tumours also have a capacity , similar to working skeletal muscle , to share lactate between hypoxic and nonhypoxic cells , so it is not extruded as a waste product . the characteristic microvascular damage caused by hyperglycemia may lead to periods of hypoxia , possibly through a nitric - oxide - mediated mechanism . the bioavailability of the vasodilator is decreased in diabetes as it is scavenged by superoxide radicals to form the highly reactive onoo molecule . transient hypoxia is thought to be one of the strongest pressures for cells to undergo transformation and is a central hypothesis explaining the glycolytic switch [ 13 , 53 ] . hypoxic conditions also increase the activity of hypoxia - inducible factor ( hif-1 ) and vegf , which are strongly associated with both tumour angiogenesis and eoc tumour aggressiveness [ 54 , 55 ] . levels of oxidative stress reflect the ability to balance production and elimination of highly reactive free radicals , which include the family of reactive oxygen species ( ros ) . because ros can also create dna damage through a number of mechanisms , it has similarly been proposed that carcinogenesis in general is caused by oxidative stress . hyperglycemia also causes increased flux of glucose through the aldose - reductase ( polyol ) pathway , which has been postulated to increase sensitivity to oxidative stress by reducing regeneration of the antioxidant glutathione . while epidemiological studies evaluating antioxidant use in diabetes [ 52 , 61 ] and ovarian cancer have not been conclusive , preliminary results suggest that this therapeutic avenue is worth further exploration . a recent study of flavonoids with antioxidant effects found that they inhibited cell growth and vegf expression in ovarian cancer cells . much of the tissue damage and cellular dysfunction associated with hyperglycemia has been attributed to advanced glycation end products ( ages ) created by the nonenzymatic glycation of proteins . rages are upregulated by presence of age ligands , and age - rage binding protects the ligands , allowing them to persist in the environment . the ovarian surface epithelium may be particularly susceptible to the effects of glycation damage because not only the tissue is well vascularized , but it is also in constant contact with peritoneal fluid , whose glucose content is reflective of blood glucose levels . mechanistically , age - rage signaling has been linked to induction of an inflammatory response in the vasculature , as well as an increase in matrix metalloproteinases ( mmps)-2 and -9 , and may , therefore , play a role in determining tumour invasiveness . because age - rage signaling seems to be part of the chronic rather than acute response , its contributions to the development of tumour formation are quite plausible . glucose enters cells by facilitated diffusion mainly through glucose transporters ( gluts ) , and the activation of glut genes is one of the earliest events in oncogenesis . because gluts have a role in glucose sensing and respond to extracellular glucose concentrations , these transporters may be very important in a hyperglycemic environment . glut1 in particular is highly expressed in ovarian cancer , where tumour status ( benign , borderline , or malignant ) is correlated with the level of glut1 expression . antibodies to glut1 decrease proliferation , induce apoptosis in nonsmall cell lung cancer and breast cancer cell lines , and appear to synergize with a number of chemotherapeutics to enhance their apoptotic effects . very recently , another class of transporters , sodium / glucose cotransporters ( sglts ) , was shown to be associated with the epidermal growth factor receptor ( egfr ) in cancer cells . the authors of the study proposed that sglts may enhance tumourigenesis by making cells independent of the glucose concentration gradient , allowing them to take up fuel in any situation . the egfr is particularly important in ovarian cancer ; it is normally expressed on ovarian surface epithelium and is often overexpressed in eoc . in both rats and humans , hyperglycemia has been shown to be a major cause of the systemic inflammatory response [ 99 , 100 ] . inflammation is thought to be associated with cancer development mechanistically because of rapid cell division , dna excision and repair , oxidative stress , and high concentrations of cytokines and prostaglandins ; all of which are promoters of mutagenesis . the high concentrations of circulating growth - promoting and inflammatory cytokines as a result of hyperglycemia may mean that factors , which normally in an autocrine or paracrine fashion are instead coming from the systemic environment and exerting an endocrine effect , potentiate tumour growth . in support of this , animal knockout studies have shown that mmp production by the host may be more important in carcinogenesis than mmp production by tumour cells themselves . cytokines can affect eoc tumour growth by acting as growth factors , increasing angiogenesis , or an immunomodulatory pathway whereby they prevent cellular recognition and destruction of the tumour . a number of cytokines that are increased as part of systemic inflammation in diabetes also have tumour promoting effects in ovarian cancer . il-1 and tnf- are thought to increase production of il-6 , which promotes cell attachment and migration and also blocks apoptosis induced by cytotoxic agents . a study evaluating human ovarian tumours in nude mice concluded that cyclooxygenase inhibitors limited tumour growth , in part through an antiangiogenic mechanism . recently , the inflammation associated with postovulatory follicle repair has received attention as a possible contributor to eoc promotion . incessant ovulation also increases the likelihood that inclusion cysts will form , trapping epithelial cells in the hormone - rich environment of the ovarian stroma [ 1 , 111 ] . lowered nitric oxide bioavailability in combination with the tissue damage caused by hyperglycemia may be partly responsible . taken together , the mutagenic risk and the risk of entrapment in inclusion cysts from repeated ovulations , combined with impaired wound healing , might mean a greater risk for ovarian cancer development in a hyperglycemic environment . this idea provides a possible mechanism by which hyperglycemia may initiate cancer , in addition to playing a role in promotion of eoc from an unrelated transforming event . most of the tumour vascularization occurs through angiogenesis , which is the development of new blood vessels from preexisting vasculature . the angiogenic process is regulated by a balance between pro- and anti - angiogenic factors and in ovarian cancer there is a concomitant overexpression of proangiogenic factors and an inhibition of anti - angiogenic molecules . granulosa cell tumours of the ovary have been shown to have increased expression of members of both the glycolytic and angiogenic pathways . glucose directly increases expression of the potent proangiogenic factor vegf , which is thought to be the mechanism involved in the vascular complications associated with diabetes ( reviewed in ) . in a similar fashion to tumour cells , endothelial cells that comprise the tumour vasculature also increase their utilization of glucose . glucose transporter expression is increased in the hypoxic environment associated with most solid tumours , and glucose increases survival of both tumour epithelial and endothelial cells . in addition to the direct effects of glucose , the effects of inflammation are likely mediated by vegf . inflammatory mediators upregulate vegf and vegf receptors , which are correlated with the clinical outcomes of ovarian cancer patients . for example , nf-b can promote angiogenesis by activating vegf and il-8 and may be central to inflammation - induced tumour growth and progression . the possible impact of hyperglycemia - related inflammation on cancer suggests that anti - angiogenic molecules such as thrombospondin-1 may be of great benefit in treating diabetic tumours . the relationship between angiogenesis , inflammation , and carcinogenesis is illustrated by the fact that a number of anti - angiogenic drugs that are promising in the treatment of cancer are also effective against chronic inflammatory diseases . because of the multitude of protumour effects of glucose , it is intuitive that glucose deprivation may be a potent antitumour treatment approach . from the literature , it is apparent that glucose is an important energy substrate , survival factor , and proangiogenic molecule . there are a number of antihyperglycemic treatments currently available for reducing serum blood glucose and these drugs may effectively inhibit glucose availability to the tumour . although the effects of antihyperglycemic drugs are well documented in diabetes , their effects in cancer are relatively unknown . preliminary reports show that these drugs may have multi - modal effects in slowing tumour growth . in an approach similar to that using anti - angiogenic drugs , the class of antihyperglycemic drugs such as metformin and rosiglitazone may reduce glucose availability to the tumour and essentially starve the tumour of nutrients . these drugs have also been shown to have direct effects on metabolic and signaling pathways that may be independent of glucose . metformin is in the biguanide class of antidiabetic drugs and decreases circulating glucose levels by suppressing hepatic production of glucose . metformin , by reducing insulin and glucose levels , reduced the size and increased latency of mammary adenocarcinomas in her-2/neu transgenic mice , demonstrating a potent antitumour effect . in vitro , metformin significantly inhibits the growth of epithelial ovarian cancer cells and may potentiate the effects of the common chemotherapy drug cisplatin . stimulation of ampk by metformin also contributes to the reduced hepatocyte production of glucose . in fact , ampk activation is associated with an inhibition of tumourigenesis through apoptosis induction , decreased cell proliferation and may be a communal molecule utilized by metformin as well as a number of anti - tumour drugs that have been shown to have effects in eoc . c93 , resveratrol [ 13 , 136 ] , 2-deoxy - d - glucose , and aicar are targeted therapies that are effective in the treatment of ovarian cancer . rosiglitazone is another antidiabetic agent in the thiazolidinedione class of drugs designed to reduce the hyperglycemia associated with this disease . one of the mechanisms by which rosiglitazone may have a significant antitumour effect is through the inhibition of angiogenesis . rosiglitazone has been shown to inhibit vegf - induced angiogenesis and is suggested as a treatment option for vascular disorders associated with diabetes such as diabetic retinopathy , macular degeneration , and so forth . as vegf expression is significantly elevated in eoc and is responsible for some of the ovarian tumour vascularization ( reviewed in ) , rosiglitazone may have a bimodal anti - tumour effect by decreasing glucose availability and also by reducing tumour angiogenesis . simply by decreasing tumour vascularity an emerging view of cancer relies on an initiation - promotion paradigm that suggests a fundamental role of the tumour environment on cancer development . new data suggests that hyperglycemia may be a contributing factor to the onset and progression of eoc through a number of complex mechanisms ( summarized in figure 1 ) . we propose that hyperglycemia has important effects on both the progression and somatic evolution of epithelial ovarian cancer . although there are a number of cellular mechanisms through which hyperglycemia may effect the promotion or initiation of ovarian cancer , there is almost no in vivo experimental data exploring the link between hyperglycemia and eoc . further research in this area not only has applications in the development of cancer therapeutics , but also will provide new insights into eoc pathogenesis , early detection , and possible prevention .

although visual acuity is an important criterion in order to measure a multidimensional function like vision , it still can not enlighten us enough to understand completely how the life styles of the patients are affected from the visual function of said patients . visual acuity will not be sufficient to define conditions such as emotionality depending on visual impairment , feeling of being incompetent , and social function loss . for this reason , several surveys have been developed in the recent years in order to investigate the effects of visual impairment to daily life . american national eye institute visual function scale ( nei - vfq 25 ) is a questionnaire that has been developed in order to reach said aim . the turkish translation of nei - vfq 25 scale and its validity and reliability have been shown [ 3 , 4 ] . diabetic retinopathy is the primary reason for newly developed blindness in developed countries . even in cases where retinopathy does not lead to decrease in visual acuity , the function of sight is affected in different ways and the living quality of patients can decrease [ 69 ] . the clinical usage soundness of nei - vfq 25 scale in ophthalmic diseases has been confirmed and publications are present in relation to its usage on patients suffering from macular degeneration dependent on age , glaucoma , optical neuritis , dry eye , uveitis , and central vein occlusion [ 1014 ] . however nowadays , the studies that evaluate the quality of life dependent on vision with clinical measurements of the central vision function in diabetics patients are limited [ 15 , 16 ] . in our study we aimed to investigate the sub scale and total scores of nei - vfq questionnaire in type 2 diabetic patients in different drp stages and the demographic and clinical characteristics that are most effective in said scores . 201 cases that have been diagnosed with type 2 diabetes mellitus have been included in the study . the cases where the patient had alzheimer 's disease or dementia , which could prevent the patient to answer the questions , or is suffering from another disease , besides drp and cataract , that could affect the visual functions in both eyes were not included in the study . all the cases participating in the study were informed regarding the contents of the study and letters of approval stating that they have accepted to participate in this study have been obtained from all patients . the age , gender , duration term of diabetes ( taken from the diagnosis date until the commencement date of the study ) , blood sugar control types ( diet , oral antidiabetic , or insulin ) , the most recent preprandial blood glucose ( values taken in the last week prior to the study ) , the hba1c levels in the last month , hypertension presence , laser treatments taken previously due to drp ( panretinal , focal , and grip photocoagulation ) , and cataract operation histories were examined and were recorded . prior to ophthalmologic examinations all patients were given the nei - vfq . if the patients were using glasses , for example , they were advised to answer the questions of the questionnaire according to their thoughts and feelings at their time of usage of said glasses . an additional number of 13 questions were asked to all patients in addition to the 25 questions of the questionnaire and their answers were recorded into the forms . the initial and best adjusted visual acuity for both eyes has been measured from 4 meters with snellen and etdrs scales . the difference between the initial visual acuity and the best adjusted visual acuity was comprised of only two sequences , the patients who did not use glasses or the patients having incompatibilities between the refraction deficiencies , and the glasses they were using were excluded from the study as it was deemed that the refraction deficiencies could affect the results of the study . it has been assumed that the quality of living was in correlation with the visual acuity of the eye that had better eyesight and as a result the initial visual acuity values of eyes having better visual acuity were used . during examination of near sight , the patients were asked to put their glasses on and the jaeger scale from 35 to 40 cm was read by the patients . those patients who did not use glasses although they needed to or those who used incompliant glasses not compliant with the refraction deficiency were excluded from the study as it was assumed that the refraction deficiencies could affect the study . the near sight acuity values that have been measured were converted to snellen equivalent values . the eyes of the patients were dilated with 1% tropicamide for lens examination and a drop in each eye was used . lens examination was carried out at a 45-degree angle , with retroillumination from the pupil center with the narrowest slit lamp measure . lens opacities classification system ( locs ii ) was used separately for nuclear ( nc ) , cortical ( cc ) , and posterior subcapsular cataracts ( psc ) in order to measure the cataract stage for both eyes . the scores with stage 2 and above values were accepted to be clinically significant cataracts ( cscat ) for nuclear , cortical , and posterior subcapsular cataracts . the drps of the patients were scaled according to the icdrs system . according to this staging system , if there is no drp which was accepted to be within stage 0 , those within the mild npdr ( nonproliferative diabetic retinopathy ) stages were accepted to be within stage 1 , the mild npdr stages were accepted to be included in stage 2 , and the severe npdr stage was accepted to be included in stage 3 and those within the proliferative drp stage were included in stage 4 group ( table 1 ) . the patients that has received grid and/or focal laser treatment previously and the patients who did not have macula edema in their ffa and/or macula edema which was not found during their ophthalmologic examinations were included as patients with clinically significant macula edema into the study . the age and gender distribution of patients according to stages have been shown in table 1 . there was no statistically significant difference in terms of the distribution of the age ( p = 0.56 ) and gender ( p = 0.51 ) groups of patients . the presence of hypertension of patients distributed according to stages has been given in table 2 . there was no statistically significant difference between the groups in terms of hypertension distribution of patients ( p = 0.53 ) . the clinical characteristics in relation to diabetes of the patients according to stages have been given in table 3 . the near vision acuity decrease ( p = 0.0001 ) results of patients according to the scales of snellen ( p = 0.0001 ) and etdrs ( p = 0.0001 ) of patients with increasing stages were statistically significant . the difference between the average intraocular pressure values was not statistically significant ( p = 0.77 ) . the increase in the number of patients having had cataract surgery as drp stage increased and the decrease in the number of patients with csc were statistically significant ( p = 0.01 ) . the increase in the number of patients with csme as drp stages increased was significantly meaningful ( p = 0.0001 ) . the nei - vfq-39 subscale and total scores of patients have been given in table 5 . the questionnaire score distribution of patients according to demographic data has been shown in table 6 . as age increased between the age groups of 5160 and 6170 , the decrease of the distance activities ( p = 0.04 ) , mental health ( p = 0.04 ) , role difficulties ( p = 0.04 ) , visual dependency ( p = 0.009 ) , and total ( 0.028 ) scores of the patients were significantly significant . the average ocular pain scores in men were 82.47 19.45 and in women were 73.30 20.63 ; and these results were significantly higher in men in comparison to women ( p = 0.001 ) . the general health scores of the group with hypertension were 44.10 16.11 , and the group without hypertension was 49.16 16.68 ; the scores of the group with hypertension were significantly lower in comparison to the group without hypertension ( p = 0.03 ) . the distribution of the questionnaire scores regarding diabetes of the patients according to clinical characteristics has been shown in table 7 . the decrease in the general vision scores as the diabetes duration increased was statistically significant ( p = 0.007 ) . the decrease in general health ( p = 0.0001 ) , general vision ( p = 0.0001 ) , and near vision ( p = 0.0001 ) scores as the drp stage increased was statistically significant . the general vision scores of the patients that used insulin to regulate blood sugar were found to be significantly lower in comparison to groups being treated with diet and oad ( oral antidiabetics ) ( p = 0.04 ) . the distribution of the questionnaire scores of patients according to ophthalmological findings which are shown in table 8 . the increase in the scores of general vision ( p = 0.0001 ) , near activities ( p = 0.0001 ) , and distance activities ( p = 0.04 ) as snellen vision acuity values increased was statistically significant . snellen vision acuity between the groups 0.2 and 0.60.8 , in terms of vision specific mental health ( p = 0.03 ) and role difficulties ( p = 0.04 ) scores , was found to be statistically significant . the increase in the scores of general health ( p = 0.04 ) , general vision ( p = 0.0001 ) , and near activities ( p = 0.0001 ) as etdrs visual acuity values increased was found to be statistically meaningful . the etdrs v.a . between 20 and 4150 groups were found to be statistically significant in ocular pain ( p = 0.01 ) and distance activities ( p = 0.01 ) scores . the general health ( p = 0.04 ) , general vision ( p = 0.001 ) , and near activities ( p = 0.008 ) were found to be significantly high in the group without csc , in comparison to the group with csc . the general health ( p = 0.03 ) , general vision ( p = 0.0001 ) , and near activities ( p = 0.0001 ) scores were significantly high in the group without csme in comparison to the group with csme . stepwise linear regression analysis has been carried out in order to determine the factor or factors that have the highest effect on the questionnaire scores from all the factors that were found to be statistically significant during univariate analysis . the most effective factors were found to be diabetics duration term in the general health score ( p = 0.029 ) ; drp stage ( p = 0.011 ) and csme presence ( p = 0.019 ) in general vision score ; near vision acuity ( p = 0.0001 ) and drp stage ( p = 0.020 ) in near activities score ; snellen v.a . ( p = 0.014 ) in distance activities score ; etdrs v.a . in role difficulties ( p = 0.034 ) and visual dependency ( p = 0.011 ) scores ; and snellen v.a . klein et al . have examined the relationship between the nei - vfq-25 subscale and total scores in relation with vision acuity , drp stage , and other demographic and clinical characteristics among patients with type 1 diabetes whose ages were averaged between 40.6 10.7 . the average general health scores of the patients in the study have been calculated as 60.5 23.8 . in our study the mean general health scores have been found to be worse than klein et al . and cusick et al . 's studies . have examined the relationship between the nei - vfq-25 near and far subscale scores with the central vision function ( contrast sensitivity and visual acuity ) in 170 patients with type 1 and type 2 diabetes patients . the mean general health scores in this study have been given as 50.0 2.0 . in our study the mean general health scores have been found to be 45.99 16.46 in our study . patients with any kind of chronic diseases that could affect their quality of living besides diabetes were not included in the study . however patients with complications due to diabetes and patients with hypertension were included within the scope of the study . in our study it has been found that the general health scores was significantly lower in patients with hypertension in comparison with the patients without hypertension , and the factors related with the general health scores have also been examined being different to the other two studies ( p = 0.039 ) . moreover it has been found out that the general health score decreased as the drp stage increased ( p = 0.04 ) . it has also been found that the general health scores of the patients significantly decreased as etdrs visual acuity values decreased ( p = 0.04 ) . it has been found that the general health score decreased again in the presence of csc ( p = 0.03 ) and csme ( p = 0.03 ) . however it has been determined that the most effective factor in the general health score as a result of the multivariate analysis was the duration of the diabetes disease ( p = 0.029 ) . it is normal to find that the most important factor in general health score is the diabetes duration as we can imagine that as the duration of the disease increases the drp stage and other systemic complications related with the disease also increase . the general vision scores of the patients included in the study of klein et al . have been found to be 79.7 16.1 . in this study it has been found that the general vision score shows decrease in vision acuity due to increased age , diabetes duration , hba1c levels , presence of hypertension , drp stage , and decrease in the presence of macular edema and cataract presence . as a result of the multivariate analysis carried out in this study it has been found that the most important factor affecting the general vision was visual acuity . in our study it has been found that the general vision scores significantly decreased as the diabetes ( p = 0.007 ) , drp stage ( p = 0.0001 ) , and hba1c levels ( p = 0.04 ) increased . we have also determined that the general vision scores significantly decreased again as the need to use insulin in order to control the blood sugar levels increased ( p = 0.04 ) . it has been determined following the multivariate analysis that the most important factors on general vision scores were drp stage ( p = 0.011 ) and csme ( p = 0.019 ) . similar to the other two studies , in our study we have also found that the general vision score was the lowest score following the general health score . as drp patients have lower visual acuity or they are worried that they will have low visual acuity even though they do not have at the moment , it is normal for the general vision score to be low . the reason that the general vision score is lower than distance acuity and near vision scores related to vision could be that the difficulty degree that forms the general vision subscale score is not questioned but it is completely left to the comments of the patients ; thereby said patients could be generally reflecting their worries regarding vision . the color vision score of the patients according to the study of klein et al . has been given as 94.8 14.9 , ocular pain score as 92.6 13.0 , and peripheral vision score as 89.3 20.9 . the ocular pain score of the patients in the study of cusick et al . has been given as 93 1.1 , color vision score as 90.0 1.6 , and peripheral vision score as 82.0 2.0 . in both studies these three scores form the highest scores amongst all scores . in our study , the color vision score ( 92.04 17.71 ) has also been given as the highest score . the average ocular pain score ( 77.77 20.54 ) and peripheral vision score ( 77.98 25.44 ) have also been determined as the highest scores in our study . we believe that these scores have been found to be high because as long as glaucoma does not develop in patients with diabetes ocular pain is not frequently seen and color vision and peripheral vision defects are not frequently faced and because said symptoms are not given too much importance by the patients in comparison to loss of vision . our pain scores are less than other studies , because our patients did not have glaucoma . it could also be cultural differences . in our study , the mean near vision activities of the patients have been determined to be 75.71 23.28 . as the drp stages increased a significant level of decrease has been noted in the near activities scores ( p = 0.0001 ) . moreover it has been determined that the near activities score significantly decreased as the snellen ( p = 0.0001 ) v.a . and etdrs v.a . it has been found out that a significant decrease was obtained in the near activities scores within the presence of cscat ( p = 0.01 ) and csme ( p = 0.0001 ) . it has been found out that the most effective factors that affected near activities scores as a result of the multivariate analysis were near vision acuity ( p = 0.0001 ) and drp stage ( p = 0.020 ) . the decrease in near activities ( p = 0.0001 ) was statistically meaningful among the increased drp stages in our study . however as a result of the multivariate analysis it has been decided that the near vision acuity value in near activities scores was more effective in comparison to the drp stage . we attribute the changes of the distance activities scores between only these two age groups to the fact that the transition from 5160 years to 6170 years age group also symbolizes the transition from said patients from a more active social life to a less active period . significant decrease has been determined in distance activities scores as drp stages increased between stage 1 drp group ( p = 0.0001 ) where drp was not found and stage 4 drp group ( p = 0.005 ) where drp was not found . values decrease it was found that distance activities score significantly decreased also ( p = 0.04 ) . values of 20 and 4150 groups ( p = 0.01 ) . as a result of the multivariate analysis however we found out that the most effective factor in distance activities scores was the snellen v.a . the significant decrease in snellen ( p = 0.0001 ) and etdrs ( p = 0.0001 ) v.a . value was more effective on the distance activities score in comparison to the drp stage . it has been determined in the study of klein et al . that a significant level of decrease was seen on vision specific mental health when the diabetes duration term was high , the hba1c level was high , hypertension was present , there was a decrease in visual acuity , drp stages increased , and macular edema and cataract were present . as a result of the multivariate analysis , it has been found however that the most effective factor in mental health was the visual acuity . in this study , the most effective factors on the social functioning , role difficulties , and visual dependency scores were not carried out . in our study age has not been determined as an effective factor on vision specific social functioning score . however we determined that the vision specific mental health ( p = 0.04 ) , role difficulties ( p = 0.04 ) , and dependency ( p = 0.009 ) scores among groups between the ages of 5160 and 6170 decreased significantly with increasing age . we believe that the patients passed onto an inactive period from a social and functional period during their passage from the 5160 age group to the 6170 age group and that the difficulty related to vision during this period caused these patients to be dependent on others and as a result we believe that the these patient 's mental health was impaired . we believe that the decrease in the visual acuities of patients must lead to difficulty in carrying out their daily activities and that this must lead to them being dependent on others . however we believe that the decrease in the visual acuities of patients does not affect their social relationships within a social community or their mental health . this could be arising from the beliefs , fatalism , and acceptance understanding of the people in our community . the study of klein et al . has determined a significant decrease in total scores ( ts ) with increased age , high hba1c levels , the presence of hypertension , reduction in visual acuity , the increase in drp stage , and the presence of macular edema and cataract . a directly proportional decrease in the total questionnaire score has been found in this study with the decrease in visual acuity after characteristics such as age , retinopathy level , and other clinical features are checked . in our study the decrease in total scores between the ages of 5160 and 6170 was significantly meaningful ( p = 0.028 ) . the decrease in ts was significant as the drp stage increased between stage 1 and stage 0 drp groups , ( p = 0.0001 ) , between stage 4 and stage 0 drp groups ( p = 0.001 ) , and between stage 3 and stage 4 drp groups ( p = 0.001 ) . it has been found out that the most effective factor in ts following multivariate analysis was the snellen v.a . value ( p = 0.026 ) . broman et al . have used the nei - vfq-25 scale in the study they carried out on 4774 people aged over 40 living in arizona , and they have examined the relationship between the decrease of visual acuity and various eye diseases on life quality dependent on vision . in the study where knudtson et al . examined the life qualities of 2670 patients , between the years of 19982000 , who had eye diseases in relation to age such as senile macular degeneration , cataract , drp , glaucoma , and macular edema , when other factors were also checked , it has been found out that the reduction in visual acuity led to the decrease of the quality of life . in the study carried out on 5119 patients with cataract , not corrected refractive disorders , glaucoma , and senile macular degeneration , by nirmalan et al . , after demographic and clinical factors were checked , it has been observed that life quality and visual function scores were related with the visual acuity of the eye that sees better . it has been stated that the slight decrease of 4.7 in the etdrs value in the study of cusick et al . had led to a 25-point decrease in near vision activities subscale scores ( p 0.001 ) . in the same study , it has been stated that the slight decrease in the etdrs value has led to a 25-point decrease in the distance activities subscale scores ( p 0.01 ) . in the study where hariprasad et al . compared quality of vision and vision - specific quality of life in type 2 diabetes patients with macular oedema versus patients with type 1 diabetic retinopathy . they concluded that type 2 diabetes patients with macular oedema experience a decreased vision - specific quality of life compared with type 1 diabetic patients with diabetic retinopathy , glaucoma , or cataracts . however , vision - specific quality of life in type 2 diabetic patients with macular oedema was similar to those individuals with age related macular degeneration . in the other study gabrielian et al . compared vision related quality of life between patients with nonproliferative diabetic retinopathy ( npdr ) and proliferative diabetic retinopathy ( pdr ) . they found that vfq is a superior measure of vision - specific quality of life for patients with diabetic retinopathy because it better captures mental and emotional aspects of the disease as well as visual function . subjects with pdr versus npdr suffer significant loss of vision - specific quality of life . in our study , as a result of the multivariate analysis , it has been found out that the most effective factor in distance activities score ( p = 0.014 ) and total score ( p = 0.026 ) was snellen visual acuity , that the most effective factor in near activities score was near vision visual acuity ( p = 0.0001 ) , and that the most effective factor in role difficulties ( p = 0.034 ) and visual dependency ( p = 0.011 ) was etdrs visual acuity . in conclusion , we can state that the vision related quality of life in type 2 diabetes patients at different drp stages is related actually to visual acuity rather than stages and other demographic and clinical factors and that nei - vfq-25 is an efficient questionnaire that can evaluate the life quality of patients depending on vision . nei - vfq questionnaire can be used to measure the efficiency of interventions regarding treatment in the ophthalmology field , to the quality of life of the patients .

aim . to examine subscale and total scores of nei - vfq questionnaire of type 2 diabetes patients at different diabetic retinopathy ( drp ) stages . methods . a total number of 201 patients have been included . prior to ophthalmological examination all patients participated in the nei - vfq questionnaire . the patients were divided into 5 groups according to the international clinical diabetic retinopathy disease severity scale ( icdrs ) . results . the diabetes duration in general health scores ( p = 0.029 ) ; the stage ( p = 0.011 ) ; and clinically significant macular edema ( csme ) ( p = 0.019 ) in general vision were found to be the most efficient factors . in near vision activities the most efficient factors were near vision acuity ( nva ) ( p = 0.0001 ) and drp stage ( p = 0.020 ) . edtrs visual acuity was found to be the most efficient factor in vision specific role difficulties ( p = 0.034 ) and dependency ( p = 0.011 ) whereas snellen visual acuity was found to be among the most effective factors in distance activities ( da ) ( p = 0.014 ) and total scores ( p = 0.026 ) . discussion . difference was based not on the diabetes duration , clinically significant cataract ( cscat ) , csme presence , and drp stage but on the visual acuity levels of the better seeing eye of the patients .

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

although visual acuity is an important criterion in order to measure a multidimensional function like vision , it still can not enlighten us enough to understand completely how the life styles of the patients are affected from the visual function of said patients . american national eye institute visual function scale ( nei - vfq 25 ) is a questionnaire that has been developed in order to reach said aim . the turkish translation of nei - vfq 25 scale and its validity and reliability have been shown [ 3 , 4 ] . the clinical usage soundness of nei - vfq 25 scale in ophthalmic diseases has been confirmed and publications are present in relation to its usage on patients suffering from macular degeneration dependent on age , glaucoma , optical neuritis , dry eye , uveitis , and central vein occlusion [ 1014 ] . in our study we aimed to investigate the sub scale and total scores of nei - vfq questionnaire in type 2 diabetic patients in different drp stages and the demographic and clinical characteristics that are most effective in said scores . 201 cases that have been diagnosed with type 2 diabetes mellitus have been included in the study . all the cases participating in the study were informed regarding the contents of the study and letters of approval stating that they have accepted to participate in this study have been obtained from all patients . the age , gender , duration term of diabetes ( taken from the diagnosis date until the commencement date of the study ) , blood sugar control types ( diet , oral antidiabetic , or insulin ) , the most recent preprandial blood glucose ( values taken in the last week prior to the study ) , the hba1c levels in the last month , hypertension presence , laser treatments taken previously due to drp ( panretinal , focal , and grip photocoagulation ) , and cataract operation histories were examined and were recorded . prior to ophthalmologic examinations all patients were given the nei - vfq . if the patients were using glasses , for example , they were advised to answer the questions of the questionnaire according to their thoughts and feelings at their time of usage of said glasses . an additional number of 13 questions were asked to all patients in addition to the 25 questions of the questionnaire and their answers were recorded into the forms . the difference between the initial visual acuity and the best adjusted visual acuity was comprised of only two sequences , the patients who did not use glasses or the patients having incompatibilities between the refraction deficiencies , and the glasses they were using were excluded from the study as it was deemed that the refraction deficiencies could affect the results of the study . it has been assumed that the quality of living was in correlation with the visual acuity of the eye that had better eyesight and as a result the initial visual acuity values of eyes having better visual acuity were used . lens opacities classification system ( locs ii ) was used separately for nuclear ( nc ) , cortical ( cc ) , and posterior subcapsular cataracts ( psc ) in order to measure the cataract stage for both eyes . the scores with stage 2 and above values were accepted to be clinically significant cataracts ( cscat ) for nuclear , cortical , and posterior subcapsular cataracts . the drps of the patients were scaled according to the icdrs system . according to this staging system , if there is no drp which was accepted to be within stage 0 , those within the mild npdr ( nonproliferative diabetic retinopathy ) stages were accepted to be within stage 1 , the mild npdr stages were accepted to be included in stage 2 , and the severe npdr stage was accepted to be included in stage 3 and those within the proliferative drp stage were included in stage 4 group ( table 1 ) . there was no statistically significant difference in terms of the distribution of the age ( p = 0.56 ) and gender ( p = 0.51 ) groups of patients . there was no statistically significant difference between the groups in terms of hypertension distribution of patients ( p = 0.53 ) . the clinical characteristics in relation to diabetes of the patients according to stages have been given in table 3 . the near vision acuity decrease ( p = 0.0001 ) results of patients according to the scales of snellen ( p = 0.0001 ) and etdrs ( p = 0.0001 ) of patients with increasing stages were statistically significant . the difference between the average intraocular pressure values was not statistically significant ( p = 0.77 ) . the increase in the number of patients having had cataract surgery as drp stage increased and the decrease in the number of patients with csc were statistically significant ( p = 0.01 ) . the increase in the number of patients with csme as drp stages increased was significantly meaningful ( p = 0.0001 ) . the nei - vfq-39 subscale and total scores of patients have been given in table 5 . as age increased between the age groups of 5160 and 6170 , the decrease of the distance activities ( p = 0.04 ) , mental health ( p = 0.04 ) , role difficulties ( p = 0.04 ) , visual dependency ( p = 0.009 ) , and total ( 0.028 ) scores of the patients were significantly significant . the average ocular pain scores in men were 82.47 19.45 and in women were 73.30 20.63 ; and these results were significantly higher in men in comparison to women ( p = 0.001 ) . the general health scores of the group with hypertension were 44.10 16.11 , and the group without hypertension was 49.16 16.68 ; the scores of the group with hypertension were significantly lower in comparison to the group without hypertension ( p = 0.03 ) . the distribution of the questionnaire scores regarding diabetes of the patients according to clinical characteristics has been shown in table 7 . the decrease in the general vision scores as the diabetes duration increased was statistically significant ( p = 0.007 ) . the decrease in general health ( p = 0.0001 ) , general vision ( p = 0.0001 ) , and near vision ( p = 0.0001 ) scores as the drp stage increased was statistically significant . the general vision scores of the patients that used insulin to regulate blood sugar were found to be significantly lower in comparison to groups being treated with diet and oad ( oral antidiabetics ) ( p = 0.04 ) . the distribution of the questionnaire scores of patients according to ophthalmological findings which are shown in table 8 . the increase in the scores of general vision ( p = 0.0001 ) , near activities ( p = 0.0001 ) , and distance activities ( p = 0.04 ) as snellen vision acuity values increased was statistically significant . snellen vision acuity between the groups 0.2 and 0.60.8 , in terms of vision specific mental health ( p = 0.03 ) and role difficulties ( p = 0.04 ) scores , was found to be statistically significant . the increase in the scores of general health ( p = 0.04 ) , general vision ( p = 0.0001 ) , and near activities ( p = 0.0001 ) as etdrs visual acuity values increased was found to be statistically meaningful . between 20 and 4150 groups were found to be statistically significant in ocular pain ( p = 0.01 ) and distance activities ( p = 0.01 ) scores . the general health ( p = 0.04 ) , general vision ( p = 0.001 ) , and near activities ( p = 0.008 ) were found to be significantly high in the group without csc , in comparison to the group with csc . the general health ( p = 0.03 ) , general vision ( p = 0.0001 ) , and near activities ( p = 0.0001 ) scores were significantly high in the group without csme in comparison to the group with csme . stepwise linear regression analysis has been carried out in order to determine the factor or factors that have the highest effect on the questionnaire scores from all the factors that were found to be statistically significant during univariate analysis . the most effective factors were found to be diabetics duration term in the general health score ( p = 0.029 ) ; drp stage ( p = 0.011 ) and csme presence ( p = 0.019 ) in general vision score ; near vision acuity ( p = 0.0001 ) and drp stage ( p = 0.020 ) in near activities score ; snellen v.a . ( p = 0.014 ) in distance activities score ; etdrs v.a . in role difficulties ( p = 0.034 ) and visual dependency ( p = 0.011 ) scores ; and snellen v.a . have examined the relationship between the nei - vfq-25 subscale and total scores in relation with vision acuity , drp stage , and other demographic and clinical characteristics among patients with type 1 diabetes whose ages were averaged between 40.6 10.7 . the average general health scores of the patients in the study have been calculated as 60.5 23.8 . in our study the mean general health scores have been found to be worse than klein et al . have examined the relationship between the nei - vfq-25 near and far subscale scores with the central vision function ( contrast sensitivity and visual acuity ) in 170 patients with type 1 and type 2 diabetes patients . in our study the mean general health scores have been found to be 45.99 16.46 in our study . in our study it has been found that the general health scores was significantly lower in patients with hypertension in comparison with the patients without hypertension , and the factors related with the general health scores have also been examined being different to the other two studies ( p = 0.039 ) . moreover it has been found out that the general health score decreased as the drp stage increased ( p = 0.04 ) . it has also been found that the general health scores of the patients significantly decreased as etdrs visual acuity values decreased ( p = 0.04 ) . it has been found that the general health score decreased again in the presence of csc ( p = 0.03 ) and csme ( p = 0.03 ) . however it has been determined that the most effective factor in the general health score as a result of the multivariate analysis was the duration of the diabetes disease ( p = 0.029 ) . it is normal to find that the most important factor in general health score is the diabetes duration as we can imagine that as the duration of the disease increases the drp stage and other systemic complications related with the disease also increase . the general vision scores of the patients included in the study of klein et al . have been found to be 79.7 16.1 . in this study it has been found that the general vision score shows decrease in vision acuity due to increased age , diabetes duration , hba1c levels , presence of hypertension , drp stage , and decrease in the presence of macular edema and cataract presence . as a result of the multivariate analysis carried out in this study it has been found that the most important factor affecting the general vision was visual acuity . in our study it has been found that the general vision scores significantly decreased as the diabetes ( p = 0.007 ) , drp stage ( p = 0.0001 ) , and hba1c levels ( p = 0.04 ) increased . we have also determined that the general vision scores significantly decreased again as the need to use insulin in order to control the blood sugar levels increased ( p = 0.04 ) . it has been determined following the multivariate analysis that the most important factors on general vision scores were drp stage ( p = 0.011 ) and csme ( p = 0.019 ) . similar to the other two studies , in our study we have also found that the general vision score was the lowest score following the general health score . as drp patients have lower visual acuity or they are worried that they will have low visual acuity even though they do not have at the moment , it is normal for the general vision score to be low . the reason that the general vision score is lower than distance acuity and near vision scores related to vision could be that the difficulty degree that forms the general vision subscale score is not questioned but it is completely left to the comments of the patients ; thereby said patients could be generally reflecting their worries regarding vision . the color vision score of the patients according to the study of klein et al . the ocular pain score of the patients in the study of cusick et al . we believe that these scores have been found to be high because as long as glaucoma does not develop in patients with diabetes ocular pain is not frequently seen and color vision and peripheral vision defects are not frequently faced and because said symptoms are not given too much importance by the patients in comparison to loss of vision . in our study , the mean near vision activities of the patients have been determined to be 75.71 23.28 . as the drp stages increased a significant level of decrease has been noted in the near activities scores ( p = 0.0001 ) . moreover it has been determined that the near activities score significantly decreased as the snellen ( p = 0.0001 ) v.a . it has been found out that a significant decrease was obtained in the near activities scores within the presence of cscat ( p = 0.01 ) and csme ( p = 0.0001 ) . it has been found out that the most effective factors that affected near activities scores as a result of the multivariate analysis were near vision acuity ( p = 0.0001 ) and drp stage ( p = 0.020 ) . the decrease in near activities ( p = 0.0001 ) was statistically meaningful among the increased drp stages in our study . however as a result of the multivariate analysis it has been decided that the near vision acuity value in near activities scores was more effective in comparison to the drp stage . we attribute the changes of the distance activities scores between only these two age groups to the fact that the transition from 5160 years to 6170 years age group also symbolizes the transition from said patients from a more active social life to a less active period . significant decrease has been determined in distance activities scores as drp stages increased between stage 1 drp group ( p = 0.0001 ) where drp was not found and stage 4 drp group ( p = 0.005 ) where drp was not found . values decrease it was found that distance activities score significantly decreased also ( p = 0.04 ) . values of 20 and 4150 groups ( p = 0.01 ) . as a result of the multivariate analysis however we found out that the most effective factor in distance activities scores was the snellen v.a . the significant decrease in snellen ( p = 0.0001 ) and etdrs ( p = 0.0001 ) v.a . value was more effective on the distance activities score in comparison to the drp stage . that a significant level of decrease was seen on vision specific mental health when the diabetes duration term was high , the hba1c level was high , hypertension was present , there was a decrease in visual acuity , drp stages increased , and macular edema and cataract were present . as a result of the multivariate analysis , it has been found however that the most effective factor in mental health was the visual acuity . in this study , the most effective factors on the social functioning , role difficulties , and visual dependency scores were not carried out . however we determined that the vision specific mental health ( p = 0.04 ) , role difficulties ( p = 0.04 ) , and dependency ( p = 0.009 ) scores among groups between the ages of 5160 and 6170 decreased significantly with increasing age . has determined a significant decrease in total scores ( ts ) with increased age , high hba1c levels , the presence of hypertension , reduction in visual acuity , the increase in drp stage , and the presence of macular edema and cataract . a directly proportional decrease in the total questionnaire score has been found in this study with the decrease in visual acuity after characteristics such as age , retinopathy level , and other clinical features are checked . in our study the decrease in total scores between the ages of 5160 and 6170 was significantly meaningful ( p = 0.028 ) . the decrease in ts was significant as the drp stage increased between stage 1 and stage 0 drp groups , ( p = 0.0001 ) , between stage 4 and stage 0 drp groups ( p = 0.001 ) , and between stage 3 and stage 4 drp groups ( p = 0.001 ) . it has been found out that the most effective factor in ts following multivariate analysis was the snellen v.a . value ( p = 0.026 ) . have used the nei - vfq-25 scale in the study they carried out on 4774 people aged over 40 living in arizona , and they have examined the relationship between the decrease of visual acuity and various eye diseases on life quality dependent on vision . examined the life qualities of 2670 patients , between the years of 19982000 , who had eye diseases in relation to age such as senile macular degeneration , cataract , drp , glaucoma , and macular edema , when other factors were also checked , it has been found out that the reduction in visual acuity led to the decrease of the quality of life . , after demographic and clinical factors were checked , it has been observed that life quality and visual function scores were related with the visual acuity of the eye that sees better . had led to a 25-point decrease in near vision activities subscale scores ( p 0.001 ) . in the same study , it has been stated that the slight decrease in the etdrs value has led to a 25-point decrease in the distance activities subscale scores ( p 0.01 ) . compared quality of vision and vision - specific quality of life in type 2 diabetes patients with macular oedema versus patients with type 1 diabetic retinopathy . they concluded that type 2 diabetes patients with macular oedema experience a decreased vision - specific quality of life compared with type 1 diabetic patients with diabetic retinopathy , glaucoma , or cataracts . compared vision related quality of life between patients with nonproliferative diabetic retinopathy ( npdr ) and proliferative diabetic retinopathy ( pdr ) . in our study , as a result of the multivariate analysis , it has been found out that the most effective factor in distance activities score ( p = 0.014 ) and total score ( p = 0.026 ) was snellen visual acuity , that the most effective factor in near activities score was near vision visual acuity ( p = 0.0001 ) , and that the most effective factor in role difficulties ( p = 0.034 ) and visual dependency ( p = 0.011 ) was etdrs visual acuity . in conclusion , we can state that the vision related quality of life in type 2 diabetes patients at different drp stages is related actually to visual acuity rather than stages and other demographic and clinical factors and that nei - vfq-25 is an efficient questionnaire that can evaluate the life quality of patients depending on vision . nei - vfq questionnaire can be used to measure the efficiency of interventions regarding treatment in the ophthalmology field , to the quality of life of the patients .

ractopamine hydrochloride ( rachcl , mw 337.85 , ( 1r*,3r*),(1r*,3s*)-4-hydroxy - r-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methyl]-benzenemethanol hydrochloride ( figure 1 ) ) is a phenethanolamine member of the family of -adrenergic agonists ( -agonists ) . these compounds are mainly used in human and veterinary medicine as tocolytic and bronchodilator agents . the advantages of feeding animals with -agonists have been reported to include the promotion of repartitioning of fat into muscles , in addition to the ability of increasing average daily weight gain , improving feed efficiency , saving on feed , and decreasing the number of days to market when higher doses are administered [ 25 ] . furthermore , as a result of better feed utilization efficiency , there are positive environmental benefits for livestock producers in terms of decreased nitrogen and phosphorus excretions , and reduced amount of total animal waste . however , meat products obtained from illegally treated animals with these compounds may pose potential risks linked to adverse cardiovascular and central nervous system effects . ractopamine , a veterinary additive drug , was approved by the us food and drug administration for the use in swine production . recently , it has become more and more popular not only in pig but also in chicken and cattle production , following clenbuterol which had been the most widely used -agonists in livestock production . due to its potential risks for human health , ractopamine is still used in a restricted manner within a low limit of dosage when used in livestock production , in many countries . therefore , there has been an increasing number of analytical methods reported to monitor ractopamine in animal urine , feeds , and tissues such as instrument methods like hplc with electrochemical detection and fluorescence , lc - ms [ 10 , 11 ] , and gc - ms . however , these analytical approaches , which use several clean - up procedures ( liquid - liquid extraction ( lle ) and solid - phase extraction ( spe ) using different sorbents ) , are quite complicated , time - consuming , and expensive . furthermore , integral production chain systems currently demand faster onsite ( farmhouses ) and/or online ( slaughterhouses ) test systems . immunoassays as a screening detection method can rapidly detect low amounts of residues in many samples . shelver et al . generated both polyclonal and monoclonal antibodies to analyze ractopamine by elisa . wang et al . also reported a monoclonal antibody immunoassay to determine ractopamine in swine feeds with a detection limit of 0.24 g / g sample . however , all of these elisa methods were mainly used for animal urine or feed samples , and complicated sample clean - up procedures ( lle and spe ) were required when animal tissues were analyzed . therefore , these tissue- ( except for urine ) extraction methods combined with rac - elisa are too complex as routine test systems . moreover , to the best of our knowledge , there is no report on the determination of ractopamine in chicken muscle . as a result , it is necessary to develop a more rapid , sensitive , and effective method for the determination of rac residues in edible animal foods including chicken muscle . bovine serum albumin ( bsa ) was obtained from merck ( darmstadt , germany ) . horseradish peroxidase ( hrp ) , ovalbumin ( ova ) , -glucuronidase from escherichia coli , and freund 's complete and incomplete adjutants were purchased from sigma chemical co. ( saint louis , mo , usa ) . reagent grade 3 , 3 , 5 , 5-tetramethyl - benzidine ( tmb ) , hydrogen peroxide , isobutyl chloroformate , butane-1 , 4-diol diglycidyl ether , and other chemicals were also from sigma . ractopamine hydrochloride was purchased from pure chemical analysis co. , ltd ( bornew , belgium ) . protein a - sepharose 4b was purchased from amersham biosciences ( uppsala , sweden ) . polystyrene 96-well microplates were from nunc ( rockilde , denmark ) , and the microplate washer was from bio - rad ( hercules , calif , usa ) . immunoassay absorbance was read with a multiskan spectrum purchased from thermo ( labsystems , vantaa , finland ) in the dual - wavelength mode ( 450650 nm ) . double deionized water ( ddw ) was prepared with a milli - q ( millipore , mass , usa ) water purification system . phosphate - buffered saline ( 1 pbs ; 38.4 mmol / l na2hpo4h2o , 11.5 mmol / l nah2po4h2o , 154 mmol / l nacl , ph 7.5 ) , phosphate - buffered saline with 0.05% tween 20 ( pbst ) , coating buffer ( cb , 50 mmol / l sodium carbonate buffer , ph 9.6 ) , blocking buffer ( 0.5% skimmed milk powder in 1 pbs ) , and tmb substrate solution ( prepared by adding 3.3 mg tmb in 250 l dmso to 25 ml phosphate - citrate buffer ( 0.1 mol / l citric acid + 0.2 mol / l na2hpo4 ; ph 4.3 ) containing 3.25 l of a 30% h2o2 solution ) , and termination solution ( 2.5 mol / l h2so4 in ddw ) were used . the hapten rac was coupled to bsa as the immunogen , and coupled to hrp as the enzyme tracer . the coupling agent , butane-1 , 4-diol diglycidyl ether , was used for coupling rac to proteins . the conjugating procedure of immunogen rac - bsa was adapted from elliott et al . . in brief , 10 mg bsa was dissolved in 0.5 ml ddw , and the ph was adjusted to 10.8 using 1.0 mol / l sodium hydroxide . coupling agent solution ( 50 l of 22 l / l butane-1 , 4-diol diglycidyl ether in ddw ) was added to the bsa solution , and the mixture was incubated for 22 hours at room temperature under nitrogen atmosphere . rachcl ( 17 mg , 50 mol ) was added to 0.5 ml naoh ( 0.5 mol / l ) containing 10% dimethylformamide . the mixture was then added to the epoxy - activated bsa solution ( precooled at 4c , in the refrigerator ) , slowly in an ice bath , and incubated for 22 hours at room temperature under nitrogen atmosphere . the conjugating method of rac - hrp was similar to that of the immunogen in the first step except for the amounts of the reagents which were 5 mg enzyme hrp instead of 10 mg , and 8.5 mg rachcl ( 25 mol ) were added to the activated hrp solution . finally , the mixture was incubated for 40 hours at room temperature under a nitrogen atmosphere . both the conjugated immunogen and enzyme tracer were dialyzed against pbs for 3 days at 4c . the conjugated immunogen was stored at 20c , and the enzyme tracer was stored at 4c with 0.01% thiomersal until required . the hapten rac was coupled to the protein ova using the method of mixed acid anhydride , as follows . rachcl ( 17 mg , 50 mol ) and 5 mg ( 50 mol ) succinic anhydride were diluted in 1 ml dry pyridine . after slow stirring for 24 hours at room temperature , the mixture was blown dry with a nitrogen flow . the residue was diluted in a 2 ml mixture of dmf and 1 , 4-dioxane ( 1 : 1 ) , and then stirred for 10 minutes in an ice bath by adding 13 l ( about 0.05 mmol ) tributylamine . after 7.2 l ( 0.05 mmol ) isobutyl chloroformate were added , the mixture was stirred for 1 hour at room temperature . subsequently , the activated rac mixture was dripped onto the precooled protein oa solution which was previously prepared with the following method . oa ( 10 mg ) was diluted in 2.5 ml phosphate buffer ( 0.1 mol / l ) . in order to avoid protein denaturation , dripping and stirring should be carried out at the same time at a low temperature , and should be finished within 30 minutes . the final mixture was stirred gently for 24 hours at room temperature , and purified using extensive dialysis against pbs buffer for 3 days at 4c . the purified coating conjugate solution was stored at 20c with 0.02% nan3 until required . antibodies were produced in rabbits using an immunization approach similar to that described by wang et al . . two white rabbits were immunized by intradermal and intramuscular injections of the emulsified rac - bsa immunogen . blood was collected from the marginal ear vein 10 days after each booster injection for antibody titer assay , and the whole blood was then collected after 6 immunizations . the anti - rac antiserum was centrifuged at 2000 rpm for 10 minutes ( eppendorf 5804r , hamburg , germany ) and stored in small tubes ( about 2 ml per tube ) at 20c . the titer for the specific antiserum was monitored by an indirect competitive elisa using the immunized hapten conjugated to ova . after dialyzing against pbs buffer for 3 days , the purified antibodies were stored at 4c with 0.02% nan3 until required . ( a ) indirect competitive elisaflat - bottom polystyrene microplates were coated with rac - oa conjugates at 1 g per well in 100 l coating buffer , and incubated overnight at room temperature . plates were washed 3 times with pbst using a 96pw microplate washer , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs per well for 1 hour at room temperature . after the plates were washed 3 times , 50 l of the appropriate antiserum / antibody dilution in pbs ( the other 50 l of rac standards in pbs were added to determine antibody specificity ) were added in each well for titer determination , and incubated for 1 hour at room temperature . after 4 additional washes , the plates were incubated for 1 hour at room temperature with 100 l peroxidase - labeled goat anti - rabbit immunoglobulins diluted 1 : 10 000 in pbs , in each well . after washing 5 times with pbst , 150 l tmb substrate solution were added to each well to measure the hrp tracer activity . the color production of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well . the absorbance in each well was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . flat - bottom polystyrene microplates were coated with rac - oa conjugates at 1 g per well in 100 l coating buffer , and incubated overnight at room temperature . plates were washed 3 times with pbst using a 96pw microplate washer , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs per well for 1 hour at room temperature . after the plates were washed 3 times , 50 l of the appropriate antiserum / antibody dilution in pbs ( the other 50 l of rac standards in pbs were added to determine antibody specificity ) were added in each well for titer determination , and incubated for 1 hour at room temperature . after 4 additional washes , the plates were incubated for 1 hour at room temperature with 100 l peroxidase - labeled goat anti - rabbit immunoglobulins diluted 1 : 10 000 in pbs , in each well . after washing 5 times with pbst , 150 l tmb substrate solution were added to each well to measure the hrp tracer activity . the color production of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well . the absorbance in each well was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . ( b ) direct competitive elisathe microplate well was coated with purified antibodies in 100 l coating buffer , and incubated overnight at room temperature . the coated plates were washed 3 times with pbst , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs for 1 hour at room temperature . after the plate was washed 4 times , 50 l of the standard solution of rac dissolved in pbs ( or diluted sample extracts ) followed by 50 l of hrp - hapten conjugate solution diluted in pbs were added to each well , and the mixture was incubated in a shaker for 1 hour at room temperature . after washing 5 times with pbst the color development of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well . the absorbance was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . the microplate well was coated with purified antibodies in 100 l coating buffer , and incubated overnight at room temperature . the coated plates were washed 3 times with pbst , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs for 1 hour at room temperature . after the plate was washed 4 times , 50 l of the standard solution of rac dissolved in pbs ( or diluted sample extracts ) followed by 50 l of hrp - hapten conjugate solution diluted in pbs were added to each well , and the mixture was incubated in a shaker for 1 hour at room temperature . after washing 5 times with pbst the color development of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well . the absorbance was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . ( c ) optimization of the rac elisa testdirect competitive elisa was used to analyze the parameters of the described method such as the amount of the antibody coating , and the ionic strength and ph of the diluting buffer . three different amounts of antibodies ( 0.5 g , 1.0 g , and 1.5 g antibody per well ) were tested . after the optimal antibody coating quantity was decided , the proper ionic strength was investigated by changing the concentration of pbs buffer ranging from 10 to 40 mmol / l . then , under the optimal conditions , the effects of different phs ( ph = 6.0 , 7.5 , 8.5 , 9.5 ) were tested . direct competitive elisa was used to analyze the parameters of the described method such as the amount of the antibody coating , and the ionic strength and ph of the diluting buffer . three different amounts of antibodies ( 0.5 g , 1.0 g , and 1.5 g antibody per well ) were tested . after the optimal antibody coating quantity was decided , the proper ionic strength was investigated by changing the concentration of pbs buffer ranging from 10 to 40 mmol / l . then , under the optimal conditions , the effects of different phs ( ph = 6.0 , 7.5 , 8.5 , 9.5 ) were tested . ( d ) sample preparationfour different samples including chicken muscle , pig muscle , pig liver , and pettitoes were chosen to evaluate the performance of elisa . for the spiking study , each sample was spiked by dropping with the rac standard solution in methanol , making the samples with well - distributed different levels of rac , the samples were thoroughly mixed and then allowed to stand at 4c overnight . all the samples were prepared for the immunoassay using the same extraction procedure as that for pig muscle as follows . the sample ( 2 g ) was chopped , and mixed with 10 ml pbs for 2 minutes using a waring blender ( omni international , marietta , ga , usa ) . then , the mixture was centrifuged for 10 minutes at 4,000 rpm ( centrifuge 5804r , eppendorf ag , hamburg , germany ) at room temperature . the fat layer was then discarded , and the upper liquid was transferred into a test tube , and analyzed directly by direct competitive elisa . four different samples including chicken muscle , pig muscle , pig liver , and pettitoes were chosen to evaluate the performance of elisa . for the spiking study , each sample was spiked by dropping with the rac standard solution in methanol , making the samples with well - distributed different levels of rac , the samples were thoroughly mixed and then allowed to stand at 4c overnight . all the samples were prepared for the immunoassay using the same extraction procedure as that for pig muscle as follows . the sample ( 2 g ) was chopped , and mixed with 10 ml pbs for 2 minutes using a waring blender ( omni international , marietta , ga , usa ) . then , the mixture was centrifuged for 10 minutes at 4,000 rpm ( centrifuge 5804r , eppendorf ag , hamburg , germany ) at room temperature . the fat layer was then discarded , and the upper liquid was transferred into a test tube , and analyzed directly by direct competitive elisa . ( a ) instrumentation for hplc analysisthe elisa results were verified using an hplc system ( shimadzu , kyoto , japan ) equipped with a lc-20ab pump and a rf-10axl fld ( excitation wavelength , 226 nm ; emission wavelength , 305 nm ) . a thermo ods-2 hypersil column ( 5 m , 4.6 mm 250 mm ) was used with a mobile phase consisting of 0.087% 1-pentanesulfonic acid sodium salt in 2% glacial acetic acid solution / acetonitrile ( 68 : 32 ) at a flow rate of 1.0 ml / min . the elisa results were verified using an hplc system ( shimadzu , kyoto , japan ) equipped with a lc-20ab pump and a rf-10axl fld ( excitation wavelength , 226 nm ; emission wavelength , 305 nm ) . a thermo ods-2 hypersil column ( 5 m , 4.6 mm 250 mm ) was used with a mobile phase consisting of 0.087% 1-pentanesulfonic acid sodium salt in 2% glacial acetic acid solution / acetonitrile ( 68 : 32 ) at a flow rate of 1.0 ml / min . ( b ) sample pretreatmenthomogenized tissue sample ( 5 g ) was extracted with acetonitrile ( 2 10 ml ) and stirred for 20 minutes on a shaker ( ika , staufen , germany ) . after centrifugation ( 1,500 g , 4c , 10 minutes ) , the supernatant was transferred into a separating funnel . the acetonitrile solution was defatted by washing with 20 ml hexane ( saturated by acetonitrile ) 3 times . the residue was redissolved in 5 ml 2% acetic acid solution , and was used for the solid - phase extraction . homogenized tissue sample ( 5 g ) was extracted with acetonitrile ( 2 10 ml ) and stirred for 20 minutes on a shaker ( ika , staufen , germany ) . after centrifugation ( 1,500 g , 4c , 10 minutes ) , the supernatant was transferred into a separating funnel . the acetonitrile solution was defatted by washing with 20 ml hexane ( saturated by acetonitrile ) 3 times . the residue was redissolved in 5 ml 2% acetic acid solution , and was used for the solid - phase extraction . ( c ) sample clean - upsolid - phase extraction was carried out using the extraction cartridges packed with multiwalled carbon nanotubes under reduced pressure . the cartridges were activated with 5 ml methanol , followed by 5 ml ddw , and then equilibrated with 5 ml 2% acetic acid solution . after the sample extracts were loaded , the cartridges were washed with 5 ml 2% acetic acid solution , and sequentially eluted with 7 ml 5% ammonia in methanol . the eluted sample was dried , and redissolved in 1 ml 2% acetic acid for hplc analysis , or redissolved in appropriate volume of pbs for elisa analysis . solid - phase extraction was carried out using the extraction cartridges packed with multiwalled carbon nanotubes under reduced pressure . the cartridges were activated with 5 ml methanol , followed by 5 ml ddw , and then equilibrated with 5 ml 2% acetic acid solution . after the sample extracts were loaded , the cartridges were washed with 5 ml 2% acetic acid solution , and sequentially eluted with 7 ml 5% ammonia in methanol . the eluted sample was dried , and redissolved in 1 ml 2% acetic acid for hplc analysis , or redissolved in appropriate volume of pbs for elisa analysis . the real food sample offered by tianjin entry - exit inspection and quarantine bureau was extracted by pbs according to sample preparation , the extracts were divided into two aliquots , one is analysed by elisa directly and the other is adjusted to ph 6.8 and incubated at 37c for 10 minutes , then dropped with -d - glucuronidase solution , deconjugated by for 60 minutes , the mixture was adjusted to ph 7.5 and analysed by elisa . after the purification of the antibody and the preparation of the enzyme tracer , direct competitive elisa was optimized . several parameters including antibody coating quantity , ionic strength , and ph of the diluting buffer were studied in details . the optimal quantities of the coating antibody and the enzyme conjugate required for the direct competitive elisa were determined by checkerboard titration . they were chosen according to the lowest ic50 with an absorbance value of 0.71.2 for the control sample during color development . the results showed that an antibody coating quantity of 1.0 g per well ( 100 l ) , and an enzyme conjugate dilution factor of 30 000 were the best among all testing conditions . it is reasonable to believe that the increased ionic strength has a detrimental effect on the interaction between the antibody and the analyte or enzyme conjugate where ionic driving forces prevail . the crystallizability of the buffer at a high salt concentration resulted in the choice of a concentration of 10 mmol / l pbs as the optimal dilution buffer . we found that the ic50 reached its minimum value at ph 7.5 , that is , the immunoassay for rac was more sensitive at ph 7.5 than at other phs . this is because acidic and alkaline solutions likely promote the denaturation of the antibody and/or enzyme conjugate , causing changes in their spatial structures with adverse effects on the reactions between the antibody and the analyte or enzyme conjugate . a 10 mmol / l pbs buffer of ph 7.5 was chosen as the optimal solvent for the rac standard ( or samples ) and enzyme conjugate dilutions , and 1.0 g of antibody per well was the optimal coating amount . a 6-point ( stepwise dilution of the rac standard solution ) calibration curve was performed in the elisa test , resulting in an average ic50 of 0.6 ng / ml and average ic15 of 0.04 ng / ml . these results showed that the obtained rac antibody was much more sensitive than those reported by previous studies [ 13 , 15 ] . crossreactions can affect analytical results by either giving false positives or by elevating the predicted concentration of the target compound when both the target and one or more structurally similar compounds are present . therefore , the specificity of the antibody toward a compound and its most probable crossreactants should be determined . the crossreactivity profile of the rac antibody was determined by comparing the dose - response curves of rac with those of 7 analogues including ractopamine , clenbuterol , salbutamol , isoproterenol , terbutaline , dobutamine , and isoxsuprine ( figure 1 ) . all these compounds showed no cross - reactivity with the rac antibody except for dobutamine . reported that if the antibody is developed against a compound with a very similar structure , crossreactivity will likely occur . in conclusion , it was reasonable that dobutamine which has a very similar structure to rac showed 7.5% crossreactivity with rac . the assay precision was studied by determining intra - assay and inter - assay reproducibilities . the variations in percent inhibition in the intra - assay for 20 , 5 , 1.25 , 0.31 , 0.08 , and 0.02 ng / ml rac tested in a microplate were 0.5 , 1.5 , 4.6 , 7.6 , 16.1 , and 29.6% , respectively . the inter - assay of the same material run over 6 months resulted in deviations from the means of 2.4 , 4.7 , 7.6 , 10.1 , 24.8 , and 32.4% , respectively . it seemed likely that antibody sensitivity to these low concentrations was poor and so reproducibility was greatly reduced . appropriate assays in accelerated trials such as the use of half - lives greater than 7 days at 37c are predictive of 612 months stability at 4c . therefore , stability trials were carried out with rac antibodies stored at 4c , room temperature , and 37c for 30 days . tables 1 and 2 show the results of the stability assays for the antibodies and enzyme conjugates , respectively . there was not a striking change in the ic50 value of the rac antibody stored at different temperatures for 30 days , and none for the enzyme conjugates stored for 7 days . moreover , color loss was not observed for both rapid assays during the experimental period . this indicated that temperature could not easily affect the activities of the rac antibody and enzyme conjugate . therefore , it is reasonable to conclude that both the antibody and enzyme tracer are stable enough to be used in subsequent tests , and even to produce a rac - elisa test kit . immunoassays are a rapid and convenient analysis method for food samples as they usually do not require sample preconcentration and clean - up steps . however , elisa methods may have high potential risks for nonspecific binding between the nontarget analytes and antibodies , and are consequently prone to matrix interferences . chemical compounds present in samples or sample extracts such as proteins , fat , and others , might nonspecifically affect the binding of the antibody and analytes , and might also affect other aspects of the assay . these so - called matrix effects can reduce the sensitivity and reliability of the competitive immunoassay . matrix effects are more pronounced in direct immunoassays where not only the specific antibodies but also the enzyme conjugate are under the influence of the sample matrix . several methods can be used to determine matrix effects . typically , interferences are quantified by comparing a standard curve prepared in buffer such as pbs with a calibration curve generated in the sample matrix known to be free of the analyte . if the 2 curves are superposable , the effect of the matrix is not significant , and the samples can be analyzed using the standard curve prepared in the matrix - free solution . in the present assay , the matrix effects of our samples were analyzed using direct competitive elisa . rac standard curves were prepared in pbs buffer , and in dilutions of extracts of rac - free samples to determine whether nonspecific interferences could be eliminated . in order to obtain a rapid , simple , and effective sample extraction method , several extraction solvents including methanol , ethanol , acetonitrile , and pbs buffer were tested . significant matrix interference was found when the sample organic solvent extracts were diluted 5 folds in pbs buffer . further dilution of the sample organic solvent extracts in pbs buffer could not reduce matrix interference . even the addition of fish skin gelatin ( fg ) , skimmed milk powder , and tween-20 to the organic solvent extracts could not reduce the nonspecific interactions , and lowered the amount of developed color in the assay . when pbs buffer was used as the extraction solvent alone without any organic solvent or enshrouding reagent , the standard curves prepared in rac - free sample extract and in pbs buffer alone were superposable , and the matrix interference problem was easily resolved . the calibration curves were created by direct competitive elisa using blank samples spiked with 6 different concentrations of rac . figure 5 shows the comparison of the standard curves of rac in extracts prepared in the 4 rac - free samples and in pbs buffer alone . considering the dilutions of the sample extracts , the detection limit of the assay in the blank samples was 0.2 g / kg which was much lower than that of the maximum residue levels ( mrls ) for rac in these matrices . thus , the loss in assay sensitivity was acceptable . to investigate the efficiency of the extraction method , 4 types of edible food samples were fortified with rac at 3 different levels , and were analyzed by the established direct competitive elisa method . it was found that all the recoveries of the rac residues in these samples were less than 100% , and those in pig liver samples were little lower than those in other samples . since the presence of water in these samples makes the amount of the whole extract bigger than that of pbs that had been added prior to extraction , the final concentration of rac is lower than that in theory . therefore , it is reasonable to conclude that pig liver with a higher quantity of water shows a lower recovery than those of other samples . due to its more complicated chemical component structure , pig liver samples were spiked with rac at 0 , 1 , 2 , 5 g / kg , respectively . after sample extraction and clean - up on a solid - phase extraction column , the purified extract was analyzed by hplc and elisa . the analytical results obtained with the two methods for the same extract were shown in figure 6 . also , the spiked pig liver was analyzed by the established elisa with a simple extraction procedure . figure 7 shows the correlations between the results of hplc with more complicated extraction and clean - up procedures and elisa with a simple extraction procedure . despite the fact that in this case the comparison was established with samples subjected to different treatments , the results correlated well ( r = 0.9517 ) . these results indicate that the developed elisa method can be used as a rapid screening method for the analysis of rac in food samples . so , it is important to detect the rac metabolites for a newly established elisa . due to the lack of rac glucuronide metabolites , they were not involved in the cross - reactivity profile of the antibody . before the analysis of the real food samples , -glucuronidase was used to deconjugate the rac glucuronide metabolites and prove indirectly if the antibody has the adequate binding of glucuronide metabolites . real food samples with precise rac concentration ( validated by hplc - ms according to china national analysis standard method , gbt 22147 - 2008 ) were offered by tianjin entry - exit inspection and quarantine bureau ( export and import samples , including pig muscle , pig liver , pettitoes ) . the results showed that there is no obvious difference in the rac concentration of the sample extracts with and without enzymatic hydrolysis , so we believe that the produced rac antibody has the ability of adequate binding with the rac glucuronide metabolites . theoretically speaking , pbs should be an appropriate extraction solution for both rac and the glucuronide metabolites due to the solubility of them in pbs . so , rac glucuronide metabolites can be analysed by the established elisa system without deconjugation by glucuronidase . the results were compared with the validated results ( shown in table 4 ) . in analysis of the 20 food samples using the direct competitive elisa resulted in 15 samples being negative ( rac concentration was less than lod of the assay ) . although the results obtained by elisa was lower than the validated results , they correlated well ( r = 0.9605 ) , indicating the precision of the elisa analytical system and the availability of it for the analysis of real food samples . the developed elisa method with a high sensitivity and specificity is suitable for the routine screening detection of ractopamine residues in chicken muscle , pettitoes , pig muscle , and liver . the sample extraction method is quite simple and rapid . although the recoveries are not very high , the detection limits ( 0.2 g / kg ) from the direct competitive elisa for these samples are low enough for the levels of mrl ( 10 g / kg for muscle , 90 g / kg for liver in japan ) . the stabilities of the rac antibody and rac enzyme tracer together with the good correlation ( r = 0.9517 ) between the analytical results of hplc and elisa demonstrate the accuracy of the developed elisa procedure , confirming its reliability for applications in the rapid screening of rac in food samples .

to determine ractopamine residues in animal food products ( chicken muscle , pettitoes , pig muscle , and pig liver ) , we established a rapid direct competitive enzyme - linked immunosorbent assay ( elisa ) using a polyclonal antibody generated from ractopamine - linker - bsa . the antibody showed high sensitivity and specificity in phosphate buffer , with an ic50 of 0.6 ng / ml , and the limit of detection was 0.04 ng / ml . the matrix effect of the samples was easily eliminated by one - step extraction with pbs , without any organic solution or clean - up procedure such as spe or liquid - liquid extraction , making it a much more simple and rapid method than previously reported ones . the detection limit in blank samples was 0.2 g / kg . to validate this new rac ( ractopamine hydrochloride ) elisa , a rac - free pig liver sample spiked at three different concentrations was prepared and analyzed by hplc and elisa . the results showed a good correlation between the data of elisa and hplc ( r2 > 0.95 ) , which proves that the established elisa is accurate enough to quantify the residue of rac in the animal derived foods .

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

ractopamine hydrochloride ( rachcl , mw 337.85 , ( 1r*,3r*),(1r*,3s*)-4-hydroxy - r-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methyl]-benzenemethanol hydrochloride ( figure 1 ) ) is a phenethanolamine member of the family of -adrenergic agonists ( -agonists ) . therefore , there has been an increasing number of analytical methods reported to monitor ractopamine in animal urine , feeds , and tissues such as instrument methods like hplc with electrochemical detection and fluorescence , lc - ms [ 10 , 11 ] , and gc - ms . however , these analytical approaches , which use several clean - up procedures ( liquid - liquid extraction ( lle ) and solid - phase extraction ( spe ) using different sorbents ) , are quite complicated , time - consuming , and expensive . also reported a monoclonal antibody immunoassay to determine ractopamine in swine feeds with a detection limit of 0.24 g / g sample . however , all of these elisa methods were mainly used for animal urine or feed samples , and complicated sample clean - up procedures ( lle and spe ) were required when animal tissues were analyzed . as a result , it is necessary to develop a more rapid , sensitive , and effective method for the determination of rac residues in edible animal foods including chicken muscle . horseradish peroxidase ( hrp ) , ovalbumin ( ova ) , -glucuronidase from escherichia coli , and freund 's complete and incomplete adjutants were purchased from sigma chemical co. ( saint louis , mo , usa ) . reagent grade 3 , 3 , 5 , 5-tetramethyl - benzidine ( tmb ) , hydrogen peroxide , isobutyl chloroformate , butane-1 , 4-diol diglycidyl ether , and other chemicals were also from sigma . polystyrene 96-well microplates were from nunc ( rockilde , denmark ) , and the microplate washer was from bio - rad ( hercules , calif , usa ) . phosphate - buffered saline ( 1 pbs ; 38.4 mmol / l na2hpo4h2o , 11.5 mmol / l nah2po4h2o , 154 mmol / l nacl , ph 7.5 ) , phosphate - buffered saline with 0.05% tween 20 ( pbst ) , coating buffer ( cb , 50 mmol / l sodium carbonate buffer , ph 9.6 ) , blocking buffer ( 0.5% skimmed milk powder in 1 pbs ) , and tmb substrate solution ( prepared by adding 3.3 mg tmb in 250 l dmso to 25 ml phosphate - citrate buffer ( 0.1 mol / l citric acid + 0.2 mol / l na2hpo4 ; ph 4.3 ) containing 3.25 l of a 30% h2o2 solution ) , and termination solution ( 2.5 mol / l h2so4 in ddw ) were used . the conjugating procedure of immunogen rac - bsa was adapted from elliott et al . in brief , 10 mg bsa was dissolved in 0.5 ml ddw , and the ph was adjusted to 10.8 using 1.0 mol / l sodium hydroxide . coupling agent solution ( 50 l of 22 l / l butane-1 , 4-diol diglycidyl ether in ddw ) was added to the bsa solution , and the mixture was incubated for 22 hours at room temperature under nitrogen atmosphere . the mixture was then added to the epoxy - activated bsa solution ( precooled at 4c , in the refrigerator ) , slowly in an ice bath , and incubated for 22 hours at room temperature under nitrogen atmosphere . the conjugating method of rac - hrp was similar to that of the immunogen in the first step except for the amounts of the reagents which were 5 mg enzyme hrp instead of 10 mg , and 8.5 mg rachcl ( 25 mol ) were added to the activated hrp solution . the conjugated immunogen was stored at 20c , and the enzyme tracer was stored at 4c with 0.01% thiomersal until required . the residue was diluted in a 2 ml mixture of dmf and 1 , 4-dioxane ( 1 : 1 ) , and then stirred for 10 minutes in an ice bath by adding 13 l ( about 0.05 mmol ) tributylamine . two white rabbits were immunized by intradermal and intramuscular injections of the emulsified rac - bsa immunogen . blood was collected from the marginal ear vein 10 days after each booster injection for antibody titer assay , and the whole blood was then collected after 6 immunizations . ( a ) indirect competitive elisaflat - bottom polystyrene microplates were coated with rac - oa conjugates at 1 g per well in 100 l coating buffer , and incubated overnight at room temperature . plates were washed 3 times with pbst using a 96pw microplate washer , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs per well for 1 hour at room temperature . after the plates were washed 3 times , 50 l of the appropriate antiserum / antibody dilution in pbs ( the other 50 l of rac standards in pbs were added to determine antibody specificity ) were added in each well for titer determination , and incubated for 1 hour at room temperature . the absorbance in each well was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . flat - bottom polystyrene microplates were coated with rac - oa conjugates at 1 g per well in 100 l coating buffer , and incubated overnight at room temperature . plates were washed 3 times with pbst using a 96pw microplate washer , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs per well for 1 hour at room temperature . after the plates were washed 3 times , 50 l of the appropriate antiserum / antibody dilution in pbs ( the other 50 l of rac standards in pbs were added to determine antibody specificity ) were added in each well for titer determination , and incubated for 1 hour at room temperature . the absorbance in each well was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . ( b ) direct competitive elisathe microplate well was coated with purified antibodies in 100 l coating buffer , and incubated overnight at room temperature . after the plate was washed 4 times , 50 l of the standard solution of rac dissolved in pbs ( or diluted sample extracts ) followed by 50 l of hrp - hapten conjugate solution diluted in pbs were added to each well , and the mixture was incubated in a shaker for 1 hour at room temperature . the absorbance was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . the microplate well was coated with purified antibodies in 100 l coating buffer , and incubated overnight at room temperature . after the plate was washed 4 times , 50 l of the standard solution of rac dissolved in pbs ( or diluted sample extracts ) followed by 50 l of hrp - hapten conjugate solution diluted in pbs were added to each well , and the mixture was incubated in a shaker for 1 hour at room temperature . the absorbance was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) . ( c ) optimization of the rac elisa testdirect competitive elisa was used to analyze the parameters of the described method such as the amount of the antibody coating , and the ionic strength and ph of the diluting buffer . three different amounts of antibodies ( 0.5 g , 1.0 g , and 1.5 g antibody per well ) were tested . direct competitive elisa was used to analyze the parameters of the described method such as the amount of the antibody coating , and the ionic strength and ph of the diluting buffer . three different amounts of antibodies ( 0.5 g , 1.0 g , and 1.5 g antibody per well ) were tested . ( d ) sample preparationfour different samples including chicken muscle , pig muscle , pig liver , and pettitoes were chosen to evaluate the performance of elisa . for the spiking study , each sample was spiked by dropping with the rac standard solution in methanol , making the samples with well - distributed different levels of rac , the samples were thoroughly mixed and then allowed to stand at 4c overnight . all the samples were prepared for the immunoassay using the same extraction procedure as that for pig muscle as follows . the sample ( 2 g ) was chopped , and mixed with 10 ml pbs for 2 minutes using a waring blender ( omni international , marietta , ga , usa ) . the fat layer was then discarded , and the upper liquid was transferred into a test tube , and analyzed directly by direct competitive elisa . four different samples including chicken muscle , pig muscle , pig liver , and pettitoes were chosen to evaluate the performance of elisa . for the spiking study , each sample was spiked by dropping with the rac standard solution in methanol , making the samples with well - distributed different levels of rac , the samples were thoroughly mixed and then allowed to stand at 4c overnight . all the samples were prepared for the immunoassay using the same extraction procedure as that for pig muscle as follows . the sample ( 2 g ) was chopped , and mixed with 10 ml pbs for 2 minutes using a waring blender ( omni international , marietta , ga , usa ) . the fat layer was then discarded , and the upper liquid was transferred into a test tube , and analyzed directly by direct competitive elisa . the residue was redissolved in 5 ml 2% acetic acid solution , and was used for the solid - phase extraction . the residue was redissolved in 5 ml 2% acetic acid solution , and was used for the solid - phase extraction . after the purification of the antibody and the preparation of the enzyme tracer , direct competitive elisa was optimized . several parameters including antibody coating quantity , ionic strength , and ph of the diluting buffer were studied in details . the optimal quantities of the coating antibody and the enzyme conjugate required for the direct competitive elisa were determined by checkerboard titration . they were chosen according to the lowest ic50 with an absorbance value of 0.71.2 for the control sample during color development . the results showed that an antibody coating quantity of 1.0 g per well ( 100 l ) , and an enzyme conjugate dilution factor of 30 000 were the best among all testing conditions . it is reasonable to believe that the increased ionic strength has a detrimental effect on the interaction between the antibody and the analyte or enzyme conjugate where ionic driving forces prevail . the crystallizability of the buffer at a high salt concentration resulted in the choice of a concentration of 10 mmol / l pbs as the optimal dilution buffer . this is because acidic and alkaline solutions likely promote the denaturation of the antibody and/or enzyme conjugate , causing changes in their spatial structures with adverse effects on the reactions between the antibody and the analyte or enzyme conjugate . a 6-point ( stepwise dilution of the rac standard solution ) calibration curve was performed in the elisa test , resulting in an average ic50 of 0.6 ng / ml and average ic15 of 0.04 ng / ml . these results showed that the obtained rac antibody was much more sensitive than those reported by previous studies [ 13 , 15 ] . therefore , the specificity of the antibody toward a compound and its most probable crossreactants should be determined . the crossreactivity profile of the rac antibody was determined by comparing the dose - response curves of rac with those of 7 analogues including ractopamine , clenbuterol , salbutamol , isoproterenol , terbutaline , dobutamine , and isoxsuprine ( figure 1 ) . the variations in percent inhibition in the intra - assay for 20 , 5 , 1.25 , 0.31 , 0.08 , and 0.02 ng / ml rac tested in a microplate were 0.5 , 1.5 , 4.6 , 7.6 , 16.1 , and 29.6% , respectively . the inter - assay of the same material run over 6 months resulted in deviations from the means of 2.4 , 4.7 , 7.6 , 10.1 , 24.8 , and 32.4% , respectively . tables 1 and 2 show the results of the stability assays for the antibodies and enzyme conjugates , respectively . there was not a striking change in the ic50 value of the rac antibody stored at different temperatures for 30 days , and none for the enzyme conjugates stored for 7 days . therefore , it is reasonable to conclude that both the antibody and enzyme tracer are stable enough to be used in subsequent tests , and even to produce a rac - elisa test kit . immunoassays are a rapid and convenient analysis method for food samples as they usually do not require sample preconcentration and clean - up steps . however , elisa methods may have high potential risks for nonspecific binding between the nontarget analytes and antibodies , and are consequently prone to matrix interferences . chemical compounds present in samples or sample extracts such as proteins , fat , and others , might nonspecifically affect the binding of the antibody and analytes , and might also affect other aspects of the assay . these so - called matrix effects can reduce the sensitivity and reliability of the competitive immunoassay . typically , interferences are quantified by comparing a standard curve prepared in buffer such as pbs with a calibration curve generated in the sample matrix known to be free of the analyte . if the 2 curves are superposable , the effect of the matrix is not significant , and the samples can be analyzed using the standard curve prepared in the matrix - free solution . in the present assay , the matrix effects of our samples were analyzed using direct competitive elisa . rac standard curves were prepared in pbs buffer , and in dilutions of extracts of rac - free samples to determine whether nonspecific interferences could be eliminated . in order to obtain a rapid , simple , and effective sample extraction method , several extraction solvents including methanol , ethanol , acetonitrile , and pbs buffer were tested . even the addition of fish skin gelatin ( fg ) , skimmed milk powder , and tween-20 to the organic solvent extracts could not reduce the nonspecific interactions , and lowered the amount of developed color in the assay . when pbs buffer was used as the extraction solvent alone without any organic solvent or enshrouding reagent , the standard curves prepared in rac - free sample extract and in pbs buffer alone were superposable , and the matrix interference problem was easily resolved . the calibration curves were created by direct competitive elisa using blank samples spiked with 6 different concentrations of rac . figure 5 shows the comparison of the standard curves of rac in extracts prepared in the 4 rac - free samples and in pbs buffer alone . considering the dilutions of the sample extracts , the detection limit of the assay in the blank samples was 0.2 g / kg which was much lower than that of the maximum residue levels ( mrls ) for rac in these matrices . to investigate the efficiency of the extraction method , 4 types of edible food samples were fortified with rac at 3 different levels , and were analyzed by the established direct competitive elisa method . it was found that all the recoveries of the rac residues in these samples were less than 100% , and those in pig liver samples were little lower than those in other samples . since the presence of water in these samples makes the amount of the whole extract bigger than that of pbs that had been added prior to extraction , the final concentration of rac is lower than that in theory . due to its more complicated chemical component structure , pig liver samples were spiked with rac at 0 , 1 , 2 , 5 g / kg , respectively . after sample extraction and clean - up on a solid - phase extraction column , the purified extract was analyzed by hplc and elisa . also , the spiked pig liver was analyzed by the established elisa with a simple extraction procedure . figure 7 shows the correlations between the results of hplc with more complicated extraction and clean - up procedures and elisa with a simple extraction procedure . these results indicate that the developed elisa method can be used as a rapid screening method for the analysis of rac in food samples . due to the lack of rac glucuronide metabolites , they were not involved in the cross - reactivity profile of the antibody . before the analysis of the real food samples , -glucuronidase was used to deconjugate the rac glucuronide metabolites and prove indirectly if the antibody has the adequate binding of glucuronide metabolites . real food samples with precise rac concentration ( validated by hplc - ms according to china national analysis standard method , gbt 22147 - 2008 ) were offered by tianjin entry - exit inspection and quarantine bureau ( export and import samples , including pig muscle , pig liver , pettitoes ) . the results showed that there is no obvious difference in the rac concentration of the sample extracts with and without enzymatic hydrolysis , so we believe that the produced rac antibody has the ability of adequate binding with the rac glucuronide metabolites . so , rac glucuronide metabolites can be analysed by the established elisa system without deconjugation by glucuronidase . in analysis of the 20 food samples using the direct competitive elisa resulted in 15 samples being negative ( rac concentration was less than lod of the assay ) . although the results obtained by elisa was lower than the validated results , they correlated well ( r = 0.9605 ) , indicating the precision of the elisa analytical system and the availability of it for the analysis of real food samples . the developed elisa method with a high sensitivity and specificity is suitable for the routine screening detection of ractopamine residues in chicken muscle , pettitoes , pig muscle , and liver . the sample extraction method is quite simple and rapid . although the recoveries are not very high , the detection limits ( 0.2 g / kg ) from the direct competitive elisa for these samples are low enough for the levels of mrl ( 10 g / kg for muscle , 90 g / kg for liver in japan ) . the stabilities of the rac antibody and rac enzyme tracer together with the good correlation ( r = 0.9517 ) between the analytical results of hplc and elisa demonstrate the accuracy of the developed elisa procedure , confirming its reliability for applications in the rapid screening of rac in food samples .

emerging demographic changes , mental health issues affecting individual and community well - being and global pressures of supply and demand present international challenges for any effort to reshape a health system , especially in countries with strained financial and human resources . primary health care 's contribution in promoting health , preventing debilitating disease and reducing disability has positioned it as a key player around the health care reform discussion table . integration , defined as the actual provision of services one needs at the time they are needed , is a key issue of contemporary primary health care . community - based integrated care is a strategic vision that promotes more joined and consistent action of the health care workforce towards improved performance , thus maximizing population health . in light of this definition , the focus is on multidisciplinary teams , rather than individuals , with partners recognizing , valuing and trusting each others ' rationales to meet shared endpoints in the provision of qualitative and comprehensive health care services . achieving integrated phc is an extremely tedious and arduous process , with inherent tension between those who advocate targeted integration of care ( vertical ) for priority conditions and those who advocate comprehensive integration ( horizontal ) that builds healthy communities . both vertical and horizontal integration are needed and without the presence of both , there is fragmentation and discontinuity in health outcomes . international organizations are currently discussing the integration of mental health care services into phc with a relevant report , developed by wonca and who , illustrating the importance and urgency of this endeavor . during the past few months , integration of phc through the development of super surgeries and polyclinics was on the front page of a daily newspaper in the uk . similarly in other countries and regions , such as australia , the usa , canada , and northern europe [ 57 ] , there is clearly the political will and a cross - governmental approach to integrate services , providing a continuum of seamless care to health care consumers . the case of greece is an example of a southern european country still striving to set health care priorities , struggling to allocate scarce resources , not always in the most cost - effective or quality assuring way . in terms of organization , the greek health care system is characterized by the co - existence of the national health system ( nhs ) , a mandatory social insurance ( si ) and a voluntary private health insurance system . the nhs provides universal coverage to the greek population , operating under the principles of equity , equal access and social cohesion . in addition , 97% of the population is covered by approximately 35 different social insurance funds ( comprising the mandatory si ) , while 8% of the population maintains complementary voluntary health insurance coverage . privately purchased health services in greece are funded , almost equally , by public and private sources . public expenditure is financed by taxes ( direct and indirect ) as well as by mandatory health insurance contributions , made by employers and insured persons . voluntary payments by individuals or employers represent 42% of total health expenditure ( 2002 ) , making the greek health care system one of the most privatized among european union ( eu ) countries . despite attempts to decentralize the governance of nhs with horizontal integration of regional health and welfare services , its main structure and orientation remains vertical , with a top - to - down approach and one central point for decision - making . primary health care in the public sector is delivered through a dual system , consisting of phc centers and hospital ambulatory ( outpatient ) services that belong to the nhs , and 350 primary care units that belong to the largest si fund ( ika ) with 5.5 million beneficiaries . as a result of the high ratio of physicians per 100,000 inhabitants , one of the highest in the eu , combined with one of the lowest ratios of nurses , there is a strong emphasis on curative services , rather than health promotion , disease prevention , rehabilitation and home care services . over the past years a few attempts , aimed at modernizing and improving the greek nhs with a specific reference to phc , have been initiated but have not been followed - up by subsequent health ministers and administrators [ 1215 ] . issues of integrated phc have not received prompt attention at a time when in the international arena policy makers and practitioners have been discussing phc reform and the potential impact on phc integration of certain barriers related to patients , health care professionals or organization . recently , the greek ministry of health and solidarity brought into consultation a legislative framework for the organization and operation of a reformed phc system . thus , we decided to systematically review the literature with the aim to identify constraints and opportunities as well as to address priorities towards the development of an integrated phc policy for greece . a full systematic review , employing both electronic and hand search techniques , was undertaken to investigate the extent to which integrated phc is considered to be a key issue in the current research , educational and policy agenda in greece . the operational definition adopted for integration included a coherent set of methods and models on the funding , administrative , organizational , service delivery and clinical levels designed to create connectivity , alignment and collaboration within and between the cure and care sectors [ 17 p. 3 ] . for the purposes of this review , integration was regarded to occur at the following four levels [ 16 , 18 ] : functional integration occurring at the macro level of the care system , i.e. through the mainstreaming of financing and regulation of cure , care , prevention , and social services . organizational integration occurring at the meso level of systems , i.e. in the form of mergers , contracting or strategic alliances between health and social care institutions . professional integration occurring at the meso level , i.e. in the form of mergers ( group practices ) , contracting or strategic alliances between health care professionals . clinical integration occurring at the micro level , i.e. by providing continuity , co - operation and coherence in the primary process of care delivery an advisory group assisted in the development of review questions and procedures as well as in the identification of the specific areas within the topic that would be most useful to scrutinize in - depth . a protocol was set out before the review , providing explicit information on the methods to be employed . the review was undertaken by a multidisciplinary team of researchers with a public health research background ( ms , cv , am , mp ) . a broad range of evidence types was addressed , including various research designs and policy , review as well as original papers , clinical practice experiences , user analytic and opinion evidence with a focus on completed studies and secondary source research . the review included all papers published in english or greek language between january 1999 and august 2008 . to deal with the heterogeneity of studies , a two - stage review was employed to assess the relevance of findings . at the first stage , a wider group of thematic fields was included to enable mapping and exploration of the whole field of policy related to integrated primary health care. at the second stage , this group was narrowed down to a sub - set of studies with a focus on policy implication , according to what extent barriers and facilitators of phc integration were addressed within the identified papers . we included all papers that contained either a statement of a decision regarding a goal or plan for implementing integrated phc , or reported on how policy for integrated primary care was made or its influence in policy markets . publications not addressing these particular areas or solely focusing on clinical topics were excluded from the review . quality assessment criteria were developed to gain an understanding of the relative strengths and weaknesses of the body of evidence to be taken into account during the process of synthesis . consensus procedures were employed to define the criteria for quality assessment and checklists were developed to facilitate this task . among the quality assessment criteria were : a ) quality of reporting ( adequacy of reporting important aspects of methodology ) , b ) quality of an intervention ( whether it has been used appropriately ) and c ) generalizability ( to what extent routine practice or the usual setting was reflected ) . the electronic search was performed during august 2008 by two researchers ( cv , ms ) in two databases ; medline and scopus from 1999 to august 2008 . the search in scopus was limited to the following pre - defined scientific fields , linked with financial , technological and social aspects of integration in phc : business and management searches were conducted using a set of pre - defined medical subject heading ( mesh ) terms , specified by the review advisory group . the terms employed were { delivery of health care } and { integrated } and { greece } or { primary health care } and { greece}. a first decision was made based on titles and , where available , abstracts , which were assessed against the inclusion criteria . for publications that appeared to meet the inclusion criteria , or in cases when a definite decision could not be made based on the title and/or abstract alone , the full paper was obtained for detailed assessment against the inclusion criteria . reference lists of all included studies were also searched for eligible studies missed by the electronic search . citation searching was also conducted selecting a number of key papers , already identified in the review , and then searching for publications that had cited these papers . this approach ensured identification of a cluster of related , and therefore , highly relevant , papers . one reviewer ( ms ) was responsible for identifying and removing duplicate references for publications that appeared to meet the inclusion criteria . since electronic databases are per definition prone to language and geographical biases , an additional hand search was performed in the following four greek journals , non - indexed in pubmed : koinoniki ergasia. the specific journals were selected as the leading scientific journals in the fields of medicine ( generic ) , primary health care , nursing and social work , respectively . two researchers ( am , mp ) appraised all volumes published during the pre - selected time period . during the first stage , the titles , abstracts and keywords were assessed against the inclusion criteria and then full text was retrieved for publications meeting inclusion criteria . at the second stage , any discrepancies in decisions were discussed in depth by the research team and the article was included or excluded , accordingly . standardized data extraction forms were used , designed according to the review objectives , capturing information required for descriptive purposes and for later analyses . recorded features of the included publications were as follows : first author 's name , year of publication , journal 's origin ( international or greek ) , level of integration , level of outcome change and study topic . three researchers ( ms , am , mp ) extracted the data and another researcher ( cl ) independently checked the data extraction forms for accuracy and completeness . disagreements noted were resolved by consensus among researchers and in certain cases by an additional independent researcher . a full systematic review , employing both electronic and hand search techniques , was undertaken to investigate the extent to which integrated phc is considered to be a key issue in the current research , educational and policy agenda in greece . the operational definition adopted for integration included a coherent set of methods and models on the funding , administrative , organizational , service delivery and clinical levels designed to create connectivity , alignment and collaboration within and between the cure and care sectors [ 17 p. 3 ] . for the purposes of this review , integration was regarded to occur at the following four levels [ 16 , 18 ] : functional integration occurring at the macro level of the care system , i.e. through the mainstreaming of financing and regulation of cure , care , prevention , and social services . organizational integration occurring at the meso level of systems , i.e. in the form of mergers , contracting or strategic alliances between health and social care institutions . professional integration occurring at the meso level , i.e. in the form of mergers ( group practices ) , contracting or strategic alliances between health care professionals . clinical integration occurring at the micro level , i.e. by providing continuity , co - operation and coherence in the primary process of care delivery an advisory group assisted in the development of review questions and procedures as well as in the identification of the specific areas within the topic that would be most useful to scrutinize in - depth . a protocol was set out before the review , providing explicit information on the methods to be employed . the review was undertaken by a multidisciplinary team of researchers with a public health research background ( ms , cv , am , mp ) . a broad range of evidence types was addressed , including various research designs and policy , review as well as original papers , clinical practice experiences , user analytic and opinion evidence with a focus on completed studies and secondary source research . the review included all papers published in english or greek language between january 1999 and august 2008 . to deal with the heterogeneity of studies , a two - stage review was employed to assess the relevance of findings . at the first stage , a wider group of thematic fields was included to enable mapping and exploration of the whole field of policy related to integrated primary health care. at the second stage , this group was narrowed down to a sub - set of studies with a focus on policy implication , according to what extent barriers and facilitators of phc integration were addressed within the identified papers . we included all papers that contained either a statement of a decision regarding a goal or plan for implementing integrated phc , or reported on how policy for integrated primary care was made or its influence in policy markets . publications not addressing these particular areas or solely focusing on clinical topics were excluded from the review . quality assessment criteria were developed to gain an understanding of the relative strengths and weaknesses of the body of evidence to be taken into account during the process of synthesis . consensus procedures were employed to define the criteria for quality assessment and checklists were developed to facilitate this task . among the quality assessment criteria were : a ) quality of reporting ( adequacy of reporting important aspects of methodology ) , b ) quality of an intervention ( whether it has been used appropriately ) and c ) generalizability ( to what extent routine practice or the usual setting was reflected ) . the electronic search was performed during august 2008 by two researchers ( cv , ms ) in two databases ; medline and scopus from 1999 to august 2008 . the search in scopus was limited to the following pre - defined scientific fields , linked with financial , technological and social aspects of integration in phc : business and management searches were conducted using a set of pre - defined medical subject heading ( mesh ) terms , specified by the review advisory group . the terms employed were { delivery of health care } and { integrated } and { greece } or { primary health care } and { greece}. a first decision was made based on titles and , where available , abstracts , which were assessed against the inclusion criteria . for publications that appeared to meet the inclusion criteria , or in cases when a definite decision could not be made based on the title and/or abstract alone , the full paper was obtained for detailed assessment against the inclusion criteria . reference lists of all included studies were also searched for eligible studies missed by the electronic search . citation searching was also conducted selecting a number of key papers , already identified in the review , and then searching for publications that had cited these papers . this approach ensured identification of a cluster of related , and therefore , highly relevant , papers . one reviewer ( ms ) was responsible for identifying and removing duplicate references for publications that appeared to meet the inclusion criteria . since electronic databases are per definition prone to language and geographical biases , an additional hand search was performed in the following four greek journals , non - indexed in pubmed : the specific journals were selected as the leading scientific journals in the fields of medicine ( generic ) , primary health care , nursing and social work , respectively . two researchers ( am , mp ) appraised all volumes published during the pre - selected time period . during the first stage , the titles , abstracts and keywords were assessed against the inclusion criteria and then full text was retrieved for publications meeting inclusion criteria . at the second stage , any discrepancies in decisions were discussed in depth by the research team and the article was included or excluded , accordingly . standardized data extraction forms were used , designed according to the review objectives , capturing information required for descriptive purposes and for later analyses . recorded features of the included publications were as follows : first author 's name , year of publication , journal 's origin ( international or greek ) , level of integration , level of outcome change and study topic . three researchers ( ms , am , mp ) extracted the data and another researcher ( cl ) independently checked the data extraction forms for accuracy and completeness . disagreements noted were resolved by consensus among researchers and in certain cases by an additional independent researcher . our electronic search of the literature identified 161 published studies . upon reviewing all of them , 27 manuscripts from medline and 26 from scopus papers when common articles were removed , a total number of 34 articles were selected for further scrutiny . upon in depth review of these electronically - searched articles , consensus was reached on 26 as meeting all inclusion criteria ( table 1 ) . manual search of four national greek journals resulted in another 37 relevant articles which , upon further scrutiny , were reduced to 27 as meeting all inclusion criteria ( table 2 ) . thus , a total of 53 electronic and paper publications were found to be within the scope of our review . a schematic view of the above search strategy and outcomes is illustrated in figure 1 . classification of articles by level and type of integration as well as by level of outcome change is presented in tables 3 and 4 , respectively . the appendix contains a complete list , alphabetical by author , of the papers reviewed for the present study . starting in the year 2000 , several editorials , proposals and reports were published with a focus on client satisfaction and improved coordination between outpatient hospital services and phc [ 12 , 2023 ] . continuity of care through the management of common episodes of care by the same health team over time has remained an unmet need within primary care delivery in greece [ 14 , 15 , 2426 ] . furthermore , the management and processing of information through the use of a common electronic medical record system with a user - friendly interface have often been the objectives of research proposals and pilot projects during the last decade [ 2733 ] . the development of an e - health care network within primary care settings has been linked with the benefits of real time access to clinical information and reliable research materials , data protection as well as community health consequently , the role of technology and telematic systems in the use of clinical information in a country with geographic and bureaucratic barriers has been viewed as a priority . although it is well - established that educational , training and awareness raising activities are required for adoption and maintenance of electronic health record services [ 33 , 35 ] , implementation of technology alone is not sufficient to cover policy or legislative gaps and other organizational deficiencies [ 20 , 31 , 36 ] . an equally significant issue is the application of scientific models to regulate health care use , eliminating waste , by supporting better utilization and allocation of resources within a national strategic health care plan [ 22 , 37 ] . coordinated care in the patient 's own environment , through a comprehensive interplay of health and social care providers is regarded as the ideal pathway , safeguarding equity , patients ' rights for care , cost - effectiveness and efficiency [ 3842 ] . the need for establishment of efficient referral pathways and services involving community nurses , social workers , psychologists and other health professionals has been underlined [ 14 , 25 , 43 ] . providing formally instituted incentives for primary care professionals towards life - long training , research capacity development and expertise building has also been emphasized in several publications [ 35 , 4448 ] . service agreements between phc providers and regional health authorities , establishment and operation of the phc team and routine evaluation of quality of services based on internationally accepted , culturally adapted tools have all been debated or even pilot - tested by greek investigators [ 13 , 14 , 38 , 39 , 49 ] . our electronic search of the literature identified 161 published studies . upon reviewing all of them , 27 manuscripts from medline and 26 from scopus papers when common articles were removed , a total number of 34 articles were selected for further scrutiny . upon in depth review of these electronically - searched articles , consensus was reached on 26 as meeting all inclusion criteria ( table 1 ) . manual search of four national greek journals resulted in another 37 relevant articles which , upon further scrutiny , were reduced to 27 as meeting all inclusion criteria ( table 2 ) . thus , a total of 53 electronic and paper publications were found to be within the scope of our review . a schematic view of the above search strategy and outcomes is illustrated in figure 1 . classification of articles by level and type of integration as well as by level of outcome change is presented in tables 3 and 4 , respectively . the appendix contains a complete list , alphabetical by author , of the papers reviewed for the present study . starting in the year 2000 , several editorials , proposals and reports were published with a focus on client satisfaction and improved coordination between outpatient hospital services and phc [ 12 , 2023 ] . continuity of care through the management of common episodes of care by the same health team over time has remained an unmet need within primary care delivery in greece [ 14 , 15 , 2426 ] . furthermore , the management and processing of information through the use of a common electronic medical record system with a user - friendly interface have often been the objectives of research proposals and pilot projects during the last decade [ 2733 ] . the development of an e - health care network within primary care settings has been linked with the benefits of real time access to clinical information and reliable research materials , data protection as well as community health consequently , the role of technology and telematic systems in the use of clinical information in a country with geographic and bureaucratic barriers has been viewed as a priority . although it is well - established that educational , training and awareness raising activities are required for adoption and maintenance of electronic health record services [ 33 , 35 ] , implementation of technology alone is not sufficient to cover policy or legislative gaps and other organizational deficiencies [ 20 , 31 , 36 ] . an equally significant issue is the application of scientific models to regulate health care use , eliminating waste , by supporting better utilization and allocation of resources within a national strategic health care plan [ 22 , 37 ] . coordinated care in the patient 's own environment , through a comprehensive interplay of health and social care providers is regarded as the ideal pathway , safeguarding equity , patients ' rights for care , cost - effectiveness and efficiency [ 3842 ] . the need for establishment of efficient referral pathways and services involving community nurses , social workers , psychologists and other health professionals has been underlined [ 14 , 25 , 43 ] . providing formally instituted incentives for primary care professionals towards life - long training , research capacity development and expertise building has also been emphasized in several publications [ 35 , 4448 ] . service agreements between phc providers and regional health authorities , establishment and operation of the phc team and routine evaluation of quality of services based on internationally accepted , culturally adapted tools have all been debated or even pilot - tested by greek investigators [ 13 , 14 , 38 , 39 , 49 ] . despite the numerous papers , reports and editorials published mostly during the last decade , development of integrated phc , based on this review , still remains a neglected subject in the current health policy agenda in greece . this is the main finding of our systematic review which , although limited to only two databases and focused mostly on health care research rather than policy literature , seems to be conclusive . it has been recognized that most of the times , national health planning has not been carried out in the context of evidence - based practice or a comprehensive health needs assessment [ 23 , 24 , 34 , 42 ] . differences across the country between urban and rural primary care provision patterns [ 43 , 50 ] as well as practice variations between nhs and major national health maintenance organizations [ 10 , 22 ] or private primary care providers have greatly contributed in the fragmentation and discontinuity of health care services [ 12 , 14 , 26 , 39 ] . although our review identified some papers calling for integration of primary care services [ 14 , 39 ] , including those reporting on attempts towards clinical governance practices , and collaborative initiatives with local authorities in establishing community outreach programs [ 26 , 42 , 51 , 52 ] , greece still operates under a conventional primary care management perspective . even the new primary health care act , introducing the concepts of a personal physician and polyclinics in greece , does not promote integrated phc measures . thus , it was not surprising that our review revealed only one study comparing integrated primary care measures vs. conventional primary care management . the study , implemented in crete in 2004 based on a pioneering health policy initiative , presented some promising outcomes but had no continuity due to withdrawal of governmental and legislative support . from a theoretical perspective , integrated phc has been seen as a solution to problems related to planning , funding , operation and effectiveness of the greek health care system . particularly , the absence of prevention and health promotion services in the community [ 48 , 53 , 54 ] as well as the shortage of phc staff and equipment in rural primary care centers [ 14 , 28 , 35 , 44 , 5557 ] , seem to contribute to the low level of integrated phc in greece . the gap observed between theory and practice [ 23 , 24 , 50 , 57 ] , as revealed from this systematic review , could be attributed to the limited research capacity in phc and the lack of evidence - based knowledge , impacting on the ability of the greek nhs to provide a seamless continuum of phc services . based on outcomes of this review , it is clear that contemporary greece is lagging behind in policy development towards integrated phc . the bold attempt towards health care reform and integration of regional health and welfare services made by the legislative act of 2001 has been negated or inactivated by recent legislation . thus , the phc policy initiative in the region of crete was short - lived , without any imitators . empirical research , implemented by the national school of public health and the department of social medicine at the university of crete , seems to have no impact on health policymakers ' intentions towards integrated phc . in order to move forward , legislators and ministry of health and solidarity administrators need to address new challenges , including poverty , population aging , increasing health care expenditures and limited resources . the rapidly changing pattern of morbidity in greece , with mental disorders and cardiovascular diseases being the predominant disease entities , signals the urgency for adopting an integrated phc policy . a more organized approach in primary care , with easily accessible community health centers ( chc ) located in neighborhoods , as suggested by han van oosterbos , seems to be suitable in the current greek setting . this approach implies transfer of resources to local authorities , an on - going request in greece , as well as a suitable legislative framework [ 24 , 26 ] . the experiences gained in crete advocate towards this direction , with geographical and cultural characteristics of the greek population favoring the development of chcs . it has been argued that the introduction of a minimum package of health care services mandated by the greek government should be introduced in the current political agenda . changes in the academic curriculum towards the introduction of interdisciplinary and problem - based undergraduate education are considered essential in establishing an integrated phc system and promoting effectiveness and efficiency of services offered [ 53 , 55 , 56 , 59 , 60 ] . research funds should also be allocated to test the effectiveness of certain organizational and administrative schemes within greek settings . integrated phc requires a multidisciplinary team approach , assigning new roles to primary care providers . development of new roles for all disciplines , and revision of existing job descriptions , is a critical task that should be undertaken at a legislative , policy and administrative level uniformly for all health regions of greece [ 15 , 25 , 45 , 48 , 50 , 53 , 57 , 61 ] . debating which roles should be assigned to phc teams or individuals in a multidisciplinary primary care setting , and how various initiatives can be integrated within different levels of care , present promising areas for future research [ 6265 ] , in line with international experience . the potential leadership role and level of involvement of greek nurses towards integrated health care has only recently been pursued as a policy topic . poor research skills and lack of interest and knowledge in evaluating outcomes of care , for the majority of greek nurses employed in phc [ 44 , 45 , 49 , 57 , 65 ] , have resulted in lack of evidence concerning nursing 's contributions to service organization management , cross - boundary working , management of resources and workforce development . similarly , social workers have been striving to claim a role within phc settings , with the discipline of social work being undervalued and unappreciated . in order to move forward , social work should become actively involved in disease prevention programs , early intervention and effective use of scarce resources . thus , possession of all necessary skills and knowledge , both from undergraduate and postgraduate education as well as continuous professional development , should become a high priority in the phc agenda . greek health policy makers and clinicians are urged to take into consideration the mounting evidence that the literature conveys , as well as the pioneering attempts of introducing clinical governance in phc settings . new educational curricula for health professionals , both at the undergraduate and graduate level , should be established with the emphasis on multidisciplinary training and collaboration . despite the numerous papers , reports and editorials published mostly during the last decade , development of integrated phc , based on this review , still remains a neglected subject in the current health policy agenda in greece . this is the main finding of our systematic review which , although limited to only two databases and focused mostly on health care research rather than policy literature , seems to be conclusive . it has been recognized that most of the times , national health planning has not been carried out in the context of evidence - based practice or a comprehensive health needs assessment [ 23 , 24 , 34 , 42 ] . differences across the country between urban and rural primary care provision patterns [ 43 , 50 ] as well as practice variations between nhs and major national health maintenance organizations [ 10 , 22 ] or private primary care providers have greatly contributed in the fragmentation and discontinuity of health care services [ 12 , 14 , 26 , 39 ] . although our review identified some papers calling for integration of primary care services [ 14 , 39 ] , including those reporting on attempts towards clinical governance practices , and collaborative initiatives with local authorities in establishing community outreach programs [ 26 , 42 , 51 , 52 ] , greece still operates under a conventional primary care management perspective . even the new primary health care act , introducing the concepts of a personal physician and polyclinics in greece , does not promote integrated phc measures . thus , it was not surprising that our review revealed only one study comparing integrated primary care measures vs. conventional primary care management . the study , implemented in crete in 2004 based on a pioneering health policy initiative , presented some promising outcomes but had no continuity due to withdrawal of governmental and legislative support . from a theoretical perspective , integrated phc has been seen as a solution to problems related to planning , funding , operation and effectiveness of the greek health care system . particularly , the absence of prevention and health promotion services in the community [ 48 , 53 , 54 ] as well as the shortage of phc staff and equipment in rural primary care centers [ 14 , 28 , 35 , 44 , 5557 ] , seem to contribute to the low level of integrated phc in greece . the gap observed between theory and practice [ 23 , 24 , 50 , 57 ] , as revealed from this systematic review , could be attributed to the limited research capacity in phc and the lack of evidence - based knowledge , impacting on the ability of the greek nhs to provide a seamless continuum of phc services . based on outcomes of this review , it is clear that contemporary greece is lagging behind in policy development towards integrated phc . the bold attempt towards health care reform and integration of regional health and welfare services made by the legislative act of 2001 has been negated or inactivated by recent legislation . thus , the phc policy initiative in the region of crete was short - lived , without any imitators . empirical research , implemented by the national school of public health and the department of social medicine at the university of crete , seems to have no impact on health policymakers ' intentions towards integrated phc . in order to move forward , legislators and ministry of health and solidarity administrators need to address new challenges , including poverty , population aging , increasing health care expenditures and limited resources . the rapidly changing pattern of morbidity in greece , with mental disorders and cardiovascular diseases being the predominant disease entities , signals the urgency for adopting an integrated phc policy . a more organized approach in primary care , with easily accessible community health centers ( chc ) located in neighborhoods , as suggested by han van oosterbos , seems to be suitable in the current greek setting . this approach implies transfer of resources to local authorities , an on - going request in greece , as well as a suitable legislative framework [ 24 , 26 ] . the experiences gained in crete advocate towards this direction , with geographical and cultural characteristics of the greek population favoring the development of chcs . it has been argued that the introduction of a minimum package of health care services mandated by the greek government should be introduced in the current political agenda . changes in the academic curriculum towards the introduction of interdisciplinary and problem - based undergraduate education are considered essential in establishing an integrated phc system and promoting effectiveness and efficiency of services offered [ 53 , 55 , 56 , 59 , 60 ] . research funds should also be allocated to test the effectiveness of certain organizational and administrative schemes within greek settings . integrated phc requires a multidisciplinary team approach , assigning new roles to primary care providers . development of new roles for all disciplines , and revision of existing job descriptions , is a critical task that should be undertaken at a legislative , policy and administrative level uniformly for all health regions of greece [ 15 , 25 , 45 , 48 , 50 , 53 , 57 , 61 ] . debating which roles should be assigned to phc teams or individuals in a multidisciplinary primary care setting , and how various initiatives can be integrated within different levels of care , present promising areas for future research [ 6265 ] , in line with international experience . the potential leadership role and level of involvement of greek nurses towards integrated health care has only recently been pursued as a policy topic . poor research skills and lack of interest and knowledge in evaluating outcomes of care , for the majority of greek nurses employed in phc [ 44 , 45 , 49 , 57 , 65 ] , have resulted in lack of evidence concerning nursing 's contributions to service organization management , cross - boundary working , management of resources and workforce development . similarly , social workers have been striving to claim a role within phc settings , with the discipline of social work being undervalued and unappreciated . in order to move forward , social work should become actively involved in disease prevention programs , early intervention and effective use of scarce resources . thus , possession of all necessary skills and knowledge , both from undergraduate and postgraduate education as well as continuous professional development , should become a high priority in the phc agenda . greek health policy makers and clinicians are urged to take into consideration the mounting evidence that the literature conveys , as well as the pioneering attempts of introducing clinical governance in phc settings . new educational curricula for health professionals , both at the undergraduate and graduate level , should be established with the emphasis on multidisciplinary training and collaboration . establishment of integrated phc in greece is still at its infancy , requiring major restructuring of the current national health system , as well as organizational culture changes that can be facilitated by the establishment of new educational curricula . moving towards a new policy - based model would bring this missing issue on the discussion table , with the hope of facilitating further development . miranda laurant , phd , senior research fellow , scientific institute for quality of healthcare , radboud university nijmegen medical center , nijmegen , the netherlands pinar topsever md , associate professor of family medicine , department of family medicine , kocaeli university faculty of medicine , izmit / kocaeli , turkey one anonymous reviewer

backgroundover the past years , greece has undergone several endeavors aimed at modernizing and improving national health care services with a focus on phc . however , the extent to which integrated primary health care has been achieved is still questioned.purposethis paper explores the extent to which integrated primary health care ( phc ) is an issue in the current agenda of policy makers in greece , reporting constraints and opportunities and highlighting the need for a policy perspective in developing integrated phc in this southern european country.methodsa systematic review in pubmed / medline and scopus , along with a hand search in selected greek biomedical journals was undertaken to identify key papers , reports , editorials or opinion letters relevant to integrated health care.resultsour systematic review identified 198 papers and 161 out of them were derived from electronic search . fifty - three papers in total served the scope of this review and are shortly reported . a key finding is that the long - standing dominance of medical perspectives in greek health policy has been paving the way towards vertical integration , pushing aside any discussions about horizontal or comprehensive integration of care.conclusionestablishment of integrated phc in greece is still at its infancy , requiring major restructuring of the current national health system , as well as organizational culture changes . moving towards a new policy - based model would bring this missing issue on the discussion table , facilitating further development .

Introduction Methods Study design Study selection and inclusion criteria Electronic search Hand search Data extraction Results Studies identified Main findings Discussion Main trends Towards policy development of integrated PHC: where does Greece stand? Conclusions Reviewers

emerging demographic changes , mental health issues affecting individual and community well - being and global pressures of supply and demand present international challenges for any effort to reshape a health system , especially in countries with strained financial and human resources . primary health care 's contribution in promoting health , preventing debilitating disease and reducing disability has positioned it as a key player around the health care reform discussion table . integration , defined as the actual provision of services one needs at the time they are needed , is a key issue of contemporary primary health care . community - based integrated care is a strategic vision that promotes more joined and consistent action of the health care workforce towards improved performance , thus maximizing population health . in light of this definition , the focus is on multidisciplinary teams , rather than individuals , with partners recognizing , valuing and trusting each others ' rationales to meet shared endpoints in the provision of qualitative and comprehensive health care services . achieving integrated phc is an extremely tedious and arduous process , with inherent tension between those who advocate targeted integration of care ( vertical ) for priority conditions and those who advocate comprehensive integration ( horizontal ) that builds healthy communities . international organizations are currently discussing the integration of mental health care services into phc with a relevant report , developed by wonca and who , illustrating the importance and urgency of this endeavor . during the past few months , integration of phc through the development of super surgeries and polyclinics was on the front page of a daily newspaper in the uk . the case of greece is an example of a southern european country still striving to set health care priorities , struggling to allocate scarce resources , not always in the most cost - effective or quality assuring way . in terms of organization , the greek health care system is characterized by the co - existence of the national health system ( nhs ) , a mandatory social insurance ( si ) and a voluntary private health insurance system . voluntary payments by individuals or employers represent 42% of total health expenditure ( 2002 ) , making the greek health care system one of the most privatized among european union ( eu ) countries . primary health care in the public sector is delivered through a dual system , consisting of phc centers and hospital ambulatory ( outpatient ) services that belong to the nhs , and 350 primary care units that belong to the largest si fund ( ika ) with 5.5 million beneficiaries . as a result of the high ratio of physicians per 100,000 inhabitants , one of the highest in the eu , combined with one of the lowest ratios of nurses , there is a strong emphasis on curative services , rather than health promotion , disease prevention , rehabilitation and home care services . over the past years a few attempts , aimed at modernizing and improving the greek nhs with a specific reference to phc , have been initiated but have not been followed - up by subsequent health ministers and administrators [ 1215 ] . issues of integrated phc have not received prompt attention at a time when in the international arena policy makers and practitioners have been discussing phc reform and the potential impact on phc integration of certain barriers related to patients , health care professionals or organization . thus , we decided to systematically review the literature with the aim to identify constraints and opportunities as well as to address priorities towards the development of an integrated phc policy for greece . a full systematic review , employing both electronic and hand search techniques , was undertaken to investigate the extent to which integrated phc is considered to be a key issue in the current research , educational and policy agenda in greece . for the purposes of this review , integration was regarded to occur at the following four levels [ 16 , 18 ] : functional integration occurring at the macro level of the care system , i.e. by providing continuity , co - operation and coherence in the primary process of care delivery an advisory group assisted in the development of review questions and procedures as well as in the identification of the specific areas within the topic that would be most useful to scrutinize in - depth . the review was undertaken by a multidisciplinary team of researchers with a public health research background ( ms , cv , am , mp ) . a broad range of evidence types was addressed , including various research designs and policy , review as well as original papers , clinical practice experiences , user analytic and opinion evidence with a focus on completed studies and secondary source research . at the first stage , a wider group of thematic fields was included to enable mapping and exploration of the whole field of policy related to integrated primary health care. at the second stage , this group was narrowed down to a sub - set of studies with a focus on policy implication , according to what extent barriers and facilitators of phc integration were addressed within the identified papers . we included all papers that contained either a statement of a decision regarding a goal or plan for implementing integrated phc , or reported on how policy for integrated primary care was made or its influence in policy markets . the electronic search was performed during august 2008 by two researchers ( cv , ms ) in two databases ; medline and scopus from 1999 to august 2008 . the terms employed were { delivery of health care } and { integrated } and { greece } or { primary health care } and { greece}. for publications that appeared to meet the inclusion criteria , or in cases when a definite decision could not be made based on the title and/or abstract alone , the full paper was obtained for detailed assessment against the inclusion criteria . citation searching was also conducted selecting a number of key papers , already identified in the review , and then searching for publications that had cited these papers . since electronic databases are per definition prone to language and geographical biases , an additional hand search was performed in the following four greek journals , non - indexed in pubmed : koinoniki ergasia. the specific journals were selected as the leading scientific journals in the fields of medicine ( generic ) , primary health care , nursing and social work , respectively . recorded features of the included publications were as follows : first author 's name , year of publication , journal 's origin ( international or greek ) , level of integration , level of outcome change and study topic . a full systematic review , employing both electronic and hand search techniques , was undertaken to investigate the extent to which integrated phc is considered to be a key issue in the current research , educational and policy agenda in greece . for the purposes of this review , integration was regarded to occur at the following four levels [ 16 , 18 ] : functional integration occurring at the macro level of the care system , i.e. in the form of mergers ( group practices ) , contracting or strategic alliances between health care professionals . by providing continuity , co - operation and coherence in the primary process of care delivery an advisory group assisted in the development of review questions and procedures as well as in the identification of the specific areas within the topic that would be most useful to scrutinize in - depth . the review was undertaken by a multidisciplinary team of researchers with a public health research background ( ms , cv , am , mp ) . a broad range of evidence types was addressed , including various research designs and policy , review as well as original papers , clinical practice experiences , user analytic and opinion evidence with a focus on completed studies and secondary source research . at the first stage , a wider group of thematic fields was included to enable mapping and exploration of the whole field of policy related to integrated primary health care. at the second stage , this group was narrowed down to a sub - set of studies with a focus on policy implication , according to what extent barriers and facilitators of phc integration were addressed within the identified papers . we included all papers that contained either a statement of a decision regarding a goal or plan for implementing integrated phc , or reported on how policy for integrated primary care was made or its influence in policy markets . the electronic search was performed during august 2008 by two researchers ( cv , ms ) in two databases ; medline and scopus from 1999 to august 2008 . the terms employed were { delivery of health care } and { integrated } and { greece } or { primary health care } and { greece}. for publications that appeared to meet the inclusion criteria , or in cases when a definite decision could not be made based on the title and/or abstract alone , the full paper was obtained for detailed assessment against the inclusion criteria . citation searching was also conducted selecting a number of key papers , already identified in the review , and then searching for publications that had cited these papers . since electronic databases are per definition prone to language and geographical biases , an additional hand search was performed in the following four greek journals , non - indexed in pubmed : the specific journals were selected as the leading scientific journals in the fields of medicine ( generic ) , primary health care , nursing and social work , respectively . our electronic search of the literature identified 161 published studies . upon reviewing all of them , 27 manuscripts from medline and 26 from scopus papers when common articles were removed , a total number of 34 articles were selected for further scrutiny . thus , a total of 53 electronic and paper publications were found to be within the scope of our review . classification of articles by level and type of integration as well as by level of outcome change is presented in tables 3 and 4 , respectively . starting in the year 2000 , several editorials , proposals and reports were published with a focus on client satisfaction and improved coordination between outpatient hospital services and phc [ 12 , 2023 ] . continuity of care through the management of common episodes of care by the same health team over time has remained an unmet need within primary care delivery in greece [ 14 , 15 , 2426 ] . furthermore , the management and processing of information through the use of a common electronic medical record system with a user - friendly interface have often been the objectives of research proposals and pilot projects during the last decade [ 2733 ] . the development of an e - health care network within primary care settings has been linked with the benefits of real time access to clinical information and reliable research materials , data protection as well as community health consequently , the role of technology and telematic systems in the use of clinical information in a country with geographic and bureaucratic barriers has been viewed as a priority . the need for establishment of efficient referral pathways and services involving community nurses , social workers , psychologists and other health professionals has been underlined [ 14 , 25 , 43 ] . upon reviewing all of them , 27 manuscripts from medline and 26 from scopus papers when common articles were removed , a total number of 34 articles were selected for further scrutiny . thus , a total of 53 electronic and paper publications were found to be within the scope of our review . classification of articles by level and type of integration as well as by level of outcome change is presented in tables 3 and 4 , respectively . starting in the year 2000 , several editorials , proposals and reports were published with a focus on client satisfaction and improved coordination between outpatient hospital services and phc [ 12 , 2023 ] . furthermore , the management and processing of information through the use of a common electronic medical record system with a user - friendly interface have often been the objectives of research proposals and pilot projects during the last decade [ 2733 ] . the development of an e - health care network within primary care settings has been linked with the benefits of real time access to clinical information and reliable research materials , data protection as well as community health consequently , the role of technology and telematic systems in the use of clinical information in a country with geographic and bureaucratic barriers has been viewed as a priority . the need for establishment of efficient referral pathways and services involving community nurses , social workers , psychologists and other health professionals has been underlined [ 14 , 25 , 43 ] . despite the numerous papers , reports and editorials published mostly during the last decade , development of integrated phc , based on this review , still remains a neglected subject in the current health policy agenda in greece . it has been recognized that most of the times , national health planning has not been carried out in the context of evidence - based practice or a comprehensive health needs assessment [ 23 , 24 , 34 , 42 ] . differences across the country between urban and rural primary care provision patterns [ 43 , 50 ] as well as practice variations between nhs and major national health maintenance organizations [ 10 , 22 ] or private primary care providers have greatly contributed in the fragmentation and discontinuity of health care services [ 12 , 14 , 26 , 39 ] . although our review identified some papers calling for integration of primary care services [ 14 , 39 ] , including those reporting on attempts towards clinical governance practices , and collaborative initiatives with local authorities in establishing community outreach programs [ 26 , 42 , 51 , 52 ] , greece still operates under a conventional primary care management perspective . even the new primary health care act , introducing the concepts of a personal physician and polyclinics in greece , does not promote integrated phc measures . from a theoretical perspective , integrated phc has been seen as a solution to problems related to planning , funding , operation and effectiveness of the greek health care system . particularly , the absence of prevention and health promotion services in the community [ 48 , 53 , 54 ] as well as the shortage of phc staff and equipment in rural primary care centers [ 14 , 28 , 35 , 44 , 5557 ] , seem to contribute to the low level of integrated phc in greece . the gap observed between theory and practice [ 23 , 24 , 50 , 57 ] , as revealed from this systematic review , could be attributed to the limited research capacity in phc and the lack of evidence - based knowledge , impacting on the ability of the greek nhs to provide a seamless continuum of phc services . based on outcomes of this review , it is clear that contemporary greece is lagging behind in policy development towards integrated phc . the bold attempt towards health care reform and integration of regional health and welfare services made by the legislative act of 2001 has been negated or inactivated by recent legislation . thus , the phc policy initiative in the region of crete was short - lived , without any imitators . the rapidly changing pattern of morbidity in greece , with mental disorders and cardiovascular diseases being the predominant disease entities , signals the urgency for adopting an integrated phc policy . a more organized approach in primary care , with easily accessible community health centers ( chc ) located in neighborhoods , as suggested by han van oosterbos , seems to be suitable in the current greek setting . this approach implies transfer of resources to local authorities , an on - going request in greece , as well as a suitable legislative framework [ 24 , 26 ] . it has been argued that the introduction of a minimum package of health care services mandated by the greek government should be introduced in the current political agenda . changes in the academic curriculum towards the introduction of interdisciplinary and problem - based undergraduate education are considered essential in establishing an integrated phc system and promoting effectiveness and efficiency of services offered [ 53 , 55 , 56 , 59 , 60 ] . the potential leadership role and level of involvement of greek nurses towards integrated health care has only recently been pursued as a policy topic . thus , possession of all necessary skills and knowledge , both from undergraduate and postgraduate education as well as continuous professional development , should become a high priority in the phc agenda . greek health policy makers and clinicians are urged to take into consideration the mounting evidence that the literature conveys , as well as the pioneering attempts of introducing clinical governance in phc settings . despite the numerous papers , reports and editorials published mostly during the last decade , development of integrated phc , based on this review , still remains a neglected subject in the current health policy agenda in greece . this is the main finding of our systematic review which , although limited to only two databases and focused mostly on health care research rather than policy literature , seems to be conclusive . it has been recognized that most of the times , national health planning has not been carried out in the context of evidence - based practice or a comprehensive health needs assessment [ 23 , 24 , 34 , 42 ] . differences across the country between urban and rural primary care provision patterns [ 43 , 50 ] as well as practice variations between nhs and major national health maintenance organizations [ 10 , 22 ] or private primary care providers have greatly contributed in the fragmentation and discontinuity of health care services [ 12 , 14 , 26 , 39 ] . although our review identified some papers calling for integration of primary care services [ 14 , 39 ] , including those reporting on attempts towards clinical governance practices , and collaborative initiatives with local authorities in establishing community outreach programs [ 26 , 42 , 51 , 52 ] , greece still operates under a conventional primary care management perspective . even the new primary health care act , introducing the concepts of a personal physician and polyclinics in greece , does not promote integrated phc measures . from a theoretical perspective , integrated phc has been seen as a solution to problems related to planning , funding , operation and effectiveness of the greek health care system . particularly , the absence of prevention and health promotion services in the community [ 48 , 53 , 54 ] as well as the shortage of phc staff and equipment in rural primary care centers [ 14 , 28 , 35 , 44 , 5557 ] , seem to contribute to the low level of integrated phc in greece . the gap observed between theory and practice [ 23 , 24 , 50 , 57 ] , as revealed from this systematic review , could be attributed to the limited research capacity in phc and the lack of evidence - based knowledge , impacting on the ability of the greek nhs to provide a seamless continuum of phc services . based on outcomes of this review , it is clear that contemporary greece is lagging behind in policy development towards integrated phc . the bold attempt towards health care reform and integration of regional health and welfare services made by the legislative act of 2001 has been negated or inactivated by recent legislation . thus , the phc policy initiative in the region of crete was short - lived , without any imitators . the rapidly changing pattern of morbidity in greece , with mental disorders and cardiovascular diseases being the predominant disease entities , signals the urgency for adopting an integrated phc policy . a more organized approach in primary care , with easily accessible community health centers ( chc ) located in neighborhoods , as suggested by han van oosterbos , seems to be suitable in the current greek setting . this approach implies transfer of resources to local authorities , an on - going request in greece , as well as a suitable legislative framework [ 24 , 26 ] . it has been argued that the introduction of a minimum package of health care services mandated by the greek government should be introduced in the current political agenda . changes in the academic curriculum towards the introduction of interdisciplinary and problem - based undergraduate education are considered essential in establishing an integrated phc system and promoting effectiveness and efficiency of services offered [ 53 , 55 , 56 , 59 , 60 ] . the potential leadership role and level of involvement of greek nurses towards integrated health care has only recently been pursued as a policy topic . thus , possession of all necessary skills and knowledge , both from undergraduate and postgraduate education as well as continuous professional development , should become a high priority in the phc agenda . greek health policy makers and clinicians are urged to take into consideration the mounting evidence that the literature conveys , as well as the pioneering attempts of introducing clinical governance in phc settings . establishment of integrated phc in greece is still at its infancy , requiring major restructuring of the current national health system , as well as organizational culture changes that can be facilitated by the establishment of new educational curricula . moving towards a new policy - based model would bring this missing issue on the discussion table , with the hope of facilitating further development .

this was a multi - institute , single - group clinical trial conducted at seoul st . approval was granted by the respective institutional review boards of each institute , and a clinical trial permit was obtained from the ministry of food and drug safety . this study was carried out in accordance with the tenets of the declaration of helsinki . at the screening visits , the investigators informed the subjects of the complete details of the clinical trial , after which the subjects signed a written consent form . we then obtained demographic information , history of ocular diseases , and current disease information from each subject and determined visual acuity , refractive error , corneal topography , slit - lamp microscopy , corneal endothelial cell count , central corneal thickness ( cct ) , intraocular pressure , schirmer 's test result , and tear break - up time . the inclusion criteria for subjects were as follows : ( 1 ) age between 7 and 49 years , ( 2 ) myopic refractive error of -0.75 to -6.0 diopters ( d ) and astigmatic refractive error of 1.25 to 4.0 d , ( 3 ) radius of corneal curvature within the range of 46.00 to 39.75 d ( 7.34 to 8.5 mm ) , ( 4 ) horizontal corneal diameter larger than 11.0 mm , ( 5 ) occupation and environment allowing wear of lenses for more than 7 hours during sleep , ( 6 ) ability to follow instructions during the clinical trial , and ( 7 ) willingness to participate in the clinical trial and provide signed written consent . the exclusion criteria were : ( 1 ) schirmer test result less than 10 mm or tear break - up time shorter than 10 seconds , ( 2 ) abnormal findings during slit - lamp microscopy ; inflammation , erosions , ulcers , or angiogenesis in the cornea after wearing the lens for a 30-minute trial period , ( 3 ) allergies , ( 4 ) eye diseases that would have made participation in the trial difficult as judged by the investigator , ( 5 ) contraindication for wearing contact lenses , ( 6 ) wearing of rgp lenses within the 2 weeks prior to the screening visit , ( 7 ) diagnosed strabismus , ( 8) past corneal refractive surgery , ( 9 ) pregnancy , nursing , or planning to become pregnant , and ( 10 ) significant disease that would have made participation in the clinical trial difficult as judged by the investigator . candidates who met any one of the above criteria were excluded from the trial . at the first visit ( day 1 ) , subjects tried the toric orthokeratology lenses prescribed at the screening visit and were informed of the precautions that must be taken when wearing the lenses , as well as the potential adverse reactions . slit - lamp microscopy was performed , the refractive error was measured , and any adverse reactions to wearing the lenses were assessed . fit was confirmed during slit - lamp microscopy , along with determination of abnormal corneal findings . to confirm fit , we verified that there was pressure on the cornea , and that a space was maintained between the back of the toric orthokeratology lens and the cornea . while monitoring the changes in the kerato - conjunctiva , the ocular status in each subject 's anterior segment moderate to severe scores in this test were considered as indicators of an adverse reaction . any other clinically significant findings in the kerato - conjunctiva were also recorded as adverse reactions . in cases of poor lens all subjects were instructed to wear the lenses for an average of 7 hours during sleep and to not wear the lenses during the day . the second visit ( day 2 ) took place 1 day after wearing the toric orthokeratology lenses overnight . both uncorrected visual acuity and refractive error were measured , and slit - lamp microscopy was performed . the same tests were repeated after 1 , 2 , and 3 weeks of lens wear . on the last visit ( week 4 ) , in addition to performing the tests described above , corneal endothelial cell count and cct were measured . the toric orthokeratology lenses used in the trial were toric lk lens ( lucid korea , bonghwa , korea ) . the primary outcome measures in this study were myopic and astigmatic refractive errors after toric orthokeratology lens use . for convenience of analysis , secondary study outcomes included visual acuity , corneal curvature , time to reach the target uncorrected vision of 16 / 20 after wearing the lenses . to assess safety , slit - lamp microscopy was performed , and the ocular status score in the anterior segment was recorded . corneal endothelial cell count and cct were also measured , and the total incidence of adverse reactions was evaluated . statistical analyses of myopic and astigmatic refractive errors were performed using a two - tailed , paired t - test . for all other outcomes we used a two - tailed , paired t - test or wilcoxon 's signed - rank test as appropriate . the change in ocular status score in the anterior segment , as determined using slit - lamp microscopy , was input into a split table ( frequency , ratio ) and analyzed using bowker 's test . for all statistics , a p - value < 0.05 was considered significant . for missing values and when subjects dropped out before the trial was completed , the data from the subject was not included in our analysis . at the screening visits , the investigators informed the subjects of the complete details of the clinical trial , after which the subjects signed a written consent form . we then obtained demographic information , history of ocular diseases , and current disease information from each subject and determined visual acuity , refractive error , corneal topography , slit - lamp microscopy , corneal endothelial cell count , central corneal thickness ( cct ) , intraocular pressure , schirmer 's test result , and tear break - up time . the inclusion criteria for subjects were as follows : ( 1 ) age between 7 and 49 years , ( 2 ) myopic refractive error of -0.75 to -6.0 diopters ( d ) and astigmatic refractive error of 1.25 to 4.0 d , ( 3 ) radius of corneal curvature within the range of 46.00 to 39.75 d ( 7.34 to 8.5 mm ) , ( 4 ) horizontal corneal diameter larger than 11.0 mm , ( 5 ) occupation and environment allowing wear of lenses for more than 7 hours during sleep , ( 6 ) ability to follow instructions during the clinical trial , and ( 7 ) willingness to participate in the clinical trial and provide signed written consent . the exclusion criteria were : ( 1 ) schirmer test result less than 10 mm or tear break - up time shorter than 10 seconds , ( 2 ) abnormal findings during slit - lamp microscopy ; inflammation , erosions , ulcers , or angiogenesis in the cornea after wearing the lens for a 30-minute trial period , ( 3 ) allergies , ( 4 ) eye diseases that would have made participation in the trial difficult as judged by the investigator , ( 5 ) contraindication for wearing contact lenses , ( 6 ) wearing of rgp lenses within the 2 weeks prior to the screening visit , ( 7 ) diagnosed strabismus , ( 8) past corneal refractive surgery , ( 9 ) pregnancy , nursing , or planning to become pregnant , and ( 10 ) significant disease that would have made participation in the clinical trial difficult as judged by the investigator . at the first visit ( day 1 ) , subjects tried the toric orthokeratology lenses prescribed at the screening visit and were informed of the precautions that must be taken when wearing the lenses , as well as the potential adverse reactions . slit - lamp microscopy was performed , the refractive error was measured , and any adverse reactions to wearing the lenses were assessed . fit was confirmed during slit - lamp microscopy , along with determination of abnormal corneal findings . to confirm fit , we verified that there was pressure on the cornea , and that a space was maintained between the back of the toric orthokeratology lens and the cornea . while monitoring the changes in the kerato - conjunctiva , the ocular status in each subject 's anterior segment moderate to severe scores in this test were considered as indicators of an adverse reaction . any other clinically significant findings in the kerato - conjunctiva were also recorded as adverse reactions . in cases of poor lens all subjects were instructed to wear the lenses for an average of 7 hours during sleep and to not wear the lenses during the day . the second visit ( day 2 ) took place 1 day after wearing the toric orthokeratology lenses overnight . both uncorrected visual acuity and refractive error were measured , and slit - lamp microscopy was performed . the same tests were repeated after 1 , 2 , and 3 weeks of lens wear . on the last visit ( week 4 ) , in addition to performing the tests described above , corneal endothelial cell count and cct were measured . the toric orthokeratology lenses used in the trial were toric lk lens ( lucid korea , bonghwa , korea ) . the primary outcome measures in this study were myopic and astigmatic refractive errors after toric orthokeratology lens use . for convenience of analysis , the astigmatic refractive error represented only corneal astigmatism not lenticular astigmatism . secondary study outcomes included visual acuity , corneal curvature , time to reach the target uncorrected vision of 16 / 20 after wearing the lenses . to assess safety , slit - lamp microscopy was performed , and the ocular status score in the anterior segment was recorded . corneal endothelial cell count and cct statistical analyses of myopic and astigmatic refractive errors were performed using a two - tailed , paired t - test . for all other outcomes we used a two - tailed , paired t - test or wilcoxon 's signed - rank test as appropriate . the change in ocular status score in the anterior segment , as determined using slit - lamp microscopy , was input into a split table ( frequency , ratio ) and analyzed using bowker 's test . for all statistics , a p - value < 0.05 was considered significant . for missing values and when subjects dropped out before the trial was completed , the data from the subject was not included in our analysis . in this clinical trial , a total of 79 eyes ( 44 subjects ) were screened at one of two trial - conducting institutes ; 40 eyes ( 21 subjects , 50.63% ) were excluded after screening , leaving a total of 39 eyes ( 23 subjects , 49.37% ) to be tested after lens use . details of the subjects ' eyes at registration are presented as a schematic diagram in fig . the mean age was 21.81 8.89 years , with a range of 7 to 39 years . there were no pregnancies among the 19 women of childbearing age that were included in the study . the mean myopic refractive error changed from -3.65 1.62 to -1.05 1.64 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 2.60 2.21 d ( p < 0.001 ) . the correction of myopia by toric or - thokeratology lenses resulted in a superior refractive state ( table 2 and fig . the mean astigmatic refractive error changed from 2.07 0.83 to 1.44 0.99 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 0.63 0.98 d ( p = 0.001 ) . the correction of astigmatism by toric orthokeratology lenses resulted in a superior refractive state ( table 2 and fig . the mean uncorrected and corrected visual acuities at the screening visit were 2.14 0.80 and 0.05 0.13 logmar , respectively . the equivalent values after wearing the toric orthokeratology lenses for 4 weeks were 0.12 0.30 and 0.01 0.04 logarithm of the minimal angle of resolution ( logmar ) , respectively . the difference in uncorrected visual acuity was -2.03 0.91 logmar , which was statistically significant ( p < 0.001 ) . the difference in corrected visual acuity was -0.04 0.14 logmar , which was not statistically significant ( p = 0.156 ) ( table 3 ) . after wearing the lenses for 1 week , corrected visual acuity was greater than 16 / 20 in 29 eyes ( 93.55% ) . all of the eyes achieved a corrected visual acuity greater than 16 / 20 after wearing the lenses for 2 weeks . at the screening visit , the mean corneal curvature was 42.54 1.06 d , and the mean corneal diameter was 11.78 0.28 mm . after wearing the toric orthokeratology lenses for 4 weeks , the mean corneal curvature was 40.92 1.38 d and the mean corneal diameter was 11.76 0.35 mm . a representative topographic pattern of corneal astigmatism is shown in fig . 4a and 4b . after treatment , the difference in corneal curvature was -1.62 0.96 d , which was statistically significant ( p < 0.001 ) . however , the difference in corneal diameter was -0.02 0.35 mm , which was not statistically significant ( p = 0.710 ) . the mean cct values before and after wearing the lenses were 560.21 38.64 and 545.76 61.37 m , respectively . the mean difference was -14.45 37.46 m , which constituted a statistically significant decrease ( p = 0.047 ) ( table 4 ) . cells / mm , which increased by 40.72 207.67 cells / mm to 3,237.28 277.25 cells / mm after 4 weeks of toric orthokeratology lens use . however , this difference was not statistically significant ( p = 0.300 ) . the difference in hexagonality was -0.07 12.07 , which was also not statistically significant ( p = 0.976 ) . the coefficient of variation before and after wearing the lenses was 0.31 0.05 and 0.33 0.07 . the difference of 0.02 0.06 was statistically significant ( p = 0.027 ) ( table 4 ) . the efron grading scale scores during the trial were normal for all eyes in all categories except for corneal staining , in which 38 eyes ( 97.44% ) were normal , and one eye ( 2.56% ) was graded as trace before wearing the lenses . after 4 weeks of lens use , 25 eyes ( 80.65% ) were normal , four eyes ( 12.90% ) were graded as trace , and two eyes ( 6.45% ) were graded as mild . the difference in grading before and after lens use was not statistically significant ( p = 0.815 ) . a total of five adverse reactions developed in five subjects , consisting of one case of conjunctival hyperemia ( 3.23% ) , one case of foreign body sensation in the eyes ( 3.23% ) , and two cases of , the adverse reactions ceased after the patients were prescribed additional artificial tears and were provided additional education regarding the proper wear and removal of the lenses . the mean myopic refractive error changed from -3.65 1.62 to -1.05 1.64 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 2.60 2.21 d ( p < 0.001 ) . the correction of myopia by toric or - thokeratology lenses resulted in a superior refractive state ( table 2 and fig . the mean astigmatic refractive error changed from 2.07 0.83 to 1.44 0.99 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 0.63 0.98 d ( p = 0.001 ) . the correction of astigmatism by toric orthokeratology lenses resulted in a superior refractive state ( table 2 and fig . the mean uncorrected and corrected visual acuities at the screening visit were 2.14 0.80 and 0.05 0.13 logmar , respectively . the equivalent values after wearing the toric orthokeratology lenses for 4 weeks were 0.12 0.30 and 0.01 0.04 logarithm of the minimal angle of resolution ( logmar ) , respectively . the difference in uncorrected visual acuity was -2.03 0.91 logmar , which was statistically significant ( p < 0.001 ) . the difference in corrected visual acuity was -0.04 0.14 logmar , which was not statistically significant ( p = 0.156 ) ( table 3 ) . after wearing the lenses for 1 week , corrected visual acuity was greater than 16 / 20 in 29 eyes ( 93.55% ) . all of the eyes achieved a corrected visual acuity greater than 16 / 20 after wearing the lenses for 2 weeks . at the screening visit , the mean corneal curvature was 42.54 1.06 d , and the mean corneal diameter was 11.78 0.28 mm . after wearing the toric orthokeratology lenses for 4 weeks , the mean corneal curvature was 40.92 1.38 d and the mean corneal diameter was 11.76 0.35 mm . a representative topographic pattern of corneal astigmatism is shown in fig . 4a and 4b . after treatment , the difference in corneal curvature was -1.62 0.96 d , which was statistically significant ( p < 0.001 ) . however , the difference in corneal diameter was -0.02 0.35 mm , which was not statistically significant ( p = 0.710 ) . the mean cct values before and after wearing the lenses were 560.21 38.64 and 545.76 61.37 m , respectively . the mean difference was -14.45 37.46 m , which constituted a statistically significant decrease ( p = 0.047 ) ( table 4 ) . the mean corneal endothelial cell count at the screening visit was 3,196.55 237.80 cells / mm , which increased by 40.72 207.67 cells / mm to 3,237.28 277.25 cells / mm after 4 weeks of toric orthokeratology lens use . the difference in hexagonality was -0.07 12.07 , which was also not statistically significant ( p = 0.976 ) . the coefficient of variation before and after wearing the lenses was 0.31 0.05 and 0.33 0.07 . the difference of 0.02 0.06 was statistically significant ( p = 0.027 ) ( table 4 ) . the efron grading scale scores during the trial were normal for all eyes in all categories except for corneal staining , in which 38 eyes ( 97.44% ) were normal , and one eye ( 2.56% ) was graded as trace before wearing the lenses . after 4 weeks of lens use , 25 eyes ( 80.65% ) were normal , four eyes ( 12.90% ) were graded as trace , and two eyes ( 6.45% ) were graded as mild . the difference in grading before and after lens use was not statistically significant ( p = 0.815 ) . a total of five adverse reactions developed in five subjects , consisting of one case of conjunctival hyperemia ( 3.23% ) , one case of foreign body sensation in the eyes ( 3.23% ) , and two cases of , the adverse reactions ceased after the patients were prescribed additional artificial tears and were provided additional education regarding the proper wear and removal of the lenses . with the introduction of orthokeratology by wesley and jessen in the early 1960s , many clinicians and researchers have attempted to correct myopia using rigid contact lenses ; however , these attempts have been limited by patient discomfort and hypoxic corneal damage . the development of rgp lens material combined with advancements in contact lens manufacturing technology has led to the development of overnight orthokeratology , which enables patients to wear myopia - correcting contact lenses during sleep . in overnight orthokeratology , the reverse - geometry lens is designed with both a flatter central curvature and steeper peripheral curvature such that a tear reservoir can be formed , and the lens is precisely placed in the corneal center . in 2000 , nichols et al . reported that overnight orthokeratology by the programmed application of rgp contact lenses with a reverse geometrical design is an effective means of temporarily reducing myopia . furthermore , they suggested a possible mechanism of corneal remodeling through central corneal thinning . a number of clinical trials to date have concluded that orthokeratology can effectively reduce moderate to high myopia , and control the progression of myopia . in addition , several reports have addressed the correction of astigmatism using orthokeratology lenses , although these effects are typically insufficient due to lens decentration . to address the issue of decentration , chen et al . examined the efficacy of toric orthokeratology in correcting myopia combined with moderate to high astigmatism . in their study , subjects aged 6 to 12 years with myopia of 0.50 to 5.00 d and with with - the - rule astigmatism of 1.25 to 3.50 d were fitted with toric orthokeratology lenses . they reported that manifest myopia was significantly reduced from 2.53 1.31 to 0.41 0.43 d ( p < 0.001 ) , and that astigmatism was also decreased from 1.91 0.64 to 0.40 0.39 d ( p < 0.001 ) after 1 month of lens wear . similarly , in 2013 , chen et al . conducted a non - randomized clinical study in which toric orthokeratology was found to effectively control myopia in children with accompanying moderate - to - high astigmatism . to our knowledge , this is the first prospective , multi - center clinical trial to have investigated the effectiveness of toric orthokeratology in korea . specifically , we evaluated the effectiveness and safety of toric orthokeratology lenses in patients with both myopia and astigmatism . patients wore the toric orthokeratology lenses during sleep for an average of 7 hours and for up to 4 weeks ( 28 days ) . the results showed than overnight toric orthokeratology reduced both myopia and astigmatism , although the degree of chance in astigmatism was relatively small . we attributed this small change to a weakened effect of the toric orthokeratology lenses to reshape the cornea during the night , as the subjects in this study visited our institute primarily in the afternoon . thus , additional studies regarding the clinical effectiveness of toric orthokeratology in correcting astigmatism are needed . unlike astigmatism , uncorrected visual acuity was significantly improved in our study , although there was no change in corrected visual acuity . thus , our results suggest that toric orthokeratology lenses do not change the ocular features associated with corrected visual acuity , which reflects the safety of this approach . a number of studies have suggested that various ocular parameters ( corneal thickness in particular ) are modifiable or predictive values for myopic reduction in orthokeratology . in 2000 , lui and edwards reported that cct is the most predictive value of myopic reduction based on the results of a randomized controlled clinical trial involving 14 subjects who wore orthokeratology lenses during the daytime . specifically , they suggested that a thicker initial central cornea is associated with a greater reduction in myopia . . conducted a meta - analysis that included a total of 10 studies with 339 eyes from 239 patients in order to assess the change to cct by orthokeratology lens use in myopic patients . they found that cct was reduced significantly between 1 day and 1 week , and that the most significant reduction occurred between 1 day and 1 month . in our study , cct was significantly decreased ( p = 0.005 ) after 4 weeks of lens wear but remained within the normal range . this might have been due to the reverse - geometry lens pressing down on the corneal center , causing rearrangement of the epithelium . among the five adverse reactions noted in five subjects during our trial , reactions exclusively concerning the lenses comprised four cases ( 12.90% ) , all of which were mild in nature and recovered fully . according to silt - lamp microscopy and other examinations of ocular parameters , there were no significant changes except for the cct and coefficient of variation , although cct remained within the normal range after 4 weeks . on the other hand , the change in coefficient of variation was considered to be a common effect of all types of contact lenses on corneal polymegathism , and the extent of the increase was not large relative to that noted in other studies . together , these data indicate that use of toric orthokeratology lenses does not cause any significant changes in ocular conditions . on the basis of these findings , our results suggest that toric orthokeratology lenses do not have any serious harmful effects . one limitation of this clinical trial was that the follow - up period was only 4 weeks . based on our results , the purpose of these studies should be to elucidate further aspects and complications of toric orthokeratology , such as maintenance of lens centration or changes in ocular features . in conclusion , the effectiveness of toric orthokeratology lenses in correcting myopia combined with astigmatism was verified statistically . orthokeratology lenses are currently limited to the correction of myopia ; however , our results provide an experimental basis for broadening the scope of orthokeratology lenses to include the correction of astigmatism . importantly , there were no notable adverse reactions or significant abnormal findings caused by use of toric orthokeratology lenses as determined by silt - lamp microscopy and other examinations . therefore , toric orthokeratology can be considered an effective and safe treatment for correcting visual acuity in patients with combined myopia and astigmatism .

purposethe purpose of this multi - institute , single - group clinical trial was to evaluate the effectiveness and safety of toric orthokeratology lenses for the treatment of patients with combined myopia and astigmatism.methodsa total of 44 patients were included in this clinical trial . the patients ranged in age from 7 to 49 years , with myopia of -0.75 to -6.0 diopters ( d ) and astigmatism of 1.25 to 4.0 d. after excluding 21 subjects , 23 subjects ( 39 eyes ) were analyzed after toric orthokeratology lens use . the subjects underwent ophthalmologic examination after 1 day and 1 , 2 , 3 , and 4 weeks of wearing overnight toric orthokeratology lenses.resultsa total of 19 subjects ( 31 eyes ) completed the trial after five subjects ( eight eyes ) dropped out . in the patients who completed the study by wearing lenses for 4 weeks , the myopic refractive error decreased significantly by 2.60 2.21 d ( p < 0.001 ) , from -3.65 1.62 to -1.05 1.64 d. the astigmatic refractive error were also significantly decreased by 0.63 0.98 d ( p = 0.001 ) , from 2.07 0.83 to 1.44 0.99 d. the mean uncorrected and corrected visual acuities before wearing the lenses were 2.14 0.80 logarithm of the logmar ( logmar ) and 0.05 0.13 logmar , respectively , which changed to 0.12 0.30 logarithm of the logmar ( p < 0.001 ) and 0.01 0.04 logmar ( p = 0.156 ) after 4 weeks . no serious adverse reactions were reported during the clinical trial.conclusionsour results suggest that toric orthokeratology is an effective and safe treatment for correcting visual acuity in patients with combined myopia and astigmatism .

Materials and Methods Subjects Examinations Statistical analysis Results Myopic and astigmatic refractive error Uncorrected and corrected visual acuity Corneal topography and thickness Corneal endothelial cell counts Slit-lamp microscopy Adverse reactions Discussion

this was a multi - institute , single - group clinical trial conducted at seoul st . approval was granted by the respective institutional review boards of each institute , and a clinical trial permit was obtained from the ministry of food and drug safety . at the screening visits , the investigators informed the subjects of the complete details of the clinical trial , after which the subjects signed a written consent form . we then obtained demographic information , history of ocular diseases , and current disease information from each subject and determined visual acuity , refractive error , corneal topography , slit - lamp microscopy , corneal endothelial cell count , central corneal thickness ( cct ) , intraocular pressure , schirmer 's test result , and tear break - up time . the inclusion criteria for subjects were as follows : ( 1 ) age between 7 and 49 years , ( 2 ) myopic refractive error of -0.75 to -6.0 diopters ( d ) and astigmatic refractive error of 1.25 to 4.0 d , ( 3 ) radius of corneal curvature within the range of 46.00 to 39.75 d ( 7.34 to 8.5 mm ) , ( 4 ) horizontal corneal diameter larger than 11.0 mm , ( 5 ) occupation and environment allowing wear of lenses for more than 7 hours during sleep , ( 6 ) ability to follow instructions during the clinical trial , and ( 7 ) willingness to participate in the clinical trial and provide signed written consent . the exclusion criteria were : ( 1 ) schirmer test result less than 10 mm or tear break - up time shorter than 10 seconds , ( 2 ) abnormal findings during slit - lamp microscopy ; inflammation , erosions , ulcers , or angiogenesis in the cornea after wearing the lens for a 30-minute trial period , ( 3 ) allergies , ( 4 ) eye diseases that would have made participation in the trial difficult as judged by the investigator , ( 5 ) contraindication for wearing contact lenses , ( 6 ) wearing of rgp lenses within the 2 weeks prior to the screening visit , ( 7 ) diagnosed strabismus , ( 8) past corneal refractive surgery , ( 9 ) pregnancy , nursing , or planning to become pregnant , and ( 10 ) significant disease that would have made participation in the clinical trial difficult as judged by the investigator . at the first visit ( day 1 ) , subjects tried the toric orthokeratology lenses prescribed at the screening visit and were informed of the precautions that must be taken when wearing the lenses , as well as the potential adverse reactions . slit - lamp microscopy was performed , the refractive error was measured , and any adverse reactions to wearing the lenses were assessed . to confirm fit , we verified that there was pressure on the cornea , and that a space was maintained between the back of the toric orthokeratology lens and the cornea . while monitoring the changes in the kerato - conjunctiva , the ocular status in each subject 's anterior segment moderate to severe scores in this test were considered as indicators of an adverse reaction . the second visit ( day 2 ) took place 1 day after wearing the toric orthokeratology lenses overnight . both uncorrected visual acuity and refractive error were measured , and slit - lamp microscopy was performed . the same tests were repeated after 1 , 2 , and 3 weeks of lens wear . the toric orthokeratology lenses used in the trial were toric lk lens ( lucid korea , bonghwa , korea ) . the primary outcome measures in this study were myopic and astigmatic refractive errors after toric orthokeratology lens use . for missing values and when subjects dropped out before the trial was completed , the data from the subject was not included in our analysis . at the screening visits , the investigators informed the subjects of the complete details of the clinical trial , after which the subjects signed a written consent form . we then obtained demographic information , history of ocular diseases , and current disease information from each subject and determined visual acuity , refractive error , corneal topography , slit - lamp microscopy , corneal endothelial cell count , central corneal thickness ( cct ) , intraocular pressure , schirmer 's test result , and tear break - up time . the inclusion criteria for subjects were as follows : ( 1 ) age between 7 and 49 years , ( 2 ) myopic refractive error of -0.75 to -6.0 diopters ( d ) and astigmatic refractive error of 1.25 to 4.0 d , ( 3 ) radius of corneal curvature within the range of 46.00 to 39.75 d ( 7.34 to 8.5 mm ) , ( 4 ) horizontal corneal diameter larger than 11.0 mm , ( 5 ) occupation and environment allowing wear of lenses for more than 7 hours during sleep , ( 6 ) ability to follow instructions during the clinical trial , and ( 7 ) willingness to participate in the clinical trial and provide signed written consent . the exclusion criteria were : ( 1 ) schirmer test result less than 10 mm or tear break - up time shorter than 10 seconds , ( 2 ) abnormal findings during slit - lamp microscopy ; inflammation , erosions , ulcers , or angiogenesis in the cornea after wearing the lens for a 30-minute trial period , ( 3 ) allergies , ( 4 ) eye diseases that would have made participation in the trial difficult as judged by the investigator , ( 5 ) contraindication for wearing contact lenses , ( 6 ) wearing of rgp lenses within the 2 weeks prior to the screening visit , ( 7 ) diagnosed strabismus , ( 8) past corneal refractive surgery , ( 9 ) pregnancy , nursing , or planning to become pregnant , and ( 10 ) significant disease that would have made participation in the clinical trial difficult as judged by the investigator . at the first visit ( day 1 ) , subjects tried the toric orthokeratology lenses prescribed at the screening visit and were informed of the precautions that must be taken when wearing the lenses , as well as the potential adverse reactions . slit - lamp microscopy was performed , the refractive error was measured , and any adverse reactions to wearing the lenses were assessed . to confirm fit , we verified that there was pressure on the cornea , and that a space was maintained between the back of the toric orthokeratology lens and the cornea . the second visit ( day 2 ) took place 1 day after wearing the toric orthokeratology lenses overnight . both uncorrected visual acuity and refractive error were measured , and slit - lamp microscopy was performed . the same tests were repeated after 1 , 2 , and 3 weeks of lens wear . the toric orthokeratology lenses used in the trial were toric lk lens ( lucid korea , bonghwa , korea ) . the primary outcome measures in this study were myopic and astigmatic refractive errors after toric orthokeratology lens use . for convenience of analysis , the astigmatic refractive error represented only corneal astigmatism not lenticular astigmatism . secondary study outcomes included visual acuity , corneal curvature , time to reach the target uncorrected vision of 16 / 20 after wearing the lenses . for missing values and when subjects dropped out before the trial was completed , the data from the subject was not included in our analysis . in this clinical trial , a total of 79 eyes ( 44 subjects ) were screened at one of two trial - conducting institutes ; 40 eyes ( 21 subjects , 50.63% ) were excluded after screening , leaving a total of 39 eyes ( 23 subjects , 49.37% ) to be tested after lens use . the mean age was 21.81 8.89 years , with a range of 7 to 39 years . there were no pregnancies among the 19 women of childbearing age that were included in the study . the mean myopic refractive error changed from -3.65 1.62 to -1.05 1.64 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 2.60 2.21 d ( p < 0.001 ) . the mean astigmatic refractive error changed from 2.07 0.83 to 1.44 0.99 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 0.63 0.98 d ( p = 0.001 ) . the mean uncorrected and corrected visual acuities at the screening visit were 2.14 0.80 and 0.05 0.13 logmar , respectively . the equivalent values after wearing the toric orthokeratology lenses for 4 weeks were 0.12 0.30 and 0.01 0.04 logarithm of the minimal angle of resolution ( logmar ) , respectively . the difference in uncorrected visual acuity was -2.03 0.91 logmar , which was statistically significant ( p < 0.001 ) . the difference in corrected visual acuity was -0.04 0.14 logmar , which was not statistically significant ( p = 0.156 ) ( table 3 ) . after wearing the lenses for 1 week , corrected visual acuity was greater than 16 / 20 in 29 eyes ( 93.55% ) . all of the eyes achieved a corrected visual acuity greater than 16 / 20 after wearing the lenses for 2 weeks . at the screening visit , the mean corneal curvature was 42.54 1.06 d , and the mean corneal diameter was 11.78 0.28 mm . after wearing the toric orthokeratology lenses for 4 weeks , the mean corneal curvature was 40.92 1.38 d and the mean corneal diameter was 11.76 0.35 mm . after treatment , the difference in corneal curvature was -1.62 0.96 d , which was statistically significant ( p < 0.001 ) . however , the difference in corneal diameter was -0.02 0.35 mm , which was not statistically significant ( p = 0.710 ) . the mean cct values before and after wearing the lenses were 560.21 38.64 and 545.76 61.37 m , respectively . the mean difference was -14.45 37.46 m , which constituted a statistically significant decrease ( p = 0.047 ) ( table 4 ) . cells / mm , which increased by 40.72 207.67 cells / mm to 3,237.28 277.25 cells / mm after 4 weeks of toric orthokeratology lens use . the difference in hexagonality was -0.07 12.07 , which was also not statistically significant ( p = 0.976 ) . the coefficient of variation before and after wearing the lenses was 0.31 0.05 and 0.33 0.07 . the efron grading scale scores during the trial were normal for all eyes in all categories except for corneal staining , in which 38 eyes ( 97.44% ) were normal , and one eye ( 2.56% ) was graded as trace before wearing the lenses . after 4 weeks of lens use , 25 eyes ( 80.65% ) were normal , four eyes ( 12.90% ) were graded as trace , and two eyes ( 6.45% ) were graded as mild . the difference in grading before and after lens use was not statistically significant ( p = 0.815 ) . a total of five adverse reactions developed in five subjects , consisting of one case of conjunctival hyperemia ( 3.23% ) , one case of foreign body sensation in the eyes ( 3.23% ) , and two cases of , the adverse reactions ceased after the patients were prescribed additional artificial tears and were provided additional education regarding the proper wear and removal of the lenses . the mean myopic refractive error changed from -3.65 1.62 to -1.05 1.64 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 2.60 2.21 d ( p < 0.001 ) . the mean astigmatic refractive error changed from 2.07 0.83 to 1.44 0.99 d after wearing the lenses for 4 weeks , which constituted a statistically significant decrease of 0.63 0.98 d ( p = 0.001 ) . the mean uncorrected and corrected visual acuities at the screening visit were 2.14 0.80 and 0.05 0.13 logmar , respectively . the equivalent values after wearing the toric orthokeratology lenses for 4 weeks were 0.12 0.30 and 0.01 0.04 logarithm of the minimal angle of resolution ( logmar ) , respectively . the difference in uncorrected visual acuity was -2.03 0.91 logmar , which was statistically significant ( p < 0.001 ) . the difference in corrected visual acuity was -0.04 0.14 logmar , which was not statistically significant ( p = 0.156 ) ( table 3 ) . after wearing the lenses for 1 week , corrected visual acuity was greater than 16 / 20 in 29 eyes ( 93.55% ) . all of the eyes achieved a corrected visual acuity greater than 16 / 20 after wearing the lenses for 2 weeks . at the screening visit , the mean corneal curvature was 42.54 1.06 d , and the mean corneal diameter was 11.78 0.28 mm . after wearing the toric orthokeratology lenses for 4 weeks , the mean corneal curvature was 40.92 1.38 d and the mean corneal diameter was 11.76 0.35 mm . after treatment , the difference in corneal curvature was -1.62 0.96 d , which was statistically significant ( p < 0.001 ) . however , the difference in corneal diameter was -0.02 0.35 mm , which was not statistically significant ( p = 0.710 ) . the mean cct values before and after wearing the lenses were 560.21 38.64 and 545.76 61.37 m , respectively . the mean difference was -14.45 37.46 m , which constituted a statistically significant decrease ( p = 0.047 ) ( table 4 ) . the mean corneal endothelial cell count at the screening visit was 3,196.55 237.80 cells / mm , which increased by 40.72 207.67 cells / mm to 3,237.28 277.25 cells / mm after 4 weeks of toric orthokeratology lens use . the difference in hexagonality was -0.07 12.07 , which was also not statistically significant ( p = 0.976 ) . the coefficient of variation before and after wearing the lenses was 0.31 0.05 and 0.33 0.07 . the difference of 0.02 0.06 was statistically significant ( p = 0.027 ) ( table 4 ) . the efron grading scale scores during the trial were normal for all eyes in all categories except for corneal staining , in which 38 eyes ( 97.44% ) were normal , and one eye ( 2.56% ) was graded as trace before wearing the lenses . after 4 weeks of lens use , 25 eyes ( 80.65% ) were normal , four eyes ( 12.90% ) were graded as trace , and two eyes ( 6.45% ) were graded as mild . the difference in grading before and after lens use was not statistically significant ( p = 0.815 ) . a total of five adverse reactions developed in five subjects , consisting of one case of conjunctival hyperemia ( 3.23% ) , one case of foreign body sensation in the eyes ( 3.23% ) , and two cases of , the adverse reactions ceased after the patients were prescribed additional artificial tears and were provided additional education regarding the proper wear and removal of the lenses . in their study , subjects aged 6 to 12 years with myopia of 0.50 to 5.00 d and with with - the - rule astigmatism of 1.25 to 3.50 d were fitted with toric orthokeratology lenses . they reported that manifest myopia was significantly reduced from 2.53 1.31 to 0.41 0.43 d ( p < 0.001 ) , and that astigmatism was also decreased from 1.91 0.64 to 0.40 0.39 d ( p < 0.001 ) after 1 month of lens wear . to our knowledge , this is the first prospective , multi - center clinical trial to have investigated the effectiveness of toric orthokeratology in korea . specifically , we evaluated the effectiveness and safety of toric orthokeratology lenses in patients with both myopia and astigmatism . patients wore the toric orthokeratology lenses during sleep for an average of 7 hours and for up to 4 weeks ( 28 days ) . the results showed than overnight toric orthokeratology reduced both myopia and astigmatism , although the degree of chance in astigmatism was relatively small . we attributed this small change to a weakened effect of the toric orthokeratology lenses to reshape the cornea during the night , as the subjects in this study visited our institute primarily in the afternoon . thus , additional studies regarding the clinical effectiveness of toric orthokeratology in correcting astigmatism are needed . thus , our results suggest that toric orthokeratology lenses do not change the ocular features associated with corrected visual acuity , which reflects the safety of this approach . in 2000 , lui and edwards reported that cct is the most predictive value of myopic reduction based on the results of a randomized controlled clinical trial involving 14 subjects who wore orthokeratology lenses during the daytime . they found that cct was reduced significantly between 1 day and 1 week , and that the most significant reduction occurred between 1 day and 1 month . in our study , cct was significantly decreased ( p = 0.005 ) after 4 weeks of lens wear but remained within the normal range . among the five adverse reactions noted in five subjects during our trial , reactions exclusively concerning the lenses comprised four cases ( 12.90% ) , all of which were mild in nature and recovered fully . on the other hand , the change in coefficient of variation was considered to be a common effect of all types of contact lenses on corneal polymegathism , and the extent of the increase was not large relative to that noted in other studies . on the basis of these findings , our results suggest that toric orthokeratology lenses do not have any serious harmful effects . one limitation of this clinical trial was that the follow - up period was only 4 weeks . based on our results , the purpose of these studies should be to elucidate further aspects and complications of toric orthokeratology , such as maintenance of lens centration or changes in ocular features . in conclusion , the effectiveness of toric orthokeratology lenses in correcting myopia combined with astigmatism was verified statistically . importantly , there were no notable adverse reactions or significant abnormal findings caused by use of toric orthokeratology lenses as determined by silt - lamp microscopy and other examinations . therefore , toric orthokeratology can be considered an effective and safe treatment for correcting visual acuity in patients with combined myopia and astigmatism .

the stratus td - oct had a scanning rate of 400 a - scans per second and generated a standard scanning pattern that consisted of six radial , concentric , 6-mm - long b - scans centered on the fovea and offset by 30. image quality often depended on the cooperation of the patient and the skill of the operator , since tracking was not yet available and higher - density scans took longer to acquire . over the past 15 years as oct technology has evolved , td - oct has given way to spectral - domain oct ( sd - oct ) . spectral - domain oct , a type of fourier - domain oct ( fd - oct ) with scanning rates of approximately 27,000 to 70,000 a - scans per second , resulted in denser , more varied scanning patterns , better image quality , better reproducibility , and large volumetric datasets that could be viewed using both the traditional cross - sectional approach and an en face approach . compared with the more advanced sd - oct technology now available in the clinics , the capabilities of the stratus td - oct instrument now seem primitive , yet at the time , the images obtained using td - oct imaging provided a view of macular neovascularization ( mnv ) and exudation that would forever change the way we managed patients . the b - scan image of a normal macula shows layers of differing reflectivity that approximate the major cellular layers of the retina and choroid . however , features of the choroidal anatomy were poorly visualized using td - oct imaging . thus , by using td - oct , it was possible to obtain a crude optical biopsy of the macula and distinguish between a normal retina / rpe contour and an abnormal one , which was all that was needed for the retinal specialist to identify exudation . moreover , one of the most commonly used quantitative parameters derived from oct datasets was the retinal thickness measurement , which was obtained by segmenting the internal limiting membrane ( ilm ) and a boundary representing the rpe . this information was used to generate retinal thickness maps , and these maps proved useful for identifying and describing deviations from normal anatomy and how these deviations changed over time . however , it was the viewing of individual b - scans that provided the retina specialist with a powerful strategy for following disease progression and response to treatment . while it came as no surprise that oct imaging was extremely useful for detecting the macular edema expected in cases of diabetes and retinal vein occlusions , it was a surprise to most retina specialists that intraretinal fluid was commonly found as an early and prominent component of the exudation associated with neovascular age - related macular degeneration ( nvamd ) . the classification system previously developed for nvamd was based on leakage patterns seen on dye - based angiography , and this leakage was thought to result in the accumulation of fluid under the retina and under the rpe . this made intuitive sense since the neovascularization was thought to arise under the retina from the choroidal circulation , so why would there be fluid in the retina ? in retrospect , the detection of intraretinal fluid turned out to be one of the most novel and important observations made when using td - oct for imaging nvamd . at the time , one explanation for the presence of intraretinal edema was that the subretinal exudation had extended into the retina , but the extension of subretinal fluid into the retina was not commonly seen in other conditions associated with subretinal fluid such as central serous chorioretinopathy ( cscr ) and retinal detachment . moreover , eyes with early cscr and subretinal fluid , prior to the onset of fibrosis and photoreceptor disorganization , tended to have good best - corrected visual acuity while eyes with nvamd and similar amounts of subretinal fluid always seemed to have worse vision due to the intraretinal fluid . an alternative explanation for the intraretinal exudation was that this fluid came from established mature retinal vessels , and the exudation was in response to vegf . after all , intraocular injections of vegf into primate eyes had previously been shown to cause retinal edema and hemorrhages . thus , the presence of macular fluid came to be appreciated as a useful surrogate measure for the presence of excess vegf . another explanation for intraretinal fluid was that the neovascularization could be arising from within the retina , and this neovascularization could be directly responsible for the intraretinal macular fluid . in the early days of oct , this hypothesis seemed far - fetched , but now we know that type 3 mnv ( known as retinal angiomatous proliferation ) can arise from within the retina and is associated with intraretinal edema . while type 3 mnv accounts for a minority of neovascular lesions and macular edema is a common feature of all mnv , the most likely explanation for the presence of intraretinal fluid is that vegf induces exudation from the mature retinal vessels in nvamd . thus , lessons learned from early oct imaging of nvamd included an appreciation that intraretinal fluid was one of the first signs of vegf - mediated exudation ( figs . late fluorescein angiographic ( fa ) images and time - domain optical coherence tomography ( oct ) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study , then followed in the extension study with observation and no additional retreatments with ranibizumab . ( c ) after eight injections over 140 days , the fa leakage had significantly diminished , and va improved to 20/80 . ( e ) after an additional 22 months without any ranibizumab injections , va improved to 20/32 and there was no evidence fa leakage . late fluorescein angiographic ( fa ) images and time - domain optical coherence tomography ( oct ) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study , then followed in the extension study with observation and retreatment with ranibizumab . ( a , c , e , g , i , k ) late fa images . ( b , d , f , h , j , l ) diagonal oct b - scans . ( c ) two weeks later , va improved to 20/160 , and the fa image showed significant reduction in leakage with focal areas of blocked fluorescence at the margin of the lesion consistent with hemorrhages . ( d ) corresponding oct b - scan with significant resolution of macular edema 2 weeks after the first injection . ( e ) after 140 days and 10 injections from baseline , va improved to 20/100 , and late fa image showed no leakage with near - complete resolution of the hemorrhages . ( f ) corresponding oct b - scan showed resolution of the macular edema with evidence of macular atrophy as seen by the hypertransmission of light into the choroid ( arrow ) . ( g ) after 10 months without treatment , va was stable at 20/100 , but the late fa image showed minimal leakage . ( h ) corresponding oct b - scan showed the appearance of small intraretinal cysts indicative of recurrent macular fluid ( i ) after 4 months without treatment , va remained unchanged at 20/100 but the patient was complaining of increasing distortion . ( j ) corresponding oct b - scan showed increased macular edema , and a ranibizumab injection was performed . ( k ) five months after retreatment , va improved to 20/63 and the fa image showed no leakage . ( l ) corresponding oct b - scan showed minimal macular thickening nasal to the fovea . overall , oct - guided retreatment improved va , decreased fa leakage , and decreased the macular fluid . not surprisingly , oct imaging also revealed that macular fluid accumulated in the space between the rpe and the neurosensory retina . serous exudate appeared on oct imaging as a homogeneous hyporeflective space , while proteinaceous exudation appeared less homogeneous and was shown to contain reflective fibrinous membranes ( fig . this proteinaceous material has been referred to as subretinal hyperreflective material ( shrm ) or subretinal hyperreflective material exudate ( shre ) and is different from the hyperreflective fibrosis that can form in more advanced neovascular lesions . the shrm / shre of particular concern is the material that accumulates between the retina and rpe as seen on oct imaging ( fig . neovascular lesions with shrm / shre have been associated with worse visual acuity ( va ) outcomes due to the formation of fibrotic scars and photoreceptor atrophy . usually , type 2 mnv , which grows in the subretinal space , has been shown to be associated with a larger volume of subretinal fluid compared with sub - rpe lesions or type 1 mnv . while these type 1 neovascular lesions have been associated with less subretinal fluid , they are associated with detachments of the rpe known as retinal pigment epithelial detachments ( peds ) ( figs . 4 , 5 ) , and early on , oct imaging was shown to be a useful technique for identifying and characterizing peds . while the presence of a ped and blood was shown to serve as a poor prognostic sign in neovascular amd , an even worse development , often associated with hemorrhage , was the formation of a rpe tear . optical coherence tomography imaging of rpe tears was shown to reveal an area of rpe discontinuity with the free edge of the rpe often curled under the ped . adjacent to the tear , oct imaging has revealed areas with increased choroidal reflectivity with improved visualization of choroidal vessels due to the absence of the rpe . typically , the overlying retina was found to be intact , but could be separated from the area of atrophy by subretinal fluid . late fluorescein angiographic ( fa ) images and time - domain optical coherence tomography ( oct ) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study , followed in the extension study by observation and then retreatment with ranibizumab . ( a , c , e , g , i , k ) late fluorescein angiographic images . ( b , d , f , h , j , l ) diagonal oct b - scans . ( b ) corresponding baseline oct image showed subretinal fluid ( arrow ) and a retinal pigment epithelial detachment ( ped , arrowhead ) . ( c ) after 140 days and six ranibizumab injections , va improved to 20/50 , and the fa image showed a marked reduction in leakage . ( d ) corresponding oct b - scan showed resolution of the subretinal fluid with a residual low - lying ped ( arrowhead ) . ( e ) after 3 months without an injection , va decreased to 20/100 , and the fa image showed a subtle increase in leakage . ( g ) three months after the ranibizumab injection , va improved to 20/80 , and the fa image showed a marked decrease in leakage . ( h ) corresponding oct b - scan showed only small intraretinal cysts , and no injection was given . ( i ) after 6 months without treatment , va slowly decreased to 20/100 and the patient then complained of distortion . ( k ) over the next 12 months , the patient received two more ranibizumab injections , and the va improved to 20/50 . ( l ) corresponding oct b - scan showed marked improvement in the macular fluid . macular neovascularization ( mnv ) with subretinal hyperreflective material / exudate ( shrm / shre ) . ( a , c , e ) horizontal spectral - domain optical coherence tomography ( sd - oct ) b - scans . ( a , b ) patient with mnv secondary to age - related macular degeneration was seen in clinic and found to have macular fluid on sd - oct imaging . visual acuity ( ( c , d ) two weeks later , the patient returned complaining of decreased vision . sd - oct imaging showed increased macular fluid and increased shrm / shre ( arrows ) . the first injection of a vascular endothelial growth factor ( vegf ) inhibitor was given . ( e , f ) one month after the third of three monthly injections , va improved to 20/30 with resolution of the macular fluid and srhm / shre . a fourth anti in all these examples of macular fluid , whether the fluid was in the retina , under the retina , or under the rpe , oct imaging was the ideal technique for detecting the excess fluid and determining whether it had changed . since most of the macular fluid occurred in response to excess vegf , the stage was now set for the convergence of an ideal imaging technique with an ideal treatment that would inhibit exudation and reduce the accumulation of fluid . at the same time that the stratus oct was introduced into the clinic , the first vegf inhibitor , previously known as rhufabv2 and now known as ranibizumab ( lucentis ; genentech / roche , south san francisco , ca , usa ) , entered into a phase i clinical trial . in this phase i single - injection , dose - escalation , safety trial , oct imaging was not available and fa imaging was used to characterize the neovascular lesions only at baseline . from this phase i trial , a maximum tolerated dose of 0.5 mg was identified based on dose - limiting inflammation that occurred at the 1.0-mg dose . while safety and va outcomes were encouraging , it was n't until the phase ii study that oct imaging was first used at the bascom palmer eye institute . one phase ii trial studied the safety and tolerability of monthly repeated dosing of 0.3- or 0.5-mg ranibizumab through 6 months , and the second phase ii trial explored a dose - escalation regimen in which the ranibizumab dose was sequentially increased from 0.3 to 2 mg and injected as frequently as every 2 weeks in eyes with nvamd . while both phase ii trials demonstrated va improvement associated with ranibizumab therapy , oct imaging unambiguously demonstrated that the improvement in va was correlated with the resolution of macular fluid ( rosenfeld pj , unpublished data , 2003 ) . moreover , in an extension program at the end of the phase ii studies , retreatment with ranibizumab was permitted at the investigators ' discretion once the fixed dosing was stopped . investigators could base their retreatment on changes in va , fa imaging , or oct imaging . as these patients were being followed off treatment , oct imaging identified the earliest recurrence of macular fluid in some patients even before fa imaging detected leakage and before va was affected ( figs . some patients needed no additional treatment for up to 2 years while some patients frequently developed recurrent macular fluid . the fluid that was initially detected by oct imaging , if untreated , would then increase over time , resulting in subsequent vision loss . thus , a small amount of fluid led to even more fluid and served as a harbinger of disease progression . as the macular fluid gradually reaccumulated in these subjects in the extension study , retreatment with ranibizumab once again resulted in resolution of the macular fluid ; and in those patients with decreased va associated with the recurrent fluid , va improved once the fluid resolved . these were the first observations of how oct imaging of macular fluid could serve as a surrogate marker for the presence of excess vegf , how oct imaging unambiguously demonstrated the profound effect of anti - vegf therapy on macular fluid , and how oct monitoring of macular fluid might serve as a strategy to determine the appropriate dosing interval used for the clinical management of nvamd . these data were presented at the retina subspecialty day before the american academy of ophthalmology meeting in 2004 and at other scientific meetings in an attempt to convince skeptical colleagues that oct imaging rather than fa imaging could be used in the management of nvamd patients . to convincingly demonstrate the benefits of oct imaging for managing nvamd patients , genentech supported an investigator - sponsored trial known as the prospective oct imaging of patients with neovascular amd treated with intra - ocular ranibizumab ( pronto ) study . in the 2-year pronto study , nvamd patients received three monthly injections of 0.5-mg ranibizumab and continued to receive monthly injections as long as there was persistent macular fluid based on oct imaging . however , once oct imaging revealed a fluid - free macula , patients were followed monthly without injection until at least one of several retreatment criteria were met . these criteria included a va loss of at least five letters with oct evidence of fluid in the macula , an increase in oct central retinal thickness of at least 100 m , new macular hemorrhage , or a new area of classic mnv identified by fa imaging . the purpose of these criteria was to determine if oct imaging was the most sensitive strategy for detecting the recurrence of macular fluid . after the first year of the study , it was obvious that oct imaging was able to detect the need for retreatment long before leakage was detected on fa imaging , before vision loss occurred , and before hemorrhage appeared . at the time , retina specialists questioned why the oct threshold for retreatment was set at an increased central retinal thickness measurement of 100 m , and the reason for this threshold was to prove a point . we wanted to show our skeptical colleagues that when a small amount of fluid appeared and it was left untreated , even more fluid and vision loss would result . once we proved our point , the threshold for retreatment was lowered in the second year of the study . a zero tolerance strategy was developed , and the protocol was amended to allow for retreatment once any fluid was detected after a dry macula was achieved . after 2 years , the va outcomes would turn out to be very similar to the outcomes observed following monthly dosing with ranibizumab in the phase ii and phase iii trials , but unlike the situation with a fixed monthly dosing regimen , these patients in the pronto study required approximately half the number of injections . the pronto study served as a model for subsequent studies investigating oct - guided retreatment using a pro re nata ( prn ) regimen for nvamd . however , several of the smaller studies failed to appreciate two key features of the pronto study : the requirement for monthly exams and the amended requirement that retreatment be given if any macular fluid was detected by oct imaging once the macula was fluid - free . moreover , retreatment was not decided by changes in va , but only by changes in oct imaging . while none of the phase iii pivotal trials that led to food and drug administration ( fda ) approval for any of the anti - vegf drugs used a pure oct - guided prn dosing regimen , monthly dosing versus oct - guided prn dosing has been studied in several large , well - controlled prospective clinical trials . the comparison of age - related macular degeneration treatments trials ( catt ) and the inhibition of vegf in age - related choroidal neovascularization ( ivan ) trial showed similar va outcomes after 2 years when the same anti - vegf drug was dosed monthly or discontinuously . however , the most convincing of these comparative trials is the phase iii , double - masked , multicenter , randomized , active treatment - controlled study of the efficacy and safety of 0.5 mg and 2.0 mg ranibizumab administered monthly or on an as - needed basis ( prn ) in patients with subfoveal neovascular age - related macular degeneration ( harbor ) trial . the harbor trial used a prn regimen similar to one employed in the pronto study . the two doses of ranibizumab were compared using a monthly versus an oct - guided prn dosing regimen in which patients received three monthly injections followed by monthly retreatment as long as there was persistent fluid . once the macula became fluid - free , patients were followed monthly and retreated if any macular fluid was detected by oct imaging . this study not only showed that monthly dosing and oct - guided prn dosing were equivalent , but did not even rely on a fundus exam when making the retreatment decision . the totality of the data suggest that a rigorously implemented oct - guided prn dosing strategy appears to be as beneficial as a fixed monthly dosing regimen , and as a result , this strategy has gained global acceptance . the greatest advantage of an oct - guided prn dosing strategy is that it improves and maintains va while significantly decreasing the number of injections needed . another dosing strategy for anti - vegf drugs that has gained global acceptance is a regimen known as an oct - guided treat - and - extend dosing . this regimen also relies on oct imaging to determine when the macula has become fluid - free , but then patients continue to be injected at each subsequent visit . however , once the macula is fluid - free , the patient is injected and the visit intervals are slowly extended to determine when and if macular fluid recurs . if oct imaging detects the recurrence of fluid at a visit , then a treatment is given and the interval is shortened . usually , visit intervals are lengthened or shortened by 2-week intervals depending on the status of macular fluid . while this oct - guided strategy mandates that an injection be given even when the macula is dry , the advantage over other strategies is that it minimizes the overall number of visits . to date , only two prospective studies , known as the lucentis compared to avastin study ( lucas ) and treat - and - extend in amd ( trex - amd ) study , have used this treat - and - extend strategy , and the results appear to be similar to the outcomes from larger monthly dosing trials . despite the limited prospective clinical trial data , clinicians and patients appear to prefer the treat - and - extend dosing regimen as a compromise between frequent monthly visits and monthly injections . on occasion , oct monitoring of neovascular lesions has shown the importance of even more frequent injections with anti - vegf drugs when monthly dosing proved unable to prevent the growth and worsening exudation of the neovascular lesions . thus , oct monitoring allows clinicians to follow a goldilocks strategy when managing patients , which is not too many injections , not too few injections , but just the right number of injections for that patient . during the early to mid-2000s when oct was being accepted into practices as an essential tool for monitoring patients with nvamd , clinicians became empowered by their ability to monitor the real - time effectiveness of different therapeutic agents , whether it was verteporfin photodynamic therapy ( pdt ) , intravitreal steroids , or anti - vegf therapy . during this time , two anti - vegf drugs for intraocular delivery were in clinical trials : ranibizumab ( lucentis , genentech / roche ) , an antibody fragment that binds all isoforms of vegf - a ; and pegaptanib sodium ( macugen ; osi / eyetech , new york , ny , usa ) , an aptamer that binds vegf165 and larger isoforms . as mentioned previously , oct imaging was useful in demonstrating the anatomic improvements that followed ranibizumab therapy , and selected sites performed oct imaging during the phase iii ranibizumab trials for neovascular amd known as the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular amd ( marina ) and the anti - vegf antibody for the treatment of predominantly classic choroidal neovascularization in age - related macular degeneration ( anchor ) . in these trials , ranibizumab was dosed monthly for 2 years , and the oct images that were obtained on a subset of subjects from these studies did confirm the drying effect of ranibizumab therapy . in contrast , oct imaging was never used in the pegaptanib trials , and after pegaptanib was approved in late 2004 and became commercially available in early 2005 , clinicians used oct imaging on patients undergoing treatment with pegaptanib . not surprisingly , the disappointing va outcomes following pegaptanib therapy were found to correlate with persistent macular fluid following treatment , thus empowering clinicians to make an objective assessment of the drug 's efficacy . as a result of this discrepancy between the oct outcomes when using pegaptanib versus other anti - vegf drugs , oct imaging confirmed the need to suppress all vegf isoforms to obtain the maximum drying effect from anti - vegf therapy . optical coherence tomography imaging in the marina and anchor trials with ranibizumab also revealed that the major cause of severe vision loss was not associated with persistent macular fluid or a scar ; rather , severe vision loss was more commonly associated with a fluid - free macula . for the first time , there was an appreciation that macular atrophy was a major cause of vision loss in treated eyes with nvamd , rather than the more typical cause of severe vision loss prior to anti - vegf therapy , which was the formation of a disciform scar . the formation of macular atrophy after anti - vegf therapy was confirmed in the catt and ivan trials after 2 years , and in these trials , the monthly dosing groups had a higher incidence of macular atrophy compared with the discontinuous treatment groups . while this observation has raised questions about whether anti - vegf therapy is responsible for the macular atrophy in some patients or whether the atrophy appears as a feature of normal disease progression and a consequence of suppressing neovascularization , it has become abundantly clear that oct imaging is an invaluable tool for not only monitoring the presence of macular fluid , but also for monitoring the anatomic integrity of the outer retina and rpe as macular atrophy evolves ( fig . macular atrophy developing after treatment with an inhibitor of vascular endothelial growth factor ( vegf ) . ( a , d , g , f ) spectral - domain optical coherence tomography ( sd - oct ) fundus images at sequential visits . ( b , e , h , k ) horizontal sd - oct b - scans . ( c , f , i , l ) vertical sd - oct b - scans . ( a , b , c ) one month after an anti - vegf injection for a hemorrhagic fibrovascular retinal pigment epithelial detachment secondary to age - related macular degeneration . the oct fundus image showed a small focus of atrophy as seen in ( a ) ( arrow ) . ( d , e , f ) two months after the second injection , va was 20/25 , but the area of atrophy was enlarging as shown in ( d ) ( arrow ) with evidence of hypertransmission shown on the horizontal ( e ) and vertical ( f ) b - scans . disruption of the outer retinal layers can be appreciated in the areas with the hypertransmission defects . ( g , h , i ) a fourth injection was given 2 months after the third injection , and 2 months after the fourth injection , the patient returned with a va of 20/25 , but an even larger area of atrophy could be appreciated as shown in ( g ) ( arrow ) with evidence of even more hypertransmission as shown on the horizontal ( h ) and vertical ( i ) b - scans . ( j , k , l ) three months after the fifth injection , the area of atrophy was slightly larger ( j ) ( arrow ) and prominent hypertransmission defects were appreciated as shown in ( k ) and ( l ) . disruption of the normal outer retinal structures was seen in the areas with the hypertransmission defect . during the clinical development of ranibizumab and pegaptanib , there were not only questions about the appropriate dose , dosing interval , and long - term effectiveness of these drugs ; there was also uncertainty about the safety of repeated intravitreal injections and whether patients would tolerate intravitreal injections as a long - term drug delivery strategy . due to these concerns about the safety and tolerability of repeated intravitreal injections , we investigated a third vegf inhibitor known as bevacizumab ( avastin ; genentech / roche ) , a full - length humanized antibody against vegf - a sharing a common clonal ancestry with an anti - vegf murine monoclonal antibody that gave rise to ranibizumab ; and like ranibizumab , bevacizumab was known to bind all the isoforms of vegf - a . since intravenous bevacizumab was being developed as a therapy for colon cancer , we decided to initiate a self - funded study using the same intravenous strategy for the treatment of nvamd , and once again , oct imaging proved pivotal in these bevacizumab studies . the systemic avastin in neovascular amd ( sana ) study was initiated once bevacizumab was approved for colon cancer in february 2004 . twenty - four hours after the first systemic dose of bevacizumab in the first patient , oct imaging unambiguously showed a profound benefit from intravenous bevacizumab . by may of 2005 , oct imaging had confirmed that systemic bevacizumab had a significant and sustained drying effect on the macula , and this drying effect was associated with va improvement . however , during the course of the sana study , the fda added a black box warning describing the small but significant risk of thromboembolic events from the use of high - dose intravenous bevacizumab . while none of the patients in the sana study experienced a thromboembolic event , they did experience mild hypertension that was easily controlled with oral medication . due to these systemic safety concerns and the appreciation from the ranibizumab and pegaptanib studies that patients were able to tolerate repeated intravitreal injections , we decided to offer patients an off - label intravitreal dose of bevacizumab as salvage therapy . this dose was 500- to 1000-fold lower than the dose given intravenously , thus minimizing the systemic risk . moreover , this intravitreal dose was capable of binding the same amount of intraocular vegf as the intravitreal dose of ranibizumab . once again , oct imaging played a pivotal role in demonstrating the benefits of bevacizumab . when first used as salvage therapy in a nvamd patient with a lesion that was enlarging despite treatments with pdt and pegaptanib , oct imaging showed a remarkable improvement in the amount of macular fluid after intravitreal bevacizumab , and this improvement was associated with va improvement . since ranibizumab had not yet been approved , and would not be commercially available for another year in july 2006 , the off - label use of intravitreal bevacizumab spread globally and became the treatment of choice in 2005 due to its perceived clinical efficacy , its widespread availability , its superiority over pegaptanib , and its low cost . in july 2006 when ranibizumab was commercially available for the treatment of nvamd , clinicians had a choice between two drugs manufactured by genentech that had a similar genetic lineage but differed with respect to size , affinity for vegf , and cost . with the widespread use of oct imaging , clinicians were now empowered to see for themselves that ranibizumab and bevacizumab were superior to pegaptanib and that ranibizumab appeared similar to bevacizumab with respect to its drying effect on the macula and improvement in va following treatment . without oct imaging , it is unlikely that bevacizumab and oct - guided retreatment would have gained worldwide popularity . in 2011 , a fourth drug , known as aflibercept ( eylea ; regeneron pharmaceutical , tarrytown , ny , usa ) , was fda approved for the treatment of nvamd . aflibercept , a fusion protein combining the fc portion of an antibody with vegf - binding domains from vegf receptors 1 and 2 , has an even higher affinity for vegf compared with ranibizumab and a molecular size in between those of bevacizumab and ranibizumab . moreover , it has the theoretical advantage of neutralizing placental growth factor , as well as all the isoforms of vegf - a . in the phase iii aflibercept trials known as the vegf trap - eye : investigation of efficacy and safety in wet amd ( view1 and view2 ) trials , intravitreal injections of aflibercept every 2 months during the first year were shown to be equivalent to monthly injections of ranibizumab . in the second year of the trials , subjects received a mandatory injection every 3 months , with oct imaging performed during the intervening monthly visits . if there was evidence of recurrent or persistent macular fluid using oct imaging , then injections could be administered during these intervening visits . this modified - prn regimen resulted in similar va and oct outcomes when aflibercept and ranibizumab were compared , but subjects receiving aflibercept required slightly fewer injections using oct - guided therapy . once again , oct imaging proved valuable for detecting macular fluid and determining the need for retreatment in this prospective clinical trial setting . since its approval , aflibercept has gained popularity as an anti - vegf drug with the ability to increase the retreatment interval while maintaining improved va and a fluid - free macula in nvamd patients . the perception that aflibercept provides increased durability arises not only from the dosing interval of every 2 months used in the phase iii studies , but also from numerous research papers showing that when patients are switched from ranibizumab or bevacizumab to aflibercept , there is a tendency for investigators to report improved macular anatomy and extended fluid - free intervals based on oct imaging . a fifth drug , known as conbercept ( lumatin ; chengdu kanghong pharmaceutical group , chengdu , china ) , gained china fda approval in 2013 . conbercept , which is a fusion protein similar to aflibercept and combines the binding domains of vegf receptors 1 and 2 with an fc portion of an antibody , has a molecular weight comparable to that of bevacizumab , but it is currently used only in china . once again , oct imaging correlated with the va benefits observed during the conbercept clinical trials . now that td - oct has been surpassed by sd - oct in the clinics , the next step in the commercial evolution of oct imaging for the management of nvamd is the development of oct angiography ( octa ) . optical coherence tomography angiography utilizes novel scanning patterns and algorithms to image blood flow in the retina and choroid without the use of exogenous dyes . not only can blood flow be imaged , but also the scans provide detailed structural information comparable to or better than the images currently available from commercial sd - oct instruments . different octa platforms are being developed that use different scanning patterns and algorithms , but they can be broadly divided into sd - oct and swept - source oct ( ss - oct ) instruments . each platform will have certain advantages and disadvantages , but whether octa has advantages over conventional oct imaging in the management of nvamd patients remains to be determined . the ability to noninvasively visualize the appearance and growth of mnv should augment the routine oct strategy of monitoring for the presence or absence of macular fluid when deciding to initiate anti - vegf therapy and when to re - treat . since exudation is the metric by which we currently measure vegf activity and exudation can arise from both the neovascularization and the normal vasculature , there should be certain advantages of using octa to directly monitor changes in the neovascular lesion rather than just monitoring the level of exudation . even if octa imaging does not influence our decision on when to treat , it will surely add to our understanding of disease onset , progression , and response to treatment . for example , octa can visualize the presence of type 1 mnv in eyes that were diagnosed with dry , intermediate amd . the presence of nonexudative neovascular lesions raises new questions about whether we can predict when exudation will arise and if there is a role for anti - vegf therapy even before exudation occurs . if octa can now replace fa and icga imaging for most mnv , then octa imaging will provide all the structural and flow information needed for the diagnosis and management of mnv . with respect to their development , oct imaging and antiangiogenic therapies have been inextricably linked . if oct imaging had not been available when anti - vegf therapies were developed , then clinicians and patients would have been chained to fixed - dosing regimens without the ability to easily and noninvasively assess whether the exudation from the neovascularization had been adequately controlled . in the era of pdt before anti - vegf therapy , patients underwent invasive , time - consuming fa imaging every 3 months to determine whether leakage was present and treatment was needed . when intravitreal pegaptanib was approved , injections were given per protocol every 6 weeks , but when patients lost vision and additional oct imaging was performed , the presence of persistent or worsening macular fluid was confirmed . in the era of oct imaging and pan - vegf blockade , oct imaging unambiguously confirmed the effectiveness of anti - vegf therapy in resolving macular fluid . optical coherence tomography imaging empowered clinicians to personalize therapy by evaluating different drugs and dosing regimens . clinicians were able to customize therapy to either minimize the number of injections or minimize the number of visits . without the confidence that came from oct imaging and the ability to demonstrate the beneficial effect of anti - vegf therapy within 24 hours of treatment , it is unlikely that intravenous and then intravitreal bevacizumab therapy for the treatment of exudative eye diseases would have been developed in 2005 , and it is unlikely that the global ophthalmologic community would have been convinced to rapidly adopt intravitreal bevacizumab as a viable treatment strategy . while significant cost savings have resulted from the use of oct compared with fa imaging , greater cost savings have resulted from the use of oct - guided retreatment to avoid monthly dosing , and even greater cost savings have resulted from the use of off - label bevacizumab in lieu of the more expensive drugs . in the united states alone , over the past 10 years , the combined medicare cost savings from the use of oct and bevacizumab have exceeded $ 20 billion when conservatively extrapolated from published utilization data for all forms of exudative eye diseases . globally , four intravitreal drugs dominate the market for anti - vegf therapy in neovascular amd , diabetic macular edema , and retinal vein occlusions : bevacizumab , ranibizumab , aflibercept , and conbercept . despite differences in molecular size , affinity for vegf , and intravitreal half - life , the most likely explanation for the effectiveness of these four drugs is that they bind all the biological isoforms of vegf - a whereas pegaptanib inhibited only vegf165 and larger isoforms . thus , this was the first example of how oct imaging was able to distinguish between different therapeutic targets . as new therapies and drug delivery strategies are developed to treat exudative eye diseases , oct will once again play an important role . for example , anti - vegf drugs are being delivered through implantable devices , as topical therapy , and as oral medication . treatments that combine intravitreal anti - vegf therapy along with inhibitors of platelet - derived growth factor ( pdgf ) , angiopoietin 2 , and tissue factor are being investigated , and topical squalamine is being combined with anti - vegf therapy in clinical trials as well . optical coherence tomography continues to be an integral part of all these trials , but the phase iii trials for the pdgf inhibitor known as e10030 ( fovista ; ophthotech corp . , princeton , nj , usa ) have incorporated a novel inclusion requirement for their treatment - nave subjects with nvamd ; that requirement is the presence of shrm ( clinicaltrials.gov identifier : nct01944839 , nct01940900 , nct01940887 ) . while improvement of va remains the ultimate outcome that will determine the success or failure of any new treatment , there 's so much more to the macula than va , which measures only central foveal function . unlike dye - based angiography , oct imaging can identify macular abnormalities involving the photoreceptors , the rpe , and the choroid that can help explain visual function changes such as reduced reading speed , reduced microperimetric thresholds , and delayed dark adaptation . while oct is clearly the gold standard imaging strategy for predicting the success of any antiangiogenic treatment , it is also a valuable strategy for studying a wide range of anatomic changes that correlate with reduced visual function and disease progression . in summary , oct is held in such high regard that clinicians will never be convinced of a treatment 's efficacy until oct imaging demonstrates an unambiguous anatomic benefit .

purposeto explain the pivotal role optical coherence tomography ( oct ) imaging had in the development of antiangiogenic therapies for the treatment of neovascular age - related macular degeneration ( nvamd).methodsa historical literature review was combined with personal perspectives from the introduction of oct imaging and the early clinical use of vascular endothelial growth factor ( vegf ) inhibitors.resultsat the time that oct emerged , the gold standard for imaging of nvamd was fluorescein angiography ( fa ) , a time - consuming , dye - based , invasive technique that provided en face images of the retina and was used to characterize leakage , perfusion status , and the types of macular neovascularization ( mnv ) . in comparison , oct imaging was a fast , safe , noninvasive technique that complemented fa imaging by providing cross - sectional images of the macula . oct was able to visualize and quantify the macular fluid that was associated with the presence of excess vegf , which was identified by intraretinal fluid , subretinal fluid , and fluid under the retinal pigment epithelium ( rpe ) . clinicians quickly appreciated the benefits of oct imaging for following macular fluid after anti - vegf therapy . by observing the qualitative and quantitative changes in macular fluid depicted by oct imaging , clinicians were empowered to compare anti - vegf drugs and move from fixed - dosing regimens to patient - specific dosing strategies requiring fewer injections.conclusionsoptical coherence tomography imaging was adopted as a vegf - meter , a method to detect excess vegf , and evolved to become the gold standard imaging strategy for diagnosing nvamd , assessing treatment responses to anti - vegf drugs , deciding when to re - treat , and evaluating disease progression .

TD-OCT and SD-OCT Imaging of Neovascular AMD OCT Imaging, the Rise of Ranibizumab, and Personalized Treatment Regimens for nvAMD OCT Imaging, the Demise of Pegaptanib, and the Rise of Bevacizumab OCT Imaging and the Rise of Aflibercept for nvAMD The Evolution of OCT Imaging for the Management of nvAMD Summary of OCT Imaging and Antiangiogenic Therapies

spectral - domain oct , a type of fourier - domain oct ( fd - oct ) with scanning rates of approximately 27,000 to 70,000 a - scans per second , resulted in denser , more varied scanning patterns , better image quality , better reproducibility , and large volumetric datasets that could be viewed using both the traditional cross - sectional approach and an en face approach . compared with the more advanced sd - oct technology now available in the clinics , the capabilities of the stratus td - oct instrument now seem primitive , yet at the time , the images obtained using td - oct imaging provided a view of macular neovascularization ( mnv ) and exudation that would forever change the way we managed patients . thus , by using td - oct , it was possible to obtain a crude optical biopsy of the macula and distinguish between a normal retina / rpe contour and an abnormal one , which was all that was needed for the retinal specialist to identify exudation . however , it was the viewing of individual b - scans that provided the retina specialist with a powerful strategy for following disease progression and response to treatment . while it came as no surprise that oct imaging was extremely useful for detecting the macular edema expected in cases of diabetes and retinal vein occlusions , it was a surprise to most retina specialists that intraretinal fluid was commonly found as an early and prominent component of the exudation associated with neovascular age - related macular degeneration ( nvamd ) . the classification system previously developed for nvamd was based on leakage patterns seen on dye - based angiography , and this leakage was thought to result in the accumulation of fluid under the retina and under the rpe . at the time , one explanation for the presence of intraretinal edema was that the subretinal exudation had extended into the retina , but the extension of subretinal fluid into the retina was not commonly seen in other conditions associated with subretinal fluid such as central serous chorioretinopathy ( cscr ) and retinal detachment . thus , the presence of macular fluid came to be appreciated as a useful surrogate measure for the presence of excess vegf . another explanation for intraretinal fluid was that the neovascularization could be arising from within the retina , and this neovascularization could be directly responsible for the intraretinal macular fluid . in the early days of oct , this hypothesis seemed far - fetched , but now we know that type 3 mnv ( known as retinal angiomatous proliferation ) can arise from within the retina and is associated with intraretinal edema . while type 3 mnv accounts for a minority of neovascular lesions and macular edema is a common feature of all mnv , the most likely explanation for the presence of intraretinal fluid is that vegf induces exudation from the mature retinal vessels in nvamd . thus , lessons learned from early oct imaging of nvamd included an appreciation that intraretinal fluid was one of the first signs of vegf - mediated exudation ( figs . late fluorescein angiographic ( fa ) images and time - domain optical coherence tomography ( oct ) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study , then followed in the extension study with observation and no additional retreatments with ranibizumab . late fluorescein angiographic ( fa ) images and time - domain optical coherence tomography ( oct ) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study , then followed in the extension study with observation and retreatment with ranibizumab . overall , oct - guided retreatment improved va , decreased fa leakage , and decreased the macular fluid . not surprisingly , oct imaging also revealed that macular fluid accumulated in the space between the rpe and the neurosensory retina . while these type 1 neovascular lesions have been associated with less subretinal fluid , they are associated with detachments of the rpe known as retinal pigment epithelial detachments ( peds ) ( figs . 4 , 5 ) , and early on , oct imaging was shown to be a useful technique for identifying and characterizing peds . while the presence of a ped and blood was shown to serve as a poor prognostic sign in neovascular amd , an even worse development , often associated with hemorrhage , was the formation of a rpe tear . optical coherence tomography imaging of rpe tears was shown to reveal an area of rpe discontinuity with the free edge of the rpe often curled under the ped . late fluorescein angiographic ( fa ) images and time - domain optical coherence tomography ( oct ) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study , followed in the extension study by observation and then retreatment with ranibizumab . ( a , c , e ) horizontal spectral - domain optical coherence tomography ( sd - oct ) b - scans . ( a , b ) patient with mnv secondary to age - related macular degeneration was seen in clinic and found to have macular fluid on sd - oct imaging . the first injection of a vascular endothelial growth factor ( vegf ) inhibitor was given . a fourth anti in all these examples of macular fluid , whether the fluid was in the retina , under the retina , or under the rpe , oct imaging was the ideal technique for detecting the excess fluid and determining whether it had changed . since most of the macular fluid occurred in response to excess vegf , the stage was now set for the convergence of an ideal imaging technique with an ideal treatment that would inhibit exudation and reduce the accumulation of fluid . at the same time that the stratus oct was introduced into the clinic , the first vegf inhibitor , previously known as rhufabv2 and now known as ranibizumab ( lucentis ; genentech / roche , south san francisco , ca , usa ) , entered into a phase i clinical trial . in this phase i single - injection , dose - escalation , safety trial , oct imaging was not available and fa imaging was used to characterize the neovascular lesions only at baseline . while both phase ii trials demonstrated va improvement associated with ranibizumab therapy , oct imaging unambiguously demonstrated that the improvement in va was correlated with the resolution of macular fluid ( rosenfeld pj , unpublished data , 2003 ) . investigators could base their retreatment on changes in va , fa imaging , or oct imaging . as these patients were being followed off treatment , oct imaging identified the earliest recurrence of macular fluid in some patients even before fa imaging detected leakage and before va was affected ( figs . the fluid that was initially detected by oct imaging , if untreated , would then increase over time , resulting in subsequent vision loss . as the macular fluid gradually reaccumulated in these subjects in the extension study , retreatment with ranibizumab once again resulted in resolution of the macular fluid ; and in those patients with decreased va associated with the recurrent fluid , va improved once the fluid resolved . these were the first observations of how oct imaging of macular fluid could serve as a surrogate marker for the presence of excess vegf , how oct imaging unambiguously demonstrated the profound effect of anti - vegf therapy on macular fluid , and how oct monitoring of macular fluid might serve as a strategy to determine the appropriate dosing interval used for the clinical management of nvamd . these data were presented at the retina subspecialty day before the american academy of ophthalmology meeting in 2004 and at other scientific meetings in an attempt to convince skeptical colleagues that oct imaging rather than fa imaging could be used in the management of nvamd patients . to convincingly demonstrate the benefits of oct imaging for managing nvamd patients , genentech supported an investigator - sponsored trial known as the prospective oct imaging of patients with neovascular amd treated with intra - ocular ranibizumab ( pronto ) study . the purpose of these criteria was to determine if oct imaging was the most sensitive strategy for detecting the recurrence of macular fluid . after the first year of the study , it was obvious that oct imaging was able to detect the need for retreatment long before leakage was detected on fa imaging , before vision loss occurred , and before hemorrhage appeared . however , several of the smaller studies failed to appreciate two key features of the pronto study : the requirement for monthly exams and the amended requirement that retreatment be given if any macular fluid was detected by oct imaging once the macula was fluid - free . the comparison of age - related macular degeneration treatments trials ( catt ) and the inhibition of vegf in age - related choroidal neovascularization ( ivan ) trial showed similar va outcomes after 2 years when the same anti - vegf drug was dosed monthly or discontinuously . however , the most convincing of these comparative trials is the phase iii , double - masked , multicenter , randomized , active treatment - controlled study of the efficacy and safety of 0.5 mg and 2.0 mg ranibizumab administered monthly or on an as - needed basis ( prn ) in patients with subfoveal neovascular age - related macular degeneration ( harbor ) trial . on occasion , oct monitoring of neovascular lesions has shown the importance of even more frequent injections with anti - vegf drugs when monthly dosing proved unable to prevent the growth and worsening exudation of the neovascular lesions . during the early to mid-2000s when oct was being accepted into practices as an essential tool for monitoring patients with nvamd , clinicians became empowered by their ability to monitor the real - time effectiveness of different therapeutic agents , whether it was verteporfin photodynamic therapy ( pdt ) , intravitreal steroids , or anti - vegf therapy . during this time , two anti - vegf drugs for intraocular delivery were in clinical trials : ranibizumab ( lucentis , genentech / roche ) , an antibody fragment that binds all isoforms of vegf - a ; and pegaptanib sodium ( macugen ; osi / eyetech , new york , ny , usa ) , an aptamer that binds vegf165 and larger isoforms . as mentioned previously , oct imaging was useful in demonstrating the anatomic improvements that followed ranibizumab therapy , and selected sites performed oct imaging during the phase iii ranibizumab trials for neovascular amd known as the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular amd ( marina ) and the anti - vegf antibody for the treatment of predominantly classic choroidal neovascularization in age - related macular degeneration ( anchor ) . in contrast , oct imaging was never used in the pegaptanib trials , and after pegaptanib was approved in late 2004 and became commercially available in early 2005 , clinicians used oct imaging on patients undergoing treatment with pegaptanib . as a result of this discrepancy between the oct outcomes when using pegaptanib versus other anti - vegf drugs , oct imaging confirmed the need to suppress all vegf isoforms to obtain the maximum drying effect from anti - vegf therapy . optical coherence tomography imaging in the marina and anchor trials with ranibizumab also revealed that the major cause of severe vision loss was not associated with persistent macular fluid or a scar ; rather , severe vision loss was more commonly associated with a fluid - free macula . for the first time , there was an appreciation that macular atrophy was a major cause of vision loss in treated eyes with nvamd , rather than the more typical cause of severe vision loss prior to anti - vegf therapy , which was the formation of a disciform scar . the formation of macular atrophy after anti - vegf therapy was confirmed in the catt and ivan trials after 2 years , and in these trials , the monthly dosing groups had a higher incidence of macular atrophy compared with the discontinuous treatment groups . while this observation has raised questions about whether anti - vegf therapy is responsible for the macular atrophy in some patients or whether the atrophy appears as a feature of normal disease progression and a consequence of suppressing neovascularization , it has become abundantly clear that oct imaging is an invaluable tool for not only monitoring the presence of macular fluid , but also for monitoring the anatomic integrity of the outer retina and rpe as macular atrophy evolves ( fig . macular atrophy developing after treatment with an inhibitor of vascular endothelial growth factor ( vegf ) . ( a , d , g , f ) spectral - domain optical coherence tomography ( sd - oct ) fundus images at sequential visits . ( a , b , c ) one month after an anti - vegf injection for a hemorrhagic fibrovascular retinal pigment epithelial detachment secondary to age - related macular degeneration . due to these concerns about the safety and tolerability of repeated intravitreal injections , we investigated a third vegf inhibitor known as bevacizumab ( avastin ; genentech / roche ) , a full - length humanized antibody against vegf - a sharing a common clonal ancestry with an anti - vegf murine monoclonal antibody that gave rise to ranibizumab ; and like ranibizumab , bevacizumab was known to bind all the isoforms of vegf - a . since intravenous bevacizumab was being developed as a therapy for colon cancer , we decided to initiate a self - funded study using the same intravenous strategy for the treatment of nvamd , and once again , oct imaging proved pivotal in these bevacizumab studies . by may of 2005 , oct imaging had confirmed that systemic bevacizumab had a significant and sustained drying effect on the macula , and this drying effect was associated with va improvement . once again , oct imaging played a pivotal role in demonstrating the benefits of bevacizumab . when first used as salvage therapy in a nvamd patient with a lesion that was enlarging despite treatments with pdt and pegaptanib , oct imaging showed a remarkable improvement in the amount of macular fluid after intravitreal bevacizumab , and this improvement was associated with va improvement . since ranibizumab had not yet been approved , and would not be commercially available for another year in july 2006 , the off - label use of intravitreal bevacizumab spread globally and became the treatment of choice in 2005 due to its perceived clinical efficacy , its widespread availability , its superiority over pegaptanib , and its low cost . in july 2006 when ranibizumab was commercially available for the treatment of nvamd , clinicians had a choice between two drugs manufactured by genentech that had a similar genetic lineage but differed with respect to size , affinity for vegf , and cost . with the widespread use of oct imaging , clinicians were now empowered to see for themselves that ranibizumab and bevacizumab were superior to pegaptanib and that ranibizumab appeared similar to bevacizumab with respect to its drying effect on the macula and improvement in va following treatment . in 2011 , a fourth drug , known as aflibercept ( eylea ; regeneron pharmaceutical , tarrytown , ny , usa ) , was fda approved for the treatment of nvamd . now that td - oct has been surpassed by sd - oct in the clinics , the next step in the commercial evolution of oct imaging for the management of nvamd is the development of oct angiography ( octa ) . optical coherence tomography angiography utilizes novel scanning patterns and algorithms to image blood flow in the retina and choroid without the use of exogenous dyes . the ability to noninvasively visualize the appearance and growth of mnv should augment the routine oct strategy of monitoring for the presence or absence of macular fluid when deciding to initiate anti - vegf therapy and when to re - treat . the presence of nonexudative neovascular lesions raises new questions about whether we can predict when exudation will arise and if there is a role for anti - vegf therapy even before exudation occurs . if oct imaging had not been available when anti - vegf therapies were developed , then clinicians and patients would have been chained to fixed - dosing regimens without the ability to easily and noninvasively assess whether the exudation from the neovascularization had been adequately controlled . in the era of pdt before anti - vegf therapy , patients underwent invasive , time - consuming fa imaging every 3 months to determine whether leakage was present and treatment was needed . when intravitreal pegaptanib was approved , injections were given per protocol every 6 weeks , but when patients lost vision and additional oct imaging was performed , the presence of persistent or worsening macular fluid was confirmed . in the era of oct imaging and pan - vegf blockade , oct imaging unambiguously confirmed the effectiveness of anti - vegf therapy in resolving macular fluid . optical coherence tomography imaging empowered clinicians to personalize therapy by evaluating different drugs and dosing regimens . without the confidence that came from oct imaging and the ability to demonstrate the beneficial effect of anti - vegf therapy within 24 hours of treatment , it is unlikely that intravenous and then intravitreal bevacizumab therapy for the treatment of exudative eye diseases would have been developed in 2005 , and it is unlikely that the global ophthalmologic community would have been convinced to rapidly adopt intravitreal bevacizumab as a viable treatment strategy . while significant cost savings have resulted from the use of oct compared with fa imaging , greater cost savings have resulted from the use of oct - guided retreatment to avoid monthly dosing , and even greater cost savings have resulted from the use of off - label bevacizumab in lieu of the more expensive drugs . in the united states alone , over the past 10 years , the combined medicare cost savings from the use of oct and bevacizumab have exceeded $ 20 billion when conservatively extrapolated from published utilization data for all forms of exudative eye diseases . despite differences in molecular size , affinity for vegf , and intravitreal half - life , the most likely explanation for the effectiveness of these four drugs is that they bind all the biological isoforms of vegf - a whereas pegaptanib inhibited only vegf165 and larger isoforms . treatments that combine intravitreal anti - vegf therapy along with inhibitors of platelet - derived growth factor ( pdgf ) , angiopoietin 2 , and tissue factor are being investigated , and topical squalamine is being combined with anti - vegf therapy in clinical trials as well . unlike dye - based angiography , oct imaging can identify macular abnormalities involving the photoreceptors , the rpe , and the choroid that can help explain visual function changes such as reduced reading speed , reduced microperimetric thresholds , and delayed dark adaptation . while oct is clearly the gold standard imaging strategy for predicting the success of any antiangiogenic treatment , it is also a valuable strategy for studying a wide range of anatomic changes that correlate with reduced visual function and disease progression .

major contrasts in the climatic preferences of grass subfamilies have been noted for more than half a century ( hartley 1950 ) , but their significance was only recognized following the discovery of c4 photosynthesis . tropical and subtropical grasslands are dominated by the predominantly c4 panicoideae and chloridoideae ( hartley 1958a , b ; hartley & slater 1960 ) , which together account for more than half of the world 's grass species ( fig . 1 ; linder & rudall 2005 ) and 20% of gross terrestrial carbon fixation ( lloyd & farquhar 1994 ) . their carbon - concentrating mechanism suppresses the energetically wasteful process of photorespiration that plagues c3 grasses at high temperatures , and significantly raises the efficiency of photosynthesis in warm climate regions . in contrast , c3 grass subfamilies such as the pooideae are largely confined to temperate climates , where photorespiration is naturally limited by lower temperatures ( hartley 1961 , 1973 ) . cladogram displaying the hypothesized relationships among subfamilies of the poaceae based on multiple markers , with the number of species shown for each , and filled circles ( ) indicating the unequivocal origins of c4 photosynthesis in independent clades ( watson & dallwitz 1992 ; gpwg 2001 ; linder & rudall 2005 ; snchez - ken et al . 2007 ) . the evolutionary history of these patterns was elucidated only in the past two decades , following the realization that c4 photosynthesis imparts a distinctive carbon isotope signature to plant materials and trophic pathways . by analysing the isotopic composition of fossilized soils ( palaeosols ) and the teeth of herbivores , geochemists uncovered a surprise ; the domination of low - latitude ecosystems by c4 grasses is a recent phenomenon in geological terms , occurring only 48 myr ago ( ma ) at the miocene pliocene boundary , when c4 grasslands expanded across at least four continents ( fig . examples of the shifts in stable carbon isotope ratio ( c ) characterizing the miocene rise of c4 plants in ( a ) pakistan ( quade & cerling 1995 ) , ( b ) the great plains ( fox & koch 2003 ) and ( c ) east africa ( cerling et al . 1997 ) . values for ( a ) and ( b ) were obtained from palaeosol carbonates , and the proportion of biomass contributed by c4 plants calculated following fox & koch ( 2003 ) . values for ( c ) are from the tooth enamel of mammalian herbivores , with the proportion of c4 plants in their diets after cerling et al . ( 1997 ) . at first , debate focused on the relative merits of co2 as a driver of c4 grassland expansion ( e.g. cerling et al . 1994 ; morgan et al . the case for co2 was persuasive , and based on the premise that selective and competitive advantages of c4 photosynthesis result from the energetic benefits of eliminating photorespiration at high temperatures and low atmospheric co2 ( ehleringer et al . these advantages over the ancestral c3 condition are reversed when photorespiration is naturally suppressed by low temperatures or high co2 , because the c4 carbon - concentrating mechanism requires energy . this leads to critical thresholds of temperature ( around 2025 c at today 's atmospheric co2 concentration ) and co2 ( around 500 p.p.m . in tropical environments ) where c3 and c4 photosynthesis have equal energy requirements and , by extension , equal competitive and selective advantages ( cerling et al . the proponents of this elegant hypothesis noted the close correspondence between the theoretical temperature threshold and mean growing season value in modern regions of equal c3 and c4 grass species richness , and postulated that declining co2 crossed an equivalent threshold at the miocene pliocene boundary ( ehleringer et al . 1991 , 1997 ; cerling et al . 1997 ) . their ideas were supported by a geochemical model of atmospheric co2 , which indicated the necessary decline during the cenozoic ( berner 1998 ) . by the late 1990s , the ideas underpinning the co2 starvation hypothesis were widely accepted , but still awaited the crucial test provided by palaeo - co2 reconstructions . this soon came with the publication of three data sets ( pagani et al . 1999 ; pearson & palmer 2000 ; royer et al . 2001 ) , each using an independent proxy for co2 , and each showing a long period of stasis in the level of atmospheric co2 during the expansion of c4 grasslands ( fig . unless these palaeo - co2 proxy records are challenged on technical or theoretical grounds , the geological evidence therefore stands firmly against the co2 starvation hypothesis for c4 grassland expansion , and new mechanisms must be sought . instead , the latest evidence suggests that atmospheric co2 dropped sharply through the c3c4crossover threshold at 2530 ma during the oligocene , when it initiated our modern icehouse era of advancing and retreating polar ice sheets ( fig . the emerging picture of palaeoenvironmental change therefore lends credence to an alternative hypothesis , proposing declining co2 concentration as a key selection pressure for the evolutionary origins of c4 photosynthesis in the grasses , rather than c4 grassland expansion ( ehleringer et al . ( a ) oxygen isotope values of deep - sea foraminifera ( o ) displayed as a locally weighted running mean calculated by zachos et al . this serves as a combined proxy for the global deep - sea temperature and continental ice volume , with increases in o indicating cooling and ice growth . the geological timescale shows ages in myr ago ( ma ) ; plio , pliocene ; plt , pleistocene . ( b ) co2 proxy data based on carbon isotope ratios of marine phytoplankton , stomatal densities of fossil leaves and the boron isotope ratios of planktonic foraminifera compiled by royer ( 2006 ) . palaeosol proxy data are not shown , because the uncertainty in this technique ( 500 p.p.m . ) here , i review two major issues currently facing geologists , physiological ecologists and ecosystem scientists with interests in this field . i first evaluate the proposed role of co2 in the evolutionary origins of c4 photosynthesis , focusing on the question of when the pathway first arose in the grasses . however , my principal focus is on the current debate surrounding the causes of c4 grassland expansion , particularly the hypothesized effects of climate change and fire . i argue that a deeper understanding of these proposed abiotic drivers could be achieved by explicitly considering their contrasting interactions within independent groups of c4 grasses . a crucial test of the co2 hypothesis for c4 grass origins is to establish whether the first appearance of the pathway coincides with the oligocene drop in atmospheric co2 . using a molecular clock , gaut & doebley ( 1997 ) calculated that divergence of two major c4 grass tribes , the paniceae and andropogoneae ( fig . 1 ) , occurred 2532 ma , leading later authors to argue that origins of c4 photosynthesis must therefore be at least this old ( e.g. kellogg 1999 ) . however , subsequent phylogenetic analysis cast doubt on this idea , suggesting that c4 origins may have post - dated the divergence event , evolving in up to eight independent subgroups of the paniceae and andropogoneae ( giussani et al . 2001 ; aliscioni et al . 2003 ; sage 2004 ) . dating the nodes of the grass phylogenetic tree ( fig . 1 ) has proved difficult because it is becoming increasingly clear that variation in the mutation rate among branches generates large errors in molecular clocks ( pulquiro & nichols 2007 ) . attempts at calibration using the fossil record have yielded dates that vary hugely because of the low sample sizes used ( linder & rudall 2005 ) . more precise dating of evolutionary events within the grass family therefore depends crucially on a better resolved fossil record . fossils only provide minimum dates for nodes of the phylogenetic tree because the geological record is incomplete . this problem is particularly acute for grasses of open habitats , where the potential for fossilization is extremely low ( cerling 1999 ) . the oldest c4 grass macrofossils date to only 12.5 ma in the middle miocene ( reviewed by osborne & beerling 2006 ) . however , the pollen record for grasses overall stretches back more than 65 myr to the late cretaceous ( jacobs et al . grass pollen can not be identified below the family level , but phytolith traits map onto the grass phylogeny at the subfamilial scale ( piperno & sues 2005 ; prasad et al . phytoliths form , to a varying degree , in all groups of living vascular plants ( piperno 2006 ) . however , grasses have a higher silica production than most other plants ( hodson et al . 2005 ) , and phytoliths in this family are more diagnostic than for most other plant groups . the interpretation of fossil phytolith assemblages is complicated by multiplicity ( each species produces more than one phytolith morphotype ) , and redundancy ( different species produce similar morphotypes ) ( piperno 1988 ) . nevertheless , the comparison of fossil assemblages with modern reference collections has allowed robust statistical inferences about the phylogenetic affinities and ecology of extinct grass communities ( strmberg 2004 , 2005 ) . phytolith analysis has been used to identify grasses with affinities to the paccmad crown group ( fig . 1 ) from 65- to 67-myr - old dinosaur dung in india ( prasad et al . 2005 ) , and from the predominantly c4 subfamily chloridoideae ( fig . although this technique can not establish when c4 photosynthesis originated in each clade , complementary methods have been developed for analysing the carbon isotopic signature of phytolith assemblages ( smith & white 2004 ) . interpretation of these data is difficult because of interspecific variation and differences in the biochemical nature of carbon compounds between c3 and c4 grass phytoliths ( reviewed by smith & white 2004 ) . however , preliminary data suggest that up to 50% of great plains grasses may have used the c4 pathway by 12 ma ( smith 2001 ) . geochemical analyses of herbivore teeth , palaeosols and the molecular markers of plant cuticles trace the c4 carbon isotope signal back to 1618 ma in the early miocene ( reviewed in tipple & pagani 2007 ) . these data are consistent with the presence of c4 grasses comprising up to 30% of the total biomass in tropical and subtropical ecosystems throughout the miocene ( fox & koch 2003 ; pagani et al . . however , the use of bulk isotope analyses to estimate c4 biomass in extinct plant communities is not a precise science . critical uncertainties remain about the isotopic composition of atmospheric co2through geological time , variation in the background signal caused by the water relationships of c3 plants and the taxonomic identity of the c4 plants ( tipple & pagani 2007 ) . partial resolution of these issues may come from a new analytical technique for measuring the carbon isotope composition of tiny samples , which currently allows c3 or c4 pathways to be identified from individual pollen grains with > 85% reliability ( nelson et al . this emerging method should enable researchers to establish directly the photosynthetic pathway of parent plants using grass pollen recovered from sediments dating to the early miocene and oligocene . unlike analyses of palaeosol or tooth carbon , this technique does not require abundant c4 plant biomass to resolve a c4 signal from the c3 background and offers the potential for identifying rare c4 plants in a predominantly c3 community , in addition to allowing identification of taxa to at least the family level . pure c4 samples , the technique is not compromised by variations in the isotopic signature of atmospheric co2 . pollen - based evidence may therefore bridge the gap between postulated c4 origins and our oldest current evidence for c4 plants . however , questions will still remain over the precise taxonomic identity of these plants , and pinning down the earliest c4 grasses is only part of the challenge confronting geologists in this field . plummeting co2 concentrations were correlated with a whole suite of climatic changes during the oligocene , including falling temperature and increasing aridity ( fig . 3 ; zachos et al . 2001 ; dupont - nivet et al . 2007 ) , and may not have been the only selection pressure for c4 photosynthesis . geological evidence therefore raises two linked questions . if co2 was not the driving force , what caused the miocene pliocene expansion of c4 grasslands ? and , if c4 grasses were present from the early miocene onwards , why did they not dominate ecosystems earlier in their evolutionary history ? the key to answering both of these questions may lie in fossil evidence that documents an abundance of woody c3 plants throughout the miocene , forming forests , woodlands or savannas in regions that subsequently became c4 grasslands ( reviewed by osborne & beerling 2006 ) . woody plant cover exerts a major limitation on c4 grass abundance , because trees and shrubs rapidly overtop herbaceous plants in the absence of disturbance , and most c4 species are intolerant of shading ( sage et al . explanations of c4 grassland expansion in the geological record therefore evoke a combination of climatic and disturbance factors that reduce tree cover , focusing primarily on changes in rainfall patterns and fire regime . the first is supported by the oxygen isotope signature of palaeosols in south asia , and proposes that the miocene replacement of c3 woody vegetation by c4 grasslands was driven by a strong increase in rainfall seasonality , caused by abrupt intensification of monsoon systems ( table 1 ; reviewed by osborne & beerling 2006 ) . a second hypothesis based on oxygen isotope data from freshwater bivalve shells in nepal suggests a decrease in the total amount of rainfall with no significant change in seasonality ( dettman et al . 2001 ) , and a similar mechanism is evoked for east africa , rooted in model simulations for the region ( table 1 ; sepulchre et al . both of these climatic drying hypotheses propose that an increased frequency and intensity of drought events killed trees and allowed the incursion of c4 grasses , to produce more open savanna or grassland vegetation ( table 1 ) . both are linked ultimately to tectonic events , including episodes of mountain building such as the uplift of the tibetan plateau and east african rift system , or changes in ocean circulation triggered , for example , by closure of the seaway between the americas ( reviewed by osborne & beerling 2006 ) . alternative hypotheses are not mutually exclusive , and overlap to a significant extent . for example , a change in the fire climate could also be the ultimate reason for an ecotone shift ( keeley & rundel 2003 , 2005 ) . an alternative viewpoint has emerged from analyses of palaeosol structure , which use the soil horizon structure to infer a vegetation type of desert , grassland or woodland , and the depth of the calcic horizon to estimate rainfall amounts ( retallack 2001 ) . application of this approach to geological sediments suggests that spatial gradients in rainfall generated parallel regional variation in vegetation ( fig . ecosystems varied from desert grassland to dry woodland throughout the early and middle miocene in south asia , the central and western united states and east africa ( fig . 4 ; reviewed by retallack 2001 ) . palaeobotanical evidence from east africa also indicates savanna and woodland vegetation during the early and middle miocene ( jacobs et al . 1999 ) . however , the inference of dry climates is not supported by plant macrofossils , phytoliths and pollen from the great plains , which indicate productive savanna or woodland vegetation with a significant c3 grass component ( jacobs et al . 1999 ; strmberg 2004 , 2005 ) . the presence of palms , gingers and bamboos ( strmberg 2004 ) , woody dicots confined to moister climates today ( axelrod 1985 ) , and giant tortoises and alligators ( hutchinson 1982 ) suggests a relatively humid , rather than arid , climate . palaeosol data suggest a major change in the ecology of these ecosystems during the late miocene , with c4 grasses displacing woodland communities in mesic regions and shifting the grassland woodland ecotone to higher rainfall areas in north america , africa and asia ( table 1 , fig . the causes of this ecotone shift are unknown , but have been attributed to the coevolution of grasses and grazers ( retallack 2001 ) , and significant changes in fire regime resulting from shifts in the seasonal distribution , but not total amount , of rainfall ( keeley & rundel 2005 ) . precipitation estimates based on depth to the calcic horizon in palaeosols from ( a ) pakistan , ( b ) the central usa and ( c ) east africa ( retallack 2001 ) . the grey - shaded area denotes the total range of values obtained for all palaeosol types ( originating in deserts , grasslands , woodlands and forests ) , and the symbols show values obtained for mollic palaeosols , which develop only beneath sod - forming grasses ( retallack 2001 ) . these data suggest that grasslands invaded wetter areas from 8 myr ago ( ma ) . evidence supporting a linkage between fire and the expansion of c4 grasslands comes from black carbon ( bc ) abundance in marine sediments , a geological proxy for fire occurrence . bc increases by 100- to 1000-fold in pacific ocean localities downwind of the indian subcontinent ( herring 1985 ; keeley & rundel 2003 ) , and 5-fold in the south china sea ( jia et al . 2003 ) , during the miocene sediments off the atlantic coast of west africa also show significant increases in charred grass cuticle and pollen abundance during the same period ( morley & richards 1993 ) . in the last 5 years , a new and compelling hypothesis has therefore added ecosystem - scale feedbacks between fire and vegetation to previous ideas about palaeoclimate ( table 1 ; keeley & rundel 2003 , 2005 ) . fire sustains c4 grasslands by killing woody plants and entrains a positive feedback because the dead foliage of grasses provides abundant fuel for fires , thereby increasing tree mortality and promoting the further spread of grasses ( keeley & rundel 2003 , 2005 ) . the fire hypothesis therefore proposes that increasing climatic seasonality during the late miocene raised fire frequency by supporting rapid biomass production and the development of a high fuel load during wet summer conditions . intensifying winter drought promoted drying , and increased the likelihood that this material would ignite , with ignition itself provided by lightening strikes at the end of the dry season . further feedbacks on the fire regime have been proposed via the hydrological cycle ( beerling & osborne 2006 ) and wind strength ( tipple & pagani 2007 ) , but these remain speculative at present . fuel remains too moist to support frequent fires in wet , aseasonal environments , whilst the low productivity of dry climate regions produces too little fuel to carry significant fires ( keeley & rundel 2005 ) . however , given sufficient periods of dry weather , fire has the potential to displace mesic forests in favour of grasslands . this is demonstrated in modern ecosystems by complementary data from : model simulations showing that fire reduces forest cover by 50% at the global scale ( bond et al . 2005 ) ; analysis of woody plant cover across africa indicating that significant areas of savanna are maintained by disturbance in regions with sufficient rainfall to support forest ( sankaran et al . 2005 ) ; fire exclusion experiments which allow the establishment of fire - sensitive trees in mesic savanna ecosystems ( bond et al . 2003 , 2005 ) ; and the fire - mediated replacement of native forests on pacific islands by invasive grasses ( dantonio & vitousek 1992 ) . these observations each provide indirect support for the hypothesized mechanism of vegetation change at the miocene pliocene boundary . climate relationships and fire frequency to explain the miocene expansion of c4 grasslands ( table 1 ; keeley & rundel 2005 ) . consensus scenario suggests that early and middle miocene landscapes were dominated by c3 forests or woodlands , with c4 grasses occupying open ground between the patches of woody plants . increasing seasonality during the late miocene concentrated rainfall into a hot growing season , creating a fire regime that removed woody vegetation and shifted the woodland grassland ecotone to wetter areas ( table 1 ) . formulated in this way , the hypothesis asserts that climates capable of supporting frequently burning mesic c4 grasslands at the expense of woodland and forests were absent during the early and middle miocene . however , application of the hypothesis to north and south america is problematic in two important respects . first , patterns of climatic change in these regions at the miocene pliocene boundary are less clear than those in asia , and an increase in rainfall seasonality has yet to be demonstrated ( passey et al . 2002 ; fox & koch 2004 ; osborne & beerling 2006 ) . secondly , pollen records , phytolith assemblages and mammalian tooth morphology suggest that a transition from closed forest to open woody vegetation with a significant c3 grass component may have occurred as early as 25 ma ( late oligocene to early miocene ) in the great plains ( strmberg 2004 , 2005 ) and 32 ma ( early oligocene ) in south america ( jacobs et al . 1999 ) . the adaptive radiation of specialist grazers ( strmberg 2006 ) and an extremely high browser diversity in these great plains ecosystems resulted in species - rich mammalian herbivore communities by 15 ma ( janis et al . carbon isotope data demonstrate that some grazers showed a dietary shift in the late miocene , subsisting almost exclusively on c4 plants for the 2 myr before c4 grassland expansion , with presumably significant , but currently unknown , effects on grass tree dynamics ( fox & koch 2004 ; beerling & osborne 2006 ) . i argue that our overall understanding and ability to test climatic and ecological hypotheses regarding miocene ecosystems would be improved by an explicit consideration of c4 grass phylogeny . to make this case , i first present biogeographical data indicating significant contrasts in the abundance of different c4 grass clades along rainfall gradients , and show how this information might be used in hypothesis testing . i re - analyse a published data set in the same context , suggesting that changes in the species richness of independent c4 grass lineages track the length of the rainy season . finally , i present intriguing preliminary evidence of fire controls on the abundance of particular c4 clades . the subfamilies panicoideae and chloridoideae together account for the majority of modern c4 grass species ( fig . 1 ; linder & rudall 2005 ) , but analyses of regional biodiversity patterns suggest a crucial difference in the climate relationships of these major clades . for the united states and argentina , the percentage of c4 species belonging to the panicoideae shows a positive correlation with annual rainfall , whereas the same relationship for the chloridoideae is negative ( fig . 5 ( a ) percentage of c4 grasses in us floras belonging to the panicoideae ( ) and the chloridoideae ) subfamilies ( taub 2000 ) . ( b ) percentage of c4 grasses in floras with the nadp - me ( ) and nad - me ( ) subtypes in the usa ( taub 2000 ) and ( c ) australia ( hattersley 1992 ) . because each flora covers a differing geographical area , values are expressed as a percentage to normalize for species area biases . ( a ) and ( b ) express the same data set on a different basis , excluding ( a ) members of the aristidoideae , and ( b ) species with the pck subtype of c4 photosynthesis . the mechanisms underpinning these relationships are unclear at present , but may be linked to the close association between biochemical subtypes of c4 photosynthesis and specific phylogenetic groups ; the majority of panicoideae species utilize the nadp - malic enzyme ( nadp - me ) pathway whereas most members of the chloridoideae use the nad - malic enzyme ( nad - me ) pathway ( taub 2000 ) . as a consequence , contrasting correlations with rainfall are also recognized for different c4 subtypes ( fig . experimental investigations suggest that these alternative forms of the pathway may be differentially adaptive in relation to water availability ( ghannoum et al . 2002 ) , or a correlated environmental variable such as soil nutrient status ( ghannoum et al . 2005 ) . however , none of these experiments have explicitly controlled for phylogeny , and the issue remains unresolved . further questions remain about how these species richness patterns translate into plant abundance along rainfall gradients . despite these uncertainties , the geographical distributions of modern c4 grasses indicate that changing rainfall patterns will drive significant shifts in the phylogenetic composition of c4 grass communities . pliocene expansion of c4 grasslands , because it allows predictions about the clades of c4 grasses that are involved . first , the range of middle miocene ecosystems along inferred rainfall gradients ( retallack 2001 ) should contain varying proportions of c4 grass subfamilies , following the qualitative patterns in fig . secondly , a shift in the woodland grassland ecotone to higher rainfall areas should increase the proportion of c4 species belonging to the panicoideae relative to the chloridoideae . finally , a decrease in the total amount of rainfall should drive the reverse pattern , favouring species of the chloridoideae relative to the panicoideae . these opposing predictions are especially critical for regions such as east africa and south asia , where alternative hypotheses are postulated . crucially , they can be tested by using emerging techniques for quantifying the relative abundance of different grass subfamilies within fossil phytolith assemblages ( e.g. prasad et al . conversely , the use of these techniques to reconstruct the phylogenetic make - up of miocene grassland communities in the great plains could offer vital clues about the environmental drivers of c4 grassland expansion in this region . finally , quantifying shifts in the abundance of different grass subfamilies could aid in the interpretation of carbon isotope signals . for example , a decrease in the proportion of chloridoideae ( nad - me ) relative to panicoideae ( nadp - me ) species would be expected to amplify the c4 signal , because discrimination against c is stronger in nad - me than nadp - me grasses ( hattersley 1982 ) , i.e. the nad - me type is slightly more c3-like. the power of these approaches would be increased greatly by a better understanding of how c4 grass distributions vary in relation to rainfall seasonality and fire frequency . previous studies suggest that species richness of the tribes andropogoneae and paniceae ( fig . 1 ; panicoideae ) may change significantly in response to the intensity and seasonality of rainfall . for southern africa , gibbs - russell ( 1988 ) used a qualitative comparison of floristic and climatic data to show that andropogoneae species account for the largest fraction of the panicoideae in monsoonal summer rainfall areas where mean annual precipitation ( map ) > 500 mm . the opposite pattern occurs in the paniceae , which account for the largest proportion of panicoideae species in more arid summer - rainfall regions ( map < 500 mm ) . hartley ( 1950 , 1958a , b ) carried out a similar analysis at the global scale , but expressed species numbers in each tribe as a fraction of the total grass flora , rather than restricting the analysis to the subfamily panicoideae . based on qualitative descriptions of maps , hartley hypothesized that centres of the highest species diversity in the andropogoneae are typically located in subtropical monsoon climates with a short rainy season ( hartley 1958a ) , whereas the highest diversity in the paniceae occurs in less seasonal wet equatorial climates ( hartley 1958b ) . to evaluate this hypothesis further , i have carried out a quantitative analysis of the same species richness data sets ( hartley 1950 , 1958a ) by using climate data recorded at nearby meteorological stations ( mller 1982 ) . a series of generalized linear models ( glims ) were fitted to the data , using a quasibinomial distribution in place of the binomial distribution when data were underdispersed [ glm ( ) , r version 2.4.1 , the r foundation for statistical computing ] . i used a forward stepwise approach , testing first the simple additive hypotheses posed in the literature , and then adding more variables and interactions . numerous previous studies have demonstrated that temperature is the primary global control on species richness for c4 grasses ( reviewed by sage et al . 1999 ) , and the andropogoneae and paniceae tribes followed this general pattern ( table 2a , b ) . glims for eurasia africa and the americas showed highly significant additive effects of mean annual temperature ( mat ) and tribe on relative species richness ( fig . relative diversity was lower in the andropogoneae than the paniceae ( table 2a , b ) , and this difference was more pronounced in the americas than eurasia and africa ( fig . generalized linear models ( glims ) explaining the global variation in relative species richness within the grass subfamily panicoideae . the models consider the interacting effects of mean annual temperature ( mat ) , rainy season length ( rsl ) and tribe ( andropogoneae or paniceae ) . global variation in relative species richness within the grass subfamily panicoideae , showing species abundance within the tribes andropogoneae and paniceae as a proportion of all grass species in a flora ( % ) , relative to climatic variables . the upper panels show variation due to mean annual temperature for ( a ) eurasia and africa and ( b ) north and south america , and the lower panels show variation due to rainy season length ( rsl ) in eurasia and africa for ( c ) the andropogoneae and ( d ) the paniceae , with lines indicating mean monthly values . i then added rainy season length ( rsl ) to the model , restricting the analysis to tropical and subtropical localities ( latitude < 35n or s ) . because the latitude criterion excluded most of the data from the americas , this analysis focused on eurasia and africa . rsl was estimated for each locality using a biologically based definition of the growing season for c4 grasses ; as the number of consecutive months in which the mean minimum temperature > 10 c and total precipitation > 30 mm ( collatz et al . the temperature threshold for growth was based upon two lines of complementary evidence : ( i ) experimental observations of chilling - mediated photoinhibition and impairment of leaf extension in c4 grasses ( long 1983 ) ; and ( ii ) the minimum summer temperature required for c4 species to persist in a grass flora ( sage et al . these criteria defined a reasonable ( non - zero ) rainy season for all localities except those in desert regions ( e.g. syria and yemen ) , where plants are probably associated with bodies of water or infrequent rains , or at high elevation ( e.g. lesotho ) , where c4 species may show an unusual resistance to low temperature extremes ( mrquez et al . the minimum adequate glim showed highly significant additive effects of mat , rsl and tribe , but not the hypothesized interaction between mat and rsl ( table 2c ) . species richness increased with rsl in both tribes , but showed a differing nonlinear response : values for the andropogoneae were low when 0 < rsl 3 months , and high when 4 rsl 12 months ( fig . 6c ) ; in contrast , values for the paniceae increased in an approximately linear response to reach a maximum at rsl 9 months ( fig . i therefore constructed a new glim attempting to account for these apparent nonlinearities by grouping rsl data into three temporal categories : 13 , 47 and 912 months ( there were no localities with rsl = 8 months ) . this model showed an additive effect of mat , and an interaction between rsl and tribe ( table 2d ) . the interaction was caused by equal species richness in climates with a rainy season of 7 months or less , and greater species richness in the panicoideae than andropogoneae in aseasonal climates with rainy seasons of 9 months or more ( fig . interaction between species richness in the andropogoneae and paniceae tribes and rainy season length ( rsl ) in eurasia and africa . species abundance within each tribe is expressed as a proportion of all grass species in a local flora ( % ) , and shown as the mean se for each rsl category . this quantitative analysis supports the hypothesis that species richness in the paniceae is highest in moist , aseasonal climates of the subtropics and tropics , but fails to show a similar association between maximal andropogoneae diversity and a short rainy season . instead , species richness in the andropogoneae remains constant across localities with rainy seasons of 412 months ( figs 6c and 7 ) . the analysis therefore highlights a phylogenetic contrast between the tribes that could prove useful for hypothesis testing in the geological record : a climatic change from moist , aseasonal conditions to a strongly seasonal climate is expected to drive a shift in the grass flora from a predominance of paniceae species towards a more equal representation of the paniceae and andropogoneae . further work is required before this expectation can be tested in the fossil record , because techniques for analysing phytolith assemblages can not yet distinguish tribes of the panicoideae . however , archaeologists already use phytolith - based diagnostics for identifying individual crop species of the andropogoneae ( piperno 2006 ) , indicating good promise for increasing the taxonomic resolution of this technique . the value of these observed patterns of species richness would be increased greatly by an understanding of the underlying mechanisms . ( 2003 ) , who hypothesize that the andropogoneae are key to the fire - mediated displacement of forests by grasslands in mesic climates of southern africa . these authors note the dominance of this group in fire - maintained and nutrient - poor mesic savannas , the obligate dependence of andropogoneae species such as themeda triandra on frequent defoliation , and their decline in the absence of fires . frequent fires are promoted by the rapid growth of andropogoneae species during summer , the accumulation of tannin - like compounds in their foliage , and the low nutritional quality of leaves during winter ( reviewed by bond et al . these latter traits retard decomposition and result in a low palatability to grazers during the dry season , leading to the build up of a high fuel load for fires . the evolution of such fire - promoting traits in one or more clades of c4 grasses during the miocene therefore provides a possible explanation for how the grassland woodland ecotone shifted to more mesic areas . but what might have selected for these characters ? one idea is suggested by the miocene radiation of grazers , and the specialization of some animals on a c4 diet prior to grassland expansion . recent studies indicate that modern grasses have evolved a suite of characteristics to resist grazing , including the accumulation of phenolic compounds ( e.g. tannins ) , a high silica content , and tough fibrous leaves with a low palatability and nutritional quality ( burt - smith et al . ecological theory proposes that selection for these traits is strongest in nutrient - poor habitats ( grime 2001 ) , and empirical evidence shows that grazer - driven selection for unpalatable species occurs in dry ecosystems with a long history of herbivory ( daz et al . speculative links may therefore be drawn between high grazing pressure in nutrient - poor savannas during the miocene , and selection for grazing - resistant ( but fire - promoting ) traits in c4 grasses . as these traits may also occur in c3 grasses ( grigulis et al . 2005 ) , this hypothesis provides an ecological explanation for miocene c4 grassland expansion that is not directly linked to photosynthetic pathway . the origin of c4 photosynthesis and expansion of c4 grasslands were major events in earth history with significant consequences for tropical and subtropical ecology . newly uncovered geological evidence has bolstered support for decreasing co2 as a selection agent for the pathway , leading to the expectation that c4 grasses first evolved during the oligocene . however , these same data cast serious doubt on the role of co2 in c4 grassland expansion . instead , current ecological understanding of vegetation fire dynamics in savannas has generated hypotheses integrating the roles of climate change and fire . i propose that the contrasting modern diversity patterns shown by independent c4 grass clades along climatic gradients generate a further expectation : that palaeoclimate change forced significant shifts in the phylogenetic composition of miocene grass communities . new techniques for the geochemical analysis of pollen and the extraction of phylogenetic information from phytolith assemblages provide the necessary tools for testing these ideas , and will help to bring the evolutionary history of c4 grasses into sharper focus .

grasses using the c4 photosynthetic pathway dominate today 's savanna ecosystems and account for 20% of terrestrial carbon fixation . however , this dominant status was reached only recently , during a period of c4 grassland expansion in the late miocene and early pliocene ( 48 myr ago ) . declining atmospheric co2 has long been considered the key driver of this event , but new geological evidence casts doubt on the idea , forcing a reconsideration of the environmental cues for c4 plant success.here , i evaluate the current hypotheses and debate in this field , beginning with a discussion of the role of co2 in the evolutionary origins , rather than expansion , of c4 grasses . atmospheric co2 starvation is a plausible selection agent for the c4 pathway , but a time gap of around 10 myr remains between major decreases in co2 during the oligocene , and the earliest current evidence of c4 plants.an emerging ecological perspective explains the miocene expansion of c4 grasslands via changes in climatic seasonality and the occurrence of fire . however , the climatic drivers of this event are debated and may vary among geographical regions.uncertainty in these areas could be reduced significantly by new directions in ecological research , especially the discovery that grass species richness along rainfall gradients shows contrasting patterns in different c4 clades . by re - evaluating a published data set , i show that increasing seasonality of rainfall is linked to changes in the relative abundance of the major c4 grass clades paniceae and andropogoneae . i propose that the explicit inclusion of these ecological patterns would significantly strengthen climate change hypotheses of miocene c4 grassland expansion . critically , they allow a new series of testable predictions to be made about the fossil record.synthesis . this paper offers a novel framework for integrating modern ecological patterns into theories about the geological history of c4 plants .

Carbon dioxide and the expansion of C Dating the origins of C Hypothesized drivers of C Contrasting climate relationships of C Re-evaluating the role of seasonal rainfall Conclusions

major contrasts in the climatic preferences of grass subfamilies have been noted for more than half a century ( hartley 1950 ) , but their significance was only recognized following the discovery of c4 photosynthesis . 1 ; linder & rudall 2005 ) and 20% of gross terrestrial carbon fixation ( lloyd & farquhar 1994 ) . the evolutionary history of these patterns was elucidated only in the past two decades , following the realization that c4 photosynthesis imparts a distinctive carbon isotope signature to plant materials and trophic pathways . by analysing the isotopic composition of fossilized soils ( palaeosols ) and the teeth of herbivores , geochemists uncovered a surprise ; the domination of low - latitude ecosystems by c4 grasses is a recent phenomenon in geological terms , occurring only 48 myr ago ( ma ) at the miocene pliocene boundary , when c4 grasslands expanded across at least four continents ( fig . examples of the shifts in stable carbon isotope ratio ( c ) characterizing the miocene rise of c4 plants in ( a ) pakistan ( quade & cerling 1995 ) , ( b ) the great plains ( fox & koch 2003 ) and ( c ) east africa ( cerling et al . at first , debate focused on the relative merits of co2 as a driver of c4 grassland expansion ( e.g. the case for co2 was persuasive , and based on the premise that selective and competitive advantages of c4 photosynthesis result from the energetic benefits of eliminating photorespiration at high temperatures and low atmospheric co2 ( ehleringer et al . the proponents of this elegant hypothesis noted the close correspondence between the theoretical temperature threshold and mean growing season value in modern regions of equal c3 and c4 grass species richness , and postulated that declining co2 crossed an equivalent threshold at the miocene pliocene boundary ( ehleringer et al . by the late 1990s , the ideas underpinning the co2 starvation hypothesis were widely accepted , but still awaited the crucial test provided by palaeo - co2 reconstructions . 2001 ) , each using an independent proxy for co2 , and each showing a long period of stasis in the level of atmospheric co2 during the expansion of c4 grasslands ( fig . unless these palaeo - co2 proxy records are challenged on technical or theoretical grounds , the geological evidence therefore stands firmly against the co2 starvation hypothesis for c4 grassland expansion , and new mechanisms must be sought . instead , the latest evidence suggests that atmospheric co2 dropped sharply through the c3c4crossover threshold at 2530 ma during the oligocene , when it initiated our modern icehouse era of advancing and retreating polar ice sheets ( fig . the emerging picture of palaeoenvironmental change therefore lends credence to an alternative hypothesis , proposing declining co2 concentration as a key selection pressure for the evolutionary origins of c4 photosynthesis in the grasses , rather than c4 grassland expansion ( ehleringer et al . here , i review two major issues currently facing geologists , physiological ecologists and ecosystem scientists with interests in this field . i first evaluate the proposed role of co2 in the evolutionary origins of c4 photosynthesis , focusing on the question of when the pathway first arose in the grasses . however , my principal focus is on the current debate surrounding the causes of c4 grassland expansion , particularly the hypothesized effects of climate change and fire . i argue that a deeper understanding of these proposed abiotic drivers could be achieved by explicitly considering their contrasting interactions within independent groups of c4 grasses . a crucial test of the co2 hypothesis for c4 grass origins is to establish whether the first appearance of the pathway coincides with the oligocene drop in atmospheric co2 . using a molecular clock , gaut & doebley ( 1997 ) calculated that divergence of two major c4 grass tribes , the paniceae and andropogoneae ( fig . however , subsequent phylogenetic analysis cast doubt on this idea , suggesting that c4 origins may have post - dated the divergence event , evolving in up to eight independent subgroups of the paniceae and andropogoneae ( giussani et al . attempts at calibration using the fossil record have yielded dates that vary hugely because of the low sample sizes used ( linder & rudall 2005 ) . however , the pollen record for grasses overall stretches back more than 65 myr to the late cretaceous ( jacobs et al . interpretation of these data is difficult because of interspecific variation and differences in the biochemical nature of carbon compounds between c3 and c4 grass phytoliths ( reviewed by smith & white 2004 ) . however , preliminary data suggest that up to 50% of great plains grasses may have used the c4 pathway by 12 ma ( smith 2001 ) . geochemical analyses of herbivore teeth , palaeosols and the molecular markers of plant cuticles trace the c4 carbon isotope signal back to 1618 ma in the early miocene ( reviewed in tipple & pagani 2007 ) . these data are consistent with the presence of c4 grasses comprising up to 30% of the total biomass in tropical and subtropical ecosystems throughout the miocene ( fox & koch 2003 ; pagani et al . critical uncertainties remain about the isotopic composition of atmospheric co2through geological time , variation in the background signal caused by the water relationships of c3 plants and the taxonomic identity of the c4 plants ( tipple & pagani 2007 ) . partial resolution of these issues may come from a new analytical technique for measuring the carbon isotope composition of tiny samples , which currently allows c3 or c4 pathways to be identified from individual pollen grains with > 85% reliability ( nelson et al . unlike analyses of palaeosol or tooth carbon , this technique does not require abundant c4 plant biomass to resolve a c4 signal from the c3 background and offers the potential for identifying rare c4 plants in a predominantly c3 community , in addition to allowing identification of taxa to at least the family level . pure c4 samples , the technique is not compromised by variations in the isotopic signature of atmospheric co2 . pollen - based evidence may therefore bridge the gap between postulated c4 origins and our oldest current evidence for c4 plants . however , questions will still remain over the precise taxonomic identity of these plants , and pinning down the earliest c4 grasses is only part of the challenge confronting geologists in this field . plummeting co2 concentrations were correlated with a whole suite of climatic changes during the oligocene , including falling temperature and increasing aridity ( fig . 2007 ) , and may not have been the only selection pressure for c4 photosynthesis . if co2 was not the driving force , what caused the miocene pliocene expansion of c4 grasslands ? the key to answering both of these questions may lie in fossil evidence that documents an abundance of woody c3 plants throughout the miocene , forming forests , woodlands or savannas in regions that subsequently became c4 grasslands ( reviewed by osborne & beerling 2006 ) . woody plant cover exerts a major limitation on c4 grass abundance , because trees and shrubs rapidly overtop herbaceous plants in the absence of disturbance , and most c4 species are intolerant of shading ( sage et al . explanations of c4 grassland expansion in the geological record therefore evoke a combination of climatic and disturbance factors that reduce tree cover , focusing primarily on changes in rainfall patterns and fire regime . the first is supported by the oxygen isotope signature of palaeosols in south asia , and proposes that the miocene replacement of c3 woody vegetation by c4 grasslands was driven by a strong increase in rainfall seasonality , caused by abrupt intensification of monsoon systems ( table 1 ; reviewed by osborne & beerling 2006 ) . both of these climatic drying hypotheses propose that an increased frequency and intensity of drought events killed trees and allowed the incursion of c4 grasses , to produce more open savanna or grassland vegetation ( table 1 ) . however , the inference of dry climates is not supported by plant macrofossils , phytoliths and pollen from the great plains , which indicate productive savanna or woodland vegetation with a significant c3 grass component ( jacobs et al . the presence of palms , gingers and bamboos ( strmberg 2004 ) , woody dicots confined to moister climates today ( axelrod 1985 ) , and giant tortoises and alligators ( hutchinson 1982 ) suggests a relatively humid , rather than arid , climate . palaeosol data suggest a major change in the ecology of these ecosystems during the late miocene , with c4 grasses displacing woodland communities in mesic regions and shifting the grassland woodland ecotone to higher rainfall areas in north america , africa and asia ( table 1 , fig . the causes of this ecotone shift are unknown , but have been attributed to the coevolution of grasses and grazers ( retallack 2001 ) , and significant changes in fire regime resulting from shifts in the seasonal distribution , but not total amount , of rainfall ( keeley & rundel 2005 ) . evidence supporting a linkage between fire and the expansion of c4 grasslands comes from black carbon ( bc ) abundance in marine sediments , a geological proxy for fire occurrence . bc increases by 100- to 1000-fold in pacific ocean localities downwind of the indian subcontinent ( herring 1985 ; keeley & rundel 2003 ) , and 5-fold in the south china sea ( jia et al . 2003 ) , during the miocene sediments off the atlantic coast of west africa also show significant increases in charred grass cuticle and pollen abundance during the same period ( morley & richards 1993 ) . the fire hypothesis therefore proposes that increasing climatic seasonality during the late miocene raised fire frequency by supporting rapid biomass production and the development of a high fuel load during wet summer conditions . climate relationships and fire frequency to explain the miocene expansion of c4 grasslands ( table 1 ; keeley & rundel 2005 ) . increasing seasonality during the late miocene concentrated rainfall into a hot growing season , creating a fire regime that removed woody vegetation and shifted the woodland grassland ecotone to wetter areas ( table 1 ) . formulated in this way , the hypothesis asserts that climates capable of supporting frequently burning mesic c4 grasslands at the expense of woodland and forests were absent during the early and middle miocene . first , patterns of climatic change in these regions at the miocene pliocene boundary are less clear than those in asia , and an increase in rainfall seasonality has yet to be demonstrated ( passey et al . carbon isotope data demonstrate that some grazers showed a dietary shift in the late miocene , subsisting almost exclusively on c4 plants for the 2 myr before c4 grassland expansion , with presumably significant , but currently unknown , effects on grass tree dynamics ( fox & koch 2004 ; beerling & osborne 2006 ) . to make this case , i first present biogeographical data indicating significant contrasts in the abundance of different c4 grass clades along rainfall gradients , and show how this information might be used in hypothesis testing . i re - analyse a published data set in the same context , suggesting that changes in the species richness of independent c4 grass lineages track the length of the rainy season . finally , i present intriguing preliminary evidence of fire controls on the abundance of particular c4 clades . the subfamilies panicoideae and chloridoideae together account for the majority of modern c4 grass species ( fig . for the united states and argentina , the percentage of c4 species belonging to the panicoideae shows a positive correlation with annual rainfall , whereas the same relationship for the chloridoideae is negative ( fig . ( b ) percentage of c4 grasses in floras with the nadp - me ( ) and nad - me ( ) subtypes in the usa ( taub 2000 ) and ( c ) australia ( hattersley 1992 ) . ( a ) and ( b ) express the same data set on a different basis , excluding ( a ) members of the aristidoideae , and ( b ) species with the pck subtype of c4 photosynthesis . the mechanisms underpinning these relationships are unclear at present , but may be linked to the close association between biochemical subtypes of c4 photosynthesis and specific phylogenetic groups ; the majority of panicoideae species utilize the nadp - malic enzyme ( nadp - me ) pathway whereas most members of the chloridoideae use the nad - malic enzyme ( nad - me ) pathway ( taub 2000 ) . however , none of these experiments have explicitly controlled for phylogeny , and the issue remains unresolved . further questions remain about how these species richness patterns translate into plant abundance along rainfall gradients . despite these uncertainties , the geographical distributions of modern c4 grasses indicate that changing rainfall patterns will drive significant shifts in the phylogenetic composition of c4 grass communities . pliocene expansion of c4 grasslands , because it allows predictions about the clades of c4 grasses that are involved . first , the range of middle miocene ecosystems along inferred rainfall gradients ( retallack 2001 ) should contain varying proportions of c4 grass subfamilies , following the qualitative patterns in fig . crucially , they can be tested by using emerging techniques for quantifying the relative abundance of different grass subfamilies within fossil phytolith assemblages ( e.g. conversely , the use of these techniques to reconstruct the phylogenetic make - up of miocene grassland communities in the great plains could offer vital clues about the environmental drivers of c4 grassland expansion in this region . for example , a decrease in the proportion of chloridoideae ( nad - me ) relative to panicoideae ( nadp - me ) species would be expected to amplify the c4 signal , because discrimination against c is stronger in nad - me than nadp - me grasses ( hattersley 1982 ) , i.e. the power of these approaches would be increased greatly by a better understanding of how c4 grass distributions vary in relation to rainfall seasonality and fire frequency . for southern africa , gibbs - russell ( 1988 ) used a qualitative comparison of floristic and climatic data to show that andropogoneae species account for the largest fraction of the panicoideae in monsoonal summer rainfall areas where mean annual precipitation ( map ) > 500 mm . the opposite pattern occurs in the paniceae , which account for the largest proportion of panicoideae species in more arid summer - rainfall regions ( map < 500 mm ) . hartley ( 1950 , 1958a , b ) carried out a similar analysis at the global scale , but expressed species numbers in each tribe as a fraction of the total grass flora , rather than restricting the analysis to the subfamily panicoideae . based on qualitative descriptions of maps , hartley hypothesized that centres of the highest species diversity in the andropogoneae are typically located in subtropical monsoon climates with a short rainy season ( hartley 1958a ) , whereas the highest diversity in the paniceae occurs in less seasonal wet equatorial climates ( hartley 1958b ) . to evaluate this hypothesis further , i have carried out a quantitative analysis of the same species richness data sets ( hartley 1950 , 1958a ) by using climate data recorded at nearby meteorological stations ( mller 1982 ) . a series of generalized linear models ( glims ) were fitted to the data , using a quasibinomial distribution in place of the binomial distribution when data were underdispersed [ glm ( ) , r version 2.4.1 , the r foundation for statistical computing ] . numerous previous studies have demonstrated that temperature is the primary global control on species richness for c4 grasses ( reviewed by sage et al . species richness increased with rsl in both tribes , but showed a differing nonlinear response : values for the andropogoneae were low when 0 < rsl 3 months , and high when 4 rsl 12 months ( fig . the interaction was caused by equal species richness in climates with a rainy season of 7 months or less , and greater species richness in the panicoideae than andropogoneae in aseasonal climates with rainy seasons of 9 months or more ( fig . this quantitative analysis supports the hypothesis that species richness in the paniceae is highest in moist , aseasonal climates of the subtropics and tropics , but fails to show a similar association between maximal andropogoneae diversity and a short rainy season . the analysis therefore highlights a phylogenetic contrast between the tribes that could prove useful for hypothesis testing in the geological record : a climatic change from moist , aseasonal conditions to a strongly seasonal climate is expected to drive a shift in the grass flora from a predominance of paniceae species towards a more equal representation of the paniceae and andropogoneae . further work is required before this expectation can be tested in the fossil record , because techniques for analysing phytolith assemblages can not yet distinguish tribes of the panicoideae . however , archaeologists already use phytolith - based diagnostics for identifying individual crop species of the andropogoneae ( piperno 2006 ) , indicating good promise for increasing the taxonomic resolution of this technique . the value of these observed patterns of species richness would be increased greatly by an understanding of the underlying mechanisms . these authors note the dominance of this group in fire - maintained and nutrient - poor mesic savannas , the obligate dependence of andropogoneae species such as themeda triandra on frequent defoliation , and their decline in the absence of fires . the evolution of such fire - promoting traits in one or more clades of c4 grasses during the miocene therefore provides a possible explanation for how the grassland woodland ecotone shifted to more mesic areas . one idea is suggested by the miocene radiation of grazers , and the specialization of some animals on a c4 diet prior to grassland expansion . speculative links may therefore be drawn between high grazing pressure in nutrient - poor savannas during the miocene , and selection for grazing - resistant ( but fire - promoting ) traits in c4 grasses . 2005 ) , this hypothesis provides an ecological explanation for miocene c4 grassland expansion that is not directly linked to photosynthetic pathway . the origin of c4 photosynthesis and expansion of c4 grasslands were major events in earth history with significant consequences for tropical and subtropical ecology . newly uncovered geological evidence has bolstered support for decreasing co2 as a selection agent for the pathway , leading to the expectation that c4 grasses first evolved during the oligocene . however , these same data cast serious doubt on the role of co2 in c4 grassland expansion . i propose that the contrasting modern diversity patterns shown by independent c4 grass clades along climatic gradients generate a further expectation : that palaeoclimate change forced significant shifts in the phylogenetic composition of miocene grass communities . new techniques for the geochemical analysis of pollen and the extraction of phylogenetic information from phytolith assemblages provide the necessary tools for testing these ideas , and will help to bring the evolutionary history of c4 grasses into sharper focus .

kareishu ( literally , distinctive body odor of the middle - aged or elderly ) , which is believed to frequently occur in men aged 40 years , is considered to be more common in modern society because of high animal fat - containing modern diets and high levels of stress . halitosis and body and fecal odor negatively affect peoples ' quality of life , and they are increasingly becoming a serious problem in caregiving settings in japan , with the rapidly increasing proportion of the elderly in the japanese society . to manufacture champignon extract , an extract boiled from the mushroom agaricus bisporus ( tsukuritake or champignon mushroom ) is mixed with dextrin and then spray - dried it into a powder . the extract contains amino acids , polyphenols , polysaccharides , flavonoids , vitamins , and minerals . currently , this extract has been patented and is available for sale in japan , six european countries , south korea , the united states , and canada . conditions such as halitosis and body and fecal odor are believed to be caused by certain toxic substances produced within the intestinal tract ; champignon extract directly inhibits their production . thus , champignon extract is widely used in candy , jellies , drinks , and other food products for maintaining both personal esthetics and health . furthermore , in an in vitro trial using gas chromatography to investigate its effects on the methyl mercaptan component of halitosis , champignon extract was found to increase the molecular weight of methyl mercaptan , thereby inhibiting its odor and causing overall decreased odor . studies regarding the function of champignon extract and the utility of its characteristic effects against methyl mercaptan are underway . in a clinical trial targeting elderly inpatients ( 70 subjects ; mean age : males 73.5 years , females 78.6 years ) and involving ingestion of 2 g of champignon extract daily for 30 days , the extract was shown to not affect gastrointestinal symptoms but improved stool characteristics , reduced fecal and body odor , and decreased blood ammonia content . in another trial involving 24 residents of an elderly care facility who ingested jelly candies containing 300 mg of champignon extract daily for 30 days , favorable effects such as improved stool characteristics and life satisfaction were observed . here we aimed to investigate whether daily champignon extract ingestion for 4 weeks improved halitosis and body and fecal odor . we included 80 men and women aged 5079 years with problematic halitosis and body and fecal odor in this placebo - controlled double - blind parallel - group comparative study . it involved four study groups : consisting of 3 test foods and a placebo , as shown in table 1 . ( 1 ) a placebo group ( n = 20 ) , ( 2 ) 50 mg / day champignon ingestion group ( n = 20 ) , ( 3 ) 500 mg / day champignon ingestion group ( n = 20 ) , and ( 4 ) 1000 mg / day champignon ingestion group ( n = 20 ) . with regard to ingestion volume and ingestion method , the test food was ingested as one packet ( 2 g ) per day . the champignon extract bx100fpd ( ricom corporation , tokyo , japan ) used for ingestion . each subject underwent a medical interview and blood test on the day of starting ingestion of the test food and after 2 and 4 weeks of ingestion . the week before starting ingestion was set as the pre - observation period ( washout period ) . after providing the subjects with written and oral explanations of the study contents in accordance with the declaration of helsinki , we obtained written consent to participate from the subjects . after initially screening , this study included 80 consenting subjects , who fulfilled the selection criteria and not the exclusion criteria , and were believed to be suitable to participate by the principal investigator . for group allocation , an allocation manager from a third - party data center ( media education center , hokkaido institute of information technology , ebetsu , hokkaido ) referred to the subject list and equally divided the subjects via stratified randomization into four groups considering age composition , male - to - female ratio , and odor questionnaire scores . the allocation manager carefully stored the allocation - related documents containing personal information of the subjects in a locked cabinet . subjects were then notified of the date , time , and place for the clinical trial . because three subjects quit the study owing to personal reasons before the trial started , subjects ingested one packet ( 2 g ) per day of test food or placebo for 4 weeks from the day of starting ingestion . subjects arrived at the assigned facilities on the day of starting ingestion and after 2 and 4 weeks of ingestion and underwent testing regarding prescribed items . the tests were conducted at the health center , hokkaido information university , ebetsu internal medicine clinic , takahashi internal medicine 3ban - dori clinic , and taguchi clinic ( all in ebetsu , hokkaido ) . subjects began recording information in their lifestyle journals ( daily condition , whether the test food was ingested , etc . ) and also documented their bowel movements 1 week before starting ingestion and then throughout the 4-week study period until the completion of stool sampling . the journals were submitted on each clinic day and every time feces were sampled . for disclosure ( display of allocation list ) , when all relevant test results and analysis data had been prepared , the allocation manager displayed the subject allocation list . the ingestion rate was calculated with the following formula : ingestion rate ( % ) = ( number of test foods actually ingested / number of test foods planned to be ingested ) 100 . all subjects provided written informed consent before undergoing any study - related tests , and the study protocol was approved by the ethics committee of hokkaido information university ( a certificate number ; 2014 - 04 ) . the study protocol conformed to the helsinki declaration and was registered at the umin clinical trial registration system ( certificate number umin000014256 ) . whitney u and chi - square tests were used for intergroup comparisons . for vas questionnaire , 100-mm lines were prepared for each item with the left and right edges indicating worst and best states , respectively . we evaluated how subjects felt about their own state at the time of the questionnaire by having them mark an the questionnaire results were scaled by measuring the length from the left edge to the x mark . subjects and cooperating people ( those living along with the subjects ) were asked to answer the questionnaire regarding halitosis and body and fecal odor using the prescribed method . subjects were instructed to bathe and brush their teeth the night before and sleep using a clean pillow cover ( or to cover the pillow with a towel ) while wearing freshly laundered pajamas . the cooperating person evaluated halitosis after standing one handbreadth away from the subject and speaking to the subject for 12 min . for body odor evaluation , the odor of the pillow cover ( or towel ) and pajamas was evaluated by the subject and cooperating person . for the questionnaire items regarding bowel movements , subjects evaluated the bowel movement status during the days between two clinic visits . however , if subjects could not record results at the time of awakening on the test day , they were allowed to include results from up to 2 days before the test day . we investigated the following four items : ( 1 ) fecal odor , ( 2 ) bowel movement regularity , ( 3 ) strain during bowel movements , and ( 4 ) sensation of residual stools . to evaluate fecal odor , the following characteristics regarding bowel movement were recorded on a daily basis : ( a ) bowel movement frequency , ( b ) bowel movement volume ( visual estimate of number of sample containers the stools would fill ) , ( c ) stool shape , ( d ) stool color , ( e ) stool odor , and ( f ) sensation of having completely evacuated after each bowel movement . the evaluation periods were the 1-week pre - observation period ( non - ingestion period ) and 4-week test food ingestion period . on days when multiple bowel movements were experienced , subjects recorded items from ( b ) through ( f ) for the first bowel movement only . in the lifestyle journal , subjects recorded meal content , physical condition , and whether they ingested the test food daily . blood samples were taken for testing on the starting day and after 4 weeks of ingestion . in addition to a medical interview by a doctor , height , weight , body mass index ( bmi ) , and percent body fat were measured . vital signs ( blood pressure upon arrival , pulse rate , body temperature ) were also taken . general blood tests included complete blood count ( wbc , rbc , hgb , hct , and platelets ) , liver function test ( ast , alt , -gtp , alp , and ldh ) , kidney function ( bun , cre , and ua ) , blood lipid profile ( total cholesterol , tg , ldl - cholesterol , and hdl - cholesterol ) , and blood sugar test ( blood sugar and hba1c ) . for the food frequency questionnaire , we used ffqg ver4 ( kenpakusha , tokyo , japan ) . we used a questionnaire composed of 29 food groups and 10 types based on food classifications from the national health and nutrition survey . with 1-week units , we estimated volume of intake by food group and nutrient intake based on portion size and food frequency . we calculated mean values and standard deviation for the intake volume of nutrients ( calories , protein , fat , carbohydrates , dietary fiber , and salt ) on the day of starting ingestion and after 4 weeks of ingestion . table 2 presents the male - to - female ratio , mean age , weight , bmi , and percent body fat of the four subject groups : placebo group ( n = 19 ) , 50 mg / day ingestion group ( n = 18 ) , 500 mg / day ingestion group ( n = 20 ) , and 1000 mg / day ingestion group ( n = 20 ) . for the continuous variables ( age , weight , bmi , and percent body fat ) we used fisher 's exact test to calculate the number and proportion of male subjects in each group for the categorical variable of sex . with regard to the analysis groups , no statistically significant differences were observed for ingestion rates between the placebo group and all ingestion groups ( table 3 ) . mean scores and standard deviation were calculated for each group for the results of the vas questionnaire regarding the seven items of halitosis , pillow odor , pajama odor , fecal odor , bowel movement regularity , strain during bowel movements , and sensation of residual stools . we then calculated the amount of change in mean scores and standard deviation for each group for each measurement time point ( evaluation time point ) from before starting ingestion ( table 4 ) . with regard to the amount of change , we also calculated frequency distribution after dividing these results into five levels ( < 20 , 2039 , 4059 , 6079 , and 80 changes expression in mm ) . the results for each of these seven items are shown below . the respective amount of change in vas questionnaire scores regarding halitosis from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 10.95 16.99 mm and 19.11 18.89 mm ; 50 mg / day group , 19.33 20.40 mm and 22.44 22.59 mm ; 500 mg / day group , 13.75 17.59 mm and 17.85 20.78 mm ; 1000 mg / day group , 20.00 19.22 mm and 29.32 25.56 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , halitosis significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.001 , p = 0.001 ) . after 2 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . after 4 weeks of ingestion , differences were observed between the 500 and 1000 mg / day ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding pillow odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 9.00 18.10 mm and 10.82 22.36 mm ; 50 mg / day group , 11.11 10.53 mm and 12.11 17.78 mm ; 500 mg / day group , 19.05 22.02 mm and 21.10 26.00 mm ; 1000 mg / day group , 12.20 16.47 mm and 17.50 22.99 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , pillow odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.004 , p = 0.003 ) . after 2 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . after 4 weeks of ingestion , differences were observed between the 50 and 500 mg / day ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding pajama odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.05 11.13 mm and 9.37 17.36 mm ; 50 mg / day group , 8.06 13.33 mm and 7.00 17.23 mm ; 500 mg / day group , 20.75 22.19 mm and 21.70 28.25 mm ; 1000 mg / day group , 12.15 17.33 mm and 15.00 20.48 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , pajama odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.005 , p = 0.004 ) . after 2 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . after 4 weeks of ingestion , differences were observed between the 500 and 1000 mg / day ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding fecal odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 12.95 19.35 mm and 21.63 23.03 mm ; 50 mg / day group , 11.22 15.34 mm and 15.56 19.57 mm ; 500 mg / day group , 19.26 14.30 mm and 27.10 21.27 mm ; 1000 mg / day group , 18.95 21.98 mm and 30.95 22.14 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , fecal odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.001 , p = 0.001 ) . after 2 and 4 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding bowel movement regularity from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 3.26 14.67 mm and 8.74 19.38 mm ; 50 mg / day group , 1.76 21.60 mm and 6.89 13.42 mm ; 500 mg / day group , 5.00 16.06 mm and 1.85 11.80 mm ; 1000 mg / day group , 8.22 21.51 mm and 8.82 30.52 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , no significant differences were observed between values at the day of starting ingestion and at each measurement time point . the respective amount of change in vas questionnaire scores regarding strain during bowel movements from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.79 30.44 mm and 0.16 17.05 mm ; 50 mg / day group , 6.11 32.04 mm and 11.61 16.99 mm ; 500 mg / day group , 0.70 13.19 mm and 3.70 11.62 mm ; 1000 mg / day group , 8.68 11.89 mm and 5.89 27.58 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , strain during bowel movements significantly decreased after 2 weeks of ingestion compared with that before starting ingestion ( p = 0.005 ) . the respective amount of change in vas questionnaire scores regarding sensation of residual stools from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group : 2.21 15.37 mm and 2.00 13.59 mm ; 50 mg / day group , 5.89 19.58 mm and 2.94 14.74 mm ; 500 mg / day group , 1.45 14.25 mm and 0.45 21.81 mm ; 1000 mg / day group , 6.74 13.01 mm and 2.78 24.18 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , no significant differences were observed between values at the day of starting ingestion and at each measurement time point . it comprised the four items of subject halitosis , pillow odor , pajama odor , and fecal odor ( odor of feces after subject used the toilet ) . we then calculated the amount of change in mean scores and standard deviation for each group for each measurement time point ( evaluation time point ) from before starting ingestion ( table 5 ) . with regard to the amount of change , we also calculated frequency distribution after dividing subjects into five levels ( < 20 , 2039 , 4059 , 6079 , and 80 years ) . the respective amount of change in vas questionnaire scores regarding halitosis from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 6.58 20.46 mm and 11.58 22.47 mm ; 50 mg / day group , 0.53 26.42 mm and 6.94 27.92 mm ; 500 mg / day group , 12.50 33.37 mm and 19.75 17.54 mm ; 1000 mg / day group , 11.05 27.97 mm and 21.53 29.79 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , halitosis significantly improved after 2 weeks of ingestion compared with that before starting ingestion ( p = 0.005 ) . after 2 weeks of ingestion , differences were also observed in the 50 and 500 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . the respective amount of change in vas questionnaire scores regarding pillow odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.95 14.05 mm and 8.63 20.35 mm ; 50 mg / day group , 10.81 22.81 mm and 14.63 26.08 mm ; 500 mg / day group , 15.45 21.85 mm and 17.50 22.81 mm ; 1000 mg / day group , 14.16 21.88 mm and 21.11 27.06 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , body odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.011 , p = 0.003 ) . after 2 weeks of ingestion , differences were also observed in the 500 and 1000 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . the respective amount of change in vas questionnaire scores regarding pajama odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.53 15.07 mm and 10.89 17.72 mm ; 50 mg / day group , 6.94 23.10 mm and 10.81 24.68 mm ; 500 mg / day group , 13.20 19.26 mm and 20.15 22.00 mm ; 1000 mg / day group , 13.89 23.25 mm and 20.05 30.24 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , pajama odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.018 , p = 0.010 ) . after 2 weeks of ingestion , differences were also observed in the 500 and 1000 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . the respective amount of change in vas questionnaire scores regarding fecal odor ( odor after using toilet ) from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 7.37 21.68 mm and 15.05 21.65 mm ; 50 mg / day group , 10.35 26.97 mm and 17.59 28.05 mm ; 500 mg / day group , 16.40 17.97 mm and 23.30 18.50 mm ; 1000 mg / day group , 16.11 27.49 mm and 25.58 24.49 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , fecal odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.021 , p = 0.001 ) . after 2 weeks of ingestion , differences were also observed only in the 1000 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . subjects in the three ingestion groups recorded in diaries information regarding bowel movement frequency , bowel movement regularity , bowel movement volume , stool shape , stool color , stool odor , and sensation of having completely evacuated after each bowel movement . when test multiplicity was adjusted and the level of significance for each test was set at 5/3 ( = 1.67% ) for the 50 mg / day group and placebo group , the proportion of responses indicating that stool color was brown , ocher , or yellow was significantly higher in the placebo group on the day of starting ingestion and after 4 weeks of ingestion ( p = 0.002 , p = 0.001 ) . no significant differences were noted for any evaluation items between the 500 mg / day group and placebo group . the following results clearly suggested the superiority of the test food when the 1000 mg / day group and placebo group were compared . first , when test multiplicity was adjusted and the level of significance for each test was set at 2.5% for bowel movement frequency , the amount of change in bowel movement frequency from before starting ingestion to after 2 and 4 weeks of ingestion was significantly higher in the ingestion groups ( p = 0.016 , p = 0.006 ) . when test multiplicity was adjusted and the level of significance for each test was set at 5/2 = 2.5% for bowel movement volume , the amount of change in bowel movement volume from before starting ingestion to after 4 weeks of ingestion was significantly higher in the ingestion group ( 1000 mg / day ) than in the placebo group ( p = 0.014 ) . when test multiplicity was adjusted and the level of significance for each test was set at 5/3 ( = 1.67% ) for stool color , the proportion of responses indicating brown , ocher , or yellow was significantly higher in the placebo group after 4 weeks of ingestion ( p = 0.001 ) . when test multiplicity was adjusted and the level of significance for each test was set at 5/3 ( = 1.67% ) for sensation of having completely evacuated after each bowel movement , the proportion of responses indicating smooth or pleasantly smooth was significantly higher in the all ingestion group after 2 weeks of ingestion ( p = 0.001 ) . we conducted a survey using the food frequency questionnaire to confirm that there was no bias related to food ingestion during the trial . no significant differences related to caloric intake , carbohydrates , protein , fat , dietary fiber , or salt were observed . we also did not observe any significant differences with regards to blood sugar or hba1c levels during fasting . champignon mushrooms , which have originated from europe and north america , are currently grown in over 70 countries worldwide . they are the most widely eaten of mushroom variety and are known to have many beneficial nutritional properties . first , they contain large amounts of vitamin d and ergosterol , which are important for bone metabolism . therefore , they are effective for preventing onset and improving symptoms of osteoporosis.3 , 4 they are also rich in minerals such as sodium , calcium , and phosphorus and contain linoleic acid and antioxidants , which have been associated with prevention of arteriosclerosis.5 , 6 the foaming components of champignon mushrooms show aromatase - inhibiting7 , 8 and anticarcinogenic9 , 10 effects and can activate natural killer cells , thus promoting the innate immune system . in vitro studies moreover , they show anti - inflammatory properties and have been suggested to inhibit inflammation and cancer cell proliferation through macrophages . notably , because they inhibit the development of fatty liver , champignon mushrooms can possibly prevent lifestyle - related diseases . in the present placebo - controlled double - blind parallel - group comparative study targeting 80 men and women aged 5079 years with problematic halitosis and body and fecal odor , the effects of 4-week daily ingestion of 50 , 500 , and 1000 mg / day champignon extract on the aforementioned conditions were investigated . we also conducted a questionnaire survey regarding bowel movements and found that champignon extract also exhibited bowel movement - related effects . moreover , no marked differences were observed between the placebo group and all ingestion groups with regard to vital signs , blood test findings , liver function , kidney function , lipid profile , blood glucose levels , or physical measurement during the ingestion of low , medium , or high doses of champignon extract ( data not shown ) . furthermore , because most findings were within the normal range , this trial was considered safe . the following conclusions were drawn from the results of the vas questionnaire regarding halitosis and body and fecal odor . for each of the champignon extract ingestion groups , improvement or improvement tendencies were observed for halitosis and body and fecal odor compared with the placebo group , even in case of cooperating people 's data . considering the bowel movement journals , for the 50 mg / day ingestion and placebo groups , the proportion of responses indicating that stool color was brown , ocher , or yellow was significantly higher in the placebo group after 2 and 4 weeks of ingestion . bowel movement volume significantly improved in the 1000 mg / day group after 4 weeks of ingestion . for sensation of having completely evacuated after each bowel movement , there was a significantly high proportion of responses indicating cleared out or very cleared out in the 1000 mg / day group after 2 weeks of ingestion . these results demonstrate that although no clear improvement was observed in bowel movements with 50 mg / day champignon extract ingestion , whereas they clearly improved with a 500 mg / day ingestion . in particular , results showed that bowel movement improvement could be anticipated by the ingestion of a high dose of champignon extract . our results strongly suggest that the ingestion of 501000 mg / day of our test food may improve halitosis and body and fecal odor . in particular , ingestion of the high dose of 1000 mg / day may result in reduction in the odor and favorable intestinal tract environment . conducting component analysis of champignon extract to identify the main components causing the deodorizing effects and determining the optimal dosage and other variables in detail is necessary . vas questionnaire regarding bowel movements also showed that ingestion of a high dose of champignon extract showed clear improvements . thus , efficacy in a wide range of areas , from body odor to bowel movements , can be anticipated . although the safety of champignon mushrooms has been highly reported , there have been no reports on their adverse or toxic effects in humans . kareishu appears with increasing age , but its incidence is increasing in japan as a result of the higher animal fat intake through increasingly westernized diets as well as increase social stress . halitosis and body and fecal odor not only decrease a person 's quality of life but also can become serious problems in caregiving settings in japan because the proportion of the elderly population in japanese society is rapidly increasing . in conclusion , champignon extract is efficacious against halitosis and body and fecal odor , and it can also improve the intestinal environment . these findings not only provide extremely helpful data that can contribute to the maintenance and improvement of health of the people of japan but also encourage further development of champignon extract as a dietary supplement . champignon extract is efficacious against halitosis and body and fecal odor , and it can also improve the intestinal environment . these findings not only provide extremely helpful data that can contribute to the maintenance and improvement of health of the people of japan but also encourage further development of champignon extract as a dietary supplement .

this was placebo - controlled double - blind parallel - group comparative clinical trial targeting 80 men and women aged 5079 years with halitosis and body and fecal odor . we investigated whether daily champignon extract ingestion for 4 weeks improved these conditions . subjects were divided into four groups : a placebo group and 50 , 500 , and 1000 mg / day ingestion groups . no severe adverse events or side effects were noted during the study period . compared with the placebo group , all champignon extract ingestion groups showed improvement or tendency toward improvement in halitosis and body and fecal odor . furthermore , our results suggested that the effectiveness of champignon extract in alleviating odors is dose - dependent , i.e. , it increases with the dosage .

Introduction Material and methods Results Discussion Conclusion

halitosis and body and fecal odor negatively affect peoples ' quality of life , and they are increasingly becoming a serious problem in caregiving settings in japan , with the rapidly increasing proportion of the elderly in the japanese society . conditions such as halitosis and body and fecal odor are believed to be caused by certain toxic substances produced within the intestinal tract ; champignon extract directly inhibits their production . thus , champignon extract is widely used in candy , jellies , drinks , and other food products for maintaining both personal esthetics and health . in a clinical trial targeting elderly inpatients ( 70 subjects ; mean age : males 73.5 years , females 78.6 years ) and involving ingestion of 2 g of champignon extract daily for 30 days , the extract was shown to not affect gastrointestinal symptoms but improved stool characteristics , reduced fecal and body odor , and decreased blood ammonia content . in another trial involving 24 residents of an elderly care facility who ingested jelly candies containing 300 mg of champignon extract daily for 30 days , favorable effects such as improved stool characteristics and life satisfaction were observed . here we aimed to investigate whether daily champignon extract ingestion for 4 weeks improved halitosis and body and fecal odor . we included 80 men and women aged 5079 years with problematic halitosis and body and fecal odor in this placebo - controlled double - blind parallel - group comparative study . it involved four study groups : consisting of 3 test foods and a placebo , as shown in table 1 . ( 1 ) a placebo group ( n = 20 ) , ( 2 ) 50 mg / day champignon ingestion group ( n = 20 ) , ( 3 ) 500 mg / day champignon ingestion group ( n = 20 ) , and ( 4 ) 1000 mg / day champignon ingestion group ( n = 20 ) . for group allocation , an allocation manager from a third - party data center ( media education center , hokkaido institute of information technology , ebetsu , hokkaido ) referred to the subject list and equally divided the subjects via stratified randomization into four groups considering age composition , male - to - female ratio , and odor questionnaire scores . subjects were then notified of the date , time , and place for the clinical trial . because three subjects quit the study owing to personal reasons before the trial started , subjects ingested one packet ( 2 g ) per day of test food or placebo for 4 weeks from the day of starting ingestion . the study protocol conformed to the helsinki declaration and was registered at the umin clinical trial registration system ( certificate number umin000014256 ) . subjects and cooperating people ( those living along with the subjects ) were asked to answer the questionnaire regarding halitosis and body and fecal odor using the prescribed method . we investigated the following four items : ( 1 ) fecal odor , ( 2 ) bowel movement regularity , ( 3 ) strain during bowel movements , and ( 4 ) sensation of residual stools . table 2 presents the male - to - female ratio , mean age , weight , bmi , and percent body fat of the four subject groups : placebo group ( n = 19 ) , 50 mg / day ingestion group ( n = 18 ) , 500 mg / day ingestion group ( n = 20 ) , and 1000 mg / day ingestion group ( n = 20 ) . with regard to the analysis groups , no statistically significant differences were observed for ingestion rates between the placebo group and all ingestion groups ( table 3 ) . mean scores and standard deviation were calculated for each group for the results of the vas questionnaire regarding the seven items of halitosis , pillow odor , pajama odor , fecal odor , bowel movement regularity , strain during bowel movements , and sensation of residual stools . the respective amount of change in vas questionnaire scores regarding halitosis from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 10.95 16.99 mm and 19.11 18.89 mm ; 50 mg / day group , 19.33 20.40 mm and 22.44 22.59 mm ; 500 mg / day group , 13.75 17.59 mm and 17.85 20.78 mm ; 1000 mg / day group , 20.00 19.22 mm and 29.32 25.56 mm . after 2 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . after 4 weeks of ingestion , differences were observed between the 500 and 1000 mg / day ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding pillow odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 9.00 18.10 mm and 10.82 22.36 mm ; 50 mg / day group , 11.11 10.53 mm and 12.11 17.78 mm ; 500 mg / day group , 19.05 22.02 mm and 21.10 26.00 mm ; 1000 mg / day group , 12.20 16.47 mm and 17.50 22.99 mm . after 2 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . after 4 weeks of ingestion , differences were observed between the 50 and 500 mg / day ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding pajama odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.05 11.13 mm and 9.37 17.36 mm ; 50 mg / day group , 8.06 13.33 mm and 7.00 17.23 mm ; 500 mg / day group , 20.75 22.19 mm and 21.70 28.25 mm ; 1000 mg / day group , 12.15 17.33 mm and 15.00 20.48 mm . after 2 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . after 4 weeks of ingestion , differences were observed between the 500 and 1000 mg / day ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding fecal odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 12.95 19.35 mm and 21.63 23.03 mm ; 50 mg / day group , 11.22 15.34 mm and 15.56 19.57 mm ; 500 mg / day group , 19.26 14.30 mm and 27.10 21.27 mm ; 1000 mg / day group , 18.95 21.98 mm and 30.95 22.14 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , fecal odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.001 , p = 0.001 ) . after 2 and 4 weeks of ingestion , differences were also observed between all ingestion groups and the placebo group . the respective amount of change in vas questionnaire scores regarding bowel movement regularity from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 3.26 14.67 mm and 8.74 19.38 mm ; 50 mg / day group , 1.76 21.60 mm and 6.89 13.42 mm ; 500 mg / day group , 5.00 16.06 mm and 1.85 11.80 mm ; 1000 mg / day group , 8.22 21.51 mm and 8.82 30.52 mm . the respective amount of change in vas questionnaire scores regarding strain during bowel movements from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.79 30.44 mm and 0.16 17.05 mm ; 50 mg / day group , 6.11 32.04 mm and 11.61 16.99 mm ; 500 mg / day group , 0.70 13.19 mm and 3.70 11.62 mm ; 1000 mg / day group , 8.68 11.89 mm and 5.89 27.58 mm . the respective amount of change in vas questionnaire scores regarding sensation of residual stools from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group : 2.21 15.37 mm and 2.00 13.59 mm ; 50 mg / day group , 5.89 19.58 mm and 2.94 14.74 mm ; 500 mg / day group , 1.45 14.25 mm and 0.45 21.81 mm ; 1000 mg / day group , 6.74 13.01 mm and 2.78 24.18 mm . it comprised the four items of subject halitosis , pillow odor , pajama odor , and fecal odor ( odor of feces after subject used the toilet ) . the respective amount of change in vas questionnaire scores regarding halitosis from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 6.58 20.46 mm and 11.58 22.47 mm ; 50 mg / day group , 0.53 26.42 mm and 6.94 27.92 mm ; 500 mg / day group , 12.50 33.37 mm and 19.75 17.54 mm ; 1000 mg / day group , 11.05 27.97 mm and 21.53 29.79 mm . after 2 weeks of ingestion , differences were also observed in the 50 and 500 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . the respective amount of change in vas questionnaire scores regarding pillow odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.95 14.05 mm and 8.63 20.35 mm ; 50 mg / day group , 10.81 22.81 mm and 14.63 26.08 mm ; 500 mg / day group , 15.45 21.85 mm and 17.50 22.81 mm ; 1000 mg / day group , 14.16 21.88 mm and 21.11 27.06 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , body odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.011 , p = 0.003 ) . after 2 weeks of ingestion , differences were also observed in the 500 and 1000 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . the respective amount of change in vas questionnaire scores regarding pajama odor from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 5.53 15.07 mm and 10.89 17.72 mm ; 50 mg / day group , 6.94 23.10 mm and 10.81 24.68 mm ; 500 mg / day group , 13.20 19.26 mm and 20.15 22.00 mm ; 1000 mg / day group , 13.89 23.25 mm and 20.05 30.24 mm . after 2 weeks of ingestion , differences were also observed in the 500 and 1000 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . the respective amount of change in vas questionnaire scores regarding fecal odor ( odor after using toilet ) from before starting ingestion to after 2 weeks of ingestion and from before starting ingestion to after 4 weeks of ingestion was as follows : placebo group , 7.37 21.68 mm and 15.05 21.65 mm ; 50 mg / day group , 10.35 26.97 mm and 17.59 28.05 mm ; 500 mg / day group , 16.40 17.97 mm and 23.30 18.50 mm ; 1000 mg / day group , 16.11 27.49 mm and 25.58 24.49 mm . when test multiplicity was adjusted and the level of significance for each test was set at 2.5% , fecal odor significantly improved after 2 and 4 weeks of ingestion compared with that before starting ingestion ( p = 0.021 , p = 0.001 ) . after 2 weeks of ingestion , differences were also observed only in the 1000 mg / day ingestion groups compared with the placebo group . after 4 weeks of ingestion , differences were observed in all ingestion groups compared with the placebo group . subjects in the three ingestion groups recorded in diaries information regarding bowel movement frequency , bowel movement regularity , bowel movement volume , stool shape , stool color , stool odor , and sensation of having completely evacuated after each bowel movement . when test multiplicity was adjusted and the level of significance for each test was set at 5/3 ( = 1.67% ) for the 50 mg / day group and placebo group , the proportion of responses indicating that stool color was brown , ocher , or yellow was significantly higher in the placebo group on the day of starting ingestion and after 4 weeks of ingestion ( p = 0.002 , p = 0.001 ) . no significant differences were noted for any evaluation items between the 500 mg / day group and placebo group . the following results clearly suggested the superiority of the test food when the 1000 mg / day group and placebo group were compared . first , when test multiplicity was adjusted and the level of significance for each test was set at 2.5% for bowel movement frequency , the amount of change in bowel movement frequency from before starting ingestion to after 2 and 4 weeks of ingestion was significantly higher in the ingestion groups ( p = 0.016 , p = 0.006 ) . when test multiplicity was adjusted and the level of significance for each test was set at 5/2 = 2.5% for bowel movement volume , the amount of change in bowel movement volume from before starting ingestion to after 4 weeks of ingestion was significantly higher in the ingestion group ( 1000 mg / day ) than in the placebo group ( p = 0.014 ) . when test multiplicity was adjusted and the level of significance for each test was set at 5/3 ( = 1.67% ) for stool color , the proportion of responses indicating brown , ocher , or yellow was significantly higher in the placebo group after 4 weeks of ingestion ( p = 0.001 ) . in the present placebo - controlled double - blind parallel - group comparative study targeting 80 men and women aged 5079 years with problematic halitosis and body and fecal odor , the effects of 4-week daily ingestion of 50 , 500 , and 1000 mg / day champignon extract on the aforementioned conditions were investigated . moreover , no marked differences were observed between the placebo group and all ingestion groups with regard to vital signs , blood test findings , liver function , kidney function , lipid profile , blood glucose levels , or physical measurement during the ingestion of low , medium , or high doses of champignon extract ( data not shown ) . the following conclusions were drawn from the results of the vas questionnaire regarding halitosis and body and fecal odor . for each of the champignon extract ingestion groups , improvement or improvement tendencies were observed for halitosis and body and fecal odor compared with the placebo group , even in case of cooperating people 's data . considering the bowel movement journals , for the 50 mg / day ingestion and placebo groups , the proportion of responses indicating that stool color was brown , ocher , or yellow was significantly higher in the placebo group after 2 and 4 weeks of ingestion . bowel movement volume significantly improved in the 1000 mg / day group after 4 weeks of ingestion . for sensation of having completely evacuated after each bowel movement , there was a significantly high proportion of responses indicating cleared out or very cleared out in the 1000 mg / day group after 2 weeks of ingestion . these results demonstrate that although no clear improvement was observed in bowel movements with 50 mg / day champignon extract ingestion , whereas they clearly improved with a 500 mg / day ingestion . our results strongly suggest that the ingestion of 501000 mg / day of our test food may improve halitosis and body and fecal odor . in particular , ingestion of the high dose of 1000 mg / day may result in reduction in the odor and favorable intestinal tract environment . halitosis and body and fecal odor not only decrease a person 's quality of life but also can become serious problems in caregiving settings in japan because the proportion of the elderly population in japanese society is rapidly increasing . in conclusion , champignon extract is efficacious against halitosis and body and fecal odor , and it can also improve the intestinal environment . these findings not only provide extremely helpful data that can contribute to the maintenance and improvement of health of the people of japan but also encourage further development of champignon extract as a dietary supplement . champignon extract is efficacious against halitosis and body and fecal odor , and it can also improve the intestinal environment . these findings not only provide extremely helpful data that can contribute to the maintenance and improvement of health of the people of japan but also encourage further development of champignon extract as a dietary supplement .

a list of organisms has been identified by the infectious disease society of america as being responsible for the majority of healthcare associated drug resistant infections . ( enterococcus faecium , staphylococcus aureus , klebsiella pneumoniae , acinetobacter baumannii , pseudomonas aeruginosa , and enterobacter species ) ( 1 ) . among these problematic organisms , a. baumannii has nowadays emerged as a particular concern worldwide ; because it is responsible for a variety of infections , such as blood infection , meningitis , ventilator associated pneumoniae ( vap ) , and wound infections , especially among patients admitted in burn and intensive care units ( 2 , 3 ) . in addition , this organism is a successful pathogen to escape the effects of several classes of antimicrobials , including fluoroquinolones and represents a significant challenge to infectious disease specialists ( 4 ) . the antimicrobial activity of fluoroquinolones is to form ternary complexes , including enzymes , such as dna gyrase or topoisomerase iv , drug , and dna which can block dna replication and transcription , probably before dna cleavage occurs ( 5 ) . there is remarkable conservation of protein sequences between the dna gyrase subunit a ( gyra ) and topoisomerase iv subunit c ( parc ) in the quinolone resistance determining region ( qrdr ) . resistance to fluoroquinolone in bacteria is mediated mainly by spontaneous mutations in the qrdr of gyra and parc genes ( 8) . in escherichia coli , the most important mutations leading to a quinolone - resistant phenotype are ser83leu and asp87asn in the gyra gene , and ser80arg and glu84val in the parc gene ( 6 , 7 ) . many fluoroquinolone - resistant a. baumannii clinical isolates have been reported in iran ( 8 - 9 ) . there are , however , few studies about the prevalence of mutations in the genes for dna gyrase and topoisomerase iv in this organism . so , the present study aimed to specifically assess the presence of mutations in the gyra and parc genes and their effects on resistance to fluoroquinolones in a. baumannii isolates from patients at a teaching hospital in tehran . motahari hospital is a 120-bed university affiliated burn and reconstruction center located in tehran , iran . a total of 50 non - repetitive a. baumannii isolates were recovered between april 2012 and march 2013 from burn wounds of patients admitted in this hospital . all isolates were assigned to a. baumannii by bacteriologic and traditional phenotypic methods , including gram s staining , oxidase and catalase tests , motility , oxidative - fermentative ( of ) test , and growth at 37 c and 44 c . the pcr of the intrinsic blaoxa-51-like gene was done to confirm a. baumannii species ( 10 ) . the susceptibility of a. baumannii isolates to ciprofloxacin disk ( 5 g ) ( mast , merseyside , u.k ) was assessed by the standard disk agar diffusion method on mueller hinton agar plates . the mic for ciprofloxacin was determined by the broth macrodilution method in accordance to the clinical and laboratory standards institute ( clsi ) criteria ( 11 ) . briefly , a serial dilutions of ciprofloxacin was prepared in mueller hinton broth tubes with the bacteria at a density of 510 cfu / ml . microbial tubes were incubated at 37 c for 18 hr and finally , the lowest concentration of antibiotic with no visible bacterial growth was defined as the mic . quality control was done using pseudomonas aeruginosa atcc 27853 strain , and results were compared to mic ranges of clsi . a. baumannii isolates were considered as intermediate - resistant and full - resistant to ciprofloxacin when the mic was 2 g / ml and 4 g / ml , respectively . the qrdrs of the gyra and parc genes in a. baumannii clinical isolates were amplified by pcr assay . two pairs of oligonucleotide primers used for the pcr reactions were gyra primer : forward , 5-aaatctgctcgtgtcgttgg-3 ; reverse , 5-gccatacctacagcaatacc-3 , and parc primer : forward , 5-aagcccgtacagcgccgtatt-3 ; reverse , 5-aaagttatcttgccattcgct-3. extraction of genomic dna from a. baumannii colonies was performed using genomic dna purification kit ( fermentas , germany ) , based on the manufacturer s instructions . for all amplification reactions , a pcr mixture was used that contained 12.5 l of 2 master mix ( ampliqon , denmark ) , including 1 pcr buffer , 1.5 mmol / l mgcl2 , dntps at a concentration of 0.15 mmol / l each dntp , 1.25 u of taq dna polymerase , 0.5 l of 0.8 m of each primer , 1 l of template dna ( 0.5 g ) , and sterile distilled water up to 25 l . dna amplification was performed in the mastercycler gradient instrument ( eppendorf , germany ) . for gyra , pcr conditions consisted of an initial denaturation at 95 c for 1 min ; 35 cycles of denaturation at 95 c for of 30 sec , 30 sec of annealing at 52 c , and 2 min of extension at 72 c ; ending with a final extension at 72 c for 10 min . the temperature profile for parc gene was comprised of 95 c for 2 min , followed by 35 cycles of 1 min at 95 c , 1 min at 60 c and 2 min at 72 c , with 10 min at 72 c for final extension step . dna fragments were analyzed by electrophoresis on agarose gel ( 2% , wt / vol ) containing 0.5 mg of ethidium bromide per liter and photographed with ultraviolet illumination . direct sequencing of the pcr products in both directions was performed by using an abi3730xl dna analyzer ( applied biosystems , forster , usa ) . nucleotide sequences data were analyzed at the national center for biotechnology information ( ncbi ) , available at the website ( http:/www.ncbi.nlm.nih.gov / blast/ ) . motahari hospital is a 120-bed university affiliated burn and reconstruction center located in tehran , iran . a total of 50 non - repetitive a. baumannii isolates were recovered between april 2012 and march 2013 from burn wounds of patients admitted in this hospital . all isolates were assigned to a. baumannii by bacteriologic and traditional phenotypic methods , including gram s staining , oxidase and catalase tests , motility , oxidative - fermentative ( of ) test , and growth at 37 c and 44 c . the pcr of the intrinsic blaoxa-51-like gene was done to confirm a. baumannii species ( 10 ) . the susceptibility of a. baumannii isolates to ciprofloxacin disk ( 5 g ) ( mast , merseyside , u.k ) was assessed by the standard disk agar diffusion method on mueller hinton agar plates . the mic for ciprofloxacin was determined by the broth macrodilution method in accordance to the clinical and laboratory standards institute ( clsi ) criteria ( 11 ) . briefly , a serial dilutions of ciprofloxacin was prepared in mueller hinton broth tubes with the bacteria at a density of 510 cfu / ml . microbial tubes were incubated at 37 c for 18 hr and finally , the lowest concentration of antibiotic with no visible bacterial growth was defined as the mic . quality control was done using pseudomonas aeruginosa atcc 27853 strain , and results were compared to mic ranges of clsi . a. baumannii isolates were considered as intermediate - resistant and full - resistant to ciprofloxacin when the mic was 2 g / ml and 4 g / ml , respectively . the qrdrs of the gyra and parc genes in a. baumannii clinical isolates were amplified by pcr assay . two pairs of oligonucleotide primers used for the pcr reactions were gyra primer : forward , 5-aaatctgctcgtgtcgttgg-3 ; reverse , 5-gccatacctacagcaatacc-3 , and parc primer : forward , 5-aagcccgtacagcgccgtatt-3 ; reverse , 5-aaagttatcttgccattcgct-3. extraction of genomic dna from a. baumannii colonies was performed using genomic dna purification kit ( fermentas , germany ) , based on the manufacturer s instructions . for all amplification reactions , a pcr mixture was used that contained 12.5 l of 2 master mix ( ampliqon , denmark ) , including 1 pcr buffer , 1.5 mmol / l mgcl2 , dntps at a concentration of 0.15 mmol / l each dntp , 1.25 u of taq dna polymerase , 0.5 l of 0.8 m of each primer , 1 l of template dna ( 0.5 g ) , and sterile distilled water up to 25 l . dna amplification was performed in the mastercycler gradient instrument ( eppendorf , germany ) . for gyra , pcr conditions consisted of an initial denaturation at 95 c for 1 min ; 35 cycles of denaturation at 95 c for of 30 sec , 30 sec of annealing at 52 c , and 2 min of extension at 72 c ; ending with a final extension at 72 c for 10 min . the temperature profile for parc gene was comprised of 95 c for 2 min , followed by 35 cycles of 1 min at 95 c , 1 min at 60 c and 2 min at 72 c , with 10 min at 72 c for final extension step . dna fragments were analyzed by electrophoresis on agarose gel ( 2% , wt / vol ) containing 0.5 mg of ethidium bromide per liter and photographed with ultraviolet illumination . direct sequencing of the pcr products in both directions was performed by using an abi3730xl dna analyzer ( applied biosystems , forster , usa ) . nucleotide sequences data were analyzed at the national center for biotechnology information ( ncbi ) , available at the website ( http:/www.ncbi.nlm.nih.gov / blast/ ) . motahari hospital is a 120-bed university affiliated burn and reconstruction center located in tehran , iran . a total of 50 non - repetitive a. baumannii isolates were recovered between april 2012 and march 2013 from burn wounds of patients admitted in this hospital . all isolates were assigned to a. baumannii by bacteriologic and traditional phenotypic methods , including gram s staining , oxidase and catalase tests , motility , oxidative - fermentative ( of ) test , and growth at 37 c and 44 c . the pcr of the intrinsic blaoxa-51-like gene was done to confirm a. baumannii species ( 10 ) . the susceptibility of a. baumannii isolates to ciprofloxacin disk ( 5 g ) ( mast , merseyside , u.k ) was assessed by the standard disk agar diffusion method on mueller hinton agar plates . the mic for ciprofloxacin was determined by the broth macrodilution method in accordance to the clinical and laboratory standards institute ( clsi ) criteria ( 11 ) . briefly , a serial dilutions of ciprofloxacin was prepared in mueller hinton broth tubes with the bacteria at a density of 510 cfu / ml . microbial tubes were incubated at 37 c for 18 hr and finally , the lowest concentration of antibiotic with no visible bacterial growth was defined as the mic . quality control was done using pseudomonas aeruginosa atcc 27853 strain , and results were compared to mic ranges of clsi . a. baumannii isolates were considered as intermediate - resistant and full - resistant to ciprofloxacin when the mic was 2 g / ml and 4 g / ml , respectively . the qrdrs of the gyra and parc genes in a. baumannii clinical isolates were amplified by pcr assay . two pairs of oligonucleotide primers used for the pcr reactions were gyra primer : forward , 5-aaatctgctcgtgtcgttgg-3 ; reverse , 5-gccatacctacagcaatacc-3 , and parc primer : forward , 5-aagcccgtacagcgccgtatt-3 ; reverse , 5-aaagttatcttgccattcgct-3. extraction of genomic dna from a. baumannii colonies was performed using genomic dna purification kit ( fermentas , germany ) , based on the manufacturer s instructions . for all amplification reactions , a pcr mixture was used that contained 12.5 l of 2 master mix ( ampliqon , denmark ) , including 1 pcr buffer , 1.5 mmol / l mgcl2 , dntps at a concentration of 0.15 mmol / l each dntp , 1.25 u of taq dna polymerase , 0.5 l of 0.8 m of each primer , 1 l of template dna ( 0.5 g ) , and sterile distilled water up to 25 l . dna amplification was performed in the mastercycler gradient instrument ( eppendorf , germany ) . for gyra , pcr conditions consisted of an initial denaturation at 95 c for 1 min ; 35 cycles of denaturation at 95 c for of 30 sec , 30 sec of annealing at 52 c , and 2 min of extension at 72 c ; ending with a final extension at 72 c for 10 min . the temperature profile for parc gene was comprised of 95 c for 2 min , followed by 35 cycles of 1 min at 95 c , 1 min at 60 c and 2 min at 72 c , with 10 min at 72 c for final extension step . dna fragments were analyzed by electrophoresis on agarose gel ( 2% , wt / vol ) containing 0.5 mg of ethidium bromide per liter and photographed with ultraviolet illumination . direct sequencing of the pcr products in both directions was performed by using an abi3730xl dna analyzer ( applied biosystems , forster , usa ) . nucleotide sequences data were analyzed at the national center for biotechnology information ( ncbi ) , available at the website ( http:/www.ncbi.nlm.nih.gov / blast/ ) . descriptive statistics and pearson s chi - square tests were used to evaluate correlation between mutation and ciprofloxacin resistance . the susceptibility testing by disk diffusion method determined that 4% , 8% , and 88% of a. baumannii clinical isolates were susceptible , intermediate - resistant and full - resistant to cipro - floxacin , respectively . the mic range of ciprofloxacin in 44 full - resistant isolates was 4 to 128 g / ml . to determine the changes in the structure of dna gyrase and topoisomerase iv enzymes , the qrdrs of corresponding genes , gyra and parc , were analyzed by pcr sequencing technique in all 50 clinical isolates with intermediate - resistant and full - resistant to ciprofloxacin . amplification of gyra and parc genes yielded pcr products of 349 and 327 bp , respectively ( figure 1 ) . the nucleotide sequencing results revealed that 45 ( 90% ) of the 50 isolates had amino acid alteration in gyra and parc , as follow : 1 ( 2.2% ) isolate in gyra only , 2 ( 4.4% ) isolates in parc only , and 42 ( 93.3% ) isolates in gyra and parc , concurrently . the a. baumannii isolates were divided into five groups based on the amino acid substitutions associated with resistance to fluoroquinolone ( table 1 ) . single mutations encoding ser83leu and gly81asp were found in the qrdr of gyra in 37 ( 82.2% ) and 6 ( 13.3% ) of the 45 mutated isolates , respectively . forty four of the 45 acinetobacter isolates ( 95.5% ) had a single mutation in parc encoding ser80leu ( 23 ; 51.1% ) , glu84lys ( 19 ; 42.2% ) , and gly78cys ( 2 ; 4.4% ) . pcr amplification of the qrdrs of the gyra ( a ) and parc ( b ) genes in acinetobacter baumannii isolates . lanes : 1- 7 , pcr products of the corresponding genes ; m , 100 bp plus dna ladder point mutations in the qrdrs of the gyra and parc genes of acinetobacter baumannii isolates , and the ciprofloxacin mic in the isolates of mutation groups groups i and ii mutants had a single mutation in gyra and parc , respectively ; but their ciprofloxacin mic ranged from 2 to 4 g / ml . acinetobacter isolates present in the mutation groups iii , iv , and v showed double mutations in gyra and parc and were resistant to ciprofloxacin with a mic range of 8 to 128 g / ml . overall , isolates with double mutations of gyra and parc genes showed a higher level of ciprofloxacin resistance than isolates with single mutations of gyra or parc ( p < 0.05 ) . the nucleotide sequences data reported in the present study have been deposited in the pubmed / ncbi / genbank nucleotide sequence database under accession numbers for gyra ( kj195830.1 and kj195831.1 ) and parc ( kj756512 , kj756513 and kj756514 ) genes . the susceptibility testing by disk diffusion method determined that 4% , 8% , and 88% of a. baumannii clinical isolates were susceptible , intermediate - resistant and full - resistant to cipro - floxacin , respectively . the mic range of ciprofloxacin in 44 full - resistant isolates was 4 to 128 g / ml . to determine the changes in the structure of dna gyrase and topoisomerase iv enzymes , the qrdrs of corresponding genes , gyra and parc , were analyzed by pcr sequencing technique in all 50 clinical isolates with intermediate - resistant and full - resistant to ciprofloxacin . amplification of gyra and parc genes yielded pcr products of 349 and 327 bp , respectively ( figure 1 ) . the nucleotide sequencing results revealed that 45 ( 90% ) of the 50 isolates had amino acid alteration in gyra and parc , as follow : 1 ( 2.2% ) isolate in gyra only , 2 ( 4.4% ) isolates in parc only , and 42 ( 93.3% ) isolates in gyra and parc , concurrently . the a. baumannii isolates were divided into five groups based on the amino acid substitutions associated with resistance to fluoroquinolone ( table 1 ) . single mutations encoding ser83leu and gly81asp were found in the qrdr of gyra in 37 ( 82.2% ) and 6 ( 13.3% ) of the 45 mutated isolates , respectively . forty four of the 45 acinetobacter isolates ( 95.5% ) had a single mutation in parc encoding ser80leu ( 23 ; 51.1% ) , glu84lys ( 19 ; 42.2% ) , and gly78cys ( 2 ; 4.4% ) . pcr amplification of the qrdrs of the gyra ( a ) and parc ( b ) genes in acinetobacter baumannii isolates . lanes : 1- 7 , pcr products of the corresponding genes ; m , 100 bp plus dna ladder point mutations in the qrdrs of the gyra and parc genes of acinetobacter baumannii isolates , and the ciprofloxacin mic in the isolates of mutation groups groups i and ii mutants had a single mutation in gyra and parc , respectively ; but their ciprofloxacin mic ranged from 2 to 4 g / ml . acinetobacter isolates present in the mutation groups iii , iv , and v showed double mutations in gyra and parc and were resistant to ciprofloxacin with a mic range of 8 to 128 g / ml . overall , isolates with double mutations of gyra and parc genes showed a higher level of ciprofloxacin resistance than isolates with single mutations of gyra or parc ( p < 0.05 ) . the nucleotide sequences data reported in the present study have been deposited in the pubmed / ncbi / genbank nucleotide sequence database under accession numbers for gyra ( kj195830.1 and kj195831.1 ) and parc ( kj756512 , kj756513 and kj756514 ) genes . the susceptibility testing by disk diffusion method determined that 4% , 8% , and 88% of a. baumannii clinical isolates were susceptible , intermediate - resistant and full - resistant to cipro - floxacin , respectively . the mic range of ciprofloxacin in 44 full - resistant isolates was 4 to 128 g / ml . to determine the changes in the structure of dna gyrase and topoisomerase iv enzymes , the qrdrs of corresponding genes , gyra and parc , were analyzed by pcr sequencing technique in all 50 clinical isolates with intermediate - resistant and full - resistant to ciprofloxacin . amplification of gyra and parc genes yielded pcr products of 349 and 327 bp , respectively ( figure 1 ) . the nucleotide sequencing results revealed that 45 ( 90% ) of the 50 isolates had amino acid alteration in gyra and parc , as follow : 1 ( 2.2% ) isolate in gyra only , 2 ( 4.4% ) isolates in parc only , and 42 ( 93.3% ) isolates in gyra and parc , concurrently . the a. baumannii isolates were divided into five groups based on the amino acid substitutions associated with resistance to fluoroquinolone ( table 1 ) . single mutations encoding ser83leu and gly81asp were found in the qrdr of gyra in 37 ( 82.2% ) and 6 ( 13.3% ) of the 45 mutated isolates , respectively . forty four of the 45 acinetobacter isolates ( 95.5% ) had a single mutation in parc encoding ser80leu ( 23 ; 51.1% ) , glu84lys ( 19 ; 42.2% ) , and gly78cys ( 2 ; 4.4% ) . pcr amplification of the qrdrs of the gyra ( a ) and parc ( b ) genes in acinetobacter baumannii isolates . lanes : 1- 7 , pcr products of the corresponding genes ; m , 100 bp plus dna ladder point mutations in the qrdrs of the gyra and parc genes of acinetobacter baumannii isolates , and the ciprofloxacin mic in the isolates of mutation groups groups i and ii mutants had a single mutation in gyra and parc , respectively ; but their ciprofloxacin mic ranged from 2 to 4 g / ml . acinetobacter isolates present in the mutation groups iii , iv , and v showed double mutations in gyra and parc and were resistant to ciprofloxacin with a mic range of 8 to 128 g / ml . overall , isolates with double mutations of gyra and parc genes showed a higher level of ciprofloxacin resistance than isolates with single mutations of gyra or parc ( p < 0.05 ) . the nucleotide sequences data reported in the present study have been deposited in the pubmed / ncbi / genbank nucleotide sequence database under accession numbers for gyra ( kj195830.1 and kj195831.1 ) and parc ( kj756512 , kj756513 and kj756514 ) genes . previous studies have shown that fluoroquino - lones are one of the first line therapies for a. baumannii infections ( 12 , 13 ) . however , our results in agreement with other studies revealed a considerable increase in ciprofloxacin resistance in iran ( 8 , 9 ) . resistance to ciprofloxacin in acinetobacter isolates is alarming ; since many of such strains are usually multi - drug resistant ( mdr ) , and at these circumstances , the remaining therapeutic options are colistin ( a relatively toxic drug ) , tigecycline ( a bacteriostatic agent , i.e. a disadvantage when using in immunocompromised patients ) , and sulbactam ( with a limited antibacterial spectrum only ) ( 14 ) . it is accepted that changes in the structure of the antibiotic targets dna gyrase and dna topoisomerase iv are one of the most significant mechanisms in conferring a resistance to fluoroquinolone in gram negative bacilli ( 7 ) . in e. coli , three or four mutations in both gyra and parc genes are necessary to obtain high - level resistance to ciprofloxacin , even as double mutations at positions 83 ( ser83 ) of gyra and 80 ( ser80 ) of parc cause only moderate - level resistance ( 6 , 7 ) . nevertheless , the situation in a. baumannii is rather different from that in e. coli , since we found in the present study that only a double mutation could lead to high - level resistant phenotype . so that , among 44 ciprofloxacin - resistant isolates , 11 ( 25% ) had mic range of 8 - 32 g / ml and the majority ( 31 ; 70.4% ) of isolates had mics of 128 g / ml or higher , that indicate these isolates are highly resistant to fluoroquinolones . our sequencing results revealed serine to leucine mutation at position 83 of gyra subunit in 38 of 44 ciprofloxacin - resistant acinetobacter isolates . similar to study by park et al ( 15 ) , this is indicative of the fact that ser83leu substitution is the principal mutation in a. baumannii for resistance to fluoroquinolones . other mutations in the gyra gene resulting in ala84pro or gly81val reported in ciprofloxacin resistant isolates of previous study ( 16 ) were not observed in our included isolates . instead , we found a novel mutation in the gyra gene , leading to gly81asp substitution dna gyrase , in six resistant isolates with a mic range at 8 to 128 g / ml . in a. baumannii , topoisomerase iv is a target of quinolones and mutations at residues ser80 and glu84 of parc contribute to decreased fluoroquino - lone susceptibility ( 16 ) . although parc mutations always along with mutations in gyra are needed to acquire a high - level resistance to quinolones ( 4 ) , two clinical isolates in our study had mutations in parc without gyra , suggesting that parc might not only be a secondary target for quinolones but is really as important as gyra to cause a decreased susceptibility to fluoroquinolones in a. baumannii . on the other hand , qrdrs of the three isolates with intermediate - resistance to ciprofloxacin in the current study did not possess alterations associated with fluoroquino - lones resistance in the sequence of either genes , indicating other resistance mechanisms , such as efflux systems should be considered in these isolates ( 17 ) . although a single point mutation in dna gyrase is enough for resistance to fluoroquinolone in a. baumannii , the concurrent mutations within qrdr regions of the gyra and parc genes are expected to significantly contribute to high - level fluoroquinolone resistance . further studies are required to elucidate mechanisms , other than alterations in gyra and parc , leading to decreased susceptibility to quinolones in a. baumannii isolates .

objective(s):we investigated the contribution of gyra and parc mutational mechanism in decreased ciprofloxacin susceptibility of acinetobacter baumannii isolated from burn wound infections.materials and methods : ciprofloxacin susceptibility of 50 a. baumannii isolates was evaluated by disk diffusion and agar dilution methods . pcr and sequencing were performed for detection of mutation in gyra and parc genes.results:the 44 and 4 isolates of a. baumannii exhibited full and intermediate - resistant to ciprofloxacin , respectively . overall , the 42 isolates with double mutations of gyra and parc genes showed a higher level of ciprofloxacin resistance than the 3 isolates with single mutations of gyra or parc.conclusion:simultaneous mutations in gyra and parc genes are expected to play a major role in high - level fluoroquinolone resistance in a. baumannii ; albeit a single mutation in dna topoisomerase iv could occasionally be associated with intermediate - resistance to these antimicrobials .

Introduction Materials and Methods None Bacterial isolates Antimicrobial susceptibility testing PCR amplification and sequencing Statistical data analysis Results None Ciprofloxacin susceptibility and amino acid substitutions MIC for ciprofloxacin in the QRDR mutants Nucleotide sequence accession number Discussion Conclusion

among these problematic organisms , a. baumannii has nowadays emerged as a particular concern worldwide ; because it is responsible for a variety of infections , such as blood infection , meningitis , ventilator associated pneumoniae ( vap ) , and wound infections , especially among patients admitted in burn and intensive care units ( 2 , 3 ) . resistance to fluoroquinolone in bacteria is mediated mainly by spontaneous mutations in the qrdr of gyra and parc genes ( 8) . in escherichia coli , the most important mutations leading to a quinolone - resistant phenotype are ser83leu and asp87asn in the gyra gene , and ser80arg and glu84val in the parc gene ( 6 , 7 ) . many fluoroquinolone - resistant a. baumannii clinical isolates have been reported in iran ( 8 - 9 ) . there are , however , few studies about the prevalence of mutations in the genes for dna gyrase and topoisomerase iv in this organism . so , the present study aimed to specifically assess the presence of mutations in the gyra and parc genes and their effects on resistance to fluoroquinolones in a. baumannii isolates from patients at a teaching hospital in tehran . a total of 50 non - repetitive a. baumannii isolates were recovered between april 2012 and march 2013 from burn wounds of patients admitted in this hospital . all isolates were assigned to a. baumannii by bacteriologic and traditional phenotypic methods , including gram s staining , oxidase and catalase tests , motility , oxidative - fermentative ( of ) test , and growth at 37 c and 44 c . the susceptibility of a. baumannii isolates to ciprofloxacin disk ( 5 g ) ( mast , merseyside , u.k ) was assessed by the standard disk agar diffusion method on mueller hinton agar plates . a. baumannii isolates were considered as intermediate - resistant and full - resistant to ciprofloxacin when the mic was 2 g / ml and 4 g / ml , respectively . the qrdrs of the gyra and parc genes in a. baumannii clinical isolates were amplified by pcr assay . extraction of genomic dna from a. baumannii colonies was performed using genomic dna purification kit ( fermentas , germany ) , based on the manufacturer s instructions . a total of 50 non - repetitive a. baumannii isolates were recovered between april 2012 and march 2013 from burn wounds of patients admitted in this hospital . all isolates were assigned to a. baumannii by bacteriologic and traditional phenotypic methods , including gram s staining , oxidase and catalase tests , motility , oxidative - fermentative ( of ) test , and growth at 37 c and 44 c . the susceptibility of a. baumannii isolates to ciprofloxacin disk ( 5 g ) ( mast , merseyside , u.k ) was assessed by the standard disk agar diffusion method on mueller hinton agar plates . microbial tubes were incubated at 37 c for 18 hr and finally , the lowest concentration of antibiotic with no visible bacterial growth was defined as the mic . a. baumannii isolates were considered as intermediate - resistant and full - resistant to ciprofloxacin when the mic was 2 g / ml and 4 g / ml , respectively . the qrdrs of the gyra and parc genes in a. baumannii clinical isolates were amplified by pcr assay . extraction of genomic dna from a. baumannii colonies was performed using genomic dna purification kit ( fermentas , germany ) , based on the manufacturer s instructions . a total of 50 non - repetitive a. baumannii isolates were recovered between april 2012 and march 2013 from burn wounds of patients admitted in this hospital . all isolates were assigned to a. baumannii by bacteriologic and traditional phenotypic methods , including gram s staining , oxidase and catalase tests , motility , oxidative - fermentative ( of ) test , and growth at 37 c and 44 c . the susceptibility of a. baumannii isolates to ciprofloxacin disk ( 5 g ) ( mast , merseyside , u.k ) was assessed by the standard disk agar diffusion method on mueller hinton agar plates . briefly , a serial dilutions of ciprofloxacin was prepared in mueller hinton broth tubes with the bacteria at a density of 510 cfu / ml . a. baumannii isolates were considered as intermediate - resistant and full - resistant to ciprofloxacin when the mic was 2 g / ml and 4 g / ml , respectively . the qrdrs of the gyra and parc genes in a. baumannii clinical isolates were amplified by pcr assay . extraction of genomic dna from a. baumannii colonies was performed using genomic dna purification kit ( fermentas , germany ) , based on the manufacturer s instructions . descriptive statistics and pearson s chi - square tests were used to evaluate correlation between mutation and ciprofloxacin resistance . the susceptibility testing by disk diffusion method determined that 4% , 8% , and 88% of a. baumannii clinical isolates were susceptible , intermediate - resistant and full - resistant to cipro - floxacin , respectively . the mic range of ciprofloxacin in 44 full - resistant isolates was 4 to 128 g / ml . to determine the changes in the structure of dna gyrase and topoisomerase iv enzymes , the qrdrs of corresponding genes , gyra and parc , were analyzed by pcr sequencing technique in all 50 clinical isolates with intermediate - resistant and full - resistant to ciprofloxacin . amplification of gyra and parc genes yielded pcr products of 349 and 327 bp , respectively ( figure 1 ) . the nucleotide sequencing results revealed that 45 ( 90% ) of the 50 isolates had amino acid alteration in gyra and parc , as follow : 1 ( 2.2% ) isolate in gyra only , 2 ( 4.4% ) isolates in parc only , and 42 ( 93.3% ) isolates in gyra and parc , concurrently . the a. baumannii isolates were divided into five groups based on the amino acid substitutions associated with resistance to fluoroquinolone ( table 1 ) . single mutations encoding ser83leu and gly81asp were found in the qrdr of gyra in 37 ( 82.2% ) and 6 ( 13.3% ) of the 45 mutated isolates , respectively . forty four of the 45 acinetobacter isolates ( 95.5% ) had a single mutation in parc encoding ser80leu ( 23 ; 51.1% ) , glu84lys ( 19 ; 42.2% ) , and gly78cys ( 2 ; 4.4% ) . pcr amplification of the qrdrs of the gyra ( a ) and parc ( b ) genes in acinetobacter baumannii isolates . lanes : 1- 7 , pcr products of the corresponding genes ; m , 100 bp plus dna ladder point mutations in the qrdrs of the gyra and parc genes of acinetobacter baumannii isolates , and the ciprofloxacin mic in the isolates of mutation groups groups i and ii mutants had a single mutation in gyra and parc , respectively ; but their ciprofloxacin mic ranged from 2 to 4 g / ml . acinetobacter isolates present in the mutation groups iii , iv , and v showed double mutations in gyra and parc and were resistant to ciprofloxacin with a mic range of 8 to 128 g / ml . overall , isolates with double mutations of gyra and parc genes showed a higher level of ciprofloxacin resistance than isolates with single mutations of gyra or parc ( p < 0.05 ) . the nucleotide sequences data reported in the present study have been deposited in the pubmed / ncbi / genbank nucleotide sequence database under accession numbers for gyra ( kj195830.1 and kj195831.1 ) and parc ( kj756512 , kj756513 and kj756514 ) genes . the susceptibility testing by disk diffusion method determined that 4% , 8% , and 88% of a. baumannii clinical isolates were susceptible , intermediate - resistant and full - resistant to cipro - floxacin , respectively . the mic range of ciprofloxacin in 44 full - resistant isolates was 4 to 128 g / ml . to determine the changes in the structure of dna gyrase and topoisomerase iv enzymes , the qrdrs of corresponding genes , gyra and parc , were analyzed by pcr sequencing technique in all 50 clinical isolates with intermediate - resistant and full - resistant to ciprofloxacin . amplification of gyra and parc genes yielded pcr products of 349 and 327 bp , respectively ( figure 1 ) . the nucleotide sequencing results revealed that 45 ( 90% ) of the 50 isolates had amino acid alteration in gyra and parc , as follow : 1 ( 2.2% ) isolate in gyra only , 2 ( 4.4% ) isolates in parc only , and 42 ( 93.3% ) isolates in gyra and parc , concurrently . the a. baumannii isolates were divided into five groups based on the amino acid substitutions associated with resistance to fluoroquinolone ( table 1 ) . single mutations encoding ser83leu and gly81asp were found in the qrdr of gyra in 37 ( 82.2% ) and 6 ( 13.3% ) of the 45 mutated isolates , respectively . forty four of the 45 acinetobacter isolates ( 95.5% ) had a single mutation in parc encoding ser80leu ( 23 ; 51.1% ) , glu84lys ( 19 ; 42.2% ) , and gly78cys ( 2 ; 4.4% ) . pcr amplification of the qrdrs of the gyra ( a ) and parc ( b ) genes in acinetobacter baumannii isolates . lanes : 1- 7 , pcr products of the corresponding genes ; m , 100 bp plus dna ladder point mutations in the qrdrs of the gyra and parc genes of acinetobacter baumannii isolates , and the ciprofloxacin mic in the isolates of mutation groups groups i and ii mutants had a single mutation in gyra and parc , respectively ; but their ciprofloxacin mic ranged from 2 to 4 g / ml . acinetobacter isolates present in the mutation groups iii , iv , and v showed double mutations in gyra and parc and were resistant to ciprofloxacin with a mic range of 8 to 128 g / ml . overall , isolates with double mutations of gyra and parc genes showed a higher level of ciprofloxacin resistance than isolates with single mutations of gyra or parc ( p < 0.05 ) . the nucleotide sequences data reported in the present study have been deposited in the pubmed / ncbi / genbank nucleotide sequence database under accession numbers for gyra ( kj195830.1 and kj195831.1 ) and parc ( kj756512 , kj756513 and kj756514 ) genes . the susceptibility testing by disk diffusion method determined that 4% , 8% , and 88% of a. baumannii clinical isolates were susceptible , intermediate - resistant and full - resistant to cipro - floxacin , respectively . the mic range of ciprofloxacin in 44 full - resistant isolates was 4 to 128 g / ml . to determine the changes in the structure of dna gyrase and topoisomerase iv enzymes , the qrdrs of corresponding genes , gyra and parc , were analyzed by pcr sequencing technique in all 50 clinical isolates with intermediate - resistant and full - resistant to ciprofloxacin . amplification of gyra and parc genes yielded pcr products of 349 and 327 bp , respectively ( figure 1 ) . the nucleotide sequencing results revealed that 45 ( 90% ) of the 50 isolates had amino acid alteration in gyra and parc , as follow : 1 ( 2.2% ) isolate in gyra only , 2 ( 4.4% ) isolates in parc only , and 42 ( 93.3% ) isolates in gyra and parc , concurrently . the a. baumannii isolates were divided into five groups based on the amino acid substitutions associated with resistance to fluoroquinolone ( table 1 ) . single mutations encoding ser83leu and gly81asp were found in the qrdr of gyra in 37 ( 82.2% ) and 6 ( 13.3% ) of the 45 mutated isolates , respectively . forty four of the 45 acinetobacter isolates ( 95.5% ) had a single mutation in parc encoding ser80leu ( 23 ; 51.1% ) , glu84lys ( 19 ; 42.2% ) , and gly78cys ( 2 ; 4.4% ) . pcr amplification of the qrdrs of the gyra ( a ) and parc ( b ) genes in acinetobacter baumannii isolates . lanes : 1- 7 , pcr products of the corresponding genes ; m , 100 bp plus dna ladder point mutations in the qrdrs of the gyra and parc genes of acinetobacter baumannii isolates , and the ciprofloxacin mic in the isolates of mutation groups groups i and ii mutants had a single mutation in gyra and parc , respectively ; but their ciprofloxacin mic ranged from 2 to 4 g / ml . acinetobacter isolates present in the mutation groups iii , iv , and v showed double mutations in gyra and parc and were resistant to ciprofloxacin with a mic range of 8 to 128 g / ml . overall , isolates with double mutations of gyra and parc genes showed a higher level of ciprofloxacin resistance than isolates with single mutations of gyra or parc ( p < 0.05 ) . the nucleotide sequences data reported in the present study have been deposited in the pubmed / ncbi / genbank nucleotide sequence database under accession numbers for gyra ( kj195830.1 and kj195831.1 ) and parc ( kj756512 , kj756513 and kj756514 ) genes . previous studies have shown that fluoroquino - lones are one of the first line therapies for a. baumannii infections ( 12 , 13 ) . however , our results in agreement with other studies revealed a considerable increase in ciprofloxacin resistance in iran ( 8 , 9 ) . resistance to ciprofloxacin in acinetobacter isolates is alarming ; since many of such strains are usually multi - drug resistant ( mdr ) , and at these circumstances , the remaining therapeutic options are colistin ( a relatively toxic drug ) , tigecycline ( a bacteriostatic agent , i.e. it is accepted that changes in the structure of the antibiotic targets dna gyrase and dna topoisomerase iv are one of the most significant mechanisms in conferring a resistance to fluoroquinolone in gram negative bacilli ( 7 ) . in e. coli , three or four mutations in both gyra and parc genes are necessary to obtain high - level resistance to ciprofloxacin , even as double mutations at positions 83 ( ser83 ) of gyra and 80 ( ser80 ) of parc cause only moderate - level resistance ( 6 , 7 ) . nevertheless , the situation in a. baumannii is rather different from that in e. coli , since we found in the present study that only a double mutation could lead to high - level resistant phenotype . so that , among 44 ciprofloxacin - resistant isolates , 11 ( 25% ) had mic range of 8 - 32 g / ml and the majority ( 31 ; 70.4% ) of isolates had mics of 128 g / ml or higher , that indicate these isolates are highly resistant to fluoroquinolones . our sequencing results revealed serine to leucine mutation at position 83 of gyra subunit in 38 of 44 ciprofloxacin - resistant acinetobacter isolates . similar to study by park et al ( 15 ) , this is indicative of the fact that ser83leu substitution is the principal mutation in a. baumannii for resistance to fluoroquinolones . other mutations in the gyra gene resulting in ala84pro or gly81val reported in ciprofloxacin resistant isolates of previous study ( 16 ) were not observed in our included isolates . instead , we found a novel mutation in the gyra gene , leading to gly81asp substitution dna gyrase , in six resistant isolates with a mic range at 8 to 128 g / ml . in a. baumannii , topoisomerase iv is a target of quinolones and mutations at residues ser80 and glu84 of parc contribute to decreased fluoroquino - lone susceptibility ( 16 ) . although parc mutations always along with mutations in gyra are needed to acquire a high - level resistance to quinolones ( 4 ) , two clinical isolates in our study had mutations in parc without gyra , suggesting that parc might not only be a secondary target for quinolones but is really as important as gyra to cause a decreased susceptibility to fluoroquinolones in a. baumannii . on the other hand , qrdrs of the three isolates with intermediate - resistance to ciprofloxacin in the current study did not possess alterations associated with fluoroquino - lones resistance in the sequence of either genes , indicating other resistance mechanisms , such as efflux systems should be considered in these isolates ( 17 ) . although a single point mutation in dna gyrase is enough for resistance to fluoroquinolone in a. baumannii , the concurrent mutations within qrdr regions of the gyra and parc genes are expected to significantly contribute to high - level fluoroquinolone resistance . further studies are required to elucidate mechanisms , other than alterations in gyra and parc , leading to decreased susceptibility to quinolones in a. baumannii isolates .

denmark s national health service provides free and equal access to health care for the entire population . health services related events are recorded in national databases , including the danish hospital register and the danish prescription database . information from both registries can be linked to each other by the use of the unique 10-digit central population registry number applied to all residents in denmark . parkinson s disease cases were ascertained from the computerized danish hospital register , with all hospitalizations with parkinson s disease diagnoses registered since 1977 and all clinic visits , including outpatient clinics , since 1995 . individual information on the date of death , disappearance , or emigration was obtained from the central population registry . we identified 82,140 subjects ( 13,695 patients with parkinson s disease and 68,445 individuals without parkinson s disease ) in the danish hospital register in the period 19862006 who 1 ) had a valid central population registry number , 2 ) were aged > 35 years at the time of diagnosis , and 3 ) had not emigrated from denmark . however , to allow for long enough lag times between diabetes and parkinson s disease , only individuals who were registered for the first time with a primary diagnosis of parkinson s disease ( icd-10 code g20 ) between january 2001 and december 2006 ( or had no previous hospitalization ) were considered for inclusion as case subjects in our analyses . earliest date of a parkinson s disease diagnosis , we dated the primary parkinson s disease diagnoses back to either the first hospital or outpatient record that ever mentioned parkinson s disease or the first prescription of parkinson s disease medications ( anatomical therapeutic chemical [ atc ] code n04b ) since inception of the danish prescription database ( 1995 ) , whichever came first . patients with parkinson s disease whose backdated diagnosis date fell into the period prior to 2001 were excluded , leaving 2,188 patients with parkinson s disease . of these , we further excluded patients with parkinson s disease who had never received a parkinson s disease drug prescription ( 257 case subjects ) . to more efficiently control for confounding by the two most important risk factors for parkinson s disease , we then individually matched five randomly selected control subjects ( i.e. , individuals without parkinson s disease at the index date ) from the central population registry by sex and year of birth to each case subject , using incidence - density sampling ( 17 ) . the date of parkinson s disease diagnosis served as the index date for control selection . this left a total of 1,931 case subjects with parkinson s disease and 9,651 control subjects for our primary analyses . in secondary analyses , to further examine the potential for disease misclassification , we excluded parkinson s disease case subjects diagnosed with any type of dementia ( both alzheimer s type and unspecified for icd-8 codes 2900929011 , 2901829019 , and 29309 and for icd-10 codes f00.x , f03 , and g30 ) or cerebrovascular disease ( icd-8 codes 430438 and icd-10 codes i60i69 and g45g46 ) in the 2 years preceding their parkinson s disease diagnosis ( 135 case subjects ) and case subjects who used neuroleptics ( atc codes n05aa , n05ab , n05ac , n05ad , n05af , and n05ag ) in the 6 months preceding parkinson s disease diagnosis ( 194 case subjects ) . finally , we obtained a full history of hospitalization backdating to 1977 for all case and control subjects and calculated the charlson comorbidity score , a weighted index of 19 medical conditions ( 18 ) , with a lag - time of 5 years prior to index date as an indicator of baseline morbidity . 2002 - 41 - 2112 ) and the university of california los angeles human subject review board . information on whether parkinson s disease case and control subjects had a diabetes diagnosis prior to the index date was extracted by use of the central population registry number from the national danish hospital register using the icd codes for diabetes ( 249 and 250.x for icd-8 and e10e14 for icd-10 ) . because icd misclassification of insulin - dependent diabetes ( also known as type 1 diabetes , icd-8 code 249 and icd-10 code e10 ) and non insulin - dependent diabetes ( also known as type 2 diabetes , icd-8 code 250 and icd-10 codes e11e14 ) is prevalent ( 19,20 ) , and because of inconsistencies in diabetes codes between icd-8 and icd-10 , we chose not to present stratified analyses by subtype of diabetes . since 1 january 1995 , the danish prescription database has received data on all dispensed prescriptions from pharmacies in denmark , including the individual s central population registry number , drug type by atc code , and prescription dispensing date . use of antidiabetes drugs by study subjects prior to the index date was extracted from this database , including atc groups a10a ( insulins and analogs ) , a10b ( oral blood glucose lowering drugs , including sulfonylureas [ e.g. , gliclazide ] and biguanides [ e.g. , metformin ] ) , and a10x ( other drugs used in diabetes ) . we defined antidiabetes drug use as any filling of one or more prescriptions during the relevant period prior to the index date ; nonusers never filled a single prescription . we also categorized study participants into insulin users versus oral diabetes medication users according to atc code . we used unconditional logistic regression to calculate odds ratios ( ors ) and 95% cis for diabetes , while adjusting for age ( continuous ) , sex , and chronic obstructive pulmonary disease ( copd ; as a proxy for heavy smoking ) diagnosis ( icd-8 codes 490.00 , 491.00 , 491.01 , and 491.03 and icd-10 code j44 ) identified in the danish hospital register . comorbidities registered before the index date ( using the charlson index ) were based on icd codes for 19 chronic disease categories recorded in the hospital records , including diabetes ( 18 ) . parameter estimates changed by < 10% when controlling for the charlson index in multivariate models , so it was excluded from additional analyses . to further preclude the possibility of having included prevalent cases , for our primary analyses , we advanced ( lagged ) the index date for antidiabetes drug prescriptions or diagnosis of diabetes ( using icd codes ) by 2 years ( i.e. , we excluded all first treatments and diagnoses of diabetes within a 2-year period prior to the index date ) . in addition , we performed nonlagged and 5-year - lagged analyses of parkinson s disease . we lagged copd by 5 years to capture the general health status of subjects prior to the index date . in secondary analyses , we stratified by age at first diagnosis ( aged 60 and > 60 years ) and sex . in sensitivity analyses aimed at reducing parkinson s disease misclassification , we excluded case subjects ( and their matched control subjects ) and all control subjects diagnosed with dementia or cerebrovascular disease 2 years prior to parkinson s disease diagnosis of the index case . information on whether parkinson s disease case and control subjects had a diabetes diagnosis prior to the index date was extracted by use of the central population registry number from the national danish hospital register using the icd codes for diabetes ( 249 and 250.x for icd-8 and e10e14 for icd-10 ) . because icd misclassification of insulin - dependent diabetes ( also known as type 1 diabetes , icd-8 code 249 and icd-10 code e10 ) and non insulin - dependent diabetes ( also known as type 2 diabetes , icd-8 code 250 and icd-10 codes e11e14 ) is prevalent ( 19,20 ) , and because of inconsistencies in diabetes codes between icd-8 and icd-10 , we chose not to present stratified analyses by subtype of diabetes . since 1 january 1995 , the danish prescription database has received data on all dispensed prescriptions from pharmacies in denmark , including the individual s central population registry number , drug type by atc code , and prescription dispensing date . use of antidiabetes drugs by study subjects prior to the index date was extracted from this database , including atc groups a10a ( insulins and analogs ) , a10b ( oral blood glucose lowering drugs , including sulfonylureas [ e.g. , gliclazide ] and biguanides [ e.g. , metformin ] ) , and a10x ( other drugs used in diabetes ) . we defined antidiabetes drug use as any filling of one or more prescriptions during the relevant period prior to the index date ; nonusers never filled a single prescription . we also categorized study participants into insulin users versus oral diabetes medication users according to atc code . we used unconditional logistic regression to calculate odds ratios ( ors ) and 95% cis for diabetes , while adjusting for age ( continuous ) , sex , and chronic obstructive pulmonary disease ( copd ; as a proxy for heavy smoking ) diagnosis ( icd-8 codes 490.00 , 491.00 , 491.01 , and 491.03 and icd-10 code j44 ) identified in the danish hospital register . comorbidities registered before the index date ( using the charlson index ) were based on icd codes for 19 chronic disease categories recorded in the hospital records , including diabetes ( 18 ) . parameter estimates changed by < 10% when controlling for the charlson index in multivariate models , so it was excluded from additional analyses . to further preclude the possibility of having included prevalent cases , for our primary analyses , we advanced ( lagged ) the index date for antidiabetes drug prescriptions or diagnosis of diabetes ( using icd codes ) by 2 years ( i.e. , we excluded all first treatments and diagnoses of diabetes within a 2-year period prior to the index date ) . in addition , we performed nonlagged and 5-year - lagged analyses of parkinson s disease . we lagged copd by 5 years to capture the general health status of subjects prior to the index date . in secondary analyses , we stratified by age at first diagnosis ( aged 60 and > 60 years ) and sex . in sensitivity analyses aimed at reducing parkinson s disease misclassification , we excluded case subjects ( and their matched control subjects ) and all control subjects diagnosed with dementia or cerebrovascular disease 2 years prior to parkinson s disease diagnosis of the index case . case subjects and their control subjects combined were on average 72.2 years of age ( sd 10.2 ) at the index date . five years prior to the index date , case subjects had less copd than control subjects , although this difference was not statistically significant ( table 1 ) . at the index date , 6.4% of our study population had received a prescription for any type of oral antidiabetes medication or insulin ; antidiabetes drug prescriptions included oral blood glucose lowering drugs , including sulfonylureas ( e.g. , gliclazide [ 43.4% ] ) and biguanides ( e.g. , metformin [ 25.3% ] ) as well as insulin ( 26.6% ) , and 53% of subjects had received more than one type of antidiabetes prescription . the median length of time for receiving prescriptions for oral antidiabetes medication since 1995 and prior to the index date was 5.5 years for case subjects and 5.2 years for control subjects and for insulin was 6.0 years among the case subjects versus 4.9 years in the control subjects . characteristics of the danish study population , 20012006 data are n ( % ) or means sd . * five - year lag : variables are ascertained 5 years prior to the index date or date of parkinson s disease diagnosis ; copd is a proxy for heavy smoking ; charlson comorbidity score is a weighted index of 19 medical conditions . in our main logistic regression analyses using a 2-year lag and relying on diabetes diagnoses based on icd codes , we estimated an overall 36% increase in the risk of developing parkinson s disease ( 95% ci 1.081.71 ) among those with diabetes ( data not shown ) . in stratified analyses ( with a 2-year lag ) , associations were slightly stronger in women ( or 1.50 [ 95% ci 1.022.22 ] ) than in men ( 1.29 [ 0.971.72 ] ) and mainly seen in all patients with early - onset parkinson s disease ( i.e. , aged < 60 years at parkinson s disease diagnosis ; 2.68 [ 1.046.91 ] ) compared with those with late - onset parkinson s disease ( i.e. , aged 60 years at parkinson s disease diagnosis ; 1.16 [ 0.851.57 ] ) . relying on antidiabetes drug use to identify participants with diabetes , we found strikingly similar associations , suggesting a positive association for parkinson s disease for those who had ever used antidiabetes drugs > 2 years prior to parkinson s disease onset ( or 1.35 [ 95% ci 1.101.65 ] ) ( table 2 ) . in analyses based on 0-year and 5-year lags instead , our risk estimates were comparable in size ( 0-year lag : 1.21 [ 1.001.47 ] ; 5-year lag : 1.35 [ 1.071.72 ] ) . overall , these associations were somewhat stronger for users of oral diabetes medication than for insulin users ( or 1.37 vs. 1.22 ) ( see table 2 ) . association between diabetes ( 2-year lag , i.e. , diabetes was present at least 2 years prior to the index date ) and parkinson s disease * data are n or or ( 95% ci ) . model 1 : adjusted for age and sex ; model 2 : adjusted for age , sex , and copd ( lagged 5 years ) . * diagnosis of diabetes based on any prescription of antidiabetes drugs ( atc codes ) . in stratified analyses ( with a 2-year lag ) , risk estimates differed for men and women and also for those younger ( aged < 60 years ) or older ( aged 60 years ) at diagnosis / index date , whereas the risk of developing parkinson s disease associated with the use of any type of antidiabetes drug was very similar for men and women ( men : or 1.33 [ 95% ci 1.031.72 ] ; women : 1.38 [ 0.991.92 ] ) ; among those using oral diabetes medication , only women were at higher risk of developing parkinson s disease ( men : 0.74 [ 0.371.50 ] ; women : 2.92 [ 1.346.36 ] ) . by contrast , men with diabetes who use insulin experienced a 48% higher risk of developing parkinson s disease ( 1.48 [ 1.131.95 ] ) , whereas the risk was slightly lower in women ( 1.20 [ 0.831.74 ] ) . similar to our icd code based results , when stratifying by age at onset of parkinson s disease ( aged < 60 years vs. 60 years at onset ) , the risk of parkinson s disease was much higher among patients with diabetes using antidiabetes drugs in early onset ( 3.07 [ 1.655.70 ] ) than in late - onset parkinson s disease ( 1.24 [ 0.991.53 ] ) . on closer examination , this difference tended to be largely attributed to insulin use , as opposed to the use of oral antidiabetes medications : for early - onset parkinson s disease ( albeit based on two cases only ) , the risk associated with insulin use was 3.74 ( 1.897.38 ) and 1.32 ( 0.286.26 ) for use of oral antidiabetes medication , whereas these risks were 1.24 ( 0.981.57 ) and 1.21 ( 0.712.06 ) , respectively , for late - onset parkinson s disease . generally , these associations tended to be attenuated in 0-lag analyses and slightly stronger in the 5-year lag analyses ( data not shown ) . when we further stratified by sex , albeit based on small case numbers , we found these associations particularly for oral antidiabetes medications ( we did not have enough power to examine this for insulin use ) , again stronger for early - onset parkinson s disease , regardless of sex . in sensitivity analyses , we excluded parkinson s disease patients and control subjects diagnosed with dementia or cerebrovascular diseases 2 years prior to the index date , but the risk estimates changed only minimally ( data not shown ) . similarly , excluding case subjects with neuroleptic use within 6 months prior to parkinson s disease diagnoses did not alter our findings substantially ( or for any use of antidiabetic drugs 1.26 [ 95% ci 1.011.56 ] ) . we identified all case subjects with a primary diagnosis of parkinson s disease in denmark from hospital and outpatient clinic records between 2001 and 2006 . in our analyses , both icd code based hospital and outpatient clinic reports of diagnoses of diabetes ( which does not allow us to distinguish between type 1 and type 2 diabetes ) , as well as the use of antidiabetes drugs ( which , to some degree , allows us to distinguish between type 1 and type 2 diabetes , as individuals using oral diabetes medications only are likely to have type 2 diabetes ) , these associations became stronger with longer lag times and showed differences in subgroup analyses ( i.e. , the associations were markedly stronger for early - onset parkinson s disease , particularly for insulin use , vs. the risk for early - onset diabetes and was more modest when oral antidiabetes drugs were used to treat diabetes ) . among female subjects , oral antidiabetes drugs were more strongly associated with overall risk of parkinson s disease than among men , and oral antidiabetes drugs also appeared to drive the association with early - onset diabetes in men . observational studies that previously examined associations between diabetes and parkinson s disease have produced mixed results . although most of the published epidemiologic studies reported a positive association between diabetes and parkinson s disease ( 2,9,10,13 ) or parkinsonian signs ( 5 ) and drug - induced parkinsonism ( 4 ) , some studies reported no association ( 6,11 ) and others even inverse associations ( 7,12 ) . a cross - sectional study of 791 patients with parkinsonism that had linked u.s . national survey data of 24,831 elderly to 1.9 million medicare claims found that those with parkinsonism had a 50% ( men ) to 70% ( women ) higher risk of concurrent diabetes ( 13 ) . in a selected group of 1,030 u.s . elderly subjects without a diagnosis of parkinson s disease , diabetes was found to be associated with a more severe score of global parkinsonian signs , which was used to determine the degree of motor dysfunction ( 5 ) . similar to our results , these researchers also found the association of diabetes with parkinsonian signs to be similar among men and women but stronger in younger compared with older patients ( 5 ) . by contrast , in a case - control study of 490 parkinson s disease patients derived from a neurologist s private - practice database , the prevalence of diabetes was found to be similar in those with and without parkinson s disease ( 12.9 vs. 12.1% ) ( 11 ) . three small case - control studies of 352 ( 12 ) , 318 ( 7 ) , and 178 ( 14 ) newly diagnosed patients with parkinson s disease reported inverse associations with diabetes prior to parkinson s disease ( or 0.52 for men with diabetes vs. or 0.80 for women with diabetes in the first , or 0.40 in the second , and or 0.30 in the third study ) . moreover , a study based on the u.k . general practice research database reported the diabetes prevalence to be similar in patients with and without parkinson s disease and the risk of developing diabetes to be lower in parkinson s disease patients ( 6 ) . in the most recent retrospective study , a hospital - based case - control study conducted in japan ( 16 ) and comprising 250 new - onset parkinson s disease ( within 6 years of diagnosis ) , a decreased risk of parkinson s disease was observed among individuals with a history of diabetes ( or 0.38 [ 95% ci 0.170.79 ] ) . this risk did not vary by sex ; however , neither was age at onset of parkinson s disease nor type of medication used taken into account in this retrospective analysis . the few prospective studies that evaluated associations between diabetes and parkinson s disease published to date tended to report positive associations between the two conditions . in a prospective study of 51,552 finnish men and women , a diabetes diagnosis at baseline was associated with an 85% increased risk of developing parkinson s disease ( 9 ) . based on data from two large prospective cohorts , the nurses health study cohort and the health professionals follow - up study ( 15 ) , the relative risk for developing parkinson s disease was 1.12 ( 95% ci 0.691.81 ) among those with a baseline history of diabetes . ( 8) , who used data from a large male prospective cohort , the physicians health study . the authors describe an overall increased risk of parkinson s disease associated with diabetes ( 1.34 [ 1.011.77 ] ) , a relative risk strikingly similar in magnitude to our own findings . however , in contrast to our own results , they reported that most of the excess diabetes risk occurred in the year prior to and the year of parkinson s disease diagnosis and speculated that this positive association may therefore either be driven by surveillance bias or by a common underlying biologic mechanism causing both diseases and yet to be determined . in our study , we excluded both first diabetes diagnoses based on icd codes as well as antidiabetes drug use by up to 5 years prior to index date and found that associations tended to become stronger with such lagging , especially when using antidiabetes medications to identify diabetic case subjects . in a previous publication based on the same danish dataset , we explored associations between autoimmune diseases , including insulin - dependent diabetes defined by icd-8 code 249 and parkinson s disease risk during a longer period of observation ( 19862006 ) ( 21 ) . on the basis of icd codes alone , we observed a higher risk of parkinson s disease ( or 1.6 [ 95% ci 1.022.5 ] ) only in women , whereas no association was apparent among men using insulin ( 0.8 [ 0.51.2 ] ) when lagging the diabetes diagnoses by 5 years . differences in the present results are likely attributed to the different types of diabetes included in the studies ( only insulin - dependent diabetes in the previous study and both insulin and non insulin dependent diabetes in this study ) and possibly also our use of a more refined parkinson s disease definition based on a combination of parkinson s disease icd codes and parkinson s disease drug prescriptions in the current study . insulin has been implicated in alzheimer s disease risk . whether this is primarily a result of vessel damage caused by long - term elevated blood glucose levels as the primary mechanism behind these associations is currently unknown . however , given that parkinson s disease is not primarily a vascular disease , we speculate that an association between diabetes and parkinson s disease , as found in our study , could suggest a different pathway , especially because the associations seemed to be stronger in younger - onset parkinson s disease ( i.e. , patients less likely to be affected by cerebrovascular disease ) . one such pathway might be related to vitamin d levels , which have been implicated in both diseases ( i.e. , it has been suggested that vitamin d lowers both the risk of parkinson s disease and type 2 diabetes ) ( 2224 ) . we found substantial differences in estimated effect size between diabetes and parkinson s disease by age at onset of parkinson s disease , although this difference is based on small numbers of patients with early onset . only one previous study , examining the association between diabetes and parkinsonian signs , was able to address this , and they too report the association between diabetes with parkinsonian signs to be stronger in younger compared with older patients ( 5 ) . although there also were differences for insulin users versus oral diabetes medication users in relation to parkinson s disease by sex and age at onset , statistical power issues limited our ability to explore these substrata in greater detail . overall , the stronger associations with early - onset parkinson s disease are suggestive of a common genetic origin and might serve as an explanation as to why previous studies were contradictory , as they were not able to stratify according to early- or late - onset parkinson s disease . recently , an intriguing hypothesis emerged that may link parkinson s disease and diabetes via the mitochondrial dysfunction pathway . neurons in the substantia nigra ( parkinson s disease ) as well as pancreatic islet -cells ( affected in diabetes ) have been described as cells with low respiratory capacity / low mitochondrial capacity and therefore have a greater sensitivity to defects or impairments in mitochondrial respiratory chain enzymes than other cells with higher respiratory capacity . thus , both cell types ( substantia nigra and islet -cells ) are more vulnerable to defects or toxins that further diminish the capacity to generate atp when the mitochondrial genome accumulates genetic lesions during aging . in a study comparing 64 japanese centenarians to 61 patients with parkinson s disease ( 25 ) , tanaka ( 25 ) found a by - far - greater frequency of deleterious mitochondrial variants and a much greater variety of amino acid replacements among parkinson s disease patients ; in contrast , such variations were absent in centenarians . given the above - mentioned bioenergetic similarity of substantia nigra and pancreatic islet -cells in terms of their ability to respond to mitochondrial impairment , this could provide a pathogenetic link between parkinson s disease and diabetes . strengths of our study are that control subjects were selected at random from a population registry and did not have to volunteer information for our study , thus avoiding bias attributed to selective nonparticipation . we required that all parkinson s disease case subjects had been admitted at least once with a primary diagnosis of parkinson s disease , and , whenever possible , we dated diagnoses back to the likely earliest diagnosis ( e.g. , a first prescription of parkinson s disease medications ) . limitations include some disease misclassification because primary parkinson s disease diagnoses were identified from hospital records that may have included some cases of nonidiopathic parkinsonism . sensitivity analyses in which we excluded parkinson s disease case subjects with prior diagnoses of dementia and cerebrovascular diseases in the 2 years before parkinson s disease diagnosis , and parkinson s disease case subjects with neuroleptic use in the 6 months preceding diagnosis suggested the bias to be minimal . we might have selected less healthy parkinson s disease case subjects more likely to be hospitalized than danish parkinson s disease patients seen exclusively by private practitioners without ever attending a specialty clinic before 2007 . the slightly higher charlson index and greater number of cardiovascular disease drug prescriptions among our study s parkinson s disease patients compared with control subjects 2 years prior to and at the index date ( data not shown ) supports , albeit only modestly , the possibility for such a selection bias . however , differences in general health status were not evident 5 years prior to parkinson s disease diagnosis / index date . the universal coverage of most health care expenses in denmark , including partial reimbursement of costs for prescribed drugs , makes it less likely that antidiabetes drug prescriptions or parkinson s disease diagnoses were influenced by factors determining access to care . in addition , we may have missed diabetes case subjects who never took any antidiabetes drugs ; however , results from icd - based diabetes diagnoses were very similar to those based on antidiabetes drug use . we were unable to fully control for smoking , known for its strong negative association with parkinson s disease , although our adjustment for copd as a proxy for heavy smoking may have , at least partly , controlled for smoking . in summary , evidence is accruing for an association between diabetes and parkinson s disease . whether this association is causal or a result of a common pathophysiologic pathway still needs to be determined , although a common biologic pathway appears to be the most plausible explanation at this point . future studies of the association between diabetes and parkinson s disease should take age at onset of parkinson s disease into account .

objectiveinsulin contributes to normal brain function . previous studies have suggested associations between midlife diabetes and neurodegenerative diseases , including parkinson s disease . using danish population registers , we investigated whether a history of diabetes or the use of antidiabetes drugs was associated with parkinson s disease.research design and methodsfrom the nationwide danish hospital register hospital records , we identified 1,931 patients with a first - time diagnosis of parkinson s disease between 2001 and 2006 . we randomly selected 9,651 population control subjects from the central population registry and density matched them by birth year and sex . pharmacy records comprising all antidiabetes and anti - parkinson drug prescriptions in denmark were available . odds ratios ( ors ) were estimated by logistic regression models.resultshaving diabetes , as defined by one or more hospitalizations and/or outpatient visits for the condition , was associated with a 36% increased risk of developing parkinson s disease ( or 1.36 [ 95% ci 1.081.71 ] ) . similarly , diabetes defined by the use of any antidiabetes medications was associated with a 35% increased parkinson s disease risk ( 1.35 [ 1.101.65 ] ) . when diabetes was defined as the use of oral antidiabetes medications , effect estimates were stronger in women ( 2.92 [ 1.346.36 ] ) , whereas when diabetes was defined as any antidiabetes drug prescription , patients with early - onset parkinson s disease were at highest risk ( i.e. , parkinson s disease diagnosed before the age of 60 years ; 3.07 [ 1.655.70]).conclusionswe found that a diagnosis of , or treatment received for , diabetes was significantly associated with an increased risk of developing parkinson s disease , especially younger - onset parkinson s disease . our results suggest a common pathophysiologic pathway between the two diseases . future studies should take age at parkinson s disease onset into account .

RESEARCH DESIGN AND METHODS Assessment of diabetes diagnosis and antidiabetes drug use Statistical analysis RESULTS CONCLUSIONS

information from both registries can be linked to each other by the use of the unique 10-digit central population registry number applied to all residents in denmark . parkinson s disease cases were ascertained from the computerized danish hospital register , with all hospitalizations with parkinson s disease diagnoses registered since 1977 and all clinic visits , including outpatient clinics , since 1995 . individual information on the date of death , disappearance , or emigration was obtained from the central population registry . we identified 82,140 subjects ( 13,695 patients with parkinson s disease and 68,445 individuals without parkinson s disease ) in the danish hospital register in the period 19862006 who 1 ) had a valid central population registry number , 2 ) were aged > 35 years at the time of diagnosis , and 3 ) had not emigrated from denmark . however , to allow for long enough lag times between diabetes and parkinson s disease , only individuals who were registered for the first time with a primary diagnosis of parkinson s disease ( icd-10 code g20 ) between january 2001 and december 2006 ( or had no previous hospitalization ) were considered for inclusion as case subjects in our analyses . earliest date of a parkinson s disease diagnosis , we dated the primary parkinson s disease diagnoses back to either the first hospital or outpatient record that ever mentioned parkinson s disease or the first prescription of parkinson s disease medications ( anatomical therapeutic chemical [ atc ] code n04b ) since inception of the danish prescription database ( 1995 ) , whichever came first . patients with parkinson s disease whose backdated diagnosis date fell into the period prior to 2001 were excluded , leaving 2,188 patients with parkinson s disease . of these , we further excluded patients with parkinson s disease who had never received a parkinson s disease drug prescription ( 257 case subjects ) . to more efficiently control for confounding by the two most important risk factors for parkinson s disease , we then individually matched five randomly selected control subjects ( i.e. , individuals without parkinson s disease at the index date ) from the central population registry by sex and year of birth to each case subject , using incidence - density sampling ( 17 ) . in secondary analyses , to further examine the potential for disease misclassification , we excluded parkinson s disease case subjects diagnosed with any type of dementia ( both alzheimer s type and unspecified for icd-8 codes 2900929011 , 2901829019 , and 29309 and for icd-10 codes f00.x , f03 , and g30 ) or cerebrovascular disease ( icd-8 codes 430438 and icd-10 codes i60i69 and g45g46 ) in the 2 years preceding their parkinson s disease diagnosis ( 135 case subjects ) and case subjects who used neuroleptics ( atc codes n05aa , n05ab , n05ac , n05ad , n05af , and n05ag ) in the 6 months preceding parkinson s disease diagnosis ( 194 case subjects ) . finally , we obtained a full history of hospitalization backdating to 1977 for all case and control subjects and calculated the charlson comorbidity score , a weighted index of 19 medical conditions ( 18 ) , with a lag - time of 5 years prior to index date as an indicator of baseline morbidity . information on whether parkinson s disease case and control subjects had a diabetes diagnosis prior to the index date was extracted by use of the central population registry number from the national danish hospital register using the icd codes for diabetes ( 249 and 250.x for icd-8 and e10e14 for icd-10 ) . since 1 january 1995 , the danish prescription database has received data on all dispensed prescriptions from pharmacies in denmark , including the individual s central population registry number , drug type by atc code , and prescription dispensing date . use of antidiabetes drugs by study subjects prior to the index date was extracted from this database , including atc groups a10a ( insulins and analogs ) , a10b ( oral blood glucose lowering drugs , including sulfonylureas [ e.g. we used unconditional logistic regression to calculate odds ratios ( ors ) and 95% cis for diabetes , while adjusting for age ( continuous ) , sex , and chronic obstructive pulmonary disease ( copd ; as a proxy for heavy smoking ) diagnosis ( icd-8 codes 490.00 , 491.00 , 491.01 , and 491.03 and icd-10 code j44 ) identified in the danish hospital register . comorbidities registered before the index date ( using the charlson index ) were based on icd codes for 19 chronic disease categories recorded in the hospital records , including diabetes ( 18 ) . to further preclude the possibility of having included prevalent cases , for our primary analyses , we advanced ( lagged ) the index date for antidiabetes drug prescriptions or diagnosis of diabetes ( using icd codes ) by 2 years ( i.e. in addition , we performed nonlagged and 5-year - lagged analyses of parkinson s disease . in sensitivity analyses aimed at reducing parkinson s disease misclassification , we excluded case subjects ( and their matched control subjects ) and all control subjects diagnosed with dementia or cerebrovascular disease 2 years prior to parkinson s disease diagnosis of the index case . information on whether parkinson s disease case and control subjects had a diabetes diagnosis prior to the index date was extracted by use of the central population registry number from the national danish hospital register using the icd codes for diabetes ( 249 and 250.x for icd-8 and e10e14 for icd-10 ) . because icd misclassification of insulin - dependent diabetes ( also known as type 1 diabetes , icd-8 code 249 and icd-10 code e10 ) and non insulin - dependent diabetes ( also known as type 2 diabetes , icd-8 code 250 and icd-10 codes e11e14 ) is prevalent ( 19,20 ) , and because of inconsistencies in diabetes codes between icd-8 and icd-10 , we chose not to present stratified analyses by subtype of diabetes . since 1 january 1995 , the danish prescription database has received data on all dispensed prescriptions from pharmacies in denmark , including the individual s central population registry number , drug type by atc code , and prescription dispensing date . use of antidiabetes drugs by study subjects prior to the index date was extracted from this database , including atc groups a10a ( insulins and analogs ) , a10b ( oral blood glucose lowering drugs , including sulfonylureas [ e.g. we defined antidiabetes drug use as any filling of one or more prescriptions during the relevant period prior to the index date ; nonusers never filled a single prescription . we used unconditional logistic regression to calculate odds ratios ( ors ) and 95% cis for diabetes , while adjusting for age ( continuous ) , sex , and chronic obstructive pulmonary disease ( copd ; as a proxy for heavy smoking ) diagnosis ( icd-8 codes 490.00 , 491.00 , 491.01 , and 491.03 and icd-10 code j44 ) identified in the danish hospital register . comorbidities registered before the index date ( using the charlson index ) were based on icd codes for 19 chronic disease categories recorded in the hospital records , including diabetes ( 18 ) . to further preclude the possibility of having included prevalent cases , for our primary analyses , we advanced ( lagged ) the index date for antidiabetes drug prescriptions or diagnosis of diabetes ( using icd codes ) by 2 years ( i.e. in sensitivity analyses aimed at reducing parkinson s disease misclassification , we excluded case subjects ( and their matched control subjects ) and all control subjects diagnosed with dementia or cerebrovascular disease 2 years prior to parkinson s disease diagnosis of the index case . at the index date , 6.4% of our study population had received a prescription for any type of oral antidiabetes medication or insulin ; antidiabetes drug prescriptions included oral blood glucose lowering drugs , including sulfonylureas ( e.g. in our main logistic regression analyses using a 2-year lag and relying on diabetes diagnoses based on icd codes , we estimated an overall 36% increase in the risk of developing parkinson s disease ( 95% ci 1.081.71 ) among those with diabetes ( data not shown ) . in stratified analyses ( with a 2-year lag ) , associations were slightly stronger in women ( or 1.50 [ 95% ci 1.022.22 ] ) than in men ( 1.29 [ 0.971.72 ] ) and mainly seen in all patients with early - onset parkinson s disease ( i.e. , aged < 60 years at parkinson s disease diagnosis ; 2.68 [ 1.046.91 ] ) compared with those with late - onset parkinson s disease ( i.e. , aged 60 years at parkinson s disease diagnosis ; 1.16 [ 0.851.57 ] ) . relying on antidiabetes drug use to identify participants with diabetes , we found strikingly similar associations , suggesting a positive association for parkinson s disease for those who had ever used antidiabetes drugs > 2 years prior to parkinson s disease onset ( or 1.35 [ 95% ci 1.101.65 ] ) ( table 2 ) . in analyses based on 0-year and 5-year lags instead , our risk estimates were comparable in size ( 0-year lag : 1.21 [ 1.001.47 ] ; 5-year lag : 1.35 [ 1.071.72 ] ) . , diabetes was present at least 2 years prior to the index date ) and parkinson s disease * data are n or or ( 95% ci ) . in stratified analyses ( with a 2-year lag ) , risk estimates differed for men and women and also for those younger ( aged < 60 years ) or older ( aged 60 years ) at diagnosis / index date , whereas the risk of developing parkinson s disease associated with the use of any type of antidiabetes drug was very similar for men and women ( men : or 1.33 [ 95% ci 1.031.72 ] ; women : 1.38 [ 0.991.92 ] ) ; among those using oral diabetes medication , only women were at higher risk of developing parkinson s disease ( men : 0.74 [ 0.371.50 ] ; women : 2.92 [ 1.346.36 ] ) . by contrast , men with diabetes who use insulin experienced a 48% higher risk of developing parkinson s disease ( 1.48 [ 1.131.95 ] ) , whereas the risk was slightly lower in women ( 1.20 [ 0.831.74 ] ) . similar to our icd code based results , when stratifying by age at onset of parkinson s disease ( aged < 60 years vs. 60 years at onset ) , the risk of parkinson s disease was much higher among patients with diabetes using antidiabetes drugs in early onset ( 3.07 [ 1.655.70 ] ) than in late - onset parkinson s disease ( 1.24 [ 0.991.53 ] ) . on closer examination , this difference tended to be largely attributed to insulin use , as opposed to the use of oral antidiabetes medications : for early - onset parkinson s disease ( albeit based on two cases only ) , the risk associated with insulin use was 3.74 ( 1.897.38 ) and 1.32 ( 0.286.26 ) for use of oral antidiabetes medication , whereas these risks were 1.24 ( 0.981.57 ) and 1.21 ( 0.712.06 ) , respectively , for late - onset parkinson s disease . when we further stratified by sex , albeit based on small case numbers , we found these associations particularly for oral antidiabetes medications ( we did not have enough power to examine this for insulin use ) , again stronger for early - onset parkinson s disease , regardless of sex . similarly , excluding case subjects with neuroleptic use within 6 months prior to parkinson s disease diagnoses did not alter our findings substantially ( or for any use of antidiabetic drugs 1.26 [ 95% ci 1.011.56 ] ) . we identified all case subjects with a primary diagnosis of parkinson s disease in denmark from hospital and outpatient clinic records between 2001 and 2006 . in our analyses , both icd code based hospital and outpatient clinic reports of diagnoses of diabetes ( which does not allow us to distinguish between type 1 and type 2 diabetes ) , as well as the use of antidiabetes drugs ( which , to some degree , allows us to distinguish between type 1 and type 2 diabetes , as individuals using oral diabetes medications only are likely to have type 2 diabetes ) , these associations became stronger with longer lag times and showed differences in subgroup analyses ( i.e. , the associations were markedly stronger for early - onset parkinson s disease , particularly for insulin use , vs. the risk for early - onset diabetes and was more modest when oral antidiabetes drugs were used to treat diabetes ) . among female subjects , oral antidiabetes drugs were more strongly associated with overall risk of parkinson s disease than among men , and oral antidiabetes drugs also appeared to drive the association with early - onset diabetes in men . although most of the published epidemiologic studies reported a positive association between diabetes and parkinson s disease ( 2,9,10,13 ) or parkinsonian signs ( 5 ) and drug - induced parkinsonism ( 4 ) , some studies reported no association ( 6,11 ) and others even inverse associations ( 7,12 ) . elderly subjects without a diagnosis of parkinson s disease , diabetes was found to be associated with a more severe score of global parkinsonian signs , which was used to determine the degree of motor dysfunction ( 5 ) . by contrast , in a case - control study of 490 parkinson s disease patients derived from a neurologist s private - practice database , the prevalence of diabetes was found to be similar in those with and without parkinson s disease ( 12.9 vs. 12.1% ) ( 11 ) . three small case - control studies of 352 ( 12 ) , 318 ( 7 ) , and 178 ( 14 ) newly diagnosed patients with parkinson s disease reported inverse associations with diabetes prior to parkinson s disease ( or 0.52 for men with diabetes vs. or 0.80 for women with diabetes in the first , or 0.40 in the second , and or 0.30 in the third study ) . general practice research database reported the diabetes prevalence to be similar in patients with and without parkinson s disease and the risk of developing diabetes to be lower in parkinson s disease patients ( 6 ) . in the most recent retrospective study , a hospital - based case - control study conducted in japan ( 16 ) and comprising 250 new - onset parkinson s disease ( within 6 years of diagnosis ) , a decreased risk of parkinson s disease was observed among individuals with a history of diabetes ( or 0.38 [ 95% ci 0.170.79 ] ) . this risk did not vary by sex ; however , neither was age at onset of parkinson s disease nor type of medication used taken into account in this retrospective analysis . the few prospective studies that evaluated associations between diabetes and parkinson s disease published to date tended to report positive associations between the two conditions . in a prospective study of 51,552 finnish men and women , a diabetes diagnosis at baseline was associated with an 85% increased risk of developing parkinson s disease ( 9 ) . based on data from two large prospective cohorts , the nurses health study cohort and the health professionals follow - up study ( 15 ) , the relative risk for developing parkinson s disease was 1.12 ( 95% ci 0.691.81 ) among those with a baseline history of diabetes . the authors describe an overall increased risk of parkinson s disease associated with diabetes ( 1.34 [ 1.011.77 ] ) , a relative risk strikingly similar in magnitude to our own findings . in our study , we excluded both first diabetes diagnoses based on icd codes as well as antidiabetes drug use by up to 5 years prior to index date and found that associations tended to become stronger with such lagging , especially when using antidiabetes medications to identify diabetic case subjects . in a previous publication based on the same danish dataset , we explored associations between autoimmune diseases , including insulin - dependent diabetes defined by icd-8 code 249 and parkinson s disease risk during a longer period of observation ( 19862006 ) ( 21 ) . on the basis of icd codes alone , we observed a higher risk of parkinson s disease ( or 1.6 [ 95% ci 1.022.5 ] ) only in women , whereas no association was apparent among men using insulin ( 0.8 [ 0.51.2 ] ) when lagging the diabetes diagnoses by 5 years . differences in the present results are likely attributed to the different types of diabetes included in the studies ( only insulin - dependent diabetes in the previous study and both insulin and non insulin dependent diabetes in this study ) and possibly also our use of a more refined parkinson s disease definition based on a combination of parkinson s disease icd codes and parkinson s disease drug prescriptions in the current study . however , given that parkinson s disease is not primarily a vascular disease , we speculate that an association between diabetes and parkinson s disease , as found in our study , could suggest a different pathway , especially because the associations seemed to be stronger in younger - onset parkinson s disease ( i.e. we found substantial differences in estimated effect size between diabetes and parkinson s disease by age at onset of parkinson s disease , although this difference is based on small numbers of patients with early onset . overall , the stronger associations with early - onset parkinson s disease are suggestive of a common genetic origin and might serve as an explanation as to why previous studies were contradictory , as they were not able to stratify according to early- or late - onset parkinson s disease . in a study comparing 64 japanese centenarians to 61 patients with parkinson s disease ( 25 ) , tanaka ( 25 ) found a by - far - greater frequency of deleterious mitochondrial variants and a much greater variety of amino acid replacements among parkinson s disease patients ; in contrast , such variations were absent in centenarians . we required that all parkinson s disease case subjects had been admitted at least once with a primary diagnosis of parkinson s disease , and , whenever possible , we dated diagnoses back to the likely earliest diagnosis ( e.g. , a first prescription of parkinson s disease medications ) . the universal coverage of most health care expenses in denmark , including partial reimbursement of costs for prescribed drugs , makes it less likely that antidiabetes drug prescriptions or parkinson s disease diagnoses were influenced by factors determining access to care . in addition , we may have missed diabetes case subjects who never took any antidiabetes drugs ; however , results from icd - based diabetes diagnoses were very similar to those based on antidiabetes drug use . we were unable to fully control for smoking , known for its strong negative association with parkinson s disease , although our adjustment for copd as a proxy for heavy smoking may have , at least partly , controlled for smoking . future studies of the association between diabetes and parkinson s disease should take age at onset of parkinson s disease into account .

data are drawn from the whitehall ii cohort study , established in 19851988 on 10,308 individuals ( 67% men ) , aged 3555 years . individuals gave written consent to participate in the study , and the university college london ethics committee approved the study . since inception , sociodemographic , behavioral , anthropometric , and health - related factors have been assessed approximately every 5 years ( 1985/1988 , 1991/1994 , 1997/1999 , 2002/2004 , 2007/2009 , and 2012/2013 ) . accelerometer measurement was added to the study at the 2012/2013 wave of data collection for participants seen at the central london clinic and for those living in the south - eastern regions of england who were screened at home . whitehall ii data , protocols , and other metadata are available to the scientific community . the instructions were as follows : we would like to know about your activities at work and in your free time that involve physical activity . the questionnaire included 20 items on the amount of time spent in the following activities : walking , sport ( cycling , soccer , golf , swimming , and 2 open - ended questions on other sports ) , gardening ( weeding , mowing , and 1 open - ended question on other gardening activities ) , housework ( carrying heavy shopping , cooking , hanging out washing , and 2 open - ended questions on other housework ) , do - it - yourself activity ( washing a car , painting , or decorating , and 1 open - ended question on other do - it - yourself activity ) , and 2 open - ended questions on other activities . for each item , the participants were required to take into account activity patterns over the past 4 weeks to give an indication of usual activity and provide the total number of hours spent in that activity per week ( see question 82 of the health survey questionnaire online available at http://www.ucl.ac.uk/whitehallii/pdf/s7_hsq.pdf ) . for each physical activity , including the open - ended items , we assigned a metabolic equivalent ( met ) value using a compendium of energy costs . total physical activity was estimated as met.hour/week , the sum of the product of the intensity ( met ) and weekly duration ( hours / week ) of all reported activities . in addition , we calculated the total number of hours per week in moderate - and - vigorous physical activity ( mvpa ) defined as activities with associated met 3 ( eg , cycling , weeding , swimming , mowing ) . participants with no contraindications ( that is , allergies to plastic or metal , traveling abroad the following week ) were asked to wear a triaxial accelerometer ( geneactiv ; activinsights ltd , kimbolton , cambs , uk , http://www.geneactiv.org/ ) on their nondominant wrist for 9 consecutive ( 24-hour ) days . the accelerometer was sampled at 85.7 hz and as in previous studies using new generation accelerometers , acceleration was expressed relative to gravity ( g units ; 1 g = 9.81 m.s ) to reflect the fact that sensors are calibrated relative to gravity . calibration error was estimated based on static periods in the data and corrected if necessary . the euclidean norm ( magnitude ) of the 3 raw signals minus 1 g , with negative numbers rounded to zero , was used to quantify the acceleration related to the movement registered and expressed in milligravity ( mg , 1 mg = 0.00981 m.s ) . accelerometer data were processed in r ( cran.r-project.org/ ) using the ggir package and executed on moveecloud ( http://movelab.org/research/moveecloud/ ) , a computing facility for physical activity research . data extracted between the first and last midnight were retained for the analysis leading to a maximum of 24-hour measurements for 8 days . participants were included in the analysis if they had valid data ( 16 hour / day ) for at least 2 weekdays and 2 weekend days . nonwear time was estimated on the basis of the standard deviation ( sd ) and value range of each accelerometer axis , calculated for moving windows of 60 minutes with 15-minute increments . for each 15-minute period of time detected as nonwear time over the valid days , missing data were replaced by the mean value calculated from measurement on other days at the same time of day . besides accelerometer - assessed total activity , for each participant duration in moderate - and - vigorous physical activity was also calculated . because a validated threshold to define mvpa in older adults does not yet exist , we chose the 100 mg threshold based on the fact that walking at 4 km / hour is classified as moderate physical activity and is equivalent to an acceleration of 100 mg in a laboratory based study on 30 adults . in order to qualify as mvpa , at least 80% of the activity needed to be at 100 mg , for at least a period ( bout ) of 10 minutes , using moving 10-minute windows . in sensitivity analyses , a more stringent cut - point of 120 mg was chosen to define mvpa . as the observation period covered 8 days , the data were recoded so that our measure reflected physical activity over 1 week to match the questionnaire - assessed physical activity . if a participant had 3 valid weekend days or 6 weekdays , the wrist acceleration of the first and last full day of measurement ( for example , 2 tuesdays a week apart ) were averaged to represent 1 unique day . then , the mean accelerometer - assessed total physical activity ( mg ) over a week was calculated as : [ ( 5 mean daily weekday wrist acceleration + 2 mean daily week - end wrist acceleration)]/7 . the same rescaling was undertaken for time spent in mvpa per week ( hour / week ) . bmi was calculated as weight ( kg ) divided by height ( m ) squared . weight was measured in light clothing , using an electronic soehnle scale with digital readout ( leifheit as , nassau , germany ) . height was measured using a stadiometer with the participant standing completely erect with their head in the frankfort plane . fmi was calculated as the ratio of fat mass ( kg ) by height ( m ) squared . fat mass was estimated by bioimpedance using the tanita tbf-300 body composition analyzer ( tanita , arlington heights , ill , www.tanita.com/en/ ) . bioimpedance data were available on participants seen at the central london clinic ( n = 4524 ) but not at home . as this measure is influenced by the total amount of body fluid , those with renal insufficiency ( assessed by estimated glomerular filtration rate < 15 ) were excluded from the analysis on fmi ( n = 1 ) . sociodemographic variables included age , sex , ethnicity ( white , south asian , black , other ) , marital status ( married / cohabiting , single , widowed , divorced / separated ) , and socioeconomic status , measured by the highest qualification on leaving full - time education ( university / higher university degree , higher secondary school , lower secondary school , and lower primary school or below ) and occupational position at age 50 years ( high , intermediate and low , representing income , and status at work ) . alcohol consumption was assessed using a question on the quantity of alcohol consumed in the previous week and was classified as no alcohol consumption in the previous week , moderate alcohol consumption ( 114 units / week in women and 121 units / week in men ) , and heavy drinkers ( 15 + units in women and 21 + units in men ) . the frequency of fruit and vegetable consumption was assessed on a 9-point scale , ranging from seldom or never to 4 or more times a day . the associations of the physical activity measures ( total physical activity and mvpa , both assessed by questionnaire and accelerometer ) with adiposity markers were examined using linear regression with adiposity markers as the outcomes . the linearity assumption was tested by comparing the fit of a model including physical activity measures as linear terms to the fit of models including degree 1 and 2 fractional polynomial terms using the deviance differences between the models ( fracpoly command in stata 13 ; statacorp lp , college station , tx ) . subsequently , models were adjusted for sociodemographic and behavioral variables in order to control for potential confounding , including those because of clustering of health behaviors . we first assessed the association of adiposity markers with total physical activity , assessed using the questionnaire ( met.h / week ) and accelerometer ( mean acceleration ) , using quartiles as the metric for these 2 measures was different . then , total physical activity ( continuous ) and mvpa ( categories ) were entered simultaneously into the model , and percentage reduction in the associations was calculated to allow the contribution on mvpa to be estimated . then , the association of adiposity markers with mvpa was investigated using the following categories for both questionnaire and accelerometer measures : < 1.0 hour / week , 1.02.5 hours / week , 2.54.0 hours / week , and 4 hours / week . analyses were repeated using sex - specific standardized measures of adiposity in order to compare whether the associations differed by adiposity markers . to compare the fit of the models for questionnaire vs accelerometer measures , the interactions of physical activity measures with sex and age ( continuous and median - based classes ) were also tested . in sensitivity analyses , we repeated the analysis for the association with mvpa using a more stringent ( 120 mg instead of 100 mg ) cut - off for accelerometry . the main analyses were performed using sas v 9.3 ( sas institute , inc , cary , nc ) . analyses based on fractional polynomial models and figures were undertaken with stata 13 statistical software ( statacorp lp ) . data are drawn from the whitehall ii cohort study , established in 19851988 on 10,308 individuals ( 67% men ) , aged 3555 years . individuals gave written consent to participate in the study , and the university college london ethics committee approved the study . since inception , sociodemographic , behavioral , anthropometric , and health - related factors have been assessed approximately every 5 years ( 1985/1988 , 1991/1994 , 1997/1999 , 2002/2004 , 2007/2009 , and 2012/2013 ) . accelerometer measurement was added to the study at the 2012/2013 wave of data collection for participants seen at the central london clinic and for those living in the south - eastern regions of england who were screened at home . whitehall ii data , protocols , and other metadata are available to the scientific community . the instructions were as follows : we would like to know about your activities at work and in your free time that involve physical activity . the questionnaire included 20 items on the amount of time spent in the following activities : walking , sport ( cycling , soccer , golf , swimming , and 2 open - ended questions on other sports ) , gardening ( weeding , mowing , and 1 open - ended question on other gardening activities ) , housework ( carrying heavy shopping , cooking , hanging out washing , and 2 open - ended questions on other housework ) , do - it - yourself activity ( washing a car , painting , or decorating , and 1 open - ended question on other do - it - yourself activity ) , and 2 open - ended questions on other activities . for each item , the participants were required to take into account activity patterns over the past 4 weeks to give an indication of usual activity and provide the total number of hours spent in that activity per week ( see question 82 of the health survey questionnaire online available at http://www.ucl.ac.uk/whitehallii/pdf/s7_hsq.pdf ) . for each physical activity , including the open - ended items , we assigned a metabolic equivalent ( met ) value using a compendium of energy costs . total physical activity was estimated as met.hour/week , the sum of the product of the intensity ( met ) and weekly duration ( hours / week ) of all reported activities . in addition , we calculated the total number of hours per week in moderate - and - vigorous physical activity ( mvpa ) defined as activities with associated met 3 ( eg , cycling , weeding , swimming , mowing ) . participants with no contraindications ( that is , allergies to plastic or metal , traveling abroad the following week ) were asked to wear a triaxial accelerometer ( geneactiv ; activinsights ltd , kimbolton , cambs , uk , http://www.geneactiv.org/ ) on their nondominant wrist for 9 consecutive ( 24-hour ) days . the accelerometer was sampled at 85.7 hz and as in previous studies using new generation accelerometers , acceleration was expressed relative to gravity ( g units ; 1 g = 9.81 m.s ) to reflect the fact that sensors are calibrated relative to gravity . calibration error was estimated based on static periods in the data and corrected if necessary . the euclidean norm ( magnitude ) of the 3 raw signals minus 1 g , with negative numbers rounded to zero , was used to quantify the acceleration related to the movement registered and expressed in milligravity ( mg , 1 mg = 0.00981 m.s ) . accelerometer data were processed in r ( cran.r-project.org/ ) using the ggir package and executed on moveecloud ( http://movelab.org/research/moveecloud/ ) , a computing facility for physical activity research . data extracted between the first and last midnight were retained for the analysis leading to a maximum of 24-hour measurements for 8 days . participants were included in the analysis if they had valid data ( 16 hour / day ) for at least 2 weekdays and 2 weekend days . nonwear time was estimated on the basis of the standard deviation ( sd ) and value range of each accelerometer axis , calculated for moving windows of 60 minutes with 15-minute increments . for each 15-minute period of time detected as nonwear time over the valid days , missing data were replaced by the mean value calculated from measurement on other days at the same time of day . besides accelerometer - assessed total activity , for each participant duration in moderate - and - vigorous physical activity was also calculated . because a validated threshold to define mvpa in older adults does not yet exist , we chose the 100 mg threshold based on the fact that walking at 4 km / hour is classified as moderate physical activity and is equivalent to an acceleration of 100 mg in a laboratory based study on 30 adults . in order to qualify as mvpa , at least 80% of the activity needed to be at 100 mg , for at least a period ( bout ) of 10 minutes , using moving 10-minute windows . in sensitivity analyses , a more stringent cut - point of 120 mg was chosen to define mvpa . as the observation period covered 8 days , the data were recoded so that our measure reflected physical activity over 1 week to match the questionnaire - assessed physical activity . if a participant had 3 valid weekend days or 6 weekdays , the wrist acceleration of the first and last full day of measurement ( for example , 2 tuesdays a week apart ) were averaged to represent 1 unique day . then , the mean accelerometer - assessed total physical activity ( mg ) over a week was calculated as : [ ( 5 mean daily weekday wrist acceleration + 2 mean daily week - end wrist acceleration)]/7 . the same rescaling was undertaken for time spent in mvpa per week ( hour / week ) . bmi was calculated as weight ( kg ) divided by height ( m ) squared . weight was measured in light clothing , using an electronic soehnle scale with digital readout ( leifheit as , nassau , germany ) . height was measured using a stadiometer with the participant standing completely erect with their head in the frankfort plane . fmi was calculated as the ratio of fat mass ( kg ) by height ( m ) squared . fat mass was estimated by bioimpedance using the tanita tbf-300 body composition analyzer ( tanita , arlington heights , ill , www.tanita.com/en/ ) . bioimpedance data were available on participants seen at the central london clinic ( n = 4524 ) but not at home . as this measure is influenced by the total amount of body fluid , those with renal insufficiency ( assessed by estimated glomerular filtration rate < 15 ) were excluded from the analysis on fmi ( n = 1 ) . sociodemographic variables included age , sex , ethnicity ( white , south asian , black , other ) , marital status ( married / cohabiting , single , widowed , divorced / separated ) , and socioeconomic status , measured by the highest qualification on leaving full - time education ( university / higher university degree , higher secondary school , lower secondary school , and lower primary school or below ) and occupational position at age 50 years ( high , intermediate and low , representing income , and status at work ) . alcohol consumption was assessed using a question on the quantity of alcohol consumed in the previous week and was classified as no alcohol consumption in the previous week , moderate alcohol consumption ( 114 units / week in women and 121 units / week in men ) , and heavy drinkers ( 15 + units in women and 21 + units in men ) . the frequency of fruit and vegetable consumption was assessed on a 9-point scale , ranging from seldom or never to 4 or more times a day . the associations of the physical activity measures ( total physical activity and mvpa , both assessed by questionnaire and accelerometer ) with adiposity markers were examined using linear regression with adiposity markers as the outcomes . the linearity assumption was tested by comparing the fit of a model including physical activity measures as linear terms to the fit of models including degree 1 and 2 fractional polynomial terms using the deviance differences between the models ( fracpoly command in stata 13 ; statacorp lp , college station , tx ) . subsequently , models were adjusted for sociodemographic and behavioral variables in order to control for potential confounding , including those because of clustering of health behaviors . we first assessed the association of adiposity markers with total physical activity , assessed using the questionnaire ( met.h / week ) and accelerometer ( mean acceleration ) , using quartiles as the metric for these 2 measures was different . then , total physical activity ( continuous ) and mvpa ( categories ) were entered simultaneously into the model , and percentage reduction in the associations was calculated to allow the contribution on mvpa to be estimated . then , the association of adiposity markers with mvpa was investigated using the following categories for both questionnaire and accelerometer measures : < 1.0 hour / week , 1.02.5 hours / week , 2.54.0 hours / week , and 4 hours / week . analyses were repeated using sex - specific standardized measures of adiposity in order to compare whether the associations differed by adiposity markers . to compare the fit of the models for questionnaire vs accelerometer measures , the interactions of physical activity measures with sex and age ( continuous and median - based classes ) were also tested . in sensitivity analyses , we repeated the analysis for the association with mvpa using a more stringent ( 120 mg instead of 100 mg ) cut - off for accelerometry . the main analyses were performed using sas v 9.3 ( sas institute , inc , cary , nc ) . analyses based on fractional polynomial models and figures were undertaken with stata 13 statistical software ( statacorp lp ) . among the 4880 participants to whom the accelerometer was proposed , 210 had contraindications , 4282 agreed to wear it , and 4040 participants had valid accelerometer data ( 16 hour / day ) for at least 2 weekdays and 2 weekend days . of those , 3940 participants also had data on questionnaire - assessed physical activity , bmi , waist circumference , and all covariates , constituting the main analytic sample of this study . compared with the 940 participants not included in the analysis , the analytic sample did not differ by age [ mean age = 69.3 , sd = 5.7 ; 95% confidence interval ( ci ) = 69.2 , 69.5 ] vs 69.1 years [ sd = 5.7 ; 95% ci = 68.7 , 69.5 ) , p = .20 ] but was composed of more men ( 74.2% vs 66.8% , p < .0001 ) and fewer participants from the lowest occupational position ( 10.7% vs 13.3% , p = .02 ) . among these 3940 participants , 3828 ( 97.1% ) had valid data for the 8-day observation period , 76 ( 1.9% ) for 67 days , and 36 ( 0.9% ) for 54 days . in all , missing data were replaced for 12 hours for 26.3% of the participants , > 25 hours for 1.6% of the participants , > 510 hours for 1.1% of the participants , and > 1025 hours for 0.4% of the participants . participants included in analyses on bmi and waist circumference but not on fmi ( n = 324 ) had on average higher bmi ( 28.5 ( 95% ci 27.9 , 29.1 ; sd = 5.5 ) vs 26.4 ( 95%ci 26.3 , 26.6 ; sd = 4.2 ) kg / m , p < .0001 ) and waist circumference [ 100.0 ( 95% ci 98.5 , 101.6 ; sd = 14.0 ) vs 96.0 ( 95% ci 95.6 , 96.4 ; sd = 12.0 ) cm , p < .0001 ] as interactions were found between physical activity measures and sex [ all p < .05 , except for questionnaire - assessed mvpa with waist circumference ( p = .09 ) and fmi ( p = .12 ) ] , all analyses were performed separately for men and women . no interactions were found between physical activity and age ( p between .18 and .91 ) . the mean total physical activity assessed by questionnaire was 46.7 ( 95% ci 45.7 , 47.7 ; sd = 27.0 ) met.h / week in men and 44.5 ( 95% ci 42.7 , 46.3 ; sd = 29.0 ) met.h / week in women . for accelerometer data , the mean acceleration over a week was 23.4 ( 95% ci 23.2 , 23.7 ; sd = 6.8 ) mg in men and 23.1 ( 95% ci 22.7 , 23.5 ; sd = 6.7 ) mg in women . analyses on the shape of the association between total physical activity and adiposity revealed linear dose - response associations with all adiposity markers ( figure 1 ) . in men , differences in adiposity markers were 2.42.8 ( 1.92.3 for women ) times higher for an increase of 1sd in the accelerometer measure compared with an increase of 1sd in the questionnaire measure . in models adjusted for sociodemographic and behavioral factors , in men , being in the highest quartile of accelerometer - assessed total physical activity was associated with a 2.68 ( 95% ci 2.28 , 3.08 ) kg / m lower bmi compared with being in the lowest quartile ( table 2 ) . the corresponding figure for questionnaire data , at 1.14 ( 95% ci 0.74 , 1.53 ) associations between total physical activity and all adiposity markers were larger in women than in men . for example , the mean difference in bmi between the highest and the lowest quartiles of accelerometer - assessed total physical activity was 4.61 ( 95% ci 3.65 , 5.57 ) kg / m in women compared with 2.68 ( 95% ci 2.28 , 3.08 ) kg / m in men . after adjustment for mvpa , the associations between total physical activity and adiposity markers were reduced by 7%29% but remained statistically significant . in addition , analyses using standardized adiposity markers showed that the effect sizes of the associations were similar for all measures of adiposity ( appendix table a1 ) . for questionnaire data , differences in adiposity markers were mainly observed between men who reported < 1 hour / week of mvpa compared with those reporting more ( appendix figure a1 , table 3 ) , but no differences in adiposity markers were found for longer durations ( all p > .05 for comparison across 12.5 hours / week , 2.54 hours / week , and 4 hours / week duration categories ) . in contrast , for accelerometer - assessed physical activity , adiposity markers decreased progressively with increasing time spent in mvpa . for example , compared with men performing < 1 hour / week of mvpa , waist circumference was 3.06 ( 95% ci 2.06 , 4.06 ) cm lower in those performing 12.5 hours / week , 4.69 ( 95% ci 3.47 , 5.91 ) cm lower in those performing 2.54 hours / week , and 7.11 ( 95% ci 5.93 , 8.29 ) cm lower in those performing 4 hours / week of mvpa . in women , the differences between the association of questionnaire and accelerometer data with adiposity markers were observed but tended to attenuate for longer time spent in mvpa ( appendix figure a1 ) . standardized measures of adiposity markers showed that associations between physical activity and all 3 adiposity markers were similar ( appendix table a2 ) . in both men and women , the fit of the models based on the accelerometer data for total physical activity and mvpa was much better than for the models based on questionnaire data ( all akaike information criteria differences > 32 , data not tabulated ) . a more stringent cut - off for mvpa ( 120 mg instead of 100 mg ) showed similar results ( appendix table a3 ) . the associations between adiposity and accelerometer were stronger than that with questionnaire in men than in women leading us to examine whether the correlation between questionnaire and accelerometer measures differed by sex . these results showed it to be similar in men ( spearman correlation = 0.33 ; 95%ci 0.300.36 ) and women ( spearman correlation = 0.32 ; 0.270.37 ; p for difference by sex = .70 ) for total physical activity but stronger for mvpa in women ( spearman correlation = 0.28 ; 95% ci 0.220.33 ) than in men ( spearman correlation = 0.16 ; 95% ci 0.120.19 ; p for difference by sex = .0006 ) . on average , men reported spending 1.7 ( 95% ci1.5 , 1.9 ; sd = 5.4 ) hours / week more in mvpa than estimated by the accelerometer while women reported 0.9 ( 95% ci 0.5 , 1.3 ; sd = 6.0 ) hour / week more ( p for difference by sex < .0001 ) . among the 4880 participants to whom the accelerometer was proposed , 210 had contraindications , 4282 agreed to wear it , and 4040 participants had valid accelerometer data ( 16 hour / day ) for at least 2 weekdays and 2 weekend days . of those , 3940 participants also had data on questionnaire - assessed physical activity , bmi , waist circumference , and all covariates , constituting the main analytic sample of this study . compared with the 940 participants not included in the analysis , the analytic sample did not differ by age [ mean age = 69.3 , sd = 5.7 ; 95% confidence interval ( ci ) = 69.2 , 69.5 ] vs 69.1 years [ sd = 5.7 ; 95% ci = 68.7 , 69.5 ) , p = .20 ] but was composed of more men ( 74.2% vs 66.8% , p < .0001 ) and fewer participants from the lowest occupational position ( 10.7% vs 13.3% , p = .02 ) . among these 3940 participants , 3828 ( 97.1% ) had valid data for the 8-day observation period , 76 ( 1.9% ) for 67 days , and 36 ( 0.9% ) for 54 days . in all , missing data were replaced for 12 hours for 26.3% of the participants , > 25 hours for 1.6% of the participants , > 510 hours for 1.1% of the participants , and > 1025 hours for 0.4% of the participants . participants included in analyses on bmi and waist circumference but not on fmi ( n = 324 ) had on average higher bmi ( 28.5 ( 95% ci 27.9 , 29.1 ; sd = 5.5 ) vs 26.4 ( 95%ci 26.3 , 26.6 ; sd = 4.2 ) kg / m , p < .0001 ) and waist circumference [ 100.0 ( 95% ci 98.5 , 101.6 ; sd = 14.0 ) vs 96.0 ( 95% ci 95.6 , 96.4 ; sd = 12.0 ) cm , p < .0001 ] as interactions were found between physical activity measures and sex [ all p < .05 , except for questionnaire - assessed mvpa with waist circumference ( p = .09 ) and fmi ( p = .12 ) ] , all analyses were performed separately for men and women . no interactions were found between physical activity and age ( p between .18 and .91 ) . the mean total physical activity assessed by questionnaire was 46.7 ( 95% ci 45.7 , 47.7 ; sd = 27.0 ) met.h / week in men and 44.5 ( 95% ci 42.7 , 46.3 ; sd = 29.0 ) met.h / week in women . for accelerometer data , the mean acceleration over a week was 23.4 ( 95% ci 23.2 , 23.7 ; sd = 6.8 ) mg in men and 23.1 ( 95% ci 22.7 , 23.5 ; sd = 6.7 ) mg in women . analyses on the shape of the association between total physical activity and adiposity revealed linear dose - response associations with all adiposity markers ( figure 1 ) . in men , differences in adiposity markers were 2.42.8 ( 1.92.3 for women ) times higher for an increase of 1sd in the accelerometer measure compared with an increase of 1sd in the questionnaire measure . in models adjusted for sociodemographic and behavioral factors , in men , being in the highest quartile of accelerometer - assessed total physical activity was associated with a 2.68 ( 95% ci 2.28 , 3.08 ) kg / m lower bmi compared with being in the lowest quartile ( table 2 ) . the corresponding figure for questionnaire data , at 1.14 ( 95% ci 0.74 , 1.53 ) kg / m , was 2.4 times smaller . associations between total physical activity and all adiposity markers were larger in women than in men . for example , the mean difference in bmi between the highest and the lowest quartiles of accelerometer - assessed total physical activity was 4.61 ( 95% ci 3.65 , 5.57 ) kg / m in women compared with 2.68 ( 95% ci 2.28 , 3.08 ) kg / m in men . after adjustment for mvpa , the associations between total physical activity and adiposity markers were reduced by 7%29% but remained statistically significant . in addition , analyses using standardized adiposity markers showed that the effect sizes of the associations were similar for all measures of adiposity ( appendix table a1 ) . for questionnaire data , differences in adiposity markers were mainly observed between men who reported < 1 hour / week of mvpa compared with those reporting more ( appendix figure a1 , table 3 ) , but no differences in adiposity markers were found for longer durations ( all p > .05 for comparison across 12.5 hours / week , 2.54 hours / week , and 4 hours / week duration categories ) . in contrast , for accelerometer - assessed physical activity , adiposity markers decreased progressively with increasing time spent in mvpa . for example , compared with men performing < 1 hour / week of mvpa , waist circumference was 3.06 ( 95% ci 2.06 , 4.06 ) cm lower in those performing 12.5 hours / week , 4.69 ( 95% ci 3.47 , 5.91 ) cm lower in those performing 2.54 hours / week , and 7.11 ( 95% ci 5.93 , 8.29 ) cm lower in those performing 4 hours / week of mvpa . in women , the differences between the association of questionnaire and accelerometer data with adiposity markers were observed but tended to attenuate for longer time spent in mvpa ( appendix figure a1 ) . standardized measures of adiposity markers showed that associations between physical activity and all 3 adiposity markers were similar ( appendix table a2 ) . in both men and women , the fit of the models based on the accelerometer data for total physical activity and mvpa was much better than for the models based on questionnaire data ( all akaike information criteria differences > 32 , data not tabulated ) . a more stringent cut - off for mvpa ( 120 mg instead of 100 mg ) showed similar results ( appendix table a3 ) . the associations between adiposity and accelerometer were stronger than that with questionnaire in men than in women leading us to examine whether the correlation between questionnaire and accelerometer measures differed by sex . these results showed it to be similar in men ( spearman correlation = 0.33 ; 95%ci 0.300.36 ) and women ( spearman correlation = 0.32 ; 0.270.37 ; p for difference by sex = .70 ) for total physical activity but stronger for mvpa in women ( spearman correlation = 0.28 ; 95% ci 0.220.33 ) than in men ( spearman correlation = 0.16 ; 95% ci 0.120.19 ; p for difference by sex = .0006 ) . on average , men reported spending 1.7 ( 95% ci1.5 , 1.9 ; sd = 5.4 ) hours / week more in mvpa than estimated by the accelerometer while women reported 0.9 ( 95% ci 0.5 , 1.3 ; sd = 6.0 ) hour / week more ( p for difference by sex < .0001 ) . this study of british adults aged 60 to 83 years presents 3 key findings . ( 1 ) total and moderate - and - vigorous physical activity , assessed by questionnaire and accelerometer , were associated with bmi , waist circumference , and fmi , effect sizes being similar across the adiposity markers . ( 2 ) associations between total physical activity and adiposity markers were up to 2.8 times larger for accelerometer- than questionnaire - assessed physical activity . ( 3 ) in men , the association of adiposity markers with accelerometer - assessed moderate - and - vigorous physical activity compared with questionnaire data was much stronger , possibly because of greater measurement error in the reporting of time spent in moderate - and - vigorous physical activity by men . as both physical activity patterns and body fat distribution change with age , results based on data on younger adults might not apply to older adults . it has been hypothesized that the association between physical activity and adiposity markers reverses at older ages because of the attenuation in age - related weight loss in those engaging in more physical activity . some studies reported greater physical activity to be associated with lower bmi , waist circumference , and fat mass , but not fat - free mass . another study based on individuals aged 7082 years found that higher energy expenditure was associated with greater total body weight and fat - free mass , but no association was found with fat mass . it has also been suggested that the association between moderate physical activity and fat mass would attenuate with age . in the present study , we used multiple adiposity markers , the commonly used measures of bmi and waist circumference , but also fmi , which is the ratio of fat mass by height squared . fmi , like bmi , has the advantage of taking into account height differences and facilitates comparison between age and ethnic groups . we found that higher physical activity was associated with lower bmi , waist circumference , and fmi , and these associations did not differ by age . in our data , simple measures of adiposity such as bmi or waist there were large differences in adiposity markers between the most and least active individuals according to the accelerometer , up to 2.7 kg / m for bmi in men and 4.6 kg / m in women , and up to 8 cm for waist circumference in men and 11 cm in women . in older adults , a difference of 4 to 5 kg / m in bmi and of 11 to 13 cm in waist circumference is associated with a doubling of the risk of diabetes , a 30% increase in risk of heart failure , and more than 2-fold greater odds of functional limitations . the extent to which there are increasing benefits for weight control at greater physical activity remains unclear . we found a linear association of adiposity markers with total physical activity , which was reduced by less than 30% after taking the effects of moderate - and - vigorous physical activity into account . however , there were large differences in adiposity between those practicing none or little moderate - and - vigorous physical activity compared with the others even though the effects were attenuated at higher levels of activity . in addition , as also shown in some but not all previous studies , associations between physical activity and adiposity markers in our study were stronger in women than in men . potential reasons for these differences include sexual dimorphisms in energy metabolism and the type of physical activity undertaken , with women being more likely to choose activities that help maintain their weight . at least 2 previous studies have examined associations between physical activity and adiposity markers in older adults using both questionnaire and accelerometer measures . our study is based on a much larger sample ( previous analyses were on 238 and 636 persons ) and shows a consistently stronger association of all adiposity markers with accelerometer rather than questionnaire assessed - physical activity . we used a waterproof accelerometer worn on the wrist instead of the waist or the hip , allowing us to assess physical activity during 8 full days ( 24 hours ) . furthermore , compliance of a wrist - worn accelerometer is better than one worn on the waist or the hip , thus , reducing misclassification because of missing data over nonwear periods . there are several possible explanations for stronger associations with adiposity using accelerometer- rather than questionnaire - based assessments . the questionnaire we used focusses on 20 activities that do not cover all possible activities in a day . in addition , questionnaire - based assessments are likely to have a degree of measurement error as the duration of each activity relies on reports from the participant . although the accelerometer measures the movement of only one body part with inferences that apply to the whole body , it has the advantage of being free of reporting bias . in our data , measurement error of self - reported physical activity appears to be nondifferential with respect to adiposity markers as there was no difference in the correlation between accelerometer and questionnaire assessed physical activity measures across bmi categories ( data not tabulated ) . thus , the expected bias of the estimate for the physical activity - adiposity association is toward the null , which may explain the weaker association with questionnaire - based assessments . physical activity will continue to be assessed by questionnaire in many large - scale studies because of the ease of administration and lower costs . increasing recognition of the importance of physical activity for health suggests that studies will attempt to measure it better , making accelerometers an important research tool . our study has several strengths including the large study sample , analysis of raw rather than counts data ( metric generated by the monitor based on algorithm specific to each accelerometer brand ) , and high compliance for accelerometer wear among participants . first , no causal inferences can be drawn because of the cross - sectional nature of the study . second , although the sample covered a wide socioeconomic range , data are from an occupational cohort and can not be assumed to be representative of the general population . in addition , less than 5% of the analytic sample had a bmi greater or equal to 35 kg / m so the results can not be extended to the highest levels of adiposity . third , food habits , associated with both body weight and physical activity , are likely to explain part of the association between physical activity and adiposity markers . in the present analysis , we used fruit and vegetable consumption as a marker of healthy diet . it explained between 4% and 9% of the associations with questionnaire - assessed physical activity and around 2% of the associations with accelerometer data . analyses on a subset of participants for whom we had data on dietary patterns showed a reduction of 3% to 15% of the associations with questionnaire data and around 4% of the associations with accelerometer data . as there was no substantial difference in results , we retained the analysis using the measure of fruit and vegetable consumption as it allowed analyses on a larger number of participants . fourth , although our results are in accordance with previous studies that have used different instruments , such as the physical activity scale for the elderly questionnaire or other types of accelerometer ( eg , actigraph ) , they are specific to the instruments used and might not be generalizable across instruments . further studies are needed to examine the generalizability of our findings across different populations and accelerometer types . although the extent to which physical activity is associated with adiposity in older adults continues to be debated , the present study shows strong inverse associations of total and moderate - and - vigorous physical activity , both questionnaire- and accelerometer - assessed , with all adiposity markers . associations were up to 2.8 times larger using accelerometer- assessed instead of questionnaire - assessed physical activity , suggesting that beneficial effects of physical activity for adiposity at older ages are much greater than previously estimated .

objectivephysical activity is critically important for successful aging , but its effect on adiposity markers at older ages is unclear as much of the evidence comes from self - reported data on physical activity . we assessed the associations of questionnaire - assessed and accelerometer - assessed physical activity with adiposity markers in older adults.design/setting/participantsthis was a cross - sectional study on 3940 participants ( age range 60 - 83 years ) of the whitehall ii study who completed a 20-item physical activity questionnaire and wore a wrist - mounted accelerometer for 9 days in 2012 and 2013.measurementstotal physical activity was estimated using metabolic equivalent hours / week for the questionnaire and mean acceleration for the accelerometer . time spent in moderate - and - vigorous physical activity ( mvpa ) was also assessed by questionnaire and accelerometer . adiposity assessment included body mass index , waist circumference , and fat mass index . fat mass index was calculated as fat mass / height ( kg / m ) , with fat mass estimated using bioimpedance.resultsgreater total physical activity was associated with lower adiposity for all adiposity markers in a dose - response manner . in men , the strength of this association was 2.4 to 2.8 times stronger with the accelerometer than with questionnaire data . in women , it was 1.9 to 2.3 times stronger . for mvpa , questionnaire data in men suggested no further benefit for adiposity markers past 1 hour / week of activity . this was not the case for accelerometer - assessed mvpa where , for example , compared with men undertaking < 1 hour / week of accelerometer - assessed mvpa , waist circumference was 3.06 ( 95% confidence interval 2.064.06 ) cm lower in those performing mvpa 12.5 hours / week , 4.69 ( 3.475.91 ) cm lower in those undertaking 2.54 hours / week , and 7.11 ( 5.938.29 ) cm lower in those performing 4 hours / week.conclusionsthe association of physical activity with adiposity markers in older adults was stronger when physical activity was assessed by accelerometer compared with questionnaire , suggesting that physical activity might be more important for adiposity than previously estimated .

Methods Study Population Questionnaire-Based Assessment of Physical Activity Accelerometer-Assessed Physical Activity Adiposity Markers Covariates Statistical Analysis Results Sample Description Associations Between Total Physical Activity and Adiposity Markers Associations Between MVPA and Adiposity Markers Sensitivity Analyses Discussion Conclusions Supplementary Data

the questionnaire included 20 items on the amount of time spent in the following activities : walking , sport ( cycling , soccer , golf , swimming , and 2 open - ended questions on other sports ) , gardening ( weeding , mowing , and 1 open - ended question on other gardening activities ) , housework ( carrying heavy shopping , cooking , hanging out washing , and 2 open - ended questions on other housework ) , do - it - yourself activity ( washing a car , painting , or decorating , and 1 open - ended question on other do - it - yourself activity ) , and 2 open - ended questions on other activities . total physical activity was estimated as met.hour/week , the sum of the product of the intensity ( met ) and weekly duration ( hours / week ) of all reported activities . in addition , we calculated the total number of hours per week in moderate - and - vigorous physical activity ( mvpa ) defined as activities with associated met 3 ( eg , cycling , weeding , swimming , mowing ) . besides accelerometer - assessed total activity , for each participant duration in moderate - and - vigorous physical activity was also calculated . as the observation period covered 8 days , the data were recoded so that our measure reflected physical activity over 1 week to match the questionnaire - assessed physical activity . then , the mean accelerometer - assessed total physical activity ( mg ) over a week was calculated as : [ ( 5 mean daily weekday wrist acceleration + 2 mean daily week - end wrist acceleration)]/7 . alcohol consumption was assessed using a question on the quantity of alcohol consumed in the previous week and was classified as no alcohol consumption in the previous week , moderate alcohol consumption ( 114 units / week in women and 121 units / week in men ) , and heavy drinkers ( 15 + units in women and 21 + units in men ) . the associations of the physical activity measures ( total physical activity and mvpa , both assessed by questionnaire and accelerometer ) with adiposity markers were examined using linear regression with adiposity markers as the outcomes . we first assessed the association of adiposity markers with total physical activity , assessed using the questionnaire ( met.h / week ) and accelerometer ( mean acceleration ) , using quartiles as the metric for these 2 measures was different . then , total physical activity ( continuous ) and mvpa ( categories ) were entered simultaneously into the model , and percentage reduction in the associations was calculated to allow the contribution on mvpa to be estimated . then , the association of adiposity markers with mvpa was investigated using the following categories for both questionnaire and accelerometer measures : < 1.0 hour / week , 1.02.5 hours / week , 2.54.0 hours / week , and 4 hours / week . total physical activity was estimated as met.hour/week , the sum of the product of the intensity ( met ) and weekly duration ( hours / week ) of all reported activities . in addition , we calculated the total number of hours per week in moderate - and - vigorous physical activity ( mvpa ) defined as activities with associated met 3 ( eg , cycling , weeding , swimming , mowing ) . besides accelerometer - assessed total activity , for each participant duration in moderate - and - vigorous physical activity was also calculated . as the observation period covered 8 days , the data were recoded so that our measure reflected physical activity over 1 week to match the questionnaire - assessed physical activity . then , the mean accelerometer - assessed total physical activity ( mg ) over a week was calculated as : [ ( 5 mean daily weekday wrist acceleration + 2 mean daily week - end wrist acceleration)]/7 . fmi was calculated as the ratio of fat mass ( kg ) by height ( m ) squared . alcohol consumption was assessed using a question on the quantity of alcohol consumed in the previous week and was classified as no alcohol consumption in the previous week , moderate alcohol consumption ( 114 units / week in women and 121 units / week in men ) , and heavy drinkers ( 15 + units in women and 21 + units in men ) . the associations of the physical activity measures ( total physical activity and mvpa , both assessed by questionnaire and accelerometer ) with adiposity markers were examined using linear regression with adiposity markers as the outcomes . we first assessed the association of adiposity markers with total physical activity , assessed using the questionnaire ( met.h / week ) and accelerometer ( mean acceleration ) , using quartiles as the metric for these 2 measures was different . then , the association of adiposity markers with mvpa was investigated using the following categories for both questionnaire and accelerometer measures : < 1.0 hour / week , 1.02.5 hours / week , 2.54.0 hours / week , and 4 hours / week . of those , 3940 participants also had data on questionnaire - assessed physical activity , bmi , waist circumference , and all covariates , constituting the main analytic sample of this study . compared with the 940 participants not included in the analysis , the analytic sample did not differ by age [ mean age = 69.3 , sd = 5.7 ; 95% confidence interval ( ci ) = 69.2 , 69.5 ] vs 69.1 years [ sd = 5.7 ; 95% ci = 68.7 , 69.5 ) , p = .20 ] but was composed of more men ( 74.2% vs 66.8% , p < .0001 ) and fewer participants from the lowest occupational position ( 10.7% vs 13.3% , p = .02 ) . participants included in analyses on bmi and waist circumference but not on fmi ( n = 324 ) had on average higher bmi ( 28.5 ( 95% ci 27.9 , 29.1 ; sd = 5.5 ) vs 26.4 ( 95%ci 26.3 , 26.6 ; sd = 4.2 ) kg / m , p < .0001 ) and waist circumference [ 100.0 ( 95% ci 98.5 , 101.6 ; sd = 14.0 ) vs 96.0 ( 95% ci 95.6 , 96.4 ; sd = 12.0 ) cm , p < .0001 ] as interactions were found between physical activity measures and sex [ all p < .05 , except for questionnaire - assessed mvpa with waist circumference ( p = .09 ) and fmi ( p = .12 ) ] , all analyses were performed separately for men and women . the mean total physical activity assessed by questionnaire was 46.7 ( 95% ci 45.7 , 47.7 ; sd = 27.0 ) met.h / week in men and 44.5 ( 95% ci 42.7 , 46.3 ; sd = 29.0 ) met.h / week in women . analyses on the shape of the association between total physical activity and adiposity revealed linear dose - response associations with all adiposity markers ( figure 1 ) . in men , differences in adiposity markers were 2.42.8 ( 1.92.3 for women ) times higher for an increase of 1sd in the accelerometer measure compared with an increase of 1sd in the questionnaire measure . in models adjusted for sociodemographic and behavioral factors , in men , being in the highest quartile of accelerometer - assessed total physical activity was associated with a 2.68 ( 95% ci 2.28 , 3.08 ) kg / m lower bmi compared with being in the lowest quartile ( table 2 ) . the corresponding figure for questionnaire data , at 1.14 ( 95% ci 0.74 , 1.53 ) associations between total physical activity and all adiposity markers were larger in women than in men . for example , the mean difference in bmi between the highest and the lowest quartiles of accelerometer - assessed total physical activity was 4.61 ( 95% ci 3.65 , 5.57 ) kg / m in women compared with 2.68 ( 95% ci 2.28 , 3.08 ) kg / m in men . after adjustment for mvpa , the associations between total physical activity and adiposity markers were reduced by 7%29% but remained statistically significant . for questionnaire data , differences in adiposity markers were mainly observed between men who reported < 1 hour / week of mvpa compared with those reporting more ( appendix figure a1 , table 3 ) , but no differences in adiposity markers were found for longer durations ( all p > .05 for comparison across 12.5 hours / week , 2.54 hours / week , and 4 hours / week duration categories ) . in contrast , for accelerometer - assessed physical activity , adiposity markers decreased progressively with increasing time spent in mvpa . for example , compared with men performing < 1 hour / week of mvpa , waist circumference was 3.06 ( 95% ci 2.06 , 4.06 ) cm lower in those performing 12.5 hours / week , 4.69 ( 95% ci 3.47 , 5.91 ) cm lower in those performing 2.54 hours / week , and 7.11 ( 95% ci 5.93 , 8.29 ) cm lower in those performing 4 hours / week of mvpa . in women , the differences between the association of questionnaire and accelerometer data with adiposity markers were observed but tended to attenuate for longer time spent in mvpa ( appendix figure a1 ) . in both men and women , the fit of the models based on the accelerometer data for total physical activity and mvpa was much better than for the models based on questionnaire data ( all akaike information criteria differences > 32 , data not tabulated ) . the associations between adiposity and accelerometer were stronger than that with questionnaire in men than in women leading us to examine whether the correlation between questionnaire and accelerometer measures differed by sex . these results showed it to be similar in men ( spearman correlation = 0.33 ; 95%ci 0.300.36 ) and women ( spearman correlation = 0.32 ; 0.270.37 ; p for difference by sex = .70 ) for total physical activity but stronger for mvpa in women ( spearman correlation = 0.28 ; 95% ci 0.220.33 ) than in men ( spearman correlation = 0.16 ; 95% ci 0.120.19 ; p for difference by sex = .0006 ) . on average , men reported spending 1.7 ( 95% ci1.5 , 1.9 ; sd = 5.4 ) hours / week more in mvpa than estimated by the accelerometer while women reported 0.9 ( 95% ci 0.5 , 1.3 ; sd = 6.0 ) hour / week more ( p for difference by sex < .0001 ) . of those , 3940 participants also had data on questionnaire - assessed physical activity , bmi , waist circumference , and all covariates , constituting the main analytic sample of this study . participants included in analyses on bmi and waist circumference but not on fmi ( n = 324 ) had on average higher bmi ( 28.5 ( 95% ci 27.9 , 29.1 ; sd = 5.5 ) vs 26.4 ( 95%ci 26.3 , 26.6 ; sd = 4.2 ) kg / m , p < .0001 ) and waist circumference [ 100.0 ( 95% ci 98.5 , 101.6 ; sd = 14.0 ) vs 96.0 ( 95% ci 95.6 , 96.4 ; sd = 12.0 ) cm , p < .0001 ] as interactions were found between physical activity measures and sex [ all p < .05 , except for questionnaire - assessed mvpa with waist circumference ( p = .09 ) and fmi ( p = .12 ) ] , all analyses were performed separately for men and women . the mean total physical activity assessed by questionnaire was 46.7 ( 95% ci 45.7 , 47.7 ; sd = 27.0 ) met.h / week in men and 44.5 ( 95% ci 42.7 , 46.3 ; sd = 29.0 ) met.h / week in women . for accelerometer data , the mean acceleration over a week was 23.4 ( 95% ci 23.2 , 23.7 ; sd = 6.8 ) mg in men and 23.1 ( 95% ci 22.7 , 23.5 ; sd = 6.7 ) mg in women . analyses on the shape of the association between total physical activity and adiposity revealed linear dose - response associations with all adiposity markers ( figure 1 ) . in models adjusted for sociodemographic and behavioral factors , in men , being in the highest quartile of accelerometer - assessed total physical activity was associated with a 2.68 ( 95% ci 2.28 , 3.08 ) kg / m lower bmi compared with being in the lowest quartile ( table 2 ) . the corresponding figure for questionnaire data , at 1.14 ( 95% ci 0.74 , 1.53 ) kg / m , was 2.4 times smaller . for example , the mean difference in bmi between the highest and the lowest quartiles of accelerometer - assessed total physical activity was 4.61 ( 95% ci 3.65 , 5.57 ) kg / m in women compared with 2.68 ( 95% ci 2.28 , 3.08 ) kg / m in men . after adjustment for mvpa , the associations between total physical activity and adiposity markers were reduced by 7%29% but remained statistically significant . for questionnaire data , differences in adiposity markers were mainly observed between men who reported < 1 hour / week of mvpa compared with those reporting more ( appendix figure a1 , table 3 ) , but no differences in adiposity markers were found for longer durations ( all p > .05 for comparison across 12.5 hours / week , 2.54 hours / week , and 4 hours / week duration categories ) . in contrast , for accelerometer - assessed physical activity , adiposity markers decreased progressively with increasing time spent in mvpa . for example , compared with men performing < 1 hour / week of mvpa , waist circumference was 3.06 ( 95% ci 2.06 , 4.06 ) cm lower in those performing 12.5 hours / week , 4.69 ( 95% ci 3.47 , 5.91 ) cm lower in those performing 2.54 hours / week , and 7.11 ( 95% ci 5.93 , 8.29 ) cm lower in those performing 4 hours / week of mvpa . in women , the differences between the association of questionnaire and accelerometer data with adiposity markers were observed but tended to attenuate for longer time spent in mvpa ( appendix figure a1 ) . in both men and women , the fit of the models based on the accelerometer data for total physical activity and mvpa was much better than for the models based on questionnaire data ( all akaike information criteria differences > 32 , data not tabulated ) . on average , men reported spending 1.7 ( 95% ci1.5 , 1.9 ; sd = 5.4 ) hours / week more in mvpa than estimated by the accelerometer while women reported 0.9 ( 95% ci 0.5 , 1.3 ; sd = 6.0 ) hour / week more ( p for difference by sex < .0001 ) . ( 1 ) total and moderate - and - vigorous physical activity , assessed by questionnaire and accelerometer , were associated with bmi , waist circumference , and fmi , effect sizes being similar across the adiposity markers . ( 2 ) associations between total physical activity and adiposity markers were up to 2.8 times larger for accelerometer- than questionnaire - assessed physical activity . ( 3 ) in men , the association of adiposity markers with accelerometer - assessed moderate - and - vigorous physical activity compared with questionnaire data was much stronger , possibly because of greater measurement error in the reporting of time spent in moderate - and - vigorous physical activity by men . it has been hypothesized that the association between physical activity and adiposity markers reverses at older ages because of the attenuation in age - related weight loss in those engaging in more physical activity . some studies reported greater physical activity to be associated with lower bmi , waist circumference , and fat mass , but not fat - free mass . another study based on individuals aged 7082 years found that higher energy expenditure was associated with greater total body weight and fat - free mass , but no association was found with fat mass . in the present study , we used multiple adiposity markers , the commonly used measures of bmi and waist circumference , but also fmi , which is the ratio of fat mass by height squared . we found that higher physical activity was associated with lower bmi , waist circumference , and fmi , and these associations did not differ by age . in our data , simple measures of adiposity such as bmi or waist there were large differences in adiposity markers between the most and least active individuals according to the accelerometer , up to 2.7 kg / m for bmi in men and 4.6 kg / m in women , and up to 8 cm for waist circumference in men and 11 cm in women . in older adults , a difference of 4 to 5 kg / m in bmi and of 11 to 13 cm in waist circumference is associated with a doubling of the risk of diabetes , a 30% increase in risk of heart failure , and more than 2-fold greater odds of functional limitations . we found a linear association of adiposity markers with total physical activity , which was reduced by less than 30% after taking the effects of moderate - and - vigorous physical activity into account . however , there were large differences in adiposity between those practicing none or little moderate - and - vigorous physical activity compared with the others even though the effects were attenuated at higher levels of activity . at least 2 previous studies have examined associations between physical activity and adiposity markers in older adults using both questionnaire and accelerometer measures . thus , the expected bias of the estimate for the physical activity - adiposity association is toward the null , which may explain the weaker association with questionnaire - based assessments . it explained between 4% and 9% of the associations with questionnaire - assessed physical activity and around 2% of the associations with accelerometer data . although the extent to which physical activity is associated with adiposity in older adults continues to be debated , the present study shows strong inverse associations of total and moderate - and - vigorous physical activity , both questionnaire- and accelerometer - assessed , with all adiposity markers . associations were up to 2.8 times larger using accelerometer- assessed instead of questionnaire - assessed physical activity , suggesting that beneficial effects of physical activity for adiposity at older ages are much greater than previously estimated .

ibandronic acid 1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid ) is a nitrogen - containing bisphosphonate ( atc m05ba06 ; cas 114084 - 78 - 5 ) acting as an inhibitor of osteoclast - mediated bone resorption . ibandronic acid is effective for the treatment and prevention of osteoporosis in postmenopausal women with increased risk of fractures , and a reduction in the risk of vertebral fractures has been demonstrated . the absorption of ibandronic acid in the upper gastrointestinal tract is rapid after oral administration . in fasted state , the maximum observed plasma concentration ( cmax ) is reached within 0.52 hours ( median 1 hour ) . the oral bioavailability after oral administration is low ( ~0.6 % ) and highly variable . bioavailability is reduced by 90 % in the presence of a standard breakfast and by approximately 75 and 30 % when is administered 2 hours after a standard meal and 30 minutes before a meal , respectively . there is no meaningful reduction in bioavailability provided ibandronic acid is taken 60 minutes before a meal [ 1 , 2 ] . there is no evidence of dose - dependent pharmacokinetics in the range of 2.550 mg oral dosage . the exposure following administration of 50 , 100 or 150 mg was not dose proportional , with area under the serum concentration time curve ( auc ) and cmax presenting greater increase in exposure with increasing dose . the reason for these dose - dependent pharmacokinetics is not fully elucidated [ 1 , 2 ] . in humans , the apparent terminal volume of distribution is high ( ~90 l ) , which is most likely related to substantial distribution within skeletal tissue : the amount of dose removed from the circulation via the bone is estimated to be 4050 % and the remainder is eliminated unchanged by the kidney . protein binding in human plasma is approximately 87 % at therapeutic concentrations , and drug drug interaction due to displacement is unlikely . the observed apparent elimination half - life ( t el ) for ibandronic acid is generally in the range of 1072 hours . total clearance of ibandronic acid is low with average values in the range of 84160 ml / min . renal clearance ( about 60 ml / min in healthy postmenopausal females ) accounts for 5060 % of total clearance and is related to creatinine clearance . the difference between the apparent total and renal clearances is considered to reflect the uptake by bone [ 1 , 2 ] . the present study aimed to compare the rate and extent of absorption of ibandronate acid ( as sodium ibandronate ) 150 mg from a test medicinal product ( test formulation ; treatment a ) , manufactured by tecnimede ( sintra , portugal ) and that of the reference medicinal product ( reference formulation ; treatment b ; bonviva ) , a surrogate for therapeutic equivalence . the clinical study protocol and related documents were approved by an independent ethics committee ( international review board services ) and a no objection letter ( nol ) was obtained from canadian authorities . the study was conducted in accordance with the most recent version of the helsinki declaration and good clinical practice guideline . the clinical and analytical parts of the study were conducted at inventive health s facility ( qubec city , qc , canada ) . pharmacokinetic and statistical analyses were also performed by inventive health s facility ( qubec city , qc , canada ) . the 153 subjects were recruited from the community at large and considered eligible for enrolment as per protocol inclusion and exclusion criteria . subjects included were males or females of non - childbearing potential , nonsmokers or moderate smokers ( no more than nine cigarettes daily ) , aged 18 years of age and older ( 18 years ) and with body mass indices ( bmi ) greater than 18.5 kg / m ( > 18.5 ) and less than 30.0 kg / m females of non - childbearing potential included post - menopausal females or surgically sterile females . the screening procedures included collection of anamnesis and demographic data ( gender , age , race , body weight [ kg ] , height [ cm ] and bmi ) , a physical examination , a resting 12-lead electrocardiogram ( ecg ) , urine illicit drug screen , urine pregnancy test ( female subjects ) and clinical laboratory tests ( haematology , biochemistry , urinalysis , human immunodeficiency virus [ hiv ] , hepatitis c [ hcv ] antibodies and hepatitis b surface antigen [ hbsag ] ) . the baseline demographic characteristics of the pharmacokinetic population are depicted in table 1.table 1demographic data for the pharmacokinetic population descriptive statisticsvariableage ( years)height ( cm)weight ( kg)bmi ( kg / m)mean sd46 13170.0 9.473.86 10.3825.50 2.37range1873146.0195.049.00106.1019.5529.70median48170.075.0025.63 bmi body mass index , sd standard deviation demographic data for the pharmacokinetic population descriptive statistics bmi body mass index , sd standard deviation a total of 153 healthy subjects were randomly assigned to a treatment in accordance with the computer - generated blocks randomization scheme ( block size 6 , randomly variable ) . the randomization scheme was generated using statistical analysis system ( sas ) program version 9.2 ( sas institute inc . , this program used the randomized block design to ensure an equal distribution of sequences at multiples of 6 in the list of subject assignment . based on results of a previous pilot study , the within - subject coefficients of variance ( cvs ) should be approximately 39 and 48 % for auc and cmax , respectively . thus , with these expected cvs and an expected ratio of auc and cmax within 0.90 and 1.11 , the study should have a power of at least 90 % to show bioequivalence with 138 subjects . in order to account for possible dropouts this study was a single - centre , randomized , single - dose , open - label , three - way , three - sequence , reference formulation - replicated , crossover bioequivalence study to compare the rate and extent of absorption of tecnimede s test formulation of ibandronic acid ( batch number 15044 ; expiration date : 04 - 2013 ; manufactured by west pharma , sa , portugal ) with the reference formulation ( batch number b1176b01 ; expiration date : 11 - 2015 ; manufactured by roche pharma ag , germany ) , acquired in the polish market , under fasting conditions , in healthy volunteers . after an overnight fast of at least 10 hours , subjects were dosed in the mornings . subjects were administered the test or the reference formulation , as per the randomization scheme , as a single oral dose of one film - coated tablet containing 150 mg of study medication , with 240 ml of water . subjects were dosed as specified in the protocol , and subsequently fasted for a period of at least 4 hours . subjects were served a controlled meal not less than 4 hours post - dose , and at appropriate times thereafter , in each period . subjects were served standardized post - dose meals similar in composition in each period . with the exception of the volume administered at the time of dosing , fluids were not permitted from 1 hour before dosing to 1 hour after dosing , but , after that period , plain water was permitted ad libitum . according with a reference formulation - replicated design , the study had three periods ( period 1 , period 2 and period 3 ) and the subjects were randomized to three sequences ( test - reference - reference [ trr ] ; reference - reference - test [ rrt ] and reference - test - reference [ rtr ] ) . in each study period , subjects were administered the test formulation ( treatment a ) or the reference formulation ( treatment b ) as per the randomization scheme . the randomization scheme was kept unavailable to the bioanalytical division until completion of the clinical and analytical phases . a dead - volume intravenous catheter was used for blood collection , which occurred prior to drug administration and 0.167 , 0.333 , 0.500 , 0.750 , 1.00 , 1.25 , 1.50 , 1.75 , 2.00 , 3.00 , 4.00 , 6.00 , 8.00 , 12.0 , 24.0 and 48.0 hours post - dose in each period . blood samples were cooled in an ice bath and were centrifuged at 3,000 rpm ( corresponding to approximately 1,900 g ) for at least 10 minutes at approximately 4 c ( no more than 110 minutes passed between the time of each blood draw and the start of centrifugation ) . the aliquots were transferred to a 20 c freezer , pending transfer to the bioanalytical facility . pharmacokinetic analyses were performed using pharsight knowledgebase server ( version 4.0.2 ) and winnonlin ( version 5.3 ) , which are validated for bioequivalence / bioavailability studies by inventive health . inferential statistical analyses were performed using sas ( release 9.2 ) according to the food and drug administration ( fda ) , health product and food branch of health canada and european medicines agency ( ema ) guidance . the number of observations ( n ) , mean , standard deviation ( sd ) , cv% , range ( minimum and maximum ) , median and geometric mean were calculated for plasma concentrations of ibandronic acid for each sampling time and treatment . these descriptive statistics were also presented for the auc from time zero to time of the last non - zero concentration ( auc0t ) , the auc from time zero to infinity ( extrapolated ) ( auc0inf ) , the cmax , the residual area calculated through the equation ( 1 auc0t / auc0inf ) 100 % , time to cmax ( tmax ) , the t el and the elimination rate constant ( kel ) . auc0inf was calculated through the following equation : auc0t + ( ct / kel ) , where ct is the fitted last non - zero concentration for that treatment . adverse events were listed and coded using medical dictionary for regulatory activities ( meddra ) , version 15.0 . treatment - emergent adverse events ( teaes ) were summarized descriptively in the safety population , and were tabulated by treatment group , system organ class , preferred term , causality and severity . for the purpose of statistical analyses , the safety population included the subjects who received at least one dose of the investigational medicinal product whereas the pharmacokinetic population included the subjects who completed at least two periods including one period with test formulation and other with the reference formulation and for whom the pharmacokinetic profile was characterized . individual and mean plasma concentrations , as well as the plots of the plasma levels for all subjects versus time , were graphically displayed for three treatments . ln - transformed auc0t , auc0inf and cmax were analysed using general linear model ( glm ) procedure in sas following the method a recommended by the ema ( chmp pharmacokinetics working party [ pkwp ] ema/618604/2008 rev . 3 ) . the statistical model included sequence , period , treatment and subject within sequence as fixed factors . the sequence effect was tested using the subject - within - sequence effect as the error term . within - subject coefficient of variation ( cvwr ) was calculated for the reference product using analysis of variance ( anova ) , on reference data only , with sequence , subject within sequence , and period as fixed effects . the point estimate and the 90 % geometric confidence interval for the test - to - reference geometric mean ratio ( t / r ) were calculated for auc0t , auc0inf and cmax using the least - squares means statement . all statistical tests were performed at the alpha level of 0.05 . according to the regulatory requirements translated into the study protocol , the hypothesis of bioequivalence between a generic medicinal product and a reference medicinal product is accepted if the 90 % geometric confidence intervals of the ratio of least - squares means of the test to reference product of ln - transformed auc0t is within the acceptance range of 80.00125.00 % . this approach is based on the cvwr : if the cvwr is inferior or equal to 30 % ( 30 % ) , the 90 % geometric confidence intervals of the ratio t / r of least - squares means of the ln - transformed cmax should be within the acceptable range of 80.00125.00 % to conclude bioequivalence . on the other hand , if the cvwr for the reference product was superior to 30 % ( > 30 % ) for cmax , the bioequivalence acceptance limits for this pharmacokinetic parameter had to be scaled to the within - subject variability of the reference product ( to a maximum of 69.84143.19 % ) . for scaled average bioequivalence , the applicant should justify that the calculated cvwr is a reliable estimate and that it is not the result of outliers . therefore , a box plot analysis using the studentized intra - subject residuals from the anova model including only data for the reference treatment was done using the univariate procedure in sas . a box plot was constructed from studentized intra - subject residuals corresponding to the first administration of reference product in each subject . values that were further away from the box by more than three interquartile ranges were considered outlying observations and these values are indicated by an asterisk in the box plot . the detected outlying observations were excluded , and within subject standard deviation of the log transformed parameter ( swr ) was re - estimated and used to widen bioequivalence limits ( if eligible ) as specified above . the clinical study protocol and related documents were approved by an independent ethics committee ( international review board services ) and a no objection letter ( nol ) was obtained from canadian authorities . the study was conducted in accordance with the most recent version of the helsinki declaration and good clinical practice guideline . the clinical and analytical parts of the study were conducted at inventive health s facility ( qubec city , qc , canada ) . pharmacokinetic and statistical analyses were also performed by inventive health s facility ( qubec city , qc , canada ) . the 153 subjects were recruited from the community at large and considered eligible for enrolment as per protocol inclusion and exclusion criteria . subjects included were males or females of non - childbearing potential , nonsmokers or moderate smokers ( no more than nine cigarettes daily ) , aged 18 years of age and older ( 18 years ) and with body mass indices ( bmi ) greater than 18.5 kg / m ( > 18.5 ) and less than 30.0 kg / m females of non - childbearing potential included post - menopausal females or surgically sterile females . the screening procedures included collection of anamnesis and demographic data ( gender , age , race , body weight [ kg ] , height [ cm ] and bmi ) , a physical examination , a resting 12-lead electrocardiogram ( ecg ) , urine illicit drug screen , urine pregnancy test ( female subjects ) and clinical laboratory tests ( haematology , biochemistry , urinalysis , human immunodeficiency virus [ hiv ] , hepatitis c [ hcv ] antibodies and hepatitis b surface antigen [ hbsag ] ) . the baseline demographic characteristics of the pharmacokinetic population are depicted in table 1.table 1demographic data for the pharmacokinetic population descriptive statisticsvariableage ( years)height ( cm)weight ( kg)bmi ( kg / m)mean sd46 13170.0 9.473.86 10.3825.50 2.37range1873146.0195.049.00106.1019.5529.70median48170.075.0025.63 bmi body mass index , sd standard deviation demographic data for the pharmacokinetic population descriptive statistics bmi body mass index , sd standard deviation a total of 153 healthy subjects were randomly assigned to a treatment in accordance with the computer - generated blocks randomization scheme ( block size 6 , randomly variable ) . the randomization scheme was generated using statistical analysis system ( sas ) program version 9.2 ( sas institute inc . this program used the randomized block design to ensure an equal distribution of sequences at multiples of 6 in the list of subject assignment . based on results of a previous pilot study , the within - subject coefficients of variance ( cvs ) should be approximately 39 and 48 % for auc and cmax , respectively . thus , with these expected cvs and an expected ratio of auc and cmax within 0.90 and 1.11 , the study should have a power of at least 90 % to show bioequivalence with 138 subjects . in order to account for possible dropouts , this study was a single - centre , randomized , single - dose , open - label , three - way , three - sequence , reference formulation - replicated , crossover bioequivalence study to compare the rate and extent of absorption of tecnimede s test formulation of ibandronic acid ( batch number 15044 ; expiration date : 04 - 2013 ; manufactured by west pharma , sa , portugal ) with the reference formulation ( batch number b1176b01 ; expiration date : 11 - 2015 ; manufactured by roche pharma ag , germany ) , acquired in the polish market , under fasting conditions , in healthy volunteers . after an overnight fast of at least 10 hours , subjects were dosed in the mornings . subjects were administered the test or the reference formulation , as per the randomization scheme , as a single oral dose of one film - coated tablet containing 150 mg of study medication , with 240 ml of water . subjects were dosed as specified in the protocol , and subsequently fasted for a period of at least 4 hours . subjects were served a controlled meal not less than 4 hours post - dose , and at appropriate times thereafter , in each period . subjects were served standardized post - dose meals similar in composition in each period . with the exception of the volume administered at the time of dosing , fluids were not permitted from 1 hour before dosing to 1 hour after dosing , but , after that period , plain water was permitted ad libitum . according with a reference formulation - replicated design , the study had three periods ( period 1 , period 2 and period 3 ) and the subjects were randomized to three sequences ( test - reference - reference [ trr ] ; reference - reference - test [ rrt ] and reference - test - reference [ rtr ] ) . in each study period , subjects were administered the test formulation ( treatment a ) or the reference formulation ( treatment b ) as per the randomization scheme . the randomization scheme was kept unavailable to the bioanalytical division until completion of the clinical and analytical phases . a dead - volume intravenous catheter was used for blood collection , which occurred prior to drug administration and 0.167 , 0.333 , 0.500 , 0.750 , 1.00 , 1.25 , 1.50 , 1.75 , 2.00 , 3.00 , 4.00 , 6.00 , 8.00 , 12.0 , 24.0 and 48.0 hours post - dose in each period . blood samples were cooled in an ice bath and were centrifuged at 3,000 rpm ( corresponding to approximately 1,900 g ) for at least 10 minutes at approximately 4 c ( no more than 110 minutes passed between the time of each blood draw and the start of centrifugation ) . the aliquots were transferred to a 20 c freezer , pending transfer to the bioanalytical facility . pharmacokinetic analyses were performed using pharsight knowledgebase server ( version 4.0.2 ) and winnonlin ( version 5.3 ) , which are validated for bioequivalence / bioavailability studies by inventive health . inferential statistical analyses were performed using sas ( release 9.2 ) according to the food and drug administration ( fda ) , health product and food branch of health canada and european medicines agency ( ema ) guidance . the number of observations ( n ) , mean , standard deviation ( sd ) , cv% , range ( minimum and maximum ) , median and geometric mean were calculated for plasma concentrations of ibandronic acid for each sampling time and treatment . these descriptive statistics were also presented for the auc from time zero to time of the last non - zero concentration ( auc0t ) , the auc from time zero to infinity ( extrapolated ) ( auc0inf ) , the cmax , the residual area calculated through the equation ( 1 auc0t / auc0inf ) 100 % , time to cmax ( tmax ) , the t el and the elimination rate constant ( kel ) . auc0inf was calculated through the following equation : auc0t + ( ct / kel ) , where ct is the fitted last non - zero concentration for that treatment . adverse events were listed and coded using medical dictionary for regulatory activities ( meddra ) , version 15.0 . treatment - emergent adverse events ( teaes ) were summarized descriptively in the safety population , and were tabulated by treatment group , system organ class , preferred term , causality and severity . for the purpose of statistical analyses , the safety population included the subjects who received at least one dose of the investigational medicinal product whereas the pharmacokinetic population included the subjects who completed at least two periods including one period with test formulation and other with the reference formulation and for whom the pharmacokinetic profile was characterized . individual and mean plasma concentrations , as well as the plots of the plasma levels for all subjects versus time , were graphically displayed for three treatments . ln - transformed auc0t , auc0inf and cmax were analysed using general linear model ( glm ) procedure in sas following the method a recommended by the ema ( chmp pharmacokinetics working party [ pkwp ] ema/618604/2008 rev . 3 ) . the statistical model included sequence , period , treatment and subject within sequence as fixed factors . the sequence effect was tested using the subject - within - sequence effect as the error term . the treatment and period effects were tested against the residual mean square error . within - subject coefficient of variation ( cvwr ) was calculated for the reference product using analysis of variance ( anova ) , on reference data only , with sequence , subject within sequence , and period as fixed effects . the point estimate and the 90 % geometric confidence interval for the test - to - reference geometric mean ratio ( t / r ) were calculated for auc0t , auc0inf and cmax using the least - squares means statement . all statistical tests were performed at the alpha level of 0.05 . according to the regulatory requirements translated into the study protocol , the hypothesis of bioequivalence between a generic medicinal product and a reference medicinal product is accepted if the 90 % geometric confidence intervals of the ratio of least - squares means of the test to reference product of ln - transformed auc0t is within the acceptance range of 80.00125.00 % . for cmax , the protocol established a scaled average bioequivalence approach . this approach is based on the cvwr : if the cvwr is inferior or equal to 30 % ( 30 % ) , the 90 % geometric confidence intervals of the ratio t / r of least - squares means of the ln - transformed cmax should be within the acceptable range of 80.00125.00 % to conclude bioequivalence . on the other hand , if the cvwr for the reference product was superior to 30 % ( > 30 % ) for cmax , the bioequivalence acceptance limits for this pharmacokinetic parameter had to be scaled to the within - subject variability of the reference product ( to a maximum of 69.84143.19 % ) . for scaled average bioequivalence , the applicant should justify that the calculated cvwr is a reliable estimate and that it is not the result of outliers . therefore , a box plot analysis using the studentized intra - subject residuals from the anova model including only data for the reference treatment was done using the univariate procedure in sas . a box plot was constructed from studentized intra - subject residuals corresponding to the first administration of reference product in each subject . values that were further away from the box by more than three interquartile ranges were considered outlying observations and these values are indicated by an asterisk in the box plot . the detected outlying observations were excluded , and within subject standard deviation of the log transformed parameter ( swr ) was re - estimated and used to widen bioequivalence limits ( if eligible ) as specified above . one hundred and fifty - three subjects ( 47 females and 106 males ) were randomized to three sequences of treatment ( trr , rtr and rrt ) , and received at least one dose of the investigational medicinal products under study . this sample size was considered according to the protocol for safety evaluation ( safety population ) . nevertheless , as previously stated in the protocol , the subjects used for pharmacokinetic and statistical analysis , the pharmacokinetic population , are those that completed at least two periods including one test and one administration of the reference product and for whom the pharmacokinetic profile was adequately characterized ( n = 146 ) . a ( test ) = tecnimede sociedade tcnico medicinal s.a . , portugal , ibandronic acid 1 150-mg film - coated tablet . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet disposition of subjects . a ( test ) = tecnimede sociedade tcnico medicinal s.a . , portugal , ibandronic acid 1 150-mg film - coated tablet . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet after the test formulation ( t ) and first and second bonviva ( r ) dosing , the cmax was 96.71 90.19 ng / ml , 92.67 91.48 ng / ml and 87.94 60.20 ng / ml and the auc0t was 390.83 287.27 ngh / ml , 388.54 356.76 ngh / ml and 383.53 246.72 ( 64.33 ) , respectively ( table 2 ) . no statistically significant difference between treatments was detected using anova for ln - transformed auc0t , auc0inf and cmax . a statistically significant period effect was detected for auc0t and auc0inf ( table 3 ) . the mean residual area was less than 20 % for the aucs obtained after administration of the test formulation ( 3.41 0.84 % ) as well as after the first and second administrations of bonviva ( 3.30 0.70 and 3.57 0.95 % , respectively ) . 2.table 2pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv%)]test formulationbonviva ( first administration)bonviva ( second administration ) n 146146142auc0t ( ngh / ml)390.83 287.27 ( 73.50)388.54 356.76 ( 91.82)383.53 246.72 ( 64.33)auc0inf ( ngh / ml)404.49 296.72 ( 73.36)401.48 366.54 ( 91.30)397.65 255.75 ( 64.31)residual area ( % ) 3.41 0.84 ( 24.61)3.30 0.70 ( 21.03)3.57 0.95 ( 26.74 ) c max ( ng / ml)96.71 90.19 ( 93.25)92.67 91.48 ( 98.72)87.94 60.20 ( 68.46 ) t max ( h)1.17 ( 0.3338.00)1.25 ( 0.3334.00)1.01 ( 0.3338.02 ) k el ( 1/h)0.0851 0.0663 ( 77.89)0.0847 0.0679 ( 80.15)0.0734 0.0450 ( 61.32 ) t el ( h)10.91 4.25 ( 38.92)10.76 3.93 ( 36.51)11.49 3.90 ( 33.97 ) median ( min max ) auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max table 3 p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid p valuestreatmentperiodsequenceauc0t 0.82270.01270.9091auc0inf 0.82550.00990.9010 c max 0.58350.12910.8606 auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentrationfig . 2mean plasma ibandronic acid concentrations obtained for the test and reference formulations following a 150-mg dose ( log scale ) . n = 146 for ibandronic acid , n = 146 for bonviva ( first administration ) , n = 142 for bonviva ( second administration ) , edta ethylene diaminetetraacetic acid pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv% ) ] auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration mean plasma ibandronic acid concentrations obtained for the test and reference formulations following a 150-mg dose ( log scale ) . n = 146 for ibandronic acid , n = 146 for bonviva ( first administration ) , n = 142 for bonviva ( second administration ) , edta ethylene diaminetetraacetic acid the cvwr for auc0t , auc0inf and cmax were 39.77 , 39.45 and 43.23 % , respectively . the limits of the acceptance range based upon the within - subject variability seen in the bioequivalence study using scaled average bioequivalence were 73.01136.97 % . no statistical outliers were detected for the reference formulation following examination of the distribution of the ln - transformed cmax . the 90 % confidence intervals were 95.05110.67 for cmax , 94.35107.94 for auc0t and 94.37107.88 for auc0inf , which are within the predefined bioequivalence acceptance range of 80.00125.00 % . for cmax , the observed ratio and confidence intervals were also within the limits of acceptance obtained using the scaled average bioequivalence approach . wilcoxon s test performed on the tmax data showed no statistically significant difference between treatments ( p = 0.1382 ) . the least - squares means ratios , the 90 % geometric confidence intervals , and the cvwr for the reference product are presented in table 4.table 4ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva variabletreatment comparisonsratio ( % ) 90 % ci ( % ) intra - subject cv ( bonviva ) ( % ) auc0t test ( a)reference ( b)100.9294.35107.9439.77auc0inf test ( a)reference ( b)100.9094.37107.8839.45 c max test ( a)reference ( b)102.5695.05110.6743.23 calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance a total of 279 teaes were reported by 91 of the 153 subjects who received at least one dose of the medicinal product ( safety population ) : 96 teaes were reported by 28.8 % ( 42/146 ) of the subjects when they received the test medicinal product ( treatment a ) and 183 teaes were reported by 47.7 % ( 73/153 ) of the 153 subjects when they received the reference medicinal product ( treatment b ) . myalgia was reported by 38 subjects , diarrhoea by 22 subjects and abdominal pain by 16 subjects , corresponding to 24.8 , 17.6 and 10.5 % of the safety population ( n = 153 ) , respectively . after the causality assessment of the 279 teaes , 70 were judged as probable / likely , 176 as unlikely. when comparing the number of subjects for each meddra preferred term , there are no relevant differences between treatments with the exception of the headache and myalgia teaes , which were reported by 11 and 19 subjects , respectively , after the administration of treatment a and by 21 and 29 subjects , respectively , after the administration of treatment b. the severity of each teae was graded as mild ( n = 223 ) , moderate ( n = 50 ) or severe ( n = 6 ) . one hundred and fifty - three subjects ( 47 females and 106 males ) were randomized to three sequences of treatment ( trr , rtr and rrt ) , and received at least one dose of the investigational medicinal products under study . this sample size was considered according to the protocol for safety evaluation ( safety population ) . nevertheless , as previously stated in the protocol , the subjects used for pharmacokinetic and statistical analysis , the pharmacokinetic population , are those that completed at least two periods including one test and one administration of the reference product and for whom the pharmacokinetic profile was adequately characterized ( n = 146 ) . a ( test ) = tecnimede sociedade tcnico medicinal s.a . , portugal , ibandronic acid 1 150-mg film - coated tablet . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet disposition of subjects . a ( test ) = tecnimede sociedade tcnico medicinal s.a . , portugal , ibandronic acid 1 150-mg film - coated tablet . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet after the test formulation ( t ) and first and second bonviva ( r ) dosing , the cmax was 96.71 90.19 ng / ml , 92.67 91.48 ng / ml and 87.94 60.20 ng / ml and the auc0t was 390.83 287.27 ngh / ml , 388.54 356.76 ngh / ml and 383.53 246.72 ( 64.33 ) , respectively ( table 2 ) . no statistically significant difference between treatments was detected using anova for ln - transformed auc0t , auc0inf and cmax . a statistically significant period effect was detected for auc0t and auc0inf ( table 3 ) . the mean residual area was less than 20 % for the aucs obtained after administration of the test formulation ( 3.41 0.84 % ) as well as after the first and second administrations of bonviva ( 3.30 0.70 and 3.57 0.95 % , respectively ) . 2.table 2pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv%)]test formulationbonviva ( first administration)bonviva ( second administration ) n 146146142auc0t ( ngh / ml)390.83 287.27 ( 73.50)388.54 356.76 ( 91.82)383.53 246.72 ( 64.33)auc0inf ( ngh / ml)404.49 296.72 ( 73.36)401.48 366.54 ( 91.30)397.65 255.75 ( 64.31)residual area ( % ) 3.41 0.84 ( 24.61)3.30 0.70 ( 21.03)3.57 0.95 ( 26.74 ) c max ( ng / ml)96.71 90.19 ( 93.25)92.67 91.48 ( 98.72)87.94 60.20 ( 68.46 ) t max ( h)1.17 ( 0.3338.00)1.25 ( 0.3334.00)1.01 ( 0.3338.02 ) k el ( 1/h)0.0851 0.0663 ( 77.89)0.0847 0.0679 ( 80.15)0.0734 0.0450 ( 61.32 ) t el ( h)10.91 4.25 ( 38.92)10.76 3.93 ( 36.51)11.49 3.90 ( 33.97 ) median ( min max ) auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max table 3 p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid p valuestreatmentperiodsequenceauc0t 0.82270.01270.9091auc0inf 0.82550.00990.9010 c max 0.58350.12910.8606 auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentrationfig . 2mean plasma ibandronic acid concentrations obtained for the test and reference formulations following a 150-mg dose ( log scale ) . n = 146 for ibandronic acid , n = 146 for bonviva ( first administration ) , n = 142 for bonviva ( second administration ) , edta ethylene diaminetetraacetic acid pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv% ) ] auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration mean plasma ibandronic acid concentrations obtained for the test and reference formulations following a 150-mg dose ( log scale ) . n = 146 for ibandronic acid , n = 146 for bonviva ( first administration ) , n = 142 for bonviva ( second administration ) , edta ethylene diaminetetraacetic acid the cvwr for auc0t , auc0inf and cmax were 39.77 , 39.45 and 43.23 % , respectively . the limits of the acceptance range based upon the within - subject variability seen in the bioequivalence study using scaled average bioequivalence were 73.01136.97 % . no statistical outliers were detected for the reference formulation following examination of the distribution of the ln - transformed cmax . the 90 % confidence intervals were 95.05110.67 for cmax , 94.35107.94 for auc0t and 94.37107.88 for auc0inf , which are within the predefined bioequivalence acceptance range of 80.00125.00 % . for cmax , the observed ratio and confidence intervals were also within the limits of acceptance obtained using the scaled average bioequivalence approach . wilcoxon s test performed on the tmax data showed no statistically significant difference between treatments ( p = 0.1382 ) . the least - squares means ratios , the 90 % geometric confidence intervals , and the cvwr for the reference product are presented in table 4.table 4ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva variabletreatment comparisonsratio ( % ) 90 % ci ( % ) intra - subject cv ( bonviva ) ( % ) auc0t test ( a)reference ( b)100.9294.35107.9439.77auc0inf test ( a)reference ( b)100.9094.37107.8839.45 c max test ( a)reference ( b)102.5695.05110.6743.23 calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance one hundred and fifty - three subjects ( 47 females and 106 males ) were randomized to three sequences of treatment ( trr , rtr and rrt ) , and received at least one dose of the investigational medicinal products under study . this sample size was considered according to the protocol for safety evaluation ( safety population ) . nevertheless , as previously stated in the protocol , the subjects used for pharmacokinetic and statistical analysis , the pharmacokinetic population , are those that completed at least two periods including one test and one administration of the reference product and for whom the pharmacokinetic profile was adequately characterized ( n = 146 ) . a ( test ) = tecnimede sociedade tcnico medicinal s.a . , portugal , ibandronic acid 1 150-mg film - coated tablet . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet disposition of subjects . a ( test ) = tecnimede sociedade tcnico medicinal s.a . , portugal , ibandronic acid 1 150-mg film - coated tablet . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet after the test formulation ( t ) and first and second bonviva ( r ) dosing , the cmax was 96.71 90.19 ng / ml , 92.67 91.48 ng / ml and 87.94 60.20 ng / ml and the auc0t was 390.83 287.27 ngh / ml , 388.54 356.76 ngh / ml and 383.53 246.72 ( 64.33 ) , respectively ( table 2 ) . no statistically significant difference between treatments was detected using anova for ln - transformed auc0t , auc0inf and cmax . a statistically significant period effect was detected for auc0t and auc0inf ( table 3 ) . the mean residual area was less than 20 % for the aucs obtained after administration of the test formulation ( 3.41 0.84 % ) as well as after the first and second administrations of bonviva ( 3.30 0.70 and 3.57 0.95 % , respectively ) . 2.table 2pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv%)]test formulationbonviva ( first administration)bonviva ( second administration ) n 146146142auc0t ( ngh / ml)390.83 287.27 ( 73.50)388.54 356.76 ( 91.82)383.53 246.72 ( 64.33)auc0inf ( ngh / ml)404.49 296.72 ( 73.36)401.48 366.54 ( 91.30)397.65 255.75 ( 64.31)residual area ( % ) 3.41 0.84 ( 24.61)3.30 0.70 ( 21.03)3.57 0.95 ( 26.74 ) c max ( ng / ml)96.71 90.19 ( 93.25)92.67 91.48 ( 98.72)87.94 60.20 ( 68.46 ) t max ( h)1.17 ( 0.3338.00)1.25 ( 0.3334.00)1.01 ( 0.3338.02 ) k el ( 1/h)0.0851 0.0663 ( 77.89)0.0847 0.0679 ( 80.15)0.0734 0.0450 ( 61.32 ) t el ( h)10.91 4.25 ( 38.92)10.76 3.93 ( 36.51)11.49 3.90 ( 33.97 ) median ( min max ) auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max table 3 p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid p valuestreatmentperiodsequenceauc0t 0.82270.01270.9091auc0inf 0.82550.00990.9010 c max 0.58350.12910.8606 auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentrationfig . 2mean plasma ibandronic acid concentrations obtained for the test and reference formulations following a 150-mg dose ( log scale ) . n = 146 for ibandronic acid , n = 146 for bonviva ( first administration ) , n = 142 for bonviva ( second administration ) , edta ethylene diaminetetraacetic acid pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv% ) ] auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration mean plasma ibandronic acid concentrations obtained for the test and reference formulations following a 150-mg dose ( log scale ) . n = 146 for ibandronic acid , n = 146 for bonviva ( first administration ) , n = 142 for bonviva ( second administration ) , edta ethylene diaminetetraacetic acid the cvwr for auc0t , auc0inf and cmax were 39.77 , 39.45 and 43.23 % , respectively . the limits of the acceptance range based upon the within - subject variability seen in the bioequivalence study using scaled average bioequivalence were 73.01136.97 % . no statistical outliers were detected for the reference formulation following examination of the distribution of the ln - transformed cmax . the 90 % confidence intervals were 95.05110.67 for cmax , 94.35107.94 for auc0t and 94.37107.88 for auc0inf , which are within the predefined bioequivalence acceptance range of 80.00125.00 % . for cmax , the observed ratio and confidence intervals were also within the limits of acceptance obtained using the scaled average bioequivalence approach . wilcoxon s test performed on the tmax data showed no statistically significant difference between treatments ( p = 0.1382 ) . the least - squares means ratios , the 90 % geometric confidence intervals , and the cvwr for the reference product are presented in table 4.table 4ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva variabletreatment comparisonsratio ( % ) 90 % ci ( % ) intra - subject cv ( bonviva ) ( % ) auc0t test ( a)reference ( b)100.9294.35107.9439.77auc0inf test ( a)reference ( b)100.9094.37107.8839.45 c max test ( a)reference ( b)102.5695.05110.6743.23 calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance a total of 279 teaes were reported by 91 of the 153 subjects who received at least one dose of the medicinal product ( safety population ) : 96 teaes were reported by 28.8 % ( 42/146 ) of the subjects when they received the test medicinal product ( treatment a ) and 183 teaes were reported by 47.7 % ( 73/153 ) of the 153 subjects when they received the reference medicinal product ( treatment b ) . myalgia was reported by 38 subjects , diarrhoea by 22 subjects and abdominal pain by 16 subjects , corresponding to 24.8 , 17.6 and 10.5 % of the safety population ( n = 153 ) , respectively . after the causality assessment of the 279 teaes , 70 were judged as probable / likely , 176 as unlikely. when comparing the number of subjects for each meddra preferred term , there are no relevant differences between treatments with the exception of the headache and myalgia teaes , which were reported by 11 and 19 subjects , respectively , after the administration of treatment a and by 21 and 29 subjects , respectively , after the administration of treatment b. the severity of each teae was graded as mild ( n = 223 ) , moderate ( n = 50 ) or severe ( n = 6 ) . ibandronic acid is a bisphosphonate compound indicated for the treatment and prevention of osteoporosis in post - menopausal women and the reduction of skeletal complications of malignant disease . the absorption in the upper gastrointestinal tract is rapid after oral administration with an absolute bioavailability of about 0.6 % . a generic medicinal product is considered to be bioequivalent to a reference medicinal product when the 90 % confidence interval around the estimated ratio of geometric means of auc and cmax is between 0.80 and 1.25 . as per regulatory and scientific requirements , when a generic medicinal product and a reference medicinal product are compared , a single - dose , crossover design is recommended . in studies with crossover design , the amplitude of the confidence interval is proportional to the within - subject sd of the pharmacokinetic parameter and reciprocally proportional to the square - root of the number of subjects . consequently , the regulatory bioequivalence limits of 0.80 and 1.25 are frequently penetrated when the intra - individual variation is high unless the number of subjects is also large . ibandronic acid is a highly variable drug and , although the reference literature confirms acceptance of widening of confidence intervals in europe , based on non - replicate designs , the latest update in the bioequivalence guideline requires that , in order to widen the intervals for cmax , a replicate design must be used . besides the fact of allowing for the widening of the intervals for cmax , replicate designs possess the advantage of reducing the sample size of subjects required to demonstrate bioequivalence between the two formulations . however , one of the main issues with a replicate design is the increased duration of the clinical phase of the study , since a full replicate study with four periods will require three washout intervals and a partial replicate with three periods will require two washout intervals . this represents more than three or two times , respectively , the required amount of time to conduct a simple crossover study . therefore , by increasing the duration of the study and the number of dosing periods , replicate designs normally exhibit a higher dropout rate , which impacts negatively on the required sample size . the issue with the higher dropout rates is evident by analysing the bibliographic references , which shows 15.8 and 12.5 % dropout rates for full replicate studies [ 6 , 7 ] , while , according to our experience , we achieved a dropout rate of 7.2 % for this partial replicate study and a 4.2 % dropout rate in a pilot crossover study ( data on file ) . so , in trying to achieve a compromise between an extended duration of the clinical phase and reducing the sample size without much impact from the dropout rate , we decided to conduct this study as a partial replicate design with three periods , including two administrations of the reference formulation in each sequence . this turned out to be a favourable decision since , according to the guidelines , the replicate design allowed for the scaled bioequivalence approach for cmax and the duration of the clinical phase was contained and acceptable ( 37 days as opposed to the required 54 days in a four - period full replicate design ) , which led to a dropout rate lower than the one observed for full replicate studies . further to this , the results of the study demonstrated that the within - subject variability for cmax of the reference formulation was more than 30 % and this value was not the result of the presence of outliers . however , it is important to point out that a replicate design may not be the solution if high within - subject variability is observed for the auc parameter , which was not the case for ibandronic acid , since the bioequivalence guideline does not allow for the widening of intervals for that pharmacokinetic parameter . the treatment periods should be separated by a washout period of at least five t el in order to guarantee that the drug concentrations are below the lower limit of quantification at the beginning of each period . in this study , when reviewing the published data on ibandronic acid pharmacokinetic properties , the authors noticed that the published half - life of ibandronic acid ranges from 10 to 60 hours and , in one study in postmenopausal women that received a single oral dose of ibandronic acid150 mg , a mean t el of 72 hours was observed . in the current study , the t el of ibandronic acid was approximately 10 hours for both formulations , which is in line with published studies but also in the lower limit of the range of values published . this may be explained by the fact that the participants in the study were healthy volunteers since similar half - life values were observed in phase i pharmacokinetic studies with the reference product in healthy subjects . the mean residual area was less than 20 % for all treatments indicating that a sampling over a period of 48 hours was sufficient . however , since there was no detectable pre - dose concentration at any of the study periods and there was no sequence effect , there is no indication of carryover effect . as the intra - subject variability was smaller for the aucs as compared with cmax , the power of the study was higher for these parameters . consequently , small differences between periods could be detected which should not be clinically meaningful . in this bioequivalence study , all the ratios and 90 % geometric confidence intervals were within the acceptance ranges . the conventional acceptance range of 0.80 and 1.25 was even met for cmax ( table 4 ) . based on these results , it can be concluded that the test formulation of ibandronic acid is bioequivalent to the test reference bonviva following a 1 150-mg dose under fasting conditions . the number of subjects reporting teae and the number of teae reported after intake of reference medicinal product ( treatment b bonviva ) is higher than the number of subjects reporting teae and the number of teae reported following intake of the test medicinal product ( treatment a test formulation ) . these differences between treatments can be explained by study design , a reference - replicate crossover study , since all subjects who completed the study received two doses of the reference medicinal product and only one dose of the test medicinal product . augusto filipe , pedro pedroso , susana almeida and rita neves are employees of the sponsor of this study . sylvie boudreault is an employee of the contract research organization contracted to perform this study .

aimsthis bioequivalence study aimed to compare rate and extent of absorption of a generic medicinal product of ibandronic acid 150-mg film - coated tablet versus bonviva.methodsthis was a single - centre , open - label , randomized , three - way , three - sequence , reference - replicated , crossover bioequivalence study , under fasting conditions . a single oral dose of ibandronic acid as one 150-mg film - coated tablet was administered in each study period . each washout period lasted 14 days . blood samples were collected according to a predefined sampling schedule and up to 48.0 hours after administraton in each period . plasma concentrations of ibandronic acid were measured using a liquid chromatograph mass spectrometry / mass spectrometry method . bioequivalence between generic and reference medicinal products is acceptable if the 90 % confidence intervals ( ci ) of ratio of least - squares means between the test and the reference product of ln - transformed area under the serum concentration time curve from time zero to time of last measurable concentration ( auc0t ) is within the 80.00125.00 % interval . prospectively , a scaled average bioequivalence approach for maximum serum concentration ( cmax ) was established.results153 healthy volunteers were enrolled and randomized . after the test formulation ( t ) and first and second bonviva ( r ) dosing , the cmax was 96.71 90.19 ng / ml , 92.67 91.48 ng / ml and 87.94 60.20 ng / ml and the auc0t was 390.83 287.27 ngh / ml , 388.54 356.76 ngh / ml and 383.53 246.72 , respectively . ratios of t / r and 90 % ci were 100.92 % ( 94.35107.94 ) for auc0t , 100.90 % ( 94.37107.88 ) for auc0inf and 102.56 % ( 95.05110.67 ) for cmax.conclusionstest formulation of ibandronic acid is bioequivalent in rate and extent of absorption to bonviva following a 150-mg dose , under fasting conditions .

Introduction Volunteers and Methods Study Protocol Volunteers Study Design Drug Analysis Pharmacokinetic Analysis Safety Analysis Statistical Analysis Results Drug Analysis Pharmacokinetic Analysis Safety Analysis Discussion and Conclusions Conflict of Interest

in order to account for possible dropouts this study was a single - centre , randomized , single - dose , open - label , three - way , three - sequence , reference formulation - replicated , crossover bioequivalence study to compare the rate and extent of absorption of tecnimede s test formulation of ibandronic acid ( batch number 15044 ; expiration date : 04 - 2013 ; manufactured by west pharma , sa , portugal ) with the reference formulation ( batch number b1176b01 ; expiration date : 11 - 2015 ; manufactured by roche pharma ag , germany ) , acquired in the polish market , under fasting conditions , in healthy volunteers . in order to account for possible dropouts , this study was a single - centre , randomized , single - dose , open - label , three - way , three - sequence , reference formulation - replicated , crossover bioequivalence study to compare the rate and extent of absorption of tecnimede s test formulation of ibandronic acid ( batch number 15044 ; expiration date : 04 - 2013 ; manufactured by west pharma , sa , portugal ) with the reference formulation ( batch number b1176b01 ; expiration date : 11 - 2015 ; manufactured by roche pharma ag , germany ) , acquired in the polish market , under fasting conditions , in healthy volunteers . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet after the test formulation ( t ) and first and second bonviva ( r ) dosing , the cmax was 96.71 90.19 ng / ml , 92.67 91.48 ng / ml and 87.94 60.20 ng / ml and the auc0t was 390.83 287.27 ngh / ml , 388.54 356.76 ngh / ml and 383.53 246.72 ( 64.33 ) , respectively ( table 2 ) . 2.table 2pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv%)]test formulationbonviva ( first administration)bonviva ( second administration ) n 146146142auc0t ( ngh / ml)390.83 287.27 ( 73.50)388.54 356.76 ( 91.82)383.53 246.72 ( 64.33)auc0inf ( ngh / ml)404.49 296.72 ( 73.36)401.48 366.54 ( 91.30)397.65 255.75 ( 64.31)residual area ( % ) 3.41 0.84 ( 24.61)3.30 0.70 ( 21.03)3.57 0.95 ( 26.74 ) c max ( ng / ml)96.71 90.19 ( 93.25)92.67 91.48 ( 98.72)87.94 60.20 ( 68.46 ) t max ( h)1.17 ( 0.3338.00)1.25 ( 0.3334.00)1.01 ( 0.3338.02 ) k el ( 1/h)0.0851 0.0663 ( 77.89)0.0847 0.0679 ( 80.15)0.0734 0.0450 ( 61.32 ) t el ( h)10.91 4.25 ( 38.92)10.76 3.93 ( 36.51)11.49 3.90 ( 33.97 ) median ( min max ) auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max table 3 p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid p valuestreatmentperiodsequenceauc0t 0.82270.01270.9091auc0inf 0.82550.00990.9010 c max 0.58350.12910.8606 auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentrationfig . the least - squares means ratios , the 90 % geometric confidence intervals , and the cvwr for the reference product are presented in table 4.table 4ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva variabletreatment comparisonsratio ( % ) 90 % ci ( % ) intra - subject cv ( bonviva ) ( % ) auc0t test ( a)reference ( b)100.9294.35107.9439.77auc0inf test ( a)reference ( b)100.9094.37107.8839.45 c max test ( a)reference ( b)102.5695.05110.6743.23 calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance a total of 279 teaes were reported by 91 of the 153 subjects who received at least one dose of the medicinal product ( safety population ) : 96 teaes were reported by 28.8 % ( 42/146 ) of the subjects when they received the test medicinal product ( treatment a ) and 183 teaes were reported by 47.7 % ( 73/153 ) of the 153 subjects when they received the reference medicinal product ( treatment b ) . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet after the test formulation ( t ) and first and second bonviva ( r ) dosing , the cmax was 96.71 90.19 ng / ml , 92.67 91.48 ng / ml and 87.94 60.20 ng / ml and the auc0t was 390.83 287.27 ngh / ml , 388.54 356.76 ngh / ml and 383.53 246.72 ( 64.33 ) , respectively ( table 2 ) . 2.table 2pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv%)]test formulationbonviva ( first administration)bonviva ( second administration ) n 146146142auc0t ( ngh / ml)390.83 287.27 ( 73.50)388.54 356.76 ( 91.82)383.53 246.72 ( 64.33)auc0inf ( ngh / ml)404.49 296.72 ( 73.36)401.48 366.54 ( 91.30)397.65 255.75 ( 64.31)residual area ( % ) 3.41 0.84 ( 24.61)3.30 0.70 ( 21.03)3.57 0.95 ( 26.74 ) c max ( ng / ml)96.71 90.19 ( 93.25)92.67 91.48 ( 98.72)87.94 60.20 ( 68.46 ) t max ( h)1.17 ( 0.3338.00)1.25 ( 0.3334.00)1.01 ( 0.3338.02 ) k el ( 1/h)0.0851 0.0663 ( 77.89)0.0847 0.0679 ( 80.15)0.0734 0.0450 ( 61.32 ) t el ( h)10.91 4.25 ( 38.92)10.76 3.93 ( 36.51)11.49 3.90 ( 33.97 ) median ( min max ) auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max table 3 p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid p valuestreatmentperiodsequenceauc0t 0.82270.01270.9091auc0inf 0.82550.00990.9010 c max 0.58350.12910.8606 auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentrationfig . the least - squares means ratios , the 90 % geometric confidence intervals , and the cvwr for the reference product are presented in table 4.table 4ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva variabletreatment comparisonsratio ( % ) 90 % ci ( % ) intra - subject cv ( bonviva ) ( % ) auc0t test ( a)reference ( b)100.9294.35107.9439.77auc0inf test ( a)reference ( b)100.9094.37107.8839.45 c max test ( a)reference ( b)102.5695.05110.6743.23 calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance one hundred and fifty - three subjects ( 47 females and 106 males ) were randomized to three sequences of treatment ( trr , rtr and rrt ) , and received at least one dose of the investigational medicinal products under study . b ( reference ) = roche registration limited , united kingdom ( bonviva ) , ibandronic acid 1 150-mg film - coated tablet after the test formulation ( t ) and first and second bonviva ( r ) dosing , the cmax was 96.71 90.19 ng / ml , 92.67 91.48 ng / ml and 87.94 60.20 ng / ml and the auc0t was 390.83 287.27 ngh / ml , 388.54 356.76 ngh / ml and 383.53 246.72 ( 64.33 ) , respectively ( table 2 ) . 2.table 2pharmacokinetic variables for ibandronic acid for each treatment / period [ mean sd and ( cv%)]test formulationbonviva ( first administration)bonviva ( second administration ) n 146146142auc0t ( ngh / ml)390.83 287.27 ( 73.50)388.54 356.76 ( 91.82)383.53 246.72 ( 64.33)auc0inf ( ngh / ml)404.49 296.72 ( 73.36)401.48 366.54 ( 91.30)397.65 255.75 ( 64.31)residual area ( % ) 3.41 0.84 ( 24.61)3.30 0.70 ( 21.03)3.57 0.95 ( 26.74 ) c max ( ng / ml)96.71 90.19 ( 93.25)92.67 91.48 ( 98.72)87.94 60.20 ( 68.46 ) t max ( h)1.17 ( 0.3338.00)1.25 ( 0.3334.00)1.01 ( 0.3338.02 ) k el ( 1/h)0.0851 0.0663 ( 77.89)0.0847 0.0679 ( 80.15)0.0734 0.0450 ( 61.32 ) t el ( h)10.91 4.25 ( 38.92)10.76 3.93 ( 36.51)11.49 3.90 ( 33.97 ) median ( min max ) auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv% coefficient of variance , k el elimination rate constant , n number of observations , sd standard deviation , t el elimination half - life , t max time to c max table 3 p values for treatment , period and sequence effects for the pharmacokinetic variables of ibandronic acid p valuestreatmentperiodsequenceauc0t 0.82270.01270.9091auc0inf 0.82550.00990.9010 c max 0.58350.12910.8606 auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentrationfig . the least - squares means ratios , the 90 % geometric confidence intervals , and the cvwr for the reference product are presented in table 4.table 4ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva variabletreatment comparisonsratio ( % ) 90 % ci ( % ) intra - subject cv ( bonviva ) ( % ) auc0t test ( a)reference ( b)100.9294.35107.9439.77auc0inf test ( a)reference ( b)100.9094.37107.8839.45 c max test ( a)reference ( b)102.5695.05110.6743.23 calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance ibandronic acid : ratios , 90 % geometric confidence intervals ( ci ) for auc0t , auc0inf and c max and intra - subject cv for bonviva calculated using least - squares means 90 % geometric confidence interval using ln - transformed data the scaled average bioequivalence approach was used for c max and the widened limits obtained were 73.01136.97 % auc 0inf area under the serum concentration time curve from time zero to infinity auc 0t area under the serum concentration time curve from time zero to time of last measurable concentration , c max maximum serum concentration , cv coefficient of variance a total of 279 teaes were reported by 91 of the 153 subjects who received at least one dose of the medicinal product ( safety population ) : 96 teaes were reported by 28.8 % ( 42/146 ) of the subjects when they received the test medicinal product ( treatment a ) and 183 teaes were reported by 47.7 % ( 73/153 ) of the 153 subjects when they received the reference medicinal product ( treatment b ) .

autoimmune hepatitis ( aih ) is a chronic inflammatory disease of unknown etiology characterized by the presence of circulating autoantibodies , hypergammaglobulinemia , necroinflammatory changes on hepatic histology , and a dramatic response to immunosuppressive therapy . earliest descriptions include those by amberg in 1942 and leber in 1950 describing a form of chronic liver disease prevalent among young women and characterized by an excessive increase in serum protein and gamma - globulins . in 1951 , kunkel et al . termed the condition hypergammaglobulinemic chronic hepatitis . since then , it has been known by various names including chronic active hepatitis , chronic aggressive hepatitis , plasma cell hepatitis , and autoimmune chronic active hepatitis . cowling and mackay coined the term lupoid hepatitis after they noted the association of this entity with autoimmune syndromes and the le cell phenomenon . the disease is rare with a mean incidence of 1 - 2 per 100,000 and a point prevalence of 1117 per 100,000 [ 5 , 6 ] . although more frequently seen in young women ( sex ratio 3.6 : 1 ) , it can affect children and adults of all ages and ethnicities [ 7 , 8 ] . a minority of patients may present with acute liver failure and need liver transplantation , but for the majority , the prognosis of aih is good and mostly determined by response to corticosteroid therapy . in general , long - term survival and average life expectancy are excellent and estimated to be comparable to the normal population . type i aih is characterized by the presence of antinuclear antibody ( ana ) , anti - smooth muscle antibody ( sma ) , or both and constitutes 80% of aih cases . about 25% have cirrhosis at presentation , and association with other autoimmune diseases is common ( celiac disease , ulcerative colitis , autoimmune thyroid disease ) [ 10 , 11 ] . type 2 aih is characterized by the presence of anti - liver kidney microsomal ( lkm ) 1 and/or anti - lkm3 and/or anti - liver cytosol 1 ( lc1 ) [ 12 , 13 ] antibodies . most patients are children , acute severe presentation can occur , and progression to cirrhosis commonly ensues . in patients who are negative for conventional antibodies and aih is strongly suspected , additional tests can be done including perinuclear antineutrophil cytoplasmic antibodies ( panca ) , actin ( anti - actin ) , soluble liver antigen ( anti - sla ) , asialoglycoprotein receptor ( anti - asgpr ) , chromatin , and liver cytosol type 1 ( anti - lc1 ) . in our experience , 1015% patients do not have either ana , sma , or anti - lkm1 at presentation , but 25% of these will have detectable conventional antibodies later in their course . another 1020% of the seronegative patients at presentation will have panca or anti - sla . although the exact etiopathogenesis is unknown , aih , like many autoimmune diseases , is thought to be caused by environmental triggers and failure of immune tolerance mechanisms in a genetically susceptible host . these triggers may be of viral or drug etiology , but most cases have an unknown trigger . triggers may share epitopes that resemble self - antigens , and molecular mimicry between foreign antigens and self - antigens is the most frequently proposed initiating mechanism in type 2 aih where the autoantigen is known . repeated exposures to the triggering antigen , in turn , may trigger autoreactive organ - specific responses . aih is a complex polygenic disease and different populations may have different genetic and environmental triggers and genetic association varies in study populations . the human leukocyte antigen ( hla ) genes on chromosome 6 are the most commonly described association with aih . hla may be associated with age at presentation , disease severity , and response to therapy . how the hla genes predispose to disease is not exactly known but is likely due to their role in autoreactive t cell selection and autoantigenic peptide presentation . different susceptibility alleles like hla dr1 * 0301 , dr1 * 0401 , dr1 * 0404 , and dr1 * 0405 share a common motif , namely , amino acids lleqkr or lleqrr at position 67 - 72 of class ii hla , whereas the resistant alleles dr11501 encodes ileqar [ 20 , 21 ] . in contrast , hla - dr1 * 1501 , encodes for the ileqar motif and is associated with protection from aih . substitution of a lysine or arginine to alanine at position 71 is postulated to change the polarity and charge of the peptide binding groove of the major histocompatibility complex thereby influencing autoantigenic peptide presentation . however , these associations are not absolute and significant geographic differences exist , for example , in japan dr2 ( drb1 * 1501 ) is a weak susceptibility rather than a resistance allele and in south american children drb1 * 1301 is a strong susceptibility allele . . a form of aih can be seen in 20% of patients with autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy ( apeced ) syndrome . apeced is a monogenic , autosomal recessive disorder characterized by hypoparathyroidism , adrenal insufficiency , and chronic mucocutaneous candidiasis . apeced is caused by mutations in a transcription factor relevant to immune tolerance called aire ( autoimmune regulator ) on chromosome 21q223 . aire is expressed in medullary epithelial and dendritic cells within the thymus and regulates clonal deletion of autoreactive t cells . the liver autoantigens associated with apeced are cytochrome p450 ( cyp ) 1a2 , cyp2a6 , and cyp2d6 [ 2426 ] . this is the only syndrome involving aih that exhibits a mendelian pattern of inheritance , and genetic testing and counseling for the patient and family members are warranted . the liver is part of the lymphoid system with the normal lymphocyte population mainly residing in the portal tracts . aih is an inflammatory disorder of the liver involving multiple components of the immune system including t cells , b cells , and cytokines . hepatocytes isolated from aih patients are coated with immunoglobulins and are susceptible to antibody - dependent cellular cytotoxicity ( adcc ) when exposed to autologous mononuclear cells bearing fc receptors . cyp2d6 , an important cytoplasmic enzyme is targeted by anti - lkm1 antibodies and plays a crucial role in liver damage . mice immunized with plasmid containing human cyp2d6 antigenic region and human formiminotransferase cyclodeaminase ( another autoantigen ) , have established a murine model for autoimmune hepatitis type 2 . these mice develop autoantibodies , elevation in transaminases , along with portal and periportal inflammatory infiltrate . another model using adenovirus vector containing human cyp2d6 infection of cyp2d6 transgenic mice had focal hepatocyte necrosis and hepatic fibrosis . these models will aid the development of more therapeutic options in the management of autoimmune hepatitis . studies have demonstrated presence of cytotoxic cells in both t and non - t cell compartment of peripheral blood from aih patients . this cytotoxic activity is higher in patients with active disease but seen in only 40% of patients in remission . patients with aih have a ten fold higher frequency of liver - specific t cells compared to normal subjects . in patients with predisposing hla allele dr1 * 0701 , cd4 t cells are able to recognize autoantigen cyp2d6 and secrete interferon- . cd8 t cells have cytotoxic capability , are capable of secreting ifn- , and their responses correlate with disease activity . defects in numbers and function of regulatory cells ( t regs ) have been demonstrated in aih . t regulatory cells normally control or limit immune responses by acting as immunoregulators , preventing the proliferation and effector function of autoreactive t cells . in patients with aih , the number of t regs is decreased more so at disease presentation than at drug - induced remission . their level correlates inversely with levels of anti - sla and anti - lkm-1 autoantibody titers . longhi et al . in their study demonstrated that tregs generated under cyp2d6-specific conditions and cocultured with semimature dendritic cells are highly effective at controlling autoreactive t cells , thus providing a potential tool for immunotherapy in type 2 aih . t regs may , therefore , be an attractive therapeutic target , but more studies are needed to elucidate this better . several drugs have also been implicated as triggers for aih including infliximab , minocycline , atorvastatin , diclofenac , isoniazid , -methyldopa , nitrofurantoin , and propylthiouracil and hepatitis a vaccine . herbal agents such as black cohosh and dai - saiko - to have been proposed to induced aih . the exact reason for drug - induced aih is not known but may be due to hepatotoxic effect of these chemicals , upregulation of proteins expression ( p450s , immunoregulatory proteins ) , or related to the drug acting as a hapten by modifying the hepatic protein , making them immunogenic . drug - induced aih may improve after discontinuation of offending agent , thus initial observation is warranted . viruses such as hepatitis a , b , or c , in addition to measles have been implicated as triggers for aih . ana and sma can occur in diverse causes of acute and chronic hepatitis including alcoholic , nonalcoholic fatty liver disease , and viral hepatitis . they are usually low titer , background reactivities that should not alter diagnosis or management . if clinical concern for autoimmune hepatitis exists , antiactin antibodies can be checked as they increase the specificity of sma testing for diagnosing aih . anti - lkm1 has been found in as many as 10% of patients with chronic hepatitis c and is different from the anti - lkm1 found in classic autoimmune hepatitis . molecular mimicry at the b - cell level between a structural motif of cyp2d6 and hcv proteins could explain the production of anti - lkm1 antibodies in hcv - infected patients . women constitute at least 70% of cases , and 50% are younger than 40 years ; however , age at onset may range from infancy to the elderly . forty percent of patients may present with an acute onset , but the presentation of severe fulminant hepatic failure is rare . presenting symptoms may include fatigue , lethargy , malaise , arthralgia of small joints , anorexia , nausea , abdominal pain , and dark urine . clinical manifestations may vary by ethnicity ; the presentation is acute and icteric in alaskan native patients , cholestatic in aboriginal north american , african , asian , and arab patients , mild in japanese patients , but severe and rapidly progressive in somali patients . physical examination may be normal , but may also reveal hepatomegaly , splenomegaly , jaundice , and stigmata of chronic liver disease . findings such as acne , hirsutism , obesity , and amenorrhea in young women are rarely seen . other autoimmune diseases such as hashimoto thyroiditis , type 1 diabetes , rheumatoid arthritis , systemic lupus erythematosus , ulcerative colitis / crohn 's disease , and celiac disease can be seen in 20% of patients . this subtype is associated with higher serum levels of igg , aih severity scores , and , more importantly , an excellent response to prednisone therapy for induction and maintenance of remission . positive igg4 staining is suggested by the authors as a surrogate marker for the subtype of aih that may respond well to corticosteroid therapy alone . whether this is a form of classic aih or a distinct entity awaits a more extensive description of its clinical and immunohistological features . the diagnosis of aih requires the presence of characteristic clinical features and exclusion of other chronic liver conditions , such as viral hepatitis , drug - induced hepatitis , fatty liver disease , alcohol related liver disease , wilson 's disease , alpha 1 antitrypsin deficiency , or hemochromatosis . laboratory studies typically show elevation of aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) levels , but levels are generally < 500 u / l , but on rare occasions can range between 5001000 some patients may have high conjugated bilirubin and alkaline phosphatase necessitating exclusion of extrahepatic biliary obstruction , cholestatic forms of viral hepatitis , drug - induced disease , primary biliary cirrhosis ( pbc ) , and primary sclerosing cholangitis ( psc ) . the alkaline phosphatase rarely exceeds 4 x normal and generally remains < 2 times normal . another characteristic laboratory feature of aih is hypergammaglobulinemia , with a selective increase in igg , which is 1.23.0 times higher than the upper level of normal . it should be noted that hla typing has not been endorsed as a diagnostic or prognostic tool . the characteristic circulating autoantibodies seen in aih include ana , sma , and ( lkm-1 ) autoantibodies . a list of the important autoantibodies and their autoantigenic targets is summarized in table 2 . they are helpful in diagnosis as well as for classification of aih into type 1 and type 2 diseases . the reader is referred to excellent reviews for description of methodology , sensitivity , and assay performance [ 27 , 39 , 64 ] . except for panca , which is readily available and can be positive in 5090% of type i aih , only antiactin can be easily in measured in some laboratories . recently , antibodies to cyclic citrullinated peptides ( ccp ) have been described in 911% of patients with aih in absence of rheumatoid arthritis . antimitochondrial antibodies are sometimes present in patients with aih and an overlap syndrome of aih and pbc should be considered in these patients . a diagnostic system was proposed by the international autoimmune hepatitis group ( iaihg ) in 1993 and subsequently updated in 1999 [ 66 , 67 ] . in 2008 , they proposed a simplified set of diagnostic criteria to facilitate early recognition and initiation of adequate immunosuppressive treatment . these included the presence of specific autoantibodies ( ana , sma , lkm antibody , sla antibody ) in moderate to high titers , hypergammaglobulinemia , typical histological pattern on liver biopsy , and exclusion of viral hepatitis . these criteria have a lower sensitivity ( 85% versus 100% ) but higher specificity ( 99% versus 93% ) than the original criteria and are good at identifying patients with all typical characteristics of a classic case of aih [ 69 , 70 ] . however , miyake et al . showed that 30% of males , 23% of patients with acute clinical presentation , and 46% patients negative for ana were not diagnosed with aih by simplified criteria even though they met the original criteria . therefore , it fails to adequately identify cases with atypical features which is an important point to keep in mind . the histologic hallmark of aih is a lymphoplasmacytic periportal infiltrate invading the limiting plate , also called piecemeal necrosis or it causes distortion of the hepatic lobule and the appearance of regenerative nodules , resulting in cirrhosis . many patients with acute presentation may have chronic features on liver biopsy indicating a subclinical phase of disease and several patients with mild clinical disease may have advanced fibrosis on biopsy . of important note is the fact that the fibrosis and even cirrhosis in aih is reversible to a significant degree with immunosuppressive therapy unlike in other chronic liver diseases . there are no specific imaging techniques to confirm the diagnosis of autoimmune hepatitis . in adults with both aih and ibd , cholangiographic changes suggestive of psc are present in up to 44% patients and may affect therapy and prognosis . in children with aih , autoimmune sclerosing cholangitis can be present with or without inflammatory bowel disease . histologic presence of bridging or multilobular necrosis is associated with progression to cirrhosis in 82% cases and a 5-year mortality of 45% in untreated patients . in asymptomatic patients with inactive cirrhosis ( defined as no or limited inflammation ) patients without cirrhosis who undergo treatment have a 1020 year survival probability more than 80% , similar to the general population . retrospective analysis of patients with mild disease has demonstrated the possibility of long - term survival without treatment , but very careful follow - up is required . untreated patients may , rarely , recover spontaneously , but improvement is less common than treated patients , and long - term survival is lower . aih can have unpredictable and varying disease activity and ultimately the majority of patients with active inflammation will warrant therapy . indications for treatment are listed in table 3 and are based on the presence and severity of hepatic inflammation . the indications are also reflective of risk factors for disease progression as severely abnormal liver enzyme elevation , incapacitating symptoms , histological presence of interface hepatitis , bridging necrosis , or multiacinar collapse portend a worse prognosis without treatment . prednisone alone ( 60 mg daily with taper down to 20 mg daily in 4 weeks ) or at a lower dose ( 30 mg with taper down to 10 mg daily in 4 weeks ) in combination with azathioprine ( 50 mg daily ) is the most effective treatment regimen studied in randomized clinical trials . histologic improvement lags behind clinical and laboratory resolution by 3 to 8 months , and therapy should be continued for at least 36 months beyond this point of improvement . treatment is often maintained for at least 2 years before withdrawal of drug therapy is considered . the end points for treatment include remission , treatment failure , incomplete response , or development of drug toxicity . prednisone is used alone in patients with severe cytopenias , active malignancy , pregnant or contemplating pregnancy , and those with complete thiomethylpurine transferase ( tpmt ) enzyme deficiency . combination therapy is associated with lesser side effects and is preferred when treatment is expected to be more than 6 months and in patients at risk of side effects including postmenopausal women , brittle diabetics , labile hypertensive , and osteoporotic patients . therapy may span over several years and hence treatment side effects must be taken into consideration . corticosteroids can cause weight gain , central obesity , moon facies , prominent supraclavicular fat pad , acne , bruising , cutaneous striae , cataracts , glaucoma , peptic ulcers , deterioration of hypertension and diabetic control . long - term side effects include increased risk of fractures secondary to osteoporosis and avascular necrosis of bone . patients with brittle diabetes , severe osteoporosis , vertebral compression fractures , psychosis , obesity , and uncontrolled hypertension should be carefully evaluated for a treatment benefit before starting corticosteroids . if severity of disease necessitates corticosteroid therapy , adequate measures should be instituted to control the comorbid conditions . in patients with mild disease or relative contraindications to prednisone , budesonide 3 mg tid ( in place of prednisone ) is an option to reduce overall treatment side effects with no impairment of efficacy [ 77 , 78 ] . its benefits are derived from the 90% first pass metabolism which results in less steroid - induced side effects while maintaining efficacy . alternative regimens must be considered in several circumstances : after treatment failure with prednisone ( 60 mg daily ) or prednisone ( 30 mg daily ) and azathioprine ( 150 mg daily ) , incomplete response to conventional therapy , or intolerance to conventional therapy . mycophenolate mofetil ( 2 g daily ) has shown improvement in 3984% patients who were unable to tolerate azathioprine but use was limited by side effects ( nausea , vomiting , rash , pancreatitis , diarrhea , cytopenia ) [ 7981 ] . there are studies demonstrating benefit to the use of cyclosporine ( in conjunction with prednisone ) for patients refractory to standard therapy . in addition , a report suggests some benefit to tacrolimus , but has not been evaluated in randomized clinical trials . ursodeoxycholic acid has been studied in randomized trials and unfortunately was not found to be of benefit . relapse is characterized by an increase in the serum aminotransferase levels to at least threefold normal . relapse occurs in 50% to 86% of patients , most often during the first 6 months after the termination of therapy ( 50% ) . the first relapse after drug withdrawal should be retreated with a combination of prednisone plus azathioprine at the same treatment regimen as with the initial course of therapy and then tapered to monotherapy with either azathioprine ( 2 mg / kg daily ) as a long - term maintenance therapy or indefinite low - dose prednisone ( 10 mg daily ) in patients intolerant of azathioprine . gradual withdrawal from maintenance therapy may be attempted again after at least 24 months of treatment and continued normal serum ast or alt level only after careful benefit risk evaluation in patients who had previously relapsed . aih is the underlying cause for 4%6% cases of liver transplants done in the western world [ 85 , 86 ] . it usually results from a failure to diagnose and treat aih as an etiology of cirrhosis , inadequate response or intolerance to immunosuppressive therapy , or noncompliance with treatment . treatment failure requiring transplant is more often associated with the hla genotype drb1 * 0301 . liver transplantation should be considered in patients with aih and acute liver failure , decompensated cirrhosis with a meld score 15 , or hepatocellular carcinoma meeting criteria for transplantation . a combination of prednisone and a calcineurin inhibitor ( tacrolimus more frequently than cyclosporine ) is the most common immunosuppression regimen after lt . despite this , aih can recur in transplanted livers or occur de novo in liver transplants done for non - aih conditions , but discussion of this is beyond the scope of this review article . the greatest risk is prematurity , but fetal mortality has been reported to be as high as 21% . maternal antibodies to sla and extractable nuclear antigens ( ro / ssa ) have been associated with a more complicated pregnancy . azathioprine is an fda category d drug and safety in pregnancy has not been well established in human studies . although increased number of birth defects have not been reported in neonates of women receiving this treatment and no adverse consequences of breast feeding have been noted by treated mothers [ 91 , 92 ] , congenital malformations have been reported in pregnant mice , and , thus there is a potential risk for teratogenicity . this justifies exercising caution when using in pregnancy , thus the mainstay of treatment in pregnancy is prednisone at as low dose as possible . aih commonly exacerbates following delivery , therefore therapy must be resumed ( if stopped ) or increased 2 weeks prior to anticipated delivery and continued in the postpartum period . women with advanced cirrhosis and portal hypertension are at high risk for variceal hemorrhage during pregnancy . aih is associated with chronic inflammation that may proceed to cirrhosis and end - stage liver disease which also puts aih patients at risk of developing hcc . however , unlike other cohorts of cirrhotic patients , the majority of patients with aih respond well to immunosuppression and in those whom enter a sustained remission , the potential exists to retain a near normal life expectancy . however , the interactions of disease activity , response to treatment , and other factors in relation to the risk of hcc development in aih are unknown . although the development of hcc in patients with aih and cirrhosis is considered a rare occurrence , the true incidence remains unknown due to the paucity of published data addressing this issue . a large prospectively obtained cohort at a single center ( n = 243 ) determined a rate of hcc development of 1.1% per year , with equal proportions among men and women . the median duration from time of confirmed cirrhosis to not surprisingly , hcc was found to occur more frequently in patients with cirrhosis at presentation ( 9.3% versus 3.4% , p = .048 ) or history of variceal bleed as the index presentation of aih ( 20% versus 5.3% , p = .003 ) . median survival in patients whose hcc was diagnosed on surveillance was higher ( 19 months versus 2 months ) compared with patients presenting symptomatically ( p = .042 ) . the majority of patients develop hcc after having cirrhosis for an average of 9 years , and although the incidence of hcc is less common than in other chronic liver diseases , the risk may be sufficient to undertake surveillance in all patients with cirrhosis with aih who are candidates to undergo curative therapies . autoimmune hepatitis is one of the few liver diseases with excellent response to therapy . on the other hand , it still remains a liver disease with many unanswered questions , particularly in respect to its etiology and pathogenesis . there is significant heterogeneity in its presentation that may mask its identity , affect its clinical behavior , and confound its management . it may start with a fulminant course , and the diagnosis should not be overlooked when dealing with patients with acute liver failure . alternatively , it may behave as a slowly progressing disease , and it is still controversial whether those patients need immunosuppressive treatment at all . there is no prescribed minimum or maximum duration of treatment . over the last decade , remarkable progress has been made in understanding and clarifying the areas of diagnosis with introduction of classification criteria , and broadening therapeutic options , with trial of several new medications like budesonide and mycophenolate mofetil , and more in the pipeline . management , however , still faces several other important issues , such as in children , the elderly , in males , and during the preconception period , pregnancy , and lactation . a key to successful management is thinking of it , recognizing the nonclassical presentations , and individualizing therapy .

autoimmune hepatitis ( aih ) is a chronic inflammatory disorder characterized by periportal inflammation , elevated immunoglobulins , autoantibodies , and a dramatic response to immunosuppression . an environmental agent is hypothesized to trigger an immune - mediated attack directed against liver antigens in genetically predisposed individuals . a plethora of clinical presentations can be seen ranging from chronic indolent disease to fulminant hepatic failure , and diagnosis requires exclusion of other causes of liver disease . corticosteroid therapy must be instituted early and modified in an individualized fashion . treatment decisions are often complicated by the diverse clinical manifestations , uncertainty about natural history , evolving ideas about treatment end points , and a multitude of alternative immunosuppressive agents . achieving normal liver tests and tissue is the ideal treatment end point , but needs to be weighed against the risk of side effects . decompensated patients may benefit from early liver transplantation . long - term prognosis is excellent with early and aggressive initiation of therapy . our paper discusses aih , giving a detailed overview of its clinical presentation , risk factors , immunopathogenesis , up - to - date diagnostic criteria , current updates in therapy with a brief discussion of aih in pregnancy , and long - term implications for cirrhosis and hepatocellular carcinoma in aih patients .

1. Background 2. Classification 3. Clinical Manifestations 4. Diagnosis of Autoimmune Hepatitis 5. Therapy 6. Other Important Dimensions of AIH 7. Conclusion and Future Perspectives

autoimmune hepatitis ( aih ) is a chronic inflammatory disease of unknown etiology characterized by the presence of circulating autoantibodies , hypergammaglobulinemia , necroinflammatory changes on hepatic histology , and a dramatic response to immunosuppressive therapy . earliest descriptions include those by amberg in 1942 and leber in 1950 describing a form of chronic liver disease prevalent among young women and characterized by an excessive increase in serum protein and gamma - globulins . the disease is rare with a mean incidence of 1 - 2 per 100,000 and a point prevalence of 1117 per 100,000 [ 5 , 6 ] . a minority of patients may present with acute liver failure and need liver transplantation , but for the majority , the prognosis of aih is good and mostly determined by response to corticosteroid therapy . in general , long - term survival and average life expectancy are excellent and estimated to be comparable to the normal population . type i aih is characterized by the presence of antinuclear antibody ( ana ) , anti - smooth muscle antibody ( sma ) , or both and constitutes 80% of aih cases . about 25% have cirrhosis at presentation , and association with other autoimmune diseases is common ( celiac disease , ulcerative colitis , autoimmune thyroid disease ) [ 10 , 11 ] . type 2 aih is characterized by the presence of anti - liver kidney microsomal ( lkm ) 1 and/or anti - lkm3 and/or anti - liver cytosol 1 ( lc1 ) [ 12 , 13 ] antibodies . most patients are children , acute severe presentation can occur , and progression to cirrhosis commonly ensues . in patients who are negative for conventional antibodies and aih is strongly suspected , additional tests can be done including perinuclear antineutrophil cytoplasmic antibodies ( panca ) , actin ( anti - actin ) , soluble liver antigen ( anti - sla ) , asialoglycoprotein receptor ( anti - asgpr ) , chromatin , and liver cytosol type 1 ( anti - lc1 ) . in our experience , 1015% patients do not have either ana , sma , or anti - lkm1 at presentation , but 25% of these will have detectable conventional antibodies later in their course . although the exact etiopathogenesis is unknown , aih , like many autoimmune diseases , is thought to be caused by environmental triggers and failure of immune tolerance mechanisms in a genetically susceptible host . these triggers may be of viral or drug etiology , but most cases have an unknown trigger . triggers may share epitopes that resemble self - antigens , and molecular mimicry between foreign antigens and self - antigens is the most frequently proposed initiating mechanism in type 2 aih where the autoantigen is known . aih is a complex polygenic disease and different populations may have different genetic and environmental triggers and genetic association varies in study populations . hla may be associated with age at presentation , disease severity , and response to therapy . different susceptibility alleles like hla dr1 * 0301 , dr1 * 0401 , dr1 * 0404 , and dr1 * 0405 share a common motif , namely , amino acids lleqkr or lleqrr at position 67 - 72 of class ii hla , whereas the resistant alleles dr11501 encodes ileqar [ 20 , 21 ] . however , these associations are not absolute and significant geographic differences exist , for example , in japan dr2 ( drb1 * 1501 ) is a weak susceptibility rather than a resistance allele and in south american children drb1 * 1301 is a strong susceptibility allele . a form of aih can be seen in 20% of patients with autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy ( apeced ) syndrome . apeced is a monogenic , autosomal recessive disorder characterized by hypoparathyroidism , adrenal insufficiency , and chronic mucocutaneous candidiasis . this is the only syndrome involving aih that exhibits a mendelian pattern of inheritance , and genetic testing and counseling for the patient and family members are warranted . aih is an inflammatory disorder of the liver involving multiple components of the immune system including t cells , b cells , and cytokines . hepatocytes isolated from aih patients are coated with immunoglobulins and are susceptible to antibody - dependent cellular cytotoxicity ( adcc ) when exposed to autologous mononuclear cells bearing fc receptors . mice immunized with plasmid containing human cyp2d6 antigenic region and human formiminotransferase cyclodeaminase ( another autoantigen ) , have established a murine model for autoimmune hepatitis type 2 . these mice develop autoantibodies , elevation in transaminases , along with portal and periportal inflammatory infiltrate . these models will aid the development of more therapeutic options in the management of autoimmune hepatitis . studies have demonstrated presence of cytotoxic cells in both t and non - t cell compartment of peripheral blood from aih patients . patients with aih have a ten fold higher frequency of liver - specific t cells compared to normal subjects . cd8 t cells have cytotoxic capability , are capable of secreting ifn- , and their responses correlate with disease activity . defects in numbers and function of regulatory cells ( t regs ) have been demonstrated in aih . in patients with aih , the number of t regs is decreased more so at disease presentation than at drug - induced remission . t regs may , therefore , be an attractive therapeutic target , but more studies are needed to elucidate this better . herbal agents such as black cohosh and dai - saiko - to have been proposed to induced aih . ana and sma can occur in diverse causes of acute and chronic hepatitis including alcoholic , nonalcoholic fatty liver disease , and viral hepatitis . if clinical concern for autoimmune hepatitis exists , antiactin antibodies can be checked as they increase the specificity of sma testing for diagnosing aih . anti - lkm1 has been found in as many as 10% of patients with chronic hepatitis c and is different from the anti - lkm1 found in classic autoimmune hepatitis . women constitute at least 70% of cases , and 50% are younger than 40 years ; however , age at onset may range from infancy to the elderly . forty percent of patients may present with an acute onset , but the presentation of severe fulminant hepatic failure is rare . presenting symptoms may include fatigue , lethargy , malaise , arthralgia of small joints , anorexia , nausea , abdominal pain , and dark urine . clinical manifestations may vary by ethnicity ; the presentation is acute and icteric in alaskan native patients , cholestatic in aboriginal north american , african , asian , and arab patients , mild in japanese patients , but severe and rapidly progressive in somali patients . physical examination may be normal , but may also reveal hepatomegaly , splenomegaly , jaundice , and stigmata of chronic liver disease . findings such as acne , hirsutism , obesity , and amenorrhea in young women are rarely seen . other autoimmune diseases such as hashimoto thyroiditis , type 1 diabetes , rheumatoid arthritis , systemic lupus erythematosus , ulcerative colitis / crohn 's disease , and celiac disease can be seen in 20% of patients . this subtype is associated with higher serum levels of igg , aih severity scores , and , more importantly , an excellent response to prednisone therapy for induction and maintenance of remission . positive igg4 staining is suggested by the authors as a surrogate marker for the subtype of aih that may respond well to corticosteroid therapy alone . whether this is a form of classic aih or a distinct entity awaits a more extensive description of its clinical and immunohistological features . the diagnosis of aih requires the presence of characteristic clinical features and exclusion of other chronic liver conditions , such as viral hepatitis , drug - induced hepatitis , fatty liver disease , alcohol related liver disease , wilson 's disease , alpha 1 antitrypsin deficiency , or hemochromatosis . laboratory studies typically show elevation of aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) levels , but levels are generally < 500 u / l , but on rare occasions can range between 5001000 some patients may have high conjugated bilirubin and alkaline phosphatase necessitating exclusion of extrahepatic biliary obstruction , cholestatic forms of viral hepatitis , drug - induced disease , primary biliary cirrhosis ( pbc ) , and primary sclerosing cholangitis ( psc ) . another characteristic laboratory feature of aih is hypergammaglobulinemia , with a selective increase in igg , which is 1.23.0 times higher than the upper level of normal . the characteristic circulating autoantibodies seen in aih include ana , sma , and ( lkm-1 ) autoantibodies . they are helpful in diagnosis as well as for classification of aih into type 1 and type 2 diseases . the reader is referred to excellent reviews for description of methodology , sensitivity , and assay performance [ 27 , 39 , 64 ] . except for panca , which is readily available and can be positive in 5090% of type i aih , only antiactin can be easily in measured in some laboratories . recently , antibodies to cyclic citrullinated peptides ( ccp ) have been described in 911% of patients with aih in absence of rheumatoid arthritis . antimitochondrial antibodies are sometimes present in patients with aih and an overlap syndrome of aih and pbc should be considered in these patients . a diagnostic system was proposed by the international autoimmune hepatitis group ( iaihg ) in 1993 and subsequently updated in 1999 [ 66 , 67 ] . in 2008 , they proposed a simplified set of diagnostic criteria to facilitate early recognition and initiation of adequate immunosuppressive treatment . these included the presence of specific autoantibodies ( ana , sma , lkm antibody , sla antibody ) in moderate to high titers , hypergammaglobulinemia , typical histological pattern on liver biopsy , and exclusion of viral hepatitis . these criteria have a lower sensitivity ( 85% versus 100% ) but higher specificity ( 99% versus 93% ) than the original criteria and are good at identifying patients with all typical characteristics of a classic case of aih [ 69 , 70 ] . showed that 30% of males , 23% of patients with acute clinical presentation , and 46% patients negative for ana were not diagnosed with aih by simplified criteria even though they met the original criteria . the histologic hallmark of aih is a lymphoplasmacytic periportal infiltrate invading the limiting plate , also called piecemeal necrosis or it causes distortion of the hepatic lobule and the appearance of regenerative nodules , resulting in cirrhosis . of important note is the fact that the fibrosis and even cirrhosis in aih is reversible to a significant degree with immunosuppressive therapy unlike in other chronic liver diseases . there are no specific imaging techniques to confirm the diagnosis of autoimmune hepatitis . in children with aih , autoimmune sclerosing cholangitis can be present with or without inflammatory bowel disease . histologic presence of bridging or multilobular necrosis is associated with progression to cirrhosis in 82% cases and a 5-year mortality of 45% in untreated patients . retrospective analysis of patients with mild disease has demonstrated the possibility of long - term survival without treatment , but very careful follow - up is required . untreated patients may , rarely , recover spontaneously , but improvement is less common than treated patients , and long - term survival is lower . the indications are also reflective of risk factors for disease progression as severely abnormal liver enzyme elevation , incapacitating symptoms , histological presence of interface hepatitis , bridging necrosis , or multiacinar collapse portend a worse prognosis without treatment . prednisone alone ( 60 mg daily with taper down to 20 mg daily in 4 weeks ) or at a lower dose ( 30 mg with taper down to 10 mg daily in 4 weeks ) in combination with azathioprine ( 50 mg daily ) is the most effective treatment regimen studied in randomized clinical trials . histologic improvement lags behind clinical and laboratory resolution by 3 to 8 months , and therapy should be continued for at least 36 months beyond this point of improvement . the end points for treatment include remission , treatment failure , incomplete response , or development of drug toxicity . prednisone is used alone in patients with severe cytopenias , active malignancy , pregnant or contemplating pregnancy , and those with complete thiomethylpurine transferase ( tpmt ) enzyme deficiency . combination therapy is associated with lesser side effects and is preferred when treatment is expected to be more than 6 months and in patients at risk of side effects including postmenopausal women , brittle diabetics , labile hypertensive , and osteoporotic patients . therapy may span over several years and hence treatment side effects must be taken into consideration . long - term side effects include increased risk of fractures secondary to osteoporosis and avascular necrosis of bone . patients with brittle diabetes , severe osteoporosis , vertebral compression fractures , psychosis , obesity , and uncontrolled hypertension should be carefully evaluated for a treatment benefit before starting corticosteroids . if severity of disease necessitates corticosteroid therapy , adequate measures should be instituted to control the comorbid conditions . in patients with mild disease or relative contraindications to prednisone , budesonide 3 mg tid ( in place of prednisone ) is an option to reduce overall treatment side effects with no impairment of efficacy [ 77 , 78 ] . its benefits are derived from the 90% first pass metabolism which results in less steroid - induced side effects while maintaining efficacy . alternative regimens must be considered in several circumstances : after treatment failure with prednisone ( 60 mg daily ) or prednisone ( 30 mg daily ) and azathioprine ( 150 mg daily ) , incomplete response to conventional therapy , or intolerance to conventional therapy . mycophenolate mofetil ( 2 g daily ) has shown improvement in 3984% patients who were unable to tolerate azathioprine but use was limited by side effects ( nausea , vomiting , rash , pancreatitis , diarrhea , cytopenia ) [ 7981 ] . in addition , a report suggests some benefit to tacrolimus , but has not been evaluated in randomized clinical trials . ursodeoxycholic acid has been studied in randomized trials and unfortunately was not found to be of benefit . relapse occurs in 50% to 86% of patients , most often during the first 6 months after the termination of therapy ( 50% ) . the first relapse after drug withdrawal should be retreated with a combination of prednisone plus azathioprine at the same treatment regimen as with the initial course of therapy and then tapered to monotherapy with either azathioprine ( 2 mg / kg daily ) as a long - term maintenance therapy or indefinite low - dose prednisone ( 10 mg daily ) in patients intolerant of azathioprine . aih is the underlying cause for 4%6% cases of liver transplants done in the western world [ 85 , 86 ] . liver transplantation should be considered in patients with aih and acute liver failure , decompensated cirrhosis with a meld score 15 , or hepatocellular carcinoma meeting criteria for transplantation . a combination of prednisone and a calcineurin inhibitor ( tacrolimus more frequently than cyclosporine ) is the most common immunosuppression regimen after lt . despite this , aih can recur in transplanted livers or occur de novo in liver transplants done for non - aih conditions , but discussion of this is beyond the scope of this review article . the greatest risk is prematurity , but fetal mortality has been reported to be as high as 21% . maternal antibodies to sla and extractable nuclear antigens ( ro / ssa ) have been associated with a more complicated pregnancy . azathioprine is an fda category d drug and safety in pregnancy has not been well established in human studies . although increased number of birth defects have not been reported in neonates of women receiving this treatment and no adverse consequences of breast feeding have been noted by treated mothers [ 91 , 92 ] , congenital malformations have been reported in pregnant mice , and , thus there is a potential risk for teratogenicity . this justifies exercising caution when using in pregnancy , thus the mainstay of treatment in pregnancy is prednisone at as low dose as possible . aih commonly exacerbates following delivery , therefore therapy must be resumed ( if stopped ) or increased 2 weeks prior to anticipated delivery and continued in the postpartum period . women with advanced cirrhosis and portal hypertension are at high risk for variceal hemorrhage during pregnancy . aih is associated with chronic inflammation that may proceed to cirrhosis and end - stage liver disease which also puts aih patients at risk of developing hcc . however , unlike other cohorts of cirrhotic patients , the majority of patients with aih respond well to immunosuppression and in those whom enter a sustained remission , the potential exists to retain a near normal life expectancy . however , the interactions of disease activity , response to treatment , and other factors in relation to the risk of hcc development in aih are unknown . the median duration from time of confirmed cirrhosis to not surprisingly , hcc was found to occur more frequently in patients with cirrhosis at presentation ( 9.3% versus 3.4% , p = .048 ) or history of variceal bleed as the index presentation of aih ( 20% versus 5.3% , p = .003 ) . the majority of patients develop hcc after having cirrhosis for an average of 9 years , and although the incidence of hcc is less common than in other chronic liver diseases , the risk may be sufficient to undertake surveillance in all patients with cirrhosis with aih who are candidates to undergo curative therapies . autoimmune hepatitis is one of the few liver diseases with excellent response to therapy . on the other hand , it still remains a liver disease with many unanswered questions , particularly in respect to its etiology and pathogenesis . there is significant heterogeneity in its presentation that may mask its identity , affect its clinical behavior , and confound its management . it may start with a fulminant course , and the diagnosis should not be overlooked when dealing with patients with acute liver failure . alternatively , it may behave as a slowly progressing disease , and it is still controversial whether those patients need immunosuppressive treatment at all . over the last decade , remarkable progress has been made in understanding and clarifying the areas of diagnosis with introduction of classification criteria , and broadening therapeutic options , with trial of several new medications like budesonide and mycophenolate mofetil , and more in the pipeline . management , however , still faces several other important issues , such as in children , the elderly , in males , and during the preconception period , pregnancy , and lactation . a key to successful management is thinking of it , recognizing the nonclassical presentations , and individualizing therapy .

the solar wind is a highly ionized , magnetized plasma which flows at supersonic and superalfvnic speeds away from the sun . in this medium there have been many observations of enhanced magnetic fluctuations at frequencies near the proton cyclotron frequency . there are two hypotheses as to the source of these enhanced fluctuation events [ jian et al . , one school holds that these largeamplitude waves are generated close to the sun and are then transported by the solar wind to observation at planetary distances . the alternative picture is that the expanding solar wind flow leads to nonthermal electron and ion velocity distributions which locally excite shortwavelength instabilities which are observed near their source . this manuscript uses plasma and magnetic field measurements with kinetic linear dispersion theory to demonstrate that several enhanced magnetic fluctuations events observed by the wind spacecraft on 19 march 2005 are unstable to one or more kinetic instabilities , thereby providing evidence that these particular events are locally generated . the hot ( tens of ev ) , tenuous ( few particles per cubic centimeter near 1 au ) character of the solar wind implies that it is a collisionless plasma in which a variety of different kinetic instabilities can arise if appropriate sources of free energy are available . kinetic instabilities are those which are driven by velocityspace anisotropies and which typically have maximum growth rates arising in the short wavelength regime corresponding to wavelengths the order of or shorter than either the thermal ion gyroradius or the ion inertial length [ gary , 1993 ] . if a plasma is in equilibrium , the velocity distributions of the constituent species are maxwellian . all fluctuations in such plasmas either are damped or propagate with no change in amplitude . for frequencies near the proton cyclotron frequency and propagation quasiparallel to b o , the cases of interest here , there are three fundamental normal modes [ gary , 1993 , chapter 6 ] : predominantly electrostatic ion acoustic waves , predominantly electromagnetic alfvncyclotron waves with lefthand circular polarization in the limit of k x b o = 0 , and predominantly electromagnetic magnetosonic waves with righthand circular polarization at k x b o = 0 . ion acoustic waves propagate with weak damping only if te > > tp ; as this is not a typical condition in the solar wind , we do not consider such modes further . at very long wavelengths and low frequencies alfvn waves at k x bo = 0 are nonresonant and essentially undamped . but as the frequencies of such modes approach p , the proton cyclotron frequency , the waves become dispersive ; the resulting alfvncyclotron fluctuations undergo proton cyclotron damping and propagate with real frequencies only at r < p . longwavelength magnetosonic fluctuations k x bo = 0 are also nonresonant and very weakly damped , but because of their righthand polarization , they do not suffer proton cyclotron damping and propagate at frequencies through and well above p where they become strongly dispersive whistler modes . although various types of nonthermal ion and electron velocity distributions are observed in the interplanetary medium [ feldman et al . , 1973 ; marsch , 1991 ] , especially in the fast wind , for purposes of instability analysis nonequilibrium distributions are usually characterized as having temperature anisotropies and/or multiple components with relative flow speeds between component pairs . here , 1973 ; leubner and vias , 1986 ; marsch and livi , 1987 ; goldstein et al . , 2000 ; he et al . , 2015 ] and assume that proton velocity distributions can be fit as the sum of two components , a more dense , slower core ( denoted by subscript c ) and a less dense , faster beam ( subscript b ) . we further assume that both proton components , as well as the electrons ( subscript e ) and the alpha particles ( subscript ) , can each be represented as a bimaxwellian distribution with t t|| ( where the subscripts represent directions relative to b o ) and relative flow velocities parallel to b o. we denote the relative beam / core flow velocity as v o = v ob v oc where v oj is the average flow velocity of the jth component in the plasma ( center of mass ) frame and the relative alpha / core flow velocity as v oc = v ov oc . we define the ion ( electron ) inertial length i = c/pi ( e = c/pe ) where pj is the plasma frequency of the jth species , and the ion ( electron ) thermal gyroradius i = vi/i ( e = ve/e ) where vj is the thermal speed of the jth species , and j is the cyclotron frequency of the jth species . the jth component parallel plasma beta is defined as ||j = 8njkbt||j / bo , and the alfvn speed is va = bo/(4nemp ) where ne = np + 2n. anisotropies in solar wind ion velocity distributions are a rich source of diverse kinetic plasma instabilities . the enhanced fluctuations that arise from such growing modes scatter the nonthermal species , reducing their anisotropies and driving them toward ( but not completely to ) the maxwellian distributions of thermal equilibrium [ e.g. , gary et al . [ 2009 , 2010 , 2014 ] ( see also s. a. boardsen et al . , mercury surface , space environment , geochemistry , and ranging ( messenger ) survey of in situ lowfrequency wave storms between 0.3 and 0.7 au , submitted to journal of geophysical research , 2015 ) have used highresolution magnetometer observations from the stereo , messenger , and helios spacecraft to study solar wind events of enhanced magnetic field fluctuations with frequencies near p at propagation quasiparallel to the background magnetic field b o. by comparing lefthand versus righthand polarizations in the spacecraft frame , they found that the lefthand waves are generally stronger and appear slightly more often . however , their analyses utilized proton data reduction algorithms which were less general than the beam / core bimaxwellian velocity distributions used in this paper , thereby limiting their ability to compare temperature anisotropy and beam / core distributions as sources of protondriven instabilities . here we follow these earlier papers by identifying events of interest as corresponding to enhanced field fluctuations with frequencies near the proton cyclotron frequency . the new element here is that we correlate these events with particle species observations under the more general and more accurate assumption that the proton distributions can be described as the sum of beam and core components with separate densities , flow speeds , temperatures , and temperature anisotropies . using this model in kinetic linear dispersion theory , we determine which types of iondriven instabilities are the likely sources of these enhanced fluctuation events . [ 2009 , 2010 , 2014 ] , our concerns here are iondriven instabilities which propagate at directions quasiparallel to b o. this condition corresponds to three categories of growing modes : alfvncyclotron instabilities driven by ion component anisotropies such that t / t|| > 1 [ gary et al . , 2001 ] , parallel firehose instabilities driven by ion component anisotropies such that t / t|| < 1 [ hellinger et al . , 2006 ] , and ion / ion magnetosonic instabilities driven by the relative flow speed of two ion components [ goldstein et al . , 2000 ] . under the condition k x b o = 0 alfvncyclotron waves and instabilities have lefthand circular polarization , whereas the parallel firehose instability , magnetosonic waves , and magnetosonic proton / proton instabilities all have righthand circular polarization in the solar wind frame . if the proton velocity distribution may be approximated as a single bimaxwellian and heavy ions may be ignored , growth rate predictions of kinetic linear theory of electromagnetic instabilities driven by proton temperature anisotropies may be expressed in terms of two parameters , tp / t||p and ||p . figure 7.3 of gary shows that these two parameters characterize the linear theory thresholds of such instabilities . gary et al . used hybrid simulations to show that the alfvncyclotron instability threshold condition represents an upper bound for tp / t||p > 1 , and gary et al . used the same type of simulations to show that the protonparallel firehose instability threshold constitutes an upper bound for the opposite sense of the proton anisotropy . considering only solar wind conditions in which the proton velocity distribution may be approximated as a single bimaxwellian component , kasper et al . [ 2002 , 2006 ] , hellinger et al . , bale et al . , maruca et al . [ 2011 , 2012 ] , osman et al . , and hellinger and trvnek used measurements from the wind spacecraft to show that instability thresholds in tp / t||p versus ||p parameter space indeed correspond to observed proton anisotropy bounds . however , thresholds of the proton mirror and the oblique proton firehose instabilities , which have wavevectors at substantially oblique angles relative to b o , provide better constraints to the observations than do the thresholds of the anisotropydriven instabilities with maximum growth at parallel propagation . furthermore , as discussed above , proton velocity distributions in the relatively fast , relatively collisionless solar wind are usually not well represented as a single bimaxwellian , but rather require at the minimum a two component core / beam description . linear dispersion theory analyses have demonstrated that a sufficiently large beam / core relative flow speed ( typically greater than about 1.5 va ) can drive both parallel magnetosonic and oblique alfvn instabilities [ montgomery et al . , 1975 , 1976 ; abrahamshrauner et al . , 1979 ; dum et al . , 1980 ; marsch and livi , 1987 ; daughton and gary , 1998 ; goldstein et al . , 2000 ] . however , kinetic linear theories of proton beam / core instabilities require at least six independent proton parameters , e.g. , the proton core || , the proton core relative density nc / ne , the dimensionless beam / core relative flow speed vo / va , t||b / t||c , and the two proton component temperature anisotropies tc / t||c and tb / t||b , as well as one or more electron parameters [ daughton and gary , 1998 ] and at least four additional parameters characterizing each minor ion species such as the alpha particles . this parametric plethora makes it difficult to present linear theory threshold conditions of beam / core instabilities as twodimensional plots , requiring instead a large number of multidimensional figures to clearly expose the plasma physics of these growing modes . our approach here is to constrain this large parameter space through the use of detailed ion and electron measurements from the plasma instruments on board the wind spacecraft and to calculate dispersion properties of the enhanced fluctuations for specific conditions observed in the solar wind . the first is that undetermined processes in the collisionless solar wind ( e.g. , shocks , diverging flows , and/or largescale compressions ) act locally to push plasma species velocity distributions away from the maxwellian condition . the second is that the resulting nonmaxwellian distributions drive shortwavelength instabilities which generate enhanced field fluctuations which , in turn , act to stabilize the growing modes by reducing the distribution anisotropies . in this scenario the largeamplitude wave events we study should correspond to marginally stable or weakly unstable modes . our conclusion that the plasma observations are indeed subject to modest instability growth the wind spacecraft carries a fast magnetometer [ lepping et al . , 1995 ] , a pair of faraday cup instruments as part of the solar wind experiment ( swe ) [ ogilvie et al . , 1995 ] which measures velocity distributions of protons and alpha particles at a 92 s cadence and an electron electrostatic analyzer [ lin et al . , 1995 ] providing a full 3d electron velocity distribution at a 97 s cadence . after completing a survey of enhanced magnetic fluctuation events in the observed frequency range 0.01 hz < f < 5 hz using 0.092 s magnetic field data , we chose observations of such events in the solar wind on 19 march 2005 to be the subject of a comprehensive study as described below . on this date the wind spacecraft detected a number of events with wave propagation quasiparallel to the background magnetic field . we chose the events on this date because both lefthand and righthand polarized waves in the spacecraft frame are observed within a relatively short time interval , and the individual ion spectra from this period have been examined in detail by the swe coauthors on this manuscript . figure 1 illustrates the dynamic spectrum of the magnetic field data from 19 march 2005 . this analysis utilizes a fast fourier transform based on the means quadrature spectrum technique , which is explained in jian et al . . as shown in figure 1 , we found seven events of enhanced magnetic fluctuations , with the following common characteristics : ( 1 ) the coherence between the two transverse magnetic field components by and bz is enhanced , ( 2 ) the transverse power is substantially increased over the background power at earlier and later times , ( 3 ) the transverse power is much stronger than the compressional power , ( 4 ) the magnetic field fluctuations are nearly circularly polarized as indicated by the high absolute ellipticity , and ( 5 ) the propagation direction is within 10 of the background magnetic field direction . given these seven candidate events , we performed linear dispersion theory analyses for most of these intervals , using the method described below . the events meeting the criteria in jian et al . are marked in figure 2 , cyan for lefthand polarized in the spacecraft frame and yellow for righthand polarized in the spacecraft frame . the intervals for the seven events indicated in figure 1 are as follows : event # 1 ( 78.380 < doy < 78.389 ) , event # 2 ( 78.488 < doy < 78.494 ) , event # 3 ( 78.506 < doy < 78.515 ) , event # 4 ( 78.672 < doy < 78.685 ) , event # 5 ( 78.741 < doy < 78.748 ) , event # 6 ( 78.786 < doy < 78.798 ) , and event # 7 ( 78.804 < doy < 78.811 ) . magnetic field fluctuation measurements from the wind spacecraft on 19 march 2005 . ( a ) coherence between the two transverse components , by and bz ; ( b ) transverse power spectral density ; ( c ) the ellipticity in the spacecraft frame where + 1 corresponds to righthand circular polarization , and 1 corresponds to lefthand circular polarization ; and ( d ) the propagation angle with respect to b o. the yellow lines represent the local proton cyclotron frequency . the white solid and dashed lines labeled fsc+ and fsc represent the dopplershifted proton cyclotron frequency if the wave propagates parallel or antiparallel to the solar wind velocity under the assumption that the phase speed of a wave at the proton cyclotron frequency is v a. the fast fourier transform is performed for every 2560 points with a shift of 320 points and a bandwidth of 29 points . in figures 1b through 1d the color black represents parameter values for which the by and bz coherence is less than 0.5 . magnetic field and plasma measurements from the wind spacecraft as a function of time for 19 march 2005 . ( a ) the magnetic field vector in gse coordinates , where black represents the field magnitude , and red , green and blue represent bx , by , and bz , respectively . for the plasma properties shown in the subsequent five panels proton core parameters are indicated by red dots , proton beam parameters as blue dots , alpha particle parameters as magenta dots , and electron parameters as green dots . here are shown ( b ) the component number densities , ( c ) the average velocities in the x gse direction , ( d ) the average flow speed parallel to b o relative to the core protons and normalized to the alfvn speed , ( e ) the component || , and ( f ) the component t/t|| values . the seven events corresponding to enhanced proton cyclotron fluctuations discussed in the text are shown as vertical bands with cyan corresponding to lefthand polarization and yellow representing righthand polarization as measured in the spacecraft frame . for one 92 s measurement cycle of the ions in each event , the proton core , the proton beam , the electrons , and the alpha particles were each fit to bimaxwellian velocity distributions with relative flow velocities parallel to b o. the proton beam and alpha particle measurements during event # 5 are noisy and poorly defined , so this event is not further considered here . table 1 presents field and plasma parameters from the other six events analyzed in section 3 . due to weak signaltonoise ratios and/or strongly nonmaxwellian distributions , the fitting procedures from events # 2 through # 7 yield relatively large error bars for many of the alpha parameters . the values of the alpha temperature anisotropies are particularly suspect , so that because of these large uncertainties , we have assumed t/t|| = 1 for events # 2 , # 3 , # 4 , # 6 , and # 7 in table 1 and in the linear dispersion theory analyses described in section 3 . field and plasma parameters for six enhanced magnetic fluctuation events observed from wind spacecraft on 19 march 2005a values in parenthesis represent the assumption of isotropic alpha particles because of poor fits to the model of flowing bimaxwellian alpha particle velocity distributions . figure 2 displays the magnetic field vector in geocentric solar equatorial ( gse ) coordinates , the number density , speed in the x direction ( antisunward ) , the dimensionless beam / core and alpha / core relative flow speeds , ||j , and temperature anisotropy of core proton ( in red ) , beam proton ( in blue ) , alpha particles ( in magenta ) , and electrons ( in green ) . for all seven events , bx > 0 , implying that this component of the magnetic field points sunward . also for all seven events vocx > 0 , vobx < 0 , and vox < 0 so that the beam / core relative flow points in the antisunward direction . figure 3 illustrates the magnetic fluctuation power spectra for four of these events as labeled . as is typical for solar wind events , 2014 , figure 7b ] , the energy density of field fluctuations transverse to b o is 1 to 2 orders of magnitude greater than that of the compressional fluctuations parallel / antiparallel to the background magnetic field . polarization analysis shows that the fluctuations of events # 1 , # 6 , and # 7 are predominantly lefthand polarized , whereas event # 4 corresponds primarily to righthand polarization fluctuations as measured in the spacecraft frame . the magnetic fluctuation power spectra during events # 1 , # 4 , # 6 , and # 7 observed from the wind spacecraft on 19 march 2005 . figure 4 shows selected ion measurements from the swe instrument during event # 1 . here bn indicates the angle between the magnetic field vector and the projection axis on which the distribution was measured ( a selection of 5 out of the 40 spin angles that make up the fit ) . the proton beam / core separation is clear here , as is the alpha / core relative flow . ( first column ) the stepwise black lines show the relative flux measurements from the swe instrument as functions of energy / charge for five different values of bn , the angle between b o and the projection axis on which the distribution was measured . the smooth curves display the ion distribution fits for proton core ( red ) , proton beam , ( blue ) , and alpha particle ( violet ) components . ( second column ) the proton and alpha particle velocity distributions in v||v space as represented by the proton core , proton beam , and alpha particle fits . this section describes results from kinetic linear dispersion theory of waves and instabilities in collisionless , homogeneous , magnetized plasmas [ gary , 1993 ] , using parameters as measured from wind during the events of 19 march 2005 . under the assumption that the enhanced fluctuations from each of these events were generated in the local solar wind , we have used magnetic field and plasma measurements as listed in table 1 . inserting these parameters into the kinetic linear dispersion theory for waves and instabilities at k x b o = 0 , we computed and plotted the dispersion properties , that is , the complex frequency = r + i as a function of parallel wave number k|| , of the four distinct lowfrequency waves at k x b o = 0 for each event . all of the linear theory calculations presented here are carried out in the plasma centerofmass frame where bo > 0 corresponds to the sunward direction . the parameters for event # 1 given in table 1 yield figure 5 which illustrates the dispersion relations ( r and versus k|| ) for each of the modes which propagate near the proton cyclotron frequency . the two lefthand polarized alfvncyclotron modes are constrained to |r| < p with one such mode stable and one unstable . if the proton velocity distribution were to consist of a single bimaxwellian component , the sunward and antisunward alfvncyclotron modes would be unstable with the same growth rates . however , the beam / core character of the observed proton distributions breaks the leftright symmetry of the system for all the events studied here . in this particular case the positive frequency sunward propagating alfvncyclotron mode is stable , whereas the negative frequency antisunward propagating alfvncyclotron mode is moderately unstable ( maximum /p ~ 5 10 ) at k||c/pp ~ 0.60 , where it has a strong proton core cyclotron resonance and is driven by the strong core temperature anisotropy . the two righthand polarized magnetosonic modes , in contrast , extend to frequencies well above p where , in the absence of proton cyclotron resonances , they remain weakly damped and exhibit the beginnings of whistler dispersion . the dispersion properties ( ( a ) real frequencies and ( b ) damping / growth rates as functions of k|| ) of the four lowfrequency electromagnetic normal modes of the plasma computed from the kinetic dispersion equation at k x b o = 0 for fluctuations in a homogeneous , collisionless plasma . parameters are those as given in table 1 for event # 1 on 19 march 2005 . red and orange represent properties of the righthand circularly polarized magnetosonic modes , whereas black and blue illustrate dispersion of the lefthand circularly polarized alfvncyclotron modes . the parameters for event # 2 yield figure 6 which illustrates the dispersion relations for each of the modes that propagate near the proton cyclotron frequency . the two lefthand polarized alfvncyclotron modes are constrained to |r| < p and become strongly damped at k||c/pp > 0.40 . the righthand polarized magnetosonic modes again extend to higher frequencies with weak damping and whistler dispersion . here the negative frequency magnetosonic branch is moderately unstable ( maximum /p ~ 2 10 ) at k||c/pp ~ 0.80 and r/p ~ 1.0 , with a strong proton beam cyclotron resonance and is driven by the superalfvnic beam / core relative flow ( this growing mode is also known as the proton / proton righthand resonant instability [ gary , 1993 , section 8.2 ] ) . the negative phase speed again indicates that unstable modes here propagate in the antisunward direction . the dispersion properties ( ( a ) real frequencies and ( b ) damping / growth rates as functions of k|| ) of the four low frequency electromagnetic normal modes of the plasma computed from the kinetic dispersion equation at k x b o = 0 for fluctuations in a homogeneous , collisionless plasma . parameters are those as given in table 1 for event # 2 on 19 march 2005 . red and orange represent properties of the righthand circularly polarized magnetosonic modes , whereas black and blue illustrate dispersion of the lefthand circularly polarized alfvncyclotron modes . the parameters for event # 3 yield r(k|| ) dispersion plots very similar to those for the two preceding events . beam / core and alpha / core relative flow speeds are not large enough to excite the ion / ion magnetosonic instabilities , so that all four modes are stable for this event and we do not illustrate this case . the parameters for event # 4 as given in table 1 yield dispersion plots r(k|| ) with two distinct unstable modes as shown in figure 7 . again the righthand polarized magnetosonic mode is unstable with maximum growth rate /p ~ 7 10 and corresponding wave number k||c/pp ~ 0.35 and frequency r/p ~ 0.33 , whereas the lefthand polarized alfvncyclotron mode is more weakly unstable with maximum growth rate /p ~ 2 10 and corresponding wavenumber k||c/pp ~ 0.24 and frequency r/p ~ 0.12 . it appears that both the beam / core and the alpha / core relative flows contribute to the magnetosonic instability and that the alfvncyclotron instability is driven primarily by the beam temperature anisotropy . the other two modes are stable , being undamped at long wavelengths and becoming strongly damped at sufficiently large wave numbers . the dispersion properties ( ( a ) real frequencies and ( b ) damping / growth rates as functions of k|| ) of the four low frequency electromagnetic normal modes of the plasma computed from the kinetic dispersion equation at k x b o = 0 for fluctuations in a homogeneous , collisionless plasma . parameters are those as given in table 1 for event # 4 on 19 march 2005 . red and orange represent properties of the righthand circularly polarized magnetosonic modes , whereas black and blue illustrate dispersion of the lefthand circularly polarized alfvncyclotron modes . for the parameters of event # 6 , the righthand polarized magnetosonic mode is again unstable , apparently driven by the beam / core relative flow . dispersion of both r(k|| ) and (k|| ) is similar to previous cases and is not shown here ; the maximum growth rate is very weak with /p < 1 10 and corresponds to k||c/pp ~ 0.63 and r/p ~ 0.72 . again , the unstable modes propagate in the antisunward direction . finally , for the parameters of event # 7 , both the alfvncyclotron and the magnetosonic modes are unstable . dispersion of both r(k|| ) and (k|| ) is similar to previous cases and is not shown here . the maximum growth rate of the alfvncyclotron instability is weak with /p ~ 1.3 10 and corresponds to k||c/pp ~ 0.40 and r/p ~ 0.20 , and the magnetosonic mode has very weak growth with /p < 2 10 at k||c/pp ~ 0.25 and r/p ~ 0.23 . we have used wind spacecraft magnetic field and plasma observations in the slow solar wind ( vsw ~ 390 km / s ) on 19 march 2005 to examine the linear dispersion properties for events of enhanced magnetic fluctuations near the proton cyclotron frequency at propagation parallel / antiparallel to the background magnetic field . we analyze the velocity distributions in terms of two proton components , a more dense , cooler core and a more tenuous , warmer beam with a relative beam / core flow parallel to b o , and a very tenuous , flowing alpha particle component . we then solve the kinetic linear dispersion equation at k x bo = 0 and find for two events the most unstable mode is the lefthand polarized alfvncyclotron instability driven by a proton component temperature anisotropy t/t|| > 1 , and for three events the most unstable mode is the righthand polarized magnetosonic instability driven primarily by ion component relative flows . jian et al . argued that , for waves propagating parallel to the solar wind flow velocity v sw , the spacecraft frame polarization should be the same as the polarization in the solar wind frame , whereas for waves at propagation antiparallel to v sw , the spacecraft frame polarization should be opposite to the intrinsic polarization . our dispersion analysis predicts that , for all five events with unstable modes , the instabilities propagated parallel to the direction of the proton beam , i.e. , in the antisunward direction . thus , as summarized in table 2 , we get agreement between polarizations inferred from observations and polarizations predicted by linear theory for four out of the five events . the single case of disagreement between observations and theory ( event # 6 ) may be due to the fact that in this case theory predicts very weak growth for the righthand plasma frame instabilities , so that this disagreement is not as robust as the four cases of agreement . polarization table for the six events of 19 march 2005 analyzed herea observed polarizations are as given in figure 2 , inferred polarizations follow from table a1 assuming antisolar propagation , and theoretical polarizations are obtained from the most unstable modes as predicted by the linear dispersion theory analyses described in section 3 . the observations of enhanced electromagnetic fluctuations near the proton cyclotron frequency in the solar wind have been credited to two distinct sources . the nonlocal hypothesis holds that these waves are generated near the sun and are then transported by the solar wind flow to observation at planetary distances . an alternate scenario is that the expanding solar wind flow leads to nonthermal particle velocity distributions which excite kinetic instabilities throughout the heliosphere so that the enhanced waves are generated locally , near the source of their observation . our work demonstrates a clear correlation between such enhanced fluctuations and proton velocity distributions sufficiently anisotropic to locally drive kinetic instabilities . this work does not disprove the nonlocal hypothesis but does demonstrate that local sources of fluctuations should be taken into account in studies of solar wind turbulence and its evolution with distance from the sun , as suggested by bale et al . . other topics worthy of future study are the sources of the ion anisotropies which drive the kinetic instabilities . what creates the solar wind t/t|| > 1 condition which , in turn , drives the alfvncyclotron instability ? what is the source of the beam / core proton distribution [ see , for example , maneva et al . , these results should not be construed as fully representative of enhanced fluctuation activity near the proton cyclotron frequency in the solar wind . as discussed in jian et al . , such events could also include both intrinsically righthand polarized waves driven by the alpha / proton magnetosonic instability [ gomberoff et al . 2000a , 2000b ; li and habbal , 2000 ; araneda et al . , 2002 ; lu et al . , 2006 ; verscharen et al . , 2013 ; verscharen and chandran , 2013 ] , and the intrinsically lefthand polarized waves driven by the t/t|| > 1 anisotropy on heavy ion velocity distributions . understanding which events correspond to which free energies and to which normal modes will require substantial additional studies of proton cyclotron events under a wide variety of solar wind conditions including fast as well as slow wind , both high and low proton , and a broad range of beam / core relative flow speeds . however , we believe that the present work clearly demonstrates that the sometimesinvoked assumption that all such events are due to the alfvncyclotron instability is invalid .

abstractintervals of enhanced magnetic fluctuations have been frequently observed in the solar wind . but it remains an open question as to whether these waves are generated at the sun and then transported outward by the solar wind or generated locally in the interplanetary medium . magnetic field and plasma measurements from the wind spacecraft under slow solar wind conditions on 19 march 2005 demonstrate seven events of enhanced magnetic fluctuations at spacecraftframe frequencies somewhat above the proton cyclotron frequency and propagation approximately parallel or antiparallel to the background magnetic field b o. the proton velocity distributions during these events are characterized by two components : a more dense , slower core and a less dense , faster beam . observed plasma parameters are used in a kinetic linear dispersion equation analysis for electromagnetic fluctuations at k x b o = 0 ; for two events the most unstable mode is the alfvncyclotron instability driven by a proton component temperature anisotropy t/t|| > 1 ( where the subscripts denote directions relative to b o ) , and for three events the most unstable mode is the righthand polarized magnetosonic instability driven primarily by ion component relative flows . thus , both types of ion anisotropies and both types of instabilities are likely to be local sources of these enhanced fluctuation events in the solar wind .

Introduction Magnetic Field and Plasma Observations on 19 March 2005 Kinetic Dispersion Theory Analyses of Six Events on 19 March 2005 Summary and Conclusions

the solar wind is a highly ionized , magnetized plasma which flows at supersonic and superalfvnic speeds away from the sun . in this medium there have been many observations of enhanced magnetic fluctuations at frequencies near the proton cyclotron frequency . there are two hypotheses as to the source of these enhanced fluctuation events [ jian et al . , one school holds that these largeamplitude waves are generated close to the sun and are then transported by the solar wind to observation at planetary distances . this manuscript uses plasma and magnetic field measurements with kinetic linear dispersion theory to demonstrate that several enhanced magnetic fluctuations events observed by the wind spacecraft on 19 march 2005 are unstable to one or more kinetic instabilities , thereby providing evidence that these particular events are locally generated . kinetic instabilities are those which are driven by velocityspace anisotropies and which typically have maximum growth rates arising in the short wavelength regime corresponding to wavelengths the order of or shorter than either the thermal ion gyroradius or the ion inertial length [ gary , 1993 ] . for frequencies near the proton cyclotron frequency and propagation quasiparallel to b o , the cases of interest here , there are three fundamental normal modes [ gary , 1993 , chapter 6 ] : predominantly electrostatic ion acoustic waves , predominantly electromagnetic alfvncyclotron waves with lefthand circular polarization in the limit of k x b o = 0 , and predominantly electromagnetic magnetosonic waves with righthand circular polarization at k x b o = 0 . although various types of nonthermal ion and electron velocity distributions are observed in the interplanetary medium [ feldman et al . , 2015 ] and assume that proton velocity distributions can be fit as the sum of two components , a more dense , slower core ( denoted by subscript c ) and a less dense , faster beam ( subscript b ) . we further assume that both proton components , as well as the electrons ( subscript e ) and the alpha particles ( subscript ) , can each be represented as a bimaxwellian distribution with t t|| ( where the subscripts represent directions relative to b o ) and relative flow velocities parallel to b o. we denote the relative beam / core flow velocity as v o = v ob v oc where v oj is the average flow velocity of the jth component in the plasma ( center of mass ) frame and the relative alpha / core flow velocity as v oc = v ov oc . , mercury surface , space environment , geochemistry , and ranging ( messenger ) survey of in situ lowfrequency wave storms between 0.3 and 0.7 au , submitted to journal of geophysical research , 2015 ) have used highresolution magnetometer observations from the stereo , messenger , and helios spacecraft to study solar wind events of enhanced magnetic field fluctuations with frequencies near p at propagation quasiparallel to the background magnetic field b o. by comparing lefthand versus righthand polarizations in the spacecraft frame , they found that the lefthand waves are generally stronger and appear slightly more often . however , their analyses utilized proton data reduction algorithms which were less general than the beam / core bimaxwellian velocity distributions used in this paper , thereby limiting their ability to compare temperature anisotropy and beam / core distributions as sources of protondriven instabilities . here we follow these earlier papers by identifying events of interest as corresponding to enhanced field fluctuations with frequencies near the proton cyclotron frequency . using this model in kinetic linear dispersion theory , we determine which types of iondriven instabilities are the likely sources of these enhanced fluctuation events . [ 2009 , 2010 , 2014 ] , our concerns here are iondriven instabilities which propagate at directions quasiparallel to b o. this condition corresponds to three categories of growing modes : alfvncyclotron instabilities driven by ion component anisotropies such that t / t|| > 1 [ gary et al . under the condition k x b o = 0 alfvncyclotron waves and instabilities have lefthand circular polarization , whereas the parallel firehose instability , magnetosonic waves , and magnetosonic proton / proton instabilities all have righthand circular polarization in the solar wind frame . if the proton velocity distribution may be approximated as a single bimaxwellian and heavy ions may be ignored , growth rate predictions of kinetic linear theory of electromagnetic instabilities driven by proton temperature anisotropies may be expressed in terms of two parameters , tp / t||p and ||p . used hybrid simulations to show that the alfvncyclotron instability threshold condition represents an upper bound for tp / t||p > 1 , and gary et al . considering only solar wind conditions in which the proton velocity distribution may be approximated as a single bimaxwellian component , kasper et al . , and hellinger and trvnek used measurements from the wind spacecraft to show that instability thresholds in tp / t||p versus ||p parameter space indeed correspond to observed proton anisotropy bounds . however , thresholds of the proton mirror and the oblique proton firehose instabilities , which have wavevectors at substantially oblique angles relative to b o , provide better constraints to the observations than do the thresholds of the anisotropydriven instabilities with maximum growth at parallel propagation . furthermore , as discussed above , proton velocity distributions in the relatively fast , relatively collisionless solar wind are usually not well represented as a single bimaxwellian , but rather require at the minimum a two component core / beam description . , the proton core || , the proton core relative density nc / ne , the dimensionless beam / core relative flow speed vo / va , t||b / t||c , and the two proton component temperature anisotropies tc / t||c and tb / t||b , as well as one or more electron parameters [ daughton and gary , 1998 ] and at least four additional parameters characterizing each minor ion species such as the alpha particles . our approach here is to constrain this large parameter space through the use of detailed ion and electron measurements from the plasma instruments on board the wind spacecraft and to calculate dispersion properties of the enhanced fluctuations for specific conditions observed in the solar wind . after completing a survey of enhanced magnetic fluctuation events in the observed frequency range 0.01 hz < f < 5 hz using 0.092 s magnetic field data , we chose observations of such events in the solar wind on 19 march 2005 to be the subject of a comprehensive study as described below . on this date the wind spacecraft detected a number of events with wave propagation quasiparallel to the background magnetic field . we chose the events on this date because both lefthand and righthand polarized waves in the spacecraft frame are observed within a relatively short time interval , and the individual ion spectra from this period have been examined in detail by the swe coauthors on this manuscript . as shown in figure 1 , we found seven events of enhanced magnetic fluctuations , with the following common characteristics : ( 1 ) the coherence between the two transverse magnetic field components by and bz is enhanced , ( 2 ) the transverse power is substantially increased over the background power at earlier and later times , ( 3 ) the transverse power is much stronger than the compressional power , ( 4 ) the magnetic field fluctuations are nearly circularly polarized as indicated by the high absolute ellipticity , and ( 5 ) the propagation direction is within 10 of the background magnetic field direction . the intervals for the seven events indicated in figure 1 are as follows : event # 1 ( 78.380 < doy < 78.389 ) , event # 2 ( 78.488 < doy < 78.494 ) , event # 3 ( 78.506 < doy < 78.515 ) , event # 4 ( 78.672 < doy < 78.685 ) , event # 5 ( 78.741 < doy < 78.748 ) , event # 6 ( 78.786 < doy < 78.798 ) , and event # 7 ( 78.804 < doy < 78.811 ) . magnetic field fluctuation measurements from the wind spacecraft on 19 march 2005 . ( a ) coherence between the two transverse components , by and bz ; ( b ) transverse power spectral density ; ( c ) the ellipticity in the spacecraft frame where + 1 corresponds to righthand circular polarization , and 1 corresponds to lefthand circular polarization ; and ( d ) the propagation angle with respect to b o. the yellow lines represent the local proton cyclotron frequency . the white solid and dashed lines labeled fsc+ and fsc represent the dopplershifted proton cyclotron frequency if the wave propagates parallel or antiparallel to the solar wind velocity under the assumption that the phase speed of a wave at the proton cyclotron frequency is v a. the fast fourier transform is performed for every 2560 points with a shift of 320 points and a bandwidth of 29 points . magnetic field and plasma measurements from the wind spacecraft as a function of time for 19 march 2005 . here are shown ( b ) the component number densities , ( c ) the average velocities in the x gse direction , ( d ) the average flow speed parallel to b o relative to the core protons and normalized to the alfvn speed , ( e ) the component || , and ( f ) the component t/t|| values . for one 92 s measurement cycle of the ions in each event , the proton core , the proton beam , the electrons , and the alpha particles were each fit to bimaxwellian velocity distributions with relative flow velocities parallel to b o. the proton beam and alpha particle measurements during event # 5 are noisy and poorly defined , so this event is not further considered here . table 1 presents field and plasma parameters from the other six events analyzed in section 3 . the values of the alpha temperature anisotropies are particularly suspect , so that because of these large uncertainties , we have assumed t/t|| = 1 for events # 2 , # 3 , # 4 , # 6 , and # 7 in table 1 and in the linear dispersion theory analyses described in section 3 . field and plasma parameters for six enhanced magnetic fluctuation events observed from wind spacecraft on 19 march 2005a values in parenthesis represent the assumption of isotropic alpha particles because of poor fits to the model of flowing bimaxwellian alpha particle velocity distributions . figure 2 displays the magnetic field vector in geocentric solar equatorial ( gse ) coordinates , the number density , speed in the x direction ( antisunward ) , the dimensionless beam / core and alpha / core relative flow speeds , ||j , and temperature anisotropy of core proton ( in red ) , beam proton ( in blue ) , alpha particles ( in magenta ) , and electrons ( in green ) . as is typical for solar wind events , 2014 , figure 7b ] , the energy density of field fluctuations transverse to b o is 1 to 2 orders of magnitude greater than that of the compressional fluctuations parallel / antiparallel to the background magnetic field . the magnetic fluctuation power spectra during events # 1 , # 4 , # 6 , and # 7 observed from the wind spacecraft on 19 march 2005 . ( second column ) the proton and alpha particle velocity distributions in v||v space as represented by the proton core , proton beam , and alpha particle fits . this section describes results from kinetic linear dispersion theory of waves and instabilities in collisionless , homogeneous , magnetized plasmas [ gary , 1993 ] , using parameters as measured from wind during the events of 19 march 2005 . under the assumption that the enhanced fluctuations from each of these events were generated in the local solar wind , we have used magnetic field and plasma measurements as listed in table 1 . inserting these parameters into the kinetic linear dispersion theory for waves and instabilities at k x b o = 0 , we computed and plotted the dispersion properties , that is , the complex frequency = r + i as a function of parallel wave number k|| , of the four distinct lowfrequency waves at k x b o = 0 for each event . in this particular case the positive frequency sunward propagating alfvncyclotron mode is stable , whereas the negative frequency antisunward propagating alfvncyclotron mode is moderately unstable ( maximum /p ~ 5 10 ) at k||c/pp ~ 0.60 , where it has a strong proton core cyclotron resonance and is driven by the strong core temperature anisotropy . the two righthand polarized magnetosonic modes , in contrast , extend to frequencies well above p where , in the absence of proton cyclotron resonances , they remain weakly damped and exhibit the beginnings of whistler dispersion . the dispersion properties ( ( a ) real frequencies and ( b ) damping / growth rates as functions of k|| ) of the four lowfrequency electromagnetic normal modes of the plasma computed from the kinetic dispersion equation at k x b o = 0 for fluctuations in a homogeneous , collisionless plasma . parameters are those as given in table 1 for event # 1 on 19 march 2005 . here the negative frequency magnetosonic branch is moderately unstable ( maximum /p ~ 2 10 ) at k||c/pp ~ 0.80 and r/p ~ 1.0 , with a strong proton beam cyclotron resonance and is driven by the superalfvnic beam / core relative flow ( this growing mode is also known as the proton / proton righthand resonant instability [ gary , 1993 , section 8.2 ] ) . the dispersion properties ( ( a ) real frequencies and ( b ) damping / growth rates as functions of k|| ) of the four low frequency electromagnetic normal modes of the plasma computed from the kinetic dispersion equation at k x b o = 0 for fluctuations in a homogeneous , collisionless plasma . parameters are those as given in table 1 for event # 2 on 19 march 2005 . again the righthand polarized magnetosonic mode is unstable with maximum growth rate /p ~ 7 10 and corresponding wave number k||c/pp ~ 0.35 and frequency r/p ~ 0.33 , whereas the lefthand polarized alfvncyclotron mode is more weakly unstable with maximum growth rate /p ~ 2 10 and corresponding wavenumber k||c/pp ~ 0.24 and frequency r/p ~ 0.12 . it appears that both the beam / core and the alpha / core relative flows contribute to the magnetosonic instability and that the alfvncyclotron instability is driven primarily by the beam temperature anisotropy . the dispersion properties ( ( a ) real frequencies and ( b ) damping / growth rates as functions of k|| ) of the four low frequency electromagnetic normal modes of the plasma computed from the kinetic dispersion equation at k x b o = 0 for fluctuations in a homogeneous , collisionless plasma . parameters are those as given in table 1 for event # 4 on 19 march 2005 . for the parameters of event # 6 , the righthand polarized magnetosonic mode is again unstable , apparently driven by the beam / core relative flow . we have used wind spacecraft magnetic field and plasma observations in the slow solar wind ( vsw ~ 390 km / s ) on 19 march 2005 to examine the linear dispersion properties for events of enhanced magnetic fluctuations near the proton cyclotron frequency at propagation parallel / antiparallel to the background magnetic field . we analyze the velocity distributions in terms of two proton components , a more dense , cooler core and a more tenuous , warmer beam with a relative beam / core flow parallel to b o , and a very tenuous , flowing alpha particle component . we then solve the kinetic linear dispersion equation at k x bo = 0 and find for two events the most unstable mode is the lefthand polarized alfvncyclotron instability driven by a proton component temperature anisotropy t/t|| > 1 , and for three events the most unstable mode is the righthand polarized magnetosonic instability driven primarily by ion component relative flows . argued that , for waves propagating parallel to the solar wind flow velocity v sw , the spacecraft frame polarization should be the same as the polarization in the solar wind frame , whereas for waves at propagation antiparallel to v sw , the spacecraft frame polarization should be opposite to the intrinsic polarization . polarization table for the six events of 19 march 2005 analyzed herea observed polarizations are as given in figure 2 , inferred polarizations follow from table a1 assuming antisolar propagation , and theoretical polarizations are obtained from the most unstable modes as predicted by the linear dispersion theory analyses described in section 3 . the observations of enhanced electromagnetic fluctuations near the proton cyclotron frequency in the solar wind have been credited to two distinct sources . the nonlocal hypothesis holds that these waves are generated near the sun and are then transported by the solar wind flow to observation at planetary distances . an alternate scenario is that the expanding solar wind flow leads to nonthermal particle velocity distributions which excite kinetic instabilities throughout the heliosphere so that the enhanced waves are generated locally , near the source of their observation . this work does not disprove the nonlocal hypothesis but does demonstrate that local sources of fluctuations should be taken into account in studies of solar wind turbulence and its evolution with distance from the sun , as suggested by bale et al . what creates the solar wind t/t|| > 1 condition which , in turn , drives the alfvncyclotron instability ? , these results should not be construed as fully representative of enhanced fluctuation activity near the proton cyclotron frequency in the solar wind . , such events could also include both intrinsically righthand polarized waves driven by the alpha / proton magnetosonic instability [ gomberoff et al . , 2013 ; verscharen and chandran , 2013 ] , and the intrinsically lefthand polarized waves driven by the t/t|| > 1 anisotropy on heavy ion velocity distributions . understanding which events correspond to which free energies and to which normal modes will require substantial additional studies of proton cyclotron events under a wide variety of solar wind conditions including fast as well as slow wind , both high and low proton , and a broad range of beam / core relative flow speeds . however , we believe that the present work clearly demonstrates that the sometimesinvoked assumption that all such events are due to the alfvncyclotron instability is invalid .

the type i phosphoinositide 3-kinases ( pi3ks ) are critical signaling proteins involved in the regulation of cell growth , survival , motility , and metabolism . in mammals , there exist four isoforms of the type i pi3k catalytic p110 subunits : , , , and . of these , and are ubiquitously expressed , whereas and have more limited distribution , most notably in hematopoietic cells ( vanhaesebroeck et al . , 2010 ) . the lipid kinase activity of p110 is regulated downstream of receptor tyrosine kinases by the binding of tyrosine - phosphorylated proteins to its regulatory p85 subunit , resulting in attenuation of its autoinhibitory activity . in addition , activated ras proteins bind directly to an n - terminal ras - binding domain ( rbd ) on p110 , acting synergistically with the input from tyrosine - phosphorylated proteins to optimally activate lipid kinase activity ( rodriguez - viciana et al . , 1994 , 1996 ) . proof of the pathophysiological importance of the direct interaction of ras with p110 came from the generation of mice bearing germline mutations in the rbd of p110 , which were found to be highly resistant to mutant - ras - induced lung and skin cancer formation ( gupta et al . , 2007 ) . the 3d structure of ras bound to p110 has been determined , and ras has been shown to activate the lipid kinase activity of p110 cooperatively with input from g subunits via the regulatory p101 subunit ( pacold et al . , 2000 ) . mice with mutations in the rbd of p110 show neutrophil defects in the regulation of pi3k activity by some g - protein - coupled receptors ( gpcrs ) ( suire et al . , 2006 ) . ras also has been reported to bind and activate p110 in vitro ( vanhaesebroeck et al . , 1997 ) . in addition , rbd mutations have been used to demonstrate that input of ras binding to the single drosophila type i pi3k is critical in insulin - pathway - controlled developmental growth ( orme et al . , 2006 ) and that ras binding is required for pi3k activation by chemoattractants in dictyostelium ( funamoto et al . , 2002 ) . p110 has been much less thoroughly studied than p110. it appears to be relatively insensitive to activation by growth factor receptor tyrosine kinase signaling but important downstream of certain gpcrs , including those for lysophosphatidic acid ( lpa ) and sphingosine 1-phosphate ( s1p ) , making p110 the only gpcr - regulated type i pi3k isoform outside the hematopoietic system ( ciraolo et al . , 2008 ; guillermet - guibert et al . , 2008 ; jia et al . , 2008 ) . p110 may also play an important role in cancer because mouse models of breast and prostate cancer , as well as a number of human cancer cell lines , depend on p110 , particularly in the setting of pten loss ( ciraolo et al . , 2008 ; jia et al . , 2008 ) . in platelets , p110 is essential for integrin - dependent adhesion and clot formation ( jackson et al . , 2005 ; martin et al . , 2010 ) , leading to the intense effort to develop isoform - specific p110 inhibitors , some of which are now in clinical trials as antiplatelet and anticancer agents ( nct01458067 , nct00688714 ) . the molecular basis of how p110 can exert these distinct functions is poorly understood . p110 is overall structurally similar to other p110 catalytic subunits and engages the very same p85 type regulatory subunits as p110 , albeit in a somewhat different way ( zhang et al . , 2011 ) . early reports have found p110 to associate with g subunits from heterotrimeric g proteins , which can directly stimulate its lipid kinase activity in vitro ( kurosu et al . , 1997 ; maier et al . , 1999 ) . it has , however , remained entirely unclear whether the p110 rbd contributes to p110 activation and function , and despite the apparently similar level of relatedness between the rbds across the four isoforms , a systematic analysis of ras effector proteins failed to detect any activation of p110 by ras in cotransfected cells ( rodriguez - viciana et al . , 2004 ) . in this report , we explore the role of p110 regulation through its rbd for pi3k signaling and function . we present extensive in vitro work to show that p110 is the only type i pi3k isoform not regulated by ras and to identify the rho family gtpases rac and cdc42 as direct isoform - specific rbd interactors and activators of p110. we go on to show that gpcrs couple to pi3k via dock180/elmo1-mediated rac activation and subsequent interaction with p110. mouse embryonic fibroblasts ( mefs ) from p110 rbd mutant mice show reduced pi3k activity and mice are resistant to bleomycin - induced lung fibrosis , a pathology that has been linked with lpa signaling . these findings explain longstanding inconsistencies and revise our understanding of type i pi3k regulation by small gtp - binding proteins , providing molecular insight into the regulation and function of the ubiquitous p110 isoform . to investigate the role of ras in regulating p110 , we set out to characterize the biochemical interaction between the two in vitro . in glutathione s - transferase ( gst ) pull - down studies using recombinant , gtps - loaded hras , kras , and nras as baits , we found strong and specific interaction between all three ras proteins and p110 ( figure 1a ) . in contrast , p110 bound to none of the ras proteins , but did bind to rab5 , a previously identified gtpase interactor of p110 ( christoforidis et al . , 1999 ) . mutating key residues within the rbd of p110 ( t208d / k227a ) abrogated ras binding , whereas introduction of analogous mutations ( see below for details ) into p110 did not affect rab5 binding . similar results were obtained when we used recombinant full - length gst - p110/p85 complexes to pull down active ras or rab5 proteins ( figure 1b ) . moreover , when we expressed constitutively active ras or rab5 , along with p110/p85 or p110/p85 in cos7 cells , and measured pip3-levels ( figure 1c ) or steady - state phospho - akt ( figure s1a available online ) as indicators of pi3k activity , hras and kras strongly enhanced p110 activity , whereas p110 was not stimulated by either ras proteins or rab5 . the modest rbd sequence similarity among the four paralogs of type i pi3k is shown in figure 1d . even though the overall structural organization of the p110 rbd is conserved ( zhang et al . , 2011 ) , we speculated that because we can not detect any interaction with ras , it might have lost or altered its function . we therefore mutated two highly conserved key residues within the p110 rbd to generate a p110-s205d / k224a double mutant ( p110-rbd - dm ; figure 1e ) . analogous mutations in p110 and p110 disrupt ras binding ( gupta et al . , 2007 ; pacold et al . , 2000 ) . in vitro , the basal lipid kinase activity of purified recombinant p110-rbd - dm protein was indistinguishable from its wild - type counterpart prepared in parallel ( figure 1f ) . moreover , p110-rbd - dm was still stimulated by the addition of purified recombinant g subunits , alone or in combination with a platelet - derived growth factor receptor ( pdgfr)-derived phosphotyrosine peptide ( py740 ) , indicating that the rbd mutations had no intrinsic effect on p110 lipid kinase activity or rbd - independent stimulatory input ( figure 1 g ) . however , rbd mutant p110 was much less active than wild - type when expressed in cos7 cells ( figures 1h and s1b ) even when g subunits were coexpressed or a myristoylation signal was added , pointing to a critical role of the rbd for p110 activity in living cells . to identify rbd interactors of p110 , we probed all 34 murine members of the ras subfamily of small gtpases ( rfgs ) for binding to p110/p85 ( figure s1c ) . parallel experiments were performed with p110/p85 , p110/p101 , and p110/p85 , respectively ( figure s1d ) . strikingly , whereas all non- isoforms interacted with the three prototypical ras proteins and a partially overlapping subset of closely related rfgs ( rras1 , rras2 , mras , and eras ) , p110 bound to none of those ( figure 2a ) . instead , p110 exclusively bound to the more distantly related diras1 and diras2 proteins in a gtp - dependent manner ( figure s1c ) . diras selectively bound wild - type and not rbd mutant p110 ( figure s2b ) , suggesting binding to the rbd . however , diras failed to stimulate p110 lipid kinase activity in vitro ( figure 2c ) and in cells , where constitutively active diras proteins seemed to repress rather than elevate phospho - akt when coexpressed along with p110 ( figure 2b ) , making diras an unlikely in vivo activator of p110. when comparing diras with ras , an obvious difference is the substitution of asp33 within the g2 box of ras with ile37 in diras ( figure 2d ) . this substitution is relevant to pi3k binding because an hras - d33i mutant showed attenuated binding to p110 and diras1-i37d showed reduced binding to p110 , even though exchange of this residue did not enable ras binding to p110 or diras binding to p110 ( figure s2a ) , pointing to additional , g2-box - independent determinants of pi3k isoform specificity . several members of the rho subfamily of small gtpases harbor a hydrophobic isoleucine or valine residue in this position ( figure s2c ) , which prompted us to test p110 for binding to representative rho family gtpases ( figure s2d ) . surprisingly , p110 bound to both rac1 and cdc42 in a gtp - dependent manner . weaker binding to rhog and minimal binding to rhoa was also observed ( figure 2e ) . importantly , rac1 , cdc42 , rhog , and rhoa preparations bound similar amounts of gtp , indicating proper folding and functionality ( figure 2e , right lower graph ) , and a rac1-i33d mutant showed reduced binding to p110 ( figure s2e ) , confirming a key role of this residue in gtpase binding to p110. the rac1/cdc42-p110 interaction was isoform specific because neither rac1 nor cdc42 significantly bound non- isoforms under parallel conditions ( figures 2f and s2f ) . strikingly , gtps - loaded rac1 or cdc42 strongly stimulated p110 lipid kinase activity in vitro ( figure 2 g ) , alone and in cooperation with a phosphotyrosine peptide ( py740 ) , or when p110 was complexed with a less inhibitory , truncated p85 ( p85 schematic in figure 3c ) . stimulation of p110 by active rac1 and cdc42 was dose dependent ( figure 2h ) . coexpression of constitutively active rac1 or cdc42 ( figure s2 g , lanes 46 ) , but not rhoa ( figure s2h ) , along with p110/p85 in cos7 cells strongly elevated cellular phospho - akt and pip3 levels ( figure 2i ) , indicating that both gtpases activate p110 in transfected cells . pip3 levels were further enhanced by coexpression of g1/g2 subunits or by myristoylation of p110 ( figure 2i ) . in contrast , gtps - loaded rac1/cdc42 did not stimulate p110 in vitro ( figure s2i ) , nor did v12-rac1/cdc42 cooperate with p110 , p110 , or p110 to elevate cellular phospho - akt levels ( figure s2j ) . taken together , these data show that the rho family gtpases rac1 and cdc42 bind to p110 in an isoform - specific manner and potently and directly stimulate its lipid kinase activity . we next aimed to confirm that rac and cdc42 are rbd interactors of p110. purified recombinant wild - type p110/p85 bound to rac1 and cdc42 in a concentration - dependent manner , whereas p110-rbd - dm / p85 complexes showed no binding ( figure 3a ) . similarly , rbd mutant p110 was not stimulated by active rac1 or cdc42 in vitro ( figure 3b ) or in cells ( figure s2e , lanes 79 ) . to test whether the bcr homology domain ( bhd ) on p85 , which had previously been shown to bind rac and cdc42 ( bokoch et al . , 1996 ; zheng et al . , 1994 ) , is required for rac / cdc42 binding to p110 , we truncated p85 ( p85 schematic in figure 3c ) and probed for binding of p110/p85 to rac1 and cdc42 in vitro . intriguingly , binding was unaffected by removal of the bhd but completely disrupted when full - length p85 was in complex with rbd mutant p110 , strongly arguing for the rbd as the rac / cdc42-binding site . to further corroborate these findings , we generated 43 single point mutations covering 37 residues across the p110 rbd and assayed these mutants for binding to rac1 and cdc42 ( figures 3d , s3a , and s3b ) . of those , 17 mutations of 14 rbd residues affected binding to both gtpases without affecting p110 protein stability . several of these residues were part of the rbd1 and 2 sheets or the loop adjacent to the rbd1 helix ( zhang et al . , 2011 ) , areas known to be important for ras binding in non- isoforms ( pacold et al . , 2000 ) . finally , we employed isothermal titration calorimetry ( itc ) to study thermodynamics of the rac1/cdc42-p110 interaction . in solution , rac1gtps bound to p110/p85 with a molar ratio close to 1 and an average kd of 1.42 m , whereas the affinity measured for cdc42gtps was 3.1 m ( figures 3e and 3f ) . similar affinities have been reported for the ras - p110 and ras - p110 interactions ( pacold et al . , 2000 ; , 1996 ) , indicating that rac1 and cdc42 are plausible rbd interactors of p110. no binding was observed between gtps - loaded rac1/cdc42 and p110 or p110-rbd - dm , respectively ( figure s3c ) . to study the role of interactor binding to the p110 rbd for pi3k signaling in vivo , we generated mice harboring the two p110 rbd point mutations ( s205d / k224a ) within their germline . homologous recombination in embryonic stem ( es ) cells was employed to replace exon 6 of the murine pik3cb gene ( figure s4a ) , and germline transmission was achieved by eight - cell embryo injection ( figures s4b and s4c ) . p110-rbd - dm mice were viable and fertile , although numbers of homozygous animals at the time of biopsy ( around day 14 ) were moderately reduced ( 73 where 105 were expected ; p < 0.02 ; figure 4a ) , indicating incomplete lethality for undetermined reasons . newborn homozygous p110-rbd - dm pups were smaller than their wild - type littermates ( figure 4b ) . the size difference in adult mice was subtle but remained significant when same - sex litter- and cage - mates were compared ( figure 4c ) . mefs homozygous for the p110 rbd mutation proliferated at a significantly slower rate than their wild - type counterparts ( figure 4d ) , which was reflected by a higher percentage of cells in g1 ( 1% fetal calf serum [ fcs ] : 56.7% 0.32% versus 64.2% 0.53% , n = 4 , p < 0.001 ; 10% fcs : 47.5% 3.0% versus 52.2% 3.9% , n = 4 , p < 0.05 ) , fewer cells in g2 ( 1% fcs : 24.5% 1.4% versus 21.0% 1.9% , n = 4 , p < 0.05 ; 10% fcs : 23.9% 1.4% versus 21.1% 1.6% , n = 4 , p < 0.05 ) and fewer cells in s phase for 1% fcs ( 1% fcs : 13.3% 1.0% versus 11.0% 169% , n , p110-rbd - dm mefs showed lower steady - state phospho - akt levels ( figure 4f ) , suggesting that stimulatory signaling to p110 via its rbd contributes to pi3k activity in vivo . expression levels of p110 , p110 , and p85 were indistinguishable among the genotypes ( figure s4d ) , and the stoichiometry of p110 subunit binding to p85 was undisturbed ( figure s4e ) . to determine whether rac and cdc42 are upstream activators of p110 in vivo , we transfected wild - type and p110-rbd - dm mefs with small interfering rna ( sirna ) pools ( dharmacon on - target plus ) targeting these gtpases ( figure 4 g ) . although single knockdowns had only minor effects , combined knockdown of rac1 and cdc42 significantly lowered phospho - akt levels in wild - type , but not in p110-rbd - dm cells , closing the gap in steady - state phospho - akt levels between the genotypes and suggesting that endogenous rac1 and cdc42 cooperatively activate p110 via its rbd . we next acutely expressed constitutively active mutants of rac1 and cdc42 in wild - type , p110- , and p110-knockout mefs ( figures 4h and s4f ) . both rac1 and cdc42 increased steady - state phospho - akt levels in wild - type and p110-deleted cells but not in p110-knockout mefs . moreover , expression of v12-rac1 and v12-cdc42 failed to elevate phospho - akt levels in p110-rbd - dm mefs , whereas v12-hras did so in both wild - type and p110-rbd - dm mefs ( figure 4i ) . taken together , these findings indicate that rac1 and cdc42 activate pi3k in living cells by isoform - specific regulation of p110 through its rbd . to study whether the p110 rbd is required for coupling p110 to gpcrs , we stimulated wild - type and p110-rbd - dm mefs with the lipid growth factors and gpcr agonists lpa and s1p . lpa and s1p dose - dependently induced both akt and erk phosphorylation in wild - type cells ( figures 5a and s5a ) . these responses were sensitive to pertussis toxin ( figure s5b ) , confirming gpcr involvement . in p110-rbd - dm mefs , lpa- and s1p - induced phosphorylation of akt was strongly diminished , whereas erk phosphorylation was undisturbed ( figures 5a and s5a ) . also , in time course experiments , akt phosphorylation was more transient when the p110 rbd was mutated ( figure 5b and not shown ) . in contrast , p110-rbd - dm mefs responded normally to epidermal growth factor ( egf ) , platelet - derived growth factor ( pdgf ) , and insulin in dose - response ( figure s5c ) and time course experiments ( data not shown ) . notably , in p110-knockout cells , akt phosphorylation in response to lpa was completely abolished ( figure 5c ) , indicating that the rbd is essential for much but not all p110 activation downstream of gpcrs . to test whether the identified p110 rbd interactors are required for linking p110 to gpcrs , we knocked down rac1 and cdc42 in wild - type mefs . knockdown of rac1 strongly impacted lpa / s1p - induced akt phosphorylation , knockdown of cdc42 had only minor effects , and combination knockdown of both rac1 and cdc42 had little additional effect compared to rac1 knockdown alone ( figure 5d ) . deconvolution experiments using single sirna oligonucleotides confirmed the leading role of rac1 in this pathway ( figure s5d ) . neither rac1 nor cdc42 knockdown affected lpa / s1p - induced phosphorylation of erk or activation of either pathway induced by tyrosine kinase receptor agonists ( egf , pdgf , and insulin ; figure s5e ) . similarly , eht1864 , a direct inhibitor of rac but not cdc42 activation , dose - dependently inhibited akt phosphorylation induced by lpa / s1p ( figure 5e ) , but not egf , pdgf , or insulin ( figure s5f ) . therefore , acute loss or inhibition of rac phenocopied the signaling defect observed in p110-rbd - dm mefs . in line with this , rac was activated very rapidly upon lpa stimulation , reaching its peak activity within 20 s ( figure 5f ) . to provide further mechanistic insight into the gpcr - rac - p110 pathway , we performed a small candidate sirna screen to identify the guanine nucleotide exchange factor ( rac - gef ) involved . transfection of wild - type mefs with sirna pools targeting the dbl family rac - gefs vav1 - 3 , prex1/2 , and -/-pix had no clear effect on lpa / s1p - induced akt phosphorylation ( figures 6a and s6a ) . in contrast , knockdown of the dock family rac - gef dock180 or its adaptor protein elmo1 interfered with akt phosphorylation induced by lpa and s1p ( figure 6b and s6b ) , but not by egf , pdgf , and insulin ( figure s6c ) . specificity of results was confirmed in deconvolution experiments using individual sirna oligonucleotides targeting dock180 and elmo1 ( figures s6d and s6e ) . moreover , knockdown of dock180 abolished lpa- but not egf - induced rac activation ( figure 6c ) , firmly placing dock180/elmo1 downstream of the lpa receptor and upstream of rac and p110. the dhr-1 domain of dock180 has been shown to bind pip3 ( ct et al . , 2005 ) , raising the possibility of a pip3-driven feedback loop , in which rac would be upstream and downstream of p110. however , whereas sensitive to pertussis toxin , lpa - induced rac activation was entirely insensitive to pi3k inhibition by gdc0941 , a pan type i pi3k inhibitor , placing all detectable rac activation upstream of p110 ( figure 6d ) . recently , dictyostelium elmoe has been reported to be a direct g effector ( yan et al . , we thus probed g subunits for direct binding to purified recombinant elmo1 ( figure 6e ) and found g to strongly bind to full - length elmo and several n - terminal fragments . this altogether suggests a model in which dock180 is recruited downstream of gpcrs , possibly through binding of g to the n terminus of elmo1 . at the same time , p110 is directly recruited by g. rac is activated in proximity to p110 and binds to the rbd to fully activate p110 lipid kinase activity ( schematic in figure 6f ) . we could not detect any tyrosine phosphorylation on p85 in response to lpa ( figure s6 g ) , and the tyrosine kinase inhibitors dasatinib , erlotinib , and pp2 ( all at 1 m ) had no effect on the signaling pathway studied ( data not shown ) . to assess the functional importance of the dock180/elmo1-rac - p110 signaling axis for fibroblast chemotaxis , we employed transwell filter assays to study migration in gradients of lpa and pdgf . p110-rbd - dm mefs showed significantly reduced migration in lpa but not pdgf gradients ( figure 7a ) . similarly , wild - type mefs transfected with sirna pools targeting dock180 , elmo1 , or rac1 showed defective migration in gradients of lpa but not pdgf ( figure 7b ) , pointing to the specificity of this pathway for gpcr - induced chemotaxis . in contrast , pi3k activity was required for normal migration in either gradient , because pretreatment of wild - type cells with gdc0941 strongly affected migration toward lpa and pdgf , whereas pretreatment of cells with pertussis toxin selectively blocked migration in lpa gradients ( figure s7a ) . in agreement with a key role of rac upstream of p110 in fibroblast migration , acute expression of constitutively active rac ( v12-rac1 ) stimulated migration of wild - type but not p110-rbd - dm mefs in the absence of chemoattractant and in the presence of a low concentration of lpa ( 10 nm ) in the lower chamber , whereas migration toward 1% fcs was largely unaffected ( figure s7b ) . lpa has been identified as important fibroblast chemoattractant in bleomycin - induced lung fibrosis , a well - studied mouse model of human fibrotic lung disease , and was found to be elevated in patients with idiopathic lung fibrosis ( tager et al . , 2008 ) . we therefore wondered whether the disruption of p110 activation by lpa in p110-rbd - dm mice would be sufficient to affect experimental lung fibrosis . following a single intratracheal application of bleomycin , a quarter of all wild - type animals died or had to be culled according to local animal welfare regulations , whereas all p110-rbd - dm mice survived ( figure 7c ) . also , wild - type but not p110-rbd - dm mice significantly lost body weight ( figure 7d ) upon bleomycin treatment . lung weights increased in both groups , but to a significantly lesser extent in p110-rbd - dm mice ( figure s7c ) . histology of lungs 14 days after bleomycin challenge revealed extended areas of fibrotic changes in wild - type animals ( figure 7e , hematoxylin and eosin staining [ h&e ] , top ) , characterized by accumulation of activated , smooth muscle antigen - positive fibroblasts ( figure 7e , middle ) and deposition of crosslinked collagen fibers ( figure 7e , sirius red , bottom ) . changes in p110-rbd - dm mice were milder , with some mice showing almost normal lungs and others showing more limited areas of fibrosis . morphometric analysis of multiple nonoverlapping lung areas confirmed the differences between the genotypes : transparent lung areas were significantly reduced ( figures 7f and s7d ) and sma - positive areas significantly increased ( figure 7 g and s7e ) in wild - type but not p110-rbd - dm mice when compared to saline controls . in this study , we show that , in contrast to what has been widely presumed , ras is not a general regulator of type i pi3ks . we find that out of the two ubiquitously expressed pi3k isoforms , only p110 is regulated by ras , whereas p110 is a direct rac and cdc42 target protein , indicating that key members of the pivotal ras and rho families of small g proteins directly regulate type i pi3ks , with each family controlling their own distinct ubiquitous p110 isoform . that p110 proved unable to physically and functionally interact with ras is unexpected given the presence of a moderately conserved pi3k - type rbd in all four type i pi3k p110 catalytic subunits ( pacold et al . , 2000 ) . comparison between the published structures of the four rbds in their interactor - free states reveals little pointing to the distinct interactor specificity of p110 ( r. chaleil and p. bates , personal communication ) . p110 is not only unable to interact with ras under conditions readily revealing the transient , low - affinity interactions of ras with other isoforms , but it also has an entirely distinct ras superfamily gtpase interactor profile with a subfamily switch from ras to rho at the core of it , making any , for whatever reason , undetectable interaction with ras unlikely . rbds , classified on grounds of a ubiquitin fold structure with interactor specificities distinct from ras , are not uncommon , as exemplified by the human formin fhod1 , a rac interactor ( schulte et al . , 2008 ) , or the n terminus of elmo1 , shown to bind rhog and the arf family member arl4a ( patel et al . , 2011 ) . our biochemical experimentation identifies rac1 and cdc42 as rbd interactors of p110. an association of pi3k with rac and cdc42 was first noticed nearly 20 years ago ( tolias et al . , 1995 ) , but was attributed to rac / cdc42 binding to the amino - terminal bhd on p85 , which has sequence homology to rho - gap domains ( bokoch et al . , 1996 ; zheng et al . , 1994 these studies left the epistasis of information transfer between rac / cdc42 and pi3k unclear . in retrospect , all functional data from these studies can be explained by the presence of p110 in the cell lysates and pi3k preparations used . we did not study monomeric p85 , which has not been found in living cells ( geering et al . , 2007 ) , but our biochemical data strongly argue against an involvement of p85 in the rac / cdc42-p110 interaction , because ( 1 ) rac1 and cdc42 do not interact with p110/p85 or p110/p85 , ( 2 ) rac1 and cdc42 bind normally to p110 in the absence of the p85 bh domain , and ( 3 ) rbd point mutations abrogate rac1 and cdc42 binding to p110 in complex with full - length p85 . a body of literature has accumulated identifying rac or cdc42 as essential upstream activators of pi3k in various systems ( keely et al . , 1997 ; 2002 ) , but straightforward analysis of the relationship between rho family gtpases and pi3k has been difficult , mainly because rac and cdc42 also act downstream of pi3k , activated through pip3-dependent gefs ( welch et al . , a very recent study using microscopy - based assays in transfected cells revisited the interaction of small gtpases and pi3k , confirming that both active ras and rho family gtpases can activate pi3k in living cells . however , based on experimentation exclusively with a p110 reporter construct , the authors interpreted pi3k regulation by rho family members as indirect ( yang et al . , 2012 ) . our findings in mefs suggest that rac1 and cdc42 cooperatively control steady - state pi3k activity in living cells by isoform - specific activation of p110 and that this requires the p110 rbd . a model in which p110 provides basal , low - level pi3k activity has been proposed in the context of insulin signaling ( knight et al . , 2006 ) , and data showing pten - loss - driven prostate cancers to be entirely dependent on p110 , as well as metabolic findings in p110 kinase - dead mice , have been interpreted in the same way ( ciraolo et al . , 2008 ; jia et al . , 2008 ) our data point to rac1 and cdc42 as drivers of such a basal , p110-controlled activity , an idea consistent with a previous report finding that ectopic expression of wild - type but not rbd mutant p110 is sufficient to transform chicken embryo fibroblasts ( kang et al . , 2006 ) . it will be interesting to explore the oncogenic potential of the rac / cdc42-p110 interaction in the setting of pten loss and also in the context of the recently discovered activating mutations in rac ( hodis et al . , 2012 ; krauthammer et al . , 2012 ) in human melanomas , where p110 could be an important downstream target . , 2008 ; guillermet - guibert et al . , 2008 ; jia et al . , 2008 ) we find that mutation of the p110 rbd strongly attenuates p110 activation downstream of gpcrs , highlighting the importance of the rbd for p110 key signaling functions . the residual p110 activity in p110-rbd - dm mefs argues for a second , rbd - independent activation route , for which direct binding of g to p110 ( dbouk et al . , 2012 ) a puzzling question is whether a cooperative effect of rbd interactor and g binding to p110 can suffice to fully activate p110 , or if additional phosphotyrosine input is required to overcome the strong inhibition of lipid kinase activity imposed by p85 ( zhang et al . , 2011 ) . transactivation of receptor tyrosine kinases has been suggested to activate p110 downstream of gpcrs ( yart et al . , 2002 ) . although we found no effect of tyrosine kinase inhibitors and no p85 tyrosine phosphorylation in response to lpa in support of such a mechanism in mefs , there is evidence for cooperative gpcr and phosphotyrosine signaling to p110 in leukocytes ( kulkarni et al . , 2011 ) , and such a scenario appears possible in thrombocytes , where p110 is activated by integrins , itam - bearing receptors , and gpcrs ( martin et al . , 2010 ) . rac1 is essential for p110 activation downstream of the gpcrs for lpa and s1p , and the rac - gef dock180/elmo1 is upstream of both rac and p110 in this pathway . the dock / elmo - rac signaling axis is a highly conserved pathway controlling rac - dependent key functions such as actin remodeling , migration , and phagocytosis ( ct et al . , 2005 ) . recent findings in dictyostelium have directly linked g subunits from gpcrs to dock / elmo - rac and the cytoskeleton ( yan et al . , 2012 ) . reminiscent of dictyostelium elmoe , we find the n terminus of human elmo1 to directly bind g subunits , suggesting conservation of this pathway in mammals . in line with this , pi3k activity is not required for rac activation by lpa , placing p110 entirely downstream of rac in fibroblasts , which contrasts with a proposed pip3-driven feedback loop controlling rac activity upstream and downstream of pi3k in leukocytes ( weiner et al . , 2002 ) . although pi3k is not required to activate rac , it is still essential for fibroblast migration in gradients of lpa , indicating that pip3-regulated pathways distinct from rac activation contribute to gpcr - driven chemotaxis . importantly , whereas our findings identify dock180/elmo1 downstream of lpa / s1p in mefs , our experiments can not rule out involvement of other rac - gefs within this pathway . we also can not directly prove that the role of dock180/elmo1 in controlling fibroblast migration is exclusively and directly through activation of rac upstream of p110. finally , different gpcrs and other cell types may signal through rac - gefs other than dock180/elmo1 , and further studies will be required to determine whether the dock180/elmo1-rac1-p110 axis is a fixed signaling module or just one example of how rac / cdc42 is activated upstream of p110. overall , the picture that emerges from these studies is one in which p110 regulation by gpcrs operates through a two - track signaling pathway with direct and indirect input into p110. the direct route involves g interaction with p110 , whereas the indirect route goes through stimulation of rac via dock180/elmo1 , possibly also recruited through g ( see figure 6f ) . such two - track wiring is reminiscent of receptor tyrosine kinase regulation of p110 via p85 interaction with tyrosine - phosphorylated receptor or adaptor protein and grb2-sos - ras - p110 interaction . it can be speculated that this signaling logic might provide an improved ability to amplify a weak signal input or might increase the possibility for fine - tuning the signal through crosstalk of other pathways onto components such as dock180/elmo1 , rac itself , or rac - gaps terminating rac activity . another interesting issue is whether such g-elmo1 and g-p110 interactions are mutually exclusive or can occur in a heterotrimeric complex . at present , we can not distinguish between a model in which one g protein heterodimer binds directly to both elmo1 and p110 simultaneously and one in which elmo and p110 are engaged by two different g protein heterodimers ( as shown in figure 6f ) . see supplemental information online for the extended discussion , including information on other gtpase interactors of p110 , the phenotype of p110-rbd mutant mice , and resistance of these mice to bleomycin - induced lung fibrosis . extended discussionother gtpase interactors of p110the only small gtpase previously found to interact with p110 is rab5 . p110/p85 copurified with rab5 in a study searching for rab5 interactors ( christoforidis et al . , 1999 ) and rab5 was identified in a yeast two hybrid screen searching for p110 interactors ( kurosu and katada , 2001 ) . p110 appears to associate with rab5 in the early endocytic pathway , where it has been proposed to indirectly contribute to ptdins(3)p formation ( shin et al . , 2005 ) , and rab5 and p110 have further been suggested to be part of the autophagy - promoting vps34vps15beclin1atg14l complex ( dou et al . , 2010 ) . the rab5-binding site on p110 remains to be determined , yet several findings argue against rab5 binding to p110 through the rbd : ( 1 ) binding of rab5 is entirely unaffected by p110 rbd mutations ( figure 3c ) ; ( 2 ) rab5 does not appear to stimulate p110 ( figure 1c , ( christoforidis et al . , 1999 ; rodriguez - viciana et al . , 2004 ) ; ( 3 ) the affinity between rab5 and p110 in solution appears to be significantly higher ( data not shown ) than for typical pi3k rbd interactors , suggesting a more stable interaction , allowing to effectively localize p110 to rab5-positve subcellular compartments.out of the murine 34 members of the ras subfamily of small gtpases , only diras-1 and diras-2 showed binding to p110 ( figures s1c and s2b ) . human diras-3 , also termed arhi or noey2 , a tumor suppressor in ovarian cancer and putative autophagy regulator ( lu et al . , 2008 ) , showed no interaction ( data not shown ) . diras-1 and -2 are relatively understudied ras subfamily gtpases with low endogenous gtpase activity , whose expression appears to be limited to brain and heart ( ellis et al . whereas the relatedness to ras and the inability to bind to rbd mutant p110 ( figure s2b ) argues for binding to the rbd in vitro , the absence of any detectable stimulatory effect on p110 , the limited tissue distribution and growth - inhibitory effects make the role of diras proteins as rbd interactors of p110 in vivo unclear , and further studies will be dedicated to this subject.p110-rbd - dm micedirect analysis of signaling complexes regulating type i pi3k isoforms in living cells is notoriously difficult due to the transient , low - affinity nature of these interactions and their instability in solution . we therefore generated p110-rbd - dm mice as a tool to study the impact of interactor binding to p110 for pi3k signaling in vivo . p110-rbd - dm pups and mice are slightly smaller than wild - type littermates , and are born in slightly sub - mendelian ratios , both features reported to an overall more severe extent from p110 knockout and p110 kinase - dead knock in mice ( -kd ) ( ciraolo et al . , 2010 ; guillermet - guibert et al . , 2008 ; kulkarni et al . , 2011 ) . a straightforward comparison between rbd mutant mice and these models is , however , complicated not only by different genetic backgrounds , but also by the different genetics of these models . complete loss of p110 catalytic subunit will inadvertently lead to some disturbance of subunit composition and/or recruitment to signaling complexes , an artifact noticeable in recently published work ( utermark et al . , 2008 ) has been found to have reduced p110 expression , both in embryos and adult tissues , placing these animals between knockout and other -kd mice . a milder phenotype of p110-rbd - dm mice is after all not unexpected , given that the rbd mutations do not disrupt basal p110 activity or rbd - independent activation routes , and some p110 activity might be enough for normal development and life under controlled conditions . we also saw moderately reduced proliferation of p110-rbd - dm primary mefs , indicating that p110 regulation through its rbd interactors is essential for normal cell proliferation , which appears to contradict findings from -kd mefs and reconstituted p110-ko mefs suggesting that p110 lipid kinase activity is not required for proliferation . although no role of p110 lipid kinase activity for cell proliferation appears surprising in light of the reported growth defect in these mice , the discrepancies between these studies could well be down to technical reasons , such as the use of short term assays against the 3t3 protocol employed in our study and at least for reconstituted ko cells - the use of immortalized and repeatedly manipulated cells against the use of early passage primary p110-rbd - dm mefs.p110-rbd - dm mice are resistant to bleomycin - induced lung fibrosisfibroblasts are motile cells with proposed key roles in the development and progression of a wide range of diseases , including cancer and fibrosis ( wynn and ramalingam , 2012 ) . lpa has been identified as a critical fibroblast chemoattractant in experimental lung fibrosis , and lpa1 receptor knockout mice are protected in this model ( tager et al . , 2008 ) . our findings establish p110 as critical downstream target of lpa in vivo . by inference , the protection of p110-rbd - dm mice against bleomycin - induced lung fibrosis , together with the reduced migration of p110-rbd - dm fibroblasts in lpa - gradients , suggests that p110 activation downstream of lpa might be critical for fibroblast chemotaxis to sites of tissue damage in vivo . however , the rac - p110 axis might also be required for other fibroblast - specific functions involved in fibrogenesis such as secretion and remodelling of extracellular matrix components . further studies will be required to decipher the precise role of p110 in the development of fibrosis in the lung and potentially other organ systems , and to learn whether a similar mechanism exists to recruit fibroblasts to sites of primary tumors and metastases , processes that could then be targeted by isoform - specific p110 inhibitors or strategies specifically disrupting the dock180/elmo1-rac - p110 signaling axis . copurified with rab5 in a study searching for rab5 interactors ( christoforidis et al . , 1999 ) and rab5 was identified in a yeast two hybrid screen searching for p110 interactors ( kurosu and katada , 2001 ) . p110 appears to associate with rab5 in the early endocytic pathway , where it has been proposed to indirectly contribute to ptdins(3)p formation ( shin et al . , 2005 ) , and rab5 and p110 have further been suggested to be part of the autophagy - promoting vps34vps15beclin1atg14l complex ( dou et al . , 2010 ) . the rab5-binding site on p110 remains to be determined , yet several findings argue against rab5 binding to p110 through the rbd : ( 1 ) binding of rab5 is entirely unaffected by p110 rbd mutations ( figure 3c ) ; ( 2 ) rab5 does not appear to stimulate p110 ( figure 1c , ( christoforidis et al . , 1999 ; rodriguez - viciana et al . , 2004 ) ; ( 3 ) the affinity between rab5 and p110 in solution appears to be significantly higher ( data not shown ) than for typical pi3k rbd interactors , suggesting a more stable interaction , allowing to effectively localize p110 to rab5-positve subcellular compartments . out of the murine 34 members of the ras subfamily of small gtpases , only diras-1 and diras-2 showed binding to p110 ( figures s1c and s2b ) . human diras-3 , also termed arhi or noey2 , a tumor suppressor in ovarian cancer and putative autophagy regulator ( lu et al . , 2008 ) , showed no interaction ( data not shown ) . diras-1 and -2 are relatively understudied ras subfamily gtpases with low endogenous gtpase activity , whose expression appears to be limited to brain and heart ( ellis et al . whereas the relatedness to ras and the inability to bind to rbd mutant p110 ( figure s2b ) argues for binding to the rbd in vitro , the absence of any detectable stimulatory effect on p110 , the limited tissue distribution and growth - inhibitory effects make the role of diras proteins as rbd interactors of p110 in vivo unclear , and further studies will be dedicated to this subject . direct analysis of signaling complexes regulating type i pi3k isoforms in living cells is notoriously difficult due to the transient , low - affinity nature of these interactions and their instability in solution . we therefore generated p110-rbd - dm mice as a tool to study the impact of interactor binding to p110 for pi3k signaling in vivo . p110-rbd - dm pups and mice are slightly smaller than wild - type littermates , and are born in slightly sub - mendelian ratios , both features reported to an overall more severe extent from p110 knockout and p110 kinase - dead knock in mice ( -kd ) ( ciraolo et al . , 2010 ; guillermet - guibert et al . , 2008 ; kulkarni et al . , 2011 ) . a straightforward comparison between rbd mutant mice and these models is , however , complicated not only by different genetic backgrounds , but also by the different genetics of these models . complete loss of p110 catalytic subunit will inadvertently lead to some disturbance of subunit composition and/or recruitment to signaling complexes , an artifact noticeable in recently published work ( utermark et al . , 2008 ) has been found to have reduced p110 expression , both in embryos and adult tissues , placing these animals between knockout and other -kd mice . a milder phenotype of p110-rbd - dm mice is after all not unexpected , given that the rbd mutations do not disrupt basal p110 activity or rbd - independent activation routes , and some p110 activity might be enough for normal development and life under controlled conditions . we also saw moderately reduced proliferation of p110-rbd - dm primary mefs , indicating that p110 regulation through its rbd interactors is essential for normal cell proliferation , which appears to contradict findings from -kd mefs and reconstituted p110-ko mefs suggesting that p110 lipid kinase activity is not required for proliferation . although no role of p110 lipid kinase activity for cell proliferation appears surprising in light of the reported growth defect in these mice , the discrepancies between these studies could well be down to technical reasons , such as the use of short term assays against the 3t3 protocol employed in our study and at least for reconstituted ko cells - the use of immortalized and repeatedly manipulated cells against the use of early passage primary p110-rbd - dm mefs . fibroblasts are motile cells with proposed key roles in the development and progression of a wide range of diseases , including cancer and fibrosis ( wynn and ramalingam , 2012 ) . lpa has been identified as a critical fibroblast chemoattractant in experimental lung fibrosis , and lpa1 receptor knockout mice are protected in this model ( tager et al . , 2008 ) . our findings establish p110 as critical downstream target of lpa in vivo . by inference , the protection of p110-rbd - dm mice against bleomycin - induced lung fibrosis , together with the reduced migration of p110-rbd - dm fibroblasts in lpa - gradients , suggests that p110 activation downstream of lpa might be critical for fibroblast chemotaxis to sites of tissue damage in vivo . however , the rac - p110 axis might also be required for other fibroblast - specific functions involved in fibrogenesis such as secretion and remodelling of extracellular matrix components . further studies will be required to decipher the precise role of p110 in the development of fibrosis in the lung and potentially other organ systems , and to learn whether a similar mechanism exists to recruit fibroblasts to sites of primary tumors and metastases , processes that could then be targeted by isoform - specific p110 inhibitors or strategies specifically disrupting the dock180/elmo1-rac - p110 signaling axis . purified recombinant soluble pi3k protein complexes were loaded into the cell of a microcal itc200 microcalorimeter at concentrations of approximately 20 m . nucleotide - loaded gtpases were loaded into the syringe at 200 m . in a typical experiment , 16 injections of gtpase into the cell were recorded at 15c , and relevant thermodynamic parameters were analyzed and calculated using the instrument s software ( origin ) . all animal experimentation was carried out in compliance with uk home office animal welfare regulations . age- and sex - matched wild - type and homozygous p110-rbd - dm mice from intercrosses of heterozygous p110-rbd - dm animals were used to study bleomycin - induced lung fibrosis . mice ( 812 weeks old ) were anaesthetized with isoflurane , and bleomycin in sterile normal saline ( 1.25 u / kg ) or saline alone ( 50 l ) was given by intratracheal instillation . after 14 days , mice were culled and lungs were recovered for further analysis . extended experimental proceduresreagents and antibodiespertussis toxin , eht1864 , sphingosine 1-phosphate and nsc23766 were from tocris bioscience ; lysophosphatidic acid , gdp , gtps , insulin , bleomycin and protease inhibitors were from sigma aldrich ; egf and pdgf were from peprotech . antibodies to p110 , p110 , p85 , phospho - akt ( t308 ) , phospho - akt ( s473 ) , total akt , phospho - erk , total erk and p - tyr were from cell signaling technology . antibodies to dock180 were from santa cruz biotechnologies , antibodies to flag , -tubulin and vinculin from sigma aldrich . monoclonal antibody to myc and gst tags were made in house.plasmids and cloningcdnas for p110 , p110 , p110 and p110 were pcr amplified from a murine cdna library introducing an n - terminal flag or myr - flag tag ( containing a myristoylation signal ) . murine p85 , p85 , g1 and g2 were amplified untagged , and all were cloned into psg5 and fully sequenced . rbd mutations were introduced by site - directed mutagenesis and final constructs were fully sequenced . ras and rho subfamily gtpases as well as rab5 were pcr - amplified from murine cdna , cloned into pgex-2 t and sequenced . rab5 , diras1 and diras2 were subcloned into pcdna3 introducing an n - terminal myc tag and mutations were generated by site - directed mutagenesis . pef - v12-rac , pef - v12-cdc42 and pef - v14-rhoa were gifts from erik sahai , pcdna - myc - v12-hras , -kras and -nras were from umr cdna resource center . human elmo1 and fragments were pcr amplified from cdna , cloned into pgex-2 t and sequenced.protein purificationto produce purified recombinant gtpases on beads , cdnas in pgex-2 t were transformed into bl21 e. coli ( stratagene ) . protein expression was induced by addition of isopropyl -d thiogalactoside ( iptg , 0.1 mm ) overnight at 16c . bacterial lysates were made and gst - fusion proteins were recovered on glutathione agarose beads . to produce soluble untagged gtpases , cdnas encoding rac1 , cdc42 , hras and diras1 were subcloned into pgex-6p and gtpases were cleaved off gst tag and beads by overnight incubation with hrv 3c protease ( in - house made ) at 4c . when required , soluble gtpases were loaded with nucleotide in solution by addition of gdp or gtps ( 20 molar excess ) and edta ( 5 mm final concentration ) , followed by incubation at 30c for 30 min and addition of mgcl2 to a final concentration of 10 mm on ice . loaded gtpases were gel - filtrated , concentrated ( all at 4c ) and snap-frozen.to express full - length gst - p110/p85 protein complexes , cdnas were subcloned into pbacpak - his3-gst , preserving the gst tag for p110 and removing it for p85/p85 . high 5 ( life technologies ) insect cells were co infected with baculoviruses encoding the respective pi3k subunits , lysed after 72 hr , and complexes were recovered on glutathione agarose , evaluated by gel electrophoresis and either used for pull down experiments on beads , or cleaved off overnight with hrv 3c protease , gel - filtrated and snap frozen.for production of recombinant g , cdnas for murine g2 and g1 were subcloned into pbacpak - his3-tev , removing the his tag for g and preserving it for g. g complexes were purified from high 5 cell lysates on nta agarose , gel - filtrated and snap frozen.protein-binding assaysfor gst pulldown assays , approximately 10 g of gst fusion protein were immobilized on 20 l glutathione sepharose beads . immobilized gtpases were loaded with nucleotide by adding 50 l of gtpase loading buffer ( 20 mm tris - hcl ph 7.4 , 5 mm edta , 25 mm nacl ) containing gdp or gtps at 2 mm . after 20 min at 37c , mgcl2 was added to a final concentration of 10 mm on ice . proteins on beads were incubated with either 1 ml of cell lysate made from a confluent 60 mm dish of transfected cos7 cells , or with recombinant purified protein diluted in gst - binding buffer ( 20 mm tris - hcl [ ph 7.4 ] , 150 mm nacl , 5 mm mgcl2 , 1% triton x-100 , 5 mg / ml bsa ) . after 1 hr , beads were washed and proteins solubilized by boiling in lds sample buffer ( life science technologies).[h]-gtp uptake of small gtpasesto compare gtp uptake across different preparations of purified recombinant gtpases , each sample was loaded on beads with 2 mm gtp containing 1 ci [ h]-gtp , following the procedure detailed above . unbound gtp was removed by rigorous washes , gtpases on beads were resuspended in scintillation fluid , and counts per minutes were recorded in a scintillation counter over a gst only background . data are presented as counts per minute normalized to protein amounts , estimated by western blot for gst and quantification on the li - cor odyssey system.lipid kinase assayspurified recombinant , untagged and soluble p110/p85 protein complexes were incubated in kinase assay buffer ( 50 mm hepes , [ ph 7.4 ] , 50 mm nacl , 5 mm mgcl2 , 0.03% chaps , 2 mm dtt , 25 m atp ) with 4 m pi(4,5)p2-dic8 as substrate for 1 hr at 25c . reactions were terminated by adding 6 mm edta , and pi(3,4,5)p3 was quantified using a commercial competitive elisa ( echelon biosciences ) . for some reactions , recombinant g21 dimers were added a 1 m or a pdgf receptor - derived recombinant phospho - tyrosine peptide ( py740 : dgg(py)mdmskde ) was present at 10 m.lipid extraction and pip3 quantificationfor quantification of cellular pi(3,4,5)p3-levels , acidic lipids were extracted from overnight serum - starved cos7 cells following a standard lipid extraction protocol . in brief , cells were lysed in ice - cold trichloroacetic acid ( tca , 0.5 m ) . pellets were washed in 5%tca/1 mm edta and neutral lipids were extracted with meoh : chcl3 ( 2:1 ) and discarded . acidic lipids were extracted with meoh : chcl3:hcl ( 80:40:1 ) , recovered by phase - split , dried and resuspended . pi(3,4,5)p3-levels were quantified by competitive elisa ( echelon biosciences).isothermal titration calorimetrypurified recombinant soluble pi3k protein complexes and soluble nucleotide - loaded gtpases were gel - filtrated into itc buffer ( phosphate - buffered saline [ ph 7.4 ] , 5 mm mgcl2 , 1 mm tris(2-carboxyethyl)phosphine hydrochloride ) at 4c . pi3k was loaded into the cell of a microcal itc200 microcalorimeter at concentrations of approximately 20 m . gtpases were loaded into the syringe at 200 m . in a typical experiment , 16 injections of gtpase into the cell were recorded at 15c , and relevant thermodynamic parameters were analyzed and calculated using the instrument s software ( origin ) . gtpase injections into buffer only were done to determine heats of dilution.generation of p110-rbd - dm micea gene targeting vector was designed to replace exon 6 of the murine pik3cb gene , encoding the p110 catalytic subunit , with an engineered version bearing two single point mutations leading to the exchange of two rbd key residues ( s205d , k224a ) . an frt - flanked neomycin selection cassette ( genebridges ) was pcr - amplified and inserted into murine genomic dna immediately downstream of pik3cb exon 6 in a murine bac clone , using red / et recombination technology ( genebridges ) . selection cassette and flanking arms of genomic dna ( arms of homology , 4 kb upstream and 2 kb downstream of exon 6 , respectively ) were subcloned into a targeting vector backbone taken from pgkneolox2dta.2 ( phil soriano ) , again by using red / et - based recombination in e. coli . plasmid dna was isolated and mutations were introduced by site - directed mutagenesis ( quikchange xl , stratagene ) . the targeting construct was fully sequenced , linearized , and electroporated into c57bl/6 es cells . neomycin - resistant es cell clones were screened for homologous recombination by genomic pcr bridging the short arm of the construct and confirmed by pcrs bridging the long arm of homology and the entire targeting region ( biallelic pcr ) . several correctly targeted es clones were injected into blastocysts or 8-cell embryos and implanted into pseudopregnant foster mothers following standard techniques . the neomycin selection cassette was removed by crosses of heterozygous p110-rbd - dm mice with flpe deleter mice , leaving a single frt site in intron 6/7 , which was used for pcr genotyping . genotyping was later outsourced to an automated genotyping provider ( transnetyx).mouse embryonic fibroblaststo generate wild - type and homozygous p110-rbd - dm mefs , e13.5 mouse embryos from intercrosses of heterozygous p110-rbd - dm mice were collected , minced and trypsinized . mefs from each four wild - type and homozygous embryos were made and used throughout this study . for immortalization , mefs were transduced with sv40 large t antigen . to generate p110 and p110 knockout mefs , mice harboring conditional floxed alleles for p110 ( zhao et al . , 2006 ) or p110 ( jia et al . , 2008 conditional mefs made from these embryos were immortalized , expanded and treated with 4-hydroxytamoxifen ( 1 m ) for 3 consecutive days , prior to use in experiments . deletion of p110 or p110 was assessed by western blot.cell culture , plasmid , and sirna transfectionscos7 cells and mouse embryonic fibroblasts ( mefs ) were maintained in dmem , supplemented with 10% fetal calf serum ( fcs ) . for serum starvation , mefs were washed twice with sterile phosphate buffered saline ( pbs ) and incubated in serum - free dmem , supplemented with 0.5% fatty acid - free bovine serum albumin ( bsa , sigma aldrich ) . plasmids were transfected into cos7 cells with lipofectamine 2000 ( life science technologies ) , following the manufacturer s protocol . mefs were plasmid transfected by electroporation using the amaxa nucleofector system.sirna was transfected into immortalized mefs using dharmafect 4 ( dharmacon ) , following manufacturer s protocols . sigenome sirna pools were used to target rac - gefs , on - targetplus pools for rac1 and cdc42 , and non - targeting pool 2 for control transfections ( all from dharmacon ) . sirna sequences are listed in table s1.immunoprecipitation and western blottingwestern blotting was performed following standard methods . in brief , cells were washed twice in ice - cold pbs , before they were lysed directly in preheated lds sample buffer ( life science technologies ) . samples were heated to 98c for 3 min , run on nupage bis - tris gels ( life science technologies ) and transferred to pvdf membranes ( millipore ) . immunodetection of proteins was performed either by conventional hrp - based chemiluminescence or by fluorescence - based detection using the li - cor odyssey system and therefore recommended protocols and reagents.for immunoprecipitation of p85 , mefs were lysed in ip buffer ( 20 mm tris - hcl [ ph 7.4 ] , 150 mmol nacl , 5 mmol mgcl2 , 1% triton x-100 , protease inhibitor cocktail [ sigma aldrich ] , 10 mm -glycerol phosphate , 10 mm naf , 10 mm sodium pyrophosphate ) , and lysates were cleared by centrifugation . anti - p85 antibody was precoupled to protein a sepharose beads ( sigma aldrich ) and was incubated with lysates for 2 hr . lysates were run in parallel.quantitative real - time pcrto compare elmo1/elmo2 mrna abundance after sirna transfections , total rna was extracted from transfected immortalized wild - type mefs ( qiagen rna mini kit ) , and 1 g of total mrna was reverse - transcribed into cdna . quantitative real - time pcr ( qpcr ) was performed following standard protocols , elmo1/2 mrna levels were normalized to beta - actin and changes estimated using the ct method . primers used were qiagen quantitect primer assays for elmo1 , elmo2 and beta-actin.growth factor and lysophospholipid signaling assaysfor growth factor and lysophospholipid stimulation experiments , early passage primary mefs were seeded at 3 10 cells in 6 cm cell culture dishes , starved in serum - free medium , supplemented with 0.5% bsa , overnight and stimulated as desired . cells were rapidly washed in ice - cold pbs and directly lysed in preheated lds sample buffer , prior to analysis by western blot.proliferation assaysto study proliferation of mefs in culture , early passage primary mefs ( p2 ) from each two wild - type and homozygous p110-rbd - dm embryos , derived from het x het crosses , were seeded at a defined number , grown in full serum for 3 days , trypsinized , counted in triplicate and reseeded at original numbers ( modified 3t3 protocol ) . population doubling rate was calculated as pdr = log(n(day3)/n(seeded))xlog(2 ) . for cell cycle analysis , primary mefs growing in 1% or 10% fcs were digested off , fixed in 70% ethanol , stained with propidium iodide , and analyzed by flow cytometry.migration assaysfor migration assays , serum - starved immortalized mefs were seeded onto 8 m pore size cell culture inserts , coated with 10 g / ml fibronectin . inserts were placed into wells containing 500 l serum - free dmem , supplemented with 0.5% fatty - acid - free bsa and chemoattractant as required . cells were allowed to migrate for 6 hr , before they were fixed in 4% paraformaldehyde for 10 min . nonmigrated cells were scraped off using cotton wool tips , and membranes were recovered and mounted onto microscopic slides in vectashield hardset mounting medium with dapi ( vector laboratories ) . several nonoverlapping low - magnification fields were photographed on a nikon fluorescence microscope , and cells were semiautomatically counted using the object count function of nikon nis elements . for studies with sirna - transfected mefs , cells were transfected 48 hr prior to migration experiment . to assess migration of plasmid transfected mefs , cells were cotransfected with pmaxgfp and migrated gfp - positive cells were counted.rac activation assayfor rac activation assays , the gst - tagged active rac and cdc42-binding domain of pak1 ( gst - pak - pbd ) was expressed in e. coli and purified on glutathione agarose beads ( 10 g protein on 20 l bead volume per reaction ) . immortalized wild - type and p110-rbd - dm mefs were seeded on 150 mm tissue culture dishes , grown to 50% confluency , serum - starved overnight , inhibitor - treated as desired and stimulated one at a time . for sirna experiments , cells were harvested 48 hr after transfection and following an overnight starvation . for harvest , cells were rapidly washed twice in ice - cold pbs and lysed in 1 ml gst lysis buffer ( 20 mm tris - hcl [ ph 7.4 ] , 150 mmol nacl , 5 mmol mgcl2 , 1% triton x-100 , 1 mm dtt , protease inhibitors , 10 mm -glycerol phosphate , 10 mm naf ) . cells were scraped off and lysates were cleared at full speed for 2 min in a cooled microcentrifuge . supernatants were snap frozen in liquid nitrogen . once all dishes were harvested , lysates were carefully thawed and incubated with gst - pak - pbd for 1 hr . beads were washed several times and bound rac was identified and quantified by western blot ( li - cor odyssey ) . lysates were analyzed in parallel and rac - gtp / total rac ratios were determined.histology and morphometric analysislungs were processed for histology after fixation in neutral - buffered formalin . frontal sections through central levels of each lung were stained with h&e , sirius red or immunohistochemistry ( ihc ) for smooth muscle actin ( anti--sma , dako ) following standard protocols . to quantify fibrotic changes , as many as possible nonoverlapping low - magnification ( 4 ) microscopic fields from representative sections of each lung stained with h&e , the transparent area of each section , made up mainly by alveolar spaces and small airways was semiautomatically quantified using nikon nis elements . pertussis toxin , eht1864 , sphingosine 1-phosphate and nsc23766 were from tocris bioscience ; lysophosphatidic acid , gdp , gtps , insulin , bleomycin and protease inhibitors were from sigma aldrich ; egf and pdgf were from peprotech . antibodies to p110 , p110 , p85 , phospho - akt ( t308 ) , phospho - akt ( s473 ) , total akt , phospho - erk , total erk and p - tyr were from cell signaling technology . antibodies to dock180 were from santa cruz biotechnologies , antibodies to flag , -tubulin and vinculin from sigma aldrich . cdnas for p110 , p110 , p110 and p110 were pcr amplified from a murine cdna library introducing an n - terminal flag or myr - flag tag ( containing a myristoylation signal ) . murine p85 , p85 , g1 and g2 were amplified untagged , and all were cloned into psg5 and fully sequenced . rbd mutations were introduced by site - directed mutagenesis and final constructs were fully sequenced . ras and rho subfamily gtpases as well as rab5 were pcr - amplified from murine cdna , cloned into pgex-2 t and sequenced . rab5 , diras1 and diras2 were subcloned into pcdna3 introducing an n - terminal myc tag and mutations were generated by site - directed mutagenesis . pef - v12-rac , pef - v12-cdc42 and pef - v14-rhoa were gifts from erik sahai , pcdna - myc - v12-hras , -kras and -nras were from umr cdna resource center . human elmo1 and fragments were pcr amplified from cdna , cloned into pgex-2 t and sequenced . to produce purified recombinant gtpases on beads , cdnas in pgex-2 t were transformed into bl21 e. coli ( stratagene ) . protein expression was induced by addition of isopropyl -d thiogalactoside ( iptg , 0.1 mm ) overnight at 16c . bacterial lysates were made and gst - fusion proteins were recovered on glutathione agarose beads . to produce soluble untagged gtpases , cdnas encoding rac1 , cdc42 , hras and diras1 were subcloned into pgex-6p and gtpases were cleaved off gst tag and beads by overnight incubation with hrv 3c protease ( in - house made ) at 4c . when required , soluble gtpases were loaded with nucleotide in solution by addition of gdp or gtps ( 20 molar excess ) and edta ( 5 mm final concentration ) , followed by incubation at 30c for 30 min and addition of mgcl2 to a final concentration of 10 mm on ice . loaded gtpases were gel - filtrated , concentrated ( all at 4c ) and snap - frozen . to express full - length gst - p110/p85 protein complexes , cdnas were subcloned into pbacpak - his3-gst , preserving the gst tag for p110 and removing it for p85/p85 . high 5 ( life technologies ) insect cells were co infected with baculoviruses encoding the respective pi3k subunits , lysed after 72 hr , and complexes were recovered on glutathione agarose , evaluated by gel electrophoresis and either used for pull down experiments on beads , or cleaved off overnight with hrv 3c protease , gel - filtrated and snap frozen . for production of recombinant g , cdnas for murine g2 and g1 were subcloned into pbacpak - his3-tev , removing the his tag for g and preserving it for g. g complexes were purified from high 5 cell lysates on nta agarose , gel - filtrated and snap frozen . for gst pulldown assays , approximately 10 g of gst fusion protein were immobilized on 20 l glutathione sepharose beads . immobilized gtpases were loaded with nucleotide by adding 50 l of gtpase loading buffer ( 20 mm tris - hcl ph 7.4 , 5 mm edta , 25 mm nacl ) containing gdp or gtps at 2 mm . after 20 min at 37c , mgcl2 was added to a final concentration of 10 mm on ice . proteins on beads were incubated with either 1 ml of cell lysate made from a confluent 60 mm dish of transfected cos7 cells , or with recombinant purified protein diluted in gst - binding buffer ( 20 mm tris - hcl [ ph 7.4 ] , 150 mm nacl , 5 mm mgcl2 , 1% triton x-100 , 5 mg / ml bsa ) . after 1 hr , beads were washed and proteins solubilized by boiling in lds sample buffer ( life science technologies ) . to compare gtp uptake across different preparations of purified recombinant gtpases , each sample was loaded on beads with 2 mm gtp containing 1 ci [ h]-gtp , following the procedure detailed above . unbound gtp was removed by rigorous washes , gtpases on beads were resuspended in scintillation fluid , and counts per minutes were recorded in a scintillation counter over a gst only background . data are presented as counts per minute normalized to protein amounts , estimated by western blot for gst and quantification on the li - cor odyssey system . purified recombinant , untagged and soluble p110/p85 protein complexes were incubated in kinase assay buffer ( 50 mm hepes , [ ph 7.4 ] , 50 mm nacl , 5 mm mgcl2 , 0.03% chaps , 2 mm dtt , 25 m atp ) with 4 m pi(4,5)p2-dic8 as substrate for 1 hr at 25c . reactions were terminated by adding 6 mm edta , and pi(3,4,5)p3 was quantified using a commercial competitive elisa ( echelon biosciences ) . for some reactions , recombinant g21 dimers were added a 1 m or a pdgf receptor - derived recombinant phospho - tyrosine peptide ( py740 : dgg(py)mdmskde ) was present at 10 m . for quantification of cellular pi(3,4,5)p3-levels , acidic lipids were extracted from overnight serum - starved cos7 cells following a standard lipid extraction protocol . in brief , cells were lysed in ice - cold trichloroacetic acid ( tca , 0.5 m ) . pellets were washed in 5%tca/1 mm edta and neutral lipids were extracted with meoh : chcl3 ( 2:1 ) and discarded . acidic lipids were extracted with meoh : chcl3:hcl ( 80:40:1 ) , recovered by phase - split , dried and resuspended . purified recombinant soluble pi3k protein complexes and soluble nucleotide - loaded gtpases were gel - filtrated into itc buffer ( phosphate - buffered saline [ ph 7.4 ] , 5 mm mgcl2 , 1 mm tris(2-carboxyethyl)phosphine hydrochloride ) at 4c . pi3k was loaded into the cell of a microcal itc200 microcalorimeter at concentrations of approximately 20 m . gtpases were loaded into the syringe at 200 m . in a typical experiment , 16 injections of gtpase into the cell were recorded at 15c , and relevant thermodynamic parameters were analyzed and calculated using the instrument s software ( origin ) . a gene targeting vector was designed to replace exon 6 of the murine pik3cb gene , encoding the p110 catalytic subunit , with an engineered version bearing two single point mutations leading to the exchange of two rbd key residues ( s205d , k224a ) . an frt - flanked neomycin selection cassette ( genebridges ) was pcr - amplified and inserted into murine genomic dna immediately downstream of pik3cb exon 6 in a murine bac clone , using red / et recombination technology ( genebridges ) . selection cassette and flanking arms of genomic dna ( arms of homology , 4 kb upstream and 2 kb downstream of exon 6 , respectively ) were subcloned into a targeting vector backbone taken from pgkneolox2dta.2 ( phil soriano ) , again by using red / et - based recombination in e. coli . plasmid dna was isolated and mutations were introduced by site - directed mutagenesis ( quikchange xl , stratagene ) . the targeting construct was fully sequenced , linearized , and electroporated into c57bl/6 es cells . neomycin - resistant es cell clones were screened for homologous recombination by genomic pcr bridging the short arm of the construct and confirmed by pcrs bridging the long arm of homology and the entire targeting region ( biallelic pcr ) . several correctly targeted es clones were injected into blastocysts or 8-cell embryos and implanted into pseudopregnant foster mothers following standard techniques . the neomycin selection cassette was removed by crosses of heterozygous p110-rbd - dm mice with flpe deleter mice , leaving a single frt site in intron 6/7 , which was used for pcr genotyping . genotyping was later outsourced to an automated genotyping provider ( transnetyx ) . to generate wild - type and homozygous p110-rbd - dm mefs , e13.5 mouse embryos from intercrosses of heterozygous p110-rbd - dm mice mefs from each four wild - type and homozygous embryos were made and used throughout this study . for immortalization , mefs were transduced with sv40 large t antigen . to generate p110 and p110 knockout mefs , mice harboring conditional floxed alleles for p110 ( zhao et al . , 2006 ) or p110 ( jia et al . , 2008 conditional mefs made from these embryos were immortalized , expanded and treated with 4-hydroxytamoxifen ( 1 m ) for 3 consecutive days , prior to use in experiments . deletion of p110 or p110 was assessed by western blot . cos7 cells and mouse embryonic fibroblasts ( mefs ) were maintained in dmem , supplemented with 10% fetal calf serum ( fcs ) . for serum starvation , mefs were washed twice with sterile phosphate buffered saline ( pbs ) and incubated in serum - free dmem , supplemented with 0.5% fatty acid - free bovine serum albumin ( bsa , sigma aldrich ) . plasmids were transfected into cos7 cells with lipofectamine 2000 ( life science technologies ) , following the manufacturer s protocol . sirna was transfected into immortalized mefs using dharmafect 4 ( dharmacon ) , following manufacturer s protocols . sigenome sirna pools were used to target rac - gefs , on - targetplus pools for rac1 and cdc42 , and non - targeting pool 2 for control transfections ( all from dharmacon ) . western blotting was performed following standard methods . in brief , cells were washed twice in ice - cold pbs , before they were lysed directly in preheated lds sample buffer ( life science technologies ) . samples were heated to 98c for 3 min , run on nupage bis - tris gels ( life science technologies ) and transferred to pvdf membranes ( millipore ) . immunodetection of proteins was performed either by conventional hrp - based chemiluminescence or by fluorescence - based detection using the li - cor odyssey system and therefore recommended protocols and reagents . for immunoprecipitation of p85 , mefs were lysed in ip buffer ( 20 mm tris - hcl [ ph 7.4 ] , 150 mmol nacl , 5 mmol mgcl2 , 1% triton x-100 , protease inhibitor cocktail [ sigma aldrich ] , 10 mm -glycerol phosphate , 10 mm naf , 10 mm sodium pyrophosphate ) , and lysates were cleared by centrifugation . anti - p85 antibody was precoupled to protein a sepharose beads ( sigma aldrich ) and was incubated with lysates for 2 hr . total rna was extracted from transfected immortalized wild - type mefs ( qiagen rna mini kit ) , and 1 g of total mrna was reverse - transcribed into cdna . quantitative real - time pcr ( qpcr ) was performed following standard protocols , elmo1/2 mrna levels were normalized to beta - actin and changes estimated using the ct method . primers used were qiagen quantitect primer assays for elmo1 , elmo2 and beta - actin . for growth factor and lysophospholipid stimulation experiments , early passage primary mefs were seeded at 3 10 cells in 6 cm cell culture dishes , starved in serum - free medium , supplemented with 0.5% bsa , overnight and stimulated as desired . cells were rapidly washed in ice - cold pbs and directly lysed in preheated lds sample buffer , prior to analysis by western blot . to study proliferation of mefs in culture , early passage primary mefs ( p2 ) from each two wild - type and homozygous p110-rbd - dm embryos , derived from het x het crosses , were seeded at a defined number , grown in full serum for 3 days , trypsinized , counted in triplicate and reseeded at original numbers ( modified 3t3 protocol ) . population doubling rate was calculated as pdr = log(n(day3)/n(seeded))xlog(2 ) . for cell cycle analysis , primary mefs growing in 1% or 10% fcs were digested off , fixed in 70% ethanol , stained with propidium iodide , and analyzed by flow cytometry . for migration assays , serum - starved immortalized mefs were seeded onto 8 m pore size cell culture inserts , coated with 10 g / ml fibronectin . inserts were placed into wells containing 500 l serum - free dmem , supplemented with 0.5% fatty - acid - free bsa and chemoattractant as required . cells were allowed to migrate for 6 hr , before they were fixed in 4% paraformaldehyde for 10 min . nonmigrated cells were scraped off using cotton wool tips , and membranes were recovered and mounted onto microscopic slides in vectashield hardset mounting medium with dapi ( vector laboratories ) . several nonoverlapping low - magnification fields were photographed on a nikon fluorescence microscope , and cells were semiautomatically counted using the object count function of nikon nis elements . for studies with sirna - transfected mefs , cells were transfected 48 hr prior to migration experiment . to assess migration of plasmid transfected mefs , cells were cotransfected with pmaxgfp and migrated gfp - positive cells were counted . for rac activation assays , the gst - tagged active rac and cdc42-binding domain of pak1 ( gst - pak - pbd ) was expressed in e. coli and purified on glutathione agarose beads ( 10 g protein on 20 l bead volume per reaction ) . immortalized wild - type and p110-rbd - dm mefs were seeded on 150 mm tissue culture dishes , grown to 50% confluency , serum - starved overnight , inhibitor - treated as desired and stimulated one at a time . for sirna experiments , cells were harvested 48 hr after transfection and following an overnight starvation . for harvest , cells were rapidly washed twice in ice - cold pbs and lysed in 1 ml gst lysis buffer ( 20 mm tris - hcl [ ph 7.4 ] , 150 mmol nacl , 5 mmol mgcl2 , 1% triton x-100 , 1 mm dtt , protease inhibitors , 10 mm -glycerol phosphate , 10 mm naf ) . cells were scraped off and lysates were cleared at full speed for 2 min in a cooled microcentrifuge . supernatants were snap frozen in liquid nitrogen . once all dishes were harvested , lysates were carefully thawed and incubated with gst - pak - pbd for 1 hr . beads were washed several times and bound rac was identified and quantified by western blot ( li - cor odyssey ) . lysates were analyzed in parallel and rac - gtp / total rac ratios were determined . frontal sections through central levels of each lung were stained with h&e , sirius red or immunohistochemistry ( ihc ) for smooth muscle actin ( anti--sma , dako ) following standard protocols . to quantify fibrotic changes , as many as possible nonoverlapping low - magnification ( 4 ) microscopic fields from representative sections of each lung stained with h&e , the transparent area of each section , made up mainly by alveolar spaces and small airways was semiautomatically quantified using nikon nis elements .

summaryras proteins are important direct activators of p110 , p110 , and p110 type i phosphoinositide 3-kinases ( pi3ks ) , interacting via an amino - terminal ras - binding domain ( rbd ) . here , we investigate the regulation of the ubiquitous p110 isoform of pi3k , implicated in g - protein - coupled receptor ( gpcr ) signaling , pten - loss - driven cancers , and thrombocyte function . unexpectedly , ras is unable to interact with p110 , but instead rac1 and cdc42 from the rho subfamily of small gtpases bind and activate p110 via its rbd . in fibroblasts , gpcrs couple to pi3k through dock180/elmo1-mediated rac activation and subsequent interaction with p110. cells from mice carrying mutations in the p110 rbd show reduced pi3k activity and defective chemotaxis , and these mice are resistant to experimental lung fibrosis . these findings revise our understanding of the regulation of type i pi3k by showing that both ras and rho family gtpases directly regulate distinct ubiquitous pi3k isoforms and that rac activates p110 downstream of gpcrs .

Introduction Results Discussion Experimental Procedures

the type i phosphoinositide 3-kinases ( pi3ks ) are critical signaling proteins involved in the regulation of cell growth , survival , motility , and metabolism . in mammals , there exist four isoforms of the type i pi3k catalytic p110 subunits : , , , and . in addition , activated ras proteins bind directly to an n - terminal ras - binding domain ( rbd ) on p110 , acting synergistically with the input from tyrosine - phosphorylated proteins to optimally activate lipid kinase activity ( rodriguez - viciana et al . proof of the pathophysiological importance of the direct interaction of ras with p110 came from the generation of mice bearing germline mutations in the rbd of p110 , which were found to be highly resistant to mutant - ras - induced lung and skin cancer formation ( gupta et al . mice with mutations in the rbd of p110 show neutrophil defects in the regulation of pi3k activity by some g - protein - coupled receptors ( gpcrs ) ( suire et al . ras also has been reported to bind and activate p110 in vitro ( vanhaesebroeck et al . it appears to be relatively insensitive to activation by growth factor receptor tyrosine kinase signaling but important downstream of certain gpcrs , including those for lysophosphatidic acid ( lpa ) and sphingosine 1-phosphate ( s1p ) , making p110 the only gpcr - regulated type i pi3k isoform outside the hematopoietic system ( ciraolo et al . it has , however , remained entirely unclear whether the p110 rbd contributes to p110 activation and function , and despite the apparently similar level of relatedness between the rbds across the four isoforms , a systematic analysis of ras effector proteins failed to detect any activation of p110 by ras in cotransfected cells ( rodriguez - viciana et al . we present extensive in vitro work to show that p110 is the only type i pi3k isoform not regulated by ras and to identify the rho family gtpases rac and cdc42 as direct isoform - specific rbd interactors and activators of p110. we go on to show that gpcrs couple to pi3k via dock180/elmo1-mediated rac activation and subsequent interaction with p110. mouse embryonic fibroblasts ( mefs ) from p110 rbd mutant mice show reduced pi3k activity and mice are resistant to bleomycin - induced lung fibrosis , a pathology that has been linked with lpa signaling . these findings explain longstanding inconsistencies and revise our understanding of type i pi3k regulation by small gtp - binding proteins , providing molecular insight into the regulation and function of the ubiquitous p110 isoform . in glutathione s - transferase ( gst ) pull - down studies using recombinant , gtps - loaded hras , kras , and nras as baits , we found strong and specific interaction between all three ras proteins and p110 ( figure 1a ) . in contrast , p110 bound to none of the ras proteins , but did bind to rab5 , a previously identified gtpase interactor of p110 ( christoforidis et al . to identify rbd interactors of p110 , we probed all 34 murine members of the ras subfamily of small gtpases ( rfgs ) for binding to p110/p85 ( figure s1c ) . strikingly , whereas all non- isoforms interacted with the three prototypical ras proteins and a partially overlapping subset of closely related rfgs ( rras1 , rras2 , mras , and eras ) , p110 bound to none of those ( figure 2a ) . this substitution is relevant to pi3k binding because an hras - d33i mutant showed attenuated binding to p110 and diras1-i37d showed reduced binding to p110 , even though exchange of this residue did not enable ras binding to p110 or diras binding to p110 ( figure s2a ) , pointing to additional , g2-box - independent determinants of pi3k isoform specificity . several members of the rho subfamily of small gtpases harbor a hydrophobic isoleucine or valine residue in this position ( figure s2c ) , which prompted us to test p110 for binding to representative rho family gtpases ( figure s2d ) . stimulation of p110 by active rac1 and cdc42 was dose dependent ( figure 2h ) . coexpression of constitutively active rac1 or cdc42 ( figure s2 g , lanes 46 ) , but not rhoa ( figure s2h ) , along with p110/p85 in cos7 cells strongly elevated cellular phospho - akt and pip3 levels ( figure 2i ) , indicating that both gtpases activate p110 in transfected cells . in contrast , gtps - loaded rac1/cdc42 did not stimulate p110 in vitro ( figure s2i ) , nor did v12-rac1/cdc42 cooperate with p110 , p110 , or p110 to elevate cellular phospho - akt levels ( figure s2j ) . taken together , these data show that the rho family gtpases rac1 and cdc42 bind to p110 in an isoform - specific manner and potently and directly stimulate its lipid kinase activity . , 1994 ) , is required for rac / cdc42 binding to p110 , we truncated p85 ( p85 schematic in figure 3c ) and probed for binding of p110/p85 to rac1 and cdc42 in vitro . to further corroborate these findings , we generated 43 single point mutations covering 37 residues across the p110 rbd and assayed these mutants for binding to rac1 and cdc42 ( figures 3d , s3a , and s3b ) . , 2000 ; , 1996 ) , indicating that rac1 and cdc42 are plausible rbd interactors of p110. homologous recombination in embryonic stem ( es ) cells was employed to replace exon 6 of the murine pik3cb gene ( figure s4a ) , and germline transmission was achieved by eight - cell embryo injection ( figures s4b and s4c ) . mefs homozygous for the p110 rbd mutation proliferated at a significantly slower rate than their wild - type counterparts ( figure 4d ) , which was reflected by a higher percentage of cells in g1 ( 1% fetal calf serum [ fcs ] : 56.7% 0.32% versus 64.2% 0.53% , n = 4 , p < 0.001 ; 10% fcs : 47.5% 3.0% versus 52.2% 3.9% , n = 4 , p < 0.05 ) , fewer cells in g2 ( 1% fcs : 24.5% 1.4% versus 21.0% 1.9% , n = 4 , p < 0.05 ; 10% fcs : 23.9% 1.4% versus 21.1% 1.6% , n = 4 , p < 0.05 ) and fewer cells in s phase for 1% fcs ( 1% fcs : 13.3% 1.0% versus 11.0% 169% , n , p110-rbd - dm mefs showed lower steady - state phospho - akt levels ( figure 4f ) , suggesting that stimulatory signaling to p110 via its rbd contributes to pi3k activity in vivo . expression levels of p110 , p110 , and p85 were indistinguishable among the genotypes ( figure s4d ) , and the stoichiometry of p110 subunit binding to p85 was undisturbed ( figure s4e ) . to determine whether rac and cdc42 are upstream activators of p110 in vivo , we transfected wild - type and p110-rbd - dm mefs with small interfering rna ( sirna ) pools ( dharmacon on - target plus ) targeting these gtpases ( figure 4 g ) . although single knockdowns had only minor effects , combined knockdown of rac1 and cdc42 significantly lowered phospho - akt levels in wild - type , but not in p110-rbd - dm cells , closing the gap in steady - state phospho - akt levels between the genotypes and suggesting that endogenous rac1 and cdc42 cooperatively activate p110 via its rbd . taken together , these findings indicate that rac1 and cdc42 activate pi3k in living cells by isoform - specific regulation of p110 through its rbd . to study whether the p110 rbd is required for coupling p110 to gpcrs , we stimulated wild - type and p110-rbd - dm mefs with the lipid growth factors and gpcr agonists lpa and s1p . notably , in p110-knockout cells , akt phosphorylation in response to lpa was completely abolished ( figure 5c ) , indicating that the rbd is essential for much but not all p110 activation downstream of gpcrs . to test whether the identified p110 rbd interactors are required for linking p110 to gpcrs , we knocked down rac1 and cdc42 in wild - type mefs . knockdown of rac1 strongly impacted lpa / s1p - induced akt phosphorylation , knockdown of cdc42 had only minor effects , and combination knockdown of both rac1 and cdc42 had little additional effect compared to rac1 knockdown alone ( figure 5d ) . in contrast , knockdown of the dock family rac - gef dock180 or its adaptor protein elmo1 interfered with akt phosphorylation induced by lpa and s1p ( figure 6b and s6b ) , but not by egf , pdgf , and insulin ( figure s6c ) . moreover , knockdown of dock180 abolished lpa- but not egf - induced rac activation ( figure 6c ) , firmly placing dock180/elmo1 downstream of the lpa receptor and upstream of rac and p110. , 2005 ) , raising the possibility of a pip3-driven feedback loop , in which rac would be upstream and downstream of p110. however , whereas sensitive to pertussis toxin , lpa - induced rac activation was entirely insensitive to pi3k inhibition by gdc0941 , a pan type i pi3k inhibitor , placing all detectable rac activation upstream of p110 ( figure 6d ) . we therefore wondered whether the disruption of p110 activation by lpa in p110-rbd - dm mice would be sufficient to affect experimental lung fibrosis . in this study , we show that , in contrast to what has been widely presumed , ras is not a general regulator of type i pi3ks . we find that out of the two ubiquitously expressed pi3k isoforms , only p110 is regulated by ras , whereas p110 is a direct rac and cdc42 target protein , indicating that key members of the pivotal ras and rho families of small g proteins directly regulate type i pi3ks , with each family controlling their own distinct ubiquitous p110 isoform . that p110 proved unable to physically and functionally interact with ras is unexpected given the presence of a moderately conserved pi3k - type rbd in all four type i pi3k p110 catalytic subunits ( pacold et al . p110 is not only unable to interact with ras under conditions readily revealing the transient , low - affinity interactions of ras with other isoforms , but it also has an entirely distinct ras superfamily gtpase interactor profile with a subfamily switch from ras to rho at the core of it , making any , for whatever reason , undetectable interaction with ras unlikely . , 1995 ) , but was attributed to rac / cdc42 binding to the amino - terminal bhd on p85 , which has sequence homology to rho - gap domains ( bokoch et al . , 2007 ) , but our biochemical data strongly argue against an involvement of p85 in the rac / cdc42-p110 interaction , because ( 1 ) rac1 and cdc42 do not interact with p110/p85 or p110/p85 , ( 2 ) rac1 and cdc42 bind normally to p110 in the absence of the p85 bh domain , and ( 3 ) rbd point mutations abrogate rac1 and cdc42 binding to p110 in complex with full - length p85 . , 1997 ; 2002 ) , but straightforward analysis of the relationship between rho family gtpases and pi3k has been difficult , mainly because rac and cdc42 also act downstream of pi3k , activated through pip3-dependent gefs ( welch et al . , a very recent study using microscopy - based assays in transfected cells revisited the interaction of small gtpases and pi3k , confirming that both active ras and rho family gtpases can activate pi3k in living cells . our findings in mefs suggest that rac1 and cdc42 cooperatively control steady - state pi3k activity in living cells by isoform - specific activation of p110 and that this requires the p110 rbd . , 2006 ) , and data showing pten - loss - driven prostate cancers to be entirely dependent on p110 , as well as metabolic findings in p110 kinase - dead mice , have been interpreted in the same way ( ciraolo et al . , 2008 ) we find that mutation of the p110 rbd strongly attenuates p110 activation downstream of gpcrs , highlighting the importance of the rbd for p110 key signaling functions . transactivation of receptor tyrosine kinases has been suggested to activate p110 downstream of gpcrs ( yart et al . rac1 is essential for p110 activation downstream of the gpcrs for lpa and s1p , and the rac - gef dock180/elmo1 is upstream of both rac and p110 in this pathway . in line with this , pi3k activity is not required for rac activation by lpa , placing p110 entirely downstream of rac in fibroblasts , which contrasts with a proposed pip3-driven feedback loop controlling rac activity upstream and downstream of pi3k in leukocytes ( weiner et al . such two - track wiring is reminiscent of receptor tyrosine kinase regulation of p110 via p85 interaction with tyrosine - phosphorylated receptor or adaptor protein and grb2-sos - ras - p110 interaction . see supplemental information online for the extended discussion , including information on other gtpase interactors of p110 , the phenotype of p110-rbd mutant mice , and resistance of these mice to bleomycin - induced lung fibrosis . , 2005 ) , and rab5 and p110 have further been suggested to be part of the autophagy - promoting vps34vps15beclin1atg14l complex ( dou et al . , 2004 ) ; ( 3 ) the affinity between rab5 and p110 in solution appears to be significantly higher ( data not shown ) than for typical pi3k rbd interactors , suggesting a more stable interaction , allowing to effectively localize p110 to rab5-positve subcellular compartments.out of the murine 34 members of the ras subfamily of small gtpases , only diras-1 and diras-2 showed binding to p110 ( figures s1c and s2b ) . whereas the relatedness to ras and the inability to bind to rbd mutant p110 ( figure s2b ) argues for binding to the rbd in vitro , the absence of any detectable stimulatory effect on p110 , the limited tissue distribution and growth - inhibitory effects make the role of diras proteins as rbd interactors of p110 in vivo unclear , and further studies will be dedicated to this subject.p110-rbd - dm micedirect analysis of signaling complexes regulating type i pi3k isoforms in living cells is notoriously difficult due to the transient , low - affinity nature of these interactions and their instability in solution . although no role of p110 lipid kinase activity for cell proliferation appears surprising in light of the reported growth defect in these mice , the discrepancies between these studies could well be down to technical reasons , such as the use of short term assays against the 3t3 protocol employed in our study and at least for reconstituted ko cells - the use of immortalized and repeatedly manipulated cells against the use of early passage primary p110-rbd - dm mefs.p110-rbd - dm mice are resistant to bleomycin - induced lung fibrosisfibroblasts are motile cells with proposed key roles in the development and progression of a wide range of diseases , including cancer and fibrosis ( wynn and ramalingam , 2012 ) . lpa has been identified as a critical fibroblast chemoattractant in experimental lung fibrosis , and lpa1 receptor knockout mice are protected in this model ( tager et al . , 2005 ) , and rab5 and p110 have further been suggested to be part of the autophagy - promoting vps34vps15beclin1atg14l complex ( dou et al . out of the murine 34 members of the ras subfamily of small gtpases , only diras-1 and diras-2 showed binding to p110 ( figures s1c and s2b ) . whereas the relatedness to ras and the inability to bind to rbd mutant p110 ( figure s2b ) argues for binding to the rbd in vitro , the absence of any detectable stimulatory effect on p110 , the limited tissue distribution and growth - inhibitory effects make the role of diras proteins as rbd interactors of p110 in vivo unclear , and further studies will be dedicated to this subject . lpa has been identified as a critical fibroblast chemoattractant in experimental lung fibrosis , and lpa1 receptor knockout mice are protected in this model ( tager et al . monoclonal antibody to myc and gst tags were made in house.plasmids and cloningcdnas for p110 , p110 , p110 and p110 were pcr amplified from a murine cdna library introducing an n - terminal flag or myr - flag tag ( containing a myristoylation signal ) . cdnas for p110 , p110 , p110 and p110 were pcr amplified from a murine cdna library introducing an n - terminal flag or myr - flag tag ( containing a myristoylation signal ) . sigenome sirna pools were used to target rac - gefs , on - targetplus pools for rac1 and cdc42 , and non - targeting pool 2 for control transfections ( all from dharmacon ) .

reverse micelles are monodisperse ensembles of surfactant - encapsulated nanoscale water pools that result from spontaneous organization of a mixture of appropriate surfactants , water , and nonpolar solvent such as the alkanes ( fig . the size of the water pool can be tightly controlled by varying the molar ratio of water to surfactant ( water loading or wo ) . proteins can be encapsulated within the water pool of reverse micelles reproducibly and with high structural fidelity and at concentrations amenable to high - resolution solution nmr methods14,15 . when prepared in low viscosity solvents , the full arsenal of solution multi - dimensional heteronuclear nmr techniques becomes accessible16,17 . reverse micelles have served as a powerful tool for nmr - based studies of protein and nucleic acid structure and biophysics under a range of conditions14,16,1823 . for water loadings such as those used here ( w0 ~ 10 ) , the reverse micelle contains approximately one thousand water molecules on average24 and the re - orientational dynamics of encapsulated water is slowed relative to bulk water by approximately one order of magnitude25,26 . this suggested that the combined effects of the protein surface and nanoscale confinement could slow the water dynamics to allow efficient detection of hydration water - protein cross - relaxation . at the same time , if reorganization of water contributes to the rate limiting step , hydrogen exchange chemistry in the reverse micelle should also be somewhat slowed , potentially minimizing complications arising from hydrogen - exchange . finally , reverse micelles also provide an appropriate experimental condition to directly assess the contributions from long - range intermolecular dipolar interactions . ubiquitin is a small 76 residue protein comprised of a five stranded mixed beta sheet , a long alpha helix and a short 310 helix27 . we first examined the hydration of ubiquitin in free aqueous solution at ph 5 and 25 c using three - dimensional n - resolved noesy29 and roesy30 experiments ( fig . only a few amide - water weak cross peaks were observed , which is generally indicative of very short residence times for protein hydration water in bulk aqueous solution3 . at this ph and temperature , the exchange of even non - hydrogen bonded amide hydrogens with solvent is generally too slow to impact these experiments . however , every amide hydrogen - water cross peak observed in the free solution spectra involves an amide hydrogen that is within the distance detectable by the noe ( see below ) of a labile side chain hydrogen . chemical exchange of side chain hydrogens with solvent water followed by intramolecular noe produces cross peaks at the water resonance that are indistinguishable from direct protein - water noes2,3 . water noe correlations seen in bulk solution are the result of such exchange - relayed interactions5 . we have previously demonstrated that the structure of ubiquitin is unperturbed by appropriate encapsulation in reverse micelles composed of bis-2-ethylhexyl sulfosuccinate ( aot)14 . water interactions revealed by the noesy spectrum of ubiquitin in free solution , the protein encapsulated in aot reverse micelles displays literally dozens of protein - water interactions under similar temperature and ph conditions ( fig . dipolar exchange is identified by the opposite phase of the cross peaks in the orthogonal frames ( shown here as positive noe , negative roe ) , while direct hydrogen exchange with solvent gives rise to peaks of identical phase ( shown as positive in both spectra)2,3 . calibration of the noe obtained at the 40 ms mixing time used here placed it in the linear regime and corresponds to a maximum noe - detected distance of approximately 4.3 . more than half of detected amide hydrogen - water cross peaks involve amides that are over 4.3 from any labile side chain hydrogen , as indicated by of the ensemble of structures determined for encapsulated ubiquitin ( pdb 1g6j)14 . these can therefore be unequivocally assigned to direct noe interactions between hydration water and the protein . in addition , the hydrogen exchange chemistry catalyzed by hydroxide ion within the reverse micelle water pool is effectively slowed by two orders of magnitude as indicated by the ph dependence of the onset of line broadening due to amide hydrogen exchange ( supplementary fig . it should be noted that the pka values of side chain hydroxyls and primary amines are low enough that general base catalysis by buffer or even the head groups of aot is also a potential concern . however , the fact that lysine side chain amine groups are observed ( fig . 2 ) and that most side chain hydroxyls could be identified by direct noe ( supplementary fig . given the relative concentrations of protein and water , this restricts the hydrogen exchange rates to the 1 s or slower rate regime . the single labile side chain hydrogen whose chemical exchange may be faster than this rate is the hydroxyl of tyr-59 , which is anticipated to have a considerably lower pka . however , contributions from hydrogen exchange - mediated pathways are sufficiently minimized to allow quantitative interpretation of the vast majority of the noe and roe cross peak intensities . interpretation of the noe / roe ratio ( see below ) as directly representative of local hydration dynamics also requires that the contributions from long - range dipolar couplings be negligible . noesy spectra of highly deuterated ubiquitin lack any measurable cross peaks to organic solvent hydrogens , demonstrating that long - range interactions beyond the exterior of the reverse micelle are minimal . it is this long - range contribution in bulk aqueous solution that has been argued to provide the vast majority of the noe intensity to the water resonance10 . it is also of note that only a handful of amide hydrogens show noes with hydrogens of the surfactant headgroup . with the exception of gln 47 and gln 49 , the grouping of such interactions suggests that they arise from local close approach of the protein to the surfactant shell rather than general long - range dipolar exchange contributions . it is important to note that these regions of the surface are not dehydrated ( fig . the spatial geometry of the reverse micelle and encapsulated protein also cause the number of water molecules to increase roughly as the square of the distance from a solvent - exposed protein hydrogen in contrast to the cubic dependence seen in bulk solution ( supplementary fig . these fundamental differences in the physical parameters of the reverse micelle system versus bulk solution combine to largely eliminate contamination of short - range dipolar interactions between protein and hydration water by contribution from long - range couplings to solvent . in summary , the residence times of water on the surface of an encapsulated ubiquitin molecule are sufficiently increased , the effective rates of specific and general base catalysis of hydrogen exchange sufficiently decreased , and the potential contribution by long - range coupling to bulk solvent sufficiently reduced to allow confident and comprehensive site - resolved measurements of dipolar cross - relaxation between hydration waters and amide hydrogens of the protein . this enables the first site - resolved analysis of relative hydration water mobilities across an entire protein surface . a quantitative measure of hydration dynamics can be obtained from the ratio of the noe to roe cross - relaxation rates , noe and roe2,3 . here ratios of the intensities of the water cross peaks are used ( supplementary tables 1 and 2 ) . in the slow correlation time limit ( 1 ) , a rigidly bound water molecule would result in a noe / roe ratio of 0.5 ( ref . we find that a minority of the resolved protein - water correlations have ratios at this limit . previous relaxation studies indicate that the effective macromolecular tumbling time for these reverse micelle particles dissolved in liquid pentane is about 10 ns16 . the majority of detected hydration sites showed a range of noe / roe values between 0 and 0.5 indicating the presence of substantial motion of the hydration water on the surface of the protein and/or a somewhat shorter residence time3,31 . in previous studies of protein - water cross relaxation , it was unclear to what degree indirect magnetization exchange contributed to noe / roe ratios more positive than 0.5 . as measurements made in the reverse micelle are generally uncomplicated by indirect exchange effects , we conclude that the range of observed noe / roe ratios arise from a distribution of hydration water dynamics that reflect both local geometrical factors and the residence times of the individual water molecules on the surface of ubiquitin31 . approximately three fourths of the amide hydrogens within the noe - detection distance limit ( 4.3 ) of solvent showed measurable cross peaks to water . no cross peaks to water from buried amide hydrogens were detected , confirming the absence of spin - diffusion or appreciable penetration of water into the interior of the protein . a detailed view of the relative hydration dynamics of ubiquitin is shown in figure 3 where the amide hydrogens within noe distance of the molecular surface are represented as spheres and are color - coded according to dynamic classes . sites that have noe / roe ratios at the bound limit , representing the slowest regions of hydration dynamics , are colored dark blue . sites with ratios between 0.47 and 0.3 ( relatively slow or spatially restricted hydration water ) or between 0.3 and 0 ( relatively fast or spatially less restricted hydration water ) are colored cyan and yellow , respectively . sites that show no cross peaks to water and are therefore representative of the fastest hydration dynamics are colored green . a clear grouping of very slow and spatially restricted hydration is evident along the c - terminal tail of the protein ( top panel ) , and a cluster of very fast hydration sites is evident along the surface of the alpha helix ( center panel ) . clusters of intermediate dynamics are also visible along the mixed beta sheet ( bottom panel ) . these groupings illustrate the relative hydration dynamics essentially across the surface of the entire protein . importantly , a minority of solvent - exposed sites lacked detectable cross - relaxation to water . these sites , again clustered along the solvent - exposed ridge of the alpha helix , also did not show measurable cross peaks to surfactant hydrogens suggesting that long - lived interactions with the inner surface of the reverse micelle shell are absent and that the fastest and least spatially restricted hydration dynamics occur at these amide sites . reverse micelles are monodisperse ensembles of surfactant - encapsulated nanoscale water pools that result from spontaneous organization of a mixture of appropriate surfactants , water , and nonpolar solvent such as the alkanes ( fig . the size of the water pool can be tightly controlled by varying the molar ratio of water to surfactant ( water loading or wo ) . proteins can be encapsulated within the water pool of reverse micelles reproducibly and with high structural fidelity and at concentrations amenable to high - resolution solution nmr methods14,15 . when prepared in low viscosity solvents , the full arsenal of solution multi - dimensional heteronuclear nmr techniques becomes accessible16,17 . reverse micelles have served as a powerful tool for nmr - based studies of protein and nucleic acid structure and biophysics under a range of conditions14,16,1823 . for water loadings such as those used here ( w0 ~ 10 ) , the reverse micelle contains approximately one thousand water molecules on average24 and the re - orientational dynamics of encapsulated water is slowed relative to bulk water by approximately one order of magnitude25,26 . this suggested that the combined effects of the protein surface and nanoscale confinement could slow the water dynamics to allow efficient detection of hydration water - protein cross - relaxation . at the same time , if reorganization of water contributes to the rate limiting step , hydrogen exchange chemistry in the reverse micelle should also be somewhat slowed , potentially minimizing complications arising from hydrogen - exchange . finally , reverse micelles also provide an appropriate experimental condition to directly assess the contributions from long - range intermolecular dipolar interactions . ubiquitin is a small 76 residue protein comprised of a five stranded mixed beta sheet , a long alpha helix and a short 310 helix27 . we first examined the hydration of ubiquitin in free aqueous solution at ph 5 and 25 c using three - dimensional n - resolved noesy29 and roesy30 experiments ( fig . only a few amide - water weak cross peaks were observed , which is generally indicative of very short residence times for protein hydration water in bulk aqueous solution3 . at this ph and temperature , the exchange of even non - hydrogen bonded amide hydrogens with solvent is generally too slow to impact these experiments . however , every amide hydrogen - water cross peak observed in the free solution spectra involves an amide hydrogen that is within the distance detectable by the noe ( see below ) of a labile side chain hydrogen . chemical exchange of side chain hydrogens with solvent water followed by intramolecular noe produces cross peaks at the water resonance that are indistinguishable from direct protein - water noes2,3 . water noe correlations seen in bulk solution are the result of such exchange - relayed interactions5 . we have previously demonstrated that the structure of ubiquitin is unperturbed by appropriate encapsulation in reverse micelles composed of bis-2-ethylhexyl sulfosuccinate ( aot)14 . water interactions revealed by the noesy spectrum of ubiquitin in free solution , the protein encapsulated in aot reverse micelles displays literally dozens of protein - water interactions under similar temperature and ph conditions ( fig . dipolar exchange is identified by the opposite phase of the cross peaks in the orthogonal frames ( shown here as positive noe , negative roe ) , while direct hydrogen exchange with solvent gives rise to peaks of identical phase ( shown as positive in both spectra)2,3 . calibration of the noe obtained at the 40 ms mixing time used here placed it in the linear regime and corresponds to a maximum noe - detected distance of approximately 4.3 . more than half of detected amide hydrogen - water cross peaks involve amides that are over 4.3 from any labile side chain hydrogen , as indicated by of the ensemble of structures determined for encapsulated ubiquitin ( pdb 1g6j)14 . these can therefore be unequivocally assigned to direct noe interactions between hydration water and the protein . in addition , the hydrogen exchange chemistry catalyzed by hydroxide ion within the reverse micelle water pool is effectively slowed by two orders of magnitude as indicated by the ph dependence of the onset of line broadening due to amide hydrogen exchange ( supplementary fig . it should be noted that the pka values of side chain hydroxyls and primary amines are low enough that general base catalysis by buffer or even the head groups of aot is also a potential concern . however , the fact that lysine side chain amine groups are observed ( fig . 2 ) and that most side chain hydroxyls could be identified by direct noe ( supplementary fig . given the relative concentrations of protein and water , this restricts the hydrogen exchange rates to the 1 s or slower rate regime . the single labile side chain hydrogen whose chemical exchange may be faster than this rate is the hydroxyl of tyr-59 , which is anticipated to have a considerably lower pka . however , contributions from hydrogen exchange - mediated pathways are sufficiently minimized to allow quantitative interpretation of the vast majority of the noe and roe cross peak intensities . interpretation of the noe / roe ratio ( see below ) as directly representative of local hydration dynamics also requires that the contributions from long - range dipolar couplings be negligible . noesy spectra of highly deuterated ubiquitin lack any measurable cross peaks to organic solvent hydrogens , demonstrating that long - range interactions beyond the exterior of the reverse micelle are minimal . it is this long - range contribution in bulk aqueous solution that has been argued to provide the vast majority of the noe intensity to the water resonance10 . it is also of note that only a handful of amide hydrogens show noes with hydrogens of the surfactant headgroup . with the exception of gln 47 and gln 49 , the grouping of such interactions suggests that they arise from local close approach of the protein to the surfactant shell rather than general long - range dipolar exchange contributions . it is important to note that these regions of the surface are not dehydrated ( fig . the spatial geometry of the reverse micelle and encapsulated protein also cause the number of water molecules to increase roughly as the square of the distance from a solvent - exposed protein hydrogen in contrast to the cubic dependence seen in bulk solution ( supplementary fig . these fundamental differences in the physical parameters of the reverse micelle system versus bulk solution combine to largely eliminate contamination of short - range dipolar interactions between protein and hydration water by contribution from long - range couplings to solvent . in summary , the residence times of water on the surface of an encapsulated ubiquitin molecule are sufficiently increased , the effective rates of specific and general base catalysis of hydrogen exchange sufficiently decreased , and the potential contribution by long - range coupling to bulk solvent sufficiently reduced to allow confident and comprehensive site - resolved measurements of dipolar cross - relaxation between hydration waters and amide hydrogens of the protein . this enables the first site - resolved analysis of relative hydration water mobilities across an entire protein surface . a quantitative measure of hydration dynamics can be obtained from the ratio of the noe to roe cross - relaxation rates , noe and roe2,3 . here ratios of the intensities of the water cross peaks are used ( supplementary tables 1 and 2 ) . in the slow correlation time limit ( 1 ) , a rigidly bound water molecule would result in a noe / roe ratio of 0.5 ( ref . we find that a minority of the resolved protein - water correlations have ratios at this limit . previous relaxation studies indicate that the effective macromolecular tumbling time for these reverse micelle particles dissolved in liquid pentane is about 10 ns16 . the majority of detected hydration sites showed a range of noe / roe values between 0 and 0.5 indicating the presence of substantial motion of the hydration water on the surface of the protein and/or a somewhat shorter residence time3,31 . in previous studies of protein - water cross relaxation , it was unclear to what degree indirect magnetization exchange contributed to noe / roe ratios more positive than 0.5 . as measurements made in the reverse micelle are generally uncomplicated by indirect exchange effects , we conclude that the range of observed noe / roe ratios arise from a distribution of hydration water dynamics that reflect both local geometrical factors and the residence times of the individual water molecules on the surface of ubiquitin31 . approximately three fourths of the amide hydrogens within the noe - detection distance limit ( 4.3 ) of solvent showed measurable cross peaks to water . no cross peaks to water from buried amide hydrogens were detected , confirming the absence of spin - diffusion or appreciable penetration of water into the interior of the protein . a detailed view of the relative hydration dynamics of ubiquitin is shown in figure 3 where the amide hydrogens within noe distance of the molecular surface are represented as spheres and are color - coded according to dynamic classes . sites that have noe / roe ratios at the bound limit , representing the slowest regions of hydration dynamics , are colored dark blue . sites with ratios between 0.47 and 0.3 ( relatively slow or spatially restricted hydration water ) or between 0.3 and 0 ( relatively fast or spatially less restricted hydration water ) are colored cyan and yellow , respectively . sites that show no cross peaks to water and are therefore representative of the fastest hydration dynamics are colored green . a clear grouping of very slow and spatially restricted hydration is evident along the c - terminal tail of the protein ( top panel ) , and a cluster of very fast hydration sites is evident along the surface of the alpha helix ( center panel ) . clusters of intermediate dynamics are also visible along the mixed beta sheet ( bottom panel ) . these groupings illustrate the relative hydration dynamics essentially across the surface of the entire protein . importantly , a minority of solvent - exposed sites lacked detectable cross - relaxation to water . these sites , again clustered along the solvent - exposed ridge of the alpha helix , also did not show measurable cross peaks to surfactant hydrogens suggesting that long - lived interactions with the inner surface of the reverse micelle shell are absent and that the fastest and least spatially restricted hydration dynamics occur at these amide sites . until now , experimental efforts to develop a site resolved understanding of the hydration layer around proteins have relied largely on crystallographic data . because most protein crystal structures are heavily hydrated and contain complex ensembles of solvent , the general view seems to be that the environment in protein crystals should be representative of the crowded conditions within the cell and that therefore crystallographic water locations should be representative of the native hydration behavior of the protein . extensive analysis of crystallographic waters has been combined with simulation data in several systems to propose a picture of the molecular nature of the hydration layer32 . two crystal structures for wild type human ubiquitin are available ( pdb codes 1ubq and 1ubi)27,33 . only about half of the defined waters in the two crystal structures were conserved to within 1 between the structures even though the proteins were crystallized in the same space group under almost identical conditions . furthermore , there is little correlation between the waters detected by solution nmr and the locations and occupancies of the crystallographic waters in these crystal structures ( supplementary fig . 4 ) . to illustrate the degree of correspondence between the long - lived hydration waters detected by nmr to the locations of the defined waters common to both crystal structures of ubiquitin , a color - coded ribbon diagram of 1ubq is shown in figure 4 . orange backbone corresponds to amides that are buried deeply enough to be outside noe detection distance from solvent . regions of the backbone that have solvent - accessible amide hydrogens but did not show cross peaks to water are colored green . as mentioned above , these sites must have only short - lived interactions with hydration water . portions of the backbone that have nmr - detected hydration water and also have water common to both crystal structures that are within noe distance are colored blue , as are the corresponding waters . crystallographic waters that do not have long - lived counterparts in the nmr hydration data are colored green . finally , yellow regions correspond to those amide hydrogens that have nmr - detected hydration water but have no counterpart in the waters common to both crystal structures . interestingly , only ~60% of the crystallographic waters that are common to both crystal structures appear within the 4.3 noe detection distance of sites where nmr - detected hydration waters are long - lived . in addition , more than half of the nmr - detected long - lived protein - water interactions involve amide hydrogens that are outside noe distance from the nearest conserved crystallographic water molecule . additionally , we have found no correlation between the noe / roe values for the nmr - detected sites of long - lived hydration and the location of the nearest conserved crystallographic water . in the case of ubiquitin , there seems to be limited correlation between the locations of the defined water molecules in the two crystal structures and the nmr - detected hydration waters of encapsulated ubiquitin . indeed , magnetic relaxation dispersion experiments have suggested one very long - lived water molecule is present in ubiquitin , which on the basis of the crystal structure ( 1ubq ) was identified with a water molecule ( water 28 ) localized near the amide nh of leu 43 ( ref . though this water is illuminated by noes in encapsulated ubiquitin , its residence time is short enough to cause complete chemical shift averaging with the bulk water resonance . in contrast , encapsulated ubiquitin shows a very long lived water molecule that is localized to a relatively deep surface pocket and is highlighted by short distance noes between the amide hydrogens of val 69 and lys 6 and a resonance with a chemical shift ( 5.4 p.p.m . ) resolved from the water resonance ( 4.32 p.p.m . ) 2 ) . these amide hydrogens are not within the noe detection distance of any hydroxyl group and we therefore identify this water , which is not resolved in the crystal structure , as the long - lived water molecule suggested by the magnetic resonance dispersion experiments . interestingly , this water resides in a pocket that is lined with hydrophobic side chains , which may account for the lack of ordered crystallographic water at this site35 . in recent years , there has been an increasing appreciation of the inherent complexity of the cellular context in which proteins must function36 . it is now well known that cells are packed with macromolecular surfaces that confine proteins to nanometer - scale volumes . confinement on this length scale can potentially alter a number of fundamental properties of proteins36 and creates an intracellular hydration environment that is potentially highly heterogeneous and distinct from bulk water1 . reverse micelle encapsulation provides a convenient way to undertake detailed studies of the effects of confinement on protein hydration , folding and stability and dynamics at the atomic - scale using solution nmr methods20,37,38 . here we have found a previously unseen range of hydration behavior across the surface of the protein . this represents an exciting alternate view for the ongoing effort to unravel the interplay between proteins and solvent that is so vital to living systems . in recent years , there has been an increasing appreciation of the inherent complexity of the cellular context in which proteins must function36 . it is now well known that cells are packed with macromolecular surfaces that confine proteins to nanometer - scale volumes . confinement on this length scale can potentially alter a number of fundamental properties of proteins36 and creates an intracellular hydration environment that is potentially highly heterogeneous and distinct from bulk water1 . reverse micelle encapsulation provides a convenient way to undertake detailed studies of the effects of confinement on protein hydration , folding and stability and dynamics at the atomic - scale using solution nmr methods20,37,38 . here we have found a previously unseen range of hydration behavior across the surface of the protein . this represents an exciting alternate view for the ongoing effort to unravel the interplay between proteins and solvent that is so vital to living systems . methods and any associated references are available in the online version of the paper at http://www.nature.com / nsmb/.

the interactions of biological macromolecules with water are fundamental to their structure , dynamics and function . historically , characterization of the location and residence times of hydration waters of proteins in solution has been quite difficult . confinement within the nanoscale interior of a reverse micelle slows water dynamics , allowing detection of global protein - water interactions using nuclear magnetic resonance techniques . complications that normally arise from hydrogen exchange and long - range dipolar coupling are overcome by the nature of the reverse micelle medium . characterization of the hydration of ubiquitin demonstrates that encapsulation within a reverse micelle allows detection of dozens of hydration waters . comparison of nuclear overhauser effects obtained in the laboratory and rotating frames indicate a considerable range of hydration water dynamics is present on the protein surface . in addition , an unprecedented clustering of different hydration dynamic classes of sites is evident .

RESULTS Encapsulation enables detection of protein hydration water The dynamical character of the protein-water interface DISCUSSION Protein solvation and nanoscale confinement METHODS Supplementary Material

the size of the water pool can be tightly controlled by varying the molar ratio of water to surfactant ( water loading or wo ) . proteins can be encapsulated within the water pool of reverse micelles reproducibly and with high structural fidelity and at concentrations amenable to high - resolution solution nmr methods14,15 . for water loadings such as those used here ( w0 ~ 10 ) , the reverse micelle contains approximately one thousand water molecules on average24 and the re - orientational dynamics of encapsulated water is slowed relative to bulk water by approximately one order of magnitude25,26 . this suggested that the combined effects of the protein surface and nanoscale confinement could slow the water dynamics to allow efficient detection of hydration water - protein cross - relaxation . at the same time , if reorganization of water contributes to the rate limiting step , hydrogen exchange chemistry in the reverse micelle should also be somewhat slowed , potentially minimizing complications arising from hydrogen - exchange . finally , reverse micelles also provide an appropriate experimental condition to directly assess the contributions from long - range intermolecular dipolar interactions . ubiquitin is a small 76 residue protein comprised of a five stranded mixed beta sheet , a long alpha helix and a short 310 helix27 . we first examined the hydration of ubiquitin in free aqueous solution at ph 5 and 25 c using three - dimensional n - resolved noesy29 and roesy30 experiments ( fig . only a few amide - water weak cross peaks were observed , which is generally indicative of very short residence times for protein hydration water in bulk aqueous solution3 . however , every amide hydrogen - water cross peak observed in the free solution spectra involves an amide hydrogen that is within the distance detectable by the noe ( see below ) of a labile side chain hydrogen . chemical exchange of side chain hydrogens with solvent water followed by intramolecular noe produces cross peaks at the water resonance that are indistinguishable from direct protein - water noes2,3 . water interactions revealed by the noesy spectrum of ubiquitin in free solution , the protein encapsulated in aot reverse micelles displays literally dozens of protein - water interactions under similar temperature and ph conditions ( fig . dipolar exchange is identified by the opposite phase of the cross peaks in the orthogonal frames ( shown here as positive noe , negative roe ) , while direct hydrogen exchange with solvent gives rise to peaks of identical phase ( shown as positive in both spectra)2,3 . calibration of the noe obtained at the 40 ms mixing time used here placed it in the linear regime and corresponds to a maximum noe - detected distance of approximately 4.3 . more than half of detected amide hydrogen - water cross peaks involve amides that are over 4.3 from any labile side chain hydrogen , as indicated by of the ensemble of structures determined for encapsulated ubiquitin ( pdb 1g6j)14 . these can therefore be unequivocally assigned to direct noe interactions between hydration water and the protein . in addition , the hydrogen exchange chemistry catalyzed by hydroxide ion within the reverse micelle water pool is effectively slowed by two orders of magnitude as indicated by the ph dependence of the onset of line broadening due to amide hydrogen exchange ( supplementary fig . however , contributions from hydrogen exchange - mediated pathways are sufficiently minimized to allow quantitative interpretation of the vast majority of the noe and roe cross peak intensities . interpretation of the noe / roe ratio ( see below ) as directly representative of local hydration dynamics also requires that the contributions from long - range dipolar couplings be negligible . noesy spectra of highly deuterated ubiquitin lack any measurable cross peaks to organic solvent hydrogens , demonstrating that long - range interactions beyond the exterior of the reverse micelle are minimal . it is this long - range contribution in bulk aqueous solution that has been argued to provide the vast majority of the noe intensity to the water resonance10 . it is also of note that only a handful of amide hydrogens show noes with hydrogens of the surfactant headgroup . with the exception of gln 47 and gln 49 , the grouping of such interactions suggests that they arise from local close approach of the protein to the surfactant shell rather than general long - range dipolar exchange contributions . it is important to note that these regions of the surface are not dehydrated ( fig . the spatial geometry of the reverse micelle and encapsulated protein also cause the number of water molecules to increase roughly as the square of the distance from a solvent - exposed protein hydrogen in contrast to the cubic dependence seen in bulk solution ( supplementary fig . these fundamental differences in the physical parameters of the reverse micelle system versus bulk solution combine to largely eliminate contamination of short - range dipolar interactions between protein and hydration water by contribution from long - range couplings to solvent . in summary , the residence times of water on the surface of an encapsulated ubiquitin molecule are sufficiently increased , the effective rates of specific and general base catalysis of hydrogen exchange sufficiently decreased , and the potential contribution by long - range coupling to bulk solvent sufficiently reduced to allow confident and comprehensive site - resolved measurements of dipolar cross - relaxation between hydration waters and amide hydrogens of the protein . this enables the first site - resolved analysis of relative hydration water mobilities across an entire protein surface . a quantitative measure of hydration dynamics can be obtained from the ratio of the noe to roe cross - relaxation rates , noe and roe2,3 . we find that a minority of the resolved protein - water correlations have ratios at this limit . previous relaxation studies indicate that the effective macromolecular tumbling time for these reverse micelle particles dissolved in liquid pentane is about 10 ns16 . the majority of detected hydration sites showed a range of noe / roe values between 0 and 0.5 indicating the presence of substantial motion of the hydration water on the surface of the protein and/or a somewhat shorter residence time3,31 . in previous studies of protein - water cross relaxation , it was unclear to what degree indirect magnetization exchange contributed to noe / roe ratios more positive than 0.5 . as measurements made in the reverse micelle are generally uncomplicated by indirect exchange effects , we conclude that the range of observed noe / roe ratios arise from a distribution of hydration water dynamics that reflect both local geometrical factors and the residence times of the individual water molecules on the surface of ubiquitin31 . approximately three fourths of the amide hydrogens within the noe - detection distance limit ( 4.3 ) of solvent showed measurable cross peaks to water . no cross peaks to water from buried amide hydrogens were detected , confirming the absence of spin - diffusion or appreciable penetration of water into the interior of the protein . a detailed view of the relative hydration dynamics of ubiquitin is shown in figure 3 where the amide hydrogens within noe distance of the molecular surface are represented as spheres and are color - coded according to dynamic classes . sites that have noe / roe ratios at the bound limit , representing the slowest regions of hydration dynamics , are colored dark blue . sites with ratios between 0.47 and 0.3 ( relatively slow or spatially restricted hydration water ) or between 0.3 and 0 ( relatively fast or spatially less restricted hydration water ) are colored cyan and yellow , respectively . a clear grouping of very slow and spatially restricted hydration is evident along the c - terminal tail of the protein ( top panel ) , and a cluster of very fast hydration sites is evident along the surface of the alpha helix ( center panel ) . these groupings illustrate the relative hydration dynamics essentially across the surface of the entire protein . these sites , again clustered along the solvent - exposed ridge of the alpha helix , also did not show measurable cross peaks to surfactant hydrogens suggesting that long - lived interactions with the inner surface of the reverse micelle shell are absent and that the fastest and least spatially restricted hydration dynamics occur at these amide sites . proteins can be encapsulated within the water pool of reverse micelles reproducibly and with high structural fidelity and at concentrations amenable to high - resolution solution nmr methods14,15 . reverse micelles have served as a powerful tool for nmr - based studies of protein and nucleic acid structure and biophysics under a range of conditions14,16,1823 . for water loadings such as those used here ( w0 ~ 10 ) , the reverse micelle contains approximately one thousand water molecules on average24 and the re - orientational dynamics of encapsulated water is slowed relative to bulk water by approximately one order of magnitude25,26 . this suggested that the combined effects of the protein surface and nanoscale confinement could slow the water dynamics to allow efficient detection of hydration water - protein cross - relaxation . at the same time , if reorganization of water contributes to the rate limiting step , hydrogen exchange chemistry in the reverse micelle should also be somewhat slowed , potentially minimizing complications arising from hydrogen - exchange . finally , reverse micelles also provide an appropriate experimental condition to directly assess the contributions from long - range intermolecular dipolar interactions . we first examined the hydration of ubiquitin in free aqueous solution at ph 5 and 25 c using three - dimensional n - resolved noesy29 and roesy30 experiments ( fig . only a few amide - water weak cross peaks were observed , which is generally indicative of very short residence times for protein hydration water in bulk aqueous solution3 . however , every amide hydrogen - water cross peak observed in the free solution spectra involves an amide hydrogen that is within the distance detectable by the noe ( see below ) of a labile side chain hydrogen . chemical exchange of side chain hydrogens with solvent water followed by intramolecular noe produces cross peaks at the water resonance that are indistinguishable from direct protein - water noes2,3 . we have previously demonstrated that the structure of ubiquitin is unperturbed by appropriate encapsulation in reverse micelles composed of bis-2-ethylhexyl sulfosuccinate ( aot)14 . water interactions revealed by the noesy spectrum of ubiquitin in free solution , the protein encapsulated in aot reverse micelles displays literally dozens of protein - water interactions under similar temperature and ph conditions ( fig . dipolar exchange is identified by the opposite phase of the cross peaks in the orthogonal frames ( shown here as positive noe , negative roe ) , while direct hydrogen exchange with solvent gives rise to peaks of identical phase ( shown as positive in both spectra)2,3 . calibration of the noe obtained at the 40 ms mixing time used here placed it in the linear regime and corresponds to a maximum noe - detected distance of approximately 4.3 . more than half of detected amide hydrogen - water cross peaks involve amides that are over 4.3 from any labile side chain hydrogen , as indicated by of the ensemble of structures determined for encapsulated ubiquitin ( pdb 1g6j)14 . these can therefore be unequivocally assigned to direct noe interactions between hydration water and the protein . in addition , the hydrogen exchange chemistry catalyzed by hydroxide ion within the reverse micelle water pool is effectively slowed by two orders of magnitude as indicated by the ph dependence of the onset of line broadening due to amide hydrogen exchange ( supplementary fig . given the relative concentrations of protein and water , this restricts the hydrogen exchange rates to the 1 s or slower rate regime . however , contributions from hydrogen exchange - mediated pathways are sufficiently minimized to allow quantitative interpretation of the vast majority of the noe and roe cross peak intensities . interpretation of the noe / roe ratio ( see below ) as directly representative of local hydration dynamics also requires that the contributions from long - range dipolar couplings be negligible . noesy spectra of highly deuterated ubiquitin lack any measurable cross peaks to organic solvent hydrogens , demonstrating that long - range interactions beyond the exterior of the reverse micelle are minimal . it is this long - range contribution in bulk aqueous solution that has been argued to provide the vast majority of the noe intensity to the water resonance10 . with the exception of gln 47 and gln 49 , the grouping of such interactions suggests that they arise from local close approach of the protein to the surfactant shell rather than general long - range dipolar exchange contributions . the spatial geometry of the reverse micelle and encapsulated protein also cause the number of water molecules to increase roughly as the square of the distance from a solvent - exposed protein hydrogen in contrast to the cubic dependence seen in bulk solution ( supplementary fig . these fundamental differences in the physical parameters of the reverse micelle system versus bulk solution combine to largely eliminate contamination of short - range dipolar interactions between protein and hydration water by contribution from long - range couplings to solvent . in summary , the residence times of water on the surface of an encapsulated ubiquitin molecule are sufficiently increased , the effective rates of specific and general base catalysis of hydrogen exchange sufficiently decreased , and the potential contribution by long - range coupling to bulk solvent sufficiently reduced to allow confident and comprehensive site - resolved measurements of dipolar cross - relaxation between hydration waters and amide hydrogens of the protein . this enables the first site - resolved analysis of relative hydration water mobilities across an entire protein surface . a quantitative measure of hydration dynamics can be obtained from the ratio of the noe to roe cross - relaxation rates , noe and roe2,3 . here ratios of the intensities of the water cross peaks are used ( supplementary tables 1 and 2 ) . we find that a minority of the resolved protein - water correlations have ratios at this limit . the majority of detected hydration sites showed a range of noe / roe values between 0 and 0.5 indicating the presence of substantial motion of the hydration water on the surface of the protein and/or a somewhat shorter residence time3,31 . in previous studies of protein - water cross relaxation , it was unclear to what degree indirect magnetization exchange contributed to noe / roe ratios more positive than 0.5 . as measurements made in the reverse micelle are generally uncomplicated by indirect exchange effects , we conclude that the range of observed noe / roe ratios arise from a distribution of hydration water dynamics that reflect both local geometrical factors and the residence times of the individual water molecules on the surface of ubiquitin31 . approximately three fourths of the amide hydrogens within the noe - detection distance limit ( 4.3 ) of solvent showed measurable cross peaks to water . no cross peaks to water from buried amide hydrogens were detected , confirming the absence of spin - diffusion or appreciable penetration of water into the interior of the protein . a detailed view of the relative hydration dynamics of ubiquitin is shown in figure 3 where the amide hydrogens within noe distance of the molecular surface are represented as spheres and are color - coded according to dynamic classes . sites that have noe / roe ratios at the bound limit , representing the slowest regions of hydration dynamics , are colored dark blue . a clear grouping of very slow and spatially restricted hydration is evident along the c - terminal tail of the protein ( top panel ) , and a cluster of very fast hydration sites is evident along the surface of the alpha helix ( center panel ) . these sites , again clustered along the solvent - exposed ridge of the alpha helix , also did not show measurable cross peaks to surfactant hydrogens suggesting that long - lived interactions with the inner surface of the reverse micelle shell are absent and that the fastest and least spatially restricted hydration dynamics occur at these amide sites . until now , experimental efforts to develop a site resolved understanding of the hydration layer around proteins have relied largely on crystallographic data . because most protein crystal structures are heavily hydrated and contain complex ensembles of solvent , the general view seems to be that the environment in protein crystals should be representative of the crowded conditions within the cell and that therefore crystallographic water locations should be representative of the native hydration behavior of the protein . extensive analysis of crystallographic waters has been combined with simulation data in several systems to propose a picture of the molecular nature of the hydration layer32 . only about half of the defined waters in the two crystal structures were conserved to within 1 between the structures even though the proteins were crystallized in the same space group under almost identical conditions . to illustrate the degree of correspondence between the long - lived hydration waters detected by nmr to the locations of the defined waters common to both crystal structures of ubiquitin , a color - coded ribbon diagram of 1ubq is shown in figure 4 . regions of the backbone that have solvent - accessible amide hydrogens but did not show cross peaks to water are colored green . portions of the backbone that have nmr - detected hydration water and also have water common to both crystal structures that are within noe distance are colored blue , as are the corresponding waters . crystallographic waters that do not have long - lived counterparts in the nmr hydration data are colored green . finally , yellow regions correspond to those amide hydrogens that have nmr - detected hydration water but have no counterpart in the waters common to both crystal structures . interestingly , only ~60% of the crystallographic waters that are common to both crystal structures appear within the 4.3 noe detection distance of sites where nmr - detected hydration waters are long - lived . in addition , more than half of the nmr - detected long - lived protein - water interactions involve amide hydrogens that are outside noe distance from the nearest conserved crystallographic water molecule . additionally , we have found no correlation between the noe / roe values for the nmr - detected sites of long - lived hydration and the location of the nearest conserved crystallographic water . in the case of ubiquitin , there seems to be limited correlation between the locations of the defined water molecules in the two crystal structures and the nmr - detected hydration waters of encapsulated ubiquitin . indeed , magnetic relaxation dispersion experiments have suggested one very long - lived water molecule is present in ubiquitin , which on the basis of the crystal structure ( 1ubq ) was identified with a water molecule ( water 28 ) localized near the amide nh of leu 43 ( ref . these amide hydrogens are not within the noe detection distance of any hydroxyl group and we therefore identify this water , which is not resolved in the crystal structure , as the long - lived water molecule suggested by the magnetic resonance dispersion experiments . in recent years , there has been an increasing appreciation of the inherent complexity of the cellular context in which proteins must function36 . reverse micelle encapsulation provides a convenient way to undertake detailed studies of the effects of confinement on protein hydration , folding and stability and dynamics at the atomic - scale using solution nmr methods20,37,38 . here we have found a previously unseen range of hydration behavior across the surface of the protein . in recent years , there has been an increasing appreciation of the inherent complexity of the cellular context in which proteins must function36 . reverse micelle encapsulation provides a convenient way to undertake detailed studies of the effects of confinement on protein hydration , folding and stability and dynamics at the atomic - scale using solution nmr methods20,37,38 . here we have found a previously unseen range of hydration behavior across the surface of the protein . methods and any associated references are available in the online version of the paper at http://www.nature.com / nsmb/.

latinos are the largest and most rapidly growing population group in the united states . although rates of childhood obesity are still high for the general child population , children of low - income , minority families are at a particularly high risk of overweight and obesity . the continuing rise of obesity within minority and immigrant populations , particularly latinos , remains a pressing public health concern with an immediate need for early prevention . children living in the united states live in a society that has changed dramatically since the obesity epidemic first developed . changes in family structure , gender roles , and families ' needs for economic security have increased the demand and reliance on child care for children at increasingly younger ages . in addition , welfare reform laws requiring employment have increased the number of employed low - income parents and have contributed to an increase in the number of children enrolled in child care programs . in 2012 , 68% of women with children under the age of 6 were either working or looking for work , and almost 11 million preschool - aged children received some form of child care while their mothers worked [ 2 , 3 ] . the child care setting is an important social environment that potentially influences the development of children 's early dietary and physical activity habits and consequently contributes to the development of child overweight [ 46 ] . given parents increasing reliance on child care settings for their children at continually younger ages , these settings are likely important venues for the implementation of programs and policies to help children develop healthful eating and physical activity habits [ 2 , 4 ] . child care providers , like parents , help establish and reinforce early healthful eating and physical activity habits among young children , and can be key players in preventing childhood obesity by developing a child care environment that fosters healthful eating and physical activity behaviors among children [ 79 ] . child care provider 's knowledge of nutrition and physical activity , the selection of food and meals , structure within their day care , and their own modeling of behaviors are all influential in young children 's development of lifelong habits that contribute to normal weight or to overweight and obesity [ 1012 ] . in fact , research suggests that child care providers may be more influential than or equally as important as parents in shaping food preferences of young children [ 13 , 14 ] . child care settings may help establish and reinforce children 's eating and physical activity habits [ 4 , 14 , 15 ] . furthermore , studies have found that latino parents who send their children to child care believe these settings are instrumental in shaping and reinforcing the eating and physical activity of their children . however , despite the growing number of studies and interventions targeting child care settings and given that more than 1.6 million children attend fcchs , there is limited research examining fcchs and their influence on the development of healthful eating and physical activity habits in latino preschool - aged children , a group at increased risk of obesity . in massachusetts , the setting for this study , the majority of children enrolled in fcchs are from minority backgrounds , including a high percentage of latino children , and most latino children attend fcchs operated by latino staff . fcchs are licensed by the massachusetts department of early education and care ( ma - eec ) . fcch providers are required to ( 1 ) have a plan for communicating with parents / guardians through various communication channels ( e.g. , handbooks , newsletters , and notes ) ; ( 2 ) provide opportunities for outdoor and indoor active play ; ( 3 ) have adequate indoor and outdoor space and equipment for active and safe play ; and ( 4 ) provide opportunities for children to develop gross and fine motor skills . state regulations do not specify the amount , frequency , and type of physical activity or regulate television use . additionally , fcch providers may be eligible to participate in the child and adult care food program ( cacfp ) , which requires that cacfp participants follow usda / cacfp guidelines for menus and feeding practices . given the growing importance of fcchs in serving a large proportion of minority , low - income children , further research is needed to examine the role that fcch providers play in establishing and reinforcing children 's early healthful eating and physical activity habits and consequently preventing childhood obesity . moreover , given the high rates of obesity among latino preschoolers , and the fact that latino families use preschools and day care centers much less than those of other ethnic groups , apparently because of cultural preferences for family - like care , there is a need to understand how family child care settings influence the development of eating , physical activity and sedentary behaviors associated with childhood obesity . preventing obesity in latino family child care homes is a multicomponent study employing qualitative methods to explore influences on eating habits , physical activity , sedentary behaviors , and ultimately risk of obesity among latino preschool - aged children attending fcchs in massachusetts . additionally , the study assessed practices , policies , and regulations of fcchs that may be associated with risk of childhood obesity among latino preschool - aged children . this current paper focuses on the results of the qualitative research examining latino fcch providers ' beliefs and practices related to nutrition and feeding , and physical activity and sedentary behaviors among low - income preschool children . we worked with ma department of early education and care ( eec ) , which develops licensing regulations and requirements for childcare providers and supports training for early educators , and the child care circuit , a nonprofit organization providing child care referrals , training , and parent and provider services , to identify cities in four regions of the state ( north shore , greater boston , central ma , and western ma ) that have a large number of fcchs run by latino providers . ma - ecc and the child care circuit , then identified two agencies that work directly with fcch providers , cacfp , and family child care systems who compiled a list of all currently licensed latino fcch providers . from this list , we randomly selected 22 names per region of the state , with a goal of recruiting 812 individuals to participate in one focus group session in each region . we mailed all selected providers a recruitment flyer in spanish that included a phone number that interested providers could call to obtain more information and/or express interest in participation . interested providers who called spoke with a native spanish speaker who explained the study and its purpose . a confirmatory / reminder phone call was made one to two days before the scheduled focus group session . focus group discussions were held in meeting rooms of public libraries , and all participants provided written informed consent . a native spanish speaker trained in qualitative research methods conducted all focus groups in spanish using a semistructured discussion guide including open - ended questions and probes . focus group discussions were audio - taped with oral consent of participants . at completion of the focus group the study protocol was approved by the internal review board at the harvard school of public health . the focus group guide explored ( 1 ) providers ' perceptions , attitudes and practices related to nutrition , and physical activity and sedentary behaviors ; ( 2 ) influences of fcch characteristics on children 's eating and physical activity behaviors ; ( 3 ) fcch providers practices related to nutrition and physical activity ; ( 4 ) educational activities offered by state and local agencies related to nutrition and physical activity and sedentary behaviors ; ( 5 ) communication between fcch providers and parents about fcch practices and policies related to nutrition and physical activity and sedentary behaviors ; and ( 6 ) barriers fcch providers face in providing an environment conducive to healthful eating and physical activity behaviors . see the following for sample of questions used in focus group discussions with latino family child care providers ( fccps ) . fcchs ' beliefs , attitudes , and practices related to child feeding practices please describe your routine , or plan , for meals that you give the children . ( e.g. , describe how you use menus , what times you serve meals ? ) please tell me about your plan or routine for giving snacks to the children . for example , frequency that you give snacks to children , and so forth . for example , books , websites , agencies , other providers , and so forth . what things affect your choice of foods that you typically serve ? please give a few of the most important reasons for your food choices . for example , cost , rules , or guidelines from food program , cultural values and tradition , good for health , family member 's advice , easy to prepare , and so forth . how much food you think a child should typically eat at a meal ? ( e.g. , what is too much , not enough)what are you and the children doing during meals ? ( e.g. , sitting together at a table , watching tv or videos during meal ) please describe your routine , or plan , for meals that you give the children . ( e.g. , describe how you use menus , what times you serve meals ? ) please tell me about your plan or routine for giving snacks to the children . for example , frequency that you give snacks to children , and so forth . please tell me about your plan or routine for giving snacks to the children . for example , frequency that you give snacks to children , and so forth . for example , books , websites , agencies , other providers , and so forth . for example , books , websites , agencies , other providers , and so forth . what things affect your choice of foods that you typically serve ? please give a few of the most important reasons for your food choices . for example , cost , rules , or guidelines from food program , cultural values and tradition , good for health , family member 's advice , easy to prepare , and so forth . for example , cost , rules , or guidelines from food program , cultural values and tradition , good for health , family member 's advice , easy to prepare , and so forth . how much food you think a child should typically eat at a meal ? ( e.g. , what is too much , not enough ) what are you and the children doing during meals ? ( e.g. , sitting together at a table , watching tv or videos during meal ) fccps ' beliefs , attitudes , and practices related to physical activity what are your ideas and thoughts about physical activity for children ( in general)?tell me about the routine you have for children to get physical activity while they are at the daycare . for example , how much time for pa daily , time of day , and so forth ? why do you have this routine?what kinds of ways are children active outdoors on a typical day ? for example , do they play games and use equipment ? for example , do they play games , use equipment , and use videos or tv ? if you wanted to get more information about how much pa children need or get ideas about how to help kids be active , how would you do this ? what are your ideas and thoughts about physical activity for children ( in general ) ? tell me about the routine you have for children to get physical activity while they are at the daycare . for example , how much time for pa daily , time of day , and so forth ? for example , how much time for pa daily , time of day , and so forth ? why do you have this routine ? for example , do they play games and use equipment ? for example , do they play games and use equipment ? for example , do they play games , use equipment , and use videos or tv ? for example , do they play games , use equipment , and use videos or tv ? if you wanted to get more information about how much pa children need or get ideas about how to help kids be active , how would you do this ? we know that all children like to watch cartoons and other shows like dora the explorer . they also enjoy playing on a computer , cell phone , ds toys , and video and nintendo games . we call these screen time.what are your thoughts in general on screen time for children aged 2 to 5?what rules or routines do you have about screen time?what do parents think about these rules ? what are your thoughts in general on screen time for children aged 2 to 5 ? providers ' beliefs , attitudes , and practices related to communication with parents how do you typically let parents know what their child did while at care , especially what they had to eat and what they did for physical activity?what , if anything , do you discuss with parents about their child 's weight?who would you talk to for advice if you thought a child was too thin or weighed too much ? ( family member , doctor , wic staff , other ) how do you typically let parents know what their child did while at care , especially what they had to eat and what they did for physical activity ? what , if anything , do you discuss with parents about their child 's weight ? who would you talk to for advice if you thought a child was too thin or weighed too much ? ( family member , doctor , wic staff , other ) audiotaped discussions were transcribed in spanish and then translated into english by a bilingual consultant . the analysis plan , which used a content analysis approach , included an initial review of all translated transcripts by two members of the study team who also developed a codebook . two coders trained in qualitative methods independently read and analyzed transcripts to identify salient convergent themes . all transcripts were then coded based on broad categories of the areas of inquiry of the focus group guide . in total , 44 providers ( 41 females , 3 males ) , all of whom are self - identified as hispanic / latino , participated in six focus groups . about one - third of participants ( n = 14 ) had graduated from high school or earned their ged , and close to forty percent ( n = 17 ) had attended some college . most ( n = 41 , 93% ) had several to up to 25 years of experience running fcchs . data analysis identified key themes related to nutrition and feeding practices , physical activity , and sedentary behaviors at latino fcchs . providers reported that foods served for breakfast often include 1% milk , fruit , yogurt , cereal , oatmeal , pancakes , and freshly squeezed or 100% juice . prohibited foods included juices that are not 100% juice , soda , hot dogs , and fried foods . providers indicated that they use guides from food programs ( e.g. , usda ) to determine portion sizes . some mentioned using measuring cups to determine portion sizes while others spoke of using informal tools such as small plates . many providers mentioned basing portion sizes on children 's ages , which they believed to be an important factor in determining the quantity of food a child should eat . providers believed that children eat a healthier diet in their fcchs than they do at home and that parents are supportive of fcch policies . the parents that i have always feel good about what i give the kids because sometimes they do not have the time to prepare food like we prepare it with all of the nutrients , like vegetables , everything a child needs in a day . most providers perceived parents as being too tired and busy to make healthy meals at home and that parents were more permissive then they were of their children eating unhealthy foods . sometimes , if parents are alone , they do not make healthy meals because they do not have the motivation . they get home and ate too tired most providers felt their role was to nurture and educate children in their care and viewed providing good nutrition and healthy diets as a priority . several providers spoke of the need to compensate for unhealthy practices at home by parents . they also felt that it is important to expose children in their care to healthy foods and eating habits . most providers reported being confident about their abilities to serve healthful foods at their fcch and viewed themselves as educators , with the knowledge needed to teach children and their families about healthful diets . i try to inform the parents if there are activities in the community geared towards healthy eating and living for families i give them pamphlets and all sorts of things that i have access to because of my work as a fcch provider and that they may not be aware of . in addition , most providers viewed themselves as educators and professionals and were vocal about wanting to discourage the perception of fcchs providers as being just babysitters . providers also spoke of enjoying seeing the children that they once cared for progressing , growing up , and being successful . it makes us feel good that we did the job we needed and those children will not have a hard time when they start school . providers across all focus groups spoke of multiple strategies they use to incorporate nutritious foods into meals and snacks . strategies included becoming familiar with foods served and meal time practices at the homes of children attending their fcch , introducing and encouraging new and healthy foods , and modeling of healthful behaviors . some providers spoke of encouraging new foods by directly involving children with healthy food choices , as they believe this gives children a sense of control and increased openness to new foods , especially if they see their peers enjoying foods they would not normally eat . i usually allow children to help pick their foods as part of a game at the beginning of the week , so that they have some choice of preferred foods . in addition , some providers mentioned that children themselves influence food choices and eating habits of other children . at first a child may not want to eat something , but when they see another child eating it , they will try . the majority of providers mentioned the importance of planning , buying , and preparing meals in advance . they felt planning ahead enabled them to serve a healthy and varied food menu on a weekly basis . planning was seen as especially important for providers who served multiple meals ( e.g. , breakfast , a morning snack , lunch , an afternoon snack , and , in some cases , dinner ) . i like to plan ahead and know what i will serve the kids for at least a week it 's just much easier that way . across all focus groups , nearly all providers mentioned using available educational resources , including minute menu for their food shopping and planning needs of the week . a few providers mentioned that minute menu made it easy to print a shopping list for their meals and allowed for some flexibility in their preplanned menus . if a child does not like one vegetable they can substitute another vegetable in its place . school - based menus and menus provided by their local food programs such as yours for children . a few providers also reported that they use pamphlets from the usda for snack and food ideas . they are always sending magazines on how to use the things to feed the children better and wo n't lead them into becoming obese children . the majority of providers mentioned participating in workshops about nutritional guidelines and using workshop resources to guide healthy eating options at their fcchs . a few stated that attending these workshops and trainings caused them to make changes to their feeding practices and meal options at their fcchs . i went to a workshop where they showed you how much sugar is in juice as measured by the number of sugar packets . after that workshop , i have just paid a lot more attention to serving juices to the children . several barriers to providing healthy foods in fcchs were mentioned , including the high cost of healthy foods , especially organic and fresh fruits and vegetables . something that affects is money , how much they [ food program ] pay you . you have to pick out what is cheaper that week , fruit or vegetables , to be able to save money . most providers acknowledged that their latino culture influences the foods they serve at their fcch as well as eating routines and the foods that children eat with their families outside of the fcchs . they learn to eat in my daycare and learn to eat food from my culture . although providers felt their latino culture influences the foods they serve , they did not see their culture as negatively impacting foods they provided at their fcchs . on the contrary , several providers noted several healthy food options that are part of the latino culture , such as beans . for example , i serve bean soup to the kids on a regular basis and that 's very healthy for them . a few providers reported having some children at risk of overweight or obesity in their care and that this influenced their feeding practices , especially in determining portion sizes . i try to reduce the portion of the one that likes to eat so he wo n't get to eat too much and later be affected by obesity . while another added , i may have the child wait to see if they are still hungry then give water or fruit if the child is still hungry . furthermore , some fccps felt that they needed to control what and how much children eat . the majority of providers was aware and supportive of eec and cacfp policies , regulations , and guidelines for both nutrition and physical activity behaviors of children attending fcchs . they felt these policies made a real difference in the health of children attending fcch . i think the eec regulations require that children engage in an hour or more a day of pa , but a minimum of an hour a day . across all focus groups , the majority of providers described physical activity as engaging in organized activities , such as throwing a ball , swinging , dancing , and climbing , or as general activity throughout their day , such as running around during free - play . the general consensus was that physical activity is an important part of children 's daily routine at fcchs . although most providers reported children being very active throughout the day and agreed on the importance of physical activity as part of children 's daily routine at their fcchs , the amount of time providers believed children should engage in physical activity ranged from 30 minutes to two hours . when asked to discuss how they ensure that children attending their fcchs are physically active , the majority of providers described creative methods such as use of small outdoor and indoor equipment including hula hoops , jump rope , small trampolines for keeping children active throughout the year including during the cold winter months . several providers described creative methods for keeping children active during cold winter months including having children use indoor equipment such as hula hoops , jump rope , and small trampolines . some providers spoke of making modifications to their homes , both indoors and outdoors to make it more conducive to physical activity . in my house we redid the basement , so the kids have a big space to play , jump , and use hula hoops . most providers felt that screen time should be regulated and that children should be allowed a maximum of one hour of screen time per day . many providers reported allowing children to have screen time during transition times such as dropoff , pickup , and meal preparation . some tv when i am preparing the food , but no more than an hour a day . a few providers seemed to make a distinction between screen time for educational purposes and screen time for entertainment and felt that as long as the tv was being used for educational purposes it was ok to let children watch 3060 minutes of tv a day . i think it 's ok for the kids to watch some educational program on tv such as the pbs programs . some of those programs are very good and teach the kids basic language skills . although most providers reported that the majority of parents do not mind their kids watch some tv while at the fcch , a few mentioned that they respect and find alternative for children whose parents do not want them to watch any tv while at fcch . i have a parent who really does not want her daughter to watch tv while at daycare . i respect that , so when the other kids are watching tv , i have her draw . although the majority of providers did not express concerns or challenges with limiting screen time , a few providers noted that some parents allowed their children to bring electronics to daycare even when there were rules against that . i have a hard time when parents do n't respect the rules that i have around children bringing and using electronics such as ds . i do n't really like to have to keep reminding them that those devices are not allowed in my fcch . nevertheless , a few providers noted that some parents allowed their children to bring electronics to daycare , even when there are rules against that , which was viewed as challenging . across all focus groups , nearly all providers noted obstacles to children being physically active with lack of space and the cold weather being the most frequently noted . it 's really hard to keep the kids active when it 's cold and they do n't have as much space to move around inside the house . in order to overcome these barriers some providers mentioned creative ways to ensure that children engage in pa while at their fcchs . my home is not that big , so we often play a game and use the stairs . i have the kids go up and down a few times my house is small , but i have a house with two garages and inside there is a ball , and things saved for winter , and we begin to throw a ball and move around in there and we maintain our activity level . furthermore , some providers reported taking children to nearby parks or going for short walks around their homes , when the weather allowed . whenever the weather is good i take the kids to a park near the house . many providers felt children were less active during the winter in comparison to the summer due to the cold weather when children spent more time indoors . a few providers noted that it can take a long time to get all children 's winter gear on and off , and that can be especially challenging if they take care of several children . i only take them out once a day [ in winter ] because it is hard to put all the coats , gloves , hats . furthermore , providers who had grown up outside the us , in warmer climates appeared to perceive the cold weather as a barrier more than providers who had grown up with cold weather . i am not used to the kind of cold weather we get here in ma . i do not think i will ever get used to it i just try to get through the winter . nearly all providers spoke of the importance of having effective , open , and ongoing communication with parents as most children spend most of the day in their care . most providers reported sharing information regularly with parents about children routines , what and how much they ate , and anything unusual such as an illness or injury . furthermore , many providers felt that ongoing communication with parents is critical , as it allows them to understand children 's home environment including family 's routines and practices , how these shape the socio - emotional and physical development of the children they care for . communication with parents is also very important because it gives us a chance to learn about the child 's home environment , the family 's routines and rules , which is really important information to have to understand and care for the child in our fcchs . providers used multiple communication channels , including notes sent home at the end of day , forms , in - person communication , emails , texts , phone calls , and bulletin boards , where parents can see when they pick up / drop off their children . in fact , bulletin boards were used at most fcchs . i tell them what the children have done , we talk about the progress of the child , what vocabulary they have learned , things like this , if the child has a necessity , how we can help the child , the resources in the community to help several providers report sharing menu information with parents . i like to give the weekly menu to parents at the beginning of the week so that they understand and know what i am feeding their child at my fcch and that i will serve a variety of foods . other providers reported giving hard copies of a weekly / monthly meal menu to parents , while others reported posting it on an online bulletin board . a few providers reported using a software program provided by their local food program to report children 's daily food consumption and physical activity . providers who used such software stated they really liked using the software for its easiness of communication with parents . i like it because all the information can be easily emailed to parents . when asked how comfortable and confident they feel about talking with parents about any concerns they might have about a child ' weight status , most providers reported feeling very comfortable and confident . however , the majority of providers reported that they did not have major concerns about weight status of children currently under their care . a couple of providers who reported having an overweight child under their care in the past mentioned that they felt comfortable and confident approaching the child 's mother , discussing their concerns and sharing resources with the mother . a year or so ago , i had a child in my home ( fcch ) who was overweight . i also told her about a training that i had attended and how during this training the instructor had stressed the importance of keeping children active , not allowing them to drink lots of sugar sweetened drinks , and having them eat plenty of fruits and vegetables a few providers stated that they did not feel comfortable discussing children 's weight status with parents . it 's kind of difficult because when i think of children i 've encountered who were overweight , usually the parents are overweight . providers who reported being reluctant to discuss child 's weight felt that parents can be very sensitive to other people 's perceptions of their children , and because of that they preferred not to talk about it with parents . additionally , some providers felt that it is hard to change families ' habits and that parents are not always open to advice . parents feed her a lot of mcdonalds so i have tried to give them information on my menus so they can take [ sic ] home . there were , however , a few providers who felt it was their responsibility and part of their job to approach parents if they had concerns about a child 's weight status . i feel it is part of my job to let parents know any concerns i have about their child , and that also goes for any concerns related to a child 's weight status . i agree that it is not always easy to talk about it , and that parents can be sensitive sometimes , but i still think it is my responsibility . in this study , theory - driven qualitative research methods were used to assess latino providers ' beliefs and practices related to promoting healthful dietary and physical activity behaviors among preschool children attending fcch . theory - driven qualitative approaches are critical to enhancing knowledge and guiding development of interventions that promote healthful behaviors related to pediatric obesity intervention [ 22 , 23 ] . study findings indicate that latino fcch providers are vested in and believe they are influential in promoting healthy eating and physical activity behaviors of the preschool children in their care . in agreement with previous studies focusing on child care centers and family child care homes , latino fcch providers participating in our study perceived their role beyond simply watching children to one that includes promotion of early healthy behaviors including nutrition and physical activity [ 13 , 24 , 25 ] . analysis revealed a few barriers and challenges faced by providers in establishing and maintaining healthful nutrition and physical activity practices in their fcchs , including financial constraints . several providers referred to high costs of fruit and vegetables , especially organic types , as a potential limiting factor . this finding is consistent with studies showing that low socioeconomic and neighborhood settings are an important factor influencing residents ' consumption of healthy food choices , such as fruits and vegetables [ 14 , 26 ] . latino fcch providers participating in our study reported using strategies such as , encouragement and role modeling to influence healthy food choice and consumption , particularly with regard to introducing new foods and increasing variety . . conducted with latino head start providers highlighting the important influence that child care providers have in the development of healthy and unhealthy eating behaviors in minority children [ 2729 ] . contrary to a recent longitudinal survey study by lanigan , latino providers in our study did not report negative practices including pressuring child to eat and rewards for eating foods , although our qualitative study has a small sample size ( n = 44 ) . although most providers were consistent regarding mealtime food choices and routines , we found varied interpretations of portion size . this suggests that providers may benefit from additional training that assesses and addresses provider 's knowledge and educates providers about evidence - based practices related to healthful eating and child feeding practices [ 3133 ] . in general , latino providers in our study perceived parents as not being aware of the importance of healthful eating practices and/or lacking the time needed to ensure that their children ate a healthful diet . in addition , most providers felt that it was part of their job to engage with and educate parents about the importance of proper child nutrition and healthy eating behaviors . this finding is consistent with findings from recent studies [ 13 , 24 , 34 ] which indicate the important role that child care providers can play in the promotion of children 's early healthy behaviors related to eating and physical activity . our findings regarding providers ' positive beliefs related to child nutrition and feeding practices suggest that regulations and resources , particularly those promulgated by cacfp , are important factors influencing latino fcch providers ' knowledge and practices related to nutrition and child feeding . providers spoke positively about educational opportunities available to them through training and workshops required for licensing of their fcchs . this finding is in agreement with that of stan et al . documenting that broad - scale , in - person training is well received by child care providers and can be effective in increasing child care providers ' knowledge of regulations to promote healthful eating and child feeding practices in child care settings , including fcchs . our results revealed that in general , fcch providers perceive physical activity as important for children 's overall health . nevertheless , we found that latino fcch providers in our study appear to have a wide range of concepts of what constitutes physical activity practices for children and reported a range of time in which they regularly implement physical for children in their fcchs . these findings are consistent with those of previous studies [ 35 , 36 ] showing a wide range of perceptions , knowledge , and practices related to physical activity among young children . previous studies [ 3739 ] have documented that caregivers ' modeling of physical activity is influential in children 's physical activity levels . providers in our focus groups did not mention the importance of caregivers ' physical activity level and modeling ; therefore future training resources for promoting physical activity practices in fcchs should highlight importance of caregiver physical activity level and modeling . our findings suggest less variation in providers ' beliefs and practices related to screen time . nearly all providers participating in our study reported believing that children should not be allowed more than one hour of screen time daily . providers in our study spoke of using screen time ( mostly educational tv programs ) only during transition times ( e.g. , at pick up , preparation of lunch , etc . ) . previous studies conducted among low - income population have shown high levels of tv - watching by children and adults [ 40 , 41 ] . it is likely that providers participating in our study have been exposed to education and training and required to comply with regulatory policies that encourage limited use of tv and other screen devices set forth by agencies working with licensed fcchs . in agreement with previous studies , our findings revealed barriers to physical activity for children in fcchs [ 35 , 36 ] including cold weather and the physical environment of the fcch that may lack appropriate indoor and outdoor spaces . this finding is consistent with previous studies , including our own [ 14 , 41 ] with latino parents , which found housing and neighborhood barriers faced by families living in low - income areas , with limited access to indoor and safe outdoor spaces [ 14 , 34 , 40 , 41 ] . this is an important finding , and as suggested by previous studies [ 35 , 36 ] , physical activity interventions targeting family child care homes must be tailored to meet the unique characteristics of this home - based child care environment . specific cultural influences related to latino fcch providers ' beliefs , attitudes , and practices related to nutrition and physical activity were not widely apparent in our study . although some providers reported serving foods typical to the latino culture such as rice and beans on a regular basis , it is possible that other cultural influences reported by previous studies conducted among latino populations such as , consumption of sugar - sweetened beverages and use of tv may be less present in licensed latino fcchs due regulatory agency requirements . finally , given the pivotal role that parents have in structuring home environment , it is important to note that the latino fcch providers in our study perceive that parental home environment is lacking in nutrition and physical activity structure . fcch providers may be well positioned given their daily and close relationship with parents to engage and educate low - income , latino parents about the importance of establishing a home environment conducive to the development of early healthy behaviors related to children 's eating and physical activity . our findings highlight the important influence and role that latino fcch providers can have as a unit of change and promotion of health in low - income , latino communities . interventions involving fcch providers may prove to be an effective way to target low - income minority families for obesity prevention efforts . given fcch providers established presence in their communities , they are well positioned to facilitate low - income families ' access to evidence - based information in a linguistic and culturally sensitive way . latino providers have established trusting and respected relationships with latino parents , which positions their family child care homes as an important venue for the delivery of long - term and sustainable efforts to prevent childhood obesity among at - risk , minority communities . the potential role of minority fcch providers should be explored in future community - based interventions aimed at promoting healthful family behaviors related to nutrition and physical activity . findings are based on a nonrandom , purposive , and relatively small sample of low - income , latino fcch providers in four selected communities in massachusetts . furthermore , fcch providers recruited to participate in this study could have been those who are more aware and concerned in general with promoting health behaviors among children in their care . future research can address these limitations by exploring influences on latino providers ' beliefs , attitudes , and practices from other communities across the us . in addition , quantitative research that builds on the qualitative findings reported here is needed to quantify latino providers ' beliefs , attitudes , and practices related to the promotion of healthy eating and physical activity behaviors among latino preschool children attending fcchs .

background . the continuing rise of obesity among latinos is a public health concern with an immediate need for early prevention . changes in family structures have increased demand and reliance for child care for young children . latino children are the fastest - growing segment of the child population in the united states , and research shows that latino families use preschools and day care centers much less than those of other ethnic groups , apparently because of cultural preferences for family - like care . objectives . given that many low income latino children attend family child care homes ( fcchs ) , there is a need to explore the role that fcch providers may play in establishing and reinforcing children 's early healthful eating and physical activity behaviors and consequently in the prevention of childhood obesity . design . using purposive sampling , six focus groups were conducted in spanish with licensed latino fcch providers ( n = 44 ) . data was analyzed to identify recurrent themes . results . latino fcch providers described how they play an influential role in promoting healthful eating and physical activity behaviors of preschool children in their care . they also identified many barriers and challenges in establishing and maintaining healthful nutrition and physical activity behaviors , including high cost of healthy foods , cold weather , and physical environment of fcch . conclusions . latino fcch providers can have a strong impact in promoting healthful behaviors in low - income , latino communities . they may be able to effectively deliver interventions targeting low - income , minority families to promote healthful eating and physical activity behaviors and prevent child obesity .

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

latinos are the largest and most rapidly growing population group in the united states . although rates of childhood obesity are still high for the general child population , children of low - income , minority families are at a particularly high risk of overweight and obesity . the continuing rise of obesity within minority and immigrant populations , particularly latinos , remains a pressing public health concern with an immediate need for early prevention . children living in the united states live in a society that has changed dramatically since the obesity epidemic first developed . changes in family structure , gender roles , and families ' needs for economic security have increased the demand and reliance on child care for children at increasingly younger ages . in addition , welfare reform laws requiring employment have increased the number of employed low - income parents and have contributed to an increase in the number of children enrolled in child care programs . in 2012 , 68% of women with children under the age of 6 were either working or looking for work , and almost 11 million preschool - aged children received some form of child care while their mothers worked [ 2 , 3 ] . the child care setting is an important social environment that potentially influences the development of children 's early dietary and physical activity habits and consequently contributes to the development of child overweight [ 46 ] . given parents increasing reliance on child care settings for their children at continually younger ages , these settings are likely important venues for the implementation of programs and policies to help children develop healthful eating and physical activity habits [ 2 , 4 ] . child care providers , like parents , help establish and reinforce early healthful eating and physical activity habits among young children , and can be key players in preventing childhood obesity by developing a child care environment that fosters healthful eating and physical activity behaviors among children [ 79 ] . child care provider 's knowledge of nutrition and physical activity , the selection of food and meals , structure within their day care , and their own modeling of behaviors are all influential in young children 's development of lifelong habits that contribute to normal weight or to overweight and obesity [ 1012 ] . in fact , research suggests that child care providers may be more influential than or equally as important as parents in shaping food preferences of young children [ 13 , 14 ] . child care settings may help establish and reinforce children 's eating and physical activity habits [ 4 , 14 , 15 ] . furthermore , studies have found that latino parents who send their children to child care believe these settings are instrumental in shaping and reinforcing the eating and physical activity of their children . however , despite the growing number of studies and interventions targeting child care settings and given that more than 1.6 million children attend fcchs , there is limited research examining fcchs and their influence on the development of healthful eating and physical activity habits in latino preschool - aged children , a group at increased risk of obesity . in massachusetts , the setting for this study , the majority of children enrolled in fcchs are from minority backgrounds , including a high percentage of latino children , and most latino children attend fcchs operated by latino staff . fcch providers are required to ( 1 ) have a plan for communicating with parents / guardians through various communication channels ( e.g. state regulations do not specify the amount , frequency , and type of physical activity or regulate television use . additionally , fcch providers may be eligible to participate in the child and adult care food program ( cacfp ) , which requires that cacfp participants follow usda / cacfp guidelines for menus and feeding practices . given the growing importance of fcchs in serving a large proportion of minority , low - income children , further research is needed to examine the role that fcch providers play in establishing and reinforcing children 's early healthful eating and physical activity habits and consequently preventing childhood obesity . moreover , given the high rates of obesity among latino preschoolers , and the fact that latino families use preschools and day care centers much less than those of other ethnic groups , apparently because of cultural preferences for family - like care , there is a need to understand how family child care settings influence the development of eating , physical activity and sedentary behaviors associated with childhood obesity . preventing obesity in latino family child care homes is a multicomponent study employing qualitative methods to explore influences on eating habits , physical activity , sedentary behaviors , and ultimately risk of obesity among latino preschool - aged children attending fcchs in massachusetts . additionally , the study assessed practices , policies , and regulations of fcchs that may be associated with risk of childhood obesity among latino preschool - aged children . this current paper focuses on the results of the qualitative research examining latino fcch providers ' beliefs and practices related to nutrition and feeding , and physical activity and sedentary behaviors among low - income preschool children . we worked with ma department of early education and care ( eec ) , which develops licensing regulations and requirements for childcare providers and supports training for early educators , and the child care circuit , a nonprofit organization providing child care referrals , training , and parent and provider services , to identify cities in four regions of the state ( north shore , greater boston , central ma , and western ma ) that have a large number of fcchs run by latino providers . ma - ecc and the child care circuit , then identified two agencies that work directly with fcch providers , cacfp , and family child care systems who compiled a list of all currently licensed latino fcch providers . a native spanish speaker trained in qualitative research methods conducted all focus groups in spanish using a semistructured discussion guide including open - ended questions and probes . at completion of the focus group the study protocol was approved by the internal review board at the harvard school of public health . the focus group guide explored ( 1 ) providers ' perceptions , attitudes and practices related to nutrition , and physical activity and sedentary behaviors ; ( 2 ) influences of fcch characteristics on children 's eating and physical activity behaviors ; ( 3 ) fcch providers practices related to nutrition and physical activity ; ( 4 ) educational activities offered by state and local agencies related to nutrition and physical activity and sedentary behaviors ; ( 5 ) communication between fcch providers and parents about fcch practices and policies related to nutrition and physical activity and sedentary behaviors ; and ( 6 ) barriers fcch providers face in providing an environment conducive to healthful eating and physical activity behaviors . see the following for sample of questions used in focus group discussions with latino family child care providers ( fccps ) . , sitting together at a table , watching tv or videos during meal ) fccps ' beliefs , attitudes , and practices related to physical activity what are your ideas and thoughts about physical activity for children ( in general)?tell me about the routine you have for children to get physical activity while they are at the daycare . for example , do they play games , use equipment , and use videos or tv ? for example , do they play games , use equipment , and use videos or tv ? for example , do they play games , use equipment , and use videos or tv ? they also enjoy playing on a computer , cell phone , ds toys , and video and nintendo games . in total , 44 providers ( 41 females , 3 males ) , all of whom are self - identified as hispanic / latino , participated in six focus groups . about one - third of participants ( n = 14 ) had graduated from high school or earned their ged , and close to forty percent ( n = 17 ) had attended some college . most ( n = 41 , 93% ) had several to up to 25 years of experience running fcchs . data analysis identified key themes related to nutrition and feeding practices , physical activity , and sedentary behaviors at latino fcchs . providers believed that children eat a healthier diet in their fcchs than they do at home and that parents are supportive of fcch policies . they get home and ate too tired most providers felt their role was to nurture and educate children in their care and viewed providing good nutrition and healthy diets as a priority . several providers spoke of the need to compensate for unhealthy practices at home by parents . they also felt that it is important to expose children in their care to healthy foods and eating habits . i try to inform the parents if there are activities in the community geared towards healthy eating and living for families i give them pamphlets and all sorts of things that i have access to because of my work as a fcch provider and that they may not be aware of . strategies included becoming familiar with foods served and meal time practices at the homes of children attending their fcch , introducing and encouraging new and healthy foods , and modeling of healthful behaviors . across all focus groups , nearly all providers mentioned using available educational resources , including minute menu for their food shopping and planning needs of the week . several barriers to providing healthy foods in fcchs were mentioned , including the high cost of healthy foods , especially organic and fresh fruits and vegetables . you have to pick out what is cheaper that week , fruit or vegetables , to be able to save money . a few providers reported having some children at risk of overweight or obesity in their care and that this influenced their feeding practices , especially in determining portion sizes . the majority of providers was aware and supportive of eec and cacfp policies , regulations , and guidelines for both nutrition and physical activity behaviors of children attending fcchs . across all focus groups , the majority of providers described physical activity as engaging in organized activities , such as throwing a ball , swinging , dancing , and climbing , or as general activity throughout their day , such as running around during free - play . the general consensus was that physical activity is an important part of children 's daily routine at fcchs . although most providers reported children being very active throughout the day and agreed on the importance of physical activity as part of children 's daily routine at their fcchs , the amount of time providers believed children should engage in physical activity ranged from 30 minutes to two hours . when asked to discuss how they ensure that children attending their fcchs are physically active , the majority of providers described creative methods such as use of small outdoor and indoor equipment including hula hoops , jump rope , small trampolines for keeping children active throughout the year including during the cold winter months . several providers described creative methods for keeping children active during cold winter months including having children use indoor equipment such as hula hoops , jump rope , and small trampolines . in my house we redid the basement , so the kids have a big space to play , jump , and use hula hoops . across all focus groups , nearly all providers noted obstacles to children being physically active with lack of space and the cold weather being the most frequently noted . i have the kids go up and down a few times my house is small , but i have a house with two garages and inside there is a ball , and things saved for winter , and we begin to throw a ball and move around in there and we maintain our activity level . a few providers noted that it can take a long time to get all children 's winter gear on and off , and that can be especially challenging if they take care of several children . nearly all providers spoke of the importance of having effective , open , and ongoing communication with parents as most children spend most of the day in their care . furthermore , many providers felt that ongoing communication with parents is critical , as it allows them to understand children 's home environment including family 's routines and practices , how these shape the socio - emotional and physical development of the children they care for . communication with parents is also very important because it gives us a chance to learn about the child 's home environment , the family 's routines and rules , which is really important information to have to understand and care for the child in our fcchs . providers used multiple communication channels , including notes sent home at the end of day , forms , in - person communication , emails , texts , phone calls , and bulletin boards , where parents can see when they pick up / drop off their children . i tell them what the children have done , we talk about the progress of the child , what vocabulary they have learned , things like this , if the child has a necessity , how we can help the child , the resources in the community to help several providers report sharing menu information with parents . a few providers reported using a software program provided by their local food program to report children 's daily food consumption and physical activity . a couple of providers who reported having an overweight child under their care in the past mentioned that they felt comfortable and confident approaching the child 's mother , discussing their concerns and sharing resources with the mother . i also told her about a training that i had attended and how during this training the instructor had stressed the importance of keeping children active , not allowing them to drink lots of sugar sweetened drinks , and having them eat plenty of fruits and vegetables a few providers stated that they did not feel comfortable discussing children 's weight status with parents . providers who reported being reluctant to discuss child 's weight felt that parents can be very sensitive to other people 's perceptions of their children , and because of that they preferred not to talk about it with parents . in this study , theory - driven qualitative research methods were used to assess latino providers ' beliefs and practices related to promoting healthful dietary and physical activity behaviors among preschool children attending fcch . theory - driven qualitative approaches are critical to enhancing knowledge and guiding development of interventions that promote healthful behaviors related to pediatric obesity intervention [ 22 , 23 ] . study findings indicate that latino fcch providers are vested in and believe they are influential in promoting healthy eating and physical activity behaviors of the preschool children in their care . in agreement with previous studies focusing on child care centers and family child care homes , latino fcch providers participating in our study perceived their role beyond simply watching children to one that includes promotion of early healthy behaviors including nutrition and physical activity [ 13 , 24 , 25 ] . analysis revealed a few barriers and challenges faced by providers in establishing and maintaining healthful nutrition and physical activity practices in their fcchs , including financial constraints . conducted with latino head start providers highlighting the important influence that child care providers have in the development of healthy and unhealthy eating behaviors in minority children [ 2729 ] . contrary to a recent longitudinal survey study by lanigan , latino providers in our study did not report negative practices including pressuring child to eat and rewards for eating foods , although our qualitative study has a small sample size ( n = 44 ) . this suggests that providers may benefit from additional training that assesses and addresses provider 's knowledge and educates providers about evidence - based practices related to healthful eating and child feeding practices [ 3133 ] . in general , latino providers in our study perceived parents as not being aware of the importance of healthful eating practices and/or lacking the time needed to ensure that their children ate a healthful diet . this finding is consistent with findings from recent studies [ 13 , 24 , 34 ] which indicate the important role that child care providers can play in the promotion of children 's early healthy behaviors related to eating and physical activity . our findings regarding providers ' positive beliefs related to child nutrition and feeding practices suggest that regulations and resources , particularly those promulgated by cacfp , are important factors influencing latino fcch providers ' knowledge and practices related to nutrition and child feeding . documenting that broad - scale , in - person training is well received by child care providers and can be effective in increasing child care providers ' knowledge of regulations to promote healthful eating and child feeding practices in child care settings , including fcchs . our results revealed that in general , fcch providers perceive physical activity as important for children 's overall health . nevertheless , we found that latino fcch providers in our study appear to have a wide range of concepts of what constitutes physical activity practices for children and reported a range of time in which they regularly implement physical for children in their fcchs . these findings are consistent with those of previous studies [ 35 , 36 ] showing a wide range of perceptions , knowledge , and practices related to physical activity among young children . previous studies [ 3739 ] have documented that caregivers ' modeling of physical activity is influential in children 's physical activity levels . providers in our focus groups did not mention the importance of caregivers ' physical activity level and modeling ; therefore future training resources for promoting physical activity practices in fcchs should highlight importance of caregiver physical activity level and modeling . previous studies conducted among low - income population have shown high levels of tv - watching by children and adults [ 40 , 41 ] . in agreement with previous studies , our findings revealed barriers to physical activity for children in fcchs [ 35 , 36 ] including cold weather and the physical environment of the fcch that may lack appropriate indoor and outdoor spaces . this finding is consistent with previous studies , including our own [ 14 , 41 ] with latino parents , which found housing and neighborhood barriers faced by families living in low - income areas , with limited access to indoor and safe outdoor spaces [ 14 , 34 , 40 , 41 ] . this is an important finding , and as suggested by previous studies [ 35 , 36 ] , physical activity interventions targeting family child care homes must be tailored to meet the unique characteristics of this home - based child care environment . specific cultural influences related to latino fcch providers ' beliefs , attitudes , and practices related to nutrition and physical activity were not widely apparent in our study . although some providers reported serving foods typical to the latino culture such as rice and beans on a regular basis , it is possible that other cultural influences reported by previous studies conducted among latino populations such as , consumption of sugar - sweetened beverages and use of tv may be less present in licensed latino fcchs due regulatory agency requirements . finally , given the pivotal role that parents have in structuring home environment , it is important to note that the latino fcch providers in our study perceive that parental home environment is lacking in nutrition and physical activity structure . fcch providers may be well positioned given their daily and close relationship with parents to engage and educate low - income , latino parents about the importance of establishing a home environment conducive to the development of early healthy behaviors related to children 's eating and physical activity . our findings highlight the important influence and role that latino fcch providers can have as a unit of change and promotion of health in low - income , latino communities . interventions involving fcch providers may prove to be an effective way to target low - income minority families for obesity prevention efforts . given fcch providers established presence in their communities , they are well positioned to facilitate low - income families ' access to evidence - based information in a linguistic and culturally sensitive way . latino providers have established trusting and respected relationships with latino parents , which positions their family child care homes as an important venue for the delivery of long - term and sustainable efforts to prevent childhood obesity among at - risk , minority communities . the potential role of minority fcch providers should be explored in future community - based interventions aimed at promoting healthful family behaviors related to nutrition and physical activity . findings are based on a nonrandom , purposive , and relatively small sample of low - income , latino fcch providers in four selected communities in massachusetts . furthermore , fcch providers recruited to participate in this study could have been those who are more aware and concerned in general with promoting health behaviors among children in their care . in addition , quantitative research that builds on the qualitative findings reported here is needed to quantify latino providers ' beliefs , attitudes , and practices related to the promotion of healthy eating and physical activity behaviors among latino preschool children attending fcchs .

we analyzed x chromosomes from 26 unrelated ( 46,xx ) individuals sequenced by completegenomics ( drmanac et al . sites were filtered , requiring that data be present ( monomorphic or variable ) in all 26 samples . human - chimpanzee ( hg19-pantro4 ) , human - macaque ( hg19-rhemac3 ) , human - dog ( hg19-canfam3 ) , and human - mouse ( hg19-mm10 ) alignments were extracted from the university of california santa cruz ( ucsc ) genome browser ( rosenbloom et al . 2015 ) . we curated the human - chimpanzee and human - macaque alignments to filter out segments that included autosomal sequences aligning to the x chromosome ( table s3 , figure s1 , and figure s2 ) . additionally , we observed several regions across the x chromosome that exhibited heightened divergence between the human and chimpanzee or the human and macaque ( figure s3 and figure s4 ) . on further inspection , these regions often contain multicopy gene families that could lead to mismapping ( table s3 ) . divergence estimates were similar with and without these regions , and here we present results with these regions of high divergence near multicopy gene families excluded . low - diversity ( dutheil et al . 2015 ) and ampliconic regions ( mueller et al . 2013 ; nam et al . 2015 ) were filtered out of the data to avoid analyzing regions potentially affected by strong selective sweeps or difficult - to - align regions . significant differences between par1 and nonpars of the x chromosome , as well as significant differences between the xtr and nonxtrs of the x chromosome persist regardless of inclusion or exclusion of ampliconic and low - diversity regions ( figure 1 , table 1 , table s4 , and table s5 ) . genetic diversity ( measured by ) is shown in 100 kb overlapping windows across the human x chromosome that includes par1 ( shown in red ) , the nonpars ( shown in black ) , the x - transposed region ( xtr ) ( shown in blue ) , and par2 ( shown in red ) for ( a ) human diversity uncorrected for divergence and then human diversity corrected for variable mutation rate using ( b ) human - chimpanzee divergence , ( c ) human - macaque divergence , ( d ) human - dog divergence , and ( e ) human - mouse divergence . the light - gray - shaded areas are the low - diversity regions , and the dark - gray - shaded areas are the ampliconic regions that were filtered out . diversity , measured as the average number of pairwise differences per site ( ) between the x chromosomes of 26 unrelated genetic females , in each region of the human x chromosome is presented first unnormalized for mutation - rate variation , then normalized using human - chimpanzee ( hg19-pantro4 ) divergence , and then separately normalized for human - macaque ( hg19-rhemac3 ) , human - dog ( hg19-canfam3 ) , and human - mouse ( hg19-mm10 ) divergence . the regions analyzed include the par1 , par2 , the xtr , and the nonpars either including the xtr ( nonpar ) or excluding the xtr ( nonparminus_xtr ) . p - values from permutation tests with 10,000 replicates are shown between each recombining region and the nonpars . all p - values are indicated in italics while bold corresponds to significant p - values . 2011 ) to filter out regions that could cause potential sequence misalignments and regions defined by the ucsc genome browser ( rosenbloom et al . 2015 ) that may be subject to selection : reference sequence ( refseq ) database genes , simple repeats , and repetitive elements . we attempted to filter out noncoding regions near genes , but doing so would leave very little analyzable sequence in par1 and par2 . we measured the diversity between the sequences as , the average pairwise nucleotide differences per site between all sequences in the sample: = 2 l kk1 i=1kpij > ikpjdijwhere l represents the number of called sites , k represents the number of dna sequences , pi and pj are the frequencies of the corresponding alleles i and j , and dij is the number of sites containing nucleotide differences . diversity was calculated within each specific region ( par1 , par2 , xtr , nonpars with xtr , and nonpars without xtr ) , as well as across sliding and nonoverlapping windows . we generated window - interval files across the human x chromosome with galaxy tools ( blankenberg et al . 2011 ) and conducted analysis in four sets of windows : ( 1 ) in a 1 mb nonoverlapping window , ( 2 ) a 1 mb window with 100 kb sliding start positions , ( 3 ) a 100 kb nonoverlapping window , and ( 4 ) a 100 kb window with 10 kb sliding start positions ( figure s5 ) . we similarly calculated human - chimpanzee , human - macaque , human - dog , and human - mouse species divergence along the x chromosome in each of the four regions and in the same windows described previously . to normalize the data , values were divided by the observed divergence within the same interval . chromosome x was divided into windows that were permuted without replacement 10,000 times to assess significant differences between diversity in each region ( par1 , xtr , and par2 ) relative to nonpar sequences . this analysis was repeated for uncorrected diversity and diversity corrected for human - chimpanzee , human - macaque , human - dog , and human - mouse divergence values . empirical p - values were calculated by computing the number of times the difference between each pair of permuted sample regions was equal to or greater than the difference in observed diversity between each pair of regions . the negative correlation along the pseudoautosomal boundary was tested using linear regressions across 100 kb windows covering a total of 3 mb for each regression ( 30 windows ) , shifting the window by 100 kb systematically ( figure 2 ) . each regression was analyzed for significance of the correlation ( p < 0.05 ) , with all data points occurring before the first nonsignificant window being included in the significant data set . the 100 kb nonoverlapping windows were permuted 10,000 times , and the correlation coefficient and the p - values of the linear regression were calculated for the first 3 mb of each permutation . the significance of the observed negative correlation was computed by comparing the 10,000 permuted linear regressions with the observed value . all the graphs were produced using r version 3.1.2 ( r core team 2015 ) . diversity in 100 kb nonoverlapping windows along the pseudoautosomal boundary is plotted across the first 6 mb of the human x chromosome , spanning the annotated pseudoautosomal boundary at 2.7 mb . a series of linear regressions was run , including 30 windows , sliding by one window across the pars to the nonpars . each 100 kb window is colored red if it is included in a regression in which distance from xp and diversity are significantly negatively correlated ; otherwise , the windows are colored black . for the entire region together , diversity is significantly negatively correlated with distance from xp ( p = 3.281 10 ; r = 0.7321563 ) and spans the pseudoautosomal boundary . the authors state that all data necessary for confirming the conclusions presented in this article are represented fully within the article . the authors state that all data necessary for confirming the conclusions presented in this article are represented fully within the article . we observe that uncorrected diversity is three times higher in par1 than in the nonpars , whereas uncorrected diversity in par2 is not significantly greater than that in the nonpars ( table 1 , figure 1 , and figure 3 ) . we studied noncoding regions across the entire x chromosome , filtering out annotated genes , to minimize the effect of selection , but given their small sizes , we could not filter out regions far from genes in the pars or xtr ( see materials and methods ) . ampliconic regions ( mueller et al . 2013 ; nam et al . 2015 ) , as well as regions of low diversity that are expected to have strong selective sweeps ( dutheil et al . 2015 ) , also were filtered out , which yielded the same result ( table s4 ) . however , mutation - rate variation across the x chromosome may account for variable levels of diversity observed in the pars and nonpars . we normalized the nucleotide diversity to correct for mutation rate using pairwise divergence between humans and several different species : pantro4 , rhemac3 , canfam3 , and mm10 ( table 1 and figure s6 ) . when we normalized with pantro4 , the difference in diversity between par1 and nonpars was not significant after filtering out the ampliconic regions , low - diversity regions , and the not applicable ( na ) values . this could be a result of large variation in divergence across regions of the x chromosome between humans and chimpanzees , potentially owing to complex speciation events ( patterson et al . we focus our interpretations on data that have been normalized using human - macaque divergence . similar to the uncorrected diversity values , when we correct for mutation rate using macaque divergence values , we observe higher nucleotide diversity across humans in par1 and par2 relative to the nonpars , with diversity being significantly higher in par1 than in nonpars ( with xtr removed ) and not significantly different between par2 and nonpars ( figure 1 , figure 3 , and table 1 ) . genetic diversity ( measured by ) is shown in box plots depicting the average diversity with error bars for the nonpars , par1 , xtr , and par2 . the p - values from a permutation test with 10,000 replicates comparing the diversity of each region to the diversity of the nonpars are shown . curiously , human - chimpanzee and human - macaque divergence are quite high in par1 relative to the nonpars in a pattern that does not reflect diversity ( figure 1 and table 1 ) . this result , predominantly , is due to high interspecies divergence in par1 and near the par boundary ( figure s3 and figure s4 ) . however , human - dog divergence roughly parallels uncorrected human diversity ( figure 1 ) . further , significantly elevated diversity in par1 relative to the nonpars can not be attributed solely to mutation - rate variation across the x chromosome because the pattern remains after correction for divergence in each region ( figure 1 and table 1 ) . the pattern we observed is consistent with several processes , including selection reducing variation more at linked sites in the nonpars than in par1 as a result of reduced rates of recombination in the nonpars relative to the pars or as a result of stronger drift in the nonpars as a result of a smaller effective population size . that we do not observe significantly elevated diversity in par2 relative to the nonpars is consistent with reports that par2 undergoes x - y recombination less frequently than par1 ( flaquer et al . 2008 ) and supports assertions that in humans only one chiasma per chromosome is needed for proper segregation rather than one per chromosome arm ( fledel - alon et al . curiously , in addition to elevated rates of diversity in the previously described par1 and par2 , we also observed that diversity was significantly higher in the recent xtr than in the nonpars ( table 1 and figure 3 ) . this increased diversity can not be attributed to mismapping between the x and y chromosome because we only analyzed individuals with two x chromosomes ( see materials and methods ) . high diversity in the xtr contrasts with initial suggestions that there is no x - y recombination in the xtr ( skaletsky et al . 2003 ) and is consistent with recent reports of x - y recombination in some human populations in this region ( veerappa et al . given the large size of the nonpars and the small size of the xtr , 5 mb ( ross et al . 2005 ) , one may wonder whether removing the xtr would make a difference to measured levels of diversity across the human x chromosome . the raw diversity of the nonpars including the xtr , measured as , is 0.000602 , while the raw diversity of the nonpars excluding the xtr is 0.000595 ( table 1 ) . removal of the xtr does decrease estimates of both diversity and divergence in the nonpars . although the xtr de facto may be removed with other filters , one should be cautious to include xtr regions because their inclusion in studies of x - specific diversity will affect inferences made when comparing x - linked and autosomal variation ( keinan and reich 2010 ; gottipati et al . recombination between the x and y chromosomes is expected to be suppressed at the pseudoautosomal boundary , where x - y sequence homology diverges owing to a y - specific inversion ( ellis et al . if diversity correlates highly with recombination rate and x - y recombination is strictly suppressed in the nonpars after the pseudoautosomal boundary , then diversity is expected to drop sharply between par1 and the nonpars . however , when we analyze patterns of human diversity in permuted windows across the x chromosome ( see materials and methods ) , we do not observe an abrupt shift in the level of diversity between par1 and the nonpars ( figure 2 ) . the lack of an observable pseudoautosomal boundary based on diversity is clear whether small or large ( 100 kb or 1 mb ) or overlapping or nonoveralapping windows are used ( figure s5 ) . in the approximately 3 mb that span the pseudoautosomal boundary , we observe a significant negative correlation between distance from xp and diversity . as we shift the window for the regression by 100 kb further from the start of par1 , we observe that the negative correlations remain independently significant and continue past the boundary ( figure 2 ) . we observe that the original linear relationship between distance from xp and diversity has a significant negative coefficient of correlation ( r = 0.6681177 ; p = 0 ) ( figure s7 ) . the significant linear relationship ( p = 3.281 10 ) that we observe in figure 2 extends nearly twice the length of par1 and supports the observation that there is no clear , abrupt drop in nucleotide diversity across the pseudoautosomal boundary . to test the significance of this correlation , we conducted a permutation test , shuffling windows ( of 100 kb ) across the x chromosome and recomputing the series of linear regressions 10,000 times ; then we computed the number of times a permuted x chromosome had a correlation that was as strong as or stronger than what we observed on the x chromosome ( figure s7 ) . we found that the negative correlation between distance from the short arm of the x chromosome and diversity is significant and spans the pseudoautosomal boundary ( see materials and methods ; p = 0 , permutation test ) . the history of gene conversion between the sex chromosomes may contribute to the increased diversity levels ( trombetta et al . 2014 ) on the nonpar side of the y - specific inversion that marks the pseudoautosomal boundary . human diversity uncorrected for divergence decreases from the proximal end of par1 through the pseudoautosomal boundary and well into the nonpar . a sex - specific map of par1 found that male recombination is higher near the telomeres and decreases near the pseudoautosomal boundary , while , in contrast , the female recombination rate reported in the same study in par1 is fairly flat throughout the region and increases near the pseudoautosomal boundary ( hinch et al . thus , genetic diversity uncorrected for divergence in par1 appears to correlate with the male recombination rate . curiously , however , a previous study of recombination rate in par1 reported an increase in the female ( but not the male ) recombination rate near the proximal end of par1 ( henke et al . 1993 ) . thus , potentially , both male and female recombination rates contribute to the linear decrease in diversity observed in par1 from the proximal end of the x chromosome through the pseudoautosomal boundary . although not yet mapped , when the data becomes available , it will be useful to compare patterns of diversity with sex - specific recombination maps across the entire x chromosome . we observe that uncorrected diversity is three times higher in par1 than in the nonpars , whereas uncorrected diversity in par2 is not significantly greater than that in the nonpars ( table 1 , figure 1 , and figure 3 ) . we studied noncoding regions across the entire x chromosome , filtering out annotated genes , to minimize the effect of selection , but given their small sizes , we could not filter out regions far from genes in the pars or xtr ( see materials and methods ) . ampliconic regions ( mueller et al . 2013 ; nam et al . 2015 ) , as well as regions of low diversity that are expected to have strong selective sweeps ( dutheil et al . 2015 ) , also were filtered out , which yielded the same result ( table s4 ) . however , mutation - rate variation across the x chromosome may account for variable levels of diversity observed in the pars and nonpars . we normalized the nucleotide diversity to correct for mutation rate using pairwise divergence between humans and several different species : pantro4 , rhemac3 , canfam3 , and mm10 ( table 1 and figure s6 ) . when we normalized with pantro4 , the difference in diversity between par1 and nonpars was not significant after filtering out the ampliconic regions , low - diversity regions , and the not applicable ( na ) values . this could be a result of large variation in divergence across regions of the x chromosome between humans and chimpanzees , potentially owing to complex speciation events ( patterson et al . we focus our interpretations on data that have been normalized using human - macaque divergence . similar to the uncorrected diversity values , when we correct for mutation rate using macaque divergence values , we observe higher nucleotide diversity across humans in par1 and par2 relative to the nonpars , with diversity being significantly higher in par1 than in nonpars ( with xtr removed ) and not significantly different between par2 and nonpars ( figure 1 , figure 3 , and table 1 ) . genetic diversity ( measured by ) is shown in box plots depicting the average diversity with error bars for the nonpars , par1 , xtr , and par2 . the p - values from a permutation test with 10,000 replicates comparing the diversity of each region to the diversity of the nonpars are shown . curiously , human - chimpanzee and human - macaque divergence are quite high in par1 relative to the nonpars in a pattern that does not reflect diversity ( figure 1 and table 1 ) . this result , predominantly , is due to high interspecies divergence in par1 and near the par boundary ( figure s3 and figure s4 ) . however , human - dog divergence roughly parallels uncorrected human diversity ( figure 1 ) . further , significantly elevated diversity in par1 relative to the nonpars can not be attributed solely to mutation - rate variation across the x chromosome because the pattern remains after correction for divergence in each region ( figure 1 and table 1 ) . the pattern we observed is consistent with several processes , including selection reducing variation more at linked sites in the nonpars than in par1 as a result of reduced rates of recombination in the nonpars relative to the pars or as a result of stronger drift in the nonpars as a result of a smaller effective population size . that we do not observe significantly elevated diversity in par2 relative to the nonpars is consistent with reports that par2 undergoes x - y recombination less frequently than par1 ( flaquer et al . 2008 ) and supports assertions that in humans only one chiasma per chromosome is needed for proper segregation rather than one per chromosome arm ( fledel - alon et al . curiously , in addition to elevated rates of diversity in the previously described par1 and par2 , we also observed that diversity was significantly higher in the recent xtr than in the nonpars ( table 1 and figure 3 ) . this increased diversity can not be attributed to mismapping between the x and y chromosome because we only analyzed individuals with two x chromosomes ( see materials and methods ) . high diversity in the xtr contrasts with initial suggestions that there is no x - y recombination in the xtr ( skaletsky et al . 2003 ) and is consistent with recent reports of x - y recombination in some human populations in this region ( veerappa et al . 2013 ) . given the large size of the nonpars and the small size of the xtr , 5 mb ( ross et al . 2005 ) , one may wonder whether removing the xtr would make a difference to measured levels of diversity across the human x chromosome . the raw diversity of the nonpars including the xtr , measured as , is 0.000602 , while the raw diversity of the nonpars excluding the xtr is 0.000595 ( table 1 ) . removal of the xtr does decrease estimates of both diversity and divergence in the nonpars . although the xtr de facto may be removed with other filters , one should be cautious to include xtr regions because their inclusion in studies of x - specific diversity will affect inferences made when comparing x - linked and autosomal variation ( keinan and reich 2010 ; gottipati et al . recombination between the x and y chromosomes is expected to be suppressed at the pseudoautosomal boundary , where x - y sequence homology diverges owing to a y - specific inversion ( ellis et al . if diversity correlates highly with recombination rate and x - y recombination is strictly suppressed in the nonpars after the pseudoautosomal boundary , then diversity is expected to drop sharply between par1 and the nonpars . however , when we analyze patterns of human diversity in permuted windows across the x chromosome ( see materials and methods ) , we do not observe an abrupt shift in the level of diversity between par1 and the nonpars ( figure 2 ) . the lack of an observable pseudoautosomal boundary based on diversity is clear whether small or large ( 100 kb or 1 mb ) or overlapping or nonoveralapping windows are used ( figure s5 ) . in the approximately 3 mb that span the pseudoautosomal boundary , as we shift the window for the regression by 100 kb further from the start of par1 , we observe that the negative correlations remain independently significant and continue past the boundary ( figure 2 ) . we observe that the original linear relationship between distance from xp and diversity has a significant negative coefficient of correlation ( r = 0.6681177 ; p = 0 ) ( figure s7 ) . the significant linear relationship ( p = 3.281 10 ) that we observe in figure 2 extends nearly twice the length of par1 and supports the observation that there is no clear , abrupt drop in nucleotide diversity across the pseudoautosomal boundary . to test the significance of this correlation , we conducted a permutation test , shuffling windows ( of 100 kb ) across the x chromosome and recomputing the series of linear regressions 10,000 times ; then we computed the number of times a permuted x chromosome had a correlation that was as strong as or stronger than what we observed on the x chromosome ( figure s7 ) . we found that the negative correlation between distance from the short arm of the x chromosome and diversity is significant and spans the pseudoautosomal boundary ( see materials and methods ; p = 0 , permutation test ) . the history of gene conversion between the sex chromosomes may contribute to the increased diversity levels ( trombetta et al . 2014 ) on the nonpar side of the y - specific inversion that marks the pseudoautosomal boundary . human diversity uncorrected for divergence decreases from the proximal end of par1 through the pseudoautosomal boundary and well into the nonpar . a sex - specific map of par1 found that male recombination is higher near the telomeres and decreases near the pseudoautosomal boundary , while , in contrast , the female recombination rate reported in the same study in par1 is fairly flat throughout the region and increases near the pseudoautosomal boundary ( hinch et al . thus , genetic diversity uncorrected for divergence in par1 appears to correlate with the male recombination rate . curiously , however , a previous study of recombination rate in par1 reported an increase in the female ( but not the male ) recombination rate near the proximal end of par1 ( henke et al . 1993 ) . thus , potentially , both male and female recombination rates contribute to the linear decrease in diversity observed in par1 from the proximal end of the x chromosome through the pseudoautosomal boundary . although not yet mapped , when the data becomes available , it will be useful to compare patterns of diversity with sex - specific recombination maps across the entire x chromosome . we show that diversity is indeed higher in the pseudoautosomal regions and lower in the regions of the x chromosome that are not known to recombine in males ( nonpars ) . diversity in par1 is significantly higher than in the nonpars regardless of normalizing the diversity with divergence between human and either macaque or dog to correct for mutation rate ( table 1 , figure 1 , and figure 3 ) . diversity also was normalized with divergence from the mouse , but there is no alignment between human and mouse in par1 because of a different evolutionary origin in par1 and no common pseudoautosomal genes being shared between them ( gianfrancesco et al . we observed that diversity is lower in par2 than expected and is not significantly different from the nonpars . we also showed that diversity is elevated in the xtr above other nonpars , verifying recent observations that the region still may undergo homologous recombination between the x and y chromosomes ( veerappa et al . finally , when analyzing patterns of genetic diversity in windows across the human x chromosome , we found that there is no strict boundary , based solely on the levels of diversity , between the recombining and putatively nonrecombining regions , which could be attributed to the evolutionary shift in the pseudoautosomal boundary over time , extending par1 as a result of a par1 length polymorphism ( mensah et al . our observations of patterns of diversity across regions of the human x chromosome with variable levels of recombination are consistent with previous reports that diversity and divergence are correlated with recombination rate in humans across the genome ( hellmann et al . elevated levels of diversity in the xtr suggest that , consistent with a recent report ( veerappa et al . curiously , we did not find a significant elevation of diversity in par2 , which , in agreement with its unusual evolution ( charchar et al . 2003 ) , indicates that it rarely recombines between x and y chromosomes during meiosis . further , the lack of a clear differentiation in diversity between par1 and the nonpars suggests that recombination suppression between the x and y chromosomes is still an actively evolving process in humans , as in other species ( bergero and charlesworth 2009 ) . this is consistent with evidence that the position of the pseudoautosomal boundary varies across mammals ( raudsepp and chowdhary 2008 ; otto et al . there is even evidence of polymorphism in the pseudoautosomal boundary in a pedigree analysis of a paternally inherited x chromosome in humans ( mensah et al . recombination spanning the pseudoautosomal boundary may account for some cases of de la chapelle syndrome ( schrander - stumpel et al . 1994 ) , in which an individual with two x chromosomes develops male gonads , and some portion of cases also have a copy of sry ( sry sits immediately proximal to the pseudoautosomal boundary in humans ) . further , it is possible that pseudoautosomal boundaries vary across populations , affecting recombination and contributing to nondisjunction of the sex chromosomes . taken together with previous inferences about the variation in pseudoautosomal boundaries , our observations suggest that assumptions should not be made of a strict suppression of x - y recombination at the proposed human pseudoautosomal boundary .

unlike the autosomes , recombination between the x chromosome and the y chromosome is often thought to be constrained to two small pseudoautosomal regions ( pars ) at the tips of each sex chromosome . par1 spans the first 2.7 mb of the proximal arm of the human sex chromosomes , whereas the much smaller par2 encompasses the distal 320 kb of the long arm of each sex chromosome . in addition to par1 and par2 , there is a human - specific x - transposed region that was duplicated from the x to the y chromosome . the x - transposed region is often not excluded from x - specific analyses , unlike the pars , because it is not thought to routinely recombine . genetic diversity is expected to be higher in recombining regions than in nonrecombining regions because recombination reduces the effect of linked selection . in this study , we investigated patterns of genetic diversity in noncoding regions across the entire x chromosome of a global sample of 26 unrelated genetic females . we found that genetic diversity in par1 is significantly greater than in the nonrecombining regions ( nonpars ) . however , rather than an abrupt drop in diversity at the pseudoautosomal boundary , there is a gradual reduction in diversity from the recombining through the nonrecombining regions , suggesting that recombination between the human sex chromosomes spans across the currently defined pseudoautosomal boundary . a consequence of recombination spanning this boundary potentially includes increasing the rate of sex - linked disorders ( e.g. , de la chapelle ) and sex chromosome aneuploidies . in contrast , diversity in par2 is not significantly elevated compared to the nonpars , suggesting that recombination is not obligatory in par2 . finally , diversity in the x - transposed region is higher than in the surrounding nonpars , providing evidence that recombination may occur with some frequency between the x and y chromosomes in the x - transposed region .

Materials and Methods Data availability Results Human Diversity is significantly higher in the XTR than in the nonPARs Pseudoautosomal boundaries cannot be inferred from patterns of diversity Discussion None

we curated the human - chimpanzee and human - macaque alignments to filter out segments that included autosomal sequences aligning to the x chromosome ( table s3 , figure s1 , and figure s2 ) . additionally , we observed several regions across the x chromosome that exhibited heightened divergence between the human and chimpanzee or the human and macaque ( figure s3 and figure s4 ) . significant differences between par1 and nonpars of the x chromosome , as well as significant differences between the xtr and nonxtrs of the x chromosome persist regardless of inclusion or exclusion of ampliconic and low - diversity regions ( figure 1 , table 1 , table s4 , and table s5 ) . genetic diversity ( measured by ) is shown in 100 kb overlapping windows across the human x chromosome that includes par1 ( shown in red ) , the nonpars ( shown in black ) , the x - transposed region ( xtr ) ( shown in blue ) , and par2 ( shown in red ) for ( a ) human diversity uncorrected for divergence and then human diversity corrected for variable mutation rate using ( b ) human - chimpanzee divergence , ( c ) human - macaque divergence , ( d ) human - dog divergence , and ( e ) human - mouse divergence . diversity , measured as the average number of pairwise differences per site ( ) between the x chromosomes of 26 unrelated genetic females , in each region of the human x chromosome is presented first unnormalized for mutation - rate variation , then normalized using human - chimpanzee ( hg19-pantro4 ) divergence , and then separately normalized for human - macaque ( hg19-rhemac3 ) , human - dog ( hg19-canfam3 ) , and human - mouse ( hg19-mm10 ) divergence . we attempted to filter out noncoding regions near genes , but doing so would leave very little analyzable sequence in par1 and par2 . we similarly calculated human - chimpanzee , human - macaque , human - dog , and human - mouse species divergence along the x chromosome in each of the four regions and in the same windows described previously . each regression was analyzed for significance of the correlation ( p < 0.05 ) , with all data points occurring before the first nonsignificant window being included in the significant data set . the 100 kb nonoverlapping windows were permuted 10,000 times , and the correlation coefficient and the p - values of the linear regression were calculated for the first 3 mb of each permutation . diversity in 100 kb nonoverlapping windows along the pseudoautosomal boundary is plotted across the first 6 mb of the human x chromosome , spanning the annotated pseudoautosomal boundary at 2.7 mb . a series of linear regressions was run , including 30 windows , sliding by one window across the pars to the nonpars . for the entire region together , diversity is significantly negatively correlated with distance from xp ( p = 3.281 10 ; r = 0.7321563 ) and spans the pseudoautosomal boundary . we observe that uncorrected diversity is three times higher in par1 than in the nonpars , whereas uncorrected diversity in par2 is not significantly greater than that in the nonpars ( table 1 , figure 1 , and figure 3 ) . we studied noncoding regions across the entire x chromosome , filtering out annotated genes , to minimize the effect of selection , but given their small sizes , we could not filter out regions far from genes in the pars or xtr ( see materials and methods ) . however , mutation - rate variation across the x chromosome may account for variable levels of diversity observed in the pars and nonpars . when we normalized with pantro4 , the difference in diversity between par1 and nonpars was not significant after filtering out the ampliconic regions , low - diversity regions , and the not applicable ( na ) values . this could be a result of large variation in divergence across regions of the x chromosome between humans and chimpanzees , potentially owing to complex speciation events ( patterson et al . similar to the uncorrected diversity values , when we correct for mutation rate using macaque divergence values , we observe higher nucleotide diversity across humans in par1 and par2 relative to the nonpars , with diversity being significantly higher in par1 than in nonpars ( with xtr removed ) and not significantly different between par2 and nonpars ( figure 1 , figure 3 , and table 1 ) . genetic diversity ( measured by ) is shown in box plots depicting the average diversity with error bars for the nonpars , par1 , xtr , and par2 . the p - values from a permutation test with 10,000 replicates comparing the diversity of each region to the diversity of the nonpars are shown . curiously , human - chimpanzee and human - macaque divergence are quite high in par1 relative to the nonpars in a pattern that does not reflect diversity ( figure 1 and table 1 ) . further , significantly elevated diversity in par1 relative to the nonpars can not be attributed solely to mutation - rate variation across the x chromosome because the pattern remains after correction for divergence in each region ( figure 1 and table 1 ) . the pattern we observed is consistent with several processes , including selection reducing variation more at linked sites in the nonpars than in par1 as a result of reduced rates of recombination in the nonpars relative to the pars or as a result of stronger drift in the nonpars as a result of a smaller effective population size . that we do not observe significantly elevated diversity in par2 relative to the nonpars is consistent with reports that par2 undergoes x - y recombination less frequently than par1 ( flaquer et al . 2008 ) and supports assertions that in humans only one chiasma per chromosome is needed for proper segregation rather than one per chromosome arm ( fledel - alon et al . curiously , in addition to elevated rates of diversity in the previously described par1 and par2 , we also observed that diversity was significantly higher in the recent xtr than in the nonpars ( table 1 and figure 3 ) . this increased diversity can not be attributed to mismapping between the x and y chromosome because we only analyzed individuals with two x chromosomes ( see materials and methods ) . high diversity in the xtr contrasts with initial suggestions that there is no x - y recombination in the xtr ( skaletsky et al . removal of the xtr does decrease estimates of both diversity and divergence in the nonpars . although the xtr de facto may be removed with other filters , one should be cautious to include xtr regions because their inclusion in studies of x - specific diversity will affect inferences made when comparing x - linked and autosomal variation ( keinan and reich 2010 ; gottipati et al . recombination between the x and y chromosomes is expected to be suppressed at the pseudoautosomal boundary , where x - y sequence homology diverges owing to a y - specific inversion ( ellis et al . if diversity correlates highly with recombination rate and x - y recombination is strictly suppressed in the nonpars after the pseudoautosomal boundary , then diversity is expected to drop sharply between par1 and the nonpars . however , when we analyze patterns of human diversity in permuted windows across the x chromosome ( see materials and methods ) , we do not observe an abrupt shift in the level of diversity between par1 and the nonpars ( figure 2 ) . in the approximately 3 mb that span the pseudoautosomal boundary , we observe a significant negative correlation between distance from xp and diversity . the significant linear relationship ( p = 3.281 10 ) that we observe in figure 2 extends nearly twice the length of par1 and supports the observation that there is no clear , abrupt drop in nucleotide diversity across the pseudoautosomal boundary . to test the significance of this correlation , we conducted a permutation test , shuffling windows ( of 100 kb ) across the x chromosome and recomputing the series of linear regressions 10,000 times ; then we computed the number of times a permuted x chromosome had a correlation that was as strong as or stronger than what we observed on the x chromosome ( figure s7 ) . we found that the negative correlation between distance from the short arm of the x chromosome and diversity is significant and spans the pseudoautosomal boundary ( see materials and methods ; p = 0 , permutation test ) . 2014 ) on the nonpar side of the y - specific inversion that marks the pseudoautosomal boundary . human diversity uncorrected for divergence decreases from the proximal end of par1 through the pseudoautosomal boundary and well into the nonpar . a sex - specific map of par1 found that male recombination is higher near the telomeres and decreases near the pseudoautosomal boundary , while , in contrast , the female recombination rate reported in the same study in par1 is fairly flat throughout the region and increases near the pseudoautosomal boundary ( hinch et al . curiously , however , a previous study of recombination rate in par1 reported an increase in the female ( but not the male ) recombination rate near the proximal end of par1 ( henke et al . thus , potentially , both male and female recombination rates contribute to the linear decrease in diversity observed in par1 from the proximal end of the x chromosome through the pseudoautosomal boundary . although not yet mapped , when the data becomes available , it will be useful to compare patterns of diversity with sex - specific recombination maps across the entire x chromosome . we observe that uncorrected diversity is three times higher in par1 than in the nonpars , whereas uncorrected diversity in par2 is not significantly greater than that in the nonpars ( table 1 , figure 1 , and figure 3 ) . we studied noncoding regions across the entire x chromosome , filtering out annotated genes , to minimize the effect of selection , but given their small sizes , we could not filter out regions far from genes in the pars or xtr ( see materials and methods ) . however , mutation - rate variation across the x chromosome may account for variable levels of diversity observed in the pars and nonpars . when we normalized with pantro4 , the difference in diversity between par1 and nonpars was not significant after filtering out the ampliconic regions , low - diversity regions , and the not applicable ( na ) values . this could be a result of large variation in divergence across regions of the x chromosome between humans and chimpanzees , potentially owing to complex speciation events ( patterson et al . similar to the uncorrected diversity values , when we correct for mutation rate using macaque divergence values , we observe higher nucleotide diversity across humans in par1 and par2 relative to the nonpars , with diversity being significantly higher in par1 than in nonpars ( with xtr removed ) and not significantly different between par2 and nonpars ( figure 1 , figure 3 , and table 1 ) . genetic diversity ( measured by ) is shown in box plots depicting the average diversity with error bars for the nonpars , par1 , xtr , and par2 . the p - values from a permutation test with 10,000 replicates comparing the diversity of each region to the diversity of the nonpars are shown . curiously , human - chimpanzee and human - macaque divergence are quite high in par1 relative to the nonpars in a pattern that does not reflect diversity ( figure 1 and table 1 ) . further , significantly elevated diversity in par1 relative to the nonpars can not be attributed solely to mutation - rate variation across the x chromosome because the pattern remains after correction for divergence in each region ( figure 1 and table 1 ) . the pattern we observed is consistent with several processes , including selection reducing variation more at linked sites in the nonpars than in par1 as a result of reduced rates of recombination in the nonpars relative to the pars or as a result of stronger drift in the nonpars as a result of a smaller effective population size . that we do not observe significantly elevated diversity in par2 relative to the nonpars is consistent with reports that par2 undergoes x - y recombination less frequently than par1 ( flaquer et al . 2008 ) and supports assertions that in humans only one chiasma per chromosome is needed for proper segregation rather than one per chromosome arm ( fledel - alon et al . curiously , in addition to elevated rates of diversity in the previously described par1 and par2 , we also observed that diversity was significantly higher in the recent xtr than in the nonpars ( table 1 and figure 3 ) . this increased diversity can not be attributed to mismapping between the x and y chromosome because we only analyzed individuals with two x chromosomes ( see materials and methods ) . high diversity in the xtr contrasts with initial suggestions that there is no x - y recombination in the xtr ( skaletsky et al . 2003 ) and is consistent with recent reports of x - y recombination in some human populations in this region ( veerappa et al . 2005 ) , one may wonder whether removing the xtr would make a difference to measured levels of diversity across the human x chromosome . removal of the xtr does decrease estimates of both diversity and divergence in the nonpars . although the xtr de facto may be removed with other filters , one should be cautious to include xtr regions because their inclusion in studies of x - specific diversity will affect inferences made when comparing x - linked and autosomal variation ( keinan and reich 2010 ; gottipati et al . recombination between the x and y chromosomes is expected to be suppressed at the pseudoautosomal boundary , where x - y sequence homology diverges owing to a y - specific inversion ( ellis et al . if diversity correlates highly with recombination rate and x - y recombination is strictly suppressed in the nonpars after the pseudoautosomal boundary , then diversity is expected to drop sharply between par1 and the nonpars . however , when we analyze patterns of human diversity in permuted windows across the x chromosome ( see materials and methods ) , we do not observe an abrupt shift in the level of diversity between par1 and the nonpars ( figure 2 ) . in the approximately 3 mb that span the pseudoautosomal boundary , as we shift the window for the regression by 100 kb further from the start of par1 , we observe that the negative correlations remain independently significant and continue past the boundary ( figure 2 ) . the significant linear relationship ( p = 3.281 10 ) that we observe in figure 2 extends nearly twice the length of par1 and supports the observation that there is no clear , abrupt drop in nucleotide diversity across the pseudoautosomal boundary . to test the significance of this correlation , we conducted a permutation test , shuffling windows ( of 100 kb ) across the x chromosome and recomputing the series of linear regressions 10,000 times ; then we computed the number of times a permuted x chromosome had a correlation that was as strong as or stronger than what we observed on the x chromosome ( figure s7 ) . we found that the negative correlation between distance from the short arm of the x chromosome and diversity is significant and spans the pseudoautosomal boundary ( see materials and methods ; p = 0 , permutation test ) . the history of gene conversion between the sex chromosomes may contribute to the increased diversity levels ( trombetta et al . 2014 ) on the nonpar side of the y - specific inversion that marks the pseudoautosomal boundary . human diversity uncorrected for divergence decreases from the proximal end of par1 through the pseudoautosomal boundary and well into the nonpar . a sex - specific map of par1 found that male recombination is higher near the telomeres and decreases near the pseudoautosomal boundary , while , in contrast , the female recombination rate reported in the same study in par1 is fairly flat throughout the region and increases near the pseudoautosomal boundary ( hinch et al . curiously , however , a previous study of recombination rate in par1 reported an increase in the female ( but not the male ) recombination rate near the proximal end of par1 ( henke et al . thus , potentially , both male and female recombination rates contribute to the linear decrease in diversity observed in par1 from the proximal end of the x chromosome through the pseudoautosomal boundary . although not yet mapped , when the data becomes available , it will be useful to compare patterns of diversity with sex - specific recombination maps across the entire x chromosome . we show that diversity is indeed higher in the pseudoautosomal regions and lower in the regions of the x chromosome that are not known to recombine in males ( nonpars ) . diversity in par1 is significantly higher than in the nonpars regardless of normalizing the diversity with divergence between human and either macaque or dog to correct for mutation rate ( table 1 , figure 1 , and figure 3 ) . diversity also was normalized with divergence from the mouse , but there is no alignment between human and mouse in par1 because of a different evolutionary origin in par1 and no common pseudoautosomal genes being shared between them ( gianfrancesco et al . we observed that diversity is lower in par2 than expected and is not significantly different from the nonpars . we also showed that diversity is elevated in the xtr above other nonpars , verifying recent observations that the region still may undergo homologous recombination between the x and y chromosomes ( veerappa et al . finally , when analyzing patterns of genetic diversity in windows across the human x chromosome , we found that there is no strict boundary , based solely on the levels of diversity , between the recombining and putatively nonrecombining regions , which could be attributed to the evolutionary shift in the pseudoautosomal boundary over time , extending par1 as a result of a par1 length polymorphism ( mensah et al . our observations of patterns of diversity across regions of the human x chromosome with variable levels of recombination are consistent with previous reports that diversity and divergence are correlated with recombination rate in humans across the genome ( hellmann et al . curiously , we did not find a significant elevation of diversity in par2 , which , in agreement with its unusual evolution ( charchar et al . 2003 ) , indicates that it rarely recombines between x and y chromosomes during meiosis . further , the lack of a clear differentiation in diversity between par1 and the nonpars suggests that recombination suppression between the x and y chromosomes is still an actively evolving process in humans , as in other species ( bergero and charlesworth 2009 ) . this is consistent with evidence that the position of the pseudoautosomal boundary varies across mammals ( raudsepp and chowdhary 2008 ; otto et al . there is even evidence of polymorphism in the pseudoautosomal boundary in a pedigree analysis of a paternally inherited x chromosome in humans ( mensah et al . recombination spanning the pseudoautosomal boundary may account for some cases of de la chapelle syndrome ( schrander - stumpel et al . taken together with previous inferences about the variation in pseudoautosomal boundaries , our observations suggest that assumptions should not be made of a strict suppression of x - y recombination at the proposed human pseudoautosomal boundary .

alternative taxon sampling strategies are known to affect many facets of phylogenetic reconstruction [ 13 ] . . conducted one of the most thorough analyses assessing the impact of taxon sampling on divergence date estimation using resampling analyses . this study found that mean estimated ages of focal nodes were significantly younger with sparser taxon sampling than when taxa from the complete dataset were included . in addition , the more distant the focal node was from the calibration point , the more sensitive estimation effects were on nodal ages if the taxa were undersampled , especially for nonparametric rate - smoothing ( nprs ) methods ; but penalized likelihood ( pl ) and bayesian methods also were prone to these effects especially if undersampling was large . this is especially important because numerous multigene studies estimate divergence times but often significantly undersample large clades using a single representative species in place of higher taxonomic groups . i argue that unless these large clades are densely sampled and calibration points are carefully chosen and spread across the tree , node divergence times in most cases will be underestimated in these studies . the sampling scheme of linder et al . did not address two important aspects of divergence time estimation . first , the effect of undersampling different numbers within specific clades could not be rigorously tested because they analyzed six smaller subsets created by selectively deleting terminals from the complete 300 species trees . second , the effect of number of fossil calibrations on estimated divergence times was not assessed as only a single basal node was used as a calibration point . use of a single fossil calibration may result in greater error in lineage rate estimates if multiple calibrations are employed . thus , there is general consensus among systematists that the more calibrations spread across the tree , the more accurate the divergence times . in addition , numerous authors have strongly emphasized the importance of critical evaluation of all fossil calibrations prior to use [ 57 ] . unfortunately , for many taxa , the fossil record is very sparse and numerous fossils may not be available given taxon sampling in a particular study . one strategy to circumvent the problem of too few fossil calibrations is to sample additional taxa outside the ingroup where additional fossils may be available . so that , even if the calibrations are a greater distance away from the focal clade , pl and bayesian methods may be able to accommodate rate heterogeneity across a large phylogeny as long as the ingroup is not drastically undersampled . for many species , this is a feasible strategy given the large amount of molecular sequence data collected over the last 30 years and their availability in public databases such as ncbi , treebase , or dryad . i employ this strategy to obtain a revised timeline of diversification within the species - rich clade of south american iguanid lizards liolaemini . the latter two groups contain the bulk of diversity with approximately 37 phymaturus species and at least 230 liolaemus species currently recognized . they are distributed throughout the southern half of south america in almost every habitat from peru to tierra del fuego . within liolaemus , two subclades ( or subgenera ) are recognized : ( 1 ) liolaemus with species mostly distributed west of the andes ; ( 2 ) eulaemus with species distributed mostly east of the andes ; however , both groups contain taxa that cross into low elevation areas on the opposite side or occupy higher elevations . liolaemini lizards also exhibit broad diversity in morphological , ecological , physiological , and life history traits that make them an ideal group to address a wide variety of evolutionary questions . to address these questions , it is essential to have an accurate estimate of diversification times within the group . this study estimated the divergence time between subgenera to be at least 12.6 million years ago based on an mtdna molecular evolutionary rate . however , this should be considered a minimum estimate with the true divergence age likely to be older . schulte ii and moreno - roark estimated crown ages of viviparous clades in liolaemini to be between 3 and 52 mya for several viviparous liolaemus clades and approximately 66 mya for phymaturus . in each of these two studies , at least 62 liolaemini taxa were sampled representing almost all major lineages . a recent analysis of 17 taxa in the subgenus eulaemus and two outgroups from the subgenus liolaemus estimated a crown eulaemus clade age of 18.08 mya with subsequent diversification of the major species groups occurring between 2.97 and 8.1 mya . these node ages were much younger than those estimated by schulte ii and moreno - roark . the discrepancies in divergence times estimated from these studies may be related to a number of factors , such as taxon sampling size differences ( 62 versus 17 ) , number of fossil calibrations ( several versus one ) , time estimation methods ( pl versus bayesian ) , or sequence data sources ( mtdna versus combined nuclear and mtdna ) . for the present study , i examine the first three issues . at present , there is no consensus whether mitochondrial or nuclear dna or a combination of both yield more accurate divergence times . first , the effect on several clade age estimates within iguanid liolaemini lizards is examined by undersampling taxa within individual clades by randomly subsampling within those clades . it is expected that when fewer ingroup species are sampled , node ages will be younger compared to complete sampling . next , i examine the effect of using different numbers of fossil calibrations on those same clade ages and alternative sampling schemes . node ages are expected to show greater variance when fewer calibrations are used and clades are drastically undersampled then with the full complement of fossil calibrations and full sampling . third , compare results of pl and bayesian methods when different numbers of fossil calibrations are used to estimate nodal ages . finally , this analysis will generate a revised timing of diversification of most major clades within liolaemini that can be used by future researchers interested in exploring the history of speciation , biogeography , and evolution of this diverse clade . this study uses a phylogeny of 209 previously published squamate reptile mtdna sequences , primarily within family iguanidae , to reconstruct divergence times and address these four goals . mitochondrial dna sequences representing 92 liolaemini taxa ( here considered the ingroup ) including almost all major lineages , as well as 110 outgroup species from all other clades within iguanidae , three representatives from acrodonta , and four additional outgroups outside iguania , are used in phylogeny and divergence time estimation . genbank accession numbers for all species are presented in supplemental material ( see the supplementary materials at http://dx.doi.org/10.1155/2013/628467 ) . these sequences represent the mitochondrial - encoded region spanning nd1 to coi . for this analysis , the protein - coding regions , part of nd1 , all of nd2 , and part of coi were used , as well as the seven intervening trna regions . dna sequences were aligned manually and then translated to amino acids using mesquite v. 2.75 for confirmation of alignment and to check for premature stop codons . trna secondary structure models were used to assess alignment of all trnas . aligned sequence base positions inferred to have ambiguous homology at the ends of nd1 , nd2 , and several loop regions in trnas were excluded from phylogenetic analyses ( 263 out of 1862 aligned positions14% ) for a final dataset of 1599 aligned base pairs . phylogenetic trees were estimated using maximum likelihood ( ml ) with raxml and mrbayes 3.2.1 in the cipres portal . partitionfinder was used to determine the best - fit model of molecular evolution and partitioning scheme with four independent runs . this program selects the best - fit partitioning scheme and dna sequence models based on predefined data blocks and using alterative information criteria . this program combines the tedious steps of performing modeltest runs and comparisons of different a priori partitions into one analysis . a priori partitions compared in partitionfinder were each codon position ( 1st , 2nd , and 3rd ) for each protein coding gene ( nd1 , nd2 , and coi ) plus one partition for all trna positions ( 10 partitions total ) . bootstrap resampling was applied using raxml with 100 pseudoreplicates and parameter values estimated for each pseudoreplicate . we considered a bootstrap value of 95% as strongly supported , < 95 to 70% as moderately supported , and < 70% as weakly supported . bayesian phylogenetic analyses were performed in mrbayes 3.2.1 using the same sequence evolution model and partitioning scheme as the ml analyses . four independent runs of 20 million generations and four markov chains with default heating values were used . parameter values for the model were estimated from the data and most were initiated with default uniform priors except that branch lengths were unconstrained ( no molecular clock ) with default exponential priors . trees and parameter values were sampled every 1000 generations resulting in 20000 saved trees per analysis , of which the first 25% were discarded as burn - in . stationarity was assessed by plotting the lnl per generation in the program tracer 1.5 and using the average standard deviation of split frequencies implemented in mrbayes . if the four runs are converging on the same tree , the average standard deviation of split frequencies is expected to approach zero . after confirming that the analysis appeared to reach stationarity , the 60000 trees ( 15000 from each run after burn - in ) were used to calculate bayesian credibility values ( bc ) for each branch in a 50% majority - rule consensus tree . clades with bc 95% were considered strongly supported with the caveat that bc may overestimate support for reasons discussed in [ 1921 ] . if significant rate variation is estimated on branches throughout the phylogenetic tree , relaxed clock methods are appropriate . to test whether relaxed clock models are a significantly better fit than a strict clock model , mrbayes was used to generate the posterior distribution of trees with and without enforcing a strict clock . analytical parameters were similar to those detailed above , and log10 bayes factors ( bfs ) calculated in tracer were used to compare the two sets of trees . because there is significant rate variation across the tree ( see bayesian results below ) , two methods were used to estimate divergence times . first , penalized likelihood ( pl ) implemented in r8s version 1.8 was used . pl has been shown to be robust to modest model violations , is easily implemented in a single software package , and performs as well or better than other rate heterogeneity methods such as bayesian and nonparametric rate smoothing in numerous empirical and simulated data sets [ 4 , 7 , 2326 ] . the overall highest maximum likelihood tree was used as a fixed , backbone tree to obtain point divergence time estimates as well as the backbone tree for bootstrapped node age estimates ( see below ) . crossvalidation analyses to determine the optimal smoothing parameter as outlined by sanderson were computationally impractical . however , a previous study found a smoothing parameter value of 0.9 to be optimal using similar taxon and nucleotide data sampling . i also tested a range of smoothing parameters from 0.5 to 2 and found that node age estimates differed by less than one million years for all nodes in the focal group ( liolaemini ) across that range of smoothing parameter values . ten internal nodes were assigned minimum age estimates corresponding to fossil calibrations determined primarily by whether a consensus exists among paleontologists on the relative position of taxa in the squamate phylogeny . minimum age estimates were assigned to stem groups , the most inclusive group of taxa that contains all extant and extinct clade members . pl requires at least one node that is either fixed or set to a maximum age , so the node age of iguania was assigned a maximum age of 218 mya and minimum age of 144 mya . these dates were chosen to span the age of iguania inferred from several previous studies dating this node 's age using mtdna , nuclear dna , or both . all other calibrations were set as minimum ages ( see the appendix for dates , fossils , and supporting references ) . divergence time confidence intervals on the highest likelihood tree were assessed using the bootstrap ( 100 replicates ) method outlined in sanderson using paup * and r8s . the second method implemented bayesian node age estimation using mrbayes 3.2.1 to compare with results from pl estimates . a uniform probability distribution on the age of each calibration node was used with the minimum set to the age of the oldest fossil that could be confidently assigned to the stem age of that clade and a maximum age of 218 million years , which is the maximum age of iguania inferred from previous studies ( see above ) . this calibration age probability distribution is preferred here because the only information that can be incorporated into node dating analysis with confidence is the minimum stem age of the fossil . a fixed distribution was not used because fossils only give information on the minimum age of a clade [ 5 , 6 ] and never its actual age of origin . an offset exponential distribution was not used because although the minimum age information is incorporated with this parameter , the rate component can not be confidently assigned a value . therefore , we prefer to use the uninformative prior of a uniform distribution age prior for all 11 calibration points . we explored three relaxed clock models implemented in mrbayes 3.2.1 to determine which was the best fit for the combination of these data and calibration points . the models are the thorne - kishino 2002 ( tk02 ) model , compound poisson process ( cpp ) model , and the independent gamma rates ( igr ) model . the tk02 model is a continuous autocorrelated model similar to the one implemented in multidivtime [ 29 , 32 ] . the cpp model is a discrete autocorrelated model similar to the model implemented in phylobayes . the igr model is a continuous uncorrelated model where branch rates are drawn independently from a gamma distribution [ 18 , 31 ] . this model is similar ( though not identical ) to the model implemented in beast . therefore , none of these mentioned software packages were used as the relaxed clock models implemented by them could be evaluated with the following mrbayes analyses . to identify which relaxed clock model was the best fit for this dataset , and 11 calibration points , i conducted analyses as in the nonclock analyses above using four independent runs of four parallel chains each for each of the three models using all calibration points . burn - in was set to 25% of samples and discarded prior to comparisons of the distribution of posterior samples using bayes factors calculated in tracer . preliminary analyses showed the cpp model to be a poorer fit than either the tk02 and igr models under a variety of conditions and is not considered further . two additional parameters implemented in the tk02 and igr models were examined to determine whether there was a better fit to the data . these parameters estimate the rate at which variance of the effective branch length increases over time under the respective models . for such a large dataset , it is be possible that large rate differences in effective branch lengths across the tree could affect divergence time estimates in different parts of the tree . for both tk02varpr and igrvarpr parameters , the exponential and uniform distributions were evaluated using the default priors of 10 and ( 0,1 ) , respectively , for each of the distributions . to examine the effect of undersampling taxa on divergence time estimates within liolaemini for each of the three sets , different numbers of taxa were randomly deleted from only the large liolaemus clade in which 85 taxa were sampled here , but all other ingroup and outgroup taxa were included as these are where most of the calibration points lie . the three sets of sampling schemes were as follows : ( 1 ) for the 85 taxon clade corresponding to genus liolaemus , 3 , 5 , 15 , 30 , 50 , 70 , 80 , and 82 taxa were randomly deleted from the clade ; ( 2 ) for the clade corresponding to subgenus eulaemus ( 49 species ) , 3 , 5 , 15 , 30 , 44 and 46 taxa were randomly deleted ; and ( 3 ) for the clade corresponding to subgenus liolaemus ( 36 species ) , 3 , 5 , 15 , 30 , and 33 taxa were randomly deleted . all subsampled datasets had to be manually manipulated to obtain summaries of results , so 25 replicates per sampling scheme ( taxon deletion set ) were produced . this number is expected to yield relatively accurate mean and standard deviation estimates of nodes ages on subsampled datasets . for each taxon deletion set , a file was created that contained the aligned sequence of the sampled taxa and the associated tree without the deleted taxa . these files were then analyzed in paup * using ml ( without a priori partitions due to computational limitations ) and the gtr++i model to obtain estimates of branch lengths on each of the subsampled trees . all trees were analyzed in r8s using the same conditions as with the full tree to obtain mean and standard deviation divergence time estimates for each subsampling scheme using the profile command in r8s . mabuya , cnemidophorus , elgaria , and varanus outgroup sequences were removed prior to pl analyses to prevent overestimation of the evolutionary rate across the phylogeny . for this part of the study , only pl in r8s was used to assess the affect of undersampling taxa on divergence time estimates as analyzing all the resampled datasets with the current implementation of mrbayes and manipulation of results files was computationally impractical . another goal of this study is to examine the effect of using fewer calibrated nodes on divergence time estimates . for this part of the study first , we reran all three sets of subsampled trees in r8s using only the node representing the common ancestor of iguania set with a maximum age of 218 mya and minimum age of 144 mya and all other nodes without calibrations . the second strategy used only two calibrations , the root node calibrated as above and the pristidactylus fossil described by albino from the early - mid miocene set at a minimum age of 16 mya ( see the appendix ) . this fossil was chosen rather than the liolaemus fossil from the same horizon because of the remaining nine sampled fossil calibrations ; it is the phylogenetically closest group , and the subsampled datasets made the use of the liolaemus fossil impractical computationally . again only pl using r8s was used to analyze all three alternative subsampling sets under the different numbers of calibrations used . however , mrbayes was used to assess the affect of different numbers of calibrations on divergence time estimates for the complete dataset . that is , mrbayes analyses performed as above using the optimal molecular clock ( see below ) were run using the complete set of 11 calibration points , only the iguanian node calibration , and the iguania plus pristidactylus fossil calibration . the best - fit partitioning scheme using the akaike information criterion in partitionfinder had a value of lnl = 98586.62 and found six partitions and a gtr++i model ( except partitions 1 and 6 which found gtr+ to be optimal ) best explained the data : ( 1 ) third codon positions in nd1 and nd2 ; ( 2 ) first codon position nd2 ; ( 3 ) second codon position nd2 ; ( 4 ) trna positions ; ( 5 ) first codon positions in nd1 and coi , third codon positions in coi ; ( 6 ) second codon positions in nd1 and coi . however , subsequent ml and bayesian analyses assumed a general time reversible sequence evolution model with gamma distributed rate variation and proportion of invariant sites ( gtr++i ) for all partitions . a single topology was found ( lnl = 98478.29 , figure 1 ) in the raxml analysis of nd1-coi data using this partitioning scheme with gtr++i for all sites . relationships among the major groups of liolamini lizards are generally consistent with previous hypotheses recovered using this region of mtdna [ 8 , 9 , 37 ] . monophyly of phymaturus and liolaemus ( 100% bootstrap ) as well as their sister taxon relationship ( 99% bootstrap ) are well supported . there is weak bootstrap support ( 76% ) for the monotypic genus ctenoblepharys as the sister taxon to the clade containing phymaturus and liolaemus . within liolaemus , the monophyly of the two subgenera , liolaemus and eulaemus , are each recovered with moderate and strong support ( 94% and 100% bootstrap , resp . ) . the first group is moderately supported ( 93% ) and contains taxa generally distributed in northern chile and includes l. cf . the sister group to this clade contains both l. tenuis and l. t. punctatissimus and a well supported ( 98% bootstrap ) clade containing l. leminscatus , l. monticola , l. nitidus , l. fuscus , and l. nigroviridis . all of the latter species are mostly distributed at a wide range of elevations in central chile . the third well - supported group is composed of taxa distributed primarily at mid and high elevations in the central and southern andes and includes l. capillitas , species in the l. elongatus complex , and l. ceii and kriegi species complexes . the sister group to the latter clade is composed of l. coeruleus as the sister taxon to two well - supported groups . a monophyletic group comprising a diverse group of small bodied taxa that occupy the highest elevations in the andes and low elevations in southern argentina includes species from the l. alticolor and bibronii species groups . the final clade is weakly supported ( 61% bootstrap ) as the sister group to the previous well - supported ( 100% ) clade and contains taxa that occupy scrub and forest habitats in central and southern chile , such as l. chiliensis , l. gravenhorstii , l. bellii , and l. pictus . subgenus eulaemus is composed of taxa primarily in the andes and eastern lowlands with five principal clades emphasized here ( figure 1 ) . a monophyletic group containing species from generally high latitudes in the eastern lowlands corresponding to the l. lineomaculatus section ( l. lineomaculatus , l. magellanicus , l. kingii , and l. somuncurae ) of schulte ii et al . is strongly supported as monophyletic and the sister group to the remaining eulaemus species ( 100% bootstrap ) . among the remaining species is a large well - supported monophyletic group corresponding to the l. montanus series and includes taxa distributed primarily at mid- to high elevations on the puna plateau . the remaining species in the eastern clade are contained mostly in three well - supported groups ( 100% bootstrap ) , the wiegmannii series , a monophyletic group corresponding to the melanops series of fontanella et al . , and a clade containing species in the l. darwinii species complex . liolaemus pseudoanomalus and two representatives of the l. rothi species group also are recovered in the remaining species with eulaemus , but their phylogenetic position is not well supported in this analysis . the harmonic mean lnl of the consensus topology of the 60000 trees from the posterior distributions of nonclock analyses using mrbayes was 102558.23 . the topology of the ingroup ( liolaemini ) was generally very similar to the nonclock ml analyses with a few minor exceptions . those exceptions were alternative positions in the two topologies of the following clades or single taxa : ( 1 ) liolaemus tenuis and l. t. punctatissimus ; ( 2 ) l. pseudoanomalus ; and ( 3 ) relationships of l. abaucan , l. koslowskyi , and l. quilmes with the l. boulengeri series . in addition , all differences between the nonclock mrbayes consensus topology and the ml topology were poorly supported with ml bootstraps and bayesian credibility ( bc ) values below 95% . the comparison of the posterior distribution of trees from bayesian analyses with and without a strict molecular clock enforced found that the distribution of trees generated from enforcing a strict molecular clock was significantly worse fit than without a strict clock enforced ( log10bf values between 99101 ) . penalized likelihood ( pl ) divergence time analyses using the overall highest ml tree and bootstrapped branch lengths and all calibrations revealed that the mean divergence time for crown clade liolaemini was 111.3 mya ( sd = 10.8 ) , which is good within the early cretaceous period . within liolaemini , crown phymaturus and liolaemus diverged 39.6 mya ( sd = 5.4 ) and 47.6 mya ( sd = 5.9 ) , respectively . the divergence time estimates for the initial splits within the crown subgenera liolaemus ( 38.9 mya ( sd = 5.6 ) ) and eulaemus ( 35.8 mya ( sd = 4.8 ) ) occurred in the late eocene . for the 10 primary clades that comprise the majority of diversity within genus liolaemus , divergences occurred almost entirely in the miocene with the earliest divergence at the oligocene - miocene boundary ( 24.09.9 mya ) . this range of node ages is contained entirely within the five clades of subgenus liolaemus with the l. nigromaculatus group species diverging earliest at 24 mya ( sd = 3.9 ) and the l. chiliensis group species diverging in the early late miocene 9.9 mya ( sd = 2.3 ) . within the subgenus eulaemus , the earliest divergence was the l. lineomaculatus series ( 18.8 mya ( sd = 3.2 ) ) in the early miocene and the remaining splits occurring in the early to middle miocene 17.1 to 11.5 mya . the igr relaxed clock model provided the best fit of these data and calibration points over the cpp and tk02 using log10bf calculated in tracer ( results not shown did not strongly favor either the exponential or uniform distribution priors for the branch length rate variance parameter . thus , both rate variance parameters were run for the igr molecular clock model and their results on molecular divergence time estimates are presented in table 6 . when all 11 calibrations and either the exponential or uniform igrvarpr was used , in all cases , divergence time estimates for the five focal nodes within liolaemini were older than pl estimates . the greatest age estimate difference between the igr - exponential model and pl was for the subgenus liolaemus . comparing the igr - exponential model to the igr - uniform model , in all cases , the uniform model yielded age estimates younger than those generated from the exponential model . however , it should be noted that given the error typically associated with divergence time estimation using any method , the standard deviations of the bootstrapped pl estimates strongly overlap with all 95% posterior credibility intervals from bayesian analyses . substantial differences were found among clade ages as taxa are increasingly undersampled . for the 85-taxon subsampling set where taxa were randomly deleted within the genus liolaemus , the mean crown age of this clade was 29 and 33.6 mya when only 3 and 5 taxa were sampled , respectively , compared to 47.6 for all 85 taxa ( table 1 ) . crown clade ages of deeper nodes in the tree such as liolaemini also were affected and estimated to have diverged 83.7 mya when only three liolaemus taxa were randomly sampled compared to 111.3 mya with the complete 85-taxon sampling . in fact , the sister taxon phymaturus , whose sampling remained the same through all subsampling schemes , also had a reduced crown clade age of 28.9 mya with three liolaemus sampled compared to a crown clade age of 39.6 mya ( table 1 ) . the trend for all five focal clades was that increasing the number of taxa deleted from the tree had the effect of making the mean divergence times younger with very few exceptions . for subsampling strategies two ( randomly deleting taxa in only subgenus eulaemus ) and three ( randomly deleting taxa in only subgenus liolaemus ) , a considerable difference in crown ages of these groups was recovered with extensive undersampling . when only three and five taxa are sampled from the full 49-taxon clade eulaemus , the estimated crown age of this group is reduced from 35.8 mya to 19.3 and 21.5 , respectively ( table 2 ) . as above , the subgenus liolaemus in subsampling set two also was affected by subsampling its sister clade showing a reduced crown clade age of 29.4 mya compared to 39 mya with a fully sampled dataset . in the final subsampling datasets when the taxa in the subgenus liolaemus are undersampled with only three and five taxa , the crown ages of this clade are 25.3 and 28 mya , respectively compared to 39 mya with complete sampling of 36 taxa ( table 3 ) . again there is an effect on the age estimates of the sister clade ( subgenus eulaemus ) with crown ages becoming younger with 35.8 mya for complete sampling of subgenus liolaemus taxa to 25.8 mya when only 3 taxa were sampled . when only the node representing the common ancestor of iguania was used as the calibration point in the complete data set , all divergence time estimates of crown clade ages across the tree were younger ( tables 4 and 5 ) . at the deeper level of the tree , the crown clade age of liolaemini was younger by between 12 and 16 million years . the genera phymaturus and liolaemus had crown clade ages that were younger by six and seven million years , respectively . the subgenera eulaemus and liolaemus each had clade ages that were 30.4 and 33.1 compared to 35.8 and 39.0 mya when the full complement of eleven calibrations was used ( table 4 ) . when all these datasets were rerun including the former single , deep calibration and a shallow , phylogenetically closer calibration fossil ( pristidactylus ) , results were very similar to one - calibration analyses with most node ages increasing in age by approximately 13 million years ( table 5 ) . i examined the combined effect of using fewer calibrations when taxa were undersampled using subsampling set 1 ( randomly deleting among 85 liolaemus taxa ) . this strategy is similar to the sampling used by fontanella et al . except that there were no taxa sampled outside liolaemini in that study . when at least 70 taxa are randomly deleted from the full complement of ingroup liolaemus taxa , the remaining 15 taxa in liolaemus yield mean crown clade estimates of 29.6 mya using one calibration point and 29.5 mya for two calibrations . this is 18 million years younger than the mean age of 47.6 mya using all calibrations and full sampling and translates to the initial diversification event in the genus liolaemus in the middle eocene to early oligocene . the mean crown clade ages of the two subgenera also are substantially lower when only 15 taxa are sampled . using one or two calibrations gives similar estimates for the crown ages of subgenera eulaemus around 19 mya and liolaemus at 23.5 mya , respectively , compared to 35.8 and 39 mya using all calibrations . the age estimate obtained by fontanella et al . for the crown age of eulaemus sampling 17 taxa and using one calibration point was 18.08 mya , which is very similar to the estimate of 19 mya here also using a single calibration , and 15 sampled taxa . these differences again move initial diversification of these groups from the late eocene to near the boundary of the early miocene and late oligocene . for bayesian analyses , when using only the single calibration of the common ancestor of iguania , all ingroup node age estimates were older for both the igr - exponential and uniform models compared to using all eleven calibrations . in contrast to the eleven calibration analysis using complete taxon sampling , the igr uniform model estimated all crown clade ages to be slightly older compared to estimates from the igr exponential model . when the ancestral iguanian node calibration was combined with the more recent calibration of the node representing the pristidactylus fossil , all age estimates were intermediate between the age estimates from the single calibration and complete eleven calibration estimates ( table 6 ) . penalized likelihood node age estimates reported here using undersampled clades and either one or two calibration fossils yield divergence times much younger compared to clades densely sampled and using eleven internal calibrations . the range of differences in mean clade ages within liolaemini spanned from more than 45% younger when subgenus eulaemus was significantly undersampled ( sampling only 3 out of 49 taxa ) while most other ages were between 20 and 30% younger using other subsampling schemes . it should be noted that node age estimates from this study should still be considered as minimum estimates as less than half the ~270 species within liolaemini were sampled as well as less than one quarter of all species in iguanidae . several studies have directly compared the results of divergence time estimates from pl and bayesian methods [ 4 , 39 , 40 ] . for two of these studies [ 39 , 40 ] , bayesian age estimates for focal nodes consistently were younger than pl divergence time estimates . however , linder et al . found that when taxa were densely sampled , bayesian node age estimates were always older than pl estimates although undersampling different numbers of taxa in this latter study did not yield a consistent pattern for either method . the results of the present work show bayesian node age estimates to be older than pl estimates when taxa are densely sampled and either all , one , or two calibration points are used . penalized likelihood is generally robust to model violations and taxon undersampling [ 4 , 7 , 25 , 26 ] , but a clear pattern can be seen in all subsampled datasets randomly removing taxa from individual clades . the more taxa removed from a particular clade , the younger age estimates of the nodes subtending those clades . this result is shown to affect node ages throughout the tree , clades directly undersampled , groups closely related to the focal clade , and node ages of clades deep in the tree . for example , the crown clade age of iguanidae was estimated to be up to 15 million years younger by undersampling liolaemus taxa ( results not shown ) . . showed no effect on divergence time estimates from undersampling individual clades but admit that this aspect was not rigorously tested since most clades were represented by less than 30% of extant species . recently , advances in sequencing technologies have facilitated acquisition of dozens to thousands of nuclear loci for a few taxa with many of these studies publishing timetrees . for example , over the last decade , there have been numerous multigene nuclear and mtdna studies published estimating divergence times within squamate reptiles [ 4043 ] . for some of these studies , divergence times among clades sampled here are generally similar but other studies attempting to estimate the age of iguanidae , and liolaemini along with its contained clades drastically undersample taxa using , in some cases , less 1% of extant species in a particular clade . as shown here and elsewhere , such extreme taxon undersampling will yield younger divergence time estimates regardless of dating methodology or data types used . numerous studies have attempted to estimate the node ages in the south american lizard clade liolaemini [ 810 , 4447 ] . with the exception of the former two studies , these studies have focused on estimating divergence times within species complexes and closely related groups shallow in liolaemus phylogeny using either a single calibration point or molecular evolutionary rate estimates . most divergence times among lineages in these latter studies were recovered as occurring in the pliocene and pleistocene and are likely to be underestimates . this led the authors of those works to conclude that diversification among species groups within liolaemus was most likely driven by glacial cycling processes over the last 3.5 million years . i agree that glacial cycling in the higher elevation regions of the andes and the southern tip of south america likely played a significant role in shaping current diversity within liolaemus , but as shown here , most divergence events among species occurred prior to the onset of pliocene - pleistocene glaciations . so that the primary impact of the glacial cycling is likely to be in shaping the current distributions and phylogeographic breaks within species rather than between species in most cases . it is recommended that future studies at any phylogenetic level utilize as many calibration points as possible to obtain accurate divergence times in all parts of the tree . in those cases where very few or no calibration points may be available for ingroup taxa sampled , this study has shown that it is better to include many closely related and distant outgroups where multiple calibrations can be applied to improve divergence time estimation . otherwise , node ages should be considered minimum ages for future studies and caution should be taken when attempting to make inferences of evolutionary processes driving diversification of modern taxa .

methods for estimating divergence times from molecular data have improved dramatically over the past decade , yet there are few studies examining alternative taxon sampling effects on node age estimates . here , i investigate the effect of undersampling species diversity on node ages of the south american lizard clade liolaemini using several alternative subsampling strategies for both time calibrations and taxa numbers . penalized likelihood ( pl ) and bayesian molecular dating analyses were conducted on a densely sampled ( 202 taxa ) mtdna - based phylogenetic hypothesis of iguanidae , including 92 liolaemini species . using all calibrations and penalized likelihood , clades with very low taxon sampling had node age estimates younger than clades with more complete taxon sampling . the effect of bayesian and pl methods differed when either one or two calibrations only were used with dense taxon sampling . bayesian node ages were always older when fewer calibrations were used , whereas pl node ages were always younger . this work reinforces two important points : ( 1 ) whenever possible , authors should strongly consider adding as many taxa as possible , including numerous outgroups , prior to node age estimation to avoid considerable node age underestimation and ( 2 ) using more , critically assessed , and accurate fossil calibrations should yield improved divergence time estimates .

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

alternative taxon sampling strategies are known to affect many facets of phylogenetic reconstruction [ 13 ] . conducted one of the most thorough analyses assessing the impact of taxon sampling on divergence date estimation using resampling analyses . this study found that mean estimated ages of focal nodes were significantly younger with sparser taxon sampling than when taxa from the complete dataset were included . in addition , the more distant the focal node was from the calibration point , the more sensitive estimation effects were on nodal ages if the taxa were undersampled , especially for nonparametric rate - smoothing ( nprs ) methods ; but penalized likelihood ( pl ) and bayesian methods also were prone to these effects especially if undersampling was large . i argue that unless these large clades are densely sampled and calibration points are carefully chosen and spread across the tree , node divergence times in most cases will be underestimated in these studies . did not address two important aspects of divergence time estimation . first , the effect of undersampling different numbers within specific clades could not be rigorously tested because they analyzed six smaller subsets created by selectively deleting terminals from the complete 300 species trees . second , the effect of number of fossil calibrations on estimated divergence times was not assessed as only a single basal node was used as a calibration point . in addition , numerous authors have strongly emphasized the importance of critical evaluation of all fossil calibrations prior to use [ 57 ] . unfortunately , for many taxa , the fossil record is very sparse and numerous fossils may not be available given taxon sampling in a particular study . for many species , this is a feasible strategy given the large amount of molecular sequence data collected over the last 30 years and their availability in public databases such as ncbi , treebase , or dryad . within liolaemus , two subclades ( or subgenera ) are recognized : ( 1 ) liolaemus with species mostly distributed west of the andes ; ( 2 ) eulaemus with species distributed mostly east of the andes ; however , both groups contain taxa that cross into low elevation areas on the opposite side or occupy higher elevations . these node ages were much younger than those estimated by schulte ii and moreno - roark . the discrepancies in divergence times estimated from these studies may be related to a number of factors , such as taxon sampling size differences ( 62 versus 17 ) , number of fossil calibrations ( several versus one ) , time estimation methods ( pl versus bayesian ) , or sequence data sources ( mtdna versus combined nuclear and mtdna ) . at present , there is no consensus whether mitochondrial or nuclear dna or a combination of both yield more accurate divergence times . first , the effect on several clade age estimates within iguanid liolaemini lizards is examined by undersampling taxa within individual clades by randomly subsampling within those clades . it is expected that when fewer ingroup species are sampled , node ages will be younger compared to complete sampling . next , i examine the effect of using different numbers of fossil calibrations on those same clade ages and alternative sampling schemes . node ages are expected to show greater variance when fewer calibrations are used and clades are drastically undersampled then with the full complement of fossil calibrations and full sampling . third , compare results of pl and bayesian methods when different numbers of fossil calibrations are used to estimate nodal ages . this study uses a phylogeny of 209 previously published squamate reptile mtdna sequences , primarily within family iguanidae , to reconstruct divergence times and address these four goals . mitochondrial dna sequences representing 92 liolaemini taxa ( here considered the ingroup ) including almost all major lineages , as well as 110 outgroup species from all other clades within iguanidae , three representatives from acrodonta , and four additional outgroups outside iguania , are used in phylogeny and divergence time estimation . for this analysis , the protein - coding regions , part of nd1 , all of nd2 , and part of coi were used , as well as the seven intervening trna regions . trna secondary structure models were used to assess alignment of all trnas . bayesian phylogenetic analyses were performed in mrbayes 3.2.1 using the same sequence evolution model and partitioning scheme as the ml analyses . analytical parameters were similar to those detailed above , and log10 bayes factors ( bfs ) calculated in tracer were used to compare the two sets of trees . because there is significant rate variation across the tree ( see bayesian results below ) , two methods were used to estimate divergence times . first , penalized likelihood ( pl ) implemented in r8s version 1.8 was used . pl has been shown to be robust to modest model violations , is easily implemented in a single software package , and performs as well or better than other rate heterogeneity methods such as bayesian and nonparametric rate smoothing in numerous empirical and simulated data sets [ 4 , 7 , 2326 ] . the overall highest maximum likelihood tree was used as a fixed , backbone tree to obtain point divergence time estimates as well as the backbone tree for bootstrapped node age estimates ( see below ) . i also tested a range of smoothing parameters from 0.5 to 2 and found that node age estimates differed by less than one million years for all nodes in the focal group ( liolaemini ) across that range of smoothing parameter values . ten internal nodes were assigned minimum age estimates corresponding to fossil calibrations determined primarily by whether a consensus exists among paleontologists on the relative position of taxa in the squamate phylogeny . minimum age estimates were assigned to stem groups , the most inclusive group of taxa that contains all extant and extinct clade members . all other calibrations were set as minimum ages ( see the appendix for dates , fossils , and supporting references ) . divergence time confidence intervals on the highest likelihood tree were assessed using the bootstrap ( 100 replicates ) method outlined in sanderson using paup * and r8s . the second method implemented bayesian node age estimation using mrbayes 3.2.1 to compare with results from pl estimates . a uniform probability distribution on the age of each calibration node was used with the minimum set to the age of the oldest fossil that could be confidently assigned to the stem age of that clade and a maximum age of 218 million years , which is the maximum age of iguania inferred from previous studies ( see above ) . therefore , none of these mentioned software packages were used as the relaxed clock models implemented by them could be evaluated with the following mrbayes analyses . to identify which relaxed clock model was the best fit for this dataset , and 11 calibration points , i conducted analyses as in the nonclock analyses above using four independent runs of four parallel chains each for each of the three models using all calibration points . burn - in was set to 25% of samples and discarded prior to comparisons of the distribution of posterior samples using bayes factors calculated in tracer . for such a large dataset , it is be possible that large rate differences in effective branch lengths across the tree could affect divergence time estimates in different parts of the tree . for both tk02varpr and igrvarpr parameters , the exponential and uniform distributions were evaluated using the default priors of 10 and ( 0,1 ) , respectively , for each of the distributions . to examine the effect of undersampling taxa on divergence time estimates within liolaemini for each of the three sets , different numbers of taxa were randomly deleted from only the large liolaemus clade in which 85 taxa were sampled here , but all other ingroup and outgroup taxa were included as these are where most of the calibration points lie . the three sets of sampling schemes were as follows : ( 1 ) for the 85 taxon clade corresponding to genus liolaemus , 3 , 5 , 15 , 30 , 50 , 70 , 80 , and 82 taxa were randomly deleted from the clade ; ( 2 ) for the clade corresponding to subgenus eulaemus ( 49 species ) , 3 , 5 , 15 , 30 , 44 and 46 taxa were randomly deleted ; and ( 3 ) for the clade corresponding to subgenus liolaemus ( 36 species ) , 3 , 5 , 15 , 30 , and 33 taxa were randomly deleted . these files were then analyzed in paup * using ml ( without a priori partitions due to computational limitations ) and the gtr++i model to obtain estimates of branch lengths on each of the subsampled trees . all trees were analyzed in r8s using the same conditions as with the full tree to obtain mean and standard deviation divergence time estimates for each subsampling scheme using the profile command in r8s . mabuya , cnemidophorus , elgaria , and varanus outgroup sequences were removed prior to pl analyses to prevent overestimation of the evolutionary rate across the phylogeny . for this part of the study , only pl in r8s was used to assess the affect of undersampling taxa on divergence time estimates as analyzing all the resampled datasets with the current implementation of mrbayes and manipulation of results files was computationally impractical . another goal of this study is to examine the effect of using fewer calibrated nodes on divergence time estimates . for this part of the study first , we reran all three sets of subsampled trees in r8s using only the node representing the common ancestor of iguania set with a maximum age of 218 mya and minimum age of 144 mya and all other nodes without calibrations . this fossil was chosen rather than the liolaemus fossil from the same horizon because of the remaining nine sampled fossil calibrations ; it is the phylogenetically closest group , and the subsampled datasets made the use of the liolaemus fossil impractical computationally . however , mrbayes was used to assess the affect of different numbers of calibrations on divergence time estimates for the complete dataset . the best - fit partitioning scheme using the akaike information criterion in partitionfinder had a value of lnl = 98586.62 and found six partitions and a gtr++i model ( except partitions 1 and 6 which found gtr+ to be optimal ) best explained the data : ( 1 ) third codon positions in nd1 and nd2 ; ( 2 ) first codon position nd2 ; ( 3 ) second codon position nd2 ; ( 4 ) trna positions ; ( 5 ) first codon positions in nd1 and coi , third codon positions in coi ; ( 6 ) second codon positions in nd1 and coi . the sister group to this clade contains both l. tenuis and l. t. punctatissimus and a well supported ( 98% bootstrap ) clade containing l. leminscatus , l. monticola , l. nitidus , l. fuscus , and l. nigroviridis . those exceptions were alternative positions in the two topologies of the following clades or single taxa : ( 1 ) liolaemus tenuis and l. t. punctatissimus ; ( 2 ) l. pseudoanomalus ; and ( 3 ) relationships of l. abaucan , l. koslowskyi , and l. quilmes with the l. boulengeri series . penalized likelihood ( pl ) divergence time analyses using the overall highest ml tree and bootstrapped branch lengths and all calibrations revealed that the mean divergence time for crown clade liolaemini was 111.3 mya ( sd = 10.8 ) , which is good within the early cretaceous period . the divergence time estimates for the initial splits within the crown subgenera liolaemus ( 38.9 mya ( sd = 5.6 ) ) and eulaemus ( 35.8 mya ( sd = 4.8 ) ) occurred in the late eocene . this range of node ages is contained entirely within the five clades of subgenus liolaemus with the l. nigromaculatus group species diverging earliest at 24 mya ( sd = 3.9 ) and the l. chiliensis group species diverging in the early late miocene 9.9 mya ( sd = 2.3 ) . thus , both rate variance parameters were run for the igr molecular clock model and their results on molecular divergence time estimates are presented in table 6 . when all 11 calibrations and either the exponential or uniform igrvarpr was used , in all cases , divergence time estimates for the five focal nodes within liolaemini were older than pl estimates . comparing the igr - exponential model to the igr - uniform model , in all cases , the uniform model yielded age estimates younger than those generated from the exponential model . however , it should be noted that given the error typically associated with divergence time estimation using any method , the standard deviations of the bootstrapped pl estimates strongly overlap with all 95% posterior credibility intervals from bayesian analyses . the trend for all five focal clades was that increasing the number of taxa deleted from the tree had the effect of making the mean divergence times younger with very few exceptions . for subsampling strategies two ( randomly deleting taxa in only subgenus eulaemus ) and three ( randomly deleting taxa in only subgenus liolaemus ) , a considerable difference in crown ages of these groups was recovered with extensive undersampling . when only three and five taxa are sampled from the full 49-taxon clade eulaemus , the estimated crown age of this group is reduced from 35.8 mya to 19.3 and 21.5 , respectively ( table 2 ) . in the final subsampling datasets when the taxa in the subgenus liolaemus are undersampled with only three and five taxa , the crown ages of this clade are 25.3 and 28 mya , respectively compared to 39 mya with complete sampling of 36 taxa ( table 3 ) . again there is an effect on the age estimates of the sister clade ( subgenus eulaemus ) with crown ages becoming younger with 35.8 mya for complete sampling of subgenus liolaemus taxa to 25.8 mya when only 3 taxa were sampled . when only the node representing the common ancestor of iguania was used as the calibration point in the complete data set , all divergence time estimates of crown clade ages across the tree were younger ( tables 4 and 5 ) . i examined the combined effect of using fewer calibrations when taxa were undersampled using subsampling set 1 ( randomly deleting among 85 liolaemus taxa ) . this is 18 million years younger than the mean age of 47.6 mya using all calibrations and full sampling and translates to the initial diversification event in the genus liolaemus in the middle eocene to early oligocene . the mean crown clade ages of the two subgenera also are substantially lower when only 15 taxa are sampled . using one or two calibrations gives similar estimates for the crown ages of subgenera eulaemus around 19 mya and liolaemus at 23.5 mya , respectively , compared to 35.8 and 39 mya using all calibrations . for the crown age of eulaemus sampling 17 taxa and using one calibration point was 18.08 mya , which is very similar to the estimate of 19 mya here also using a single calibration , and 15 sampled taxa . for bayesian analyses , when using only the single calibration of the common ancestor of iguania , all ingroup node age estimates were older for both the igr - exponential and uniform models compared to using all eleven calibrations . in contrast to the eleven calibration analysis using complete taxon sampling , the igr uniform model estimated all crown clade ages to be slightly older compared to estimates from the igr exponential model . when the ancestral iguanian node calibration was combined with the more recent calibration of the node representing the pristidactylus fossil , all age estimates were intermediate between the age estimates from the single calibration and complete eleven calibration estimates ( table 6 ) . penalized likelihood node age estimates reported here using undersampled clades and either one or two calibration fossils yield divergence times much younger compared to clades densely sampled and using eleven internal calibrations . the range of differences in mean clade ages within liolaemini spanned from more than 45% younger when subgenus eulaemus was significantly undersampled ( sampling only 3 out of 49 taxa ) while most other ages were between 20 and 30% younger using other subsampling schemes . it should be noted that node age estimates from this study should still be considered as minimum estimates as less than half the ~270 species within liolaemini were sampled as well as less than one quarter of all species in iguanidae . several studies have directly compared the results of divergence time estimates from pl and bayesian methods [ 4 , 39 , 40 ] . for two of these studies [ 39 , 40 ] , bayesian age estimates for focal nodes consistently were younger than pl divergence time estimates . found that when taxa were densely sampled , bayesian node age estimates were always older than pl estimates although undersampling different numbers of taxa in this latter study did not yield a consistent pattern for either method . the results of the present work show bayesian node age estimates to be older than pl estimates when taxa are densely sampled and either all , one , or two calibration points are used . the more taxa removed from a particular clade , the younger age estimates of the nodes subtending those clades . this result is shown to affect node ages throughout the tree , clades directly undersampled , groups closely related to the focal clade , and node ages of clades deep in the tree . showed no effect on divergence time estimates from undersampling individual clades but admit that this aspect was not rigorously tested since most clades were represented by less than 30% of extant species . for example , over the last decade , there have been numerous multigene nuclear and mtdna studies published estimating divergence times within squamate reptiles [ 4043 ] . for some of these studies , divergence times among clades sampled here are generally similar but other studies attempting to estimate the age of iguanidae , and liolaemini along with its contained clades drastically undersample taxa using , in some cases , less 1% of extant species in a particular clade . as shown here and elsewhere , such extreme taxon undersampling will yield younger divergence time estimates regardless of dating methodology or data types used . numerous studies have attempted to estimate the node ages in the south american lizard clade liolaemini [ 810 , 4447 ] . with the exception of the former two studies , these studies have focused on estimating divergence times within species complexes and closely related groups shallow in liolaemus phylogeny using either a single calibration point or molecular evolutionary rate estimates . this led the authors of those works to conclude that diversification among species groups within liolaemus was most likely driven by glacial cycling processes over the last 3.5 million years . i agree that glacial cycling in the higher elevation regions of the andes and the southern tip of south america likely played a significant role in shaping current diversity within liolaemus , but as shown here , most divergence events among species occurred prior to the onset of pliocene - pleistocene glaciations . it is recommended that future studies at any phylogenetic level utilize as many calibration points as possible to obtain accurate divergence times in all parts of the tree .

recent studies indicate that prevalence of both prediabetes and overt diabetes continues to rise in the u.s . . this increasing prevalence may be due to an increase in diabetes incidence , improved survival among individuals with diabetes , or a combination of these factors . the trends in prediabetes and diabetes prevalence have paralleled high rates of overweight and obesity , which are leading risk factors for the development of diabetes ( 4,5 ) . recent data , however , suggest the prevalence of obesity among adults , which was increasing from the 1970s through the early 2000s , may have leveled off in the past few years ( 4,6,7 ) . specifically , data from the national health and nutrition examination survey ( a large nationally representative sample ) suggested that the prevalence of obesity , while high , did not change significantly between 2003 and 2004 and 2011 and 2012 ( 6 ) . there are few estimates of trends in diabetes incidence in recent times ( 8) . some existing estimates focus on a single ethnic group or age group and thus may not apply to the general adult population ( 9,10 ) . other estimates are based on self - reported diabetes , which may underestimate the true incidence ( 11,12 ) . we previously reported diabetes incidence in our cohort in the 1970s , 1980s , and 1990s ( 8) , and in the current study , we seek to update these incidence estimates in the context of the obesity epidemic . in the framingham heart study , screening for diabetes is conducted at each examination , along with measures of adiposity such as bmi . thus , our study is able to provide reliable estimates of diabetes incidence and furthermore to assess the associations of overweight and obesity to the observed trends . the framingham heart study is a community - based cohort study of cardiovascular disease and risk factors that has been in continuous operation since 1948 beginning with the original cohort . recruitment of the offspring cohort began in 1971 ; participants were invited back for a second examination 8 years after the initial exam and subsequently every 4 years thereafter . the second examination cycle lasted from 2008 through 2011 . to ensure an overlapping age distribution across all decades , we limited the current analysis to participants ages 40 to 55 years who were free of diabetes at the baseline examination . prevalent diabetes was defined as fasting glucose 126 mg / dl or on treatment with insulin or other antidiabetes agent . offspring participants ages 4055 years and free of diabetes at exam 1 , which ran from 1971 to 1975 , formed the sample for the 1970s . using the same selection criteria , offspring participants from the second examination ( 19791983 ) formed the sample for the 1980s , and participants from the fourth examination ( 19871991 ) formed the sample for the 1990s . for the 1970s , 1980s , and 1990s , generation 3 participants from the first examination ( 20022005 ) formed the sample for the 2000s . one individual who was included in the previous report from our cohort in the 1970s was excluded from the present analysis due to missing bmi ( 8) . a total of 3,103 unique individuals from the offspring cohort ( n = 1,587 women ) contributed to 1,308 , 1,551 , and 1,663 person - examinations in the 1970s , 1980s , and 1990s , respectively . a total of 1,692 unique individuals from the generation 3 cohort ( n = 889 women ) were observed in the 2000s . thus , the overall sample consisted of a total of 4,795 unique individuals ( n = 2,476 women ) . incident diabetes was defined as a fasting plasma glucose 126 mg / dl or use of glucose - lowering medication at the follow - up examination . in a secondary analysis , cases if they were treated with a glucose - lowering agent at the time of the follow - up examination . bmi , defined as weight in kilograms ( assessed using a detecto scale ; detecto , webb city , mo ) divided by the square of the height in meters , was calculated . waist circumference was measured at the level of the umbilicus and reported to the nearest quarter inch . hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or on treatment . sex - specific mean age and bmi were calculated for each decade . to adjust for difference in follow - up time between cohorts , we used poisson regression with duration of follow - up as an offset to calculate overall and sex - specific annualized diabetes incidence rates for a 47-year - old individual in each decade . next , we stratified individuals by bmi category normal ( bmi < 25 kg / m ) , overweight ( bmi 25 to < 30 kg / m ) , class 1 obesity ( bmi 30 to < 35 kg / m ) , or class 2 obesity ( bmi 35 kg / m)and calculated diabetes incidence rates for a 47-year - old individual within each bmi category for each decade . using the 1970s as the reference period , we calculated overall and sex - specific age - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . we also calculated overall and sex - specific age- and bmi - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . additionally , we calculated the relative risk of incident diabetes in the 2000s compared with the 1990s . finally , we characterized individuals who developed diabetes and individuals who remained free of diabetes based on demographics and standard cardiovascular risk factors including smoking status , blood pressure , and hypercholesterolemia . all analyses were performed using sas version 9.3 . to evaluate the possibility that differences in incidence in the 2000s were attributable to the shorter accrual time ( 6-year follow - up vs. 8-year follow - up in other decades ) , we performed a sensitivity analysis by expanding the age range in the 2000s to include individuals from ages 40 to 57 years at baseline . to assess whether diabetes detection is improving , we calculated rates of diagnosed diabetes for each decade . because of the reported increases in bariatric surgery in the 2000s ( 13 ) , we conducted a chart review to determine the number of participants who had undergone bariatric surgery . we selected all participants who had lost > 20% of their body weight between baseline and follow - up examination . fifteen participants were identified in this manner ; we then performed a detailed chart review on these individuals to determine whether they had undergone a bariatric procedure . of these , five had had a bariatric procedure , and two of the five had developed diabetes in the interval , which then resolved after weight loss . the framingham heart study is a community - based cohort study of cardiovascular disease and risk factors that has been in continuous operation since 1948 beginning with the original cohort . recruitment of the offspring cohort began in 1971 ; participants were invited back for a second examination 8 years after the initial exam and subsequently every 4 years thereafter . the second examination cycle lasted from 2008 through 2011 . to ensure an overlapping age distribution across all decades , we limited the current analysis to participants ages 40 to 55 years who were free of diabetes at the baseline examination . prevalent diabetes was defined as fasting glucose 126 mg / dl or on treatment with insulin or other antidiabetes agent . offspring participants ages 4055 years and free of diabetes at exam 1 , which ran from 1971 to 1975 , formed the sample for the 1970s . using the same selection criteria , offspring participants from the second examination ( 19791983 ) formed the sample for the 1980s , and participants from the fourth examination ( 19871991 ) formed the sample for the 1990s . for the 1970s , 1980s , and 1990s , generation 3 participants from the first examination ( 20022005 ) formed the sample for the 2000s . one individual who was included in the previous report from our cohort in the 1970s was excluded from the present analysis due to missing bmi ( 8) . a total of 3,103 unique individuals from the offspring cohort ( n = 1,587 women ) contributed to 1,308 , 1,551 , and 1,663 person - examinations in the 1970s , 1980s , and 1990s , respectively . a total of 1,692 unique individuals from the generation 3 cohort ( n = 889 women ) were observed in the 2000s . thus , the overall sample consisted of a total of 4,795 unique individuals ( n = 2,476 women ) . incident diabetes was defined as a fasting plasma glucose 126 mg / dl or use of glucose - lowering medication at the follow - up examination . in a secondary analysis , cases if they were treated with a glucose - lowering agent at the time of the follow - up examination . bmi , defined as weight in kilograms ( assessed using a detecto scale ; detecto , webb city , mo ) divided by the square of the height in meters , was calculated . waist circumference was measured at the level of the umbilicus and reported to the nearest quarter inch . hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or on treatment . sex - specific mean age and bmi were calculated for each decade . to adjust for difference in follow - up time between cohorts , we used poisson regression with duration of follow - up as an offset to calculate overall and sex - specific annualized diabetes incidence rates for a 47-year - old individual in each decade . next , we stratified individuals by bmi category normal ( bmi < 25 kg / m ) , overweight ( bmi 25 to < 30 kg / m ) , class 1 obesity ( bmi 30 to < 35 kg / m ) , or class 2 obesity ( bmi 35 kg / m)and calculated diabetes incidence rates for a 47-year - old individual within each bmi category for each decade . using the 1970s as the reference period , we calculated overall and sex - specific age - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . we also calculated overall and sex - specific age- and bmi - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . additionally , we calculated the relative risk of incident diabetes in the 2000s compared with the 1990s . finally , we characterized individuals who developed diabetes and individuals who remained free of diabetes based on demographics and standard cardiovascular risk factors including smoking status , blood pressure , and hypercholesterolemia . to evaluate the possibility that differences in incidence in the 2000s were attributable to the shorter accrual time ( 6-year follow - up vs. 8-year follow - up in other decades ) , we performed a sensitivity analysis by expanding the age range in the 2000s to include individuals from ages 40 to 57 years at baseline . to assess whether diabetes detection is improving , we calculated rates of diagnosed diabetes for each decade . because of the reported increases in bariatric surgery in the 2000s ( 13 ) , we conducted a chart review to determine the number of participants who had undergone bariatric surgery . we selected all participants who had lost > 20% of their body weight between baseline and follow - up examination . fifteen participants were identified in this manner ; we then performed a detailed chart review on these individuals to determine whether they had undergone a bariatric procedure . of these , five had had a bariatric procedure , and two of the five had developed diabetes in the interval , which then resolved after weight loss . the mean age ranged from 46.0 years in the 1970s to 45.9 years in the 2000s . 24.8 , 25.6 , and 26.4 kg / m among women and 26.9 , 27.0 , 27.8 , and 28.0 kg / m among men in the 1970s , 1980s , 1990s , and 2000s respectively ( table 1 ) . baseline characteristics and annualized rates of incident diabetes for a 47-year - old individual data are mean ( sd ) or n unless otherwise indicated . overall sample included 2,476 unique women and 2,319 unique men ; some individuals were included in more than one decade . the overall annualized rates of diabetes per 1,000 individuals were 3.0 , 4.1 , 6.0 , and 5.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 1 ) . the age - adjusted relative risks of diabetes were 1.37 ( 95% ci 0.872.16 ; p = 0.17 ) in the 1980s , 1.99 ( 95% ci 1.303.03 ; p < 0.01 ) in the 1990s , and 1.81 ( 95% ci 1.162.82 ; p = 0.01 ) in the 2000s , using the 1970s are the reference period ( table 2 ) . relative risks of incident diabetes for individuals ages 4055 years free of diabetes in the 1980s , 1990s , and 2000s compared with the 1970s ref , referent ; rr , relative risk . compared with the 1990s , the age - adjusted relative risk of diabetes incidence in the 2000s was 0.85 ( 95% ci 0.611.20 ; p = 0.36 ) . the annualized diabetes rates for a 47-year - old woman per 1,000 individuals were 2.6 , 3.8 , 4.7 , and 3.6 in the 1970s , 1980s , 1990s , and 2000s ( table 1 ) . the corresponding rates in men were 3.4 , 4.5 , 7.4 , and 7.5 . among women the decade - specific , age - adjusted relative risks of diabetes with 1970s as a reference were 1.45 , 1.80 , and 1.37 in the 1980s , 1990s , and 2000s ( table 2 ) . among men the corresponding relative risks were 1.32 ( p = 0.36 ) , 2.16 ( p < 0.01 ) , and 2.20 ( p < 0.01 ) . to evaluate the association between the risk of diabetes and increasing adiposity , we calculated diabetes incidence stratified by normal , overweight , or obese bmi ( fig . kg / m ) , the annualized rates of diabetes for a 47-year - old individual per 1,000 people were 7.0 , 8.0 , 14.1 , and 7.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 3 ) . among class 2 obese individuals ( bmi 35 kg / m ) , the annualized rates were 11.7 , 18.8 , 21.7 , and 26.7 per 1,000 in each successive decade . thus , among class 1 obese , incidence rates were higher in the 1990s than in the 2000s , but among class 2 obese that trend was reversed . the number of individuals with class 2 obesity in each decade was relatively small , yielding wider cis . age - adjusted annualized rates of diabetes for a 47-year - old per 1,000 individuals , stratified by normal , overweight , or obese bmi . number of participants at risk in the normal , overweight , and obese categories , respectively , in the 1970s were 585 , 542 , and 181 ; in the 1980s : 721 , 604 , and 226 ; in the 1990s : 671 , 654 , and 338 ; in the 2000s : 651 , 637 , and 404 . annualized rates of diabetes by bmi category for a 47-year - old individual cardiovascular risk factor profiles describing individuals who developed incident diabetes in each decade are presented as a descriptive analysis ( table 4 ) . risk factor profiles by decade for diabetes and no diabetes ( ages 4055 years ) continuous data are shown as mean ( sd ) . we considered that the shorter follow - up time in the 2000s ( 6 years ) compared with the other decades ( 8 years ) may have led to underestimation of the true diabetes incidence in the 2000s . to assess this possibility , we conducted a sensitivity analysis expanding the baseline age range to 4057 years in the 2000s ( supplementary table 1 ) . in this analysis , the diabetes incidence rates were 3.6 per 1,000 women ( 95% ci 2.35.6 ) and 7.5 per 1,000 men ( 95% ci 5.410.4 ) . the annualized rates of diagnosed diabetes per 1,000 for a 47-year - old individual increased steadily with each decade : 0.6 ( 95% ci 0.31.3 ) in the 1970s , 1.4 ( 95% ci 0.82.2 ) in the 1980s , 2.2 ( 95% ci 1.53.2 ) in the 1990s , and 3.0 ( 95% ci 2.14.3 ) in the 2000s . the overall annualized rates of diabetes per 1,000 individuals were 3.0 , 4.1 , 6.0 , and 5.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 1 ) . the age - adjusted relative risks of diabetes were 1.37 ( 95% ci 0.872.16 ; p = 0.17 ) in the 1980s , 1.99 ( 95% ci 1.303.03 ; p < 0.01 ) in the 1990s , and 1.81 ( 95% ci 1.162.82 ; p = 0.01 ) in the 2000s , using the 1970s are the reference period ( table 2 ) . relative risks of incident diabetes for individuals ages 4055 years free of diabetes in the 1980s , 1990s , and 2000s compared with the 1970s ref , referent ; rr , relative risk . compared with the 1990s , the age - adjusted relative risk of diabetes incidence in the 2000s was 0.85 ( 95% ci 0.611.20 ; p = 0.36 ) . the annualized diabetes rates for a 47-year - old woman per 1,000 individuals were 2.6 , 3.8 , 4.7 , and 3.6 in the 1970s , 1980s , 1990s , and 2000s ( table 1 ) . the corresponding rates in men were 3.4 , 4.5 , 7.4 , and 7.5 . among women the decade - specific , age - adjusted relative risks of diabetes with 1970s as a reference were 1.45 , 1.80 , and 1.37 in the 1980s , 1990s , and 2000s ( table 2 ) . among men the corresponding relative risks were 1.32 ( p = 0.36 ) , 2.16 ( p < 0.01 ) , and 2.20 ( p < 0.01 ) . to evaluate the association between the risk of diabetes and increasing adiposity , we calculated diabetes incidence stratified by normal , overweight , or obese bmi ( fig . kg / m ) , the annualized rates of diabetes for a 47-year - old individual per 1,000 people were 7.0 , 8.0 , 14.1 , and 7.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 3 ) . among class 2 obese individuals ( bmi 35 kg / m ) , the annualized rates were 11.7 , 18.8 , 21.7 , and 26.7 per 1,000 in each successive decade . thus , among class 1 obese , incidence rates were higher in the 1990s than in the 2000s , but among class 2 obese that trend was reversed . the number of individuals with class 2 obesity in each decade was relatively small , yielding wider cis . age - adjusted annualized rates of diabetes for a 47-year - old per 1,000 individuals , stratified by normal , overweight , or obese bmi . number of participants at risk in the normal , overweight , and obese categories , respectively , in the 1970s were 585 , 542 , and 181 ; in the 1980s : 721 , 604 , and 226 ; in the 1990s : 671 , 654 , and 338 ; in the 2000s : 651 , 637 , and 404 . cardiovascular risk factor profiles describing individuals who developed incident diabetes in each decade are presented as a descriptive analysis ( table 4 ) . risk factor profiles by decade for diabetes and no diabetes ( ages 4055 years ) continuous data are shown as mean ( sd ) . we considered that the shorter follow - up time in the 2000s ( 6 years ) compared with the other decades ( 8 years ) may have led to underestimation of the true diabetes incidence in the 2000s . to assess this possibility , we conducted a sensitivity analysis expanding the baseline age range to 4057 years in the 2000s ( supplementary table 1 ) . in this analysis , the diabetes incidence rates were 3.6 per 1,000 women ( 95% ci 2.35.6 ) and 7.5 per 1,000 men ( 95% ci 5.410.4 ) . the annualized rates of diagnosed diabetes per 1,000 for a 47-year - old individual increased steadily with each decade : 0.6 ( 95% ci 0.31.3 ) in the 1970s , 1.4 ( 95% ci 0.82.2 ) in the 1980s , 2.2 ( 95% ci 1.53.2 ) in the 1990s , and 3.0 ( 95% ci 2.14.3 ) in the 2000s . in our community - based cohort , we observed that the risk of incident diabetes was higher in the 1990s compared with the 1970s . mean bmi has increased with each decade , and diabetes incidence remains highest among obese individuals . notably , even among obese individuals , diabetes incidence rates increased in the 1990s and 2000s compared with the 1970s . thus , rates of incident diabetes remained higher in the 2000s compared with the 1970s , likely related to ongoing high prevalence of obesity . while there are some published studies reporting an increase in diabetes incidence in the 1990s ( 8,10 ) , there are few other estimates of trends in u.s . data from the national health interview survey from 19972003 showed an increase in diabetes incidence from 4.9 per 1,000 individuals in 1997 to 6.9 per 1,000 individuals in 2004 ( 14 ) . there are several possible explanations for their reported increase in diabetes incidence , while we observed no increase over a similar time frame . their data are based on self - report of diabetes and thus are less reliable than data obtained through directly measured fasting plasma glucose values and physician - reviewed medication history . furthermore , the reported increase in diabetes incidence in that study could be due to improved detection of diabetes rather than a true population change in incidence . in contrast , our study followed the same individuals over time and applied a consistent definition of diabetes that included both diagnosed and undiagnosed cases . we conducted a sensitivity of diabetes incidence limited to diagnosed cases , and we observed an increase in diagnosed diabetes in each successive decade , suggesting that detection is indeed improving . finally , the national health interview survey includes a broader age range of 1879 years . the inclusion of older individuals in particular would be expected to yield a different estimate than the age range used in our study . consistent with our findings , another study reported that self - reported diabetes incidence in new york city was stabilizing , from 9.4 per 1,000 in 2002 to 8.6 per 1,000 in 2008 ( 11 ) . however as discussed above , self - reported diabetes is likely less reliable ; in this case , the incidence could be underestimated depending on patient education and access to health care . the national health and nutrition examination survey data showed that prediabetes prevalence based on fasting glucose remained unchanged from 25.4% in 19992002 to 24.6% in 20032006 to 27.5% in 20072010 ( 1 ) . because prediabetes is a precursor of overt diabetes , this report that prediabetes did not change is consistent with our findings of stable diabetes incidence in the 2000s compared with the 1990s . we also found that diabetes incidence did not change significantly in the 2000s compared with the 1990s , despite an increase in mean bmi and despite more individuals with class 2 obesity in the 2000s . there are several potential factors that may have contributed to the stable diabetes incidence that we observed between the 1990s and the 2000s . one possibility is that we underestimated diabetes in the 2000s because of the shorter accrual time ( 6 years ) compared with the prior decades ( 8 years ) . we attempted to correct for this in a secondary analysis that expanded the age range studied in the 2000s , which did not significantly alter the results . thus , the shorter accrual time is unlikely to have substantially impacted our results from the 2000s . participants for the 2000s were drawn from a different population ( third generation ) than the participants from the prior decades ( offspring cohort ) , and thus , observed differences in the 2000s may reflect a characteristic or exposure that is unique to the third generation cohort . potential factors may include differences in diet or physical activity between cohorts , both of which could affect diabetes risk . third , increased use of bariatric surgery in the 2000s may have affected diabetes incidence . bariatric procedures increased 10-fold in the u.s . from 16,200 in 1994 to 171,000 in 2005 ( 15 ) . in our population , we identified only five individuals who had had bariatric surgery , only two of whom had documented diabetes . however , even if all five individuals would have developed diabetes had they not had bariatric surgery , this would not explain the stable incidence rate between the 1990s and 2000s . finally , it is possible that the leveling of diabetes incidence in the 2000s despite an increase in mean bmi compared with the 1990s is partly due to a saturation effect , meaning that the most susceptible individuals in the 2000s may have already acquired diabetes at baseline and thus been excluded from the current analysis . this phenomenon has been described in the pima indian population , for example , in which diabetes incidence increased among ages 514 years but decreased among ages 2534 years over the same time period ( 16 ) . our descriptive analysis suggested that individuals diagnosed with diabetes in the 2000s seem to have lower rates of cigarette smoking and higher rates of treatment for other modifiable cardiovascular risk factors . strengths of our study include the large community - based cohort with directly measured bmi and fasting blood glucose as well as robust assessment of other cardiovascular risk factors . we did not rely on self - reported diabetes or height and weight , which can be susceptible to bias . moreover , diabetes trends in the framingham cohorts may not be nationally representative . due to the age range used in this study ( 4055 years ) , we would not have captured an increase in diabetes incidence at a younger age . in our community - based sample , over the past decade , despite the ongoing trend of rising obesity , diabetes incidence remained relatively stable .

objectiveobesity and type 2 diabetes continue to increase in prevalence in the u.s . whether diabetes incidence continues to increase in recent times is less well documented . we examined trends in diabetes incidence over the previous four decades.research design and methodsframingham heart study participants ages 4055 years and free of diabetes at baseline ( n = 4,795 ; mean age 45.3 years ; 51.6% women ) were followed for the development of diabetes in the 1970s , 1980s , 1990s , and 2000s . diabetes was defined as either fasting glucose 126 mg / dl or use of antidiabetes medication . poisson regression was used to calculate sex - specific diabetes incidence rates for a 47-year - old individual in each decade . rates were also calculated among obese , overweight , and normal weight individuals.resultsthe annualized rates of diabetes per 1,000 individuals were 2.6 , 3.8 , 4.7 , and 3.0 ( women ) and 3.4 , 4.5 , 7.4 , and 7.3 ( men ) in the 1970s , 1980s , 1990s , and 2000s , respectively . compared with the 1970s , the age- and sex - adjusted relative risks of diabetes were 1.37 ( 95% ci 0.872.16 ; p = 0.17 ) , 1.99 ( 95% ci 1.303.03 ; p = 0.001 ) , and 1.81 ( 95% ci 1.162.82 ; p = 0.01 ) in the 1980s , 1990s , and 2000s , respectively . compared with the 1990s , the relative risk of diabetes in the 2000s was 0.85 ( 95% ci 0.611.20 ; p = 0.36).conclusionsin our community - based sample , the risk of new - onset diabetes continued to be higher in the 2000s compared with the 1970s . in the past decade , diabetes incidence remained steady despite the ongoing trend of rising adiposity .

Introduction Research Design and Methods Study Sample Assessment of Outcome Metabolic Risk Factor Assessment Statistical Analyses Secondary Analyses Assessment of Bariatric Surgery Results Overall Diabetes Incidence Sex-Specific Diabetes Incidence Diabetes Incidence by BMI Category Characterization of Individuals With Incident Diabetes Secondary Analyses Conclusions

recent studies indicate that prevalence of both prediabetes and overt diabetes continues to rise in the u.s . this increasing prevalence may be due to an increase in diabetes incidence , improved survival among individuals with diabetes , or a combination of these factors . the trends in prediabetes and diabetes prevalence have paralleled high rates of overweight and obesity , which are leading risk factors for the development of diabetes ( 4,5 ) . recent data , however , suggest the prevalence of obesity among adults , which was increasing from the 1970s through the early 2000s , may have leveled off in the past few years ( 4,6,7 ) . there are few estimates of trends in diabetes incidence in recent times ( 8) . we previously reported diabetes incidence in our cohort in the 1970s , 1980s , and 1990s ( 8) , and in the current study , we seek to update these incidence estimates in the context of the obesity epidemic . the framingham heart study is a community - based cohort study of cardiovascular disease and risk factors that has been in continuous operation since 1948 beginning with the original cohort . to ensure an overlapping age distribution across all decades , we limited the current analysis to participants ages 40 to 55 years who were free of diabetes at the baseline examination . prevalent diabetes was defined as fasting glucose 126 mg / dl or on treatment with insulin or other antidiabetes agent . offspring participants ages 4055 years and free of diabetes at exam 1 , which ran from 1971 to 1975 , formed the sample for the 1970s . using the same selection criteria , offspring participants from the second examination ( 19791983 ) formed the sample for the 1980s , and participants from the fourth examination ( 19871991 ) formed the sample for the 1990s . for the 1970s , 1980s , and 1990s , generation 3 participants from the first examination ( 20022005 ) formed the sample for the 2000s . one individual who was included in the previous report from our cohort in the 1970s was excluded from the present analysis due to missing bmi ( 8) . a total of 3,103 unique individuals from the offspring cohort ( n = 1,587 women ) contributed to 1,308 , 1,551 , and 1,663 person - examinations in the 1970s , 1980s , and 1990s , respectively . a total of 1,692 unique individuals from the generation 3 cohort ( n = 889 women ) were observed in the 2000s . thus , the overall sample consisted of a total of 4,795 unique individuals ( n = 2,476 women ) . incident diabetes was defined as a fasting plasma glucose 126 mg / dl or use of glucose - lowering medication at the follow - up examination . sex - specific mean age and bmi were calculated for each decade . to adjust for difference in follow - up time between cohorts , we used poisson regression with duration of follow - up as an offset to calculate overall and sex - specific annualized diabetes incidence rates for a 47-year - old individual in each decade . next , we stratified individuals by bmi category normal ( bmi < 25 kg / m ) , overweight ( bmi 25 to < 30 kg / m ) , class 1 obesity ( bmi 30 to < 35 kg / m ) , or class 2 obesity ( bmi 35 kg / m)and calculated diabetes incidence rates for a 47-year - old individual within each bmi category for each decade . using the 1970s as the reference period , we calculated overall and sex - specific age - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . we also calculated overall and sex - specific age- and bmi - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . additionally , we calculated the relative risk of incident diabetes in the 2000s compared with the 1990s . to evaluate the possibility that differences in incidence in the 2000s were attributable to the shorter accrual time ( 6-year follow - up vs. 8-year follow - up in other decades ) , we performed a sensitivity analysis by expanding the age range in the 2000s to include individuals from ages 40 to 57 years at baseline . of these , five had had a bariatric procedure , and two of the five had developed diabetes in the interval , which then resolved after weight loss . the framingham heart study is a community - based cohort study of cardiovascular disease and risk factors that has been in continuous operation since 1948 beginning with the original cohort . to ensure an overlapping age distribution across all decades , we limited the current analysis to participants ages 40 to 55 years who were free of diabetes at the baseline examination . prevalent diabetes was defined as fasting glucose 126 mg / dl or on treatment with insulin or other antidiabetes agent . offspring participants ages 4055 years and free of diabetes at exam 1 , which ran from 1971 to 1975 , formed the sample for the 1970s . using the same selection criteria , offspring participants from the second examination ( 19791983 ) formed the sample for the 1980s , and participants from the fourth examination ( 19871991 ) formed the sample for the 1990s . for the 1970s , 1980s , and 1990s , generation 3 participants from the first examination ( 20022005 ) formed the sample for the 2000s . one individual who was included in the previous report from our cohort in the 1970s was excluded from the present analysis due to missing bmi ( 8) . a total of 3,103 unique individuals from the offspring cohort ( n = 1,587 women ) contributed to 1,308 , 1,551 , and 1,663 person - examinations in the 1970s , 1980s , and 1990s , respectively . a total of 1,692 unique individuals from the generation 3 cohort ( n = 889 women ) were observed in the 2000s . thus , the overall sample consisted of a total of 4,795 unique individuals ( n = 2,476 women ) . incident diabetes was defined as a fasting plasma glucose 126 mg / dl or use of glucose - lowering medication at the follow - up examination . sex - specific mean age and bmi were calculated for each decade . to adjust for difference in follow - up time between cohorts , we used poisson regression with duration of follow - up as an offset to calculate overall and sex - specific annualized diabetes incidence rates for a 47-year - old individual in each decade . next , we stratified individuals by bmi category normal ( bmi < 25 kg / m ) , overweight ( bmi 25 to < 30 kg / m ) , class 1 obesity ( bmi 30 to < 35 kg / m ) , or class 2 obesity ( bmi 35 kg / m)and calculated diabetes incidence rates for a 47-year - old individual within each bmi category for each decade . using the 1970s as the reference period , we calculated overall and sex - specific age - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . we also calculated overall and sex - specific age- and bmi - adjusted relative risks of incident diabetes in the 1980s , 1990s , and 2000s . additionally , we calculated the relative risk of incident diabetes in the 2000s compared with the 1990s . to evaluate the possibility that differences in incidence in the 2000s were attributable to the shorter accrual time ( 6-year follow - up vs. 8-year follow - up in other decades ) , we performed a sensitivity analysis by expanding the age range in the 2000s to include individuals from ages 40 to 57 years at baseline . of these , five had had a bariatric procedure , and two of the five had developed diabetes in the interval , which then resolved after weight loss . the mean age ranged from 46.0 years in the 1970s to 45.9 years in the 2000s . 24.8 , 25.6 , and 26.4 kg / m among women and 26.9 , 27.0 , 27.8 , and 28.0 kg / m among men in the 1970s , 1980s , 1990s , and 2000s respectively ( table 1 ) . baseline characteristics and annualized rates of incident diabetes for a 47-year - old individual data are mean ( sd ) or n unless otherwise indicated . the overall annualized rates of diabetes per 1,000 individuals were 3.0 , 4.1 , 6.0 , and 5.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 1 ) . the age - adjusted relative risks of diabetes were 1.37 ( 95% ci 0.872.16 ; p = 0.17 ) in the 1980s , 1.99 ( 95% ci 1.303.03 ; p < 0.01 ) in the 1990s , and 1.81 ( 95% ci 1.162.82 ; p = 0.01 ) in the 2000s , using the 1970s are the reference period ( table 2 ) . relative risks of incident diabetes for individuals ages 4055 years free of diabetes in the 1980s , 1990s , and 2000s compared with the 1970s ref , referent ; rr , relative risk . compared with the 1990s , the age - adjusted relative risk of diabetes incidence in the 2000s was 0.85 ( 95% ci 0.611.20 ; p = 0.36 ) . the annualized diabetes rates for a 47-year - old woman per 1,000 individuals were 2.6 , 3.8 , 4.7 , and 3.6 in the 1970s , 1980s , 1990s , and 2000s ( table 1 ) . the corresponding rates in men were 3.4 , 4.5 , 7.4 , and 7.5 . among women the decade - specific , age - adjusted relative risks of diabetes with 1970s as a reference were 1.45 , 1.80 , and 1.37 in the 1980s , 1990s , and 2000s ( table 2 ) . among men the corresponding relative risks were 1.32 ( p = 0.36 ) , 2.16 ( p < 0.01 ) , and 2.20 ( p < 0.01 ) . to evaluate the association between the risk of diabetes and increasing adiposity , we calculated diabetes incidence stratified by normal , overweight , or obese bmi ( fig . kg / m ) , the annualized rates of diabetes for a 47-year - old individual per 1,000 people were 7.0 , 8.0 , 14.1 , and 7.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 3 ) . among class 2 obese individuals ( bmi 35 kg / m ) , the annualized rates were 11.7 , 18.8 , 21.7 , and 26.7 per 1,000 in each successive decade . thus , among class 1 obese , incidence rates were higher in the 1990s than in the 2000s , but among class 2 obese that trend was reversed . age - adjusted annualized rates of diabetes for a 47-year - old per 1,000 individuals , stratified by normal , overweight , or obese bmi . number of participants at risk in the normal , overweight , and obese categories , respectively , in the 1970s were 585 , 542 , and 181 ; in the 1980s : 721 , 604 , and 226 ; in the 1990s : 671 , 654 , and 338 ; in the 2000s : 651 , 637 , and 404 . annualized rates of diabetes by bmi category for a 47-year - old individual cardiovascular risk factor profiles describing individuals who developed incident diabetes in each decade are presented as a descriptive analysis ( table 4 ) . we considered that the shorter follow - up time in the 2000s ( 6 years ) compared with the other decades ( 8 years ) may have led to underestimation of the true diabetes incidence in the 2000s . in this analysis , the diabetes incidence rates were 3.6 per 1,000 women ( 95% ci 2.35.6 ) and 7.5 per 1,000 men ( 95% ci 5.410.4 ) . the annualized rates of diagnosed diabetes per 1,000 for a 47-year - old individual increased steadily with each decade : 0.6 ( 95% ci 0.31.3 ) in the 1970s , 1.4 ( 95% ci 0.82.2 ) in the 1980s , 2.2 ( 95% ci 1.53.2 ) in the 1990s , and 3.0 ( 95% ci 2.14.3 ) in the 2000s . the overall annualized rates of diabetes per 1,000 individuals were 3.0 , 4.1 , 6.0 , and 5.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 1 ) . the age - adjusted relative risks of diabetes were 1.37 ( 95% ci 0.872.16 ; p = 0.17 ) in the 1980s , 1.99 ( 95% ci 1.303.03 ; p < 0.01 ) in the 1990s , and 1.81 ( 95% ci 1.162.82 ; p = 0.01 ) in the 2000s , using the 1970s are the reference period ( table 2 ) . relative risks of incident diabetes for individuals ages 4055 years free of diabetes in the 1980s , 1990s , and 2000s compared with the 1970s ref , referent ; rr , relative risk . compared with the 1990s , the age - adjusted relative risk of diabetes incidence in the 2000s was 0.85 ( 95% ci 0.611.20 ; p = 0.36 ) . the annualized diabetes rates for a 47-year - old woman per 1,000 individuals were 2.6 , 3.8 , 4.7 , and 3.6 in the 1970s , 1980s , 1990s , and 2000s ( table 1 ) . the corresponding rates in men were 3.4 , 4.5 , 7.4 , and 7.5 . among women the decade - specific , age - adjusted relative risks of diabetes with 1970s as a reference were 1.45 , 1.80 , and 1.37 in the 1980s , 1990s , and 2000s ( table 2 ) . among men the corresponding relative risks were 1.32 ( p = 0.36 ) , 2.16 ( p < 0.01 ) , and 2.20 ( p < 0.01 ) . to evaluate the association between the risk of diabetes and increasing adiposity , we calculated diabetes incidence stratified by normal , overweight , or obese bmi ( fig . kg / m ) , the annualized rates of diabetes for a 47-year - old individual per 1,000 people were 7.0 , 8.0 , 14.1 , and 7.5 in the 1970s , 1980s , 1990s , and 2000s , respectively ( table 3 ) . among class 2 obese individuals ( bmi 35 kg / m ) , the annualized rates were 11.7 , 18.8 , 21.7 , and 26.7 per 1,000 in each successive decade . thus , among class 1 obese , incidence rates were higher in the 1990s than in the 2000s , but among class 2 obese that trend was reversed . age - adjusted annualized rates of diabetes for a 47-year - old per 1,000 individuals , stratified by normal , overweight , or obese bmi . number of participants at risk in the normal , overweight , and obese categories , respectively , in the 1970s were 585 , 542 , and 181 ; in the 1980s : 721 , 604 , and 226 ; in the 1990s : 671 , 654 , and 338 ; in the 2000s : 651 , 637 , and 404 . cardiovascular risk factor profiles describing individuals who developed incident diabetes in each decade are presented as a descriptive analysis ( table 4 ) . we considered that the shorter follow - up time in the 2000s ( 6 years ) compared with the other decades ( 8 years ) may have led to underestimation of the true diabetes incidence in the 2000s . in this analysis , the diabetes incidence rates were 3.6 per 1,000 women ( 95% ci 2.35.6 ) and 7.5 per 1,000 men ( 95% ci 5.410.4 ) . the annualized rates of diagnosed diabetes per 1,000 for a 47-year - old individual increased steadily with each decade : 0.6 ( 95% ci 0.31.3 ) in the 1970s , 1.4 ( 95% ci 0.82.2 ) in the 1980s , 2.2 ( 95% ci 1.53.2 ) in the 1990s , and 3.0 ( 95% ci 2.14.3 ) in the 2000s . in our community - based cohort , we observed that the risk of incident diabetes was higher in the 1990s compared with the 1970s . mean bmi has increased with each decade , and diabetes incidence remains highest among obese individuals . notably , even among obese individuals , diabetes incidence rates increased in the 1990s and 2000s compared with the 1970s . thus , rates of incident diabetes remained higher in the 2000s compared with the 1970s , likely related to ongoing high prevalence of obesity . while there are some published studies reporting an increase in diabetes incidence in the 1990s ( 8,10 ) , there are few other estimates of trends in u.s . data from the national health interview survey from 19972003 showed an increase in diabetes incidence from 4.9 per 1,000 individuals in 1997 to 6.9 per 1,000 individuals in 2004 ( 14 ) . there are several possible explanations for their reported increase in diabetes incidence , while we observed no increase over a similar time frame . furthermore , the reported increase in diabetes incidence in that study could be due to improved detection of diabetes rather than a true population change in incidence . we conducted a sensitivity of diabetes incidence limited to diagnosed cases , and we observed an increase in diagnosed diabetes in each successive decade , suggesting that detection is indeed improving . because prediabetes is a precursor of overt diabetes , this report that prediabetes did not change is consistent with our findings of stable diabetes incidence in the 2000s compared with the 1990s . we also found that diabetes incidence did not change significantly in the 2000s compared with the 1990s , despite an increase in mean bmi and despite more individuals with class 2 obesity in the 2000s . one possibility is that we underestimated diabetes in the 2000s because of the shorter accrual time ( 6 years ) compared with the prior decades ( 8 years ) . participants for the 2000s were drawn from a different population ( third generation ) than the participants from the prior decades ( offspring cohort ) , and thus , observed differences in the 2000s may reflect a characteristic or exposure that is unique to the third generation cohort . third , increased use of bariatric surgery in the 2000s may have affected diabetes incidence . finally , it is possible that the leveling of diabetes incidence in the 2000s despite an increase in mean bmi compared with the 1990s is partly due to a saturation effect , meaning that the most susceptible individuals in the 2000s may have already acquired diabetes at baseline and thus been excluded from the current analysis . our descriptive analysis suggested that individuals diagnosed with diabetes in the 2000s seem to have lower rates of cigarette smoking and higher rates of treatment for other modifiable cardiovascular risk factors . moreover , diabetes trends in the framingham cohorts may not be nationally representative . due to the age range used in this study ( 4055 years ) , we would not have captured an increase in diabetes incidence at a younger age . in our community - based sample , over the past decade , despite the ongoing trend of rising obesity , diabetes incidence remained relatively stable .

sepsis is defined as the systemic response to infection , and it has a complex pathophysiology . while severe sepsis is manifested by organ dysfunction , septic shock is a type of severe sepsis marked by hypotension despite fluid resuscitation . however , although there are many different therapeutic modalities ; severe sepsis and septic shock are major causes of mortality in intensive care units . there is inflammatory response in the pathogenesis of sepsis and associated with excessive production of cytokines such as interleukin-6 ( il-6 ) and tumor necrosis factor- ( tnf- ) . the endothelial cells and neutrophils were activated with tnf- to induce an inflammatory response . additionally , in addition , tnf- amplifies inflammatory cascades by activating macrophages / monocytes to secrete other pro - inflammatory cytokines . while the effect of progesterone on cecal ligation and puncture ( clp)-induced tissue injury has not been studied , progesterone is reported to have a protective effect on cerebral or myocardial i / r injury in rats . in one study that was conducted to examine the inhibitory effects of progesterone on inflammatory response , progesterone treatment decreased expression of inflammatory cytokines e.g. , il-1 and tnf- in brain - injured rats . the aim of this study was investigate whether progesterone affect systemic inflammation and tissue damage in female rat sepsis models of polymicrobial peritonitis caused by clp . this study was performed in accordance with the national institute of health 's approved guidelines and the experimental protocol ( protocol number : b.30.2.ata.023.85-59 ) approved by the animal research ethics committee of ataturk university , medical faculty , erzurum , turkey . twenty - eight adult female sprague - dawley rats ( weighing 200 - 250 g ) were purchased from the ataturk university experimental animal laboratory . all rats were kept in a light- and temperature - controlled room with 14:10-hour light - dark cycles and temperature of 22 0.5c , and were fed a standard pellet diet . fifteen days before the experimental study , all rats were bilaterally ovariectomized to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels . thiopental dose of 25 mg / kg was administered and bilateral ovariectomy was performed by a midline dorsal skin incision . the peritoneal cavity was reached and ovaries were found . after the ligation of blood vessels , the connection fallopian tube and uterine horn was cut and ovaries were excised . the rats were randomly divided into four groups of seven individuals : the overiectomy group ( ovx ) , the sham operated ( control ) group , the sepsis ( clp ) group and the progesterone - treated clp group ( clp+ progesterone ) . a clp method of polymicrobial sepsis was applied to the rats in clp and clp+ progesterone groups . polymicrobial sepsis was induced by cecal ligation and two - hole puncture , as described by wichterman et al . thiopental ( 25 mg / kg ) was used to provide surgical anesthesia in rats . after shaving the rats abdomen , a midline laparotomy was performed and the cecum was ligated just below the ileocecal valve using 3 - 0 silk ligatures . using a 12-gauge needle , the cecum was perforated through the cecum distal to the point of ligation at two locations 1 cm apart . the cecum was then returned to the abdomen and the abdominal incision was closed using a 4/0 sterile synthetic absorbable suture . laparotomy was performed on the control group , and the cecum was manipulated , although not ligated or perforated . all animals were resuscitated with normal saline ( 2 ml/100 g body weight ) injected subcutaneously at the time of surgery and also at 6 hours postoperatively . injections of 2 mg / kg progesterone in peanut oil at 2 hours after sepsis induction . postoperatively , the rats were deprived of food , although they had free access to water until they were sacrificed . sixteen hours after the surgery , all rats were sacrificed with an overdose of a general anesthetic ( thiopental sodium , 50 mg / kg ) . the liver , uterus and kidney tissues of all rats were rapidly removed and washed in ice - cold saline . activities of myeloperoxidase ( mpo ) , indicating neutrophil infiltration , and glutathione peroxidase ( gpx ) , which is an antioxidant enzyme , and concentration of malondialdehyde ( mda ) , a marker of lipid peroxidation , were measured in tissue samples . a portion of tissue was homogenized in a potassium phosphate buffer ( ph : 6 ) containing 5% hexadecyltrimethyl ammonium bromide solution ( w / v ) for mpo activity measurement ; the rest of the tissue was homogenized using a solution of 1.15% kcl for gpx and mda measurements . serum tnf- and il-6 levels were measured using rat tnf- elisa kit ( invitrogen , carlsbad , ca , usa ; catalog number : krc3011 , lot number : 776995a ) and rat il-6 platinum elisa kit ( ebioscience , san diego , ca , usa ; catalog number : bms625 , lot number : 60405021 ) according to the manufacturer 's instructions . intra - assay coefficiency of variation ( cv ) was 5.8% for tnf- and 5% for il-6 . it was determined by adding 100 l of the supernatant to 1.9 ml of 10 mmol / l phosphate buffer ( ph = 6.0 ) and 1 ml of 1.5 mmol / l o - dianisidine hydrochloride containing 0.0005% ( wt / vol ) hydrogen peroxide . the changes in each sample 's absorbance at 450 nm were recorded on a uv vis spectrophotometer . then , in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate , the oxidized glutathione was converted to its reduced form with a concomitant oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate . the decrease in absorbance tissue mda levels were determined spectrophotometrically according to the method described by ohkawa et al . briefly , supernatant ( 0.5 ml ) was added to a solution containing 0.2 ml of 80 g / l sodium lauryl sulfate , 1.5 ml of 200 g / l acetic acid , 1.5 ml of 8 g the mixture was incubated at boiled water for 1 h. upon cooling , 5 ml of n - butanol : pyridine ( 15:l ) was added . this study was performed in accordance with the national institute of health 's approved guidelines and the experimental protocol ( protocol number : b.30.2.ata.023.85-59 ) approved by the animal research ethics committee of ataturk university , medical faculty , erzurum , turkey . twenty - eight adult female sprague - dawley rats ( weighing 200 - 250 g ) were purchased from the ataturk university experimental animal laboratory . all rats were kept in a light- and temperature - controlled room with 14:10-hour light - dark cycles and temperature of 22 0.5c , and were fed a standard pellet diet . fifteen days before the experimental study , all rats were bilaterally ovariectomized to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels . thiopental dose of 25 mg / kg was administered and bilateral ovariectomy was performed by a midline dorsal skin incision . the peritoneal cavity was reached and ovaries were found . after the ligation of blood vessels , the connection fallopian tube and uterine horn was cut and ovaries were excised . the rats were randomly divided into four groups of seven individuals : the overiectomy group ( ovx ) , the sham operated ( control ) group , the sepsis ( clp ) group and the progesterone - treated clp group ( clp+ progesterone ) . a clp method of polymicrobial sepsis was applied to the rats in clp and clp+ progesterone groups . polymicrobial sepsis was induced by cecal ligation and two - hole puncture , as described by wichterman et al . thiopental ( 25 mg / kg ) was used to provide surgical anesthesia in rats . after shaving the rats abdomen , a midline laparotomy was performed and the cecum was ligated just below the ileocecal valve using 3 - 0 silk ligatures . using a 12-gauge needle , the cecum was perforated through the cecum distal to the point of ligation at two locations 1 cm apart . the cecum was then returned to the abdomen and the abdominal incision was closed using a 4/0 sterile synthetic absorbable suture . laparotomy was performed on the control group , and the cecum was manipulated , although not ligated or perforated . all animals were resuscitated with normal saline ( 2 ml/100 g body weight ) injected subcutaneously at the time of surgery and also at 6 hours postoperatively . injections of 2 mg / kg progesterone in peanut oil at 2 hours after sepsis induction . postoperatively , the rats were deprived of food , although they had free access to water until they were sacrificed . sixteen hours after the surgery , all rats were sacrificed with an overdose of a general anesthetic ( thiopental sodium , 50 mg / kg ) . the liver , uterus and kidney tissues of all rats were rapidly removed and washed in ice - cold saline . activities of myeloperoxidase ( mpo ) , indicating neutrophil infiltration , and glutathione peroxidase ( gpx ) , which is an antioxidant enzyme , and concentration of malondialdehyde ( mda ) , a marker of lipid peroxidation , were measured in tissue samples . a portion of tissue was homogenized in a potassium phosphate buffer ( ph : 6 ) containing 5% hexadecyltrimethyl ammonium bromide solution ( w / v ) for mpo activity measurement ; the rest of the tissue was homogenized using a solution of 1.15% kcl for gpx and mda measurements . serum tnf- and il-6 levels were measured using rat tnf- elisa kit ( invitrogen , carlsbad , ca , usa ; catalog number : krc3011 , lot number : 776995a ) and rat il-6 platinum elisa kit ( ebioscience , san diego , ca , usa ; catalog number : bms625 , lot number : 60405021 ) according to the manufacturer 's instructions . intra - assay coefficiency of variation ( cv ) was 5.8% for tnf- and 5% for il-6 . it was determined by adding 100 l of the supernatant to 1.9 ml of 10 mmol / l phosphate buffer ( ph = 6.0 ) and 1 ml of 1.5 mmol / l o - dianisidine hydrochloride containing 0.0005% ( wt / vol ) hydrogen peroxide . the changes in each sample 's absorbance at 450 nm were recorded on a uv vis spectrophotometer . then , in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate , the oxidized glutathione was converted to its reduced form with a concomitant oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate . the decrease in absorbance tissue mda levels were determined spectrophotometrically according to the method described by ohkawa et al . briefly , supernatant ( 0.5 ml ) was added to a solution containing 0.2 ml of 80 g / l sodium lauryl sulfate , 1.5 ml of 200 g / l acetic acid , 1.5 ml of 8 g the mixture was incubated at boiled water for 1 h. upon cooling , 5 ml of n - butanol : pyridine ( 15:l ) was added . this study was performed in accordance with the national institute of health 's approved guidelines and the experimental protocol ( protocol number : b.30.2.ata.023.85-59 ) approved by the animal research ethics committee of ataturk university , medical faculty , erzurum , turkey . twenty - eight adult female sprague - dawley rats ( weighing 200 - 250 g ) were purchased from the ataturk university experimental animal laboratory . all rats were kept in a light- and temperature - controlled room with 14:10-hour light - dark cycles and temperature of 22 0.5c , and were fed a standard pellet diet . fifteen days before the experimental study , all rats were bilaterally ovariectomized to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels . thiopental dose of 25 mg / kg was administered and bilateral ovariectomy was performed by a midline dorsal skin incision . the peritoneal cavity was reached and ovaries were found . after the ligation of blood vessels , the connection fallopian tube and uterine horn was cut and ovaries were excised . the rats were randomly divided into four groups of seven individuals : the overiectomy group ( ovx ) , the sham operated ( control ) group , the sepsis ( clp ) group and the progesterone - treated clp group ( clp+ progesterone ) . a clp method of polymicrobial sepsis was applied to the rats in clp and clp+ progesterone groups . polymicrobial sepsis was induced by cecal ligation and two - hole puncture , as described by wichterman et al . thiopental ( 25 mg / kg ) was used to provide surgical anesthesia in rats . after shaving the rats abdomen , a midline laparotomy was performed and the cecum was ligated just below the ileocecal valve using 3 - 0 silk ligatures . using a 12-gauge needle , the cecum was perforated through the cecum distal to the point of ligation at two locations 1 cm apart . the cecum was then returned to the abdomen and the abdominal incision was closed using a 4/0 sterile synthetic absorbable suture . laparotomy was performed on the control group , and the cecum was manipulated , although not ligated or perforated . all animals were resuscitated with normal saline ( 2 ml/100 g body weight ) injected subcutaneously at the time of surgery and also at 6 hours postoperatively . injections of 2 mg / kg progesterone in peanut oil at 2 hours after sepsis induction . postoperatively , the rats were deprived of food , although they had free access to water until they were sacrificed . sixteen hours after the surgery , all rats were sacrificed with an overdose of a general anesthetic ( thiopental sodium , 50 mg / kg ) . the liver , uterus and kidney tissues of all rats were rapidly removed and washed in ice - cold saline . activities of myeloperoxidase ( mpo ) , indicating neutrophil infiltration , and glutathione peroxidase ( gpx ) , which is an antioxidant enzyme , and concentration of malondialdehyde ( mda ) , a marker of lipid peroxidation , were measured in tissue samples . a portion of tissue was homogenized in a potassium phosphate buffer ( ph : 6 ) containing 5% hexadecyltrimethyl ammonium bromide solution ( w / v ) for mpo activity measurement ; the rest of the tissue was homogenized using a solution of 1.15% kcl for gpx and mda measurements . serum tnf- and il-6 levels were measured using rat tnf- elisa kit ( invitrogen , carlsbad , ca , usa ; catalog number : krc3011 , lot number : 776995a ) and rat il-6 platinum elisa kit ( ebioscience , san diego , ca , usa ; catalog number : bms625 , lot number : 60405021 ) according to the manufacturer 's instructions . intra - assay coefficiency of variation ( cv ) was 5.8% for tnf- and 5% for il-6 . it was determined by adding 100 l of the supernatant to 1.9 ml of 10 mmol / l phosphate buffer ( ph = 6.0 ) and 1 ml of 1.5 mmol / l o - dianisidine hydrochloride containing 0.0005% ( wt / vol ) hydrogen peroxide . the changes in each sample 's absorbance at 450 nm were recorded on a uv vis spectrophotometer . in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate , the oxidized glutathione was converted to its reduced form with a concomitant oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate . the decrease in absorbance tissue mda levels were determined spectrophotometrically according to the method described by ohkawa et al . briefly , supernatant ( 0.5 ml ) was added to a solution containing 0.2 ml of 80 g / l sodium lauryl sulfate , 1.5 ml of 200 g / l acetic acid , 1.5 ml of 8 g / l 2-thiobarbiturate and 0.3 ml distilled water . the mixture was incubated at boiled water for 1 h. upon cooling , 5 ml of n - butanol : pyridine ( 15:l ) was added . increased plasma il-6 and tnf- levels ( pg / ml ) were found in the clp group ( 36.17 3.41 and 15.56 4.46 ) in comparison with the control group ( 26.85 2.74 and 11.5 1.72 ) ( p = 0.01 , p = 0.02 ; respectively ) . however in clp+ progesterone group , the serum levels of il-6 and tnf- ( 31.79 7.89 and 12.99 3.42 ) were similar to the control group [ figure 1 ] . mpo , gpx activities and mda levels in liver , kidney and uterine tissues of study groups are presented in table 1 . in the ovx group , while liver and uterine mda concentrations ( mol / g protein ) were similar to control , the kidney mda concentration was higher than those of the control group ( p = 0.02 ) . in the clp group , kidney mda concentration was significantly higher than in the control ( p = 0.0001 ) and ovx groups ( p = 0.02 ) . in clp+ progesterone group , mean mda concentration of kidney tissue was significantly lower than in clp group ( p = 0.003 ) . mda concentrations in the liver and uterine tissues were significantly elevated in the clp group compared to the control group ( p = 0.01 for both ) . liver mda concentration of the clp+ progesterone group was not significantly different from that of control group . while there were no significant differences among groups regarding to liver mpo ( u / g protein ) ; in the clp group , mpo activity in kidney ( p = 0.02 ) and uterine tissue ( p = 0.03 ) were found to be significantly higher compared to the control group . in clp+ progesterone group , mean mpo activities of all tissues were not different than those of control group . while the gpx activity ( u / g protein ) in uterine tissue of the clp group was lower than those of the control group ( p = 0.02 ) , the gpx activity in the clp+ progesterone group was not statistically significantly different from control group . in the kidney and liver tissues comparison of groups according to serum tnf alpha and il-6 levels . * p = 0.01 and * * p = 0.02 compared to control group mpo and gp activities and mda levels in liver , kidney and uterine tissues of control , clp , ovx and clp+prog groups excessive production of cytokines ( e.g. , il-6 , tnf- ) is reported in a part of sepsis pathogenesis . the mortality and morbidity of sepsis are high despite advances in new therapeutic agents . considering that sepsis is a very significant disease that leads to death in intensive care units , there is a great need to better understand the pathogenesis of this disease and develop effective treatment modalities . the purpose of this study was to examine the effects of progesterone on systemic inflammation and tissue damage of ovariectomized rats in a clp - induced sepsis model . progesterone , a sex steroid hormone has been reported to suppress the oxidative damage in cardiac and brain tissues , decrease oxidative damage in the colonic mucosa and reduce cell apoptosis in brain tissue . however , the potential role of progesterone in sepsis has not been studied . in our study , increased serum levels of il-6 and tnf- reduced after progesterone treatment in a rat model created sepsis with clp . also , clp increased the mpo activity in kidney and uterine tissues , but similar values to the control group were observed after treatment with progesterone . the key finding in our study was that exogenously given progesterone in sepsis resulted in a significant decrease in systemic inflammation and mpo activity , as a leukocyte activation marker , compared to rats that did not receive it . emerging evidence suggests that progesterone has the potential to influence inflammation . in a study , expression of tnf- reduced in progesterone - treated rats compared to controls and authors suggested that progesterone administration is beneficial for cerebral trauma and infarction by inhibiting inflammatory reaction . 's study , they investigated the effect of progesterone on oxidative stress and myocardial ischemia markers in the myocardial ischemia - reperfusion model . at the end of the study , they concluded that the administration of progesterone during myocardial injury reduces inflammatory reactivity and provides better cardioprotection in female rats compared to male and they suggested that progesterone therapy may be useful in myocardial injury due to a diminished inflammatory response . there is no consensus among researchers regarding the influence of gender on survival in patients with sepsis . for example , esper et al . found no gender differences , adrie et al . reported higher risk in men and dombrovskiy et al . declared higher risk in women in terms of sepsis survival . on the other hand , schroder et al . suggested that sex hormones estrogen and progesterone may be effective in providing protection against sepsis in women . before beginning the experimental study , we removed the ovaries of all rats to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels . our results demonstrated that progesterone attenuates the sepsis - induced elevations of il-6 and tnf- . we applied 2 mg / kg dose of progesterone immediately after clp . in the brain injury model , it was suggested that progesterone in inhibiting the proinflammatory cytokines is time - dependent . progesterone effectively reduced the gene expression of il-1 and tnf- at 3 hours post - injury . after 8 and 12 hours of continued administration of progesterone , cytokine elevations were not observed . it has been demonstrated that progesterone is also neuroprotective in cerebral ischemia and traumatic brain injury . researched the effects of progesterone on the intestinal pathophysiologic changes following subarachnoid hemorrhage in male rats . after progesterone administration ( 2 mg / kg i.p . ) , the concentrations of il-1 , tnf- and il-6 were found to be significantly lower in rat ileum tissue . sahin et al . examined the effect of carnosine in an experimental septic shock model . they concluded that carnosine may be an effective treatment for oxidative damage in cases of septic shock . according to our literature data , this present study is the first investigating the efficacy of progesterone on sepsis in a rat model created sepsis using clp . mda is a lipid peroxidation product and a marker for tissue damage . in a recent study , they reported both lower blood and colonic tissue levels of mda , il-6 and tnf- in rats treated with progesterone ( subcutaneously with dose of 2 mg / kg ) compared to the rats that only created colitis by intrarectal administration of 5 mg trinitrobenzene sulfonic acid . in another study , investigating possible neuroprotective effects of progesterone in rats subjected to global cerebral ischemia , progesterone administration ameliorated ischemia - induced decrease in glutathione ( major endogenous antioxidant ) and increase in mda levels in hippocampus , striatum and cortex . they evaluated the remarkable neuroprotective effect of progesterone reducing oxidative stress . in our study , the mda concentration in tissues of uterine , liver and kidney and plasma il-6 and tnf- level increased in rats after created clp , but the values after progesterone treatment were similar to the values in the control group . also while increased uterine tissue gpx activity ( an enzymatic antioxidant ) was found in rats to create sepsis , similar uterine gpx activity to the control group it may be suggested that progesterone ameliorates sepsis syndrome by reduction of the inflammatory cytokines , il-6 and tnf- , and by restoration of antioxidant defense system . further prospective and randomized clinical controlled trials are required to investigate the therapeutic role of progesterone in tissue damage resulting from sepsis .

objectives : to explore the protective effect of progesterone on inflammation and oxidative stress in a rat model of sepsis created by cecal ligation and puncture ( clp).materials and methods : rats were randomly divided into 4 groups : overiectomy group ( ovx ) , sham operated ( control ) , sepsis ( clp ) group and progesterone - treated clp group ( clp+ progesterone ) . the rats in clp+ progesterone group received intraperitoneal progesterone ( 2 mg / kg ) . cardiac blood samples were obtained for the measurement levels of interleukin-6 ( il-6 ) and tumor necrosis factor- ( tnf- ) . tissue samples , including liver , kidney and uterus of rats were prepared to determine activities of myeloperoxidase ( mpo ) , glutathione peroxidase ( gpx ) and levels of malondialdehyde ( mda).results : increased serum il-6 and tnf- levels were found in the clp group in comparison with the control group ( p = 0.01 , p = 0.02 ; respectively ) . in clp+ progesterone group , mean mda concentration of kidney tissue was significantly lower than in clp group ( p = 0.003 ) . liver mda concentration of the clp+ progesterone group was not significantly different from that of the control group . while there were no significant differences among groups regarding liver mpo ; in the clp group , mpo activity in kidney ( p = 0.02 ) and uterine tissues ( p = 0.03 ) were found to be significantly higher compared to the control group . in clp+ progesterone group , mean mpo activities of all tissues were not different than those of control group . the uterine tissue gpx activity in the clp+ progesterone group was not statistically significantly different from control group.conclusions:we suggest that progesterone ameliorates sepsis syndrome by reduction of the inflammatory cytokines il-6 and tnf- , and by restoration of antioxidant enzyme activities in some tissues .

Introduction Materials and Methods None Experimental Animals Animal Groups and Study Design Preparation of Tissues Biochemical Analyses Serum TNF- and IL-6 levels MPO activity GPx activity MDA levels Statistical Analysis Results Discussion Conclusion

there is inflammatory response in the pathogenesis of sepsis and associated with excessive production of cytokines such as interleukin-6 ( il-6 ) and tumor necrosis factor- ( tnf- ) . additionally , in addition , tnf- amplifies inflammatory cascades by activating macrophages / monocytes to secrete other pro - inflammatory cytokines . while the effect of progesterone on cecal ligation and puncture ( clp)-induced tissue injury has not been studied , progesterone is reported to have a protective effect on cerebral or myocardial i / r injury in rats . in one study that was conducted to examine the inhibitory effects of progesterone on inflammatory response , progesterone treatment decreased expression of inflammatory cytokines e.g. all rats were kept in a light- and temperature - controlled room with 14:10-hour light - dark cycles and temperature of 22 0.5c , and were fed a standard pellet diet . thiopental dose of 25 mg / kg was administered and bilateral ovariectomy was performed by a midline dorsal skin incision . after the ligation of blood vessels , the connection fallopian tube and uterine horn was cut and ovaries were excised . the rats were randomly divided into four groups of seven individuals : the overiectomy group ( ovx ) , the sham operated ( control ) group , the sepsis ( clp ) group and the progesterone - treated clp group ( clp+ progesterone ) . a clp method of polymicrobial sepsis was applied to the rats in clp and clp+ progesterone groups . polymicrobial sepsis was induced by cecal ligation and two - hole puncture , as described by wichterman et al . thiopental ( 25 mg / kg ) was used to provide surgical anesthesia in rats . laparotomy was performed on the control group , and the cecum was manipulated , although not ligated or perforated . injections of 2 mg / kg progesterone in peanut oil at 2 hours after sepsis induction . postoperatively , the rats were deprived of food , although they had free access to water until they were sacrificed . sixteen hours after the surgery , all rats were sacrificed with an overdose of a general anesthetic ( thiopental sodium , 50 mg / kg ) . the liver , uterus and kidney tissues of all rats were rapidly removed and washed in ice - cold saline . activities of myeloperoxidase ( mpo ) , indicating neutrophil infiltration , and glutathione peroxidase ( gpx ) , which is an antioxidant enzyme , and concentration of malondialdehyde ( mda ) , a marker of lipid peroxidation , were measured in tissue samples . a portion of tissue was homogenized in a potassium phosphate buffer ( ph : 6 ) containing 5% hexadecyltrimethyl ammonium bromide solution ( w / v ) for mpo activity measurement ; the rest of the tissue was homogenized using a solution of 1.15% kcl for gpx and mda measurements . serum tnf- and il-6 levels were measured using rat tnf- elisa kit ( invitrogen , carlsbad , ca , usa ; catalog number : krc3011 , lot number : 776995a ) and rat il-6 platinum elisa kit ( ebioscience , san diego , ca , usa ; catalog number : bms625 , lot number : 60405021 ) according to the manufacturer 's instructions . it was determined by adding 100 l of the supernatant to 1.9 ml of 10 mmol / l phosphate buffer ( ph = 6.0 ) and 1 ml of 1.5 mmol / l o - dianisidine hydrochloride containing 0.0005% ( wt / vol ) hydrogen peroxide . then , in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate , the oxidized glutathione was converted to its reduced form with a concomitant oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate . the decrease in absorbance tissue mda levels were determined spectrophotometrically according to the method described by ohkawa et al . twenty - eight adult female sprague - dawley rats ( weighing 200 - 250 g ) were purchased from the ataturk university experimental animal laboratory . all rats were kept in a light- and temperature - controlled room with 14:10-hour light - dark cycles and temperature of 22 0.5c , and were fed a standard pellet diet . thiopental dose of 25 mg / kg was administered and bilateral ovariectomy was performed by a midline dorsal skin incision . the rats were randomly divided into four groups of seven individuals : the overiectomy group ( ovx ) , the sham operated ( control ) group , the sepsis ( clp ) group and the progesterone - treated clp group ( clp+ progesterone ) . a clp method of polymicrobial sepsis was applied to the rats in clp and clp+ progesterone groups . polymicrobial sepsis was induced by cecal ligation and two - hole puncture , as described by wichterman et al . thiopental ( 25 mg / kg ) was used to provide surgical anesthesia in rats . laparotomy was performed on the control group , and the cecum was manipulated , although not ligated or perforated . all animals were resuscitated with normal saline ( 2 ml/100 g body weight ) injected subcutaneously at the time of surgery and also at 6 hours postoperatively . injections of 2 mg / kg progesterone in peanut oil at 2 hours after sepsis induction . postoperatively , the rats were deprived of food , although they had free access to water until they were sacrificed . sixteen hours after the surgery , all rats were sacrificed with an overdose of a general anesthetic ( thiopental sodium , 50 mg / kg ) . the liver , uterus and kidney tissues of all rats were rapidly removed and washed in ice - cold saline . activities of myeloperoxidase ( mpo ) , indicating neutrophil infiltration , and glutathione peroxidase ( gpx ) , which is an antioxidant enzyme , and concentration of malondialdehyde ( mda ) , a marker of lipid peroxidation , were measured in tissue samples . a portion of tissue was homogenized in a potassium phosphate buffer ( ph : 6 ) containing 5% hexadecyltrimethyl ammonium bromide solution ( w / v ) for mpo activity measurement ; the rest of the tissue was homogenized using a solution of 1.15% kcl for gpx and mda measurements . serum tnf- and il-6 levels were measured using rat tnf- elisa kit ( invitrogen , carlsbad , ca , usa ; catalog number : krc3011 , lot number : 776995a ) and rat il-6 platinum elisa kit ( ebioscience , san diego , ca , usa ; catalog number : bms625 , lot number : 60405021 ) according to the manufacturer 's instructions . it was determined by adding 100 l of the supernatant to 1.9 ml of 10 mmol / l phosphate buffer ( ph = 6.0 ) and 1 ml of 1.5 mmol / l o - dianisidine hydrochloride containing 0.0005% ( wt / vol ) hydrogen peroxide . then , in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate , the oxidized glutathione was converted to its reduced form with a concomitant oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate . the decrease in absorbance tissue mda levels were determined spectrophotometrically according to the method described by ohkawa et al . this study was performed in accordance with the national institute of health 's approved guidelines and the experimental protocol ( protocol number : b.30.2.ata.023.85-59 ) approved by the animal research ethics committee of ataturk university , medical faculty , erzurum , turkey . twenty - eight adult female sprague - dawley rats ( weighing 200 - 250 g ) were purchased from the ataturk university experimental animal laboratory . all rats were kept in a light- and temperature - controlled room with 14:10-hour light - dark cycles and temperature of 22 0.5c , and were fed a standard pellet diet . fifteen days before the experimental study , all rats were bilaterally ovariectomized to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels . thiopental dose of 25 mg / kg was administered and bilateral ovariectomy was performed by a midline dorsal skin incision . after the ligation of blood vessels , the connection fallopian tube and uterine horn was cut and ovaries were excised . the rats were randomly divided into four groups of seven individuals : the overiectomy group ( ovx ) , the sham operated ( control ) group , the sepsis ( clp ) group and the progesterone - treated clp group ( clp+ progesterone ) . a clp method of polymicrobial sepsis was applied to the rats in clp and clp+ progesterone groups . polymicrobial sepsis was induced by cecal ligation and two - hole puncture , as described by wichterman et al . thiopental ( 25 mg / kg ) was used to provide surgical anesthesia in rats . after shaving the rats abdomen , a midline laparotomy was performed and the cecum was ligated just below the ileocecal valve using 3 - 0 silk ligatures . using a 12-gauge needle , the cecum was perforated through the cecum distal to the point of ligation at two locations 1 cm apart . laparotomy was performed on the control group , and the cecum was manipulated , although not ligated or perforated . all animals were resuscitated with normal saline ( 2 ml/100 g body weight ) injected subcutaneously at the time of surgery and also at 6 hours postoperatively . injections of 2 mg / kg progesterone in peanut oil at 2 hours after sepsis induction . postoperatively , the rats were deprived of food , although they had free access to water until they were sacrificed . sixteen hours after the surgery , all rats were sacrificed with an overdose of a general anesthetic ( thiopental sodium , 50 mg / kg ) . the liver , uterus and kidney tissues of all rats were rapidly removed and washed in ice - cold saline . activities of myeloperoxidase ( mpo ) , indicating neutrophil infiltration , and glutathione peroxidase ( gpx ) , which is an antioxidant enzyme , and concentration of malondialdehyde ( mda ) , a marker of lipid peroxidation , were measured in tissue samples . a portion of tissue was homogenized in a potassium phosphate buffer ( ph : 6 ) containing 5% hexadecyltrimethyl ammonium bromide solution ( w / v ) for mpo activity measurement ; the rest of the tissue was homogenized using a solution of 1.15% kcl for gpx and mda measurements . serum tnf- and il-6 levels were measured using rat tnf- elisa kit ( invitrogen , carlsbad , ca , usa ; catalog number : krc3011 , lot number : 776995a ) and rat il-6 platinum elisa kit ( ebioscience , san diego , ca , usa ; catalog number : bms625 , lot number : 60405021 ) according to the manufacturer 's instructions . it was determined by adding 100 l of the supernatant to 1.9 ml of 10 mmol / l phosphate buffer ( ph = 6.0 ) and 1 ml of 1.5 mmol / l o - dianisidine hydrochloride containing 0.0005% ( wt / vol ) hydrogen peroxide . in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate , the oxidized glutathione was converted to its reduced form with a concomitant oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate . the decrease in absorbance tissue mda levels were determined spectrophotometrically according to the method described by ohkawa et al . increased plasma il-6 and tnf- levels ( pg / ml ) were found in the clp group ( 36.17 3.41 and 15.56 4.46 ) in comparison with the control group ( 26.85 2.74 and 11.5 1.72 ) ( p = 0.01 , p = 0.02 ; respectively ) . however in clp+ progesterone group , the serum levels of il-6 and tnf- ( 31.79 7.89 and 12.99 3.42 ) were similar to the control group [ figure 1 ] . mpo , gpx activities and mda levels in liver , kidney and uterine tissues of study groups are presented in table 1 . in the ovx group , while liver and uterine mda concentrations ( mol / g protein ) were similar to control , the kidney mda concentration was higher than those of the control group ( p = 0.02 ) . in the clp group , kidney mda concentration was significantly higher than in the control ( p = 0.0001 ) and ovx groups ( p = 0.02 ) . in clp+ progesterone group , mean mda concentration of kidney tissue was significantly lower than in clp group ( p = 0.003 ) . mda concentrations in the liver and uterine tissues were significantly elevated in the clp group compared to the control group ( p = 0.01 for both ) . liver mda concentration of the clp+ progesterone group was not significantly different from that of control group . while there were no significant differences among groups regarding to liver mpo ( u / g protein ) ; in the clp group , mpo activity in kidney ( p = 0.02 ) and uterine tissue ( p = 0.03 ) were found to be significantly higher compared to the control group . in clp+ progesterone group , mean mpo activities of all tissues were not different than those of control group . while the gpx activity ( u / g protein ) in uterine tissue of the clp group was lower than those of the control group ( p = 0.02 ) , the gpx activity in the clp+ progesterone group was not statistically significantly different from control group . in the kidney and liver tissues comparison of groups according to serum tnf alpha and il-6 levels . * p = 0.01 and * * p = 0.02 compared to control group mpo and gp activities and mda levels in liver , kidney and uterine tissues of control , clp , ovx and clp+prog groups excessive production of cytokines ( e.g. , il-6 , tnf- ) is reported in a part of sepsis pathogenesis . the purpose of this study was to examine the effects of progesterone on systemic inflammation and tissue damage of ovariectomized rats in a clp - induced sepsis model . however , the potential role of progesterone in sepsis has not been studied . in our study , increased serum levels of il-6 and tnf- reduced after progesterone treatment in a rat model created sepsis with clp . also , clp increased the mpo activity in kidney and uterine tissues , but similar values to the control group were observed after treatment with progesterone . the key finding in our study was that exogenously given progesterone in sepsis resulted in a significant decrease in systemic inflammation and mpo activity , as a leukocyte activation marker , compared to rats that did not receive it . in a study , expression of tnf- reduced in progesterone - treated rats compared to controls and authors suggested that progesterone administration is beneficial for cerebral trauma and infarction by inhibiting inflammatory reaction . 's study , they investigated the effect of progesterone on oxidative stress and myocardial ischemia markers in the myocardial ischemia - reperfusion model . at the end of the study , they concluded that the administration of progesterone during myocardial injury reduces inflammatory reactivity and provides better cardioprotection in female rats compared to male and they suggested that progesterone therapy may be useful in myocardial injury due to a diminished inflammatory response . before beginning the experimental study , we removed the ovaries of all rats to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels . our results demonstrated that progesterone attenuates the sepsis - induced elevations of il-6 and tnf- . we applied 2 mg / kg dose of progesterone immediately after clp . in the brain injury model , it was suggested that progesterone in inhibiting the proinflammatory cytokines is time - dependent . after 8 and 12 hours of continued administration of progesterone , cytokine elevations were not observed . researched the effects of progesterone on the intestinal pathophysiologic changes following subarachnoid hemorrhage in male rats . after progesterone administration ( 2 mg / kg i.p . ) , the concentrations of il-1 , tnf- and il-6 were found to be significantly lower in rat ileum tissue . according to our literature data , this present study is the first investigating the efficacy of progesterone on sepsis in a rat model created sepsis using clp . in a recent study , they reported both lower blood and colonic tissue levels of mda , il-6 and tnf- in rats treated with progesterone ( subcutaneously with dose of 2 mg / kg ) compared to the rats that only created colitis by intrarectal administration of 5 mg trinitrobenzene sulfonic acid . in another study , investigating possible neuroprotective effects of progesterone in rats subjected to global cerebral ischemia , progesterone administration ameliorated ischemia - induced decrease in glutathione ( major endogenous antioxidant ) and increase in mda levels in hippocampus , striatum and cortex . they evaluated the remarkable neuroprotective effect of progesterone reducing oxidative stress . in our study , the mda concentration in tissues of uterine , liver and kidney and plasma il-6 and tnf- level increased in rats after created clp , but the values after progesterone treatment were similar to the values in the control group . also while increased uterine tissue gpx activity ( an enzymatic antioxidant ) was found in rats to create sepsis , similar uterine gpx activity to the control group it may be suggested that progesterone ameliorates sepsis syndrome by reduction of the inflammatory cytokines , il-6 and tnf- , and by restoration of antioxidant defense system .

coordination polymers and especially their porous congeners , which are frequently termed mofs ( metal - organic frameworks),(1 ) are in the focus of many research groups worldwide . the interest in this class of compounds is mainly based on their easy synthetic accessibility and potential applications . one of the most frequently used linker ligands is the dianion of terephthalic acid ( h2bdc ) , from which two of the most prominent members of this class of compounds , mof-5(1 ) and mil-53,(5 ) are constructed . for its perfluorinated counterpart , that is 2,3,5,6-tetrafluoroterephthalic acid ( h2tfbdc ) , theoretical investigations predict superior h2 adsorbing properties of mofs built with this linker ligand.(6 ) by contrast , a different computational study concludes that the interaction between h2 and fluoroaromatic compounds may be even weaker compared with their non - fluorinated analogues.(7 ) the beneficial properties of fluorinated linkers are , however , supported by experimental results for a mof based on a triazolate with cf3 substituents , which shows excellent gas storage capacities for o2 and h2.(8 ) to date only few coordination polymers or mofs employing perfluorinated linking terephthalates ( tfbdc ) have been published . most of them are non - porous . in 1964 a synthesis of h2tfbdc was described starting from 1,2,4,5-tetrafluorobenzene , which was reacted with n - butyllithium and carbonated with co2.(22 ) although this route yielded 67% of pure product , it suffers from incomplete lithiation , and consequently the monosubstituted tetrafluorobenzene is always obtained as a byproduct . this disadvantage makes an extensive and time - consuming purification procedure necessary . in the following we will present an optimized synthesis , which allows the preparation of gram quantities of pure h2tfbdc by a comparatively simple procedure , also starting from 1,2,4,5-tetrafluorobenzene , which is available at a reasonable price . crystal structures of h2tfbdc ( 1 ) , h2tfbdc2h2o ( 2 ) , and ( nh4)2tfbdc ( 3 ) are presented , as well as the synthesis and crystal structure of the monosubstituted 2,3,5,6-tetrafluorobenzoic acid ( 4 ) , which can be obtained as a byproduct using a slightly modified procedure . 1,2,4,5-tetrafluorobenzene was obtained from molekula and fluorochem , n - butyllithium ( 1.6 m solution in hexane ) and magnesium sulfate from acros organics . 2.13 g of 1,2,4,5-tetrafluorobenzene ( 14.2 mmol , 1.0 equiv ) were dissolved in 250 ml of dry thf and cooled to approximately 75 c . during 30 min 25 ml of n - buli ( 40.0 mmol , 2.8 equiv ) after 4 h of stirring , co2 obtained by sublimating dry ice was bubbled through the solution , upon which the mixture became a white sludge . the solvent was removed , and the white solid residue was hydrolyzed with 100 ml of aqueous hcl ( 7.5% ) and 100 ml of et2o . the total yield after recrystallization was 3.2 g ( 13.4 mmol , 95% based on c6f4h2 ) . elemental analysis for c8o4h2f4 ( 238.096 ) : calcd c , 40.35% , h , 0.85% ; found c , 40.44 , h , 0.85% . the melting point is somewhat lower than that given in the literature ( 283284 c),(22 ) but agrees well with the melting point of the raw material before recrystallization ( 261276 c)(22 ) and the commercially available product ( 275277 c , sigma - aldrich ) . we assume that recrystallization from water(22 ) leads to the dihydrate ( 2 ) , which shows a slightly higher melting point . actually , 1 does not melt , instead it decomposes . a proof based on dta / tg investigations h nmr ( acetone - d6 ) : [ ppm ] = 11.2 ( s , 2h ; cooh ) . c nmr ( acetone - d6 ) [ ppm ] = 160.1 ( s ; cooh ) ; 145.9 ( dm ; jcf = 253.1 hz , c2,3,5,6 ) , 116.4 ( m , c1,4 ) . f - nmr ( acetone - d6 ) = 140.75 ( s ; f2,3,5,6 ) . the purity was checked by nmr and x - ray powder diffraction ( xrpd ) . as no additional signals or reflections were visible in the nmr spectra ( h , c , f ) and xrpd patterns the purity can be estimated to be > 98% . a 5.00 g portion of 1,2,4,5-tetrafluorobenzene , ( 33.3 mmol , 1.0 equiv ) , dissolved in thf ( 200 ml ) , was cooled to approximately 75 c , and 42.7 ml of n - buli ( 68.3 mmol , 2.05 equiv ) were added over a period of 30 min . the suspension was allowed to stir for 30 min at this temperature never rising over 60 c . dried co2 ( passed through a p4o10 column ) was bubbled through the solution , 10 min at about 70 c , and then continued , while the mixture was allowed to reach ambient temperature ( 1 h ) . to the recooled ( 0 c ) solution , aqueous hcl ( 1 m ) was added and stirred overnight . the solvents were removed by distillation ( the fraction boiling around 105 was collected , as it contains almost pure monocarboxylic acid , as described in literature(22 ) ) . the residue of the distillation was washed with a small amount of pentane yielding a crude product mixture of 1 and 4 in a ratio of about 7:3 . the above - described procedure , lithiation and subsequent carbonation , employing the product mixture of 1 and 4 , rather than tetrafluorobenzene was repeated two or three times giving highly pure 1 in a total yield of about 44% based on c6f4h2 ( 3.53 g , 14.8 mmol ) . single crystals of a dihydrate of 1 were obtained by recrystallization of 1 from a water / acetone mixture . a 250.0 mg portion of tetrafluoroterephthalic acid ( 1 ) ( 1.05 mmol ) was suspended in 25 ml of deionized water . water and excess ammonia were removed by distillation , and 3 was obtained as a white powder in a total yield of 98% ( 280.2 mg , 1.03 mmol ) . after recrystallization from water , colorless flaky crystals suitable for a single crystal structure determination were obtained after 3 weeks . elemental analysis for c8o4h8f4n2 ( 272.164 ) : calcd n , 10.30% , c , 35.30% , h , 2.96% ; found n , 10.24% , c , 35.87% , h , 2.94% . 4 was obtained as a byproduct of the synthesis of 1 using the synthesis protocol ( b ) as described above . the fraction boiling between 100108 c h nmr ( acetone - d6 ) : [ ppm ] = 11.7 ( s , 1h ; cooh ) 7.74 ( tt , jhf 10.2 hz , jhf 7.4 hz , c4h ) . c nmr ( acetone - d6 ) [ ppm ] = 160.5 ( s ; cooh ) ; 146.96 ( dm , jcf 243.5 hz , c3,5 ) , 145.27 ( dm , jcf 252.6 hz , c2,6 ) , 114.76 ( t , jcf 18.7 hz , c1 ) , 109.76 ( t , jcf 23.0 hz , c4 ) . f - nmr ( acetone - d6 ) = 139.82 ( m , -f2,6 ) , 142.03 ( br.s , f3,5 ) . single crystals of 15 were isolated as described above and mounted in sealed glass capillaries on a stoe four - circle ( stadi ) , stoe ipds ii , or a bruker apex - ii single crystal diffractometer ( mok radiation ) . for data collection and reduction the stoe program package was applied.(24 ) the structural models were solved using sir-92 ( 3 ) or shelxs ( 1 , 2 , 4 , 5 ) and completed using difference fourier maps calculated with shelxl-97 , which was also used for final refinements . for the structure of 3 and 5 all programs were run under the wingx system.(27 ) all non - hydrogen atoms were refined anisotropically . more details of the structural analysis are given in table 1.(28 ) selected interatomic distances and angles are listed in table 2 . xrpd data were collected on a huber g670 with a germanium monochromator and cu k1 radiation at room temperature , with exposure times of approximately 2 h. samples were sealed in capillaries ( 0.30.5 mm ) . within the winxpow software suite(29 ) the recorded patterns were compared with theoretical patterns calculated from the obtained crystal structure data . a differential thermal analysis ( dta ) and thermogravimetry ( tg ) investigation was performed on diammonium-2,3,5,6-tetrafluoroterephthalate ( 3 ) ( sample mass : 19.1 mg ) in the temperature range 24400 c using a netzsch sta 409c housed in a glovebox ( m. braun , garching / germany ) , heating rate 10 c / min . nmr spectra have been recorded on a bruker avance 300 spectrometer ( h , 300.13 mhz , c , 75.47 mhz , f , 282.35 mhz ) , with h decoupling for f and c. chemical shifts are specified relative to the residual solvent signal ( h , c ) and ccl3f as internal standard ( f ) . pure 2,3,5,6-tetrafluoroterephthalic acid ( h2tfbdc , 1 ) is obtained in high yields ( 95% ) by reacting 1,2,4,5-tetrafluorobenzene with a surplus ( > 2 equiv ) of n - buli in thf and subsequent carbonation with co2 without any extensive purification procedure . it was found that a larger surplus ( approximately 2.8 equiv ) of n - buli is needed to avoid the formation of the monosubstituted product 2,3,5,6-tetrafluorobenzoic acid ( 4 ) . a mixture of the mono- and disubstituted products makes an extensive purification procedure necessary , as was shown in the literature(22 ) and was confirmed by us ( see protocol ( b ) ) . so the synthesis described in protocol ( a ) , which exclusively leads to h2tfbdc , presents a convenient route to gram quantities of pure 1 . the unit cell constants are quite similar to those previously reported in x - ray and neutron diffraction studies of the deuterated derivative , which have been performed at room temperature.(30 ) the molecular structure of 1 ( figure 1 ) is almost identical to the reported structures from these studies , although slight deviations occur . for instance , the plane of the carboxylate group is twisted by 19.3(1) around the bond c1c7 out of the least - squares plane of the aryl ring , which is slightly smaller than reported for the structure of the perdeuterated acid ( 19.8(1) ) . this disorder is also reflected in similar c7o1 and c7o2 distances ( table 2 ) . ortep diagram of h2tfbdc ( 1 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . hydrogen atoms ( h1 , h2 ) are disordered over two sites with occupancy of 0.5 . compound 1 shows an infinite one - dimensional chain structure ( figure 2 ) connected by hydrogen bonds between neighboring carboxylate units . as mentioned the h atoms of the oh groups they were refined with site occupation factors of 0.5 , tetrahedral coh angles , enabling rotation around the co bond , oh distances of 0.84 , and with individual isotropic displacement parameters . packing diagram of h2tfbdc ( 1 ) ( c : white , o : red , f : green ) . the packing of the structures of 1 and its deuterated congener reveal , however , significant differences.(30 ) in different layers the infinite chains are arranged in a way that the carboxylate groups are above and below the aryl rings of the neighboring layers for 1 , while in the deuterated variety the aryl rings are stacked above each other . in 1 the planes of the aryl rings of different layers are related by translation along the crystallographic a - axis showing a distance c3c3 of 3.294(2 ) , which is significantly shorter than in its perdeuterated analogue ( 3.377 ) . concomitantly , the centroids of the aryl rings are closer in 1 ( 4.565(2 ) ) than in the deuterated variety ( 4.617(2 ) ) . this arrangement results in a short intramolecular contact between the layers ( o1o2 : 3.033(2 ) ) . furthermore , short fcaryl ( 2.979(2 ) 3.109(2 ) ) contacts contribute to the packing of this structure . according to a preliminary dta / tg investigation a mass loss of 1 accompanied by an exothermal signal started at approximately 220 c . this process is finished at approximately 290 c with no mass remaining . to prove whether this effect is due to decomposition or sublimation of 1 , in an additional experiment performed in an argon atmosphere some of the gases evolved upon heating thus , 1 decomposes to give 4 by releasing co2 : in the presence of water , 1 forms a 1:2 adduct with this solvent ( figure 3 ) . all oh distances were fixed to a bond length of 0.95 , but no further constraints were applied to the hydrogen atoms . structural parameters for the perfluoro aryl moiety are almost identical to the parameters of 1 ( table 2 ) . the carboxylate group shows two distinctive co distances of 1.219(1 ) and 1.304(1 ) , which is in the typical range for c = o and co bonds , respectively . the planes spanned by the aryl moiety and the carboxylate group enclose an angle of 39.9(1) , which is significantly larger than in 1 . the oho distances are 1.59(2 ) , 1.89(2 ) , and 1.95(2 ) ( table 3 ) . via these hydrogen bonds ortep diagram of h2tfbdc2h2o ( 2 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . packing diagram of h2tfbdc2h2o ( 2 ) ( c , white ; o , red ; f , green ; h , white , small spheres ) . hydrogen atoms were located in difference fourier maps and refined with fixed nh distances ( 1.00(2 ) ) ( figure 5 ) . the resulting nh bond lengths and hnh bond angles ( tables 2 and 3 ) agree well with those expected for a perfect tetrahedral nh4 cation . structural parameters for the perfluoro aryl moiety are almost identical to the parameters obtained for 1 and 2 ( table 2 ) . the carboxylate groups of one tfbdc anion are parallel to each other and enclose an angle of 44.5(2) with the plane of the aryl ring . this twist is much larger than in 1 , slightly larger than in 2 and also larger than in the non - fluorinated congener ( 18(1) and 29(2)).(31 ) but as the latter was solved and refined from powder diffraction data the reliability of these angles is somewhat lower . thus , a further comparison between the fluorinated and the non - fluorinated compound shall be omitted here . the tfbdc anions are bridged via oxygen atoms of the carboxylate groups and protons of the ammonium cations to form a 3d structure ( figure 6 ) . as each oxygen atom is connected to two nh4 cations the tfbdc anion acts as an 8-connector . the resulting ho distances of these hydrogen bonds are 1.93(2 ) and 1.98(2 ) . this makes 3 an attractive candidate for the synthesis of new coordination polymers with tfbdc as bridging ligand in aqueous solutions . ortep diagram of ( nh4)2tfbdc ( 3 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . packing diagram of ( nh4)2tfbdc ( 3 ) in a projection along ( c , white ; o , red ; f , green ; n , blue ; h , white , small spheres ) . hydrogen bonds are drawn as red broken lines . to explore the thermal stability of 3 we performed basic thermoanalytical measurements . in figure 7 starting at approximately 225 c a complete mass loss is observed , which is accompanied by a strong endothermic signal . ( nh4)2adc ( adc = acetylenedicarboxylate ) to nh3 , co2 , and c2h2 gives an exothermic signal.(32 ) to check whether 3 decomposes or sublimes , some of the gases evolved upon heating were condensed at the wall of a glass tube in an argon atmosphere . xrpd data revealed that the collected colorless solid is neither 3 ( sublimation ) nor 1 or 4 ( decomposition ) . by recrystallization from dry acetone single crystals have been obtained . according to the structure solution , the composition of this new compound ( 5 ) can be described as a 1:1 adduct of 2,3,5,6-tetrafluorobenzoic acid ( 4 ) with its ammonium salt ( figure 8) . elemental analysis of the white residue gave n , 5.98% , c , 40.90% , h , 2.28% . the calculated values for 5 are c14o4h7f8n ( 405.201 ) : n , 3.46% , c , 41.50% , h , 1.74% . the large deviation between these values proves that the collected white residue does not contain 2,3,5,6-tetrafluorobenzoic acid and ammonium ( nh4)2tfbdc ( 3 ) : dta ( blue ) and tg ( black ) curves . ortep diagram of h2tfbc(nh4)htfbc ( 5 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . compound 5 crystallizes in the orthorhombic chiral space group p212121 as colorless needles ( table 1).(33 ) all non - hydrogen atoms were refined with anisotropic displacement parameters without any constraints . the h atoms h5 and h12 were put at the external bisector of the ccc angle at a ch distance of 0.95 , but the individual isotropic displacement parameters are free to refine . h atoms of the ammonium ion are located in the difference fourier map and refined isotropically . the h atom of carboxylic acid was refined isotropically with a tetrahedral coh angle . owing to the lack of heavier atoms , determination of the absolute structure in 5 tetrafluorobenzoic acid forms a quite strong hydrogen bond(34 ) to tetrafluorobenzoate via the carboxylic oh group ( donoracceptor [ d / a ] distance 2.541(2 ) ) , which is significantly shorter than in 1 and 4 . a second hydrogen bond between the ion pairs of the tetrafluorobenzoate and the ammonium ion ( d / a distance 2.833(2 ) ) is formed , resulting in a 2-dimensional network built up by the ammonium ion acting as a 4-connector . the carboxylic groups are twisted out of the least - squares planes of the aryl rings by 40.2(1) and 50.1(1) for the tetrafluorobenzoic acid and the tetrafluorobenzoate , respectively , which is for the latter even larger than in 3 . in essence , the composition of 5 proves that the thermal decomposition of 3 involves the release of both co2 and nh3 . the h atoms h14 and h24 were put at the external bisector of the ccc angle at a ch distance of 0.95 , but the individual isotropic displacement parameters are free to refine . the disordered h atoms h15 and h25 of the cooh groups were refined with site occupation factors of 0.5 , tetrahedral coh angles , enabling rotation around the co bond , oh distances of 0.95 , and with individual isotropic displacement parameters . the molecules are lying on 2-fold axes parallel to the c - axis forming dimers held together by two hydrogen bonds ( figure 9 ) . whereas the two carboxylate groups are almost coplanar ( angle between the root mean square ( rms ) planes = 2.7(1) ) , there are considerable torsions around the cc single bonds between the phenyl rings and the carboxylate groups ( = 22.8(1) and = 40.9(1) for c11c15 and c21c25 , respectively ) resulting in a torsional angle between the two aryl groups of 60.9(1). structural parameters of the phenyl ring and the carboxylic moiety in 4 are almost identical to its disubstituted congener 1 ( table 2 ) . the f22h24 and f12h14 distances are 2.418(2 ) and 2.771(2 ) , which is for the former significantly shorter than the sum of their van der waals radii ( 2.67 ) . the axis along the dimers is perpendicular to the ( 001 ) plane ( figure 10 ) . this is in sharp contrast to crystal structures of benzoic acid , which also forms dimers in the solid state , but in the packing two distinctive orientations of the dimers are found . ortep diagram of h2tfbc ( 4 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . hydrogen atoms ( h15 , h25 ) are disordered over two sites with occupancy of 0.5 . packing diagram of h2tfbc ( 4 ) ( c , white , o , red , f , green , h , white , small spheres ) . disordered hydrogen atoms ( h15 , h25 ) have been omitted for clarity . by a simple modification of the synthesis known from the literature(22 ) we developed a convenient synthesis for pure 2,3,5,6-tetrafluoroterephthalic acid ( h2tfbdc ) . just by adding a surplus of n - buli the formation of the byproduct 2,3,5,6-tetrafluorobenzoic acid is suppressed so that an extensive purification procedure no longer is necessary . as tfbdc is expected to be an important ligand in the synthesis of coordination polymers and mofs we think that this synthesis might boost the research with this ligand . several compounds have already been synthesized with this ligand , but only few of them show permanent porosity . to the best of our knowledge an all - f - mof is still unknown , although such a compound should show interesting hydrogen storage properties and would be a perfect model compound for the investigation of h2 bonding sites in such a mof , as no deuteration of the ligand for neutron diffraction experiments is necessary . but it was stated that a second , non - fluorinated ligand is usually necessary to facilitate the incorporation of perfluorinated dicarboxylates into hybrid structures.(21 ) with gram quantities of h2tfbdc at hand at comparatively low costs , it might be worthwhile to prove whether this assumption is correct . next to h2tfbdc ( 1 ) the dihydrate h2tfbdc2h2o ( 2 ) , the ammonium salt ( nh4)2tfbdc ( 3 ) , and the monosubstituted acid h2tfbc ( 4 ) were synthesized and characterized by an x - ray single crystal structure analysis . 1 and 4 exhibit hydrogen bonding via the carboxylate groups , which leads to a dimer in 4 ( figure 9 ) and a chain - like polymer in 1 ( figure 2 ) . are included so that a complex 3d hydrogen bonding network is formed ( figure 4 ) , where , with respect to the distances , the strongest hydrogen bond is found ( o2h2o3 ; da 2.540(1 ) , table 3 ) , which are comparable to those found for 5 ( o1h1bo4 ; da 2.541(2 ) , table 3 ) . in 3 the cations ( nh4 ) and anions ( tfbdc ) are connected by hydrogen bonds again arranged in a 3d network ( figure 6 ) . for 1 , 3 , and 4 non - fluorinated congeners are known , but all of them exhibit crystal structures with distinct differences compared to the fluorinated counterparts , although the general structural arrangements of 1 ( p1 , z = 1 ) and its non - fluorinated congener ( p1 , z = 1)(37 ) , both form infinite chains of h2tfbdc and h2bdc molecules linked by double hydrogen bonds , and 4 ( fdd2 , z = 16 ) and its non - fluorinated congener ( p21/c , z = 4)(36 ) , both form dimers with disordered h atoms participating in the hydrogen bonds , are very similar . in contrast , for the fluorinated compounds the carboxylate groups are tilted with respect to the plane of the aryl rings ( 1 : 19.35(7) ; 4 : 22.8(1) and 40.9(1) , respectively ) , whereas in the non - fluorinated compounds they are almost coplanar . the crystal structure of the non - fluorinated congener ( pbc21 , z = 4)(31 ) of 3 ( c2/m , z = 2 ) was solved and refined from synchrotron powder diffraction data . here angles between the planes of the carboxylate groups and the aryl ring of 29.0(8) and 31(1) are found , which are also significantly smaller than in 3 ( 44.54(7) ) . thus , the presence of the f atoms reduces the electron density in the aryl rings and therefore conjugation between the aryl -system and the carboxyl group , which in turn allows stronger deviations from the coplanarity . furthermore , deviation from coplanarity may be a consequence of electrostatic repulsion between o and the o - fluorine atoms . both effects work synergistically and illustrate nicely that structural properties of non - fluorinated compounds can not be transferred one to one to their fluorinated counterparts . this different structural behavior might be among the reasons why perfluorinated analogues of mil-53 or mof-5 are currently still unknown .

pure 2,3,5,6-tetrafluoroterephthalic acid ( h2tfbdc ) is obtained in high yields ( 95% ) by reacting 1,2,4,5-tetrafluorobenzene with a surplus ( > 2 equiv ) of n - butyllithium in tetrahydrofuran ( thf ) and subsequent carbonation with co2 without any extensive purification procedure . a single crystal x - ray structure analysis of h2tfbdc ( 1 ) confirms former data obtained for a deuterated sample ( p1 , z = 1 ) . recrystallization from water / acetone leads to single crystals of h2tfbdc2h2o ( 2 , p21/c , z = 2 ) , where an extensive hydrogen bonding network is found . by reacting h2tfbdc with an aqueous ammonia solution , single crystals of ( nh4)2tfbdc ( 3 , c2/m , z = 2 ) are obtained . 3 is thermally stable up to 250 c and shows an enhanced solubility in water compared to h2tfbdc . monosubstituted 2,3,5,6-tetrafluorobenzoic acid ( h2tfbc , 4 ) is obtained by reacting 1,2,4,5-tetrafluorobenzene with stoichiometric amounts ( 1 equiv ) of n - butyllithium in thf . its crystal structure ( fdd2 , z = 16 ) shows dimeric units as characteristic structural feature .

Introduction Experimental Section Results and Discussion Conclusion

coordination polymers and especially their porous congeners , which are frequently termed mofs ( metal - organic frameworks),(1 ) are in the focus of many research groups worldwide . one of the most frequently used linker ligands is the dianion of terephthalic acid ( h2bdc ) , from which two of the most prominent members of this class of compounds , mof-5(1 ) and mil-53,(5 ) are constructed . for its perfluorinated counterpart , that is 2,3,5,6-tetrafluoroterephthalic acid ( h2tfbdc ) , theoretical investigations predict superior h2 adsorbing properties of mofs built with this linker ligand. (6 ) by contrast , a different computational study concludes that the interaction between h2 and fluoroaromatic compounds may be even weaker compared with their non - fluorinated analogues. (7 ) the beneficial properties of fluorinated linkers are , however , supported by experimental results for a mof based on a triazolate with cf3 substituents , which shows excellent gas storage capacities for o2 and h2. (8 ) to date only few coordination polymers or mofs employing perfluorinated linking terephthalates ( tfbdc ) have been published . in 1964 a synthesis of h2tfbdc was described starting from 1,2,4,5-tetrafluorobenzene , which was reacted with n - butyllithium and carbonated with co2. this disadvantage makes an extensive and time - consuming purification procedure necessary . crystal structures of h2tfbdc ( 1 ) , h2tfbdc2h2o ( 2 ) , and ( nh4)2tfbdc ( 3 ) are presented , as well as the synthesis and crystal structure of the monosubstituted 2,3,5,6-tetrafluorobenzoic acid ( 4 ) , which can be obtained as a byproduct using a slightly modified procedure . 1,2,4,5-tetrafluorobenzene was obtained from molekula and fluorochem , n - butyllithium ( 1.6 m solution in hexane ) and magnesium sulfate from acros organics . 2.13 g of 1,2,4,5-tetrafluorobenzene ( 14.2 mmol , 1.0 equiv ) were dissolved in 250 ml of dry thf and cooled to approximately 75 c . during 30 min 25 ml of n - buli ( 40.0 mmol , 2.8 equiv ) after 4 h of stirring , co2 obtained by sublimating dry ice was bubbled through the solution , upon which the mixture became a white sludge . the solvent was removed , and the white solid residue was hydrolyzed with 100 ml of aqueous hcl ( 7.5% ) and 100 ml of et2o . the melting point is somewhat lower than that given in the literature ( 283284 c),(22 ) but agrees well with the melting point of the raw material before recrystallization ( 261276 c)(22 ) and the commercially available product ( 275277 c , sigma - aldrich ) . we assume that recrystallization from water(22 ) leads to the dihydrate ( 2 ) , which shows a slightly higher melting point . c nmr ( acetone - d6 ) [ ppm ] = 160.1 ( s ; cooh ) ; 145.9 ( dm ; jcf = 253.1 hz , c2,3,5,6 ) , 116.4 ( m , c1,4 ) . the purity was checked by nmr and x - ray powder diffraction ( xrpd ) . as no additional signals or reflections were visible in the nmr spectra ( h , c , f ) and xrpd patterns the purity can be estimated to be > 98% . a 5.00 g portion of 1,2,4,5-tetrafluorobenzene , ( 33.3 mmol , 1.0 equiv ) , dissolved in thf ( 200 ml ) , was cooled to approximately 75 c , and 42.7 ml of n - buli ( 68.3 mmol , 2.05 equiv ) were added over a period of 30 min . dried co2 ( passed through a p4o10 column ) was bubbled through the solution , 10 min at about 70 c , and then continued , while the mixture was allowed to reach ambient temperature ( 1 h ) . to the recooled ( 0 c ) solution , aqueous hcl ( 1 m ) was added and stirred overnight . the solvents were removed by distillation ( the fraction boiling around 105 was collected , as it contains almost pure monocarboxylic acid , as described in literature(22 ) ) . the residue of the distillation was washed with a small amount of pentane yielding a crude product mixture of 1 and 4 in a ratio of about 7:3 . the above - described procedure , lithiation and subsequent carbonation , employing the product mixture of 1 and 4 , rather than tetrafluorobenzene was repeated two or three times giving highly pure 1 in a total yield of about 44% based on c6f4h2 ( 3.53 g , 14.8 mmol ) . single crystals of a dihydrate of 1 were obtained by recrystallization of 1 from a water / acetone mixture . a 250.0 mg portion of tetrafluoroterephthalic acid ( 1 ) ( 1.05 mmol ) was suspended in 25 ml of deionized water . after recrystallization from water , colorless flaky crystals suitable for a single crystal structure determination were obtained after 3 weeks . c nmr ( acetone - d6 ) [ ppm ] = 160.5 ( s ; cooh ) ; 146.96 ( dm , jcf 243.5 hz , c3,5 ) , 145.27 ( dm , jcf 252.6 hz , c2,6 ) , 114.76 ( t , jcf 18.7 hz , c1 ) , 109.76 ( t , jcf 23.0 hz , c4 ) . f - nmr ( acetone - d6 ) = 139.82 ( m , -f2,6 ) , 142.03 ( br.s , f3,5 ) . single crystals of 15 were isolated as described above and mounted in sealed glass capillaries on a stoe four - circle ( stadi ) , stoe ipds ii , or a bruker apex - ii single crystal diffractometer ( mok radiation ) . (24 ) the structural models were solved using sir-92 ( 3 ) or shelxs ( 1 , 2 , 4 , 5 ) and completed using difference fourier maps calculated with shelxl-97 , which was also used for final refinements . xrpd data were collected on a huber g670 with a germanium monochromator and cu k1 radiation at room temperature , with exposure times of approximately 2 h. samples were sealed in capillaries ( 0.30.5 mm ) . within the winxpow software suite(29 ) the recorded patterns were compared with theoretical patterns calculated from the obtained crystal structure data . a differential thermal analysis ( dta ) and thermogravimetry ( tg ) investigation was performed on diammonium-2,3,5,6-tetrafluoroterephthalate ( 3 ) ( sample mass : 19.1 mg ) in the temperature range 24400 c using a netzsch sta 409c housed in a glovebox ( m. braun , garching / germany ) , heating rate 10 c / min . nmr spectra have been recorded on a bruker avance 300 spectrometer ( h , 300.13 mhz , c , 75.47 mhz , f , 282.35 mhz ) , with h decoupling for f and c. chemical shifts are specified relative to the residual solvent signal ( h , c ) and ccl3f as internal standard ( f ) . pure 2,3,5,6-tetrafluoroterephthalic acid ( h2tfbdc , 1 ) is obtained in high yields ( 95% ) by reacting 1,2,4,5-tetrafluorobenzene with a surplus ( > 2 equiv ) of n - buli in thf and subsequent carbonation with co2 without any extensive purification procedure . it was found that a larger surplus ( approximately 2.8 equiv ) of n - buli is needed to avoid the formation of the monosubstituted product 2,3,5,6-tetrafluorobenzoic acid ( 4 ) . a mixture of the mono- and disubstituted products makes an extensive purification procedure necessary , as was shown in the literature(22 ) and was confirmed by us ( see protocol ( b ) ) . so the synthesis described in protocol ( a ) , which exclusively leads to h2tfbdc , presents a convenient route to gram quantities of pure 1 . the unit cell constants are quite similar to those previously reported in x - ray and neutron diffraction studies of the deuterated derivative , which have been performed at room temperature. (30 ) the molecular structure of 1 ( figure 1 ) is almost identical to the reported structures from these studies , although slight deviations occur . for instance , the plane of the carboxylate group is twisted by 19.3(1) around the bond c1c7 out of the least - squares plane of the aryl ring , which is slightly smaller than reported for the structure of the perdeuterated acid ( 19.8(1) ) . this disorder is also reflected in similar c7o1 and c7o2 distances ( table 2 ) . ortep diagram of h2tfbdc ( 1 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . hydrogen atoms ( h1 , h2 ) are disordered over two sites with occupancy of 0.5 . compound 1 shows an infinite one - dimensional chain structure ( figure 2 ) connected by hydrogen bonds between neighboring carboxylate units . packing diagram of h2tfbdc ( 1 ) ( c : white , o : red , f : green ) . in 1 the planes of the aryl rings of different layers are related by translation along the crystallographic a - axis showing a distance c3c3 of 3.294(2 ) , which is significantly shorter than in its perdeuterated analogue ( 3.377 ) . furthermore , short fcaryl ( 2.979(2 ) 3.109(2 ) ) contacts contribute to the packing of this structure . structural parameters for the perfluoro aryl moiety are almost identical to the parameters of 1 ( table 2 ) . the carboxylate group shows two distinctive co distances of 1.219(1 ) and 1.304(1 ) , which is in the typical range for c = o and co bonds , respectively . the oho distances are 1.59(2 ) , 1.89(2 ) , and 1.95(2 ) ( table 3 ) . via these hydrogen bonds ortep diagram of h2tfbdc2h2o ( 2 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . packing diagram of h2tfbdc2h2o ( 2 ) ( c , white ; o , red ; f , green ; h , white , small spheres ) . hydrogen atoms were located in difference fourier maps and refined with fixed nh distances ( 1.00(2 ) ) ( figure 5 ) . the resulting nh bond lengths and hnh bond angles ( tables 2 and 3 ) agree well with those expected for a perfect tetrahedral nh4 cation . structural parameters for the perfluoro aryl moiety are almost identical to the parameters obtained for 1 and 2 ( table 2 ) . this twist is much larger than in 1 , slightly larger than in 2 and also larger than in the non - fluorinated congener ( 18(1) and 29(2)). the tfbdc anions are bridged via oxygen atoms of the carboxylate groups and protons of the ammonium cations to form a 3d structure ( figure 6 ) . the resulting ho distances of these hydrogen bonds are 1.93(2 ) and 1.98(2 ) . ortep diagram of ( nh4)2tfbdc ( 3 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . packing diagram of ( nh4)2tfbdc ( 3 ) in a projection along ( c , white ; o , red ; f , green ; n , blue ; h , white , small spheres ) . ( nh4)2adc ( adc = acetylenedicarboxylate ) to nh3 , co2 , and c2h2 gives an exothermic signal. by recrystallization from dry acetone single crystals have been obtained . according to the structure solution , the composition of this new compound ( 5 ) can be described as a 1:1 adduct of 2,3,5,6-tetrafluorobenzoic acid ( 4 ) with its ammonium salt ( figure 8) . elemental analysis of the white residue gave n , 5.98% , c , 40.90% , h , 2.28% . the large deviation between these values proves that the collected white residue does not contain 2,3,5,6-tetrafluorobenzoic acid and ammonium ( nh4)2tfbdc ( 3 ) : dta ( blue ) and tg ( black ) curves . (33 ) all non - hydrogen atoms were refined with anisotropic displacement parameters without any constraints . h atoms of the ammonium ion are located in the difference fourier map and refined isotropically . the h atom of carboxylic acid was refined isotropically with a tetrahedral coh angle . owing to the lack of heavier atoms , determination of the absolute structure in 5 tetrafluorobenzoic acid forms a quite strong hydrogen bond(34 ) to tetrafluorobenzoate via the carboxylic oh group ( donoracceptor [ d / a ] distance 2.541(2 ) ) , which is significantly shorter than in 1 and 4 . a second hydrogen bond between the ion pairs of the tetrafluorobenzoate and the ammonium ion ( d / a distance 2.833(2 ) ) is formed , resulting in a 2-dimensional network built up by the ammonium ion acting as a 4-connector . whereas the two carboxylate groups are almost coplanar ( angle between the root mean square ( rms ) planes = 2.7(1) ) , there are considerable torsions around the cc single bonds between the phenyl rings and the carboxylate groups ( = 22.8(1) and = 40.9(1) for c11c15 and c21c25 , respectively ) resulting in a torsional angle between the two aryl groups of 60.9(1). structural parameters of the phenyl ring and the carboxylic moiety in 4 are almost identical to its disubstituted congener 1 ( table 2 ) . the f22h24 and f12h14 distances are 2.418(2 ) and 2.771(2 ) , which is for the former significantly shorter than the sum of their van der waals radii ( 2.67 ) . ortep diagram of h2tfbc ( 4 ) showing 50% probability thermal ellipsoids and the atom - numbering scheme . hydrogen atoms ( h15 , h25 ) are disordered over two sites with occupancy of 0.5 . packing diagram of h2tfbc ( 4 ) ( c , white , o , red , f , green , h , white , small spheres ) . by a simple modification of the synthesis known from the literature(22 ) we developed a convenient synthesis for pure 2,3,5,6-tetrafluoroterephthalic acid ( h2tfbdc ) . just by adding a surplus of n - buli the formation of the byproduct 2,3,5,6-tetrafluorobenzoic acid is suppressed so that an extensive purification procedure no longer is necessary . (21 ) with gram quantities of h2tfbdc at hand at comparatively low costs , it might be worthwhile to prove whether this assumption is correct . next to h2tfbdc ( 1 ) the dihydrate h2tfbdc2h2o ( 2 ) , the ammonium salt ( nh4)2tfbdc ( 3 ) , and the monosubstituted acid h2tfbc ( 4 ) were synthesized and characterized by an x - ray single crystal structure analysis . 1 and 4 exhibit hydrogen bonding via the carboxylate groups , which leads to a dimer in 4 ( figure 9 ) and a chain - like polymer in 1 ( figure 2 ) . are included so that a complex 3d hydrogen bonding network is formed ( figure 4 ) , where , with respect to the distances , the strongest hydrogen bond is found ( o2h2o3 ; da 2.540(1 ) , table 3 ) , which are comparable to those found for 5 ( o1h1bo4 ; da 2.541(2 ) , table 3 ) . in 3 the cations ( nh4 ) and anions ( tfbdc ) are connected by hydrogen bonds again arranged in a 3d network ( figure 6 ) . for 1 , 3 , and 4 non - fluorinated congeners are known , but all of them exhibit crystal structures with distinct differences compared to the fluorinated counterparts , although the general structural arrangements of 1 ( p1 , z = 1 ) and its non - fluorinated congener ( p1 , z = 1)(37 ) , both form infinite chains of h2tfbdc and h2bdc molecules linked by double hydrogen bonds , and 4 ( fdd2 , z = 16 ) and its non - fluorinated congener ( p21/c , z = 4)(36 ) , both form dimers with disordered h atoms participating in the hydrogen bonds , are very similar . in contrast , for the fluorinated compounds the carboxylate groups are tilted with respect to the plane of the aryl rings ( 1 : 19.35(7) ; 4 : 22.8(1) and 40.9(1) , respectively ) , whereas in the non - fluorinated compounds they are almost coplanar . the crystal structure of the non - fluorinated congener ( pbc21 , z = 4)(31 ) of 3 ( c2/m , z = 2 ) was solved and refined from synchrotron powder diffraction data . here angles between the planes of the carboxylate groups and the aryl ring of 29.0(8) and 31(1) are found , which are also significantly smaller than in 3 ( 44.54(7) ) .

nontraumatic osteonecrosis of the femoral head ( nonfh ) is a pathological process that occurs at the femoral head as a result of interruption of blood supply following ischemic insult . this progressive , multifactorial , and debilitating condition can result in significant morbidity in patients of any age . although its clinical manifestations have been well - defined , the etiology and epidemiology of nonfh have yet to be fully elucidated . although few epidemiological studies to date have reported on the incidence of nonfh in the general population , between 300,000 and 600,000 people in the united states suffer from this condition . furthermore , studies from the late 1990s showed that 10,000 to 20,000 new cases of nonfh were diagnosed in the united states each year . in japan , an estimated 25003300 new cases are reported annually while around 11,400 patients were found to have sought treatment in 2004 . another recent analysis has showed that the annual incidence rate in japan is 1.91 per 100,000 . in korea , prevalence has increased from 20.53 per 100,000 in 2002 to 37.96 in 2006 , and the average number of new cases annually has been estimated at 14,103 . few previous epidemiological studies have investigated nonfh in a chinese context , however , we therefore undertook a nationally representative survey , including a questionnaire , blood sample , a hip joint examination , and x - ray or magnetic resonance imaging ( mri ) scanning from june 2012 to august 2013 to estimate the prevalence of nonfh and characterize its associated risk factors among the chinese general population aged 15 years or over . we employed multistage random sampling to obtain a nationally representative sample of people aged 15 years and over in the general population . sampling took place in five provinces and autonomous regions in northern china ( heilongjiang , liaoning , shaanxi provinces , and inner mongolia and xinjiang uyghur autonomous region ) and four provinces in southern china ( yunnan , hunan , zhejiang , and hainan provinces ) as defined by the qinling - huaihe line ( 3234.5n ) . in the subsequent stages of sampling cities and rural counties , they were selected from the nine provinces and autonomous regions . we then randomly selected sub - districts , from which neighborhood - level sampling areas were identified . only those residents who had lived at their current address for 5 years were eligible to participate . each respondent was requested to complete a questionnaire , and undergo blood sampling , physical examination , and an x - ray in addition to mri where it was considered appropriate . all interviewers attended a training program to familiarize them with the study 's aims and methods . of a total of 31,036 respondents selected for participation in the study , 535 were excluded due to incomplete demographic data and 421 due to missing x - ray or mri results . a further 50 were removed following a diagnosis of traumatic osteonecrosis of the femoral head ( onfh ) . the study protocol was approved by the ethics committee of the affiliated zhongshan hospital of dalian university , dalian , liaoning , china . we collected information on participants place of residence ( urban or rural ) , gender , age , height , weight , clinical history of onfh and etiology ( including hip trauma , dysbarisms , blood system diseases , gaucher 's disease , radiation exposure , hyperuricemia , and other diseases ) , family history of nonfh , and history of tobacco , alcohol , and steroid use ( including type of steroid , duration of use and dose ) . a smoking index was derived by multiplying the self - reported number of standard packs of 20 cigarettes smoked per day multiplied by years of smoking . heavy drinkers were defined as those reporting an average consumption of 40 g pure alcohol per day for men and 20 g pure alcohol per day for women during the previous 12 months . systematic steroid users were defined as those with an intake of 2 g of prednisone or its equivalent within a period of 3 months . body mass index ( bmi ) was calculated by dividing weight in kilograms by height in meters squared . blood specimens were collected using a vacuum tube after at least 10 h of overnight fasting to measure participants serum total cholesterol , triglyceride , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) levels . all participants underwent a physical examination including assessments of gait , hip deformity , tenderness in the groin area , pain in the region of the greater trochanter , and range of motion of the hip joint and leg length , in addition to the flexion , abduction , and external rotation ( faber ) test , internal rotation test , and trendelenburg test . mri was used for those participants at a higher risk of onfh and reported hip pain but whose x - ray results suggested a negative diagnosis ( n = 553 ) . a team of six orthopedic surgeons and radiologists interpreted the results of these tests to determine a diagnosis of nonfh . criteria used to diagnose nonfh were based on those proposed by the research committee on idiopathic osteonecrosis of the femoral head in japan and the findings of an expert committee based in china . the major diagnostic criteria comprised ( 1 ) cystic and sclerotic changes in femoral head without joint space narrowing or acetabular abnormality , ( 2 ) collapse of the femoral head and/or presence of the crescent sign without joint space narrowing or acetabular abnormality on radiographic images , and ( 3 ) a circumscribed subchondral band - like lesion with low signal intensity on t1-weighted images and/or a double - line sign is seen on t2-weighted images . the two minor criteria were ( 1 ) pain ( groin area , greater trochanteric area , ipsilateral buttock , and knee ) and/or movement limitation of the hip and ( 2 ) positive finding on physical examination of the hip ( e.g. , faber test , internal rotation test , and trendelenburg test ) . a positive diagnosis of nonfh was given when participants fulfilled one of the three major criteria with or without one of the two minor criteria . the association research circulation osseous ( arco ) classification system was used to determine the stage of nonfh progression . we first performed a descriptive analysis to describe the demographic and metabolic characteristics of the overall sample and estimate the crude and adjusted prevalence of nonfh . means were given with 95% confidence intervals ( cis ) for continuous variables and percentages ( with 95% cis ) were given for categorical variables . statistics was then calculated according to participants gender , age ( 1524 years , 2534 years , 3544 years , 4554 years , 5564 years , 6574 years , and 75 years ) , place of residence ( urban or rural ) , and region of china ( north or south ) . the overall prevalence of nonfh in the chinese general population aged 15 years and older was then estimated using 2010 census data . participants were classified according to bmi as underweight , normal , overweight , or obese using who standard guidelines . we then classified participants non - hdl - cholesterol ( hdl - c ) and ldl - cholesterol ( ldl - c ) levels as desirable , above desirable , borderline , high or very high group , and the triglyceride levels as normal , borderline , high , or very high according to 2014 united states guidelines . we analyzed the association between nonfh and metabolic , demographic , and lifestyle - related factors using multivariate logistic regression and reported adjusted odds ratios with 95% cis . while age and bmi ( < 18.5 kg / m , 18.524.9 kg / m , 25.029.9 kg / m , or 30.0 kg / m ) were included as categorical variables , gender , family history of nonfh , residence ( urban or rural ) , smoking status ( smoker or nonsmoker ) , alcohol use ( heavy drinker or nonheavy drinker ) , and glucocorticoid intake were operationalized as binary variables . blood levels of triglyceride ( change per 50 mmol / l ) , total cholesterol ( change per 30 two - tailed p values were given and values of < 0.05 were considered statistically significant . all statistical analyses were conducted using r version 3.1.1 ( r foundation for statistical computing , vienna , austria ) . we employed multistage random sampling to obtain a nationally representative sample of people aged 15 years and over in the general population . sampling took place in five provinces and autonomous regions in northern china ( heilongjiang , liaoning , shaanxi provinces , and inner mongolia and xinjiang uyghur autonomous region ) and four provinces in southern china ( yunnan , hunan , zhejiang , and hainan provinces ) as defined by the qinling - huaihe line ( 3234.5n ) . in the subsequent stages of sampling cities and rural counties , they were selected from the nine provinces and autonomous regions . we then randomly selected sub - districts , from which neighborhood - level sampling areas were identified . only those residents who had lived at their current address for 5 years were eligible to participate . each respondent was requested to complete a questionnaire , and undergo blood sampling , physical examination , and an x - ray in addition to mri where it was considered appropriate . all interviewers attended a training program to familiarize them with the study 's aims and methods . of a total of 31,036 respondents selected for participation in the study , 535 were excluded due to incomplete demographic data and 421 due to missing x - ray or mri results . a further 50 were removed following a diagnosis of traumatic osteonecrosis of the femoral head ( onfh ) . the study protocol was approved by the ethics committee of the affiliated zhongshan hospital of dalian university , dalian , liaoning , china . we collected information on participants place of residence ( urban or rural ) , gender , age , height , weight , clinical history of onfh and etiology ( including hip trauma , dysbarisms , blood system diseases , gaucher 's disease , radiation exposure , hyperuricemia , and other diseases ) , family history of nonfh , and history of tobacco , alcohol , and steroid use ( including type of steroid , duration of use and dose ) . a smoking index was derived by multiplying the self - reported number of standard packs of 20 cigarettes smoked per day multiplied by years of smoking . heavy drinkers were defined as those reporting an average consumption of 40 g pure alcohol per day for men and 20 g pure alcohol per day for women during the previous 12 months . systematic steroid users were defined as those with an intake of 2 g of prednisone or its equivalent within a period of 3 months . body mass index ( bmi ) blood specimens were collected using a vacuum tube after at least 10 h of overnight fasting to measure participants serum total cholesterol , triglyceride , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) levels . all participants underwent a physical examination including assessments of gait , hip deformity , tenderness in the groin area , pain in the region of the greater trochanter , and range of motion of the hip joint and leg length , in addition to the flexion , abduction , and external rotation ( faber ) test , internal rotation test , and trendelenburg test . mri was used for those participants at a higher risk of onfh and reported hip pain but whose x - ray results suggested a negative diagnosis ( n = 553 ) . a team of six orthopedic surgeons and radiologists interpreted the results of these tests to determine a diagnosis of nonfh . criteria used to diagnose nonfh were based on those proposed by the research committee on idiopathic osteonecrosis of the femoral head in japan and the findings of an expert committee based in china . the major diagnostic criteria comprised ( 1 ) cystic and sclerotic changes in femoral head without joint space narrowing or acetabular abnormality , ( 2 ) collapse of the femoral head and/or presence of the crescent sign without joint space narrowing or acetabular abnormality on radiographic images , and ( 3 ) a circumscribed subchondral band - like lesion with low signal intensity on t1-weighted images and/or a double - line sign is seen on t2-weighted images . the two minor criteria were ( 1 ) pain ( groin area , greater trochanteric area , ipsilateral buttock , and knee ) and/or movement limitation of the hip and ( 2 ) positive finding on physical examination of the hip ( e.g. , faber test , internal rotation test , and trendelenburg test ) . a positive diagnosis of nonfh was given when participants fulfilled one of the three major criteria with or without one of the two minor criteria . the association research circulation osseous ( arco ) classification system was used to determine the stage of nonfh progression . we first performed a descriptive analysis to describe the demographic and metabolic characteristics of the overall sample and estimate the crude and adjusted prevalence of nonfh . means were given with 95% confidence intervals ( cis ) for continuous variables and percentages ( with 95% cis ) were given for categorical variables . statistics was then calculated according to participants gender , age ( 1524 years , 2534 years , 3544 years , 4554 years , 5564 years , 6574 years , and 75 years ) , place of residence ( urban or rural ) , and region of china ( north or south ) . the overall prevalence of nonfh in the chinese general population aged 15 years and older was then estimated using 2010 census data . participants were classified according to bmi as underweight , normal , overweight , or obese using who standard guidelines . we then classified participants non - hdl - cholesterol ( hdl - c ) and ldl - cholesterol ( ldl - c ) levels as desirable , above desirable , borderline , high or very high group , and the triglyceride levels as normal , borderline , high , or very high according to 2014 united states guidelines . we analyzed the association between nonfh and metabolic , demographic , and lifestyle - related factors using multivariate logistic regression and reported adjusted odds ratios with 95% cis . while age and bmi ( < 18.5 kg / m , 18.524.9 kg / m , 25.029.9 kg / m , or 30.0 kg / m ) were included as categorical variables , gender , family history of nonfh , residence ( urban or rural ) , smoking status ( smoker or nonsmoker ) , alcohol use ( heavy drinker or nonheavy drinker ) , and glucocorticoid intake were operationalized as binary variables . blood levels of triglyceride ( change per 50 mmol / l ) , total cholesterol ( change per 30 mmol / l ) , ldl - cholesterol ( change per 30 two - tailed p values were given and values of < 0.05 were considered statistically significant . all statistical analyses were conducted using r version 3.1.1 ( r foundation for statistical computing , vienna , austria ) . the overall prevalence of nonfh among our nationally representative sample , whose characteristics are shown in table 1 , was found to be 0.725% . the prevalence of nonfh was significantly higher among males than females ( 1.02% vs. 0.51% , = 24.997 , p < 0.001 ) . within various age groups , prevalence of nonfh consistently was higher in male ( 1524 years : 0.27% , 2534 years : 1.27% , 3544 years : 1.64% , 4554 years : 0.68% , 5564 years : 1.08% , 6574 years : 1.11% , and 75 years : 0.94% ) than females ( 1524 years : 0.15% , 2534 years : 0.27% , 3544 years : 0.49% , 4554 years : 0.33% , 5564 years : 0.59% , 6574 years : 0.86% , and 75 years : 0.85% ) [ figure 1a ] . in addition , significant difference was found in age groups of 2534 , 3544 , and 5564 years old ( value was 9.033 , 15.348 , and 4.643 , respectively , and p value was 0.003 , < 0.0001 , and 0.031 , respectively ) . among nonfh patients , urban ( female : 0.59% , male : 1.14% , = 13.272 , p = 0.0003 ) and north ( female : 0.62% , male : 1.19% , = 12.807 , p = 0.0003 ) residence were subjected to higher prevalence of nonfh than those of rural ( female : 0.41% , male : 0.90% , = 12.820 , p = 0.0003 ) ( 0.80% vs. 0.64% , = 2.534 , p = 0.111 ) [ figure 1b ] and south ( female : 0.40% ; male : 0.87% , = 12.661 , p = 0.0003 ) ( 0.85% vs. 0.61% , = 5.847 , p = 0.016 ) [ figure 1c ] . general characteristics of chinese participants enrolled in this survey * non - hdl - c = total cholesterol minus hdl - c . 95% ci : the rate of 95% confidence intervals ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; bmi : body mass index . age - specific prevalence of nontraumatic osteonecrosis of the femoral head in chinese participants 15 years and over ( a ) . prevalence of nontraumatic osteonecrosis of the femoral head among chinese adults 15 years and over , according to urban and rural residence ( b ) and north and south region ( c ) . < 0.01 , p < 0.0001 , p < 0.05 , p < 0.001 . based on chinese population data from 2010 , we estimated that there are around 8.12 million cases of nonfh among the chinese general population aged 15 years and over . we found that 36.70% of the cases in our study sample were newly diagnosed , of which 86.25% had early - to - middle stage nonfh ( 18.75% in arco stage i , 51.25% in arco stage ii , 16.25% in arco stage iii , and 13.75% in arco stage iv ) . the mean age of newly - diagnosed nonfh patients was significantly lower than that of patients previously diagnosed with the condition ( 47.19 years vs. 55.48 years , p < 0.001 ) . moreover , 19 cases of nonfh were diagnosed by mri ( 15 in acro stage i and 4 in arco stage ii ) . among participants diagnosed with nonfh , 20.96% of males and 4.42% of females were heavy smokers ( = 13.725 , p < 0.0001 ) , 32.93% of males and 7.96% of females reported being heavy drinkers ( = 22.436 , p < 0.0001 ) , 26.35% of males and 55.75% of females reported corticosteroid use ( = 23.451 , p < 0.0001 ) , and 19.76% of males and 31.86% of females reported none of these risk factors ( = 4.680 , p = 0.031 ) [ figure 2 ] . while a further seven patients were overweight or obese , sixteen were diagnosed with dyslipidemia and five with dysbarism . the percent of different causes of nontraumatic osteonecrosis of the femoral head between women and men . our results also showed that bmi and levels of non - hdl - c and triglyceride were significantly higher among both males and females diagnosed with nonfh than in males and females without the condition [ table 2 ] . contingency table of onfh prevalence stratified by bmi , non - hdl - c , ldl - c , and triglyceride levels in men and women figures are numbers of participants unless stated otherwise . nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . as shown in table 3 , elevated blood levels of triglycerides and ldl - c and non - hdl - c , male , urban residence , family history of onfh , heavy smoking , heavy drinking , glucocorticoid intake , overweight and obesity were all positively associated with an increased risk of nonfh in our multivariate model . multivariate - adjusted ors for onfh in chinese participants all covariables listed were included in the model simultaneously . p<0.05 is statistically significant . * non - hdl - c = total cholesterol minus hdl - c ; overweight was defined as a bmi between 25.0 and 29.9 ; obesity was defined as a bmi of 30.0 or more . 95% ci : the rate of 95% confidence intervals ; nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : nonhigh - density lipoprotein - cholesterol ; or : odds ratio ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . the overall prevalence of nonfh among our nationally representative sample , whose characteristics are shown in table 1 , was found to be 0.725% . the prevalence of nonfh was significantly higher among males than females ( 1.02% vs. 0.51% , = 24.997 , p < 0.001 ) . within various age groups , prevalence of nonfh consistently was higher in male ( 1524 years : 0.27% , 2534 years : 1.27% , 3544 years : 1.64% , 4554 years : 0.68% , 5564 years : 1.08% , 6574 years : 1.11% , and 75 years : 0.94% ) than females ( 1524 years : 0.15% , 2534 years : 0.27% , 3544 years : 0.49% , 4554 years : 0.33% , 5564 years : 0.59% , 6574 years : 0.86% , and 75 years : 0.85% ) [ figure 1a ] . in addition , significant difference was found in age groups of 2534 , 3544 , and 5564 years old ( value was 9.033 , 15.348 , and 4.643 , respectively , and p value was 0.003 , < 0.0001 , and 0.031 , respectively ) . among nonfh patients , urban ( female : 0.59% , male : 1.14% , = 13.272 , p = 0.0003 ) and north ( female : 0.62% , male : 1.19% , = 12.807 , p = 0.0003 ) residence were subjected to higher prevalence of nonfh than those of rural ( female : 0.41% , male : 0.90% , = 12.820 , p = 0.0003 ) ( 0.80% vs. 0.64% , = 2.534 , p = 0.111 ) [ figure 1b ] and south ( female : 0.40% ; male : 0.87% , = 12.661 , p = 0.0003 ) ( 0.85% vs. 0.61% , = 5.847 , p = 0.016 ) [ figure 1c ] . general characteristics of chinese participants enrolled in this survey * non - hdl - c = total cholesterol minus hdl - c . 95% ci : the rate of 95% confidence intervals ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; bmi : body mass index . age - specific prevalence of nontraumatic osteonecrosis of the femoral head in chinese participants 15 years and over ( a ) . prevalence of nontraumatic osteonecrosis of the femoral head among chinese adults 15 years and over , according to urban and rural residence ( b ) and north and south region ( c ) . < 0.01 , p < 0.0001 , p < 0.05 , p < 0.001 . based on chinese population data from 2010 , we estimated that there are around 8.12 million cases of nonfh among the chinese general population aged 15 years and over . we found that 36.70% of the cases in our study sample were newly diagnosed , of which 86.25% had early - to - middle stage nonfh ( 18.75% in arco stage i , 51.25% in arco stage ii , 16.25% in arco stage iii , and 13.75% in arco stage iv ) . the mean age of newly - diagnosed nonfh patients was significantly lower than that of patients previously diagnosed with the condition ( 47.19 years vs. 55.48 years , p < 0.001 ) . moreover , 19 cases of nonfh were diagnosed by mri ( 15 in acro stage i and 4 in arco stage ii ) . among participants diagnosed with nonfh , 20.96% of males and 4.42% of females were heavy smokers ( = 13.725 , p < 0.0001 ) , 32.93% of males and 7.96% of females reported being heavy drinkers ( = 22.436 , p < 0.0001 ) , 26.35% of males and 55.75% of females reported corticosteroid use ( = 23.451 , p < 0.0001 ) , and 19.76% of males and 31.86% of females reported none of these risk factors ( = 4.680 , p = 0.031 ) [ figure 2 ] . while a further seven patients were overweight or obese , sixteen were diagnosed with dyslipidemia and five with dysbarism . the percent of different causes of nontraumatic osteonecrosis of the femoral head between women and men . . our results also showed that bmi and levels of non - hdl - c and triglyceride were significantly higher among both males and females diagnosed with nonfh than in males and females without the condition [ table 2 ] . contingency table of onfh prevalence stratified by bmi , non - hdl - c , ldl - c , and triglyceride levels in men and women figures are numbers of participants unless stated otherwise . nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . as shown in table 3 , elevated blood levels of triglycerides and ldl - c and non - hdl - c , male , urban residence , family history of onfh , heavy smoking , heavy drinking , glucocorticoid intake , overweight and obesity were all positively associated with an increased risk of nonfh in our multivariate model . multivariate - adjusted ors for onfh in chinese participants all covariables listed were included in the model simultaneously . * non - hdl - c = total cholesterol minus hdl - c ; overweight was defined as a bmi between 25.0 and 29.9 ; obesity was defined as a bmi of 30.0 or more . 95% ci : the rate of 95% confidence intervals ; nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : nonhigh - density lipoprotein - cholesterol ; or : odds ratio ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . a positive diagnosis of nonfh was established on the basis of both physical examination and imaging using stringent diagnostic criteria and quality - control procedures . our results showed a high prevalence of nonfh among the chinese general population and we estimated that around 8.12 million chinese people aged 15 years and over are affected by the condition . our survey found that 36.70% of nonfh cases in our sample were newly diagnosed after x - ray or mri examination some of which were asymptomatic . the majority ( 86.25% ) of patients , however , were in the early or middle stages of disease progression . given that previous work has shown that early diagnosis and intervention for patients with nonfh can effectively delay disease progression or prevent patients from requiring total hip arthroplasty , our findings highlight the importance of active screening for nonfh to detect the disease in its early stages . while the results of our multivariate analysis show that male , family history of nonfh , and region of residence can influence disease risk , they suggest that changes in lifestyle - related factors such as smoking , alcohol use , corticosteroid intake , overweight , obesity , and dyslipidemia could be contributing to an increase in the burden of nonfh among the chinese population . furthermore , the higher prevalence of nonfh among males of all ages could be attributed to higher levels of smoking and alcohol use . greater fluctuations in temperature , in addition to cultural and lifestyle factors , may also contribute to higher rates of nonfh in northern china . although the pathophysiology of nonfh has yet to be fully elucidated , previous studies hypothesize that nonfh is induced by vascular impairment , altered bone cell physiology , oxidative stress , and insufficient blood supply among other factors . meanwhile , alcohol intake and steroid use have been identified as the major risk factors for nonfh . while this is consistent with our findings , glucocorticoid intake was found to have a stronger association with nonfh than alcohol use . numerous studies have also concluded that hyperlipidemia in the femoral head induced by steroid and alcohol use are associated with nonfh . both of these factors precipitate an increase in fat volume in bone marrow and blood lipid levels , thereby increasing deposition of fat and interrupting blood flow to the femoral head . in addition to these two major risk factors , our results and those of previous studies suggest that cigarette smoking may also be conducive to nonfh . this may be due to changes in nitric oxide bioavailability , resulting in increased oxidative stress and endothelial dysfunction . we also identified obesity as a risk factor for nonfh , and our results show that risk of nonfh is positively associated with bmi . in general , overweight and obesity are often associated with hyperlipidemia . this in turn is associated with elevated fasting and postprandial plasma insulin concentrations that can promote adipose synthesis and inhibit the decomposition of adipose tissue . given that hypercholesterolemia has also been associated with idiopathic avascular necrosis , this is consistent with our finding that elevated triglyceride and ldl and non - hdl - c levels in the blood are associated with an increased risk of nonfh . non - hdl - c is considered a more reliable indicator for assessing cholesterol - related risk and is often the first to be used when evaluating patients with nonfh . our novel findings support the hypothesis that the factors involved in the pathogenesis of atherosclerotic cardiovascular disease ( ascvd ) could also contribute to the progression of nonfh . a 3-year follow - up study found that patients were at an increased risk of developing coronary heart disease after being diagnosed with nonfh , and that this patients could be attributable to risk factors such as smoking , obesity , and dyslipidemia . further work is needed to fully elucidate the mechanisms for this association . because these risk factors for nonfh , which occurs as a result of ischemic insult and disruption of blood supply to the femoral head , have also been identified as risk factors for ascvd , this suggests that both conditions may share the same etiology and occur as a result of dyslipidemia . a previous study has suggested that statins may offer some protection against osteonecrosis when steroid treatment is indicated . we may , therefore , speculate that strategies for preventing ascvd may also be applicable to nonfh . nonfh is a multifactorial disease that is associated with both genetic susceptibility and exposure to certain risk factors , and previous work has already shown that specific genetic polymorphisms are associated with nonfh . our results identified family history of nonfh as a risk factor for the conditions , which may hint to the influence of genetic factors on the pathogenesis of nonfh . further work on specific genetic polymorphisms may prove beneficial in terms of characterizing genetic risk profiles for nonfh . one of the major strengths of the present study was the use of a large nationally representative sample of the chinese general population rather than a clinical sample , which may have led to oversampling of more severe cases of nonfh . at the same time , the use of clinical measurements in addition to self - reported data allowed us to investigate whether overweight , obesity , and dyslipidemia represented risk factors for nonfh in the chinese population and to identify previously undiagnosed cases . in summary , our results show that nonfh is highly prevalent in china . early diagnosis and treatment are essential for delaying disease progression and minimizing the need for costly and invasive procedures in younger nonfh patients , who may require multiple hip replacement surgeries during their lifetime . given that the direct cost to patients for undergoing primary total hip arthroplasty per hip without revision is between $ 8000 and $ 10,000 in china , this suggests that the overall cost of treating all outstanding cases on the national level could total at least $ 64 billion representing a substantial financial burden on the chinese healthcare system . our results indicate that nonfh has the potential to be a national public health challenge and underscores urgent need for national strategies aimed at the prevention , detection , and treatment of nonfh among the chinese general population . this work was supported by grants from nhfpc special fund for health scientific research in the public welfare ( no . this work was supported by grants from nhfpc special fund for health scientific research in the public welfare ( no .

background : nontraumatic osteonecrosis of the femoral head ( nonfh ) is a debilitating disease that represents a significant financial burden for both individuals and healthcare systems . despite its significance , however , its prevalence in the chinese general population remains unknown . this study aimed to investigate the prevalence of nonfh and its associated risk factors in the chinese population.methods:a nationally representative survey of 30,030 respondents was undertaken from june 2012 to august 2013 . all participants underwent a questionnaire investigation , physical examination of hip , and bilateral hip joint x - ray and/or magnetic resonance imaging examination . blood samples were taken after overnight fasting to test serum total cholesterol , triglyceride , and high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) levels . we then used multivariate logistic regression analysis to investigate the associations between various metabolic , demographic , and lifestyle - related variables and nonfh.results:nonfh was diagnosed in 218 subjects ( 0.725% ) and the estimated nonfh cases were 8.12 million among chinese people aged 15 years and over . the prevalence of nonfh was significantly higher in males than in females ( 1.02% vs. 0.51% , 2 = 24.997 , p < 0.001 ) . among nonfh patients , north residents were subjected to higher prevalence of nonfh than that of south residents ( 0.85% vs. 0.61% , 2 = 5.847 , p = 0.016 ) . our multivariate regression analysis showed that high blood levels of triglycerides , total cholesterol , ldl - cholesterol , and non - hdl - cholesterol , male , urban residence , family history of osteonecrosis of the femoral head , heavy smoking , alcohol abuse and glucocorticoid intake , overweight , and obesity were all significantly associated with an increased risk of nonfh.conclusions:our findings highlight that nonfh is a significant public health challenge in china and underscore the need for policy measures on the national level . furthermore , nonfh shares a number of risk factors with atherosclerosis .

I M Study design Questionnaire Blood sampling Diagnosis of nontraumatic osteonecrosis of the femoral head Outcome measures and diagnostic criteria Statistical analysis R Prevalence of nontraumatic osteonecrosis of the femoral head Risk factors for nontraumatic osteonecrosis of the femoral head D Financial support and sponsorship Conflicts of interest

nontraumatic osteonecrosis of the femoral head ( nonfh ) is a pathological process that occurs at the femoral head as a result of interruption of blood supply following ischemic insult . furthermore , studies from the late 1990s showed that 10,000 to 20,000 new cases of nonfh were diagnosed in the united states each year . few previous epidemiological studies have investigated nonfh in a chinese context , however , we therefore undertook a nationally representative survey , including a questionnaire , blood sample , a hip joint examination , and x - ray or magnetic resonance imaging ( mri ) scanning from june 2012 to august 2013 to estimate the prevalence of nonfh and characterize its associated risk factors among the chinese general population aged 15 years or over . we employed multistage random sampling to obtain a nationally representative sample of people aged 15 years and over in the general population . each respondent was requested to complete a questionnaire , and undergo blood sampling , physical examination , and an x - ray in addition to mri where it was considered appropriate . we collected information on participants place of residence ( urban or rural ) , gender , age , height , weight , clinical history of onfh and etiology ( including hip trauma , dysbarisms , blood system diseases , gaucher 's disease , radiation exposure , hyperuricemia , and other diseases ) , family history of nonfh , and history of tobacco , alcohol , and steroid use ( including type of steroid , duration of use and dose ) . blood specimens were collected using a vacuum tube after at least 10 h of overnight fasting to measure participants serum total cholesterol , triglyceride , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) levels . all participants underwent a physical examination including assessments of gait , hip deformity , tenderness in the groin area , pain in the region of the greater trochanter , and range of motion of the hip joint and leg length , in addition to the flexion , abduction , and external rotation ( faber ) test , internal rotation test , and trendelenburg test . criteria used to diagnose nonfh were based on those proposed by the research committee on idiopathic osteonecrosis of the femoral head in japan and the findings of an expert committee based in china . the major diagnostic criteria comprised ( 1 ) cystic and sclerotic changes in femoral head without joint space narrowing or acetabular abnormality , ( 2 ) collapse of the femoral head and/or presence of the crescent sign without joint space narrowing or acetabular abnormality on radiographic images , and ( 3 ) a circumscribed subchondral band - like lesion with low signal intensity on t1-weighted images and/or a double - line sign is seen on t2-weighted images . the overall prevalence of nonfh in the chinese general population aged 15 years and older was then estimated using 2010 census data . we then classified participants non - hdl - cholesterol ( hdl - c ) and ldl - cholesterol ( ldl - c ) levels as desirable , above desirable , borderline , high or very high group , and the triglyceride levels as normal , borderline , high , or very high according to 2014 united states guidelines . we analyzed the association between nonfh and metabolic , demographic , and lifestyle - related factors using multivariate logistic regression and reported adjusted odds ratios with 95% cis . while age and bmi ( < 18.5 kg / m , 18.524.9 kg / m , 25.029.9 kg / m , or 30.0 kg / m ) were included as categorical variables , gender , family history of nonfh , residence ( urban or rural ) , smoking status ( smoker or nonsmoker ) , alcohol use ( heavy drinker or nonheavy drinker ) , and glucocorticoid intake were operationalized as binary variables . we employed multistage random sampling to obtain a nationally representative sample of people aged 15 years and over in the general population . each respondent was requested to complete a questionnaire , and undergo blood sampling , physical examination , and an x - ray in addition to mri where it was considered appropriate . we collected information on participants place of residence ( urban or rural ) , gender , age , height , weight , clinical history of onfh and etiology ( including hip trauma , dysbarisms , blood system diseases , gaucher 's disease , radiation exposure , hyperuricemia , and other diseases ) , family history of nonfh , and history of tobacco , alcohol , and steroid use ( including type of steroid , duration of use and dose ) . body mass index ( bmi ) blood specimens were collected using a vacuum tube after at least 10 h of overnight fasting to measure participants serum total cholesterol , triglyceride , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) levels . all participants underwent a physical examination including assessments of gait , hip deformity , tenderness in the groin area , pain in the region of the greater trochanter , and range of motion of the hip joint and leg length , in addition to the flexion , abduction , and external rotation ( faber ) test , internal rotation test , and trendelenburg test . criteria used to diagnose nonfh were based on those proposed by the research committee on idiopathic osteonecrosis of the femoral head in japan and the findings of an expert committee based in china . the major diagnostic criteria comprised ( 1 ) cystic and sclerotic changes in femoral head without joint space narrowing or acetabular abnormality , ( 2 ) collapse of the femoral head and/or presence of the crescent sign without joint space narrowing or acetabular abnormality on radiographic images , and ( 3 ) a circumscribed subchondral band - like lesion with low signal intensity on t1-weighted images and/or a double - line sign is seen on t2-weighted images . the overall prevalence of nonfh in the chinese general population aged 15 years and older was then estimated using 2010 census data . we then classified participants non - hdl - cholesterol ( hdl - c ) and ldl - cholesterol ( ldl - c ) levels as desirable , above desirable , borderline , high or very high group , and the triglyceride levels as normal , borderline , high , or very high according to 2014 united states guidelines . we analyzed the association between nonfh and metabolic , demographic , and lifestyle - related factors using multivariate logistic regression and reported adjusted odds ratios with 95% cis . while age and bmi ( < 18.5 kg / m , 18.524.9 kg / m , 25.029.9 kg / m , or 30.0 kg / m ) were included as categorical variables , gender , family history of nonfh , residence ( urban or rural ) , smoking status ( smoker or nonsmoker ) , alcohol use ( heavy drinker or nonheavy drinker ) , and glucocorticoid intake were operationalized as binary variables . blood levels of triglyceride ( change per 50 mmol / l ) , total cholesterol ( change per 30 mmol / l ) , ldl - cholesterol ( change per 30 two - tailed p values were given and values of < 0.05 were considered statistically significant . the prevalence of nonfh was significantly higher among males than females ( 1.02% vs. 0.51% , = 24.997 , p < 0.001 ) . within various age groups , prevalence of nonfh consistently was higher in male ( 1524 years : 0.27% , 2534 years : 1.27% , 3544 years : 1.64% , 4554 years : 0.68% , 5564 years : 1.08% , 6574 years : 1.11% , and 75 years : 0.94% ) than females ( 1524 years : 0.15% , 2534 years : 0.27% , 3544 years : 0.49% , 4554 years : 0.33% , 5564 years : 0.59% , 6574 years : 0.86% , and 75 years : 0.85% ) [ figure 1a ] . among nonfh patients , urban ( female : 0.59% , male : 1.14% , = 13.272 , p = 0.0003 ) and north ( female : 0.62% , male : 1.19% , = 12.807 , p = 0.0003 ) residence were subjected to higher prevalence of nonfh than those of rural ( female : 0.41% , male : 0.90% , = 12.820 , p = 0.0003 ) ( 0.80% vs. 0.64% , = 2.534 , p = 0.111 ) [ figure 1b ] and south ( female : 0.40% ; male : 0.87% , = 12.661 , p = 0.0003 ) ( 0.85% vs. 0.61% , = 5.847 , p = 0.016 ) [ figure 1c ] . 95% ci : the rate of 95% confidence intervals ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; bmi : body mass index . age - specific prevalence of nontraumatic osteonecrosis of the femoral head in chinese participants 15 years and over ( a ) . prevalence of nontraumatic osteonecrosis of the femoral head among chinese adults 15 years and over , according to urban and rural residence ( b ) and north and south region ( c ) . based on chinese population data from 2010 , we estimated that there are around 8.12 million cases of nonfh among the chinese general population aged 15 years and over . the mean age of newly - diagnosed nonfh patients was significantly lower than that of patients previously diagnosed with the condition ( 47.19 years vs. 55.48 years , p < 0.001 ) . among participants diagnosed with nonfh , 20.96% of males and 4.42% of females were heavy smokers ( = 13.725 , p < 0.0001 ) , 32.93% of males and 7.96% of females reported being heavy drinkers ( = 22.436 , p < 0.0001 ) , 26.35% of males and 55.75% of females reported corticosteroid use ( = 23.451 , p < 0.0001 ) , and 19.76% of males and 31.86% of females reported none of these risk factors ( = 4.680 , p = 0.031 ) [ figure 2 ] . our results also showed that bmi and levels of non - hdl - c and triglyceride were significantly higher among both males and females diagnosed with nonfh than in males and females without the condition [ table 2 ] . contingency table of onfh prevalence stratified by bmi , non - hdl - c , ldl - c , and triglyceride levels in men and women figures are numbers of participants unless stated otherwise . nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . as shown in table 3 , elevated blood levels of triglycerides and ldl - c and non - hdl - c , male , urban residence , family history of onfh , heavy smoking , heavy drinking , glucocorticoid intake , overweight and obesity were all positively associated with an increased risk of nonfh in our multivariate model . 95% ci : the rate of 95% confidence intervals ; nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : nonhigh - density lipoprotein - cholesterol ; or : odds ratio ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . the prevalence of nonfh was significantly higher among males than females ( 1.02% vs. 0.51% , = 24.997 , p < 0.001 ) . within various age groups , prevalence of nonfh consistently was higher in male ( 1524 years : 0.27% , 2534 years : 1.27% , 3544 years : 1.64% , 4554 years : 0.68% , 5564 years : 1.08% , 6574 years : 1.11% , and 75 years : 0.94% ) than females ( 1524 years : 0.15% , 2534 years : 0.27% , 3544 years : 0.49% , 4554 years : 0.33% , 5564 years : 0.59% , 6574 years : 0.86% , and 75 years : 0.85% ) [ figure 1a ] . among nonfh patients , urban ( female : 0.59% , male : 1.14% , = 13.272 , p = 0.0003 ) and north ( female : 0.62% , male : 1.19% , = 12.807 , p = 0.0003 ) residence were subjected to higher prevalence of nonfh than those of rural ( female : 0.41% , male : 0.90% , = 12.820 , p = 0.0003 ) ( 0.80% vs. 0.64% , = 2.534 , p = 0.111 ) [ figure 1b ] and south ( female : 0.40% ; male : 0.87% , = 12.661 , p = 0.0003 ) ( 0.85% vs. 0.61% , = 5.847 , p = 0.016 ) [ figure 1c ] . 95% ci : the rate of 95% confidence intervals ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; bmi : body mass index . age - specific prevalence of nontraumatic osteonecrosis of the femoral head in chinese participants 15 years and over ( a ) . prevalence of nontraumatic osteonecrosis of the femoral head among chinese adults 15 years and over , according to urban and rural residence ( b ) and north and south region ( c ) . based on chinese population data from 2010 , we estimated that there are around 8.12 million cases of nonfh among the chinese general population aged 15 years and over . the mean age of newly - diagnosed nonfh patients was significantly lower than that of patients previously diagnosed with the condition ( 47.19 years vs. 55.48 years , p < 0.001 ) . among participants diagnosed with nonfh , 20.96% of males and 4.42% of females were heavy smokers ( = 13.725 , p < 0.0001 ) , 32.93% of males and 7.96% of females reported being heavy drinkers ( = 22.436 , p < 0.0001 ) , 26.35% of males and 55.75% of females reported corticosteroid use ( = 23.451 , p < 0.0001 ) , and 19.76% of males and 31.86% of females reported none of these risk factors ( = 4.680 , p = 0.031 ) [ figure 2 ] . our results also showed that bmi and levels of non - hdl - c and triglyceride were significantly higher among both males and females diagnosed with nonfh than in males and females without the condition [ table 2 ] . contingency table of onfh prevalence stratified by bmi , non - hdl - c , ldl - c , and triglyceride levels in men and women figures are numbers of participants unless stated otherwise . nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : non - high - density lipoprotein - cholesterol ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . as shown in table 3 , elevated blood levels of triglycerides and ldl - c and non - hdl - c , male , urban residence , family history of onfh , heavy smoking , heavy drinking , glucocorticoid intake , overweight and obesity were all positively associated with an increased risk of nonfh in our multivariate model . 95% ci : the rate of 95% confidence intervals ; nonfh : nontraumatic osteonecrosis of the femoral head ; ldl - c : low - density lipoprotein - cholesterol ; non - hdl - c : nonhigh - density lipoprotein - cholesterol ; or : odds ratio ; onfh : osteonecrosis of the femoral head ; bmi : body mass index . our results showed a high prevalence of nonfh among the chinese general population and we estimated that around 8.12 million chinese people aged 15 years and over are affected by the condition . while the results of our multivariate analysis show that male , family history of nonfh , and region of residence can influence disease risk , they suggest that changes in lifestyle - related factors such as smoking , alcohol use , corticosteroid intake , overweight , obesity , and dyslipidemia could be contributing to an increase in the burden of nonfh among the chinese population . furthermore , the higher prevalence of nonfh among males of all ages could be attributed to higher levels of smoking and alcohol use . given that hypercholesterolemia has also been associated with idiopathic avascular necrosis , this is consistent with our finding that elevated triglyceride and ldl and non - hdl - c levels in the blood are associated with an increased risk of nonfh . a 3-year follow - up study found that patients were at an increased risk of developing coronary heart disease after being diagnosed with nonfh , and that this patients could be attributable to risk factors such as smoking , obesity , and dyslipidemia . nonfh is a multifactorial disease that is associated with both genetic susceptibility and exposure to certain risk factors , and previous work has already shown that specific genetic polymorphisms are associated with nonfh . one of the major strengths of the present study was the use of a large nationally representative sample of the chinese general population rather than a clinical sample , which may have led to oversampling of more severe cases of nonfh . at the same time , the use of clinical measurements in addition to self - reported data allowed us to investigate whether overweight , obesity , and dyslipidemia represented risk factors for nonfh in the chinese population and to identify previously undiagnosed cases . given that the direct cost to patients for undergoing primary total hip arthroplasty per hip without revision is between $ 8000 and $ 10,000 in china , this suggests that the overall cost of treating all outstanding cases on the national level could total at least $ 64 billion representing a substantial financial burden on the chinese healthcare system . our results indicate that nonfh has the potential to be a national public health challenge and underscores urgent need for national strategies aimed at the prevention , detection , and treatment of nonfh among the chinese general population .

it has long been known that nutritional status is a critical factor in determining the timing of the onset of puberty ( kennedy , 1969 ; frisch and mcarthur , 1974 ) . classical studies suggested that a minimum amount of body fat is required to attain sexual maturation ( kennedy , 1969 ; frisch and mcarthur , 1974 ; frisch , 1985 ) . when prepubertal animals , including primates , are exposed to energy deprivation the onset of puberty is delayed or even blocked , until a favorable energy balance is achieved ( kennedy and mitra , 1963 ; kennedy , 1969 ; foster and olster , 1985 ) . in adults , severe energy deficits are a frequent cause of hypothalamic amenorrhea ( warren et al . , 1999 ; recent studies not only confirm the importance of the adiposity in influencing the onset of puberty , but also suggest that excess of body fat in children cause early onset of puberty ( biro et al . , 2010 ) . epidemiological data have indicated a high prevalence of obesity among us children and adolescents ( flegal et al . , 2010 ) . concomitantly , a higher proportion of girls have shown signs of pubertal development at earlier ages ( biro et al . , 2010 ) . a breakthrough in the field occurred after the discovery of the gene that encodes the adipocyte - derived hormone leptin ( zhang et al . , 1994 ) . soon after that , leptin became the missing piece that would complete the lipostatic theory , proposed several decades earlier ( kennedy , 1953 ) . the lipostatic theory suggests that changes in fat deposition trigger a feedback system aiming to restore the balance between food intake and energy expenditure . leptin levels reflect the body fat content in rodents and humans ( frederich et al . , 1995a ; maffei et al . , 1995 ; considine et al . , 1996 ) . overfeeding increases leptin levels , whereas food deprivation causes a strong decrease in the circulating concentration of leptin ( frederich et al . , 1995b ; ahima et al . , 1996 it has been proposed that the body interprets a high concentration of leptin as a signal of energy abundance , whereas falling levels of leptin signals starvation . therefore , low leptin levels increase hunger and decrease energy expenditure , partly because energy - demanding physiological functions are suppressed in leptin - deficient states , presumably as a way to save energy and prolong survival ( ahima et al . , 1996 ; friedman and halaas , 1998 ; rosenbaum and leibel , 1998 ) . reproduction is one of the physiological functions strongly affected by leptin ( barash et al . because lack of leptin is interpreted as a signal of starvation , leptin - deficient individuals become hyperphagic , massively obese and infertile , and exhibit a series of metabolic dysfunctions ( zhang et al . , 1994 ; campfield et al . , 1995 ; halaas et al . , 1995 ; pelleymounter et al . , 1995 ; montague et al . , 1997 ; leptin treatment rescues the alterations in body weight , metabolism , and the reproductive system ( ahima et al . , 1996 ; , 1996 ; mounzih et al . , 1997 ; farooqi et al . , 1999 ) . in addition , exogenous leptin administration causes early onset of puberty in mice ( ahima et al . 1997 ) , and a similar mechanism could account for the trend observed in overweight children ( biro et al . , 2010 ) . after the discovery of leptin , many studies focused on deciphering the mechanism by which leptin regulates the reproductive system . although leptin receptors ( lepr ) are expressed in many organs , including pituitary gland and gonads ( zamorano et al . , 1997 ) , it is now clear that the main target of leptin is the brain ( cohen et al . , 2001 ; de luca et al . , 2005 ) the expression of lepr is found in innumerous brain nuclei , particularly in specific populations of hypothalamic neurons ( schwartz et al . , 1996 ; elmquist et al . , 1998 ; scott et al . , 2009 however , defining the key neuronal population that mediates the effects of leptin on reproduction has proven to be a challenging task ( hill et al . , 2008 ; recent findings suggest that the ventral premammillary nucleus ( pmv ) is the long sought area in which leptin modulates the activity of the reproductive system ( donato et al . , 2009 , 2011b ; leshan et al . , 2009 ; louis et al . , , we will summarize the existing literature about the potential role played by pmv neurons in the regulation of the hypothalamus ventral premammillary nucleus neurons exhibit a broad expression of receptors for hormones related to the regulation of the energy balance ( table 1 ) . pmv neurons express lepr ( elmquist et al . , 1998 ; scott et al . , 2009 ) , the ghrelin receptor , in mice but not in rats ( zigman et al . , 2006 ) and the insulin receptor ( figure 1a ) . innumerous receptors for neurotransmitters involved with the regulation of energy balance are also found in the pmv , including the cannabinoid receptor 1 ( figure 1b ) , the melanocortin-4 receptor in mice ( liu et al . , 2003 ) but not in rats ( kishi et al . , 2003 ) , the orexin receptor 1 and 2 ( marcus et al . , 2001 ) , the neuropeptide y y1 receptor ( higher expression in mice than rats ; kishi et al . , 2005 ) , and the vasopressin receptor in hamsters ( sexually dimorphic and dependent upon photoperiod length ; dubois - dauphin et al . , 1991 ) . ( a , b ) darkfield photomicrographs demonstrating the distribution of insulin receptor ( insr ) mrna ( a ) and cannabinoid receptor type 1 ( cb1r ) mrna ( b ) in the mouse pmv . ( c , d ) darkfield photomicrographs demonstrating the distribution of cocaine and amphetamine regulated transcript ( cart ) mrna in the rat ( c ) and in the mouse ( d ) pmv . note the abundance of cart mrna in the pmv of rats compared to mice . expression level : + + + , high ; + + , moderate ; + , low ; , not described . species : b , birds ; ha , hamsters , hu , humans ; m , mouse ; r , rat ; s , sheep . ventral premammillary nucleus neurons are potential targets of sex hormones ( table 1 ) , as they express a dense amount of androgen receptor ( ar ) and a moderate to low amount of estrogen receptor and ( er and er ) and progesterone receptor ( simerly et al . , 1990 ; yokosuka and hayashi , 1996 ; merchenthaler et al . , 2004 ; intlekofer and petersen , 2011 ) . the relatively high ratio of ar to er and the lack of definitive data demonstrating the expression of aromatase in the pmv indicate a potential role for androgens in pmv neuronal biology ( yokosuka and hayashi , 1996 ; wu et al . , 2009 despite the strong presence of sexual steroid receptors in the pmv , it has not been determined whether changing levels of sexual hormones affect the neuronal activity or gene expression in this area . in addition to sex steroid receptors , several neurotransmitters are expressed by pmv neurons ( table 1 ) , including glutamate ( ziegler et al . , 2002 ) . the expression of glutamatergic markers , such as vesicular glutamate transporter 2 ( vglut2 ) , can be found in the entire extension of the pmv and show a 94% colocalization with neurons that express lepr ( donato et al . , 2011b ) . on the other hand , there is virtually no expression of gabaergic markers in the pmv , such as glutamic acid decarboxylase 67 ( gad67 ) or vesicular gaba transporter ( vgat ; donato et al . thus , the projections originated from pmv neurons are thought to be excitatory due to their glutamatergic component . peptidergic neurotransmitters are also expressed by pmv neurons ( table 1 ) , including cocaine and amphetamine regulated transcript ( cart ) , substance p ( a tachykinin family member ) and enkephalin ( wamsley et al . , 1980 ; shimada et al . , 1987 ; larsen , 1992 ; douglass et al . , 1995 ; sukhov et al . , however , sexually dimorphic or species - related differences exist . while the pmv of rats densely expresses cart mrna and peptide ( douglass et al . 1997 ) , the pmv of mice exhibits low cart expression ( figures 1c d ) . in addition , the number of neurons that express tachykinin peptides in the pmv is higher in male than in female rats ( akesson , 1993 ) . pmv neurons also densely express neuronal nitric oxide synthase ( nnos ) , which catalyzes the synthesis of nitric oxide ( no ; vincent and kimura , 1992 ) . many of the neurotransmitters expressed in the pmv are involved in the neuroendocrine regulation of reproduction . for example , glutamatergic inputs were shown to induce gonadotropin - releasing hormone ( gnrh ) release and subsequent activation of hpg axis ( brann and mahesh , 1994 ; gargiulo and donoso , 1995 ; dhandapani and brann , 2000 ; mahesh and brann , 2005 ) . moreover , glutamate facilitates the expression of sexual behaviors ( gargiulo and donoso , 1995 ) and has regulatory effects on the timing of puberty ( zamorano et al . , 1998 ; of note , intracerebroventricular administration of glutamate receptor agonists , such as nmda , elicits secretion of gnrh and luteinizing hormone ( lh ) , even in kiss1 and kiss1r knockout mice , suggesting a role for glutamatergic neurotransmission outside the kiss1 neuronal system ( danglemont de tassigny et al . , 2010 ) . cart peptide was shown to mediate the stimulatory effects of leptin on gnrh secretion in vitro and in vivo ( lebrethon et al . , 2000 , 2007 ; in addition , no has been implicated in the regulation of sexual behaviors and hpg axis ( moretto et al . , 1993 ; rettori et al . , 1993 ; mani et al . , 1994 ; , 1995 ; nelson et al . , 1995 ) . a complete disruption of nos1 gene results in hypogonadism and infertility ( gyurko et al . , 2002 furthermore , several studies found that no is a key neurotransmitter that mediates leptin - induced gnrh / lh secretion ( yu et al . , 1997 ; mccann et al . , 1999 ; watanobe and schioth , 2001 ; reynoso et al . , 2007 ) . recently , we reported that 73% of leptin responsive cells in the pmv express no - synthesizing enzymes ( donato et al . leptin does not affect the expression of nos1 mrna in the pmv , but low leptin levels , as in fasting or in ob / ob mice , cause a reduction in the number of pmv neurons expressing the phosphorylated form of nnos ( pnnos ) . the phosphorylation of nnos at ser1412 increases nnos enzymatic activity ( parkash et al . , 2010 ) and acute injection of leptin restores the number of pnnos neurons in the pmv of fasted mice ( donato et al . , 2010b ) . the projections of pmv neurons were first described in rats using the neurotracer phaseolus vulgaris leucoagglutinin ( canteras et al . , 1992b ) . it was demonstrated that pmv neurons project mainly to the periventricular zone of the hypothalamus , which is composed of nuclei involved in the regulation of anterior pituitary function . pmv neurons also project to major nuclei of the sexually dimorphic circuitry , including the ventrolateral part of the ventromedial nucleus of hypothalamus ( vmh ) , medial preoptic nucleus , bed nuclei of the stria terminalis ( bst ) , ventral lateral septal nucleus , posterodorsal part of the medial nucleus of the amygdala ( mea ) , and posterior nucleus of the amygdala ( canteras et al . , 1992b ) . it is interesting that the major neuronal inputs to the pmv originate from neurons located in the sexually dimorphic circuitry , highlighting the intense intercommunication between this circuitry and the pmv ( simerly and swanson , 1988 ; canteras et al . , 1992a , b , 1994 , 1995 ; coolen and wood , 1998 ) . for example , pmv is densely innervated by neurons located in the mea , including cells that express urocortin 3 ( canteras et al . , 1995 ; coolen and wood , 1998 ; cavalcante et al . more recent studies in mice and in rats using genetic tools in combination with tracing techniques highlighted a putative role of the pmv in the regulation of the hpg axis . it was shown that pmv neurons project directly to gnrh perikarya in the medial preoptic area ( mpa ; rondini et al . , 2004 ; boehm et al . , 2005 ; leshan et al . , 2009 ) and to gnrh fibers in the median eminence ( donato et al . , 2011b ) . interestingly , among all neurons that express lepr , only those in the pmv and a subpopulation of neurons in the mpa seem to project directly to gnrh neurons ( louis et al . , 2011 ) . in addition , pmv neurons project to the anteroventral periventricular nucleus ( avpv ; canteras et al . , 2004 ; hahn and coen , 2006 ) , a key site for female reproductive function ( wiegand and terasawa , 1982 ; gottsch et al . the avpv contains a subpopulation of kisspeptin neurons , which is critical for the preovulatory lh surge ( smith et al . , 2006 ; herbison , 2008 ; cravo et al . , 2011 ) . we have recently found that fibers from pmv neurons make apparent synaptic contact with kisspeptin neurons in the avpv ( donato et al . , 2011b ) . the arcuate nucleus ( arh ) also receives a dense projection from pmv neurons ( canteras et al . , 1992b ) , but whether kisspeptin neurons in the arh or a specific population of arh neurons is selectively targeted by pmv inputs is still unknown . overall , pmv neurons potentially regulate the reproductive system directly through inputs to gnrh neurons and also to upstream neuronal populations , such as kisspeptin cells . previous studies using electrolytic lesions described a potential role for pmv neurons in odor - induced lh secretion in rats ( beltramino and taleisnik , 1985 ) . olfaction is a critical sense used by rodents to discriminate socially relevant cues and to trigger social behaviors , including sexual behaviors ( romero et al . , 1990 ; halpern and martinez - marcos , 2003 ; yoon et al . , 2005 ; brennan and zufall , 2006 ) . in response to conspecific odors , males and females of different species exhibit increased circulating levels of gonadotropins and sex steroids ( maruniak and bronson , 1976 ; kamel et al . , 1977 ; beltramino and taleisnik , 1983 ; coquelin et al . , 1984 ) . rats and mice exposed to conspecific odors show a large number of neurons expressing fos immunoreactivity ( fos - ir ) in the pmv , which suggests that the pmv is involved in the neuronal circuitry that conveys olfactory information ( yokosuka et al . , 1999 ; cavalcante et al . , 2006a ; leshan et al . , 2009 ; donato et al . moreover , roughly 50% of pmv neurons activated by opposite - sex odor express cart and , in male rats , cart mrna increases after exposure to female odors ( cavalcante et al . most of the cart neurons in the pmv express the enzymes that synthesize no . besides , a parcel of nitrergic neurons is stimulated by female odors and virtually all nitrergic cells in the pmv express ars ( yokosuka and hayashi , 1996 ) . altogether , these studies indicate that pmv is apt to integrate information about circulating levels of sexual hormones and socially relevant cues ( through brain areas related to pheromonal processing , such as mea or bst ) and generate appropriate neuroendocrine responses to modulate socially relevant behaviors . the pmv may also be involved in the expression of conspecific behaviors because pmv neurons of male rats are also responsive to conspecific male odors ( donato et al . in addition , pmv neurons express fos - ir after mating or agonistic behavior ( kollack - walker and newman , 1995 ; coolen et al . previous studies showed that lesions of the premammillary area increases aggression between males of the same species ( van den berg et al . , 1983 ) . however , these studies should be interpreted with caution due to the extension of the lesion . restricted and/or selective lesions are required to determine the real contribution of the pmv in aggressive behaviors . an interesting question is whether nutritional conditions may alter the responsiveness of an individual to environmental cues . pmv neurons are the target of metabolic cues and also respond to socially relevant sensory stimulation . of note , 44% of the lepr - expressing cells in the pmv of male mice are activated by female odors , whereas in female mice , 18% of lepr cells are activated by male odors ( leshan et al . , 2009 ) . these findings suggest that food availability or energy stored affect neuronal responses to odors . however , we observed that fasting caused no changes in female odor - induced fos - ir in the pmv and in the mea of male rats compared to normally fed controls ( donato et al . , 2010a ) . although this finding may suggest a dissociation of neuronal responses to different stimuli , it is important to mention that induction of fos protein may not be the definitive indicator of changes in neuronal activity or responsiveness . further studies will be necessary to determine the influence of the nutritional state on the response to environmental stimulation . although most of the studies about the pmv have used rats and mice as experimental models , there are several pieces of evidence that the premammillary hypothalamic area ( pmh ) also plays a key role in reproductive function of seasonal breeders ( i.e. , sheep and birds ) . seasonal reproduction is a strategy used by several species to increase survival of offspring by reproducing during a period of the year when the environment offers favorable conditions . in sheep , the major environmental cue controlling reproduction is the photoperiod or day length ( duan et al . , 2007 ) . changes in day light exposure alter the synthesis and secretion of the pineal gland hormone melatonin , which in turn binds to hypothalamic nuclei and modulates the pulsatile secretion of gnrh ( emilsson et al . , 1999 ; the pmh of ewes is composed of the caudal arh , the pmv and the ventral tuberomammillary nucleus . similarly to rats , pmv neurons in ewes express cart and nnos ( sliwowska et al . , 2004 ) . bilateral microimplantation of melatonin into the pmv of ewes stimulates lh secretion ( malpaux et al . , 1998 ) , indicating that in sheep , the pmv appears to play a key role in seasonal reproduction . the reproductive cycle of a variety of avian species is regulated by circadian mechanisms driven by intrinsic oscillators ( petersen et al . , 1996 ; wikelski et al . , 2008 these mechanisms are modulated by light - sensitive neuronal populations located in the caudal hypothalamus , in a site identified as the pmh ( kang et al . , 2007 ) . in birds , a subpopulation of pmh neurons expresses dopamine , a neurotransmitter known to affect the secretion of several reproductive hormones , including lh , fsh , and prolactin . using a complex paradigm of light - induced gnrh neuronal activation , studies identified in turkeys a photosensitive subpopulation of dopaminergic pmh neurons likely involved in gnrh secretion ( thayananuphat et al . , 2007 ) . dopaminergic neurons ( immunoreactive to tyrosine hydroxylase ) in the pmh of turkeys coexpress melatonin and its synthesizing enzymes ( kang et al . , 2007 ) . melatonin neurons in the pmh exhibit high activity at the photosensitive phase , which was associated with higher dopaminergic neurotransmission and gnrh activation . additionally , these neurons express the photoreceptive molecule melanopsin , which is involved in extra - retinal photoreception in birds and non - mammalian vertebrates . in hens , the expression of melanopsin mrna in the pmh is downregulated by light in a series of models and shows a diurnal regulation ; it is high during the night and low during the day ( kang et al . , 2010 ) . the pmh of turkeys also presents a distinct circadian expression of clock genes compared to the pineal gland and the brain master clock , the suprachiasmatic nucleus . in particular , cry1 and per3 seem to mediate the photic responses associated with the control of the reproductive system ( leclerc et al . , 2010 ) . the high expression of receptors of hormones related with the regulation of the energy balance might imply that the pmv is involved in the control of energy balance . however , bilateral excitotoxic lesions of the pmv did not affect body weight , mean food intake and circulating leptin levels in adult female rats ( donato et al . , 2009 ) . nonetheless , pmv - lesioned rats exhibit an attenuated reduction in food intake between the proestrus and the estrus day ( donato et al . , 2009 ) . female rats normally show a decreased food intake in the behavioral estrus that is linked with the high estrogen levels observed during the proestrus day ( drewett , 1973 ; geary et al . , 2001 ; therefore , the regulation of food intake across the estrous cycle by pmv neurons can be an indirect consequence of changes in sexual hormone levels after lesions of the pmv . leptin exerts a pivotal role in the long - term regulation of energy balance ( schwartz , 2006 ; gautron and elmquist , 2011 ) . as mentioned , leptin administration to leptin - deficient mice ( ob / ob ) rescues all the metabolic and neuroendocrine deficits observed in these mice ( campfield et al . , 1995 ; halaas et al . , 1995 ; pelleymounter et al . , 1995 ; chehab et al . , following the same paradigm , we generated ob / ob mice with bilateral lesions of the pmv ( donato et al . , 2011b ) . upon leptin treatment , these mice showed drastic reduction in food intake and body weight , indicating that leptin may restore the metabolic deficits of ob / ob female mice in the absence of pmv neurons ( donato et al . , 2011b ) . to further investigate the role played by lepr in the pmv , we generated a lepr - null mouse model in which lepr is expressed selectively in pmv neurons . we found that endogenous expression of lepr only in the pmv did not affect food intake , body weight , and fat mass in male and female mice ( donato et al . , a recent study found that after genetic ablation of lepr expression from all glutamatergic ( vglut2-positive ) neurons , which includes pmv cells , only minor changes in body weight , food intake , and fat mass were observed in male and female mice ( vong et al . , overall , these results suggest that despite the presence of innumerous receptors involved with the regulation of energy balance , pmv neurons are not key players in the modulation of food intake and body weight . rather , pmv neurons may function as a key integrative site conveying metabolic cues to the reproductive system . accordingly , pmv lesions cause a temporary anestrus in rats ( persistent leukocytes in the vaginal smears ) . however , after a few weeks pmv - lesioned rats recover their cyclicity , although vaginal cytology continues to exhibit an atypical mixed cell profile ( donato et al . , 2009 ) . several weeks after lesions of the pmv , rats still show reduced concentration of lh and estradiol and decreased activation of avpv and gnrh neurons at the time of the preovulatory lh surge but no changes in kiss1 mrna expression ( donato et al . , 2009 ) . possibly secondary to decreased gonadotropin levels , the ovaries of pmv - lesioned rats display a lower number of antral follicles and a trend toward a reduction in the number of corpora lutea ( donato et al . , 2009 ) . these results indicate that the pmv is required for the normal activity of the hpg axis in female rats . following the same line , we hypothesized that the pmv would be apt to mediate the effects of leptin on the reproductive neuroendocrine axis . to test this model , we used a well - established paradigm in which leptin treatment can restore or increase lh levels in fasted rodents ( ahima et al . lesions of the pmv blocked the stimulatory effect of leptin on lh secretion in fasted rats ( donato et al . , 2009 ) . in order to investigate putative signaling pathways that mediate the acute effects of leptin on pmv neurons , patch - clamp recordings of hypothalamic slices were performed . leptin caused a rapid depolarization of 75% of lepr - expressing neurons in the pmv through a putative trpc channel ( leshan et al . the other 25% recorded lepr cells were hyperpolarized in response to leptin , and this response required the activation of a putative katp channel . importantly , pharmacological or genetic disruption of the phosphoinositide 3-kinase ( pi3k ) pathway prevented the leptin - induced changes in the activity of pmv lepr neurons ( williams et al . , 2011 ) . these results indicate that pi3k is required for the acute changes in biophysical properties of pmv neurons induced by leptin . whether these changes in cellular activity underlie the physiological effects of leptin are under investigation . we further assessed whether leptin signaling in pmv neurons is critical to induce the onset of puberty and restore fertility in leptin- or lepr - deficient mouse models . lesions of the pmv in female ob / ob mice reduced the capacity of exogenous leptin to induce sexual maturation . besides , acute injection of leptin did not increase lh and progesterone levels in pmv - lesioned ob / ob mice , as observed in pmv - intact ob / ob mice ( donato et al . , 2011b ) . in addition , female lepr - null mice with endogenous re - expression of lepr in pmv neurons showed unambiguous signs of sexual maturation , such as vaginal opening , increased uterus weight and size , and ovaries with corpora lutea . after a period of 6 weeks of breeding tests , 50% of mice with selective reactivation of lepr in pmv neurons became pregnant , despite their obese and diabetic phenotype ( donato et al . notably , the improvement of the infertile phenotype of the lepr - null mice following pmv lepr reactivation was only observed in females , not in males . as previously mentioned , neurotransmitters found in the pmv , such as glutamate and no , were shown to stimulate the release of gnrh . moreover , earlier studies suggested that the lack of leptin signaling causes a deficient release of gnrh because ob / ob and db / db mice have high content of gnrh in the median eminence / medial basal hypothalamus ( me / mbh ; johnson and sidman , 1979 ; batt et al . , 1982 ) . re - expression of lepr only in the pmv normalized the me / mbh gnrh content in female lepr - null mice . together , these findings have determined the pmv as a key site linking leptin action and the female reproductive physiology . overall , this review highlights a series of recent data demonstrating that pmv neurons are apt to mediate the effects of leptin on gnrh secretion . the stimulatory effect of pmv neurons on gnrh release is possibly mediated by the coordinated effects of glutamate and no on gnrh terminals in the median eminence ( figure 2 ) . although it is very likely that other neuronal populations also convey metabolic cues to modulate the hpg axis , the existing evidence suggests that the pmv is a key site relaying the effects of leptin on the reproductive neuroendocrine axis . we postulate that through pmv neurons , leptin modulates the influence of adiposity on the timing of puberty and the coordinated secretion of lh during conditions of negative energy balance . the data presented in this review provide the physiological and neuroanatomical basis underlying the effects of leptin on the hpg axis . proposed role for the ventral premammillary nucleus ( pmv ) in the female reproductive physiology . the pmv integrates environmental cues ( odors in rodents and daylight in seasonal breeders ) and signals from the internal milieu related to the reproductive status ( sex steroids ) and energy store ( leptin and insulin ) . pmv neurons express excitatory neurotransmitters ( e.g. , glutamate and nitric oxide ) and directly project to the anteroventral periventricular nucleus ( avpv ) , to the arcuate nucleus ( arh ) , and to gonadotropin - releasing hormone ( gnrh ) neurons . once stimulated , pmv neurons activate the target sites inducing gnrh release and lh secretion from the pituitary gland . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the amount of body fat and the energy balance are important factors that influence the timing of puberty and the normal reproductive function . leptin is a key hormone that conveys to the central nervous system information about the individual energy reserve and modulates the hypothalamus pituitary gonad ( hpg ) axis . recent findings suggest that the ventral premammillary nucleus ( pmv ) mediates the effects of leptin as a permissive factor for the onset of puberty and the coordinated secretion of luteinizing hormone during conditions of negative energy balance . in this review , we will summarize the existing literature about the potential role played by pmv neurons in the regulation of the hpg axis .

Introduction The Neurochemical Profile of PMV Neurons Highlights a Key Integrative Function PMV Neurons are Neurochemically and Neuroanatomically Well Positioned to Regulate Reproduction PMV Neurons are Responsive to Conspecific Behaviors and Socially Relevant Cues Premammillary Hypothalamic Area Mediates Seasonal Reproduction in Ewes and Birds PMV Neurons Integrate Metabolic Cues to Regulate Reproduction Rather than Energy Balance Concluding Remarks Conflict of Interest Statement

it has long been known that nutritional status is a critical factor in determining the timing of the onset of puberty ( kennedy , 1969 ; frisch and mcarthur , 1974 ) . classical studies suggested that a minimum amount of body fat is required to attain sexual maturation ( kennedy , 1969 ; frisch and mcarthur , 1974 ; frisch , 1985 ) . when prepubertal animals , including primates , are exposed to energy deprivation the onset of puberty is delayed or even blocked , until a favorable energy balance is achieved ( kennedy and mitra , 1963 ; kennedy , 1969 ; foster and olster , 1985 ) . , 1999 ; recent studies not only confirm the importance of the adiposity in influencing the onset of puberty , but also suggest that excess of body fat in children cause early onset of puberty ( biro et al . a breakthrough in the field occurred after the discovery of the gene that encodes the adipocyte - derived hormone leptin ( zhang et al . leptin levels reflect the body fat content in rodents and humans ( frederich et al . overfeeding increases leptin levels , whereas food deprivation causes a strong decrease in the circulating concentration of leptin ( frederich et al . , 1996 it has been proposed that the body interprets a high concentration of leptin as a signal of energy abundance , whereas falling levels of leptin signals starvation . therefore , low leptin levels increase hunger and decrease energy expenditure , partly because energy - demanding physiological functions are suppressed in leptin - deficient states , presumably as a way to save energy and prolong survival ( ahima et al . reproduction is one of the physiological functions strongly affected by leptin ( barash et al . because lack of leptin is interpreted as a signal of starvation , leptin - deficient individuals become hyperphagic , massively obese and infertile , and exhibit a series of metabolic dysfunctions ( zhang et al . , 1997 ; leptin treatment rescues the alterations in body weight , metabolism , and the reproductive system ( ahima et al . in addition , exogenous leptin administration causes early onset of puberty in mice ( ahima et al . after the discovery of leptin , many studies focused on deciphering the mechanism by which leptin regulates the reproductive system . , 1997 ) , it is now clear that the main target of leptin is the brain ( cohen et al . , 2009 however , defining the key neuronal population that mediates the effects of leptin on reproduction has proven to be a challenging task ( hill et al . , 2008 ; recent findings suggest that the ventral premammillary nucleus ( pmv ) is the long sought area in which leptin modulates the activity of the reproductive system ( donato et al . , , we will summarize the existing literature about the potential role played by pmv neurons in the regulation of the hypothalamus ventral premammillary nucleus neurons exhibit a broad expression of receptors for hormones related to the regulation of the energy balance ( table 1 ) . pmv neurons express lepr ( elmquist et al . innumerous receptors for neurotransmitters involved with the regulation of energy balance are also found in the pmv , including the cannabinoid receptor 1 ( figure 1b ) , the melanocortin-4 receptor in mice ( liu et al . , 2005 ) , and the vasopressin receptor in hamsters ( sexually dimorphic and dependent upon photoperiod length ; dubois - dauphin et al . ( a , b ) darkfield photomicrographs demonstrating the distribution of insulin receptor ( insr ) mrna ( a ) and cannabinoid receptor type 1 ( cb1r ) mrna ( b ) in the mouse pmv . ( c , d ) darkfield photomicrographs demonstrating the distribution of cocaine and amphetamine regulated transcript ( cart ) mrna in the rat ( c ) and in the mouse ( d ) pmv . note the abundance of cart mrna in the pmv of rats compared to mice . ventral premammillary nucleus neurons are potential targets of sex hormones ( table 1 ) , as they express a dense amount of androgen receptor ( ar ) and a moderate to low amount of estrogen receptor and ( er and er ) and progesterone receptor ( simerly et al . the relatively high ratio of ar to er and the lack of definitive data demonstrating the expression of aromatase in the pmv indicate a potential role for androgens in pmv neuronal biology ( yokosuka and hayashi , 1996 ; wu et al . , 2009 despite the strong presence of sexual steroid receptors in the pmv , it has not been determined whether changing levels of sexual hormones affect the neuronal activity or gene expression in this area . in addition to sex steroid receptors , several neurotransmitters are expressed by pmv neurons ( table 1 ) , including glutamate ( ziegler et al . the expression of glutamatergic markers , such as vesicular glutamate transporter 2 ( vglut2 ) , can be found in the entire extension of the pmv and show a 94% colocalization with neurons that express lepr ( donato et al . thus , the projections originated from pmv neurons are thought to be excitatory due to their glutamatergic component . peptidergic neurotransmitters are also expressed by pmv neurons ( table 1 ) , including cocaine and amphetamine regulated transcript ( cart ) , substance p ( a tachykinin family member ) and enkephalin ( wamsley et al . in addition , the number of neurons that express tachykinin peptides in the pmv is higher in male than in female rats ( akesson , 1993 ) . pmv neurons also densely express neuronal nitric oxide synthase ( nnos ) , which catalyzes the synthesis of nitric oxide ( no ; vincent and kimura , 1992 ) . many of the neurotransmitters expressed in the pmv are involved in the neuroendocrine regulation of reproduction . for example , glutamatergic inputs were shown to induce gonadotropin - releasing hormone ( gnrh ) release and subsequent activation of hpg axis ( brann and mahesh , 1994 ; gargiulo and donoso , 1995 ; dhandapani and brann , 2000 ; mahesh and brann , 2005 ) . moreover , glutamate facilitates the expression of sexual behaviors ( gargiulo and donoso , 1995 ) and has regulatory effects on the timing of puberty ( zamorano et al . , 1998 ; of note , intracerebroventricular administration of glutamate receptor agonists , such as nmda , elicits secretion of gnrh and luteinizing hormone ( lh ) , even in kiss1 and kiss1r knockout mice , suggesting a role for glutamatergic neurotransmission outside the kiss1 neuronal system ( danglemont de tassigny et al . cart peptide was shown to mediate the stimulatory effects of leptin on gnrh secretion in vitro and in vivo ( lebrethon et al . , 2000 , 2007 ; in addition , no has been implicated in the regulation of sexual behaviors and hpg axis ( moretto et al . , 2002 furthermore , several studies found that no is a key neurotransmitter that mediates leptin - induced gnrh / lh secretion ( yu et al . recently , we reported that 73% of leptin responsive cells in the pmv express no - synthesizing enzymes ( donato et al . leptin does not affect the expression of nos1 mrna in the pmv , but low leptin levels , as in fasting or in ob / ob mice , cause a reduction in the number of pmv neurons expressing the phosphorylated form of nnos ( pnnos ) . , 2010 ) and acute injection of leptin restores the number of pnnos neurons in the pmv of fasted mice ( donato et al . the projections of pmv neurons were first described in rats using the neurotracer phaseolus vulgaris leucoagglutinin ( canteras et al . it was demonstrated that pmv neurons project mainly to the periventricular zone of the hypothalamus , which is composed of nuclei involved in the regulation of anterior pituitary function . pmv neurons also project to major nuclei of the sexually dimorphic circuitry , including the ventrolateral part of the ventromedial nucleus of hypothalamus ( vmh ) , medial preoptic nucleus , bed nuclei of the stria terminalis ( bst ) , ventral lateral septal nucleus , posterodorsal part of the medial nucleus of the amygdala ( mea ) , and posterior nucleus of the amygdala ( canteras et al . it is interesting that the major neuronal inputs to the pmv originate from neurons located in the sexually dimorphic circuitry , highlighting the intense intercommunication between this circuitry and the pmv ( simerly and swanson , 1988 ; canteras et al . for example , pmv is densely innervated by neurons located in the mea , including cells that express urocortin 3 ( canteras et al . more recent studies in mice and in rats using genetic tools in combination with tracing techniques highlighted a putative role of the pmv in the regulation of the hpg axis . it was shown that pmv neurons project directly to gnrh perikarya in the medial preoptic area ( mpa ; rondini et al . , 2009 ) and to gnrh fibers in the median eminence ( donato et al . interestingly , among all neurons that express lepr , only those in the pmv and a subpopulation of neurons in the mpa seem to project directly to gnrh neurons ( louis et al . in addition , pmv neurons project to the anteroventral periventricular nucleus ( avpv ; canteras et al . , 2004 ; hahn and coen , 2006 ) , a key site for female reproductive function ( wiegand and terasawa , 1982 ; gottsch et al . the avpv contains a subpopulation of kisspeptin neurons , which is critical for the preovulatory lh surge ( smith et al . we have recently found that fibers from pmv neurons make apparent synaptic contact with kisspeptin neurons in the avpv ( donato et al . the arcuate nucleus ( arh ) also receives a dense projection from pmv neurons ( canteras et al . , 1992b ) , but whether kisspeptin neurons in the arh or a specific population of arh neurons is selectively targeted by pmv inputs is still unknown . previous studies using electrolytic lesions described a potential role for pmv neurons in odor - induced lh secretion in rats ( beltramino and taleisnik , 1985 ) . olfaction is a critical sense used by rodents to discriminate socially relevant cues and to trigger social behaviors , including sexual behaviors ( romero et al . rats and mice exposed to conspecific odors show a large number of neurons expressing fos immunoreactivity ( fos - ir ) in the pmv , which suggests that the pmv is involved in the neuronal circuitry that conveys olfactory information ( yokosuka et al . most of the cart neurons in the pmv express the enzymes that synthesize no . besides , a parcel of nitrergic neurons is stimulated by female odors and virtually all nitrergic cells in the pmv express ars ( yokosuka and hayashi , 1996 ) . altogether , these studies indicate that pmv is apt to integrate information about circulating levels of sexual hormones and socially relevant cues ( through brain areas related to pheromonal processing , such as mea or bst ) and generate appropriate neuroendocrine responses to modulate socially relevant behaviors . the pmv may also be involved in the expression of conspecific behaviors because pmv neurons of male rats are also responsive to conspecific male odors ( donato et al . in addition , pmv neurons express fos - ir after mating or agonistic behavior ( kollack - walker and newman , 1995 ; coolen et al . however , these studies should be interpreted with caution due to the extension of the lesion . restricted and/or selective lesions are required to determine the real contribution of the pmv in aggressive behaviors . pmv neurons are the target of metabolic cues and also respond to socially relevant sensory stimulation . of note , 44% of the lepr - expressing cells in the pmv of male mice are activated by female odors , whereas in female mice , 18% of lepr cells are activated by male odors ( leshan et al . these findings suggest that food availability or energy stored affect neuronal responses to odors . however , we observed that fasting caused no changes in female odor - induced fos - ir in the pmv and in the mea of male rats compared to normally fed controls ( donato et al . further studies will be necessary to determine the influence of the nutritional state on the response to environmental stimulation . although most of the studies about the pmv have used rats and mice as experimental models , there are several pieces of evidence that the premammillary hypothalamic area ( pmh ) also plays a key role in reproductive function of seasonal breeders ( i.e. seasonal reproduction is a strategy used by several species to increase survival of offspring by reproducing during a period of the year when the environment offers favorable conditions . changes in day light exposure alter the synthesis and secretion of the pineal gland hormone melatonin , which in turn binds to hypothalamic nuclei and modulates the pulsatile secretion of gnrh ( emilsson et al . , 1999 ; the pmh of ewes is composed of the caudal arh , the pmv and the ventral tuberomammillary nucleus . similarly to rats , pmv neurons in ewes express cart and nnos ( sliwowska et al . , 1998 ) , indicating that in sheep , the pmv appears to play a key role in seasonal reproduction . in birds , a subpopulation of pmh neurons expresses dopamine , a neurotransmitter known to affect the secretion of several reproductive hormones , including lh , fsh , and prolactin . melatonin neurons in the pmh exhibit high activity at the photosensitive phase , which was associated with higher dopaminergic neurotransmission and gnrh activation . the pmh of turkeys also presents a distinct circadian expression of clock genes compared to the pineal gland and the brain master clock , the suprachiasmatic nucleus . in particular , cry1 and per3 seem to mediate the photic responses associated with the control of the reproductive system ( leclerc et al . the high expression of receptors of hormones related with the regulation of the energy balance might imply that the pmv is involved in the control of energy balance . however , bilateral excitotoxic lesions of the pmv did not affect body weight , mean food intake and circulating leptin levels in adult female rats ( donato et al . , 2001 ; therefore , the regulation of food intake across the estrous cycle by pmv neurons can be an indirect consequence of changes in sexual hormone levels after lesions of the pmv . leptin exerts a pivotal role in the long - term regulation of energy balance ( schwartz , 2006 ; gautron and elmquist , 2011 ) . , following the same paradigm , we generated ob / ob mice with bilateral lesions of the pmv ( donato et al . upon leptin treatment , these mice showed drastic reduction in food intake and body weight , indicating that leptin may restore the metabolic deficits of ob / ob female mice in the absence of pmv neurons ( donato et al . to further investigate the role played by lepr in the pmv , we generated a lepr - null mouse model in which lepr is expressed selectively in pmv neurons . we found that endogenous expression of lepr only in the pmv did not affect food intake , body weight , and fat mass in male and female mice ( donato et al . , overall , these results suggest that despite the presence of innumerous receptors involved with the regulation of energy balance , pmv neurons are not key players in the modulation of food intake and body weight . rather , pmv neurons may function as a key integrative site conveying metabolic cues to the reproductive system . accordingly , pmv lesions cause a temporary anestrus in rats ( persistent leukocytes in the vaginal smears ) . these results indicate that the pmv is required for the normal activity of the hpg axis in female rats . following the same line , we hypothesized that the pmv would be apt to mediate the effects of leptin on the reproductive neuroendocrine axis . lesions of the pmv blocked the stimulatory effect of leptin on lh secretion in fasted rats ( donato et al . in order to investigate putative signaling pathways that mediate the acute effects of leptin on pmv neurons , patch - clamp recordings of hypothalamic slices were performed . leptin caused a rapid depolarization of 75% of lepr - expressing neurons in the pmv through a putative trpc channel ( leshan et al . importantly , pharmacological or genetic disruption of the phosphoinositide 3-kinase ( pi3k ) pathway prevented the leptin - induced changes in the activity of pmv lepr neurons ( williams et al . these results indicate that pi3k is required for the acute changes in biophysical properties of pmv neurons induced by leptin . whether these changes in cellular activity underlie the physiological effects of leptin are under investigation . we further assessed whether leptin signaling in pmv neurons is critical to induce the onset of puberty and restore fertility in leptin- or lepr - deficient mouse models . after a period of 6 weeks of breeding tests , 50% of mice with selective reactivation of lepr in pmv neurons became pregnant , despite their obese and diabetic phenotype ( donato et al . moreover , earlier studies suggested that the lack of leptin signaling causes a deficient release of gnrh because ob / ob and db / db mice have high content of gnrh in the median eminence / medial basal hypothalamus ( me / mbh ; johnson and sidman , 1979 ; batt et al . together , these findings have determined the pmv as a key site linking leptin action and the female reproductive physiology . overall , this review highlights a series of recent data demonstrating that pmv neurons are apt to mediate the effects of leptin on gnrh secretion . the stimulatory effect of pmv neurons on gnrh release is possibly mediated by the coordinated effects of glutamate and no on gnrh terminals in the median eminence ( figure 2 ) . although it is very likely that other neuronal populations also convey metabolic cues to modulate the hpg axis , the existing evidence suggests that the pmv is a key site relaying the effects of leptin on the reproductive neuroendocrine axis . we postulate that through pmv neurons , leptin modulates the influence of adiposity on the timing of puberty and the coordinated secretion of lh during conditions of negative energy balance . the data presented in this review provide the physiological and neuroanatomical basis underlying the effects of leptin on the hpg axis . proposed role for the ventral premammillary nucleus ( pmv ) in the female reproductive physiology . pmv neurons express excitatory neurotransmitters ( e.g. , glutamate and nitric oxide ) and directly project to the anteroventral periventricular nucleus ( avpv ) , to the arcuate nucleus ( arh ) , and to gonadotropin - releasing hormone ( gnrh ) neurons . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

meta - analyses and systematic reviews are useful , but potentially dangerous tools in the present age of evidence - based medicine . evidence presented in such studies is graded as type ia , and is more than likely to be accepted uncritically and to change clinical practice . the conduct of a systematic review or a meta - analysis is , therefore , a process of utmost scientific importance , and must be undertaken with the same care as a randomized controlled study . each study needs to be critically appraised and rated not only for its statistical methodology , but also for its clinical and physiological validity . the systematic review of the use of albumin by the cochrane injuries group has rightly been criticized for a number of reasons . first , the media hype that accompanied it was inappropriate for a subject that requires rational scientific appraisal . second , pooling data from studies on extremely heterogeneous groups of patients makes firm conclusions difficult . third , although the authors were undoubtedly expert statisticians , none of them appeared to have much understanding or experience of the underlying pathophysiological problems being studied . thus , although they were able to score the trials analyzed for their statistical validity , they appeared to take no account of the clinical and physiological validity of the studies being conducted . there was no comment , for example , on whether the fluids studied were titrated to accurate physiological end - points , or were merely given in absolute amounts , irrespective of the underlying physiology . solutions of albumin , crystalloids or plasma substitutes can not be treated like drugs of fixed dosage , and need to be titrated to desirable physiological end - points . this is because if they are given indiscriminately to individuals with a normal or excessive plasma volume , there are clearly grave risks of overloading the patient and increasing mortality . a further major condemnation was the fact that the paper was published despite adverse referee criticism , and a number of paediatricians and burns specialists criticized the inadequacy of the studies quoted from their own fields . it is common practice , for example , to use albumin in the management of shock associated with meningococcal meningitis in children , and also in the postshock phase in severe burns . none of the studies in the review were designed with mortality as the primary end - point , and there was therefore no prerandomization stratification for mortality risk . it was perhaps the messianic rather than scientific method of its public presentation that antagonized many thoughtful clinicians . if , instead , its conclusions had been presented with caution and as a stimulus to further carefully conducted studies in specific groups of patients , then despite its limitations it would have served a useful purpose . one might draw an analogy with the situation in the late 1970s and early 1980s concerning nutritional support . critics such as koretz and detsky et al were rightly able to point to the lack of clear evidence that nutritional support was beneficial , and also to the poor quality of many of the studies conducted up to that point . since that time , a whole flood of excellent studies has defined clear benefit in certain clinical circumstances and in certain groups of patients . conversely , they have shown either no benefit or even harm in other groups . the clinical indications for this modality of treatment have therefore become much better defined . in the present discussion we make a plea for a similarly thoughtful approach to the use of albumin . there are three clinical situations in which albumin has been or should be considered : hypoalbuminaemia ; resuscitation in acute hypovolaemia ; and in hypovolaemia occurring after the acute phase of critical illness . use of albumin in hypoalbuminaemia is the ultimate navet , because hypoalbuminaemia bears no direct relationship to plasma or other fluid compartment volumes . it may occur in the presence of intravascular overload or deficit ; it is also affected by dilution , disease and distributional factors . we may therefore dismiss the hypoalbuminaemia group of studies considered in the cochrane report , because there must be few , if any , clinicians who would even consider using albumin merely to correct a serum albumin concentration . one might just as well say that all patients with hyponatraemia should be given salt solutions , irrespective of the patient 's total extracellular sodium . here again , in the adult patient there is no evidence in most situations that albumin solutions are any better than crystalloids or plasma substitutes . indeed , at face value the results of the cochrane report are open to the interpretation either that there is no difference between albumin and other solutions , or that albumin gives a worse outcome , but not that albumin is potentially superior to any other solutions . another systematic review of colloids ( including albumin ) versus crystalloids included methodological quality assessment of 17 trials on 814 patients , with mortality data . that review rightly concluded that , although the mortality results tended to favour the crystalloid group , an excess mortality in the colloid group could not be inferred on statistical analysis . it also suggested that colloids caused less pulmonary oedema than crystalloids , with a relative risk ( 95% confidence interval ) of 0.84 ( 0.25 - 2.45 ) . that review excluded studies in which hypertonic crystalloids were used in order to avoid the confounding influence of these solutions . trial quality was assessed by evaluating the randomization procedure , population description , blinding , documentation of cointerventions and whether patient selection was consecutive . on this basis , only 11 out of the 17 studies had a validity score of 8 or more ( worst possible score 0 ; best possible score 16 ) . the authors of that review also assessed the use of physiological end - points such as tissue perfusion and oncotic pressure . it is argued that , during the acute phase of critical illness , the transcapillary escape rate of albumin is high , and therefore administered albumin is not retained within the circulation for a sufficient length of time to be useful , and may accumulate in the tissues , causing adverse effects . even here one should be cautious because there were some studies quoted in the cochrane report that measured the physiological consequences of giving albumin versus crystalloid solutions , and appeared to show superior physiological effects of giving albumin without any detriment in terms of mortality . the papers with the largest relative risk of death in the albumin group , and which therefore weighted the meta - analysis of the cochrane injuries group , are either open to major criticism in terms of physiological and clinical methodology or are so small and with so few deaths that mortality figures in the studies themselves were statistically insignificant . the small study by zetterstrom , with its relative risk of 5.0 , had only two deaths out of 18 in the entire study , both in the albumin group . the study of woods and kelley had only one death in 69 patients , but the relative risk was 2.61 . this paper was included by the cochrane reviewers in the hypovolaemia group even though the authors categorically state that ' thirty - four treatment group patients ( n = 37 ) received albumin for low levels postoperatively ' , clearly indicating that the study should have been analyzed in the hypoalbuminaemia group . in none of these studies was mortality cited as a primary or even a secondary end - point . the paper with the largest excess mortality , by lucas et al , showed that this was predominantly due to cardiopulmonary failure . in that study , albumin was added to a standardized fluid resuscitation regimen in the protocol group , rather than being substituted for other fluids . this meant that the protocol group received higher volumes of fluids than did the control individuals , and it is hardly surprising therefore that there was an excess mortality in a group that was clearly given excessive fluid . it is of note that the albumin group received the same salt load as the nonalbumin group in phase 1 , and a greater than average salt load in phases 2 and 3 of the resuscitation process . patients in the albumin group also received more whole blood and fresh frozen plasma than did the control individuals . in fact , not unexpectedly , the measured plasma volume in the albumin group was , on average , 1294 ml greater than the theoretically expected volume . as webb recently indicated , most of the papers discussed in the cochrane study were designed to assess the effects of various fluids on physiological variables , and were not designed to assess mortality . he pointed out that a randomized controlled trial comparing one colloid with one crystalloid would require over 6500 patients to detect an excess mortality from colloid of 4% on the basis of the data presented in the cochrane review . he concluded ' i can not see how we can make a meaningful statement on comparative mortality . ' although current evidence does not favour the use of albumin in acute resuscitation in most situations , the proper scientific view should be that the jury is still out , particularly in paediatrics . as some have pointed out , however , the way in which the cochrane report was presented and interpreted has made the setting up of careful trials more rather than less difficult . it is to be hoped that the recent paper by sort et al will be the first of many such trials that will define the indications for this expensive form of treatment in a more rational manner . they studied the effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis . they randomly assigned 126 patients with cirrhosis and spontaneous bacterial peritonitis to treatment with intravenous cefotaxime ( 63 patients ) or cefotaxime and intravenous albumin ( 63 patients ) . mortality in the first group was 41% , and 22% in the albumin group . that study did not compare the use of albumin with crystalloids or plasma substitutes . however , because both of these contain large amounts of sodium , which might be expected to have adverse effects in cirrhotic persons with ascites , this is perhaps a theoretical rather than a relevant clinical consideration . the contention that albumin should be avoided in acute hypovolaemia is therefore a dogmatic and untrue generalization . one of the problems of resuscitation with crystalloids , or even the plasma substitutes , which all have a high sodium content , is that circulatory function is maintained at the expense of interstitial salt and water overload . the effect of acute illness and injury on salt and water retention have been known for many years . acutely ill patients are unable to excrete an excess salt and water load , and the return of a sodium diuresis is the first herald of recovery and convalescence . one of the major arguments for the use of salt - poor albumin in resuscitation is that the circulation might be maintained without incurring a large interstitial salt overload . in the majority of patients , however , who make a good recovery , this overload is excreted spontaneously in the postacute period . those patients who suffer persisting complications , however , continue to retain salt and water and to become increasingly oedematous . we have shown , for example , that patients , who are referred for nutritional support in the postacute phase and who are oedematous , have an average salt and water overload equivalent to 10 l extracellular fluid . such patients are often those with major complications of gastrointestinal surgery , with dehiscent wounds , intra - abdominal abscesses , fistulae and continuing losses of serous fluids . many of these patients combine interstitial overload with a low plasma volume , leading to a low renal plasma flow . these patients are not only unable to diurese their salt and water overload because of renal responses to plasma hypovolaemia , but are also diuretic resistant . the logical treatment of such patients is the use of salt - poor concentrated albumin in a dose that is titrated carefully to physiological end - points such as central venous pressure , pulse rate , blood pressure , and urinary salt and water excretion . plasma substitutes contain high sodium concentrations and have a shorter half - life than plasma or albumin in the circulation . it seems , therefore , less appropriate to use these in the postacute phase . compounding the salt excess with crystalloid is certainly inappropriate and merely worsens the oedema . our own experience of the use of salt - poor albumin under these circumstances is that the diuresis ensues immediately with improved cardiovascular parameters . we have also found , in our clinical experience , that such patients only require 200 - 400ml of 20% salt - poor albumin over 48h . once the plasma volume has been restored , no further administration of albumin is necessary , thereby indicating that the albumin is retained within the circulation , rather than leaking out continuously as it might do in the acute phase of illness . it is common practice in some burns centres to use albumin during the postacute phase . many years ago we reported data on fluid and electrolyte balance in burned patients during the weeks after injury , and described the appropriate use of plasma and whole blood to sustain the intravascular volume at a level where salt and water diuresis occurred . the cochrane report did not even mention the postacute situation , in which there are few formal studies . doctors whose main experience is in intensive or critical care rarely have to deal with this postacute problem , and because most of the literature concerning the use of crystalloid , colloids and albumin emanates from this group , it is hardly surprising that it has received little or no attention , despite the fact that it is an important and common clinical entity . studies comparing salt - poor albumin and plasma substitutes , titrated to physiological end - points , are needed in this situation . use of albumin in hypoalbuminaemia is the ultimate navet , because hypoalbuminaemia bears no direct relationship to plasma or other fluid compartment volumes . it may occur in the presence of intravascular overload or deficit ; it is also affected by dilution , disease and distributional factors . we may therefore dismiss the hypoalbuminaemia group of studies considered in the cochrane report , because there must be few , if any , clinicians who would even consider using albumin merely to correct a serum albumin concentration . one might just as well say that all patients with hyponatraemia should be given salt solutions , irrespective of the patient 's total extracellular sodium . here again , in the adult patient there is no evidence in most situations that albumin solutions are any better than crystalloids or plasma substitutes . indeed , at face value the results of the cochrane report are open to the interpretation either that there is no difference between albumin and other solutions , or that albumin gives a worse outcome , but not that albumin is potentially superior to any other solutions . another systematic review of colloids ( including albumin ) versus crystalloids included methodological quality assessment of 17 trials on 814 patients , with mortality data . that review rightly concluded that , although the mortality results tended to favour the crystalloid group , an excess mortality in the colloid group could not be inferred on statistical analysis . it also suggested that colloids caused less pulmonary oedema than crystalloids , with a relative risk ( 95% confidence interval ) of 0.84 ( 0.25 - 2.45 ) . that review excluded studies in which hypertonic crystalloids were used in order to avoid the confounding influence of these solutions . trial quality was assessed by evaluating the randomization procedure , population description , blinding , documentation of cointerventions and whether patient selection was consecutive . on this basis , only 11 out of the 17 studies had a validity score of 8 or more ( worst possible score 0 ; best possible score 16 ) . the authors of that review also assessed the use of physiological end - points such as tissue perfusion and oncotic pressure . it is argued that , during the acute phase of critical illness , the transcapillary escape rate of albumin is high , and therefore administered albumin is not retained within the circulation for a sufficient length of time to be useful , and may accumulate in the tissues , causing adverse effects . even here one should be cautious because there were some studies quoted in the cochrane report that measured the physiological consequences of giving albumin versus crystalloid solutions , and appeared to show superior physiological effects of giving albumin without any detriment in terms of mortality . the papers with the largest relative risk of death in the albumin group , and which therefore weighted the meta - analysis of the cochrane injuries group , are either open to major criticism in terms of physiological and clinical methodology or are so small and with so few deaths that mortality figures in the studies themselves were statistically insignificant . the small study by zetterstrom , with its relative risk of 5.0 , had only two deaths out of 18 in the entire study , both in the albumin group . the study of woods and kelley had only one death in 69 patients , but the relative risk was 2.61 . this paper was included by the cochrane reviewers in the hypovolaemia group even though the authors categorically state that ' thirty - four treatment group patients ( n = 37 ) received albumin for low levels postoperatively ' , clearly indicating that the study should have been analyzed in the hypoalbuminaemia group . in none of these studies was mortality cited as a primary or even a secondary end - point . the paper with the largest excess mortality , by lucas et al , showed that this was predominantly due to cardiopulmonary failure . in that study , albumin was added to a standardized fluid resuscitation regimen in the protocol group , rather than being substituted for other fluids . this meant that the protocol group received higher volumes of fluids than did the control individuals , and it is hardly surprising therefore that there was an excess mortality in a group that was clearly given excessive fluid . it is of note that the albumin group received the same salt load as the nonalbumin group in phase 1 , and a greater than average salt load in phases 2 and 3 of the resuscitation process . patients in the albumin group also received more whole blood and fresh frozen plasma than did the control individuals . in fact , not unexpectedly , the measured plasma volume in the albumin group was , on average , 1294 ml greater than the theoretically expected volume . as webb recently indicated , most of the papers discussed in the cochrane study were designed to assess the effects of various fluids on physiological variables , and were not designed to assess mortality . he pointed out that a randomized controlled trial comparing one colloid with one crystalloid would require over 6500 patients to detect an excess mortality from colloid of 4% on the basis of the data presented in the cochrane review . he concluded ' i can not see how we can make a meaningful statement on comparative mortality . ' although current evidence does not favour the use of albumin in acute resuscitation in most situations , the proper scientific view should be that the jury is still out , particularly in paediatrics . as some have pointed out , however , the way in which the cochrane report was presented and interpreted has made the setting up of careful trials more rather than less difficult . it is to be hoped that the recent paper by sort et al will be the first of many such trials that will define the indications for this expensive form of treatment in a more rational manner . they studied the effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis . they randomly assigned 126 patients with cirrhosis and spontaneous bacterial peritonitis to treatment with intravenous cefotaxime ( 63 patients ) or cefotaxime and intravenous albumin ( 63 patients ) . mortality in the first group was 41% , and 22% in the albumin group . that study did not compare the use of albumin with crystalloids or plasma substitutes . however , because both of these contain large amounts of sodium , which might be expected to have adverse effects in cirrhotic persons with ascites , this is perhaps a theoretical rather than a relevant clinical consideration . the contention that albumin should be avoided in acute hypovolaemia is therefore a dogmatic and untrue generalization . one of the problems of resuscitation with crystalloids , or even the plasma substitutes , which all have a high sodium content , is that circulatory function is maintained at the expense of interstitial salt and water overload . the effect of acute illness and injury on salt and water retention have been known for many years . acutely ill patients are unable to excrete an excess salt and water load , and the return of a sodium diuresis is the first herald of recovery and convalescence . one of the major arguments for the use of salt - poor albumin in resuscitation is that the circulation might be maintained without incurring a large interstitial salt overload . in the majority of patients , however , who make a good recovery , this overload is excreted spontaneously in the postacute period . those patients who suffer persisting complications , however , continue to retain salt and water and to become increasingly oedematous . we have shown , for example , that patients , who are referred for nutritional support in the postacute phase and who are oedematous , have an average salt and water overload equivalent to 10 l extracellular fluid . such patients are often those with major complications of gastrointestinal surgery , with dehiscent wounds , intra - abdominal abscesses , fistulae and continuing losses of serous fluids . many of these patients combine interstitial overload with a low plasma volume , leading to a low renal plasma flow . these patients are not only unable to diurese their salt and water overload because of renal responses to plasma hypovolaemia , but are also diuretic resistant . the logical treatment of such patients is the use of salt - poor concentrated albumin in a dose that is titrated carefully to physiological end - points such as central venous pressure , pulse rate , blood pressure , and urinary salt and water excretion . plasma substitutes contain high sodium concentrations and have a shorter half - life than plasma or albumin in the circulation . it seems , therefore , less appropriate to use these in the postacute phase . compounding the salt excess with crystalloid is certainly inappropriate and merely worsens the oedema . our own experience of the use of salt - poor albumin under these circumstances is that the diuresis ensues immediately with improved cardiovascular parameters . we have also found , in our clinical experience , that such patients only require 200 - 400ml of 20% salt - poor albumin over 48h . once the plasma volume has been restored , no further administration of albumin is necessary , thereby indicating that the albumin is retained within the circulation , rather than leaking out continuously as it might do in the acute phase of illness . it is common practice in some burns centres to use albumin during the postacute phase . many years ago we reported data on fluid and electrolyte balance in burned patients during the weeks after injury , and described the appropriate use of plasma and whole blood to sustain the intravascular volume at a level where salt and water diuresis occurred . the cochrane report did not even mention the postacute situation , in which there are few formal studies . doctors whose main experience is in intensive or critical care rarely have to deal with this postacute problem , and because most of the literature concerning the use of crystalloid , colloids and albumin emanates from this group , it is hardly surprising that it has received little or no attention , despite the fact that it is an important and common clinical entity . studies comparing salt - poor albumin and plasma substitutes , titrated to physiological end - points , are needed in this situation . we make a plea for a more thoughtful approach to the therapeutic use of albumin solutions . there is no physiological logic in using albumin simply for hypoalbuminaemia , because it is plasma volume rather than protein concentration that is most clinically relevant . in acute resuscitation , we accept that the weight of literature suggests at least that albumin has no advantage over crystalloid or other colloids in most cases , although there may be specific instances in adults and in children where its use may be beneficial , and one recent paper concerning its use in peritonitis in cirrhotic persons showed a clear reduction in mortality . to regard this subject as closed by the cochrane report would be unfortunate in the extreme , and we express the hope that further careful studies will be published in this area . finally , during the postacute phase of major illness , there are many patients who are left with an interstitial salt and water overload combined with a low plasma volume , in which the use of concentrated salt - poor albumin is the most logical treatment . although clinical experience with the use of albumin in this group of patients is encouraging , formal studies are required to compare the use of albumin with that of the less expensive plasma substitutes . the question to be addressed in each clinical situation is not only whether albumin is better than other fluids , but whether cheaper substitutes are just as effective and , therefore , more cost effective . we plead for an open - minded , thoughtful and scientific approach to this field , devoid of hyperbole or messianic fervour . we suggest that the view that albumin administration should be avoided in all situations is , on current evidence , untenable .

the recent cochrane report on albumin administration is analysed and criticised on the grounds of clinical methodology , content and interpretation . although it is nave and illogical to treat hypoalbuminaemia with albumin infusions , a more balanced view on the use of albumin for resuscitation in acute hypovolaemia is necessary . once the acute phase of critical illness is past , interstitial volume is often expanded causing oedema , with a low plasma volume . we argue for the use of salt - poor albumin solutions in this situation and conclude that , on current evidence , the assertion that albumin should be avoided in all situations is irrational and untenable .

Introduction Clinical situations Hypoalbuminaemia Resuscitation in acute hypovolaemia Plasma hypovolaemia after the acute phase of critical illness Conclusion

the conduct of a systematic review or a meta - analysis is , therefore , a process of utmost scientific importance , and must be undertaken with the same care as a randomized controlled study . each study needs to be critically appraised and rated not only for its statistical methodology , but also for its clinical and physiological validity . the systematic review of the use of albumin by the cochrane injuries group has rightly been criticized for a number of reasons . first , the media hype that accompanied it was inappropriate for a subject that requires rational scientific appraisal . there was no comment , for example , on whether the fluids studied were titrated to accurate physiological end - points , or were merely given in absolute amounts , irrespective of the underlying physiology . solutions of albumin , crystalloids or plasma substitutes can not be treated like drugs of fixed dosage , and need to be titrated to desirable physiological end - points . this is because if they are given indiscriminately to individuals with a normal or excessive plasma volume , there are clearly grave risks of overloading the patient and increasing mortality . it is common practice , for example , to use albumin in the management of shock associated with meningococcal meningitis in children , and also in the postshock phase in severe burns . in the present discussion we make a plea for a similarly thoughtful approach to the use of albumin . there are three clinical situations in which albumin has been or should be considered : hypoalbuminaemia ; resuscitation in acute hypovolaemia ; and in hypovolaemia occurring after the acute phase of critical illness . use of albumin in hypoalbuminaemia is the ultimate navet , because hypoalbuminaemia bears no direct relationship to plasma or other fluid compartment volumes . it may occur in the presence of intravascular overload or deficit ; it is also affected by dilution , disease and distributional factors . we may therefore dismiss the hypoalbuminaemia group of studies considered in the cochrane report , because there must be few , if any , clinicians who would even consider using albumin merely to correct a serum albumin concentration . here again , in the adult patient there is no evidence in most situations that albumin solutions are any better than crystalloids or plasma substitutes . indeed , at face value the results of the cochrane report are open to the interpretation either that there is no difference between albumin and other solutions , or that albumin gives a worse outcome , but not that albumin is potentially superior to any other solutions . another systematic review of colloids ( including albumin ) versus crystalloids included methodological quality assessment of 17 trials on 814 patients , with mortality data . that review rightly concluded that , although the mortality results tended to favour the crystalloid group , an excess mortality in the colloid group could not be inferred on statistical analysis . it also suggested that colloids caused less pulmonary oedema than crystalloids , with a relative risk ( 95% confidence interval ) of 0.84 ( 0.25 - 2.45 ) . the authors of that review also assessed the use of physiological end - points such as tissue perfusion and oncotic pressure . it is argued that , during the acute phase of critical illness , the transcapillary escape rate of albumin is high , and therefore administered albumin is not retained within the circulation for a sufficient length of time to be useful , and may accumulate in the tissues , causing adverse effects . even here one should be cautious because there were some studies quoted in the cochrane report that measured the physiological consequences of giving albumin versus crystalloid solutions , and appeared to show superior physiological effects of giving albumin without any detriment in terms of mortality . the papers with the largest relative risk of death in the albumin group , and which therefore weighted the meta - analysis of the cochrane injuries group , are either open to major criticism in terms of physiological and clinical methodology or are so small and with so few deaths that mortality figures in the studies themselves were statistically insignificant . the small study by zetterstrom , with its relative risk of 5.0 , had only two deaths out of 18 in the entire study , both in the albumin group . this paper was included by the cochrane reviewers in the hypovolaemia group even though the authors categorically state that ' thirty - four treatment group patients ( n = 37 ) received albumin for low levels postoperatively ' , clearly indicating that the study should have been analyzed in the hypoalbuminaemia group . this meant that the protocol group received higher volumes of fluids than did the control individuals , and it is hardly surprising therefore that there was an excess mortality in a group that was clearly given excessive fluid . it is of note that the albumin group received the same salt load as the nonalbumin group in phase 1 , and a greater than average salt load in phases 2 and 3 of the resuscitation process . in fact , not unexpectedly , the measured plasma volume in the albumin group was , on average , 1294 ml greater than the theoretically expected volume . he pointed out that a randomized controlled trial comparing one colloid with one crystalloid would require over 6500 patients to detect an excess mortality from colloid of 4% on the basis of the data presented in the cochrane review . although current evidence does not favour the use of albumin in acute resuscitation in most situations , the proper scientific view should be that the jury is still out , particularly in paediatrics . as some have pointed out , however , the way in which the cochrane report was presented and interpreted has made the setting up of careful trials more rather than less difficult . it is to be hoped that the recent paper by sort et al will be the first of many such trials that will define the indications for this expensive form of treatment in a more rational manner . that study did not compare the use of albumin with crystalloids or plasma substitutes . the contention that albumin should be avoided in acute hypovolaemia is therefore a dogmatic and untrue generalization . one of the major arguments for the use of salt - poor albumin in resuscitation is that the circulation might be maintained without incurring a large interstitial salt overload . such patients are often those with major complications of gastrointestinal surgery , with dehiscent wounds , intra - abdominal abscesses , fistulae and continuing losses of serous fluids . many of these patients combine interstitial overload with a low plasma volume , leading to a low renal plasma flow . the logical treatment of such patients is the use of salt - poor concentrated albumin in a dose that is titrated carefully to physiological end - points such as central venous pressure , pulse rate , blood pressure , and urinary salt and water excretion . our own experience of the use of salt - poor albumin under these circumstances is that the diuresis ensues immediately with improved cardiovascular parameters . we have also found , in our clinical experience , that such patients only require 200 - 400ml of 20% salt - poor albumin over 48h . once the plasma volume has been restored , no further administration of albumin is necessary , thereby indicating that the albumin is retained within the circulation , rather than leaking out continuously as it might do in the acute phase of illness . it is common practice in some burns centres to use albumin during the postacute phase . the cochrane report did not even mention the postacute situation , in which there are few formal studies . doctors whose main experience is in intensive or critical care rarely have to deal with this postacute problem , and because most of the literature concerning the use of crystalloid , colloids and albumin emanates from this group , it is hardly surprising that it has received little or no attention , despite the fact that it is an important and common clinical entity . studies comparing salt - poor albumin and plasma substitutes , titrated to physiological end - points , are needed in this situation . use of albumin in hypoalbuminaemia is the ultimate navet , because hypoalbuminaemia bears no direct relationship to plasma or other fluid compartment volumes . it may occur in the presence of intravascular overload or deficit ; it is also affected by dilution , disease and distributional factors . we may therefore dismiss the hypoalbuminaemia group of studies considered in the cochrane report , because there must be few , if any , clinicians who would even consider using albumin merely to correct a serum albumin concentration . one might just as well say that all patients with hyponatraemia should be given salt solutions , irrespective of the patient 's total extracellular sodium . here again , in the adult patient there is no evidence in most situations that albumin solutions are any better than crystalloids or plasma substitutes . indeed , at face value the results of the cochrane report are open to the interpretation either that there is no difference between albumin and other solutions , or that albumin gives a worse outcome , but not that albumin is potentially superior to any other solutions . another systematic review of colloids ( including albumin ) versus crystalloids included methodological quality assessment of 17 trials on 814 patients , with mortality data . that review rightly concluded that , although the mortality results tended to favour the crystalloid group , an excess mortality in the colloid group could not be inferred on statistical analysis . it also suggested that colloids caused less pulmonary oedema than crystalloids , with a relative risk ( 95% confidence interval ) of 0.84 ( 0.25 - 2.45 ) . the authors of that review also assessed the use of physiological end - points such as tissue perfusion and oncotic pressure . it is argued that , during the acute phase of critical illness , the transcapillary escape rate of albumin is high , and therefore administered albumin is not retained within the circulation for a sufficient length of time to be useful , and may accumulate in the tissues , causing adverse effects . even here one should be cautious because there were some studies quoted in the cochrane report that measured the physiological consequences of giving albumin versus crystalloid solutions , and appeared to show superior physiological effects of giving albumin without any detriment in terms of mortality . the papers with the largest relative risk of death in the albumin group , and which therefore weighted the meta - analysis of the cochrane injuries group , are either open to major criticism in terms of physiological and clinical methodology or are so small and with so few deaths that mortality figures in the studies themselves were statistically insignificant . the small study by zetterstrom , with its relative risk of 5.0 , had only two deaths out of 18 in the entire study , both in the albumin group . this paper was included by the cochrane reviewers in the hypovolaemia group even though the authors categorically state that ' thirty - four treatment group patients ( n = 37 ) received albumin for low levels postoperatively ' , clearly indicating that the study should have been analyzed in the hypoalbuminaemia group . it is of note that the albumin group received the same salt load as the nonalbumin group in phase 1 , and a greater than average salt load in phases 2 and 3 of the resuscitation process . in fact , not unexpectedly , the measured plasma volume in the albumin group was , on average , 1294 ml greater than the theoretically expected volume . although current evidence does not favour the use of albumin in acute resuscitation in most situations , the proper scientific view should be that the jury is still out , particularly in paediatrics . as some have pointed out , however , the way in which the cochrane report was presented and interpreted has made the setting up of careful trials more rather than less difficult . it is to be hoped that the recent paper by sort et al will be the first of many such trials that will define the indications for this expensive form of treatment in a more rational manner . that study did not compare the use of albumin with crystalloids or plasma substitutes . the contention that albumin should be avoided in acute hypovolaemia is therefore a dogmatic and untrue generalization . one of the major arguments for the use of salt - poor albumin in resuscitation is that the circulation might be maintained without incurring a large interstitial salt overload . such patients are often those with major complications of gastrointestinal surgery , with dehiscent wounds , intra - abdominal abscesses , fistulae and continuing losses of serous fluids . many of these patients combine interstitial overload with a low plasma volume , leading to a low renal plasma flow . the logical treatment of such patients is the use of salt - poor concentrated albumin in a dose that is titrated carefully to physiological end - points such as central venous pressure , pulse rate , blood pressure , and urinary salt and water excretion . our own experience of the use of salt - poor albumin under these circumstances is that the diuresis ensues immediately with improved cardiovascular parameters . we have also found , in our clinical experience , that such patients only require 200 - 400ml of 20% salt - poor albumin over 48h . once the plasma volume has been restored , no further administration of albumin is necessary , thereby indicating that the albumin is retained within the circulation , rather than leaking out continuously as it might do in the acute phase of illness . it is common practice in some burns centres to use albumin during the postacute phase . the cochrane report did not even mention the postacute situation , in which there are few formal studies . doctors whose main experience is in intensive or critical care rarely have to deal with this postacute problem , and because most of the literature concerning the use of crystalloid , colloids and albumin emanates from this group , it is hardly surprising that it has received little or no attention , despite the fact that it is an important and common clinical entity . studies comparing salt - poor albumin and plasma substitutes , titrated to physiological end - points , are needed in this situation . we make a plea for a more thoughtful approach to the therapeutic use of albumin solutions . there is no physiological logic in using albumin simply for hypoalbuminaemia , because it is plasma volume rather than protein concentration that is most clinically relevant . in acute resuscitation , we accept that the weight of literature suggests at least that albumin has no advantage over crystalloid or other colloids in most cases , although there may be specific instances in adults and in children where its use may be beneficial , and one recent paper concerning its use in peritonitis in cirrhotic persons showed a clear reduction in mortality . to regard this subject as closed by the cochrane report would be unfortunate in the extreme , and we express the hope that further careful studies will be published in this area . finally , during the postacute phase of major illness , there are many patients who are left with an interstitial salt and water overload combined with a low plasma volume , in which the use of concentrated salt - poor albumin is the most logical treatment . although clinical experience with the use of albumin in this group of patients is encouraging , formal studies are required to compare the use of albumin with that of the less expensive plasma substitutes . we suggest that the view that albumin administration should be avoided in all situations is , on current evidence , untenable .

heart failure ( hf ) results in substantial morbidity , mortality , and healthcare expenditures . african americans are at increased risk for developing hf and experience worse outcomes posthf development . beyond angiotensinconverting enzyme inhibitors ( aceis ) or angiotensin receptor blockers ( arbs ) , betablockers , and aldosterone antagonists use in patients with heart failure with reduced ejection fraction ( hfref ) , clinical trials have established incremental benefit with hydralazineisosorbide dinitrate ( hisdn ) therapy in african american hfref patients in terms of mortality , morbidity , and quality of life . in addition , data in non african american hf patients with hisdn are encouraging , with proven benefits in terms of remodeling and exercise tolerance , but not on hard outcomes such as mortality and morbidity . both the american college of cardiology / american heart association and heart failure society of america guidelines recommend hisdn as a part of standard therapy for selfidentified african american patients with hfref . in addition , these guidelines also recommend hisdn therapy to be considered in non african american patients with hfref who remain symptomatic despite optimized standard therapy . despite these guideline recommendations , there are very few contemporary studies regarding the utilization of hisdn in realworld practice . heart failure ( gwtghf ) program is a national quality improvement program designed to promote adherence to guidelinebased medical therapies . using data from gwtghf , we conducted an analysis to examine contemporary use of hisdn in the overall cohort of hfref patients and then specifically in selfidentified african american patients hospitalized with hfref , temporal trends in hisdn use over time , and patient and hospital factors associated with hisdn prescription . gwtghf is an ongoing prospective registry and quality improvement program initiated in january 2005 by the american heart association . participating hospitals include institutions from all regions of the united states and represent community hospitals as well as tertiarycare referral centers . the registry enrolls adults hospitalized with new or worsening hf as the primary diagnosis or with significant hf symptoms that develop during the hospitalization for which hf is the primary discharge diagnosis . data collected include demographic and clinical characteristics , comorbidities , previous therapies and interventions , laboratory values , pharmacological and nonpharmacological interventions , contraindications to evidencebased therapies , and inhospital outcomes . patient 's selfidentified race ( american indian or alaska native , asian , african american or black , native hawaiian or pacific islander , white , or unable to be determined ) and ethnicity ( hispanic , yes , no , or unable to be determined ) is registered . all participating institutions are required to comply with local regulatory and privacy guidelines and to obtain approval from their institutional review boards before participation . as data are primarily collected for quality improvement purposes , all sites are granted a waiver for informed consent under the common rule . internetbased system checks of data quality are performed to ensure their completeness and accuracy . outcome sciences , inc ( cambridge , ma ) , serves as the data collection and coordination center , and the duke clinical research institute ( durham , nc ) serves as the data analysis center under an agreement to analyze the aggregate deidentified data for research purposes . from april 1 , 2008 , through march 24 , 2012 , 122 395 patients admitted with hf were discharged from 207 hospitals participating in the gwtghf program . the use of hisdn prior to 2008 was collected differently and could not be directly compared , and hence the study period was 2008 onward . the use of hisdn was defined as patients prescribed either ( 1 ) the fixed dose combination of hisdn or ( 2 ) hydralazine+any formulation of nitrates ( ie , mononitrate or dinitrate ) . the following groups of patients were excluded from the study analysis : patients with missing data on ejection fraction ( n=3868 ) or ejection fraction > 40% ( n=63 905 ) ; patients with unknown race or ethnicity ( n=2288 ) ; those with documented contraindication to hisdn therapy ( n=2508 ) ; those who were comfort care only ( n=2974 ) ; and those who died , left against medical advice , transferred to another hospital , discharged to hospice , or had missing information on discharge destination ( n=2954 ) . the final study population thus included 43 898 patients with hfref from 195 hospitals who were discharged home without medical contraindications to hisdn therapy . the primary outcome was prescription rate of hisdn in hfref patients at discharge in ( 1 ) african americans patients and ( 2 ) the entire study population irrespective of race / ethnicity . we also studied the temporal trends in the use of hisdn over the study period ( 20082012 ) , variation in use of hisdn across hospitals , patterns of use at various times ( eg , newly started , discontinued or continued during hospitalization , or at discharge ) , and factors associated with its use . data were compared between patients prescribed and not prescribed hisdn using chisquare tests for categorical data and wilcoxon rank sum tests for continuous variables . percentages and means with standard deviations ( sds ) were reported for categorical and normally distributed continuous variables , and medians with 25th and 75th percentiles were reported for nonnormally distributed continuous variables . cochranmantelhaenzel row mean scores statistics were used to test the trend of prescription over the study period . a multivariable logistic regression model was used to examine factors associated with hisdn use among eligible patients ( in overall population as well as among african american patients ) . the candidate variables in the initial models included demographics , medical history , vital signs , insurance status , hospital teaching status , region , and number of beds . also performed was a similar model including the variables mentioned above and use of hisdn before admission or during hospitalization as well . some patient variables , including body mass index , weight , and laboratory values and hospitallevel variables including capability of percutaneous coronary intervention , surgery , and heart transplant had > 10% to 20% missing data and thus were not included in the multivariate model . the patient characteristic variables included in the model had < 5% missing data , and their missing data were imputed to median value for continuous variables and dominant level for categorical variables . hospital region data were complete , and there was 2% missing data on teaching status and number of beds , and these missing data were excluded from the multivariable analysis . all tests were 2sided , and because of the large cohort studied , p<0.01 was considered statistically significant . analysis was performed using sas version 9.2 ( sas institute inc , cary , nc ) . gwtghf is an ongoing prospective registry and quality improvement program initiated in january 2005 by the american heart association . participating hospitals include institutions from all regions of the united states and represent community hospitals as well as tertiarycare referral centers . the registry enrolls adults hospitalized with new or worsening hf as the primary diagnosis or with significant hf symptoms that develop during the hospitalization for which hf is the primary discharge diagnosis . data collected include demographic and clinical characteristics , comorbidities , previous therapies and interventions , laboratory values , pharmacological and nonpharmacological interventions , contraindications to evidencebased therapies , and inhospital outcomes . patient 's selfidentified race ( american indian or alaska native , asian , african american or black , native hawaiian or pacific islander , white , or unable to be determined ) and ethnicity ( hispanic , yes , no , or unable to be determined ) is registered . all participating institutions are required to comply with local regulatory and privacy guidelines and to obtain approval from their institutional review boards before participation . as data are primarily collected for quality improvement purposes , all sites are granted a waiver for informed consent under the common rule . internetbased system checks of data quality are performed to ensure their completeness and accuracy . outcome sciences , inc ( cambridge , ma ) , serves as the data collection and coordination center , and the duke clinical research institute ( durham , nc ) serves as the data analysis center under an agreement to analyze the aggregate deidentified data for research purposes . from april 1 , 2008 , through march 24 , 2012 , 122 395 patients admitted with hf were discharged from 207 hospitals participating in the gwtghf program . the use of hisdn prior to 2008 was collected differently and could not be directly compared , and hence the study period was 2008 onward . the use of hisdn was defined as patients prescribed either ( 1 ) the fixed dose combination of hisdn or ( 2 ) hydralazine+any formulation of nitrates ( ie , mononitrate or dinitrate ) . the following groups of patients were excluded from the study analysis : patients with missing data on ejection fraction ( n=3868 ) or ejection fraction > 40% ( n=63 905 ) ; patients with unknown race or ethnicity ( n=2288 ) ; those with documented contraindication to hisdn therapy ( n=2508 ) ; those who were comfort care only ( n=2974 ) ; and those who died , left against medical advice , transferred to another hospital , discharged to hospice , or had missing information on discharge destination ( n=2954 ) . the final study population thus included 43 898 patients with hfref from 195 hospitals who were discharged home without medical contraindications to hisdn therapy . the primary outcome was prescription rate of hisdn in hfref patients at discharge in ( 1 ) african americans patients and ( 2 ) the entire study population irrespective of race / ethnicity . we also studied the temporal trends in the use of hisdn over the study period ( 20082012 ) , variation in use of hisdn across hospitals , patterns of use at various times ( eg , newly started , discontinued or continued during hospitalization , or at discharge ) , and factors associated with its use . data were compared between patients prescribed and not prescribed hisdn using chisquare tests for categorical data and wilcoxon rank sum tests for continuous variables . percentages and means with standard deviations ( sds ) were reported for categorical and normally distributed continuous variables , and medians with 25th and 75th percentiles were reported for nonnormally distributed continuous variables . cochranmantelhaenzel row mean scores statistics were used to test the trend of prescription over the study period . a multivariable logistic regression model was used to examine factors associated with hisdn use among eligible patients ( in overall population as well as among african american patients ) . the candidate variables in the initial models included demographics , medical history , vital signs , insurance status , hospital teaching status , region , and number of beds . also performed was a similar model including the variables mentioned above and use of hisdn before admission or during hospitalization as well . some patient variables , including body mass index , weight , and laboratory values and hospitallevel variables including capability of percutaneous coronary intervention , surgery , and heart transplant had > 10% to 20% missing data and thus were not included in the multivariate model . the patient characteristic variables included in the model had < 5% missing data , and their missing data were imputed to median value for continuous variables and dominant level for categorical variables . hospital region data were complete , and there was 2% missing data on teaching status and number of beds , and these missing data were excluded from the multivariable analysis . all tests were 2sided , and because of the large cohort studied , p<0.01 was considered statistically significant . analysis was performed using sas version 9.2 ( sas institute inc , cary , nc ) . irrespective of race / ethnicity , overall 43 898 patients from 195 hospitals treated with hfref were potentially eligible for hisdn therapy at discharge . baseline demographic and clinical characteristics of patients stratified by prescription of hisdn at the time of discharge are shown in table 1 . patients who received hisdn at discharge were younger , more likely to be male and african american , appeared to have more advanced disease , and were more likely to have comorbidities including history of chronic obstructive pulmonary disease , diabetes mellitus , hyperlipidemia , hypertension , peripheral vascular disease , stroke , anemia , renal insufficiency , chronic dialysis , lower heart rate , and higher systolic blood pressure . even more importantly , patients who received hisdn were more likely to be treated with betablockers , aldosterone antagonists , calcium channel blockers , and diuretics and less likely to be treated with aceis before admission . patient characteristics by hydralazineisosorbide dinitrate use at hospital discharge si conversion factors : to convert bun values to millimoles per liter , multiply by 0.357 ; to convert creatinine values to micromoles per liter , multiply by 88.4 . sd indicates standard deviation ; copd , chronic obstructive pulmonary disease ; icd , implantable cardioverterdefibrillator ; crtd / p , cardiac synchronization therapydefibrillator / pacemaker ; pci , percutaneous coronary intervention ; cabg , coronary artery bypass graft ; bun , blood urea nitrogen ; bnp , braintype natriuretic peptide ; ace , angiotensinconverting enzyme ; arbs , angiotenin receptor blockers ; ccbs , calcium channel blockers . hisdn use at the time of discharge differed by race / ethnic group , being more frequent in african american patients ( 2500 of 11 185 [ 22.4% ] among african american patients ; 2409 of 26 922 [ 9.0% ] among white patients , 414 of 3824 [ 10.8% ] among hispanic patients , and 192 of 1967 [ 9.8% ] among other race / ethnic group patients ) . in the entire cohort , the discharge prescription of hisdn was more common among those patients with hisdn from before admission ( 1469 of 1818 , 80.8% ) . this is in comparison with discharge prescription of hisdn in patients without hisdn before admission ( 4046 of 42 080 , 9.6% ) . in the african american subgroup , the discharge prescription of hisdn was 84.6% versus 17.3% in those with versus without hisdn before admission . table 2 shows hospital characteristics , american college of cardiology / american heart association performance measures , and gwtghf quality measures stratified by hisdn use . patients discharged with hisdn were more likely to be treated at academic medical centers , hospitals with surgical capabilities , and those with a greater number of beds . hospital characteristics and adherence to performance and quality measures by hydralazineisosorbide dinitrate use at hospital discharge ptca indicates percutaneous transluminal coronary angioplasty ; ace , angiotensinconverting enzyme ; sbp , systolic blood pressure ; dbp , diastolic blood pressure ; icd , implantable cardioverterdefibrillator ; ef , ejection fraction . * diet , medications , activity level , follow up appointment , weight monitoring , what to do if symptoms worsen . at the time of discharge , the mean rate of hisdn use in 167 hospitals with 10 eligible patients was 12.1% , with a median ( 25th , 75th percentiles ) of 9.6% ( 4.4% , 15.1% ) ( figure 1 ) . among 90 hospitals with 10 african american eligible patients , mean hisdn rate among all patients was 13.6% , with a median ( 25th , 75th percentiles ) of 11.6% ( 7.7% , 17.2% ) ( figure 2 ) and mean rate of 21.0% with a median ( 25th , 75th percentiles ) of 19.2% ( 12.1% , 26.3% ) among african american patients . hydralazineisosorbide dinitrate ( hisdn ) use in african american patients in hospitals with 10 selfidentified african american patients . table 3 shows the patterns of use at the time of admission , hospitalization , and discharge . of the patients already on hisdn before hospital admission , 61.3% had hisdn continued through the entire hospitalization , 19.5% had it stopped during hospitalization but restarted at discharge , whereas 11.6% had hisdn stopped at discharge . among african american patients on hisdn before hospitalization , 63.7% had it continued through discharge , whereas 8.9% had it discontinued at discharge . among patients not on hisdn at the time of admission , only 3.6% had hisdn initiated at the time of discharge in the entire cohort ; among african american patients , this figure was 6.5% . table 4 shows the patterns of hisdn use among hf patients with documented contraindication / intolerance to aceis / arbs . the percentage of african american hfref patients with documented contraindication / intolerance to aceis / arbs who were not receiving hisdn before hospital admission was 85.5% . in this patient group , hisdn therapy was initiated in 25.3% during hospitalization and started in another 11.8% of patients at the time of hospital discharge . . patterns of hydralazineisosorbide dinitrate use in patients with documented contraindication / intolerance to aceis / arbs aceis indicates angiotensionconverting enzyme inhibitors ; arbs , angiotension receptor blockers ; hisdn , hydralazineisosorbide dinitrate . table 5 demonstrates independent patient and hospital characteristics associated with hisdn prescription . in the overall population , factors associated with hisdn use were younger age , male sex , african american / hispanic race , and history of diabetes mellitus , hypertension , implantable cardioverter defibrillator implantation , heart failure , anemia , renal insufficiency , higher systolic blood pressure , and lower heart rate . on the other hand , patients with a history of smoking and on chronic dialysis had lower use of hisdn at discharge . in african american patients , factors associated with hisdn use were similar except that being uninsured was associated with lower use , and history of chronic obstructive pulmonary disease was associated with higher use ( table 6 ) . furthermore , when prior use of hisdn before discharge ( either before admission or during hospitalization ) was included in the model for overall population , age , history of hypertension , and chronic dialysis were no longer significantly associated factors , and the strength of association decreased for those other significant characteristics for the overall population . patient and hospital factors associated with hydralazineisosorbide dinitrate use in overall cohort candidate variables in the model included demographics ( age , sex , race ) , insurance status , medical history ( atrial fibrillation or flutter , chronic obstructive pulmonary disease , diabetes , dyslipidemia , hypertension , peripheral vascular disease , stroke , implantable cardioverter defibrillator implantation , previous history of heart failure , anemia , pacemaker , dialysis , renal insufficiency , depression , ischemic etiology of heart failure , smoker ) , vital signs ( systolic bp and heart rate ) , hospital characteristics ( region , teaching status and number of beds ) . ci indicates confidence interval ; icd , implantable cardioverterdefibrillator ; bp , blood pressure . patient and hospital factors associated with hydralazineisosorbide dinitrate use in selfidentified african american patients meeting guideline criteria candidate variables in the model included demographics ( age , sex ) , insurance status , medical history ( atrial fibrillation or flutter , chronic obstructive pulmonary disease , diabetes , dyslipidemia , hypertension , peripheral vascular disease , stroke , implantable cardioverterdefibrillator implantation , history of heart failure , anemia , pacemaker , dialysis , renal insufficiency , depression , ischemic etiology of heart failure , smoker ) , vital signs ( systolic bp and heart rate ) , hospital characteristics ( region , teaching status , and number of beds ) . ci indicates confidence interval ; copd , chronic obstructive pulmonary disease ; icd , implantable cardioverterdefibrillator ; bp , blood pressure . hisdn prescription increased over 4 years in the overall population from 10.1% in 2008 to 13.3% in 20112012 ( p<0.0001 ) . this trend was significant in both african american ( 16.6% to 25.0% , p<0.0001 ) and white ( 7.2% to 9.3% , p=0.0004 ) patients , whereas statistically nonsignificant trends were noted between hispanic and other race / ethnic groups . trends in the use of hydralazineisosorbide dinitrate ( hisdn ) at discharge in eligible patients from 2008 to 2012 . both the american college of cardiology / american heart association and the heart failure society of america hf management guidelines recommend the use of a fixeddose combination of hisdn added to a standard medical regimen for hf , including aceis and betablockers , in selfidentified african american patients with symptomatic hfref ( class i , level of evidence b ) . african american patients who remain symptomatic on optimized medical therapy ( class iib , level of evidence c ) . in this cohort of > 40 000 patients with hfref , we demonstrate that < 15% of the overall cohort and < 25% of the eligible african american patients were prescribed hisdn therapy at the time of discharge . prescription of hisdn varied at discharge widely across hospitals and according to patient characteristics . when compared with reports from prior registries , the findings of this study suggest higher rates of treatment with hisdn . reports from the organized program to initiate lifesaving treatment in hospitalized patients with heart failure registry from 2003 to 2004 showed that only 4.5% of african american patients and 2.6% of white patients hospitalized with hf were discharged with prescriptions for hisdn . in addition , improving evidencebased care for heart failure in outpatient cardiology practices registry data , which studied the use of evidencebased medical therapy in outpatient cardiology practices , demonstrated that the mean prescription rate for hisdn was only 7.3% in african american patients with hfref . together , these findings suggest that the use of hisdn in eligible patients with hfref has improved over time but still is low during hospitalization , low in the transition from the hospital to the outpatient setting , and low during longitudinal followup in outpatient practices . the small but statistically significant increase in the use of hisdn over the study period among american americans could potentially be explained by participation in the gwtghf quality improvement registry among other factors . although the african american heart failure trial proved the effectiveness of hisdn in selfidentified african american patients , there has been greater uncertainty regarding the efficacy of hisdn in other race / ethnic groups . mullen et al demonstrated that hisdn when added to aceis / arbs was associated with a favorable hemodynamic profile and longterm clinical outcomes in hfref patients with acute decompensated hf , regardless of race or ethnicity . however , prospective randomized , doubleblind , placebocontrolled outcome studies with hisdn added to other contemporary hf therapies are lacking in non african american patients with hfref . thus , the usefulness of routine use of hisdn in non african patients with hfref is far from certain . in our study , when hisdn use was stratified among different racial / ethnic groups , the prescription of hisdn at the time of discharge as expected was lower for hispanic patients , white patients , and other race / ethnic groups compared with african american patients . chronic renal failure was identified as 1 of the most important predictors of hisdn use among african american patients with hfref ( 2.33 , adjusted or , 2.02 to 2.69 ; p<0.0001 ) as well as the overall cohort . this suggests that hisdn is being applied in patients who were or are perceived to be intolerant to aceis . it is also notable that patients who were prescribed hisdn were less likely to receive aceis at discharge . although the prescription of hisdn increased over time in gwtghf , its use among african american hfref patients was unacceptably low considering the conclusive clinical trial evidence demonstrating substantial reductions in allcause mortality and hospitalizations with hisdn use . more importantly , our results indicate that the prescription of hisdn at discharge was significantly higher in patients already on hisdn before the time of admission , whereas in patients without prior hisdn , only 8.5% had this therapy initiated during the hospitalization , and 3.6% had this therapy initiated at the time of discharge . these data demonstrate the reluctance to initiate hisdn therapy in eligible patients as opposed to continuing the therapy in patients already on hisdn . however , data with multiple drugs in both patients with hf and patients with acute myocardial infarction demonstrate that by far the best predictor of longterm adherence with therapy is prescription at the time of hospital discharge . although there have been no specific clinical trials performed with hisdn therapy among hf patients hospitalized with hf , the cumulative clinical trials and observational data suggest that hospitalization provides a potential opportunity to improve adherence with this therapy . if this can not be achieved during the hospitalization , a mandatory followup visit with instructions to consider adding hisdn therapy in african american patients with hfref may be warranted . lack of monitoring of hisdn use , because it is not an hf performance measure selected by the joint commission / centers for medicare and medicaid services or the american college of cardiology / american heart association , could partially explain its limited uptake in clinical practice . despite a mean systolic blood pressure of > 120 mm hg in the treatment arm of the african american heart failure trial , this along with other side effects , which may be more prominent in the realworld population , which tends to be older , has multiple comorbidities , and is on polypharmacy , could also contribute to diminished use of hisdn in practices . in addition , many hfref patients , especially those with severely depressed ejection fraction , tend to have low baseline blood pressure , especially when receiving other guidelinesrecommended therapy , which may deter providers from prescribing hisdn because of concern for hypotension many hf patients have erectile dysfunction and desire to use medications like sildenafil , which limits the concomitant use of nitrate therapy . in the case of branded fixeddose combination of hisdn ( bidil ) , cost may be an important consideration and may explain the low use of hisdn in the uninsured african american population . finally , the use of hisdn was significantly higher in patients also receiving other evidencebased therapies , raising the possibility of increased compliance in general with all therapies if further education were imparted to providers about hf care in general . incorporation of some of these interventions in clinical practice , including , for example , hisdn as a core performance measures , appropriate documentation of contraindication of its use , further educating primary care physicians and cardiologists , and other interventions , might help to improve prescription patterns with hisdn in clinical practice . regardless of these issues , there remains a substantial gap between guideline recommendations and implementation of clinical use of hisdn in african american patients with hfref . the data collection was dependent on the accuracy and completeness of data abstraction from medical chart review , particularly because eligibility for care metrics is based on documentation . the doses of hydralazine and nitrates that patients were discharged home on were not recorded . we did not have information about why hisdn was discontinued or not started in the absence of documented contraindications or intolerance . this should be evaluated in future studies . because our data set also did not include longitudinal followup , a portion of eligible patients may have been started on hisdn as outpatients , underestimating its real use . however , previous data suggest that if a medication is not started at the time of discharge , the subsequent new prescription rate in the outpatient setting for aceis and betablockers is very low . with the large number of patients studied , some statistically significant differences may be of questionable clinical relevance and should be interpreted with caution . finally , gwtghf hospitals were selfselected and may not be representative of all hospitals in the united states . the data collection was dependent on the accuracy and completeness of data abstraction from medical chart review , particularly because eligibility for care metrics is based on documentation . the doses of hydralazine and nitrates that patients were discharged home on were not recorded . we did not have information about why hisdn was discontinued or not started in the absence of documented contraindications or intolerance . this should be evaluated in future studies . because our data set also did not include longitudinal followup , a portion of eligible patients may have been started on hisdn as outpatients , underestimating its real use . however , previous data suggest that if a medication is not started at the time of discharge , the subsequent new prescription rate in the outpatient setting for aceis and betablockers is very low . with the large number of patients studied , some statistically significant differences may be of questionable clinical relevance and should be interpreted with caution . finally , gwtghf hospitals were selfselected and may not be representative of all hospitals in the united states . hydralazineisosorbide dinitrate use in eligible african american patients with hfref remains very low in realworld practice despite clinical trial evidence and incorporation of these data into recommendations by multiple professional guidelines for many years . although its use has increased over time from 2008 through 2011 , it has nevertheless remained < 25% , even in african american patients with hfref . given the substantial morbidity and mortality faced by patients with hfref and the established efficacy of hisdn among african american patients , aggressive measures to facilitate adherence to hisdn

backgroundhydralazineisosorbide dinitrate ( hisdn ) therapy is recommended for african american patients with moderate to severe heart failure with reduced ejection fraction ( < 40% ) ( hfref ) , but use , temporal trends , and clinical characteristics associated with hisdn therapy in clinical practice are unknown.methods and resultsan observational analysis of 54 622 patients admitted with hfref and discharged home from 207 hospitals participating in the get with the guidelines heart failure registry from april 2008 to march 2012 was conducted to assess prescription , trends , and predictors of use of hisdn among eligible patients . among 11 185 african american patients eligible for hisdn therapy , only 2500 ( 22.4% ) received hisdn therapy at discharge . in the overall eligible population , 5115 of 43 498 ( 12.6% ) received hisdn at discharge . treatment rates increased over the study period from 16% to 24% among african americans and from 10% to 13% among the entire hfref population . in a multivariable model , factors associated with hisdn use among the entire cohort included younger age ; male sex ; african american / hispanic ethnicity ; and history of diabetes , hypertension , anemia , renal insufficiency , higher systolic blood pressure , and lower heart rate . in african american patients , these factors were similar ; in addition , being uninsured was associated with lower use.conclusionsoverall , few potentially eligible patients with hfref are treated with hisdn , and among africanamericans fewer than onefourth of eligible patients received guidelinerecommended hisdn therapy . improved ways to facilitate use of hisdn therapy in african american patients with hfref are needed .

Introduction Methods Data Collection Study Population Outcome and Definitions Statistical Analysis Results Discussion Limitations Conclusions

beyond angiotensinconverting enzyme inhibitors ( aceis ) or angiotensin receptor blockers ( arbs ) , betablockers , and aldosterone antagonists use in patients with heart failure with reduced ejection fraction ( hfref ) , clinical trials have established incremental benefit with hydralazineisosorbide dinitrate ( hisdn ) therapy in african american hfref patients in terms of mortality , morbidity , and quality of life . in addition , data in non african american hf patients with hisdn are encouraging , with proven benefits in terms of remodeling and exercise tolerance , but not on hard outcomes such as mortality and morbidity . both the american college of cardiology / american heart association and heart failure society of america guidelines recommend hisdn as a part of standard therapy for selfidentified african american patients with hfref . in addition , these guidelines also recommend hisdn therapy to be considered in non african american patients with hfref who remain symptomatic despite optimized standard therapy . using data from gwtghf , we conducted an analysis to examine contemporary use of hisdn in the overall cohort of hfref patients and then specifically in selfidentified african american patients hospitalized with hfref , temporal trends in hisdn use over time , and patient and hospital factors associated with hisdn prescription . data collected include demographic and clinical characteristics , comorbidities , previous therapies and interventions , laboratory values , pharmacological and nonpharmacological interventions , contraindications to evidencebased therapies , and inhospital outcomes . from april 1 , 2008 , through march 24 , 2012 , 122 395 patients admitted with hf were discharged from 207 hospitals participating in the gwtghf program . the use of hisdn prior to 2008 was collected differently and could not be directly compared , and hence the study period was 2008 onward . the following groups of patients were excluded from the study analysis : patients with missing data on ejection fraction ( n=3868 ) or ejection fraction > 40% ( n=63 905 ) ; patients with unknown race or ethnicity ( n=2288 ) ; those with documented contraindication to hisdn therapy ( n=2508 ) ; those who were comfort care only ( n=2974 ) ; and those who died , left against medical advice , transferred to another hospital , discharged to hospice , or had missing information on discharge destination ( n=2954 ) . the final study population thus included 43 898 patients with hfref from 195 hospitals who were discharged home without medical contraindications to hisdn therapy . the primary outcome was prescription rate of hisdn in hfref patients at discharge in ( 1 ) african americans patients and ( 2 ) the entire study population irrespective of race / ethnicity . we also studied the temporal trends in the use of hisdn over the study period ( 20082012 ) , variation in use of hisdn across hospitals , patterns of use at various times ( eg , newly started , discontinued or continued during hospitalization , or at discharge ) , and factors associated with its use . cochranmantelhaenzel row mean scores statistics were used to test the trend of prescription over the study period . a multivariable logistic regression model was used to examine factors associated with hisdn use among eligible patients ( in overall population as well as among african american patients ) . some patient variables , including body mass index , weight , and laboratory values and hospitallevel variables including capability of percutaneous coronary intervention , surgery , and heart transplant had > 10% to 20% missing data and thus were not included in the multivariate model . data collected include demographic and clinical characteristics , comorbidities , previous therapies and interventions , laboratory values , pharmacological and nonpharmacological interventions , contraindications to evidencebased therapies , and inhospital outcomes . from april 1 , 2008 , through march 24 , 2012 , 122 395 patients admitted with hf were discharged from 207 hospitals participating in the gwtghf program . the use of hisdn prior to 2008 was collected differently and could not be directly compared , and hence the study period was 2008 onward . the following groups of patients were excluded from the study analysis : patients with missing data on ejection fraction ( n=3868 ) or ejection fraction > 40% ( n=63 905 ) ; patients with unknown race or ethnicity ( n=2288 ) ; those with documented contraindication to hisdn therapy ( n=2508 ) ; those who were comfort care only ( n=2974 ) ; and those who died , left against medical advice , transferred to another hospital , discharged to hospice , or had missing information on discharge destination ( n=2954 ) . the final study population thus included 43 898 patients with hfref from 195 hospitals who were discharged home without medical contraindications to hisdn therapy . the primary outcome was prescription rate of hisdn in hfref patients at discharge in ( 1 ) african americans patients and ( 2 ) the entire study population irrespective of race / ethnicity . we also studied the temporal trends in the use of hisdn over the study period ( 20082012 ) , variation in use of hisdn across hospitals , patterns of use at various times ( eg , newly started , discontinued or continued during hospitalization , or at discharge ) , and factors associated with its use . cochranmantelhaenzel row mean scores statistics were used to test the trend of prescription over the study period . a multivariable logistic regression model was used to examine factors associated with hisdn use among eligible patients ( in overall population as well as among african american patients ) . some patient variables , including body mass index , weight , and laboratory values and hospitallevel variables including capability of percutaneous coronary intervention , surgery , and heart transplant had > 10% to 20% missing data and thus were not included in the multivariate model . irrespective of race / ethnicity , overall 43 898 patients from 195 hospitals treated with hfref were potentially eligible for hisdn therapy at discharge . baseline demographic and clinical characteristics of patients stratified by prescription of hisdn at the time of discharge are shown in table 1 . patients who received hisdn at discharge were younger , more likely to be male and african american , appeared to have more advanced disease , and were more likely to have comorbidities including history of chronic obstructive pulmonary disease , diabetes mellitus , hyperlipidemia , hypertension , peripheral vascular disease , stroke , anemia , renal insufficiency , chronic dialysis , lower heart rate , and higher systolic blood pressure . even more importantly , patients who received hisdn were more likely to be treated with betablockers , aldosterone antagonists , calcium channel blockers , and diuretics and less likely to be treated with aceis before admission . hisdn use at the time of discharge differed by race / ethnic group , being more frequent in african american patients ( 2500 of 11 185 [ 22.4% ] among african american patients ; 2409 of 26 922 [ 9.0% ] among white patients , 414 of 3824 [ 10.8% ] among hispanic patients , and 192 of 1967 [ 9.8% ] among other race / ethnic group patients ) . in the entire cohort , the discharge prescription of hisdn was more common among those patients with hisdn from before admission ( 1469 of 1818 , 80.8% ) . in the african american subgroup , the discharge prescription of hisdn was 84.6% versus 17.3% in those with versus without hisdn before admission . hospital characteristics and adherence to performance and quality measures by hydralazineisosorbide dinitrate use at hospital discharge ptca indicates percutaneous transluminal coronary angioplasty ; ace , angiotensinconverting enzyme ; sbp , systolic blood pressure ; dbp , diastolic blood pressure ; icd , implantable cardioverterdefibrillator ; ef , ejection fraction . at the time of discharge , the mean rate of hisdn use in 167 hospitals with 10 eligible patients was 12.1% , with a median ( 25th , 75th percentiles ) of 9.6% ( 4.4% , 15.1% ) ( figure 1 ) . among 90 hospitals with 10 african american eligible patients , mean hisdn rate among all patients was 13.6% , with a median ( 25th , 75th percentiles ) of 11.6% ( 7.7% , 17.2% ) ( figure 2 ) and mean rate of 21.0% with a median ( 25th , 75th percentiles ) of 19.2% ( 12.1% , 26.3% ) among african american patients . hydralazineisosorbide dinitrate ( hisdn ) use in african american patients in hospitals with 10 selfidentified african american patients . among african american patients on hisdn before hospitalization , 63.7% had it continued through discharge , whereas 8.9% had it discontinued at discharge . among patients not on hisdn at the time of admission , only 3.6% had hisdn initiated at the time of discharge in the entire cohort ; among african american patients , this figure was 6.5% . table 4 shows the patterns of hisdn use among hf patients with documented contraindication / intolerance to aceis / arbs . table 5 demonstrates independent patient and hospital characteristics associated with hisdn prescription . in the overall population , factors associated with hisdn use were younger age , male sex , african american / hispanic race , and history of diabetes mellitus , hypertension , implantable cardioverter defibrillator implantation , heart failure , anemia , renal insufficiency , higher systolic blood pressure , and lower heart rate . on the other hand , patients with a history of smoking and on chronic dialysis had lower use of hisdn at discharge . in african american patients , factors associated with hisdn use were similar except that being uninsured was associated with lower use , and history of chronic obstructive pulmonary disease was associated with higher use ( table 6 ) . furthermore , when prior use of hisdn before discharge ( either before admission or during hospitalization ) was included in the model for overall population , age , history of hypertension , and chronic dialysis were no longer significantly associated factors , and the strength of association decreased for those other significant characteristics for the overall population . patient and hospital factors associated with hydralazineisosorbide dinitrate use in overall cohort candidate variables in the model included demographics ( age , sex , race ) , insurance status , medical history ( atrial fibrillation or flutter , chronic obstructive pulmonary disease , diabetes , dyslipidemia , hypertension , peripheral vascular disease , stroke , implantable cardioverter defibrillator implantation , previous history of heart failure , anemia , pacemaker , dialysis , renal insufficiency , depression , ischemic etiology of heart failure , smoker ) , vital signs ( systolic bp and heart rate ) , hospital characteristics ( region , teaching status and number of beds ) . patient and hospital factors associated with hydralazineisosorbide dinitrate use in selfidentified african american patients meeting guideline criteria candidate variables in the model included demographics ( age , sex ) , insurance status , medical history ( atrial fibrillation or flutter , chronic obstructive pulmonary disease , diabetes , dyslipidemia , hypertension , peripheral vascular disease , stroke , implantable cardioverterdefibrillator implantation , history of heart failure , anemia , pacemaker , dialysis , renal insufficiency , depression , ischemic etiology of heart failure , smoker ) , vital signs ( systolic bp and heart rate ) , hospital characteristics ( region , teaching status , and number of beds ) . hisdn prescription increased over 4 years in the overall population from 10.1% in 2008 to 13.3% in 20112012 ( p<0.0001 ) . trends in the use of hydralazineisosorbide dinitrate ( hisdn ) at discharge in eligible patients from 2008 to 2012 . both the american college of cardiology / american heart association and the heart failure society of america hf management guidelines recommend the use of a fixeddose combination of hisdn added to a standard medical regimen for hf , including aceis and betablockers , in selfidentified african american patients with symptomatic hfref ( class i , level of evidence b ) . in this cohort of > 40 000 patients with hfref , we demonstrate that < 15% of the overall cohort and < 25% of the eligible african american patients were prescribed hisdn therapy at the time of discharge . reports from the organized program to initiate lifesaving treatment in hospitalized patients with heart failure registry from 2003 to 2004 showed that only 4.5% of african american patients and 2.6% of white patients hospitalized with hf were discharged with prescriptions for hisdn . in addition , improving evidencebased care for heart failure in outpatient cardiology practices registry data , which studied the use of evidencebased medical therapy in outpatient cardiology practices , demonstrated that the mean prescription rate for hisdn was only 7.3% in african american patients with hfref . together , these findings suggest that the use of hisdn in eligible patients with hfref has improved over time but still is low during hospitalization , low in the transition from the hospital to the outpatient setting , and low during longitudinal followup in outpatient practices . the small but statistically significant increase in the use of hisdn over the study period among american americans could potentially be explained by participation in the gwtghf quality improvement registry among other factors . although the african american heart failure trial proved the effectiveness of hisdn in selfidentified african american patients , there has been greater uncertainty regarding the efficacy of hisdn in other race / ethnic groups . however , prospective randomized , doubleblind , placebocontrolled outcome studies with hisdn added to other contemporary hf therapies are lacking in non african american patients with hfref . thus , the usefulness of routine use of hisdn in non african patients with hfref is far from certain . in our study , when hisdn use was stratified among different racial / ethnic groups , the prescription of hisdn at the time of discharge as expected was lower for hispanic patients , white patients , and other race / ethnic groups compared with african american patients . chronic renal failure was identified as 1 of the most important predictors of hisdn use among african american patients with hfref ( 2.33 , adjusted or , 2.02 to 2.69 ; p<0.0001 ) as well as the overall cohort . although the prescription of hisdn increased over time in gwtghf , its use among african american hfref patients was unacceptably low considering the conclusive clinical trial evidence demonstrating substantial reductions in allcause mortality and hospitalizations with hisdn use . more importantly , our results indicate that the prescription of hisdn at discharge was significantly higher in patients already on hisdn before the time of admission , whereas in patients without prior hisdn , only 8.5% had this therapy initiated during the hospitalization , and 3.6% had this therapy initiated at the time of discharge . these data demonstrate the reluctance to initiate hisdn therapy in eligible patients as opposed to continuing the therapy in patients already on hisdn . if this can not be achieved during the hospitalization , a mandatory followup visit with instructions to consider adding hisdn therapy in african american patients with hfref may be warranted . lack of monitoring of hisdn use , because it is not an hf performance measure selected by the joint commission / centers for medicare and medicaid services or the american college of cardiology / american heart association , could partially explain its limited uptake in clinical practice . despite a mean systolic blood pressure of > 120 mm hg in the treatment arm of the african american heart failure trial , this along with other side effects , which may be more prominent in the realworld population , which tends to be older , has multiple comorbidities , and is on polypharmacy , could also contribute to diminished use of hisdn in practices . in addition , many hfref patients , especially those with severely depressed ejection fraction , tend to have low baseline blood pressure , especially when receiving other guidelinesrecommended therapy , which may deter providers from prescribing hisdn because of concern for hypotension many hf patients have erectile dysfunction and desire to use medications like sildenafil , which limits the concomitant use of nitrate therapy . in the case of branded fixeddose combination of hisdn ( bidil ) , cost may be an important consideration and may explain the low use of hisdn in the uninsured african american population . incorporation of some of these interventions in clinical practice , including , for example , hisdn as a core performance measures , appropriate documentation of contraindication of its use , further educating primary care physicians and cardiologists , and other interventions , might help to improve prescription patterns with hisdn in clinical practice . regardless of these issues , there remains a substantial gap between guideline recommendations and implementation of clinical use of hisdn in african american patients with hfref . hydralazineisosorbide dinitrate use in eligible african american patients with hfref remains very low in realworld practice despite clinical trial evidence and incorporation of these data into recommendations by multiple professional guidelines for many years . although its use has increased over time from 2008 through 2011 , it has nevertheless remained < 25% , even in african american patients with hfref . given the substantial morbidity and mortality faced by patients with hfref and the established efficacy of hisdn among african american patients , aggressive measures to facilitate adherence to hisdn

in the framework of enhanced and flexible materials for optoelectronics and energy applications , organic semiconductors have been largely exploited due to their favorable charge - carrier and emission properties , and easy processing by solution methods , printing , and soft lithographies . current applications include , but are not limited to , light - emitting devices , photovoltaic cells , field effect transistors , and lasers . the optoelectronic properties of organic semiconductors are mainly determined by the behavior of excitons , quasi - particles formed by an electron tightly bounded to a hole , which can be excited either optically or electrically . such excitations are typically localized on the scale of a single or few molecules , and their migration and recombination dynamics determines both light - emission and light - harvesting properties . more specifically , short migration lengths are preferred for high fluorescence yield because this decreases the possibility of quenching excitons by encountering trap states and concentration quenching . moreover , in photovoltaics long exciton migration lengths might favor exciton dissociation at the interface between organic semiconductors and electron / hole acceptors . therefore , understanding and tailoring how excitation energy travels in organic semiconductors is an issue for improving device performance . in fact , most current applications rely on materials with randomly oriented conjugated chains , which might limit the yield of photogenerated charges or photoluminescence . in such systems , electronic energy transfer directs the excitation toward lower energy chromophores , typically associated with low - emissive aggregates . although several studies on isolated conjugated polymer chains suggested that a route to improve charge mobilities and emission yield is the development of effective processing methods which can force macromolecules to adopt extended and elongated conformations , little has been done in this direction . indeed , individual conjugated polymer chains are intrinsically anisotropic and any high - performing solid state structure must inherit such a property . guest systems by using mesoporous silica templates , which led to fast intrachain electronic energy transfer , highly polarized emission , and low - threshold amplified spontaneous emission . however , these results were limited to templates with a pore size smaller than 5 nm , hosting isolated polymer chains , whereas properties similar to those of spin - cast films are found as soon as the channel diameter increases above 5 nm . in other attempts , an alignment of specific , molecularly designed conjugated polymer chains was induced along an applied flow field , a method allowing for obtaining a three - order of magnitude faster hole mobility along the direction of chain alignment compared to the perpendicular one , as well as high absorption and emission dichroic ratios . poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene - vinylene ] ( meh - ppv ) has also been aligned using liquid crystalline hosts . while it was established that the conjugated chains were highly extended , the procedure is only possible for very dilute polymer - liquid crystal solutions . in another approach , a simple shear flow was exploited to induce an extension of the meh - ppv molecules in solution , using a coutte cell . a small increase of the polarization anisotropy was observed , together with a variation of the emission intensity and wavelength depending on the solution viscosity and solvents , which was attributed to different energy migration pathways activated by shear - induced modification of the molecular conformation . despite such efforts , solid state structures made of conjugated polymers are still far from the performances expected for extended chains . recently , micro- and nanofibers made by conjugated polymers have emerged as a novel class of nanostructured , solid state materials featuring enhanced properties compared to bulk or thin film samples . various approaches for producing polymer fibers are currently based on extensional flows or on other methods which induce extended and ordered chain configurations . as a consequence , improved charge mobilities , emission quantum yield , and polarized emission are observed in these systems . these nanostructures have already demonstrated potential as the active component of organic light - emitting diodes , solar cells , and transistors , and they can constitute a valuable benchmark system to tailor and control the fundamental properties of conjugated molecules in the solid state . here we report on fibers made of meh - ppv , realized by electrospinning . tailoring the degree of order of molecular chains through solution control is revealed by polarization spectroscopies and x - ray diffraction measurements . a comparison with thin films indicates that fibers composed of aligned and more extended polymer chains achieve a 5-fold enhancement of the emission quantum yield as well as emission polarized along the fiber axis , independent of excitation configuration . probe anisotropy measurements indicate that , in the ordered fibers , photogenerated excitons preferentially reorient along the fiber axis . we find that the time scale of the energy migration is of the order of that expected for enhanced interchain coupling . the meh - ppv nanofiber samples used in this work are shown in figure 1 . meh - ppv is chosen as a prototype conjugated polymer , whose optical properties have been investigated in different packing configurations of the macromolecules , from single molecules , to isolated and stretched individual chains , small aggregates , and thin films . uniaxially oriented arrays of fibers ( figure s1 ) are produced by using a rotating collector during electrospinning , and then they are collected onto quartz substrates and encapsulated in a photocurable polymer ( figure 1a , b ) to prevent photo - oxidation ( figure s2 ) . the encapsulation of the fibers in the resin also decreases the amount of reflected light , because of the reduced refractive index contrast with the conjugated polymer . the fibers have a ribbon shape , with an average ratio of their width and thickness of 2.5 ( figure 1c , d ) , and an average width of 300 nm . the rapid evaporation of the more volatile component ( chloroform ) of the liquid jet during the spinning process and the formation of a collapsing solid skin can be the origin of the ribbon shape . a flattening of the fibers upon substrate deposition , due to the presence of a fraction of slowly evaporating solvent ( dimethyl sulfoxide , dmso ) in the solution , may also occur . ( a b ) photographs of meh - ppv fibers encapsulated in a photocurable resin scale bars : ( c ) 30 m and ( d ) 5 m . inset scale bars : ( c ) 2 m and ( d ) 100 nm . the possibility of obtaining fibers with strongly anisotropic packing of the constituent polymer chains is related to the very high strain rate ( 10 s ) exerted in electrospinning , which typically induces molecular stretching and orientation in the jet , that is then retained in the fibers . in general , conjugated polymers are poorly viscoelastic , a property often associated with their semiflexible backbones , and the final degree of chain orientation depends on many factors . modeling of the conjugated polymer chain networks under elastic stretching evidenced that substantial axial extension is achieved within a few millimeters from the spinneret and that this depends on the volume fraction occupied by the polymer , , the applied electric field , e , the jet initial velocity , v0 , and the solution conductivity , k. typically , lower values of and v0 , and higher e and k are needed to increase the axial chain orientation . in our system , the presence of a nonsolvent ( see experimental section ) decreases the overall polymer volume fraction in the solution . moreover , increasing the nonsolvent content is expected to improve the solution conductivity as well , because of the 2 orders of magnitude higher electrical conductivity of dmso compared to chloroform . this approach is supported by comparing the molecular order of fibers produced from solutions with even slightly different nonsolvent content . figure 2a , b shows the polarized fourier transform infrared ( ftir ) spectra of meh - ppv fibers obtained with chloroform : dmso mixtures with volume ratios of 9:1 and 9:2 , respectively . the spectra show peaks typical of meh - ppv , such as the modes at 1040 cm ( ether c o c stretch ) , 1204 cm ( phenyl - oxygen stretch ) , 1415 cm , and 1500 cm ( c c ring stretch ) . in both samples , the absorption is maximized for incident light polarization parallel with the fiber longitudinal axis , which is indicative of alignment of the conjugated chains . in particular , the fibers obtained from solutions with higher content of dmso show a more pronounced difference of spectra measured with the incident light polarized parallel or perpendicular to their axis , meaning that a larger fraction of chain segments are oriented along the fiber length . this is highlighted in figure 2c , where the amplitude of the peak at 1500 cm is shown as a function of the angle , , formed by the incident polarization and the direction of fiber alignment ( figure s1a ) . this mode has a transition dipole moment forming an angle of about 9 with respect to the chain axis , and it can be therefore be exploited to investigate the degree of chain alignment . an enhanced chain alignment is highlighted by the ftir analysis upon increasing the dmso volume fraction up to 20% ( figure s3a e ) , whereas a further increase of the dmso content negatively affects both the polymer chain alignment and the stability of electrospinning . a decrease of chain alignment is found with dmso content in the range 2030% , while continuous solid state fibers can not be obtained for dmso volume fractions above 30% . overall , these and other results show that electrospinning may be exploited to finely tailor the orientation of conjugated chains in nanostructured materials . hereafter , fibers obtained by ( 9:1 ) and ( 9:2 ) content of dmso will be referred to as lower molecular order ( lmo ) and higher molecular order ( hmo ) samples , respectively . ( a b ) polarized ftir spectra of uniaxially aligned meh - ppv fibers spun from chloroform : dmso mixtures with a volume ratio of 9:1 ( a ) and 9:2 ( b ) . the spectra are obtained by using incident light with polarization parallel ( red continuous line ) or perpendicular ( black dashed line ) to the fiber alignment direction . the arrows indicate the mode at about 1500 cm , attributed to a c ( c ) amplitude of the transmission peak at 1500 cm vs the angle , , formed by the fiber axis and the incident polarization direction ( see figure s1 of the supporting information ) for fibers spun from chloroform : dmso mixtures with volume ratio of 9:1 ( circles ) and 9:2 ( squares ) . 0 corresponds to the direction of the incident light polarization parallel to the fiber axis . the molecular orientation and the structure of the hmo samples , free - standing bundles of uniaxially aligned fibers were investigated by scanning small - angle x - ray scattering ( saxs ) and wide - angle x - ray scattering ( waxs ) measurements . figure 3a shows the scanning saxs map obtained from a 4 4 mm sample area . the contrast variation in the map is related to the amount of material . the predominant color in the 2d map represents the orientation of the anisotropic saxs signal , and hence of the fibers , according to the color wheel reported in the inset of figure 3a . it follows that the greenish color is related to a basically horizontal orientation of the fibers ( i.e. , parallel to the x axis in the map ) . simultaneous ( a ) scanning saxs microscopy and ( b ) integrated waxs microscopy of a bundle of uniaxially aligned free - standing fibers . ( c ) top : azimuthal profiles along the rings of figure 3b at q 1 and q 1.5 , related to the c and a lattice parameters , respectively . bottom : azimuthal profiles along the rings of figure s4b at q 0.4 , q 1 , and q 1.5 , related to the b , c , and a lattice parameters , respectively ( data multiplied by a factor 2 for better clarity ) . ( d ) azimuthal profiles taken at q 0.4 for the b lattice parameter of the free - standing fibers , from 2d saxs patterns collected at 120 mm ( figure s4a ) and 250 mm ( not shown ) . ( e ) linear cuts taken from the waxs patterns of part b ( 28 mm detector distance ) and figure s4a ( 120 mm detector distance ) , in the directions parallel ( black line ) and perpendicular ( orange symbols ) to the fiber axis , and from the giwaxs pattern of figure s4b in the directions parallel ( black dots ) and perpendicular ( green triangles ) to the film surface . the in - plane profile has been multiplied by the factor 1.1 , in order to compensate for the larger absorption due to the larger beam path in the film . the waxs signal , collected simultaneously and averaged all over the investigated area , is shown in figure 3b . with this rotated by 90 relative to the orientation of figure 3a for graphic reasons , the qz = 0 direction ( i.e. , the qy axis ) in figure 3b corresponds to the y axis in figure 3a . the intensity modulation along the azimuthal angle for the two most intense diffraction rings is plotted in figure 3c , where the red and the blue ( top ) curves are related to the a and c parameters , respectively ( outer and inner rings in figure 3b ) . the azimuthal profiles were extracted from figure 3b , at q 1.5 and 1 , respectively , by integrating over a 0.2 q - range . it can be recognized in figure 3c that the intensity modulations of the a and c parameters are out of phase by 90 , indicating that they are partially aligned along orthogonal preferred orientations . by taking into account the direction of the orientations of the fibers as indicated by figure 3a , it can be concluded that the a and c parameters are preferentially oriented perpendicular and parallel to the fiber axis , respectively . moreover , figure 3d shows the azimuthal intensity modulation in the 0.30.5 q - range , corresponding to the periodicity of the b lattice parameter , which has been here reported in a separated plot because this signal was extracted from waxs patterns ( see figure s4a ) collected at a larger distance from the sample ( 120 or 250 mm as specified in figure 3d ) in order to access lower q - values , although with much lower intensity . notwithstanding the low signal - to - noise ratio , an overall intensity modulation along the azimuth can be recognized also for the b parameter , and well reproduced in both measurements as highlighted by the superimposed smoothed profiles , which is coherent with the azimuthal modulation of the a parameter in figure 3c . the b and a parameters are therefore expected to preferentially lie in the plane perpendicular to c , and hence to the fiber axis . the waxs pattern collected at 120 mm sample - to - detector distance is shown in figure s4a . in this case , a very thin layer of si powder ( from nist ) was deposited on the bundle of fibers , as an internal standard for calibration and correction of detector tilt . the preferred orientation is also reflected in the 1d radial profiles of figure 3e , resulting from the cuts taken along the horizontal and vertical directions ( i.e. , perpendicular and parallel to fibers , respectively ) in the 2d waxs pattern of figure 3b : the intensity ratio between the two diffraction peaks related to a and c is seen indeed to be reversed in the two cuts ( superimposed curves with symbols and continuous line ) . a significant fraction of randomly oriented crystalline domains is also expected throughout the fibers , based on the fairly small amplitude of the azimuthal intensity modulation . moreover , based on the calibration from figure s4a , and a gaussian fit , it was verified that no appreciable shift of peak positions and hence no variations of the related d - spacings occurred between in plane and out of plane directions . as a result , figure 3 indicates the presence of nanocrystals in meh - ppv electrospun fibers , with crystallographic axes preferentially oriented along the fiber ( c axis ) or perpendicular to the fiber ( a and b axes ) , determining a preferred orientation of the meh - ppv active subunits along the fiber axis . for comparison , meh - ppv films were studied by collecting the waxs signal in grazing incidence reflection geometry ( see e.g. the giwaxs pattern in figure s4b ) . by extracting linear cuts from figure s4b along the in - plane and out - of - plane directions ( 1d profiles with black spots and green triangles , respectively , in figure 3e ) , an intensity modulation results also in this case , as a difference in the ratios of diffraction peaks related to a and c. such a difference is more evident for the a parameter , which clearly features different diffraction intensities along the two directions . note that the in - plane profile has been multiplied by a factor 1.1 , in order to compensate for the larger absorption because of the larger beam path in the film . accordingly , in the azimuthal profiles extracted from figure s4b and reported in figure 3c ( bottom curves ) for comparison , a corrected intensity larger by the factor 1.1 should be considered , toward the angular interval extremes , in order to compare it with the out - of - plane intensity ( corresponding to the center of the curves ) . such a correction factor does not significantly affect the azimuthal trends for the a and b parameters ( red triangles and black line , respectively , in figure 3c ) , both featuring a clear intensity maximum in the out - of - plane direction . on the contrary , the basically isotropic intensity distribution observed for the c parameter ( blue circles ) would change to a slightly concave profile , suggesting a slight preferred orientation of the c axis parallel to the sample plane , and hence perpendicular to a. on the other hand , the b parameter also features a larger azimuthal intensity perpendicular to the sample plane . therefore , the presence of several differently oriented crystalline domains is expected , particularly with preferred orientation of either the a or the b axis perpendicular to the sample plane . overall , a lower degree of crystalline order is found in meh - ppv thin films compared to electrospun fibers . indeed , differently from fibers , crystalline domains in films develop independently , with random orientations as well as with the a or b axis preferentially oriented perpendicular to the film surface , as in a lamellar stacking , whereas the c axis of the domains lies preferentially in the sample plane , with random in - plane orientations . meh - ppv samples with varied degree of chain alignment are investigated by steady state absorption and photoluminescence ( pl ) spectroscopies . in figure 4a we compare the absorption spectra of spin - cast films and of hmo samples . as a general trend we observe a blue - shift of the absorption peak and a red - shift of the pl peak upon increasing chain alignment ( table 1 ) . interestingly , the absorption peak of the hmo fibers is comparable to the one measured in dilute solutions of meh - ppv in chloroform ( 494 nm ) , suggesting that in fibers the polymer chains adopt a conformation closer to that of a good solvent solution . furthermore , polarized absorption spectra acquired on arrays of uniaxially aligned hmo fibers show a relative red - shift of the absorption band for polarization of the incident light parallel to the fiber axis ( inset of figure 4a ) , which is indicative of a longer conjugation length associated with oriented chromophoric subunits . the observed spectral shifts are also compatible with an increasing degree of h - aggregation in more ordered samples , according to recent h- and j - aggregate models developed for conjugated polymers . in further support of this model , in the film and hmo fiber fluorescence spectra the intensity ratio between the ( 00 ) and ( 01 ) vibronic replica , rpl = i00/i01 , is less in the hmo fiber ( figure s5 ) . this ratio provides quantitative measurements of the h- or j - aggregation prevalence in conjugated polymers , being typically > 2 for dominant intrachain coupling ( j - aggregation ) , whereas values < 2 are indicative of interchain coupling ( h - aggregation ) . here we find a decrease of rpl from values in the range 1.41.7 for films to values of 1.31.5 and 11.3 for lmo and hmo fibers , respectively , consistent with increased interchain coupling and h - aggregate behavior in fibers . overall , from polarized ftir , x - ray scattering , and absorption spectroscopies we argue that during spinning a fraction of the active subchains are effectively stretched and oriented along the fiber axis , adopting a more extended chain conformation compared to amorphous films , with an increase of the associated conjugation length and a packing arrangement that supports interchain interactions with nearby subchains ( figure 4b , c ) . ( a ) absorption spectra of a meh - ppv spin - cast film ( continuous line ) and hmo fibers ( circles ) . the absorption spectrum of a dilute solution of meh - ppv in chloroform is shown for reference ( dashed line ) . inset : polarized absorption spectra of hmo fibers , acquired by incident light polarized parallel ( continuous line ) or perpendicular ( dashed line ) to the fiber axis . ( b c ) pictorial representations of the microscopic arrangement of individual conjugated polymer chains in nanofiber ( b ) and film ( c ) . , arrows indicate the c axis of the crystalline domains , whereas the a and b axes lie in a plane perpendicular to c. ( ) is the ratio between the pl intensity polarized parallel and perpendicular to the fiber axis , collected upon excitation with a laser polarized parallel with ( perpendicular to ) the fiber axis . the associated errors are obtained as the standard deviation of the distributions shown in figures 5 and 6 . ( ) is the spectral shift between the pl polarized parallel and perpendicular to the fiber axis , upon excitation with a laser polarized parallel with ( perpendicular to ) the fiber axis . more importantly , the fibers with more oriented polymer chains feature an increase of the pl quantum yield ( f ) by about a factor 5 compared to films ( table 1 ) . these results are in striking contrast to what is typically observed in conjugated polymers in the solid state , where a red - shift of the emission is associated with the formation of low - emissive aggregates . to investigate more in depth the origin of the enhanced and red - shifted emission , we perform a study of the polarization properties of a set of individual nanofibers by microphotoluminescence ( -pl ) . figures 5 and 6 display the polarized pl spectra of films and individual lmo and hmo fibers , acquired by exciting the fibers with a laser polarized either parallel with ( figure 5 ) or perpendicular to ( figure 6 ) their length . independent of excitation configuration , the emission from films is largely unpolarized , as is characteristic of systems with randomly oriented chains , while the emission from fibers is polarized along their length ( figure 5a c and 6a c ) . the degree of molecular alignment affects the amount of polarized light detected , since the ratio between the intensity of the pl polarized parallel with ( i ) and perpendicular to ( i ) the fiber axis ( = i/i ) increases by enhancing the chain orientation ( figures 5 , 6 , and s3f and table 1 ) . moreover , the emission polarized parallel to the fiber longitudinal axis is red - shifted up to about 6 nm compared to the perpendicular one , and the spectral shift is even more pronounced for excitation polarization perpendicular to the fiber axis ( figure s6 and table 1 ) . while in films energy migration is effective in randomizing the emission polarization , which causes any memory of the excitation polarization to be lost , in nanofibers energy migration funnels energy toward chromophores oriented along the fiber axis , which emit at lower energy ( i.e. , are red - shifted ) as a consequence of their more extended and conjugated network . the high stretching rate and electric field applied during the spinning process causes a prevalent orientation of chains along the jet axis but also their disentanglement , favoring h - aggregation and interchain interactions . ( a ) pl spectral components of meh - ppv films polarized either parallel with ( red continuous lines ) or perpendicular to ( black dashed lines ) the excitation laser polarization . ( b c ) pl spectra of meh - ppv fibers polarized either parallel with ( red continuous lines ) or perpendicular to ( black dashed lines ) the fiber longitudinal axis . the insets show the distributions of the nanofiber polarization ratio , , obtained by measuring several individual meh - ppv nanofibers , or different regions of the film . ( d f ) plot of the sample emission intensity vs the angle between the fiber and the polarization axis of the collection filter . the emission intensity maximum corresponds to the axis of polarization filter parallel with fiber length . ( a and d ) : films ; ( b and e ) lmo fibers ; ( c and f ) hmo fibers . polarization pl analysis with excitation polarization perpendicular to fiber axis . ( a ) pl spectral components of meh - ppv films polarized along two orthogonal directions . ( b c ) pl spectra of meh - ppv fibers polarized either parallel with ( red continuous lines ) or perpendicular to ( black dashed lines ) the fiber longitudinal axis . the insets show the distributions of the nanofiber polarization ratio , . ( d f ) plot of the sample emission intensity vs the angle between the fiber and the polarization axis of the collection filter . the emission intensity maximum corresponds to the axis of polarization filter parallel with fiber length . ( a and d ) : films ; ( b and e ) lmo fibers ; ( c and f ) hmo fibers . the increased emission quantum yield is therefore attributable to the unique processing conditions that , in contrast to other solution - based deposition processes , freeze the polymer chains in highly anisotropic configurations yet retain strong interchain interactions that promote energy migration . remarkably , the emission polarization found on hmo individual fibers is indeed comparable to the pl polarization measured in isolated meh - ppv chains , i.e. the ultimate achievable polarization properties for the emission of the studied system . to investigate the origin of the anisotropic emission properties of the meh - ppv films and fibers and more clearly understand energy migration in these systems , we perform pump probe stimulated emission anisotropy measurements ( figure 7 ) . probe anisotropy measurements has been shown to be a sensitive probe of the migration of energy in conjugated polymers . in this technique , a pump beam with a defined linear polarization state prepares a subensemble population of excitons whose transition dipole moments are aligned parallel to the laser beam . a probe beam then interrogates the state of the system with a polarization state that is variably oriented either parallel or perpendicular to that of the pump beam . the measured pump probe stimulated emission data can be used to calculate the time - dependent anisotropy of excitons in the system , that is , their polarization memory , according to the following equation:1the anisotropy of the 00 band of the stimulated emission of the meh - ppv film decays on the picosecond time scale toward a value close to zero . in the experiments on the meh - ppv nanofibers , the nanofiber axis was oriented perpendicular to the pump beam polarization . we find that the anisotropy of the 00 band of the stimulated emission of the meh - ppv hmo nanofibers also decays on the picosecond time scale but , in contrast to the meh - ppv film , continues to decay beyond zero , taking on negative values at long pump probe time delays . steady - state absorption and photoluminescence of an meh - ppv film and hmo nanofibers ( upper panels ) . probe anisotropy decay observed in the vicinity of the 00 band of the stimulated emission of an meh - ppv film and hmo fibers ( bottom panels ) . the data are plotted with seven counters in intervals of 0.1 over the range 0.25 to 0.45 . the incident pump energy density values in the case of the film and nanofiber measurements were 40 and 15 j / cm , respectively . we observe that the polarization memory of excitons in both films and hmo nanofibers of meh - ppv is lost on the few picosecond time scale ( figure 8) . a stretched exponential function with a time constant of 2 ps reasonably well describes the anisotropy decay of the meh - ppv films and overlays the data in figure 8 as a guide to the eye . these observations are generally consistent with prior measurements on meh - ppv films . in the meh - ppv hmo nanofibers , a stretched exponential function with a time constant of 1 ps adequately describes the anisotropy decay . given the relatively fast depolarization time scale , this suggests a prominent role of interchain electronic energy transfer in both systems . significantly , whereas the decay of the anisotropy of the 00 band of the stimulated emission in the meh - ppv film approaches zero at long pump probe time delay , the corresponding decay in the meh - ppv nanofibers takes on negative values at long pump probe time delay . these observations suggest a preferential alignment of excitons along the fiber axis following energy migration . probe anisotropy decay of the 00 band of the stimulated emission of an meh - ppv film and hmo nanofibers . the decay of the 00 band of the stimulated emission was taken as the mean over the spectral ranges 563592 nm and 575606 nm for the meh - ppv film and hmo nanofibers , respectively . stretched exponential functions with time constants of 2 and 1 ps , corresponding to the decay of the anisotropy of the stimulated emission observed in the meh - ppv film and nanofibers , respectively , overlay the data as a guide to the eye . in summary , meh - ppv fibers with oriented polymer chains are investigated by modulating the composition of the solution used for electrospinning . the structural characterization by x - ray diffraction measurements highlights a preferred orientation of molecules in the electrospun fibers , with the c axis parallel to the fiber axis and the a and b axes perpendicularly oriented , evidencing the formation of a well - defined 3-dimensional nanocrystalline order , differently from films , where randomly oriented crystalline domains are present as well as domains with different preferred orientations . the anisotropic packing and stretching of the meh - ppv macromolecules in the solid state fibers leads to a 5-fold enhancement of the pl quantum yield compared to samples composed of chains exhibiting disordered packing ( i.e. , spin - cast films ) . polarized steady state spectroscopy and femtosecond pump probe anisotropy measurements indicate that energy migration prevalently funnels excitations toward chromophores oriented along the fiber axis , within a characteristic picosecond time scale . these emissive chromophores feature longer conjugation lengths and increased interchain interactions , demonstrating improved emission properties of the fibers compared to films . these nanostructures with precisely tailored internal molecular order are an example of how more efficient and directed energy transfer can be achieved , and can potentially enhance performance in light - harvesting architectures . g / mol ) was purchased from american dye source , inc . and used as received . for electrospinning experiments , 36 mg of meh - ppv was dissolved in 1 ml of a chcl3:dmso mixture , with relative ratios in the range 9:1 to 9:4 ( v : v ) . experiments performed with higher content of dmso did not allow a stable electrospinning jet and uniform solid state fibers to be obtained . the solution was stirred for 24 h to achieve the complete dissolution of the conjugated polymer . afterward , the solution was stored in a 1 ml syringe , tipped with a 27 g stainless steel needle . the syringe was loaded in a microprocessor dual drive syringe pump ( 33 dual syringe pump , harvard apparatus ) , used with a constant rate of 0.5 ml / h . a rotating disk ( 4000 rpm ) positioned 10 cm far away from the needle was used as a collector , allowing uniaxially aligned arrays of fibers to be obtained . all experiments were performed by applying a positive bias of 10 kv to the needle , while the collector was biased at a negative voltage of 5 kv . quartz coverslips ( thickness 250 m ) were used as substrates . following deposition , fibers were embedded in a photocurable polymer ( noa68 , norland products inc . , refractive index , n = 1.54 ) , which was cured for 3 min . alternatively , free - standing bundles of uniaxially aligned fibers are produced by using the rotating collector and punched al foils as substrates . reference films were produced by spin coating at 6000 rpm on quartz substrates for 40 s. the reference dilute solution was produced by dissolving 0.2 mg of meh - ppv powder in 1 ml of chcl3 . the morphology of the fibers was investigated by scanning electron microscopy ( sem ) and atomic force microscopy ( afm ) . sem was performed by a nova nanosem 450 system ( fei ) , operating at an acceleration voltage of 3 kv . afm was performed with a multimode head , equipped with a nanoscope iiia electronic controller ( veeco instruments ) , imaging in tapping mode with silicon cantilevers ( resonance frequency 250 khz ) . polarized ftir spectroscopy was carried out by using a spectrophotometer ( spectrum 100 , perkinelmer inc . ) , equipped with a ir grid polarizer ( specac limited , uk ) , consisting of 0.12-m - wide strips of aluminum , here used to generate an incident polarized light beam . fluorescence imaging of the fibers arrays was carried out by using an inverted microscope ( eclipse ti , nikon ) and an a1r mp confocal system ( nikon ) . the samples were excited by an ar ion laser ( exc = 488 nm ) through an oil immersion objective with a numerical aperture ( n.a . ) of 1.4 , which also collected the light emitted by the sample . the fluorescence intensity and spectral distribution were measured by a spectral detection unit equipped with a multianode photomultiplier ( nikon ) . saxs , waxs , and giwaxs measurements were performed in a laboratory ( gi)saxs/(gi)waxs setup ( xmi - lab ) , equipped with a rigaku fr - e+ superbright microsource ( = 0.154 nm ) and a smax3000 three pinhole camera . saxs patterns were collected at a 2.2 m distance from the sample , with a 200 m step size , by using a multiwire triton detector . giwaxs patterns were collected at a 0.2 incidence angle , on an image plate ( ip ) detector with 100 m pixel size at a 87 mm sample - to - detector distance . patterns saxs/(gi)waxs collected at 87 mm , or more , from the sample were calibrated by using a standard silver behenate powder sample , or a si powder from nist as specified in the text . waxs patterns collected at a 28 mm distance were calibrated by using a standard lab6 powder from nist . the batch for the scanning saxs / waxs experiment and the composition of data in the color map were realized by using the in - house software sunbim . absorption spectra were collected by using a tungsten lamp and a monochromator equipped with a ccd camera detector ( usb4000 , ocean optics ) . the samples were mounted in an integrating sphere ( labsphere ) , in order to minimize artifacts due to light scattering . polarized absorption spectra were obtained by using incident light polarized either parallel or perpendicular to the fiber axis . similarly , pl spectra were acquired by exciting the samples , placed in the integrating sphere , with a laser ( coherent verdi , exc = 532 nm , beam diameter = 1 mm ) and collecting the emission with an optical fiber coupled to a monochromator . this system was also used for measuring the pl quantum yield following the procedure reported in ref ( 66 ) . this system includes a linearly polarized diode laser ( exc = 405 nm ) , focused onto single fibers by a 20 objective ( n.a . the vertically emitted light was then collected with a spherical lens ( f = 60 mm ) , coupled to an optical fiber and measured with a spectrometer . the polarization state of the emission was analyzed with a polarization filter , which was placed on a precision rotation stage , between the sample and the collecting lens . the samples were placed on a rotation stage , which allows the fiber axis to be precisely aligned with the polarization direction of the excitation laser . the pump probe stimulated emission anisotropy measurements were performed on a pump probe spectrometer that has been described in detail previously . briefly , a ti : sapphire - based regenerative amplifier ( spectra - physics spitfire pro ) generates about 3.5 w of 800 pulsed laser light that has a duration of 100 fs and a repetition rate of 5 khz . a portion of the output is directed toward a custom - built noncollinear optical parametric amplifier ( nopa ) that converts the 800 nm radiation into wavelengths in the visible region . in the present experiments , the nopa was tuned to encompass a spectral range from about 500 to 620 nm . the output of the nopa is directed toward a series of compressors , including a grating and prism compressor . details of the optical layout of the nopa as well as the grating and prism compressors were reported previously . the nopa spectrum is shown in figure s7 . the output of the prism compressor is then directed toward the pump probe spectrometer where a glass wedge serves to split the beam into pump and probe beam paths . both pump and probe beams pass through a waveplate and glan - thompson polarizer ( new focus ) , the combination of which allows us to variably control the beam intensity and define the polarization state of the beams at the sample position . the path length of the pump beam is variably controlled with an automated , mechanical delay stage ( newport , santa clara , ca ) . the probe beam transmitted through the sample is collected and directed toward a monochromator and camera combination ( andor ) for spectral detection . an optical chopper ( new focus ) in the pump beam path operating at 625 hz enables differential detection . balanced detection is achieved by simultaneously accounting for fluctuations in the laser beam by using a photodiode to monitor the intensity of the second beam reflected from the glass wedge . pulse compression was guided by minimizing the full - width - at - half - maximum of the coherent artifact measured from a solution of methanol in a spectrophotometer cell placed at the sample position that was subsequently replaced with a blank resin substrate . pump pulse energies were determined by measuring the beam power , and the beam spot sizes were estimated by comparing the power transmitted through a pinhole placed at the sample position . incident pump energy densities estimated in this manner were 40 and 15 j / cm in the film and nanofiber measurements , respectively .

conjugated polymers are complex multichromophore systems , with emission properties strongly dependent on the electronic energy transfer through active subunits . although the packing of the conjugated chains in the solid state is known to be a key factor to tailor the electronic energy transfer and the resulting optical properties , most of the current solution - based processing methods do not allow for effectively controlling the molecular order , thus making the full unveiling of energy transfer mechanisms very complex . here we report on conjugated polymer fibers with tailored internal molecular order , leading to a significant enhancement of the emission quantum yield . steady state and femtosecond time - resolved polarized spectroscopies evidence that excitation is directed toward those chromophores oriented along the fiber axis , on a typical time scale of picoseconds . these aligned and more extended chromophores , resulting from the high stretching rate and electric field applied during the fiber spinning process , lead to improved emission properties . conjugated polymer fibers are relevant to develop optoelectronic plastic devices with enhanced and anisotropic properties .

Introduction Results and Discussion Conclusions Experimental Section

in the framework of enhanced and flexible materials for optoelectronics and energy applications , organic semiconductors have been largely exploited due to their favorable charge - carrier and emission properties , and easy processing by solution methods , printing , and soft lithographies . such excitations are typically localized on the scale of a single or few molecules , and their migration and recombination dynamics determines both light - emission and light - harvesting properties . in such systems , electronic energy transfer directs the excitation toward lower energy chromophores , typically associated with low - emissive aggregates . guest systems by using mesoporous silica templates , which led to fast intrachain electronic energy transfer , highly polarized emission , and low - threshold amplified spontaneous emission . a small increase of the polarization anisotropy was observed , together with a variation of the emission intensity and wavelength depending on the solution viscosity and solvents , which was attributed to different energy migration pathways activated by shear - induced modification of the molecular conformation . despite such efforts , solid state structures made of conjugated polymers are still far from the performances expected for extended chains . these nanostructures have already demonstrated potential as the active component of organic light - emitting diodes , solar cells , and transistors , and they can constitute a valuable benchmark system to tailor and control the fundamental properties of conjugated molecules in the solid state . here we report on fibers made of meh - ppv , realized by electrospinning . a comparison with thin films indicates that fibers composed of aligned and more extended polymer chains achieve a 5-fold enhancement of the emission quantum yield as well as emission polarized along the fiber axis , independent of excitation configuration . probe anisotropy measurements indicate that , in the ordered fibers , photogenerated excitons preferentially reorient along the fiber axis . we find that the time scale of the energy migration is of the order of that expected for enhanced interchain coupling . meh - ppv is chosen as a prototype conjugated polymer , whose optical properties have been investigated in different packing configurations of the macromolecules , from single molecules , to isolated and stretched individual chains , small aggregates , and thin films . the encapsulation of the fibers in the resin also decreases the amount of reflected light , because of the reduced refractive index contrast with the conjugated polymer . the rapid evaporation of the more volatile component ( chloroform ) of the liquid jet during the spinning process and the formation of a collapsing solid skin can be the origin of the ribbon shape . a flattening of the fibers upon substrate deposition , due to the presence of a fraction of slowly evaporating solvent ( dimethyl sulfoxide , dmso ) in the solution , may also occur . the possibility of obtaining fibers with strongly anisotropic packing of the constituent polymer chains is related to the very high strain rate ( 10 s ) exerted in electrospinning , which typically induces molecular stretching and orientation in the jet , that is then retained in the fibers . in general , conjugated polymers are poorly viscoelastic , a property often associated with their semiflexible backbones , and the final degree of chain orientation depends on many factors . modeling of the conjugated polymer chain networks under elastic stretching evidenced that substantial axial extension is achieved within a few millimeters from the spinneret and that this depends on the volume fraction occupied by the polymer , , the applied electric field , e , the jet initial velocity , v0 , and the solution conductivity , k. typically , lower values of and v0 , and higher e and k are needed to increase the axial chain orientation . this approach is supported by comparing the molecular order of fibers produced from solutions with even slightly different nonsolvent content . in both samples , the absorption is maximized for incident light polarization parallel with the fiber longitudinal axis , which is indicative of alignment of the conjugated chains . in particular , the fibers obtained from solutions with higher content of dmso show a more pronounced difference of spectra measured with the incident light polarized parallel or perpendicular to their axis , meaning that a larger fraction of chain segments are oriented along the fiber length . an enhanced chain alignment is highlighted by the ftir analysis upon increasing the dmso volume fraction up to 20% ( figure s3a e ) , whereas a further increase of the dmso content negatively affects both the polymer chain alignment and the stability of electrospinning . overall , these and other results show that electrospinning may be exploited to finely tailor the orientation of conjugated chains in nanostructured materials . the arrows indicate the mode at about 1500 cm , attributed to a c ( c ) amplitude of the transmission peak at 1500 cm vs the angle , , formed by the fiber axis and the incident polarization direction ( see figure s1 of the supporting information ) for fibers spun from chloroform : dmso mixtures with volume ratio of 9:1 ( circles ) and 9:2 ( squares ) . 0 corresponds to the direction of the incident light polarization parallel to the fiber axis . the molecular orientation and the structure of the hmo samples , free - standing bundles of uniaxially aligned fibers were investigated by scanning small - angle x - ray scattering ( saxs ) and wide - angle x - ray scattering ( waxs ) measurements . ( e ) linear cuts taken from the waxs patterns of part b ( 28 mm detector distance ) and figure s4a ( 120 mm detector distance ) , in the directions parallel ( black line ) and perpendicular ( orange symbols ) to the fiber axis , and from the giwaxs pattern of figure s4b in the directions parallel ( black dots ) and perpendicular ( green triangles ) to the film surface . the intensity modulation along the azimuthal angle for the two most intense diffraction rings is plotted in figure 3c , where the red and the blue ( top ) curves are related to the a and c parameters , respectively ( outer and inner rings in figure 3b ) . by taking into account the direction of the orientations of the fibers as indicated by figure 3a , it can be concluded that the a and c parameters are preferentially oriented perpendicular and parallel to the fiber axis , respectively . moreover , figure 3d shows the azimuthal intensity modulation in the 0.30.5 q - range , corresponding to the periodicity of the b lattice parameter , which has been here reported in a separated plot because this signal was extracted from waxs patterns ( see figure s4a ) collected at a larger distance from the sample ( 120 or 250 mm as specified in figure 3d ) in order to access lower q - values , although with much lower intensity . the b and a parameters are therefore expected to preferentially lie in the plane perpendicular to c , and hence to the fiber axis . the preferred orientation is also reflected in the 1d radial profiles of figure 3e , resulting from the cuts taken along the horizontal and vertical directions ( i.e. , perpendicular and parallel to fibers , respectively ) in the 2d waxs pattern of figure 3b : the intensity ratio between the two diffraction peaks related to a and c is seen indeed to be reversed in the two cuts ( superimposed curves with symbols and continuous line ) . a significant fraction of randomly oriented crystalline domains is also expected throughout the fibers , based on the fairly small amplitude of the azimuthal intensity modulation . moreover , based on the calibration from figure s4a , and a gaussian fit , it was verified that no appreciable shift of peak positions and hence no variations of the related d - spacings occurred between in plane and out of plane directions . as a result , figure 3 indicates the presence of nanocrystals in meh - ppv electrospun fibers , with crystallographic axes preferentially oriented along the fiber ( c axis ) or perpendicular to the fiber ( a and b axes ) , determining a preferred orientation of the meh - ppv active subunits along the fiber axis . by extracting linear cuts from figure s4b along the in - plane and out - of - plane directions ( 1d profiles with black spots and green triangles , respectively , in figure 3e ) , an intensity modulation results also in this case , as a difference in the ratios of diffraction peaks related to a and c. such a difference is more evident for the a parameter , which clearly features different diffraction intensities along the two directions . accordingly , in the azimuthal profiles extracted from figure s4b and reported in figure 3c ( bottom curves ) for comparison , a corrected intensity larger by the factor 1.1 should be considered , toward the angular interval extremes , in order to compare it with the out - of - plane intensity ( corresponding to the center of the curves ) . on the contrary , the basically isotropic intensity distribution observed for the c parameter ( blue circles ) would change to a slightly concave profile , suggesting a slight preferred orientation of the c axis parallel to the sample plane , and hence perpendicular to a. on the other hand , the b parameter also features a larger azimuthal intensity perpendicular to the sample plane . indeed , differently from fibers , crystalline domains in films develop independently , with random orientations as well as with the a or b axis preferentially oriented perpendicular to the film surface , as in a lamellar stacking , whereas the c axis of the domains lies preferentially in the sample plane , with random in - plane orientations . furthermore , polarized absorption spectra acquired on arrays of uniaxially aligned hmo fibers show a relative red - shift of the absorption band for polarization of the incident light parallel to the fiber axis ( inset of figure 4a ) , which is indicative of a longer conjugation length associated with oriented chromophoric subunits . this ratio provides quantitative measurements of the h- or j - aggregation prevalence in conjugated polymers , being typically > 2 for dominant intrachain coupling ( j - aggregation ) , whereas values < 2 are indicative of interchain coupling ( h - aggregation ) . overall , from polarized ftir , x - ray scattering , and absorption spectroscopies we argue that during spinning a fraction of the active subchains are effectively stretched and oriented along the fiber axis , adopting a more extended chain conformation compared to amorphous films , with an increase of the associated conjugation length and a packing arrangement that supports interchain interactions with nearby subchains ( figure 4b , c ) . ( b c ) pictorial representations of the microscopic arrangement of individual conjugated polymer chains in nanofiber ( b ) and film ( c ) . , arrows indicate the c axis of the crystalline domains , whereas the a and b axes lie in a plane perpendicular to c. ( ) is the ratio between the pl intensity polarized parallel and perpendicular to the fiber axis , collected upon excitation with a laser polarized parallel with ( perpendicular to ) the fiber axis . ( ) is the spectral shift between the pl polarized parallel and perpendicular to the fiber axis , upon excitation with a laser polarized parallel with ( perpendicular to ) the fiber axis . more importantly , the fibers with more oriented polymer chains feature an increase of the pl quantum yield ( f ) by about a factor 5 compared to films ( table 1 ) . these results are in striking contrast to what is typically observed in conjugated polymers in the solid state , where a red - shift of the emission is associated with the formation of low - emissive aggregates . to investigate more in depth the origin of the enhanced and red - shifted emission , we perform a study of the polarization properties of a set of individual nanofibers by microphotoluminescence ( -pl ) . the degree of molecular alignment affects the amount of polarized light detected , since the ratio between the intensity of the pl polarized parallel with ( i ) and perpendicular to ( i ) the fiber axis ( = i/i ) increases by enhancing the chain orientation ( figures 5 , 6 , and s3f and table 1 ) . moreover , the emission polarized parallel to the fiber longitudinal axis is red - shifted up to about 6 nm compared to the perpendicular one , and the spectral shift is even more pronounced for excitation polarization perpendicular to the fiber axis ( figure s6 and table 1 ) . while in films energy migration is effective in randomizing the emission polarization , which causes any memory of the excitation polarization to be lost , in nanofibers energy migration funnels energy toward chromophores oriented along the fiber axis , which emit at lower energy ( i.e. the high stretching rate and electric field applied during the spinning process causes a prevalent orientation of chains along the jet axis but also their disentanglement , favoring h - aggregation and interchain interactions . ( d f ) plot of the sample emission intensity vs the angle between the fiber and the polarization axis of the collection filter . ( d f ) plot of the sample emission intensity vs the angle between the fiber and the polarization axis of the collection filter . the increased emission quantum yield is therefore attributable to the unique processing conditions that , in contrast to other solution - based deposition processes , freeze the polymer chains in highly anisotropic configurations yet retain strong interchain interactions that promote energy migration . to investigate the origin of the anisotropic emission properties of the meh - ppv films and fibers and more clearly understand energy migration in these systems , we perform pump probe stimulated emission anisotropy measurements ( figure 7 ) . probe anisotropy measurements has been shown to be a sensitive probe of the migration of energy in conjugated polymers . the measured pump probe stimulated emission data can be used to calculate the time - dependent anisotropy of excitons in the system , that is , their polarization memory , according to the following equation:1the anisotropy of the 00 band of the stimulated emission of the meh - ppv film decays on the picosecond time scale toward a value close to zero . in the experiments on the meh - ppv nanofibers , the nanofiber axis was oriented perpendicular to the pump beam polarization . we find that the anisotropy of the 00 band of the stimulated emission of the meh - ppv hmo nanofibers also decays on the picosecond time scale but , in contrast to the meh - ppv film , continues to decay beyond zero , taking on negative values at long pump probe time delays . given the relatively fast depolarization time scale , this suggests a prominent role of interchain electronic energy transfer in both systems . these observations suggest a preferential alignment of excitons along the fiber axis following energy migration . stretched exponential functions with time constants of 2 and 1 ps , corresponding to the decay of the anisotropy of the stimulated emission observed in the meh - ppv film and nanofibers , respectively , overlay the data as a guide to the eye . the structural characterization by x - ray diffraction measurements highlights a preferred orientation of molecules in the electrospun fibers , with the c axis parallel to the fiber axis and the a and b axes perpendicularly oriented , evidencing the formation of a well - defined 3-dimensional nanocrystalline order , differently from films , where randomly oriented crystalline domains are present as well as domains with different preferred orientations . the anisotropic packing and stretching of the meh - ppv macromolecules in the solid state fibers leads to a 5-fold enhancement of the pl quantum yield compared to samples composed of chains exhibiting disordered packing ( i.e. polarized steady state spectroscopy and femtosecond pump probe anisotropy measurements indicate that energy migration prevalently funnels excitations toward chromophores oriented along the fiber axis , within a characteristic picosecond time scale . these emissive chromophores feature longer conjugation lengths and increased interchain interactions , demonstrating improved emission properties of the fibers compared to films . these nanostructures with precisely tailored internal molecular order are an example of how more efficient and directed energy transfer can be achieved , and can potentially enhance performance in light - harvesting architectures . experiments performed with higher content of dmso did not allow a stable electrospinning jet and uniform solid state fibers to be obtained . the solution was stirred for 24 h to achieve the complete dissolution of the conjugated polymer . polarized absorption spectra were obtained by using incident light polarized either parallel or perpendicular to the fiber axis . similarly , pl spectra were acquired by exciting the samples , placed in the integrating sphere , with a laser ( coherent verdi , exc = 532 nm , beam diameter = 1 mm ) and collecting the emission with an optical fiber coupled to a monochromator . the polarization state of the emission was analyzed with a polarization filter , which was placed on a precision rotation stage , between the sample and the collecting lens . the samples were placed on a rotation stage , which allows the fiber axis to be precisely aligned with the polarization direction of the excitation laser . a portion of the output is directed toward a custom - built noncollinear optical parametric amplifier ( nopa ) that converts the 800 nm radiation into wavelengths in the visible region . the output of the nopa is directed toward a series of compressors , including a grating and prism compressor . balanced detection is achieved by simultaneously accounting for fluctuations in the laser beam by using a photodiode to monitor the intensity of the second beam reflected from the glass wedge .

1 ) is a newly approved orphan antiepileptic drug ( aed ) [ 1 , 2 ] . a characteristic feature of the drug is the presence of a chiral center at c-3 . stp is marketed as a racemic mixture of two enantiomers : r(+)-stp and s()-stp of which r(+)-stp has about 2.4-fold greater anticonvulsant potency and it is also associated with ~3-fold faster elimination rate than s()-stp . the compound has been under investigations for nearly 4 decades [ 6 , 7 ] ; its clinical development was delayed due to the inhibitory effect of stp on hepatic cytochrome - p450 ( cyp-450 ) . stiripentol , ( diacomit ; biocodex inc . ) has been approved by the european and canadian marketing authorization for the treatment of dravet syndrome or severe myoclonic epilepsy in infancy ( smei ) in conjunction with sodium valproate and clobazam when seizures are not adequately controlled with the association of these two medications [ 9 , 10 ] . stp inhibits gaba metabolism through the blockade of gaba - transaminase activity and reduces synaptosomal uptake of gaba , leading to an increase in gaba brain content . stp , at relevant clinical concentrations ( 30300 m ) , markedly increases the mean open duration of gabaa receptor dependent chloride channels by a barbiturate - like mechanism . stability testing of pharmaceutical dosage form of stp or any other drug , ( i.e. , purity and stability of the active ingredient and/or final product ) is imperative for the optimum and efficient delivery of its therapeutic activity to the patients . the purpose of stability testing is to investigate changes in the quality of a drug substance or a drug product with time under the influence of environmental factors , such as temperature , humidity , and light . moreover , stability testing establishes the recommended storage conditions and shelf life for the drug product [ 14 , 15 ] . this is actually due to the fact that presence of impurities and/or potential degradation products may alter the pharmacological and/or toxicological characteristics of the active drug [ 16 , 17 ] . in particular , pharmaceuticals are significantly sensitive to environmental conditions , which are usually varied during the different stages ( i.e. , manufacturing , transportation and storage ) of the finished products . based on the aforementioned importance of stability testing , it is vital to investigate degradation pathways and the intrinsic stability of the drug , compatibility of the drug with the excipients in the dosage form , and the long - term effects of the environment . recently , the need of establishing a stability - indicating assay method for the stability testing has become more clearly mandated in the official guidelines at the international conference on harmonization ( ich ) and united states pharmacopeia ( usp ) . a thorough literature survey revealed that only few high performance liquid chromatography ( hplc ) methods were reported for the determination of stp to study its pharmacokinetics [ 7 , 18 ] , enantioseparation , and its estimation in plasma . consequently , a stability - indicating method for determination of stp in its bulk drug and pharmaceutical capsules is important . accordingly , the present study describes for the first time a comprehensive stability - indicating study for stp . this study aims to develop and fully validate a rapid , simple , and accurate hplc - dad method to quantify stp in the presence of its degradation product in bulk and pharmaceutical dosage form . additionally , structure elucidation techniques including the h - nmr , c - nmr , and mass spectrometry were used to identify stp degradation product . stiripentol reference standard ( purity ~99.6% ) was purchased from sigma - aldrich co. ( st . louis , mo , usa ) . diacomit capsules ( biocodex , montrouge , france ) , labeled to contain 250 mg ( as the anhydrous base ) per capsule , were procured from the local market . hplc - grade solvents and reagent - grade sodium hydroxide , hydrochloric acid , hydrogen peroxide , potassium dihydrogen phosphate , and orthophosphoric acid were purchased from merck ( darmstadt , germany ) . ultrapure water of 18 was obtained from a millipore milli - q uf and purification system ( millipore , bedford , ma , usa ) was used throughout the study . hplc apparatus consists of a waters breeze system consisting of 1525 binary pump with online degasser , 717 + autosampler , 5ch thermostated column compartment , and a 2996 photodiode array detector ( dad ) ( waters corporation , milford , ma , usa ) . the chromatographic separations were performed on a symmetry c18 column ( 3.5 m , 75 mm 4.6 mm i.d ) manufactured by waters corporation , milford , ma , usa . the column temperature was kept constant at 25 2c . separations were performed in isocratic mode using a mobile phase consisting of acetonitrile and 50 mm potassium dihydrogen phosphate buffer ( 60 : 40 , v / v ) , and ph was adjusted to 4.1 0.1 with 10% phosphoric acid solution . the mobile phase was filtered through a millipore vacuum filtration system equipped with a 0.45 m filter ( millipore , bedford , ma , usa ) , degassed by ultrasonic water bath prior to its use . the flow rate of the mobile phase was 1 ml / min , and the sample injection volume was 20 l . the dad detector was set at scan range of 210400 nm to allow investigation of any impurities and/or degradation products with shorter wavelength . additionally , quantitation of stp and its acidic degradation product was performed at 262.5 nm . prior to each run , the hplc - dad system was allowed to warm up for nearly 30 min and the baseline was monitored until it was stable before the samples were injected . peak identity was confirmed by retention time comparison and comparison of the spectra obtained from the dad detector . stp stock solution ( 1 mg / ml ) was prepared by dissolving 25 mg of stp reference standard material into 25 ml methanol in a 25 ml volumetric flask and completing the volume properly . this stock solution was later diluted with methanol to produce a working standard solution of 100 g / ml . an eight - point calibration curve ( 1 , 2 , 4 , 6 , 10 , 15 , 20 , and 25 g / ml ) was constructed by plotting the peak area of stp ( y ) versus stp nominal concentration ( x ) . slope , intercept , and r values were calculated as regression parameters by linear regression . the linear regression equation was used to calculate the concentrations of stp in aqueous solutions and dosage form based on their peak area . sample solutions were prepared by diluting the working solution with mobile phase to obtain final concentrations of stp . twenty capsules ( diacomit 250 mg capsules ; batch number 2611 ) were weighed and average weight was determined . capsules were emptied and two portions ( 250 mg each ) were separated where one portion was transferred to a 500 ml volumetric flask . a volume of 250 ml of methanol was added , the contents were mechanically shaken for 10 min and ultrasonicated for 5 min , and the volume was diluted to 500 ml with methanol . this solution ( 0.5 mg / ml ) was diluted as required for analysis . the remaining 250 mg powder was used as it is for thermal and photodegradation studies . thermal degradation of the drug substance in its bulk form and capsules was carried out in both solid and solution state . powder samples of standard stp ( 10 mg ) and diacomit capsules ( 10 mg of capsule content ) were kept in a controlled - temperature oven at 80c for 48 hours . solutions were then diluted with mobile phase to obtain a concentration of 10 g / ml , and a volume of 20 l of each solution was injected into the hplc system . meanwhile , a volume of 1 ml of stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ; prepared as previously mentioned in section 2.5 . ) was transferred to small round bottom flasks and subjected to reflux at 80c for 48 hours . the solutions were cooled to room temperature ( 25 5c ) and diluted with mobile phase to yield final concentrations of 10 g / ml ; and a volume of 20 l of each solution was then injected into the hplc system . aliquots of 1 ml of stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ) were transferred to small round bottom flasks . in the preliminary forced degradation testing , it was observed that acid degradation takes place rapidly and the drug was almost fully degraded when 0.5 n hydrochloric acid was used . therefore , in later experiment , acid degradation was performed with a maximum of 0.1 n hydrochloric acid . ultimately , solutions were individually mixed with 4 ml of ( 0.005 n , 0.01 n , 0.05 n , and 0.1 n hydrochloric acid ) and/or ( 0.05 n , 0.1 n , 0.5 n , and 1 n sodium hydroxide ) . the prepared solutions were heated for 1 , 2 , and 3 hours at 60c in a water bath . the samples were cooled to room temperature ( 25 5c ) , neutralized with an amount of acid or base equivalent to that of the previously added . the resulting neutral solutions were then diluted with mobile phase to 10 g / ml and a volume of 20 l was injected into the hplc system . four aliquots of 1 ml stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ) were transferred into small round bottom flasks . the contents were then mixed separately with 4 ml of ( 1 , 6 , 10 , and 30% ) hydrogen peroxide solutions , and the reaction mixtures were kept at ambient temperature for 2 hours with intermittent shaking . the resulting solutions were diluted with mobile phase to 10 g / ml and a volume of 20 l of each solution was injected into the hplc system . powder samples of standard stp ( 10 mg ) and diacomit capsules ( 10 mg of capsule content ) were uv irradiated ( peak intensity of 254 nm ) for 72 hours . solutions were then diluted with mobile phase to obtain a concentration of 10 g / ml , and a volume of 20 l of each solution was injected into the hplc system . similarly , an aqueous solution of stp ( 1 mg / ml ) and capsule extract solution ( 1 mg / ml ) were exposed to the uv light for 72 hours and diluted with mobile phase to 10 g / ml and a volume of 20 l of each was then injected into the hplc system . acid degradation was carried out by refluxing stp ( 40 mg ) in methanol ( 15 ml ) and 8 n hcl ( 5 ml ) for 3 h at 60c using the same procedure discussed in section 2.6.2 . the acid degradation product ( dstp ) was extracted from the residue using diethyl ether which was subsequently evaporated under reduced pressure . crude dstp was then purified via column chromatography ( silica gel 60 ; 0.0630.200 mm ) using chloroform as a solvent . the collected eluate was evaporated under vacuum to give pure dstp , which was identified through h - nmr , c - nmr , and mass spectrometry . purified dstp was dissolved in deuterated chloroform and the nmr measurements were performed at 25c on a bruker ac-500 nmr spectrometer operating at 500 mhz for h and 125.76 mhz for c. tetramethylsilane ( tms ) was used as an internal standard and chemical shift values were recorded in ppm on the scale . the h - nmr data were represented in terms of chemical shifts , multiplicity , and number of protons . the c - nmr data were represented as chemical shifts and type of carbon . for structure elucidation of dstp , an agilent 6410 triple quadrupole mass spectrometer ( agilent technologies , usa ) equipped with an electrospray ionization interface ( esi ) coupled to an agilent 1200 hplc ( agilent technologies , usa ) was used . agilent 1200 series system consists of g1311a binary pump , g1322a degasser , and g1367b hip - als autosampler , and g1316a thermostated column compartment was used . purified dstp was dissolved in acetonitrile ( 5 g / ml ) , and a connector was used instead of the column to allow direct injection of the sample . mobile phase composed of two solvents : a is hplc grade water and b is acetonitrile mixed in the ratio 50 : 50 v / v% . ms parameters were optimized to scan in ultrascan mode . for screening of mass signals of dstp the source temperature was set to 350c nebulizer gas pressure of 55 psi and dry gas flow rate of 12 l / min . four sets of stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ) , 1 ml of each , were subjected to acid degradation using 4 ml of 0.005 n , 0.01 n , 0.05 n , and 0.1 n hydrochloric acid ( procedure described in section 2.6.2 ) . ( 0 , 15 , 30 , 60 , 120 , 180 , and 240 min ) . the samples were cooled to room temperature ( 25 5c ) and neutralized with an amount of base equivalent to that of the previously added acid . the resulting neutral solutions were then diluted with mobile phase to 10 g / ml and a volume of 20 l was injected into the hplc system and the peak area of stp was compared with a freshly prepared standard solution . the initial method development was conducted on pure drug using working standards solution protected from light . optimized chromatographic conditions were established after a number of preliminary experiments for selecting the most efficient mobile system , separation column , and detection wavelength range . selection of the proper system was based on its ability to give good separation between the pure stp and its possible impurities and/or degradation products . different mobile phase compositions and ph ranges were tested to achieve a more symmetric peak and shorter retention time for stp . mobile phase was modified between 20 : 80% and 70 : 30% acetonitrile : 1 mm phosphate buffer and ph was changed in the range of 37 . ultimately , optimum chromatographic separation was achieved as described in section 2.2 . under these chromatographic conditions , the run time of each sample was 6 min , and the retention time of stp was 1.80 0.07 min ( n = 3 ) ( figure 2 ) . an advantage of the current method is its simple sample preparation as the procedure did not include significant error - prone experimental manipulations ( e.g. , chemical derivatization , etc . ) which would negatively affect the results . this supported the avoidance of internal standard use , which is usually added to increase the method efficiency when there are multisteps in sample preparation . the method was fully validated in accordance with the recommendations of ich and is rugged and adequately sensitive for routine stp analysis . using the above - mentioned optimum chromatographic conditions , three independent calibration curves were constructed correlating the detector signals with the corresponding stp concentrations . each curve was generated by 8 concentration points ; each concentration was injected in triplicates . the standard deviation values of each concentration point ( triplicates ) did not exceed 2% . the method revealed a good linear calibration fit in the range of 125 g / ml . system suitability tests for the principle peak and its degradation product were evaluated using stp solution of 12 g / ml . the capacity factor k = 0.8884 indicates that the stp peak is well resolved with respect to the void volume . the asymmetry factor at 5% of peak height ( b / a ) was 1.3079 for stp peak which reflects good peak symmetry . theoretical plate number ( n ) of 5008.09 for the column used in the study ( 3.5 m , 75 mm 4.6 mm i.d ) thus demonstrates acceptable column efficiency . all previous results assured the adequacy of the proposed hplc method for routine analysis of stp . the lower limit of quantitation ( loq ) is the lowest concentration of the standard curve which can be measured with acceptable accuracy and precision for the analyte . the limit of detection ( lod ) and the lower limit of quantitation ( loq ) were calculated based on the following equations : ( 1)lod=3.3s , loq=10s , where is the standard deviation of the intercept ( 115.82 3300 ) of regression line and s is the slope ( 30875.64 248.86 ) of regression line of the calibration curve . the lod and loq values were 0.081 and 0.242 g / ml , respectively . accuracy and precision were determined by the recovery study of known amounts in accordance with ich recommendation . five consecutive injections of stp sample solutions ( 2 , 6 , 10 , 15 , and 20 g / ml ) showed excellent intraday accuracy ( 98.74101.64% ) and precision ( 0.642.32% ) ( table 2 ) . interday accuracy and precision data of back - calculated concentration of calibration samples for stp were evaluated in triplicates by recovery studies using the standard addition method ( table 3 ) . in order to measure the extent of the method robustness , the most critical parameters were interchanged while keeping the other parameters unchanged , and in parallel the chromatographic profile was observed and recorded . robustness of the proposed method was determined using stp concentration of 10 g / ml and changing the flow rate ( 0.91.1 ml / min ) and column temperature ( 2c ) and change in the ph of mobile phase ( 0.2 ) . no significant changes in assay value were observed ( table 3 ) by changing the chromatographic conditions which confirms the robustness of the method . ruggedness of the method was determined by using mobile phase components from two different manufactures and changing analysts . there was no significant change in the retention time of stp which was observed ; % rsd was 0.310.63% indicating the ruggedness of the method . the stability of the drug in solution during analysis was determined by repeated analysis of samples during the course of experimentation on the same day and also after storage of the drug solutions ( calibration samples ) for 24 , 48 , and 72 hours under laboratory bench condition ( 25 1c ) and under refrigeration ( 4.0 0.5c ) . the mean % rsd between peak areas for the samples stored under refrigeration ( 4.0 0.5c ) and at laboratory temperature ( 25 1c ) was found to be 0.58% and 0.93% , respectively , suggesting that the drug solution can be stored without any degradation over the time interval studied . the ich guideline entitled stability testing of drug substances and products requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substance and provide a rapid identification of differences that might result from changes in the manufacturing processes or source sample . an ideal stability - indicating method is one that quantifies the standard drug alone and also resolves its degradation products . as described in the experimental section , different stress conditions were applied : boiling , acid / base forced degradation , oxidation , and irradiation with uv light . from this investigation , it was clear that , in case of boiling , base - forced degradation ( 1 n naoh ) , oxidation ( h2o2 ) , and uv irradiation ( solid state and solution ) , stp was stable under the employed stress conditions as no degradation products were observed in their chromatograms ( figures 3(a ) and 3(b ) ) which confirms the data from european medicines agency ( emea ) . however , in case of acidic conditions , one degradation product was observed , dstp , at a retention time of 4.25 min ( peak resolution from stp peak was 5.89 ) ( figures 3(c)3(e ) ) . the intact stp concentration was calculated and found to be degraded by 76.03 , 83.51 , 84.87 , and 95% in case of acid degradation using 0.1 n hydrochloric acid after 1 , 2 , 3 , and 5 hours , respectively . acidic degradation of stp was studied using h - nmr , c - nmr ( figures 4 and 5 ) , and mass spectral data . h - nmr spectrum of dstp showed a signal integrating to one proton at 4.09 ppm which was assigned to be cl ch with concomitant disappearance of the signal of oh functionality at 1.56 ppm and the signal of the methine proton ( oh ch ) at 3.80 ppm . additionally , c - nmr of dstp showed signal at 74.8 ppm which was assigned to be cl ch with concomitant disappearance of the signal of oh ch at 83.9 ppm . moreover , mass spectrum ( esi ) of dstp showed peak at m / z 217 [ m-36 + h ] . uv spectra of stp ( figure 1 ) and dstp are identical indicating that there is no change in the conjugation system upon this acidic degradation of stp . structure elucidation of dstp explained its delaying in hplc elution due to its lower polarity compared to stp . accordingly , the aforementioned analytical data of dstp suggested that upon acid degradation of stp the hydroxyl moiety was protonated with hydrochloric acid and subsequently substituted with chloride ion to give the halogenated derivative dstp ( scheme 1 ) . h - nmr ( cdcl3 ) of stp : ( ppm ) = 0.88 ( s , 9h , t - butyl ) , 1.56 ( s , 1h , oh ) , 3.80 ( d , j = 7.0 hz , 1h , choh ) , 5.86 ( s , 2h , och2o ) , 6.05 ( dd , j = 15.7 , 7.85 hz , 1h , ch = ch c6h3 ) , 6.39 ( d , j = 15.5 hz , 1h , ch = ch c6h3 ) , 6.67 ( d , j = 8.0 hz , 1h , ar h ) , 6.72 ( d , j = 7.95 hz , 1h , ar c - nmr ( cdcl3 ) of stp : ( ppm ) = 26.5 ( c(ch3)3 ) , 32.8 ( c(ch3)3 ) , 81.9 ( ch oh ) , 101.1 ( och2o ) , 107.1 , 108.3 , 121.1 ( ar ch ) , 126.9 , 131.5 ( ch = ch c6h3 , ch = ch c6h3 , ar ms m / z ( esi ) : 217 [ m-18 + h ] . h - nmr ( cdcl3 ) of dstp : ( ppm ) = 0.83 ( s , 9h , t - butyl ) , 4.09 ( d , j = 9.5 hz , 1h , ch cl ) , 5.74 ( s , 2h , och2o ) , 5.92 ( dd , j = 15.4 , 7.95 hz , 1h , ch = ch c6h3 ) , 6.23 ( d , j = 15.5 hz , 1h , ch = ch c6h3 ) , 6.54 ( d , j = 8.0 hz , 1h , ar h ) , 6.60 ( d , j = 7.96 hz , 1h , ar c - nmr ( cdcl3 ) of dstp : ( ppm ) = 26.8 ( c(ch3)3 ) , 36.4 ( c(ch3)3 ) , 74.8 ( ch cl ) , 101.2 ( och2o ) , 105.9 , 108.3 , 121.6 ( ar ch ) , 125.7 , 130.6 , 132.5 ( ch = ch c6h3 , ch = ch c6h3 , ar c ) , 147.8 , 148.1 ( ar c ) . ms m / z ( esi ) : 217 [ m-36 + h ] . the degradation of stp was investigated in acidic condition using 0.005 , 0.01 , 0.05 , 0.1 n hcl . the drug was found to be extremely labile for higher concentrations of hcl ( 0.05 and 0.1 n ) . a gradual decrease in stp concentration was observed with increasing time at lower hcl concentrations ( 0.005 and 0.01 n ) ( figure 6(a ) ) . a linear relationship was observed by plotting the 1og of residual molar concentration of stp versus time ( figure 6(b ) ) which indicates first - order kinetics according to the following equation : ( 2)lnct=kt+lnc0 . here , ct is the remaining stp molar concentration , c0 is the initial molar concentration of stp , k is the apparent first order rate constant with a negative sign , and t is the time . the half - life ( t1/2 ) of the first - order reaction was found to be t1/2 = 1.42 h according to the following equation : ( 3)t1/2=ln(2)k . it is evident from the results obtained previously that the proposed method gave satisfactory results with the analysis of stp in bulk . thus , stp - containing capsules were subjected to the analysis by the proposed method . the same procedure for determination of drug dosage form the peak area of five injections was compared by a standard solution of stp with the same concentration level and the mean recovery percentage was calculated to be 99.34 0.59% . this acceptable value indicated the applicability of the method for the routine quality control of stp capsules without interference from the excipients . this was evidenced from the good label claim percentage as well as the absence of any peaks in the chromatogram of the capsule extract solution ( figure 2 ) . the present study represents the first report that deals with the development and validation of a stability - indicating hplc - dad method for determination of stp . this study is a typical example of development of a stability - indicating assay , established following the recommendations of ich guidelines . the proposed method showed acceptable accuracy , precision , and selectivity . from the economical point of view , the method involved the native uv - absorbing property of stp , rather than expensive derivatizing analytical reagents . stp showed great stability at the various stress conditions ( thermal , alkaline , oxidative , and photodegradation ) . a single degradation product , dstp , was only formed with the exposure of stp to acidic degradation using hcl . structure elucidation of the acidic degradation product using h - nmr , c - nmr , and ion trap mass spectrometry revealed substitution of oh functionality of stp with chloride following treatment of stp with hydrochloric acid . additionally , kinetic study of the acidic degradation reaction demonstrated that stp undergoes first - order kinetics . the method is suitable for the determination of stp in bulk and capsule form without any interference from the degradation products , and it is recommended for routine use in quality control industry laboratories .

a rapid , simple , sensitive , and accurate isocratic reversed - phase stability - indicating high performance liquid chromatography method has been developed and validated for the determination of stiripentol and its degradation product in its bulk form and pharmaceutical dosage form . chromatographic separation was achieved on a symmetry c18 column and quantification was achieved using photodiode array detector ( dad ) . the method was validated in accordance with the ich requirements showing specificity , linearity ( r2 = 0.9996 , range of 125 g / ml ) , precision ( relative standard deviation lower than 2% ) , accuracy ( mean recovery 100.08 1.73 ) , limits of detection and quantitation ( lod = 0.024 and loq = 0.081 g / ml ) , and robustness . stiripentol was subjected to various stress conditions and it has shown marked stability under alkaline hydrolytic stress conditions , thermal , oxidative , and photolytic conditions . stiripentol degraded only under acidic conditions , forming a single degradation product which was well resolved from the pure drug with significantly different retention time values . this degradation product was characterized by 1h - nmr and 13c - nmr spectroscopy as well as ion trap mass spectrometry . the results demonstrated that the method would have a great value when applied in quality control and stability studies for stiripentol .

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

stp is marketed as a racemic mixture of two enantiomers : r(+)-stp and s()-stp of which r(+)-stp has about 2.4-fold greater anticonvulsant potency and it is also associated with ~3-fold faster elimination rate than s()-stp . has been approved by the european and canadian marketing authorization for the treatment of dravet syndrome or severe myoclonic epilepsy in infancy ( smei ) in conjunction with sodium valproate and clobazam when seizures are not adequately controlled with the association of these two medications [ 9 , 10 ] . stp , at relevant clinical concentrations ( 30300 m ) , markedly increases the mean open duration of gabaa receptor dependent chloride channels by a barbiturate - like mechanism . stability testing of pharmaceutical dosage form of stp or any other drug , ( i.e. , purity and stability of the active ingredient and/or final product ) is imperative for the optimum and efficient delivery of its therapeutic activity to the patients . the purpose of stability testing is to investigate changes in the quality of a drug substance or a drug product with time under the influence of environmental factors , such as temperature , humidity , and light . moreover , stability testing establishes the recommended storage conditions and shelf life for the drug product [ 14 , 15 ] . based on the aforementioned importance of stability testing , it is vital to investigate degradation pathways and the intrinsic stability of the drug , compatibility of the drug with the excipients in the dosage form , and the long - term effects of the environment . recently , the need of establishing a stability - indicating assay method for the stability testing has become more clearly mandated in the official guidelines at the international conference on harmonization ( ich ) and united states pharmacopeia ( usp ) . a thorough literature survey revealed that only few high performance liquid chromatography ( hplc ) methods were reported for the determination of stp to study its pharmacokinetics [ 7 , 18 ] , enantioseparation , and its estimation in plasma . consequently , a stability - indicating method for determination of stp in its bulk drug and pharmaceutical capsules is important . accordingly , the present study describes for the first time a comprehensive stability - indicating study for stp . this study aims to develop and fully validate a rapid , simple , and accurate hplc - dad method to quantify stp in the presence of its degradation product in bulk and pharmaceutical dosage form . additionally , structure elucidation techniques including the h - nmr , c - nmr , and mass spectrometry were used to identify stp degradation product . diacomit capsules ( biocodex , montrouge , france ) , labeled to contain 250 mg ( as the anhydrous base ) per capsule , were procured from the local market . hplc - grade solvents and reagent - grade sodium hydroxide , hydrochloric acid , hydrogen peroxide , potassium dihydrogen phosphate , and orthophosphoric acid were purchased from merck ( darmstadt , germany ) . hplc apparatus consists of a waters breeze system consisting of 1525 binary pump with online degasser , 717 + autosampler , 5ch thermostated column compartment , and a 2996 photodiode array detector ( dad ) ( waters corporation , milford , ma , usa ) . the chromatographic separations were performed on a symmetry c18 column ( 3.5 m , 75 mm 4.6 mm i.d ) manufactured by waters corporation , milford , ma , usa . separations were performed in isocratic mode using a mobile phase consisting of acetonitrile and 50 mm potassium dihydrogen phosphate buffer ( 60 : 40 , v / v ) , and ph was adjusted to 4.1 0.1 with 10% phosphoric acid solution . additionally , quantitation of stp and its acidic degradation product was performed at 262.5 nm . peak identity was confirmed by retention time comparison and comparison of the spectra obtained from the dad detector . stp stock solution ( 1 mg / ml ) was prepared by dissolving 25 mg of stp reference standard material into 25 ml methanol in a 25 ml volumetric flask and completing the volume properly . this stock solution was later diluted with methanol to produce a working standard solution of 100 g / ml . an eight - point calibration curve ( 1 , 2 , 4 , 6 , 10 , 15 , 20 , and 25 g / ml ) was constructed by plotting the peak area of stp ( y ) versus stp nominal concentration ( x ) . the linear regression equation was used to calculate the concentrations of stp in aqueous solutions and dosage form based on their peak area . this solution ( 0.5 mg / ml ) was diluted as required for analysis . thermal degradation of the drug substance in its bulk form and capsules was carried out in both solid and solution state . solutions were then diluted with mobile phase to obtain a concentration of 10 g / ml , and a volume of 20 l of each solution was injected into the hplc system . meanwhile , a volume of 1 ml of stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ; prepared as previously mentioned in section 2.5 . ) the solutions were cooled to room temperature ( 25 5c ) and diluted with mobile phase to yield final concentrations of 10 g / ml ; and a volume of 20 l of each solution was then injected into the hplc system . aliquots of 1 ml of stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ) were transferred to small round bottom flasks . the prepared solutions were heated for 1 , 2 , and 3 hours at 60c in a water bath . the samples were cooled to room temperature ( 25 5c ) , neutralized with an amount of acid or base equivalent to that of the previously added . the resulting neutral solutions were then diluted with mobile phase to 10 g / ml and a volume of 20 l was injected into the hplc system . four aliquots of 1 ml stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ) were transferred into small round bottom flasks . the contents were then mixed separately with 4 ml of ( 1 , 6 , 10 , and 30% ) hydrogen peroxide solutions , and the reaction mixtures were kept at ambient temperature for 2 hours with intermittent shaking . the resulting solutions were diluted with mobile phase to 10 g / ml and a volume of 20 l of each solution was injected into the hplc system . solutions were then diluted with mobile phase to obtain a concentration of 10 g / ml , and a volume of 20 l of each solution was injected into the hplc system . similarly , an aqueous solution of stp ( 1 mg / ml ) and capsule extract solution ( 1 mg / ml ) were exposed to the uv light for 72 hours and diluted with mobile phase to 10 g / ml and a volume of 20 l of each was then injected into the hplc system . the acid degradation product ( dstp ) was extracted from the residue using diethyl ether which was subsequently evaporated under reduced pressure . the collected eluate was evaporated under vacuum to give pure dstp , which was identified through h - nmr , c - nmr , and mass spectrometry . the h - nmr data were represented in terms of chemical shifts , multiplicity , and number of protons . purified dstp was dissolved in acetonitrile ( 5 g / ml ) , and a connector was used instead of the column to allow direct injection of the sample . four sets of stp working solution ( 100 g / ml ) and capsule extract solution ( 100 g / ml ) , 1 ml of each , were subjected to acid degradation using 4 ml of 0.005 n , 0.01 n , 0.05 n , and 0.1 n hydrochloric acid ( procedure described in section 2.6.2 ) . ( 0 , 15 , 30 , 60 , 120 , 180 , and 240 min ) . the resulting neutral solutions were then diluted with mobile phase to 10 g / ml and a volume of 20 l was injected into the hplc system and the peak area of stp was compared with a freshly prepared standard solution . optimized chromatographic conditions were established after a number of preliminary experiments for selecting the most efficient mobile system , separation column , and detection wavelength range . selection of the proper system was based on its ability to give good separation between the pure stp and its possible impurities and/or degradation products . different mobile phase compositions and ph ranges were tested to achieve a more symmetric peak and shorter retention time for stp . ultimately , optimum chromatographic separation was achieved as described in section 2.2 . under these chromatographic conditions , the run time of each sample was 6 min , and the retention time of stp was 1.80 0.07 min ( n = 3 ) ( figure 2 ) . which would negatively affect the results . this supported the avoidance of internal standard use , which is usually added to increase the method efficiency when there are multisteps in sample preparation . the method was fully validated in accordance with the recommendations of ich and is rugged and adequately sensitive for routine stp analysis . using the above - mentioned optimum chromatographic conditions , three independent calibration curves were constructed correlating the detector signals with the corresponding stp concentrations . the standard deviation values of each concentration point ( triplicates ) did not exceed 2% . the method revealed a good linear calibration fit in the range of 125 g / ml . system suitability tests for the principle peak and its degradation product were evaluated using stp solution of 12 g / ml . the capacity factor k = 0.8884 indicates that the stp peak is well resolved with respect to the void volume . the lower limit of quantitation ( loq ) is the lowest concentration of the standard curve which can be measured with acceptable accuracy and precision for the analyte . the limit of detection ( lod ) and the lower limit of quantitation ( loq ) were calculated based on the following equations : ( 1)lod=3.3s , loq=10s , where is the standard deviation of the intercept ( 115.82 3300 ) of regression line and s is the slope ( 30875.64 248.86 ) of regression line of the calibration curve . the lod and loq values were 0.081 and 0.242 g / ml , respectively . accuracy and precision were determined by the recovery study of known amounts in accordance with ich recommendation . five consecutive injections of stp sample solutions ( 2 , 6 , 10 , 15 , and 20 g / ml ) showed excellent intraday accuracy ( 98.74101.64% ) and precision ( 0.642.32% ) ( table 2 ) . in order to measure the extent of the method robustness , the most critical parameters were interchanged while keeping the other parameters unchanged , and in parallel the chromatographic profile was observed and recorded . robustness of the proposed method was determined using stp concentration of 10 g / ml and changing the flow rate ( 0.91.1 ml / min ) and column temperature ( 2c ) and change in the ph of mobile phase ( 0.2 ) . no significant changes in assay value were observed ( table 3 ) by changing the chromatographic conditions which confirms the robustness of the method . ruggedness of the method was determined by using mobile phase components from two different manufactures and changing analysts . there was no significant change in the retention time of stp which was observed ; % rsd was 0.310.63% indicating the ruggedness of the method . the stability of the drug in solution during analysis was determined by repeated analysis of samples during the course of experimentation on the same day and also after storage of the drug solutions ( calibration samples ) for 24 , 48 , and 72 hours under laboratory bench condition ( 25 1c ) and under refrigeration ( 4.0 0.5c ) . the mean % rsd between peak areas for the samples stored under refrigeration ( 4.0 0.5c ) and at laboratory temperature ( 25 1c ) was found to be 0.58% and 0.93% , respectively , suggesting that the drug solution can be stored without any degradation over the time interval studied . the ich guideline entitled stability testing of drug substances and products requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substance and provide a rapid identification of differences that might result from changes in the manufacturing processes or source sample . an ideal stability - indicating method is one that quantifies the standard drug alone and also resolves its degradation products . as described in the experimental section , different stress conditions were applied : boiling , acid / base forced degradation , oxidation , and irradiation with uv light . from this investigation , it was clear that , in case of boiling , base - forced degradation ( 1 n naoh ) , oxidation ( h2o2 ) , and uv irradiation ( solid state and solution ) , stp was stable under the employed stress conditions as no degradation products were observed in their chromatograms ( figures 3(a ) and 3(b ) ) which confirms the data from european medicines agency ( emea ) . however , in case of acidic conditions , one degradation product was observed , dstp , at a retention time of 4.25 min ( peak resolution from stp peak was 5.89 ) ( figures 3(c)3(e ) ) . the intact stp concentration was calculated and found to be degraded by 76.03 , 83.51 , 84.87 , and 95% in case of acid degradation using 0.1 n hydrochloric acid after 1 , 2 , 3 , and 5 hours , respectively . acidic degradation of stp was studied using h - nmr , c - nmr ( figures 4 and 5 ) , and mass spectral data . h - nmr spectrum of dstp showed a signal integrating to one proton at 4.09 ppm which was assigned to be cl ch with concomitant disappearance of the signal of oh functionality at 1.56 ppm and the signal of the methine proton ( oh ch ) at 3.80 ppm . additionally , c - nmr of dstp showed signal at 74.8 ppm which was assigned to be cl ch with concomitant disappearance of the signal of oh ch at 83.9 ppm . h - nmr ( cdcl3 ) of stp : ( ppm ) = 0.88 ( s , 9h , t - butyl ) , 1.56 ( s , 1h , oh ) , 3.80 ( d , j = 7.0 hz , 1h , choh ) , 5.86 ( s , 2h , och2o ) , 6.05 ( dd , j = 15.7 , 7.85 hz , 1h , ch = ch c6h3 ) , 6.39 ( d , j = 15.5 hz , 1h , ch = ch c6h3 ) , 6.67 ( d , j = 8.0 hz , 1h , ar h ) , 6.72 ( d , j = 7.95 hz , 1h , ar c - nmr ( cdcl3 ) of stp : ( ppm ) = 26.5 ( c(ch3)3 ) , 32.8 ( c(ch3)3 ) , 81.9 ( ch oh ) , 101.1 ( och2o ) , 107.1 , 108.3 , 121.1 ( ar ch ) , 126.9 , 131.5 ( ch = ch c6h3 , ch = ch c6h3 , ar ms m / z ( esi ) : 217 [ m-18 + h ] . h - nmr ( cdcl3 ) of dstp : ( ppm ) = 0.83 ( s , 9h , t - butyl ) , 4.09 ( d , j = 9.5 hz , 1h , ch cl ) , 5.74 ( s , 2h , och2o ) , 5.92 ( dd , j = 15.4 , 7.95 hz , 1h , ch = ch c6h3 ) , 6.23 ( d , j = 15.5 hz , 1h , ch = ch c6h3 ) , 6.54 ( d , j = 8.0 hz , 1h , ar h ) , 6.60 ( d , j = 7.96 hz , 1h , ar c - nmr ( cdcl3 ) of dstp : ( ppm ) = 26.8 ( c(ch3)3 ) , 36.4 ( c(ch3)3 ) , 74.8 ( ch cl ) , 101.2 ( och2o ) , 105.9 , 108.3 , 121.6 ( ar ch ) , 125.7 , 130.6 , 132.5 ( ch = ch c6h3 , ch = ch c6h3 , ar c ) , 147.8 , 148.1 ( ar c ) . it is evident from the results obtained previously that the proposed method gave satisfactory results with the analysis of stp in bulk . the same procedure for determination of drug dosage form the peak area of five injections was compared by a standard solution of stp with the same concentration level and the mean recovery percentage was calculated to be 99.34 0.59% . this acceptable value indicated the applicability of the method for the routine quality control of stp capsules without interference from the excipients . this was evidenced from the good label claim percentage as well as the absence of any peaks in the chromatogram of the capsule extract solution ( figure 2 ) . the present study represents the first report that deals with the development and validation of a stability - indicating hplc - dad method for determination of stp . this study is a typical example of development of a stability - indicating assay , established following the recommendations of ich guidelines . the proposed method showed acceptable accuracy , precision , and selectivity . from the economical point of view , the method involved the native uv - absorbing property of stp , rather than expensive derivatizing analytical reagents . stp showed great stability at the various stress conditions ( thermal , alkaline , oxidative , and photodegradation ) . a single degradation product , dstp , was only formed with the exposure of stp to acidic degradation using hcl . structure elucidation of the acidic degradation product using h - nmr , c - nmr , and ion trap mass spectrometry revealed substitution of oh functionality of stp with chloride following treatment of stp with hydrochloric acid . additionally , kinetic study of the acidic degradation reaction demonstrated that stp undergoes first - order kinetics . the method is suitable for the determination of stp in bulk and capsule form without any interference from the degradation products , and it is recommended for routine use in quality control industry laboratories .

the cancer genome atlas ( tcga ) project included comprehensive genomic characterization of glioblastoma genes and core pathways . this pilot project confirmed that an atlas of changes could be created for specific cancer types . the tcga research network identified 19 nf1 ( neurofibromin 1 ) somatic mutations , egfr ( epidermal growth factor receptor ) alterations , erbb2 ( 11 somatic v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 ) mutations , and somatic mutations in the pi3k ( phosphatidylinositide 3-kinase ) complex in glioblastoma . parsons et al . found a novel idh1 ( isocitrate dehydrogenase 1 ) candidate cancer gene in 12 % of glioblastoma multiforme ( gbm ) patients who had distinct clinical characteristics : younger age and an improved clinical prognosis . furthermore , the median survival of patients with idh1 c.g395a ; p.r132h was 3.8 years compared with 1.1 years in patients with wild - type ( wt ) idh1 . interestingly , all mutations in the idh1 gene resulted in amino acid substitutions at position 132 , an evolutionarily conserved residue located within the isocitrate binding site [ 3 , 4 ] . patients with wt idh1 often had a mutation at codon 172 of the idh2 ( isocitrate dehydrogenase 2 ) gene . mutations in both the idh1 and idh2 genes reduced the enzymatic catalytic activity of the encoded protein . the idh1 protein is localized in the cytoplasm and peroxisomes , whereas the idh2 enzyme is localized in mitochondria . both idh1 and idh2 catalyze the oxidative decarboxylation of isocitrate for alpha - ketoglutarate ( alpha - kg ) production . studies using a transformed human embryonic kidney ( hek ) 293 t cell line expressing idh1 mutants ( r132h , r132c , or 132s ) found at least an 80 % reduction in activity compared with that observed for wt idh1 . the presence of five common mutations at the same codon ( 132 ) simplifies the use of several molecular methods , such as direct sequencing and use of an r132h mutation - specific anti - idh1 antibody , for diagnostic purposes . recent methylation data from parental , idh1 wt , and mutant idh1 astrocytes have indicated an important role of mutant idh1 in primary human astrocytes that alters specific histone markers , induces extensive dna hypermethylation , and reshapes the methylome in a fashion that mirrors the changes observed in cpg island methylator phenotype ( cimp)-positive lower - grade gliomas . these data demonstrate that the idh1 c.g395a ; p.r132h mutation is the molecular basis of cimp in gliomas and represents an advancement in the understanding of oncogenesis and the correlation between genomic and epigenomic changes in gliomas . on the other hand , recent studies on the role of mutant idh1 , in which a selective r132h - idh1 inhibitor ( agi-5198 ) delayed growth and promoted differentiation of glioma cells , showed that mutant idh1 may promote glioma growth through mechanisms beyond its well - characterized epigenetic effects . we assessed the presence of the somatic idh1 c.g395a ; p.r132h mutation in polish glioma patients . we analyzed mutation status using immunohistochemistry and molecular analyses , and established the prognostic value of idh1 status in correlation with the clinical outcome . we retrospectively reviewed a cohort of 139 patients with histopathologically confirmed glioma diagnosed from 2000 to 2011 at the 10th military clinical hospital and the franciszek lukaszczyk oncology center ( both in bydgoszcz , poland ) . the ages of the patients ranged from 18 to 76 years ( average 36 years ) . patients were divided into groups according to the histological type of the tumor , whether the tumor was primary ( meaning detected primarily ) or secondary ( meaning a tumor recurrence after previously performed surgery ) , the average age at diagnosis , and the presence or absence of mutations in the idh1 gene ( table 1).table 1detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymomatumornumber of patientstumor primary / secondaryidh1 r132h / primaryidh1 r132h / secondaryaverage age at diagnosis ( years)idh1 r132h positive / total ( % ) iwho grade i astrocytoma33/03/30/028.30/3 ( 0)iiwho grade ii astrocytoma7568/738/684/739.342/75 ( 56.0)iiiwho grade iii astrocytoma170/170/016/173416/17 ( 94.1)ivwho grade iv glioblastoma multiforme161/150/18/15438/16 ( 50)vwho grade ii oligoastrocytoma2415/912/157/936.719/24 ( 79.1 ) who grade ii ganglioglioma32/12/21/133.63/3 ( 100 ) oligoastrocytoma2113/810/136/837.116/21 ( 76.2)viwho grade ii oligodendroglioma20/00/00/042.52/2 ( 100)viiwho grade ii ependymoma20/00/00/042.50/2 ( 0 ) who world health organization detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymoma who world health organization all samples were analyzed as a blind samples by immunohistochemistry and co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) high - resolution melting - curve analysis ( hrm ) . idh1-p.r132h expression was assessed on formalin - fixed , paraffin - embedded ( ffpe ) tissue , using the immunoperoxidase method based on the two - step visualization system en vision / hrp ( dako polska sp . z o.o . , warsaw , poland ) and the anti - human idh1 r132h astrocytoma , oligodendroglioma tumor cell marker mouse monoclonal antibody clone h09 ( dianova gmbh , hamburg , germany ) . the signal of the anti - idh1 r132h antibody was estimated under an olympus bx41 microscope ( magnification 10 and 20 ) , in accordance with the dianova instructions . two clinical pathomorphologists ( t.s . and w.j . ) , who were unaware of the patient characteristics , examined the idh1 expression in a blinded manner . previous comparison of two idh1 monoclonal antibodies , dia - h09 and imab-1 , had shown a higher signal - to - background ratio for dia - h09 than for imab-1 . samples with < 10 % positively stained cells were rated as negative , in accordance with previous data . idh1 expression was marked both in the stroma and in all histologically identifiable tumor cells . genomic dna was isolated automatically from the ffpe tissue using a magna dna isolation kit , in accordance with the manufacturer s instructions ( roche applied science , penzberg , germany ) . all sanger sequencing was performed bidirectionally , using published primers , and was analyzed using chromas software . hrm analysis was performed on all samples , using the cold pcr method , hrm guidance , and primers for amplification on a light cycler 480 ( roche ) . fluorescent melting curves were evaluated using light cycler 480 software release 1.5.0 sp4 ( roche ) , and group sensitivity was set at 0.2 for experiments . all idh1 results were expressed as means , using statsoft software ( 2009 ; statsoft , inc . , the association between overall survival and the presence of the idh1 c.g395a ; p.r132h mutation and wt idh1 based on immunohistochemical analysis was estimated using the kaplan meier method and assessed using the log - rank test . overall survival was calculated from the day of the first surgery until death or the end of the follow - up period . we retrospectively reviewed a cohort of 139 patients with histopathologically confirmed glioma diagnosed from 2000 to 2011 at the 10th military clinical hospital and the franciszek lukaszczyk oncology center ( both in bydgoszcz , poland ) . the ages of the patients ranged from 18 to 76 years ( average 36 years ) . patients were divided into groups according to the histological type of the tumor , whether the tumor was primary ( meaning detected primarily ) or secondary ( meaning a tumor recurrence after previously performed surgery ) , the average age at diagnosis , and the presence or absence of mutations in the idh1 gene ( table 1).table 1detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymomatumornumber of patientstumor primary / secondaryidh1 r132h / primaryidh1 r132h / secondaryaverage age at diagnosis ( years)idh1 r132h positive / total ( % ) iwho grade i astrocytoma33/03/30/028.30/3 ( 0)iiwho grade ii astrocytoma7568/738/684/739.342/75 ( 56.0)iiiwho grade iii astrocytoma170/170/016/173416/17 ( 94.1)ivwho grade iv glioblastoma multiforme161/150/18/15438/16 ( 50)vwho grade ii oligoastrocytoma2415/912/157/936.719/24 ( 79.1 ) who grade ii ganglioglioma32/12/21/133.63/3 ( 100 ) oligoastrocytoma2113/810/136/837.116/21 ( 76.2)viwho grade ii oligodendroglioma20/00/00/042.52/2 ( 100)viiwho grade ii ependymoma20/00/00/042.50/2 ( 0 ) who world health organization detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymoma who world health organization all samples were analyzed as a blind samples by immunohistochemistry and co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) high - resolution melting - curve analysis ( hrm ) . idh1-p.r132h expression was assessed on formalin - fixed , paraffin - embedded ( ffpe ) tissue , using the immunoperoxidase method based on the two - step visualization system en vision / hrp ( dako polska sp . warsaw , poland ) and the anti - human idh1 r132h astrocytoma , oligodendroglioma tumor cell marker mouse monoclonal antibody clone h09 ( dianova gmbh , hamburg , germany ) . the signal of the anti - idh1 r132h antibody was estimated under an olympus bx41 microscope ( magnification 10 and 20 ) , in accordance with the dianova instructions . two clinical pathomorphologists ( t.s . and w.j . ) , who were unaware of the patient characteristics , examined the idh1 expression in a blinded manner . previous comparison of two idh1 monoclonal antibodies , dia - h09 and imab-1 , had shown a higher signal - to - background ratio for dia - h09 than for imab-1 . samples with < 10 % positively stained cells were rated as negative , in accordance with previous data . idh1 expression was marked both in the stroma and in all histologically identifiable tumor cells . genomic dna was isolated automatically from the ffpe tissue using a magna dna isolation kit , in accordance with the manufacturer s instructions ( roche applied science , penzberg , germany ) . all sanger sequencing was performed bidirectionally , using published primers , and was analyzed using chromas software . hrm analysis was performed on all samples , using the cold pcr method , hrm guidance , and primers for amplification on a light cycler 480 ( roche ) . fluorescent melting curves were evaluated using light cycler 480 software release 1.5.0 sp4 ( roche ) , and group sensitivity was set at 0.2 for experiments . all idh1 results were expressed as means , using statsoft software ( 2009 ; statsoft , inc . , tulsa , ok , usa ) . the association between overall survival and the presence of the idh1 c.g395a ; p.r132h mutation and wt idh1 based on immunohistochemical analysis was estimated using the kaplan meier method and assessed using the log - rank test . overall survival was calculated from the day of the first surgery until death or the end of the follow - up period . the clinical characteristics of 137 samples derived from glioma patients , principally with astrocytomas , are listed in table 1 . all cases had standard surgery , and the pathological material was ffpe tissue , using the same procedure at the 10th military clinical hospital of bydgoszcz . we evaluated 87 primary tumors and 48 recurrences in 135 glioma samples ; four samples ( oligodendroglioma and ependymoma ) were not classified as primary tumors or recurrences . the average age at diagnosis was 28.3 years for world health organization ( who ) grade i astrocytoma and increased for who grade ii , iii , and iv astrocytomas , up to 43 years . the average age at diagnosis of patients with ependymoma and oligodendroglioma was 42.5 years ( table 1 ) . idh1 gene mutations were assessed via immunohistochemistry on all 139 samples ( table 1 ) . in addition , amplicons ( including exon 4 ) were checked using the cold pcr hrm method ( fig . 1 ) , or selected ffpe tissue samples were sequenced . we analyzed 87 samples that were positive for idh1 p.r132h , and 52 negative tumor samples ( table 1 ) . idh1 p.r132h staining was strong in the cytoplasm and weaker in the nucleus ( fig . 2 ) . hrm analysis distinguished two major groups of amplicons : with idh1 wt status in codon 132 , and the r132h mutation . in addition , we found a few different melting curves , which we pooled into a third group : negative for r132h and negative for wt status.fig . a and b : a representative idh1 wild - type ( wt ) sample ( no . 212 ) from a world health organization ( who ) grade ii fibrillary astrocytoma , as analyzed using a sanger sequencing and b immunohistochemistry . c and d : a representative idh1 c.g395a ; p.r132h sample ( no . 68 ) from an oligodendroglioma , as analyzed using c sanger sequencing and d immunohistochemistry ( which was strongly positive for idh1 with the p.r132h mutation ) . e detection of idh1 wt in a fibrillary astrocytoma sample ( no . 212 ) , a gemistocytic astrocytoma sample ( no . 224 ) , and a secondary glioblastoma multiforme ( gbm ) sample ( no . 94 ) ; and detection of idh1 p.r132h in a secondary gbm sample ( no . 92 ) , a protoplasmatic astrocytoma sample ( no . 221 ) , a fibrillary astrocytoma sample ( no . 68 ) , and an oligodendroglioma sample ( no . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . ; immunohistochemistry ( ihc ) magnification 400 . the green and red stars represent wild - type and p.r132h idh1 , respectively ; difference plot normalized and temperature ( temp)-shifted with melting curve for heterozygous mutation ( above the baseline ) and idh1 wt ( below the baseline ) ; the baseline ( in red ) represents the no - template control ( ntc)fig . 2immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma analysis of idh1 ( isocitrate dehydrogenase 1 ) mutation status . a and b : a representative idh1 wild - type ( wt ) sample ( no . 212 ) from a world health organization ( who ) grade ii fibrillary astrocytoma , as analyzed using a sanger sequencing and b immunohistochemistry . c and d : a representative idh1 c.g395a ; p.r132h sample ( no . 68 ) from an oligodendroglioma , as analyzed using c sanger sequencing and d immunohistochemistry ( which was strongly positive for idh1 with the p.r132h mutation ) . e detection of idh1 wt in a fibrillary astrocytoma sample ( no . 212 ) , a gemistocytic astrocytoma sample ( no . 224 ) , and a secondary glioblastoma multiforme ( gbm ) sample ( no . 94 ) ; and detection of idh1 p.r132h in a secondary gbm sample ( no . 92 ) , a protoplasmatic astrocytoma sample ( no . 221 ) , a fibrillary astrocytoma sample ( no . 68 ) , and an oligodendroglioma sample ( no . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . ; immunohistochemistry ( ihc ) magnification 400 . the green and red stars represent wild - type and p.r132h idh1 , respectively ; difference plot normalized and temperature ( temp)-shifted with melting curve for heterozygous mutation ( above the baseline ) and idh1 wt ( below the baseline ) ; the baseline ( in red ) represents the no - template control ( ntc ) immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma the correlation between expression of the idh1 r132h mutant protein , as analyzed using an intensity scale in relation to the percentage of positive reactions , is presented in fig . 4 . interestingly , the median survival of 30 grade ii astrocytoma patients with lower idh1 p.r132h expression of 1 + or 2 + ( measured on a scale of 0 to 4 , according to the intensity of the immunohistochemical reaction ) [ fig . 4a ] was 12 months longer than that of the idh1 p.r132h group , with a score of 3+/4 + ( grade ii astrocytoma , n = 12 ) . this association was estimated using the kaplan meier method and the log - rank test , and was significant ( p < 0.05 ) [ fig . in contrast , the anti - idh1 r132h antibody did not stain who grade i pilocytic astrocytoma and , as expected , no positive staining was observed in who grade ii ependymoma . representative results of the immunohistochemistry of idh1 p.r132h in who grade i astrocytoma ( pilocytic ; n = 3 ) , who grade ii astrocytoma ( diffuse , fibrillary , and gemistocytic ; n = 75 ) , who grade iii astrocytoma ( astrocytoma and anaplastic ; n = 17 ) , and who grade iv glioblastoma multiforme ( n = 16 ) are presented in fig . the average age at diagnosis of astrocytoma ranged from 28.3 years for grade i to 43 years for gbm . in contrast , the r132h mutation was observed in 94.1 % of the anaplastic astrocytoma samples , all in secondary tumors ( table 1 ) . the idh1 c.g395a ; p.r132h mutation was frequent in tumors with higher who grades : grade iii astrocytoma ( 94.1 % ) , grade ii anaplastic oligodendroglioma ( 76.5 % ) , and grade iii anaplastic oligodendroglioma ( 75 % ) . evaluation of idh1 status in our gbm group indicated that half of the samples had the r132h mutation and the other half had idh1 wt ; however , the mutation was present in 100 % of the secondary tumors [ table 1 ] . in selected samples ( 10 % ) , idh1 mutation status was determined using three methods : immunohistochemistry , direct dna sequencing , and cold pcr hrm . in mutated samples , we confirmed the presence of a heterozygous c.g395a ; p.r132h mutation . in samples with no idh1 r132h staining , we confirmed the absence of the c.g395a ; p.r132h mutation , and dna sequencing did not reveal the presence of any additional mutations , such as idh1 r132c , r132 g , or r132s in the evaluated 10 % of the entire cohort . also , we tested whether cold pcr hrm could be used as a potential screening methodology for quick detection of idh1 mutations within codon 132 . most of the hrm data were in concordance with immunohistochemistry ; in a few cases , we detected the r132h mutation in a dna sample isolated from the sample analyzed by immunohistochemistry as idh wt . also , a few cases had different melting curves , which we pooled into a third group but , unfortunately , sanger sequencing of those samples was too unclear to provide an unambiguous answer . figure 1 presents the results of the immunohistochemical analysis , sequencing , and hrm , which yielded consistent results for mutation analysis purposes . 3 show the patient characteristics regarding the presence or absence of the idh1 c.g395a ; p.r132h mutation in primary and secondary tumors , and overall survival in a cohort of glioma patients according to histology and idh1 status . the idh1 c.g395a ; p.r132h mutation was associated with longer survival in the grade ii astrocytoma , gbm , and pooled groups of patients with who grade ii glioma : oligoastrocytoma , oligodendroglioma , and ganglioglioma . a grade iii astrocytoma with idh1 wt status was found in only one patient , whereas the p.r132h mutation was detected in 16 secondary tumors ( 94.1 % of grade iii astrocytomas ) . a kaplan meier survival analysis showed that grade ii astrocytoma patients ( fig . 3a ) and who grade ii glioma patients [ oligoastrocytoma / oligodendroglioma / ganglioglioma ] ( fig . 3c ) with the p.r132h idh1 mutation had significantly longer overall survival ( up to 25 months ; p = 0.05 , log - rank test ) . conversely , kaplan meier curves of overall survival ( p = 0.05 ) in secondary gbm indicated an overall survival that was 9 months shorter in the idh1 wt cohort ( fig . 3kaplan meier curves of overall survival ( p = 0.05 ) according to idh1 ( isocitrate dehydrogenase 1 ) status in a grade ii astrocytoma ; b glioblastoma multiforme ; and c oligoastrocytoma , oligodendroglioma , and ganglioglioma samples . wt wild - type kaplan meier curves of overall survival ( p = 0.05 ) according to idh1 ( isocitrate dehydrogenase 1 ) status in a grade ii astrocytoma ; b glioblastoma multiforme ; and c oligoastrocytoma , oligodendroglioma , and ganglioglioma samples . wt wild - type another kaplan meier analysis of grade ii astrocytoma patients revealed that those who had low p.r132h idh1 expression had significantly longer overall survival than did patients with high expression of the protein ( 42 versus 30 months ; p = 0.05 , log - rank test ) [ fig . although the number of patients was small , a similar trend was observed in the glioblastoma group of patients ( data not shown).fig . 4overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . a intensity scale in relation to the percentage of positive reactions ; b kaplan meier curves of overall survival between the two groups of grade ii astrocytoma patients overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . a intensity scale in relation to the percentage of positive reactions ; b kaplan meier curves of overall survival between the two groups of grade ii astrocytoma patients the clinical characteristics of 137 samples derived from glioma patients , principally with astrocytomas , are listed in table 1 . all cases had standard surgery , and the pathological material was ffpe tissue , using the same procedure at the 10th military clinical hospital of bydgoszcz . we evaluated 87 primary tumors and 48 recurrences in 135 glioma samples ; four samples ( oligodendroglioma and ependymoma ) were not classified as primary tumors or recurrences . the average age at diagnosis was 28.3 years for world health organization ( who ) grade i astrocytoma and increased for who grade ii , iii , and iv astrocytomas , up to 43 years . the average age at diagnosis of patients with ependymoma and oligodendroglioma was 42.5 years ( table 1 ) . idh1 gene mutations were assessed via immunohistochemistry on all 139 samples ( table 1 ) . in addition , amplicons ( including exon 4 ) were checked using the cold pcr hrm method ( fig . 1 ) , or selected ffpe tissue samples were sequenced . we analyzed 87 samples that were positive for idh1 p.r132h , and 52 negative tumor samples ( table 1 ) . idh1 p.r132h staining was strong in the cytoplasm and weaker in the nucleus ( fig . 2 ) . hrm analysis distinguished two major groups of amplicons : with idh1 wt status in codon 132 , and the r132h mutation . in addition , we found a few different melting curves , which we pooled into a third group : negative for r132h and negative for wt status.fig . a and b : a representative idh1 wild - type ( wt ) sample ( no . 212 ) from a world health organization ( who ) grade ii fibrillary astrocytoma , as analyzed using a sanger sequencing and b immunohistochemistry . c and d : a representative idh1 c.g395a ; p.r132h sample ( no . 68 ) from an oligodendroglioma , as analyzed using c sanger sequencing and d immunohistochemistry ( which was strongly positive for idh1 with the p.r132h mutation ) . e detection of idh1 wt in a fibrillary astrocytoma sample ( no . 212 ) , a gemistocytic astrocytoma sample ( no . 224 ) , and a secondary glioblastoma multiforme ( gbm ) sample ( no . 94 ) ; and detection of idh1 p.r132h in a secondary gbm sample ( no . 92 ) , a protoplasmatic astrocytoma sample ( no . 221 ) , a fibrillary astrocytoma sample ( no . 68 ) , and an oligodendroglioma sample ( no . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . ; immunohistochemistry ( ihc ) magnification 400 . the green and red stars represent wild - type and p.r132h idh1 , respectively ; difference plot normalized and temperature ( temp)-shifted with melting curve for heterozygous mutation ( above the baseline ) and idh1 wt ( below the baseline ) ; the baseline ( in red ) represents the no - template control ( ntc)fig . 2immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma analysis of idh1 ( isocitrate dehydrogenase 1 ) mutation status . a and b : a representative idh1 wild - type ( wt ) sample ( no . 212 ) from a world health organization ( who ) grade ii fibrillary astrocytoma , as analyzed using a sanger sequencing and b immunohistochemistry . c and d : a representative idh1 c.g395a ; p.r132h sample ( no . 68 ) from an oligodendroglioma , as analyzed using c sanger sequencing and d immunohistochemistry ( which was strongly positive for idh1 with the p.r132h mutation ) . e detection of idh1 wt in a fibrillary astrocytoma sample ( no . 212 ) , a gemistocytic astrocytoma sample ( no . 224 ) , and a secondary glioblastoma multiforme ( gbm ) sample ( no . 94 ) ; and detection of idh1 p.r132h in a secondary gbm sample ( no . 92 ) , a protoplasmatic astrocytoma sample ( no . 221 ) , a fibrillary astrocytoma sample ( no . 68 ) , and an oligodendroglioma sample ( no . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . ; immunohistochemistry ( ihc ) magnification 400 . the green and red stars represent wild - type and p.r132h idh1 , respectively ; difference plot normalized and temperature ( temp)-shifted with melting curve for heterozygous mutation ( above the baseline ) and idh1 wt ( below the baseline ) ; the baseline ( in red ) represents the no - template control ( ntc ) immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma the correlation between expression of the idh1 r132h mutant protein , as analyzed using an intensity scale in relation to the percentage of positive reactions , is presented in fig . 4 . interestingly , the median survival of 30 grade ii astrocytoma patients with lower idh1 p.r132h expression of 1 + or 2 + ( measured on a scale of 0 to 4 , according to the intensity of the immunohistochemical reaction ) [ fig . 4a ] was 12 months longer than that of the idh1 p.r132h group , with a score of 3+/4 + ( grade ii astrocytoma , n = 12 ) . this association was estimated using the kaplan meier method and the log - rank test , and was significant ( p < 0.05 ) [ fig . in contrast , the anti - idh1 r132h antibody did not stain who grade i pilocytic astrocytoma and , as expected , no positive staining was observed in who grade ii ependymoma . representative results of the immunohistochemistry of idh1 p.r132h in who grade i astrocytoma ( pilocytic ; n = 3 ) , who grade ii astrocytoma ( diffuse , fibrillary , and gemistocytic ; n = 75 ) , who grade iii astrocytoma ( astrocytoma and anaplastic ; n = 17 ) , and who grade iv glioblastoma multiforme ( n = 16 ) are presented in fig . the average age at diagnosis of astrocytoma ranged from 28.3 years for grade i to 43 years for gbm . in contrast , the r132h mutation was observed in 94.1 % of the anaplastic astrocytoma samples , all in secondary tumors ( table 1 ) . the idh1 c.g395a ; p.r132h mutation was frequent in tumors with higher who grades : grade iii astrocytoma ( 94.1 % ) , grade ii anaplastic oligodendroglioma ( 76.5 % ) , and grade iii anaplastic oligodendroglioma ( 75 % ) . evaluation of idh1 status in our gbm group indicated that half of the samples had the r132h mutation and the other half had idh1 wt ; however , the mutation was present in 100 % of the secondary tumors [ table 1 ] . in selected samples ( 10 % ) , idh1 mutation status was determined using three methods : immunohistochemistry , direct dna sequencing , and cold pcr hrm . in mutated samples , we confirmed the presence of a heterozygous c.g395a ; p.r132h mutation . in samples with no idh1 r132h staining , we confirmed the absence of the c.g395a ; p.r132h mutation , and dna sequencing did not reveal the presence of any additional mutations , such as idh1 r132c , r132 g , or r132s in the evaluated 10 % of the entire cohort . also , we tested whether cold pcr hrm could be used as a potential screening methodology for quick detection of idh1 mutations within codon 132 . most of the hrm data were in concordance with immunohistochemistry ; in a few cases , we detected the r132h mutation in a dna sample isolated from the sample analyzed by immunohistochemistry as idh wt . also , a few cases had different melting curves , which we pooled into a third group but , unfortunately , sanger sequencing of those samples was too unclear to provide an unambiguous answer . figure 1 presents the results of the immunohistochemical analysis , sequencing , and hrm , which yielded consistent results for mutation analysis purposes . 3 show the patient characteristics regarding the presence or absence of the idh1 c.g395a ; p.r132h mutation in primary and secondary tumors , and overall survival in a cohort of glioma patients according to histology and idh1 status . the idh1 c.g395a ; p.r132h mutation was associated with longer survival in the grade ii astrocytoma , gbm , and pooled groups of patients with who grade ii glioma : oligoastrocytoma , oligodendroglioma , and ganglioglioma . a grade iii astrocytoma with idh1 wt status was found in only one patient , whereas the p.r132h mutation was detected in 16 secondary tumors ( 94.1 % of grade iii astrocytomas ) . a kaplan meier survival analysis showed that grade ii astrocytoma patients ( fig . 3a ) and who grade ii glioma patients [ oligoastrocytoma / oligodendroglioma / ganglioglioma ] ( fig . 3c ) with the p.r132h idh1 mutation had significantly longer overall survival ( up to 25 months ; p = 0.05 , log - rank test ) . conversely , kaplan meier curves of overall survival ( p = 0.05 ) in secondary gbm indicated an overall survival that was 9 months shorter in the idh1 wt cohort ( fig . 3b).fig . 3kaplan meier curves of overall survival ( p = 0.05 ) according to idh1 ( isocitrate dehydrogenase 1 ) status in a grade ii astrocytoma ; b glioblastoma multiforme ; and c oligoastrocytoma , oligodendroglioma , and ganglioglioma samples . wt wild - type kaplan meier curves of overall survival ( p = 0.05 ) according to idh1 ( isocitrate dehydrogenase 1 ) status in a grade ii astrocytoma ; b glioblastoma multiforme ; and c oligoastrocytoma , oligodendroglioma , and ganglioglioma samples . wt wild - type another kaplan meier analysis of grade ii astrocytoma patients revealed that those who had low p.r132h idh1 expression had significantly longer overall survival than did patients with high expression of the protein ( 42 versus 30 months ; p = 0.05 , log - rank test ) [ fig . although the number of patients was small , a similar trend was observed in the glioblastoma group of patients ( data not shown).fig . 4overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . a intensity scale in relation to the percentage of positive reactions ; b kaplan meier curves of overall survival between the two groups of grade ii astrocytoma patients overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . a intensity scale in relation to the percentage of positive reactions ; b kaplan meier curves of overall survival between the two groups of grade ii astrocytoma patients replacement of arginine at position 132 by histidine is an evolutionarily conserved residue located within the isocitrate binding site . idh1 c.g395a ; p.r132h is more frequent in who grades ii and iii than it is in who grade iv ( up to 60 and 80 % , respectively ) , which is in accordance with our data and supports the hypothesis of a role of idh1 mutation in the early steps of oncogenesis ( table 1 ) . in the same study , only 510 % of primary gbm tumors had idh1 mutated status , versus 50 % in secondary gbm . our findings were similar to an observation by ichimura et al . that the p.r132h mutation was present in 53.3 % of secondary gbm cases ( table 1 ) . those data suggest that idh1 mutation status can be used as a diagnostic tool to distinguish primary gbm from other malignant astrocytomas . idh1 mutation has never been described in who grade i astrocytoma , which is in agreement with our observation ( table 1 ; fig . conversely , somatic mutation analysis in grade i pilocytic astrocytoma over the past 10 years has indicated the presence of a 3 bp insertion within codon 598 of the braf ( v - raf murine sarcoma viral oncogene homolog b ) gene ; this resulted in a constitutively active braf and a g - to - c transversion at codon 13 of the kras ( kirsten rat sarcoma viral oncogene homolog ) gene , which led to increased activation of the ras pathway . such a distinct molecular pattern and the presence of the idh1 c.g395a ; p.r132h mutation have practical implications and can be used directly to help pathologists distinguish pilocytic astrocytomas ( with idh1 wt status ) from diffuse ones [ 4 , 16 ] . the presence of the idh1 c.g395a ; p.r132h mutation correlated not only with the who grade but also with the frequency of secondary tumors [ table 1 ] and increased median overall survival ( up to 25 months ) [ fig . , we also evaluated the methods of detection of idh1 c.g395a ; p.r132h mutation in gliomas . recent molecular diagnostic methods have used not only gold - standard sanger sequencing but also a broad spectrum of molecular biology techniques [ 18 , 19 , 20 ] . detection of somatic mutations using antibodies specific for the mutation of interest has been applied not only in the case of the idh1 c.g395a ; p.r132h mutation but also in the case of deletions ( anti - dele746-a750 antibody ) and substitutions ( anti - l858r antibody ) in the egfr gene . although dozens of mutations located in the kinase domain of the egfr gene need to be evaluated before introduction of targeted therapy in lung cancer patients , a single mutation - specific anti - egfr antibody does not seem to be an appropriate diagnostic tool ; however , it may be the right choice for idh1 status analysis . we correlated sequencing , hrm , and immunohistochemistry results , and found that they were mostly concordant . any discrepancies in somatic mutation detection arose from the limit of detection , as was seen in glioblastoma , where immunohistochemistry detected idh1 r132h expression in 1125 % cells , while a sequencing chromatogram presented the peak for idh1 conversely , because of the fragmentation of the dna derived from ffpe glioma samples , dna quality and quantity were low , as the samples had been formalin - fixed since 2000 . sanger sequencing done on 20 % of samples had to be performed at least in duplicate or in triplicate ( both strands ) to confirm idh1 status and was diagnostically successful only in 10 % of cases . moreover , cold pcr hrm , which was assessed as a potential screening methodology for quick idh1 diagnostic testing , is a more sensitive method for detection of the idh1 c.g395a ; p.r132h mutation at tumor edges than immunohistochemistry or simple pcr hrm . in fact , we detected mutations in a few cases where idh1 wt status was stated by immunohistochemistry , which confirms the usefulness of this method and its particular importance during the evaluation of biopsy samples or ffpe tissue with a low percentage of tumor cells . furthermore , different melting curves that were other than positive for r132h or for wt idh1 status were detected . we assume that they might represent amplicons with r132c , r132l , r132s , or r132 g mutations . however , we found that immunohistochemistry was the most practical method for assessment of idh1 status in tissue samples . 4 ) allowed classification of all patients with somatic mutations in the idh1 gene into two groups : low and high expression of the mutant r132h protein . we observed that patients with low idh1 mutant protein expression levels in all tumor cells had a better prognosis than did those with high levels of expression of the mutant protein . this type of analysis was performed for the first time here , and suggests that the presence of idh1 mutations has prognostic value in glioma patients . as the previous comparison by preusser et al . of dia - h09 and imab-1 showed concordance of those two antibodies in 98.9 % , we did not perform the same analysis with another monoclonal antibody . moreover , measurement of protein expression levels using immunohistochemistry or real - time pcr can provide additional information that can be used to establish the prognosis . studies performed in cell lines have shown that idh1 c.g395a ; p.r132h overexpression decreased cell proliferation and akt phosphorylation and altered cell morphology . further studies performed in mice have confirmed longer survival in the presence of the mutant protein . the c.g395a ; p.r132h mutation leads to slow tumor growth and a better outcome ; however , in the higher expression range ( 51100 % ) , a better prognosis can still be expected ( in comparison with patients with idh1 wt ) , albeit with shorter overall survival , than in the group with lower expression of the mutant protein . we evaluated the presence of the r132h mutation as a prognostic biomarker in polish patients with astrocytoma , glioblastoma , oligoastrocytoma , ganglioglioma , oligodendroglioma , and ependymoma , using immunohistochemistry , sanger sequencing , and cold pcr hrm . from the economic point of view , immunohistochemistry seems to be superior to real - time pcr or sanger sequencing , as there is no need to isolate dna for further steps . immunohistochemistry is not only more cost effective but also has a shorter turnover time , especially considering that the low quality of fragmented dna from ffpe samples often necessitates additional repetition . the p.r132h idh1 mutation was observed in 56 % of grade ii astrocytomas and in 94 % of grade iii astrocytomas . meier survival analysis showed that grade ii astrocytoma patients and who grade ii glioma patients with the p.r132h idh1 mutation had significantly longer overall survival , up to 25 months . on the other hand , grade ii astrocytoma patients with low p.r132h idh1 expression had a significantly longer overall survival than did patients with high expression of the mutated protein . the authors have no conflicts of interest that are directly relevant to the content of this article .

background and objectiveidh1 ( isocitrate dehydrogenase 1 ) is a potential biomarker and drug target . genomic and epigenetic data on astrocytoma have demonstrated that the idh1 mutation is sufficient to establish the glioma hypermethylator phenotype . furthermore , recent studies have also indicated that a mutant idh1 inhibitor induced demethylation of histone h3k9me3 and expression of genes associated with gliogenic differentiation . as the presence of the p.r132h mutation in the idh1 gene seems to be a more powerful prognostic marker than o6-methylguanine - dna methyltransferase promoter status , we evaluated the presence of idh1 mutation in polish patients with astrocytoma , glioblastoma , oligoastrocytoma , ganglioglioma , oligodendroglioma , and ependymoma.methodsthe idh1 mutation status at codon 132 was determined using a mouse monoclonal antibody specific for the r132h mutation , direct sequencing , and co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) high - resolution melting - curve analysis ( hrm).resultswild - type ( wt ) idh1 was detected in cases with a world health organization ( who ) grade i astrocytoma . the idh1 c.g395a ; p.r132h mutation was observed in 56 and 94 % of grade ii and grade iii astrocytoma cases , respectively . significant differences in the median overall survival were observed in astrocytoma patients grouped on the basis of the presence of idh1 mutation : survival was 24 months longer in grade ii astrocytoma and 12 months longer in glioblastoma . overall survival was compared between grade ii astrocytoma patients with low or high expression of the mutant protein . interestingly , lower r132h expression correlated with better overall survival.conclusionour results indicate the usefulness of assessing the r132h idh1 mutation in glioma patients : the presence or absence of the r132h mutation can help pathologists to distinguish pilocytic astrocytomas ( idh1 wt ) from diffuse ones ( r132h idh1/wt ) . moreover , low idh1 p.r132h expression was related to better prognosis . this clinical implication appears to be important for personalization of prognosis and treatment by oncologists .

Introduction Materials and Methods Patient Specimens IDH1 Mutation Analysis (Immunohistochemistry, Sanger Sequencing, and COLD PCR HRM) Statistical Analyses Results Patient Characteristics Assessment of IDH1 Gene Mutations Correlation Between IDH1 Mutation, R132H Protein Expression, and Clinical Data Discussion Conclusion Conflicts of interest

furthermore , the median survival of patients with idh1 c.g395a ; p.r132h was 3.8 years compared with 1.1 years in patients with wild - type ( wt ) idh1 . these data demonstrate that the idh1 c.g395a ; p.r132h mutation is the molecular basis of cimp in gliomas and represents an advancement in the understanding of oncogenesis and the correlation between genomic and epigenomic changes in gliomas . we assessed the presence of the somatic idh1 c.g395a ; p.r132h mutation in polish glioma patients . patients were divided into groups according to the histological type of the tumor , whether the tumor was primary ( meaning detected primarily ) or secondary ( meaning a tumor recurrence after previously performed surgery ) , the average age at diagnosis , and the presence or absence of mutations in the idh1 gene ( table 1).table 1detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymomatumornumber of patientstumor primary / secondaryidh1 r132h / primaryidh1 r132h / secondaryaverage age at diagnosis ( years)idh1 r132h positive / total ( % ) iwho grade i astrocytoma33/03/30/028.30/3 ( 0)iiwho grade ii astrocytoma7568/738/684/739.342/75 ( 56.0)iiiwho grade iii astrocytoma170/170/016/173416/17 ( 94.1)ivwho grade iv glioblastoma multiforme161/150/18/15438/16 ( 50)vwho grade ii oligoastrocytoma2415/912/157/936.719/24 ( 79.1 ) who grade ii ganglioglioma32/12/21/133.63/3 ( 100 ) oligoastrocytoma2113/810/136/837.116/21 ( 76.2)viwho grade ii oligodendroglioma20/00/00/042.52/2 ( 100)viiwho grade ii ependymoma20/00/00/042.50/2 ( 0 ) who world health organization detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymoma who world health organization all samples were analyzed as a blind samples by immunohistochemistry and co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) high - resolution melting - curve analysis ( hrm ) . patients were divided into groups according to the histological type of the tumor , whether the tumor was primary ( meaning detected primarily ) or secondary ( meaning a tumor recurrence after previously performed surgery ) , the average age at diagnosis , and the presence or absence of mutations in the idh1 gene ( table 1).table 1detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymomatumornumber of patientstumor primary / secondaryidh1 r132h / primaryidh1 r132h / secondaryaverage age at diagnosis ( years)idh1 r132h positive / total ( % ) iwho grade i astrocytoma33/03/30/028.30/3 ( 0)iiwho grade ii astrocytoma7568/738/684/739.342/75 ( 56.0)iiiwho grade iii astrocytoma170/170/016/173416/17 ( 94.1)ivwho grade iv glioblastoma multiforme161/150/18/15438/16 ( 50)vwho grade ii oligoastrocytoma2415/912/157/936.719/24 ( 79.1 ) who grade ii ganglioglioma32/12/21/133.63/3 ( 100 ) oligoastrocytoma2113/810/136/837.116/21 ( 76.2)viwho grade ii oligodendroglioma20/00/00/042.52/2 ( 100)viiwho grade ii ependymoma20/00/00/042.50/2 ( 0 ) who world health organization detailed patient characteristics ( n = 139 ) and idh1 ( isocitrate dehydrogenase 1 ) c.g395a ; p.r132h mutation analysis in a cohort of patients with astrocytoma , glioblastoma , oligodendroglioma , ganglioglioma , oligoastrocytoma , or ependymoma who world health organization all samples were analyzed as a blind samples by immunohistochemistry and co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) high - resolution melting - curve analysis ( hrm ) . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . 2immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma analysis of idh1 ( isocitrate dehydrogenase 1 ) mutation status . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . the green and red stars represent wild - type and p.r132h idh1 , respectively ; difference plot normalized and temperature ( temp)-shifted with melting curve for heterozygous mutation ( above the baseline ) and idh1 wt ( below the baseline ) ; the baseline ( in red ) represents the no - template control ( ntc ) immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma the correlation between expression of the idh1 r132h mutant protein , as analyzed using an intensity scale in relation to the percentage of positive reactions , is presented in fig . representative results of the immunohistochemistry of idh1 p.r132h in who grade i astrocytoma ( pilocytic ; n = 3 ) , who grade ii astrocytoma ( diffuse , fibrillary , and gemistocytic ; n = 75 ) , who grade iii astrocytoma ( astrocytoma and anaplastic ; n = 17 ) , and who grade iv glioblastoma multiforme ( n = 16 ) are presented in fig . the idh1 c.g395a ; p.r132h mutation was frequent in tumors with higher who grades : grade iii astrocytoma ( 94.1 % ) , grade ii anaplastic oligodendroglioma ( 76.5 % ) , and grade iii anaplastic oligodendroglioma ( 75 % ) . in samples with no idh1 r132h staining , we confirmed the absence of the c.g395a ; p.r132h mutation , and dna sequencing did not reveal the presence of any additional mutations , such as idh1 r132c , r132 g , or r132s in the evaluated 10 % of the entire cohort . 3 show the patient characteristics regarding the presence or absence of the idh1 c.g395a ; p.r132h mutation in primary and secondary tumors , and overall survival in a cohort of glioma patients according to histology and idh1 status . the idh1 c.g395a ; p.r132h mutation was associated with longer survival in the grade ii astrocytoma , gbm , and pooled groups of patients with who grade ii glioma : oligoastrocytoma , oligodendroglioma , and ganglioglioma . wt wild - type kaplan meier curves of overall survival ( p = 0.05 ) according to idh1 ( isocitrate dehydrogenase 1 ) status in a grade ii astrocytoma ; b glioblastoma multiforme ; and c oligoastrocytoma , oligodendroglioma , and ganglioglioma samples . wt wild - type another kaplan meier analysis of grade ii astrocytoma patients revealed that those who had low p.r132h idh1 expression had significantly longer overall survival than did patients with high expression of the protein ( 42 versus 30 months ; p = 0.05 , log - rank test ) [ fig . 4overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . a intensity scale in relation to the percentage of positive reactions ; b kaplan meier curves of overall survival between the two groups of grade ii astrocytoma patients overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . 2immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma analysis of idh1 ( isocitrate dehydrogenase 1 ) mutation status . 81 ) , using co - amplification at lower denaturation temperature ( cold ) polymerase chain reaction ( pcr ) with high - resolution melting ( hrm ) . the green and red stars represent wild - type and p.r132h idh1 , respectively ; difference plot normalized and temperature ( temp)-shifted with melting curve for heterozygous mutation ( above the baseline ) and idh1 wt ( below the baseline ) ; the baseline ( in red ) represents the no - template control ( ntc ) immunohistochemical expression of idh1 ( isocitrate dehydrogenase 1 ) p.r132h in different grades of gliomas , demonstrated using haematoxylin and eosin ( h&e ) staining ( on the left ) and immunohistochemical staining with the anti - human idh1 r132h mouse monoclonal antibody ( on the right ) : a ependymoma ; b world health organization ( who ) grade i pilocytic astrocytoma ; c who grade ii fibrillary astrocytoma ; d who grade iii anaplastic astrocytoma ; e who grade iv glioblastoma multiforme ; f who grade ii oligodendroglioma the correlation between expression of the idh1 r132h mutant protein , as analyzed using an intensity scale in relation to the percentage of positive reactions , is presented in fig . representative results of the immunohistochemistry of idh1 p.r132h in who grade i astrocytoma ( pilocytic ; n = 3 ) , who grade ii astrocytoma ( diffuse , fibrillary , and gemistocytic ; n = 75 ) , who grade iii astrocytoma ( astrocytoma and anaplastic ; n = 17 ) , and who grade iv glioblastoma multiforme ( n = 16 ) are presented in fig . the idh1 c.g395a ; p.r132h mutation was frequent in tumors with higher who grades : grade iii astrocytoma ( 94.1 % ) , grade ii anaplastic oligodendroglioma ( 76.5 % ) , and grade iii anaplastic oligodendroglioma ( 75 % ) . in samples with no idh1 r132h staining , we confirmed the absence of the c.g395a ; p.r132h mutation , and dna sequencing did not reveal the presence of any additional mutations , such as idh1 r132c , r132 g , or r132s in the evaluated 10 % of the entire cohort . 3 show the patient characteristics regarding the presence or absence of the idh1 c.g395a ; p.r132h mutation in primary and secondary tumors , and overall survival in a cohort of glioma patients according to histology and idh1 status . the idh1 c.g395a ; p.r132h mutation was associated with longer survival in the grade ii astrocytoma , gbm , and pooled groups of patients with who grade ii glioma : oligoastrocytoma , oligodendroglioma , and ganglioglioma . wt wild - type kaplan meier curves of overall survival ( p = 0.05 ) according to idh1 ( isocitrate dehydrogenase 1 ) status in a grade ii astrocytoma ; b glioblastoma multiforme ; and c oligoastrocytoma , oligodendroglioma , and ganglioglioma samples . wt wild - type another kaplan meier analysis of grade ii astrocytoma patients revealed that those who had low p.r132h idh1 expression had significantly longer overall survival than did patients with high expression of the protein ( 42 versus 30 months ; p = 0.05 , log - rank test ) [ fig . 4overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . a intensity scale in relation to the percentage of positive reactions ; b kaplan meier curves of overall survival between the two groups of grade ii astrocytoma patients overall survival of grade ii astrocytoma patients divided into two subgroups : low ( 1+/2 + ) or high ( 3+/4 + ) idh1 ( isocitrate dehydrogenase 1 ) p.r132h expression , as measured using the intensity of the immunohistochemical reaction . idh1 c.g395a ; p.r132h is more frequent in who grades ii and iii than it is in who grade iv ( up to 60 and 80 % , respectively ) , which is in accordance with our data and supports the hypothesis of a role of idh1 mutation in the early steps of oncogenesis ( table 1 ) . such a distinct molecular pattern and the presence of the idh1 c.g395a ; p.r132h mutation have practical implications and can be used directly to help pathologists distinguish pilocytic astrocytomas ( with idh1 wt status ) from diffuse ones [ 4 , 16 ] . moreover , cold pcr hrm , which was assessed as a potential screening methodology for quick idh1 diagnostic testing , is a more sensitive method for detection of the idh1 c.g395a ; p.r132h mutation at tumor edges than immunohistochemistry or simple pcr hrm . the c.g395a ; p.r132h mutation leads to slow tumor growth and a better outcome ; however , in the higher expression range ( 51100 % ) , a better prognosis can still be expected ( in comparison with patients with idh1 wt ) , albeit with shorter overall survival , than in the group with lower expression of the mutant protein . we evaluated the presence of the r132h mutation as a prognostic biomarker in polish patients with astrocytoma , glioblastoma , oligoastrocytoma , ganglioglioma , oligodendroglioma , and ependymoma , using immunohistochemistry , sanger sequencing , and cold pcr hrm . the p.r132h idh1 mutation was observed in 56 % of grade ii astrocytomas and in 94 % of grade iii astrocytomas . on the other hand , grade ii astrocytoma patients with low p.r132h idh1 expression had a significantly longer overall survival than did patients with high expression of the mutated protein .

anesthesia / analgesia for critical airway and choice of the most appropriate technique of emergency surgical airway are issues left to individual experience or institutional protocols . following anecdotal experience with cases needing analgesia / anesthesia before an emergency surgical airway and a discrete personal experience with different aspects of critical airway management , suggestions as to safe and effective methods of analgesia / anesthesia and surgical airway are given . critical airway is a life - threatening scenario of hypoxemia about to become hypoxia , following failed or inadequate ventilation , where emergency surgical access must be obtained to avoid catastrophic consequences or death . the key indication is a scenario of impossible or inadequate ventilation . in the last two decades a new philosophy of airway control has been established that has shifted emphasis from mask ventilation to supraglottic ventilation with the introduction of supra - glottic ventilatory devices ( sgvd ) . protection from stomach contents aspiration is not guaranteed but the risk has been reduced with the introduction of extra channels for nasogastric tube ( ngt ) and endotracheal tube ( ett ) . other drawbacks are oropharyngeal decubitus effects , decrease of lung compliance with increased airway resistance , and the need to be inserted in unconscious patient . sgvd obviate to cervical manipulations and can be used as a bridge during eti attempts or by untrained or inexperienced personnel outdoor . svgd are optimally placed for airway control outdoors in the instances where maxillofacial injuries can not be intubated , with the added advantage of tamponading intraoral bleeding , and cervical spine injury ( csi ) is certain or likely . combined with the right technique of continuous intravenous anesthesia , sgvd are invaluable presidiums in some scenarios of critical airway secondary to trauma . it is imperative therefore that any anesthetic or analgesic be titrated to response and for the required duration only . the agents used should have immediate effect on injection , be suitable for continuous intravenous infusion , nonetheless effective and safe with no effect on blood pressure and ventilation at physiological doses . these targets can only be met with a short duration rapid acting opioids in continuous intravenous anesthesia like alfentanyl and remifentanyl or drugs not depressing the cardiovascular and respiratory systems like ketamine.[35 ] two scenarios , personally encountered by the author , of maxillofacial trauma compounded by head injury , hemorrhagic shock and critical airway , epitomize with its potential pitfalls and caveat the challenge some patients with post - traumatic critical airway represent . the approach proposed as ideal management was retrospectively worked out by combining a svgd with a method of total intravenous anesthesia extrapolated by a consolidated technique [ table 1 ] . anaesthesia for critical airway it is the one of a patient being shot to the face , presenting with severe maxillofacial injury bleeding into the airways and outside , a neck collar for possible cervical spine injury ( csi ) , associated head injury with some deterioration of consciousness but still conscious , some degrees of hemorrhagic shock from oropharyngeal hemorrhage , and staying in obligatory sitting position in the attempt to relieve the dyspnea by avoiding the effect of deformity and blood on the breathing . this type of scenario would require , in the author 's view , in order : i ) no face - mask ventilation but free high - oxygen concentration via high flow mask and reservoir bag kept near but at some distance over the face ; ii ) rapid iodine painting on anterior neck skin in preparation of a surgical airway - local anesthesia ( la ) with lignocaine infiltration would risk confuse the landmarks ! - ; iii ) the collar removed with the head kept carefully in manual - in - line - stabilization ( mils ) on sitting / semi - sitting position by an assistant ; iv ) ketamine iv as bolus is then administered ; v ) once anesthesia is reached ( fixed gaze plus a possible or spontaneous eye closure and miosis ) , the patient is rapidly positioned supine with mils maintained and continuous intravenous anesthesia ( civa ) with infusion of ketamine or remifentanyl started up ; vi ) a diagnostic laryngoscopy ( dl ) is done to see the feasibility of a rapid endotracheal intubation ( eti ) ( visible laryngeal inlet and vocal cords ( vc ) or not , plus aspiration of blood debris and foreign bodies in the laryngeal inlet ) ; vii ) eti is done ; viii ) if eti not feasible due to deformity and view - obstructing debris and blood , a supraglottic ventilatory device like an intubating laryngeal mask airway ( ilma ) fast - trach or c - trach , or a laryngeal tube ( lt ) like a king ls ii , should rapidly be inserted ; ix ) ett is inserted through the sgvd ; ix ) a tracheostomy is then done if ett can not be inserted through the svgd or done at some stage with the svgd in situ before maxillofacial reconstruction . insufficient ventilation or rapid deterioration during the above steps calls at any time for an emergency cricothyroidotomy ( cty ) , or tracheostomy if landmarks for cty are obscured by swelling ) . pulse oximeter and an arterial blood gas analyzer in perfect order should be at hand . no formal sedative or paralytic agent should be given to this type of patient until the airway is secured . the preliminary steps ending with the sedation with ketamine should be explained to the patient in a reassuring / relaxed and cool confident voice and demeanor despite he or she may not be in enough concentration but in severe distress for the dyspnea , gurgling sounds and all the hectic and frantic preparations around that should be absolutely carried out in silence - " you will go to sleep now so i can put a tube in your windpipe for you to breathe better . " to watch the patient breathing and taking a chance before intervening is a big error as deterioration and hypoxic damages are about to happen , likewise it is a big error to ventilate without looking first to retrieve and remove material in the upper airways that can be pushed inside the airways . another extreme scenario is the one of an unconscious patient following blunt trauma to face and neck after car accident , who comes ventilated manually due to hypoventilation , manifesting indirect signs of impending upper airways obstruction , impalpable anterior neck landmarks , hypoxemia and severely bleeding massive facial injury , already supine and with a neck collar on for possible or suspected csi . similar decision - making as above : do not face - mask ventilate ; give oxygen by mask at distance from face ; do dl ; insert an ett or a svgd ; if svgd , try passing ett through it ; if eti can not be passed then obtain an emergency surgical airway ( esa ) with svgd in situ ; esa at anytime if rapid deterioration of sao2 or patient incapable to be ventilated satisfactorily with svgd . in a cardiac or respiratory / cardiac arrest by upper airways obstruction sedation or myoparalysis are also contraindicated in patients requiring a surgical airway who are still awake , breathing spontaneously and sitting to prevent or relief their hypoxemia in severe face and neck injury with respiratory compromise - a posture often observed also in patients with cardiac tamponade or post - traumatic diaphragmatic hernia.th when analgesia is required in a conscious patient candidate for an esa other than transtracheal or percutaneous transtracheal jet ventilation ( ttjv / ptjv ) or stab cricothyroidotomy , both feasible with simple la infiltration , rapid onset induction agents and rapidly reversible anesthetic agents not interfering with spontaneous ventilation or blood pressure must be used for civa . ketamine , remifentanyl or alfentanyl stat followed by continuous infusions are effective and safe drugs . the choice and dosages for the different anaesthetic drugs should be institutionalized in protocols ready to use . combinations ketamine - opioid work well too . in tachycardic patients with heart rate ( hr ) < 120 bpm , alfentanyl , or ketamine , are the induction drugs of choice ; in patients with coronary heart disease or hr 120 bpm ketamine is contraindicated . alfentanyl would be then the most optimal induction agent as for its fastest onset of action , with remifentanyl the more optimal for civa due to its shortest half - life and rapid metabolism such as its effects terminate synchronously with termination of the infusion . ketamine is a unique anaesthetic in that it does not depress myocardium , vasomotor and airways tone . for these reasons it has been successfully used in war - outdoors scenarios for civa or as induction agent . despite being an excellent for induction and maintenance , nonetheless as analgesic and sedative , ketamine has the important side effect of increasing oxygen consumption ( vo2 ) and heart rate ( hr ) in an organ , the heart , which is dependent on flow to increase oxygen delivery and at basic regimen extracts 75% ( o2er ) of the oxygen delivered ( do2 ) . other side - effects of the classical racemic mixture are the occasional extrapyramidal signs , increased salivation and secretions in general , and the frequent hallucinations on emergence , which are all dose dependent and bufferable with a benzodiazepine like midazolam or diazepam and an antisialogue as anticholinergic that does not cross the blood - brain barrier and with the lowest psychotropic and chronotropic effects , namely glycopyrrolate . ketamine left isomer , ( s+)-ketamine , is to become the drug par excellence for anesthesia or sedation in critical illness due to the much superior analgesic power and much lesser chronotropic and neurotropic effects when compared to the racemic mixture.[1012 ] midazolam should not be administered before airway control and normalized bp as for its cardiovascular and ventilatory depression effect , and only with a view to decrease extrapyramidal and neurotropic effects ( hallucinations ) of the racemic mixture . laryngospasm attributable to ketamine or remifentanyl administration is counteracted by a short acting myorelaxant like suxamethonium . glycopyrrolate should not be administered before airway control despite its potential benefits , due to the possibility of distracting the team of three from their respective tasks , not being an essential drug . each second counts ! fighting patients ( agitated , confused and restless ) with critical airway must be induced with ketamine , and so patients in obligatory sitting position and in severe shock . as far as the techniques with ketamine , all pct and some ost can be done under one ketamine induction dose whereas most ost require both induction and maintenance dosages of any combination proposed in table 1 . despite the wave of enthusiasm accompanying percutaneous tracheostomy ( pct ) in the last two decades,[1317 ] there is no evidence that the closed technique is superior or more convenient than the open standard tracheostomy ( ost ) in election,[1823 ] even less in emergency . although it is acceptable for operators to use the techniques where they are more prone and acquainted in order to save lives , the corollary of all reliable comparative studies among the techniques in election done by teams of ear - nose - throat , maxillofacial or cardio - thoracic surgeons experienced in both procedures , is that ost is to be considered safer and preferable than pct in emergencies . the time factor is crucial for patients outcome during a ventilatory crisis and during a conversion from failed or complicated cty or pct to open tracheostomy in desperate situations . although in trained hands pct takes an average of 4 - 5 min , while the open technique is reported with a wider performance time between 5 and 15 min,[18192427 ] in extreme emergency such a cardiac arrest ( ca ) or respiratory - cardiac ( r - ca ) arrest a pct is not as fast as the open technique . only unquestionable advantage of pct over the open technique is the aesthetic one , in view of the small incision ( 1 - 1.5 cm vs 4 - 4.5 cm ) . if the sequential method has a characteristic peril in the high risk of posterior tracheal wall penetration , the single dilator method has a high risk of anterior wall damage , risk diminished when the griggs technique is used . both griggs and single dilator methods are now standard , being faster and simpler and with lesser complications rates than the original multi - dilators technique , due to the continuum control of pressure and stability in manouvering , a very important and crucial factor for success and safety . the risk of complications is much lessened in the open technique , which allows positioning the tracheostomy centrally and in the most optimal space between second and third ring without unduly damage to the adjacent cartilaginous rings , and to be fashioned for a perfectly matching cannula . the site and the centrality of the initial needle puncture / tracheotomy are crucial in avoiding complications . a0 plane of dissection not strictly on the midline inevitably provokes or accentuates peri - tracheal bleeding ; a high / very high tracheotomy for its proximity to the larynx is a good recipe for laryngeal stenosis ; a low tracheotomy causes decubitus of the cannula on the trachea during neck flexion / extension movements predisposing to fistulae ; a not central ostomy with lateral decubitus of the cannula can damage the wall ; an erroneous estimation of the depth of the trachea during maneuvering risks posterior wall damage ; a lack of open vision risks to unduly damage the anterior tracheal rings ; are the six mechanisms accounting for the complications of tracheostomies.[133035 ] other considerations make pct unsuitable or contraindicated in emergency : it is impossible before procedure to predict the need of a long cannula in cases of deep trachea , problem easily solvable in the open situation ; intraoperative and perioperative complications such as peritracheal and intratracheal bleeding , cannula malposition , cannula dislodgement , tracheal damage , airway loss with sudden fall of airways pressures and hypoxia can not be dealt with during the procedure without rapid conversion to ost . it follows that the technique can not be recommended in ca or r - ca despite successful anecdotal cases . the open procedure as a matter of fact represents the only possible treatment for cty and pct intraoperative complications . the vertical skin incision is preferred in emergency as well as in urgencies because makes the procedure safer , easier and faster . a semiopen tracheostomy ( sot ) with 2 - 3-cm skin incision down to visualize the pretracheal fascia followed by a percutaneous method , is a valid alternative to ost . sot offers the best option in cases of cervical spine injury ( csi ) with impalpable trachea . sot requires less dissection , though at risk of not being able to control the occurrence of intraoperative complications , and often the help of hookers and traction stitches , but it obviates to hazardous head / neck manipulations . while ptjv and cty , likewise ett and sgvd , can be managed by a not medic , tracheostomies and improvised airway device ( iad ) should be inserted only by medics . the trachea is a marvelous and unique piece of engineering , result of the necessity to combine semi - rigid flexibility and not collapsible property during neck movements . it is a relatively fragile and essential structure , witness of a unique metaphysical ideation for the vital function to equilibrate atmospheric pressure with the alveolar one . nevertheless it may occur in exceptional circumstances that it must be violated mercilessly in order to get life saving oxygen . virtually any iad like any not collapsible cylindrical device e.g. , a pen cover broken on the side of the conical tip end to avoid stenosis and inserted religiously centrally through a hole done with a simple pointed- edge knife rotated for 180 - 360 degrees suffices the purpose . advantages , scenario - indications , contraindications and disadvantages of the different methods are outlined in table 2.[3945 ] pros and cons of surgical airways the author 's personal recommendations for esa in critical airway are outlined in table 3.[4653 ] recommendations on which esa to use in critical airway critical airway is a life - threatening scenario of hypoxemia about to become hypoxia , following failed or inadequate ventilation , where emergency surgical access must be obtained to avoid catastrophic consequences or death . the key indication is a scenario of impossible or inadequate ventilation . in the last two decades a new philosophy of airway control has been established that has shifted emphasis from mask ventilation to supraglottic ventilation with the introduction of supra - glottic ventilatory devices ( sgvd ) . protection from stomach contents aspiration is not guaranteed but the risk has been reduced with the introduction of extra channels for nasogastric tube ( ngt ) and endotracheal tube ( ett ) . other drawbacks are oropharyngeal decubitus effects , decrease of lung compliance with increased airway resistance , and the need to be inserted in unconscious patient . sgvd obviate to cervical manipulations and can be used as a bridge during eti attempts or by untrained or inexperienced personnel outdoor . svgd are optimally placed for airway control outdoors in the instances where maxillofacial injuries can not be intubated , with the added advantage of tamponading intraoral bleeding , and cervical spine injury ( csi ) is certain or likely . combined with the right technique of continuous intravenous anesthesia , sgvd are invaluable presidiums in some scenarios of critical airway secondary to trauma . it is imperative therefore that any anesthetic or analgesic be titrated to response and for the required duration only . the agents used should have immediate effect on injection , be suitable for continuous intravenous infusion , nonetheless effective and safe with no effect on blood pressure and ventilation at physiological doses . these targets can only be met with a short duration rapid acting opioids in continuous intravenous anesthesia like alfentanyl and remifentanyl or drugs not depressing the cardiovascular and respiratory systems like ketamine.[35 ] two scenarios , personally encountered by the author , of maxillofacial trauma compounded by head injury , hemorrhagic shock and critical airway , epitomize with its potential pitfalls and caveat the challenge some patients with post - traumatic critical airway represent . the approach proposed as ideal management was retrospectively worked out by combining a svgd with a method of total intravenous anesthesia extrapolated by a consolidated technique [ table 1 ] . anaesthesia for critical airway it is the one of a patient being shot to the face , presenting with severe maxillofacial injury bleeding into the airways and outside , a neck collar for possible cervical spine injury ( csi ) , associated head injury with some deterioration of consciousness but still conscious , some degrees of hemorrhagic shock from oropharyngeal hemorrhage , and staying in obligatory sitting position in the attempt to relieve the dyspnea by avoiding the effect of deformity and blood on the breathing . this type of scenario would require , in the author 's view , in order : i ) no face - mask ventilation but free high - oxygen concentration via high flow mask and reservoir bag kept near but at some distance over the face ; ii ) rapid iodine painting on anterior neck skin in preparation of a surgical airway - local anesthesia ( la ) with lignocaine infiltration would risk confuse the landmarks ! - ; iii ) the collar removed with the head kept carefully in manual - in - line - stabilization ( mils ) on sitting / semi - sitting position by an assistant ; iv ) ketamine iv as bolus is then administered ; v ) once anesthesia is reached ( fixed gaze plus a possible or spontaneous eye closure and miosis ) , the patient is rapidly positioned supine with mils maintained and continuous intravenous anesthesia ( civa ) with infusion of ketamine or remifentanyl started up ; vi ) a diagnostic laryngoscopy ( dl ) is done to see the feasibility of a rapid endotracheal intubation ( eti ) ( visible laryngeal inlet and vocal cords ( vc ) or not , plus aspiration of blood debris and foreign bodies in the laryngeal inlet ) ; vii ) eti is done ; viii ) if eti not feasible due to deformity and view - obstructing debris and blood , a supraglottic ventilatory device like an intubating laryngeal mask airway ( ilma ) fast - trach or c - trach , or a laryngeal tube ( lt ) like a king ls ii , should rapidly be inserted ; ix ) ett is inserted through the sgvd ; ix ) a tracheostomy is then done if ett can not be inserted through the svgd or done at some stage with the svgd in situ before maxillofacial reconstruction . insufficient ventilation or rapid deterioration during the above steps calls at any time for an emergency cricothyroidotomy ( cty ) , or tracheostomy if landmarks for cty are obscured by swelling ) . pulse oximeter and an arterial blood gas analyzer in perfect order should be at hand . no formal sedative or paralytic agent should be given to this type of patient until the airway is secured . the preliminary steps ending with the sedation with ketamine should be explained to the patient in a reassuring / relaxed and cool confident voice and demeanor despite he or she may not be in enough concentration but in severe distress for the dyspnea , gurgling sounds and all the hectic and frantic preparations around that should be absolutely carried out in silence - " you will go to sleep now so i can put a tube in your windpipe for you to breathe better . " to watch the patient breathing and taking a chance before intervening is a big error as deterioration and hypoxic damages are about to happen , likewise it is a big error to ventilate without looking first to retrieve and remove material in the upper airways that can be pushed inside the airways . another extreme scenario is the one of an unconscious patient following blunt trauma to face and neck after car accident , who comes ventilated manually due to hypoventilation , manifesting indirect signs of impending upper airways obstruction , impalpable anterior neck landmarks , hypoxemia and severely bleeding massive facial injury , already supine and with a neck collar on for possible or suspected csi . similar decision - making as above : do not face - mask ventilate ; give oxygen by mask at distance from face ; do dl ; insert an ett or a svgd ; if svgd , try passing ett through it ; if eti can not be passed then obtain an emergency surgical airway ( esa ) with svgd in situ ; esa at anytime if rapid deterioration of sao2 or patient incapable to be ventilated satisfactorily with svgd . in a cardiac or respiratory / cardiac arrest by upper airways obstruction there is no need for any analgesia or anesthesia . sedation or myoparalysis are also contraindicated in patients requiring a surgical airway who are still awake , breathing spontaneously and sitting to prevent or relief their hypoxemia in severe face and neck injury with respiratory compromise - a posture often observed also in patients with cardiac tamponade or post - traumatic diaphragmatic hernia.th when analgesia is required in a conscious patient candidate for an esa other than transtracheal or percutaneous transtracheal jet ventilation ( ttjv / ptjv ) or stab cricothyroidotomy , both feasible with simple la infiltration , rapid onset induction agents and rapidly reversible anesthetic agents not interfering with spontaneous ventilation or blood pressure must be used for civa . ketamine , remifentanyl or alfentanyl stat followed by continuous infusions are effective and safe drugs . the choice and dosages for the different anaesthetic drugs should be institutionalized in protocols ready to use . combinations ketamine - opioid work well too . in tachycardic patients with heart rate ( hr ) < 120 bpm , alfentanyl , or ketamine , are the induction drugs of choice ; in patients with coronary heart disease or hr 120 bpm ketamine is contraindicated . alfentanyl would be then the most optimal induction agent as for its fastest onset of action , with remifentanyl the more optimal for civa due to its shortest half - life and rapid metabolism such as its effects terminate synchronously with termination of the infusion . ketamine is a unique anaesthetic in that it does not depress myocardium , vasomotor and airways tone . for these reasons it has been successfully used in war - outdoors scenarios for civa or as induction agent . despite being an excellent for induction and maintenance , nonetheless as analgesic and sedative , ketamine has the important side effect of increasing oxygen consumption ( vo2 ) and heart rate ( hr ) in an organ , the heart , which is dependent on flow to increase oxygen delivery and at basic regimen extracts 75% ( o2er ) of the oxygen delivered ( do2 ) . other side - effects of the classical racemic mixture are the occasional extrapyramidal signs , increased salivation and secretions in general , and the frequent hallucinations on emergence , which are all dose dependent and bufferable with a benzodiazepine like midazolam or diazepam and an antisialogue as anticholinergic that does not cross the blood - brain barrier and with the lowest psychotropic and chronotropic effects , namely glycopyrrolate . ketamine left isomer , ( s+)-ketamine , is to become the drug par excellence for anesthesia or sedation in critical illness due to the much superior analgesic power and much lesser chronotropic and neurotropic effects when compared to the racemic mixture.[1012 ] midazolam should not be administered before airway control and normalized bp as for its cardiovascular and ventilatory depression effect , and only with a view to decrease extrapyramidal and neurotropic effects ( hallucinations ) of the racemic mixture . laryngospasm attributable to ketamine or remifentanyl administration is counteracted by a short acting myorelaxant like suxamethonium . glycopyrrolate should not be administered before airway control despite its potential benefits , due to the possibility of distracting the team of three from their respective tasks , not being an essential drug . each second counts ! fighting patients ( agitated , confused and restless ) with critical airway must be induced with ketamine , and so patients in obligatory sitting position and in severe shock . as far as the techniques with ketamine , all pct and some ost can be done under one ketamine induction dose whereas most ost require both induction and maintenance dosages of any combination proposed in table 1 . it is the one of a patient being shot to the face , presenting with severe maxillofacial injury bleeding into the airways and outside , a neck collar for possible cervical spine injury ( csi ) , associated head injury with some deterioration of consciousness but still conscious , some degrees of hemorrhagic shock from oropharyngeal hemorrhage , and staying in obligatory sitting position in the attempt to relieve the dyspnea by avoiding the effect of deformity and blood on the breathing . this type of scenario would require , in the author 's view , in order : i ) no face - mask ventilation but free high - oxygen concentration via high flow mask and reservoir bag kept near but at some distance over the face ; ii ) rapid iodine painting on anterior neck skin in preparation of a surgical airway - local anesthesia ( la ) with lignocaine infiltration would risk confuse the landmarks ! - ; iii ) the collar removed with the head kept carefully in manual - in - line - stabilization ( mils ) on sitting / semi - sitting position by an assistant ; iv ) ketamine iv as bolus is then administered ; v ) once anesthesia is reached ( fixed gaze plus a possible or spontaneous eye closure and miosis ) , the patient is rapidly positioned supine with mils maintained and continuous intravenous anesthesia ( civa ) with infusion of ketamine or remifentanyl started up ; vi ) a diagnostic laryngoscopy ( dl ) is done to see the feasibility of a rapid endotracheal intubation ( eti ) ( visible laryngeal inlet and vocal cords ( vc ) or not , plus aspiration of blood debris and foreign bodies in the laryngeal inlet ) ; vii ) eti is done ; viii ) if eti not feasible due to deformity and view - obstructing debris and blood , a supraglottic ventilatory device like an intubating laryngeal mask airway ( ilma ) fast - trach or c - trach , or a laryngeal tube ( lt ) like a king ls ii , should rapidly be inserted ; ix ) ett is inserted through the sgvd ; ix ) a tracheostomy is then done if ett can not be inserted through the svgd or done at some stage with the svgd in situ before maxillofacial reconstruction . insufficient ventilation or rapid deterioration during the above steps calls at any time for an emergency cricothyroidotomy ( cty ) , or tracheostomy if landmarks for cty are obscured by swelling ) . pulse oximeter and an arterial blood gas analyzer in perfect order should be at hand . no formal sedative or paralytic agent should be given to this type of patient until the airway is secured . the preliminary steps ending with the sedation with ketamine should be explained to the patient in a reassuring / relaxed and cool confident voice and demeanor despite he or she may not be in enough concentration but in severe distress for the dyspnea , gurgling sounds and all the hectic and frantic preparations around that should be absolutely carried out in silence - " you will go to sleep now so i can put a tube in your windpipe for you to breathe better . " to watch the patient breathing and taking a chance before intervening is a big error as deterioration and hypoxic damages are about to happen , likewise it is a big error to ventilate without looking first to retrieve and remove material in the upper airways that can be pushed inside the airways . another extreme scenario is the one of an unconscious patient following blunt trauma to face and neck after car accident , who comes ventilated manually due to hypoventilation , manifesting indirect signs of impending upper airways obstruction , impalpable anterior neck landmarks , hypoxemia and severely bleeding massive facial injury , already supine and with a neck collar on for possible or suspected csi . similar decision - making as above : do not face - mask ventilate ; give oxygen by mask at distance from face ; do dl ; insert an ett or a svgd ; if svgd , try passing ett through it ; if eti can not be passed then obtain an emergency surgical airway ( esa ) with svgd in situ ; esa at anytime if rapid deterioration of sao2 or patient incapable to be ventilated satisfactorily with svgd . in a cardiac or respiratory / cardiac arrest by upper airways obstruction there is no need for any analgesia or anesthesia . sedation or myoparalysis are also contraindicated in patients requiring a surgical airway who are still awake , breathing spontaneously and sitting to prevent or relief their hypoxemia in severe face and neck injury with respiratory compromise - a posture often observed also in patients with cardiac tamponade or post - traumatic diaphragmatic hernia.th when analgesia is required in a conscious patient candidate for an esa other than transtracheal or percutaneous transtracheal jet ventilation ( ttjv / ptjv ) or stab cricothyroidotomy , both feasible with simple la infiltration , rapid onset induction agents and rapidly reversible anesthetic agents not interfering with spontaneous ventilation or blood pressure must be used for civa . ketamine , remifentanyl or alfentanyl stat followed by continuous infusions are effective and safe drugs . the choice and dosages for the different anaesthetic drugs should be institutionalized in protocols ready to use . combinations ketamine - opioid work well too . in tachycardic patients with heart rate ( hr ) < 120 bpm , alfentanyl , or ketamine , are the induction drugs of choice ; in patients with coronary heart disease or hr 120 bpm ketamine is contraindicated . alfentanyl would be then the most optimal induction agent as for its fastest onset of action , with remifentanyl the more optimal for civa due to its shortest half - life and rapid metabolism such as its effects terminate synchronously with termination of the infusion . ketamine is a unique anaesthetic in that it does not depress myocardium , vasomotor and airways tone . for these reasons it has been successfully used in war - outdoors scenarios for civa or as induction agent . despite being an excellent for induction and maintenance , nonetheless as analgesic and sedative , ketamine has the important side effect of increasing oxygen consumption ( vo2 ) and heart rate ( hr ) in an organ , the heart , which is dependent on flow to increase oxygen delivery and at basic regimen extracts 75% ( o2er ) of the oxygen delivered ( do2 ) . other side - effects of the classical racemic mixture are the occasional extrapyramidal signs , increased salivation and secretions in general , and the frequent hallucinations on emergence , which are all dose dependent and bufferable with a benzodiazepine like midazolam or diazepam and an antisialogue as anticholinergic that does not cross the blood - brain barrier and with the lowest psychotropic and chronotropic effects , namely glycopyrrolate . ketamine left isomer , ( s+)-ketamine , is to become the drug par excellence for anesthesia or sedation in critical illness due to the much superior analgesic power and much lesser chronotropic and neurotropic effects when compared to the racemic mixture.[1012 ] midazolam should not be administered before airway control and normalized bp as for its cardiovascular and ventilatory depression effect , and only with a view to decrease extrapyramidal and neurotropic effects ( hallucinations ) of the racemic mixture . laryngospasm attributable to ketamine or remifentanyl administration is counteracted by a short acting myorelaxant like suxamethonium . glycopyrrolate should not be administered before airway control despite its potential benefits , due to the possibility of distracting the team of three from their respective tasks , not being an essential drug . each second counts ! fighting patients ( agitated , confused and restless ) with critical airway must be induced with ketamine , and so patients in obligatory sitting position and in severe shock . as far as the techniques with ketamine , all pct and some ost can be done under one ketamine induction dose whereas most ost require both induction and maintenance dosages of any combination proposed in table 1 . despite the wave of enthusiasm accompanying percutaneous tracheostomy ( pct ) in the last two decades,[1317 ] there is no evidence that the closed technique is superior or more convenient than the open standard tracheostomy ( ost ) in election,[1823 ] even less in emergency . although it is acceptable for operators to use the techniques where they are more prone and acquainted in order to save lives , the corollary of all reliable comparative studies among the techniques in election done by teams of ear - nose - throat , maxillofacial or cardio - thoracic surgeons experienced in both procedures , is that ost is to be considered safer and preferable than pct in emergencies . the time factor is crucial for patients outcome during a ventilatory crisis and during a conversion from failed or complicated cty or pct to open tracheostomy in desperate situations . although in trained hands pct takes an average of 4 - 5 min , while the open technique is reported with a wider performance time between 5 and 15 min,[18192427 ] in extreme emergency such a cardiac arrest ( ca ) or respiratory - cardiac ( r - ca ) arrest a pct is not as fast as the open technique . only unquestionable advantage of pct over the open technique is the aesthetic one , in view of the small incision ( 1 - 1.5 cm vs 4 - 4.5 cm ) . if the sequential method has a characteristic peril in the high risk of posterior tracheal wall penetration , the single dilator method has a high risk of anterior wall damage , risk diminished when the griggs technique is used . both griggs and single dilator methods are now standard , being faster and simpler and with lesser complications rates than the original multi - dilators technique , due to the continuum control of pressure and stability in manouvering , a very important and crucial factor for success and safety . the risk of complications is much lessened in the open technique , which allows positioning the tracheostomy centrally and in the most optimal space between second and third ring without unduly damage to the adjacent cartilaginous rings , and to be fashioned for a perfectly matching cannula . the site and the centrality of the initial needle puncture / tracheotomy are crucial in avoiding complications . a0 plane of dissection not strictly on the midline inevitably provokes or accentuates peri - tracheal bleeding ; a high / very high tracheotomy for its proximity to the larynx is a good recipe for laryngeal stenosis ; a low tracheotomy causes decubitus of the cannula on the trachea during neck flexion / extension movements predisposing to fistulae ; a not central ostomy with lateral decubitus of the cannula can damage the wall ; an erroneous estimation of the depth of the trachea during maneuvering risks posterior wall damage ; a lack of open vision risks to unduly damage the anterior tracheal rings ; are the six mechanisms accounting for the complications of tracheostomies.[133035 ] other considerations make pct unsuitable or contraindicated in emergency : it is impossible before procedure to predict the need of a long cannula in cases of deep trachea , problem easily solvable in the open situation ; intraoperative and perioperative complications such as peritracheal and intratracheal bleeding , cannula malposition , cannula dislodgement , tracheal damage , airway loss with sudden fall of airways pressures and hypoxia can not be dealt with during the procedure without rapid conversion to ost . it follows that the technique can not be recommended in ca or r - ca despite successful anecdotal cases . the open procedure as a matter of fact represents the only possible treatment for cty and pct intraoperative complications . the vertical skin incision is preferred in emergency as well as in urgencies because makes the procedure safer , easier and faster . a semiopen tracheostomy ( sot ) with 2 - 3-cm skin incision down to visualize the pretracheal fascia followed by a percutaneous method , is a valid alternative to ost . sot offers the best option in cases of cervical spine injury ( csi ) with impalpable trachea . sot requires less dissection , though at risk of not being able to control the occurrence of intraoperative complications , and often the help of hookers and traction stitches , but it obviates to hazardous head / neck manipulations . while ptjv and cty , likewise ett and sgvd , can be managed by a not medic , tracheostomies and improvised airway device ( iad ) should be inserted only by medics . the trachea is a marvelous and unique piece of engineering , result of the necessity to combine semi - rigid flexibility and not collapsible property during neck movements . it is a relatively fragile and essential structure , witness of a unique metaphysical ideation for the vital function to equilibrate atmospheric pressure with the alveolar one . nevertheless it may occur in exceptional circumstances that it must be violated mercilessly in order to get life saving oxygen . virtually any iad like any not collapsible cylindrical device e.g. , a pen cover broken on the side of the conical tip end to avoid stenosis and inserted religiously centrally through a hole done with a simple pointed- edge knife rotated for 180 - 360 degrees suffices the purpose . advantages , scenario - indications , contraindications and disadvantages of the different methods are outlined in table 2.[3945 ] pros and cons of surgical airways the author 's personal recommendations for esa in critical airway are outlined in table 3.[4653 ] recommendations on which esa to use in critical airway analgesia / anesthesia for critical airway or during a situation of critical airway is a highly skilled task to be accomplished with sheer accuracy and a reasonable safety margin , due to the side - effects of anesthetic or analgesic drugs on airway and ventilation . because of intrinsic difficulties in doing studies , perils of its implementation and rarity of requirement , protocols must be established before - hand and possibly studied in situations where an anesthetic is required with the patient kept breathing spontaneously . safety and efficacy in those circumstances proves reliability of the method . following analog ethical and realistic considerations , specific indications , timing and type of emergency surgical airway can therefore only be given as result of axioms and corollaries , personal experience and reliable comparative studies among the techniques in election by specialist teams experienced in both techniques .

which anesthesia for patients with critical airway ? safe and effective analgesia and anesthesia in critical airway is a skilled task especially after severe maxillofacial injury combined with head injury and hemorrhagic shock . if on one side sedation is wanted , on the other hand it may worsen the airway and hemodynamic situation to a point where hypoventilation and decrease of blood pressure , common side - effect of many opioids , may prejudice the patient 's level of consciousness and hemodynamic compensation , compounding an already critical situation . what to do when endotracheal intubation fails and blood is trickling down the airways in an unconscious patient or when a conscious patient has to sit up to breathe ? which surgical airway in critical airway ? comparative studies among the various methods of emergency surgical airway would be unethical ; furthermore , operator 's training and experience is relevant for indications and performance .

INTRODUCTION Which anesthesia for critical airway? Scenario 1 Scenario 2 Comments Which surgical airway? CONCLUSIONS

anesthesia / analgesia for critical airway and choice of the most appropriate technique of emergency surgical airway are issues left to individual experience or institutional protocols . following anecdotal experience with cases needing analgesia / anesthesia before an emergency surgical airway and a discrete personal experience with different aspects of critical airway management , suggestions as to safe and effective methods of analgesia / anesthesia and surgical airway are given . critical airway is a life - threatening scenario of hypoxemia about to become hypoxia , following failed or inadequate ventilation , where emergency surgical access must be obtained to avoid catastrophic consequences or death . other drawbacks are oropharyngeal decubitus effects , decrease of lung compliance with increased airway resistance , and the need to be inserted in unconscious patient . combined with the right technique of continuous intravenous anesthesia , sgvd are invaluable presidiums in some scenarios of critical airway secondary to trauma . [35 ] two scenarios , personally encountered by the author , of maxillofacial trauma compounded by head injury , hemorrhagic shock and critical airway , epitomize with its potential pitfalls and caveat the challenge some patients with post - traumatic critical airway represent . anaesthesia for critical airway it is the one of a patient being shot to the face , presenting with severe maxillofacial injury bleeding into the airways and outside , a neck collar for possible cervical spine injury ( csi ) , associated head injury with some deterioration of consciousness but still conscious , some degrees of hemorrhagic shock from oropharyngeal hemorrhage , and staying in obligatory sitting position in the attempt to relieve the dyspnea by avoiding the effect of deformity and blood on the breathing . - ; iii ) the collar removed with the head kept carefully in manual - in - line - stabilization ( mils ) on sitting / semi - sitting position by an assistant ; iv ) ketamine iv as bolus is then administered ; v ) once anesthesia is reached ( fixed gaze plus a possible or spontaneous eye closure and miosis ) , the patient is rapidly positioned supine with mils maintained and continuous intravenous anesthesia ( civa ) with infusion of ketamine or remifentanyl started up ; vi ) a diagnostic laryngoscopy ( dl ) is done to see the feasibility of a rapid endotracheal intubation ( eti ) ( visible laryngeal inlet and vocal cords ( vc ) or not , plus aspiration of blood debris and foreign bodies in the laryngeal inlet ) ; vii ) eti is done ; viii ) if eti not feasible due to deformity and view - obstructing debris and blood , a supraglottic ventilatory device like an intubating laryngeal mask airway ( ilma ) fast - trach or c - trach , or a laryngeal tube ( lt ) like a king ls ii , should rapidly be inserted ; ix ) ett is inserted through the sgvd ; ix ) a tracheostomy is then done if ett can not be inserted through the svgd or done at some stage with the svgd in situ before maxillofacial reconstruction . no formal sedative or paralytic agent should be given to this type of patient until the airway is secured . the preliminary steps ending with the sedation with ketamine should be explained to the patient in a reassuring / relaxed and cool confident voice and demeanor despite he or she may not be in enough concentration but in severe distress for the dyspnea , gurgling sounds and all the hectic and frantic preparations around that should be absolutely carried out in silence - " you will go to sleep now so i can put a tube in your windpipe for you to breathe better . " to watch the patient breathing and taking a chance before intervening is a big error as deterioration and hypoxic damages are about to happen , likewise it is a big error to ventilate without looking first to retrieve and remove material in the upper airways that can be pushed inside the airways . another extreme scenario is the one of an unconscious patient following blunt trauma to face and neck after car accident , who comes ventilated manually due to hypoventilation , manifesting indirect signs of impending upper airways obstruction , impalpable anterior neck landmarks , hypoxemia and severely bleeding massive facial injury , already supine and with a neck collar on for possible or suspected csi . similar decision - making as above : do not face - mask ventilate ; give oxygen by mask at distance from face ; do dl ; insert an ett or a svgd ; if svgd , try passing ett through it ; if eti can not be passed then obtain an emergency surgical airway ( esa ) with svgd in situ ; esa at anytime if rapid deterioration of sao2 or patient incapable to be ventilated satisfactorily with svgd . in a cardiac or respiratory / cardiac arrest by upper airways obstruction sedation or myoparalysis are also contraindicated in patients requiring a surgical airway who are still awake , breathing spontaneously and sitting to prevent or relief their hypoxemia in severe face and neck injury with respiratory compromise - a posture often observed also in patients with cardiac tamponade or post - traumatic diaphragmatic hernia.th when analgesia is required in a conscious patient candidate for an esa other than transtracheal or percutaneous transtracheal jet ventilation ( ttjv / ptjv ) or stab cricothyroidotomy , both feasible with simple la infiltration , rapid onset induction agents and rapidly reversible anesthetic agents not interfering with spontaneous ventilation or blood pressure must be used for civa . despite being an excellent for induction and maintenance , nonetheless as analgesic and sedative , ketamine has the important side effect of increasing oxygen consumption ( vo2 ) and heart rate ( hr ) in an organ , the heart , which is dependent on flow to increase oxygen delivery and at basic regimen extracts 75% ( o2er ) of the oxygen delivered ( do2 ) . fighting patients ( agitated , confused and restless ) with critical airway must be induced with ketamine , and so patients in obligatory sitting position and in severe shock . although it is acceptable for operators to use the techniques where they are more prone and acquainted in order to save lives , the corollary of all reliable comparative studies among the techniques in election done by teams of ear - nose - throat , maxillofacial or cardio - thoracic surgeons experienced in both procedures , is that ost is to be considered safer and preferable than pct in emergencies . a0 plane of dissection not strictly on the midline inevitably provokes or accentuates peri - tracheal bleeding ; a high / very high tracheotomy for its proximity to the larynx is a good recipe for laryngeal stenosis ; a low tracheotomy causes decubitus of the cannula on the trachea during neck flexion / extension movements predisposing to fistulae ; a not central ostomy with lateral decubitus of the cannula can damage the wall ; an erroneous estimation of the depth of the trachea during maneuvering risks posterior wall damage ; a lack of open vision risks to unduly damage the anterior tracheal rings ; are the six mechanisms accounting for the complications of tracheostomies. [3945 ] pros and cons of surgical airways the author 's personal recommendations for esa in critical airway are outlined in table 3. [4653 ] recommendations on which esa to use in critical airway critical airway is a life - threatening scenario of hypoxemia about to become hypoxia , following failed or inadequate ventilation , where emergency surgical access must be obtained to avoid catastrophic consequences or death . combined with the right technique of continuous intravenous anesthesia , sgvd are invaluable presidiums in some scenarios of critical airway secondary to trauma . [35 ] two scenarios , personally encountered by the author , of maxillofacial trauma compounded by head injury , hemorrhagic shock and critical airway , epitomize with its potential pitfalls and caveat the challenge some patients with post - traumatic critical airway represent . anaesthesia for critical airway it is the one of a patient being shot to the face , presenting with severe maxillofacial injury bleeding into the airways and outside , a neck collar for possible cervical spine injury ( csi ) , associated head injury with some deterioration of consciousness but still conscious , some degrees of hemorrhagic shock from oropharyngeal hemorrhage , and staying in obligatory sitting position in the attempt to relieve the dyspnea by avoiding the effect of deformity and blood on the breathing . - ; iii ) the collar removed with the head kept carefully in manual - in - line - stabilization ( mils ) on sitting / semi - sitting position by an assistant ; iv ) ketamine iv as bolus is then administered ; v ) once anesthesia is reached ( fixed gaze plus a possible or spontaneous eye closure and miosis ) , the patient is rapidly positioned supine with mils maintained and continuous intravenous anesthesia ( civa ) with infusion of ketamine or remifentanyl started up ; vi ) a diagnostic laryngoscopy ( dl ) is done to see the feasibility of a rapid endotracheal intubation ( eti ) ( visible laryngeal inlet and vocal cords ( vc ) or not , plus aspiration of blood debris and foreign bodies in the laryngeal inlet ) ; vii ) eti is done ; viii ) if eti not feasible due to deformity and view - obstructing debris and blood , a supraglottic ventilatory device like an intubating laryngeal mask airway ( ilma ) fast - trach or c - trach , or a laryngeal tube ( lt ) like a king ls ii , should rapidly be inserted ; ix ) ett is inserted through the sgvd ; ix ) a tracheostomy is then done if ett can not be inserted through the svgd or done at some stage with the svgd in situ before maxillofacial reconstruction . no formal sedative or paralytic agent should be given to this type of patient until the airway is secured . the preliminary steps ending with the sedation with ketamine should be explained to the patient in a reassuring / relaxed and cool confident voice and demeanor despite he or she may not be in enough concentration but in severe distress for the dyspnea , gurgling sounds and all the hectic and frantic preparations around that should be absolutely carried out in silence - " you will go to sleep now so i can put a tube in your windpipe for you to breathe better . " to watch the patient breathing and taking a chance before intervening is a big error as deterioration and hypoxic damages are about to happen , likewise it is a big error to ventilate without looking first to retrieve and remove material in the upper airways that can be pushed inside the airways . another extreme scenario is the one of an unconscious patient following blunt trauma to face and neck after car accident , who comes ventilated manually due to hypoventilation , manifesting indirect signs of impending upper airways obstruction , impalpable anterior neck landmarks , hypoxemia and severely bleeding massive facial injury , already supine and with a neck collar on for possible or suspected csi . similar decision - making as above : do not face - mask ventilate ; give oxygen by mask at distance from face ; do dl ; insert an ett or a svgd ; if svgd , try passing ett through it ; if eti can not be passed then obtain an emergency surgical airway ( esa ) with svgd in situ ; esa at anytime if rapid deterioration of sao2 or patient incapable to be ventilated satisfactorily with svgd . sedation or myoparalysis are also contraindicated in patients requiring a surgical airway who are still awake , breathing spontaneously and sitting to prevent or relief their hypoxemia in severe face and neck injury with respiratory compromise - a posture often observed also in patients with cardiac tamponade or post - traumatic diaphragmatic hernia.th when analgesia is required in a conscious patient candidate for an esa other than transtracheal or percutaneous transtracheal jet ventilation ( ttjv / ptjv ) or stab cricothyroidotomy , both feasible with simple la infiltration , rapid onset induction agents and rapidly reversible anesthetic agents not interfering with spontaneous ventilation or blood pressure must be used for civa . despite being an excellent for induction and maintenance , nonetheless as analgesic and sedative , ketamine has the important side effect of increasing oxygen consumption ( vo2 ) and heart rate ( hr ) in an organ , the heart , which is dependent on flow to increase oxygen delivery and at basic regimen extracts 75% ( o2er ) of the oxygen delivered ( do2 ) . fighting patients ( agitated , confused and restless ) with critical airway must be induced with ketamine , and so patients in obligatory sitting position and in severe shock . it is the one of a patient being shot to the face , presenting with severe maxillofacial injury bleeding into the airways and outside , a neck collar for possible cervical spine injury ( csi ) , associated head injury with some deterioration of consciousness but still conscious , some degrees of hemorrhagic shock from oropharyngeal hemorrhage , and staying in obligatory sitting position in the attempt to relieve the dyspnea by avoiding the effect of deformity and blood on the breathing . - ; iii ) the collar removed with the head kept carefully in manual - in - line - stabilization ( mils ) on sitting / semi - sitting position by an assistant ; iv ) ketamine iv as bolus is then administered ; v ) once anesthesia is reached ( fixed gaze plus a possible or spontaneous eye closure and miosis ) , the patient is rapidly positioned supine with mils maintained and continuous intravenous anesthesia ( civa ) with infusion of ketamine or remifentanyl started up ; vi ) a diagnostic laryngoscopy ( dl ) is done to see the feasibility of a rapid endotracheal intubation ( eti ) ( visible laryngeal inlet and vocal cords ( vc ) or not , plus aspiration of blood debris and foreign bodies in the laryngeal inlet ) ; vii ) eti is done ; viii ) if eti not feasible due to deformity and view - obstructing debris and blood , a supraglottic ventilatory device like an intubating laryngeal mask airway ( ilma ) fast - trach or c - trach , or a laryngeal tube ( lt ) like a king ls ii , should rapidly be inserted ; ix ) ett is inserted through the sgvd ; ix ) a tracheostomy is then done if ett can not be inserted through the svgd or done at some stage with the svgd in situ before maxillofacial reconstruction . no formal sedative or paralytic agent should be given to this type of patient until the airway is secured . the preliminary steps ending with the sedation with ketamine should be explained to the patient in a reassuring / relaxed and cool confident voice and demeanor despite he or she may not be in enough concentration but in severe distress for the dyspnea , gurgling sounds and all the hectic and frantic preparations around that should be absolutely carried out in silence - " you will go to sleep now so i can put a tube in your windpipe for you to breathe better . " to watch the patient breathing and taking a chance before intervening is a big error as deterioration and hypoxic damages are about to happen , likewise it is a big error to ventilate without looking first to retrieve and remove material in the upper airways that can be pushed inside the airways . another extreme scenario is the one of an unconscious patient following blunt trauma to face and neck after car accident , who comes ventilated manually due to hypoventilation , manifesting indirect signs of impending upper airways obstruction , impalpable anterior neck landmarks , hypoxemia and severely bleeding massive facial injury , already supine and with a neck collar on for possible or suspected csi . similar decision - making as above : do not face - mask ventilate ; give oxygen by mask at distance from face ; do dl ; insert an ett or a svgd ; if svgd , try passing ett through it ; if eti can not be passed then obtain an emergency surgical airway ( esa ) with svgd in situ ; esa at anytime if rapid deterioration of sao2 or patient incapable to be ventilated satisfactorily with svgd . sedation or myoparalysis are also contraindicated in patients requiring a surgical airway who are still awake , breathing spontaneously and sitting to prevent or relief their hypoxemia in severe face and neck injury with respiratory compromise - a posture often observed also in patients with cardiac tamponade or post - traumatic diaphragmatic hernia.th when analgesia is required in a conscious patient candidate for an esa other than transtracheal or percutaneous transtracheal jet ventilation ( ttjv / ptjv ) or stab cricothyroidotomy , both feasible with simple la infiltration , rapid onset induction agents and rapidly reversible anesthetic agents not interfering with spontaneous ventilation or blood pressure must be used for civa . despite being an excellent for induction and maintenance , nonetheless as analgesic and sedative , ketamine has the important side effect of increasing oxygen consumption ( vo2 ) and heart rate ( hr ) in an organ , the heart , which is dependent on flow to increase oxygen delivery and at basic regimen extracts 75% ( o2er ) of the oxygen delivered ( do2 ) . fighting patients ( agitated , confused and restless ) with critical airway must be induced with ketamine , and so patients in obligatory sitting position and in severe shock . although it is acceptable for operators to use the techniques where they are more prone and acquainted in order to save lives , the corollary of all reliable comparative studies among the techniques in election done by teams of ear - nose - throat , maxillofacial or cardio - thoracic surgeons experienced in both procedures , is that ost is to be considered safer and preferable than pct in emergencies . a0 plane of dissection not strictly on the midline inevitably provokes or accentuates peri - tracheal bleeding ; a high / very high tracheotomy for its proximity to the larynx is a good recipe for laryngeal stenosis ; a low tracheotomy causes decubitus of the cannula on the trachea during neck flexion / extension movements predisposing to fistulae ; a not central ostomy with lateral decubitus of the cannula can damage the wall ; an erroneous estimation of the depth of the trachea during maneuvering risks posterior wall damage ; a lack of open vision risks to unduly damage the anterior tracheal rings ; are the six mechanisms accounting for the complications of tracheostomies. [3945 ] pros and cons of surgical airways the author 's personal recommendations for esa in critical airway are outlined in table 3. [4653 ] recommendations on which esa to use in critical airway analgesia / anesthesia for critical airway or during a situation of critical airway is a highly skilled task to be accomplished with sheer accuracy and a reasonable safety margin , due to the side - effects of anesthetic or analgesic drugs on airway and ventilation . following analog ethical and realistic considerations , specific indications , timing and type of emergency surgical airway can therefore only be given as result of axioms and corollaries , personal experience and reliable comparative studies among the techniques in election by specialist teams experienced in both techniques .

it describes the uneven distribution of population groups defined by a special characteristic among the subunits of a social space . population groups can be defined as subgroups of race / ethnicity , socioeconomic status , education , gender , age , etc . , social space can be a geographic social space like city , province , municipality district , or school , or an economic social space like labor market or other groups . since 1955 , for 20 years , index of dissimilarity was the standard index of residential segregation until it was criticized by charles cortes and some new indices were invented . in 1988 , massey and denton did a systematic analysis on 19 segregation indices which they extracted from literature and claimed that segregation is not a one - dimensional construct . they described five dimensions for residential segregation named as evenness , exposure , clustering , centralization , and concentration . evenness and exposure are aspatial dimensions , whereas clustering , concentration , and centralization are spatial dimensions of segregation and require information on the locations and areas of census tracts to compute . these resources might be institutional like health care facilities and hospitals , job opportunities , different types of schools , labor markets ; social like social networks , cultural capital ; contextual like different proportions of individuals with different educational levels , married versus single and divorced individuals , people of different socioeconomic status , and even environmental and social hazards like lead poisoning , pollution , higher violence , and crime rates . many recent studies have documented association between segregation and health outcomes like overall health , infant mortality , tuberculosis , cardiovascular disease , high body mass index , and non - health outcomes like access to employment and education inequalities , early adolescent sexual activity , black homicidal rates , and health service use . in many developing and underdeveloped countries , the patterns of residential segregation are well described , e.g. , south africa , great britain , singapore , united stated . first , most of the work in measuring segregation was limited to measuring racial segregation and segregation of other aspects of residential place like the demographic characteristics of residents , and the structural features of neighborhood or the socioeconomic characteristics of people living in a neighborhood have been neglected so far . second , so far segregation has been measured mostly for dichotomous variables and few papers have used multigroup indices of segregation . third , measures of segregation have been reported mostly for western contexts and few articles exist in the literature about these measures in eastern developing countries . considering the above - mentioned limitations in the existing literature and with the help of recent advances in measuring segregation for multi - group and ordinal variables , we are going to answer the following questions . how segregated are the iran provinces with regard to socioeconomic factors and some health indices , and is there any association between these indices ? residential segregation indices measure distribution of different population dimensions across residential units within larger geographic areas . thus , to measure segregation , one must first define the larger area and its subunits . second , the population dimension along which one wishes to measure segregation must be defined since measuring segregation among two , multiple unordered , or multiple ordered categories of a variable needs a different set of measurement tools . third , the dimension of segregation which is going to be measured should also be defined . in this paper , segregation was measured for each province of the country across its municipality spaces . according to country divisions , islamic republic of iran in 2006 had 30 provinces , 1012 cities ; but among these cities , there were metropolises like tehran which contains about 10% of the population . it would not be appropriate to consider these cities as one subunit in the province . therefore , like the work of ethington for los angles city , we divided each major city into its municipality districts and considered them as subunits of analysis in each province . in this way , a large number of indices of residential segregation have been used in researches on segregation and its consequences . among these indices , measures of evenness the most commonly used index for measuring this dimension of segregation is dissimilarity index ( denoted as d ) . it can be interpreted as the proportion of all people who should be transferred among residential units to have the same group proportions across residential units . it can be written as : where r refers to municipality spaces in each province , tr is the population of municipality space r , rm is the proportion of group m in municipality space r , m is the proportion of group m in the province , and t is the total population of the province . in 2002 , a revised index of dissimilarity named generalized dissimilarity index was proposed by reardon et al . , which is used for measuring segregation among multiple unordered categories of a variable ( e.g. , multiple age groups ) . it is interpreted as difference between diversity ( entropy ) of the system and the weighted average diversity of individual units , expressed as a fraction of total diversity of the system . diversity ( entropy ) of the population ( denoted as e ) is defined as : information theory index can be used for measuring segregation for multiple categorical variables . besides , this index satisfies the principles of exchange , transfer , additive group decomposability , and additive organizational decomposability , while the dissimilarity index does not satisfy these properties . we used seg command in stata software to compute segregation indices and double checked the results in excel software . since these two indices are not appropriate where the variable is an ordinal , for measuring segregation among ordered groups of ordinal variables ( e.g. , education attainment ) using the work of reardon in 2009 , we used ordinal information theory index ( denote as h0 ) . this census is one of the biggest data gathering projects in iran , which is conducted every 10 years and the latest one was in 2006 . different data about socioeconomic , demographic , and health variables from both individuals and family units were gathered through this census . a random sample containing 20% of data was presented in two databases by iranian national statistic center : an individual database , which each record of it contained the data of one person and a family database which each record of it contained the aggregated data of one family . these databases were used for measuring segregation . generalized dissimilarity index and information theory index were measured for the demographic , socioeconomic , and health variables defined in tables 1 and 2 . one of the special features of the latest census was adding some questions about health outcomes including infant mortality , existence of disability in individuals , existence of disability in the family , and death of offspring of the mother , to the standard questions of the census . according to the definition mentioned in technical guideline of the census , any person with at least one of the following problems was considered a disabled person : blindness , deafness , speech disorders , upper or lower extremities amputation at any level , any physical or functional disorder in upper extremities , lower extremities , or trunk , and any mental disorder . description of demographic , socioeconomic , and health variables used for measuring segregation ( individual dataset ) description of demographic , socioeconomic , and health variables used for measuring segregation ( family dataset ) for interpreting the values of dissimilarity index and information theory index according to the sociology literature , we used the following cut - off points : extreme segregation ( 40 - 100 ) , high segregation ( 25 - 39 ) , moderate segregation ( 10 - 24 ) , and low segregation ( 0 - 9 ) for information theory index ; and extreme segregation ( 70 - 100 ) , high segregation ( 40 - 69 ) , moderate segregation ( 30 - 39 ) , and low segregation ( 0 - 29 ) for dissimilarity index . if a measured index for a variable was classified in one level according to dissimilarity index and in another level according to information theory index , the segregation was classified in the higher level . to assess the association of segregation of health variables with segregation of other socioeconomic and demographic variables , matrix of correlations was applied and significant correlations between 0.3 and 0.7 were reported as weak correlation and correlations more than 0.7 were reported as strong correlation . residential segregation indices measure distribution of different population dimensions across residential units within larger geographic areas . thus , to measure segregation , one must first define the larger area and its subunits . second , the population dimension along which one wishes to measure segregation must be defined since measuring segregation among two , multiple unordered , or multiple ordered categories of a variable needs a different set of measurement tools . third , the dimension of segregation which is going to be measured should also be defined . in this paper , segregation was measured for each province of the country across its municipality spaces . according to country divisions , islamic republic of iran in 2006 had 30 provinces , 1012 cities ; but among these cities , there were metropolises like tehran which contains about 10% of the population . it would not be appropriate to consider these cities as one subunit in the province . therefore , like the work of ethington for los angles city , we divided each major city into its municipality districts and considered them as subunits of analysis in each province . in this way , a large number of indices of residential segregation have been used in researches on segregation and its consequences . among these indices , measures of evenness the most commonly used index for measuring this dimension of segregation is dissimilarity index ( denoted as d ) . it can be interpreted as the proportion of all people who should be transferred among residential units to have the same group proportions across residential units . it can be written as : where r refers to municipality spaces in each province , tr is the population of municipality space r , rm is the proportion of group m in municipality space r , m is the proportion of group m in the province , and t is the total population of the province . in 2002 , a revised index of dissimilarity named generalized dissimilarity index was proposed by reardon et al . , which is used for measuring segregation among multiple unordered categories of a variable ( e.g. , multiple age groups ) . it is interpreted as difference between diversity ( entropy ) of the system and the weighted average diversity of individual units , expressed as a fraction of total diversity of the system . diversity ( entropy ) of the population ( denoted as e ) is defined as : information theory index can be used for measuring segregation for multiple categorical variables . besides , this index satisfies the principles of exchange , transfer , additive group decomposability , and additive organizational decomposability , while the dissimilarity index does not satisfy these properties . we used seg command in stata software to compute segregation indices and double checked the results in excel software . since these two indices are not appropriate where the variable is an ordinal , for measuring segregation among ordered groups of ordinal variables ( e.g. , education attainment ) using the work of reardon in 2009 , we used ordinal information theory index ( denote as h0 ) . this census is one of the biggest data gathering projects in iran , which is conducted every 10 years and the latest one was in 2006 . different data about socioeconomic , demographic , and health variables from both individuals and family units were gathered through this census . a random sample containing 20% of data was presented in two databases by iranian national statistic center : an individual database , which each record of it contained the data of one person and a family database which each record of it contained the aggregated data of one family . generalized dissimilarity index and information theory index were measured for the demographic , socioeconomic , and health variables defined in tables 1 and 2 . one of the special features of the latest census was adding some questions about health outcomes including infant mortality , existence of disability in individuals , existence of disability in the family , and death of offspring of the mother , to the standard questions of the census . according to the definition mentioned in technical guideline of the census , any person with at least one of the following problems was considered a disabled person : blindness , deafness , speech disorders , upper or lower extremities amputation at any level , any physical or functional disorder in upper extremities , lower extremities , or trunk , and any mental disorder . description of demographic , socioeconomic , and health variables used for measuring segregation ( individual dataset ) description of demographic , socioeconomic , and health variables used for measuring segregation ( family dataset ) for interpreting the values of dissimilarity index and information theory index according to the sociology literature , we used the following cut - off points : extreme segregation ( 40 - 100 ) , high segregation ( 25 - 39 ) , moderate segregation ( 10 - 24 ) , and low segregation ( 0 - 9 ) for information theory index ; and extreme segregation ( 70 - 100 ) , high segregation ( 40 - 69 ) , moderate segregation ( 30 - 39 ) , and low segregation ( 0 - 29 ) for dissimilarity index . if a measured index for a variable was classified in one level according to dissimilarity index and in another level according to information theory index , the segregation was classified in the higher level . to assess the association of segregation of health variables with segregation of other socioeconomic and demographic variables , matrix of correlations was applied and significant correlations between 0.3 and 0.7 were reported as weak correlation and correlations more than 0.7 were reported as strong correlation . according to the results of iranian national census in 2006 , the population in the country was 70,495,782 ; 50.54% of the population was males and 49.46% was females . 25.14% of the population was under 15 years old , 69.62% was between 15 and 65 years , and 5.24% were more than 65 years . literacy rate of the population of more 6 years of age was 84.2% . about 59.5% of the population was living in their birthplace , and 16.15% of the population had history of migration in the 10 years preceding the census . in the population older than 10 years , 39.9% was active and 60.1% was inactive , 5.26% did not have a job , 21.66% were students , 27.64% were housewives , 5.33% had income without job , 34.61% had job , and 5.5% were in other categories . regarding marital status , 38.8% of the population older than 10 years was single , 56.8% was married , and 3.4% was widow or divorced . considering the number of marriages in the population older than 10 years , the number of marriages was zero in 40.17% , one in 57.89% , and two or more in 1.94% . the proportion of disability according to the medical definition presented by scientific committee of census in the total population was 1.56% ( 1.44 - 1.69 ) , in the population less than 10 years old was 0.65 ( 0.59 - 0.71 ) , and in the population older than 10 years was 1.75% ( 1.61 - 1.89 ) . according to the census data , 23.5% ( 20.9 - 26 ) of mothers who had given birth to at least one child had lost at least one of their offspring and 0.048% ( 0.40 - 0.055 ) of mothers who had given birth to a live infant in the 365-day period preceding the census had lost their infant . generalized dissimilarity segregation index and information theory index measured for all the individual socioeconomic and health indices are presented in table 1 . all the provinces were in the category of low segregation for age groups , sex , literacy , education level , type of activity , activity status , employment type , marital status , existence of disability in individuals , death of at least one offspring of mother , and number of remarriages . for birthplace , tehran , qazvin , isfahan , and chahar mahal and bakhtiari provinces were moderately segregated . for immigration history in the previous 10 years , just tehran province was moderately segregated . for infant mortality , 13 provinces including qazvin , khorasan razavi , khuzestan , w. azarbaijan , lorestan , markazi , kurdistan , zanjan , hamedan , kohgiluyeh and buyer ahmad , bushehr , chahar mahal and bakhtiari , and fars were moderately segregated ( in order of increasing segregation ) and semnan and mazandaran were highly segregated [ figure 1 ] . classification of provinces according to segregation of infant mortality generalized dissimilarity segregation index and information theory index were also measured for family socioeconomic and health indices which are defined in table 2 . all the provinces were in the category of low segregation for existence of disability in the family , number of family members , number of literate individuals in the family , number of illiterate individuals in the family , number of individuals with job in the family , number of individuals without job in the family , number of students in the family , existence of vehicle in the family , owning a house , and numbers of rooms per capita . for household size , just tehran province was moderately segregated . for existence of motorcycle in the family , 10 provinces including hamedan , s. khorasan , gilan , chahar mahal and bakhtiari , hormozgan , e. azarbaijan , kurdistan , khuzestan , ilam , and qazvin ( in order of increasing segregation ) were in the category of moderate segregation and two provinces including fars and kermanshah were in the category of high segregation . for existence of computer in the family , two provinces including khorasan razavi and tehran were moderately segregated . for access to internet by the family , three provinces including sistan va baluchestan , khorasan razavi , and tehran were moderately segregated . for existence of telephone in the family , qom , kerman , and tehran provinces were moderately segregated . for use of natural gas for cooking , hamedan , qazvin , gilan , and e. azarbaijan were moderately segregated , isfahan , w. azarbaijan , kohgiluyeh and buyer ahmad , lorestan , semnan , golestan , and mazandaran provinces were highly segregated , and fars , ardebil , khuzestan , yazd , n. khorasan , tehran , chahar mahal and bakhtiari , markazi , kerman , khorasan razavi kermanshah , kurdistan , zanjan , and bushehr provinces were extremely segregated . for use of natural gas for heating , markazi , qazvin , khuzestan , hormozgan , and tehran provinces were moderately segregated and bushehr was highly segregated . for main source of drinking water , yazd , markazi , s. khorasan , gilan , ardebil , bushehr sistan va baluchestan , khorasan razavi , w. azarbaijan were moderately segregated , mazandaran , e. azarbaijan , ch . and bakhtiari , tehran , kermanshah , semnan , isfahan , kurdistan , golestan , and hamedan provinces were highly segregated , and kerman , hormozgan , khuzestan , fars , and kohgiluyeh and buyer ahmad were extremely segregated . for existence of bathroom in the household , khorasan razavi and chahar mahal and bakhtiari provinces were moderately segregated and kohgiluyeh and buyer ahmad and qazvin provinces were highly segregated . for type of bathroom effluent disposal , just kerman province had low segregation , ilam , mazandaran , kohgiluyeh and buyer ahmad , zanjan , and yazd were moderately segregated , golestan , n. khorasan , tehran , fars , s. khorasan , hormozgan , w. azarbaijan , e. azarbaijan , qom , markazi , ardebil , kermanshah , kurdistan , bushehr , and semnan were highly segregated , and gilan , khuzestan , qazvin , khorasan razavi , sistan va baluchestan , chahar mahal and bakhtiari , isfahan , lorestan , and hamedan were extremely segregated . for existence of separate kitchen in the household , golestan , sistan va baluchestan , kurdistan , ilam , e. azarbaijan , mazandaran , and w. azarbaijan were moderately segregated and hormozgan , kermanshah , s. khorasan , khuzestan , khorasan razavi , bushehr , hamedan , isfahan , kerman , lorestan , qazvin , yazd , fars , chahar mahal and bakhtiari , semnan , gilan , kohgiluyeh and buyer ahmad , and markazi were highly segregated . to answer the question whether segregation of health indices is correlated with segregation of other socioeconomic variables , we used pair wise correlation of existence of disability in individual , existence of disability in family , infant mortality , and death of at least one offspring of mother with other socioeconomic variables . for example , sex segregation across different occupations can explain the gender gap in earnings . residential segregation has been thought to contribute to black poverty and higher black mortality among whites . racial segregation among schools has been blamed for low educational achievement among minority groups ; besides , it is considered as the prime suspect in explaining the gap in human capital between racial groups in western countries . in some other contexts , segregation might be useful . a segregated place for a social characteristic means accumulation of people with that characteristic in smaller places . it is debated that this formation of homogenous living residential areas might be a solution for highly polarized conflicts in the middle east . in many countries , measures of segregation residential racial segregation indices are available since 1980 and many articles in the literature have investigated different effects of segregation on social and health outcomes . in this study , for the first time in iran , we computed the measures of segregation for socioeconomic and health variables using the national census data of 2006 . for infant mortality , mazandaran and semnan provinces were highly segregated ; in other words , the incidence of infant death was not evenly distributed across the municipality units in these provinces . in 12 provinces , the segregation wad moderate . this finding is in accordance with the results of the study on socioeconomic inequality in infant mortality across the provinces in iran , which was performed on data of infant mortality in iran from 1995 to 2000 . in that study , mazandaran province was one of the five provinces in iran which had the highest inequality in infant mortality in iran . of the 12 provinces which were in the category of moderate segregation in our study , khuzestan and zanjan had high inequality , and khorasan razavi , w. azarbaijan , markazi , kurdistan , kohgiluyeh and buyer ahmad , bushehr , fars , and qazvin had moderate inequality in infant mortality in that study . lorestan , hamedan , and ch . and bakhtiari were moderately segregated for infant mortality in 2006 , but in that study they had low inequality . this province was also segregated for birthplace . considering that tehran is the capital city of the country and the rate of immigration to this province is high , this means that immigrant people are not evenly distributed across residential areas in this province . of the socioeconomic variables , segregation indices measured for housing conditions like main source of drinking water and existence of separate kitchen in the household were correlated with segregation of all four health indices in this study . in the provinces where access to tap water and housing conditions of the families are not evenly distributed across the municipality units , there is more probability of finding hot zones for infant mortality , disability , and offspring death . segregation of activity level and job characteristic of individuals , economic situation of family presented by house ownership and existence of motorcycle in family , number of literate individuals in the family , and number of students in the family were also correlated with segregation of two or more health indices in this study . sex segregation was correlated with segregation of infant mortality , existence of at least one disabled person in the family , and death of at least one offspring , and literacy segregation was correlated with existence of one disabled person in the family and death of one offspring , but all the provinces were in the category of low segregation for these tow characteristics . it can be concluded the even small changes in segregation of sex and literacy proportions in provinces might result in uneven distribution of health outcomes and creates hot zones for these health outcomes . these findings are consistent with the results of some other studies in different parts of the world . in a study of inequality in nine developing countries , levels of inequality in consumption , levels of socioeconomic status composed of income , education , housing condition , and house ownership were associated negatively with child death rates . in a study in a brazilian city , geo - economic classification of city was correlated negatively with infant mortality . the critical question about the importance of segregation is on the causal pathways that segregation acts through them . the third and fourth hypothesized mechanisms are modification of social capital of a residential place and changing individual risk behaviors and exposures to stressful situations . degree of social trust between individuals in a neighborhood , extent of social networks , and tendency for mutual aid and support are the determining factors of social capital . information theory index and generalized dissimilarity index were used in the study because of their unique statistical characteristics and high use in other studies of segregation , but in some studies , isolation index has been used . further studies are necessary to determine the best segregation index for measuring segregation of markers of socioeconomic status and health this approach makes comparison of measured indices across periods of census and checking the trend of changes feasible . in this study , segregation was measured in each province across municipality spaces , and provinces were compared according to these measures . it is possible to measure segregation across smaller geographic units for each city in every province to compare the degree of segregation of major cities inside each province . this method might show better correlation of segregation of heath indices and determinants of socioeconomic status . the choice of health variables in this study was very limited because of limited health variables measured in the national census . it is recommended that by using data of national health surveys , segregation of other health variables is measured . aside from these limitations , this study is of high value since it is the first study in iran which measures segregation and one of the first studies in the literature to measure segregation of socioeconomic status variables in national level . information theory index and generalized dissimilarity index were used in the study because of their unique statistical characteristics and high use in other studies of segregation , but in some studies , isolation index has been used . further studies are necessary to determine the best segregation index for measuring segregation of markers of socioeconomic status and health . this approach makes comparison of measured indices across periods of census and checking the trend of changes feasible . in this study , segregation was measured in each province across municipality spaces , and provinces were compared according to these measures . it is possible to measure segregation across smaller geographic units for each city in every province to compare the degree of segregation of major cities inside each province . this method might show better correlation of segregation of heath indices and determinants of socioeconomic status . the choice of health variables in this study was very limited because of limited health variables measured in the national census . it is recommended that by using data of national health surveys , segregation of other health variables is measured . aside from these limitations , this study is of high value since it is the first study in iran which measures segregation and one of the first studies in the literature to measure segregation of socioeconomic status variables in national level . in this study , segregation of iranian provinces for major determinants of socioeconomic status and also some health indices were reviewed . for infant mortality and some of the socioeconomic factors , considering the effect of segregation on health outcomes which have been supported by large body of evidence in the literature , investigating why segregation is higher in some provinces deserves special attention .

background : measures of segregation are essential tools for evaluation of social equality . they describe complex structural patterns by single quantities and allow the comparison of inequalities over time or between residential places . in many countries , patterns of residential segregation are well described ( e.g. , south africa , great britain , united states of america ) . in this study , for the first time in iran , we measured residential segregation for some socioeconomic and health variables and described their pair wise correlation.methods:we measured evenness dimension of segregation by generalized dissimilarity segregation index and information theory index and its ordinal equivalent for some determinants of socioeconomic status and health variables using data of last national census in iran . segregation indices were computed for 31 socioeconomic variables and four health indices.results:all the provinces were in the category of low segregation for individual and family disability and death of at least one offspring of mother , but for infant mortality half of the provinces were moderately or highly segregated . for some of socioeconomic variables , many provinces were in the category of moderate , high , or extreme segregation . there was significant correlation between segregation of heath indices and some socioeconomic variables.conclusions:correlation of segregation of determinants of socioeconomic status with segregation of health indices is an indicator of existence of hot zones of health problems across some provinces . further studies using multilevel modeling and individual data in health outcomes at individual level and segregation measures at appropriate geographic levels are required to confirm these relations .

INTRODUCTION METHODS Segregation indices Database Variables Analysis RESULTS DISCUSSION Limitations CONCLUSIONS

since 1955 , for 20 years , index of dissimilarity was the standard index of residential segregation until it was criticized by charles cortes and some new indices were invented . many recent studies have documented association between segregation and health outcomes like overall health , infant mortality , tuberculosis , cardiovascular disease , high body mass index , and non - health outcomes like access to employment and education inequalities , early adolescent sexual activity , black homicidal rates , and health service use . in many developing and underdeveloped countries , the patterns of residential segregation are well described , e.g. , south africa , great britain , singapore , united stated . first , most of the work in measuring segregation was limited to measuring racial segregation and segregation of other aspects of residential place like the demographic characteristics of residents , and the structural features of neighborhood or the socioeconomic characteristics of people living in a neighborhood have been neglected so far . second , so far segregation has been measured mostly for dichotomous variables and few papers have used multigroup indices of segregation . third , measures of segregation have been reported mostly for western contexts and few articles exist in the literature about these measures in eastern developing countries . considering the above - mentioned limitations in the existing literature and with the help of recent advances in measuring segregation for multi - group and ordinal variables , we are going to answer the following questions . how segregated are the iran provinces with regard to socioeconomic factors and some health indices , and is there any association between these indices ? residential segregation indices measure distribution of different population dimensions across residential units within larger geographic areas . third , the dimension of segregation which is going to be measured should also be defined . in this paper , segregation was measured for each province of the country across its municipality spaces . in this way , a large number of indices of residential segregation have been used in researches on segregation and its consequences . among these indices , measures of evenness the most commonly used index for measuring this dimension of segregation is dissimilarity index ( denoted as d ) . , which is used for measuring segregation among multiple unordered categories of a variable ( e.g. diversity ( entropy ) of the population ( denoted as e ) is defined as : information theory index can be used for measuring segregation for multiple categorical variables . we used seg command in stata software to compute segregation indices and double checked the results in excel software . since these two indices are not appropriate where the variable is an ordinal , for measuring segregation among ordered groups of ordinal variables ( e.g. , education attainment ) using the work of reardon in 2009 , we used ordinal information theory index ( denote as h0 ) . this census is one of the biggest data gathering projects in iran , which is conducted every 10 years and the latest one was in 2006 . different data about socioeconomic , demographic , and health variables from both individuals and family units were gathered through this census . generalized dissimilarity index and information theory index were measured for the demographic , socioeconomic , and health variables defined in tables 1 and 2 . one of the special features of the latest census was adding some questions about health outcomes including infant mortality , existence of disability in individuals , existence of disability in the family , and death of offspring of the mother , to the standard questions of the census . according to the definition mentioned in technical guideline of the census , any person with at least one of the following problems was considered a disabled person : blindness , deafness , speech disorders , upper or lower extremities amputation at any level , any physical or functional disorder in upper extremities , lower extremities , or trunk , and any mental disorder . description of demographic , socioeconomic , and health variables used for measuring segregation ( individual dataset ) description of demographic , socioeconomic , and health variables used for measuring segregation ( family dataset ) for interpreting the values of dissimilarity index and information theory index according to the sociology literature , we used the following cut - off points : extreme segregation ( 40 - 100 ) , high segregation ( 25 - 39 ) , moderate segregation ( 10 - 24 ) , and low segregation ( 0 - 9 ) for information theory index ; and extreme segregation ( 70 - 100 ) , high segregation ( 40 - 69 ) , moderate segregation ( 30 - 39 ) , and low segregation ( 0 - 29 ) for dissimilarity index . if a measured index for a variable was classified in one level according to dissimilarity index and in another level according to information theory index , the segregation was classified in the higher level . to assess the association of segregation of health variables with segregation of other socioeconomic and demographic variables , matrix of correlations was applied and significant correlations between 0.3 and 0.7 were reported as weak correlation and correlations more than 0.7 were reported as strong correlation . residential segregation indices measure distribution of different population dimensions across residential units within larger geographic areas . third , the dimension of segregation which is going to be measured should also be defined . in this way , a large number of indices of residential segregation have been used in researches on segregation and its consequences . among these indices , measures of evenness the most commonly used index for measuring this dimension of segregation is dissimilarity index ( denoted as d ) . it can be written as : where r refers to municipality spaces in each province , tr is the population of municipality space r , rm is the proportion of group m in municipality space r , m is the proportion of group m in the province , and t is the total population of the province . diversity ( entropy ) of the population ( denoted as e ) is defined as : information theory index can be used for measuring segregation for multiple categorical variables . we used seg command in stata software to compute segregation indices and double checked the results in excel software . since these two indices are not appropriate where the variable is an ordinal , for measuring segregation among ordered groups of ordinal variables ( e.g. , education attainment ) using the work of reardon in 2009 , we used ordinal information theory index ( denote as h0 ) . this census is one of the biggest data gathering projects in iran , which is conducted every 10 years and the latest one was in 2006 . different data about socioeconomic , demographic , and health variables from both individuals and family units were gathered through this census . generalized dissimilarity index and information theory index were measured for the demographic , socioeconomic , and health variables defined in tables 1 and 2 . one of the special features of the latest census was adding some questions about health outcomes including infant mortality , existence of disability in individuals , existence of disability in the family , and death of offspring of the mother , to the standard questions of the census . according to the definition mentioned in technical guideline of the census , any person with at least one of the following problems was considered a disabled person : blindness , deafness , speech disorders , upper or lower extremities amputation at any level , any physical or functional disorder in upper extremities , lower extremities , or trunk , and any mental disorder . description of demographic , socioeconomic , and health variables used for measuring segregation ( individual dataset ) description of demographic , socioeconomic , and health variables used for measuring segregation ( family dataset ) for interpreting the values of dissimilarity index and information theory index according to the sociology literature , we used the following cut - off points : extreme segregation ( 40 - 100 ) , high segregation ( 25 - 39 ) , moderate segregation ( 10 - 24 ) , and low segregation ( 0 - 9 ) for information theory index ; and extreme segregation ( 70 - 100 ) , high segregation ( 40 - 69 ) , moderate segregation ( 30 - 39 ) , and low segregation ( 0 - 29 ) for dissimilarity index . if a measured index for a variable was classified in one level according to dissimilarity index and in another level according to information theory index , the segregation was classified in the higher level . to assess the association of segregation of health variables with segregation of other socioeconomic and demographic variables , matrix of correlations was applied and significant correlations between 0.3 and 0.7 were reported as weak correlation and correlations more than 0.7 were reported as strong correlation . according to the results of iranian national census in 2006 , the population in the country was 70,495,782 ; 50.54% of the population was males and 49.46% was females . the proportion of disability according to the medical definition presented by scientific committee of census in the total population was 1.56% ( 1.44 - 1.69 ) , in the population less than 10 years old was 0.65 ( 0.59 - 0.71 ) , and in the population older than 10 years was 1.75% ( 1.61 - 1.89 ) . according to the census data , 23.5% ( 20.9 - 26 ) of mothers who had given birth to at least one child had lost at least one of their offspring and 0.048% ( 0.40 - 0.055 ) of mothers who had given birth to a live infant in the 365-day period preceding the census had lost their infant . generalized dissimilarity segregation index and information theory index measured for all the individual socioeconomic and health indices are presented in table 1 . all the provinces were in the category of low segregation for age groups , sex , literacy , education level , type of activity , activity status , employment type , marital status , existence of disability in individuals , death of at least one offspring of mother , and number of remarriages . for birthplace , tehran , qazvin , isfahan , and chahar mahal and bakhtiari provinces were moderately segregated . for infant mortality , 13 provinces including qazvin , khorasan razavi , khuzestan , w. azarbaijan , lorestan , markazi , kurdistan , zanjan , hamedan , kohgiluyeh and buyer ahmad , bushehr , chahar mahal and bakhtiari , and fars were moderately segregated ( in order of increasing segregation ) and semnan and mazandaran were highly segregated [ figure 1 ] . classification of provinces according to segregation of infant mortality generalized dissimilarity segregation index and information theory index were also measured for family socioeconomic and health indices which are defined in table 2 . all the provinces were in the category of low segregation for existence of disability in the family , number of family members , number of literate individuals in the family , number of illiterate individuals in the family , number of individuals with job in the family , number of individuals without job in the family , number of students in the family , existence of vehicle in the family , owning a house , and numbers of rooms per capita . for existence of motorcycle in the family , 10 provinces including hamedan , s. khorasan , gilan , chahar mahal and bakhtiari , hormozgan , e. azarbaijan , kurdistan , khuzestan , ilam , and qazvin ( in order of increasing segregation ) were in the category of moderate segregation and two provinces including fars and kermanshah were in the category of high segregation . for existence of computer in the family , two provinces including khorasan razavi and tehran were moderately segregated . for existence of telephone in the family , qom , kerman , and tehran provinces were moderately segregated . for use of natural gas for cooking , hamedan , qazvin , gilan , and e. azarbaijan were moderately segregated , isfahan , w. azarbaijan , kohgiluyeh and buyer ahmad , lorestan , semnan , golestan , and mazandaran provinces were highly segregated , and fars , ardebil , khuzestan , yazd , n. khorasan , tehran , chahar mahal and bakhtiari , markazi , kerman , khorasan razavi kermanshah , kurdistan , zanjan , and bushehr provinces were extremely segregated . for use of natural gas for heating , markazi , qazvin , khuzestan , hormozgan , and tehran provinces were moderately segregated and bushehr was highly segregated . and bakhtiari , tehran , kermanshah , semnan , isfahan , kurdistan , golestan , and hamedan provinces were highly segregated , and kerman , hormozgan , khuzestan , fars , and kohgiluyeh and buyer ahmad were extremely segregated . for existence of bathroom in the household , khorasan razavi and chahar mahal and bakhtiari provinces were moderately segregated and kohgiluyeh and buyer ahmad and qazvin provinces were highly segregated . for type of bathroom effluent disposal , just kerman province had low segregation , ilam , mazandaran , kohgiluyeh and buyer ahmad , zanjan , and yazd were moderately segregated , golestan , n. khorasan , tehran , fars , s. khorasan , hormozgan , w. azarbaijan , e. azarbaijan , qom , markazi , ardebil , kermanshah , kurdistan , bushehr , and semnan were highly segregated , and gilan , khuzestan , qazvin , khorasan razavi , sistan va baluchestan , chahar mahal and bakhtiari , isfahan , lorestan , and hamedan were extremely segregated . for existence of separate kitchen in the household , golestan , sistan va baluchestan , kurdistan , ilam , e. azarbaijan , mazandaran , and w. azarbaijan were moderately segregated and hormozgan , kermanshah , s. khorasan , khuzestan , khorasan razavi , bushehr , hamedan , isfahan , kerman , lorestan , qazvin , yazd , fars , chahar mahal and bakhtiari , semnan , gilan , kohgiluyeh and buyer ahmad , and markazi were highly segregated . to answer the question whether segregation of health indices is correlated with segregation of other socioeconomic variables , we used pair wise correlation of existence of disability in individual , existence of disability in family , infant mortality , and death of at least one offspring of mother with other socioeconomic variables . in many countries , measures of segregation residential racial segregation indices are available since 1980 and many articles in the literature have investigated different effects of segregation on social and health outcomes . in this study , for the first time in iran , we computed the measures of segregation for socioeconomic and health variables using the national census data of 2006 . for infant mortality , mazandaran and semnan provinces were highly segregated ; in other words , the incidence of infant death was not evenly distributed across the municipality units in these provinces . this finding is in accordance with the results of the study on socioeconomic inequality in infant mortality across the provinces in iran , which was performed on data of infant mortality in iran from 1995 to 2000 . in that study , mazandaran province was one of the five provinces in iran which had the highest inequality in infant mortality in iran . of the 12 provinces which were in the category of moderate segregation in our study , khuzestan and zanjan had high inequality , and khorasan razavi , w. azarbaijan , markazi , kurdistan , kohgiluyeh and buyer ahmad , bushehr , fars , and qazvin had moderate inequality in infant mortality in that study . and bakhtiari were moderately segregated for infant mortality in 2006 , but in that study they had low inequality . considering that tehran is the capital city of the country and the rate of immigration to this province is high , this means that immigrant people are not evenly distributed across residential areas in this province . of the socioeconomic variables , segregation indices measured for housing conditions like main source of drinking water and existence of separate kitchen in the household were correlated with segregation of all four health indices in this study . in the provinces where access to tap water and housing conditions of the families are not evenly distributed across the municipality units , there is more probability of finding hot zones for infant mortality , disability , and offspring death . segregation of activity level and job characteristic of individuals , economic situation of family presented by house ownership and existence of motorcycle in family , number of literate individuals in the family , and number of students in the family were also correlated with segregation of two or more health indices in this study . sex segregation was correlated with segregation of infant mortality , existence of at least one disabled person in the family , and death of at least one offspring , and literacy segregation was correlated with existence of one disabled person in the family and death of one offspring , but all the provinces were in the category of low segregation for these tow characteristics . it can be concluded the even small changes in segregation of sex and literacy proportions in provinces might result in uneven distribution of health outcomes and creates hot zones for these health outcomes . in a study of inequality in nine developing countries , levels of inequality in consumption , levels of socioeconomic status composed of income , education , housing condition , and house ownership were associated negatively with child death rates . information theory index and generalized dissimilarity index were used in the study because of their unique statistical characteristics and high use in other studies of segregation , but in some studies , isolation index has been used . further studies are necessary to determine the best segregation index for measuring segregation of markers of socioeconomic status and health this approach makes comparison of measured indices across periods of census and checking the trend of changes feasible . in this study , segregation was measured in each province across municipality spaces , and provinces were compared according to these measures . this method might show better correlation of segregation of heath indices and determinants of socioeconomic status . the choice of health variables in this study was very limited because of limited health variables measured in the national census . it is recommended that by using data of national health surveys , segregation of other health variables is measured . aside from these limitations , this study is of high value since it is the first study in iran which measures segregation and one of the first studies in the literature to measure segregation of socioeconomic status variables in national level . information theory index and generalized dissimilarity index were used in the study because of their unique statistical characteristics and high use in other studies of segregation , but in some studies , isolation index has been used . further studies are necessary to determine the best segregation index for measuring segregation of markers of socioeconomic status and health . in this study , segregation was measured in each province across municipality spaces , and provinces were compared according to these measures . it is possible to measure segregation across smaller geographic units for each city in every province to compare the degree of segregation of major cities inside each province . this method might show better correlation of segregation of heath indices and determinants of socioeconomic status . the choice of health variables in this study was very limited because of limited health variables measured in the national census . it is recommended that by using data of national health surveys , segregation of other health variables is measured . aside from these limitations , this study is of high value since it is the first study in iran which measures segregation and one of the first studies in the literature to measure segregation of socioeconomic status variables in national level . in this study , segregation of iranian provinces for major determinants of socioeconomic status and also some health indices were reviewed . for infant mortality and some of the socioeconomic factors , considering the effect of segregation on health outcomes which have been supported by large body of evidence in the literature , investigating why segregation is higher in some provinces deserves special attention .

in 2000 , the population aged 60 years or over numbered 600 million , triples the number presented in 1950 . in 2009 , by 2050 , two billion old people are projected to be alive , implying that their number will once again triple over a span of 50 years . in developed countries , the percentage of the elderly population is even higher ( 15% ) , and still growing . in the islamic republic of iran , the proportion of elderly people is increasing due to a decreasing birth rate and access to a better health care . the proportion of the population aged 60 years and older in 2005 was approximately 7.3% , and is projected to rise to 11.6% in 2025 and 30.8% by 2050 . people over 65 years use health services more than others , and up to one - thirds of this age group has a health problem that limits activities of daily living . congruently in iran , the rate of transferring the elderly to nursing homes is also increasing . aging is not a disease , but a natural process that can not be stopped or reversed . many chronic diseases are preventable , and their prevention and early management mean reduction of enormous human , social , and economic cost to the country . regular physical activity is ranked as a leading health indicator , and has been shown to have many health benefits for all age groups . according to the world health organization , physical activity is the single most useful thing that individuals can do to maintain their health , daily function and quality of life . the challenge of the public health is to promote the awareness of physical activity and its implementation as an important aspect of a healthy lifestyle among older adults . physical activity may also offer a useful alternative to drug management by reducing the need for medication in some conditions , such as hypertension and type 2 diabetes in elder people . unfortunately , despite the extensive benefits of physical activity , the population of older people is much less active than desired . therefore , the need to understand physical activity behavior and implement effective intervention strategies is paramount . this study was designed to examine to predictors of physical activity intention and behavior in a sample of elderly men of nursing home residents using theory of planned behavior ( tpb ) and the self - efficacy construct . theory of planned behavior theories can be used to plan these programs by providing a method of understanding why people are or are not following public health and medical advice . existing theories can help pinpoint what you need to know before developing an intervention program and provide insight into how to shape program strategies to effectively reach people and organizations . they can also help identify what should be monitored , measured and or compared to during program evaluations . the tpb suggests that the proximal determinant of a volitional behavior is one s intention to engage in that behavior . subjective norm and attitude are suggested to exert their effects upon a behavior through its influence on one s intentions . subjective norm assesses the social pressures on an individual to perform or not to perform a particular behavior . attitude is an individual s positive or negative evaluation of self - performance of a particular behavior . currently , there are few studies on the behavioral theories in elder men . in 2002 , semi - structured pilot interviews were carried out with a small sample of elder men to elicit the behavioral , normative and control beliefs associated with the target behavior . self - efficacy is an individual s confidence or belief in his own capability of performing an action , and is a salient predictor of health behavior change and maintenance . self - efficacy is a key factor because it operates based on motivation and action both directly and through its impact on the other determinants . studies adopting the tpb for physical activity behavior have catalogued the independent influence of self - efficacy on intention and behavior . moreover , a number of studies successfully paired self - efficacy with the tpb in various behavioral settings . self - efficacy is more concerned with cognitive perceptions of the control based on internal control factors . further , in a comparison of the theories of reasoned action , planned behavior and social cognitive theory , self - efficacy rather than perceived behavioral control ( pbc ) , had a direct impact on behavior . consequently , this current study used an expanded tbp model which incorporates the two constructs of attitude , subjective norm and pbc as well as self - efficacy , to investigate physical activity intention and behavior in elderly men . this study aims to identify the relationship of the tpb and self - efficacy constructs associated with self - reported physical activity behavior and physical activity intention in elder men . lastly , we attempt to identify and compare the effectiveness of the tpb with self - efficacy as predictors of physical activity and intention . participants and procedure the study was a cross - sectional study using a census sample of 120 elder men aged 60 to 85 years in a population of elderly men , who constituted whole resident of kahrizak nursing home in tehran , iran . the institutional review board of the approved and supported the study . after institutional ethical approval , the objectives and methodology of the study was explained to the management of kahrizak nursing home , and its approval was obtained . the sample size , calculated using an of 0.05 and a power of 0.95 , was found to be 120 individuals . the inclusion criteria for the study were an age of 60 years or older , independent living ( no assistance from paid or unpaid persons for personal care ) , no suffering from several diseases including osteoarthritis , heart diseases , osteoporosis , pulmonary diseases , and ability for independent mobility ( moving without canes , etc . ) , and ability of verbal communication . each participant was given a packet of questionnaires on physical activity that contained questions in regards to the assessment of physical activity intention , behavior , attitude , subjective norm , pbc , and self - efficacy . participants were individually interviewed for 45 minutes using questions in regards to their demography , physical activity beliefs , and physical activity behavior , respectively . the interviewer explained questionnaires used for data collection to the participants to prevent illiteracy or vision difficulties from affecting the study participation or findings . in order to assure the validity of the persian version of the questionnaire , it was translated into persian and then back into english . the translations were then compared and the questionnaire was corrected accordingly . the questionnaire was then given to 10 professionals in health education , sociology and gerontology at to examine the item clarity , face validity , and content validity . reliability of the questionnaire was evaluated using a sample of 20 subjects over 10 days using test - retest for physical activity behavior , and cronbach s alpha for other items . participants were instructed to answer all questions based on the definition of regular physical activity . regular physical activity was defined as a moderately intense physical activity ( such as brisk walking ) that is performed ideally every day for a minimum of 30 minutes . the duration of regular physical activity may be fulfilled either in a single session or accumulated in multiple bouts of at least 810 minutes throughout the day . questions of tpb were based on previously used measures of tpb constructs , and were all measured on 7-point scales . physical activity intention physical activity intention was measured with a single item modeled after ajzen s work in 1999 . subjects were asked about the extent of their agreement with the statement that they intend to perform regular physical activity . physical activity behavior physical activity was measured using the physical activity scale for the elderly ( pase ) . the pase is a brief instrument designed specifically to assess the frequency and duration of recreational , leisure , and occupational physical activity in older adults over a 7-day period . frequency was categorized by as never , seldom ( 1 - 2 days / week ) , sometimes ( 3 - 4 days / week ) , and often ( 5 - 7 days / week ) . duration was categorized as less than 1 hour , between 1 - 2 hours , 2 - 4 hours , and more than 4 hours . the total pase score was computed by multiplying the duration of time spent in each activity or participation ( yes / no ) by the empirically - derived item weights , and summing up all activities . reliability , evaluated in 20 subjects over 10 days , was ( correlation coefficient=0.76 ) . physical activity attitude the participants ' attitude was assessed using the statement for me , participating in regular physical activity would be . the answers assessed components of both instrumental attitude ( useful / useless , healthy / unhealthy , bad / good ) and affective attitude ( enjoyable / unenjoyable , boring / interesting , pleasant / unpleasant , stressful / relaxing ) . alpha for affective attitude ( =0.74 ) and instrumental attitude ( =0.81 ) were good . subjective norm was operationalized by three statements : people close to me think that i should participate in regular physical activity , people who are important to me think that i should participate in regular physical activity , and these items were scored using seven - point scales from 1 ( strongly disagree ) to 7 ( strongly agree ) . perceived behavioral control perceived behavioral control ( pbc ) was measured by four questions . the first question was how much control the subjects had over participating in regular physical activity scored from 1 ( very little control ) to 7 ( complete control ) . the second question was whether or not the subjects could easily participate in regular physical activity if they wanted . the answer to this question was scored from 1 ( strongly disagree ) to 7 ( strongly agree ) . the next question was how confident were the subjects that they were capable of participating in regular physical activity . the level of confidence was ranked from 1 ( not at all confident ) to 7 ( extremely confident ) . the fourth question was about extent of control that the subjects had over the amount of time they had for physical activity . the extent of the control was ranked from 1 ( very little control ) to 7 ( complete control ) . this 5-item instrument was designed to assess confidence in the ability to overcome the barriers for increasing physical activity in various situations . a 4-point likert scale from 1 ( very uncertain ) to 4 ( very certain ) was used for scoring . statistical analysis data were analyzed using the statistical package for social sciences ( spss , v. 13 ) . kolmogorov - smirnov test was used to examine the normality of distribution of quantitative data . most men were married ( n=62 , 51.6% ) , had a mean of 2.33 illnesses ( sd=1.95 , range 010 ) , moderate level of socioeconomic status ( 71.9% ) , body mass index ( bmi ) of 24.743.46 ) and instrumental activities of daily living ( iadl ) of 77.138.46 . intention was most strongly correlated with affective attitude and perceived behavior control ( r=0.573 , p<0.01 ; r=0.507 , p<0.01 ) , and was most weakly correlated with subjective norm ( r=0.339 , p<0.01 ) . behavior was most strongly correlated to self - efficacy ( r=0.428 , p<0.01 ) and was most weakly associated with perceived behavior control and subjective norm ( r=0.311 , p<0.01 ; r=0.319 , p<0.01 ) . self - efficacy was most correlated to instrumental and affective attitude of the tpb variables ( r=0.603 , p<0.01 ; r=0.616 , p<0.01 ) . prediction of intention the stepwise regression results for intention in relation to the tpb variables and self - efficacy are shown in tables 2 and 3 . instrumental and affective attitude , subjective norm and pbc were entered in the first step of the regression ( step 1 , table 2 ) and the total variance in physical activity intention explained was 32.8% . the affective attitude has significant beta weight in the regression equation ( b=0.146 , p<0.0001 ) , and was the only significant predictor of intention . in step two , self - efficacy was entered in the regression ( table 2 ) . self - efficacy accounted for an additional 2.7% of the variance in intention ( b=0.071 , p<0.02 ) . affective attitude ( b=0.113 , p<0.0001 ) remained significant in step two of the regression equation . in a reverse regression ( table 3 ) , self - efficacy was entered in the first step of the regression , and the explained total variance in physical activity intention was 23.4% . self - efficacy had a significant beta weight in the regression equation ( b=0.164 , p<0.0001 ) . instrumental and affective attitude , subjective norm and pbc were entered in the second step of the regression and accounted for an additional 12.2% of the variance in intention . affective attitude has a significant beta weight in the regression equation ( b=0.113 , p<0.0001 ) , and was the most important predictor of intention . self - efficacy ( b=0.071 , p<0.027 ) remained significant in the second step of the regression equation . a total of 35.6% of the variance in physical activity intention was explained by all variables . hierarchical multiple regression analysis to predict intention from the theory of planned behavior variables first and then self - efficacy ( n=120 ) note . pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic hierarchical multiple regression analysis to predict intention from self - efficacy first and then the theory of planned behavior variables ( n=120 ) pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic prediction of behavior in a hierarchical regression for predicting behavior , intention , instrumental and affective attitude , subjective norm and pbc were entered on step one ( table 4 ) . a part ( 15.7% ) of the variance in physical activity behavior instrumental attitude ( b=4.79 , p<0.0001 ) had a significant beta weight in the regression . self - efficacy entered in step two of the regression ( table 4 ) accounted for an additional 5.6% of the variance in behavior , and had a significant beta weight ( b=3.853 , p<0.005 ) . instrumental attitude ( b=2.623 , p<0.037 ) remained significant in the regression equation in step 2 . in the reverse regression , self - efficacy self - efficacy explained 18.3% of the variance in physical activity behavior and had a significant beta weight ( b=0.428 , p<0.0001 ) . subjective norm , instrumental and affective attitude , intention and pbc were entered on step two ( table 5 ) . instrumental attitude had a significant beta weight in the regression equation ( b=2.623 , p<0.037 ) , and explained an additional of 3.0% . self - efficacy ( b=3.853 , p<0.005 ) remained significant in the second step of the regression equation . a total of 21.3% of the variance in physical activity behavior was explained by all variables . there have been a few studies that have used the tpb to explain physical activity in a general population of older adults ( > 60 years of age ) , but results are varied . the present study of the physical activity in an older adult population nursing home resident showed that the tpb model that included self - efficacy explained more variance in physical activity intention and behavior than did the tpb alone . according to our step wise regression data ( table 2 - 5 ) , variables of the tpb predicted 32.8% of variance in the physical activity intention in older adult . a combination of tpb variables and self - efficacy explained a higher percentage ( 35.6% ) of the variance in physical activity intention . while tpb alone explained 15.7% of variance in behavior physical activity , a combination with self - efficacy explained 21.3% of it . affective attitude and self - efficacy were the significant predictors of intention to physical activity . interestingly , self - efficacy was a more effective predictor of behavior by contributing an additional 5.6% in the second step of the regression compared to the tpb variable contribution of 3.0% . however , the tpb variable , affective attitude , remains the stronger predictor of intention as shown by its second step contribution of 12.2% compared to the 2.7% of self - efficacy . in general , our data showed that self - efficacy does add to the effectiveness of tpb and provides additional support for the proposition that tpb is a multidimensional theory that can be expanded upon . the results of this study have several important consequences for both theory and practice . from a theoretical perspective , they highlight the importance of self - efficacy in relation to the tpb . the combination of tpb with self - efficacy not only explained more of the variance in intention and behavior than tpb alone , but made a greater contribution to the prediction of behavior than any other independent tpb variable . such results suggest that future model construction and studies on physical activity among older adults nursing home residents should incorporate self - efficacy as a distinct construct that was confirmed with by another study . hierarchical multiple regression analysis to predict behavior first from the theory of planned behavior variables and then from self - efficacy ( n=120 ) note . pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic hierarchical multiple regression analysis to predict behavior first from self - efficacy and then from the theory of planned behavior variables ( n=120 ) pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic in common with previous research , affective attitudes explain unique variance in intention above and beyond that explained by standard tpb variables . according to narrative reviews , the majority of studies using the tpb in physical activity behavior research have reported that attitudes have the most pervasive influence on intentions . for example , when estabrooks and carron used the tpb to predict attendance in a physical activity program for older adults , they found that although intention predicted attendance , neither attitude or subjective norm predicted intention or attendance in the physical activity program . courneya and colleagues reported that older adults intended to do physical activity when they held a positive attitude toward physical activity , had perceptions of control over their physical activity , or perceived pressure from important others . our results show that affective attitude explained a considerable amount of unique variance in intention ( table 2 ) , whereas this was not the case for instrumental attitude . our study and a previous study , have shown that affective attitude was the stronger predictor of physical activity intention than is instrumental attitude . this suggests that interventions aimed at improving affective attitudes toward physical activity among older adults nursing home residents may lead to successful increases in physical activity intention . perhaps people who had a more negative attitude were less likely to intend to perform physical activity . this study further shows that instrumental attitude was the stronger tpb predictor of physical activity behavior . this suggests that interventions designed to emphasize aspects of pleasure in physical activity may be more effective to build intention whereas instrumental attitude should be addressed when translating intention to behavior . an explanation for the modest amount of variance is the restriction in the range of intentions and behavior . ajzen indicates that the magnitude of attitudes , subjective norm and pbc , on intention could vary with situational conditions ( 1991 ) . most of our elderly people in the nursing home spent most of their time in their residences , and did not engage in social or recreational activities . when using such participants , intentions are not likely to be a significant mediator in this model . direct paths from attitudes , subjective norms and perceived behavioral control to behavior should instead be tested when there are apparent restrictors preventing intention - behavior relationships . a previous study also shows that intention was not itself significantly predictive of reported activity levels . perceived behavioral control did not add significantly to the prediction of intention and behavior that is confirmed with other study . this may be due to the possibility that older adults with several years of experience already take into account the actual control they have over the target behavior . or perhaps certain behavior control were also limited by situational conditions that conflict with what subjects perceive as their own control versus what the institutions in tehran may encourage . this study also reveals that subjective norm did not add significantly to the prediction of intention and behavior . although the elder adults of nursing home in this study believe physical activity is beneficial , they appear to be less influenced by others to change their physical activity behavior as evidenced by the small impact of subjective norm on intention and physical activity behavior . a previous study also shows that subjective norm did not add significantly to the prediction of intention and behavior predictor of physical activity intention compared to attitude and perceived behavioral control . this may be consistent with the notion that participation in physical activity relies more on personal motivational judgments than on outside influence in the case of older adults . perhaps these consistent results point to some potential culture - specific protective factors against these physical activity changes . or perhaps similar to the case of intention and pbc , for example , in , there are few fitness centers , which few can afford , thus discouraging the elderly from going to these fitness centers and increasing the priority to stay in their nursing home . this financial hurdle would definitely affect the relationships between intention - behavior , pbc - actual behavior , and subjective norm - behavior . another explanation may be that one s subjective norms and pbc are less susceptible to change by means of communication alone than is the case for one s attitude because subjective norms and pbc comprise external as well as internal dimensions . considerable evidence has consistently linked physical activity self - efficacy with actual performance of activity among samples of healthy adults of all ages . in this study , self - efficacy has been successful in explaining additional variance in physical activity intention and behavior in this study . future physical activity interventions may prove to be more effective by focusing on a social - cognitive design that emphasizes internal aspects of confidence towards physical activity . the present findings provide further support for the tpb in predicting physical activity intention and behavior . the present study suggests that people s attitudes and self - efficacy seem to be the key influences in forming interventions to improve participation in physical activity . in practical terms this suggests that interventions based on the enhancement of attitudes and self efficacy toward physical activity may lead to a concomitant increase in physical activity behavior . clearly , as the research community reaches a consensus on defining and measuring the tpb construct , the understanding of its contribution to the explanation of behavior and the need for further expansion on the construct will become more transparent . limitations of the present study were that it used a convenient sample of older adults ' nursing home residents therefore , we do nt generalize the results to all of elderly . we also employed a very brief questionnaire with fewer item measures of tpb constructs because it is difficult for older people to complete a full tpb questionnaire . another limitation of this study was its use of a single item to measure physical activity intention . although single items for measuring this construct predominate in research based on the tpb , a multiple - item assessment would allow researchers to estimate internal consistency . the findings of this study indicate that physical activity behavior in elderly men of nursing home residents was largely predicted by self - efficacy and instrumental attitude , and physical activity intention was predicted by self - efficacy and affective attitude . they also showed that compared to tpb variables , self - efficacy was the stronger predictor of physical activity behavior in these subjects , while affective attitude was the stronger predictor of intention .

background : regular physical activity is ranked as a leading health indicator . despite the extensive benefits of physical activity , elder people are much less active than desired . using theory of planned behavior ( tpb ) and the self - efficacy construct , this study examined the prediction of physical activity intention and behavior in a sample of elderly male resident of a nursing home . methods : in a cross - sectional study of the residents of kahrizak nursing home in tehran , iran , elderly men who were 60 years or older , capable of independent living , mobility , and verbal communication were asked to complete measures of the tpb , self - efficacy and physical activity behavior . results : a hierarchical step - wise multiple regression analysis indicated that affective / instrumental attitude , subjective norm , and perceived behavioral control ( pbc ) explained 32.8% of the variance in physical activity intention , and self - efficacy provided an additional 2.7% . in a reverse step regression , the tpb variables explained an additional 12.2% of physical activity intention . in a multiple regression analysis on physical activity behavior , affective / instrumental attitude , subjective norm , perceived behavioral control ( pbc ) and intention explained 15.7% of the variance in physical activity behavior while self - efficacy contributed an additional 5.6% . in the reverse step regression , tpb predictors contributed an additional 3.0% in explaining the variance in physical activity behavior . conclusion : the results indicate that in addition to the tpb , self - efficacy may also play an important role in the prediction of behavior , and should be included in the design of physical activity programs for elderly men of nursing home residents .

Introduction Materials and Methods Results Discussion Conclusion None

in 2000 , the population aged 60 years or over numbered 600 million , triples the number presented in 1950 . in 2009 , by 2050 , two billion old people are projected to be alive , implying that their number will once again triple over a span of 50 years . in developed countries , the percentage of the elderly population is even higher ( 15% ) , and still growing . in the islamic republic of iran , the proportion of elderly people is increasing due to a decreasing birth rate and access to a better health care . the proportion of the population aged 60 years and older in 2005 was approximately 7.3% , and is projected to rise to 11.6% in 2025 and 30.8% by 2050 . congruently in iran , the rate of transferring the elderly to nursing homes is also increasing . many chronic diseases are preventable , and their prevention and early management mean reduction of enormous human , social , and economic cost to the country . regular physical activity is ranked as a leading health indicator , and has been shown to have many health benefits for all age groups . according to the world health organization , physical activity is the single most useful thing that individuals can do to maintain their health , daily function and quality of life . the challenge of the public health is to promote the awareness of physical activity and its implementation as an important aspect of a healthy lifestyle among older adults . physical activity may also offer a useful alternative to drug management by reducing the need for medication in some conditions , such as hypertension and type 2 diabetes in elder people . unfortunately , despite the extensive benefits of physical activity , the population of older people is much less active than desired . therefore , the need to understand physical activity behavior and implement effective intervention strategies is paramount . this study was designed to examine to predictors of physical activity intention and behavior in a sample of elderly men of nursing home residents using theory of planned behavior ( tpb ) and the self - efficacy construct . theory of planned behavior theories can be used to plan these programs by providing a method of understanding why people are or are not following public health and medical advice . the tpb suggests that the proximal determinant of a volitional behavior is one s intention to engage in that behavior . subjective norm assesses the social pressures on an individual to perform or not to perform a particular behavior . attitude is an individual s positive or negative evaluation of self - performance of a particular behavior . self - efficacy is an individual s confidence or belief in his own capability of performing an action , and is a salient predictor of health behavior change and maintenance . self - efficacy is a key factor because it operates based on motivation and action both directly and through its impact on the other determinants . studies adopting the tpb for physical activity behavior have catalogued the independent influence of self - efficacy on intention and behavior . moreover , a number of studies successfully paired self - efficacy with the tpb in various behavioral settings . self - efficacy is more concerned with cognitive perceptions of the control based on internal control factors . further , in a comparison of the theories of reasoned action , planned behavior and social cognitive theory , self - efficacy rather than perceived behavioral control ( pbc ) , had a direct impact on behavior . consequently , this current study used an expanded tbp model which incorporates the two constructs of attitude , subjective norm and pbc as well as self - efficacy , to investigate physical activity intention and behavior in elderly men . this study aims to identify the relationship of the tpb and self - efficacy constructs associated with self - reported physical activity behavior and physical activity intention in elder men . lastly , we attempt to identify and compare the effectiveness of the tpb with self - efficacy as predictors of physical activity and intention . participants and procedure the study was a cross - sectional study using a census sample of 120 elder men aged 60 to 85 years in a population of elderly men , who constituted whole resident of kahrizak nursing home in tehran , iran . the institutional review board of the approved and supported the study . after institutional ethical approval , the objectives and methodology of the study was explained to the management of kahrizak nursing home , and its approval was obtained . the inclusion criteria for the study were an age of 60 years or older , independent living ( no assistance from paid or unpaid persons for personal care ) , no suffering from several diseases including osteoarthritis , heart diseases , osteoporosis , pulmonary diseases , and ability for independent mobility ( moving without canes , etc . ) , and ability of verbal communication . each participant was given a packet of questionnaires on physical activity that contained questions in regards to the assessment of physical activity intention , behavior , attitude , subjective norm , pbc , and self - efficacy . participants were individually interviewed for 45 minutes using questions in regards to their demography , physical activity beliefs , and physical activity behavior , respectively . the questionnaire was then given to 10 professionals in health education , sociology and gerontology at to examine the item clarity , face validity , and content validity . reliability of the questionnaire was evaluated using a sample of 20 subjects over 10 days using test - retest for physical activity behavior , and cronbach s alpha for other items . participants were instructed to answer all questions based on the definition of regular physical activity . regular physical activity was defined as a moderately intense physical activity ( such as brisk walking ) that is performed ideally every day for a minimum of 30 minutes . the duration of regular physical activity may be fulfilled either in a single session or accumulated in multiple bouts of at least 810 minutes throughout the day . questions of tpb were based on previously used measures of tpb constructs , and were all measured on 7-point scales . physical activity intention physical activity intention was measured with a single item modeled after ajzen s work in 1999 . subjects were asked about the extent of their agreement with the statement that they intend to perform regular physical activity . physical activity behavior physical activity was measured using the physical activity scale for the elderly ( pase ) . the pase is a brief instrument designed specifically to assess the frequency and duration of recreational , leisure , and occupational physical activity in older adults over a 7-day period . duration was categorized as less than 1 hour , between 1 - 2 hours , 2 - 4 hours , and more than 4 hours . the total pase score was computed by multiplying the duration of time spent in each activity or participation ( yes / no ) by the empirically - derived item weights , and summing up all activities . physical activity attitude the participants ' attitude was assessed using the statement for me , participating in regular physical activity would be . the answers assessed components of both instrumental attitude ( useful / useless , healthy / unhealthy , bad / good ) and affective attitude ( enjoyable / unenjoyable , boring / interesting , pleasant / unpleasant , stressful / relaxing ) . alpha for affective attitude ( =0.74 ) and instrumental attitude ( =0.81 ) were good . subjective norm was operationalized by three statements : people close to me think that i should participate in regular physical activity , people who are important to me think that i should participate in regular physical activity , and these items were scored using seven - point scales from 1 ( strongly disagree ) to 7 ( strongly agree ) . perceived behavioral control perceived behavioral control ( pbc ) was measured by four questions . the first question was how much control the subjects had over participating in regular physical activity scored from 1 ( very little control ) to 7 ( complete control ) . the second question was whether or not the subjects could easily participate in regular physical activity if they wanted . the next question was how confident were the subjects that they were capable of participating in regular physical activity . the fourth question was about extent of control that the subjects had over the amount of time they had for physical activity . the extent of the control was ranked from 1 ( very little control ) to 7 ( complete control ) . this 5-item instrument was designed to assess confidence in the ability to overcome the barriers for increasing physical activity in various situations . intention was most strongly correlated with affective attitude and perceived behavior control ( r=0.573 , p<0.01 ; r=0.507 , p<0.01 ) , and was most weakly correlated with subjective norm ( r=0.339 , p<0.01 ) . behavior was most strongly correlated to self - efficacy ( r=0.428 , p<0.01 ) and was most weakly associated with perceived behavior control and subjective norm ( r=0.311 , p<0.01 ; r=0.319 , p<0.01 ) . self - efficacy was most correlated to instrumental and affective attitude of the tpb variables ( r=0.603 , p<0.01 ; r=0.616 , p<0.01 ) . prediction of intention the stepwise regression results for intention in relation to the tpb variables and self - efficacy are shown in tables 2 and 3 . instrumental and affective attitude , subjective norm and pbc were entered in the first step of the regression ( step 1 , table 2 ) and the total variance in physical activity intention explained was 32.8% . the affective attitude has significant beta weight in the regression equation ( b=0.146 , p<0.0001 ) , and was the only significant predictor of intention . in step two , self - efficacy was entered in the regression ( table 2 ) . self - efficacy accounted for an additional 2.7% of the variance in intention ( b=0.071 , p<0.02 ) . in a reverse regression ( table 3 ) , self - efficacy was entered in the first step of the regression , and the explained total variance in physical activity intention was 23.4% . self - efficacy had a significant beta weight in the regression equation ( b=0.164 , p<0.0001 ) . instrumental and affective attitude , subjective norm and pbc were entered in the second step of the regression and accounted for an additional 12.2% of the variance in intention . affective attitude has a significant beta weight in the regression equation ( b=0.113 , p<0.0001 ) , and was the most important predictor of intention . self - efficacy ( b=0.071 , p<0.027 ) remained significant in the second step of the regression equation . a total of 35.6% of the variance in physical activity intention was explained by all variables . hierarchical multiple regression analysis to predict intention from the theory of planned behavior variables first and then self - efficacy ( n=120 ) note . pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic hierarchical multiple regression analysis to predict intention from self - efficacy first and then the theory of planned behavior variables ( n=120 ) pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic prediction of behavior in a hierarchical regression for predicting behavior , intention , instrumental and affective attitude , subjective norm and pbc were entered on step one ( table 4 ) . a part ( 15.7% ) of the variance in physical activity behavior instrumental attitude ( b=4.79 , p<0.0001 ) had a significant beta weight in the regression . self - efficacy entered in step two of the regression ( table 4 ) accounted for an additional 5.6% of the variance in behavior , and had a significant beta weight ( b=3.853 , p<0.005 ) . instrumental attitude ( b=2.623 , p<0.037 ) remained significant in the regression equation in step 2 . in the reverse regression , self - efficacy self - efficacy explained 18.3% of the variance in physical activity behavior and had a significant beta weight ( b=0.428 , p<0.0001 ) . subjective norm , instrumental and affective attitude , intention and pbc were entered on step two ( table 5 ) . instrumental attitude had a significant beta weight in the regression equation ( b=2.623 , p<0.037 ) , and explained an additional of 3.0% . self - efficacy ( b=3.853 , p<0.005 ) remained significant in the second step of the regression equation . a total of 21.3% of the variance in physical activity behavior was explained by all variables . there have been a few studies that have used the tpb to explain physical activity in a general population of older adults ( > 60 years of age ) , but results are varied . the present study of the physical activity in an older adult population nursing home resident showed that the tpb model that included self - efficacy explained more variance in physical activity intention and behavior than did the tpb alone . according to our step wise regression data ( table 2 - 5 ) , variables of the tpb predicted 32.8% of variance in the physical activity intention in older adult . a combination of tpb variables and self - efficacy explained a higher percentage ( 35.6% ) of the variance in physical activity intention . while tpb alone explained 15.7% of variance in behavior physical activity , a combination with self - efficacy explained 21.3% of it . affective attitude and self - efficacy were the significant predictors of intention to physical activity . interestingly , self - efficacy was a more effective predictor of behavior by contributing an additional 5.6% in the second step of the regression compared to the tpb variable contribution of 3.0% . however , the tpb variable , affective attitude , remains the stronger predictor of intention as shown by its second step contribution of 12.2% compared to the 2.7% of self - efficacy . in general , our data showed that self - efficacy does add to the effectiveness of tpb and provides additional support for the proposition that tpb is a multidimensional theory that can be expanded upon . the results of this study have several important consequences for both theory and practice . from a theoretical perspective , they highlight the importance of self - efficacy in relation to the tpb . the combination of tpb with self - efficacy not only explained more of the variance in intention and behavior than tpb alone , but made a greater contribution to the prediction of behavior than any other independent tpb variable . such results suggest that future model construction and studies on physical activity among older adults nursing home residents should incorporate self - efficacy as a distinct construct that was confirmed with by another study . hierarchical multiple regression analysis to predict behavior first from the theory of planned behavior variables and then from self - efficacy ( n=120 ) note . pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic hierarchical multiple regression analysis to predict behavior first from self - efficacy and then from the theory of planned behavior variables ( n=120 ) pbc = perceived behavioral control ; b : unstandarized coefficients ; t : test statistic in common with previous research , affective attitudes explain unique variance in intention above and beyond that explained by standard tpb variables . according to narrative reviews , the majority of studies using the tpb in physical activity behavior research have reported that attitudes have the most pervasive influence on intentions . for example , when estabrooks and carron used the tpb to predict attendance in a physical activity program for older adults , they found that although intention predicted attendance , neither attitude or subjective norm predicted intention or attendance in the physical activity program . courneya and colleagues reported that older adults intended to do physical activity when they held a positive attitude toward physical activity , had perceptions of control over their physical activity , or perceived pressure from important others . our results show that affective attitude explained a considerable amount of unique variance in intention ( table 2 ) , whereas this was not the case for instrumental attitude . our study and a previous study , have shown that affective attitude was the stronger predictor of physical activity intention than is instrumental attitude . this suggests that interventions aimed at improving affective attitudes toward physical activity among older adults nursing home residents may lead to successful increases in physical activity intention . this study further shows that instrumental attitude was the stronger tpb predictor of physical activity behavior . this suggests that interventions designed to emphasize aspects of pleasure in physical activity may be more effective to build intention whereas instrumental attitude should be addressed when translating intention to behavior . an explanation for the modest amount of variance is the restriction in the range of intentions and behavior . ajzen indicates that the magnitude of attitudes , subjective norm and pbc , on intention could vary with situational conditions ( 1991 ) . most of our elderly people in the nursing home spent most of their time in their residences , and did not engage in social or recreational activities . direct paths from attitudes , subjective norms and perceived behavioral control to behavior should instead be tested when there are apparent restrictors preventing intention - behavior relationships . perceived behavioral control did not add significantly to the prediction of intention and behavior that is confirmed with other study . this study also reveals that subjective norm did not add significantly to the prediction of intention and behavior . although the elder adults of nursing home in this study believe physical activity is beneficial , they appear to be less influenced by others to change their physical activity behavior as evidenced by the small impact of subjective norm on intention and physical activity behavior . a previous study also shows that subjective norm did not add significantly to the prediction of intention and behavior predictor of physical activity intention compared to attitude and perceived behavioral control . this may be consistent with the notion that participation in physical activity relies more on personal motivational judgments than on outside influence in the case of older adults . or perhaps similar to the case of intention and pbc , for example , in , there are few fitness centers , which few can afford , thus discouraging the elderly from going to these fitness centers and increasing the priority to stay in their nursing home . this financial hurdle would definitely affect the relationships between intention - behavior , pbc - actual behavior , and subjective norm - behavior . considerable evidence has consistently linked physical activity self - efficacy with actual performance of activity among samples of healthy adults of all ages . in this study , self - efficacy has been successful in explaining additional variance in physical activity intention and behavior in this study . future physical activity interventions may prove to be more effective by focusing on a social - cognitive design that emphasizes internal aspects of confidence towards physical activity . the present findings provide further support for the tpb in predicting physical activity intention and behavior . the present study suggests that people s attitudes and self - efficacy seem to be the key influences in forming interventions to improve participation in physical activity . in practical terms this suggests that interventions based on the enhancement of attitudes and self efficacy toward physical activity may lead to a concomitant increase in physical activity behavior . clearly , as the research community reaches a consensus on defining and measuring the tpb construct , the understanding of its contribution to the explanation of behavior and the need for further expansion on the construct will become more transparent . limitations of the present study were that it used a convenient sample of older adults ' nursing home residents therefore , we do nt generalize the results to all of elderly . we also employed a very brief questionnaire with fewer item measures of tpb constructs because it is difficult for older people to complete a full tpb questionnaire . another limitation of this study was its use of a single item to measure physical activity intention . although single items for measuring this construct predominate in research based on the tpb , a multiple - item assessment would allow researchers to estimate internal consistency . the findings of this study indicate that physical activity behavior in elderly men of nursing home residents was largely predicted by self - efficacy and instrumental attitude , and physical activity intention was predicted by self - efficacy and affective attitude . they also showed that compared to tpb variables , self - efficacy was the stronger predictor of physical activity behavior in these subjects , while affective attitude was the stronger predictor of intention .

hepatitis c virus ( hcv ) is a ribonucleic acid ( rna ) virus that causes chronic hepatitis and liver failure , worldwide.1,2 it consists of six different genotypes that are differentially distributed geographically.3,4 success of treatment varies greatly depending on the genotype.4,5 the genome contains cis - acting replication elements ( cres ) that are critical for hcv rna replication and translation.6,7 rna structural elements present in 5- and 3-untranslated regions ( 3utr ) of the hcv genome interact with viral and cellular proteins to initiate and facilitate the replication and translation processes.811 in our previous studies , we showed that rna secondary structure of the nonstructural ( ns)5b coding region of the hcv genome was required for hcv rna replication , and hence viral particle production.1215 it has been shown that the x region in the 3-utr of the hcv rna genome contains a highly conserved sequence.16 the latter have also been found to form stable secondary stem - loop structures that require physical contact between its rna - dependent rna polymerase for hcv replication.1719 we hypothesized that rna structural analogs resembling secondary stem - loop structure of the x region could be created that can compete with natural hcv genomic structure for binding to proteins and inhibit hcv replication . the aim of this study was to introduce rna structural analogs resembling these cres into human liver cells and to identify secondary structural elements of the hcv x - region involved in hcv replication and infection . in order to identify important secondary structural elements , the effects of hcv structural analogs on hcv replication in two model cell lines were studied : a constitutive replication model and an infection model . in addition , to evaluate whether differences in viral genotype could affect the possible interactions of structural analogs , viruses representing two different genotypes 1 and 2 , were studied . replicon cells , bb7 , a cell culture system containing the hcv genotype 1b genome , were obtained from apath ( st . louis , usa).20,21 cells were maintained in dulbecco 's modified eagle medium ( dmem ) supplemented with antibiotic / antimycotic solution ( invitrogen , usa ) , 10% fetal bovine serum ( fbs ) and 0.5 mg / ml g418 . for jfh-1 hcv genotype 2a studies , huh7.5 cells ( human hepatoma cell line ) ( obtained from dr . charles rice , rockefeller university , ny , usa ) were maintained in dmem supplemented with antibiotic / antimycotic solution and 10% fbs . jfh-1 complementary deoxyribonucleic acid ( cdna ) , an hcv genotype 2a strain , from dr . takaji wakita ( national institute of infectious diseases , tokyo , japan)2224 was used to produce infectious hcv viral particles in huh7.5.25,26 to make jfh-1 hcv stocks for infection , huh 7.5 cells were infected with jfh-1 hcv . media were collected 5 days post - infection and centrifuged at 1000g for 20 min to remove debris . jfh-1 hcv in media was concentrated with a centrifugal device ( 100k nmwl , amicon ultra , millipore , usa ) . infectivity levels of viral stocks were checked by quantitation of jfh-1 hcv rna in media 48 h post - infection of huh7.5 cells with hcv genotype 2a specific primers ( table 1 ) using real time reverse transcriptase polymerase chain reaction ( rt - pcr ) . the pcr conditions were : one cycle of 2 min at 50 c and 10 min at 95 c followed by 40 cycles of 15 sec at 95 c and 1 min at 60 c . specificity of all designed primers was determined with pcr amplification and sequencing of amplified product ( data not shown ) . melt curve analysis was performed following each rt - pcr to identify the presence of primer dimers and analyze the specificity of the reaction . cmv , cytomegalovirus ; fw , forward ; hcv , hepatitis c virus ; jfh-1 , japanese fulminant hepatitis virus-1 ; ldha , lactate dehydrogenase a ; rv , reverse . rna structural analog x-94 was designed to be 100% identical to the ( + ) strand of x - region ( 9508 - 9605 nucleotides ( nt ) ) on the 3-end of the hcv genotype 1b ( 94% identical to genotype 2a ) genome ( fig . 1a ) . to determine whether stem - loop structures versus specific sequences of hcv of rna were most important in hcv replication , base pair changes , described below , were made in stems ( analog x-12 ) and loops ( analog x-12c ) of analog x-94 ( fig . 1b and 1d ) . several software products are available for prediction of secondary structures of rna based on thermodynamic parameters . because of its high reliability and reproducibility , mfold ver 3.2 was used in the current study.2729 rna structural analogs x-12 and x-12c were predicted to adopt stem - loop structure identical to analog x-94 ( fig . table 3 shows the percent identity of the various rna structural analogs with hcv genotypes 1b and 2a . to determine the minimal structure of the x - region that could inhibit hcv rna replication , shorter rna structural analogs x-12a and x-12b ( fig . 1c ) were designed that were predicted to retain individual stem - loop structures of analog x-94.2729 all analogs were named based on hcv genome region studied and sequence homology of the analogues relative to the jfh-1 hcv genotype 2a genome . for expression studies , the x-94 sequence was subcloned into a psilencer 4.1 cytomegalovirus ( cmv ) puro plasmid ( ambion , usa ) , as previously described.12 psilencer 4.1 cmv puro plasmid enables high level expression of cloned hairpin short rna templates . expression vectors for rna structural analogs x-12 and x-12c were constructed from a plasmid expressing the rna analog x-94 using a quikchange ii site - directed mutagenesis kit ( agilent technologies , inc . 1b shows nucleotide replacements made in stems of analog x-94 resulting in analog x-12 , and fig . 1d illustrates nucleotide replacements made in loops of analog x-12 to construct analog x-12c . shorter rna structural analogs x-12a and x-12b ( fig . 1c ) were constructed from a plasmid expressing rna analog x-12 using a quikchange ii site - directed mutagenesis kit . these sequences were selected as non - overlapping fragments of x-12 that were individually predicted by mfold to retain all the structural elements present in the parent x-12 analog . 5b-74 rna analog , which was previously shown to inhibit viral genome replication12 was used as a positive control for inhibition of viral replication , and a plasmid expressing an unrelated sequence , hb , from hepatitis b virus was used as negative control . an rna structural analog 5b-46 was predicted to adopt stem - loop structures identical to analog 5b-74 as determined by mfold , and was constructed using a plasmid expressing rna analog 5b-74 with quikchange ii site - directed mutagenesis kit . sequences of each analog in psilencer 4.1 cmv puro plasmid ( ambion ) were verified with cmv puro primers ( table 1 ) as recommended by the manufacturer . for bb7 hcv genotype 1b replicon studies , cells were plated in 6-well plates 2 days before transfection . seventy - five percent confluent cells were transfected with various amounts of each plasmid to generate rna structural analogs individually or in combinations using lipofectamine ( life technologies ) according to manufacturer 's instructions . in brief , lipofectamine and plasmid dna were separately diluted in opti - mem i medium ( invitrogen ) without serum . after 15 min incubation , they were combined , incubated for 20 min at room temperature , and added to cells in varying concentrations . cells were harvested 48 h post - transfection with trizol ( invitrogen ) , and hcv rna levels quantitated in the cell lysates . for hcv genotype 2a infection studies , two models were used : transfection into cells with a pre - existing infection and transfection into cells before infection . for pre - existing jfh-1 hcv infection studies , 75% confluent huh7.5 cells were infected with jfh-1 hcv for 8 h and then transfected with plasmids expressing rna structural analogs with lipofectamine , as described above . in brief , cells were washed with phosphate buffered saline ( pbs ) . lipofectamine and plasmid dna were separately diluted in opti - mem i medium without serum . after a 15 min incubation , they were combined , and incubated for 20 min at room temperature before being added to cells . culture medium , 200 l , was collected for quantification of jfh-1 hcv levels , and replaced with fresh 200 l of cell culture medium at 0 , 4 , 8 , 12 , 24 , 36 , 48 , and 72 h of transfection . for before infection studies , 75% confluent huh7.5 cells in 6-well plates were transfected with 16 g of each plasmid expressing rna structural analogs , as described above . after 8 h of infection , cells were washed twice with pbs to remove input jfh-1 hcv . culture medium , 200 l , was collected for quantification of jfh-1 hcv levels and replaced with fresh 200 l of cell culture medium at 0 , 4 , 8 , 12 , 24 , 36 , 48 , and 72 h of infection . for bb7 hcv genotype 1b studies , whole cell rna was isolated from replicon cell lysates with an rneasy kit ( qiagen , germany ) according to manufacturer 's instructions and treated with rnase free dnase ( invitrogen ) . cdna was synthesized using 4 g dnase treated rna with superscript iii first - strand kit ( invitrogen ) and quantified by real time rt - pcr with power sybr green pcr master mix ( applied biosystems , usa ) using hcv genotype 1b specific primers ( table 1 ) according to manufacturer 's instructions . human lactate dehydrogenase a ( ldha ) mrna levels were quantified in each sample to normalize hcv rna levels using human ldha specific primers ( table 1 ) . assays were done in quadruplicate , and results expressed as mean standard error of hcv rna levels in cells transfected with analogs compared to untreated controls . for jfh-1 hcv infection studies , viral rna was extracted from 200 l media collected from infected cells with a qiaamp viral rna kit ( qiagen , germany ) according to manufacturer 's instructions . cdna was synthesized using 4 g rna with superscript iii first - strand kit ( invitrogen ) , and quantified by real - time rt - pcr with power sybr green pcr master mix ( applied biosystems ) using jfh-1 hcv rna specific primers ( table 1 ) according to manufacturer 's instructions . assays were repeated with three independent replicates , and results are expressed as means standard error of jfh-1 hcv rna levels in media from infected cells transfected with analogs before or after infection compared to untreated controls . in order to identify important secondary structural elements , the effects of hcv structural analogs on hcv replication in two model cell lines were studied : a constitutive replication model and an infection model . in addition , to evaluate whether differences in viral genotype could affect the possible interactions of structural analogs , viruses representing two different genotypes 1 and 2 , were studied . replicon cells , bb7 , a cell culture system containing the hcv genotype 1b genome , were obtained from apath ( st . louis , usa).20,21 cells were maintained in dulbecco 's modified eagle medium ( dmem ) supplemented with antibiotic / antimycotic solution ( invitrogen , usa ) , 10% fetal bovine serum ( fbs ) and 0.5 mg / ml g418 . for jfh-1 hcv genotype 2a studies , huh7.5 cells ( human hepatoma cell line ) ( obtained from dr . charles rice , rockefeller university , ny , usa ) jfh-1 complementary deoxyribonucleic acid ( cdna ) , an hcv genotype 2a strain , from dr . takaji wakita ( national institute of infectious diseases , tokyo , japan)2224 was used to produce infectious hcv viral particles in huh7.5.25,26 to make jfh-1 hcv stocks for infection , huh 7.5 cells were infected with jfh-1 hcv . media were collected 5 days post - infection and centrifuged at 1000g for 20 min to remove debris . jfh-1 hcv in media was concentrated with a centrifugal device ( 100k nmwl , amicon ultra , millipore , usa ) . infectivity levels of viral stocks were checked by quantitation of jfh-1 hcv rna in media 48 h post - infection of huh7.5 cells with hcv genotype 2a specific primers ( table 1 ) using real time reverse transcriptase polymerase chain reaction ( rt - pcr ) . the pcr conditions were : one cycle of 2 min at 50 c and 10 min at 95 c followed by 40 cycles of 15 sec at 95 c and 1 min at 60 c . specificity of all designed primers was determined with pcr amplification and sequencing of amplified product ( data not shown ) . melt curve analysis was performed following each rt - pcr to identify the presence of primer dimers and analyze the specificity of the reaction . cmv , cytomegalovirus ; fw , forward ; hcv , hepatitis c virus ; jfh-1 , japanese fulminant hepatitis virus-1 ; ldha , lactate dehydrogenase a ; rv , reverse . rna structural analog x-94 was designed to be 100% identical to the ( + ) strand of x - region ( 9508 - 9605 nucleotides ( nt ) ) on the 3-end of the hcv genotype 1b ( 94% identical to genotype 2a ) genome ( fig . 1a ) . to determine whether stem - loop structures versus specific sequences of hcv of rna were most important in hcv replication , base pair changes , described below , were made in stems ( analog x-12 ) and loops ( analog x-12c ) of analog x-94 ( fig . 1b and 1d ) . several software products are available for prediction of secondary structures of rna based on thermodynamic parameters . because of its high reliability and reproducibility , mfold ver 3.2 was used in the current study.2729 rna structural analogs x-12 and x-12c were predicted to adopt stem - loop structure identical to analog x-94 ( fig . 1b and 1d ) . table 3 shows the percent identity of the various rna structural analogs with hcv genotypes 1b and 2a . to determine the minimal structure of the x - region that could inhibit hcv rna replication , shorter rna structural analogs x-12a and x-12b ( fig . 1c ) were designed that were predicted to retain individual stem - loop structures of analog x-94.2729 all analogs were named based on hcv genome region studied and sequence homology of the analogues relative to the jfh-1 hcv genotype 2a genome . for expression studies , the x-94 sequence was subcloned into a psilencer 4.1 cytomegalovirus ( cmv ) puro plasmid ( ambion , usa ) , as previously described.12 psilencer 4.1 cmv puro plasmid enables high level expression of cloned hairpin short rna templates . expression vectors for rna structural analogs x-12 and x-12c were constructed from a plasmid expressing the rna analog x-94 using a quikchange ii site - directed mutagenesis kit ( agilent technologies , inc . 1b shows nucleotide replacements made in stems of analog x-94 resulting in analog x-12 , and fig . 1d illustrates nucleotide replacements made in loops of analog x-12 to construct analog x-12c . shorter rna structural analogs x-12a and x-12b ( fig . 1c ) were constructed from a plasmid expressing rna analog x-12 using a quikchange ii site - directed mutagenesis kit . these sequences were selected as non - overlapping fragments of x-12 that were individually predicted by mfold to retain all the structural elements present in the parent x-12 analog . 5b-74 rna analog , which was previously shown to inhibit viral genome replication12 was used as a positive control for inhibition of viral replication , and a plasmid expressing an unrelated sequence , hb , from hepatitis b virus was used as negative control . an rna structural analog 5b-46 was predicted to adopt stem - loop structures identical to analog 5b-74 as determined by mfold , and was constructed using a plasmid expressing rna analog 5b-74 with quikchange ii site - directed mutagenesis kit . sequences of each analog in psilencer 4.1 cmv puro plasmid ( ambion ) were verified with cmv puro primers ( table 1 ) as recommended by the manufacturer . for bb7 hcv genotype 1b replicon studies , cells were plated in 6-well plates 2 days before transfection . seventy - five percent confluent cells were transfected with various amounts of each plasmid to generate rna structural analogs individually or in combinations using lipofectamine ( life technologies ) according to manufacturer 's instructions . in brief , lipofectamine and plasmid dna were separately diluted in opti - mem i medium ( invitrogen ) without serum . after 15 min incubation , they were combined , incubated for 20 min at room temperature , and added to cells in varying concentrations . cells were harvested 48 h post - transfection with trizol ( invitrogen ) , and hcv rna levels quantitated in the cell lysates . for hcv genotype 2a infection studies , two models were used : transfection into cells with a pre - existing infection and transfection into cells before infection . for pre - existing jfh-1 hcv infection studies , 75% confluent huh7.5 cells were infected with jfh-1 hcv for 8 h and then transfected with plasmids expressing rna structural analogs with lipofectamine , as described above . in brief , cells were washed with phosphate buffered saline ( pbs ) . lipofectamine and plasmid dna were separately diluted in opti - mem i medium without serum . after a 15 min incubation , they were combined , and incubated for 20 min at room temperature before being added to cells . culture medium , 200 l , was collected for quantification of jfh-1 hcv levels , and replaced with fresh 200 l of cell culture medium at 0 , 4 , 8 , 12 , 24 , 36 , 48 , and 72 h of transfection . for before infection studies , 75% confluent huh7.5 cells in 6-well plates were transfected with 16 g of each plasmid expressing rna structural analogs , as described above . cells were then infected with jfh-1 hcv 48 h post - transfection . after 8 h of infection , cells were washed twice with pbs to remove input jfh-1 hcv . culture medium , 200 l , was collected for quantification of jfh-1 hcv levels and replaced with fresh 200 l of cell culture medium at 0 , 4 , 8 , 12 , 24 , 36 , 48 , and 72 h of infection . for bb7 hcv genotype 1b studies , whole cell rna was isolated from replicon cell lysates with an rneasy kit ( qiagen , germany ) according to manufacturer 's instructions and treated with rnase free dnase ( invitrogen ) . cdna was synthesized using 4 g dnase treated rna with superscript iii first - strand kit ( invitrogen ) and quantified by real time rt - pcr with power sybr green pcr master mix ( applied biosystems , usa ) using hcv genotype 1b specific primers ( table 1 ) according to manufacturer 's instructions . human lactate dehydrogenase a ( ldha ) mrna levels were quantified in each sample to normalize hcv rna levels using human ldha specific primers ( table 1 ) . assays were done in quadruplicate , and results expressed as mean standard error of hcv rna levels in cells transfected with analogs compared to untreated controls . for jfh-1 hcv infection studies , viral rna was extracted from 200 l media collected from infected cells with a qiaamp viral rna kit ( qiagen , germany ) according to manufacturer 's instructions . cdna was synthesized using 4 g rna with superscript iii first - strand kit ( invitrogen ) , and quantified by real - time rt - pcr with power sybr green pcr master mix ( applied biosystems ) using jfh-1 hcv rna specific primers ( table 1 ) according to manufacturer 's instructions . assays were repeated with three independent replicates , and results are expressed as means standard error of jfh-1 hcv rna levels in media from infected cells transfected with analogs before or after infection compared to untreated controls . analogs x-94 and x-12 were 100% and 59% identical to hcv genotype 1b , respectively , but were only 94% and 12% identical to the jfh-1 hcv genome , respectively . analog x-12c was 50% identical to hcv genotype 1b , and 0% identical to the jfh-1 hcv genome . to determine whether short rna sequences predicted to fold into secondary structural analogs of the x region of hcv rna genome could inhibit hcv replication , plasmids expressing rna analogs x-94 , x-12 , and x-12c were transfected in replicon cells . 2 shows that the effects of transfection of plasmids expressing rna structural analogs were dose - dependent and most effective at 16 g of plasmid . higher doses did not increase effects beyond those at 16 g , and based on this information , 16 g of each plasmid was used for transfection for subsequent experiments . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assays showed no significant toxic effects due to dna transfection ( data not shown ) . replicon cells were transfected with various concentrations of plasmids expressing hb , 5b-74 , x-94 , and x-12 . hcv rna levels were quantified in cell lysates by qrt - pcr 48 h post - transfection . the values represent hcv1b rna levels in transfected cells compared to untreated controls ( n=4 ) , * p<0.001 . 3 shows that transfection of plasmids generating rna analogs affected hcv rna levels in replicon cells . after transfection of 5b-74 , 5b-46 , x-94 , x-12 , and x-12c at 16 g , hcv rna levels were decreased to 42% , 55% , 52% , 53% , and 54% , respectively , compared to levels of untreated controls . these differences were significant ( p<0.001 for all ) . an unrelated control plasmid ( hb ) generating an hbv sequence had no significant effect under identical conditions . furthermore , combinations of 5b-74 plus x-94 and 5b-46 plus x-12 administered at the same total dose , and under identical conditions , decreased hcv rna levels to 21% and 30% , ( p<0.001 ) , respectively , compared to untreated controls ( fig . these levels of inhibition for the combinations were greater than that for any individual analog alone . replicon cells were transfected with hb , 5b-74 , x-94 , x-12 , x-12c , 5b-74 plus x-94 , and 5b-46 plus x-12 , and hcv rna levels were quantified in cell lysates by qrt - pcr 48 h post - transfection . the figure represents hcv 1b rna levels in transfected cells compared to untreated controls ( n=4 ) , * p<0.001 . we wondered whether smaller structural elements could be produced that retained inhibitory effects . to test this hypothesis , two smaller analogs , x-12a and x-12b , were created that corresponded to nt 1 - 55 and nt 56 - 95 regions , respectively , of analog x-12 ( fig . transfection with x-12a decreased hcv rna to 58% , about the same as intact x-12 ( p<0.001 ) . however , the other fragment , x-12b , was much less effective , resulting in a level of only 78% and not significantly different from untreated control . a combination of x-12a plus x-12b inhibited levels to 60% of untreated controls , which was similar to the effects of x-12a alone ( fig . the data suggested that the x-12a region was the portion of the x analog that was responsible for the majority of the inhibitory activity . replicon cells were transfected with hb , 5b-74 , x-12a , x-12b , and x-12a plus x-12b , and hcv rna levels were quantified in cell lysates by qrt - pcr 48 h post - transfection . the figure represents hcv 1b rna levels in transfected cells compared to untreated controls ( n=4 ) , * p<0.001 . to determine whether rna structural analogs were also effective in an infection model and against a different hcv genotype , 5 shows that the levels in untreated controls increased progressively , and by 12 h , exceeded the level of input hcv rna levels by 6-fold ( extreme left bars ) at 72 h. in contrast , huh7.5 cells transfected with analogs 5b-74 , 5b-46 , x-94 , and x-12 and infected with jfh-1 48 h later , inhibited hcv rna levels to 5.9% , 6.2% , 6.6% , and 1.8% , respectively , compared to untreated controls ( p<0.001 ) . even after 72 h , no cells treated with analogs had hcv rna levels that exceeded more than 30% of input levels . huh7.5 cells were transfected with analogs hb , 5b-74 , 5b-46 , x-94 , and x-12 and then infected with jfh-1 hcv 48 h post - transfection . hcv rna levels in media were quantified by qrt - pcr at various time points post - infection . the values represent jfh-1 hcv rna levels in media from cells transfected with analogs 48 h before infection compared to untreated controls at various time points ( n=3 ) , * p<0.001 . to determine whether rna structural analogs could inhibit a pre - existing hcv infection , cells were infected with jfh-1 hcv for 8 h and then transfected with analogs hb , 5b-74 , 5b-46 , x-94 , and x-12 . 6 shows that there was a progressive increase in jfh-1 hcv rna levels with time in the media compared to uninfected controls . rna levels exceeded those of input virus at 12 h post - transfection and were four - fold higher by 72 h. however , 72 h after transfection with analogs 5b-74 , 5b-46 , x-94 , and x-12 ; hcv rna levels were 8.8% , 10.5% , 9.0% , and 11.6% , respectively , compared to untreated controls . huh7.5 cells infected with jfh-1 hcv , and then transfected with analogs hb , 5b-74 , 5b-46 , x-94 and x-12 8 h post - infection . hcv rna levels in media from infected cells were quantified by qrt - pcr at various time points post - transfection . the figure represents jfh-1 hcv rna levels in media from infected cells transfected with analogs 8 h post - infection compared to untreated controls at various time points ( n=3 ) , * p<0.001 . many previous studies , including our own , have shown that specific domains of the genomes of some rna viruses are critical for viral translation and replication.3032 the ns5b coding region of the hcv genome adopts a stem - loop structure that is involved in the replication of hcv . expression of rna structural analogs predicted to mimic the stem - loop structure identical to the ns5b region of the hcv genome was able to inhibit replication of hcv genotype 2a.12 the current study confirms previous reports that rna secondary structure is important for hcv rna replication.16,33 conserved genomic rna sequences have been shown to fold to adopt stem - loop motifs that interact with other rna motifs and/or proteins required for translation and replication.10,31,3437 identification of such rna sequences and determination of secondary structure formed by these sequences are challenging because structural motifs depend on various parameters , including host cellular microenvironments and the presence of other host and viral interacting molecules.29,3840 several types of software have been developed to predict the stable structures formed by rna sequences based on thermodynamic parameters . the current data generated by mfold software confirm our previous findings that structures predicted using these two - dimensional models do have substantial inhibitory activity against hcv replication . it is also clear that the actual molecules exist not in a two - dimensional but a three - dimensional state , and it is the latter that causes the inhibitory activity . nevertheless , the data support the notion that two - dimensional structures are related to and can predict the activity of analogs in three - dimensions . the structural analogs were effective in models of both genotype 1 and 2 viruses , suggesting that because the design of the molecules was based on secondary rather than primary structure , the effects are more likely to be multigenotypic . this may be clinically relevant as it has already been demonstrated that some current direct acting antiviral agents vary in efficacy against hcv genotypes41,42 and even subtypes.43 design of novel anti - hcv agents based on secondary structural considerations may offer a strategy to develop new agents that are independent of viral genotype or subtype . for hcv genotype 1b studies , a replicon cell model with an integrated hcv genome was used to constitutively generate subgenomic hcv1b replicons in huh7.5 cells.20 this system has been used extensively to determine the effects of various drugs and proteins on viral replication and infection.44,45 however , because hcv rna replication in this model is constitutive , it is not a simulation of hcv infection . for this reason , we examined here the effects of structural analogs on jfh1 , to provide a more realistic hcv infection model system.24,46,47 the results from the jfh-1 infection model systems offered insight into the differences in efficacy among structural analogs depending on whether they were introduced before or after viral infection . the jfh-1 hcv infection system has been used to determine the anti - hcv activity of several proteins and inhibitors ( example : raloxifene , nsc compounds ) before or after infection of hepatic cells.48,49 introduction of rna structural analogs after viral infection resembles treatment strategies for hepatocytes already infected with hcv , while exposure of cells before viral infection represents a potential prophylactic approach . expression of the stem - loop structure of x - region in the 3-utr of hcv genome ( using rna analogs x-94 , x-12 , and x-12c ) was found to be effective against hcv replication , regardless of the sequence of rna . furthermore , we have identified a small portion , one of the stem - loop structures of the x - region , x-12a , as the smallest identified portion of the x - region analog that retains inhibitory activity . the other stem - loop structure , x-12b of approximately the same length ; which also possesses natural hcv sequences , was virtually ineffective . these data confirmed that a specific structure , the stem - loop conformation , was involved , and that the observed inhibitory effects were not due a nonspecific interaction of hcv sequences . the studies on nucleotide base substitution in the x - region showing reduction in the identity to the natural hcv sequence to less than 50% , while retaining secondary structure , indicated that the observed inhibitory effects did not likely involve anti - sense mechanisms . the data indicate that conformation of secondary structures in 3-utr of hcv rna genome is required for hcv replication . stable expression of rna analogs predicted to have identical stem - loop structures , but sequences vastly different from hcv genomic rna , might inhibit hcv infection of hepatocytes in liver , and may represent a novel approach to design anti - hcv agents .

background and aimsthe noncoding regions in the 3-untranslated region ( utr ) of the hepatitis c virus ( hcv ) genome contain secondary structures that are important for replication . the aim of this study was to identify detailed conformational elements of the x - region involved in hcv replication.methodsribonucleic acid ( rna ) structural analogs x94 , x12 , and x12c were constructed to have identical conformation but 94% , 12% , and 0% sequence identity , respectively , to the x region of hcv genotype 2a . effects of structural analogs on replication of hcv genotypes 1b and 2a hcv rna were studied by quantitative reverse transcriptase polymerase chain reaction.resultsin replicon bb7 cells , a constitutive replication model , hcv rna levels decreased to 55% , 52% , 53% , and 54% after transfection with expression plasmids generating rna structural analogs 5b-46 , x-94 , x-12 , and x-12c , respectively ( p<0.001 for all ) . in an hcv genotype 2a infection model , rna analogs 5b-46 , x-94 , and x-12 in hepatic cells inhibited replication to 11% , 9% , and 12% , respectively . because the x-12 analog was only 12% identical to the corresponding sequence of hcv genotype 2a , the sequence per se , or antisense effects were unlikely to be involved.conclusionsthe data suggest that conformation of secondary structures in 3-utr of hcv rna genome is required for hcv replication . stable expression of rna analogs predicted to have identical stem - loop structures might inhibit hcv infection of hepatocytes in liver and may represent a novel approach to design anti - hcv agents .

Introduction Materials and methods Cell culture A genotype 1b BB7 constitutive replication system A Japanese Fulminant Hepatitis Virus-1 (JFH-1) HCV infection system RNA structural analogs Cloning and expression Quantitative RT-PCR (qRT-PCR) Results Discussion Conclusions

hepatitis c virus ( hcv ) is a ribonucleic acid ( rna ) virus that causes chronic hepatitis and liver failure , worldwide.1,2 it consists of six different genotypes that are differentially distributed geographically.3,4 success of treatment varies greatly depending on the genotype.4,5 the genome contains cis - acting replication elements ( cres ) that are critical for hcv rna replication and translation.6,7 rna structural elements present in 5- and 3-untranslated regions ( 3utr ) of the hcv genome interact with viral and cellular proteins to initiate and facilitate the replication and translation processes.811 in our previous studies , we showed that rna secondary structure of the nonstructural ( ns)5b coding region of the hcv genome was required for hcv rna replication , and hence viral particle production.1215 it has been shown that the x region in the 3-utr of the hcv rna genome contains a highly conserved sequence.16 the latter have also been found to form stable secondary stem - loop structures that require physical contact between its rna - dependent rna polymerase for hcv replication.1719 we hypothesized that rna structural analogs resembling secondary stem - loop structure of the x region could be created that can compete with natural hcv genomic structure for binding to proteins and inhibit hcv replication . the aim of this study was to introduce rna structural analogs resembling these cres into human liver cells and to identify secondary structural elements of the hcv x - region involved in hcv replication and infection . in order to identify important secondary structural elements , the effects of hcv structural analogs on hcv replication in two model cell lines were studied : a constitutive replication model and an infection model . in addition , to evaluate whether differences in viral genotype could affect the possible interactions of structural analogs , viruses representing two different genotypes 1 and 2 , were studied . replicon cells , bb7 , a cell culture system containing the hcv genotype 1b genome , were obtained from apath ( st . jfh-1 complementary deoxyribonucleic acid ( cdna ) , an hcv genotype 2a strain , from dr . infectivity levels of viral stocks were checked by quantitation of jfh-1 hcv rna in media 48 h post - infection of huh7.5 cells with hcv genotype 2a specific primers ( table 1 ) using real time reverse transcriptase polymerase chain reaction ( rt - pcr ) . rna structural analog x-94 was designed to be 100% identical to the ( + ) strand of x - region ( 9508 - 9605 nucleotides ( nt ) ) on the 3-end of the hcv genotype 1b ( 94% identical to genotype 2a ) genome ( fig . to determine whether stem - loop structures versus specific sequences of hcv of rna were most important in hcv replication , base pair changes , described below , were made in stems ( analog x-12 ) and loops ( analog x-12c ) of analog x-94 ( fig . several software products are available for prediction of secondary structures of rna based on thermodynamic parameters . because of its high reliability and reproducibility , mfold ver 3.2 was used in the current study.2729 rna structural analogs x-12 and x-12c were predicted to adopt stem - loop structure identical to analog x-94 ( fig . table 3 shows the percent identity of the various rna structural analogs with hcv genotypes 1b and 2a . to determine the minimal structure of the x - region that could inhibit hcv rna replication , shorter rna structural analogs x-12a and x-12b ( fig . 1c ) were designed that were predicted to retain individual stem - loop structures of analog x-94.2729 all analogs were named based on hcv genome region studied and sequence homology of the analogues relative to the jfh-1 hcv genotype 2a genome . expression vectors for rna structural analogs x-12 and x-12c were constructed from a plasmid expressing the rna analog x-94 using a quikchange ii site - directed mutagenesis kit ( agilent technologies , inc . an rna structural analog 5b-46 was predicted to adopt stem - loop structures identical to analog 5b-74 as determined by mfold , and was constructed using a plasmid expressing rna analog 5b-74 with quikchange ii site - directed mutagenesis kit . cells were harvested 48 h post - transfection with trizol ( invitrogen ) , and hcv rna levels quantitated in the cell lysates . for hcv genotype 2a infection studies , two models were used : transfection into cells with a pre - existing infection and transfection into cells before infection . for pre - existing jfh-1 hcv infection studies , 75% confluent huh7.5 cells were infected with jfh-1 hcv for 8 h and then transfected with plasmids expressing rna structural analogs with lipofectamine , as described above . assays were done in quadruplicate , and results expressed as mean standard error of hcv rna levels in cells transfected with analogs compared to untreated controls . assays were repeated with three independent replicates , and results are expressed as means standard error of jfh-1 hcv rna levels in media from infected cells transfected with analogs before or after infection compared to untreated controls . in order to identify important secondary structural elements , the effects of hcv structural analogs on hcv replication in two model cell lines were studied : a constitutive replication model and an infection model . in addition , to evaluate whether differences in viral genotype could affect the possible interactions of structural analogs , viruses representing two different genotypes 1 and 2 , were studied . replicon cells , bb7 , a cell culture system containing the hcv genotype 1b genome , were obtained from apath ( st . charles rice , rockefeller university , ny , usa ) jfh-1 complementary deoxyribonucleic acid ( cdna ) , an hcv genotype 2a strain , from dr . infectivity levels of viral stocks were checked by quantitation of jfh-1 hcv rna in media 48 h post - infection of huh7.5 cells with hcv genotype 2a specific primers ( table 1 ) using real time reverse transcriptase polymerase chain reaction ( rt - pcr ) . rna structural analog x-94 was designed to be 100% identical to the ( + ) strand of x - region ( 9508 - 9605 nucleotides ( nt ) ) on the 3-end of the hcv genotype 1b ( 94% identical to genotype 2a ) genome ( fig . to determine whether stem - loop structures versus specific sequences of hcv of rna were most important in hcv replication , base pair changes , described below , were made in stems ( analog x-12 ) and loops ( analog x-12c ) of analog x-94 ( fig . several software products are available for prediction of secondary structures of rna based on thermodynamic parameters . because of its high reliability and reproducibility , mfold ver 3.2 was used in the current study.2729 rna structural analogs x-12 and x-12c were predicted to adopt stem - loop structure identical to analog x-94 ( fig . table 3 shows the percent identity of the various rna structural analogs with hcv genotypes 1b and 2a . to determine the minimal structure of the x - region that could inhibit hcv rna replication , shorter rna structural analogs x-12a and x-12b ( fig . 1c ) were designed that were predicted to retain individual stem - loop structures of analog x-94.2729 all analogs were named based on hcv genome region studied and sequence homology of the analogues relative to the jfh-1 hcv genotype 2a genome . expression vectors for rna structural analogs x-12 and x-12c were constructed from a plasmid expressing the rna analog x-94 using a quikchange ii site - directed mutagenesis kit ( agilent technologies , inc . 1b shows nucleotide replacements made in stems of analog x-94 resulting in analog x-12 , and fig . an rna structural analog 5b-46 was predicted to adopt stem - loop structures identical to analog 5b-74 as determined by mfold , and was constructed using a plasmid expressing rna analog 5b-74 with quikchange ii site - directed mutagenesis kit . cells were harvested 48 h post - transfection with trizol ( invitrogen ) , and hcv rna levels quantitated in the cell lysates . for hcv genotype 2a infection studies , two models were used : transfection into cells with a pre - existing infection and transfection into cells before infection . for pre - existing jfh-1 hcv infection studies , 75% confluent huh7.5 cells were infected with jfh-1 hcv for 8 h and then transfected with plasmids expressing rna structural analogs with lipofectamine , as described above . assays were done in quadruplicate , and results expressed as mean standard error of hcv rna levels in cells transfected with analogs compared to untreated controls . cdna was synthesized using 4 g rna with superscript iii first - strand kit ( invitrogen ) , and quantified by real - time rt - pcr with power sybr green pcr master mix ( applied biosystems ) using jfh-1 hcv rna specific primers ( table 1 ) according to manufacturer 's instructions . assays were repeated with three independent replicates , and results are expressed as means standard error of jfh-1 hcv rna levels in media from infected cells transfected with analogs before or after infection compared to untreated controls . analogs x-94 and x-12 were 100% and 59% identical to hcv genotype 1b , respectively , but were only 94% and 12% identical to the jfh-1 hcv genome , respectively . analog x-12c was 50% identical to hcv genotype 1b , and 0% identical to the jfh-1 hcv genome . to determine whether short rna sequences predicted to fold into secondary structural analogs of the x region of hcv rna genome could inhibit hcv replication , plasmids expressing rna analogs x-94 , x-12 , and x-12c were transfected in replicon cells . 2 shows that the effects of transfection of plasmids expressing rna structural analogs were dose - dependent and most effective at 16 g of plasmid . replicon cells were transfected with various concentrations of plasmids expressing hb , 5b-74 , x-94 , and x-12 . 3 shows that transfection of plasmids generating rna analogs affected hcv rna levels in replicon cells . after transfection of 5b-74 , 5b-46 , x-94 , x-12 , and x-12c at 16 g , hcv rna levels were decreased to 42% , 55% , 52% , 53% , and 54% , respectively , compared to levels of untreated controls . these differences were significant ( p<0.001 for all ) . furthermore , combinations of 5b-74 plus x-94 and 5b-46 plus x-12 administered at the same total dose , and under identical conditions , decreased hcv rna levels to 21% and 30% , ( p<0.001 ) , respectively , compared to untreated controls ( fig . replicon cells were transfected with hb , 5b-74 , x-94 , x-12 , x-12c , 5b-74 plus x-94 , and 5b-46 plus x-12 , and hcv rna levels were quantified in cell lysates by qrt - pcr 48 h post - transfection . transfection with x-12a decreased hcv rna to 58% , about the same as intact x-12 ( p<0.001 ) . replicon cells were transfected with hb , 5b-74 , x-12a , x-12b , and x-12a plus x-12b , and hcv rna levels were quantified in cell lysates by qrt - pcr 48 h post - transfection . to determine whether rna structural analogs were also effective in an infection model and against a different hcv genotype , 5 shows that the levels in untreated controls increased progressively , and by 12 h , exceeded the level of input hcv rna levels by 6-fold ( extreme left bars ) at 72 h. in contrast , huh7.5 cells transfected with analogs 5b-74 , 5b-46 , x-94 , and x-12 and infected with jfh-1 48 h later , inhibited hcv rna levels to 5.9% , 6.2% , 6.6% , and 1.8% , respectively , compared to untreated controls ( p<0.001 ) . huh7.5 cells were transfected with analogs hb , 5b-74 , 5b-46 , x-94 , and x-12 and then infected with jfh-1 hcv 48 h post - transfection . the values represent jfh-1 hcv rna levels in media from cells transfected with analogs 48 h before infection compared to untreated controls at various time points ( n=3 ) , * p<0.001 . to determine whether rna structural analogs could inhibit a pre - existing hcv infection , cells were infected with jfh-1 hcv for 8 h and then transfected with analogs hb , 5b-74 , 5b-46 , x-94 , and x-12 . 6 shows that there was a progressive increase in jfh-1 hcv rna levels with time in the media compared to uninfected controls . rna levels exceeded those of input virus at 12 h post - transfection and were four - fold higher by 72 h. however , 72 h after transfection with analogs 5b-74 , 5b-46 , x-94 , and x-12 ; hcv rna levels were 8.8% , 10.5% , 9.0% , and 11.6% , respectively , compared to untreated controls . huh7.5 cells infected with jfh-1 hcv , and then transfected with analogs hb , 5b-74 , 5b-46 , x-94 and x-12 8 h post - infection . the figure represents jfh-1 hcv rna levels in media from infected cells transfected with analogs 8 h post - infection compared to untreated controls at various time points ( n=3 ) , * p<0.001 . many previous studies , including our own , have shown that specific domains of the genomes of some rna viruses are critical for viral translation and replication.3032 the ns5b coding region of the hcv genome adopts a stem - loop structure that is involved in the replication of hcv . expression of rna structural analogs predicted to mimic the stem - loop structure identical to the ns5b region of the hcv genome was able to inhibit replication of hcv genotype 2a.12 the current study confirms previous reports that rna secondary structure is important for hcv rna replication.16,33 conserved genomic rna sequences have been shown to fold to adopt stem - loop motifs that interact with other rna motifs and/or proteins required for translation and replication.10,31,3437 identification of such rna sequences and determination of secondary structure formed by these sequences are challenging because structural motifs depend on various parameters , including host cellular microenvironments and the presence of other host and viral interacting molecules.29,3840 several types of software have been developed to predict the stable structures formed by rna sequences based on thermodynamic parameters . the structural analogs were effective in models of both genotype 1 and 2 viruses , suggesting that because the design of the molecules was based on secondary rather than primary structure , the effects are more likely to be multigenotypic . this may be clinically relevant as it has already been demonstrated that some current direct acting antiviral agents vary in efficacy against hcv genotypes41,42 and even subtypes.43 design of novel anti - hcv agents based on secondary structural considerations may offer a strategy to develop new agents that are independent of viral genotype or subtype . for hcv genotype 1b studies , a replicon cell model with an integrated hcv genome was used to constitutively generate subgenomic hcv1b replicons in huh7.5 cells.20 this system has been used extensively to determine the effects of various drugs and proteins on viral replication and infection.44,45 however , because hcv rna replication in this model is constitutive , it is not a simulation of hcv infection . for this reason , we examined here the effects of structural analogs on jfh1 , to provide a more realistic hcv infection model system.24,46,47 the results from the jfh-1 infection model systems offered insight into the differences in efficacy among structural analogs depending on whether they were introduced before or after viral infection . the jfh-1 hcv infection system has been used to determine the anti - hcv activity of several proteins and inhibitors ( example : raloxifene , nsc compounds ) before or after infection of hepatic cells.48,49 introduction of rna structural analogs after viral infection resembles treatment strategies for hepatocytes already infected with hcv , while exposure of cells before viral infection represents a potential prophylactic approach . expression of the stem - loop structure of x - region in the 3-utr of hcv genome ( using rna analogs x-94 , x-12 , and x-12c ) was found to be effective against hcv replication , regardless of the sequence of rna . furthermore , we have identified a small portion , one of the stem - loop structures of the x - region , x-12a , as the smallest identified portion of the x - region analog that retains inhibitory activity . these data confirmed that a specific structure , the stem - loop conformation , was involved , and that the observed inhibitory effects were not due a nonspecific interaction of hcv sequences . the studies on nucleotide base substitution in the x - region showing reduction in the identity to the natural hcv sequence to less than 50% , while retaining secondary structure , indicated that the observed inhibitory effects did not likely involve anti - sense mechanisms . the data indicate that conformation of secondary structures in 3-utr of hcv rna genome is required for hcv replication . stable expression of rna analogs predicted to have identical stem - loop structures , but sequences vastly different from hcv genomic rna , might inhibit hcv infection of hepatocytes in liver , and may represent a novel approach to design anti - hcv agents .

in diagnostic medical imaging the performance of imaging modalities needs to be tested and evaluated on a regular basis in order to ensure correct functionality and optimal image quality . systems are analyzed by a variety of methods [ 1 , 2 ] . for pet scanners in particular , the national electrical manufacturers association ( nema ) has defined a standard to assess the performance of the tomographic system . such image quality control measurements also need to be conducted in pet / mr hybrid imaging for pet performance measurements when introducing a new system [ 1 , 2 ] or , on a regular basis , when monitoring quality of a specific pet system over time . comparability in clinical studies evaluating pet / ct and pet / mr imaging performance in patient studies also relies on nema iq phantom measurements [ 4 , 5 ] . furthermore , accurate nema iq phantom measurements are a precondition for studies investigating the attenuating influence of new hardware components such as radiofrequency ( rf ) coils [ 6 , 7 ] and radio therapy planning equipment that are designed for use in pet / mr systems . dose optimization studies that have been reported for pet / mr hybrid imaging also rely on nema iq phantom measurements . all these studies have in common that they build on accurate methods for attenuation correction ( ac ) of the phantoms involved . to obtain quantitative pet images that can be used to determine the scanner performance parameters , the acquired pet data need to be corrected for attenuation of the photons caused by the scanned object as well as by the attenuating hardware components of the system . in pet / ct imaging , information about the attenuating characteristics of the scanned object is derived from the ct scan itself . in pet / mr patient imaging , mr - based attenuation correction ( ac ) methods are applied to correct for attenuation caused by human tissue , however , the applicability in phantom imaging has not yet been quantified . current mr - based ac only considers the fluid phantom filling as it provides sufficient mr signal , but it does not correct for plastic or glass materials commonly used in pet phantom housings , as these can not be reliably detected in standard mr imaging ( fig . b mr imaging ( in - phase image ) of the nema iq phantom using the mr ac dixon vibe sequence showing only fluid phantom filling ( water ) . the phantom housing ( plexiglas ) is not visible in mr imaging and thus , also not considered in the mr - based -map of the phantom ( c ) . note that the glass spheres are displayed as signal voids ( dark rims in b ) that are segmented as water in the mr - based -map ( c ) a the nema iq phantom . b mr imaging ( in - phase image ) of the nema iq phantom using the mr ac dixon vibe sequence showing only fluid phantom filling ( water ) . the phantom housing ( plexiglas ) is not visible in mr imaging and thus , also not considered in the mr - based -map of the phantom ( c ) . note that the glass spheres are displayed as signal voids ( dark rims in b ) that are segmented as water in the mr - based -map ( c ) in this study , therefore , an alternative strategy for performing nema iq phantom measurements is evaluated . the use of ct - based ac maps and templates for attenuation correction of hardware components such as the pet / mr system patient table and various stationary and mechanically rigid radiofrequency ( rf ) coils can be considered as the current standard approach for hardware component ac in combined pet / mr systems across all vendors . consequently , this study proposes to use a pre - acquired ct - based template -map of the filled nema iq phantom in conjunction with a phantom holder and to install this template -map on the pet / mr system to be used for ac whenever nema iq phantom experiments are performed . this study investigates the standard nema iq test utilizing ct - based ac for the biograph mmr pet / mr hybrid system ( siemens healthcare , erlangen , germany ) . contrast recovery , background variability and signal - to - noise ratio are determined as a function of different reconstruction parameters . in comparison , the effect of mr - based ac is evaluated and the impact on the image quality parameters assessed . all phantom experiments were performed on an integrated pet / mr whole - body hybrid system ( biograph mmr , siemens ag ) . the mr component of the hybrid system consists of a 3.0 tesla static magnetic field , a radiofrequency ( rf ) transmit body coil and a gradient coil system which provides a maximum amplitude of 45 mt / m and a maximum slew rate of 200 t / m / s . the pet component is comprised of 8 detector rings , each consisting of 56 detector blocks . one detector block consists of 8x8 lutetium oxyorthosilicate ( lso ) scintillator crystal elements connected to 3 x 3 avalanche photodiodes ( apds ) . according to the nema nu 22007 standard , image quality parameters of pet scanners are obtained by measuring a specific international electrotechnical commission ( iec ) 616751 emission phantom ( nema image quality phantom ) ( fig . 1a , ptw , freiburg , germany ) . this image quality phantom mimics the shape of an upper human body and is built of acrylic glass material . it comprises 6 hollow glass spheres ( inner diameters 37 , 28 , 22 , 17 , 13 , and 10 mm ) which can be inserted into the large phantom compartment . additionally a cylindrical insert containing styrofoam with an average density of 0.3 0.1 g ml ( simulates patient lung tissue ( lung - insert ~ 0.026 cm ) and is positioned in the center of the phantom . the volume of the tested phantom ( ptw , freiburg , germany , fig . 1a ) was measured to be 9.5 l 1 % when the spheres and lung cylinder are inserted . the phantom housing has a thickness of approximately 3 mm along the phantom body , and 10 mm ( in few parts 20 mm ) at the lids at both ends of the phantom . the glass material ( 0.118 cm ) of the spheres has a thickness of around 1 mm . when nema iq measurements with a ct - based phantom template -map are performed , the phantom needs to be placed at a pre - defined position in the pet field of view ( fov ) with a known reference to the patient table , to ensure alignment between the -map and the position of the phantom . to guarantee a reproducible phantom placement , a defined set of phantom holders a spacer is positioned adjacent to the rf head coil connection port on the patient table in order to create a defined and reproducible distance of the nema iq phantom to the stronger photon - attenuating rf coil port . the nema iq phantom is placed next to the spacer on a foam block , which was designed in order to align the phantom at a pre - defined patient table position . as required by the nema standard , a scatter phantom ( a 70 cm long plastic cylinder with an activated line source ) is positioned contiguously to the nema iq phantom to generate scattered and random coincidences from outside the fov , such as in a patient examination ( fig . 2).fig . 2the nema iq phantom imaging setup as performed in this study . the schematic drawing in ( a ) and the image in ( b ) show the spacer ( 1 ) , the nema iq phantom ( 2 ) , and the scatter phantom ( 3 ) arranged on top of the pet / mr system patient table the spacer ( 1 ) ensures a predefined and reproducible position of the phantom ( 2 ) on the patient table the nema iq phantom imaging setup as performed in this study . the schematic drawing in ( a ) and the image in ( b ) show the spacer ( 1 ) , the nema iq phantom ( 2 ) , and the scatter phantom ( 3 ) arranged on top of the pet / mr system patient table . the spacer ( 1 ) ensures a predefined and reproducible position of the phantom ( 2 ) on the patient table the background volume of the nema iq phantom and the four smallest spheres were filled with f - fdg mixed with pure water using a 4:1 sphere - to - background activity concentration ratio , as specified by nema . the initial tracer activity concentration was specifically calibrated to the start of the measurement : 5.3 kbq / ml 1 % in the phantom background and 21.2 kbq / ml 5 % in the four smallest spheres . the non - radioactive cylindrical insert simulating lung tissue was placed in the center of the phantom . the line source contained in the large scatter phantom was injected with 110 mbq of f - fdg . the defined measurement time by the nema nu 22007 standard is dependent on the axial imaging distance of the pet system and amounted to 12 min in one bed position for the biograph mmr . this imaging time resulted from the specification of nema nu 22007 that 100 cm of axial imaging distance shall be covered in 60 min . the pet images were reconstructed using the 3d ordinary poisson ordered - subset - expectation - maximization ( op - osem ) reconstruction algorithm as implemented in the system . according to the nema nu 22007 protocol , pet image quality is analyzed by means of two parameters : contrast recovery and background variability [ 3 , 15 ] . these parameters are calculated by evaluation of various regions of interest ( rois ) in the transverse image slice that contains the centers of the spheres , as well as in adjacent slices ( fig . 3position of the regions of interest ( rois ) placed over the active spheres ( red ) , nonradioactive spheres ( blue ) and the phantom background ( green ) of the reconstructed pet images , which are used for analysisdrawn over the spheres as well as in the background region as is illustrated in fig . 3 . in this study the evaluation of the rois was performed with the open - source image analysis tool imagej ( fiji ) . position of the regions of interest ( rois ) placed over the active spheres ( red ) , nonradioactive spheres ( blue ) and the phantom background ( green ) of the reconstructed pet images , which are used for analysis the percentage contrast recovery ( in an ideal case = 100 % ) is determined for each hot sphere j by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { q}_{h , j}=\frac{\raisebox{1ex}{${c}_{h , j}$}\!\left/ \!\raisebox{-1ex}{${c}_{b , j}$}\right . - 1}{\raisebox{1ex}{${a}_h$}\!\left/ \!\raisebox{-1ex}{${a}_b$}\right . - 1}*100\ \left[\%\right ] $ $ \end{document}qh , j = ch , jcb , j1ahab1 * 100% ch , j = average counts in the roi for sphere j cb , j = average counts in the background roi for sphere j ah = activity concentration in the hot spheres ab = activity concentration in the background for each nonradioactive sphere j the percentage contrast recovery qc , j is given by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { q}_{c , j } = \left(1 - \frac{c_{c , j}}{c_{b , j}}\right)*100\ \left[\%\right ] $ $ \end{document}qc , j=1cc , jcb , j*100% cc , j = average counts in the roi for sphere j cb , j = average of all background roi counts for sphere j in order to determine the percentage background variability nj as a measure for the image noise for sphere j ( in an ideal case = 0 % ) , the following equation is used:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { n}_j = \frac{s{d}_j}{c_{b , j}}*100\ \left[\%\right ] $ $ \end{document}nj = sdjcb , j*100% sdj = standard deviation of the background roi counts for sphere j cb , j = average of all background roi counts for sphere j to evaluate the effect of neglecting acrylic glass and glass material in the attenuation correction of the nema iq phantom emission data , reconstructions with an mr - based -map containing only the fluid phantom filling were performed and nema iq parameters were calculated . linear attenuation coefficients of water were assigned to the whole phantom content including phantom liquid and glass inserts , despite of higher attenuation of the glass material of the spheres . for reconstruction , the above - mentioned op - osem algorithm was used with 3 iterations , 21 subsets , 172 matrix size , and 4 mm gaussian post - smoothing filter . mr - based ac was performed by a 3d dixon vibe - based approach with the following imaging sequence parameters : tr : 4.07 ms , te in - phase : 2.46 ms , te opposed - phase : 1.23 ms , flip angle : 10 , slice thickness : 3.12 mm , field of view : 500 mm x 328 mm and matrix size : 128 x 84 . the system s built - in rf transmit body coil was used for rf transmission and signal reception . all other rf coils were removed from the patient table to avoid additional pet photon attenuation . the assigned -values were restricted to two -values for water and air for the performed phantom measurements . in general , imaging the nema iq phantom filled with pure water on a 3.0 tesla mr system leads to artifacts and signal inhomogeneities due to standing - rf - wave phenomena and t1 effects , thus affecting mr - based ac of the nema phantom in pet / mr hybrid imaging ( as shown in ) . manually reducing the initial voltage of the rf transmitter adjustment algorithm led to a lower adjusted transmitter voltage of 92.7 volts , instead of the default value for patient imaging ( ~300 v ) , and resulted in fairly homogeneous -maps of the nema iq phantom filling ( fig . alternatively the addition of substances ( e.g. niso4 ) could be considered that decrease the t1 relaxation time of water and as a consequence decrease the mentioned image artefacts , as discussed in . 4 a mr - based -map in transversal and coronal orientation only contains discrete attenuation values for water and air , and therefore only corrects for photon attenuation caused by the water content of the phantom and not by the phantom housing materials , as these materials can not be detected with standard mr imaging . b , c ct - based -map contains continuous attenuation values including -values for the phantom housing , glass spheres , and styrofoam block used as phantom holder ( displayed in c ) . b and c visualize the same content , however windowing properties were adjusted individually in order to visualize either the phantom content ( b ) or the styrofoam holder , on which the phantom is placed ( c ) a mr - based -map in transversal and coronal orientation only contains discrete attenuation values for water and air , and therefore only corrects for photon attenuation caused by the water content of the phantom and not by the phantom housing materials , as these materials can not be detected with standard mr imaging . b , c ct - based -map contains continuous attenuation values including -values for the phantom housing , glass spheres , and styrofoam block used as phantom holder ( displayed in c ) . b and c visualize the same content , however windowing properties were adjusted individually in order to visualize either the phantom content ( b ) or the styrofoam holder , on which the phantom is placed ( c ) the ct image of the phantom , which was used to generate the ct - based template -map ( fig . 4b and c ) , was acquired on a 128-slice pet / ct system ( biograph 128 , siemens ag ) with the following parameters : 500 effective mas and 140 kvp . images were reconstructed with 0.6 mm slice thickness , 512 x 512 image matrix size and a b30f convolution kernel . following a 2 mm gaussian filtering and bilinear scaling of the ct hounsfield units to linear attenuation coefficients at the pet energy level of 511 kev , the ct - based -map was installed on the pet / mr system in order to be used for ac whenever nema iq measurements are performed . the phantom holder minimizes misalignment between the actual phantom position and the ct - based template ac map of the phantom as much as possible . in case of remaining slight misplacement between ct - based phantom template and actual phantom position , a practical use of a ct - ac method as proposed in this study by potential future pet / mr users will require the registration to be performed based solely on the images available . thus the registration in this study was also performed by means of the available information from the pet and ct images in order to simulate a realistic case . this was performed by using the inherent landmarks of the phantom such as the glass spheres , the lung insert and the phantom outer boundary that function as orientation when performing manual image registration . for the pet measurements utilizing ct - based ac , reconstruction parameters were set to 3d op - osem with 3 iterations , 21 subsets , 172 matrix size and 4 mm gaussian filtering , as also used with mr - based ac . pet performance parameters , as well as the visibility of lesions in phantom and patient images , in general strongly depend on the applied reconstruction algorithm parameters . to investigate this effect in the specific context of nema iq phantom imaging particularly for the biograph mmr system and its pet detector geometry and reconstruction algorithm , different reconstructions of the same pet phantom data acquisition using ct - based attenuation correction were evaluated . hereby the number of iterations ( 15 iterations ) , the image matrix size ( 172 x 172 , 344 x 344 matrix ) and the gaussian filter ( 2 mm , 4 mm ) were varied and the resulting impact on the image quality was investigated , as has been discussed often in the literature and is described e.g. in . the number of subsets was kept constant at 21 as this is the default setting on the scanner console . to determine the optimal reconstruction parameters for best nema iq phantom image quality with high lesion contrast and low background noise , the signal - to - noise ratio ( snr ) was selected as a representative image quality measure and was calculated for each active sphere and for all listed reconstruction parameter combinations . the snr for sphere j was determined , as described in the literature when performing similar evaluations for pet / ct imaging [ 20 , 21 ] , according to the following equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ sn{r}_j = \frac{signa{l}_j- background}{\sigma_b\ } $ $ \end{document}snrj = signaljbackgroundb signalj = average counts in the roi for sphere j background = average counts in a roi placed in a uniform area outside the b = standard deviation of the background roi counts , corresponding to noise in the image all phantom experiments were performed on an integrated pet / mr whole - body hybrid system ( biograph mmr , siemens ag ) . the mr component of the hybrid system consists of a 3.0 tesla static magnetic field , a radiofrequency ( rf ) transmit body coil and a gradient coil system which provides a maximum amplitude of 45 mt / m and a maximum slew rate of 200 t / m / s . the pet component is comprised of 8 detector rings , each consisting of 56 detector blocks . one detector block consists of 8x8 lutetium oxyorthosilicate ( lso ) scintillator crystal elements connected to 3 x 3 avalanche photodiodes ( apds ) . according to the nema nu 22007 standard , image quality parameters of pet scanners are obtained by measuring a specific international electrotechnical commission ( iec ) 616751 emission phantom ( nema image quality phantom ) ( fig . 1a , ptw , freiburg , germany ) . this image quality phantom mimics the shape of an upper human body and is built of acrylic glass material . it comprises 6 hollow glass spheres ( inner diameters 37 , 28 , 22 , 17 , 13 , and 10 mm ) which can be inserted into the large phantom compartment . additionally a cylindrical insert containing styrofoam with an average density of 0.3 0.1 g ml ( simulates patient lung tissue ( lung - insert ~ 0.026 cm ) and is positioned in the center of the phantom . the volume of the tested phantom ( ptw , freiburg , germany , fig . 1a ) was measured to be 9.5 l 1 % when the spheres and lung cylinder are inserted . the phantom housing has a thickness of approximately 3 mm along the phantom body , and 10 mm ( in few parts 20 mm ) at the lids at both ends of the phantom . the glass material ( ~ 0.118 cm ) of the spheres has a thickness of around 1 mm . when nema iq measurements with a ct - based phantom template -map are performed , the phantom needs to be placed at a pre - defined position in the pet field of view ( fov ) with a known reference to the patient table , to ensure alignment between the -map and the position of the phantom . to guarantee a reproducible phantom placement , a defined set of phantom holders a spacer is positioned adjacent to the rf head coil connection port on the patient table in order to create a defined and reproducible distance of the nema iq phantom to the stronger photon - attenuating rf coil port . the nema iq phantom is placed next to the spacer on a foam block , which was designed in order to align the phantom at a pre - defined patient table position . as required by the nema standard , a scatter phantom ( a 70 cm long plastic cylinder with an activated line source ) is positioned contiguously to the nema iq phantom to generate scattered and random coincidences from outside the fov , such as in a patient examination ( fig . 2the nema iq phantom imaging setup as performed in this study . the schematic drawing in ( a ) and the image in ( b ) show the spacer ( 1 ) , the nema iq phantom ( 2 ) , and the scatter phantom ( 3 ) arranged on top of the pet / mr system patient table . the spacer ( 1 ) ensures a predefined and reproducible position of the phantom ( 2 ) on the patient table the nema iq phantom imaging setup as performed in this study . the schematic drawing in ( a ) and the image in ( b ) show the spacer ( 1 ) , the nema iq phantom ( 2 ) , and the scatter phantom ( 3 ) arranged on top of the pet / mr system patient table . the spacer ( 1 ) ensures a predefined and reproducible position of the phantom ( 2 ) on the patient table the background volume of the nema iq phantom and the four smallest spheres were filled with f - fdg mixed with pure water using a 4:1 sphere - to - background activity concentration ratio , as specified by nema . the initial tracer activity concentration was specifically calibrated to the start of the measurement : 5.3 kbq / ml 1 % in the phantom background and 21.2 kbq / ml 5 % in the four smallest spheres . the non - radioactive cylindrical insert simulating lung tissue was placed in the center of the phantom . the line source contained in the large scatter phantom was injected with 110 mbq of f - fdg . the defined measurement time by the nema nu 22007 standard is dependent on the axial imaging distance of the pet system and amounted to 12 min in one bed position for the biograph mmr . this imaging time resulted from the specification of nema nu 22007 that 100 cm of axial imaging distance shall be covered in 60 min . the pet images were reconstructed using the 3d ordinary poisson ordered - subset - expectation - maximization ( op - osem ) reconstruction algorithm as implemented in the system . according to the nema nu 22007 protocol , pet image quality is analyzed by means of two parameters : contrast recovery and background variability [ 3 , 15 ] . these parameters are calculated by evaluation of various regions of interest ( rois ) in the transverse image slice that contains the centers of the spheres , as well as in adjacent slices ( fig . 3position of the regions of interest ( rois ) placed over the active spheres ( red ) , nonradioactive spheres ( blue ) and the phantom background ( green ) of the reconstructed pet images , which are used for analysisdrawn over the spheres as well as in the background region as is illustrated in fig . 3 . in this study the evaluation of the rois was performed with the open - source image analysis tool imagej ( fiji ) . position of the regions of interest ( rois ) placed over the active spheres ( red ) , nonradioactive spheres ( blue ) and the phantom background ( green ) of the reconstructed pet images , which are used for analysis the percentage contrast recovery ( in an ideal case = 100 % ) is determined for each hot sphere j by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { q}_{h , j}=\frac{\raisebox{1ex}{${c}_{h , j}$}\!\left/ \!\raisebox{-1ex}{${c}_{b , j}$}\right . - 1}{\raisebox{1ex}{${a}_h$}\!\left/ \!\raisebox{-1ex}{${a}_b$}\right . - 1}*100\ \left[\%\right ] $ $ \end{document}qh , j = ch , jcb , j1ahab1 * 100% ch , j = average counts in the roi for sphere j cb , j = average counts in the background roi for sphere j ah = activity concentration in the hot spheres ab = activity concentration in the background for each nonradioactive sphere j the percentage contrast recovery qc , j is given by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { q}_{c , j } = \left(1 - \frac{c_{c , j}}{c_{b , j}}\right)*100\ \left[\%\right ] $ $ \end{document}qc , j=1cc , jcb , j*100% cc , j = average counts in the roi for sphere j cb , j = average of all background roi counts for sphere j in order to determine the percentage background variability nj as a measure for the image noise for sphere j ( in an ideal case = 0 % ) , the following equation is used:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { n}_j = \frac{s{d}_j}{c_{b , j}}*100\ \left[\%\right ] $ $ \end{document}nj = sdjcb , j*100% sdj = standard deviation of the background roi counts for sphere j cb , j = average of all background roi counts for sphere j to evaluate the effect of neglecting acrylic glass and glass material in the attenuation correction of the nema iq phantom emission data , reconstructions with an mr - based -map containing only the fluid phantom filling were performed and nema iq parameters were calculated . linear attenuation coefficients of water were assigned to the whole phantom content including phantom liquid and glass inserts , despite of higher attenuation of the glass material of the spheres . for reconstruction , the above - mentioned op - osem algorithm was used with 3 iterations , 21 subsets , 172 matrix size , and 4 mm gaussian post - smoothing filter . mr - based ac was performed by a 3d dixon vibe - based approach with the following imaging sequence parameters : tr : 4.07 ms , te in - phase : 2.46 ms , te opposed - phase : 1.23 ms , flip angle : 10 , slice thickness : 3.12 mm , field of view : 500 mm x 328 mm and matrix size : 128 x 84 . the system s built - in rf transmit body coil was used for rf transmission and signal reception . all other rf coils were removed from the patient table to avoid additional pet photon attenuation . the assigned -values were restricted to two -values for water and air for the performed phantom measurements . attenuation caused by the styrofoam material in the lung insert was not accounted for . in general , imaging the nema iq phantom filled with pure water on a 3.0 tesla mr system leads to artifacts and signal inhomogeneities due to standing - rf - wave phenomena and t1 effects , thus affecting mr - based ac of the nema phantom in pet / mr hybrid imaging ( as shown in ) . manually reducing the initial voltage of the rf transmitter adjustment algorithm led to a lower adjusted transmitter voltage of 92.7 volts , instead of the default value for patient imaging ( ~300 v ) , and resulted in fairly homogeneous -maps of the nema iq phantom filling ( fig . 1c , fig . alternatively the addition of substances ( e.g. niso4 ) could be considered that decrease the t1 relaxation time of water and as a consequence decrease the mentioned image artefacts , as discussed in . 4 a mr - based -map in transversal and coronal orientation only contains discrete attenuation values for water and air , and therefore only corrects for photon attenuation caused by the water content of the phantom and not by the phantom housing materials , as these materials can not be detected with standard mr imaging . b , c ct - based -map contains continuous attenuation values including -values for the phantom housing , glass spheres , and styrofoam block used as phantom holder ( displayed in c ) . b and c visualize the same content , however windowing properties were adjusted individually in order to visualize either the phantom content ( b ) or the styrofoam holder , on which the phantom is placed ( c ) a mr - based -map in transversal and coronal orientation only contains discrete attenuation values for water and air , and therefore only corrects for photon attenuation caused by the water content of the phantom and not by the phantom housing materials , as these materials can not be detected with standard mr imaging . b , c ct - based -map contains continuous attenuation values including -values for the phantom housing , glass spheres , and styrofoam block used as phantom holder ( displayed in c ) . b and c visualize the same content , however windowing properties were adjusted individually in order to visualize either the phantom content ( b ) or the styrofoam holder , on which the phantom is placed ( c ) the ct image of the phantom , which was used to generate the ct - based template -map ( fig . 4b and c ) , was acquired on a 128-slice pet / ct system ( biograph 128 , siemens ag ) with the following parameters : 500 effective mas and 140 kvp . images were reconstructed with 0.6 mm slice thickness , 512 x 512 image matrix size and a b30f convolution kernel . following a 2 mm gaussian filtering and bilinear scaling of the ct hounsfield units to linear attenuation coefficients at the pet energy level of 511 kev , the ct - based -map was installed on the pet / mr system in order to be used for ac whenever nema iq measurements are performed . the phantom holder minimizes misalignment between the actual phantom position and the ct - based template ac map of the phantom as much as possible . in case of remaining slight misplacement between ct - based phantom template and actual phantom position , a practical use of a ct - ac method as proposed in this study by potential future pet / mr users will require the registration to be performed based solely on the images available . thus the registration in this study was also performed by means of the available information from the pet and ct images in order to simulate a realistic case . this was performed by using the inherent landmarks of the phantom such as the glass spheres , the lung insert and the phantom outer boundary that function as orientation when performing manual image registration . for the pet measurements utilizing ct - based ac , reconstruction parameters were set to 3d op - osem with 3 iterations , 21 subsets , 172 matrix size and 4 mm gaussian filtering , as also used with mr - based ac . pet performance parameters , as well as the visibility of lesions in phantom and patient images , in general strongly depend on the applied reconstruction algorithm parameters . to investigate this effect in the specific context of nema iq phantom imaging particularly for the biograph mmr system and its pet detector geometry and reconstruction algorithm , different reconstructions of the same pet phantom data acquisition using ct - based attenuation correction hereby the number of iterations ( 15 iterations ) , the image matrix size ( 172 x 172 , 344 x 344 matrix ) and the gaussian filter ( 2 mm , 4 mm ) were varied and the resulting impact on the image quality was investigated , as has been discussed often in the literature and is described e.g. in . the number of subsets was kept constant at 21 as this is the default setting on the scanner console . to determine the optimal reconstruction parameters for best nema iq phantom image quality with high lesion contrast and low background noise , the signal - to - noise ratio ( snr ) was selected as a representative image quality measure and the snr for sphere j was determined , as described in the literature when performing similar evaluations for pet / ct imaging [ 20 , 21 ] , according to the following equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ sn{r}_j = \frac{signa{l}_j- background}{\sigma_b\ } $ $ \end{document}snrj = signaljbackgroundb signalj = average counts in the roi for sphere j background = average counts in a roi placed in a uniform area outside the b = standard deviation of the background roi counts , corresponding to noise in the image the resulting nema iq parameters utilizing mr - based ac are listed in table 1 . regarding the four smallest spheres filled with tracer activity ( radioactive spheres ) the performance values using mr - based ac are lower than the expected values for the underlying pet component when comparing as a general orientation to results reported in the literature for the biograph mmr system . the contrast recovery values for the two largest spheres filled with non - radioactive water only ( non - radioactive spheres ) ( table 1 ) show higher values when compared to the results reported in the literature for the biograph mmr system .table 1contrast recovery and background variability parameters when applying mr - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive16.8 1.94.8 0.813radioactive31.7 2.94.0 0.417radioactive52.7 3.03.6 0.322radioactive64.8 4.13.4 0.428non - radioactive68.9 1.53.2 0.437non - radioactive76.1 0.63.1 0.3 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying mr - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering table 2 presents the resulting nema iq parameters when applying ct - based ac of four independent measurements with separate fillings on four different days . in comparison to values obtained when applying mr - based ac ( table 1 ) , contrast recovery was , for instance for the smallest sphere , almost approximately twice as high when using ct - based ac . the larger the size of the spheres , the smaller the deviation in contrast recovery to mr - based ac becomes . the results using ct - based ac are in the same range as results obtained in the literature .table 2contrast recovery and background variability parameters when applying ct - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive30.5 1.33.8 0.813radioactive50.5 2.43.0 0.517radioactive72.9 1.92.5 0.222radioactive74.5 3.32.3 0.228non - radioactive56.6 2.32.0 0.237non - radioactive64.8 0.91.8 0.1 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying ct - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering the influence of varying reconstruction parameters is demonstrated for the four smallest spheres in table 3 and plotted in fig . it can be determined that contrast recovery increases with an increasing number of iterations at the cost of higher background variability , which is essentially noise.table 3contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac contrast recovery [ % ] background variability [ % ] sphere 10 mmsphere 13 mmsphere 17 mmsphere 22 mmiteration172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344113.8 2.515.8 2.828.1 2.329.6 2.548.5 2.151.3 2.353.7 2.156.0 2.2224.3 3.228.1 3.543.8 2.645.3 2.866.8 2.369.7 2.370.0 1.972.0 2.2330.5 3.835.0 4.350.5 3.051.1 3.272.9 2.575.2 2.674.5 2.376.3 2.4434.3 4.438.7 4.953.4 3.353.3 3.575.4 2.777.5 2.876.5 2.578.2 2.6536.8 4.840.8 5.354.8 3.554.3 3.876.9 2.978.7 3.077.6 2.679.2 2.7 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . 5contrast recovery vs. background variability using ct - based ac shown for the four smallest spheres with 172x172 matrix size ( hollow symbols ) and 344x344 matrix size ( solid symbols ) and 4 mm gaussian filter . the values from left to right represent the results for an increasing number of iterations ( 15 ) of the reconstruction algorithm contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . the values represent the mean of four independent measurements contrast recovery vs. background variability using ct - based ac shown for the four smallest spheres with 172x172 matrix size ( hollow symbols ) and 344x344 matrix size ( solid symbols ) and 4 mm gaussian filter . the values from left to right represent the results for an increasing number of iterations ( 15 ) of the reconstruction algorithm when comparing two different matrix sizes ( 344 x 344 and 172 x 172 ) , it can be observed that with higher image matrix the contrast increases but at the same time it causes an increase in noise . these effects can also be perceived in the phantom images in fig . 6images of the first five iterations reconstructed with 3d op - osem for different matrix and gaussian filter sizes and using ct - based ac . note : increasing the number of iterations is associated with increasing lesion sharpness while also increasing background noise . this also applies when increasing the image matrix size from 172 x 172 to 344 x 344 images of the first five iterations reconstructed with 3d op - osem for different matrix and gaussian filter sizes and using ct - based ac . note : increasing the number of iterations is associated with increasing lesion sharpness while also increasing background noise . this also applies when increasing the image matrix size from 172 x 172 to 344 x 344 the mean snr of four independent measurements as a function of iterations for all investigated reconstruction parameter combinations is plotted for each of the four smallest spheres in fig . the best choice of matrix size , gaussian filter size and number of iterations can be determined from the peak snr value for each sphere in the graphs . as can be seen , highest snr is achieved for all spheres using a 4 mm gaussian filter . for the two larger spheres ( 17 and 22 mm ) 2 iterations led to highest snr , whereas for the two smallest spheres ( 10 and 13 mm ) the peak snr was achieved at 3 iterations . no significant difference in resulting snr values can be observed when comparing 344 x 344 to 172 x 172 matrix size . 7the signal - to - noise ratio ( snr ) calculated for different reconstruction parameter combinations for the four active spheres : 10 mm ( a ) , 13 mm ( b ) , 17 mm ( c ) and 22 mm ( d ) . the values represent the mean of four independent nema iq measurements using ct - based ac the signal - to - noise ratio ( snr ) calculated for different reconstruction parameter combinations for the four active spheres : 10 mm ( a ) , 13 mm ( b ) , 17 mm ( c ) and 22 mm ( d ) . the values represent the mean of four independent nema iq measurements using ct - based ac the resulting nema iq parameters utilizing mr - based ac are listed in table 1 . regarding the four smallest spheres filled with tracer activity ( radioactive spheres ) the performance values using mr - based ac are lower than the expected values for the underlying pet component when comparing as a general orientation to results reported in the literature for the biograph mmr system . the contrast recovery values for the two largest spheres filled with non - radioactive water only ( non - radioactive spheres ) ( table 1 ) show higher values when compared to the results reported in the literature for the biograph mmr system .table 1contrast recovery and background variability parameters when applying mr - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive16.8 1.94.8 0.813radioactive31.7 2.94.0 0.417radioactive52.7 3.03.6 0.322radioactive64.8 4.13.4 0.428non - radioactive68.9 1.53.2 0.437non - radioactive76.1 0.63.1 0.3 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying mr - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering table 2 presents the resulting nema iq parameters when applying ct - based ac of four independent measurements with separate fillings on four different days . in comparison to values obtained when applying mr - based ac ( table 1 ) , contrast recovery was , for instance for the smallest sphere , almost approximately twice as high when using ct - based ac . the larger the size of the spheres , the smaller the deviation in contrast recovery to mr - based ac becomes . the results using ct - based ac are in the same range as results obtained in the literature .table 2contrast recovery and background variability parameters when applying ct - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive30.5 1.33.8 0.813radioactive50.5 2.43.0 0.517radioactive72.9 1.92.5 0.222radioactive74.5 3.32.3 0.228non - radioactive56.6 2.32.0 0.237non - radioactive64.8 0.91.8 0.1 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying ct - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering the influence of varying reconstruction parameters is demonstrated for the four smallest spheres in table 3 and plotted in fig . it can be determined that contrast recovery increases with an increasing number of iterations at the cost of higher background variability , which is essentially noise.table 3contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac contrast recovery [ % ] background variability [ % ] sphere 10 mmsphere 13 mmsphere 17 mmsphere 22 mmiteration172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344113.8 2.515.8 2.828.1 2.329.6 2.548.5 2.151.3 2.353.7 2.156.0 2.2224.3 3.228.1 3.543.8 2.645.3 2.866.8 2.369.7 2.370.0 1.972.0 2.2330.5 3.835.0 4.350.5 3.051.1 3.272.9 2.575.2 2.674.5 2.376.3 2.4434.3 4.438.7 4.953.4 3.353.3 3.575.4 2.777.5 2.876.5 2.578.2 2.6536.8 4.840.8 5.354.8 3.554.3 3.876.9 2.978.7 3.077.6 2.679.2 2.7 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . 5contrast recovery vs. background variability using ct - based ac shown for the four smallest spheres with 172x172 matrix size ( hollow symbols ) and 344x344 matrix size ( solid symbols ) and 4 mm gaussian filter . the values from left to right represent the results for an increasing number of iterations ( 15 ) of the reconstruction algorithm contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . the values represent the mean of four independent measurements contrast recovery vs. background variability using ct - based ac shown for the four smallest spheres with 172x172 matrix size ( hollow symbols ) and 344x344 matrix size ( solid symbols ) and 4 mm gaussian filter . the values from left to right represent the results for an increasing number of iterations ( 15 ) of the reconstruction algorithm when comparing two different matrix sizes ( 344 x 344 and 172 x 172 ) , it can be observed that with higher image matrix the contrast increases but at the same time it causes an increase in noise . these effects can also be perceived in the phantom images in fig 6images of the first five iterations reconstructed with 3d op - osem for different matrix and gaussian filter sizes and using ct - based ac . note : increasing the number of iterations is associated with increasing lesion sharpness while also increasing background noise . this also applies when increasing the image matrix size from 172 x 172 to 344 x 344 images of the first five iterations reconstructed with 3d op - osem for different matrix and gaussian filter sizes and using ct - based ac . note : increasing the number of iterations is associated with increasing lesion sharpness while also increasing background noise . this also applies when increasing the image matrix size from 172 x 172 to 344 x 344 the mean snr of four independent measurements as a function of iterations for all investigated reconstruction parameter combinations is plotted for each of the four smallest spheres in fig . the best choice of matrix size , gaussian filter size and number of iterations can be determined from the peak snr value for each sphere in the graphs . as can be seen , highest snr is achieved for all spheres using a 4 mm gaussian filter . for the two larger spheres ( 17 and 22 mm ) 2 iterations led to highest snr , whereas for the two smallest spheres ( 10 and 13 mm ) the peak snr was achieved at 3 iterations . no significant difference in resulting snr values can be observed when comparing 344 x 344 to 172 x 172 matrix size . 7the signal - to - noise ratio ( snr ) calculated for different reconstruction parameter combinations for the four active spheres : 10 mm ( a ) , 13 mm ( b ) , 17 mm ( c ) and 22 mm ( d ) . the values represent the mean of four independent nema iq measurements using ct - based ac the signal - to - noise ratio ( snr ) calculated for different reconstruction parameter combinations for the four active spheres : 10 mm ( a ) , 13 mm ( b ) , 17 mm ( c ) and 22 mm ( d ) . the values represent the mean of four independent nema iq measurements using ct - based ac in this study , a strategy for using ct - based phantom attenuation correction in the context of nema iq measurements in pet / mr hybrid imaging has been evaluated . the proposed strategy features a pre - acquired ct - based 3d attenuation template of the nema iq phantom in conjunction with a phantom holder providing exact and reproducible repositioning of the phantom on the systems patient table . this strategy for phantom ac is straightforward and in line with currently implemented ac methods for hardware component ac such as rf coils on the available sequential and integrated pet / mr systems . respective image quality and activity quantification parameters were systematically investigated using mr - based and ct - based ac of the nema iq phantom . furthermore , reconstruction parameters for nema iq phantom measurements were optimized for the biograph mmr system by evaluating image quality parameters using the ct - based approach . it was demonstrated that the tested mr - based ac leads to insufficient quantification results and to degraded image quality as compared to ct - based ac for the evaluated nema iq phantom . specifically , the activity quantification values for the four radioactive spheres were lower than activity values reported in the literature for nema iq measurements on the biograph mmr system . the low values indicate insufficient ac of the phantom , which is associated with the fact that mr - based ac only considers the fluid phantom filling but neglects the attenuating phantom housing made from acrylic glass and glass components . on the other hand , the contrast recovery values for the two non - radioactive spheres show higher values when compared to the values reported in the literature . because these spheres are filled with non - radioactive water only , the corresponding contrast recovery values are highest when no counts are detected from inside these spheres . applying an insufficient attenuation correction , such as the mr - based ac , leads to less counts being detected from inside the non - radioactive spheres resulting in a higher contrast recovery value than when using ct - based ac . based on the resulting contrast recovery parameters for radioactive and non - radioactive spheres it can be concluded that mr - based attenuation correction performed with the current ac sequence is inadequate to determine representative pet image quality parameters in the tested nema iq phantom . the proposed method , using pre - acquired ct - based nema iq phantom attenuation templates instead of mr - based ac , takes the complete phantom into account , including the fluid filling , phantom housing , lung insert and foam phantom holder . using this approach leads to nema pet image quality parameters similar to values reported in the literature . the ct - based approach can thus be considered adequate for representing the performance characteristics of the biograph mmr pet component . the suggested ct - based ac requires the nema iq phantom to be located at a known position on the patient table during the measurements . this was achieved by a foam block that carries the nema iq phantom and fits the shape of the systems patient table . to allow for exact and reproducible positioning of the foam block in z - direction on the patient table , the proposed approach can theoretically be implemented on any current pet / mr system ( tri - modality , sequential or integrated ) as a practical method to perform pet nema image quality measurements . in theory , other methods are conceivable to obtain improved mr - based ac of the nema iq phantom . in principle this requires that the phantom fluid is displayed in homogeneous manner across the entire phantom . another precondition is that the phantom housing made e.g. of plexiglas is also considered in mr - based ac . this may be achieved by using mr imaging sequences capable of displaying structures made of plastic , e.g. ultrashort echo time ( ute ) sequences . alternatively , the virtual addition of a defined rim of attenuating pixels to the outer borders of the phantom fluid visible in mr imaging may provide a method for consideration of the phantom housing in mr - based ac . additionally , a 511 kev transmission scan of the phantom may also be a possibility to generate a phantom ac map . however , a very long scan would be required in order to limit the noise properties , which are much higher in comparison to ct , and achieve acceptable spatial resolution . in addition , since the advent of combined pet / ct scanners access to pet only scanners with transmission sources is very limited . in the present study we have focused on the ct - based ac strategy since it is straightforward , accurate , and can be implemented on current pet / mr systems in the same manner hardware component ac is performed for rf coils . a goal of this study was to assess a solution to be able to perform nema pet image quality evaluations on a pet / mr system as closely to the nema nu 22007 standard as possible . nema nu 22007 specifies that the iq phantom shall be positioned in the isocenter of the pet fov . since the patient table does not accommodate this vertical phantom position , a suggested phantom holder ensures the required positioning in a reproducible manner . it reduces misplacement of the phantom in all three spatial dimensions as much as possible and consequently , the position of the phantom during a nema measurement matches the position of the ct - based template ac map of the phantom . in case of remaining slight misalignment , the phantom -map was registered manually to the non - attenuation corrected pet data . possibly , manual or automatic registration to mr data may be another alternative , however , due to suboptimal image quality of the mr data acquired with the rf body coil , this was not performed for the presented data . for the 3d op - osem reconstruction in this nema iq phantom study , an optimized lesion snr was achieved by reconstructing with 3 iterations , 21 subsets and 4 mm gaussian filtering . every lesion in patient data sets has a different optimal reconstruction parameter setting to obtain the highest individual lesion snr . therefore , the parameter optimization in this study can be considered only a general guideline when performing pet patient measurements on the biograph mmr hybrid system . this study validates nema ( nu 22007 ) pet image quality performance measurements by applying ct - based attenuation correction for an integrated pet / mr hybrid system . the necessity and superiority of ct - based nema iq phantom ac is demonstrated by a comparison to results using mr - based ac . furthermore , optimized image reconstruction parameters are provided for highest lesion snr in the context of nema iq phantom measurements on the biograph mmr system . the results of this study can thus be seen as an important step towards standardization and image quality control of pet measurements in the context of pet / mr hybrid imaging .

backgroundin integrated pet / mr hybrid imaging the evaluation of pet performance characteristics according to the nema standard nu 22007 is challenging because of incomplete mr - based attenuation correction ( ac ) for phantom imaging . in this study , a strategy for ct - based ac of the nema image quality ( iq ) phantom is assessed . the method is systematically evaluated in nema iq phantom measurements on an integrated pet / mr system.methodsnema iq measurements were performed on the integrated 3.0 tesla pet / mr hybrid system ( biograph mmr , siemens healthcare ) . ac of the nema iq phantom was realized by an mr - based and by a ct - based method . the suggested ct - based ac uses a template -map of the nema iq phantom and a phantom holder for exact repositioning of the phantom on the systems patient table . the pet image quality parameters contrast recovery , background variability , and signal - to - noise ratio ( snr ) were determined and compared for both phantom ac methods . reconstruction parameters of an iterative 3d op - osem reconstruction were optimized for highest lesion snr in nema iq phantom imaging.resultsusing a ct - based nema iq phantom -map on the pet / mr system is straightforward and allowed performing accurate nema iq measurements on the hybrid system . mr - based ac was determined to be insufficient for pet quantification in the tested nema iq phantom because only photon attenuation caused by the mr - visible phantom filling but not the phantom housing is considered . using the suggested ct - based ac , the highest snr in this phantom experiment for small lesions ( < = 13 mm ) was obtained with 3 iterations , 21 subsets and 4 mm gaussian filtering.conclusionthis study suggests ct - based ac for the nema iq phantom when performing pet nema iq measurements on an integrated pet / mr hybrid system . the superiority of ct - based ac for this phantom is demonstrated by comparison to measurements using mr - based ac . furthermore , optimized pet image reconstruction parameters are provided for the highest lesion snr in nema iq phantom measurements .

Background Methods PET/MR hybrid scanner NEMA image quality phantom Phantom measurement setup PET phantom preparation and data acquisition NEMA image quality parameters NEMA IQ measurement with MR-based AC NEMA IQ measurement with CT-based AC Optimization of PET reconstruction parameters in phantom images Results NEMA IQ measurement with MR-based AC NEMA IQ measurement with CT-based AC Optimization of PET reconstruction parameters of 3D OP-OSEM Discussion Conclusion

consequently , this study proposes to use a pre - acquired ct - based template -map of the filled nema iq phantom in conjunction with a phantom holder and to install this template -map on the pet / mr system to be used for ac whenever nema iq phantom experiments are performed . the schematic drawing in ( a ) and the image in ( b ) show the spacer ( 1 ) , the nema iq phantom ( 2 ) , and the scatter phantom ( 3 ) arranged on top of the pet / mr system patient table the spacer ( 1 ) ensures a predefined and reproducible position of the phantom ( 2 ) on the patient table the nema iq phantom imaging setup as performed in this study . in general , imaging the nema iq phantom filled with pure water on a 3.0 tesla mr system leads to artifacts and signal inhomogeneities due to standing - rf - wave phenomena and t1 effects , thus affecting mr - based ac of the nema phantom in pet / mr hybrid imaging ( as shown in ) . for the pet measurements utilizing ct - based ac , reconstruction parameters were set to 3d op - osem with 3 iterations , 21 subsets , 172 matrix size and 4 mm gaussian filtering , as also used with mr - based ac . the snr for sphere j was determined , as described in the literature when performing similar evaluations for pet / ct imaging [ 20 , 21 ] , according to the following equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ sn{r}_j = \frac{signa{l}_j- background}{\sigma_b\ } $ $ \end{document}snrj = signaljbackgroundb signalj = average counts in the roi for sphere j background = average counts in a roi placed in a uniform area outside the b = standard deviation of the background roi counts , corresponding to noise in the image all phantom experiments were performed on an integrated pet / mr whole - body hybrid system ( biograph mmr , siemens ag ) . - 1}*100\ \left[\%\right ] $ $ \end{document}qh , j = ch , jcb , j1ahab1 * 100% ch , j = average counts in the roi for sphere j cb , j = average counts in the background roi for sphere j ah = activity concentration in the hot spheres ab = activity concentration in the background for each nonradioactive sphere j the percentage contrast recovery qc , j is given by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { q}_{c , j } = \left(1 - \frac{c_{c , j}}{c_{b , j}}\right)*100\ \left[\%\right ] $ $ \end{document}qc , j=1cc , jcb , j*100% cc , j = average counts in the roi for sphere j cb , j = average of all background roi counts for sphere j in order to determine the percentage background variability nj as a measure for the image noise for sphere j ( in an ideal case = 0 % ) , the following equation is used:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { n}_j = \frac{s{d}_j}{c_{b , j}}*100\ \left[\%\right ] $ $ \end{document}nj = sdjcb , j*100% sdj = standard deviation of the background roi counts for sphere j cb , j = average of all background roi counts for sphere j to evaluate the effect of neglecting acrylic glass and glass material in the attenuation correction of the nema iq phantom emission data , reconstructions with an mr - based -map containing only the fluid phantom filling were performed and nema iq parameters were calculated . in general , imaging the nema iq phantom filled with pure water on a 3.0 tesla mr system leads to artifacts and signal inhomogeneities due to standing - rf - wave phenomena and t1 effects , thus affecting mr - based ac of the nema phantom in pet / mr hybrid imaging ( as shown in ) . for the pet measurements utilizing ct - based ac , reconstruction parameters were set to 3d op - osem with 3 iterations , 21 subsets , 172 matrix size and 4 mm gaussian filtering , as also used with mr - based ac . to investigate this effect in the specific context of nema iq phantom imaging particularly for the biograph mmr system and its pet detector geometry and reconstruction algorithm , different reconstructions of the same pet phantom data acquisition using ct - based attenuation correction hereby the number of iterations ( 15 iterations ) , the image matrix size ( 172 x 172 , 344 x 344 matrix ) and the gaussian filter ( 2 mm , 4 mm ) were varied and the resulting impact on the image quality was investigated , as has been discussed often in the literature and is described e.g. to determine the optimal reconstruction parameters for best nema iq phantom image quality with high lesion contrast and low background noise , the signal - to - noise ratio ( snr ) was selected as a representative image quality measure and the snr for sphere j was determined , as described in the literature when performing similar evaluations for pet / ct imaging [ 20 , 21 ] , according to the following equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ sn{r}_j = \frac{signa{l}_j- background}{\sigma_b\ } $ $ \end{document}snrj = signaljbackgroundb signalj = average counts in the roi for sphere j background = average counts in a roi placed in a uniform area outside the b = standard deviation of the background roi counts , corresponding to noise in the image the resulting nema iq parameters utilizing mr - based ac are listed in table 1 . the contrast recovery values for the two largest spheres filled with non - radioactive water only ( non - radioactive spheres ) ( table 1 ) show higher values when compared to the results reported in the literature for the biograph mmr system .table 1contrast recovery and background variability parameters when applying mr - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive16.8 1.94.8 0.813radioactive31.7 2.94.0 0.417radioactive52.7 3.03.6 0.322radioactive64.8 4.13.4 0.428non - radioactive68.9 1.53.2 0.437non - radioactive76.1 0.63.1 0.3 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying mr - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering table 2 presents the resulting nema iq parameters when applying ct - based ac of four independent measurements with separate fillings on four different days . the results using ct - based ac are in the same range as results obtained in the literature .table 2contrast recovery and background variability parameters when applying ct - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive30.5 1.33.8 0.813radioactive50.5 2.43.0 0.517radioactive72.9 1.92.5 0.222radioactive74.5 3.32.3 0.228non - radioactive56.6 2.32.0 0.237non - radioactive64.8 0.91.8 0.1 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying ct - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering the influence of varying reconstruction parameters is demonstrated for the four smallest spheres in table 3 and plotted in fig . it can be determined that contrast recovery increases with an increasing number of iterations at the cost of higher background variability , which is essentially noise.table 3contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac contrast recovery [ % ] background variability [ % ] sphere 10 mmsphere 13 mmsphere 17 mmsphere 22 mmiteration172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344113.8 2.515.8 2.828.1 2.329.6 2.548.5 2.151.3 2.353.7 2.156.0 2.2224.3 3.228.1 3.543.8 2.645.3 2.866.8 2.369.7 2.370.0 1.972.0 2.2330.5 3.835.0 4.350.5 3.051.1 3.272.9 2.575.2 2.674.5 2.376.3 2.4434.3 4.438.7 4.953.4 3.353.3 3.575.4 2.777.5 2.876.5 2.578.2 2.6536.8 4.840.8 5.354.8 3.554.3 3.876.9 2.978.7 3.077.6 2.679.2 2.7 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . the values from left to right represent the results for an increasing number of iterations ( 15 ) of the reconstruction algorithm contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . the values represent the mean of four independent nema iq measurements using ct - based ac the signal - to - noise ratio ( snr ) calculated for different reconstruction parameter combinations for the four active spheres : 10 mm ( a ) , 13 mm ( b ) , 17 mm ( c ) and 22 mm ( d ) . the contrast recovery values for the two largest spheres filled with non - radioactive water only ( non - radioactive spheres ) ( table 1 ) show higher values when compared to the results reported in the literature for the biograph mmr system .table 1contrast recovery and background variability parameters when applying mr - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive16.8 1.94.8 0.813radioactive31.7 2.94.0 0.417radioactive52.7 3.03.6 0.322radioactive64.8 4.13.4 0.428non - radioactive68.9 1.53.2 0.437non - radioactive76.1 0.63.1 0.3 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying mr - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering table 2 presents the resulting nema iq parameters when applying ct - based ac of four independent measurements with separate fillings on four different days . the results using ct - based ac are in the same range as results obtained in the literature .table 2contrast recovery and background variability parameters when applying ct - based acsphere size [ mm]radioactive / non - radioactive contrast recovery [ % ] background variability [ % ] 10radioactive30.5 1.33.8 0.813radioactive50.5 2.43.0 0.517radioactive72.9 1.92.5 0.222radioactive74.5 3.32.3 0.228non - radioactive56.6 2.32.0 0.237non - radioactive64.8 0.91.8 0.1 the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering contrast recovery and background variability parameters when applying ct - based ac the values represent the mean values and standard deviation of four independent measurements for the reconstruction : 3 iterations , 21 subsets , 172 matrix and 4 mm gaussian filtering the influence of varying reconstruction parameters is demonstrated for the four smallest spheres in table 3 and plotted in fig . it can be determined that contrast recovery increases with an increasing number of iterations at the cost of higher background variability , which is essentially noise.table 3contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac contrast recovery [ % ] background variability [ % ] sphere 10 mmsphere 13 mmsphere 17 mmsphere 22 mmiteration172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344172 x 172344 x 344113.8 2.515.8 2.828.1 2.329.6 2.548.5 2.151.3 2.353.7 2.156.0 2.2224.3 3.228.1 3.543.8 2.645.3 2.866.8 2.369.7 2.370.0 1.972.0 2.2330.5 3.835.0 4.350.5 3.051.1 3.272.9 2.575.2 2.674.5 2.376.3 2.4434.3 4.438.7 4.953.4 3.353.3 3.575.4 2.777.5 2.876.5 2.578.2 2.6536.8 4.840.8 5.354.8 3.554.3 3.876.9 2.978.7 3.077.6 2.679.2 2.7 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . the values from left to right represent the results for an increasing number of iterations ( 15 ) of the reconstruction algorithm contrast recovery and background variability values in % for each active sphere for different reconstruction parameters of the 3d op - osem reconstruction algorithm when using ct - based ac 4 mm gaussian filtering and 21 subsets were kept constant for all reconstructions . the values represent the mean of four independent nema iq measurements using ct - based ac the signal - to - noise ratio ( snr ) calculated for different reconstruction parameter combinations for the four active spheres : 10 mm ( a ) , 13 mm ( b ) , 17 mm ( c ) and 22 mm ( d ) . the values represent the mean of four independent nema iq measurements using ct - based ac in this study , a strategy for using ct - based phantom attenuation correction in the context of nema iq measurements in pet / mr hybrid imaging has been evaluated . for the 3d op - osem reconstruction in this nema iq phantom study , an optimized lesion snr was achieved by reconstructing with 3 iterations , 21 subsets and 4 mm gaussian filtering . furthermore , optimized image reconstruction parameters are provided for highest lesion snr in the context of nema iq phantom measurements on the biograph mmr system .

a gap exists between evidence - based knowledge and the care that is actually delivered to our patients [ 1 , 2 ] . practice guideline development , a pivotal step in this process , has limited effect on changing physician practice behavior [ 36 ] . this issue holds true in emergency medicine as well [ 79 ] . the american college of emergency physicians ( acep ) clinical policies have been shown to be safe and effective , and are even cited by other specialties [ 10 , 11 ] . in spite of the benefits of the acep clinical policies , implementation of these clinical practice guidelines into physician practice continues to be a challenge . even when physicians are aware of the evidence , they may not adhere to it [ 3 , 12 ] . . found that knowledge of the acep clinical policy on hypertension did not translate into changes in physician practice . clinical decision support systems ( cdss ) are systems designed to aid directly in clinical decision - making , in which characteristics of individual patients are used to generate patient - specific assessments or recommendations that are then presented to clinicians for consideration . clinical decision support systems ( cdsss ) can significantly improve clinical practice [ 1416 ] . found that clinical practice was improved with provision of cdsss : ( 1 ) as part of clinician workflow , ( 2 ) with recommendations rather than assessments , ( 3 ) at the time and location of decision - making , and ( 4 ) if computer - based . realizing the potential efficacy of cdsss as emergency department documentation increasingly becomes computerized , napoli and jagoda and this study aimed to improve knowledge translation from evidence - based emergency medicine practice guidelines by creating a cdss for implementation of the recommendations from an acep clinical policy in an urban academic emergency department . we specifically chose the 2007 acep clinical policy on syncope because it included newly published recommendations on a frequently encountered diagnosis . we also hypothesized that there would be room for change in previously accepted physician practice behavior for the reasons outlined below . we sought to identify the presence of a change in physician ordering of cranial imaging and admission practices due to implementation of a hpi - based cdss in patients with a final diagnosis of syncope . in our retrospective control population , due to the high costs of admission for syncope ( estimated at $ 2 billion annually ) , there may be potential for cost savings if syncope admission guidelines are more closely followed . the 2007 acep clinical policy on syncope provides new recommendations on decision - making regarding need for head computed tomography ( ct ) imaging and hospital admission in adult patients presenting to the emergency department with syncope . we hypothesized that incorporating these recommendations into a computerized cdss embedded in the physician workflow of an emergency department information system ( edis ) would influence physician behavior . a change in behavior would suggest that this point - of - care decision support tool helps improve practitioner awareness , adoption , and adherence to acep practice guidelines and bridge the gap from evidence to practice . this study was designed as a pre - post intervention design with a prospective cohort and retrospective controls . we conducted a medical chart review with 6-month retrospective baseline analysis and prospective data collection following implementation of a cdss based on the 2007 acep clinical policy on syncope into the edis . the mount sinai school of medicine institutional review board reviewed and approved this study . the mount sinai medical center ( new york , ny ) is an 1,171-bed tertiary care academic medical center located in manhattan , bordering the upper east side and east harlem . the ed adopted a comprehensive edis in 2004 ( picis , ed pulsecheck , wakefield , ma , formerly ibex ) that provides triage , patient tracking , physician and nurse documentation , retrieval of charts from prior ed encounters and inpatient data , computerized provider order entry , results review , discharge instructions and prescription writing . attendings and/or residents could complete charts and final diagnosis on every ed patient , including a history of present illness ( hpi ) and final emergency department diagnosis ( chosen from a drop - down list of international classification of diseases , 9th revision approved diagnoses or free - text entries ) . the intervention assessed behavior of physicians caring for consecutive syncope patients over 18 years of age in the emergency department . the medical records of 410 patients were reviewed prior to implementation , and 301 records were reviewed after implementation . physicians included 34 attending physicians board - certified or board - eligible in emergency medicine , with additional information - gathering and decision - making provided by residents primarily in emergency medicine ( em ) , as well as occasional rotators from the departments of internal medicine , psychiatry , and obstetrics and gynecology . a three - item module based on the 2007 acep clinical policy on syncope was added to the syncope hpi template , for completion by attending or mid - level providers ( see fig . 1 , table 1 ) . each item served a dual role in : ( 1 ) reminding physicians of the strength of the policy s recommendations and ( 2 ) prompting physicians to document their clinical decision - making . 1a screen capture of the cdss module as it appeared in the syncope hpi templatetable 1drop - down menu options for physicians charting syncope presentations . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . cranial ct scanning need not be routinely performed unless guided by specific findings in the history or physical examination . consider older age , structural heart disease , or a history of coronary artery disease as risk factors for adverse outcome . admit patients with syncope and evidence of heart failure or structural heart disease . admit patients with older age and associated comorbidities , abnormal ecg , hct < 30 ( if obtained ) , history or presence of heart failure , coronary artery disease , or structural heart diseaseekg risk stratificationreason head ct orderedreason admission consideredno acute / concerning changessyncope led to head traumaolder age with comorbiditiessigns of ischemiasuspected seizureanemia with hct < 30 , if obtainedsigns of dysrhythmia ( brugada syndrome , wpw)focal neurologic deficitabnormal ekgconduction abnormalities ( lbbb , prolonged intervals)incomplete resolution of altered mssigns of heart failure / cad / cardiomyopathyrequested by other providerfamily history of sudden deathother concerning history or physical findings a screen capture of the cdss module as it appeared in the syncope hpi template drop - down menu options for physicians charting syncope presentations . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . cranial ct scanning need not be routinely performed unless guided by specific findings in the history or physical examination . consider older age , structural heart disease , or a history of coronary artery disease as risk factors for adverse outcome . admit patients with syncope and evidence of heart failure or structural heart disease . admit patients with older age and associated comorbidities , abnormal ecg , hct < 30 ( if obtained ) , history or presence of heart failure , coronary artery disease , or structural heart disease the items included in the module prompted physicians to risk - stratify adult syncope patients based on ekg findings , and to explain the rationale for head ct and admission . each item included a drop - down list based on recommendations and phrasings from the acep clinical policy , whose guidelines state that head cts should not be ordered in patients presenting with syncope unless suggested by specific findings in the patient s history or physical , and that hospital admission be reserved for specific high - risk patients . coinciding with the appearance in the electronic chart , the module s presence as a decision support tool was announced via e - mail by a non - investigator to the em residents and faculty . the announcement only brought to the physicians attention that the tool had become available and did not include details of this study . there was no additional marketing strategy as the objective of the study was to assess the impact of the cdss alone . a retrospective chart review was employed to quantify baseline rates for ordering head ct and admission of adult patients with syncope presenting to the ed . patients aged 18 years or older were identified by searching the electronic archive of all ed visits by patients in the periods 6 months pre - intervention and 20 weeks post - intervention , using a search of final diagnoses that included the word our edis stores final diagnoses in text format . two abstractors ( nc and ng , who were not blinded to the study purpose ) then de - identified these records and exported them to excel ( microsoft , redmond , wa ) for analysis . patient disposition ( discharge from ed or admission to any inpatient service ) was tabulated . additionally , patient records were cross - matched against a radiology requisition record to determine which syncope patients had a head ct ordered and performed during their ed course . using sas ( version 9.2 : sas institute inc . , cary , nc ) , descriptive statistics were calculated ( mean and standard deviation for continuous variables and proportions with the corresponding two - sided 95% confidence interval for categorical variables ) . comparability of the pre - intervention group and the post - intervention group was analyzed using the two - sample t - test for age and the chi - square test for categorical variables , such as gender , admission rate , and head ct scan rate . following the initial analysis , subset analyses for admission rate and head ct imaging rate were performed : ( 1 ) comparing the behavior of seven physicians who cared for ten or more patients in both the pre- or post - intervention groups and ( 2 ) comparing groups completing and bypassing the cdss in the post - intervention group using the two - tail z - test . in the patient population studied in the san francisco syncope rule derivation , application of the rule might have reduced admissions by 10% . due to this finding , a 10% absolute reduction in admissions and head ct scans was used for the sample size justification . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention admission rate of 0.68 and a post - intervention rate of 0.58 when the sample size is 177 . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention head ct scan rate of 0.40 and a post - intervention rate of 0.30 when the sample size is 182 . as age was found to have a possible confounding effect on the outcome of ordering a head ct and admission in the univariate screen , it was included in a multivariable analysis . this study was designed as a pre - post intervention design with a prospective cohort and retrospective controls . we conducted a medical chart review with 6-month retrospective baseline analysis and prospective data collection following implementation of a cdss based on the 2007 acep clinical policy on syncope into the edis . the mount sinai school of medicine institutional review board reviewed and approved this study . the mount sinai medical center ( new york , ny ) is an 1,171-bed tertiary care academic medical center located in manhattan , bordering the upper east side and east harlem . the ed adopted a comprehensive edis in 2004 ( picis , ed pulsecheck , wakefield , ma , formerly ibex ) that provides triage , patient tracking , physician and nurse documentation , retrieval of charts from prior ed encounters and inpatient data , computerized provider order entry , results review , discharge instructions and prescription writing . attendings and/or residents could complete charts and final diagnosis on every ed patient , including a history of present illness ( hpi ) and final emergency department diagnosis ( chosen from a drop - down list of international classification of diseases , 9th revision approved diagnoses or free - text entries ) . the intervention assessed behavior of physicians caring for consecutive syncope patients over 18 years of age in the emergency department . the medical records of 410 patients were reviewed prior to implementation , and 301 records were reviewed after implementation . physicians included 34 attending physicians board - certified or board - eligible in emergency medicine , with additional information - gathering and decision - making provided by residents primarily in emergency medicine ( em ) , as well as occasional rotators from the departments of internal medicine , psychiatry , and obstetrics and gynecology . a three - item module based on the 2007 acep clinical policy on syncope was added to the syncope hpi template , for completion by attending or mid - level providers ( see fig . 1 , table 1 ) . each item served a dual role in : ( 1 ) reminding physicians of the strength of the policy s recommendations and ( 2 ) prompting physicians to document their clinical decision - making . 1a screen capture of the cdss module as it appeared in the syncope hpi templatetable 1drop - down menu options for physicians charting syncope presentations . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . cranial ct scanning need not be routinely performed unless guided by specific findings in the history or physical examination . consider older age , structural heart disease , or a history of coronary artery disease as risk factors for adverse outcome . admit patients with syncope and evidence of heart failure or structural heart disease . admit patients with older age and associated comorbidities , abnormal ecg , hct < 30 ( if obtained ) , history or presence of heart failure , coronary artery disease , or structural heart diseaseekg risk stratificationreason head ct orderedreason admission consideredno acute / concerning changessyncope led to head traumaolder age with comorbiditiessigns of ischemiasuspected seizureanemia with hct < 30 , if obtainedsigns of dysrhythmia ( brugada syndrome , wpw)focal neurologic deficitabnormal ekgconduction abnormalities ( lbbb , prolonged intervals)incomplete resolution of altered mssigns of heart failure / cad / cardiomyopathyrequested by other providerfamily history of sudden deathother concerning history or physical findings a screen capture of the cdss module as it appeared in the syncope hpi template drop - down menu options for physicians charting syncope presentations . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . cranial ct scanning need not be routinely performed unless guided by specific findings in the history or physical examination . consider older age , structural heart disease , or a history of coronary artery disease as risk factors for adverse outcome . admit patients with syncope and evidence of heart failure or structural heart disease . admit patients with older age and associated comorbidities , abnormal ecg , hct < 30 ( if obtained ) , history or presence of heart failure , coronary artery disease , or structural heart disease the items included in the module prompted physicians to risk - stratify adult syncope patients based on ekg findings , and to explain the rationale for head ct and admission . each item included a drop - down list based on recommendations and phrasings from the acep clinical policy , whose guidelines state that head cts should not be ordered in patients presenting with syncope unless suggested by specific findings in the patient s history or physical , and that hospital admission be reserved for specific high - risk patients . coinciding with the appearance in the electronic chart , the module s presence as a decision support tool was announced via e - mail by a non - investigator to the em residents and faculty . the announcement only brought to the physicians attention that the tool had become available and did not include details of this study . there was no additional marketing strategy as the objective of the study was to assess the impact of the cdss alone . a retrospective chart review was employed to quantify baseline rates for ordering head ct and admission of adult patients with syncope presenting to the ed . patients aged 18 years or older were identified by searching the electronic archive of all ed visits by patients in the periods 6 months pre - intervention and 20 weeks post - intervention , using a search of final diagnoses that included the word syncope or our edis stores final diagnoses in text format . two abstractors ( nc and ng , who were not blinded to the study purpose ) then de - identified these records and exported them to excel ( microsoft , redmond , wa ) for analysis . patient disposition ( discharge from ed or admission to any inpatient service ) was tabulated . additionally , patient records were cross - matched against a radiology requisition record to determine which syncope patients had a head ct ordered and performed during their ed course . using sas ( version 9.2 : sas institute inc . , cary , nc ) , descriptive statistics were calculated ( mean and standard deviation for continuous variables and proportions with the corresponding two - sided 95% confidence interval for categorical variables ) . comparability of the pre - intervention group and the post - intervention group was analyzed using the two - sample t - test for age and the chi - square test for categorical variables , such as gender , admission rate , and head ct scan rate . following the initial analysis , subset analyses for admission rate and head ct imaging rate were performed : ( 1 ) comparing the behavior of seven physicians who cared for ten or more patients in both the pre- or post - intervention groups and ( 2 ) comparing groups completing and bypassing the cdss in the post - intervention group using the two - tail z - test . in the patient population studied in the san francisco syncope rule derivation , application of the rule might have reduced admissions by 10% . due to this finding , a 10% absolute reduction in admissions and head ct scans was used for the sample size justification . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention admission rate of 0.68 and a post - intervention rate of 0.58 when the sample size is 177 . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention head ct scan rate of 0.40 and a post - intervention rate of 0.30 when the sample size is 182 . as age was found to have a possible confounding effect on the outcome of ordering a head ct and admission in the univariate screen , it was included in a multivariable analysis . in the 6-month pre - implementation period between 1 june and 30 november 2007 , a total of 410 patients aged 18 years or older presented to the mount sinai ed with a final diagnosis of syncope ( see table 2 ) . two hundred forty - four of these 410 patients were female ( 59.5% ) , and the average age was 64.9 21.3 years . table 2patient demographics , pre- and post - cdss interventionpre - interventionpost - interventionp - valuen410301sex , % female59.561.70.562average age , years64.9 21.361.9 22.50.067 patient demographics , pre- and post - cdss intervention the decision support module was added to the mount sinai emergency department information system ( edis ) on 4 february 2008 , and between that date and 22 june 2008 , a total of 301 patients aged 18 years or older presented to the mount sinai ed with a final diagnosis of syncope ( see table 2 ) . one hundred eighty - five of these 301 patients were female ( 61% ) , and the average age was 61.9 22.5 years . there was no significant difference in gender or age between the pre- and post - intervention groups . there was no significant interaction between intervention and age for admission rate ( p < 0.258 ) or for head ct rate ( p < 0.420 ) . in the pre - intervention cohort , 68.1% of patients were admitted [ 95% ci : ( 63.3 to 72.5 ) ] ( see table 3 and fig . 2 ) . the post - intervention admission rate was 60.5% [ 95% ci : ( 54.7 to 66.0 ) ] . there was a significant difference in admission rate pre- and post - intervention ( p = 0.036 ) . the pre - intervention rate of obtaining a head ct was 39.8% [ 95% ci : ( 35.0 to 44.7 ) ] compared to 43.2% [ 95% ci : ( 37.5 to 49.0 ) ] post - intervention ( see table 3 and fig . 2 ) . there was no significant difference in the head ct scan rate pre- and post - intervention ( p = 0.358 ) . table 3percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periodspre - interventionpost - interventionp - valuen410301% admitted ( 95% ci)68.1 ( 63.3 , 72.5)60.5 ( 54.7 , 66.0)0.036% with ct head ( 95% ci)39.8 ( 35.0 , 44.7)43.2 ( 37.5 , 49.0)0.358fig . 2percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods there were seven physicians who saw ten or more patients during the pre- and post - intervention . a subset analysis for the admission rate and head ct scan rate was performed on these seven physicians . there was a slight significant difference in the admission rate pre- and post - intervention for the subset of seven physicians who saw ten or more patients pre- and post - intervention ( 74.3% vs. 63.9% , respectively , p = 0.0495 ; see table 4 and fig . 3 ) . there was no significant difference in the head ct scan rate pre- and post - intervention for the subset of seven physicians who saw ten or more patients pre- and post - intervention ( 42.9% vs. 45.4% , respectively , p = 0.660 ) . table 4subset analysis of percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - interventionpre - interventionpost - interventionp - valuen175130% admitted ( 95% ci)74.3 ( 67.1 , 80.6)63.9 ( 55.0 , 72.1)0.0495% with ct head ( 95% ci)42.9 ( 35.4 , 50.5)45.4 ( 36.6 , 54.3)0.660fig . 3subset analysis : percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - intervention subset analysis of percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - intervention subset analysis : percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - intervention in the post - cdss intervention group comparing admission and ct head rates when the cdss was completed versus when it was not completed , subset analysis revealed an admission rate of 51.7% when the cdss was completed compared to 64.0% when it was not ( z - value 1.96 , statistically significant to 95.0% confidence level ) and a ct head rate of 43.7% when the cdss was completed compared to 43.0% when it was not ( z - value 0.03 , not statistically significant ; see table 5 ) . in the post - cdss intervention group comparing admission and ct head rates when the cdss was visible versus when it was not visible , subset analysis revealed an admission rate of 54.0% when the cdss was visible compared to 73.3% when it was not ( z - value 3.06 , statistically significant to 99.8% confidence level ) and a ct head rate of 38.5% when the cdss was visible compared to 52.5% when it was not ( z - value 2.19 , statistically significant to 97.2% confidence level ; see table 6 ) . table 5subset analysis in the post - cdss intervention group comparing admission and ct head rates when the cdss was completed versus when it was not completed . a two - tailed z - value is provided along with the confidence level ( cl% ) at which the two rates are deemed to be statistically significanttotalcdss completedcdss not completedz - value ( cl%)n30187214no . admitted ( % ) 182 ( 60.5 5.5)45 ( 51.7 10.6)137 ( 64.0 6.4)1.96 ( 95.0% ) # cts done ( % ) 130 ( 43.2 5.6)38 ( 43.7 10.5)92 ( 43.0 6.6)0.03 ( 2.4%)table 6subset analysis in the post - cdss intervention group comparing admission and ct head rates when the cdss was visible versus when it was not visible . a two - tailed z - value is provided along with the confidence level ( cl% ) at which the two rates are deemed to be statistically significanttotalcdss visiblecdss not visiblez - value ( cl%)n301200101no . admitted ( % ) 182 ( 60.5 5.5)108 ( 54.0 6.9)74 ( 73.3 8.7)3.06 ( 99.8%)no . cts done ( % ) 130 ( 43.2 5.6)77 ( 38.5 6.8)53 ( 52.5 9.8)2.19 ( 97.2% ) subset analysis in the post - cdss intervention group comparing admission and ct head rates when the cdss was completed versus when it was not completed . a two - tailed z - value is provided along with the confidence level ( cl% ) at which the two rates are deemed to be statistically significant subset analysis in the post - cdss intervention group comparing admission and ct head rates when the cdss was visible versus when it was not visible . a two - tailed z - value is provided along with the confidence level ( cl% ) at which the two rates are deemed to be statistically significant this study assessed the impact on physician management of syncope patients after implementation of a cdss based upon the 2007 acep clinical policy on syncope into an edis . the admission rate for syncope patients was significantly lower in the post - intervention period compared to the pre - intervention period . the head ct imaging rate for syncope patients was not significantly different during the pre- and post - intervention periods . since there was no significant interaction between intervention and age for admission rate ( p < 0.258 ) or for head ct rate ( p < 0.420 ) , we can therefore conclude that age in conjunction with pre-/post - intervention does not affect the admission rate or ordering head ct scan rate in our cohort . subset analysis of physicians seeing more than ten patients in both the pre- and post - intervention periods showed similar changes and did not suggest cluster - associated phenomenon . observed changes in admission rates in adult syncope patients may be indicative of improved awareness , adoption , and adherence to acep practice guidelines . subset analysis of physician behavior when the cdss was completed versus not completed and visible versus not visible revealed significant differences in admission and ct head rates when the cdss was visible and significant differences in admission when the cdss was completed . these findings suggest that although this intervention was a passive one , the cdss s presence may have had significant effect on physician practice behavior . an alternative conclusion might be that physicians who are likely to ignore cdss may also be less likely to adhere to evidence - based clinical practice guidelines . it is well - established in the literature that even when physicians are aware of evidence , they may not adhere to it [ 3 , 12 ] . found that increased knowledge of the acep clinical policy on hypertension following distribution of the guidelines did not translate into changes in physician practice . identified knowledge , attitudes , and behavior as barriers to physician adherence to clinical practice guidelines . kirkpatrick s hierarchy of levels of evaluation proposes that complexity of behavioral change increases as evaluation of intervention ascends the hierarchy . as evaluation ascends from ( 1 ) reactions to ( 2 ) learning to ( 3 ) behavior to ( 4 ) results , the impact of the intervention strengthens from ( 1 ) learner satisfaction to ( 2 ) knowledge to ( 3 ) transfer of learning to the workplace to ( 4 ) impact on society , respectively . since acquired knowledge of acep clinical policy on hypertension did not translate into changes in physician practice in lehrmann s trial , we approached the problem of adopting evidence - based guidelines in clinical practice at the next level in the hierarchy of interventions , namely , transfer of learning to the workplace via evaluation of behavior . while developing the cdss , we focused on following the provisions for improved clinical practice outlined by kawamoto et al . namely , the cdss was included in the clinician workflow in our computer - based edis . we also developed a cdss that used recommendations rather than assessments . instead of explicitly assessing for compliance to acep clinical policy recommendations , we sought a measurable change in physician behavior . although our population demographics were similar to previously studied populations , this admission rate is considerably higher than previously studied populations [ 10 , 20 , 22 ] . we chose head ct imaging as our other outcome because in the absence of focal neurologic findings , head ct imaging is of low yield in determining the etiology of syncope [ 2325 ] . we suspected that clinicians were ordering more cranial imaging in syncope patients than necessary . to our knowledge this is the first study to demonstrate a significant change in physician behavior after implementation of a cdss based upon acep clinical policy . while the body of medical research and literature grows rapidly , practice guidelines provide a means to educate , summarize , and distill evidence - based medicine to the practicing physician . however , implementation and utilization of the acep clinical policies has historically been a challenge . given the increased adoption of robust ediss , these results will encourage further experimentation and implementation of cdsss based upon evidence - based clinical practice guidelines into ediss . the next step for research and development of such cdsss could include : ( 1 ) assessment of our cdss closer to the point of decision - making or in a different practice environment , ( 2 ) integration of other acep clinical policies into similar cdsss in an effort to create an edis with comprehensive decision support , or ( 3 ) focus on more complex outcome measures such as compliance with guidelines or patient outcome . first , instead of explicitly assessing for compliance to acep clinical policy recommendations , we assessed for a measurable change in physician behavior . consequently , admissions or head cts could have increased in one type of patient and decreased in another , leaving the global rate unchanged . second , the edis in our institution did not have the capability to incorporate decision support at the precise time and location of decision - making in physician work flow . specifically , our edis provides decision support in the documentation template for history of present illness ( hpi ) . therefore , the cdss was more passive than active , meaning that cdss use did not actually result in the action of ordering a head ct or admitting a patient . although our chart extraction selected patients with a diagnosis of or including the term syncope , the physician documenting on such a patient had the option to choose the patient s hpi template independently of the patient s diagnosis . furthermore , as is common in unpredictable environments like the ed , hpi documentation may occur before or after the decision to obtain imaging or admit the patient . since this study was completed at a teaching hospital , the physician documenting the hpi was not always the physician deciding the patient s diagnosis , need for imaging , or admission . next , to prevent delaying or disrupting physician work flow , we decided the hpi documentation could not be made to require use of the cdss , so the cdss could be bypassed without reading or completing it . in order to minimize interruption of work flow , we abbreviated the language of the recommendations from the acep clinical policy on syncope to fit within the format of the drop - down menus seen in fig . 1 . we assume that the practice guideline was not originally authored with the intention of implementation in an abbreviated form . thus , it is possible that rewording the recommendations creates a simplified or modified recommendation for clinical decision - making . in a separate performance improvement project , however , we had markedly improved documentation of aspirin and beta - blocker use in chest pain patients using a cdss with a single reminder phrase just above the enter button of a given hpi template . we found the structure and placement of the chest pain cdss provided a quick and accessible yet not prohibitive reminder . it is important to note that the 2007 version of acep clinical policy on syncope was revised to include new recommendations using the findings from the derivation and validation of the san francisco syncope rule . two studies have had problems validating the san francisco syncope rule [ 26 , 27 ] . a final limitation of this study was that the cdss was trialed at the practice site of the cdss s creators . . found that studies in which authors also created the cdss reported better performance compared with those in which the trialists were independent of the cdss development process . bias by having a third party announce the implementation of the cdss without mention of the fact that we would be gathering data on physician behavior associated with the cdss . additionally , within our department there was no specific notification or implementation of new acep clinical policies . first , instead of explicitly assessing for compliance to acep clinical policy recommendations , we assessed for a measurable change in physician behavior . consequently , admissions or head cts could have increased in one type of patient and decreased in another , leaving the global rate unchanged . second , the edis in our institution did not have the capability to incorporate decision support at the precise time and location of decision - making in physician work flow . specifically , our edis provides decision support in the documentation template for history of present illness ( hpi ) . therefore , the cdss was more passive than active , meaning that cdss use did not actually result in the action of ordering a head ct or admitting a patient . although our chart extraction selected patients with a diagnosis of or including the term syncope , the physician documenting on such a patient had the option to choose the patient s hpi template independently of the patient s diagnosis . furthermore , as is common in unpredictable environments like the ed , hpi documentation may occur before or after the decision to obtain imaging or admit the patient . since this study was completed at a teaching hospital , the physician documenting the hpi was not always the physician deciding the patient s diagnosis , need for imaging , or admission . next , to prevent delaying or disrupting physician work flow , we decided the hpi documentation could not be made to require use of the cdss , so the cdss could be bypassed without reading or completing it . in order to minimize interruption of work flow , we abbreviated the language of the recommendations from the acep clinical policy on syncope to fit within the format of the drop - down menus seen in fig . 1 . we assume that the practice guideline was not originally authored with the intention of implementation in an abbreviated form . thus , it is possible that rewording the recommendations creates a simplified or modified recommendation for clinical decision - making . in a separate performance improvement project , however , we had markedly improved documentation of aspirin and beta - blocker use in chest pain patients using a cdss with a single reminder phrase just above the enter button of a given hpi template . we found the structure and placement of the chest pain cdss provided a quick and accessible yet not prohibitive reminder . it is important to note that the 2007 version of acep clinical policy on syncope was revised to include new recommendations using the findings from the derivation and validation of the san francisco syncope rule . two studies have had problems validating the san francisco syncope rule [ 26 , 27 ] . a final limitation of this study was that the cdss was trialed at the practice site of the cdss s creators . . found that studies in which authors also created the cdss reported better performance compared with those in which the trialists were independent of the cdss development process . bias by having a third party announce the implementation of the cdss without mention of the fact that we would be gathering data on physician behavior associated with the cdss . additionally , within our department there was no specific notification or implementation of new acep clinical policies . in conclusion , in our urban academic emergency department the introduction of an evidence - based cdss based upon acep clinical policy recommendations on syncope correlated with a change in physician practice behavior in terms of admission but not for head ct imaging . when the cdss was visible but not used , it had significant effect on both admission and head ct imaging rates . this change suggests emergency medicine clinical practice guideline recommendations can be incorporated into the physician workflow of an edis to enhance the quality of practice . a more active cdss implemented at the point of medical decision - making and whose use resulted in physician order entry might have a greater impact on behavior .

aimsto influence physician practice behavior after implementation of a computerized clinical decision support system ( cdss ) based upon the recommendations from the 2007 acep clinical policy on syncope.methodsthis was a pre - post intervention with a prospective cohort and retrospective controls . we conducted a medical chart review of consecutive adult patients with syncope . a computerized cdss prompting physicians to explain their decision - making regarding imaging and admission in syncope patients based upon acep clinical policy recommendations was embedded into the emergency department information system ( edis ) . the medical records of 410 consecutive adult patients presenting with syncope were reviewed prior to implementation , and 301 records were reviewed after implementation . primary outcomes were physician practice behavior demonstrated by admission rate and rate of head computed tomography ( ct ) imaging before and after implementation.resultsthere was a significant difference in admission rate pre- and post - intervention ( 68.1% vs. 60.5% respectively , p = 0.036 ) . there was no significant difference in the head ct imaging rate pre- and post - intervention ( 39.8% vs. 43.2% , p = 0.358 ) . there were seven physicians who saw ten or more patients during the pre- and post - intervention . subset analysis of these seven physicians practice behavior revealed a slight significant difference in the admission rate pre- and post - intervention ( 74.3% vs. 63.9% , p = 0.0495 ) and no significant difference in the head ct scan rate pre- and post - intervention ( 42.9% vs. 45.4% , p = 0.660).conclusionsthe introduction of an evidence - based cdss based upon acep clinical policy recommendations on syncope correlated with a change in physician practice behavior in an urban academic emergency department . this change suggests emergency medicine clinical practice guideline recommendations can be incorporated into the physician workflow of an edis to enhance the quality of practice .

Introduction Methods Study design Study setting Study population Study protocol Measurements Data analysis Results Discussion Limitations Conclusions

in spite of the benefits of the acep clinical policies , implementation of these clinical practice guidelines into physician practice continues to be a challenge . found that knowledge of the acep clinical policy on hypertension did not translate into changes in physician practice . clinical decision support systems ( cdss ) are systems designed to aid directly in clinical decision - making , in which characteristics of individual patients are used to generate patient - specific assessments or recommendations that are then presented to clinicians for consideration . found that clinical practice was improved with provision of cdsss : ( 1 ) as part of clinician workflow , ( 2 ) with recommendations rather than assessments , ( 3 ) at the time and location of decision - making , and ( 4 ) if computer - based . realizing the potential efficacy of cdsss as emergency department documentation increasingly becomes computerized , napoli and jagoda and this study aimed to improve knowledge translation from evidence - based emergency medicine practice guidelines by creating a cdss for implementation of the recommendations from an acep clinical policy in an urban academic emergency department . we specifically chose the 2007 acep clinical policy on syncope because it included newly published recommendations on a frequently encountered diagnosis . we sought to identify the presence of a change in physician ordering of cranial imaging and admission practices due to implementation of a hpi - based cdss in patients with a final diagnosis of syncope . the 2007 acep clinical policy on syncope provides new recommendations on decision - making regarding need for head computed tomography ( ct ) imaging and hospital admission in adult patients presenting to the emergency department with syncope . we hypothesized that incorporating these recommendations into a computerized cdss embedded in the physician workflow of an emergency department information system ( edis ) would influence physician behavior . a change in behavior would suggest that this point - of - care decision support tool helps improve practitioner awareness , adoption , and adherence to acep practice guidelines and bridge the gap from evidence to practice . this study was designed as a pre - post intervention design with a prospective cohort and retrospective controls . we conducted a medical chart review with 6-month retrospective baseline analysis and prospective data collection following implementation of a cdss based on the 2007 acep clinical policy on syncope into the edis . the intervention assessed behavior of physicians caring for consecutive syncope patients over 18 years of age in the emergency department . the medical records of 410 patients were reviewed prior to implementation , and 301 records were reviewed after implementation . physicians included 34 attending physicians board - certified or board - eligible in emergency medicine , with additional information - gathering and decision - making provided by residents primarily in emergency medicine ( em ) , as well as occasional rotators from the departments of internal medicine , psychiatry , and obstetrics and gynecology . a three - item module based on the 2007 acep clinical policy on syncope was added to the syncope hpi template , for completion by attending or mid - level providers ( see fig . each item served a dual role in : ( 1 ) reminding physicians of the strength of the policy s recommendations and ( 2 ) prompting physicians to document their clinical decision - making . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . admit patients with older age and associated comorbidities , abnormal ecg , hct < 30 ( if obtained ) , history or presence of heart failure , coronary artery disease , or structural heart disease the items included in the module prompted physicians to risk - stratify adult syncope patients based on ekg findings , and to explain the rationale for head ct and admission . each item included a drop - down list based on recommendations and phrasings from the acep clinical policy , whose guidelines state that head cts should not be ordered in patients presenting with syncope unless suggested by specific findings in the patient s history or physical , and that hospital admission be reserved for specific high - risk patients . a retrospective chart review was employed to quantify baseline rates for ordering head ct and admission of adult patients with syncope presenting to the ed . patients aged 18 years or older were identified by searching the electronic archive of all ed visits by patients in the periods 6 months pre - intervention and 20 weeks post - intervention , using a search of final diagnoses that included the word our edis stores final diagnoses in text format . comparability of the pre - intervention group and the post - intervention group was analyzed using the two - sample t - test for age and the chi - square test for categorical variables , such as gender , admission rate , and head ct scan rate . following the initial analysis , subset analyses for admission rate and head ct imaging rate were performed : ( 1 ) comparing the behavior of seven physicians who cared for ten or more patients in both the pre- or post - intervention groups and ( 2 ) comparing groups completing and bypassing the cdss in the post - intervention group using the two - tail z - test . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention admission rate of 0.68 and a post - intervention rate of 0.58 when the sample size is 177 . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention head ct scan rate of 0.40 and a post - intervention rate of 0.30 when the sample size is 182 . as age was found to have a possible confounding effect on the outcome of ordering a head ct and admission in the univariate screen , it was included in a multivariable analysis . this study was designed as a pre - post intervention design with a prospective cohort and retrospective controls . we conducted a medical chart review with 6-month retrospective baseline analysis and prospective data collection following implementation of a cdss based on the 2007 acep clinical policy on syncope into the edis . the intervention assessed behavior of physicians caring for consecutive syncope patients over 18 years of age in the emergency department . the medical records of 410 patients were reviewed prior to implementation , and 301 records were reviewed after implementation . physicians included 34 attending physicians board - certified or board - eligible in emergency medicine , with additional information - gathering and decision - making provided by residents primarily in emergency medicine ( em ) , as well as occasional rotators from the departments of internal medicine , psychiatry , and obstetrics and gynecology . a three - item module based on the 2007 acep clinical policy on syncope was added to the syncope hpi template , for completion by attending or mid - level providers ( see fig . each item served a dual role in : ( 1 ) reminding physicians of the strength of the policy s recommendations and ( 2 ) prompting physicians to document their clinical decision - making . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . menu options are adapted from the following recommendations in the 2007 acep clinical policy of syncope . admit patients with older age and associated comorbidities , abnormal ecg , hct < 30 ( if obtained ) , history or presence of heart failure , coronary artery disease , or structural heart disease the items included in the module prompted physicians to risk - stratify adult syncope patients based on ekg findings , and to explain the rationale for head ct and admission . each item included a drop - down list based on recommendations and phrasings from the acep clinical policy , whose guidelines state that head cts should not be ordered in patients presenting with syncope unless suggested by specific findings in the patient s history or physical , and that hospital admission be reserved for specific high - risk patients . a retrospective chart review was employed to quantify baseline rates for ordering head ct and admission of adult patients with syncope presenting to the ed . patients aged 18 years or older were identified by searching the electronic archive of all ed visits by patients in the periods 6 months pre - intervention and 20 weeks post - intervention , using a search of final diagnoses that included the word syncope or our edis stores final diagnoses in text format . comparability of the pre - intervention group and the post - intervention group was analyzed using the two - sample t - test for age and the chi - square test for categorical variables , such as gender , admission rate , and head ct scan rate . following the initial analysis , subset analyses for admission rate and head ct imaging rate were performed : ( 1 ) comparing the behavior of seven physicians who cared for ten or more patients in both the pre- or post - intervention groups and ( 2 ) comparing groups completing and bypassing the cdss in the post - intervention group using the two - tail z - test . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention admission rate of 0.68 and a post - intervention rate of 0.58 when the sample size is 177 . a one group test with a 0.05 two - sided significance level will have 80% power to detect a difference between a pre - intervention head ct scan rate of 0.40 and a post - intervention rate of 0.30 when the sample size is 182 . in the 6-month pre - implementation period between 1 june and 30 november 2007 , a total of 410 patients aged 18 years or older presented to the mount sinai ed with a final diagnosis of syncope ( see table 2 ) . table 2patient demographics , pre- and post - cdss interventionpre - interventionpost - interventionp - valuen410301sex , % female59.561.70.562average age , years64.9 21.361.9 22.50.067 patient demographics , pre- and post - cdss intervention the decision support module was added to the mount sinai emergency department information system ( edis ) on 4 february 2008 , and between that date and 22 june 2008 , a total of 301 patients aged 18 years or older presented to the mount sinai ed with a final diagnosis of syncope ( see table 2 ) . there was no significant difference in gender or age between the pre- and post - intervention groups . there was no significant interaction between intervention and age for admission rate ( p < 0.258 ) or for head ct rate ( p < 0.420 ) . in the pre - intervention cohort , 68.1% of patients were admitted [ 95% ci : ( 63.3 to 72.5 ) ] ( see table 3 and fig . the post - intervention admission rate was 60.5% [ 95% ci : ( 54.7 to 66.0 ) ] . there was a significant difference in admission rate pre- and post - intervention ( p = 0.036 ) . the pre - intervention rate of obtaining a head ct was 39.8% [ 95% ci : ( 35.0 to 44.7 ) ] compared to 43.2% [ 95% ci : ( 37.5 to 49.0 ) ] post - intervention ( see table 3 and fig . there was no significant difference in the head ct scan rate pre- and post - intervention ( p = 0.358 ) . table 3percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periodspre - interventionpost - interventionp - valuen410301% admitted ( 95% ci)68.1 ( 63.3 , 72.5)60.5 ( 54.7 , 66.0)0.036% with ct head ( 95% ci)39.8 ( 35.0 , 44.7)43.2 ( 37.5 , 49.0)0.358fig . 2percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods there were seven physicians who saw ten or more patients during the pre- and post - intervention . a subset analysis for the admission rate and head ct scan rate was performed on these seven physicians . there was a slight significant difference in the admission rate pre- and post - intervention for the subset of seven physicians who saw ten or more patients pre- and post - intervention ( 74.3% vs. 63.9% , respectively , p = 0.0495 ; see table 4 and fig . there was no significant difference in the head ct scan rate pre- and post - intervention for the subset of seven physicians who saw ten or more patients pre- and post - intervention ( 42.9% vs. 45.4% , respectively , p = 0.660 ) . table 4subset analysis of percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - interventionpre - interventionpost - interventionp - valuen175130% admitted ( 95% ci)74.3 ( 67.1 , 80.6)63.9 ( 55.0 , 72.1)0.0495% with ct head ( 95% ci)42.9 ( 35.4 , 50.5)45.4 ( 36.6 , 54.3)0.660fig . 3subset analysis : percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - intervention subset analysis of percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - intervention subset analysis : percentage of syncope patients admitted and receiving head ct in the pre- and post - cdss intervention periods among seven physicians who saw ten or more patients during the pre- and post - intervention in the post - cdss intervention group comparing admission and ct head rates when the cdss was completed versus when it was not completed , subset analysis revealed an admission rate of 51.7% when the cdss was completed compared to 64.0% when it was not ( z - value 1.96 , statistically significant to 95.0% confidence level ) and a ct head rate of 43.7% when the cdss was completed compared to 43.0% when it was not ( z - value 0.03 , not statistically significant ; see table 5 ) . in the post - cdss intervention group comparing admission and ct head rates when the cdss was visible versus when it was not visible , subset analysis revealed an admission rate of 54.0% when the cdss was visible compared to 73.3% when it was not ( z - value 3.06 , statistically significant to 99.8% confidence level ) and a ct head rate of 38.5% when the cdss was visible compared to 52.5% when it was not ( z - value 2.19 , statistically significant to 97.2% confidence level ; see table 6 ) . a two - tailed z - value is provided along with the confidence level ( cl% ) at which the two rates are deemed to be statistically significant this study assessed the impact on physician management of syncope patients after implementation of a cdss based upon the 2007 acep clinical policy on syncope into an edis . the admission rate for syncope patients was significantly lower in the post - intervention period compared to the pre - intervention period . the head ct imaging rate for syncope patients was not significantly different during the pre- and post - intervention periods . since there was no significant interaction between intervention and age for admission rate ( p < 0.258 ) or for head ct rate ( p < 0.420 ) , we can therefore conclude that age in conjunction with pre-/post - intervention does not affect the admission rate or ordering head ct scan rate in our cohort . subset analysis of physicians seeing more than ten patients in both the pre- and post - intervention periods showed similar changes and did not suggest cluster - associated phenomenon . found that increased knowledge of the acep clinical policy on hypertension following distribution of the guidelines did not translate into changes in physician practice . since acquired knowledge of acep clinical policy on hypertension did not translate into changes in physician practice in lehrmann s trial , we approached the problem of adopting evidence - based guidelines in clinical practice at the next level in the hierarchy of interventions , namely , transfer of learning to the workplace via evaluation of behavior . instead of explicitly assessing for compliance to acep clinical policy recommendations , we sought a measurable change in physician behavior . to our knowledge this is the first study to demonstrate a significant change in physician behavior after implementation of a cdss based upon acep clinical policy . given the increased adoption of robust ediss , these results will encourage further experimentation and implementation of cdsss based upon evidence - based clinical practice guidelines into ediss . the next step for research and development of such cdsss could include : ( 1 ) assessment of our cdss closer to the point of decision - making or in a different practice environment , ( 2 ) integration of other acep clinical policies into similar cdsss in an effort to create an edis with comprehensive decision support , or ( 3 ) focus on more complex outcome measures such as compliance with guidelines or patient outcome . first , instead of explicitly assessing for compliance to acep clinical policy recommendations , we assessed for a measurable change in physician behavior . second , the edis in our institution did not have the capability to incorporate decision support at the precise time and location of decision - making in physician work flow . in order to minimize interruption of work flow , we abbreviated the language of the recommendations from the acep clinical policy on syncope to fit within the format of the drop - down menus seen in fig . thus , it is possible that rewording the recommendations creates a simplified or modified recommendation for clinical decision - making . it is important to note that the 2007 version of acep clinical policy on syncope was revised to include new recommendations using the findings from the derivation and validation of the san francisco syncope rule . additionally , within our department there was no specific notification or implementation of new acep clinical policies . first , instead of explicitly assessing for compliance to acep clinical policy recommendations , we assessed for a measurable change in physician behavior . second , the edis in our institution did not have the capability to incorporate decision support at the precise time and location of decision - making in physician work flow . in order to minimize interruption of work flow , we abbreviated the language of the recommendations from the acep clinical policy on syncope to fit within the format of the drop - down menus seen in fig . thus , it is possible that rewording the recommendations creates a simplified or modified recommendation for clinical decision - making . it is important to note that the 2007 version of acep clinical policy on syncope was revised to include new recommendations using the findings from the derivation and validation of the san francisco syncope rule . additionally , within our department there was no specific notification or implementation of new acep clinical policies . in conclusion , in our urban academic emergency department the introduction of an evidence - based cdss based upon acep clinical policy recommendations on syncope correlated with a change in physician practice behavior in terms of admission but not for head ct imaging . this change suggests emergency medicine clinical practice guideline recommendations can be incorporated into the physician workflow of an edis to enhance the quality of practice .

it is the second most common cause of morbidity and mortality globally , especially in children aged 3 years , and mortality is most frequent in low - income regions . rehydration is a key intervention for acute diarrhea and should be introduced as soon as possible ; a reduced osmolality oral rehydration solution ( ors ) should be started promptly ( i.e. , within 34 hours of symptom onset ) and used frequently . drug treatment is usually not required , although some probiotics may decrease symptom duration and severity . antibacterial therapy is usually not needed , as it can lead to carrier status for salmonella spp . however , some drugs used in combination with ors may effectively reduce symptom duration and severity , although further investigations are warranted . these agents form a protective biofilm in the intestinal mucosa and enhance mucosal resistance to pathological insults . indeed , pathogenic microorganisms adhere to the intestinal mucosa , weaken cellular tight - junction function , and reduce transepithelial electrical resistance ( teer ) . they can then penetrate the mucosa , and become internalized and proliferate within intestinal cells . conversely , mucoprotective agents such as gelatin tannate and xyloglucan help to re - establish normal intestinal function . additional randomized studies are now needed to clearly define the clinical profiles of these mucoprotectors in patients with acute diarrhea . besides its mucoprotective activity , additional evidence suggests that gelatin tannate reduces inflammation , prevents growth of some bacterial species , and preserves the intestinal mucous layer [ 6,1013 ] . thus , in line with the need for additional assessment of mucoprotectors , the present single - center , randomized , controlled , double - blind trial was designed to assess the efficacy and safety of gelatin tannate in pediatric patients with acute diarrhea . this randomized , controlled , double - blind , parallel - group , single - center clinical trial was conducted to determine the efficacy and safety of gelatin tannate plus ors compared with ors plus placebo in pediatric patients ( aged 3 months to 12 years ) with infectious or noninfectious acute diarrhea . the study protocol was approved by the local ethics committee and the turkish medicines and medical devices agency ( online follow - up number 1997575 ) , and procedures were carried out in accordance with ethical standards of the declaration of helsinki ( revised 2000 ) . written informed consent was obtained from each child s parents or legal guardian . potential study participants were excluded for the following reasons : chronic or toxic diarrhea ; celiac disease ; diarrhea due to milk or protein intolerance ; immune disorders ; infantile colic ; other gastrointestinal disorders ( e.g. , crohn s disease ) ; or the use of oral antidiarrheal or other treatments during the study period . patients who could not be followed - up for at least 48 hours in hospital were also excluded . patients were randomly assigned to receive gelatin tannate plus ors , or ors plus placebo at a ratio of 1 : 1 . gelatin tannate was administered in the form of 250 mg oral sachets ( tasectan ; onko & kosel lalari , istanbul , turkey ) at a dosage of 1 sachet every 6 hours . the sachet contents could be mixed with a milk feed , water , fruit juice , or yoghurt . ors ( ge - oral ; kansuk laboratuari , istanbul , turkey ) 50 ml / kg ad libitum was administered as a powder for oral solution , containing sodium chloride , trisodium citrate , potassium chloride , and glucose . patients were divided into 4 age groups : 3<12 months ; 1<3 years ; 3<7 years ; and 7 years and older . regardless of the clinical service , newly hospitalized patients were randomized to placebo or gelatin tannate . every 23 weeks , patient numbers were calculated and recruitment adjustments made , if necessary , to try to equalize group numbers . as such , the study was conducted on a triple - blind basis : physicians and nurses did not know whether the study drug administered was placebo or gelatin tannate ; patients were not informed about whether they were being given placebo or gelatin tannate ; and physicians and nurses did not know from which clinical service each patient had been referred . during the first study visit , patients were randomized into 2 groups ( gelatin tannate plus ors , or ors plus placebo ) to receive at least 48-hour treatment in hospital ; some patients continued treatment after discharge from hospital . during treatment , all patients were reviewed every 24 hours in hospital , or by telephone or call back to the hospital after hospital discharge . at the time of hospitalization , baseline demographic ( bodyweight ) and clinical characteristics ( comorbidities , symptoms of acute diarrhea during the previous 3 days , and vital signs ) were recorded . symptoms of acute diarrhea were evaluated during patient interview and included : abdominal pain ; anorexia ; dehydration ( abnormal skin turgor , and bodyweight reduction ) ; fever ; flatulence ; nausea ; signs of peritonitis and/or sepsis ; and stools ( duration of diarrhea , presence of blood / mucus / pus in feces , stool frequency , and stool type ) . investigators recorded details about stool consistency and total time to resolution of diarrhea ( primary study endpoint ) . stool production ( number in the previous 24 hours ) , with mucus and/or blood , was recorded , as was stool consistency , according to the bristol stool scale ( bss ) : e.g. , type 6 refers to diarrhea with soft stools ; type 7 refers to watery stools , no solid pieces , and entirely liquid . at study evaluation was subsequently made of the stool decrease index ( sdi ) : the proportion of patients whose stool consistency improved from diarrhea ( bss 6 or 7 ) to non - diarrhea ( bss 5 ) . secondary study endpoints comprised symptoms of acute diarrhea at 12 , 24 , 36 , 48 , and 72 hours after the first dose of study medication , and adverse events throughout the trial . in addition , the following biochemical tests could be performed ( at study start ) if required according to patient condition : blood culture ( e.g. , for patients with fever for > 2 days ) ; complete blood count ; serum c - reactive protein ; and serum electrolyte measurement ( calcium , chlorine , potassium , and sodium ) . based on findings of a previous trial , it was determined that a total of 240 children ( approximately 120 per group ) would be sufficient to identify clinically significant differences between the 2 treatment arms , and a period of 6 months would be sufficient to enroll this number of patients . data were classified as numerical or categorical , and were checked by the shapiro - wilk test to ascertain whether they conformed to a normal distribution ( numerical data ) . an independent - sample t test was used for data conforming to a normal distribution , whereas the mann - whitney u test was used for data not conforming to a normal distribution ( independent numerical data ) . a dependent - sample t test was used for data conforming to a normal distribution , whereas the wilcoxon test was used for data not conforming to a normal distribution ( dependent numerical data ) . fisher s chi - squared test was used for comparison of categorical data in independent groups , whereas the mcnemar test was used for comparison of categorical data in dependent groups . for all results , this randomized , controlled , double - blind , parallel - group , single - center clinical trial was conducted to determine the efficacy and safety of gelatin tannate plus ors compared with ors plus placebo in pediatric patients ( aged 3 months to 12 years ) with infectious or noninfectious acute diarrhea . the study protocol was approved by the local ethics committee and the turkish medicines and medical devices agency ( online follow - up number 1997575 ) , and procedures were carried out in accordance with ethical standards of the declaration of helsinki ( revised 2000 ) . written informed consent was obtained from each child s parents or legal guardian . potential study participants were excluded for the following reasons : chronic or toxic diarrhea ; celiac disease ; diarrhea due to milk or protein intolerance ; immune disorders ; infantile colic ; other gastrointestinal disorders ( e.g. , crohn s disease ) ; or the use of oral antidiarrheal or other treatments during the study period . patients who could not be followed - up for at least 48 hours in hospital were also excluded . patients were randomly assigned to receive gelatin tannate plus ors , or ors plus placebo at a ratio of 1 : 1 . gelatin tannate was administered in the form of 250 mg oral sachets ( tasectan ; onko & kosel lalari , istanbul , turkey ) at a dosage of 1 sachet every 6 hours . the sachet contents could be mixed with a milk feed , water , fruit juice , or yoghurt . ors ( ge - oral ; kansuk laboratuari , istanbul , turkey ) 50 ml / kg ad libitum was administered as a powder for oral solution , containing sodium chloride , trisodium citrate , potassium chloride , and glucose . patients were divided into 4 age groups : 3<12 months ; 1<3 years ; 3<7 years ; and 7 years and older . regardless of the clinical service , newly hospitalized patients were randomized to placebo or gelatin tannate . every 23 weeks , patient numbers were calculated and recruitment adjustments made , if necessary , to try to equalize group numbers . triple - blind basis : physicians and nurses did not know whether the study drug administered was placebo or gelatin tannate ; patients were not informed about whether they were being given placebo or gelatin tannate ; and physicians and nurses did not know from which clinical service each patient had been referred . during the first study visit , patients were randomized into 2 groups ( gelatin tannate plus ors , or ors plus placebo ) to receive at least 48-hour treatment in hospital ; some patients continued treatment after discharge from hospital . during treatment , all patients were reviewed every 24 hours in hospital , or by telephone or call back to the hospital after hospital discharge . at the time of hospitalization , baseline demographic ( bodyweight ) and clinical characteristics ( comorbidities , symptoms of acute diarrhea during the previous 3 days , and vital signs ) were recorded . symptoms of acute diarrhea were evaluated during patient interview and included : abdominal pain ; anorexia ; dehydration ( abnormal skin turgor , and bodyweight reduction ) ; fever ; flatulence ; nausea ; signs of peritonitis and/or sepsis ; and stools ( duration of diarrhea , presence of blood / mucus / pus in feces , stool frequency , and stool type ) . investigators recorded details about stool consistency and total time to resolution of diarrhea ( primary study endpoint ) . stool production ( number in the previous 24 hours ) , with mucus and/or blood , was recorded , as was stool consistency , according to the bristol stool scale ( bss ) : e.g. , type 6 refers to diarrhea with soft stools ; type 7 refers to watery stools , no solid pieces , and entirely liquid . at study evaluation was subsequently made of the stool decrease index ( sdi ) : the proportion of patients whose stool consistency improved from diarrhea ( bss 6 or 7 ) to non - diarrhea ( bss 5 ) . secondary study endpoints comprised symptoms of acute diarrhea at 12 , 24 , 36 , 48 , and 72 hours after the first dose of study medication , and adverse events throughout the trial . in addition , the following biochemical tests could be performed ( at study start ) if required according to patient condition : blood culture ( e.g. , for patients with fever for > 2 days ) ; complete blood count ; serum c - reactive protein ; and serum electrolyte measurement ( calcium , chlorine , potassium , and sodium ) . based on findings of a previous trial , it was determined that a total of 240 children ( approximately 120 per group ) would be sufficient to identify clinically significant differences between the 2 treatment arms , and a period of 6 months would be sufficient to enroll this number of patients . data were classified as numerical or categorical , and were checked by the shapiro - wilk test to ascertain whether they conformed to a normal distribution ( numerical data ) . an independent - sample t test was used for data conforming to a normal distribution , whereas the mann - whitney u test was used for data not conforming to a normal distribution ( independent numerical data ) . a dependent - sample t test was used for data conforming to a normal distribution , whereas the wilcoxon test was used for data not conforming to a normal distribution ( dependent numerical data ) . fisher s chi - squared test was used for comparison of categorical data in independent groups , whereas the mcnemar test was used for comparison of categorical data in dependent groups . for all results , children were aged between 3 months and 12 years , and had acute diarrhea of infectious origin ( bacterial , parasitic , or viral ) or noninfectious origin ( antibiotic - related ) for 72 hours . the diagnosis of acute diarrhea was made based on investigators judgement of the clinical picture of objective ( e.g. , stools , vomiting , or fever ) and subjective e.g. , ( abdominal pain , or nausea ) symptoms . however , 48 patients were excluded for the following reasons : early discharge from hospital ( n=6 ) ; loss of contact with patient after discharge from hospital ( 7 ) ; duty physician not familiar with the study ( 20 ) ; patients received additional antidiarrheal medication ( 8) ; patients refused to take oral medication ( 4 ) ; and increased severity of vomiting or diarrhea ( 3 ) . thus , 203 patients completed the study ( gelatin tannate plus ors , n=103 ; ors alone , n=100 ) and received at least 1 dose of study medication ( per - protocol population ) . results were recorded and data analyzed for the per - protocol population . table 1 shows demographic and clinical characteristics for these patients . mean patient age was 40 months , 117 patients were male ( 57.6% ) , and mean ( standard deviation ) stool frequency in the previous 24 hours was 7.75.0 . more than one - third of patients ( 36.2% ) received antibacterial therapy during the trial . at all study assessment timepoints from 24 hours onwards , the incidence of nausea was significantly lower in the gelatin tannate plus ors group than in the ors group alone ( at 24 hours : 11.7% vs. 26.0% of patients ; p=0.01 ; figure 1a ) . the same was true for abdominal pain ( at 24 hours : 10.7% vs. 24.0% of patients ; p=0.02 ; figure 1b ) . from 12 hours onwards , the incidence of watery stools was significantly lower in the gelatin tannate plus ors than ors - alone group ( at 12 hours : 59.2% vs. 77.0% ; p=0.01 ; figure 1c ) . significantly more patients in the combination than ors - alone group had dehydration at baseline ( 35.0% vs. 16.0% ; p<0.01 ) . subsequently , no significant difference in the occurrence of dehydration was noted between the 2 groups , since all patients in both groups were treated with ors . nonetheless , from 36 hours onwards , a nonsignificant trend ( p=0.05 ) was evident towards a lower incidence of dehydration in the combination group than in the ors - alone group ( figure 1d ) . after 36- and 72-hours hospitalization , fever was recorded in significantly fewer patients treated with gelatin tannate plus ors rather than in the ors - alone group ( at 36 hours : 13.6% vs. 65.0% ; p<0.01 ; figure 1e ) . as shown in figure 2 , from 12 hours onwards , stool frequency was significantly lower in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : mean 2 vs. 3 stool productions in the previous 12 hours ; p<0.01 ) . at all timepoints during the study , the proportion of patients with sdi improvement , indicating resolution of diarrhea , was significantly greater ( p<0.01 ) in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : 66.6% vs. 33.3% ; p<0.01 ; children were aged between 3 months and 12 years , and had acute diarrhea of infectious origin ( bacterial , parasitic , or viral ) or noninfectious origin ( antibiotic - related ) for 72 hours . the diagnosis of acute diarrhea was made based on investigators judgement of the clinical picture of objective ( e.g. , stools , vomiting , or fever ) and subjective e.g. , ( abdominal pain , or nausea ) symptoms . however , 48 patients were excluded for the following reasons : early discharge from hospital ( n=6 ) ; loss of contact with patient after discharge from hospital ( 7 ) ; duty physician not familiar with the study ( 20 ) ; patients received additional antidiarrheal medication ( 8) ; patients refused to take oral medication ( 4 ) ; and increased severity of vomiting or diarrhea ( 3 ) . thus , 203 patients completed the study ( gelatin tannate plus ors , n=103 ; ors alone , n=100 ) and received at least 1 dose of study medication ( per - protocol population ) . results were recorded and data analyzed for the per - protocol population . table 1 shows demographic and clinical characteristics for these patients . mean patient age was 40 months , 117 patients were male ( 57.6% ) , and mean ( standard deviation ) stool frequency in the previous 24 hours was 7.75.0 . more than one - third of patients ( 36.2% ) received antibacterial therapy during the trial . at all study assessment timepoints from 24 hours onwards , the incidence of nausea was significantly lower in the gelatin tannate plus ors group than in the ors group alone ( at 24 hours : 11.7% vs. 26.0% of patients ; p=0.01 ; figure 1a ) . the same was true for abdominal pain ( at 24 hours : 10.7% vs. 24.0% of patients ; p=0.02 ; figure 1b ) . from 12 hours onwards , the incidence of watery stools was significantly lower in the gelatin tannate plus ors than ors - alone group ( at 12 hours : 59.2% vs. 77.0% ; p=0.01 ; figure 1c ) . significantly more patients in the combination than ors - alone group had dehydration at baseline ( 35.0% vs. 16.0% ; p<0.01 ) . subsequently , no significant difference in the occurrence of dehydration was noted between the 2 groups , since all patients in both groups were treated with ors . nonetheless , from 36 hours onwards , a nonsignificant trend ( p=0.05 ) was evident towards a lower incidence of dehydration in the combination group than in the ors - alone group ( figure 1d ) . after 36- and 72-hours hospitalization , fever was recorded in significantly fewer patients treated with gelatin tannate plus ors rather than in the ors - alone group ( at 36 hours : 13.6% vs. 65.0% ; p<0.01 ; figure 1e ) . as shown in figure 2 , from 12 hours onwards , stool frequency was significantly lower in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : mean 2 vs. 3 stool productions in the previous 12 hours ; p<0.01 ) . at all timepoints during the study , the proportion of patients with sdi improvement , indicating resolution of diarrhea , was significantly greater ( p<0.01 ) in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : 66.6% vs. 33.3% ; p<0.01 ; figure 3 ) . the principal intervention for pediatric patients with acute diarrhea is rehydration , which should be used as soon as possible after symptoms occur . in this way , complications and risks can be avoided , such as serious dehydration , electrolyte disturbances , and altered nutrient absorption and digestion with worsening nutritional status . such complications can lead to increased requirements for enteral or parenteral rehydration and nutrition , and hospitalization . thus , treatments such as gelatin tannate , with the potential to enhance efficacy of oral rehydration therapy and obviate the need for enteral or parenteral intervention , clearly warrant detailed investigation . in the present study , gelatin tannate plus ors was significantly more effective than ors alone in reducing symptoms ( e.g. , nausea , abdominal pain , fever , and watery stools ) and stool frequency in children hospitalized with acute diarrhea . the symptom reduction was particularly evident for nausea and abdominal pain at 2472 hours of hospitalization , and for watery stools after 1272 hours of treatment . another interesting aspect of the present trial is that average direct costs per patient ( e.g. , total costs of drugs , diagnostic tests , and consultations ) were approximately 40% lower in the gelatin tannate plus ors group than in the ors - placebo group ( 229.26 vs. 386.56 turkish lira ) . this creates significant scope for future , more - detailed cost - utility analyses of gelatin tannate in children with acute diarrhea . a potential limitation of the trial is that the exact proportions of patients who completed the 72-hour study period in hospital or at home were unclear . clearly , patients in the hospital environment are more likely to have adhered to the gelatin tannate plus ors schedule than patients in the home setting . in addition , we did not manage to include the anticipated number of children during the study period . however , this was not materially important since the numbers were sufficient to demonstrate both clinically and statistically significant differences between the 2 treatments . altogether , our results are consistent with findings from other clinical trials of mucoprotective agents . for example , in an observational study involving 239 children ( aged 3 months to 12 years ) with acute diarrhea , gelatin tannate plus ors versus ors alone significantly reduced stool frequency at 12 hours post - treatment . in a randomized study in adults with acute diarrhea , another mucoprotective agent , xyloglucan , was shown to significantly reduce symptoms of diarrhea and to have faster onset of action than diosmectite or saccharomyces boulardii . other well - controlled studies of gelatin tannate are ongoing : for example , this mucoprotective agent is currently being compared with placebo in up to 158 children aged < 5 years with acute gastroenteritis ; the primary study endpoint is duration of diarrhea , and study results are soon expected to be released ( study number nct02280759 ) . in vitro findings also endorse the mucoprotective activity of gelatin tannate , which is considered to adhere to apical epithelial cells in the intestinal mucosa and interact favorably with tight junctions , strongly increasing teer , and thereby maintaining intestinal wall integrity . overall , it appears that sufficient data now exist to clearly endorse the use of film - forming , mucoprotective agents , such as gelatin tannate or xyloglucan , in combination with ors to stop diarrhea , especially in the pediatric population with acute diarrhea . this is particularly pertinent given the significantly favorable action of gelatin tannate on symptoms other than diarrhea ( e.g. , abdominal pain , fever , and nausea ) in the current trial . potentially , amelioration of these additional symptoms was also due to the beneficial effect of gelatin tannate on the intestinal mucosa . no adverse events were reported during the current trial , thereby further corroborating the positive safety profiles of mucoprotective agents . administration of gelatin tannate in combination with ors is an effective and safe option for the treatment of acute diarrhea in children . clearly , results from this trial validate the use of gelatin tannate as an addition to ors in children with acute diarrhea .

backgroundacute diarrhea is the second most common cause of morbidity and mortality worldwide , especially in children aged 3 years . some drugs ( e.g. , the mucoprotector gelatin tannate ) plus a reduced osmolality oral rehydration solution ( ors ) may effectively reduce symptom duration and severity . the current trial was therefore designed to assess the efficacy and safety of gelatin tannate in pediatric patients with acute diarrhea.material/methodsthis was a randomized , controlled , double - blind , parallel - group , single - center study comparing gelatin tannate plus ors ( 103 patients ) with ors plus placebo ( 100 patients ) in children aged 3 months to 12 years with infectious or noninfectious acute diarrhea . details about stool consistency and total time to resolution of diarrhea comprised the primary study endpoints . secondary study endpoints included symptoms of diarrhea at 12 , 24 , 36 , 48 , and 72 hours after the first dose of study medication.resultsfrom 12 hours onwards , the incidence of watery stools was significantly lower in the gelatin tannate group than in the ors group ( at 12 hours : 59.2% vs. 77.0% ; p=0.01 ) . the same was true for stool frequency ( at 12 hours : mean 2 vs. 3 stool productions in the previous 12 hours ; p<0.01 ) . at all timepoints during the study , the proportion of patients with stool decrease index improvement was significantly greater ( p<0.01 ) in the gelatin tannate group than in the placebo group ( at 12 hours : 66.6% vs. 33.3% ; p<0.01).conclusionsgelatin tannate plus ors is an effective and safe option for the treatment of acute diarrhea in children . significant symptom relief is evident 12 hours after starting treatment .

Background Material and Methods Study design Exclusion criteria Treatment and randomization Study procedure Statistical analyses Results Patient characteristics Effects on symptoms of acute diarrhea Effects on stool frequency Effects on secondary study endpoints Discussion Conclusions

it is the second most common cause of morbidity and mortality globally , especially in children aged 3 years , and mortality is most frequent in low - income regions . rehydration is a key intervention for acute diarrhea and should be introduced as soon as possible ; a reduced osmolality oral rehydration solution ( ors ) should be started promptly ( i.e. drug treatment is usually not required , although some probiotics may decrease symptom duration and severity . however , some drugs used in combination with ors may effectively reduce symptom duration and severity , although further investigations are warranted . these agents form a protective biofilm in the intestinal mucosa and enhance mucosal resistance to pathological insults . additional randomized studies are now needed to clearly define the clinical profiles of these mucoprotectors in patients with acute diarrhea . besides its mucoprotective activity , additional evidence suggests that gelatin tannate reduces inflammation , prevents growth of some bacterial species , and preserves the intestinal mucous layer [ 6,1013 ] . thus , in line with the need for additional assessment of mucoprotectors , the present single - center , randomized , controlled , double - blind trial was designed to assess the efficacy and safety of gelatin tannate in pediatric patients with acute diarrhea . this randomized , controlled , double - blind , parallel - group , single - center clinical trial was conducted to determine the efficacy and safety of gelatin tannate plus ors compared with ors plus placebo in pediatric patients ( aged 3 months to 12 years ) with infectious or noninfectious acute diarrhea . the study protocol was approved by the local ethics committee and the turkish medicines and medical devices agency ( online follow - up number 1997575 ) , and procedures were carried out in accordance with ethical standards of the declaration of helsinki ( revised 2000 ) . potential study participants were excluded for the following reasons : chronic or toxic diarrhea ; celiac disease ; diarrhea due to milk or protein intolerance ; immune disorders ; infantile colic ; other gastrointestinal disorders ( e.g. , crohn s disease ) ; or the use of oral antidiarrheal or other treatments during the study period . patients were randomly assigned to receive gelatin tannate plus ors , or ors plus placebo at a ratio of 1 : 1 . gelatin tannate was administered in the form of 250 mg oral sachets ( tasectan ; onko & kosel lalari , istanbul , turkey ) at a dosage of 1 sachet every 6 hours . ors ( ge - oral ; kansuk laboratuari , istanbul , turkey ) 50 ml / kg ad libitum was administered as a powder for oral solution , containing sodium chloride , trisodium citrate , potassium chloride , and glucose . as such , the study was conducted on a triple - blind basis : physicians and nurses did not know whether the study drug administered was placebo or gelatin tannate ; patients were not informed about whether they were being given placebo or gelatin tannate ; and physicians and nurses did not know from which clinical service each patient had been referred . during the first study visit , patients were randomized into 2 groups ( gelatin tannate plus ors , or ors plus placebo ) to receive at least 48-hour treatment in hospital ; some patients continued treatment after discharge from hospital . at the time of hospitalization , baseline demographic ( bodyweight ) and clinical characteristics ( comorbidities , symptoms of acute diarrhea during the previous 3 days , and vital signs ) were recorded . symptoms of acute diarrhea were evaluated during patient interview and included : abdominal pain ; anorexia ; dehydration ( abnormal skin turgor , and bodyweight reduction ) ; fever ; flatulence ; nausea ; signs of peritonitis and/or sepsis ; and stools ( duration of diarrhea , presence of blood / mucus / pus in feces , stool frequency , and stool type ) . investigators recorded details about stool consistency and total time to resolution of diarrhea ( primary study endpoint ) . stool production ( number in the previous 24 hours ) , with mucus and/or blood , was recorded , as was stool consistency , according to the bristol stool scale ( bss ) : e.g. , type 6 refers to diarrhea with soft stools ; type 7 refers to watery stools , no solid pieces , and entirely liquid . at study evaluation was subsequently made of the stool decrease index ( sdi ) : the proportion of patients whose stool consistency improved from diarrhea ( bss 6 or 7 ) to non - diarrhea ( bss 5 ) . secondary study endpoints comprised symptoms of acute diarrhea at 12 , 24 , 36 , 48 , and 72 hours after the first dose of study medication , and adverse events throughout the trial . in addition , the following biochemical tests could be performed ( at study start ) if required according to patient condition : blood culture ( e.g. , for patients with fever for > 2 days ) ; complete blood count ; serum c - reactive protein ; and serum electrolyte measurement ( calcium , chlorine , potassium , and sodium ) . based on findings of a previous trial , it was determined that a total of 240 children ( approximately 120 per group ) would be sufficient to identify clinically significant differences between the 2 treatment arms , and a period of 6 months would be sufficient to enroll this number of patients . for all results , this randomized , controlled , double - blind , parallel - group , single - center clinical trial was conducted to determine the efficacy and safety of gelatin tannate plus ors compared with ors plus placebo in pediatric patients ( aged 3 months to 12 years ) with infectious or noninfectious acute diarrhea . the study protocol was approved by the local ethics committee and the turkish medicines and medical devices agency ( online follow - up number 1997575 ) , and procedures were carried out in accordance with ethical standards of the declaration of helsinki ( revised 2000 ) . potential study participants were excluded for the following reasons : chronic or toxic diarrhea ; celiac disease ; diarrhea due to milk or protein intolerance ; immune disorders ; infantile colic ; other gastrointestinal disorders ( e.g. , crohn s disease ) ; or the use of oral antidiarrheal or other treatments during the study period . patients were randomly assigned to receive gelatin tannate plus ors , or ors plus placebo at a ratio of 1 : 1 . gelatin tannate was administered in the form of 250 mg oral sachets ( tasectan ; onko & kosel lalari , istanbul , turkey ) at a dosage of 1 sachet every 6 hours . ors ( ge - oral ; kansuk laboratuari , istanbul , turkey ) 50 ml / kg ad libitum was administered as a powder for oral solution , containing sodium chloride , trisodium citrate , potassium chloride , and glucose . triple - blind basis : physicians and nurses did not know whether the study drug administered was placebo or gelatin tannate ; patients were not informed about whether they were being given placebo or gelatin tannate ; and physicians and nurses did not know from which clinical service each patient had been referred . during the first study visit , patients were randomized into 2 groups ( gelatin tannate plus ors , or ors plus placebo ) to receive at least 48-hour treatment in hospital ; some patients continued treatment after discharge from hospital . at the time of hospitalization , baseline demographic ( bodyweight ) and clinical characteristics ( comorbidities , symptoms of acute diarrhea during the previous 3 days , and vital signs ) were recorded . symptoms of acute diarrhea were evaluated during patient interview and included : abdominal pain ; anorexia ; dehydration ( abnormal skin turgor , and bodyweight reduction ) ; fever ; flatulence ; nausea ; signs of peritonitis and/or sepsis ; and stools ( duration of diarrhea , presence of blood / mucus / pus in feces , stool frequency , and stool type ) . investigators recorded details about stool consistency and total time to resolution of diarrhea ( primary study endpoint ) . stool production ( number in the previous 24 hours ) , with mucus and/or blood , was recorded , as was stool consistency , according to the bristol stool scale ( bss ) : e.g. , type 6 refers to diarrhea with soft stools ; type 7 refers to watery stools , no solid pieces , and entirely liquid . at study evaluation was subsequently made of the stool decrease index ( sdi ) : the proportion of patients whose stool consistency improved from diarrhea ( bss 6 or 7 ) to non - diarrhea ( bss 5 ) . secondary study endpoints comprised symptoms of acute diarrhea at 12 , 24 , 36 , 48 , and 72 hours after the first dose of study medication , and adverse events throughout the trial . in addition , the following biochemical tests could be performed ( at study start ) if required according to patient condition : blood culture ( e.g. , for patients with fever for > 2 days ) ; complete blood count ; serum c - reactive protein ; and serum electrolyte measurement ( calcium , chlorine , potassium , and sodium ) . based on findings of a previous trial , it was determined that a total of 240 children ( approximately 120 per group ) would be sufficient to identify clinically significant differences between the 2 treatment arms , and a period of 6 months would be sufficient to enroll this number of patients . for all results , children were aged between 3 months and 12 years , and had acute diarrhea of infectious origin ( bacterial , parasitic , or viral ) or noninfectious origin ( antibiotic - related ) for 72 hours . the diagnosis of acute diarrhea was made based on investigators judgement of the clinical picture of objective ( e.g. however , 48 patients were excluded for the following reasons : early discharge from hospital ( n=6 ) ; loss of contact with patient after discharge from hospital ( 7 ) ; duty physician not familiar with the study ( 20 ) ; patients received additional antidiarrheal medication ( 8) ; patients refused to take oral medication ( 4 ) ; and increased severity of vomiting or diarrhea ( 3 ) . thus , 203 patients completed the study ( gelatin tannate plus ors , n=103 ; ors alone , n=100 ) and received at least 1 dose of study medication ( per - protocol population ) . mean patient age was 40 months , 117 patients were male ( 57.6% ) , and mean ( standard deviation ) stool frequency in the previous 24 hours was 7.75.0 . more than one - third of patients ( 36.2% ) received antibacterial therapy during the trial . at all study assessment timepoints from 24 hours onwards , the incidence of nausea was significantly lower in the gelatin tannate plus ors group than in the ors group alone ( at 24 hours : 11.7% vs. 26.0% of patients ; p=0.01 ; figure 1a ) . the same was true for abdominal pain ( at 24 hours : 10.7% vs. 24.0% of patients ; p=0.02 ; figure 1b ) . from 12 hours onwards , the incidence of watery stools was significantly lower in the gelatin tannate plus ors than ors - alone group ( at 12 hours : 59.2% vs. 77.0% ; p=0.01 ; figure 1c ) . significantly more patients in the combination than ors - alone group had dehydration at baseline ( 35.0% vs. 16.0% ; p<0.01 ) . subsequently , no significant difference in the occurrence of dehydration was noted between the 2 groups , since all patients in both groups were treated with ors . nonetheless , from 36 hours onwards , a nonsignificant trend ( p=0.05 ) was evident towards a lower incidence of dehydration in the combination group than in the ors - alone group ( figure 1d ) . after 36- and 72-hours hospitalization , fever was recorded in significantly fewer patients treated with gelatin tannate plus ors rather than in the ors - alone group ( at 36 hours : 13.6% vs. 65.0% ; p<0.01 ; figure 1e ) . as shown in figure 2 , from 12 hours onwards , stool frequency was significantly lower in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : mean 2 vs. 3 stool productions in the previous 12 hours ; p<0.01 ) . at all timepoints during the study , the proportion of patients with sdi improvement , indicating resolution of diarrhea , was significantly greater ( p<0.01 ) in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : 66.6% vs. 33.3% ; p<0.01 ; children were aged between 3 months and 12 years , and had acute diarrhea of infectious origin ( bacterial , parasitic , or viral ) or noninfectious origin ( antibiotic - related ) for 72 hours . the diagnosis of acute diarrhea was made based on investigators judgement of the clinical picture of objective ( e.g. however , 48 patients were excluded for the following reasons : early discharge from hospital ( n=6 ) ; loss of contact with patient after discharge from hospital ( 7 ) ; duty physician not familiar with the study ( 20 ) ; patients received additional antidiarrheal medication ( 8) ; patients refused to take oral medication ( 4 ) ; and increased severity of vomiting or diarrhea ( 3 ) . thus , 203 patients completed the study ( gelatin tannate plus ors , n=103 ; ors alone , n=100 ) and received at least 1 dose of study medication ( per - protocol population ) . results were recorded and data analyzed for the per - protocol population . mean patient age was 40 months , 117 patients were male ( 57.6% ) , and mean ( standard deviation ) stool frequency in the previous 24 hours was 7.75.0 . more than one - third of patients ( 36.2% ) received antibacterial therapy during the trial . at all study assessment timepoints from 24 hours onwards , the incidence of nausea was significantly lower in the gelatin tannate plus ors group than in the ors group alone ( at 24 hours : 11.7% vs. 26.0% of patients ; p=0.01 ; figure 1a ) . the same was true for abdominal pain ( at 24 hours : 10.7% vs. 24.0% of patients ; p=0.02 ; figure 1b ) . from 12 hours onwards , the incidence of watery stools was significantly lower in the gelatin tannate plus ors than ors - alone group ( at 12 hours : 59.2% vs. 77.0% ; p=0.01 ; figure 1c ) . significantly more patients in the combination than ors - alone group had dehydration at baseline ( 35.0% vs. 16.0% ; p<0.01 ) . subsequently , no significant difference in the occurrence of dehydration was noted between the 2 groups , since all patients in both groups were treated with ors . nonetheless , from 36 hours onwards , a nonsignificant trend ( p=0.05 ) was evident towards a lower incidence of dehydration in the combination group than in the ors - alone group ( figure 1d ) . after 36- and 72-hours hospitalization , fever was recorded in significantly fewer patients treated with gelatin tannate plus ors rather than in the ors - alone group ( at 36 hours : 13.6% vs. 65.0% ; p<0.01 ; figure 1e ) . as shown in figure 2 , from 12 hours onwards , stool frequency was significantly lower in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : mean 2 vs. 3 stool productions in the previous 12 hours ; p<0.01 ) . at all timepoints during the study , the proportion of patients with sdi improvement , indicating resolution of diarrhea , was significantly greater ( p<0.01 ) in the gelatin tannate plus ors group than in the ors - alone group ( at 12 hours : 66.6% vs. 33.3% ; p<0.01 ; figure 3 ) . the principal intervention for pediatric patients with acute diarrhea is rehydration , which should be used as soon as possible after symptoms occur . thus , treatments such as gelatin tannate , with the potential to enhance efficacy of oral rehydration therapy and obviate the need for enteral or parenteral intervention , clearly warrant detailed investigation . in the present study , gelatin tannate plus ors was significantly more effective than ors alone in reducing symptoms ( e.g. , nausea , abdominal pain , fever , and watery stools ) and stool frequency in children hospitalized with acute diarrhea . the symptom reduction was particularly evident for nausea and abdominal pain at 2472 hours of hospitalization , and for watery stools after 1272 hours of treatment . another interesting aspect of the present trial is that average direct costs per patient ( e.g. , total costs of drugs , diagnostic tests , and consultations ) were approximately 40% lower in the gelatin tannate plus ors group than in the ors - placebo group ( 229.26 vs. 386.56 turkish lira ) . this creates significant scope for future , more - detailed cost - utility analyses of gelatin tannate in children with acute diarrhea . clearly , patients in the hospital environment are more likely to have adhered to the gelatin tannate plus ors schedule than patients in the home setting . in addition , we did not manage to include the anticipated number of children during the study period . for example , in an observational study involving 239 children ( aged 3 months to 12 years ) with acute diarrhea , gelatin tannate plus ors versus ors alone significantly reduced stool frequency at 12 hours post - treatment . in a randomized study in adults with acute diarrhea , another mucoprotective agent , xyloglucan , was shown to significantly reduce symptoms of diarrhea and to have faster onset of action than diosmectite or saccharomyces boulardii . other well - controlled studies of gelatin tannate are ongoing : for example , this mucoprotective agent is currently being compared with placebo in up to 158 children aged < 5 years with acute gastroenteritis ; the primary study endpoint is duration of diarrhea , and study results are soon expected to be released ( study number nct02280759 ) . in vitro findings also endorse the mucoprotective activity of gelatin tannate , which is considered to adhere to apical epithelial cells in the intestinal mucosa and interact favorably with tight junctions , strongly increasing teer , and thereby maintaining intestinal wall integrity . overall , it appears that sufficient data now exist to clearly endorse the use of film - forming , mucoprotective agents , such as gelatin tannate or xyloglucan , in combination with ors to stop diarrhea , especially in the pediatric population with acute diarrhea . this is particularly pertinent given the significantly favorable action of gelatin tannate on symptoms other than diarrhea ( e.g. , abdominal pain , fever , and nausea ) in the current trial . potentially , amelioration of these additional symptoms was also due to the beneficial effect of gelatin tannate on the intestinal mucosa . no adverse events were reported during the current trial , thereby further corroborating the positive safety profiles of mucoprotective agents . administration of gelatin tannate in combination with ors is an effective and safe option for the treatment of acute diarrhea in children . clearly , results from this trial validate the use of gelatin tannate as an addition to ors in children with acute diarrhea .

a common complication in patients on long - term dialysis is secondary hyperparathyroidism ( shpt ) , which causes mineral bone disorder of chronic kidney disease ( ckd - mbd ) . active vitamin d analogues are widely used as standard medical therapy for shpt , but it can be difficult to maintain serum calcium and phosphate levels within the normal range , and response to medical therapy declines as shpt progresses . in the advanced stage , the parathyroid glands ( ptgs ) exhibit nodular hyperplasia , there is a reduced therapeutic effect on the secretion of parathyroid hormone ( pth ) and the patient is considered to be resistant to medical treatment . in such cases , surgical parathyroidectomy ( ptx ) is the conventional treatment choice and has been widely performed in japan . in the 1980s , percutaneous ethanol injection therapy ( peit ) under ultrasound guidance was developed in italy for ptg tumours and because the glands are physically destroyed , peit is also considered to be an effective treatment for resistant shpt . in 1994 , kitaoka et al . reported on the use of peit for shpt , and it has since been widely used in japan as a new intervention . standardized guidelines for indications , equipment and postoperative management were produced in 2003 , consolidating the place of peit as a tool for managing advanced shpt in japan . in the 1990s when peit was first introduced , some reports from europe indicated that pth levels were reduced by > 30% in only 4060% of cases [ 68 ] , whereas in japan the success rate was comparatively high . in kitaoka et al 's 1994 report , serum intact pth levels decreased from 727 pg / ml to < 200 pg / ml in seven of nine patients who underwent peit , and in the remaining two patients levels were maintained within the target range by postoperative medical therapy ( i.e. vitamin d analogues ) . those good results were obtained because not only the target gland but also secondary and tertiary glands in size were treated , and because of improvements in equipment , such as the addition of three small holes in the side of the needle to improve ethanol infiltration . furthermore , another paper from japan reported that the mean serum pth levels were reduced to 226 pg / ml after one session of peit targeting all glands > 5 mm in size on ultrasonography , and their ratio of pth reduction was equal to that of the previous report . the results of the two japanese reports indicate that if all hyperplastic glands are destroyed by peit , serum pth levels can be reduced to the target range . however , as the number of interventions increases , incidences of injection - related complications also increase . therefore , the japanese guidelines also recommend target glands of peit as > 0.5 cm or > 1 cm in size , but not all glands were detectable by ultrasonography . moreover , other studies state that the efficacy of peit is decreased or is nullified when the preoperative pth level is > 1000 pg / ml and there is more than one gland > 0.5 cm on ultrasonography . therefore , various factors , such as the stage of shpt and the number of enlarged ptg , influence the efficacy of peit . as the ptgs become > 0.5 cm , or 1 cm in diameter , they became resistant to medical therapy , so the presence of enlarged glands is a strong indicator of responsiveness to conventional treatment . furthermore , histological analysis of surgically removed glands has confirmed that > 90% of glands heavier than 0.5 g have nodular hyperplasia , so for peit to be effective all glands > 0.5 cm have to be destroyed . according to the japanese guidelines for peit of the ptg , glands with nodular hyperplasia are selectively destroyed and glands with diffuse hyperplasia are then managed by medical therapy . if there is only one enlarged gland , a single session of peit will usually be successful , but if there are more than two , it becomes difficult to destroy all target glands with certainty in one session of peit and the number of interventions increases . in a study comparing the number of enlarged glands in haemodialysis patients with intact pth levels < 300 pg / ml after peit , the efficacy was 66.7% with one gland , but only 7.7% if there were more than two . in another study , there was a negative correlation between the number of glands > 0.5 cm and the efficacy of peit , although there was no correlation between the total number of enlarged glands and the efficacy ( figure 1a , b ) . nakamura also reported that the efficacy of peit diminishes as the number of hyperplastic glands increases . these reports confirm that superior results will be obtained with peit when there is only one ptg that is > 0.5 cm . influence on the efficacy of percutaneous ethanol injection therapy ( peit ) of ( a ) the number of parathyroid glands and ( b ) the number of parathyroid glands with a volume 0.5 cm . a recent clinical study reported that the serum pth level was not reduced in the case of advanced shpt with preoperative intact pth levels > 1000 pg / ml , even though peit was performed as described in other reports . the authors considered that the reason for treatment failure was that the subject of their study had advanced shpt , in which most of the enlarged glands are resistant to medical therapy , so multiple injections over a prolonged period are required to achieve an effect . this is undesirable because of the increased incidence of the side effects of ethanol leakage , such as pain , haemorrhage and recurrent laryngeal nerve palsy . moreover , adhesion of surrounding tissue is exacerbated with repeated peit and thus the therapeutic efficacy diminishes . we have recently shown that the odds ratio for success versus failure by multivariate regression analysis is 0.29 for preoperative intact pth levels > 500 pg / ml compared with < 500 pg / ml . in europe and the united states , calcimimetics have shown superior efficacy as medical treatment for shpt resistant to conventional treatment , but as of the end of 2007 they are not available in japan , so interventions such as peit and ptx are the choices for shpt resistant to medical therapy . particularly , peit is desirable , and still effective , in the early phase when the incidence of ckd - mbd is thought to be low . in other words , peit is indicated at the stage when the patient is resistant to medical therapy , with only one enlarged ptg , and the indication for ptx is more advanced shpt , or more than two enlarged ptgs . at the time peit was first introduced , it was considered that subsequent treatment with active vitamin d analogues strongly influenced long - term post - precedural prognosis . when peit is successful , serum ca and p levels , as well as the pth level , decrease immediately after peit , enabling a strong inhibition of the pth level with medical therapy . however , if this becomes difficult after peit , then the remaining ptgs have advanced hyperplasia and , in such cases , serum ca and p levels increase , promoting a recurrence of shpt . one group has reported that it is possible to maintain a low pth level for 1 year after peit , if it is performed correctly , and furthermore , they also reported that pth was maintained at an adequate level for at least 3 years with a combination of after - treatment and additional peit . in both reports , the pth level was decreased to the target level by the first session of peit , which suggests that the long - term prognosis is influenced by the rate of pth decrease immediately after peit . for peit to be considered an effective therapy for shpt , serum pth and ca levels must be decreased to their target ranges by the first intervention , and then maintained in that range for a long period . however , the therapeutic effect of peit is greatly influenced by the skill of the operator , so a full assessment of outcomes is difficult in small - scale , single - centre studies , and instead requires large numbers of peit procedures performed in several centres under the same therapeutic guidelines to minimize the influence of variations in skill level between institutions . in addition , there were no standardized therapeutic guidelines for peit in the early 1990s , and therapeutic outcomes depend on correct indications , equipment , technique and postoperative management . furthermore , improvements in mineral metabolism after peit have not been fully investigated . in our study of a retrospective cohort of > 300 subjects , based on the japanese peit guidelines , patients were divided into an effective group that achieved the specified levels of serum corrected ca < 10.5 mg / dl and serum intact pth < 250 pg / ml , and an ineffective group that failed to achieve these target levels after one session of peit . active vitamin d analogues were used as after - treatment in all cases . in the effective group , the response period was defined as the period from the day on which target levels were reached until the day on which either target level was exceeded , and the remission period was defined as the period from the day on which target levels were reached until the day of repeat peit or ptx because of recurrent disease . peit was effective in 208 patients ( 66.2% ) , in whom serum intact pth levels decreased from 603 292 to 183 62 pg / ml ( ng / l ) , and serum corrected ca levels from 10.7 0.8 to 10.1 0.5 mg / dl . the risk of relapse was evaluated by multivariate analysis using a logistic regression model , with the event of exceeding target levels ( relapse ) as a dependent variable . the odds of relapse versus non - relapse throughout the follow - up period was 2.37 in the presence of more than two hyperplastic glands versus one or no glands larger > 0.5 cm . in addition , the event - free survival of the remission period was evaluated using kaplan meier survival analysis and the remission period for the group with only one hyperplastic gland showed a significant extension by log - rank analysis . nodular hyperplasia can be presented if ptgs are enlarged by between 0.25 g and 0.5 g . in the case of only one enlarged gland < 0.5 cm , this ptg may have nodular hyperplasia if it does not respond to medical therapy . in such a case , peit becomes an effective treatment if peit is possible technically . therefore , as shown in figure 2 , superior prognosis including high efficacy , low recurrence , and long - term remission period can be obtained with peit when there is only one enlarged gland > 0.5 cm or there is only one enlarged gland on which we can perform peit . preferable indication of peit achieving good prognosis including high efficacy , low recurrence and long - term remission period . in the 1990s when peit was first introduced , some reports from europe indicated that pth levels were reduced by > 30% in only 4060% of cases [ 68 ] , whereas in japan the success rate was comparatively high . in kitaoka et al 's 1994 report , serum intact pth levels decreased from 727 pg / ml to < 200 pg / ml in seven of nine patients who underwent peit , and in the remaining two patients levels were maintained within the target range by postoperative medical therapy ( i.e. vitamin d analogues ) . those good results were obtained because not only the target gland but also secondary and tertiary glands in size were treated , and because of improvements in equipment , such as the addition of three small holes in the side of the needle to improve ethanol infiltration . furthermore , another paper from japan reported that the mean serum pth levels were reduced to 226 pg / ml after one session of peit targeting all glands > 5 mm in size on ultrasonography , and their ratio of pth reduction was equal to that of the previous report . the results of the two japanese reports indicate that if all hyperplastic glands are destroyed by peit , serum pth levels can be reduced to the target range . however , as the number of interventions increases , incidences of injection - related complications also increase . therefore , the japanese guidelines also recommend target glands of peit as > 0.5 cm or > 1 cm in size , but not all glands were detectable by ultrasonography . moreover , other studies state that the efficacy of peit is decreased or is nullified when the preoperative pth level is > 1000 pg / ml and there is more than one gland > 0.5 cm on ultrasonography . therefore , various factors , such as the stage of shpt and the number of enlarged ptg , influence the efficacy of peit . as the ptgs become > 0.5 cm , or 1 cm in diameter , they became resistant to medical therapy , so the presence of enlarged glands is a strong indicator of responsiveness to conventional treatment . furthermore , histological analysis of surgically removed glands has confirmed that > 90% of glands heavier than 0.5 g have nodular hyperplasia , so for peit to be effective all glands > 0.5 cm have to be destroyed . according to the japanese guidelines for peit of the ptg , glands with nodular hyperplasia are selectively destroyed and glands with diffuse hyperplasia are then managed by medical therapy . if there is only one enlarged gland , a single session of peit will usually be successful , but if there are more than two , it becomes difficult to destroy all target glands with certainty in one session of peit and the number of interventions increases . in a study comparing the number of enlarged glands in haemodialysis patients with intact pth levels < 300 pg / ml after peit , the efficacy was 66.7% with one gland , but only 7.7% if there were more than two . in another study , there was a negative correlation between the number of glands > 0.5 cm and the efficacy of peit , although there was no correlation between the total number of enlarged glands and the efficacy ( figure 1a , b ) . nakamura also reported that the efficacy of peit diminishes as the number of hyperplastic glands increases . these reports confirm that superior results will be obtained with peit when there is only one ptg that is > 0.5 cm . influence on the efficacy of percutaneous ethanol injection therapy ( peit ) of ( a ) the number of parathyroid glands and ( b ) the number of parathyroid glands with a volume 0.5 cm . a recent clinical study reported that the serum pth level was not reduced in the case of advanced shpt with preoperative intact pth levels > 1000 pg / ml , even though peit was performed as described in other reports . the authors considered that the reason for treatment failure was that the subject of their study had advanced shpt , in which most of the enlarged glands are resistant to medical therapy , so multiple injections over a prolonged period are required to achieve an effect . this is undesirable because of the increased incidence of the side effects of ethanol leakage , such as pain , haemorrhage and recurrent laryngeal nerve palsy . moreover , adhesion of surrounding tissue is exacerbated with repeated peit and thus the therapeutic efficacy diminishes . we have recently shown that the odds ratio for success versus failure by multivariate regression analysis is 0.29 for preoperative intact pth levels > 500 pg / ml compared with < 500 pg / ml . in europe and the united states , calcimimetics have shown superior efficacy as medical treatment for shpt resistant to conventional treatment , but as of the end of 2007 they are not available in japan , so interventions such as peit and ptx are the choices for shpt resistant to medical therapy . particularly , peit is desirable , and still effective , in the early phase when the incidence of ckd - mbd is thought to be low . in other words , peit is indicated at the stage when the patient is resistant to medical therapy , with only one enlarged ptg , and the indication for ptx is more advanced shpt , or more than two enlarged ptgs . as the ptgs become > 0.5 cm , or 1 cm in diameter , they became resistant to medical therapy , so the presence of enlarged glands is a strong indicator of responsiveness to conventional treatment . furthermore , histological analysis of surgically removed glands has confirmed that > 90% of glands heavier than 0.5 g have nodular hyperplasia , so for peit to be effective all glands > 0.5 cm have to be destroyed . according to the japanese guidelines for peit of the ptg , glands with nodular hyperplasia are selectively destroyed and glands with diffuse hyperplasia are then managed by medical therapy . if there is only one enlarged gland , a single session of peit will usually be successful , but if there are more than two , it becomes difficult to destroy all target glands with certainty in one session of peit and the number of interventions increases . in a study comparing the number of enlarged glands in haemodialysis patients with intact pth levels < 300 pg / ml after peit , the efficacy was 66.7% with one gland , but only 7.7% if there were more than two . in another study , there was a negative correlation between the number of glands > 0.5 cm and the efficacy of peit , although there was no correlation between the total number of enlarged glands and the efficacy ( figure 1a , b ) . nakamura also reported that the efficacy of peit diminishes as the number of hyperplastic glands increases . these reports confirm that superior results will be obtained with peit when there is only one ptg that is > 0.5 cm . influence on the efficacy of percutaneous ethanol injection therapy ( peit ) of ( a ) the number of parathyroid glands and ( b ) the number of parathyroid glands with a volume 0.5 cm . a recent clinical study reported that the serum pth level was not reduced in the case of advanced shpt with preoperative intact pth levels > 1000 pg / ml , even though peit was performed as described in other reports . the authors considered that the reason for treatment failure was that the subject of their study had advanced shpt , in which most of the enlarged glands are resistant to medical therapy , so multiple injections over a prolonged period are required to achieve an effect . this is undesirable because of the increased incidence of the side effects of ethanol leakage , such as pain , haemorrhage and recurrent laryngeal nerve palsy . moreover , adhesion of surrounding tissue is exacerbated with repeated peit and thus the therapeutic efficacy diminishes . we have recently shown that the odds ratio for success versus failure by multivariate regression analysis is 0.29 for preoperative intact pth levels > 500 pg / ml compared with < 500 pg / ml . in europe and the united states , calcimimetics have shown superior efficacy as medical treatment for shpt resistant to conventional treatment , but as of the end of 2007 they are not available in japan , so interventions such as peit and ptx are the choices for shpt resistant to medical therapy . particularly , peit is desirable , and still effective , in the early phase when the incidence of ckd - mbd is thought to be low . in other words , peit is indicated at the stage when the patient is resistant to medical therapy , with only one enlarged ptg , and the indication for ptx is more advanced shpt , or more than two enlarged ptgs . at the time peit was first introduced , it was considered that subsequent treatment with active vitamin d analogues strongly influenced long - term post - precedural prognosis . when peit is successful , serum ca and p levels , as well as the pth level , decrease immediately after peit , enabling a strong inhibition of the pth level with medical therapy . however , if this becomes difficult after peit , then the remaining ptgs have advanced hyperplasia and , in such cases , serum ca and p levels increase , promoting a recurrence of shpt . one group has reported that it is possible to maintain a low pth level for 1 year after peit , if it is performed correctly , and furthermore , they also reported that pth was maintained at an adequate level for at least 3 years with a combination of after - treatment and additional peit . in both reports , the pth level was decreased to the target level by the first session of peit , which suggests that the long - term prognosis is influenced by the rate of pth decrease immediately after peit . for peit to be considered an effective therapy for shpt , serum pth and ca levels must be decreased to their target ranges by the first intervention , and then maintained in that range for a long period . however , the therapeutic effect of peit is greatly influenced by the skill of the operator , so a full assessment of outcomes is difficult in small - scale , single - centre studies , and instead requires large numbers of peit procedures performed in several centres under the same therapeutic guidelines to minimize the influence of variations in skill level between institutions . in addition , there were no standardized therapeutic guidelines for peit in the early 1990s , and therapeutic outcomes depend on correct indications , equipment , technique and postoperative management . furthermore , improvements in mineral metabolism after peit have not been fully investigated . in our study of a retrospective cohort of > 300 subjects , based on the japanese peit guidelines , patients were divided into an effective group that achieved the specified levels of serum corrected ca < 10.5 mg / dl and serum intact pth < 250 pg / ml , and an ineffective group that failed to achieve these target levels after one session of peit . active vitamin d analogues were used as after - treatment in all cases . in the effective group , the response period was defined as the period from the day on which target levels were reached until the day on which either target level was exceeded , and the remission period was defined as the period from the day on which target levels were reached until the day of repeat peit or ptx because of recurrent disease . peit was effective in 208 patients ( 66.2% ) , in whom serum intact pth levels decreased from 603 292 to 183 62 pg / ml ( ng / l ) , and serum corrected ca levels from 10.7 0.8 to 10.1 0.5 mg / dl . the risk of relapse was evaluated by multivariate analysis using a logistic regression model , with the event of exceeding target levels ( relapse ) as a dependent variable . the odds of relapse versus non - relapse throughout the follow - up period was 2.37 in the presence of more than two hyperplastic glands versus one or no glands larger > 0.5 cm . in addition , the event - free survival of the remission period was evaluated using kaplan meier survival analysis and the remission period for the group with only one hyperplastic gland showed a significant extension by log - rank analysis . nodular hyperplasia can be presented if ptgs are enlarged by between 0.25 g and 0.5 g . in the case of only one enlarged gland < 0.5 cm , this ptg may have nodular hyperplasia if it does not respond to medical therapy . in such a case , peit becomes an effective treatment if peit is possible technically . therefore , as shown in figure 2 , superior prognosis including high efficacy , low recurrence , and long - term remission period can be obtained with peit when there is only one enlarged gland > 0.5 cm or there is only one enlarged gland on which we can perform peit . preferable indication of peit achieving good prognosis including high efficacy , low recurrence and long - term remission period . in japan , where at present calcicimetics are unavailable , the presence of hyperplastic ptgs is a good indication for peit , even if the serum pth level is < 500 pg / ml . superior results for peit , in terms of efficacy and superior prognosis , can be obtained when it is restricted to patients with not more than one ptg > 0.5 cm .

control of secondary hyperparathyroidism ( shpt ) using active vitamin d analogues becomes difficult in advanced shpt , because the enlarged parathyroid glands ( ptgs ) are resistant to medical therapy . percutaneous ethanol injection therapy ( peit ) has been widely used in japan since the 1990s as a surgical intervention for advanced shpt , by selectively destroying only the enlarged glands with nodular hyperplasia ( i.e. > 0.5 cm3 , measured by ultrasonography ) . if there is only one ptg with nodular hyperplasia , peit will be successful with a small number of injections , and it then becomes possible to maintain target levels of parathyroid hormone by treatment with active vitamin d analogues . recent studies have demonstrated that in the advanced phase of shpt , it is desirable to perform peit when it is restricted to patients with not more than one ptg larger than 0.5 cm3 in terms of superior prognosis can be obtained including efficacy , low recurrence , and long - term remission period .

Introduction Efficacy of PEIT Factors contributing to the efficacy of PEIT Number of enlarged glands. Serum PTH levels Good prognosis after PEIT Conclusion

a common complication in patients on long - term dialysis is secondary hyperparathyroidism ( shpt ) , which causes mineral bone disorder of chronic kidney disease ( ckd - mbd ) . active vitamin d analogues are widely used as standard medical therapy for shpt , but it can be difficult to maintain serum calcium and phosphate levels within the normal range , and response to medical therapy declines as shpt progresses . in the advanced stage , the parathyroid glands ( ptgs ) exhibit nodular hyperplasia , there is a reduced therapeutic effect on the secretion of parathyroid hormone ( pth ) and the patient is considered to be resistant to medical treatment . in such cases , surgical parathyroidectomy ( ptx ) is the conventional treatment choice and has been widely performed in japan . in the 1980s , percutaneous ethanol injection therapy ( peit ) under ultrasound guidance was developed in italy for ptg tumours and because the glands are physically destroyed , peit is also considered to be an effective treatment for resistant shpt . reported on the use of peit for shpt , and it has since been widely used in japan as a new intervention . standardized guidelines for indications , equipment and postoperative management were produced in 2003 , consolidating the place of peit as a tool for managing advanced shpt in japan . in the 1990s when peit was first introduced , some reports from europe indicated that pth levels were reduced by > 30% in only 4060% of cases [ 68 ] , whereas in japan the success rate was comparatively high . in kitaoka et al 's 1994 report , serum intact pth levels decreased from 727 pg / ml to < 200 pg / ml in seven of nine patients who underwent peit , and in the remaining two patients levels were maintained within the target range by postoperative medical therapy ( i.e. vitamin d analogues ) . those good results were obtained because not only the target gland but also secondary and tertiary glands in size were treated , and because of improvements in equipment , such as the addition of three small holes in the side of the needle to improve ethanol infiltration . therefore , the japanese guidelines also recommend target glands of peit as > 0.5 cm or > 1 cm in size , but not all glands were detectable by ultrasonography . moreover , other studies state that the efficacy of peit is decreased or is nullified when the preoperative pth level is > 1000 pg / ml and there is more than one gland > 0.5 cm on ultrasonography . as the ptgs become > 0.5 cm , or 1 cm in diameter , they became resistant to medical therapy , so the presence of enlarged glands is a strong indicator of responsiveness to conventional treatment . furthermore , histological analysis of surgically removed glands has confirmed that > 90% of glands heavier than 0.5 g have nodular hyperplasia , so for peit to be effective all glands > 0.5 cm have to be destroyed . according to the japanese guidelines for peit of the ptg , glands with nodular hyperplasia are selectively destroyed and glands with diffuse hyperplasia are then managed by medical therapy . if there is only one enlarged gland , a single session of peit will usually be successful , but if there are more than two , it becomes difficult to destroy all target glands with certainty in one session of peit and the number of interventions increases . in a study comparing the number of enlarged glands in haemodialysis patients with intact pth levels < 300 pg / ml after peit , the efficacy was 66.7% with one gland , but only 7.7% if there were more than two . in another study , there was a negative correlation between the number of glands > 0.5 cm and the efficacy of peit , although there was no correlation between the total number of enlarged glands and the efficacy ( figure 1a , b ) . these reports confirm that superior results will be obtained with peit when there is only one ptg that is > 0.5 cm . influence on the efficacy of percutaneous ethanol injection therapy ( peit ) of ( a ) the number of parathyroid glands and ( b ) the number of parathyroid glands with a volume 0.5 cm . a recent clinical study reported that the serum pth level was not reduced in the case of advanced shpt with preoperative intact pth levels > 1000 pg / ml , even though peit was performed as described in other reports . the authors considered that the reason for treatment failure was that the subject of their study had advanced shpt , in which most of the enlarged glands are resistant to medical therapy , so multiple injections over a prolonged period are required to achieve an effect . in europe and the united states , calcimimetics have shown superior efficacy as medical treatment for shpt resistant to conventional treatment , but as of the end of 2007 they are not available in japan , so interventions such as peit and ptx are the choices for shpt resistant to medical therapy . particularly , peit is desirable , and still effective , in the early phase when the incidence of ckd - mbd is thought to be low . in other words , peit is indicated at the stage when the patient is resistant to medical therapy , with only one enlarged ptg , and the indication for ptx is more advanced shpt , or more than two enlarged ptgs . at the time peit was first introduced , it was considered that subsequent treatment with active vitamin d analogues strongly influenced long - term post - precedural prognosis . however , if this becomes difficult after peit , then the remaining ptgs have advanced hyperplasia and , in such cases , serum ca and p levels increase , promoting a recurrence of shpt . one group has reported that it is possible to maintain a low pth level for 1 year after peit , if it is performed correctly , and furthermore , they also reported that pth was maintained at an adequate level for at least 3 years with a combination of after - treatment and additional peit . in both reports , the pth level was decreased to the target level by the first session of peit , which suggests that the long - term prognosis is influenced by the rate of pth decrease immediately after peit . however , the therapeutic effect of peit is greatly influenced by the skill of the operator , so a full assessment of outcomes is difficult in small - scale , single - centre studies , and instead requires large numbers of peit procedures performed in several centres under the same therapeutic guidelines to minimize the influence of variations in skill level between institutions . in addition , there were no standardized therapeutic guidelines for peit in the early 1990s , and therapeutic outcomes depend on correct indications , equipment , technique and postoperative management . in our study of a retrospective cohort of > 300 subjects , based on the japanese peit guidelines , patients were divided into an effective group that achieved the specified levels of serum corrected ca < 10.5 mg / dl and serum intact pth < 250 pg / ml , and an ineffective group that failed to achieve these target levels after one session of peit . active vitamin d analogues were used as after - treatment in all cases . in the effective group , the response period was defined as the period from the day on which target levels were reached until the day on which either target level was exceeded , and the remission period was defined as the period from the day on which target levels were reached until the day of repeat peit or ptx because of recurrent disease . peit was effective in 208 patients ( 66.2% ) , in whom serum intact pth levels decreased from 603 292 to 183 62 pg / ml ( ng / l ) , and serum corrected ca levels from 10.7 0.8 to 10.1 0.5 mg / dl . the risk of relapse was evaluated by multivariate analysis using a logistic regression model , with the event of exceeding target levels ( relapse ) as a dependent variable . the odds of relapse versus non - relapse throughout the follow - up period was 2.37 in the presence of more than two hyperplastic glands versus one or no glands larger > 0.5 cm . in addition , the event - free survival of the remission period was evaluated using kaplan meier survival analysis and the remission period for the group with only one hyperplastic gland showed a significant extension by log - rank analysis . nodular hyperplasia can be presented if ptgs are enlarged by between 0.25 g and 0.5 g . in the case of only one enlarged gland < 0.5 cm , this ptg may have nodular hyperplasia if it does not respond to medical therapy . therefore , as shown in figure 2 , superior prognosis including high efficacy , low recurrence , and long - term remission period can be obtained with peit when there is only one enlarged gland > 0.5 cm or there is only one enlarged gland on which we can perform peit . preferable indication of peit achieving good prognosis including high efficacy , low recurrence and long - term remission period . in the 1990s when peit was first introduced , some reports from europe indicated that pth levels were reduced by > 30% in only 4060% of cases [ 68 ] , whereas in japan the success rate was comparatively high . in kitaoka et al 's 1994 report , serum intact pth levels decreased from 727 pg / ml to < 200 pg / ml in seven of nine patients who underwent peit , and in the remaining two patients levels were maintained within the target range by postoperative medical therapy ( i.e. those good results were obtained because not only the target gland but also secondary and tertiary glands in size were treated , and because of improvements in equipment , such as the addition of three small holes in the side of the needle to improve ethanol infiltration . therefore , the japanese guidelines also recommend target glands of peit as > 0.5 cm or > 1 cm in size , but not all glands were detectable by ultrasonography . moreover , other studies state that the efficacy of peit is decreased or is nullified when the preoperative pth level is > 1000 pg / ml and there is more than one gland > 0.5 cm on ultrasonography . therefore , various factors , such as the stage of shpt and the number of enlarged ptg , influence the efficacy of peit . as the ptgs become > 0.5 cm , or 1 cm in diameter , they became resistant to medical therapy , so the presence of enlarged glands is a strong indicator of responsiveness to conventional treatment . furthermore , histological analysis of surgically removed glands has confirmed that > 90% of glands heavier than 0.5 g have nodular hyperplasia , so for peit to be effective all glands > 0.5 cm have to be destroyed . according to the japanese guidelines for peit of the ptg , glands with nodular hyperplasia are selectively destroyed and glands with diffuse hyperplasia are then managed by medical therapy . if there is only one enlarged gland , a single session of peit will usually be successful , but if there are more than two , it becomes difficult to destroy all target glands with certainty in one session of peit and the number of interventions increases . in a study comparing the number of enlarged glands in haemodialysis patients with intact pth levels < 300 pg / ml after peit , the efficacy was 66.7% with one gland , but only 7.7% if there were more than two . in another study , there was a negative correlation between the number of glands > 0.5 cm and the efficacy of peit , although there was no correlation between the total number of enlarged glands and the efficacy ( figure 1a , b ) . these reports confirm that superior results will be obtained with peit when there is only one ptg that is > 0.5 cm . influence on the efficacy of percutaneous ethanol injection therapy ( peit ) of ( a ) the number of parathyroid glands and ( b ) the number of parathyroid glands with a volume 0.5 cm . a recent clinical study reported that the serum pth level was not reduced in the case of advanced shpt with preoperative intact pth levels > 1000 pg / ml , even though peit was performed as described in other reports . the authors considered that the reason for treatment failure was that the subject of their study had advanced shpt , in which most of the enlarged glands are resistant to medical therapy , so multiple injections over a prolonged period are required to achieve an effect . in europe and the united states , calcimimetics have shown superior efficacy as medical treatment for shpt resistant to conventional treatment , but as of the end of 2007 they are not available in japan , so interventions such as peit and ptx are the choices for shpt resistant to medical therapy . particularly , peit is desirable , and still effective , in the early phase when the incidence of ckd - mbd is thought to be low . in other words , peit is indicated at the stage when the patient is resistant to medical therapy , with only one enlarged ptg , and the indication for ptx is more advanced shpt , or more than two enlarged ptgs . as the ptgs become > 0.5 cm , or 1 cm in diameter , they became resistant to medical therapy , so the presence of enlarged glands is a strong indicator of responsiveness to conventional treatment . furthermore , histological analysis of surgically removed glands has confirmed that > 90% of glands heavier than 0.5 g have nodular hyperplasia , so for peit to be effective all glands > 0.5 cm have to be destroyed . according to the japanese guidelines for peit of the ptg , glands with nodular hyperplasia are selectively destroyed and glands with diffuse hyperplasia are then managed by medical therapy . if there is only one enlarged gland , a single session of peit will usually be successful , but if there are more than two , it becomes difficult to destroy all target glands with certainty in one session of peit and the number of interventions increases . in a study comparing the number of enlarged glands in haemodialysis patients with intact pth levels < 300 pg / ml after peit , the efficacy was 66.7% with one gland , but only 7.7% if there were more than two . in another study , there was a negative correlation between the number of glands > 0.5 cm and the efficacy of peit , although there was no correlation between the total number of enlarged glands and the efficacy ( figure 1a , b ) . these reports confirm that superior results will be obtained with peit when there is only one ptg that is > 0.5 cm . influence on the efficacy of percutaneous ethanol injection therapy ( peit ) of ( a ) the number of parathyroid glands and ( b ) the number of parathyroid glands with a volume 0.5 cm . the authors considered that the reason for treatment failure was that the subject of their study had advanced shpt , in which most of the enlarged glands are resistant to medical therapy , so multiple injections over a prolonged period are required to achieve an effect . in europe and the united states , calcimimetics have shown superior efficacy as medical treatment for shpt resistant to conventional treatment , but as of the end of 2007 they are not available in japan , so interventions such as peit and ptx are the choices for shpt resistant to medical therapy . particularly , peit is desirable , and still effective , in the early phase when the incidence of ckd - mbd is thought to be low . in other words , peit is indicated at the stage when the patient is resistant to medical therapy , with only one enlarged ptg , and the indication for ptx is more advanced shpt , or more than two enlarged ptgs . at the time peit was first introduced , it was considered that subsequent treatment with active vitamin d analogues strongly influenced long - term post - precedural prognosis . one group has reported that it is possible to maintain a low pth level for 1 year after peit , if it is performed correctly , and furthermore , they also reported that pth was maintained at an adequate level for at least 3 years with a combination of after - treatment and additional peit . in both reports , the pth level was decreased to the target level by the first session of peit , which suggests that the long - term prognosis is influenced by the rate of pth decrease immediately after peit . for peit to be considered an effective therapy for shpt , serum pth and ca levels must be decreased to their target ranges by the first intervention , and then maintained in that range for a long period . however , the therapeutic effect of peit is greatly influenced by the skill of the operator , so a full assessment of outcomes is difficult in small - scale , single - centre studies , and instead requires large numbers of peit procedures performed in several centres under the same therapeutic guidelines to minimize the influence of variations in skill level between institutions . in addition , there were no standardized therapeutic guidelines for peit in the early 1990s , and therapeutic outcomes depend on correct indications , equipment , technique and postoperative management . in our study of a retrospective cohort of > 300 subjects , based on the japanese peit guidelines , patients were divided into an effective group that achieved the specified levels of serum corrected ca < 10.5 mg / dl and serum intact pth < 250 pg / ml , and an ineffective group that failed to achieve these target levels after one session of peit . active vitamin d analogues were used as after - treatment in all cases . in the effective group , the response period was defined as the period from the day on which target levels were reached until the day on which either target level was exceeded , and the remission period was defined as the period from the day on which target levels were reached until the day of repeat peit or ptx because of recurrent disease . peit was effective in 208 patients ( 66.2% ) , in whom serum intact pth levels decreased from 603 292 to 183 62 pg / ml ( ng / l ) , and serum corrected ca levels from 10.7 0.8 to 10.1 0.5 mg / dl . the risk of relapse was evaluated by multivariate analysis using a logistic regression model , with the event of exceeding target levels ( relapse ) as a dependent variable . the odds of relapse versus non - relapse throughout the follow - up period was 2.37 in the presence of more than two hyperplastic glands versus one or no glands larger > 0.5 cm . in addition , the event - free survival of the remission period was evaluated using kaplan meier survival analysis and the remission period for the group with only one hyperplastic gland showed a significant extension by log - rank analysis . nodular hyperplasia can be presented if ptgs are enlarged by between 0.25 g and 0.5 g . in the case of only one enlarged gland < 0.5 cm , this ptg may have nodular hyperplasia if it does not respond to medical therapy . therefore , as shown in figure 2 , superior prognosis including high efficacy , low recurrence , and long - term remission period can be obtained with peit when there is only one enlarged gland > 0.5 cm or there is only one enlarged gland on which we can perform peit . preferable indication of peit achieving good prognosis including high efficacy , low recurrence and long - term remission period . superior results for peit , in terms of efficacy and superior prognosis , can be obtained when it is restricted to patients with not more than one ptg > 0.5 cm .

tumors are considered congenital when detected during pregnancy or in the first 3 months of life [ 1 , 2 ] . congenital tumors represent only 1.52% of all pediatric tumors , with a prevalence of 1:12,500 to 1:27,500 live births [ 35 ] . recent studies demonstrate an increase in the incidence of congenital tumors , probably as a consequence of both the generalization of routine prenatal and neonatal medical controls and of the improvement of prenatal imaging techniques . congenital tumors differ in many aspects from tumors presenting in older children , with respect to histology , behavior and prognosis . the most frequent congenital tumor types are shown in table 1 [ 2 , 3 , 58 ] . the diagnosis of a congenital tumor involves a tremendous emotional impact for a family and implies ethical questions about prognosis , therapy possibilities and long - term consequences of the treatment . prognosis is related not only to the histological type , but also to the size and location of the tumor and the presence or not of associated congenital anomalies , which are found in 1520.8% of cases [ 5 , 9 ] . another prognostic factor is the age at which the lesion is detected , with most histological tumor types showing a worse prognosis for cases detected during pregnancy [ 1014 ] . table 1distribution of main congenital tumors [ 2 , 3 , 58]type of tumor%extracranial teratomas23.529%neuroblastomas22.530%soft tissue tumors8.112%cns tumors5.910%leukemia5.9%12.35%renal tumors57.1%hepatic tumors5%cardiopulmonary tumors<3% distribution of main congenital tumors [ 2 , 3 , 58 ] over the last few years , we have witnessed significant technical improvements in prenatal imaging diagnostic tools . ultrasound is usually the first imaging method used , but antenatal magnetic resonance imaging ( mri ) is increasingly being used as a complementary study . standard postnatal imaging methods include conventional x - ray , ultrasound , ct and mri . the purposes of this review are to describe the most frequent types of congenital tumors and their prognosis , to review the main imaging characteristics of this heterogeneous group of neoplasms , and to define the role of the different pre- and postnatal imaging methods in the characterisation of congenital tumors , with special emphasis on prenatal mri . they may contain components arising from all three germ layers and therefore may present different tissue types . the main location of these tumors is the sacrococcygeal region ( 45% of cases ) , followed by the cervicofacial area ( 28% ) and the thorax ( 11% ) . sacrococcygeal teratomas ( sct ) are one of the most common overall congenital neoplasms , with an incidence of 1:40,000 infants and a female predominance of 4:1 [ 15 , 16 ] . they arise from rests of pluripotent cells at the caudal end of the notochord , originating from the coccyx . of all teratomas , these tumors are associated with the highest incidence of malignancy ( 10% ) and of associated anomalies ( 1015% ) . according to the tumor extension , scts are classified into four categories by the american pediatric surgery association : type i , lesions almost completely external ; type ii , predominantly external masses with pelvic extension ; type iii , external tumors with intra - abdominal extension ; type iv , exclusively intra - abdominal lesions . 1a , b ) with a high incidence of complications , such as polyhydramnios , fetal cardiac failure , fetal hydrops , tumoral hemorrhage and prematurity , which are related to the tumor size and to the effect of the intrapelvic and intra - abdominal tumor components over the developing organs . coronal ( a ) and sagittal t2-trufi ( b ) fetal mris ( 29th week ) show the large , complex sct type ii with both cystic ( arrow ) and solid components . the images clearly illustrate the extension of the tumor into the pelvis , the abdomen and the spinal canal ( block arrow ) . the ct topogram ( c ) and the axial nect image ( d ) demonstrate the heterogenicity of the tumor , with calcifications ( white arrows ) , fat , solid and liquid ( star ) components extracranial teratomas . coronal ( a ) and sagittal t2-trufi ( b ) fetal mris ( 29th week ) show the large , complex sct type ii with both cystic ( arrow ) and solid components . the images clearly illustrate the extension of the tumor into the pelvis , the abdomen and the spinal canal ( block arrow ) . the ct topogram ( c ) and the axial nect image ( d ) demonstrate the heterogenicity of the tumor , with calcifications ( white arrows ) , fat , solid and liquid ( star ) components cervicofacial teratomas ( fig . they may originate in different anatomical structures , including the orbit , the naso- and oropharynx , the tongue , the palate and the anterior neck . cervical teratomas in utero may not only cause hydrops secondary to fetal swallowing disturbances , but also fetal pulmonary hypoplasia . ex utero intrapartum treatment , or exit , has been performed in recent years in cases of giant neck masses in highly specialized neonatal medical centers , improving the initial fetal survival [ 21 , 22 ] . indeed , in cases of fetal pericardial effusion , teratoma should be seriously considered in the differential diagnosis . congenital sacrococcygeal and cervicofacial teratomas present an overall mortality of 3033.5% , mainly related to large tumors with extensive local infiltration . the main role of pre- and postnatal imaging for teratomas is to recognize any associated anomalies and to determine the full extent of the mass for prognosis and evaluation of surgical planning . on fetal ultrasound , teratomas are seen as large , heterogeneous masses due to their different tissue components . in some cases of sct , an accurate ultrasound evaluation may be insufficient because of shadowing by the fetal pelvic bones , which may cause an underestimation of the tumor . fetal mri is therefore considered the method of choice for evaluating the extent of the internal mass ( fig . indeed , the method can serve as a roadmap before surgery , as ultrasound images are sometimes difficult to interpret for clinicians and may fail to provide a good overview of the limits of the relation between the tumor and the surrounding anatomy . post - natal ct and mri studies show a heterogeneous mass with solid , liquid and fat components ( fig . calcifications and bone tissue are often found and are better recognized on ct ( figs . 1 , 2b ) . the abdominal ultrasound ( a ) and the axial fat - saturated t2-weighted images ( b ) show the well - defined , homogeneous mass arising from the right adrenal gland ( white arrow ) and displacing the kidney in this 2-day - old baby . axial contrast - enhanced ct ( cect ) ( c ) shows the enhancing retroperitoneal tumor ( white arrow ) originating from the organ of zckerkandl in this 10-day - old girl . axial cect ( d ) shows the mass arising from the right paraspinal sympathetic chain and extending into the spinal canal in this 3-day - old baby boy . contrast medium administration ( f ) show the retroperitoneal , lobulated mass infiltrating the right kidney and causing hydronephrosis ( arrow ) in this 5-week - old baby . the lesion was initially considered to be a renal tumor , but histology revealed neuroblastoma neuroblastoma . the abdominal ultrasound ( a ) and the axial fat - saturated t2-weighted images ( b ) show the well - defined , homogeneous mass arising from the right adrenal gland ( white arrow ) and displacing the kidney in this 2-day - old baby . axial contrast - enhanced ct ( cect ) ( c ) shows the enhancing retroperitoneal tumor ( white arrow ) originating from the organ of zckerkandl in this 10-day - old girl . axial cect ( d ) shows the mass arising from the right paraspinal sympathetic chain and extending into the spinal canal in this 3-day - old baby boy . contrast medium administration ( f ) show the retroperitoneal , lobulated mass infiltrating the right kidney and causing hydronephrosis ( arrow ) in this 5-week - old baby . the neuroblastoma is the second most common pediatric malignancy overall , after teratoma , in most published series [ 3 , 5 ] and the most common malignancy of the first month of life ( 3050% ] . the tumor arises from primordial neural crest cells , anywhere along the sympathetic chains , at the adrenal medulla ( fig . 2b ) , the extra - adrenal retroperitoneum ( fig . 2c , d ) or the posterior mediastinum . the prognosis is excellent , with a survival rate of 8890% [ 1 , 24 ] , metastases are rare and spontaneous involution in utero has been described . on ultrasound , the tumor is mostly seen as a solid , heterogeneous echogenic mass that displaces the adjacent kidney inferiorly and laterally . calcifications are present in 30% of all cases . on mri , neuroblastomas are typically heterogeneous , with relatively low signal intensity on t1- and high signal intensity on t2-weighted images . 2b ) and of extra - adrenal origin in 55% of all cases ( fig . 2c , d ] . 2c ) , bone and skin ] , it has an overall survival rate of 64% [ 1 , 24 ] . on ct , the tumor is heterogeneous , with calcifications , necrosis and areas of hemorrhage . 2d ) and in 28% of thoracic masses , and is clearly visualized on mri studies . metaiodobenzylguanidine ( mibg - i ) scintigraphy is an excellent method for determining the presence of metastases or tumor recidivation , but 30% of neonatal tumors are mibg - negative . the international neuroblastoma staging system is mainly based on the degree of surgical resection and shows some difficulties for application in congenital cases . recently , a new international neuroblastoma risk group staging system ( inrgss ) , based on imaging findings , was designed to stage the patients at the time of diagnosis . in this classification , the extent of locoregional disease is determined by the absence ( l1 ) or presence ( l2 ) of imaging - defined risk factors , with stage m used for widely disseminated disease and stage ms for metastatic disease limited to liver , skin and bone narrow in children younger than 18 months . the main role of imaging methods for both fetal and neonatal neuroblastoma is then to determine the origin of the mass , its locoregional extent and the presence or not of metastases for staging before therapy . congenital soft - tissue tumors constitute a heterogeneous group of lesions , including mainly vascular and muscular tumors . the international society for the study of vascular anomalies ( issva ) differentiates between vascular tumors and vascular malformations ( capillary , venous , lymphatic or mixed ) , which are not discussed in this review . infantile hemangiomas ( ih ) are true neoplasms that originate from the cellular proliferation of vascular endothelial cells . they account for a significant number of prenatally diagnosed neoplasms , but because most of them are small or absent at birth , they are often not included in the register of congenital tumors . the most frequent location is the head and neck ( 60% ) ( fig . ih usually present a biphasic cycle , with initial rapid growth followed by an involution over months or years . on ultrasound , they appear as lobulated , hypo- or hyperechoic , well - defined masses with increased doppler flow . fig . coronal ( a ) and axial ( b ) fetal t2-haste mri ( 21st week ) demonstrate the cephalic , but completely extracranial heterogeneous mass ( star ) . the axial ( c ) and coronal ( d ) t2-weighted images show the homogeneous hyperintense mass in the left posterior mediastinum , extending into the spinal canal ( arrow ) in this newborn . axial cect image ( e ) shows the large , infiltrating left cervical muscular mass , with intense enhancement after contrast medium administration soft - tissue tumors . coronal ( a ) and axial ( b ) fetal t2-haste mri ( 21st week ) demonstrate the cephalic , but completely extracranial heterogeneous mass ( star ) . the axial ( c ) and coronal ( d ) t2-weighted images show the homogeneous hyperintense mass in the left posterior mediastinum , extending into the spinal canal ( arrow ) in this newborn . axial cect image ( e ) shows the large , infiltrating left cervical muscular mass , with intense enhancement after contrast medium administration the notion of congenital hemangiomas has been introduced recently , differentiating between non - involuting congenital hemangiomas , or nich , and rapidly involuting congenital hemangiomas , or rich . although both rich and nich groups show similar imaging findings to ih on ultrasound , they may also present vascular aneurysms , intravascular thrombi and arteriovenous shunting , features not usually observed in ih . large congenital tumors may cause compression of vital structures or cardiovascular complications , and in these cases , mri is the method of choice to determine the mass extension and its relationship to the adjacent anatomical structures ( fig . hemangiomas are usually hyperintense on t2-weighted and isointense on t1-weighted images related to muscle , with prominent draining veins and intense enhancement after contrast medium administration , but no perilesional edema . fibromatosis and myofibromatosis are rare benign disorders with a tendency to infiltrate adjacent tissues but no metastases . they present an increment of the size and the number of lesions during the first year of life with later regression . 3d ) , with low signal intensity on both t1-weighted and t2-weighted images , infiltration of the fat and muscle , and intense enhancement after i.v . the tumor represents > 50% of all soft tissue sarcomas in children , but its congenital presentation is extremely rare . it can arise in any anatomical region of the body ( except bone ) whether there is skeletal muscle or not , but most often it presents in the head and neck ( 2840% ) and in the genitourinary region ( 20% ) . the ultrasound shows a soft - tissue mass , similar to muscle , whereas mri reveals a mass isointense to muscle on t1-weighted and hyperintense on t2-weighted images , with heterogeneous enhancement after gadolinium administration . immunochemistry tests show that rhabdoid tumors typically lack staining for baf47 in tumor cells because of a clonal mutation in the ini1 gene . this anomaly now allows correct differentiation between rhabdoid tumors usually associated with a bad prognosis and rhabdomyosarcomas . the main role of imaging in the case of congenital soft tissue tumors is to determine the extent of the mass and to identify the invasion of adjacent structures in order to optimize the surgical approach . they represent only 0.51.9% of all pediatric brain tumors , but are responsible for 520% of deaths secondary to neoplasms in this age group [ 6 , 38 ] . congenital cns tumors differ in histology , location , biological behavior and response to therapy compared with cns tumors in older children . most congenital tumors ( about 60% ) are supratentorial , arising from the pineal gland , the suprasellar area or the cerebral hemispheres . increased intracranial pressure signs are not often observed because of the great capacity for accommodation to the increased volume in the fetus and in neonates with opened sutures . intratumoral hemorrhage is relatively common , varying between 3 and 18% [ 3840 ] . histological type and mortality rate of main cns tumors [ 3741]cns tumors%mortality rateintracranial teratomas26.648%88%astrocytomas7.428.8%6468%choroid plexus papilloma3.713.2%27%pnet313%88%craniopharyngioma5.66.8%76.5%ependymoma4.4%91% cns tumors . histological type and mortality rate of main cns tumors [ 3741 ] teratoma is the most common congenital brain tumor and represents 26.6 to 48% [ 3841 ] ( table 2 ) . congenital teratomas are predominantly supratentorial , with the cerebral hemispheres being the main primary site , followed by the third ventricle and the pineal region . on ultrasound , they are seen as typically large , mid - line heterogeneous tumors with solid areas replacing much of the brain . cystic components are often found and probably represent necrotic areas in tumors with a rapid growth rate . the cerebral hemispheres are the main primary site , followed by the optic nerve , the thalamus and the mesencephalus . the choroid plexus papilloma is composed of mature epithelial cells derived from choroid plexus epithelium . it presents clinically as macrocephaly , with rapid onset of hydrocephalus , secondary to the overproduction and/or mechanical obstruction to cerebrospinal fluid ( csf ) circulation . these are well defined , intraventricular masses , hyperechoic on ultrasound and mostly hyperattenuating on ct ( fig . 4a , b ) , with intense contrast enhancement . on mri , they present as homogeneous masses on t1-weighted images , with the central tumor areas hypointense compared with grey matter on t2-weighted images . complete surgical resection is curative , but intraoperative hemorrhage is common and may have serious consequences . they are often associated with marked vasogenic edema and may be hyperdense on ct because of the high cellularity . axial t2-weighted fetal mri ( 34th week ) ( a ) shows an intraventricular , solid mass in the posterior horn of the right lateral ventricle ( white arrow ) . postnatal axial t2-weighted image ( b ) 3 days after birth reveals an increase in the tumor size . sagittal t1-weighted image after i.v contrast medium administration ( c ) and coronal t2-weighted image ( d ) in this 6-week - old boy presenting with macrocephaly show the heterogeneous tumor in the posterior fossa ( arrow ) , with both solid and liquid ( block arrow ) components . axial t2-weighted fetal mri ( 34th week ) ( a ) shows an intraventricular , solid mass in the posterior horn of the right lateral ventricle ( white arrow ) . postnatal axial t2-weighted image ( b ) 3 days after birth reveals an increase in the tumor size . sagittal t1-weighted image after i.v contrast medium administration ( c ) and coronal t2-weighted image ( d ) in this 6-week - old boy presenting with macrocephaly show the heterogeneous tumor in the posterior fossa ( arrow ) , with both solid and liquid ( block arrow ) components . the tumor causes a secondary hydrocephalus primitive neuroectodermal tumors ( pnet ) ( fig . 4c , d ) are small - cell malignant tumors arising from the neural crest . congenital tumors have a poor prognosis as a consequence of the rapid tumor growth , with early extension throughout the csf pathways . the diagnosis of fetal and neonatal brain tumors is associated with high mortality rates ( table 2 ) . recent advances in mri technology include the development of magnetic resonance diffusion sequences ( dwi ) , which may identify areas of restricted water movement and of magnetic resonance spectroscopy ( mrs ) , which recognizes changes in metabolite concentration . these technical improvements have opened up exciting perspectives , suggesting the possibility of differentiating between histological tumor types [ 20 , 45 ] , but preliminary reports have not yet been confirmed for congenital brain tumors . the main role of imaging studies is still to determine the extent of the tumor in order to evaluate therapy challenges and to identify potentially curable tumors , such as plexus papillomas , differentiating them from rapidly fatal ones . they are mostly benign , with the most frequent histological types being mesoblastic nephroma ( msn ) , nephroblastomatosis and the multilocular cystic nephroma . wilms tumor and renal rhabdoid tumors are rare in neonates [ 1 , 14 , 46 ] . they present in utero as large , solid , infiltrative , unilateral renal masses , sometimes associated with polyhydramnios . ct and mri show a solid , relatively homogeneous renal tumor , typically involving the renal sinus and with variable contrast enhancement ( fig . contrast medium administration ( b ) show a solid , focal renal mass replacing the normal right renal parenchyma ( block arrows ) in this newborn boy . axial cect ( c ) shows the well - defined bilateral renal masses ( stars ) in this 3-month - old baby , corresponding to a histologically proven bilateral wilms tumor in a child with areas of nephroblastomatosis . t2-haste mr image ( d ) shows a multilocular cystic mass in the right kidney with tumor septae . observe a similar but smaller left renal lesion ( white arrow ) in this 10-week - old boy with multiple congenital tumors ( same patient as in fig . contrast medium administration ( b ) show a solid , focal renal mass replacing the normal right renal parenchyma ( block arrows ) in this newborn boy . axial cect ( c ) shows the well - defined bilateral renal masses ( stars ) in this 3-month - old baby , corresponding to a histologically proven bilateral wilms tumor in a child with areas of nephroblastomatosis . t2-haste mr image ( d ) shows a multilocular cystic mass in the right kidney with tumor septae . observe a similar but smaller left renal lesion ( white arrow ) in this 10-week - old boy with multiple congenital tumors ( same patient as in fig . it is found incidentally in 1% of normal children at post - mortem studies and may cause a wilms tumor in 3040% of the cases ( fig . there is a well - known association with beckwith - wiedemann syndrome , trisomy 18 and sporadic aniridia . typical ultrasound images show multifocal , subcapsular renal nodules , hypo- or isoechoic , related to renal parenchyma . the nodules present low attenuation on ct and low signal intensity on both t1- and t2-weighted images on mri relative to normal parenchyma , with reduced contrast enhancement . multilocular cystic nephroma ( mcn ) is a benign cystic renal neoplasm , arising from metanephric blastema . imaging shows a large multilocular cystic renal mass , with septa as the only solid component ( fig . the role of imaging studies for congenital renal tumors is to determine the extent of the mass and its relationship to the renal vascular structures before surgical therapy . hepatic tumors comprise 5% of all congenital neoplasms ( table 1 ) . excluding metastases , principally from leukemia and neuroblastoma , most primary hepatic tumors are hemangiomas ( 60.3% ) , followed by mesenchymal hamartoma ( 23.2% ) and hepatoblastoma ( 16.5% ) . the infantile hepatic hemangioma , frequently referred to as hepatic hemangioendothelioma , is the most common congenital vascular hepatic tumor [ 13 , 49 ] . hepatic hemangiomas may be focal ( solitary ) or multifocal , confluent lesions ( fig . although spontaneous involution is possible , rapid growth may instigate fetal anasarca ( fig . 6b ) , thrombocytopenia resulting from consumptive coagulopathies ( kasabach - merritt syndrome ) or postnatal death due to cardiac failure . on ultrasound , focal hemangioma is classically a well - defined , hypo- , iso- or hyperechogenic lesion with a prominent high flow on doppler ultrasound . . large tumors present as well - defined , solid masses , often with central necrosis . most lesions are hypodense compared with the rest of the liver on non - enhanced ct . after contrast medium administration , they show a centripetal enhancement that acquires uniformity on delayed ct . on mri , the lesions usually show low t1 and high t2 signal intensities , but may be heterogeneous because of hemorrhage and calcification , with variable enhancement patterns . necrotic or thrombotic regions show a heterogeneous signal both before and after gadolinium administration . coronal t2-haste mri ( a ) shows the multiple hyperintense hepatic lesions ( white arrows ) . observe the right hemihypertrophy in this newborn with beckwith - wiedemann syndrome ( vertical arrows ) . coronal ( b ) t2-haste mri ( 27th week ) shows a large and infiltrating mass arising from the left hepatic lobe with predominantly high signal intensity . observe the signs of fetal congestive heart failure with marked hydrops fetalis ( white block arrows ) . axial t2-weighted image ( c ) shows a centrally located , hepatic hyperintense cystic mass with internal septations in this 10-week - old boy ( same patient as in fig . hepatoblastoma . abdominal ultrasound ( d ) shows the large , heterogeneous mass in the right hepatic lobe of this 2-day - old girl hepatic tumors . coronal t2-haste mri ( a ) shows the multiple hyperintense hepatic lesions ( white arrows ) . observe the right hemihypertrophy in this newborn with beckwith - wiedemann syndrome ( vertical arrows ) . coronal ( b ) t2-haste mri ( 27th week ) shows a large and infiltrating mass arising from the left hepatic lobe with predominantly high signal intensity . observe the signs of fetal congestive heart failure with marked hydrops fetalis ( white block arrows ) . axial t2-weighted image ( c ) shows a centrally located , hepatic hyperintense cystic mass with internal septations in this 10-week - old boy ( same patient as in fig . hepatoblastoma . abdominal ultrasound ( d ) shows the large , heterogeneous mass in the right hepatic lobe of this 2-day - old girl the mesenchymal hamartoma is a developmental cystic tumor , resulting from the benign overgrowth of mature hepatic tissue , with variable loose mesenchyme . solid tumors have been described , but they are atypical [ 47 , 51 ] . on mri congenital hepatoblastoma is a malignant embryonic tumor composed of only epithelial cells or a mixture of epithelial and mesenchymal cells . they present as lobulated , sometimes multifocal large solid masses , most often in the right hepatic lobe ( 60% ) . an association with hemihypertrophy ( 2% ) , beckwith - wiedemann and intestinal polyposis is well established . the lesion is heterogeneous , with calcifications ( 50% ) , cystic and necrotic areas . the most extensive published series about congenital hepatic tumors describes a poor prognosis for hepatoblastomas , with common early metastases and high mortality rates , but this series probably includes some cases of congenital rhabdoid tumors , an aggressive lesion previously classified as hepatoblastoma with negative -fetoprotein . as mentioned elsewhere in this article , immunochemistry tests for ini1/baf47 protein , which is abnormally low in all rhabdoid tumors , have recently allowed a correct differentiation between the entities [ 36 , 37 , 53 , 54 ] , which present similar findings on imaging studies . the main role of imaging methods for congenital hepatic tumors is to evaluate the anatomical extent of the tumor and to clarify the relationship with hepatic lobar anatomy before surgical planning . the heart is one of the most common organs of origin of congenital tumors , with a reported incidence of 0.14 to 0.2% . the incidence has been increasing in recent years because of the technical advances of imaging studies , especially ultrasound cardiography and cardiac mri . cardiac tumors may be related to genetic disorders such as neurofibromatosis , beckwith - wiedemann syndrome , familial myxoma syndrome and tuberous sclerosis . the most frequent cardiac tumor is the myocardial rhabdomyoma ( 78% ) , followed by the pericardial teratoma ( 18% ) and the cardiac fibroma ( 12% ) . the rhabdomyoma is a hamartoma , and as such it is not considered a true neoplasm . its incidence oscillates between 0.12% in prenatal fetal studies and 0.020.08% in live - born infants [ 55 , 56 ] . the tumor is multiple in > 90% of cases , not invasive and not metastasizing . rhabdomyoma shows a high association with tuberous sclerosis ( 86% ) and may manifest as the earliest clinical sign of this pathological condition in utero . indeed , the detection of multiple cardiac rhabdomyomas on prenatal ultrasound is considered sufficient to establish the diagnosis of tuberous sclerosis and makes mri evaluation of the fetal cns mandatory . clinical findings oscillate from asymptomatic to congestive heart failure , arrhythmias , valvular dysfunction and sudden death , which cause a mortality of 40% . surgical intervention is only recommended in cases of persistent arrhythmias or severe hemodynamic compromise because rhabdomyomas usually increase in size until late pregnancy and then tend to regress progressively during the first year of life . cardio - ultrasound is the imaging method of choice and provides a dynamic evaluation of the myocardial wall motion and the valvular functionality . rhabdomyoma . the cardio - ultrasound image ( a ) shows the multiple hyperechoic tumors originating from the ventricular septum ( arrows ) and the ventricular walls . observe the large mass in the right ventricle ( star ) of this asymptomatic 3-day - old newborn boy with tuberous sclerosis . axial fetal ultrasound ( b , 22nd week ) and fetal t2-wi ( c , 24th week ) demonstrate a well - defined , hypoechogenic , solid mass , isointense to lung on mri ( block arrow ) . rhabdomyoma . the cardio - ultrasound image ( a ) shows the multiple hyperechoic tumors originating from the ventricular septum ( arrows ) and the ventricular walls . observe the large mass in the right ventricle ( star ) of this asymptomatic 3-day - old newborn boy with tuberous sclerosis . axial fetal ultrasound ( b , 22nd week ) and fetal t2-wi ( c , 24th week ) demonstrate a well - defined , hypoechogenic , solid mass , isointense to lung on mri ( block arrow ) . the pleuropulmonary blastoma is a primary mesenchymal neoplasm with a very poor prognosis . on prenatal ultrasound and mri , it presents as a solid heterogeneous mass originating from the pleura or the pulmonary parenchyma ( fig . after birth , the tumor may manifest as a benign - appearing , air - filled cystic lesion or as large heterogeneous solid masses . congenital tumors are a unique , heterogeneous group of neoplasms that differ in many aspects from tumors presenting later in life . congenital fetal and neonatal neuroblastomas are associated with a better prognosis than tumors detected later in life , but they remain an exception . although mostly benign , the therapeutic possibilities of most congenital tumors may be limited because of the tumor size , its location or the stage of the pregnancy at the time of diagnosis . moreover , the low incidence and the great histological diversity of these tumors reduces the number of published articles about them . indeed , most of the published reference series are retrospective studies , including cases diagnosed and treated for the last 2030 years and giving mortality values that are far away from actual reality in pediatric hospitals [ 2 , 3 , 1014 ] . although prediction of the possible prognosis and postnatal outcome is complicated , perinatal , anesthetic and surgical care is constantly improving nowadays such that more infants survive in general . in recent years , imaging methods have acquired a relevant role in the diagnosis of fetal tumors . ultrasound is the standard method for fetal evaluation : it is ideal for screening , safe for mother and child , widely accessible and relatively inexpensive , but its effectiveness may be limited by factors such as oligoamnios , maternal body habitus , fetal position and fetal shadow bone artifacts . prenatal mri is increasingly being used as a complementary imaging technique , but its real utility for congenital tumors has not yet been widely evaluated . in general , fetal mri should only be performed if it is considered that additional results might influence the management of the pregnancy and/or the therapeutic approach . recent articles have shown a more accurate evaluation of mri compared with ultrasound in some cases of congenital tumors , including the local anatomical extent of sacrococcygeal and cervicofacial teratomas , which may be underestimated on ultrasound because of fetal bone shadowing [ 18 , 23 ] . mri may also discriminate between cystic and hemorrhagic areas with t1 or t2 * sequences , information that may be relevant for evaluating the therapeutic challenges for sct [ 18 , 23 ] and some intracranial tumors . mri also seems to be better in the evaluation of the deep extension of large tumors that do not always respect anatomical planes , such as hemangiomas or cervicofacial teratomas [ 20 , 32 ] . moreover , mri allows a complete examination of the spinal canal in paravertebrally located tumors , such as neuroblastomas or sct . finally , mri performed during late pregnancy provides excellent anatomical details in a safe intrauterine environment , which may delay the need for immediate postnatal imaging . the detection of a congenital brain tumor in a fetus is followed by serious ethical considerations about the real prognosis , postnatal quality of life , therapy challenges and long - term consequences of the applied therapy . the low incidence , great diversity and different behaviors of congenital brain tumors compared with tumors in older patients mean that published results , obtained from older children and adults , can not be directly extrapolated . overall prognosis is poor and therapeutic options limited , with an evident discrepancy between our diagnostic capacities and our therapy possibilities . a confident identification of the individual tumor types based on imaging is almost impossible , and therefore , in countries in which a legal medical interruption of pregnancy is possible , this diagnosis may lead to a high rate of legal abortions . in conclusion , ultrasound and mri have increased the rate of detection of congenital tumors in prenatal life . during pregnancy and after birth , the main role of imaging is to provide accurate information about the origin and the extent of the tumor , the invasion of adjacent organs , and the presence or absence of metastasis . this information is crucial for reducing the differential diagnosis and to evaluate and optimize the pre- and postnatal therapeutic challenges . a correct knowledge of the possibilities of the different imaging techniques in fetal and neonatal studies would improve all obtainable information , helping the medical team to make the most appropriate decisions about therapy . prenatal ultrasound remains the standard diagnostic method , but fetal mri seems to be an excellent complementary imaging technique , and its real utility should be evaluated in larger , multicentric series .

backgroundthe technical developments of imaging methods over the last 2 decades are changing our knowledge of perinatal oncology . fetal ultrasound is usually the first imaging method used and thus constitutes the reference prenatal study , but mri seems to be an excellent complementary method for evaluating the fetus . the widespread use of both techniques has increased the diagnosis rates of congenital tumors . during pregnancy and after birth , an accurate knowledge of the possibilities and limits of the different imaging techniques available would improve the information obtainable , thus helping the medical team to make the most appropriate decisions about therapy and to inform the family about the prognosis.conclusionin this review article , we describe the main congenital neoplasms , their prognosis and their imaging characteristics with the different pre- and postnatal imaging methods available .

Introduction Extracranial teratomas Neuroblastoma Soft-tissue tumors Central nervous system tumors Renal tumors Hepatic tumors Cardiopulmonary tumors Conclusion

tumors are considered congenital when detected during pregnancy or in the first 3 months of life [ 1 , 2 ] . congenital tumors represent only 1.52% of all pediatric tumors , with a prevalence of 1:12,500 to 1:27,500 live births [ 35 ] . recent studies demonstrate an increase in the incidence of congenital tumors , probably as a consequence of both the generalization of routine prenatal and neonatal medical controls and of the improvement of prenatal imaging techniques . congenital tumors differ in many aspects from tumors presenting in older children , with respect to histology , behavior and prognosis . the most frequent congenital tumor types are shown in table 1 [ 2 , 3 , 58 ] . the diagnosis of a congenital tumor involves a tremendous emotional impact for a family and implies ethical questions about prognosis , therapy possibilities and long - term consequences of the treatment . prognosis is related not only to the histological type , but also to the size and location of the tumor and the presence or not of associated congenital anomalies , which are found in 1520.8% of cases [ 5 , 9 ] . another prognostic factor is the age at which the lesion is detected , with most histological tumor types showing a worse prognosis for cases detected during pregnancy [ 1014 ] . table 1distribution of main congenital tumors [ 2 , 3 , 58]type of tumor%extracranial teratomas23.529%neuroblastomas22.530%soft tissue tumors8.112%cns tumors5.910%leukemia5.9%12.35%renal tumors57.1%hepatic tumors5%cardiopulmonary tumors<3% distribution of main congenital tumors [ 2 , 3 , 58 ] over the last few years , we have witnessed significant technical improvements in prenatal imaging diagnostic tools . ultrasound is usually the first imaging method used , but antenatal magnetic resonance imaging ( mri ) is increasingly being used as a complementary study . standard postnatal imaging methods include conventional x - ray , ultrasound , ct and mri . the purposes of this review are to describe the most frequent types of congenital tumors and their prognosis , to review the main imaging characteristics of this heterogeneous group of neoplasms , and to define the role of the different pre- and postnatal imaging methods in the characterisation of congenital tumors , with special emphasis on prenatal mri . the main location of these tumors is the sacrococcygeal region ( 45% of cases ) , followed by the cervicofacial area ( 28% ) and the thorax ( 11% ) . sacrococcygeal teratomas ( sct ) are one of the most common overall congenital neoplasms , with an incidence of 1:40,000 infants and a female predominance of 4:1 [ 15 , 16 ] . they arise from rests of pluripotent cells at the caudal end of the notochord , originating from the coccyx . of all teratomas , these tumors are associated with the highest incidence of malignancy ( 10% ) and of associated anomalies ( 1015% ) . 1a , b ) with a high incidence of complications , such as polyhydramnios , fetal cardiac failure , fetal hydrops , tumoral hemorrhage and prematurity , which are related to the tumor size and to the effect of the intrapelvic and intra - abdominal tumor components over the developing organs . the images clearly illustrate the extension of the tumor into the pelvis , the abdomen and the spinal canal ( block arrow ) . the ct topogram ( c ) and the axial nect image ( d ) demonstrate the heterogenicity of the tumor , with calcifications ( white arrows ) , fat , solid and liquid ( star ) components extracranial teratomas . the images clearly illustrate the extension of the tumor into the pelvis , the abdomen and the spinal canal ( block arrow ) . the ct topogram ( c ) and the axial nect image ( d ) demonstrate the heterogenicity of the tumor , with calcifications ( white arrows ) , fat , solid and liquid ( star ) components cervicofacial teratomas ( fig . cervical teratomas in utero may not only cause hydrops secondary to fetal swallowing disturbances , but also fetal pulmonary hypoplasia . the main role of pre- and postnatal imaging for teratomas is to recognize any associated anomalies and to determine the full extent of the mass for prognosis and evaluation of surgical planning . on fetal ultrasound , teratomas are seen as large , heterogeneous masses due to their different tissue components . in some cases of sct , an accurate ultrasound evaluation may be insufficient because of shadowing by the fetal pelvic bones , which may cause an underestimation of the tumor . fetal mri is therefore considered the method of choice for evaluating the extent of the internal mass ( fig . indeed , the method can serve as a roadmap before surgery , as ultrasound images are sometimes difficult to interpret for clinicians and may fail to provide a good overview of the limits of the relation between the tumor and the surrounding anatomy . the lesion was initially considered to be a renal tumor , but histology revealed neuroblastoma neuroblastoma . the neuroblastoma is the second most common pediatric malignancy overall , after teratoma , in most published series [ 3 , 5 ] and the most common malignancy of the first month of life ( 3050% ] . the prognosis is excellent , with a survival rate of 8890% [ 1 , 24 ] , metastases are rare and spontaneous involution in utero has been described . metaiodobenzylguanidine ( mibg - i ) scintigraphy is an excellent method for determining the presence of metastases or tumor recidivation , but 30% of neonatal tumors are mibg - negative . in this classification , the extent of locoregional disease is determined by the absence ( l1 ) or presence ( l2 ) of imaging - defined risk factors , with stage m used for widely disseminated disease and stage ms for metastatic disease limited to liver , skin and bone narrow in children younger than 18 months . the main role of imaging methods for both fetal and neonatal neuroblastoma is then to determine the origin of the mass , its locoregional extent and the presence or not of metastases for staging before therapy . they account for a significant number of prenatally diagnosed neoplasms , but because most of them are small or absent at birth , they are often not included in the register of congenital tumors . the most frequent location is the head and neck ( 60% ) ( fig . coronal ( a ) and axial ( b ) fetal t2-haste mri ( 21st week ) demonstrate the cephalic , but completely extracranial heterogeneous mass ( star ) . coronal ( a ) and axial ( b ) fetal t2-haste mri ( 21st week ) demonstrate the cephalic , but completely extracranial heterogeneous mass ( star ) . axial cect image ( e ) shows the large , infiltrating left cervical muscular mass , with intense enhancement after contrast medium administration the notion of congenital hemangiomas has been introduced recently , differentiating between non - involuting congenital hemangiomas , or nich , and rapidly involuting congenital hemangiomas , or rich . large congenital tumors may cause compression of vital structures or cardiovascular complications , and in these cases , mri is the method of choice to determine the mass extension and its relationship to the adjacent anatomical structures ( fig . hemangiomas are usually hyperintense on t2-weighted and isointense on t1-weighted images related to muscle , with prominent draining veins and intense enhancement after contrast medium administration , but no perilesional edema . they present an increment of the size and the number of lesions during the first year of life with later regression . 3d ) , with low signal intensity on both t1-weighted and t2-weighted images , infiltration of the fat and muscle , and intense enhancement after i.v . the tumor represents > 50% of all soft tissue sarcomas in children , but its congenital presentation is extremely rare . it can arise in any anatomical region of the body ( except bone ) whether there is skeletal muscle or not , but most often it presents in the head and neck ( 2840% ) and in the genitourinary region ( 20% ) . this anomaly now allows correct differentiation between rhabdoid tumors usually associated with a bad prognosis and rhabdomyosarcomas . the main role of imaging in the case of congenital soft tissue tumors is to determine the extent of the mass and to identify the invasion of adjacent structures in order to optimize the surgical approach . they represent only 0.51.9% of all pediatric brain tumors , but are responsible for 520% of deaths secondary to neoplasms in this age group [ 6 , 38 ] . most congenital tumors ( about 60% ) are supratentorial , arising from the pineal gland , the suprasellar area or the cerebral hemispheres . increased intracranial pressure signs are not often observed because of the great capacity for accommodation to the increased volume in the fetus and in neonates with opened sutures . histological type and mortality rate of main cns tumors [ 3741 ] teratoma is the most common congenital brain tumor and represents 26.6 to 48% [ 3841 ] ( table 2 ) . congenital teratomas are predominantly supratentorial , with the cerebral hemispheres being the main primary site , followed by the third ventricle and the pineal region . on ultrasound , they are seen as typically large , mid - line heterogeneous tumors with solid areas replacing much of the brain . the cerebral hemispheres are the main primary site , followed by the optic nerve , the thalamus and the mesencephalus . on mri , they present as homogeneous masses on t1-weighted images , with the central tumor areas hypointense compared with grey matter on t2-weighted images . complete surgical resection is curative , but intraoperative hemorrhage is common and may have serious consequences . they are often associated with marked vasogenic edema and may be hyperdense on ct because of the high cellularity . axial t2-weighted fetal mri ( 34th week ) ( a ) shows an intraventricular , solid mass in the posterior horn of the right lateral ventricle ( white arrow ) . postnatal axial t2-weighted image ( b ) 3 days after birth reveals an increase in the tumor size . axial t2-weighted fetal mri ( 34th week ) ( a ) shows an intraventricular , solid mass in the posterior horn of the right lateral ventricle ( white arrow ) . postnatal axial t2-weighted image ( b ) 3 days after birth reveals an increase in the tumor size . congenital tumors have a poor prognosis as a consequence of the rapid tumor growth , with early extension throughout the csf pathways . the diagnosis of fetal and neonatal brain tumors is associated with high mortality rates ( table 2 ) . these technical improvements have opened up exciting perspectives , suggesting the possibility of differentiating between histological tumor types [ 20 , 45 ] , but preliminary reports have not yet been confirmed for congenital brain tumors . the main role of imaging studies is still to determine the extent of the tumor in order to evaluate therapy challenges and to identify potentially curable tumors , such as plexus papillomas , differentiating them from rapidly fatal ones . they are mostly benign , with the most frequent histological types being mesoblastic nephroma ( msn ) , nephroblastomatosis and the multilocular cystic nephroma . observe a similar but smaller left renal lesion ( white arrow ) in this 10-week - old boy with multiple congenital tumors ( same patient as in fig . observe a similar but smaller left renal lesion ( white arrow ) in this 10-week - old boy with multiple congenital tumors ( same patient as in fig . it is found incidentally in 1% of normal children at post - mortem studies and may cause a wilms tumor in 3040% of the cases ( fig . the role of imaging studies for congenital renal tumors is to determine the extent of the mass and its relationship to the renal vascular structures before surgical therapy . hepatic tumors comprise 5% of all congenital neoplasms ( table 1 ) . the infantile hepatic hemangioma , frequently referred to as hepatic hemangioendothelioma , is the most common congenital vascular hepatic tumor [ 13 , 49 ] . most lesions are hypodense compared with the rest of the liver on non - enhanced ct . on mri , the lesions usually show low t1 and high t2 signal intensities , but may be heterogeneous because of hemorrhage and calcification , with variable enhancement patterns . necrotic or thrombotic regions show a heterogeneous signal both before and after gadolinium administration . solid tumors have been described , but they are atypical [ 47 , 51 ] . the most extensive published series about congenital hepatic tumors describes a poor prognosis for hepatoblastomas , with common early metastases and high mortality rates , but this series probably includes some cases of congenital rhabdoid tumors , an aggressive lesion previously classified as hepatoblastoma with negative -fetoprotein . as mentioned elsewhere in this article , immunochemistry tests for ini1/baf47 protein , which is abnormally low in all rhabdoid tumors , have recently allowed a correct differentiation between the entities [ 36 , 37 , 53 , 54 ] , which present similar findings on imaging studies . the main role of imaging methods for congenital hepatic tumors is to evaluate the anatomical extent of the tumor and to clarify the relationship with hepatic lobar anatomy before surgical planning . the heart is one of the most common organs of origin of congenital tumors , with a reported incidence of 0.14 to 0.2% . the incidence has been increasing in recent years because of the technical advances of imaging studies , especially ultrasound cardiography and cardiac mri . the most frequent cardiac tumor is the myocardial rhabdomyoma ( 78% ) , followed by the pericardial teratoma ( 18% ) and the cardiac fibroma ( 12% ) . indeed , the detection of multiple cardiac rhabdomyomas on prenatal ultrasound is considered sufficient to establish the diagnosis of tuberous sclerosis and makes mri evaluation of the fetal cns mandatory . surgical intervention is only recommended in cases of persistent arrhythmias or severe hemodynamic compromise because rhabdomyomas usually increase in size until late pregnancy and then tend to regress progressively during the first year of life . cardio - ultrasound is the imaging method of choice and provides a dynamic evaluation of the myocardial wall motion and the valvular functionality . axial fetal ultrasound ( b , 22nd week ) and fetal t2-wi ( c , 24th week ) demonstrate a well - defined , hypoechogenic , solid mass , isointense to lung on mri ( block arrow ) . axial fetal ultrasound ( b , 22nd week ) and fetal t2-wi ( c , 24th week ) demonstrate a well - defined , hypoechogenic , solid mass , isointense to lung on mri ( block arrow ) . after birth , the tumor may manifest as a benign - appearing , air - filled cystic lesion or as large heterogeneous solid masses . congenital tumors are a unique , heterogeneous group of neoplasms that differ in many aspects from tumors presenting later in life . congenital fetal and neonatal neuroblastomas are associated with a better prognosis than tumors detected later in life , but they remain an exception . although mostly benign , the therapeutic possibilities of most congenital tumors may be limited because of the tumor size , its location or the stage of the pregnancy at the time of diagnosis . indeed , most of the published reference series are retrospective studies , including cases diagnosed and treated for the last 2030 years and giving mortality values that are far away from actual reality in pediatric hospitals [ 2 , 3 , 1014 ] . although prediction of the possible prognosis and postnatal outcome is complicated , perinatal , anesthetic and surgical care is constantly improving nowadays such that more infants survive in general . in recent years , imaging methods have acquired a relevant role in the diagnosis of fetal tumors . ultrasound is the standard method for fetal evaluation : it is ideal for screening , safe for mother and child , widely accessible and relatively inexpensive , but its effectiveness may be limited by factors such as oligoamnios , maternal body habitus , fetal position and fetal shadow bone artifacts . prenatal mri is increasingly being used as a complementary imaging technique , but its real utility for congenital tumors has not yet been widely evaluated . in general , fetal mri should only be performed if it is considered that additional results might influence the management of the pregnancy and/or the therapeutic approach . recent articles have shown a more accurate evaluation of mri compared with ultrasound in some cases of congenital tumors , including the local anatomical extent of sacrococcygeal and cervicofacial teratomas , which may be underestimated on ultrasound because of fetal bone shadowing [ 18 , 23 ] . mri may also discriminate between cystic and hemorrhagic areas with t1 or t2 * sequences , information that may be relevant for evaluating the therapeutic challenges for sct [ 18 , 23 ] and some intracranial tumors . mri also seems to be better in the evaluation of the deep extension of large tumors that do not always respect anatomical planes , such as hemangiomas or cervicofacial teratomas [ 20 , 32 ] . moreover , mri allows a complete examination of the spinal canal in paravertebrally located tumors , such as neuroblastomas or sct . finally , mri performed during late pregnancy provides excellent anatomical details in a safe intrauterine environment , which may delay the need for immediate postnatal imaging . the detection of a congenital brain tumor in a fetus is followed by serious ethical considerations about the real prognosis , postnatal quality of life , therapy challenges and long - term consequences of the applied therapy . the low incidence , great diversity and different behaviors of congenital brain tumors compared with tumors in older patients mean that published results , obtained from older children and adults , can not be directly extrapolated . a confident identification of the individual tumor types based on imaging is almost impossible , and therefore , in countries in which a legal medical interruption of pregnancy is possible , this diagnosis may lead to a high rate of legal abortions . in conclusion , ultrasound and mri have increased the rate of detection of congenital tumors in prenatal life . during pregnancy and after birth , the main role of imaging is to provide accurate information about the origin and the extent of the tumor , the invasion of adjacent organs , and the presence or absence of metastasis . this information is crucial for reducing the differential diagnosis and to evaluate and optimize the pre- and postnatal therapeutic challenges . a correct knowledge of the possibilities of the different imaging techniques in fetal and neonatal studies would improve all obtainable information , helping the medical team to make the most appropriate decisions about therapy . prenatal ultrasound remains the standard diagnostic method , but fetal mri seems to be an excellent complementary imaging technique , and its real utility should be evaluated in larger , multicentric series .

the treatment of heart failure patients has been found to entail tremendous medical costs in many countries [ 1 , 2 ] . although heart failure treatment has improved greatly , the 5-year mortality rate continues to be 4050 % [ 2 , 3 ] . myocardial infarction ( mi ) is recognized as a prime cause of systolic heart failure [ 4 , 5 ] . an acute mi will result in changes in left ventricular ( lv ) size , shape , and wall thickness that involve the infarcted and non - infarcted regions ; these changes are referred to as lv remodeling [ 69 ] . various myocardial disorders , including dilated cardiomyopathy , also cause severe lv dilatation with heart failure . a number of non - invasive cardiovascular imaging modalities are currently used to assess the severity of heart failure . among them , molecular imaging has recently been a focus of interest on account of its ability to characterize tissues involved in myocardial disorders at different levels : molecular , subcellular , and cellular [ 1012 ] . molecular imaging promises to not only deepen our understanding of already known biological processes , but also to uncover unknown molecular and cellular events that are at the center of the initiation and evolution of disease . compared to traditional in vitro tissue / cell culture and ex vivo animal studies , molecular imaging permits the non - invasive and repetitive imaging of targeted biological processes at both cellular and subcellular levels in live organs . molecular imaging thus offers a means to visualize specific abnormal biological processes for diagnosis and treatment . radionuclide imaging , using either positron emission tomography ( pet ) or single - photon emission computed tomography ( spect ) , has been the modality most commonly used for human molecular imaging , mainly due to its quite high sensitivity to radiolabeled ligands with nano- or micro - order levels compared to other methods of molecular imaging . even though molecular imaging has low spatial and temporal resolutions and high background activity and radiation , radionuclide imaging has been safely used in a wide range of clinical applications , including evaluations of various disease processes . as a result of advances in vivo biochemical imaging using radionuclide techniques , the modulation of functional and electrophysiological properties of the heart by the autonomic nervous system has become a focus of interest in the field of cardiovascular research . cardiac neuronal function is compromised in various cardiac diseases , including congestive heart failure , ischemia , arrhythmia , and some types of cardiomyopathy . tracer approaches are considered uniquely suited for radionuclide imaging - based in vivo characterization of neuronal function in the myocardium . their major transmitters are norepinephrine and acetylcholine , respectively , which define the stimulatory and inhibitory physiological effects of each system . sympathetic innervation originates mainly from the right and left stellate ganglia , which provide the sympathetic nerves to form the cardiac plexus of the heart . the sympathetic nerve fibers travel parallel to the vascular structures on the epicardial surface of the heart and then penetrate the underlying myocardium in a manner similar to that seen in the coronary vessels . as regard to tissue levels of norepinephrine , the mammalian heart is characterized by dense adrenergic innervation with a norepinephrine concentration gradient from the atria to the base of the heart and from the base to the apex of the left ventricle [ 15 , 16 ] . parasympathetic innervation , by contrast , originates from the medulla and passes through the right and left vagal nerves , which further divide into the superior and inferior cardiac nerves . parasympathetic nerve fibers primarily modulate sinoatrial nodal and atrioventricular nodal function and innervate the atria , whereas , vagal fibers to the ventricles are rather sparse . these autonomic nervous systems are involved in the synthesis and storage of neurotransmitters and their release , reuptake , metabolism , and interaction with presynaptic and postsynaptic receptor sites . there are a number of radiotracers that can be used to probe each step of autonomic neuronal functions . table 1 shows representative radiotracers used to probe sympathetic and parasympathetic nerve functions , both presynaptic and postsynaptic . figure 1 illustrates a number of adrenergic presynaptic and postsynaptic ( receptor ) functions , indicating the radiopharmaceuticals that have been used to probe those functions . [ 1820 ] used radiolabeled norepinephrine for the assessment of sympathetic nerve function and norepinephrine kinetics.table 1radiotracers used for the evaluation of autonomic nervous system functionssympathetic nervesparasympathetic nervespresynaptic i - metaiodobenzylguanidine ( mibg ) i - iodobenzoversamicol c - hydroxyephedrine f - fluorobenzyl - benzovesamicol f - metaraminol f - dopamine c - threohydroxyepinephrine c - epinephrinepostsynaptici - iodocyanopindolol ( icyp ) i - quinuclidinyl benzylate ( qnb ) c - practolol c - methyl qnb c - propranolol c - cgp 12177 c - prazocinfig . 1schema of myocardial nerve terminals and various radioligands used to probe adrenergic functions ( color figure online ) radiotracers used for the evaluation of autonomic nervous system functions schema of myocardial nerve terminals and various radioligands used to probe adrenergic functions ( color figure online ) among these radiopharmaceuticals , the norepinephrine analog metaiodobenzylguanidine ( mibg ) has been widely used in experimental and clinical studies for sympathetic nerve imaging . in the early 1980s , university of michigan researchers developed i mibg as a norepinephrine analog for the selective mapping of sympathetic nerve endings in the heart [ 21 , 22 ] . it is taken up by sympathetic nerves in a similar manner to norepinephrine , but is not metabolized . most mibg , following its administration , is actively taken up into neuronal vesicles by means of a na - dependent specific process ( uptake 1 ) , and the remaining mibg enters the nerve terminals by passive diffusion ( uptake 2 ) [ 23 , 24 ] . although uptake 1 accounts for most mibg distribution in the myocardium , uptake 2 may play a minor role [ 25 , 26 ] . mibg is stored by the neurons and is released along with endogenous norepinephrine upon nerve stimulation , but it has a low affinity for postsynaptic adrenergic receptors . generally , mibg distribution correlates with that of tissue norepinephrine , but the ability of sympathetic nerve terminals to take up catecholamine is a more sensitive index of nerve function and viability than catecholamine content . based on the mechanisms of uptake of this spect agent , it is recommended to temporarily discontinue medications and substrates that may interfere with norepinephrine uptake , including opioids , antidepressants , reserpine , and others . i - labeled mibg is used in clinical settings , as it provides better myocardial images than the i - labeled compound . a clinical study showed that increased sympathetic tone may be associated with increased washout of mibg [ 23 , 29 ] . following the administration of 111222 mbq ( 36 mci ) of i - labeled mibg at rest , myocardial images are usually obtained twice ( at 10 min and 4 h ) to calculate the myocardial uptake and washout . sometimes only the delayed images are obtained to estimate delayed myocardial uptake as an index of adrenergic neuron function . in each acquisition , the planar images are obtained in the anterior position to assess the global uptake of mibg in the myocardium . the general acquisition time is 35 min for planar imaging and 1530 min for spect imaging . a suitable collimator ( either a medium - energy collimator or a i collimator ) should be used for i imaging . some low - energy collimators are also suitable for i energy , showing higher sensitivity than medium - energy collimators . resting administration is most commonly used for the resting state [ 28 , 30 ] . the radiation dose with the use of 111 mbq of mibg is 1.2 mgy to the myocardium and 7.9 mgy to the liver . to minimize radiation to the thyroid gland , thyroid blocking is recommended . although i - labeled mibg yields high - quality images of myocardial neuronal function , high activity in the liver may be superimposed on myocardial activity in planar images , and thus affect the interpretation of myocardial distribution , particularly in the inferior region . to minimize such superimposition , spect is preferred for the assessment of regional mibg distribution . planar imaging is used to assess the global uptake of the tracer in the myocardium . two regions of interest are considered : an irregular region in the whole myocardium and a rectangular region in the upper mediastinum to measure the myocardium - to - mediastinum count rate , which is commonly used as an index of myocardial uptake of mibg ( fig . 2 ) [ 28 , 30 ] . a semi - automatic algorithm for calculating the heart - to - mediastinum ( h / m ) ratio on mibg imaging was recently introduced to obtain highly reproducible values . however , there is no established spect - based method for estimating or scoring mibg uptake.fig . 2anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart in reports concerning imaging standards and a relevant japanese database , the normal myocardium - to - mediastinum count rate ranged from 2.0 to 2.7 for the early scan and from 2.1 to 2.9 for the late scan [ 32 , 33 ] , but these values seemed to be dependent on the collimator used for acquisition and the specific activity of the mibg . nakajima and colleagues , using different energy collimators , recently developed a calibration phantom as a tool for standardizing mibg h / m ratio calculation for a normal - values database . the washout from the early to the late scan ranged from 21 to 33 % . in a 1993 clinical study , the normal distribution of mibg in the myocardium was not quite homogeneous , showing a slight reduction in the inferior region . this heterogeneity seems to be more enhanced with age . in view of this physiological heterogeneity of the tracer distribution in the myocardium , mibg images should be interpreted cautiously . it is well - known that adrenergic dysfunction plays a key role in heart failure , and the plasma catecholamine level has been recognized as a valuable prognostic tool . basic studies have indicated that an increased neuronal release of norepinephrine and decreased efficiency of norepinephrine reuptake both contribute to increased cardiac adrenergic drive in congestive heart failure . in addition , decreased vesicular leakage of norepinephrine limits the increase in its cardiac turnover . thus , analyses of norepinephrine kinetics have a key role in the assessment of the severity of congestive heart failure . there are a number of reports showing a reduction of mibg retention in patients with idiopathic dilated cardiomyopathy [ 3740 ] . first indicated that the h / m ratio for mibg is correlated with the plasma norepinephrine level and the left ventricular ejection fraction ( lvef ) in patients with dilated cardiomyopathy . their mibg results showed that uptake and vesicular storage of norepinephrine were reduced in these patients , similar to the findings in experimental heart failure . a number of papers have highlighted the h / m ratio and/or the washout rate of mibg as new parameters of heart failure severity and independent prognostic indicators [ 4150 ] . merlet et al . , in 1999 , were the first to indicate the prognostic value of the h / m ratio in patients with heart failure . thereafter , many prognostic studies using this imaging technique were performed in european countries and in japan ; there thus emerged a close association between the h / m ratio and/or the washout rate of mibg as markers of impaired cardiac adrenergic innervation and mortality in patients with heart failure [ 4150 ] . a meta - analysis of 18 studies including 1,755 patients with heart failure confirmed that heart failure patients showing a reduced h / m ratio on late mibg imaging had a worse prognosis as compared to those with a normal h / m ratio . a similar meta - analysis was reported from japan , indicating that both decreased mibg uptake and its increased washout are indicative of a poor prognosis in heart failure patients . implantable cardioverter - defibrillator ( icd ) treatment has become well established for preventive use in patients at high risk of arrhythmic death . autonomic dysfunction assessed by mibg is thought to play an important role in the detection of high - risk ventricular arrhythmia [ 14 , 53 , 54 ] . in particular , mibg can identify areas of denervation hypersensitivity which may be likely to cause ventricular arrhythmias . a number of pilot studies have indicated mibg as a potential predictor of ventricular arrhythmias in patients with an icd [ 5560 ] . nishisato et al . , in a prospective study using both mibg and resting myocardial perfusion imaging in patients who received icd treatment , showed that icd discharge was documented in 30 of the total 60 patients ( 50 % ) . they reported that among the various clinical and scintigraphic variables , low h / m ratio on mibg and a large perfusion defect were the most powerful predictors of icd discharge in a cox multivariate analysis . this report was the first to suggest an incremental benefit for the assessment of sympathetic nerve function in combination with myocardial perfusion , and a role for this combined assessment in the risk stratification of patients who may need prophylactic icd therapy . however , it remains unknown whether simple perfusion imaging is enough , or whether both mibg and perfusion imaging are required to predict such fatal arrhythmias . these authors provided consistent findings showing that i - labeled mibg may predict icd discharge and sudden cardiac death independently of conventional parameters . examined the ability of mibg imaging to predict sudden cardiac death in comparison with ecg parameters such as t - wave alteration . they found that the mibg washout rate was independent of lvef in risk analyses of patients with heart failure . a multivariate cox analysis suggested that the mibg washout rate and the lvef but not ecg parameters were significant and independent predictors of sudden cardiac death . jacobson et al . reported the results of a prospective mibg imaging trial of 961 patients with heart failure recruited at 96 sites in north america and europe . their admire - hf study enrolled patients with new york heart association ( nyha ) class ii and iii heart failure and lvef 35 % and revealed that an h / m ratio of < 1.6 measured at 4 h after an mibg administration provided prognostic data beyond that available from the lvef , b - type natriuretic peptide assay , and nyha class at the time of enrollment . their multicenter prospective study confirmed the adrenergic neuronal functional parameter obtained by mibg scan as an important parameter for predicting sudden death , independent of the commonly used lv functional parameters . it is important to determine whether adrenergic function can predict the recovery of lv function or an improvement in outcome after treatment in patients with severe heart failure . there are a number of studies indicating that cardiac mibg uptake improves after -blocker or other therapy [ 6371 ] . both showed improvements of mibg uptake and washout after -blocker therapy in patients with dilated cardiomyopathy . the h / m ratio on the delayed images was a good predictor of the response to the -blocker therapy . takeishi et al . also reported improvement of mibg uptake in relation to lvef recovery after treatment with angiotensin - converting enzyme inhibitor in patients with congestive heart failure . gerson et al . showed that the h / m ratio improved significantly after carvedilol therapy , especially in patients with an h / m ratio < 1.40 . reported favorable changes in symptoms , nyha functional class , lv function , and the h / m ratio on mibg imaging following treatment with the 1-blocker metoprolol . observed a therapeutic effect of carvedilol on mibg parameters and lv remodeling in patients with dilated cardiomyopathy . these data indicate that mibg imaging may provide valuable information for the selection and optimization of treatment for congestive heart failure . pet offers great advantages in terms of higher spatial resolution and higher sensitivity with better quantification of tracer concentration compared to the commonly performed spect ( fig . 3 ) . with the use of suitable tracer kinetic models , various molecular as well as functional parameters have been estimated in vivo . various sympathetic neuronal pet tracers have been introduced which closely resemble the endogenous neurotransmitters , and these may allow more detailed analyses of neuronal signaling than mibg can [ 72 , 73].fig . 3 c - hydroxyephedrine ( he d ) pet and mibg spect of a normal subject . the he d pet obtained better - quality images in the myocardium as compared to the mibg spect ( color figure online ) c - hydroxyephedrine ( he d ) pet and mibg spect of a normal subject . the he d pet obtained better - quality images in the myocardium as compared to the mibg spect ( color figure online ) c - epinephrine is a radiolabeled true neurotransmitter , and the uptake and storage of this tracer resemble those of norepinephrine [ 74 , 75 ] . c - phenylephrine is another pet tracer that is trapped in neuronal vesicles and metabolized by neuronal monoamine oxidase ( mao ) . c - hydroxyephedrine ( he d ) is the most widely used pet tracer for cardiac neuronal imaging . it has high affinity for presynaptic neuronal catecholamine transporter ( uptake 1 ) without being metabolized by mao or catechol - o - methyl - transferase [ 77 , 78 ] . denervation and reinnervation have been investigated using he d in various disorders , such as diabetes [ 7981 ] and heart transplantation [ 8284 ] . a study of patients with heart failure suggested that they had reduced he d uptake , particularly in the apical and inferoapical regions ( fig . 4 ) . in a study of heart failure patients before heart transplantation , the he d uptake in the myocardium closely correlated with uptake 1 and the norepinephrine content , confirming the value of he d imaging as a marker of sympathetic neuronal imaging .fig . 4a series of short - axis slices of mibg spect and he d pet images of a patient with heart failure . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a series of short - axis slices of mibg spect and he d pet images of a patient with heart failure . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a reduction of he d uptake may be a prognostic marker for heart failure patients . described he d reduction as an independent risk factor , together with left ventricular end diastolic volume and peak vo2 . another study compared he d retention with oxidative metabolism , but found no significant association in heart failure patients . most of these studies using he d seem to give findings similar to those that used mibg imaging , suggesting autonomic function as an important new parameter for risk analysis and treatment monitoring , independent of other imaging and/or biomarkers . since he d pet provides higher spatial resolution and has a higher quantitative capability , it may hold promise for assessing patients with fatal arrhythmias . a global reduction in he d uptake in the myocardium was not observed in patients with brugada syndrome or those with long qt syndrome . since most of these studies are preliminary , more clinical studies with high - resolution pet are warranted . , 1 adrenoreceptor plays an important role in the regulation of myocardial cell function . compared to the many presynaptic radioligands used for imaging , there are only a few radioligands that are suitable for postsynaptic neuron imaging and probing of neuronal functions . c - cgp 12177 is a non - selective hydrophilic -receptor antagonist that provides high - quality pet images with low background activity ( fig . 5 ) . two sequential pet images after an injection of high - specific - activity tracer followed by low - specific - activity tracer allow quantification of -receptor density using a graphical analysis method . two other radiolabeled ligands were introduced for adrenoreceptor study : c - cgp 12388 for -receptor imaging , and c gb-67 for 1-receptor imaging .fig . 5coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) cgp 12177 has been used to demonstrate -receptor downregulation in many heart failure patients [ 9698 ] . our data suggested that myocardial -receptor density is inversely correlated with the mibg washout rate and positively correlated with a delayed h / m ratio , indicating that decreased -receptor density was due to the increased presynaptic sympathetic tone . these results suggest that myocardial -receptor density may be directly associated with the beneficial response obtained by -blocker treatment in heart failure patients . in addition , there was a close relationship between myocardial -receptor density assessed by c - cgp 12177 pet and the improvement in left ventricular function after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy ( fig . we thus suggested that patients with advanced downregulation of myocardial -receptor density have higher resting adrenergic drive and may derive greater benefits from the anti - adrenergic effects of -blocker treatment . further studies with many more patients with heart failure are warranted to test this finding.fig . 6correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy it is taken up by sympathetic nerves in a similar manner to norepinephrine , but is not metabolized . most mibg , following its administration , is actively taken up into neuronal vesicles by means of a na - dependent specific process ( uptake 1 ) , and the remaining mibg enters the nerve terminals by passive diffusion ( uptake 2 ) [ 23 , 24 ] . although uptake 1 accounts for most mibg distribution in the myocardium , uptake 2 may play a minor role [ 25 , 26 ] . mibg is stored by the neurons and is released along with endogenous norepinephrine upon nerve stimulation , but it has a low affinity for postsynaptic adrenergic receptors . generally , mibg distribution correlates with that of tissue norepinephrine , but the ability of sympathetic nerve terminals to take up catecholamine is a more sensitive index of nerve function and viability than catecholamine content . based on the mechanisms of uptake of this spect agent , it is recommended to temporarily discontinue medications and substrates that may interfere with norepinephrine uptake , including opioids , antidepressants , reserpine , and others . i - labeled mibg is used in clinical settings , as it provides better myocardial images than the i - labeled compound . a clinical study showed that increased sympathetic tone may be associated with increased washout of mibg [ 23 , 29 ] . following the administration of 111222 mbq ( 36 mci ) of i - labeled mibg at rest , myocardial images are usually obtained twice ( at 10 min and 4 h ) to calculate the myocardial uptake and washout . sometimes only the delayed images are obtained to estimate delayed myocardial uptake as an index of adrenergic neuron function . in each acquisition , the planar images are obtained in the anterior position to assess the global uptake of mibg in the myocardium . the general acquisition time is 35 min for planar imaging and 1530 min for spect imaging . a suitable collimator ( either a medium - energy collimator or a i collimator ) should be used for i imaging . some low - energy collimators are also suitable for i energy , showing higher sensitivity than medium - energy collimators . resting administration is most commonly used for the resting state [ 28 , 30 ] . the radiation dose with the use of 111 mbq of mibg is 1.2 mgy to the myocardium and 7.9 mgy to the liver . to minimize radiation to the thyroid gland , thyroid blocking is recommended . although i - labeled mibg yields high - quality images of myocardial neuronal function , high activity in the liver may be superimposed on myocardial activity in planar images , and thus affect the interpretation of myocardial distribution , particularly in the inferior region . to minimize such superimposition , spect is preferred for the assessment of regional mibg distribution . planar imaging is used to assess the global uptake of the tracer in the myocardium . two regions of interest are considered : an irregular region in the whole myocardium and a rectangular region in the upper mediastinum to measure the myocardium - to - mediastinum count rate , which is commonly used as an index of myocardial uptake of mibg ( fig . 2 ) [ 28 , 30 ] . a semi - automatic algorithm for calculating the heart - to - mediastinum ( h / m ) ratio on mibg imaging was recently introduced to obtain highly reproducible values . however , there is no established spect - based method for estimating or scoring mibg uptake.fig . 2anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart in reports concerning imaging standards and a relevant japanese database , the normal myocardium - to - mediastinum count rate ranged from 2.0 to 2.7 for the early scan and from 2.1 to 2.9 for the late scan [ 32 , 33 ] , but these values seemed to be dependent on the collimator used for acquisition and the specific activity of the mibg . nakajima and colleagues , using different energy collimators , recently developed a calibration phantom as a tool for standardizing mibg h / m ratio calculation for a normal - values database . the washout from the early to the late scan ranged from 21 to 33 % . in a 1993 clinical study , the normal distribution of mibg in the myocardium was not quite homogeneous , showing a slight reduction in the inferior region . this heterogeneity seems to be more enhanced with age . in view of this physiological heterogeneity of the tracer distribution in the myocardium , mibg images should be interpreted cautiously . it is well - known that adrenergic dysfunction plays a key role in heart failure , and the plasma catecholamine level has been recognized as a valuable prognostic tool . basic studies have indicated that an increased neuronal release of norepinephrine and decreased efficiency of norepinephrine reuptake both contribute to increased cardiac adrenergic drive in congestive heart failure . in addition , decreased vesicular leakage of norepinephrine limits the increase in its cardiac turnover . thus , analyses of norepinephrine kinetics have a key role in the assessment of the severity of congestive heart failure . there are a number of reports showing a reduction of mibg retention in patients with idiopathic dilated cardiomyopathy [ 3740 ] . first indicated that the h / m ratio for mibg is correlated with the plasma norepinephrine level and the left ventricular ejection fraction ( lvef ) in patients with dilated cardiomyopathy . their mibg results showed that uptake and vesicular storage of norepinephrine were reduced in these patients , similar to the findings in experimental heart failure . a number of papers have highlighted the h / m ratio and/or the washout rate of mibg as new parameters of heart failure severity and independent prognostic indicators [ 4150 ] . merlet et al . , in 1999 , were the first to indicate the prognostic value of the h / m ratio in patients with heart failure . thereafter , many prognostic studies using this imaging technique were performed in european countries and in japan ; there thus emerged a close association between the h / m ratio and/or the washout rate of mibg as markers of impaired cardiac adrenergic innervation and mortality in patients with heart failure [ 4150 ] . a meta - analysis of 18 studies including 1,755 patients with heart failure confirmed that heart failure patients showing a reduced h / m ratio on late mibg imaging had a worse prognosis as compared to those with a normal h / m ratio . a similar meta - analysis was reported from japan , indicating that both decreased mibg uptake and its increased washout are indicative of a poor prognosis in heart failure patients . implantable cardioverter - defibrillator ( icd ) treatment has become well established for preventive use in patients at high risk of arrhythmic death . autonomic dysfunction assessed by mibg is thought to play an important role in the detection of high - risk ventricular arrhythmia [ 14 , 53 , 54 ] . in particular , mibg can identify areas of denervation hypersensitivity which may be likely to cause ventricular arrhythmias . a number of pilot studies have indicated mibg as a potential predictor of ventricular arrhythmias in patients with an icd [ 5560 ] . nishisato et al . , in a prospective study using both mibg and resting myocardial perfusion imaging in patients who received icd treatment , showed that icd discharge was documented in 30 of the total 60 patients ( 50 % ) . they reported that among the various clinical and scintigraphic variables , low h / m ratio on mibg and a large perfusion defect were the most powerful predictors of icd discharge in a cox multivariate analysis . this report was the first to suggest an incremental benefit for the assessment of sympathetic nerve function in combination with myocardial perfusion , and a role for this combined assessment in the risk stratification of patients who may need prophylactic icd therapy . however , it remains unknown whether simple perfusion imaging is enough , or whether both mibg and perfusion imaging are required to predict such fatal arrhythmias . these authors provided consistent findings showing that i - labeled mibg may predict icd discharge and sudden cardiac death independently of conventional parameters . examined the ability of mibg imaging to predict sudden cardiac death in comparison with ecg parameters such as t - wave alteration . they found that the mibg washout rate was independent of lvef in risk analyses of patients with heart failure . a multivariate cox analysis suggested that the mibg washout rate and the lvef but not ecg parameters were significant and independent predictors of sudden cardiac death . jacobson et al . reported the results of a prospective mibg imaging trial of 961 patients with heart failure recruited at 96 sites in north america and europe . their admire - hf study enrolled patients with new york heart association ( nyha ) class ii and iii heart failure and lvef 35 % and revealed that an h / m ratio of < 1.6 measured at 4 h after an mibg administration provided prognostic data beyond that available from the lvef , b - type natriuretic peptide assay , and nyha class at the time of enrollment . their multicenter prospective study confirmed the adrenergic neuronal functional parameter obtained by mibg scan as an important parameter for predicting sudden death , independent of the commonly used lv functional parameters . it is important to determine whether adrenergic function can predict the recovery of lv function or an improvement in outcome after treatment in patients with severe heart failure . there are a number of studies indicating that cardiac mibg uptake improves after -blocker or other therapy [ 6371 ] . fukuoka et al . both showed improvements of mibg uptake and washout after -blocker therapy in patients with dilated cardiomyopathy . the h / m ratio on the delayed images was a good predictor of the response to the -blocker therapy . takeishi et al . also reported improvement of mibg uptake in relation to lvef recovery after treatment with angiotensin - converting enzyme inhibitor in patients with congestive heart failure . gerson et al . showed that the h / m ratio improved significantly after carvedilol therapy , especially in patients with an h / m ratio < 1.40 . reported favorable changes in symptoms , nyha functional class , lv function , and the h / m ratio on mibg imaging following treatment with the 1-blocker metoprolol . observed a therapeutic effect of carvedilol on mibg parameters and lv remodeling in patients with dilated cardiomyopathy . these data indicate that mibg imaging may provide valuable information for the selection and optimization of treatment for congestive heart failure . pet offers great advantages in terms of higher spatial resolution and higher sensitivity with better quantification of tracer concentration compared to the commonly performed spect ( fig . 3 ) . with the use of suitable tracer kinetic models , various molecular as well as functional parameters various sympathetic neuronal pet tracers have been introduced which closely resemble the endogenous neurotransmitters , and these may allow more detailed analyses of neuronal signaling than mibg can [ 72 , 73].fig . 3 c - hydroxyephedrine ( he d ) pet and mibg spect of a normal subject . the he d pet obtained better - quality images in the myocardium as compared to the mibg spect ( color figure online ) c - hydroxyephedrine ( he d ) pet and mibg spect of a normal subject . the he d pet obtained better - quality images in the myocardium as compared to the mibg spect ( color figure online ) c - epinephrine is a radiolabeled true neurotransmitter , and the uptake and storage of this tracer resemble those of norepinephrine [ 74 , 75 ] . c - phenylephrine is another pet tracer that is trapped in neuronal vesicles and metabolized by neuronal monoamine oxidase ( mao ) . c - hydroxyephedrine ( he d ) is the most widely used pet tracer for cardiac neuronal imaging . it has high affinity for presynaptic neuronal catecholamine transporter ( uptake 1 ) without being metabolized by mao or catechol - o - methyl - transferase [ 77 , 78 ] . denervation and reinnervation have been investigated using he d in various disorders , such as diabetes [ 7981 ] and heart transplantation [ 8284 ] . a study of patients with heart failure suggested that they had reduced he d uptake , particularly in the apical and inferoapical regions ( fig . 4 ) . in a study of heart failure patients before heart transplantation , the he d uptake in the myocardium closely correlated with uptake 1 and the norepinephrine content , confirming the value of he d imaging as a marker of sympathetic neuronal imaging .fig . 4a series of short - axis slices of mibg spect and he d pet images of a patient with heart failure . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a series of short - axis slices of mibg spect and he d pet images of a patient with heart failure . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a reduction of he d uptake may be a prognostic marker for heart failure patients . described he d reduction as an independent risk factor , together with left ventricular end diastolic volume and peak vo2 . another study compared he d retention with oxidative metabolism , but found no significant association in heart failure patients . most of these studies using he d seem to give findings similar to those that used mibg imaging , suggesting autonomic function as an important new parameter for risk analysis and treatment monitoring , independent of other imaging and/or biomarkers . since he d pet provides higher spatial resolution and has a higher quantitative capability , it may hold promise for assessing patients with fatal arrhythmias . a global reduction in he d uptake in the myocardium was not observed in patients with brugada syndrome or those with long qt syndrome . since most of these studies are preliminary , more clinical studies with high - resolution pet are warranted . , 1 adrenoreceptor plays an important role in the regulation of myocardial cell function . compared to the many presynaptic radioligands used for imaging , there are only a few radioligands that are suitable for postsynaptic neuron imaging and probing of neuronal functions . c - cgp 12177 is a non - selective hydrophilic -receptor antagonist that provides high - quality pet images with low background activity ( fig . 5 ) . two sequential pet images after an injection of high - specific - activity tracer followed by low - specific - activity tracer allow quantification of -receptor density using a graphical analysis method . two other radiolabeled ligands were introduced for adrenoreceptor study : c - cgp 12388 for -receptor imaging , and c gb-67 for 1-receptor imaging .fig . 5coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) cgp 12177 has been used to demonstrate -receptor downregulation in many heart failure patients [ 9698 ] . our data suggested that myocardial -receptor density is inversely correlated with the mibg washout rate and positively correlated with a delayed h / m ratio , indicating that decreased -receptor density was due to the increased presynaptic sympathetic tone . these results suggest that myocardial -receptor density may be directly associated with the beneficial response obtained by -blocker treatment in heart failure patients . in addition , there was a close relationship between myocardial -receptor density assessed by c - cgp 12177 pet and the improvement in left ventricular function after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy ( fig . we thus suggested that patients with advanced downregulation of myocardial -receptor density have higher resting adrenergic drive and may derive greater benefits from the anti - adrenergic effects of -blocker treatment . further studies with many more patients with heart failure are warranted to test this finding.fig . 6correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy cardiovascular molecular imaging allows tissue characterization in myocardial disorders at molecular , subcellular , and cellular levels . it has potential for assessing the severity of heart failure and for monitoring treatment effects . i - labeled mibg , as a marker of adrenergic neuron function , could play an important role in the risk stratification of heart failure patients . presynaptic and postsynaptic neuron imaging using pet can potentially allow better quantifications of autonomic function . more clinical experience is needed to confirm the clinical impact of these imaging modalities for the management of heart failure patients .

this review focuses on molecular imaging using various radioligands for the tissue characterization of patients with heart failure . 123i - labeled metaiodobenzylguanidine ( mibg ) , as a marker of adrenergic neuron function , plays an important role in risk stratification in heart failure and may be useful for predicting fatal arrhythmias that may require implantable cardioverter - defibrillator treatment . mibg has also been used for monitoring treatment effects under various medications . various positron emission tomography ( pet ) radioligands have been introduced for the quantitative assessment of presynaptic and postsynaptic neuronal function in vivo . 11c - hydroxyephedrine , like mibg , has potential for assessing the severity of heart failure . our pet study using the -receptor antagonist 11c - cgp 12177 in patients with heart failure showed a reduction of -receptor density , indicating downregulation , in most of the patients . more studies are needed to confirm the clinical utility of these molecular imaging modalities for the management of heart failure patients .

Introduction Imaging autonomic functions Fundamental aspects of Clinical aspects of Sympathetic neuronal imaging using PET Adrenoreceptor imaging using PET Conclusions

the treatment of heart failure patients has been found to entail tremendous medical costs in many countries [ 1 , 2 ] . a number of non - invasive cardiovascular imaging modalities are currently used to assess the severity of heart failure . radionuclide imaging , using either positron emission tomography ( pet ) or single - photon emission computed tomography ( spect ) , has been the modality most commonly used for human molecular imaging , mainly due to its quite high sensitivity to radiolabeled ligands with nano- or micro - order levels compared to other methods of molecular imaging . as a result of advances in vivo biochemical imaging using radionuclide techniques , the modulation of functional and electrophysiological properties of the heart by the autonomic nervous system has become a focus of interest in the field of cardiovascular research . tracer approaches are considered uniquely suited for radionuclide imaging - based in vivo characterization of neuronal function in the myocardium . figure 1 illustrates a number of adrenergic presynaptic and postsynaptic ( receptor ) functions , indicating the radiopharmaceuticals that have been used to probe those functions . [ 1820 ] used radiolabeled norepinephrine for the assessment of sympathetic nerve function and norepinephrine kinetics.table 1radiotracers used for the evaluation of autonomic nervous system functionssympathetic nervesparasympathetic nervespresynaptic i - metaiodobenzylguanidine ( mibg ) i - iodobenzoversamicol c - hydroxyephedrine f - fluorobenzyl - benzovesamicol f - metaraminol f - dopamine c - threohydroxyepinephrine c - epinephrinepostsynaptici - iodocyanopindolol ( icyp ) i - quinuclidinyl benzylate ( qnb ) c - practolol c - methyl qnb c - propranolol c - cgp 12177 c - prazocinfig . 1schema of myocardial nerve terminals and various radioligands used to probe adrenergic functions ( color figure online ) radiotracers used for the evaluation of autonomic nervous system functions schema of myocardial nerve terminals and various radioligands used to probe adrenergic functions ( color figure online ) among these radiopharmaceuticals , the norepinephrine analog metaiodobenzylguanidine ( mibg ) has been widely used in experimental and clinical studies for sympathetic nerve imaging . sometimes only the delayed images are obtained to estimate delayed myocardial uptake as an index of adrenergic neuron function . although i - labeled mibg yields high - quality images of myocardial neuronal function , high activity in the liver may be superimposed on myocardial activity in planar images , and thus affect the interpretation of myocardial distribution , particularly in the inferior region . 2anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart in reports concerning imaging standards and a relevant japanese database , the normal myocardium - to - mediastinum count rate ranged from 2.0 to 2.7 for the early scan and from 2.1 to 2.9 for the late scan [ 32 , 33 ] , but these values seemed to be dependent on the collimator used for acquisition and the specific activity of the mibg . it is well - known that adrenergic dysfunction plays a key role in heart failure , and the plasma catecholamine level has been recognized as a valuable prognostic tool . thus , analyses of norepinephrine kinetics have a key role in the assessment of the severity of congestive heart failure . there are a number of reports showing a reduction of mibg retention in patients with idiopathic dilated cardiomyopathy [ 3740 ] . , in 1999 , were the first to indicate the prognostic value of the h / m ratio in patients with heart failure . thereafter , many prognostic studies using this imaging technique were performed in european countries and in japan ; there thus emerged a close association between the h / m ratio and/or the washout rate of mibg as markers of impaired cardiac adrenergic innervation and mortality in patients with heart failure [ 4150 ] . a meta - analysis of 18 studies including 1,755 patients with heart failure confirmed that heart failure patients showing a reduced h / m ratio on late mibg imaging had a worse prognosis as compared to those with a normal h / m ratio . a similar meta - analysis was reported from japan , indicating that both decreased mibg uptake and its increased washout are indicative of a poor prognosis in heart failure patients . implantable cardioverter - defibrillator ( icd ) treatment has become well established for preventive use in patients at high risk of arrhythmic death . autonomic dysfunction assessed by mibg is thought to play an important role in the detection of high - risk ventricular arrhythmia [ 14 , 53 , 54 ] . a number of pilot studies have indicated mibg as a potential predictor of ventricular arrhythmias in patients with an icd [ 5560 ] . , in a prospective study using both mibg and resting myocardial perfusion imaging in patients who received icd treatment , showed that icd discharge was documented in 30 of the total 60 patients ( 50 % ) . this report was the first to suggest an incremental benefit for the assessment of sympathetic nerve function in combination with myocardial perfusion , and a role for this combined assessment in the risk stratification of patients who may need prophylactic icd therapy . they found that the mibg washout rate was independent of lvef in risk analyses of patients with heart failure . reported the results of a prospective mibg imaging trial of 961 patients with heart failure recruited at 96 sites in north america and europe . their admire - hf study enrolled patients with new york heart association ( nyha ) class ii and iii heart failure and lvef 35 % and revealed that an h / m ratio of < 1.6 measured at 4 h after an mibg administration provided prognostic data beyond that available from the lvef , b - type natriuretic peptide assay , and nyha class at the time of enrollment . their multicenter prospective study confirmed the adrenergic neuronal functional parameter obtained by mibg scan as an important parameter for predicting sudden death , independent of the commonly used lv functional parameters . it is important to determine whether adrenergic function can predict the recovery of lv function or an improvement in outcome after treatment in patients with severe heart failure . also reported improvement of mibg uptake in relation to lvef recovery after treatment with angiotensin - converting enzyme inhibitor in patients with congestive heart failure . a study of patients with heart failure suggested that they had reduced he d uptake , particularly in the apical and inferoapical regions ( fig . in a study of heart failure patients before heart transplantation , the he d uptake in the myocardium closely correlated with uptake 1 and the norepinephrine content , confirming the value of he d imaging as a marker of sympathetic neuronal imaging .fig . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a series of short - axis slices of mibg spect and he d pet images of a patient with heart failure . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a reduction of he d uptake may be a prognostic marker for heart failure patients . another study compared he d retention with oxidative metabolism , but found no significant association in heart failure patients . most of these studies using he d seem to give findings similar to those that used mibg imaging , suggesting autonomic function as an important new parameter for risk analysis and treatment monitoring , independent of other imaging and/or biomarkers . since he d pet provides higher spatial resolution and has a higher quantitative capability , it may hold promise for assessing patients with fatal arrhythmias . since most of these studies are preliminary , more clinical studies with high - resolution pet are warranted . , 1 adrenoreceptor plays an important role in the regulation of myocardial cell function . c - cgp 12177 is a non - selective hydrophilic -receptor antagonist that provides high - quality pet images with low background activity ( fig . 5coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) cgp 12177 has been used to demonstrate -receptor downregulation in many heart failure patients [ 9698 ] . these results suggest that myocardial -receptor density may be directly associated with the beneficial response obtained by -blocker treatment in heart failure patients . in addition , there was a close relationship between myocardial -receptor density assessed by c - cgp 12177 pet and the improvement in left ventricular function after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy ( fig . we thus suggested that patients with advanced downregulation of myocardial -receptor density have higher resting adrenergic drive and may derive greater benefits from the anti - adrenergic effects of -blocker treatment . further studies with many more patients with heart failure are warranted to test this finding.fig . 6correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy it is taken up by sympathetic nerves in a similar manner to norepinephrine , but is not metabolized . sometimes only the delayed images are obtained to estimate delayed myocardial uptake as an index of adrenergic neuron function . although i - labeled mibg yields high - quality images of myocardial neuronal function , high activity in the liver may be superimposed on myocardial activity in planar images , and thus affect the interpretation of myocardial distribution , particularly in the inferior region . 2anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart anterior images at 4 h after i - labeled mibg administration in a normal control ( left ) , a patient with moderate ( middle ) , and a patient with severe heart failure ( right ) . the heart - to - mediastinal count ratio ( h / m ) is a marker of mibg uptake in the heart in reports concerning imaging standards and a relevant japanese database , the normal myocardium - to - mediastinum count rate ranged from 2.0 to 2.7 for the early scan and from 2.1 to 2.9 for the late scan [ 32 , 33 ] , but these values seemed to be dependent on the collimator used for acquisition and the specific activity of the mibg . it is well - known that adrenergic dysfunction plays a key role in heart failure , and the plasma catecholamine level has been recognized as a valuable prognostic tool . thus , analyses of norepinephrine kinetics have a key role in the assessment of the severity of congestive heart failure . there are a number of reports showing a reduction of mibg retention in patients with idiopathic dilated cardiomyopathy [ 3740 ] . , in 1999 , were the first to indicate the prognostic value of the h / m ratio in patients with heart failure . thereafter , many prognostic studies using this imaging technique were performed in european countries and in japan ; there thus emerged a close association between the h / m ratio and/or the washout rate of mibg as markers of impaired cardiac adrenergic innervation and mortality in patients with heart failure [ 4150 ] . a meta - analysis of 18 studies including 1,755 patients with heart failure confirmed that heart failure patients showing a reduced h / m ratio on late mibg imaging had a worse prognosis as compared to those with a normal h / m ratio . a similar meta - analysis was reported from japan , indicating that both decreased mibg uptake and its increased washout are indicative of a poor prognosis in heart failure patients . implantable cardioverter - defibrillator ( icd ) treatment has become well established for preventive use in patients at high risk of arrhythmic death . autonomic dysfunction assessed by mibg is thought to play an important role in the detection of high - risk ventricular arrhythmia [ 14 , 53 , 54 ] . a number of pilot studies have indicated mibg as a potential predictor of ventricular arrhythmias in patients with an icd [ 5560 ] . , in a prospective study using both mibg and resting myocardial perfusion imaging in patients who received icd treatment , showed that icd discharge was documented in 30 of the total 60 patients ( 50 % ) . this report was the first to suggest an incremental benefit for the assessment of sympathetic nerve function in combination with myocardial perfusion , and a role for this combined assessment in the risk stratification of patients who may need prophylactic icd therapy . they found that the mibg washout rate was independent of lvef in risk analyses of patients with heart failure . their admire - hf study enrolled patients with new york heart association ( nyha ) class ii and iii heart failure and lvef 35 % and revealed that an h / m ratio of < 1.6 measured at 4 h after an mibg administration provided prognostic data beyond that available from the lvef , b - type natriuretic peptide assay , and nyha class at the time of enrollment . their multicenter prospective study confirmed the adrenergic neuronal functional parameter obtained by mibg scan as an important parameter for predicting sudden death , independent of the commonly used lv functional parameters . it is important to determine whether adrenergic function can predict the recovery of lv function or an improvement in outcome after treatment in patients with severe heart failure . also reported improvement of mibg uptake in relation to lvef recovery after treatment with angiotensin - converting enzyme inhibitor in patients with congestive heart failure . a study of patients with heart failure suggested that they had reduced he d uptake , particularly in the apical and inferoapical regions ( fig . in a study of heart failure patients before heart transplantation , the he d uptake in the myocardium closely correlated with uptake 1 and the norepinephrine content , confirming the value of he d imaging as a marker of sympathetic neuronal imaging .fig . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a series of short - axis slices of mibg spect and he d pet images of a patient with heart failure . a moderate reduction of tracer uptake is noted , particularly in the inferior and posterolateral regions ( color figure online ) a reduction of he d uptake may be a prognostic marker for heart failure patients . another study compared he d retention with oxidative metabolism , but found no significant association in heart failure patients . most of these studies using he d seem to give findings similar to those that used mibg imaging , suggesting autonomic function as an important new parameter for risk analysis and treatment monitoring , independent of other imaging and/or biomarkers . since he d pet provides higher spatial resolution and has a higher quantitative capability , it may hold promise for assessing patients with fatal arrhythmias . since most of these studies are preliminary , more clinical studies with high - resolution pet are warranted . , 1 adrenoreceptor plays an important role in the regulation of myocardial cell function . c - cgp 12177 is a non - selective hydrophilic -receptor antagonist that provides high - quality pet images with low background activity ( fig . 5coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) coronal slice ( left ) and transaxial slice ( right ) of pet images after the administration of c - cgp 12177 in a patient with idiopathic dilated cardiomyopathy ( color figure online ) cgp 12177 has been used to demonstrate -receptor downregulation in many heart failure patients [ 9698 ] . these results suggest that myocardial -receptor density may be directly associated with the beneficial response obtained by -blocker treatment in heart failure patients . in addition , there was a close relationship between myocardial -receptor density assessed by c - cgp 12177 pet and the improvement in left ventricular function after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy ( fig . we thus suggested that patients with advanced downregulation of myocardial -receptor density have higher resting adrenergic drive and may derive greater benefits from the anti - adrenergic effects of -blocker treatment . further studies with many more patients with heart failure are warranted to test this finding.fig . 6correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy correlation between pretreatment -receptor density estimated by c - cgp 12177 pet and the improvement in left ventricular ejection fraction ( lvef ) after long - term -blocker treatment in patients with idiopathic dilated cardiomyopathy cardiovascular molecular imaging allows tissue characterization in myocardial disorders at molecular , subcellular , and cellular levels . it has potential for assessing the severity of heart failure and for monitoring treatment effects . i - labeled mibg , as a marker of adrenergic neuron function , could play an important role in the risk stratification of heart failure patients . presynaptic and postsynaptic neuron imaging using pet can potentially allow better quantifications of autonomic function . more clinical experience is needed to confirm the clinical impact of these imaging modalities for the management of heart failure patients .

on may 8 , 2014 , the united states court of appeals for the federal circuit issued an opinion , in re roslin institute , rejecting patent claims to mammals cloned from somatic cells . the patent at issue covered animals such as the famous cloned sheep , dolly , which had been produced without sexual reproduction from the cells of an existing adult sheep . although dolly was widely hailed as a scientific breakthrough in cellular and reproductive technology , the federal circuit panel held that such a cloned mammal was not sufficiently inventive to constitute patentable subject matter . because of her genetic identity with a progenitor animal , dolly was deemed a patent ineligible product of nature . the roslin opinion was rendered about a month before the united states supreme court 's decision in alice corp . v. cls bank international , which explicitly sets out the standard for determining whether an invention falls within statutory patentable subject matter . consequently , the federal circuit panel deciding roslin attempted its subject matter analysis following the supreme court 's prior opinion on dna patentability in myriad genetics , without the benefit of the supreme court 's later guidance in alice . the standard applied to determine patentable subject matter in alice is not found or discussed in the myriad opinion , and although the alice standard is derived from the earlier supreme court decision mayo collaborative services v. prometheus laboratories , inc . , the proto - standard from mayo is discussed in neither myriad nor in roslin . thus , one is thus left to wonder what the roslin opinion might have looked like had it been decided only a few months later , after the alice decision was published , with the benefit of the supreme court 's further direction on patentable subject matter . it may be that the subject matter analysis made explicit in alice would have changed the outcome , or at least the reasoning , of the roslin decision . in this essay , i explore whether in hindsight the alice standard might have dictated a different outcome in roslin , suggesting how the two - part test articulated by the supreme court in alice might apply to a products of nature analysis for cloned mammals . drawing on that analysis , i then use the roslin case as a vehicle to highlight certain serious issues with the supreme court 's current subject matter jurisprudence as applied to biotechnology . i conclude that the alice test demands a different view of dolly 's patent eligibility , but that significant issues remain to be resolved before the alice test can be coherently applied to products of nature analysis . determining the application of alice to the roslin situation is not a simple exercise , as it requires making some sense of the relationship between the myriad and alice opinions . roslin follows myriad in implicating the product of nature prohibition on patentable subject matter . thus , understanding how alice applies to roslin requires understanding how alice applies to myriad . but since myriad preceded alice , and alice nonetheless largely ignores it , we are left to speculate somewhat regarding myriad 's fit to the alice test . i therefore begin by laying some groundwork regarding the reasoning in myriad and its relationship to the supreme court 's other subject matter cases . i then turn to roslin , discussing how it might have been approached after alice , in light of myriad . all of the cases considered here struggle with long - standing uncertainties in the law of patentable subject matter . section 101 of the american patent statute sets forth four categories of patent eligible subject matter : processes , machines , articles of manufacture , and compositions of matter . these categories have been read by the supreme court as broad , inclusive , and representative . the statute is silent as to what does not constitute patentable subject matter ; presumably we can infer that inventions that lie outside the four broad and inclusive categories fail the requirements for subject matter . but , given that the explicit categories are highly inclusive , relatively little should fall outside their scope . some excluded categories have over the years been supplied by the courts as a judicial gloss on the statute : abstract ideas , laws of nature , products of nature , and perhaps one or two related categories such as mental processes , are deemed to fall outside even the broadest reading of the statutory language . the myriad genetics opinion is included among the most recent suite of supreme court cases attempting to define the scope of the judicially created exclusions . in addition to being the most recent decision on subject matter prior to roslin , the myriad opinion is probably the closest to roslin in subject matter ; the supreme court 's other opinions largely concern software or other processes . myriad also is the only supreme court case to squarely address the products of nature subject matter exclusion . so if the roslin opinion seems a bit muddled ( and we will see that it does ) , the federal circuit panel deciding the case perhaps can not be entirely blamed for the outcome , as they were bound to follow the supreme court 's recent subject matter guidance in myriad , and that guidance is far from illuminating . as i have shown at greater length in previous work , the opinion considers the patent eligibility of nucleotide sequences for certain cancer causing genes , designated brca1 and brca2 . myriad genetics had claimed two kinds of nucleotides for the genes , genomic dna sequences or gdna , and complementary dna sequences , or cdna . the opinion holds that , as between these two particular types of dna sequences encoding the genes , one type constitutes a patent ineligible product of nature , and the other does not . the sequence held not to be patent eligible was the ( gdna ) , which followed the native chromosomal sequence ; the sequence held to be patent eligible was a complementary dna sequence ( cdna ) , synthesized in the laboratory via a process termed reverse transcription . the gdna sequence was held to constitute prohibited subject matter because it contained the same information as sequences found in the wild ; the court dismissed as unimportant the molecule 's structural differences from that found in the cell . but the court held the cdna sequence to constitute patent eligible subject matter due to its structural differences from that found in the cell despite the fact that it carried the same genetic information . it is thus difficult , and perhaps impossible , to divine from the myriad opinion what exactly distinguishes a product of nature from patentable subject matter , at least so far as nucleotide sequences go the holdings regarding the two types of nucleotides seem diametrically opposed regarding the importance of differences in structure or coding sequence . given such unhelpful analytical distinctions , the federal circuit panel reviewing the roslin patent had only the general outline of the myriad result to work with in considering dolly and other genetic clones . without the specific framework articulated in alice ( and indeed even with it ) myriad may not have been the most trustworthy guide to determining the patent eligibility of the roslin clones . myriad 's relationship to the supreme court 's other subject matter cases is also a bit of a muddle . the myriad opinion is sandwiched in time between the supreme court 's previous opinion in mayo v. prometheus and the later alice corp . alice skips backwards over myriad to draw on mayo for its analysis , attempting to distill the logic of mayo into a workable standard . but alice makes only a passing mention of myriad , just as myriad make little mention of mayo . this could perhaps be attributed to the difference in the inventions at issue in the cases . like the majority of the court 's subject matter decisions , whether contemporary or classical , both alice and mayo concern process claims , where the prohibited subject matter is either an abstract idea or a law of nature . the two categories are themselves sufficiently abstract that it is often hard to tell which is which , or which is being invoked in a given instance to exclude an invention from section 101 . alice sets forth a two - part test , ostensibly derived from mayo v. prometheus . according the supreme court , a subject matter analysis under section 101 must first inquire as to whether the claimed invention entails one of the species of prohibited subject matter found in its prior opinions : an abstract idea , a law of nature , or ( presumably ) a product of nature . if the claimed invention involves a prohibited category , then under the second prong of the alice test , analysis shifts to determining whether the patent is simply an attempt to claim the prohibited entity , or whether the inventor instead has added something more. the something more must constitute an inventive concept beyond an abstract idea , law of nature , or ( presumably ) a product of nature . presumably in discussing products of nature in the alice test , because unlike the other supreme court subject matter cases , myriad concerns a product or material . among the subject matter opinions , it is the court 's sole foray into products claims , in which products of nature rather than laws of nature are the prohibited category at issue . indeed , prior to the myriad decision , it would not have been irrational to question whether products of nature were in fact part of the court 's subject matter jurisprudence . given the almost complete lack of reference between myriad and the process subject matter cases , one might still plausibly wonder whether the alice test is meant to apply to products of nature analyses . but the alice opinion speaks broadly of the two - part analysis as the proper test for subject matter , intimating that it applies to all the judicially prohibited categories , including products of nature . indeed , there is no suggestion in any of the subject matter cases that products are treated differently than processes in assessing patent eligibility . but since the test set forth in mayo is not explicitly followed in myriad , we are left to guess a bit as to how the nucleotide sequences in myriad failed the test . if in fact myriad follows the proto - alice analysis of mayo , and myriad holds that cdna is patent eligible but gdna is not , then presumably cdna passes the alice test and gdna fails it . the question then is how each of each kind of nucleotide polymer fits the two - part test , as the myriad case itself is not explicit about following the mayo / alice test at all . considering the myriad outcome as a result of the mayo / alice test , one has to conclude that cdna passes the first prong of the test and never reaches the second . it seems implausible that cdna could pass the second prong of the test ; there is no palpable inventive concept in reverse transcribing the mrna sequence derived from the brca1 or brca2 genes . the reverse transcription process that produces the cdnas uses messenger rna as a template to generate a cdna transcript ; creating cdnas from this method is today the most routine and unimaginative of laboratory procedures . neither is there anything surprising about the structure of the myriad cdnas ; they appear to follow the genomic nucleotide sequence minus non - coding introns or intervening sequences that would be edited out of the mrna transcript from which the cdna was derived . this is exactly what one would expect given either the genomic sequence or the mrna sequences . one has to conclude , then , that within the alice framework the myriad cdnas stand or fall on the first prong ; that is , they must not entail a prohibited category , and so never reach the second prong of the test , and do not require an inventive concept that they rather clearly lack . the myriad cdnas are apparently not products of nature ( nor laws of nature and abstract ideas ) . conversely , given their failure to meet subject matter eligibility , the myriad gdnas apparently fail both prongs : they must be products of nature , and must they lack an inventive concept as well . informational identity with native dna apparently classifies the gdna as a product of nature , and one must conclude that the chemical and structural differences attending isolation of the molecule are insufficiently inventive to imbue the sequence with the something more required under mayo . armed with what we can glean of the relationship between alice and myriad , particularly the application of alice 's subject matter test to products of nature , we are prepared to consider how the roslin clone patent might have fared under the test . we must unfortunately begin by recognizing that , even allowing for the lack of the supreme court 's subsequent guidance in alice , the roslin opinion is hardly a model of coherent judicial reasoning , either on its own terms or with regard to the supreme court 's subject matter jurisprudence to that point . for example , a substantial chunk of the opinion consists of a somewhat bizarre interlude discussing the sears and compco line of supreme court constitutional supremacy clause cases , which bar state law from prohibiting copying of subject matter that federal policy mandates remain in the public domain . the court suggests that the holdings in these cases somehow support the conclusion that cloning produces unpatentable copies . the discussion of these cases seems ( to put it mildly ) misplaced , first because roslin involves no conflict between state and federal law , and second because the argument essentially assumes that clones are unpatentable as part of an argument leading to the conclusion that they are indeed unpatentable . the remainder of the opinion relies on cases of greater relevance , although much of the reasoning remains problematic . the federal circuit panel looks to myriad primarily for a dubious comparison between dolly 's nuclear genetic material and the ineligible genomic dna sequences claimed in the earlier case . dolly 's primary claim to fame was that her cellular nuclei were derived from an existing adult sheep . mammalian cells can be divided into somatic cells that are constituent of the body and have a full genetic complement , and gametes such as oocytes or spermatozoa that conjoin for reproduction and have only half the genetic complement of mature somatic cells . dolly was created by a process of transferring the nuclear material of a somatic cell into an enucleated oocyte , which then developed into an embryo and gestated under the direction of the transferred genes . the identity between dolly 's nuclear material and that of her donor was the basis for the conclusion in roslin that clones are patent ineligible products of nature . the supreme court characterized the dna sequences in myriad as having the same unaltered genetic information as found in human cells ; so too the federal circuit characterized dolly 's cell nuclei as having the same unaltered genetic information found in her donor sheep 's cells . tellingly , although it compares dolly to the gdna molecules found patent ineligible in myriad , the cafc panel did not attempt to distinguish the clone from the cdna molecules found patent eligible in that case . to advance the comparison of dolly to myriad 's gdna sequences , the federal circuit adopted a line of argument from the myriad opinion rejecting as inconsequential or irrelevant clear distinctions between the claimed invention and its natural analog , due to the absence of those distinctions as limitations in the patent claims . in myriad , justice thomas opinion rejects structural changes in the myriad gdna molecules as differentiating the claimed invention from materials found in nature , because he says the claims do not recite those differences as limitations . this reasoning is somewhat troubling because the supreme court has repeatedly declaimed that subject matter determinations should not be driven by the draftsman 's art , yet reliance on such arguments makes the subject matter determination very much a product of the draftsman 's art . the clear implication seems to be that a claim drafted to include such differences might qualify as patentable subject matter . the roslin opinion travels the same road , rejecting the differences between cloned animals and those gestated by conventional means as distinctions between the claimed invention and products of nature because , the court says , those differences are not recited in the claims . perhaps the most striking distinction raised by the patentees , and rejected by the panel , is the difference between nuclear and non - nuclear genetic sequences in each type of animal . the cloned animal claimed in the patent is created by transferring to a zygote a new nucleus , with its complement of genetic material , taken from the somatic cells of an existing animal . eukaryotic cell cytoplasm contains energy - producing organelles called mitochondria , and mitochondria carry their own dna , separate from the nuclear dna of the cell . indeed , mitochondria reproduce themselves separately from the replication cycle followed by the rest of the cell . in conventional mammalian reproduction , cytoplasmic mitochondria come entirely from the mother , via her ovum ; male gametes contribute no mitochondria to the zygote . thus , the genetic complement in cellular mitochondria descends through an entirely matriarchal line , accompanying a random selection of half the mother 's nuclear genetic complement . consequently , in conventionally gestated zygotes , a particular complement of mitochondrial dna accompanies the nuclear complement of maternal dna into the new organism . in the case of the roslin clones , the mitochondria found in the cells of the clone would be derived from those in the cytoplasm of the recipient cell , with no relationship to the nuclear dna . this seems a fairly clear distinction between the cellular composition of cloned animals and other conventionally reproduced animals . cloned which in reference to the process disclosed in the patent would necessarily entail this limitation . the specification of the roslin patents in fact explicitly discusses the difference in mitochondrial genetic material . it is of course a general rule of claim interpretation that one is not to read limitations from the specification into the claims , but at the same time it is equally the rule that claims are to be read in light of the specification . i shall return later to the question of claims interpretation in relation to a subject matter analysis , but assuming the conventional rules apply , the difference between the dna complement of a conventionally gestated animal and that of a cloned animal seems at least inherent in the term clone as that is explained in the roslin specification . in a related passage , the opinion dismisses this same distinction on the grounds that the patentee had not explained what effect differences in mitochondrial dna might have . this might be relevant for purpose of other sections of the patent act , for example if dolly were purportedly non - obvious over prior art clones on the basis of the mitochondrial difference , or if there were some question as to whether the patent enabled the claimed clones . but it is not at all clear that for purposes patent eligibility the patentee is required to justify a structural or physical distinctions from the native analog of the invention the existence of such distinctions by themselves sets the invention apart from what is found in nature . thus , for example , in myriad , the patentee was not required to explain what effect or advantage came from the structural differences between the patent eligible cdnas and native dna ; the presence of the difference was itself sufficient to satisfy the section 101 inquiry . nonetheless , ignoring such differences at the cellular and organism levels , the panel in roslin asserts that dolly is an identical genetic replica of a naturally occurring sheep . the paradox in that assertion is of course that a sheep with a genetic complement identical to that found in nature is not what one would find in nature . nuclear genetic identity is of course a major difference , if not the major difference between the clone and anything found in nature ; genetically identical mammals are not what one finds in the wild . this might not be true for certain simpler organisms that pass their exact genetic complement on to their progeny , or for plants , where genetic identity is a characteristic of some forms of vegetable propagation . but mammals such as sheep propagate via sexual recombination which typically renders them not genetically identical . nuclear genetic identity can occur rarely in mammals due to gestational twinning , but as explained above twins are not clones unlike any naturally occurring animal , dolly might be said to have two genetic mothers : one who donated the cellular cytoplasm and another who donated a diploid nucleus . much of the panel 's reasoning in the case remains mired in this type of category error , assuming that because dolly is supposedly identical to her donor sheep , the category of claimed mammalian clones is identical to the category of conventionally gestated sheep . but even if the former is assumed to be true , it does not make the latter true , as demonstrated by dolly 's genetic age . mammalian chromosomes end with repetitive sequences called telomeres , which become degraded and shorter with each cycle of cell replication . shortened telomeres are thus causally related to the cellular aging process . by virtue of inheriting a mature set of somatic cell chromosomes , rather than the freshly recombined set of germ - line chromosomes that would accompany natural conception , dolly began life with shortened telomeres . thus , dolly was in a genetic sense born old and lived a shortened life as a result . this difference , too , was brushed aside by the federal circuit as a characteristic true of any copy. but it is no argument to simply label dolly a copy . a photograph of a tree , a sculpture of a tree , and a model of a tree are all copies of an item found in nature , but nonetheless are not naturally occurring artifacts , being clearly the handiwork of a human creator . they each resemble a natural object but differ from it in significant respects . similarly , dolly or other clones may resemble an existing animal , but are not naturally occurring organisms , instead bearing the mark of human handiwork . specifically , naturally conceived zygotes do not have dolly 's unnaturally shortened telomeres , nor do new - born lambs , and nor even adult sheep of dolly 's chronological age when she died . the question is not whether dolly was a copy , but whether such a copy existed in nature . in addition to the rather strained analogies to myriad , the roslin opinion offers some strained analogies to other supreme court opinions dealing with patents on biological inventions . the federal circuit panel compares the claimed clones to the inventions considered by the supreme court in funk brothers v. kalo inoculant and in diamond v. chakrabarty . the former decision concerned a mixture of agriculturally beneficial bacteria , which was held unpatentable under the pre-1952 patent statute . the chakrabarty case concerned a modified microorganism , capable of digesting petroleum , which was held patentable under the current statute . although often cited with regard to patentable subject matter , neither of these decisions is an especially good guide to determining the relevant exceptions to patent eligibility under section 101 . as i have pointed out elsewhere , funk bros . was decided prior to the enactment of the current statute , under a different standard , and chakrabarty held that living organisms are patentable subject matter , not that the claimed invention was ( or was not ) a product of nature . indeed , it is worth noting that much of what passes for analysis in the roslin opinion dwells on the supreme court 's dicta from chakrabarty that the organism under consideration there was markedly different from those found in the wild . this comment in chakrabarty was part of the court 's discussion as to whether living organisms could be patentable subject matter , but even were it a holding as to what constitutes a product of nature , the opinion offers little guidance as to what the federal circuit appears to have forgotten as perhaps has the supreme court that ananda chakrabarty 's petroleum - digesting pseudomonas species bacterium was created by moving existing genetic sequences from different strains of bacteria into a single strain by means of the bacteria 's chakrabarty carefully selected the bacterial strains he wanted to cross , and stabilized the exchanged genetic components after conjugation . inventively distinguishable from that in roslin : bacteria normally exchange genes by means of conjugation in the wild without human intervention , but sheep ova never naturally lose their nuclei and acquire the nucleus of a different animal 's somatic cell without determined human manipulation . markedly different standard , alice frames the subject matter inquiry as the two - step test described above . applying the first step , asking whether the patent encompasses a prohibited subject matter category , either seems to disfavor a finding of a prohibited category or are at worst indeterminate . the roslin patent encompasses product claims , so the relevant prohibited category would be products of nature . given the characteristics i have reviewed thus far , it is questionable that the roslin clones would constitute products of nature , any more than the myriad cdna molecules did . like cdna , the roslin clones are the product of synthetic laboratory processes , and entail differences from their natural counterparts at least as distinctive as the missing intervening sequences in the myriad cdnas . the fact that one portion of the clones their nuclear material was identical to that of a natural counterpart seems no more subject matter disqualifying than the fact that the coding sequences of the myriad cdnas were identical to the coding sequences of naturally occurring brca1 and brca2 gdna , or for that matter that the nuclear material of chakrabarty 's bacterium was identical to the nuclear material of pseudomonas species found in the wild . one might nonetheless expect that , had the federal circuit panel in roslin applied the alice test to the clones , the claims would have failed the first prong . the panel seemed determined to analogize the clones to the myriad gdnas rather than the myriad cdnas , and given that the myriad opinion offers little in the way of principled distinction between its gdna and cdna holdings , the federal circuit panel may have felt compelled to force the roslin clones into the first prong 's products of nature box . in some senses , the roslin panel 's analysis might be viewed as encompassing only the first prong of the alice test ; the analysis looks for a product of nature and then stops . to the extent that roslin follows myriad , this might be expected ; as i have indicated above , the myriad outcome seems to rest entirely on the first step of the alice test . but , even assuming that the cloned sheep failed the first prong of the alice test , the analysis would then move to the second prong to look for an inventive concept that takes the claimed invention beyond an attempt to merely capture the prohibited category of subject matter identified in the first step . following the claiming logic of myriad , as the federal circuit did the roslin patent claims surely entail such an inventive concept in the method of creating the sheep . the claims recite clones , which the specification discloses were produced by a novel method that is universally acknowledged to have been a highly significant and difficult advance in reproductive technology an inventive concept if there ever was one . both alice and mayo hold that implementing prohibited subject matter by means of conventional , routine , or off - the - shelf technology fails the inventive concept requirement of step two . but the creation of the roslin clones was not achieved via conventional , routine , or readily available techniques ; rather , biologically reprogramming somatic cell dna to generate a clone constituted a novel technical breakthrough . to the extent that the roslin opinion attempts to follow the markedly different dictum from chakrabarty , the alice decision perhaps puts some flesh on the bones of that standard : where the invention incorporates the disfavored category , product of nature , the marked difference necessary to place a claimed invention within patentable subject matter is an inventive concept that separates the claims from what might be found in the wild . marked difference. but the former criteria at least indicates the type and nature of the difference , suggests what differences might be relevant , and points toward a substantive distinction between products of nature and products of human ingenuity . the search for an inventive concept can not be quite the same exercise for product claims as it is for process claims . initial step determination as to whether an invention falls into one of the forbidden categories of excluded subject matter has something to do with the breadth of the attendant claims , but also much to do with the degree of generality with which it is regarded by a reviewing judge or examiner . much as in copyright law 's famous level of abstractions test , the first prong of the alice test asks the reviewer of a patent to assign the invention to some level of an increasingly abstract scale of interpretations . thus , in mayo , the supreme court views the claimed invention as highly abstract , effectively as a claim to a naturally occurring correlation between illness and symptoms . it is therefore not surprising that one can hardly tell if the mayo patent fails for claiming a law of nature or an abstract idea , since viewing the patent as a claim to a law of nature requires viewing it as an abstraction . similarly , in alice , the court views the financial transaction process at issue as entirely conceptual , abstracting away the disclosure ( and claims ) that discuss the apparatus and media implementing the method small wonder the opinion concludes that the method is an ineligible abstract idea ; the court chose to view it at a high level of abstraction . the method claims in such cases lend themselves to such abstraction , because processes are by their nature relational even when the claims recite devices or apparatus , the invention lies in the relationships between such objects , and not in the objects themselves . but it is far more difficult to adopt an abstract level of interpretation for composition claims , because some concrete structure or formulation will typically be used to define the invention . hence , the products of nature category , rather than the abstract idea , will typically be the relevant category against which to measure compositions of matter one might try to view chakrabarty 's bacterium as the idea of a microorganism that digests petroleum , but properly enabled claims are more likely to require consideration of the invention as a particular strain of bacterium imbued with particular genetic elements . the analytical move toward abstraction may be one reason that justice thomas in myriad emphasizes the informational similarities between the isolated gdna molecule and chromosomal nucleotide sequences , and refuses to view the invention at the more concrete structural level adopted in the federal circuit holding below . focusing on the sequence rather than the structure allows thomas to treat the genomic dna sequence as the idea of a molecule . informational level , but at the level of its particular instantiation , as a structure lacking intervening sequences . while it is not entirely clear what principled distinction causes the court to adopt these different levels for analysis , the outcome once they adopted is fairly clear : by ignoring structural features , compositions of matter can be treated the abstract way that processes are treated in mayo . it is this abstractive sleight of hand that the federal circuit mimics , somewhat ineptly , for its analysis in roslin . the federal circuit opinion essentially abstracts away the concrete instantiation of the roslin invention ignoring the invention 's relative chronological and genetic age , non - nuclear genetic elements , and the non - natural identity between donor and offspring sheep . informational character for the roslin panel to rely upon for a high level of abstraction as thomas does in myriad . instead the panel relies only on blunt and implausible dismissals of physiology to pare away dolly 's non - native differences . the criteria for deciding that differences such as dna introns do matter , but that those such as non - identical mitochondrial dna do not , remain as elusive as the degree of abstraction to be chosen when considering a diagnostic test . the alice opinion holds out the hope that abstract or naturally occurring process claims might , at least in theory , be saved on the second prong of the test by something more that transforms the claims into patent eligibility by means of an inventive concept . but this second prong recovery seems an unlikely and perhaps impossible occurrence for composition claims : if the invention is insufficiently distinguishable from its native counterpart that it fails the first prong of the test , what might be left to render it inventive on the second ? presumably any unique structural features have already been taken into account in the initial inquiry and found wanting . if the alice test is not to collapse into a single question , the inventive concept for compositions of matter must lie elsewhere . if the inventive concept is not found in the product 's structure , then it must lie in its function , with the processes surrounding its making or use . our understanding of the inventive concept necessary to take claims outside the prohibited product of nature category might additionally be clarified by reference to the guiding principles articulated by the supreme court in all of its recent subject matter decisions . stepping away for a moment from the formalisms of the alice framework , one might ask how well the federal circuit 's analysis fits with the purpose of the judicially created subject matter exceptions . the court has been clear that the purpose of its judicially created subject matter restrictions is to prevent fundamental concepts and materials , on which all inventors must draw , from being caught up in patent claims . avoiding such preemption of future inventive activity is the guiding purpose by which subject matter restrictions are judged . there is little evidence that the roslin patent attempts to capture fundamental or basic science on which future invention will depend , or if it does so , there is no indication in the roslin opinion that this informed the analysis of patentable subject matter . the federal circuit opinion spends no time in discussing the potential for preemption of downstream invention by the patent claims . this is perhaps unsurprising , given that the federal circuit has shown extreme reluctance to inquire into preemption in its post - myriad jurisprudence . preemption is brushed aside on the rationale that where a patent 's claims are deemed only to disclose patent ineligible subject matter under the mayo framework , as they are in this case , preemption concerns are fully addressed and made moot. this may be true , but conceives the issue entirely backwards . given that pre - emption of downstream innovations is one of the few markers the supreme court has offered regarding the substance of prohibited subject matter categories , preemption seems antecedent to the issue is not that a subject matter exclusion needs to be recognized in order to avoid preemption ; it is that preemption needs to be recognized in order to know when to impose a subject matter exclusion . i have suggested previously that the court 's subject matter jurisprudence is largely an attempt to drive patent holder toward narrower claiming , as a proxy for excluding fundamental building block developments from patentability . probably there is some correlation between breadth of claiming and fundamentality of inventions , despite the court 's warning against determining patent eligibility according to the draftsman 's art. and here again , taking seriously the stated purposes of the mayo / alice test points toward patent eligibility . plainly the roslin patent is not attempting to claim all sheep , nor even all genetically identical sheep . the claims only extend to sheep produced by the cloning process , a limitation that constrains the patent to the specific and novel implementation disclosed by the applicant . the alice test casts dolly the sheep in a different light , suggesting the potential for a different outcome in roslin . but at the same time , the roslin opinion highlights certain problematic features of alice as applied to biotechnology . in particular , roslin raises questions from the early days of biotechnology patenting that were thought laid to rest : the question of product - by - process claims , and the relationship between patent and process , known to patent lawyers as the durden problem. in their original incarnations , these issues were long ago resolved , or at least relegated to a position of doctrinal dtente ; but as the roslin opinion shows , they now threaten to reappear in the context of the alice subject matter test . the implication of the cloning process in the roslin claims raises the question as to whether this type of product patent might be treated under a specialized form of claiming , known as product by process claiming , and whether that characterization would make a difference to the outcome . the patentee specifically disclaimed this approach in its arguments to the patent office , so that the question remains unaddressed on appeal . nonetheless , the roslin claims implicitly entail the cloning process , and could well have been drafted to explicitly recite the process and that path may seem inviting to future applicants seeking patents on similar biological inventions . claims of this type were originally formulated as a claiming strategy for products that were difficult to describe ; rather than attempt to capture in language the elusive limitations of a problematic product , the invention could be claimed as the product of its more easily described process . it was generally understood that , since the product was the subject of a patent application , it was the product that would be examined for novelty and non - obviousness against identical or similar prior art products , no matter what process produced such products . this followed from the long - standing rule that preparation of a known product by a new process does not confer patentable novelty on the product , and posed a significant impediment to early biotechnology patents , which frequently generated known materials from novel processes . the patent office continues to follow this rule in examination of claims having the product by process format . but federal circuit decisions became fragmented on the proper scope of such claims when judging infringement : having been described as the product of a certain process was the claim limited to the product only when produced by that process ? or was the product covered by the patent when produced by any process , as it would have been if claimed in terms of its own characteristics ? current federal circuit jurisprudence has adopted the latter position , treating the process recited in the claims as a limitation on the claims , rather than as a descriptive mechanism . a section 101 subject matter inquiry is not a section 102 novelty analysis , nor a section 271 infringement analysis . but the history of product - by - process claims under these provisions leaves the question as to how a product by process claim would be treated under section 101 : would the process recited be treated as a limitation , and would such a limitation matter ? to the extent that current subject matter jurisprudence seems to recapitulate a novelty inquiry , the answer might well be that a process limitation would be immaterial : a product that exists in nature remains ineligible for a patent , no matter how produced . if one ignores the genetic , cytological , and physiological differences between dolly and her predecessor sheep as the federal circuit did to conclude that she was identical to existing sheep , then her origins might not matter . on the other hand , given the federal circuit panel 's repeated references to the claim limitation in determining subject matter eligibility , and its apparent invitation to draft clone claims in terms of their originating process , it might be that a process limitation would have been treated under 101 as it might under an infringement analysis , limiting the subject matter to non - naturally occurring products . this might matter from an alice / mayo preemption standpoint ; the process limitation would then demarcate claims to dolly from claims to conventional sheep that must remain accessible to the public and to future inventors . this would also appear consonant with the treatment of the dna sequences in myriad ; the major distinction between the cdna and gdna claims appears to be the method of producing the former . if one takes seriously justice thomas assertion in myriad that informational identity is what matters as between a gdna molecule and its native analog , then cdna and gdna are informationally the same , but produced by different processes ; that is , the cdna was synthesized via an all of these considerations are overshadowed by an additional troubling problem in the alice framework that some other commentators have already noticed . the supreme court rejected arguments regarding the structure of gdna because these characteristics were not incorporated into the myriad claims ; the federal circuit rejected arguments regarding dolly 's non - nuclear genetic complement because that was not incorporated into the roslin claims . the federal circuit treated the roslin claims as product rather than product - by - process claims because the patentee declined to make a product - by - process argument . the alice opinion directs lower courts to look for an inventive concept that adds something more than a bare claim to prohibited subject matter . all of this subject matter analysis with reference to the patent claims requires courts to construe the claims , but apparently without formal claim construction , which normally takes place at a particular time and in a particular context for patent adjudication . the treatment of product - by - process claims within the alice test leads to another , perhaps even more striking question , which is thrown into sharp relief by the roslin facts . i have argued in previous work that the question of patentable subject matter has no independent analytical content , but is rather forced to borrow from other sections of the patent statute for its substance . thus , the analysis of subject matter at different times takes on the aspects of a sort of pseudo - novelty , pseudo - obviousness , or even pseudo - utility assessment , asking whether the claimed invention is something different than what is found in nature , whether it has uses or applications that distinguish it from its natural counterparts , or whether it is only a trivial advance over what can be found in the wild . indeed , one might characterize the analysis in roslin as a type of pseudo - novelty analysis , asking in effect whether every element of the claimed invention is found in naturally occurring animals . this means that the relationship between section 101 and other limiting statutory sections is at best unclear . as presently employed , section 101 seems to duplicate doctrinal filters associated with other patentability provisions , leaving one to wonder what purpose is served by such redundancy . in particular , i have previously argued that and that the inventive concept language of the alice test is an attempt to screen out undesirable subject matter by importing into section 101 a kind of obviousness analysis that since the 1952 incorporation of obviousness into the patent statute had been reserved for assessment under section 103 . since the articulation of the alice test , patent eligibility doctrine appears to constitute a kind of pseudo - obviousness inquiry . thus , the introduction of the alice inventive concept requirement into section 101 might leave some question as to what work section 103 is now doing , or whether an invention that is found the standards for the two are of course not necessarily identical ; section 103 specifies that obviousness is to be assessed at a particular time ( the time an application is filed ) against particular prior art ( the categories contemplated in section 102 ) according to a particular standard ( the knowledge and skill of the person having ordinary skill ) . section 101 sets forth no such criteria for inventive concepts since this requirement is entirely a judicial creation , rather than the product of statutory language . following an aberrant and discredited bit of analysis in the 1979 parker v. flook decision , the supreme court in mayo has hinted that nature itself is the standard against which inventiveness is assessed , meaning that the outcome of a 101 inventive concept assessment might be quite different than a 103 obviousness assessment . at the same time , the two standards are at least conceptually similar ; both are directed to the determination of the result of such similarity is that importing obviousness concepts into section 101 brings with it baggage formerly associated with section 103 . if section 101 can be distinguished from section 103 because it lacks the criteria that define obviousness under section 103 , so too it lacks the accumulated precedent that has settled many of the problems that have historically cropped up in the analysis of obviousness , leaving it vulnerable to controversies that have haunted section 103 . although a careful consideration of the myriad decision hinted at the potential for populating section 101 with such doctrinal revenants , the roslin case squarely raises at least one of them , arising from the relationship between the patent eligibility of product and process . section 101 recites three categories of patentable subject matter that constitute product patents : machines , compositions , and manufactures . these two general categories are intimately related ; products are the results of processes , the inputs into processes , and the substrates of processes . given these causal relationships , there have been numerous past questions regarding the ontological status of processes and products when one or the other was judged patentably novel and non - obvious : does a novel or non - obvious substrate necessarily give rise to a novel or non - obvious process ? does a novel or non - obvious end product necessarily arise out of a novel or non - obvious process ? logically , one can argue that a novel substrate , to a process , must by definition give rise to a novel process if the substrate is previously unknown , the process in which it is involved can not have previously been known . as one opinion reasoned , without possession of the novel starting material it can not be obvious to use the starting material in the process , for one can not choose from the unknown. similarly , if a product would not have been obvious to one of ordinary skill , then logically the process that produced it can not have been obvious to one of ordinary skill ; if the output is non - obvious , then it can not have been produced by obvious means . courts struggled over decades to determine whether such logical strictures could lead to predictable patent doctrine when novel materials were incorporated into known processes , or when known processes were used to produce novel materials . the decisions attempting to rectify the product and process relationship came to be known as the durden line of cases , from the key decision rendered with regard to a chemical process . in general , the durden cases appeared to hold that where a starting material was known in the art but the end product was non - obvious , employing a known process to generate the product should be deemed obvious and unpatentable ; whereas if the end product was known and obvious , but the starting material was patentably novel and non - obvious , then the process should be deemed patentable as well . the former type of cases often were often grouped together as involving a method of making a patentable compound , whereas the latter type of cases became grouped together as involving a method of using a patentable compound , and attempts were made to distinguish the outcomes of the two sets of cases on that basis . these opinions suggested that patentable end products might be made from old and obvious processes , but patentable starting materials imbued old processes with their non - obvious qualities . the cases repeatedly focus on the question of whether in an obviousness analysis a court or the patent office was effectively treating a novel product as if it were prior art in determining the obviousness of its production . indeed , the treatment of such biotechnology applications by the patent office , under what seemed to be a per se hindsight rule , resulted in a successful lobbying effort to amend section 103 to add a specialized provision for biotechnology , allowing applicants to elect prosecution of product and process claims in the same application , and statutorily requiring under such circumstances that the patentability of the one be imputed to the other . at the same time , the federal circuit became increasingly insistent that bright - line rules were unworkable in such obviousness determinations . one could not assume the existence of a novel product in order to ask whether one of ordinary skill might be motivated to prepare it via an existing process , or alternatively would be motivated to use it in an existing process to produce some end product . ultimately , this became the judicial resolution for the recurring problem ; rejecting bright - line rules regarding methods of making and methods of using , the federal circuit held that , as provided by the language of section 103 , obviousness must of products or processes must be determined on a case - by - case basis against the relevant prior art , notwithstanding their relationship to related inventions . while that approach provides an answer for sorting out the obviousness standard in section 103 , the supreme court has now created an analogous problem within section 101 . importing pseudo - obviousness into section 101 resurrects the durden question , but in the context of patentable subject matter . the patents at issue in roslin concerned particular products , that is , dolly or other clones . the process of producing such clones was not at issue in the case , but there seems little dispute that the cloning process would constitute patentable subject matter , and indeed the federal circuit suggests in passing that any innovation attributable to dolly 's inventors should and would be better protected by process claims . the durden cases plumb the relationship between product and process , exploring the impact of substrate novelty or non - obviousness on those qualities in attendant processes . the alice test contemplates inventive concepts , not quite the same issue as novelty or non - obviousness , but clearly related . as in the durden cases , inventiveness of process may be reflected in its products , and vice versa . as i suggested above , under some conceptions of inventive , an inventive process such as that in roslin something that is a decidedly unnatural manipulation of physical materials must logically give rise to an conversely , in parallel to the durden obviousness cases , one would expect that there will be subject matter cases in which the inventive nature of products or substrates implies inventiveness of attendant processes . there is no reason to believe that this issue is unique to roslin ; rather , as in the section 103 durden cases , the question will arise wherever there are companion products and processes , one or the other of which qualifies as patent eligibly inventive under the alice test . for reasons that i have articulated in previous work , biotechnology is an inherently fertile field for the confluence of product and process inventions ; so as a practical matter , this means that the issue will routinely come up in biotechnology and related wet sciences . one is tempted to say that the new section 101 analog of the durden problem should be solved the way that the old section 103 version of the problem was solved . if one imports section 103 problems into section 101 , perhaps one can import the section 103 solutions as well . but one can not escape the quandary by saying , as the federal circuit did in the context of section 103 , that one must simply decide on a case - by - case basis , by following the statutory criteria . in section 101 , there are no statutory criteria to save us . but as a question of subject matter eligibility , the federal circuit 's treatment of patents covering dolly the sheep might have benefitted from the framework outlined in the later alice opinion , and considering how alice might have changed the roslin analysis offers a worthwhile exercise in determining how the alice framework operates in the context of products of nature . at the same time , reconsideration of dolly 's situation simultaneously reveals how , as applied to biotechnology , the alice approach creates confusion across a range of doctrines , including both product - by - process claims and a section 101 analog of the old durden analysis . and , although the question extends well beyond my brief in this particular essay , it is worth at least noting that , given the technological subject matter of the alice opinion , and of most of the other supreme court subject matter opinions , one might expect the framework 's inventive concept requirement to raise similarly intractable issues in the context of data processing . this makes clear that , far from settling ongoing questions about patentable subject matter , the supreme court 's alice opinion leaves unsettled questions that will haunt us for years to come .

the opinion of the united states court of appeals for the federal circuit , in re roslin institute , rejecting patent claims to mammals cloned from somatic cells , was rendered about a month before the united states supreme court 's decision in alice corp . v. cls bank international . the alice opinion explicitly sets out the standard for determining whether an invention falls within statutory patentable subject matter . thus one is thus left to wonder what the roslin opinion might have looked like had it been decided only a few weeks later , after the alice decision was published , with the benefit of the supreme court 's further direction on patentable subject matter . in this essay i explore whether in hindsight the alice standard might have dictated a different outcome in roslin , suggesting how the two - part test articulated by the supreme court in alice might apply to a products of nature analysis for cloned mammals . drawing on that analysis , i then use the roslin case as a vehicle to highlight certain issues with the supreme court 's current subject matter jurisprudence as applied to biotechnology . by juxtaposing dolly with alice , it becomes clear that the supreme court has revivified a number of dormant biotechnology patent problems in the guise of subject matter analysis .

INTRODUCTION SITUATING MAKING SENSE OF APPLYING THE IMPLICATIONS FOR THE CONCLUSION

on may 8 , 2014 , the united states court of appeals for the federal circuit issued an opinion , in re roslin institute , rejecting patent claims to mammals cloned from somatic cells . although dolly was widely hailed as a scientific breakthrough in cellular and reproductive technology , the federal circuit panel held that such a cloned mammal was not sufficiently inventive to constitute patentable subject matter . the roslin opinion was rendered about a month before the united states supreme court 's decision in alice corp . v. cls bank international , which explicitly sets out the standard for determining whether an invention falls within statutory patentable subject matter . consequently , the federal circuit panel deciding roslin attempted its subject matter analysis following the supreme court 's prior opinion on dna patentability in myriad genetics , without the benefit of the supreme court 's later guidance in alice . the standard applied to determine patentable subject matter in alice is not found or discussed in the myriad opinion , and although the alice standard is derived from the earlier supreme court decision mayo collaborative services v. prometheus laboratories , inc . thus , one is thus left to wonder what the roslin opinion might have looked like had it been decided only a few months later , after the alice decision was published , with the benefit of the supreme court 's further direction on patentable subject matter . it may be that the subject matter analysis made explicit in alice would have changed the outcome , or at least the reasoning , of the roslin decision . in this essay , i explore whether in hindsight the alice standard might have dictated a different outcome in roslin , suggesting how the two - part test articulated by the supreme court in alice might apply to a products of nature analysis for cloned mammals . drawing on that analysis , i then use the roslin case as a vehicle to highlight certain serious issues with the supreme court 's current subject matter jurisprudence as applied to biotechnology . i conclude that the alice test demands a different view of dolly 's patent eligibility , but that significant issues remain to be resolved before the alice test can be coherently applied to products of nature analysis . roslin follows myriad in implicating the product of nature prohibition on patentable subject matter . i therefore begin by laying some groundwork regarding the reasoning in myriad and its relationship to the supreme court 's other subject matter cases . i then turn to roslin , discussing how it might have been approached after alice , in light of myriad . all of the cases considered here struggle with long - standing uncertainties in the law of patentable subject matter . some excluded categories have over the years been supplied by the courts as a judicial gloss on the statute : abstract ideas , laws of nature , products of nature , and perhaps one or two related categories such as mental processes , are deemed to fall outside even the broadest reading of the statutory language . in addition to being the most recent decision on subject matter prior to roslin , the myriad opinion is probably the closest to roslin in subject matter ; the supreme court 's other opinions largely concern software or other processes . myriad also is the only supreme court case to squarely address the products of nature subject matter exclusion . so if the roslin opinion seems a bit muddled ( and we will see that it does ) , the federal circuit panel deciding the case perhaps can not be entirely blamed for the outcome , as they were bound to follow the supreme court 's recent subject matter guidance in myriad , and that guidance is far from illuminating . it is thus difficult , and perhaps impossible , to divine from the myriad opinion what exactly distinguishes a product of nature from patentable subject matter , at least so far as nucleotide sequences go the holdings regarding the two types of nucleotides seem diametrically opposed regarding the importance of differences in structure or coding sequence . given such unhelpful analytical distinctions , the federal circuit panel reviewing the roslin patent had only the general outline of the myriad result to work with in considering dolly and other genetic clones . myriad 's relationship to the supreme court 's other subject matter cases is also a bit of a muddle . the myriad opinion is sandwiched in time between the supreme court 's previous opinion in mayo v. prometheus and the later alice corp . like the majority of the court 's subject matter decisions , whether contemporary or classical , both alice and mayo concern process claims , where the prohibited subject matter is either an abstract idea or a law of nature . according the supreme court , a subject matter analysis under section 101 must first inquire as to whether the claimed invention entails one of the species of prohibited subject matter found in its prior opinions : an abstract idea , a law of nature , or ( presumably ) a product of nature . presumably in discussing products of nature in the alice test , because unlike the other supreme court subject matter cases , myriad concerns a product or material . among the subject matter opinions , it is the court 's sole foray into products claims , in which products of nature rather than laws of nature are the prohibited category at issue . indeed , prior to the myriad decision , it would not have been irrational to question whether products of nature were in fact part of the court 's subject matter jurisprudence . given the almost complete lack of reference between myriad and the process subject matter cases , one might still plausibly wonder whether the alice test is meant to apply to products of nature analyses . but the alice opinion speaks broadly of the two - part analysis as the proper test for subject matter , intimating that it applies to all the judicially prohibited categories , including products of nature . the question then is how each of each kind of nucleotide polymer fits the two - part test , as the myriad case itself is not explicit about following the mayo / alice test at all . informational identity with native dna apparently classifies the gdna as a product of nature , and one must conclude that the chemical and structural differences attending isolation of the molecule are insufficiently inventive to imbue the sequence with the something more required under mayo . armed with what we can glean of the relationship between alice and myriad , particularly the application of alice 's subject matter test to products of nature , we are prepared to consider how the roslin clone patent might have fared under the test . we must unfortunately begin by recognizing that , even allowing for the lack of the supreme court 's subsequent guidance in alice , the roslin opinion is hardly a model of coherent judicial reasoning , either on its own terms or with regard to the supreme court 's subject matter jurisprudence to that point . for example , a substantial chunk of the opinion consists of a somewhat bizarre interlude discussing the sears and compco line of supreme court constitutional supremacy clause cases , which bar state law from prohibiting copying of subject matter that federal policy mandates remain in the public domain . the identity between dolly 's nuclear material and that of her donor was the basis for the conclusion in roslin that clones are patent ineligible products of nature . to advance the comparison of dolly to myriad 's gdna sequences , the federal circuit adopted a line of argument from the myriad opinion rejecting as inconsequential or irrelevant clear distinctions between the claimed invention and its natural analog , due to the absence of those distinctions as limitations in the patent claims . this reasoning is somewhat troubling because the supreme court has repeatedly declaimed that subject matter determinations should not be driven by the draftsman 's art , yet reliance on such arguments makes the subject matter determination very much a product of the draftsman 's art . the roslin opinion travels the same road , rejecting the differences between cloned animals and those gestated by conventional means as distinctions between the claimed invention and products of nature because , the court says , those differences are not recited in the claims . the cloned animal claimed in the patent is created by transferring to a zygote a new nucleus , with its complement of genetic material , taken from the somatic cells of an existing animal . in the case of the roslin clones , the mitochondria found in the cells of the clone would be derived from those in the cytoplasm of the recipient cell , with no relationship to the nuclear dna . i shall return later to the question of claims interpretation in relation to a subject matter analysis , but assuming the conventional rules apply , the difference between the dna complement of a conventionally gestated animal and that of a cloned animal seems at least inherent in the term clone as that is explained in the roslin specification . this difference , too , was brushed aside by the federal circuit as a characteristic true of any copy. the federal circuit panel compares the claimed clones to the inventions considered by the supreme court in funk brothers v. kalo inoculant and in diamond v. chakrabarty . was decided prior to the enactment of the current statute , under a different standard , and chakrabarty held that living organisms are patentable subject matter , not that the claimed invention was ( or was not ) a product of nature . indeed , it is worth noting that much of what passes for analysis in the roslin opinion dwells on the supreme court 's dicta from chakrabarty that the organism under consideration there was markedly different from those found in the wild . this comment in chakrabarty was part of the court 's discussion as to whether living organisms could be patentable subject matter , but even were it a holding as to what constitutes a product of nature , the opinion offers little guidance as to what the federal circuit appears to have forgotten as perhaps has the supreme court that ananda chakrabarty 's petroleum - digesting pseudomonas species bacterium was created by moving existing genetic sequences from different strains of bacteria into a single strain by means of the bacteria 's chakrabarty carefully selected the bacterial strains he wanted to cross , and stabilized the exchanged genetic components after conjugation . given the characteristics i have reviewed thus far , it is questionable that the roslin clones would constitute products of nature , any more than the myriad cdna molecules did . the fact that one portion of the clones their nuclear material was identical to that of a natural counterpart seems no more subject matter disqualifying than the fact that the coding sequences of the myriad cdnas were identical to the coding sequences of naturally occurring brca1 and brca2 gdna , or for that matter that the nuclear material of chakrabarty 's bacterium was identical to the nuclear material of pseudomonas species found in the wild . one might nonetheless expect that , had the federal circuit panel in roslin applied the alice test to the clones , the claims would have failed the first prong . the panel seemed determined to analogize the clones to the myriad gdnas rather than the myriad cdnas , and given that the myriad opinion offers little in the way of principled distinction between its gdna and cdna holdings , the federal circuit panel may have felt compelled to force the roslin clones into the first prong 's products of nature box . in some senses , the roslin panel 's analysis might be viewed as encompassing only the first prong of the alice test ; the analysis looks for a product of nature and then stops . but , even assuming that the cloned sheep failed the first prong of the alice test , the analysis would then move to the second prong to look for an inventive concept that takes the claimed invention beyond an attempt to merely capture the prohibited category of subject matter identified in the first step . following the claiming logic of myriad , as the federal circuit did the roslin patent claims surely entail such an inventive concept in the method of creating the sheep . to the extent that the roslin opinion attempts to follow the markedly different dictum from chakrabarty , the alice decision perhaps puts some flesh on the bones of that standard : where the invention incorporates the disfavored category , product of nature , the marked difference necessary to place a claimed invention within patentable subject matter is an inventive concept that separates the claims from what might be found in the wild . initial step determination as to whether an invention falls into one of the forbidden categories of excluded subject matter has something to do with the breadth of the attendant claims , but also much to do with the degree of generality with which it is regarded by a reviewing judge or examiner . thus , in mayo , the supreme court views the claimed invention as highly abstract , effectively as a claim to a naturally occurring correlation between illness and symptoms . similarly , in alice , the court views the financial transaction process at issue as entirely conceptual , abstracting away the disclosure ( and claims ) that discuss the apparatus and media implementing the method small wonder the opinion concludes that the method is an ineligible abstract idea ; the court chose to view it at a high level of abstraction . hence , the products of nature category , rather than the abstract idea , will typically be the relevant category against which to measure compositions of matter one might try to view chakrabarty 's bacterium as the idea of a microorganism that digests petroleum , but properly enabled claims are more likely to require consideration of the invention as a particular strain of bacterium imbued with particular genetic elements . the federal circuit opinion essentially abstracts away the concrete instantiation of the roslin invention ignoring the invention 's relative chronological and genetic age , non - nuclear genetic elements , and the non - natural identity between donor and offspring sheep . the alice opinion holds out the hope that abstract or naturally occurring process claims might , at least in theory , be saved on the second prong of the test by something more that transforms the claims into patent eligibility by means of an inventive concept . our understanding of the inventive concept necessary to take claims outside the prohibited product of nature category might additionally be clarified by reference to the guiding principles articulated by the supreme court in all of its recent subject matter decisions . stepping away for a moment from the formalisms of the alice framework , one might ask how well the federal circuit 's analysis fits with the purpose of the judicially created subject matter exceptions . the court has been clear that the purpose of its judicially created subject matter restrictions is to prevent fundamental concepts and materials , on which all inventors must draw , from being caught up in patent claims . there is little evidence that the roslin patent attempts to capture fundamental or basic science on which future invention will depend , or if it does so , there is no indication in the roslin opinion that this informed the analysis of patentable subject matter . the federal circuit opinion spends no time in discussing the potential for preemption of downstream invention by the patent claims . given that pre - emption of downstream innovations is one of the few markers the supreme court has offered regarding the substance of prohibited subject matter categories , preemption seems antecedent to the issue is not that a subject matter exclusion needs to be recognized in order to avoid preemption ; it is that preemption needs to be recognized in order to know when to impose a subject matter exclusion . i have suggested previously that the court 's subject matter jurisprudence is largely an attempt to drive patent holder toward narrower claiming , as a proxy for excluding fundamental building block developments from patentability . the alice test casts dolly the sheep in a different light , suggesting the potential for a different outcome in roslin . but at the same time , the roslin opinion highlights certain problematic features of alice as applied to biotechnology . in their original incarnations , these issues were long ago resolved , or at least relegated to a position of doctrinal dtente ; but as the roslin opinion shows , they now threaten to reappear in the context of the alice subject matter test . on the other hand , given the federal circuit panel 's repeated references to the claim limitation in determining subject matter eligibility , and its apparent invitation to draft clone claims in terms of their originating process , it might be that a process limitation would have been treated under 101 as it might under an infringement analysis , limiting the subject matter to non - naturally occurring products . the supreme court rejected arguments regarding the structure of gdna because these characteristics were not incorporated into the myriad claims ; the federal circuit rejected arguments regarding dolly 's non - nuclear genetic complement because that was not incorporated into the roslin claims . i have argued in previous work that the question of patentable subject matter has no independent analytical content , but is rather forced to borrow from other sections of the patent statute for its substance . thus , the analysis of subject matter at different times takes on the aspects of a sort of pseudo - novelty , pseudo - obviousness , or even pseudo - utility assessment , asking whether the claimed invention is something different than what is found in nature , whether it has uses or applications that distinguish it from its natural counterparts , or whether it is only a trivial advance over what can be found in the wild . in particular , i have previously argued that and that the inventive concept language of the alice test is an attempt to screen out undesirable subject matter by importing into section 101 a kind of obviousness analysis that since the 1952 incorporation of obviousness into the patent statute had been reserved for assessment under section 103 . thus , the introduction of the alice inventive concept requirement into section 101 might leave some question as to what work section 103 is now doing , or whether an invention that is found the standards for the two are of course not necessarily identical ; section 103 specifies that obviousness is to be assessed at a particular time ( the time an application is filed ) against particular prior art ( the categories contemplated in section 102 ) according to a particular standard ( the knowledge and skill of the person having ordinary skill ) . following an aberrant and discredited bit of analysis in the 1979 parker v. flook decision , the supreme court in mayo has hinted that nature itself is the standard against which inventiveness is assessed , meaning that the outcome of a 101 inventive concept assessment might be quite different than a 103 obviousness assessment . ultimately , this became the judicial resolution for the recurring problem ; rejecting bright - line rules regarding methods of making and methods of using , the federal circuit held that , as provided by the language of section 103 , obviousness must of products or processes must be determined on a case - by - case basis against the relevant prior art , notwithstanding their relationship to related inventions . while that approach provides an answer for sorting out the obviousness standard in section 103 , the supreme court has now created an analogous problem within section 101 . the process of producing such clones was not at issue in the case , but there seems little dispute that the cloning process would constitute patentable subject matter , and indeed the federal circuit suggests in passing that any innovation attributable to dolly 's inventors should and would be better protected by process claims . but as a question of subject matter eligibility , the federal circuit 's treatment of patents covering dolly the sheep might have benefitted from the framework outlined in the later alice opinion , and considering how alice might have changed the roslin analysis offers a worthwhile exercise in determining how the alice framework operates in the context of products of nature . at the same time , reconsideration of dolly 's situation simultaneously reveals how , as applied to biotechnology , the alice approach creates confusion across a range of doctrines , including both product - by - process claims and a section 101 analog of the old durden analysis . and , although the question extends well beyond my brief in this particular essay , it is worth at least noting that , given the technological subject matter of the alice opinion , and of most of the other supreme court subject matter opinions , one might expect the framework 's inventive concept requirement to raise similarly intractable issues in the context of data processing . this makes clear that , far from settling ongoing questions about patentable subject matter , the supreme court 's alice opinion leaves unsettled questions that will haunt us for years to come .

catalytic reactions aided by directing groups are now ubiquitous in the chemical literature . for example , great strides have been made in the area of hydroxyl - directed epoxidation , with enantioselective methods for allylic , homoallylic , and even bis - homoallylic alcohols now recorded . more rare , however , are catalytic reactions directed by a remote chemical moiety that is , a functional group that is several bonds distal to the reactive site of the substrate . often , these general synthetic strategies rely on conformational restriction or strong molecular interactions to achieve selectivity . in contrast , we recently discovered a peptide - based catalyst ( 1 ) capable of oxidizing an olefin positioned five rotatable bonds ( in addition to one c notably , the selectivity achieved by 1 differs from that observed with farnesol ( 2 ) when m - chloroperoxybenzoic acid ( mcpba ) or , to our knowledge , any other previously known site - selective oxidation catalyst is used . despite the remarkable site selectivity exhibited by catalyst 1 ( 1:8:1 for the 2,3- , 6,7- , and 10,11-positions of 2 , shown in green , blue , and red , respectively , in scheme 1a ) , given the peculiar selectivity imparted by this remotely directed catalyst , we sought to understand the molecular underpinnings of the catalyst substrate interaction . catalyst 1 was developed using an on - bead screening protocol used to evolve combinatorial libraries for site selectivity in the epoxidation of farnesol ( scheme 1a ) . each peptide belonging to the initial screening libraries contained a boc - protected aspartic acid residue at the n - terminus , which possesses a catalytic carboxylic acid moiety in the side chain . in the catalytic cycle ( abbreviated in scheme 1b ) , the carboxylic acid of 1 is activated with n , n-diisopropylcarbodiimide ( dic ) and , in the presence of h2o2 , forms a peptidyl peracid that selectively epoxidizes the 6,7-olefin of either 2 or geranylgeraniol ( 3 ) . critically , we had established previously that the reaction catalyzed by 1 was predominantly hydroxyl - directed through the observation of selective 6,7-epoxidation of both 2and3 . in addition , our preliminary experiments showed that catalysts of this type deliver substantially lower selectivity for the 6,7-olefin of farnesyl methyl ether , comparable to that exhibited by propionic acid [ supporting information ( si ) figure 9 ] . other than the apparently fortuitous choice of the amino acids and their position within our screening libraries , we developed 1 without any preconceived notion of how such a catalyst would deliver selectivity . therefore , an understanding of the mechanism for the selectivity displayed by catalyst 1 demanded further studies , which are presented herein . simultaneously , a detailed retrospective analysis of the arrival at the best peptide sequence appeared to be inextricably related and has not been described previously . a limited understanding of the importance of the residues within peptide 1 was initially derived from studies of the libraries leading to the discovery of 1 . the observation of a peptide that exhibited a modicum of selectivity for the 6,7-position of 2 ( relative to the 2,3- or 10,11-positions ) was unexpected . in fact , the library from which this initial 6,7-selective hit derived was generated in order to study 2,3-selectivity . this library had been designed such that two internal residues had been omitted from the original hexameric sequences . this shortened library of tetramers was biased to contain a choice of d - amino acids adjacent to the catalytic n - terminal aspartic acid residue and two additional c - terminal variable residues . the first peptide that was found to exhibit the alternate 6,7-selectivity , boc - asp - d - pro - thr(bn)-leu ( boc = tert - butoxycarbonyl , bn = benzyl ) , exhibited only modest selectivity ( 4:5:6 = 1.3:1.5:1.0 , point shown in figure 1a ) . a subsequent library biased toward this initial hit was immediately prepared . split and pool libraries biased for this study were prepared such that at each varied position in the peptide design , roughly half the library members possess the amino acid of the parent sequence while the remaining library members would have one of six or seven alternative residues . additionally , each successive library was designed to possess a longer sequence length than the previous generation , with an additional variable residue added to the c - terminal side . at the same time that the first directed library was being prepared ( second generation ) , further screening of the initial library ( first generation ) ultimately led to another more 6,7-selective peptide with an i + 3 trityl ( trt)-protected asparagine [ asn(trt ) ] ( 4:5:6 = 1.2:2.1:1.0 ) . the data from the first - generation library are presented graphically in figure 1a and discussed in more detail below . figure 1 contains ternary plots that illustrate the percentage distribution of the monoepoxides formed by catalysts during on - bead screening experiments . in these plots , thus , points appearing higher on the graph represent product mixtures with a higher proportion of product 5 . points that appear closer to the other vertices represent peptides that produce a higher proportion of 4 or 6 . ( a c ) ternary plots showing an overlay of peptide selectivities from successive generations of 6,7-selective peptides , where each axis represents the fraction of the total monoepoxide : ( a ) the first library demonstrating 6,7-selectivity ( first generation , red ) ; ( b ) the first biased library for 6,7-selectivity ( second generation , green ) ; points that are higher ( further away from the triangle base ) are more 6,7-selective . the highlighted generation from each plot is in the solid color in the foreground atop the other library generations in the background . ( d ) list of a portion of sequenced peptides from each library generation ( shown with solid markers in a residues shown in blue indicate were picked from the pool of variable residues from their particular library . ( e ) selectivity with amino acids at the i + 2 position , which can be compared to the library with i + 2 thr(bn ) directly to the left ( c , third generation , blue ) . product ratios were measured by gc . in all of the on - bead screening studies , the formation of diepoxides was intentionally limited through the use of 0.3 equiv of dic , resulting in analysis of low - conversion reaction mixtures . screening of the second generation of catalysts biased toward sequences containing an i + 3 leu revealed two additional sequences that performed better than the others ( 4:5:6 = 1.0:1.7:1.1 and 1.5:2.6:1.0 ) . both of these second - generation hits contained an i + 2 thr(bn ) and an i + 3 asn(trt ) ( figure 1b ) . finally , a third - generation library was studied wherein the i + 2 thr(bn ) was fixed and the i + 3 position was biased toward sequences containing asn(trt ) . a number of peptides from this library were found to yield favorable selectivity ( figure 1c ) , and we subjected many to sequencing . while it is difficult to draw conclusions from any individual peptide sequenced from these libraries without validation , all of the high - performing sequences contained an i + 3 asn(trt ) , suggesting that the asn(trt ) conferred advantageous selectivity . however , a variety of other residues was found in the i + 4 and i + 5 positions in these high - performing sequences ( figure 1d ) . it is interesting to note that the best catalysts from each library generation seem to yield similar positional selectivity when examined on solid supports and under the initial screening conditions ( figure 1a c ) . while many of the catalysts found from the later library generations performed similarly , these on - bead screens yielded several leads for peptide validation and optimization in solution , which ultimately led to 1 nonetheless , the on - bead screening data suggest that a tetramer is sufficient to achieve 6,7-selectivity on - bead ( from the first - generation library , observed 4:5:6 = 1.2:2.1:1.0 ) and that the best sequenced peptides from each on - bead generation contained common elements : an i + 2 thr(bn ) and an i + 3 asn(trt ) . with no other structural data but with knowledge of the amino acid sequence of peptide 1 , and no expectation that the full diversity of each peptide library had been sampled , we hypothesized that the embedded dipeptide d - pro - thr(bn ) might enforce a -turn . notably , d - pro adjacent to a -branched amino acid is suggestive of a type ii -turn , an oft - studied motif in a number of peptide - based catalysts from our lab and others . to examine the existence of this structural element within 1 , we resorted to the examination of further altered sequences within on - bead peptide libraries . if peptide 1 were to exhibit a turn , then perhaps on - bead libraries with different turn - promoting residues at the i + 2 position , such as val or aib , might also work at this position . such substitutions were thought to be a potential avenue for the identification of more selective catalysts . four distinct biased split - and - pool libraries of on - bead peptides derived from libraries that initially led to 1 were therefore compared for site selectivity in the epoxidation of 2 ( compare panels c and e of figure 1 ) . each library possessed either val , aib , thr(tbu ) , or thr(bn ) in the i + 2 position . although the data in figure 1e represent only a limited sampling of three libraries , the testing of different i + 2 residues while simultaneously evaluating other residues at adjacent positions can potentially reveal epistatic interactions . that is , in theory , this experiment allows for the possibility of finding better peptides that might only be found through multiple amino acid substitutions , where the better selectivity results from some combination of changes . nonetheless , peptides containing protected thr derivatives [ thr(bn ) or thr(tbu ) ] were more 6,7-selective than those containing either val or aib . these findings suggest that even if the i + 2 thr(bn ) is turn - promoting , it likely plays an additional role in delivering selectivity for the 6,7-olefin of 2 . peptides with either an i + 2 thr(bn ) or thr(tbu ) demonstrated favorable 6,7-selectivity on - bead and in solution . during the optimization studies of the 6,7-selective catalysts in solution , we generally observed that the site and enantioselectivity afforded by the catalysts for the 6,7-position were uncorrelated . for example , i + 2 thr(bn)-containing peptide 7 gave 4:5:6 = 1.0:4.1:1.3 with 5 in 1.6:1.0 er , yet the thr(tbu)-containing analogue of 7 gave 4:5:6 = 1.0:3.7:1.1 with 5 in 1.9:1.0 er . these findings underline the potentially complicated relationship between the different rates of epoxidation leading to either site and/or enantioselectivity . substitutions of other residues within 1 were studied by examining specific analogues on - resin . these peptide analogues were examined on - resin for convenience of synthesis and ease of screening , and they yielded reduced selectivity in comparison with their solution - phase counterparts . additionally , the on - bead reactions were performed under unoptimized conditions with substoichiometric oxidant , restricting the conversion . an on - bead analogue of 1 furnished a 4:5:6 product mixture of 1.2:2.3:1.0 ( table 1 , entry 1 ) . modifications to the i + 2 thr side chains , such as replacement of the benzyl protecting group with a trityl group ( entry 2 ) or removal of the -methyl ( entry 3 ) resulted in diminished selectivity . replacement of the ethereal o - benzyl with an ethyl group ( ile ; entry 4 ) still furnished some selectivity for 5 , although it was attenuated . reactions were conducted with 2 ( 1 mol , 1.0 equiv ) , dic ( 0.3 equiv ) , 1-hydroxybenzotriazole ( hobt ) ( 0.1 equiv ) , 4-dimethylaminopyridine ( dmap ) ( 0.1 equiv ) , h2o2 ( 1.0 equiv ) , dichloromethane ( dcm ) ( 0.2 m ) , and a single bead bound to peptide . 2-nal = 2-naphthylalanine , cha = cyclohexylalanine , hphe = homophenylalanine , hse = homoserine . when other trityl - containing amino acids [ hse(trt ) and ser(trt ) ; table 1 , entries 6 and 7 ] or asp(tbu ) ( entry 5 ) were studied in this position , the selectivity was nearly entirely lost . in comparison with other substitutions , leu yielded a decent level of selectivity ( entry 8) , though less than the parent peptide , which is potentially meaningful because leu is sometimes thought to be an asn isostere , though without a trityl protecting group . these observations are consistent with those made during the screening of on - bead libraries : both the i + 2 thr(bn ) and i + 3 asn(trt ) are important and sensitive to substitution . substitution of the i + 4 tyr(tbu ) side chain with other aryl- ( table 1 , entries 911 ) and alkyl - containing ( entry 12 ) side chains did not seem to lead to drastic changes in selectivity . these observations largely agree with those made from studies of substitutions of the i + 4 and i + 5 residues with peptides under more homogeneous conditions ( i.e. , not resin - bound ) . to explore the necessity of each amino acid , the selectivities of a number of truncated analogues of 1 ( 712 ) the results , presented in figure 2 , underline the complicated function that each amino acid of 1 plays in delivering optimal selectivity for the 6,7-olefin . 5 c lower than the previously reported epoxidation conditions . at 20 c , 1 ( b ) abbreviated 1d h nmr spectra of 1 and its truncated analogues acquired at 20 c at 20 mm . the truncated peptides reveal the interesting roles played by the i + 3 asn(trt ) and i + 4 tyr(tbu ) . replacement of the c - terminal gly - och3 of 1 with a methyl ester , as in 7 , results in roughly the same 6,7:10,11 selectivity but decreases the 6,7:2,3 selectivity by about half . deletion of each sequential amino acid from the c - terminus seems to decrease the overall selectivity for the 6,7-olefin relative to the 10,11-olefin . moreover , through each truncation , the selectivity for 6 increases relative to 5 , yet peptides 7 , 8 , and 9 display roughly the same 5:6 selectivity of 3:1 to 4:1 . consistent with the hypothesis of an interaction of the thr(bn ) region of the peptide with farnesol , tripeptide 9 with a c - terminal thr(bn)-nhch3 ( entry 4 ) is sufficient to furnish some 6,7-selectivity , though reduced from the parent peptide ( entry 1 ) . however , tripeptide 10 with a c - terminal methyl ester lacks selectivity entirely . additionally , the selectivity actually decreases substantially and begins to favor the 2,3-olefin within farnesol in the case of 11 and 12 , where the thr(bn ) is lacking . we were surprised to find that the simple aspartic acid derivative 12 slightly favors 6 given that simpler peracids ( e.g. , propionic acid ) favor oxidation of the most electron - rich olefin to make 4 . perhaps these observations underscore the surprising frequency with which catalysts selective for the olefin most proximal to a directing group are found . indeed , our experience exploring catalysts for the oxidation of 2 has been rife with allylic epoxidation catalysts . the intriguing transition from the observed 6,7-selectivity to an absence of selectivity with the change of an amide to an ester in the trimer may result from a variety of scenarios . the nh of the methyl amide in 9 may interact with farnesol to deliver selectivity . alternatively , the change from amide to ester ( 9 to 10 ) may result in a number of conformational changes , which are manifest in the h nmr spectrum . compared with the other peptides , some proton signals in the spectra of 10 are doubled , whereas the longer truncated variants seem to display less conformational heterogeneity . one possible conformation available to 9 but not 10 is a -turn with a 10-membered ring formed through hydrogen bonding of the methyl amide . the selectivity data from the truncated analogues combined with the 1d h nmr spectra ( figure 2b ) suggest that a -turn may form in trimer 9 and the longer peptides . in particular , the h nmr spectrum of tetramer 8 reveals significant downfield shifts of the boc - nh and nh - asn signals , corresponding to protons that would participate in the turn , while the nh - thr signal shifts upfield . while the nh - asn signals shift back upfield in the spectra of pentamer 7 and hexamer 1 ( relative to 8) , the observed boc - nh shifts appear further downfield for both . indeed , the spectra of the longer peptides are difficult to interpret given the broadening of some peaks at this low temperature . h rotating - frame nuclear overhauser effect spectroscopy ( roesy ) data corroborate the interpretation of a -turn ( figure 3a ) . the spectra suggest a through - space correlation between the -protons of the i asp and i + 3 asn(trt ) in spectra of 1 and 7 acquired at 25 and 20 c , respectively . additionally , these data suggest that the h-d - pro is close to the amide protons of both the i + 2 thr(bn ) and notably , many of the correlations found in 1 at 25 c are also found in truncated peptide 7 at 20 c , indicating that the room - temperature data for 1 are likely relevant . h roesy correlations found in 1 at 25 c and truncated peptide 7 at 20 c . ( b ) two structural ensembles from the 20 cns - generated structures of 1 computed using 25 c roesy data . ( c ) one structure chosen from the 20 structures shown with side chains . to determine whether the nmr data fit our qualitative inferences about secondary structure , we estimated and binned distance restraints from the roesy data for 1 acquired at 25 c for structure calculations using the program cns . for example , as with any structural study of a catalyst , observations of the resting or intermediate state may differ completely from those of the active catalyst . additionally , calibration of distance restraints is usually performed by comparison to an experimentally determined distance ( e.g. , two ortho aryl protons ) , but we were unable to find pairs of protons that could confidently be used for this purpose , so we looked at two different calibration sets between h-asp and h-d - pro ( see the supporting information ) . from each calibrated distance , the other correlations were assigned to different bins as appropriate , and 10 structures that best fit the data were analyzed from each data set . an overlay of the combined 20 structures suggested the existence of two structural ensembles , each of which contained members derived from both of the distance restraint sets . the calculated structures agree with other experimental observations presented herein and provide insight into some possible structural features . many of the structures that best fit the data are suggestive of a -turn characterized by an apparent hydrogen bond between the i asp - co and the i + 3 nh - asn(trt ) ( the distance between the backbone i co and i + 3 n ranged from 2.8 to 3.7 across the 20 structures ) . indeed , these observations are not surprising given the correlation between the -protons of those residues in the nmr spectra . a smaller set of the calculated structures may also contain a hydrogen bond between the i + 2 nh - thr(bn ) and the i co , indicative of a -turn . additionally , the data suggest that the i + 5 tyr(tbu ) side chain is oriented underneath the turn , as is evident from a representative structure picked from the ensembles ( figure 3c ) . on the basis of the aforementioned experimental evidence , we hypothesized that the interactions of 1 and 2 defined in figure 4 are consistent with the selective oxidation of the 6,7-olefin . we consider on the basis of analogue and structural studies that the nh of the i + 2 thr(bn ) could interact with the hydroxyl ( as shown ) . the data suggest that a type ii -turn can form , which orients this nh of the i + 2 thr(bn ) in the same direction as the aspartic acid side chain ( upward as in figure 4a , b ) . a -turn may also form in addition to or in combination with the -turn ( figure 4c ) . furthermore , the i + 2 thr(bn ) side chain o and the backbone nh may serve as hydrogen - bond acceptor and donor , respectively , with the hydroxyl of farnesol , leading to the proper proximal position of the 6,7-olefin relative to the modeled peracid ( figure 4a c ) . while the nmr experiments suggest a -turn , which is often accompanied by the discussed i i + 3 hydrogen bond , it is possible that under the reaction conditions , the nh of the i + 3 asn(trt ) is oriented such that a hydrogen bond forms with the substrate hydroxyl as well ( figure 4b ) . it is not clear whether the intramolecular i i + 3 hydrogen bond is beneficial to the selectivity or whether the i + 3 nh is important for binding with the substrate , or both , a detail that is difficult to discern experimentally . regardless , it appears that this nh is important for one or both hydrogen - bonding interactions . of note , our data support models wherein at least two loci contribute to the hydrogen - bonding network between the catalyst and substrate . that is , ablation of the ethereal hydrogen - bond acceptor in the i + 2 position is not enough to induce a complete loss of 6,7-selectivity ( table 1 , entry 4 ; see also table 2 , entry 4 ) . analysis of the other libraries is also internally consistent ( figure 1e ; val and aib libraries ) . hypothesized models for selectivity toward 5 . the hypothetical transition - state interactions between the catalyst and substrate in figure 4 are consistent with the observed selectivity with trimer 9 and are supported by the nmr studies of the longer peptides . interestingly , the minimal enantioselectivity in the formation of 5 can be explained by the accessibility of either face of the olefin through a number of possible -bond rotations in the substrate ( different rotational configurations are shown in figure 4a,b ) . it is not clear from these data what the additional amino acids that are potentially disposed farther away from the thr(bn ) and catalytic asp residue are doing to improve the 6,7-selectivity of 1 . farnesol interactions that lead to allylic alcohol epoxidation , as the 2,3-position is most proximal to the putative directing group . our studies have suggested that the mode by which these catalysts achieve selectivity is quite delicate , which is perhaps underlined by the limited solvent compatibility . whereas other peptidyl peracids that our group has studied have generally yielded some selectivity in a range of solvents , we have observed substantial selectivity of 1 toward 5 only in halogenated solvents . moreover , our data are consistent with several possible transition - state models ( e.g. , figure 4 ) that can not be resolved at this time . to test our hypothetical models , we re - examined a substrate that we had studied earlier , farnesyl methyl ether ( 13 ) . as noted in the introduction , we found that a 6,7-selective pentamer affords no appreciable selectivity under a previous set of reaction conditions , commensurate with loss of a hydroxyl - group - directed mechanism . under the updated reaction conditions of table 2 , we found once again that peptide 1 affords no selectivity between the 10,11-epoxide 14 and the 6,7-epoxide 15 ( entry 1 ) . moreover , when the terminus is functionalized ( e.g. , 13 ) , we observe little formation of the 2,3-epoxide ( e.g. , 16 ) ; the reactivity of the allylic olefin in the electrophilic epoxidation is attenuated by the electron - withdrawing oxygen - containing substituent , and there is no compensatory hydroxyl direction . however , the lack of selectivity between the 10,11-epoxide 14 and the 6,7-epoxide 15 observed with catalyst 1 allows a test of our models with an opportunity to enhance selectivity for one isomer . the models in figure 4 are consistent with the methyl group of farnesyl methyl ether ( 13 ) obstructing the highly orchestrated hydrogen - bonding array that accommodates the hydroxyl of farnesol ( one scenario is depicted in figure 5a ) . notably , each of the arrays in figure 4 is buttressed by an attractive interaction between the thr(bn ) -oxygen atom and the oh group . substitution of the i + 2 thr(bn ) of 1 with an ile , as in peptide 17 , eradicates this attractive interaction . its absence could then make space available for the methyl group while retaining a less elaborate but still functional group - directing array for ether - directed epoxidation ( figure 5b ) . consistent with this notion , catalyst 17 delivers an increase in the product ratio to nearly 1:4 for the 6,7-position ( 14:15 ; table 2 , entry 2 ) , instead of the 1:1 ratio observed with catalyst 1 ( entry 1 ) . as emphasized in figure 5a , it is possible that with 1 , unfavorable steric or electronic interactions exclude ether - directed chemistry . however , with the alkyl side chain of ile in 17 , the peptide is still able to accommodate hydrogen - bonding interactions with the ethereal oxygen of the substrate ( figure 5b ) , albeit with a perhaps reduced efficiency as one of several hydrogen - bond loci is removed . in this vein , catalyst 17 is indeed quite a bit less selective than catalyst 1 for farnesol itself ( 4:5:6 = 1.1:8.6:1.0 for 1 and 1.7:2.1:1.1 for 17 ; table 2 , entries 3 and 4 ) . taken together , the picture that emerges as farnesol ( 2 ) and its derived methyl ether ( 13 ) are evaluated with catalysts 1 and 17 is one that links the models in figures 4 and 5 in a self - consistent manner . hypothesized interactions of 13 with i + 3 side - chain variants of 1 and 17 . a thought - provoking and also consistent experiment was performed with difarnesyl ether ( 18 ) . of note , this compound is an oxide of the venerable natural product squalene , with a sole oxygen atom inserted within its central c c bond . when catalyst 17 was examined with substrate 18 , once again we observed that the catalytic formation of monoxides was appreciably enhanced in the anticipated internal olefin 19 ( 19:20 > 3:1 ; scheme 2 inset ) , with minimal allylic oxidation detected . this result contrasts with the virtual lack of selectivity observed with mcpba ( 19:20 = 1:1 ) or catalyst 1 ( 19:20 = 1:1 ) . the selective formation of 19 also differs markedly from the apparent statistical distribution of epoxides observed for squalene itself under the various conditions we have examined . taken together , these results represent a self - consistent data set for the models presented in figures 4 and 5 , ultimately derived from nmr structural data , extensive sar , and challenging of the model with a new substrate type . although we employed a diversity - based approach to catalyst discovery , we found that the catalyst that provides selectivity for the 6,7-position of farnesol exhibits a propensity for -turn formation , a motif that our lab has studied on numerous occasions . however , the structural analysis of 1 and its large extended family of variants led to new insights . notably , an interaction between the backbone nh and the side - chain oxygen of thr is thought to form part of the oxyanion hole of lipases . indeed , the comparative behavior of catalysts 1 and 17 provides some analogy for this feature . indeed , since our experiments with catalysts 1 and 17 provide evidence for remote directing effects with different substrate types , the observations highlight the tunability of catalyst hydrogen - bond arrays to accommodate different substrate - directing groups . substrates like farnesol , its derived ethers , and many other polyenes remain great challenges for total control of site selectivity through catalysis . nevertheless , the mechanistic hypotheses derived from the experimental data presented herein have suggested several opportunities for further catalyst development . future efforts in our lab will be focused on developing analogues of catalyst 1 ( and 17 ) for remote oxidation of other substrates and other positions .

we describe mechanistic investigations of a catalyst ( 1 ) that leads to selective epoxidation of farnesol at the 6,7-position , remote from the hydroxyl directing group . the experimental lineage of peptide 1 and a number of resin - bound peptide analogues were examined to reveal the importance of four n - terminal residues . we examined the selectivity of truncated analogues to find that a trimer is sufficient to furnish the remote selectivity . both 1d and 2d 1h nmr studies were used to determine possible catalyst conformations , culminating in proposed models showing possible interactions of farnesol with a protected thr side chain and backbone nh . the models were used to rationalize the selectivity of a modified catalyst ( 17 ) for the 6,7-position relative to an ether moiety in two related substrates .

Introduction Results and Discussion

in contrast , we recently discovered a peptide - based catalyst ( 1 ) capable of oxidizing an olefin positioned five rotatable bonds ( in addition to one c notably , the selectivity achieved by 1 differs from that observed with farnesol ( 2 ) when m - chloroperoxybenzoic acid ( mcpba ) or , to our knowledge , any other previously known site - selective oxidation catalyst is used . despite the remarkable site selectivity exhibited by catalyst 1 ( 1:8:1 for the 2,3- , 6,7- , and 10,11-positions of 2 , shown in green , blue , and red , respectively , in scheme 1a ) , given the peculiar selectivity imparted by this remotely directed catalyst , we sought to understand the molecular underpinnings of the catalyst substrate interaction . catalyst 1 was developed using an on - bead screening protocol used to evolve combinatorial libraries for site selectivity in the epoxidation of farnesol ( scheme 1a ) . each peptide belonging to the initial screening libraries contained a boc - protected aspartic acid residue at the n - terminus , which possesses a catalytic carboxylic acid moiety in the side chain . other than the apparently fortuitous choice of the amino acids and their position within our screening libraries , we developed 1 without any preconceived notion of how such a catalyst would deliver selectivity . therefore , an understanding of the mechanism for the selectivity displayed by catalyst 1 demanded further studies , which are presented herein . simultaneously , a detailed retrospective analysis of the arrival at the best peptide sequence appeared to be inextricably related and has not been described previously . a limited understanding of the importance of the residues within peptide 1 was initially derived from studies of the libraries leading to the discovery of 1 . the observation of a peptide that exhibited a modicum of selectivity for the 6,7-position of 2 ( relative to the 2,3- or 10,11-positions ) was unexpected . this library had been designed such that two internal residues had been omitted from the original hexameric sequences . this shortened library of tetramers was biased to contain a choice of d - amino acids adjacent to the catalytic n - terminal aspartic acid residue and two additional c - terminal variable residues . at the same time that the first directed library was being prepared ( second generation ) , further screening of the initial library ( first generation ) ultimately led to another more 6,7-selective peptide with an i + 3 trityl ( trt)-protected asparagine [ asn(trt ) ] ( 4:5:6 = 1.2:2.1:1.0 ) . the data from the first - generation library are presented graphically in figure 1a and discussed in more detail below . ( a c ) ternary plots showing an overlay of peptide selectivities from successive generations of 6,7-selective peptides , where each axis represents the fraction of the total monoepoxide : ( a ) the first library demonstrating 6,7-selectivity ( first generation , red ) ; ( b ) the first biased library for 6,7-selectivity ( second generation , green ) ; points that are higher ( further away from the triangle base ) are more 6,7-selective . ( d ) list of a portion of sequenced peptides from each library generation ( shown with solid markers in a residues shown in blue indicate were picked from the pool of variable residues from their particular library . a number of peptides from this library were found to yield favorable selectivity ( figure 1c ) , and we subjected many to sequencing . while many of the catalysts found from the later library generations performed similarly , these on - bead screens yielded several leads for peptide validation and optimization in solution , which ultimately led to 1 nonetheless , the on - bead screening data suggest that a tetramer is sufficient to achieve 6,7-selectivity on - bead ( from the first - generation library , observed 4:5:6 = 1.2:2.1:1.0 ) and that the best sequenced peptides from each on - bead generation contained common elements : an i + 2 thr(bn ) and an i + 3 asn(trt ) . with no other structural data but with knowledge of the amino acid sequence of peptide 1 , and no expectation that the full diversity of each peptide library had been sampled , we hypothesized that the embedded dipeptide d - pro - thr(bn ) might enforce a -turn . notably , d - pro adjacent to a -branched amino acid is suggestive of a type ii -turn , an oft - studied motif in a number of peptide - based catalysts from our lab and others . if peptide 1 were to exhibit a turn , then perhaps on - bead libraries with different turn - promoting residues at the i + 2 position , such as val or aib , might also work at this position . such substitutions were thought to be a potential avenue for the identification of more selective catalysts . four distinct biased split - and - pool libraries of on - bead peptides derived from libraries that initially led to 1 were therefore compared for site selectivity in the epoxidation of 2 ( compare panels c and e of figure 1 ) . that is , in theory , this experiment allows for the possibility of finding better peptides that might only be found through multiple amino acid substitutions , where the better selectivity results from some combination of changes . nonetheless , peptides containing protected thr derivatives [ thr(bn ) or thr(tbu ) ] were more 6,7-selective than those containing either val or aib . these findings suggest that even if the i + 2 thr(bn ) is turn - promoting , it likely plays an additional role in delivering selectivity for the 6,7-olefin of 2 . during the optimization studies of the 6,7-selective catalysts in solution , we generally observed that the site and enantioselectivity afforded by the catalysts for the 6,7-position were uncorrelated . these peptide analogues were examined on - resin for convenience of synthesis and ease of screening , and they yielded reduced selectivity in comparison with their solution - phase counterparts . an on - bead analogue of 1 furnished a 4:5:6 product mixture of 1.2:2.3:1.0 ( table 1 , entry 1 ) . modifications to the i + 2 thr side chains , such as replacement of the benzyl protecting group with a trityl group ( entry 2 ) or removal of the -methyl ( entry 3 ) resulted in diminished selectivity . reactions were conducted with 2 ( 1 mol , 1.0 equiv ) , dic ( 0.3 equiv ) , 1-hydroxybenzotriazole ( hobt ) ( 0.1 equiv ) , 4-dimethylaminopyridine ( dmap ) ( 0.1 equiv ) , h2o2 ( 1.0 equiv ) , dichloromethane ( dcm ) ( 0.2 m ) , and a single bead bound to peptide . when other trityl - containing amino acids [ hse(trt ) and ser(trt ) ; table 1 , entries 6 and 7 ] or asp(tbu ) ( entry 5 ) were studied in this position , the selectivity was nearly entirely lost . substitution of the i + 4 tyr(tbu ) side chain with other aryl- ( table 1 , entries 911 ) and alkyl - containing ( entry 12 ) side chains did not seem to lead to drastic changes in selectivity . , not resin - bound ) . to explore the necessity of each amino acid , the selectivities of a number of truncated analogues of 1 ( 712 ) the results , presented in figure 2 , underline the complicated function that each amino acid of 1 plays in delivering optimal selectivity for the 6,7-olefin . at 20 c , 1 ( b ) abbreviated 1d h nmr spectra of 1 and its truncated analogues acquired at 20 c at 20 mm . the truncated peptides reveal the interesting roles played by the i + 3 asn(trt ) and i + 4 tyr(tbu ) . replacement of the c - terminal gly - och3 of 1 with a methyl ester , as in 7 , results in roughly the same 6,7:10,11 selectivity but decreases the 6,7:2,3 selectivity by about half . deletion of each sequential amino acid from the c - terminus seems to decrease the overall selectivity for the 6,7-olefin relative to the 10,11-olefin . moreover , through each truncation , the selectivity for 6 increases relative to 5 , yet peptides 7 , 8 , and 9 display roughly the same 5:6 selectivity of 3:1 to 4:1 . consistent with the hypothesis of an interaction of the thr(bn ) region of the peptide with farnesol , tripeptide 9 with a c - terminal thr(bn)-nhch3 ( entry 4 ) is sufficient to furnish some 6,7-selectivity , though reduced from the parent peptide ( entry 1 ) . however , tripeptide 10 with a c - terminal methyl ester lacks selectivity entirely . additionally , the selectivity actually decreases substantially and begins to favor the 2,3-olefin within farnesol in the case of 11 and 12 , where the thr(bn ) is lacking . we were surprised to find that the simple aspartic acid derivative 12 slightly favors 6 given that simpler peracids ( e.g. perhaps these observations underscore the surprising frequency with which catalysts selective for the olefin most proximal to a directing group are found . indeed , our experience exploring catalysts for the oxidation of 2 has been rife with allylic epoxidation catalysts . the intriguing transition from the observed 6,7-selectivity to an absence of selectivity with the change of an amide to an ester in the trimer may result from a variety of scenarios . alternatively , the change from amide to ester ( 9 to 10 ) may result in a number of conformational changes , which are manifest in the h nmr spectrum . one possible conformation available to 9 but not 10 is a -turn with a 10-membered ring formed through hydrogen bonding of the methyl amide . the selectivity data from the truncated analogues combined with the 1d h nmr spectra ( figure 2b ) suggest that a -turn may form in trimer 9 and the longer peptides . h rotating - frame nuclear overhauser effect spectroscopy ( roesy ) data corroborate the interpretation of a -turn ( figure 3a ) . the spectra suggest a through - space correlation between the -protons of the i asp and i + 3 asn(trt ) in spectra of 1 and 7 acquired at 25 and 20 c , respectively . ( b ) two structural ensembles from the 20 cns - generated structures of 1 computed using 25 c roesy data . to determine whether the nmr data fit our qualitative inferences about secondary structure , we estimated and binned distance restraints from the roesy data for 1 acquired at 25 c for structure calculations using the program cns . for example , as with any structural study of a catalyst , observations of the resting or intermediate state may differ completely from those of the active catalyst . , two ortho aryl protons ) , but we were unable to find pairs of protons that could confidently be used for this purpose , so we looked at two different calibration sets between h-asp and h-d - pro ( see the supporting information ) . additionally , the data suggest that the i + 5 tyr(tbu ) side chain is oriented underneath the turn , as is evident from a representative structure picked from the ensembles ( figure 3c ) . on the basis of the aforementioned experimental evidence , we hypothesized that the interactions of 1 and 2 defined in figure 4 are consistent with the selective oxidation of the 6,7-olefin . the data suggest that a type ii -turn can form , which orients this nh of the i + 2 thr(bn ) in the same direction as the aspartic acid side chain ( upward as in figure 4a , b ) . furthermore , the i + 2 thr(bn ) side chain o and the backbone nh may serve as hydrogen - bond acceptor and donor , respectively , with the hydroxyl of farnesol , leading to the proper proximal position of the 6,7-olefin relative to the modeled peracid ( figure 4a c ) . while the nmr experiments suggest a -turn , which is often accompanied by the discussed i i + 3 hydrogen bond , it is possible that under the reaction conditions , the nh of the i + 3 asn(trt ) is oriented such that a hydrogen bond forms with the substrate hydroxyl as well ( figure 4b ) . it is not clear whether the intramolecular i i + 3 hydrogen bond is beneficial to the selectivity or whether the i + 3 nh is important for binding with the substrate , or both , a detail that is difficult to discern experimentally . the hypothetical transition - state interactions between the catalyst and substrate in figure 4 are consistent with the observed selectivity with trimer 9 and are supported by the nmr studies of the longer peptides . interestingly , the minimal enantioselectivity in the formation of 5 can be explained by the accessibility of either face of the olefin through a number of possible -bond rotations in the substrate ( different rotational configurations are shown in figure 4a,b ) . it is not clear from these data what the additional amino acids that are potentially disposed farther away from the thr(bn ) and catalytic asp residue are doing to improve the 6,7-selectivity of 1 . farnesol interactions that lead to allylic alcohol epoxidation , as the 2,3-position is most proximal to the putative directing group . whereas other peptidyl peracids that our group has studied have generally yielded some selectivity in a range of solvents , we have observed substantial selectivity of 1 toward 5 only in halogenated solvents . , figure 4 ) that can not be resolved at this time . as noted in the introduction , we found that a 6,7-selective pentamer affords no appreciable selectivity under a previous set of reaction conditions , commensurate with loss of a hydroxyl - group - directed mechanism . under the updated reaction conditions of table 2 , we found once again that peptide 1 affords no selectivity between the 10,11-epoxide 14 and the 6,7-epoxide 15 ( entry 1 ) . the models in figure 4 are consistent with the methyl group of farnesyl methyl ether ( 13 ) obstructing the highly orchestrated hydrogen - bonding array that accommodates the hydroxyl of farnesol ( one scenario is depicted in figure 5a ) . consistent with this notion , catalyst 17 delivers an increase in the product ratio to nearly 1:4 for the 6,7-position ( 14:15 ; table 2 , entry 2 ) , instead of the 1:1 ratio observed with catalyst 1 ( entry 1 ) . however , with the alkyl side chain of ile in 17 , the peptide is still able to accommodate hydrogen - bonding interactions with the ethereal oxygen of the substrate ( figure 5b ) , albeit with a perhaps reduced efficiency as one of several hydrogen - bond loci is removed . taken together , the picture that emerges as farnesol ( 2 ) and its derived methyl ether ( 13 ) are evaluated with catalysts 1 and 17 is one that links the models in figures 4 and 5 in a self - consistent manner . hypothesized interactions of 13 with i + 3 side - chain variants of 1 and 17 . of note , this compound is an oxide of the venerable natural product squalene , with a sole oxygen atom inserted within its central c c bond . the selective formation of 19 also differs markedly from the apparent statistical distribution of epoxides observed for squalene itself under the various conditions we have examined . taken together , these results represent a self - consistent data set for the models presented in figures 4 and 5 , ultimately derived from nmr structural data , extensive sar , and challenging of the model with a new substrate type . although we employed a diversity - based approach to catalyst discovery , we found that the catalyst that provides selectivity for the 6,7-position of farnesol exhibits a propensity for -turn formation , a motif that our lab has studied on numerous occasions . however , the structural analysis of 1 and its large extended family of variants led to new insights . notably , an interaction between the backbone nh and the side - chain oxygen of thr is thought to form part of the oxyanion hole of lipases . indeed , the comparative behavior of catalysts 1 and 17 provides some analogy for this feature . indeed , since our experiments with catalysts 1 and 17 provide evidence for remote directing effects with different substrate types , the observations highlight the tunability of catalyst hydrogen - bond arrays to accommodate different substrate - directing groups . nevertheless , the mechanistic hypotheses derived from the experimental data presented herein have suggested several opportunities for further catalyst development . future efforts in our lab will be focused on developing analogues of catalyst 1 ( and 17 ) for remote oxidation of other substrates and other positions .

epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitors are well - established treatments for advanced non - small cell lung cancer ( nsclc ) in many countries . they are generally well tolerated and not associated with the side effects typical of cytotoxic chemotherapy . gefitinib ( iressa , astrazeneca , london , uk ) is an orally administered , reversible tyrosine kinase inhibitor of egfr . in 2002 , it was approved for the treatment of inoperable or recurrent nsclc based on the results of two large - scale multicenter phase ii studies that yielded favorable responses and low toxicities . the iressa pan - asia study ( ipass ) found that gefitinib produced longer progression - free survival compared to carboplatin plus paclitaxel in patients with advanced pulmonary adenocarcinoma harboring egfr mutations . a more recent phase iii trial , conducted in metastatic nsclc patients with mutated egfr , confirmed these findings . common adverse events associated with gefitinib treatment are diarrhea , skin rashes , and nausea , but most of these are mild in severity and manageable . however , since the first report of gefitinib - induced interstitial lung disease ( ild ) from japan , ild associated with molecularly targeted agents has drawn considerable attention . the incidence of gefitinibinduced ild was approximately 1% in worldwide populations , while the frequency of ild in the japanese series was reported to be much higher than that in the rest of the world . in korean patients , several small prospective studies reported a high incidence ( 1.3%-3.7% ) of ild during gefitinib treatment [ 6 - 8 ] . gefitinib - induced ild is often life - threatening ; its mortality is approximately 30%-40% . however , investigation of predictive and prognostic factors for gefitinib - induced ild is limited . , we estimate the incidence of gefitinibinduced ild in a large korean population and describe the major clinical findings . a retrospective cohort study was performed with histology proven nsclc patients who were treated with gefitinib at seoul national university hospital from january 2002 through december 2011 . patient clinical data , including medical records , radiographic findings and laboratory results were reviewed . this study protocol was approved by the institutional review board ( irb ) of the seoul national university hospital ( irb protocol number : h 1308 - 047 - 511 ) . the following demographic data were abstracted : age , sex , comorbidities , smoking history , eastern cooperative oncology group ( ecog ) performance status , histologic type , previous anticancer treatment and concurrent pulmonary disease ( e.g. , pulmonary emphysema or interstitial pneumonitis ) . adverse events from gefitinib treatment were evaluated using the common terminology criteria for adverse events ( ctcae ) from the national cancer institute ver . 4.0 and a fatal adverse event was defined as being ctcae grade 4 or grade 5 . treatment response to gefitinib was assessed according to the criteria of the response evaluation criteria in solid tumors ( recist ) ver . laboratory results , including complete blood cell and differential counts , chemistry tests , and oxyhemoglobin saturation measured by pulse oximetry ( spo2 ) performed when gefitinib treatment began and when ild occurred were collected . overall survival was calculated from the initiation of gefitinib treatment to the date of death or last follow - up . new abnormal radiologic findings with respiratory symptoms after gefitinib treatment were defined as possible adverse pulmonary reactions . to identify the cause of an adverse pulmonary reaction , two of the investigators ( s .- h.b and s.h.s ) reviewed the data independently . if their opinions differed regarding the cause of an adverse pulmonary reaction , another investigator ( d .- w.k . ) made the decision . adverse pulmonary reactions were classified as non - infectious and infectious complications ; the causes of non - infectious complications were categorized using published classifications . the diagnosis of gefitinib - induced ild was based on published , generally accepted clinical criteria . in brief , for the diagnosis of ild , four specific findings were required : ( 1 ) progressive dyspnea with or without cough or fever , ( 2 ) lack of evidence of infection , ( 3 ) radiologic findings consistent with drug - induced ild ( i.e. , bilateral , diffuse , or patchy interstitial and/or alveolar opacifications without evidence of marked progression of underlying lung cancer ) , and ( 4 ) consistent pathologic findings , if available . categorical clinical variables were compared using a chi - squared test or fisher exact test . continuous variables were compared using an independent unpaired t test or the mann - whitney u test if the variables were not normally distributed . univariate analysis was performed to evaluate associations between the outcomes of gefitinib - induced ild and patient characteristics . variables with a p - value in the univariate analysis of less than 0.2 were selected for the multivariate analysis . overall survival was assessed by kaplan - meier method after onset of ild in different patient groups . two - sided null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 . all statistical analyses were performed with spss ver . 21.0 ( ibm spss statistics , ibm co. , armonk , ny ) . a retrospective cohort study was performed with histology proven nsclc patients who were treated with gefitinib at seoul national university hospital from january 2002 through december 2011 . patient clinical data , including medical records , radiographic findings and laboratory results were reviewed . this study protocol was approved by the institutional review board ( irb ) of the seoul national university hospital ( irb protocol number : h 1308 - 047 - 511 ) . the following demographic data were abstracted : age , sex , comorbidities , smoking history , eastern cooperative oncology group ( ecog ) performance status , histologic type , previous anticancer treatment and concurrent pulmonary disease ( e.g. , pulmonary emphysema or interstitial pneumonitis ) . adverse events from gefitinib treatment were evaluated using the common terminology criteria for adverse events ( ctcae ) from the national cancer institute ver . 4.0 and a fatal adverse event was defined as being ctcae grade 4 or grade 5 . treatment response to gefitinib was assessed according to the criteria of the response evaluation criteria in solid tumors ( recist ) ver . laboratory results , including complete blood cell and differential counts , chemistry tests , and oxyhemoglobin saturation measured by pulse oximetry ( spo2 ) performed when gefitinib treatment began and when ild occurred were collected . overall survival was calculated from the initiation of gefitinib treatment to the date of death or last follow - up . new abnormal radiologic findings with respiratory symptoms after gefitinib treatment were defined as possible adverse pulmonary reactions . to identify the cause of an adverse pulmonary reaction , two of the investigators ( s .- h.b and s.h.s ) reviewed the data independently . if their opinions differed regarding the cause of an adverse pulmonary reaction , another investigator ( d .- w.k . ) made the decision . adverse pulmonary reactions were classified as non - infectious and infectious complications ; the causes of non - infectious complications were categorized using published classifications . the diagnosis of gefitinib - induced ild was based on published , generally accepted clinical criteria . in brief , for the diagnosis of ild , four specific findings were required : ( 1 ) progressive dyspnea with or without cough or fever , ( 2 ) lack of evidence of infection , ( 3 ) radiologic findings consistent with drug - induced ild ( i.e. , bilateral , diffuse , or patchy interstitial and/or alveolar opacifications without evidence of marked progression of underlying lung cancer ) , and ( 4 ) consistent pathologic findings , if available . categorical clinical variables were compared using a chi - squared test or fisher exact test . continuous variables were compared using an independent unpaired t test or the mann - whitney u test if the variables were not normally distributed . univariate analysis was performed to evaluate associations between the outcomes of gefitinib - induced ild and patient characteristics . variables with a p - value in the univariate analysis of less than 0.2 were selected for the multivariate analysis . overall survival was assessed by kaplan - meier method after onset of ild in different patient groups . two - sided null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 . all statistical analyses were performed with spss ver . 21.0 ( ibm spss statistics , ibm co. , armonk , ny ) . the medical records of the 1,114 patients who received gefitinib from january 2002 through december 2011 were reviewed and abstracted ( fig . 1 ) . among these patients , 128 patients ( 11.5% ) developed symptomatic pulmonary infiltrates after treatment initiation . an infectious pulmonary complication occurred in 98 patients ( 8.8% ) , and 30 patients ( 2.7% ) experienced a non - infectious pulmonary complication . fifteen patients ( 1.3% ) with gefitinib - induced ild were identified . in multivariate analysis , only a lower serum albumin level was significantly associated with the development of gefitinib - induced ild after adjusting for sex , hypertension and coincidence of ild ( odds ratio , 3.91 ; 95% ci , 1.20 to 12.71 ) ( tables 1 and 2 ) . the association between the coincidence of ild before treatment and the development of gefitinibinduced ild was not significant statistically . the model fit was confirmed with the hosmer - lemeshow goodness of fitness test ( p=0.419 ) . the median time interval from the start of gefitinib administration to onset of ild was 29.0 days ( range , 3 to 1,953 days ) ( fig . gefitinib was used as second or later line treatment . in three patients ( 20.0% ) , the computed tomography ( ct ) findings at the time of ild diagnosis revealed combined lung cancer progression . the most common clinical presentation of gefitinib - induced ild was dyspnea ( 100% ) , followed by cough ( 60.0% ) . leukocytosis and elevated level of c - reactive protein ( crp ) were common ; mean leukocytes count was 13.9010/l and mean crp level was 12.01 mg / dl . the mean albumin level at the time of ild development was 3.03 g / dl . in two patients ( 13.3% ) , the patient treated with erlotinib began medication 3 months after development of gefitinib - induced ild . she received erlotinib treatment for 1 month but had to stop the medication due to newly appeared multifocal patch ground - glass opacities in both lungs on chest ct . in microbiologic examinations , sputum pneumocystis jirovecii polymerase chain reaction was positive , and the patient was treated with intravenous sulfamethoxazole , trimethoprim , and steroid . after pneumocystis pneumonia developed , she was transferred to another hospital near her hometown for hospice care because of worsened general condition . the mean dose was equivalent to 0.87 mg / kg / day methylprednisolone initially and was gradually tapered . among the 15 patients with gefitinib - induced ild , nine patients ( 60.0% ) had a fatal clinical course and six patients ( 40.0% ) died directly from ild . the other three patients ( 20.0% ) recovered from the potentially fatal ild condition after stopping the medication or steroid treatment . however , nosocomial respiratory superinfection developed in two patients and uncontrolled infection caused their death . one patient expired from sudden cardiac arrest of unknown cause when he under observation in a general ward as gefitinib - induced ild improving after steroid treatment . we compared the clinical and laboratory variables of the fatal and non - fatal cases of ild and found that patients in the fatal group developed ild after a shorter median length of gefitinib administration and had lower mean spo2 and lower albumin level at the time of ild development than did the non - fatal group ( table 3 ) . however , the fatal and nonfatal groups did not differ significantly in underlying comorbidities , pulmonary disease , types of previous cancer treatment , time interval from onset of ild to cessation of gefitinib or administration of corticosteroids , and methylprednisolone dosage . the medical records of the 1,114 patients who received gefitinib from january 2002 through december 2011 were reviewed and abstracted ( fig . 1 ) . among these patients , 128 patients ( 11.5% ) developed symptomatic pulmonary infiltrates after treatment initiation . an infectious pulmonary complication occurred in 98 patients ( 8.8% ) , and 30 patients ( 2.7% ) experienced a non - infectious pulmonary complication . in multivariate analysis , only a lower serum albumin level was significantly associated with the development of gefitinib - induced ild after adjusting for sex , hypertension and coincidence of ild ( odds ratio , 3.91 ; 95% ci , 1.20 to 12.71 ) ( tables 1 and 2 ) . the association between the coincidence of ild before treatment and the development of gefitinibinduced ild was not significant statistically . the model fit was confirmed with the hosmer - lemeshow goodness of fitness test ( p=0.419 ) . the median time interval from the start of gefitinib administration to onset of ild was 29.0 days ( range , 3 to 1,953 days ) ( fig . gefitinib was used as second or later line treatment . in three patients ( 20.0% ) , the computed tomography ( ct ) findings at the time of ild diagnosis revealed combined lung cancer progression . the most common clinical presentation of gefitinib - induced ild was dyspnea ( 100% ) , followed by cough ( 60.0% ) . leukocytosis and elevated level of c - reactive protein ( crp ) were common ; mean leukocytes count was 13.9010/l and mean crp level was 12.01 mg / dl . the mean albumin level at the time of ild development was 3.03 g / dl . in two patients ( 13.3% ) , the patient treated with erlotinib began medication 3 months after development of gefitinib - induced ild . she received erlotinib treatment for 1 month but had to stop the medication due to newly appeared multifocal patch ground - glass opacities in both lungs on chest ct . in microbiologic examinations , sputum pneumocystis jirovecii polymerase chain reaction was positive , and the patient was treated with intravenous sulfamethoxazole , trimethoprim , and steroid . after pneumocystis pneumonia developed , she was transferred to another hospital near her hometown for hospice care because of worsened general condition . the mean dose was equivalent to 0.87 mg / kg / day methylprednisolone initially and was gradually tapered . among the 15 patients with gefitinib - induced ild , nine patients ( 60.0% ) had a fatal clinical course and six patients ( 40.0% ) died directly from ild . the other three patients ( 20.0% ) recovered from the potentially fatal ild condition after stopping the medication or steroid treatment . however , nosocomial respiratory superinfection developed in two patients and uncontrolled infection caused their death . one patient expired from sudden cardiac arrest of unknown cause when he under observation in a general ward as gefitinib - induced ild improving after steroid treatment . we compared the clinical and laboratory variables of the fatal and non - fatal cases of ild and found that patients in the fatal group developed ild after a shorter median length of gefitinib administration and had lower mean spo2 and lower albumin level at the time of ild development than did the non - fatal group ( table 3 ) . however , the fatal and nonfatal groups did not differ significantly in underlying comorbidities , pulmonary disease , types of previous cancer treatment , time interval from onset of ild to cessation of gefitinib or administration of corticosteroids , and methylprednisolone dosage . gefitinib is recognized as an acceptably safe oral agent , given that the most common toxicities associated with its use are mild and self - limiting , such as skin toxicities and diarrhea . however , the development of ild during gefitinib treatment is a drug - related toxicity that sometimes has serious consequences . in 2003 , an food and drug administration ( fda ) analysis of 50,000 patients who received gefitinib reported a 1% worldwide incidence of ild . notably , the incidence of ild in japanese populations was reported to range between 2.4% and 5.4% in clinical trials [ 9,13 - 18 ] . in other asian studies , the incidence of ild also exceeds the global incidence ; in taiwanese patients , it was reported at 2.3% . however , the incidence of ild in our study was 1.3% , similar to values reported worldwide . this is much lower than reported in other asian populations , including studies of japanese and taiwanese patients ( table 4 ) . risk factors for ild have been identified in several japanese studies ( table 4 ) , but the findings from the reported studies are inconsistent . in the present study , we attempted to identify risk factors for ild in gefitinib - treated nsclc patients by collecting all possible risk factors , including laboratory results and clinical factors that were evaluated in previous studies . unlike the previous results , a lower serum albumin level at the time of gefitinib treatment initiation was the only risk factor for developing gefitinib - induced ild . a smaller japanese study evaluated the association between ild and several laboratory values including albumin level , but did not find a significant association . however , decreased egfr phosphorylation resulting in a decrease in regenerative epithelial proliferation could augment pulmonary fibrosis . we found that a lower serum albumin level might be an important surrogate marker for predicting gefitinib - induced ild . notably , the albumin level at the time of ild development decreased compared to that before gefitinib administration ( from a mean of 3.51 to 3.03 g / dl ) . hypoalbuminemia is a predisposing factor for methotrexate - induced pulmonary toxicity ( either before or during therapy ) . it has been hypothesized that a lower degree of protein binding of methotrexate and higher levels of free methotrexate resulting from hypoalbuminemia could enhance drug - induced pulmonary toxicity in methotrexate - treated patients . gefitinib is also primarily bound to plasma proteins including human serum albumin ( 90% ) and alpha-1 acid glycoprotein ( 78% ) . ter heine et al . reported a case of fatal ild associated with another egfr tyrosine kinase inhibitor erlotinib , with high erlotinib and metabolite plasma levels , and suggested that high drug and metabolite levels result in severe adverse pulmonary reactions . with these drugs , a lower albumin level might lead to higher levels of free gefitinib and enhance pulmonary toxicity . in a study of another target agent , monoclonal antibodies , low albumin level was identified as a risk factor for developing adverse pulmonary reactions in cancer patients . however , the cause for gefitinib - associated ild is multifactorial and it is difficult to demonstrate causation between hypoalbuminemia and the development of gefitinib - induced ild from the results in our study . in addition , hypoalbuminemia in itself is a marker for illness severity and malnutrition , and many confounding clinical factors can influence the development of hypoalbuminemia . another possible predisposing factor for gefitinib - induced ild is ethnic differences in susceptibility to ild . notably , the incidence of gefitinib - induced ild in japanese patients was higher than the incidence found in our study ; the incidence in our study was similar to values reported in worldwide populations . forsythe and faulkner suggest that the reason for the observed difference in susceptibility to ild is related to population or environmental differences or differences in diagnostic or clinical practice . a higher risk of ild has been noted in japan than elsewhere with other medications . in fact , the frequency of coincidence with interstitial pneumonitis before gefitinib treatment was much higher ( 5.0%-13.6% ) [ 13 - 17 ] than that observed in our korean population ( 1.6% ) . of note , comorbid interstitial pneumonitis was found to be a major risk factor for gefitinib - induced ild in many japanese studies ( table 4 ) . gefitinib - induced ild usually occurs during the first 3 months of treatment ; the range to onset is 24 to 42 days . in our study , the median time to development of ild was 29 days , and 80.0% of ild cases presented within the first 8 weeks of starting gefitinib treatment ( fig . the main manifestations of gefitinib - induced ild were dyspnea ( 100.0% ) and cough ( 60.0% ) , sequentially , while only two patients ( 13.3% ) presented with fever , a commonly recognized sign of infectious pneumonia . the prognosis for gefitinib - induced ild was consistent with other studies ( table 4 ) . in the present study , among patients diagnosed with ild , six patients had an ild related death ( mortality , 40.0% ) . treatment of gefitinib - induced ild is largely supportive , including supplemental oxygen , empirical antibiotics and mechanical ventilation . immediate discontinuation of the drug is strongly recommended and systemic corticosteroids are usually prescribed , although no controlled trials have been conducted to evaluate their efficacy . we investigated the association between clinical factors at the time of onset and fatality from gefitinib - induced ild . in this study , the treatment for gefitinib - induced ild was not significantly different between the fatal and non - fatal groups . the time interval from onset of symptoms to cessation of gefitinib was similar between groups . among the 15 patients who developed ild , 12 patients ( 80.0% ) the median dose of corticosteroids at the time of onset of ild , treatment duration and the time interval from ild onset to administration of corticosteroids were not significantly associated with the prognosis of ild . no patient was rechallenged with gefitinib after recovery from ild . in univariate analysis , patients with fatal ild were more likely to develop ild within a shorter interval , to have lower spo2 and lower serum albumin compared to those with non - fatal ild . although not confirmed by multivariate analysis , due to small sample size , ild with abrupt onset after gefitinib treatment and severe pneumonitis requiring high o2 demand apparently has a poor prognosis despite appropriate treatment . also lower albumin level might be a prognostic factor as well as a predictive factor . this is the first study that evaluated predictive factors for gefitinib - induced ild and examined prognostic factors for ild with the same data set in a large scale of korean patients treated with gefitinib . another concern was misdiagnosis of ild , but we believe that the independent review of clinical data by investigators minimized this problem . it was based on ct findings and clinical characteristics , and biopsies generally considered the gold standard for ild diagnosis were not performed in all cases . nevertheless the clinical features of gefitinib - induced ild found in our study were similar to those previously reported . in summary , the incidence of gefitinib - induced ild in korean nsclc patients was similar to that of worldwide reports but lower than values reported for japanese population . a lower serum albumin level ( 3.0 g / dl ) at baseline was the only predictive factor for gefitinib - induced ild . ild is a serious adverse effect and clinicians should give attention to the possibility of gefitinib - induced ild , particularly among patients with a lower serum albumin level .

purposeinterstitial lung disease ( ild ) is a serious adverse effect of gefitinib . we examined the incidence and clinical characteristics of drug - induced ild in korean non - small cell lung carcinoma patients treated with gefitinib.materials and methodsa retrospective cohort study was performed in non - small cell lung cancer ( nsclc ) patients who started gefitinib treatment at seoul national university hospital from january 2002 through december 2011 . patients who developed new abnormal radiologic findings with respiratory symptoms after gefitinib treatment were defined as having possible adverse pulmonary reactions . the patients medical records were reviewed independently by investigators to identify the causes of pulmonary toxicities.resultsamong the 1,114 patients evaluated , 128 patients ( 11.5% ) developed pulmonary adverse reactions after taking gefitinib . an infectious complication occurred in 98 patients ( 8.8% ) and 15 patients ( 1.3% ) developed ild . nine of the 15 patients ( 60.0% ) with gefitinib - induced ild experienced a fatal clinical course that met either the common terminology criteria for adverse events grade 4 ( n=3 ) or grade 5 ( n=6 ) . in the multivariate analysis , a lower serum albumin level ( 3.0 g / dl ) at baseline was significantly associated with the development of gefitinib - induced ild ( odds ratio , 3.91 ; 95% confidence interval , 1.20 to 12.71).conclusionthe incidence of gefitinib - induced ild in korean nsclc patients was similar to that reported worldwide , but lower than values reported for japanese population . ild was usually a life - threatening adverse effect of gefitinib , and the development of ild was significantly associated with a lower baseline serum albumin level .

Introduction Materials and Methods 1. Study populations 2. Clinical data collection 3. Confirmation of adverse pulmonary reaction and gefitinib-induced ILD 4. Statistical analysis Results 1. Incidence of adverse pulmonary reaction and gefitinibinduced ILD 2. Risk factors for gefitinib-induced ILD 3. Clinical characteristics of gefitinib-induced ILD 4. Treatment and outcomes of gefitinib-induced ILD Discussion Conclusion

epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitors are well - established treatments for advanced non - small cell lung cancer ( nsclc ) in many countries . they are generally well tolerated and not associated with the side effects typical of cytotoxic chemotherapy . common adverse events associated with gefitinib treatment are diarrhea , skin rashes , and nausea , but most of these are mild in severity and manageable . however , since the first report of gefitinib - induced interstitial lung disease ( ild ) from japan , ild associated with molecularly targeted agents has drawn considerable attention . the incidence of gefitinibinduced ild was approximately 1% in worldwide populations , while the frequency of ild in the japanese series was reported to be much higher than that in the rest of the world . in korean patients , several small prospective studies reported a high incidence ( 1.3%-3.7% ) of ild during gefitinib treatment [ 6 - 8 ] . gefitinib - induced ild is often life - threatening ; its mortality is approximately 30%-40% . however , investigation of predictive and prognostic factors for gefitinib - induced ild is limited . , we estimate the incidence of gefitinibinduced ild in a large korean population and describe the major clinical findings . a retrospective cohort study was performed with histology proven nsclc patients who were treated with gefitinib at seoul national university hospital from january 2002 through december 2011 . patient clinical data , including medical records , radiographic findings and laboratory results were reviewed . this study protocol was approved by the institutional review board ( irb ) of the seoul national university hospital ( irb protocol number : h 1308 - 047 - 511 ) . adverse events from gefitinib treatment were evaluated using the common terminology criteria for adverse events ( ctcae ) from the national cancer institute ver . 4.0 and a fatal adverse event was defined as being ctcae grade 4 or grade 5 . laboratory results , including complete blood cell and differential counts , chemistry tests , and oxyhemoglobin saturation measured by pulse oximetry ( spo2 ) performed when gefitinib treatment began and when ild occurred were collected . overall survival was calculated from the initiation of gefitinib treatment to the date of death or last follow - up . new abnormal radiologic findings with respiratory symptoms after gefitinib treatment were defined as possible adverse pulmonary reactions . to identify the cause of an adverse pulmonary reaction , two of the investigators ( s .- h.b and s.h.s ) reviewed the data independently . adverse pulmonary reactions were classified as non - infectious and infectious complications ; the causes of non - infectious complications were categorized using published classifications . the diagnosis of gefitinib - induced ild was based on published , generally accepted clinical criteria . in brief , for the diagnosis of ild , four specific findings were required : ( 1 ) progressive dyspnea with or without cough or fever , ( 2 ) lack of evidence of infection , ( 3 ) radiologic findings consistent with drug - induced ild ( i.e. , bilateral , diffuse , or patchy interstitial and/or alveolar opacifications without evidence of marked progression of underlying lung cancer ) , and ( 4 ) consistent pathologic findings , if available . univariate analysis was performed to evaluate associations between the outcomes of gefitinib - induced ild and patient characteristics . variables with a p - value in the univariate analysis of less than 0.2 were selected for the multivariate analysis . overall survival was assessed by kaplan - meier method after onset of ild in different patient groups . a retrospective cohort study was performed with histology proven nsclc patients who were treated with gefitinib at seoul national university hospital from january 2002 through december 2011 . patient clinical data , including medical records , radiographic findings and laboratory results were reviewed . this study protocol was approved by the institutional review board ( irb ) of the seoul national university hospital ( irb protocol number : h 1308 - 047 - 511 ) . adverse events from gefitinib treatment were evaluated using the common terminology criteria for adverse events ( ctcae ) from the national cancer institute ver . 4.0 and a fatal adverse event was defined as being ctcae grade 4 or grade 5 . laboratory results , including complete blood cell and differential counts , chemistry tests , and oxyhemoglobin saturation measured by pulse oximetry ( spo2 ) performed when gefitinib treatment began and when ild occurred were collected . overall survival was calculated from the initiation of gefitinib treatment to the date of death or last follow - up . new abnormal radiologic findings with respiratory symptoms after gefitinib treatment were defined as possible adverse pulmonary reactions . to identify the cause of an adverse pulmonary reaction , two of the investigators ( s .- h.b and s.h.s ) reviewed the data independently . adverse pulmonary reactions were classified as non - infectious and infectious complications ; the causes of non - infectious complications were categorized using published classifications . the diagnosis of gefitinib - induced ild was based on published , generally accepted clinical criteria . in brief , for the diagnosis of ild , four specific findings were required : ( 1 ) progressive dyspnea with or without cough or fever , ( 2 ) lack of evidence of infection , ( 3 ) radiologic findings consistent with drug - induced ild ( i.e. , bilateral , diffuse , or patchy interstitial and/or alveolar opacifications without evidence of marked progression of underlying lung cancer ) , and ( 4 ) consistent pathologic findings , if available . univariate analysis was performed to evaluate associations between the outcomes of gefitinib - induced ild and patient characteristics . variables with a p - value in the univariate analysis of less than 0.2 were selected for the multivariate analysis . overall survival was assessed by kaplan - meier method after onset of ild in different patient groups . the medical records of the 1,114 patients who received gefitinib from january 2002 through december 2011 were reviewed and abstracted ( fig . among these patients , 128 patients ( 11.5% ) developed symptomatic pulmonary infiltrates after treatment initiation . an infectious pulmonary complication occurred in 98 patients ( 8.8% ) , and 30 patients ( 2.7% ) experienced a non - infectious pulmonary complication . fifteen patients ( 1.3% ) with gefitinib - induced ild were identified . in multivariate analysis , only a lower serum albumin level was significantly associated with the development of gefitinib - induced ild after adjusting for sex , hypertension and coincidence of ild ( odds ratio , 3.91 ; 95% ci , 1.20 to 12.71 ) ( tables 1 and 2 ) . the association between the coincidence of ild before treatment and the development of gefitinibinduced ild was not significant statistically . the median time interval from the start of gefitinib administration to onset of ild was 29.0 days ( range , 3 to 1,953 days ) ( fig . in three patients ( 20.0% ) , the computed tomography ( ct ) findings at the time of ild diagnosis revealed combined lung cancer progression . the most common clinical presentation of gefitinib - induced ild was dyspnea ( 100% ) , followed by cough ( 60.0% ) . the mean albumin level at the time of ild development was 3.03 g / dl . in two patients ( 13.3% ) , the patient treated with erlotinib began medication 3 months after development of gefitinib - induced ild . in microbiologic examinations , sputum pneumocystis jirovecii polymerase chain reaction was positive , and the patient was treated with intravenous sulfamethoxazole , trimethoprim , and steroid . among the 15 patients with gefitinib - induced ild , nine patients ( 60.0% ) had a fatal clinical course and six patients ( 40.0% ) died directly from ild . the other three patients ( 20.0% ) recovered from the potentially fatal ild condition after stopping the medication or steroid treatment . one patient expired from sudden cardiac arrest of unknown cause when he under observation in a general ward as gefitinib - induced ild improving after steroid treatment . we compared the clinical and laboratory variables of the fatal and non - fatal cases of ild and found that patients in the fatal group developed ild after a shorter median length of gefitinib administration and had lower mean spo2 and lower albumin level at the time of ild development than did the non - fatal group ( table 3 ) . however , the fatal and nonfatal groups did not differ significantly in underlying comorbidities , pulmonary disease , types of previous cancer treatment , time interval from onset of ild to cessation of gefitinib or administration of corticosteroids , and methylprednisolone dosage . the medical records of the 1,114 patients who received gefitinib from january 2002 through december 2011 were reviewed and abstracted ( fig . among these patients , 128 patients ( 11.5% ) developed symptomatic pulmonary infiltrates after treatment initiation . an infectious pulmonary complication occurred in 98 patients ( 8.8% ) , and 30 patients ( 2.7% ) experienced a non - infectious pulmonary complication . in multivariate analysis , only a lower serum albumin level was significantly associated with the development of gefitinib - induced ild after adjusting for sex , hypertension and coincidence of ild ( odds ratio , 3.91 ; 95% ci , 1.20 to 12.71 ) ( tables 1 and 2 ) . the association between the coincidence of ild before treatment and the development of gefitinibinduced ild was not significant statistically . the median time interval from the start of gefitinib administration to onset of ild was 29.0 days ( range , 3 to 1,953 days ) ( fig . in three patients ( 20.0% ) , the computed tomography ( ct ) findings at the time of ild diagnosis revealed combined lung cancer progression . the most common clinical presentation of gefitinib - induced ild was dyspnea ( 100% ) , followed by cough ( 60.0% ) . leukocytosis and elevated level of c - reactive protein ( crp ) were common ; mean leukocytes count was 13.9010/l and mean crp level was 12.01 mg / dl . the mean albumin level at the time of ild development was 3.03 g / dl . in two patients ( 13.3% ) , the patient treated with erlotinib began medication 3 months after development of gefitinib - induced ild . in microbiologic examinations , sputum pneumocystis jirovecii polymerase chain reaction was positive , and the patient was treated with intravenous sulfamethoxazole , trimethoprim , and steroid . among the 15 patients with gefitinib - induced ild , nine patients ( 60.0% ) had a fatal clinical course and six patients ( 40.0% ) died directly from ild . the other three patients ( 20.0% ) recovered from the potentially fatal ild condition after stopping the medication or steroid treatment . one patient expired from sudden cardiac arrest of unknown cause when he under observation in a general ward as gefitinib - induced ild improving after steroid treatment . we compared the clinical and laboratory variables of the fatal and non - fatal cases of ild and found that patients in the fatal group developed ild after a shorter median length of gefitinib administration and had lower mean spo2 and lower albumin level at the time of ild development than did the non - fatal group ( table 3 ) . however , the fatal and nonfatal groups did not differ significantly in underlying comorbidities , pulmonary disease , types of previous cancer treatment , time interval from onset of ild to cessation of gefitinib or administration of corticosteroids , and methylprednisolone dosage . however , the development of ild during gefitinib treatment is a drug - related toxicity that sometimes has serious consequences . in 2003 , an food and drug administration ( fda ) analysis of 50,000 patients who received gefitinib reported a 1% worldwide incidence of ild . notably , the incidence of ild in japanese populations was reported to range between 2.4% and 5.4% in clinical trials [ 9,13 - 18 ] . in other asian studies , the incidence of ild also exceeds the global incidence ; in taiwanese patients , it was reported at 2.3% . however , the incidence of ild in our study was 1.3% , similar to values reported worldwide . this is much lower than reported in other asian populations , including studies of japanese and taiwanese patients ( table 4 ) . in the present study , we attempted to identify risk factors for ild in gefitinib - treated nsclc patients by collecting all possible risk factors , including laboratory results and clinical factors that were evaluated in previous studies . unlike the previous results , a lower serum albumin level at the time of gefitinib treatment initiation was the only risk factor for developing gefitinib - induced ild . a smaller japanese study evaluated the association between ild and several laboratory values including albumin level , but did not find a significant association . we found that a lower serum albumin level might be an important surrogate marker for predicting gefitinib - induced ild . notably , the albumin level at the time of ild development decreased compared to that before gefitinib administration ( from a mean of 3.51 to 3.03 g / dl ) . hypoalbuminemia is a predisposing factor for methotrexate - induced pulmonary toxicity ( either before or during therapy ) . it has been hypothesized that a lower degree of protein binding of methotrexate and higher levels of free methotrexate resulting from hypoalbuminemia could enhance drug - induced pulmonary toxicity in methotrexate - treated patients . gefitinib is also primarily bound to plasma proteins including human serum albumin ( 90% ) and alpha-1 acid glycoprotein ( 78% ) . reported a case of fatal ild associated with another egfr tyrosine kinase inhibitor erlotinib , with high erlotinib and metabolite plasma levels , and suggested that high drug and metabolite levels result in severe adverse pulmonary reactions . with these drugs , a lower albumin level might lead to higher levels of free gefitinib and enhance pulmonary toxicity . in a study of another target agent , monoclonal antibodies , low albumin level was identified as a risk factor for developing adverse pulmonary reactions in cancer patients . however , the cause for gefitinib - associated ild is multifactorial and it is difficult to demonstrate causation between hypoalbuminemia and the development of gefitinib - induced ild from the results in our study . in addition , hypoalbuminemia in itself is a marker for illness severity and malnutrition , and many confounding clinical factors can influence the development of hypoalbuminemia . another possible predisposing factor for gefitinib - induced ild is ethnic differences in susceptibility to ild . notably , the incidence of gefitinib - induced ild in japanese patients was higher than the incidence found in our study ; the incidence in our study was similar to values reported in worldwide populations . in fact , the frequency of coincidence with interstitial pneumonitis before gefitinib treatment was much higher ( 5.0%-13.6% ) [ 13 - 17 ] than that observed in our korean population ( 1.6% ) . of note , comorbid interstitial pneumonitis was found to be a major risk factor for gefitinib - induced ild in many japanese studies ( table 4 ) . gefitinib - induced ild usually occurs during the first 3 months of treatment ; the range to onset is 24 to 42 days . in our study , the median time to development of ild was 29 days , and 80.0% of ild cases presented within the first 8 weeks of starting gefitinib treatment ( fig . the main manifestations of gefitinib - induced ild were dyspnea ( 100.0% ) and cough ( 60.0% ) , sequentially , while only two patients ( 13.3% ) presented with fever , a commonly recognized sign of infectious pneumonia . the prognosis for gefitinib - induced ild was consistent with other studies ( table 4 ) . in the present study , among patients diagnosed with ild , six patients had an ild related death ( mortality , 40.0% ) . treatment of gefitinib - induced ild is largely supportive , including supplemental oxygen , empirical antibiotics and mechanical ventilation . we investigated the association between clinical factors at the time of onset and fatality from gefitinib - induced ild . in this study , the treatment for gefitinib - induced ild was not significantly different between the fatal and non - fatal groups . the time interval from onset of symptoms to cessation of gefitinib was similar between groups . among the 15 patients who developed ild , 12 patients ( 80.0% ) the median dose of corticosteroids at the time of onset of ild , treatment duration and the time interval from ild onset to administration of corticosteroids were not significantly associated with the prognosis of ild . in univariate analysis , patients with fatal ild were more likely to develop ild within a shorter interval , to have lower spo2 and lower serum albumin compared to those with non - fatal ild . although not confirmed by multivariate analysis , due to small sample size , ild with abrupt onset after gefitinib treatment and severe pneumonitis requiring high o2 demand apparently has a poor prognosis despite appropriate treatment . this is the first study that evaluated predictive factors for gefitinib - induced ild and examined prognostic factors for ild with the same data set in a large scale of korean patients treated with gefitinib . another concern was misdiagnosis of ild , but we believe that the independent review of clinical data by investigators minimized this problem . it was based on ct findings and clinical characteristics , and biopsies generally considered the gold standard for ild diagnosis were not performed in all cases . nevertheless the clinical features of gefitinib - induced ild found in our study were similar to those previously reported . in summary , the incidence of gefitinib - induced ild in korean nsclc patients was similar to that of worldwide reports but lower than values reported for japanese population . a lower serum albumin level ( 3.0 g / dl ) at baseline was the only predictive factor for gefitinib - induced ild . ild is a serious adverse effect and clinicians should give attention to the possibility of gefitinib - induced ild , particularly among patients with a lower serum albumin level .

muscarinic receptors are the main mediator of physiological contraction of the urinary bladder . while m2 and m3 subtypes exist in an about 4:1 ratio in the bladder of humans and many other mammals , direct contraction responses to exogenous agonists or endogenous agonist as released by field stimulation are mediated predominantly if not exclusively by the minor fraction of m3 receptors ( hegde 2006 ) . while it has been questioned on theoretical grounds that subtype - selective antagonists are sufficient to support this conclusion ( ehlert 2003 ) , it should be noted that it is consistent with findings from m2 and m3 receptor knockout mice ( matsui et al . m3 receptors , including those in the urinary bladder , couple to phospholipase c ( plc ) stimulation , but the direct contractile effect via m3 receptors occurs largely independent of plc in the bladder ( frazier et al . bladder smooth muscle relaxation is mediated by -adrenoceptors , in humans and many species mostly their 3 subtype ( michel and vrydag 2006 ) . while -adrenoceptors , including those in the bladder , couple to stimulation of an adenylyl cyclase , bladder relaxation by -adrenergic agonists occurs largely independent of adenylyl cyclase stimulation ( frazier et al . thus , bladder smooth muscle tone is under a dual control of m3 muscarinic receptors and -adrenoceptors . this is similar to smooth muscle tone regulation in many other tissues , e.g. , the airways , except relaxation responses in most cases outside the bladder are mediated by the 2 subtype ( michel and parra 2008 ) . the physiological control of bladder function involves activation of -adrenoceptors by neuronally released noradrenaline during the storage phase of the micturition cycle , whereas neuronally released acetylcholine acting on m3 receptors mediates detrusor contraction during the voiding phase ( andersson et al . however , under pathophysiological conditions nonneuronal acetylcholine release from the urothelium may also play a role and can be present not only during the voiding but also the storage phase ( andersson 2011 ) . the concomitant exposure of the detrusor to noradrenaline and acetylcholine during the storage phase may have important implications for bladder smooth muscle function . thus , it has been shown that the potency and efficacy of a muscarinic agonist to elicit detrusor contraction is attenuated in the presence of a -adrenoceptor agonist ( yamanishi et al . perhaps even more important , muscarinic agonists can also attenuate the relaxing effects of -adrenoceptor stimulation in the bladder ( ehlert et al . 2007 ; longhurst and levendusky 1999 ; michel and sand 2009 ) , airways ( matsui et al . 2003 ; naline et al . 2007 ; sarria et al . 2002 ) , and ileum ( matsui et al . 2003 ) . such attenuation has been shown across multiple species , i.e. , rat ( longhurst and levendusky 1999 ; michel and sand 2009 ) , mouse ( ehlert et al . 2007 ; matsui et al . 2003 ) , and humans ( naline et al . 2007 ; sarria et al . 2002 ) , indicating that it is a general principle in the regulation of smooth muscle tone upon concomitant exposure to muscarinic and -adrenoceptor agonists . while all studies agree on the existence of attenuation of -adrenoceptor - mediated relaxation by muscarinic agonists , reduced potency and/or efficacy have been reported to underlie such attenuation . of note , the degree of attenuation of -adrenoceptor - mediated relaxation by muscarinic agonists exceeds that by other contractile stimuli in all cases where this has been studied , e.g. , that by kcl , bradykinin , or serotonin in the bladder ( michel and sand 2009 ) or that of histamine in the airways ( naline et al . this differential degree of attenuation can not be explained by a comparison between weak and strong contractile agonists , indicating that it may involve a specific property of muscarinic receptors rather than purely reflecting functional antagonism . the interaction between muscarinic receptors and -adrenoceptors may also shed light on the physiological role of the numerous m2 receptors in bladder and airways . thus , m2 receptor knockout mice exhibited a mitigated attenuation of relaxation responses to isoprenaline ( ehlert et al . 2007 ; matsui et al . 2003 ) and receptor - independent elevation of cellular camp content such by the adenylyl cyclase activator forskolin ( pak et al . 2010 ) . as m2 receptors largely signal via pertussis toxin - sensitive g proteins ( caulfield and birdsall 1998 ) , further support for a role of m2 receptors in the attenuation response comes from studies in which pertussis toxin treatment enhanced the relaxant effects of forskolin on oxotremorine - m - mediated contractions in the ileum and trachea ( ostrom and ehlert 1998 , 1999 ) . in a similar vein , it has been reported that under conditions of preferential alkylation of m3 receptors , the ability of a muscarinic agonist to reverse isoprenaline - induced relaxation is largely maintained ( ehlert et al . such alkylation protocols have been employed by several investigators to achieve reasonable subtype selectivity . while this has been successful in some cases under very specific conditions ( griffin et al . 2009 ) , other alkylation protocols have proven to be poorly selective ( braverman and ruggieri 1999 ) . moreover , in some cases , m3 responses have also been pertussis toxin sensitive ( offermanns et al . 1994 ) , making pertussis toxin an unreliable witness of m2 involvement . while the knockout mice circumvent selectivity problems , they are prone to other complications such as compensatory regulation of other receptors or signal transduction pathways . recently , an agonist with considerable selectivity for m2 over m3 receptors became available , but this compound exhibits a lower degree of selectivity , if any , over other subtypes ; in the bladder , where muscarinic receptor subtypes other than m2 and m3 are largely absent , this compound produces selective m2 stimulation ( jasper et al . moreover , we have recently discovered an m2 receptor antagonist with unprecedented selectivity for this subtype , which we report here for the first time ( fig . 1 ) . these two tools have enabled us to explore the role of m2 and m3 receptors in the attenuation of isoprenaline - induced bladder relaxation . moreover , we have used this approach to characterize the role of plc and protein kinase c ( pkc ) activation in the attenuation response.fig . 1structures of thrx-199874 ( bzi ) and thrx-182087 structures of thrx-199874 ( bzi ) and thrx-182087 radioligand binding assays were conducted with 1 nm [ h]n - methyl scopolamine ( ge healthcare , piscataway , nj , usa ) in a buffer consisting of 10 mm hepes , 100 mm nacl , 10 mm mgcl2 , and 0.025% bovine serum albumin , ph 7.4 at 37c . membrane fractions from cho - k1 cells expressing human recombinant m1 , m2 , m3 , m4 , or m5 muscarinic receptors were incubated with radioligand and unlabelled drugs for 1 h at 37c in a volume of 100 l . receptor expression levels ( bmax ) measured by saturation binding were determined to be 2.7 , 2.5 , 2.4 , 2.0 , and 3.2 pmol / mg protein for human recombinant m1 , m2 , m3 , m4 , or m5 muscarinic receptors , respectively . after separation by vacuum filtration onto gf / b filter plates presoaked with 0.3% polyethyleneimine , the quantity of membrane - bound radioligand was measured by scintillation counting . tissue preparation adult male wistar rats weighing 300 22 g were purchased from charles river ( maastricht , the netherlands ) . animals were anesthetized using pentobarbital ( 75 mg / kg , i.p . ) and sacrificed by decapitation . after removal of the dome and the trigonum , the middle parts of the cleaned bladders ( weight of 96 15 mg ) were cut transversally in four equal strips . the strips had a length of 19.9 3.6 mm and a weight of 9.5 2.7 mg ( n = 76 ) . all experimental procedures were in line with european union guidelines for the use of laboratory animals and approved by the animal care committee of academisch medisch centrum . briefly , the bladder strips were mounted under a resting tension of 10 mn in organ baths containing 7 ml of krebs henseleit buffer of the following composition : 118.5 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 0.025 mm na4edta , 2.5 mm cacl2 , 1.2 mm kh2po4 , 25 mm nahco3 , and 5.6 mm glucose at a temperature of 37c , yielding a total potassium concentration of 5.9 mm . the organ baths were continually gassed 95% o2/5% co2 to maintain a ph of 7.4 . the bladder strips were equilibrated for 120 min , during which the buffer solution was refreshed every 15 min until a steady contractile state had been reached . following the equilibration , the tissues were challenged with 50 mm kcl for 6 min ( maintaining isoosmolarity by reducing nacl concentration from 116.8 to 68.5 mm ) . after the first challenge , we equilibrated the strips again at passive tension of 10 mn for 90 min and challenged the strips again with 50 mm kcl for 6 min . after 60 min equilibration at 10 mn after the second kcl challenge , we added vehicle or substance of interest . carbachol was used at a concentration of 1 m , bzi and thrx at 100 nm . the enzyme inhibitors u 73,122 ( 10 m ) and chelerythrine ( 1 m ) were added 5 min prior to the administration of thrx . ten minutes later carbachol ( 1 m ) was added , which was given an incubation time of 15 min to reach steady contractile stadium . as isoprenaline - induced rat bladder relaxation can exhibit desensitization ( vrydag and michel 2009 ) , only one isoprenaline concentration response curve was constructed per bladder strip ; however , conditions being compared were always tested in parallel using strips from the same animal , and those paired comparisons were the basis of our statistical analysis ( see below ) . the m3-sparing agonist bzi [ also known as thrx-199874 , 4-(2-oxo-2,3-dihydro - benzimidazol-1-yl)-1,4-bipiperidinyl-1-carboxylic acid ethyl ester ] was synthesized at theravance as described ( yamakawa et al . the m2-selective antagonist thrx-182087 [ n-(3-{(r)-1-[1-(1h - imidazole-4-carbonyl)-piperidin-4-ylmethyl]-piperidin-2-ylmethyl}-phenyl)-4-methoxy - benzamide ] was synthesized in house as follows : preparation of thrx-182087 was initiated from the condensation of 4-methoxyl benzoyl chloride and 3-bromo aniline . metal halogen exchange with n - butyllithium in the presence of triethylchlorosilane and addition to ( r)-2-formyl-1-cbz - piperidine , followed by decarboxylation with palladium on carbon and potassium formate . reductive alkylation with 4-formyl-1-cbz - piperidine and cbz removal with palladium on carbon , followed by acylation with 1h - imidazole-4-carbonyl chloride provided thrx-182087 ( for detailed synthetic route see stangeland et al . , manuscript in preparation ) . u 73,122 [ 1-(6-[([17]-3-methoxyestra-1,3,5-trien-17-yl)-amino]hexyl)-1h - pyrrole-2,5-dione ] was obtained from sigma - aldrich ( zwijndrecht , the netherlands ) and chelerythrine hcl from calbiochem ( via vwr , amsterdam , the netherlands ) . bound radioactivity in counts - per - minute data was normalized to percent specific binding and analyzed using a four - parameter logistic equation in prism 3.0 ( graphpad software , san diego , ca , usa ) . because hill coefficients did not significantly differ from unity , ic50s binding constants ( ki ) for test compounds were calculated from the ic50 values using the cheng and prusoff correction ( cheng and prusoff 1973 ) and reported as the mean negative logarithm of the inhibition binding constants ( pki ) sem . nonlinear regression was used to fit sigmoidal curves to the isoprenaline concentration response curves to determine agonist potency ( pec50 ) and maximum effects ( emax ) using prism 5.0 . the force of contraction immediately prior to addition of the first isoprenaline concentration within a given experiment was defined as 0% relaxation , and a force of contraction of 0 mn was defined as 100% relaxation . statistical significance of inhibitor effects on the emax or pec50 of isoprenaline was assessed by paired two - tailed t tests as compared to the indicated control condition , with treated and control conditions being measured in paired strips prepared from the same bladder . all statistical analyses were calculated using the prism program , and a p < 0.05 was considered statistically significant . radioligand binding assays were conducted with 1 nm [ h]n - methyl scopolamine ( ge healthcare , piscataway , nj , usa ) in a buffer consisting of 10 mm hepes , 100 mm nacl , 10 mm mgcl2 , and 0.025% bovine serum albumin , ph 7.4 at 37c . membrane fractions from cho - k1 cells expressing human recombinant m1 , m2 , m3 , m4 , or m5 muscarinic receptors were incubated with radioligand and unlabelled drugs for 1 h at 37c in a volume of 100 l . receptor expression levels ( bmax ) measured by saturation binding were determined to be 2.7 , 2.5 , 2.4 , 2.0 , and 3.2 pmol / mg protein for human recombinant m1 , m2 , m3 , m4 , or m5 muscarinic receptors , respectively . after separation by vacuum filtration onto gf / b filter plates presoaked with 0.3% polyethyleneimine , the quantity of membrane - bound radioligand was measured by scintillation counting . tissue preparation adult male wistar rats weighing 300 22 g were purchased from charles river ( maastricht , the netherlands ) . animals were anesthetized using pentobarbital ( 75 mg / kg , i.p . ) and sacrificed by decapitation . after removal of the dome and the trigonum , the middle parts of the cleaned bladders ( weight of 96 15 mg ) were cut transversally in four equal strips . the strips had a length of 19.9 3.6 mm and a weight of 9.5 2.7 mg ( n = 76 ) . all experimental procedures were in line with european union guidelines for the use of laboratory animals and approved by the animal care committee of academisch medisch centrum . briefly , the bladder strips were mounted under a resting tension of 10 mn in organ baths containing 7 ml of krebs henseleit buffer of the following composition : 118.5 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 0.025 mm na4edta , 2.5 mm cacl2 , 1.2 mm kh2po4 , 25 mm nahco3 , and 5.6 mm glucose at a temperature of 37c , yielding a total potassium concentration of 5.9 mm . the organ baths were continually gassed 95% o2/5% co2 to maintain a ph of 7.4 . the bladder strips were equilibrated for 120 min , during which the buffer solution was refreshed every 15 min until a steady contractile state had been reached . following the equilibration , the tissues were challenged with 50 mm kcl for 6 min ( maintaining isoosmolarity by reducing nacl concentration from 116.8 to 68.5 mm ) . after the first challenge , we equilibrated the strips again at passive tension of 10 mn for 90 min and challenged the strips again with 50 mm kcl for 6 min . after 60 min equilibration at 10 mn after the second kcl challenge , we added vehicle or substance of interest . carbachol was used at a concentration of 1 m , bzi and thrx at 100 nm . the enzyme inhibitors u 73,122 ( 10 m ) and chelerythrine ( 1 m ) were added 5 min prior to the administration of thrx . ten minutes later carbachol ( 1 m ) was added , which was given an incubation time of 15 min to reach steady contractile stadium . thereafter , cumulative concentration response curves were generated for the -adrenoceptor agonist isoprenaline . as isoprenaline - induced rat bladder relaxation can exhibit desensitization ( vrydag and michel 2009 ) , only one isoprenaline concentration response curve was constructed per bladder strip ; however , conditions being compared were always tested in parallel using strips from the same animal , and those paired comparisons were the basis of our statistical analysis ( see below ) . the m3-sparing agonist bzi [ also known as thrx-199874 , 4-(2-oxo-2,3-dihydro - benzimidazol-1-yl)-1,4-bipiperidinyl-1-carboxylic acid ethyl ester ] was synthesized at theravance as described ( yamakawa et al . the m2-selective antagonist thrx-182087 [ n-(3-{(r)-1-[1-(1h - imidazole-4-carbonyl)-piperidin-4-ylmethyl]-piperidin-2-ylmethyl}-phenyl)-4-methoxy - benzamide ] was synthesized in house as follows : preparation of thrx-182087 was initiated from the condensation of 4-methoxyl benzoyl chloride and 3-bromo aniline . metal halogen exchange with n - butyllithium in the presence of triethylchlorosilane and addition to ( r)-2-formyl-1-cbz - piperidine , followed by decarboxylation with palladium on carbon and potassium formate . reductive alkylation with 4-formyl-1-cbz - piperidine and cbz removal with palladium on carbon , followed by acylation with 1h - imidazole-4-carbonyl chloride provided thrx-182087 ( for detailed synthetic route see stangeland et al . , manuscript in preparation ) . u 73,122 [ 1-(6-[([17]-3-methoxyestra-1,3,5-trien-17-yl)-amino]hexyl)-1h - pyrrole-2,5-dione ] was obtained from sigma - aldrich ( zwijndrecht , the netherlands ) and chelerythrine hcl from calbiochem ( via vwr , amsterdam , the netherlands ) . bound radioactivity in counts - per - minute data was normalized to percent specific binding and analyzed using a four - parameter logistic equation in prism 3.0 ( graphpad software , san diego , ca , usa ) . because hill coefficients did not significantly differ from unity , ic50s were determined with slopes fixed to 1 . inhibition binding constants ( ki ) for test compounds were calculated from the ic50 values using the cheng and prusoff correction ( cheng and prusoff 1973 ) and reported as the mean negative logarithm of the inhibition binding constants ( pki ) sem . nonlinear regression was used to fit sigmoidal curves to the isoprenaline concentration response curves to determine agonist potency ( pec50 ) and maximum effects ( emax ) using prism 5.0 . the force of contraction immediately prior to addition of the first isoprenaline concentration within a given experiment was defined as 0% relaxation , and a force of contraction of 0 mn was defined as 100% relaxation . statistical significance of inhibitor effects on the emax or pec50 of isoprenaline was assessed by paired two - tailed t tests as compared to the indicated control condition , with treated and control conditions being measured in paired strips prepared from the same bladder . all statistical analyses were calculated using the prism program , and a p < 0.05 was considered statistically significant . the affinities of bzi and thrx-182087 for human recombinant m1 , m2 , m3 , m4 , and m5 muscarinic receptors as determined in competition radioligand binding experiments are shown in table 1 , most notably demonstrating a > 100-fold selectivity for m2 over m3 receptors for both compounds.table 1affinity estimates of bzi and thrx-182087 at human muscarinic receptor subtypes as determined in competition radioligand binding studiesbzithrx-182087m17.27 0.046.82 0.02m28.59 0.059.06 0.02m3<56.61 0.02m48.01 0.057.34 0.02m56.11 0.055.46 0.02data are meanssem of 1927 experiments and shown as pki values affinity estimates of bzi and thrx-182087 at human muscarinic receptor subtypes as determined in competition radioligand binding studies data are meanssem of 1927 experiments and shown as pki values five series of functional experiments were performed . in the first series , we reinvestigated the role of muscarinic receptor stimulation vs. receptor - independent bladder contraction , induced by kcl , for isoprenaline - induced relaxation . the maximum relaxation by isoprenaline was significantly smaller in carbachol- than kcl - precontracted strips , whereas the potency of isoprenaline did not differ significantly between the two conditions ( fig . 2).table 2starting tension of relaxation experiments in the absence and presence of muscarinic agonists and antagonists and/or signal transduction inhibitorsseries 1 : kcl vs. mixed muscarinic stimulation kcl , 50 mm2.86 0.22n = 8 carbachol , 1 m2.46 0.12n = 8series 2 : passive tension vs. m2 stimulation passive0.95 0.15n = 6 bzi , 100 nm1.08 0.10n = 6series 3 : mixed vs. m3 stimulation carbachol , 1 m2.60 0.13n = 6 carbachol , 1 m + thrx-182087 , 100 nm2.14 0.22n = 6series 4 : m3 stimulation in absence and presence of phospholipase c inhibitor carbachol , 1 m + thrx-182087 , 100 nm2.90 0.21n = 6 carbachol , 1 m + thrx-182087 , 100 nm + u 73,122 , 10 m2.33 0.12*n = 6series 5 : m3 stimulation in absence and presence of protein kinase c inhibitor carbachol , 1 m + thrx-182087 , 100 nm2.89 0.51n = 6 carbachol , 1 m + thrx-182087 , 100 nm + chelerythrine , 1 m2.90 0.22n = 6data are meanssem of the indicated number of experiments and shown in millinewtons per milligram strip weight*p < 0.05 vs. test condition ( kcl , carbachol , or passive tension ) in a paired , two - tailed t testfig . 2comparison of isoprenaline - induced relaxation against tension induced by 50 mm kcl and 1 m carbachol . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. kcl in a paired t test starting tension of relaxation experiments in the absence and presence of muscarinic agonists and antagonists and/or signal transduction inhibitors data are meanssem of the indicated number of experiments and shown in millinewtons per milligram strip weight * p < 0.05 vs. test condition ( kcl , carbachol , or passive tension ) in a paired , two - tailed t test comparison of isoprenaline - induced relaxation against tension induced by 50 mm kcl and 1 m carbachol . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. kcl in a paired t test the second series of experiments explored whether selective m2 receptor stimulation mimics the effect of carbachol . based upon previous data that bzi alone causes little bladder contraction ( schneider et al . 2005 ) , we compared bzi with passive tension and confirmed the lack of effect of bzi on detrusor tone ( table 2 ) . while bzi did not affect maximum isoprenaline - induced relaxation , it significantly reduced its potency ( fig . 3comparison of passive tension and m2-selective muscarinic stimulation by 100 nm bzi on isoprenaline - induced relaxation . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. passive tension in a paired t test comparison of passive tension and m2-selective muscarinic stimulation by 100 nm bzi on isoprenaline - induced relaxation . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. passive tension in a paired t test the third series addressed the reverse question , i.e. , whether selective m3 receptor stimulation ( carbachol in presence of thrx-182087 ) mimics the carbachol effect . the potency and efficacy of isoprenaline were significantly greater upon m3 selective as compared to general muscarinic receptor stimulation , confirming a contribution of m2 receptors to the attenuation of the isoprenaline response ( fig . 2 ) , indicating that both subtypes contribute to the attenuation of isoprenaline responses by carbachol.fig . 4comparison of mixed ( 1 m carbachol ) vs. m3-selective muscarinic stimulation ( 1 m carbachol + 100 nm thrx-182087 ) on isoprenaline - induced relaxation . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. carbachol alone in a paired t test comparison of mixed ( 1 m carbachol ) vs. m3-selective muscarinic stimulation ( 1 m carbachol + 100 nm thrx-182087 ) on isoprenaline - induced relaxation . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. carbachol alone in a paired t test the fourth and fifth series of experiments explored whether plc and/or pkc contribute to the attenuation of isoprenaline responses by m3-selective stimulation . in the presence of the plc inhibitor u 73,122 starting tension was significantly smaller than with m3-selective stimulation alone ( table 2 ) . potency and efficacy of isoprenaline were significantly greater in the presence of u 73,122 ( fig . in contrast , the pkc inhibitor chelerythrine affected neither starting tension ( table 2 ) nor potency or efficacy of isoprenaline - induced relaxation ( fig . 5effect of the phospholipase c inhibitor u 73,122 ( 10 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx ) . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. data in absence of signalling inhibitor in a paired t testfig . 6effect of the the protein kinase c inhibitor chelerythrine ( 1 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx-182087 ) . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . data in the absence and presence of the signalling inhibitor were not significantly different in a paired t test ( p > 0.05 ) effect of the phospholipase c inhibitor u 73,122 ( 10 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx ) . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . * p < 0.05 vs. data in absence of signalling inhibitor in a paired t test effect of the the protein kinase c inhibitor chelerythrine ( 1 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx-182087 ) . middle and lower panel bars showing emax and pec50 as derived from the curves in the upper panel indicate meanssem , whereas filled circles represent individual experiments . data in the absence and presence of the signalling inhibitor were not significantly different in a paired t test ( p > 0.05 ) our study introduces a novel highly m2-selective antagonist ( thrx-182087 ) and uses it together with the m3-sparing agonist bzi to explore the muscarinic receptor subtypes involved in the attenuation of isoprenaline effects in rat urinary bladder as well as the signalling pathways mediating such attenuation . according to our competition binding data , this compares favorably to the limited m2 selectivity of other compounds which have been used in this field such as methoctramine which is only 30-fold selective ( hegde et al . while the selectivity of thrx-182087 over m4 receptors is less pronounced , these receptors are of little importance in the regulation of bladder smooth muscle tone ( hegde 2006 ) . hence , for practical purposes , thrx-182087 is a highly selective m2 antagonist in our experimental setting . the thrx-182087 concentration of 100 nm used in our studies produces an almost complete occupancy of m2 receptors , and accordingly the combination of carbachol with thrx-182087 provides selective m3 agonism . 2002 ; yamakawa et al . 2001a ) , and our binding data show a more than 3,000-fold selectivity for m2 over m3 receptors . accordingly , bzi provides selective stimulation of m2 receptors in bladder smooth muscle , where only m2 and m3 receptors are functionally relevant ( hegde 2006 ) . the combined use of thrx-182087 and bzi has enabled us to explore the relative roles of m2 and m3 receptors in the attenuation of isoprenaline - induced relaxation . our data on starting tension demonstrate that inhibition of the m2 receptors did not attenuate carbachol responses , whereas selective activation of m2 receptors did not induce contraction as also observed in previous studies ( schneider et al . 2005 ) . these data confirm a large body of evidence that despite the much larger presence of m2 receptors in the urinary bladder , direct contractile responses are mediated predominantly if not exclusively by the m3 receptor ( hegde 2006 ) . studies in multiple tissues and species had demonstrated that muscarinic receptors can attenuate relaxation responses to the -adrenoceptor agonist isoprenaline ( see introduction section ) , and this is confirmed in the present data . while such attenuation was found very consistently , those previous studies had been inconsistent with regard to the question whether such attenuation affects the potency and/or efficacy of isoprenaline , and our data also are not fully consistent in this regard . studies based on knockout mice had indicated that the m2 subtype , which contributes little to direct detrusor contraction , plays a role in the attenuation of the relaxation response ( ehlert et al . our finding that selective inhibition of m2 receptors by thrx-182087 in the presence of carbachol enhances relaxation by isoprenaline further corroborates this idea . however , it should be noted that neither the effect of bzi nor that of thrx-182087 can fully explain the attenuation obtained by mixed muscarinic stimulation using carbachol alone , suggesting that the attenuation response may also contain an m3 component . stimulation of the plc / pkc pathway is a prototypical signalling response of m3 receptors ( caulfield and birdsall 1998 ; ehlert et al . 1997 ) which also was detected in the bladder as being mediated predominantly if not exclusively via m3 receptors ( kories et al . nevertheless , m3 receptor - mediated bladder contraction has been shown to be insensitive to inhibition of plc or pkc in rats , mice , and humans ( frazier et al . 2008 ) . in the present study , we have used the plc inhibitor u 73,122 in a concentration where it fully suppresses inositol phosphate formation in the bladder but does not affect rat or human bladder contraction ( schneider et al . 2004a , b ) . interestingly , u 73,122 significantly enhanced isoprenaline - induced relaxation in the presence of m3-selective stimulation . thus , plc may be involved in the m3 component of attenuation of relaxation but not in direct bladder contraction mediated by the same receptor subtype . according to our data , pkc is not involved in either response , indicating that the involvement of plc in the attenuation of relaxation occurs via a pkc - independent pathway . in conclusion , we have introduced a novel antagonist with very high selectivity for m2 over m3 receptors , thrx-182087 . using this compound as well as the m3-sparing agonist bzi , we confirm a role for m2 receptors in the attenuation of isoprenaline - induced bladder relaxation , which previously was mainly supported by genetic evidence , by a pharmacological approach . thus , muscarinic receptors cause direct contraction and inhibition of relaxation in the bladder , but the two responses involve different subtypes and , at least for m3 receptors , different signalling pathways . this interaction may become clinically relevant under pathophysiological conditions when acetylcholine is being released in the bladder during the storage phase of the micturition cycle .

-adrenoceptors are important mediators of smooth muscle relaxation in the urinary bladder , but the concomitant presence of a muscarinic agonist , e.g. , carbachol , can attenuate relaxation responses by reducing potency and/or efficacy of -adrenoceptor agonists such as isoprenaline . therefore , the present study was designed to explore the subtypes and signalling pathways of muscarinic receptors involved in the attenuation of isoprenaline - induced isolated rat detrusor preparations using novel subtype - selective receptor ligands . in radioligand binding studies , we characterized bzi to be a m3-sparing muscarinic agonist , providing selective m2 stimulation in rat bladder , and thrx-182087 as a highly m2-selective antagonist . the use of bzi and of thrx-182087 in the presence of carbachol enabled experimental conditions with a selective stimulation of only m2 or m3 receptors , respectively . confirming previous findings , carbachol attenuated isoprenaline - induced detrusor relaxation . m2-selective stimulation partly mimicked this attenuation , indicating that both m2 and m3 receptors are involved . during m3-selective stimulation , the attenuation of isoprenaline responses was reduced by the phospholipase c inhibitor u 73,122 but not by the protein kinase c inhibitor chelerythrine . we conclude that both m2 and m3 receptors contribute to attenuation of -adrenoceptor - mediated relaxation of rat urinary bladder ; the signal transduction pathway involved in the m3 component of this attenuation differs from that mediating direct contractile effects of m3 receptors .

Introduction Methods Radioligand binding experiments Organ bath experiments Chemicals Data analysis Results Discussion

muscarinic receptors are the main mediator of physiological contraction of the urinary bladder . while m2 and m3 subtypes exist in an about 4:1 ratio in the bladder of humans and many other mammals , direct contraction responses to exogenous agonists or endogenous agonist as released by field stimulation are mediated predominantly if not exclusively by the minor fraction of m3 receptors ( hegde 2006 ) . while it has been questioned on theoretical grounds that subtype - selective antagonists are sufficient to support this conclusion ( ehlert 2003 ) , it should be noted that it is consistent with findings from m2 and m3 receptor knockout mice ( matsui et al . m3 receptors , including those in the urinary bladder , couple to phospholipase c ( plc ) stimulation , but the direct contractile effect via m3 receptors occurs largely independent of plc in the bladder ( frazier et al . bladder smooth muscle relaxation is mediated by -adrenoceptors , in humans and many species mostly their 3 subtype ( michel and vrydag 2006 ) . while -adrenoceptors , including those in the bladder , couple to stimulation of an adenylyl cyclase , bladder relaxation by -adrenergic agonists occurs largely independent of adenylyl cyclase stimulation ( frazier et al . thus , bladder smooth muscle tone is under a dual control of m3 muscarinic receptors and -adrenoceptors . this is similar to smooth muscle tone regulation in many other tissues , e.g. , the airways , except relaxation responses in most cases outside the bladder are mediated by the 2 subtype ( michel and parra 2008 ) . thus , it has been shown that the potency and efficacy of a muscarinic agonist to elicit detrusor contraction is attenuated in the presence of a -adrenoceptor agonist ( yamanishi et al . perhaps even more important , muscarinic agonists can also attenuate the relaxing effects of -adrenoceptor stimulation in the bladder ( ehlert et al . 2002 ) , indicating that it is a general principle in the regulation of smooth muscle tone upon concomitant exposure to muscarinic and -adrenoceptor agonists . while all studies agree on the existence of attenuation of -adrenoceptor - mediated relaxation by muscarinic agonists , reduced potency and/or efficacy have been reported to underlie such attenuation . of note , the degree of attenuation of -adrenoceptor - mediated relaxation by muscarinic agonists exceeds that by other contractile stimuli in all cases where this has been studied , e.g. this differential degree of attenuation can not be explained by a comparison between weak and strong contractile agonists , indicating that it may involve a specific property of muscarinic receptors rather than purely reflecting functional antagonism . as m2 receptors largely signal via pertussis toxin - sensitive g proteins ( caulfield and birdsall 1998 ) , further support for a role of m2 receptors in the attenuation response comes from studies in which pertussis toxin treatment enhanced the relaxant effects of forskolin on oxotremorine - m - mediated contractions in the ileum and trachea ( ostrom and ehlert 1998 , 1999 ) . in a similar vein , it has been reported that under conditions of preferential alkylation of m3 receptors , the ability of a muscarinic agonist to reverse isoprenaline - induced relaxation is largely maintained ( ehlert et al . recently , an agonist with considerable selectivity for m2 over m3 receptors became available , but this compound exhibits a lower degree of selectivity , if any , over other subtypes ; in the bladder , where muscarinic receptor subtypes other than m2 and m3 are largely absent , this compound produces selective m2 stimulation ( jasper et al . these two tools have enabled us to explore the role of m2 and m3 receptors in the attenuation of isoprenaline - induced bladder relaxation . moreover , we have used this approach to characterize the role of plc and protein kinase c ( pkc ) activation in the attenuation response.fig . 1structures of thrx-199874 ( bzi ) and thrx-182087 structures of thrx-199874 ( bzi ) and thrx-182087 radioligand binding assays were conducted with 1 nm [ h]n - methyl scopolamine ( ge healthcare , piscataway , nj , usa ) in a buffer consisting of 10 mm hepes , 100 mm nacl , 10 mm mgcl2 , and 0.025% bovine serum albumin , ph 7.4 at 37c . receptor expression levels ( bmax ) measured by saturation binding were determined to be 2.7 , 2.5 , 2.4 , 2.0 , and 3.2 pmol / mg protein for human recombinant m1 , m2 , m3 , m4 , or m5 muscarinic receptors , respectively . all experimental procedures were in line with european union guidelines for the use of laboratory animals and approved by the animal care committee of academisch medisch centrum . as isoprenaline - induced rat bladder relaxation can exhibit desensitization ( vrydag and michel 2009 ) , only one isoprenaline concentration response curve was constructed per bladder strip ; however , conditions being compared were always tested in parallel using strips from the same animal , and those paired comparisons were the basis of our statistical analysis ( see below ) . metal halogen exchange with n - butyllithium in the presence of triethylchlorosilane and addition to ( r)-2-formyl-1-cbz - piperidine , followed by decarboxylation with palladium on carbon and potassium formate . radioligand binding assays were conducted with 1 nm [ h]n - methyl scopolamine ( ge healthcare , piscataway , nj , usa ) in a buffer consisting of 10 mm hepes , 100 mm nacl , 10 mm mgcl2 , and 0.025% bovine serum albumin , ph 7.4 at 37c . receptor expression levels ( bmax ) measured by saturation binding were determined to be 2.7 , 2.5 , 2.4 , 2.0 , and 3.2 pmol / mg protein for human recombinant m1 , m2 , m3 , m4 , or m5 muscarinic receptors , respectively . all experimental procedures were in line with european union guidelines for the use of laboratory animals and approved by the animal care committee of academisch medisch centrum . briefly , the bladder strips were mounted under a resting tension of 10 mn in organ baths containing 7 ml of krebs henseleit buffer of the following composition : 118.5 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 0.025 mm na4edta , 2.5 mm cacl2 , 1.2 mm kh2po4 , 25 mm nahco3 , and 5.6 mm glucose at a temperature of 37c , yielding a total potassium concentration of 5.9 mm . following the equilibration , the tissues were challenged with 50 mm kcl for 6 min ( maintaining isoosmolarity by reducing nacl concentration from 116.8 to 68.5 mm ) . as isoprenaline - induced rat bladder relaxation can exhibit desensitization ( vrydag and michel 2009 ) , only one isoprenaline concentration response curve was constructed per bladder strip ; however , conditions being compared were always tested in parallel using strips from the same animal , and those paired comparisons were the basis of our statistical analysis ( see below ) . metal halogen exchange with n - butyllithium in the presence of triethylchlorosilane and addition to ( r)-2-formyl-1-cbz - piperidine , followed by decarboxylation with palladium on carbon and potassium formate . the affinities of bzi and thrx-182087 for human recombinant m1 , m2 , m3 , m4 , and m5 muscarinic receptors as determined in competition radioligand binding experiments are shown in table 1 , most notably demonstrating a > 100-fold selectivity for m2 over m3 receptors for both compounds.table 1affinity estimates of bzi and thrx-182087 at human muscarinic receptor subtypes as determined in competition radioligand binding studiesbzithrx-182087m17.27 0.046.82 0.02m28.59 0.059.06 0.02m3<56.61 0.02m48.01 0.057.34 0.02m56.11 0.055.46 0.02data are meanssem of 1927 experiments and shown as pki values affinity estimates of bzi and thrx-182087 at human muscarinic receptor subtypes as determined in competition radioligand binding studies data are meanssem of 1927 experiments and shown as pki values five series of functional experiments were performed . in the first series , we reinvestigated the role of muscarinic receptor stimulation vs. receptor - independent bladder contraction , induced by kcl , for isoprenaline - induced relaxation . 2).table 2starting tension of relaxation experiments in the absence and presence of muscarinic agonists and antagonists and/or signal transduction inhibitorsseries 1 : kcl vs. mixed muscarinic stimulation kcl , 50 mm2.86 0.22n = 8 carbachol , 1 m2.46 0.12n = 8series 2 : passive tension vs. m2 stimulation passive0.95 0.15n = 6 bzi , 100 nm1.08 0.10n = 6series 3 : mixed vs. m3 stimulation carbachol , 1 m2.60 0.13n = 6 carbachol , 1 m + thrx-182087 , 100 nm2.14 0.22n = 6series 4 : m3 stimulation in absence and presence of phospholipase c inhibitor carbachol , 1 m + thrx-182087 , 100 nm2.90 0.21n = 6 carbachol , 1 m + thrx-182087 , 100 nm + u 73,122 , 10 m2.33 0.12*n = 6series 5 : m3 stimulation in absence and presence of protein kinase c inhibitor carbachol , 1 m + thrx-182087 , 100 nm2.89 0.51n = 6 carbachol , 1 m + thrx-182087 , 100 nm + chelerythrine , 1 m2.90 0.22n = 6data are meanssem of the indicated number of experiments and shown in millinewtons per milligram strip weight*p < 0.05 vs. test condition ( kcl , carbachol , or passive tension ) in a paired , two - tailed t testfig . 2comparison of isoprenaline - induced relaxation against tension induced by 50 mm kcl and 1 m carbachol . * p < 0.05 vs. kcl in a paired t test starting tension of relaxation experiments in the absence and presence of muscarinic agonists and antagonists and/or signal transduction inhibitors data are meanssem of the indicated number of experiments and shown in millinewtons per milligram strip weight * p < 0.05 vs. test condition ( kcl , carbachol , or passive tension ) in a paired , two - tailed t test comparison of isoprenaline - induced relaxation against tension induced by 50 mm kcl and 1 m carbachol . 2005 ) , we compared bzi with passive tension and confirmed the lack of effect of bzi on detrusor tone ( table 2 ) . 3comparison of passive tension and m2-selective muscarinic stimulation by 100 nm bzi on isoprenaline - induced relaxation . * p < 0.05 vs. passive tension in a paired t test comparison of passive tension and m2-selective muscarinic stimulation by 100 nm bzi on isoprenaline - induced relaxation . , whether selective m3 receptor stimulation ( carbachol in presence of thrx-182087 ) mimics the carbachol effect . the potency and efficacy of isoprenaline were significantly greater upon m3 selective as compared to general muscarinic receptor stimulation , confirming a contribution of m2 receptors to the attenuation of the isoprenaline response ( fig . 2 ) , indicating that both subtypes contribute to the attenuation of isoprenaline responses by carbachol.fig . * p < 0.05 vs. carbachol alone in a paired t test the fourth and fifth series of experiments explored whether plc and/or pkc contribute to the attenuation of isoprenaline responses by m3-selective stimulation . in the presence of the plc inhibitor u 73,122 starting tension was significantly smaller than with m3-selective stimulation alone ( table 2 ) . potency and efficacy of isoprenaline were significantly greater in the presence of u 73,122 ( fig . in contrast , the pkc inhibitor chelerythrine affected neither starting tension ( table 2 ) nor potency or efficacy of isoprenaline - induced relaxation ( fig . 5effect of the phospholipase c inhibitor u 73,122 ( 10 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx ) . 6effect of the the protein kinase c inhibitor chelerythrine ( 1 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx-182087 ) . data in the absence and presence of the signalling inhibitor were not significantly different in a paired t test ( p > 0.05 ) effect of the phospholipase c inhibitor u 73,122 ( 10 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx ) . * p < 0.05 vs. data in absence of signalling inhibitor in a paired t test effect of the the protein kinase c inhibitor chelerythrine ( 1 m ) on isoprenaline - induced relaxation during m3-selective stimulation ( 1 m carbachol + 100 nm thrx-182087 ) . data in the absence and presence of the signalling inhibitor were not significantly different in a paired t test ( p > 0.05 ) our study introduces a novel highly m2-selective antagonist ( thrx-182087 ) and uses it together with the m3-sparing agonist bzi to explore the muscarinic receptor subtypes involved in the attenuation of isoprenaline effects in rat urinary bladder as well as the signalling pathways mediating such attenuation . while the selectivity of thrx-182087 over m4 receptors is less pronounced , these receptors are of little importance in the regulation of bladder smooth muscle tone ( hegde 2006 ) . the thrx-182087 concentration of 100 nm used in our studies produces an almost complete occupancy of m2 receptors , and accordingly the combination of carbachol with thrx-182087 provides selective m3 agonism . accordingly , bzi provides selective stimulation of m2 receptors in bladder smooth muscle , where only m2 and m3 receptors are functionally relevant ( hegde 2006 ) . the combined use of thrx-182087 and bzi has enabled us to explore the relative roles of m2 and m3 receptors in the attenuation of isoprenaline - induced relaxation . these data confirm a large body of evidence that despite the much larger presence of m2 receptors in the urinary bladder , direct contractile responses are mediated predominantly if not exclusively by the m3 receptor ( hegde 2006 ) . studies in multiple tissues and species had demonstrated that muscarinic receptors can attenuate relaxation responses to the -adrenoceptor agonist isoprenaline ( see introduction section ) , and this is confirmed in the present data . while such attenuation was found very consistently , those previous studies had been inconsistent with regard to the question whether such attenuation affects the potency and/or efficacy of isoprenaline , and our data also are not fully consistent in this regard . studies based on knockout mice had indicated that the m2 subtype , which contributes little to direct detrusor contraction , plays a role in the attenuation of the relaxation response ( ehlert et al . our finding that selective inhibition of m2 receptors by thrx-182087 in the presence of carbachol enhances relaxation by isoprenaline further corroborates this idea . however , it should be noted that neither the effect of bzi nor that of thrx-182087 can fully explain the attenuation obtained by mixed muscarinic stimulation using carbachol alone , suggesting that the attenuation response may also contain an m3 component . stimulation of the plc / pkc pathway is a prototypical signalling response of m3 receptors ( caulfield and birdsall 1998 ; ehlert et al . nevertheless , m3 receptor - mediated bladder contraction has been shown to be insensitive to inhibition of plc or pkc in rats , mice , and humans ( frazier et al . in the present study , we have used the plc inhibitor u 73,122 in a concentration where it fully suppresses inositol phosphate formation in the bladder but does not affect rat or human bladder contraction ( schneider et al . interestingly , u 73,122 significantly enhanced isoprenaline - induced relaxation in the presence of m3-selective stimulation . thus , plc may be involved in the m3 component of attenuation of relaxation but not in direct bladder contraction mediated by the same receptor subtype . according to our data , pkc is not involved in either response , indicating that the involvement of plc in the attenuation of relaxation occurs via a pkc - independent pathway . in conclusion , we have introduced a novel antagonist with very high selectivity for m2 over m3 receptors , thrx-182087 . using this compound as well as the m3-sparing agonist bzi , we confirm a role for m2 receptors in the attenuation of isoprenaline - induced bladder relaxation , which previously was mainly supported by genetic evidence , by a pharmacological approach . thus , muscarinic receptors cause direct contraction and inhibition of relaxation in the bladder , but the two responses involve different subtypes and , at least for m3 receptors , different signalling pathways .

in the wide spectrum of congenital hemoglobin disorders , two entities have always attracted attention because of their severity and extensive geographic distribution , ie , beta thalassemia major and sickle cell disease ( scd ) . in the present review , attention is focused on the latter disease , characterized by a qualitative defect in beta - globin production , due to replacement of a single amino acid ( valine for glutamic acid ) in the beta - globin chain and formation of an anomalous hemoglobin , called hemoglobin s. this induces severe deformity of red cells upon deoxygenation , hampering microcirculation , and leading to vascular occlusion and critical organ damage.1 however , it has been noted that the natural history of this disease shows considerable heterogeneity in signs and symptoms , due to a variety of concomitant situations . actually , the main factor in the variable severity of scd is the level of fetal hemoglobin ( hbf ) produced by patients . there are indeed many disparate conditions , in which a higher level of hbf than expected can be found . they range from hereditary disorders to acquired ones , as well as from blood diseases to nonhematological situations ( table 1 ) . it is well known that populations showing a genetically determined presence of hbf have a milder form of scd , including a reduced incidence of severe clinical complications.2 it is therefore understandable that many efforts have been directed to restore the production of hbf in adults.3 as for the background of this process , recent studies have produced a good amount of information , particularly on gamma - globin gene control . in the present paper , we therefore propose a brief initial survey of these genetic factors , and then focus on new ways of drug treatment for reactivation of hbf level , as well as attempts at correction of the genetic defect by cell engineering . a brief update on recent methods for hbf assay is also included . it is well known that only a tiny fraction of hbf is still present in the majority of adults , as a result of the switch to hba in early life . however , the switch may be impaired by a number of mutations in the beta - globin cluster,4 leading to uniform increase of hbf ( pancellular hereditary persistence of fetal hemoglobin [ hpfh ] ) , a condition allowing a normal way of life , or to a nonuniform distribution of hbf , based on the coexistence of two populations of red cells , one with a high content of hbf ( so - called f cells ) and another with a negligible amount of the same . in this case , the condition is called heterocellular hpfh , which is regarded as a multifactorial quantitative trait , quite distinct from the classical form of pancellular hereditary persistence of fetal hemoglobin.5 a number of interesting studies have proposed different mechanisms which may be responsible for such persistence of hbf in adults , and can be either mutations in critical genic regions involved in the activation of globin genes or deletions of the globin gene cluster . examples of the former event are single base substitutions in the promoter region of the gamma - globin genes , found in adults with high hbf production,6 or t to c substitution in the gamma - globin gene , leading to disruption of the assembly of a repressive chromatin structure , which normally silences the expression of the gamma - globin gene in adult erythrocytes.7 in the latter case , ( ie , deletional persistence of hbf ) , gazouli et al,8 attempting to explain the production of the hbf phenotype in adult red cells , carried out an investigation in transgenic mice lacking two elements of the a-to- gene region , suggesting that a deletion of silencer elements in that gene region may account for persistent expression of hbf in the adult stage . previous experiments by the same authors had shown that a juxtaposition of downstream enhancers may also contribute to that effect , but different models are not mutually exclusive . according to another study , a downregulation of the transcriptional repressor , zhx2 , or some chromatin remodelling factors may also be involved in deletional hereditary persistence of fetal hemoglobin.9 anyway , there is an obvious benefit from the analysis of multiple mechanisms of hbf persistence in adults , namely a better understanding of the molecular basis for the perinatal hemoglobin switch . as far as possible genetic determinants of hbf production an early hypothesis suggested that variability in hbf was mainly due to independent genetic factors , an effect particularly evident in patients with scd.10 a seminal study showed that a variation of genetic determinants on chromosome 11p16 was involved in the expression of hbf , and precisely at position 158.11 however , the effect of such variation is modest in normal people , and its presence is not always associated with high hbf , but variants at this locus have a considerable frequency in certain populations like asian indians , in whom it was first studied and found to be associated with the presence of moderate levels of hbf in normal individuals.12 a second determinant was found in a large indian family with beta thalassemia and hereditary persistence of fetal hemoglobin , situated in a region on chromosome 6q23q24 , between the hbs1l and myb genes.13 this locus was therefore termed hmip ( hbs1l - myb intergenic polymorphism ) , and is an important component ( about 19% ) of the f cell trait in the general population . a strong association with hbf level was eventually observed with a third locus , situated on chromosome 2p15 , namely the bcl11a , leading to the hypothesis that a product of this locus , a multizinc finger transcription factor , may encode a stage - specific regulator of hbf expression.14 on the clinical side , extensive research carried out among patients in sardinia and other groups showed that a variant of bcl11a was strongly associated with individuals having high hbf levels , as well as with patients having mild forms of thalassemia and scd.15 a very recent study , based on genome - wide association , has also identified a regulatory region in the olfactory receptor gene cluster ( on chromosome 11 ) , which may play a role in gamma - globin gene expression.16 finally , it should be stressed that , although recent progress in the genetics of hbf has not yet promoted therapeutic applications , every contribution to the understanding of genetic mechanisms presiding over hbf synthesis and to the hbf - to - hba switch is a necessary premise for the formulation of treatment strategies for severe hemoglobin disorders . agents capable of inducing production of fetal hemoglobin have been known for many years , and have also been used in clinical trials ( table 3 ) . 1 ) it was in the early 1980s that 5-azacytidine was shown to be able to reinduce production of hbf in adults , as well as in experimental animals . a few patients with beta thalassemia and scd treated with this drug showed a modest correction of globin chain imbalance and had lower transfusion needs.17 however , the possibility that such treatment could have an oncogenic effect discouraged the undertaking of these trials . it was only years later that a derivative of azacytidine , namely deoxy - azacytidine , or decitabine , was found to be effective in reactivating production of fetal hemoglobin , and was therefore started in regular trials on scd patients.18 as for possible toxic effects from this drug , only reversible neutropenia was observed , while tests on experimental animals not only showed absence of tumorigenic action , but even suggested a decrease of tumor formation in mice predisposed to cancer.19 parenteral administration of decitabine can produce a notable increase of hbf levels as well as stimulate erythroid differentiation in scd patients.20 oral preparations have also been tested in animals , with the aim of making them more acceptable to patients.21 more investigations are needed to study possible long - term adverse effects of this compound . great interest was devoted to the study of the mechanism of action of different compounds in the induction of hbf . early suggestions of a relevant effect on dna hypomethylation at the promoters of the gamma - globin gene3 were followed by the realization that different mechanisms may be involved . a unifying theory has been proposed , based on the assumption that a variety of cellular stresses and stimuli can promote coordinated stress responses , including gamma - globin gene activation . different signaling pathways , like camp , p38mapk , and others may be involved.22 2 ) hydroxyurea ( hydroxycarbamide [ hc ] ) has been known for a long time as a well tolerated oral treatment for some myeloproliferative disorders . its action on hbf production , first observed in baboons , was then tested and demonstrated in clinical trials on scd patients . predictably , bone marrow suppression was observed after long - term treatment , but this effect was reversible.3 the mechanism of action of this compound seems to be mediated through the induction of a gtp - binding protein sar , which modulates expression of the gamma - globin gene in erythroid cells.23 very good results have been reported recently in a 17-year trial carried out in a variety of sickle cell syndromes . a dramatic reduction of severe pain crises , transfusion requirements and hospital admissions was obtained , showing how prolonged and tolerable treatment with hydroxyurea can profoundly modify the natural history of the disease.24 as for the mechanism of action of hydroxyurea , a series of investigations point to an effect on genes which are involved in hemoglobin synthesis . one of these genes is the transcription factor , egr1 , which is one of the most upregulated genes following hc treatment . another group of genes , represented by centb1 ( centaurin , beta 1 ) , arhgap4 ( rho gtpase activating protein 4 ) , and rin3 ( ras and rab interactor 3 ) , were found to be induced after hydroxyurea administration.25 another study , carried out in reticulocytes from scd patients after hydroxyurea treatment showed an altered expression of genes associated with the regulation of globin expression , like sud53 , fzd5 , and phc3.26 these results show that hydroxyurea produces significant changes in the gene expression pattern , with activation of transcription factors and pathways involved in signal transduction , eventually leading to an increase of globin gene expression . the search continues to find agents able to enhance the action of hydroxyurea in scd patients . in this context , a compound which has been known for a long time as an hbf inducer is erythropoietin , which is capable of increasing the amount of f cells and hbf concentration . a synergistic action was seen when erythropoietin was associated with hydroxyurea , either simultaneously or sequentially , and of special interest is the finding of a good erythropoietin response in patients who were hydroxyurea - intolerant . in this case , erythropoietin treatment made it possible to tolerate hydroxyurea dosage escalation , with an increase in f cells and only minor side effects.27 more studies are in progress to investigate the potential of such a drug combination . 3 ) butyrate was the earliest of a number of compounds used in this context because of their action as inhibitors of histone deacetylase . it is indeed known that by promoting histone acetylation , butyrate increases the transcription rate of the gamma - globin gene , as well as the translation of gamma - globin mrna.28 it has also been demonstrated that transcription factors like gata1 and nf - e32 are involved.29 other histone deacetylase inhibitors , and butyrate itself , have also shown a different mechanism of action , namely through activation of the p38map kinase pathway . the effector molecules have been identified by sangerman et al as creb1 and atf2 , acting via the aforementioned pathway , which therefore appears to be one of the main mediators of gamma - globin gene regulation.30 many more compounds , acting as histone deacetylase inhibitors , have been mentioned as beneficial agents in sickle cell disease.31 new classes of synthetic histone deacetylase inhibitors , defined as aroyl - pyrrolyl hydroxyamides and uracil - based hydroxyamides , have also been tested . of 24 agents , two were found to be active for their ability to induce hbf in different models of erythroid differentiation . interestingly , both compounds were also effective in correcting the impaired in vitro maturation of beta thalassemic erythroblasts.32 these results and those obtained with other types of histone deacetylase inhibitors seem to suggest that compounds acting as epigenetic modifiers , namely capable of modulating gene expression , may be a promising area of investigation . 4 ) a number of disparate compounds has been suggested as endowed with stimulating activity on the production of gamma - globin and hbf . one of them is valproate , which was found to induce hemoglobin synthesis in erythroid cells , again by activation of the p38 pathway.33 an interesting compound tried in this context is the kit ligand , an important cytokine for the initiation of hemopoiesis . in unilineage erythroid cultures of 20 patients with major beta thalassemia or thalassemia intermedia , addition of kit ligand induced a marked increase of gamma - globin synthesis , thus reaching hbf levels three - fold higher than in control cultures.34 worth mentioning is the effect of thalidomide , which seems capable of inducing hbf expression via activation of the same p38mapk signaling pathway , as well as the action of two thalidomide analogs , used as immunomodulators , which have been shown to possess , among other well known activities , a stimulating effect on hbf production . such compounds , ie , pomalidomide and lenalinamide , recently tested on early erythroid progenitors in vitro , caused increased proliferation of immature erythroid cells , regulated hemoglobin transcription , and induction of hbf without cytotoxicity . an effective strategy employed in scd is the transplantation of hemopoietic bone marrow or cord blood , thus delegating to transplanted stem cells the task of providing normal levels of hemoglobin a. however , it has been calculated that , up to 2008 , more than 1600 allogeneic transplants had been performed in beta thalassemia , but many fewer were done in scd , perhaps because fewer patients in the advanced stages were eligible , due to the presence of severe vascular damage.36 this option is moreover restricted by limited availability of suitable donors and the severe effects of marrow ablation treatment , including reduced fertility , especially in women . to overcome such limitations , unrelated donors have been used with some success and nonablative regimens have also been employed , while , in order to preserve reproductive potential in women , ovarian tissue preservation and subsequent implantation have been employed , obtaining successful embryo development.37 more recently , sophisticated strategies , based on cell engineering , have been developed to transfer correcting genes in patients with a single gene defect . it is certainly fair to mention some remarkable success obtained by gene therapy in genetic immunodeficiency diseases.38 as far as scd is concerned , a search has been going on for a long time to find an effective gene vector which would allow transfer of a desired gene into hemopoietic stem cells . after early attempts using gamma retroviral vectors to transduce globin genes into dividing stem cells,39 lentiviral vectors , a subclass of retroviruses capable of transducing not only proliferating but also quiescent cells , proved more effective.40 it has been stressed that lentiviral vectors should be erythroid - specific , differentiation- and stage - restricted , position - independent , and sustainable over time.41 this approach can be adopted for scd , forcing the expression of fetal hemoglobin , which , even at low concentration , has been shown to exert a potent antisickling effect.42 on the other side , it has been noted that gene therapy for scd , to be effective , should induce a therapeutic gene in the greatest part of the red cell population , because even a small proportion of remaining sickle cells may cause vaso - occlusion and severe ischemia.43 as for the cell type to be used for such correction , the most primitive progenitors , namely embryonic stem cells , were used initially in mice with scd by the technique of gene targeting and homologous recombination.44 the clinical limitation of this procedure is at present the restriction on human embryonic stem cell research . however , great attention has been devoted recently to a special class of hemopoietic stem cells , which can be used for cell engineering , namely induced pluripotent stem cells , which can be obtained by reprogramming somatic cells and restoring the potential to develop new differentiated cells.45 however , many questions are still unsolved about possible applications of human induced pluripotent stem cells in cell engineering and regenerative medicine , like the permanence of foreign dna in the host genome . new techniques have therefore been developed to remove the integrated dna from the genome of induced pluripotent stem cells,46 making them safer for clinical use . it is apparent that a number of modalities are now available to try to modify the genetic defects in hemoglobinopathies , including the sickle mutation . it is indeed well known that , in a few cases of genetic immunodeficiency , a neoplastic proliferation was triggered by insertional mutagenesis of a proto - oncogene , raising the possibility that recombinant retroviral vectors could influence the expression of nearby genes.47 although gamma - globin gene vectors seem less prone to induce the expression of dangerous genes , this is a problem which requires great attention and is presently the object of intensive research . unfortunately , there is no international standardization program for hbf , and no quality specifications have been reported so far , although these are available for glycated hemoglobin and total hemoglobin . therefore , it is important to outline criteria used for measurement units and the reference interval for the expression of hbf . a uniform measurement unit , used worldwide , is the relative percentage of total hemoglobin , although this is not in line with the international si system . every professional laboratory should build its own reference interval , by measuring hbf in at least 100 adults , who are not iron - depleted and not carriers of alpha or beta thalassemia . it is generally assumed that hbf above 1% in a healthy adult individual could be due either to a genetic defect or to some acquired condition . a review on this topic has been proposed recently as a guide for the interpretation of clinical hbf data and a valuable aid to correct diagnosis.48 among the more sophisticated methods for the assessment of hbf levels , we may include the determination of single nucleotide polymorphisms , whose variations have received wide application in molecular genetics.49 it is to be expected that studies of genomic regions associated with hbf levels will provide interesting indications for the diagnosis and perhaps the therapeutic approach to hemoglobinopathies . identification of the full set of genes controlling gamma - globin synthesis and hbf production optimization of the efficacy of compounds already used for activating hbf production and exploration of new drug combinations reduction of the risk - benefit ratio of stem cell transplants , making it possible to treat a larger number of patients attempt to translate gene therapy from the experimental arena to clinical reality , keeping into account its dangers and limitations . the first point implies further efforts along a pathway which has already produced promising results . it has been stressed , however , that only about half of the genetic factors influencing hbf control has been identified and that multiple loci , perhaps of limited relevance , still have to be located.5 environmental factors also await to be clarified and in this context more comparisons between affected populations in different parts of the world and groups exposed to different life situations should be profitable.50 as for the second point , the list of compounds which have been proposed as therapeutic agents is quite impressive , but so far hydroxyurea is the only one subjected to extensive clinical trials and shown to produce satisfactory results , although not in all cases . more research is needed to elucidate the mechanism of action of hydroxyurea and the other compounds , because a better pharmacological treatment will reduce the need for the most difficult and expensive forms of therapy . more combination treatments will undoubtedly be tried ( keeping hydroxyurea as the main component ) with the aim to formulate the best scheme for any individual patient , according to age , gender , and severity of disease . further investigations will also be centered on practical aspects of drug presentation , eg , the possibility to give decitabine in an oral form,21 which has been already tried in monkeys and is likely to prove more acceptable in human patients . stem cell transplantation is commonly considered the only curative treatment for scd and , as such , is receiving special attention . a number of reports have highlighted the problems involved using reduced intensity preparative regimens for treating these patients and many failures have been reported , due in most cases to lack of sustained donor engraftment.51 however , in a recent study of nonmyeloablative allogeneic hematopoietic stem cell transplantation , including total body irradiation and treatment with immune suppressors , a stable , mixed donor recipient chimerism was achieved , with reversal of the sickle cell phenotype.52 furthermore , in addition to technical problems , there is a daunting issue of ethical impact pending on hemopoietic stem cell transplantation , namely the opportunity of giving birth to a sibling in order to provide a suitable donor for a severely affected child . the morality of this practice has been indeed questioned , particularly if programming a donor is performed exclusively as a utilitarian operation.53 the problems involved in gene therapy designs are still a great challenge and more research is certainly needed . as for human trials , worries have been raised very recently , because it was shown that a patient with beta thalassemia , treated successfully with a lentivirus - modified beta - globin gene in 2007 , and presently transfusion - free , now produces about 10% of blood cells with an insertion which may give a growth advantage and thus tumorigenic potential.54 one area of investigation is therefore the search for new types of vectors , possibly nonviral . in this respect , it is worthwhile to mention very recent progress obtained using biodegradable polymeric vectors , and a class of these vectors has recently been developed and optimized for high transfection efficiency.55 this may encourage more investigation along this line . further developments are awaited following the previously quoted attempts to correct the scd mutation by homologous recombination . actually , this procedure , proposed earlier in a mouse model,56 was performed recently in a patient with beta thalassemia , with skin fibroblasts removed , transformed into pluripotent stem cells , and then differentiated into hemopoietic stem cells , capable of producing normal adult hemoglobin . it was even suggested to collect cells from amniotic fluid or chorionic villus sampling , used for prenatal diagnosis , reprogram them into induced pluripotent stem cells , correct the mutation , and reinfuse them during the perinatal period , ie , an option for very early treatment before organ damage takes place.57 as for the tools necessary to introduce specific changes into the genome of patient - derived induced pluripotent stem cells , a recent approach is based on zinc finger nuclease technology , which enables targeting of a specific dna binding domain to a preselected chromosomal site.58,59 both approaches allowed the introduction of defined genetic modifications in highly sensitive induced pluripotent stem cells , without reported side effects on the pluripotency of these cells or their genetic stability . mention was made before of an encouraging improvement obtained with severe thalassemia by gene therapy.54 however , as far as scd is concerned , 100 years have passed since the first description of the disease and , despite remarkable progress in the knowledge of pathogenetic and clinical aspects , a consistently effective treatment remains elusive and requires further investigations.60

the natural history of severe hemoglobinopathies like sickle cell disease ( scd ) is rather variable , depending on the circumstances , but the main influence on such variability is the level of fetal hemoglobin ( hbf ) in the patient s red cells . it is well known that a significant hbf level is associated with a milder course of disease and fewer complications . therefore , attempts have been made to reactivate using various means the hbf production , which is normally switched off perinatally . a pharmacological approach has been attempted since the 1980s , ranging from drugs like 5-azacytidine and its derivative , decitabine , to a series of compounds like hydroxyurea and a number of histone deacetylase inhibitors like butyrate , which seem to act as epigenetic modifiers . many other disparate agents have been tried with mixed results , but hydroxyurea remains the most effective compound so far available . combinations of different compounds have also been tried with some success . established treatments like bone marrow or cord blood transplantation are so far the only real cure for a limited number of patients with severe hemoglobinopathies . improved chemotherapy regimens of milder toxicity than those employed in the past have made it possible recently to obtain a stable , mixed donor - recipient chimerism , with reversal of the scd phenotype . however , great effort is directed to cell engineering , searching for an effective gene vector by which a desired gene can be transferred into new classes of vectors for autologous hemopoietic stem cells . recent studies are also aiming at targeted insertion of the therapeutic gene into hemopoietic cells , which can also be induced human stem cells , obtained from somatic dedifferentiated cells . attention in this area must be paid to the possibility of undesired effects , like the emergence of potentially oncogenic cell populations . finally , an update is presented on improved hbf determination methods , because common international standards are becoming mandatory .

Introduction Genetics of HbF Inducers of fetal hemoglobin Cell transplant and cell engineering Progress in methods for fetal hemoglobin determination Future directions

in the wide spectrum of congenital hemoglobin disorders , two entities have always attracted attention because of their severity and extensive geographic distribution , ie , beta thalassemia major and sickle cell disease ( scd ) . in the present review , attention is focused on the latter disease , characterized by a qualitative defect in beta - globin production , due to replacement of a single amino acid ( valine for glutamic acid ) in the beta - globin chain and formation of an anomalous hemoglobin , called hemoglobin s. this induces severe deformity of red cells upon deoxygenation , hampering microcirculation , and leading to vascular occlusion and critical organ damage.1 however , it has been noted that the natural history of this disease shows considerable heterogeneity in signs and symptoms , due to a variety of concomitant situations . actually , the main factor in the variable severity of scd is the level of fetal hemoglobin ( hbf ) produced by patients . it is well known that populations showing a genetically determined presence of hbf have a milder form of scd , including a reduced incidence of severe clinical complications.2 it is therefore understandable that many efforts have been directed to restore the production of hbf in adults.3 as for the background of this process , recent studies have produced a good amount of information , particularly on gamma - globin gene control . in the present paper , we therefore propose a brief initial survey of these genetic factors , and then focus on new ways of drug treatment for reactivation of hbf level , as well as attempts at correction of the genetic defect by cell engineering . it is well known that only a tiny fraction of hbf is still present in the majority of adults , as a result of the switch to hba in early life . however , the switch may be impaired by a number of mutations in the beta - globin cluster,4 leading to uniform increase of hbf ( pancellular hereditary persistence of fetal hemoglobin [ hpfh ] ) , a condition allowing a normal way of life , or to a nonuniform distribution of hbf , based on the coexistence of two populations of red cells , one with a high content of hbf ( so - called f cells ) and another with a negligible amount of the same . in this case , the condition is called heterocellular hpfh , which is regarded as a multifactorial quantitative trait , quite distinct from the classical form of pancellular hereditary persistence of fetal hemoglobin.5 a number of interesting studies have proposed different mechanisms which may be responsible for such persistence of hbf in adults , and can be either mutations in critical genic regions involved in the activation of globin genes or deletions of the globin gene cluster . examples of the former event are single base substitutions in the promoter region of the gamma - globin genes , found in adults with high hbf production,6 or t to c substitution in the gamma - globin gene , leading to disruption of the assembly of a repressive chromatin structure , which normally silences the expression of the gamma - globin gene in adult erythrocytes.7 in the latter case , ( ie , deletional persistence of hbf ) , gazouli et al,8 attempting to explain the production of the hbf phenotype in adult red cells , carried out an investigation in transgenic mice lacking two elements of the a-to- gene region , suggesting that a deletion of silencer elements in that gene region may account for persistent expression of hbf in the adult stage . according to another study , a downregulation of the transcriptional repressor , zhx2 , or some chromatin remodelling factors may also be involved in deletional hereditary persistence of fetal hemoglobin.9 anyway , there is an obvious benefit from the analysis of multiple mechanisms of hbf persistence in adults , namely a better understanding of the molecular basis for the perinatal hemoglobin switch . as far as possible genetic determinants of hbf production an early hypothesis suggested that variability in hbf was mainly due to independent genetic factors , an effect particularly evident in patients with scd.10 a seminal study showed that a variation of genetic determinants on chromosome 11p16 was involved in the expression of hbf , and precisely at position 158.11 however , the effect of such variation is modest in normal people , and its presence is not always associated with high hbf , but variants at this locus have a considerable frequency in certain populations like asian indians , in whom it was first studied and found to be associated with the presence of moderate levels of hbf in normal individuals.12 a second determinant was found in a large indian family with beta thalassemia and hereditary persistence of fetal hemoglobin , situated in a region on chromosome 6q23q24 , between the hbs1l and myb genes.13 this locus was therefore termed hmip ( hbs1l - myb intergenic polymorphism ) , and is an important component ( about 19% ) of the f cell trait in the general population . a strong association with hbf level was eventually observed with a third locus , situated on chromosome 2p15 , namely the bcl11a , leading to the hypothesis that a product of this locus , a multizinc finger transcription factor , may encode a stage - specific regulator of hbf expression.14 on the clinical side , extensive research carried out among patients in sardinia and other groups showed that a variant of bcl11a was strongly associated with individuals having high hbf levels , as well as with patients having mild forms of thalassemia and scd.15 a very recent study , based on genome - wide association , has also identified a regulatory region in the olfactory receptor gene cluster ( on chromosome 11 ) , which may play a role in gamma - globin gene expression.16 finally , it should be stressed that , although recent progress in the genetics of hbf has not yet promoted therapeutic applications , every contribution to the understanding of genetic mechanisms presiding over hbf synthesis and to the hbf - to - hba switch is a necessary premise for the formulation of treatment strategies for severe hemoglobin disorders . agents capable of inducing production of fetal hemoglobin have been known for many years , and have also been used in clinical trials ( table 3 ) . a few patients with beta thalassemia and scd treated with this drug showed a modest correction of globin chain imbalance and had lower transfusion needs.17 however , the possibility that such treatment could have an oncogenic effect discouraged the undertaking of these trials . it was only years later that a derivative of azacytidine , namely deoxy - azacytidine , or decitabine , was found to be effective in reactivating production of fetal hemoglobin , and was therefore started in regular trials on scd patients.18 as for possible toxic effects from this drug , only reversible neutropenia was observed , while tests on experimental animals not only showed absence of tumorigenic action , but even suggested a decrease of tumor formation in mice predisposed to cancer.19 parenteral administration of decitabine can produce a notable increase of hbf levels as well as stimulate erythroid differentiation in scd patients.20 oral preparations have also been tested in animals , with the aim of making them more acceptable to patients.21 more investigations are needed to study possible long - term adverse effects of this compound . great interest was devoted to the study of the mechanism of action of different compounds in the induction of hbf . predictably , bone marrow suppression was observed after long - term treatment , but this effect was reversible.3 the mechanism of action of this compound seems to be mediated through the induction of a gtp - binding protein sar , which modulates expression of the gamma - globin gene in erythroid cells.23 very good results have been reported recently in a 17-year trial carried out in a variety of sickle cell syndromes . a dramatic reduction of severe pain crises , transfusion requirements and hospital admissions was obtained , showing how prolonged and tolerable treatment with hydroxyurea can profoundly modify the natural history of the disease.24 as for the mechanism of action of hydroxyurea , a series of investigations point to an effect on genes which are involved in hemoglobin synthesis . one of these genes is the transcription factor , egr1 , which is one of the most upregulated genes following hc treatment . another group of genes , represented by centb1 ( centaurin , beta 1 ) , arhgap4 ( rho gtpase activating protein 4 ) , and rin3 ( ras and rab interactor 3 ) , were found to be induced after hydroxyurea administration.25 another study , carried out in reticulocytes from scd patients after hydroxyurea treatment showed an altered expression of genes associated with the regulation of globin expression , like sud53 , fzd5 , and phc3.26 these results show that hydroxyurea produces significant changes in the gene expression pattern , with activation of transcription factors and pathways involved in signal transduction , eventually leading to an increase of globin gene expression . in this context , a compound which has been known for a long time as an hbf inducer is erythropoietin , which is capable of increasing the amount of f cells and hbf concentration . in this case , erythropoietin treatment made it possible to tolerate hydroxyurea dosage escalation , with an increase in f cells and only minor side effects.27 more studies are in progress to investigate the potential of such a drug combination . 3 ) butyrate was the earliest of a number of compounds used in this context because of their action as inhibitors of histone deacetylase . it is indeed known that by promoting histone acetylation , butyrate increases the transcription rate of the gamma - globin gene , as well as the translation of gamma - globin mrna.28 it has also been demonstrated that transcription factors like gata1 and nf - e32 are involved.29 other histone deacetylase inhibitors , and butyrate itself , have also shown a different mechanism of action , namely through activation of the p38map kinase pathway . the effector molecules have been identified by sangerman et al as creb1 and atf2 , acting via the aforementioned pathway , which therefore appears to be one of the main mediators of gamma - globin gene regulation.30 many more compounds , acting as histone deacetylase inhibitors , have been mentioned as beneficial agents in sickle cell disease.31 new classes of synthetic histone deacetylase inhibitors , defined as aroyl - pyrrolyl hydroxyamides and uracil - based hydroxyamides , have also been tested . interestingly , both compounds were also effective in correcting the impaired in vitro maturation of beta thalassemic erythroblasts.32 these results and those obtained with other types of histone deacetylase inhibitors seem to suggest that compounds acting as epigenetic modifiers , namely capable of modulating gene expression , may be a promising area of investigation . 4 ) a number of disparate compounds has been suggested as endowed with stimulating activity on the production of gamma - globin and hbf . one of them is valproate , which was found to induce hemoglobin synthesis in erythroid cells , again by activation of the p38 pathway.33 an interesting compound tried in this context is the kit ligand , an important cytokine for the initiation of hemopoiesis . in unilineage erythroid cultures of 20 patients with major beta thalassemia or thalassemia intermedia , addition of kit ligand induced a marked increase of gamma - globin synthesis , thus reaching hbf levels three - fold higher than in control cultures.34 worth mentioning is the effect of thalidomide , which seems capable of inducing hbf expression via activation of the same p38mapk signaling pathway , as well as the action of two thalidomide analogs , used as immunomodulators , which have been shown to possess , among other well known activities , a stimulating effect on hbf production . an effective strategy employed in scd is the transplantation of hemopoietic bone marrow or cord blood , thus delegating to transplanted stem cells the task of providing normal levels of hemoglobin a. however , it has been calculated that , up to 2008 , more than 1600 allogeneic transplants had been performed in beta thalassemia , but many fewer were done in scd , perhaps because fewer patients in the advanced stages were eligible , due to the presence of severe vascular damage.36 this option is moreover restricted by limited availability of suitable donors and the severe effects of marrow ablation treatment , including reduced fertility , especially in women . to overcome such limitations , unrelated donors have been used with some success and nonablative regimens have also been employed , while , in order to preserve reproductive potential in women , ovarian tissue preservation and subsequent implantation have been employed , obtaining successful embryo development.37 more recently , sophisticated strategies , based on cell engineering , have been developed to transfer correcting genes in patients with a single gene defect . it is certainly fair to mention some remarkable success obtained by gene therapy in genetic immunodeficiency diseases.38 as far as scd is concerned , a search has been going on for a long time to find an effective gene vector which would allow transfer of a desired gene into hemopoietic stem cells . after early attempts using gamma retroviral vectors to transduce globin genes into dividing stem cells,39 lentiviral vectors , a subclass of retroviruses capable of transducing not only proliferating but also quiescent cells , proved more effective.40 it has been stressed that lentiviral vectors should be erythroid - specific , differentiation- and stage - restricted , position - independent , and sustainable over time.41 this approach can be adopted for scd , forcing the expression of fetal hemoglobin , which , even at low concentration , has been shown to exert a potent antisickling effect.42 on the other side , it has been noted that gene therapy for scd , to be effective , should induce a therapeutic gene in the greatest part of the red cell population , because even a small proportion of remaining sickle cells may cause vaso - occlusion and severe ischemia.43 as for the cell type to be used for such correction , the most primitive progenitors , namely embryonic stem cells , were used initially in mice with scd by the technique of gene targeting and homologous recombination.44 the clinical limitation of this procedure is at present the restriction on human embryonic stem cell research . however , great attention has been devoted recently to a special class of hemopoietic stem cells , which can be used for cell engineering , namely induced pluripotent stem cells , which can be obtained by reprogramming somatic cells and restoring the potential to develop new differentiated cells.45 however , many questions are still unsolved about possible applications of human induced pluripotent stem cells in cell engineering and regenerative medicine , like the permanence of foreign dna in the host genome . it is apparent that a number of modalities are now available to try to modify the genetic defects in hemoglobinopathies , including the sickle mutation . it is indeed well known that , in a few cases of genetic immunodeficiency , a neoplastic proliferation was triggered by insertional mutagenesis of a proto - oncogene , raising the possibility that recombinant retroviral vectors could influence the expression of nearby genes.47 although gamma - globin gene vectors seem less prone to induce the expression of dangerous genes , this is a problem which requires great attention and is presently the object of intensive research . a review on this topic has been proposed recently as a guide for the interpretation of clinical hbf data and a valuable aid to correct diagnosis.48 among the more sophisticated methods for the assessment of hbf levels , we may include the determination of single nucleotide polymorphisms , whose variations have received wide application in molecular genetics.49 it is to be expected that studies of genomic regions associated with hbf levels will provide interesting indications for the diagnosis and perhaps the therapeutic approach to hemoglobinopathies . identification of the full set of genes controlling gamma - globin synthesis and hbf production optimization of the efficacy of compounds already used for activating hbf production and exploration of new drug combinations reduction of the risk - benefit ratio of stem cell transplants , making it possible to treat a larger number of patients attempt to translate gene therapy from the experimental arena to clinical reality , keeping into account its dangers and limitations . it has been stressed , however , that only about half of the genetic factors influencing hbf control has been identified and that multiple loci , perhaps of limited relevance , still have to be located.5 environmental factors also await to be clarified and in this context more comparisons between affected populations in different parts of the world and groups exposed to different life situations should be profitable.50 as for the second point , the list of compounds which have been proposed as therapeutic agents is quite impressive , but so far hydroxyurea is the only one subjected to extensive clinical trials and shown to produce satisfactory results , although not in all cases . a number of reports have highlighted the problems involved using reduced intensity preparative regimens for treating these patients and many failures have been reported , due in most cases to lack of sustained donor engraftment.51 however , in a recent study of nonmyeloablative allogeneic hematopoietic stem cell transplantation , including total body irradiation and treatment with immune suppressors , a stable , mixed donor recipient chimerism was achieved , with reversal of the sickle cell phenotype.52 furthermore , in addition to technical problems , there is a daunting issue of ethical impact pending on hemopoietic stem cell transplantation , namely the opportunity of giving birth to a sibling in order to provide a suitable donor for a severely affected child . as for human trials , worries have been raised very recently , because it was shown that a patient with beta thalassemia , treated successfully with a lentivirus - modified beta - globin gene in 2007 , and presently transfusion - free , now produces about 10% of blood cells with an insertion which may give a growth advantage and thus tumorigenic potential.54 one area of investigation is therefore the search for new types of vectors , possibly nonviral . actually , this procedure , proposed earlier in a mouse model,56 was performed recently in a patient with beta thalassemia , with skin fibroblasts removed , transformed into pluripotent stem cells , and then differentiated into hemopoietic stem cells , capable of producing normal adult hemoglobin . it was even suggested to collect cells from amniotic fluid or chorionic villus sampling , used for prenatal diagnosis , reprogram them into induced pluripotent stem cells , correct the mutation , and reinfuse them during the perinatal period , ie , an option for very early treatment before organ damage takes place.57 as for the tools necessary to introduce specific changes into the genome of patient - derived induced pluripotent stem cells , a recent approach is based on zinc finger nuclease technology , which enables targeting of a specific dna binding domain to a preselected chromosomal site.58,59 both approaches allowed the introduction of defined genetic modifications in highly sensitive induced pluripotent stem cells , without reported side effects on the pluripotency of these cells or their genetic stability . mention was made before of an encouraging improvement obtained with severe thalassemia by gene therapy.54 however , as far as scd is concerned , 100 years have passed since the first description of the disease and , despite remarkable progress in the knowledge of pathogenetic and clinical aspects , a consistently effective treatment remains elusive and requires further investigations.60

primary malignant liver tumors are rare in childhood with an incidence of about 1.6 cases per million children ( 014 years).1,2 while hepatoblastoma ( hb ) represents 80% of the hepatic - related cancer affecting children predominantly between 6 months and 3 years , hepatocellular carcinoma ( hcc ) is more uncommon , with incidence increasing with age . only about 0.5%1% of all pediatric tumors are hcc.1,2 in hepatoblastoma , event - free survival ( efs ) and overall survival ( os ) was increased from roughly 30% in the 1970s to 70%90% these days , especially due to advances of chemotherapy regimens and surgical approaches.3 in hcc , the results with unresectable tumor especially are rather dismal . contrary to adults , in the majority of children or adolescents , no etiologic factors can be detected . however , in areas with a high prevalence of hepatitis b virus ( hbv ) infection rate , the lifetime risk of hcc in chronic hbv carriers is estimated to be 10%25%.4 for example , in taiwan and hong kong , 100% and 64% , respectively , of all children with hcc were chronic hbv carriers . it can be expected that universal newborn vaccination will have an effect in reducing the incidence of hcc.5,6 only a minority of hcc cases are associated with cirrhosis or other chronic liver diseases such as glycogen storage disease type iii , tyrosinemia type i , wilson disease , or biliary atresia . this indicates that the pathogenesis of hcc in childhood is different compared with that in adults.79 management of hcc remains difficult since complete surgical resection is fundamental for cure . however , in pedi - atric hcc , < 20% of the patients are considered eligible for initial resection . various studies have been conducted using different combinations of chemotherapeutic agents to reduce tumor load , thereby helping patients become suitable candidates for resection . historically , hcc patients were treated with the same protocols as hb patients , and so , primarily , cisplatin , doxorubicin , carboplatin , 5-fluorouracil , and vin - cristine were used.1,10 however , to date , there is no convincing data that this approach results in a benefit for survival . the 3-year efs and os for children with complete tumor excision upfront and two courses of carboplatin ( 200 mg / m / d 4 ) and etoposide ( 100 mg / m / d 4 ) were 72% and 89% , respectively , in the hb99 study ( 19992008)11 conducted by the german society for pediatric oncology and hematology ( gpoh ) . however , the prognosis remained poor , with 3-year efs and os rates of 12% and 20% in those patients who had inoperable or metastatic disease . intensifying preoperative chemotherapy with two courses of carboplatin and etoposide followed by two courses of high - dose carboplatin ( 500 mg / m / d 4 ) and etoposide ( 300 mg / m / d 4 ) with autologous stem cell transplantation did not translate into a satisfactory operability rate , efs , or os . similar findings were reported by the north american intergroup hepatoblastoma study ( int-0098 ) ( 19891992).12 the 5-year efs ( os ) for pediatric patients ( 26/46 patients 10 years ) with inoperable tumor upfront was 8% ( 23% ) , and for those with metastases it was 0% ( 19% ) . there was no difference based on whether the children received cisplatin ( 90 mg / m on d0 ) and doxorubicin ( 20 mg / m / d 4 from d2 ) or cisplatin ( 90 mg / m on d0 ) , 5-fluorouracil ( 600 mg / m on d2 ) , and vincristine ( 1.5 mg / m on d2 ) . importantly , the first international society of pediatric oncology liver tumor study ( siopel-1 study , 19901994)1 demonstrated that hcc in childhood ( 415 years , median : 12 years ) can be chemotherapy sensitive . they proved this by showing that 49% of the children responded to cisplatin ( 80 mg / m on d1 ) and doxorubicin ( 30 mg / m / d on d2 + 3 ) ( plado ) . however , taking into consideration that complete resection is the cornerstone of cure , only 36% had complete tumor excision , and so the 5-year efs was only 17% . the next attempt ( siopel-2 study , 19951998)10,13 tried was rapidly switching between cisplatin ( 80 mg / m on d1 ) and carboplatin ( 500 mg / m on d1)/doxorubicin ( 30 mg / m / d on d2 + 3 ) ( superplado ) every 14 days , but this did not improve the response rate after preoperative chemotherapy ( 46% ) , and therefore also not 3-year os ( 22% ) . thus , to date , for children with inoperable liver tumor and/or with metastases , the complete resection ( and so the efs and os ) have not improved although different strategies have been attempted . sorafenib is an inhibitor of several tyrosine protein kinases such as vegfr , pdgfr , and raf kinases.1416 in preclinical models , sorafenib demonstrated antitumor activity alone and in combination with , for instance , doxorubicin , gemcitabine , and cisplatin.17,18 in adult patients with advanced hcc , sorafenib significantly improved both time to tumor progression and os from a median of 2.8 to 5.5 months and from 7.9 to 10.7 months , respectively , compared with placebo . the most important grade 3 adverse effects were diarrhea , hand foot skin reaction , and fatigue.19 therefore , sorafenib has become the standard therapy for adult patients with hcc.20 furthermore , in a randomized , double - blind , phase ii study combining sorafenib with doxorubicin , the progression - free survival ( pfs ) was significantly better in patients receiving sorafenib and doxorubicin than in those receiving doxorubicin and placebo ( median : 4.8 vs 8.6 months).21,22 moreover , tumor reduction was achieved in 62% vs 29% of the patients . this effect could be due to the fact that combining sorafenib with doxorubicin translated into an increased mean values of doxorubicin cmax and area under the curve by 33% and 21% , respectively.23 in the recent study with 12 children ( seven with unresectable liver tumor , age 716 years ) , it was demonstrated that sorafenib ( 244602 mg / m / d , median : 288 mg / m / d ) added to plado is a promising new therapeutic option with hand foot skin reaction being the most relevant toxicity.24 with this combination , four of the seven children with inoperable liver tumor achieved a partial response ( pr ) , two a stable disease , and one a progression . three patients were alive without evidence of tumor after complete tumor excision at 12 months ( with second - line chemotherapy after two courses sorafenib and plado ) , 12 months , and 18 months ( both patients had six courses sorafenib and plado ) , respectively , after primary diagnosis . the elevated -fetoprotein levels seen in four patients at diagnosis markedly declined after two courses of therapy . since then , some pediatric liver tumor specialists have recommended plado with sorafenib as a standard chemotherapy . thus , there are big challenges to be solved for pediatric patients with hcc , namely : 1 ) what is the standard - of - care in children with newly diagnosed hcc with complete resection upfront : observation vs sorafenib vs plado vs plado and sorafenib ? 2 ) what are the therapeutic options in newly diagnosed patients with unresectable tumors and/or metastatic disease ? 3 ) are there new approaches on the horizon for hcc in children ? 4 ) must the milan criteria for a liver transplantation be strictly adhered to ? and 5 ) does transarterial chemoembolization ( tace ) play a role in pediatric patients ? katzenstein et al12 reported an 88% 5-year efs in patients with completely resected hcc receiving either cisplatin , 5-fluorouracil , and vincristine or plado ( n=8 ) . the german hb99 study11 used two cycles of carboplatin / eto - poside postoperatively , which translated into 5-year efs and os probabilities of 72% and 89% ( n=14 ) , respectively . thus , there seems to be no difference in survival based on the chemotherapy used . in adults , it was recently shown that sorafenib is not effective as an adjuvant treatment following resection or ablation.25 an enhanced chemotherapeutic response to sorafenib and plado was demonstrated in the small series of patients with advanced hcc ( pr in four out of seven ) . the problem is that it is impossible to realize phase ii to iii studies in an entity as rare as hcc in childhood . in the siopel 1 , 2 , and 3 studies1,10 recruiting patients between 1990 and 2004 , 15/121 had an hcc with complete resection at diagnosis . internationally , the estimated number of primary resectable patients would be about 10/year . with such low numbers , a study randomizing patients after upfront complete surgical resection to observation vs sorafenib vs plado vs plado and sorafenib , and even to a two - arm study , will never be feasible within an adequate amount of time . pediatric liver tumor specialists currently recommend that children with hcc should receive plado with or without sorafenib , as more intensive regimens have not yielded better results . but the role of postoperative chemotherapy and the amount ( plado for two or four cycles ? sorafenib at all or for 6 or 12 months ? ) for a stage i disease that has demonstrated chemotherapeutic sensitivity in pediatric patients are unknown . pediatric patients with unresectable or primarily metastatic hcc do not survive unless the disease can be rendered resectable . given preexisting evidence that pediatric hcc is chemotherapy responsive in nearly 50% of the patients ( table 1 ) intensification of platinum and doxorubicin agents , as in the siopel 2 and 3 studies,13 did not result in improved survival . however , 5-year efs rates still remain between 10%34% since response mostly does not translate into complete surgical resection . hopefully , sorafenib in addition to plado improves the resectability rate , efs , and os.24 in a recently published multicenter study from france , 204 adults with advanced hcc received gemcitabine ( 1,000 mg / m on d1 ) and oxaliplatin ( 100 mg / m on d2 ) ( gemox ) , with promising response and tumor control rates of 22% and 66% , respectively.26 in 44% of the patients , grade 34 toxicities were reported , especially neutropenia , thrombocytopenia , neurotoxicity , and diarrhea . in a retrospective survey within the international liver tumor community , the response to gemox was 30% in heavily pretreated pediatric patients ( personal communication ) . adding gemox to sorafenib ( n=83 ) increased 4-month pfs rate from 54% to 61% and median os from 13 to 13.5 months.27 williet et al28 described a 61 year old man with hcc and lymph node metastasis treated with sorafenib and gemox , who achieved a pr and drop of -fetoprotein to normal levels . the experience within the gpoh with gemox given every 14 days with sorafenib in - between further supports that this regimen is worth being evaluated in a prospective study in pediatric patients . but still , new effective drugs besides the conventional chemotherapeutic ones ( eg , plado , gemox , and sorafenib ) are definitely needed with the goal to achieve a higher response rate , thus translating into a higher surgical resection rate . since it is strongly believed that hcc in children is a different biologic disease , results from studies in the adult population can not simply be translated to children.7,8 the better response to chemotherapy in pediatric patients may be due to the much higher rate of de novo tumors and normal liver function . in addition , in older children and in young adolescents , an entity called transitional liver cell tumor has been observed , which is made up of chemotherapy - sensitive hepatoblastoma - like cells , cells similar to those of hcc , and intermediate cell forms.29 young people more often present the fibrolamellar histologic variant.30 it was thought that this variant has a more favorable prognosis , but recently it was shown that the long - term os is similar to that for hcc.31 since hccs are highly vascularized tumors with increased levels of vegf , antiangiogenic approaches represent a potential new therapeutic strategy . in adults , but not in children , different antiangiogenic agents besides sorafenib have been tested in clinical studies , eg , sunitinib , brivanib , bevacizumab , and ramucirumab.32 for example , sunitinib was proven to be nonsuperior when randomized with sorafenib and was highly toxic , with side effects including thrombo - cytopenia and neutropenia.33 bevacizumab was the most promising agent , showing an objective response in six of 46 patients ( 13% ) and a pfs rate of 65% at 6 months.34 in combination with erlotinib ( egfr inhibitor ) , a response rate of 25% was reported.35 despite the initial promising results , there were no plans for a phase iii study with bevacizumab . egfr inhibitors ( eg , erlotinib or cetuximab),32 mtor inhibitors ( eg , sirolimus),36 and mek inhibitors ( eg , selumetinib)37 as single agents have not demonstrated significant antitumor activity . the hgf / c - met pathway has been identified as having an important role in tumor progression , angiogenesis , and appearance of metastases in hcc . silencing the c - met expression in cell lines and preclinical models was shown to inhibit hcc growth.38 however , only in patients with a high met expression , tivantinib , a selective tyrosine kinase inhibitor , achieved a significantly longer median time to progression ( 1.4 vs 2.7 months ) , pfs ( 1.4 vs 2.2 months ) , and overall survival ( 3.8 vs 7.2 months).39 cabozantinib ( xl184 ) , a dual tyrosine kinase inhibitor with activity against c - met , vegfr2 , and ret , demonstrated a tumor control rate at week 12 of 71% in a randomized phase ii study.40 a 10 year old child was treated with relapsed hcc in complete remission with cabozantinib as maintenance therapy for 12 months . immune checkpoint blockade has been presented as a new encouraging therapeutic option for various malignancies including hcc . blocking of pd-1 with a specific antibody has been shown to amplify t - cell function and enhance antitumor effects.41 hcc is known to be an inflammation - associated cancer and can therefore be immunogenic.42 upregulation of pd-1 and the pd-1 immune checkpoint ligand ( pd - l1 ) in hcc is associated with poor prognosis.43,44 in a phase i / ii study , pd-1 blockade with nivolumab showed complete responses in two out of 39 patients ( 5% ) and prs in seven ( 18% ) patients . os rate was 72% at 6 months.45 these initial data support the continued exploration of nivolumab in hcc . the abovementioned 10 year old boy was treated for the second relapse with nivolumab and achieved a significant but temporary clinical response . current studies in adult cancer are ongoing , combining immune checkpoint inhibitors with targeted therapy or chemotherapeutic agents such as anthracyclines or gemcitabine since these can also modulate t - cell proliferation and promote immunogenic cell death.41,46,47 this makes sense as monotherapy with targeted agents demonstrated an improved response rate but a limited time to tumor progression , whereas checkpoint blockade monotherapy seems to have a lower response rate but an extended pfs.48 unfortunately , information about the feasibility or effects of those new agents is not available for childhood hcc . the indication for liver transplantation in adults is restricted to the milan criteria , ie , the evidence of a single tumor < 5 cm in size or no more than three foci with each not exceeding 3 cm and no vascular invasion or extrahepatic involvement.49 the practice guidelines of the american association for transplantation and the north american society for pediatric gastroeneterology , hepatology , and nutrition recommend that the indication for liver transplantation in childhood hcc must be discussed individually for each patient . in principle , liver transplantation should be considered in children with no extrahepatic tumor or gross vascular invasion on radiological imaging , irrespective of the size of the lesions or the number of the lesions.50 successful transplantations have been done in children with more liberal criteria , even in patients with large , multifocal hcc , microscopic blood vessel involvement , or limited extrahepatic tumor.5153 a meta - analysis showed lower relapse rate and longer efs and os in patients treated with sirolimus compared with calcineurin inhibitor following liver transplantation for hcc.54 to conclude , the milan criteria are not applicable for children with hcc . palliative tace is a standard procedure in adults with solitary or multifocal hcc without extrahepatic metastases . back in 2,000 , malogolowkin et al55 reported that all eleven children ( 18 months14 years old ) with unresectable , chemotherapy - resistant liver tumors ( three with hcc ) responded five ( one with hcc ) went on to surgical resection and three survived . the conclusion was that tace with a suspension of cisplatin , doxorubicin , and mitomycin mixed with lipiodol is feasible , well - tolerated , and effective in achieving surgical resectability in pediatric patients . these encouraging results were confirmed by czauderna et al56 ( five patients , 112 years old , one with hcc ) . thus , tace could be offered to patients with chemotherapy - resistant liver tumor for palliative care or even with the goal of achieving surgical resectability and cure . katzenstein et al12 reported an 88% 5-year efs in patients with completely resected hcc receiving either cisplatin , 5-fluorouracil , and vincristine or plado ( n=8 ) . the german hb99 study11 used two cycles of carboplatin / eto - poside postoperatively , which translated into 5-year efs and os probabilities of 72% and 89% ( n=14 ) , respectively . thus , there seems to be no difference in survival based on the chemotherapy used . whether postoperative sorafenib has a survival benefit remains unclear . in adults , it was recently shown that sorafenib is not effective as an adjuvant treatment following resection or ablation.25 an enhanced chemotherapeutic response to sorafenib and plado was demonstrated in the small series of patients with advanced hcc ( pr in four out of seven ) . however , further data regarding sorafenib are urgently needed in the pediatric hcc population.24 the problem is that it is impossible to realize phase ii to iii studies in an entity as rare as hcc in childhood . in the siopel 1 , 2 , and 3 studies1,10 recruiting patients between 1990 and 2004 , 15/121 had an hcc with complete resection at diagnosis . internationally , the estimated number of primary resectable patients would be about 10/year . with such low numbers , a study randomizing patients after upfront complete surgical resection to observation vs sorafenib vs plado vs plado and sorafenib , and even to a two - arm study , will never be feasible within an adequate amount of time . pediatric liver tumor specialists currently recommend that children with hcc should receive plado with or without sorafenib , as more intensive regimens have not yielded better results . but the role of postoperative chemotherapy and the amount ( plado for two or four cycles ? sorafenib at all or for 6 or 12 months ? ) for a stage i disease that has demonstrated chemotherapeutic sensitivity in pediatric patients are unknown . pediatric patients with unresectable or primarily metastatic hcc do not survive unless the disease can be rendered resectable . given preexisting evidence that pediatric hcc is chemotherapy responsive in nearly 50% of the patients ( table 1 ) intensification of platinum and doxorubicin agents , as in the siopel 2 and 3 studies,13 did not result in improved survival . however , 5-year efs rates still remain between 10%34% since response mostly does not translate into complete surgical resection . hopefully , sorafenib in addition to plado improves the resectability rate , efs , and os.24 in a recently published multicenter study from france , 204 adults with advanced hcc received gemcitabine ( 1,000 mg / m on d1 ) and oxaliplatin ( 100 mg / m on d2 ) ( gemox ) , with promising response and tumor control rates of 22% and 66% , respectively.26 in 44% of the patients , grade 34 toxicities were reported , especially neutropenia , thrombocytopenia , neurotoxicity , and diarrhea . in a retrospective survey within the international liver tumor community , the response to gemox was 30% in heavily pretreated pediatric patients ( personal communication ) . adding gemox to sorafenib ( n=83 ) increased 4-month pfs rate from 54% to 61% and median os from 13 to 13.5 months.27 williet et al28 described a 61 year old man with hcc and lymph node metastasis treated with sorafenib and gemox , who achieved a pr and drop of -fetoprotein to normal levels . the experience within the gpoh with gemox given every 14 days with sorafenib in - between further supports that this regimen is worth being evaluated in a prospective study in pediatric patients . but still , new effective drugs besides the conventional chemotherapeutic ones ( eg , plado , gemox , and sorafenib ) are definitely needed with the goal to achieve a higher response rate , thus translating into a higher surgical resection rate . since it is strongly believed that hcc in children is a different biologic disease , results from studies in the adult population can not simply be translated to children.7,8 the better response to chemotherapy in pediatric patients may be due to the much higher rate of de novo tumors and normal liver function . in addition , in older children and in young adolescents , an entity called transitional liver cell tumor has been observed , which is made up of chemotherapy - sensitive hepatoblastoma - like cells , cells similar to those of hcc , and intermediate cell forms.29 young people more often present the fibrolamellar histologic variant.30 it was thought that this variant has a more favorable prognosis , but recently it was shown that the long - term os is similar to that for hcc.31 since hccs are highly vascularized tumors with increased levels of vegf , antiangiogenic approaches represent a potential new therapeutic strategy . in adults , but not in children , different antiangiogenic agents besides sorafenib have been tested in clinical studies , eg , sunitinib , brivanib , bevacizumab , and ramucirumab.32 for example , sunitinib was proven to be nonsuperior when randomized with sorafenib and was highly toxic , with side effects including thrombo - cytopenia and neutropenia.33 bevacizumab was the most promising agent , showing an objective response in six of 46 patients ( 13% ) and a pfs rate of 65% at 6 months.34 in combination with erlotinib ( egfr inhibitor ) , a response rate of 25% was reported.35 despite the initial promising results , there were no plans for a phase iii study with bevacizumab . egfr inhibitors ( eg , erlotinib or cetuximab),32 mtor inhibitors ( eg , sirolimus),36 and mek inhibitors ( eg , selumetinib)37 as single agents have not demonstrated significant antitumor activity . the hgf / c - met pathway has been identified as having an important role in tumor progression , angiogenesis , and appearance of metastases in hcc . silencing the c - met expression in cell lines and preclinical models was shown to inhibit hcc growth.38 however , only in patients with a high met expression , tivantinib , a selective tyrosine kinase inhibitor , achieved a significantly longer median time to progression ( 1.4 vs 2.7 months ) , pfs ( 1.4 vs 2.2 months ) , and overall survival ( 3.8 vs 7.2 months).39 cabozantinib ( xl184 ) , a dual tyrosine kinase inhibitor with activity against c - met , vegfr2 , and ret , demonstrated a tumor control rate at week 12 of 71% in a randomized phase ii study.40 a 10 year old child was treated with relapsed hcc in complete remission with cabozantinib as maintenance therapy for 12 months . immune checkpoint blockade has been presented as a new encouraging therapeutic option for various malignancies including hcc . blocking of pd-1 with a specific antibody has been shown to amplify t - cell function and enhance antitumor effects.41 hcc is known to be an inflammation - associated cancer and can therefore be immunogenic.42 upregulation of pd-1 and the pd-1 immune checkpoint ligand ( pd - l1 ) in hcc is associated with poor prognosis.43,44 in a phase i / ii study , pd-1 blockade with nivolumab showed complete responses in two out of 39 patients ( 5% ) and prs in seven ( 18% ) patients . os rate was 72% at 6 months.45 these initial data support the continued exploration of nivolumab in hcc . the abovementioned 10 year old boy was treated for the second relapse with nivolumab and achieved a significant but temporary clinical response . current studies in adult cancer are ongoing , combining immune checkpoint inhibitors with targeted therapy or chemotherapeutic agents such as anthracyclines or gemcitabine since these can also modulate t - cell proliferation and promote immunogenic cell death.41,46,47 this makes sense as monotherapy with targeted agents demonstrated an improved response rate but a limited time to tumor progression , whereas checkpoint blockade monotherapy seems to have a lower response rate but an extended pfs.48 unfortunately , information about the feasibility or effects of those new agents is not available for childhood hcc . therefore , phase i / ii trials are urgently needed in childhood hcc the indication for liver transplantation in adults is restricted to the milan criteria , ie , the evidence of a single tumor < 5 cm in size or no more than three foci with each not exceeding 3 cm and no vascular invasion or extrahepatic involvement.49 the practice guidelines of the american association for transplantation and the north american society for pediatric gastroeneterology , hepatology , and nutrition recommend that the indication for liver transplantation in childhood hcc must be discussed individually for each patient . in principle , liver transplantation should be considered in children with no extrahepatic tumor or gross vascular invasion on radiological imaging , irrespective of the size of the lesions or the number of the lesions.50 successful transplantations have been done in children with more liberal criteria , even in patients with large , multifocal hcc , microscopic blood vessel involvement , or limited extrahepatic tumor.5153 a meta - analysis showed lower relapse rate and longer efs and os in patients treated with sirolimus compared with calcineurin inhibitor following liver transplantation for hcc.54 to conclude , the milan criteria are not applicable for children with hcc . palliative tace is a standard procedure in adults with solitary or multifocal hcc without extrahepatic metastases . back in 2,000 , malogolowkin et al55 reported that all eleven children ( 18 months14 years old ) with unresectable , chemotherapy - resistant liver tumors ( three with hcc ) responded five ( one with hcc ) went on to surgical resection and three survived . the conclusion was that tace with a suspension of cisplatin , doxorubicin , and mitomycin mixed with lipiodol is feasible , well - tolerated , and effective in achieving surgical resectability in pediatric patients . these encouraging results were confirmed by czauderna et al56 ( five patients , 112 years old , one with hcc ) . thus , tace could be offered to patients with chemotherapy - resistant liver tumor for palliative care or even with the goal of achieving surgical resectability and cure . research has to be done to characterize the molecular and genomic mechanisms of pediatric hcc to support the development of novel therapeutic approaches and the implementation of personalized medicine . at the moment , it would be worth initiating clinical studies to evaluate bevacizumab combined with standard chemotherapy ( plado or gemox with sorafenib ) , c - met inhibitors like cabozantinib in tumors with high met expression , and immune checkpoint blockade agents like nivolumab .

hepatocellular carcinoma ( hcc ) is a very rare entity in children , making it nearly impossible to orchestrate phase ii / iii studies even as multinational cooperative trials . in contrast to adults , nearly 50% of the children have a response ( -fetoprotein decline and/or tumor shrinkage ) to chemotherapeutic agents such as cisplatin and doxorubicin ( plado ) , demonstrating that hcc in childhood can be chemotherapy sensitive . as a result , the main treatment options in pediatric hcc focus on systemic drug therapies and resection as the central therapy . in nonmetastatic patients with complete resection upfront , the 5-year event - free survival and overall survival has reached 80%90% . in almost all reported studies , children received adjuvant chemotherapy ( mostly plado ) , but it has never been proven that postoperative chemotherapy is superior to observation . no data are available for the effects of sorafenib . the 3-year survival is < 20% in children with unresectable hcc independent of the chemotherapy given preoperatively . currently , plado in combination with sorafenib is recommended with the goal of achieving operability status . alternatively , data are promising for the combination of sorafenib with gemcitabine and oxaliplatin . for children with nonresectable and nonmetastastic liver tumors , it has been shown that the milan criteria regarding liver transplantation are not applicable individual decisions have to be made . transarterial chemoembolization could be offered to patients with chemotherapy - resistant liver tumors for palliative care or potentially to achieve surgical resectability , and therefore cure . information about the feasibility or effects of new agents or approaches as discussed in adult hcc patients is not available for childhood hcc . research has to be done for characterizing the molecular and genomic mechanisms of pediatric hcc to support the development of novel therapeutic approaches and the implementation of personalized medicine .

Introduction Standard-of-care with complete resection upfront: observation vs sorafenib vs PLADO vs PLADO and sorafenib? Current therapeutic options in newly diagnosed patients with unresectable tumors and/or metastatic disease New agents in pediatric HCC Liver transplantation across the Milan criteria? TACE in pediatric HCC patients Conclusion

primary malignant liver tumors are rare in childhood with an incidence of about 1.6 cases per million children ( 014 years).1,2 while hepatoblastoma ( hb ) represents 80% of the hepatic - related cancer affecting children predominantly between 6 months and 3 years , hepatocellular carcinoma ( hcc ) is more uncommon , with incidence increasing with age . only about 0.5%1% of all pediatric tumors are hcc.1,2 in hepatoblastoma , event - free survival ( efs ) and overall survival ( os ) was increased from roughly 30% in the 1970s to 70%90% these days , especially due to advances of chemotherapy regimens and surgical approaches.3 in hcc , the results with unresectable tumor especially are rather dismal . the 3-year efs and os for children with complete tumor excision upfront and two courses of carboplatin ( 200 mg / m / d 4 ) and etoposide ( 100 mg / m / d 4 ) were 72% and 89% , respectively , in the hb99 study ( 19992008)11 conducted by the german society for pediatric oncology and hematology ( gpoh ) . there was no difference based on whether the children received cisplatin ( 90 mg / m on d0 ) and doxorubicin ( 20 mg / m / d 4 from d2 ) or cisplatin ( 90 mg / m on d0 ) , 5-fluorouracil ( 600 mg / m on d2 ) , and vincristine ( 1.5 mg / m on d2 ) . importantly , the first international society of pediatric oncology liver tumor study ( siopel-1 study , 19901994)1 demonstrated that hcc in childhood ( 415 years , median : 12 years ) can be chemotherapy sensitive . they proved this by showing that 49% of the children responded to cisplatin ( 80 mg / m on d1 ) and doxorubicin ( 30 mg / m / d on d2 + 3 ) ( plado ) . the next attempt ( siopel-2 study , 19951998)10,13 tried was rapidly switching between cisplatin ( 80 mg / m on d1 ) and carboplatin ( 500 mg / m on d1)/doxorubicin ( 30 mg / m / d on d2 + 3 ) ( superplado ) every 14 days , but this did not improve the response rate after preoperative chemotherapy ( 46% ) , and therefore also not 3-year os ( 22% ) . thus , to date , for children with inoperable liver tumor and/or with metastases , the complete resection ( and so the efs and os ) have not improved although different strategies have been attempted . sorafenib is an inhibitor of several tyrosine protein kinases such as vegfr , pdgfr , and raf kinases.1416 in preclinical models , sorafenib demonstrated antitumor activity alone and in combination with , for instance , doxorubicin , gemcitabine , and cisplatin.17,18 in adult patients with advanced hcc , sorafenib significantly improved both time to tumor progression and os from a median of 2.8 to 5.5 months and from 7.9 to 10.7 months , respectively , compared with placebo . the most important grade 3 adverse effects were diarrhea , hand foot skin reaction , and fatigue.19 therefore , sorafenib has become the standard therapy for adult patients with hcc.20 furthermore , in a randomized , double - blind , phase ii study combining sorafenib with doxorubicin , the progression - free survival ( pfs ) was significantly better in patients receiving sorafenib and doxorubicin than in those receiving doxorubicin and placebo ( median : 4.8 vs 8.6 months).21,22 moreover , tumor reduction was achieved in 62% vs 29% of the patients . this effect could be due to the fact that combining sorafenib with doxorubicin translated into an increased mean values of doxorubicin cmax and area under the curve by 33% and 21% , respectively.23 in the recent study with 12 children ( seven with unresectable liver tumor , age 716 years ) , it was demonstrated that sorafenib ( 244602 mg / m / d , median : 288 mg / m / d ) added to plado is a promising new therapeutic option with hand foot skin reaction being the most relevant toxicity.24 with this combination , four of the seven children with inoperable liver tumor achieved a partial response ( pr ) , two a stable disease , and one a progression . thus , there are big challenges to be solved for pediatric patients with hcc , namely : 1 ) what is the standard - of - care in children with newly diagnosed hcc with complete resection upfront : observation vs sorafenib vs plado vs plado and sorafenib ? in adults , it was recently shown that sorafenib is not effective as an adjuvant treatment following resection or ablation.25 an enhanced chemotherapeutic response to sorafenib and plado was demonstrated in the small series of patients with advanced hcc ( pr in four out of seven ) . the problem is that it is impossible to realize phase ii to iii studies in an entity as rare as hcc in childhood . given preexisting evidence that pediatric hcc is chemotherapy responsive in nearly 50% of the patients ( table 1 ) intensification of platinum and doxorubicin agents , as in the siopel 2 and 3 studies,13 did not result in improved survival . hopefully , sorafenib in addition to plado improves the resectability rate , efs , and os.24 in a recently published multicenter study from france , 204 adults with advanced hcc received gemcitabine ( 1,000 mg / m on d1 ) and oxaliplatin ( 100 mg / m on d2 ) ( gemox ) , with promising response and tumor control rates of 22% and 66% , respectively.26 in 44% of the patients , grade 34 toxicities were reported , especially neutropenia , thrombocytopenia , neurotoxicity , and diarrhea . but still , new effective drugs besides the conventional chemotherapeutic ones ( eg , plado , gemox , and sorafenib ) are definitely needed with the goal to achieve a higher response rate , thus translating into a higher surgical resection rate . since it is strongly believed that hcc in children is a different biologic disease , results from studies in the adult population can not simply be translated to children.7,8 the better response to chemotherapy in pediatric patients may be due to the much higher rate of de novo tumors and normal liver function . in addition , in older children and in young adolescents , an entity called transitional liver cell tumor has been observed , which is made up of chemotherapy - sensitive hepatoblastoma - like cells , cells similar to those of hcc , and intermediate cell forms.29 young people more often present the fibrolamellar histologic variant.30 it was thought that this variant has a more favorable prognosis , but recently it was shown that the long - term os is similar to that for hcc.31 since hccs are highly vascularized tumors with increased levels of vegf , antiangiogenic approaches represent a potential new therapeutic strategy . in adults , but not in children , different antiangiogenic agents besides sorafenib have been tested in clinical studies , eg , sunitinib , brivanib , bevacizumab , and ramucirumab.32 for example , sunitinib was proven to be nonsuperior when randomized with sorafenib and was highly toxic , with side effects including thrombo - cytopenia and neutropenia.33 bevacizumab was the most promising agent , showing an objective response in six of 46 patients ( 13% ) and a pfs rate of 65% at 6 months.34 in combination with erlotinib ( egfr inhibitor ) , a response rate of 25% was reported.35 despite the initial promising results , there were no plans for a phase iii study with bevacizumab . silencing the c - met expression in cell lines and preclinical models was shown to inhibit hcc growth.38 however , only in patients with a high met expression , tivantinib , a selective tyrosine kinase inhibitor , achieved a significantly longer median time to progression ( 1.4 vs 2.7 months ) , pfs ( 1.4 vs 2.2 months ) , and overall survival ( 3.8 vs 7.2 months).39 cabozantinib ( xl184 ) , a dual tyrosine kinase inhibitor with activity against c - met , vegfr2 , and ret , demonstrated a tumor control rate at week 12 of 71% in a randomized phase ii study.40 a 10 year old child was treated with relapsed hcc in complete remission with cabozantinib as maintenance therapy for 12 months . blocking of pd-1 with a specific antibody has been shown to amplify t - cell function and enhance antitumor effects.41 hcc is known to be an inflammation - associated cancer and can therefore be immunogenic.42 upregulation of pd-1 and the pd-1 immune checkpoint ligand ( pd - l1 ) in hcc is associated with poor prognosis.43,44 in a phase i / ii study , pd-1 blockade with nivolumab showed complete responses in two out of 39 patients ( 5% ) and prs in seven ( 18% ) patients . current studies in adult cancer are ongoing , combining immune checkpoint inhibitors with targeted therapy or chemotherapeutic agents such as anthracyclines or gemcitabine since these can also modulate t - cell proliferation and promote immunogenic cell death.41,46,47 this makes sense as monotherapy with targeted agents demonstrated an improved response rate but a limited time to tumor progression , whereas checkpoint blockade monotherapy seems to have a lower response rate but an extended pfs.48 unfortunately , information about the feasibility or effects of those new agents is not available for childhood hcc . the indication for liver transplantation in adults is restricted to the milan criteria , ie , the evidence of a single tumor < 5 cm in size or no more than three foci with each not exceeding 3 cm and no vascular invasion or extrahepatic involvement.49 the practice guidelines of the american association for transplantation and the north american society for pediatric gastroeneterology , hepatology , and nutrition recommend that the indication for liver transplantation in childhood hcc must be discussed individually for each patient . in principle , liver transplantation should be considered in children with no extrahepatic tumor or gross vascular invasion on radiological imaging , irrespective of the size of the lesions or the number of the lesions.50 successful transplantations have been done in children with more liberal criteria , even in patients with large , multifocal hcc , microscopic blood vessel involvement , or limited extrahepatic tumor.5153 a meta - analysis showed lower relapse rate and longer efs and os in patients treated with sirolimus compared with calcineurin inhibitor following liver transplantation for hcc.54 to conclude , the milan criteria are not applicable for children with hcc . back in 2,000 , malogolowkin et al55 reported that all eleven children ( 18 months14 years old ) with unresectable , chemotherapy - resistant liver tumors ( three with hcc ) responded five ( one with hcc ) went on to surgical resection and three survived . thus , tace could be offered to patients with chemotherapy - resistant liver tumor for palliative care or even with the goal of achieving surgical resectability and cure . in adults , it was recently shown that sorafenib is not effective as an adjuvant treatment following resection or ablation.25 an enhanced chemotherapeutic response to sorafenib and plado was demonstrated in the small series of patients with advanced hcc ( pr in four out of seven ) . however , further data regarding sorafenib are urgently needed in the pediatric hcc population.24 the problem is that it is impossible to realize phase ii to iii studies in an entity as rare as hcc in childhood . given preexisting evidence that pediatric hcc is chemotherapy responsive in nearly 50% of the patients ( table 1 ) intensification of platinum and doxorubicin agents , as in the siopel 2 and 3 studies,13 did not result in improved survival . hopefully , sorafenib in addition to plado improves the resectability rate , efs , and os.24 in a recently published multicenter study from france , 204 adults with advanced hcc received gemcitabine ( 1,000 mg / m on d1 ) and oxaliplatin ( 100 mg / m on d2 ) ( gemox ) , with promising response and tumor control rates of 22% and 66% , respectively.26 in 44% of the patients , grade 34 toxicities were reported , especially neutropenia , thrombocytopenia , neurotoxicity , and diarrhea . but still , new effective drugs besides the conventional chemotherapeutic ones ( eg , plado , gemox , and sorafenib ) are definitely needed with the goal to achieve a higher response rate , thus translating into a higher surgical resection rate . since it is strongly believed that hcc in children is a different biologic disease , results from studies in the adult population can not simply be translated to children.7,8 the better response to chemotherapy in pediatric patients may be due to the much higher rate of de novo tumors and normal liver function . in addition , in older children and in young adolescents , an entity called transitional liver cell tumor has been observed , which is made up of chemotherapy - sensitive hepatoblastoma - like cells , cells similar to those of hcc , and intermediate cell forms.29 young people more often present the fibrolamellar histologic variant.30 it was thought that this variant has a more favorable prognosis , but recently it was shown that the long - term os is similar to that for hcc.31 since hccs are highly vascularized tumors with increased levels of vegf , antiangiogenic approaches represent a potential new therapeutic strategy . in adults , but not in children , different antiangiogenic agents besides sorafenib have been tested in clinical studies , eg , sunitinib , brivanib , bevacizumab , and ramucirumab.32 for example , sunitinib was proven to be nonsuperior when randomized with sorafenib and was highly toxic , with side effects including thrombo - cytopenia and neutropenia.33 bevacizumab was the most promising agent , showing an objective response in six of 46 patients ( 13% ) and a pfs rate of 65% at 6 months.34 in combination with erlotinib ( egfr inhibitor ) , a response rate of 25% was reported.35 despite the initial promising results , there were no plans for a phase iii study with bevacizumab . silencing the c - met expression in cell lines and preclinical models was shown to inhibit hcc growth.38 however , only in patients with a high met expression , tivantinib , a selective tyrosine kinase inhibitor , achieved a significantly longer median time to progression ( 1.4 vs 2.7 months ) , pfs ( 1.4 vs 2.2 months ) , and overall survival ( 3.8 vs 7.2 months).39 cabozantinib ( xl184 ) , a dual tyrosine kinase inhibitor with activity against c - met , vegfr2 , and ret , demonstrated a tumor control rate at week 12 of 71% in a randomized phase ii study.40 a 10 year old child was treated with relapsed hcc in complete remission with cabozantinib as maintenance therapy for 12 months . blocking of pd-1 with a specific antibody has been shown to amplify t - cell function and enhance antitumor effects.41 hcc is known to be an inflammation - associated cancer and can therefore be immunogenic.42 upregulation of pd-1 and the pd-1 immune checkpoint ligand ( pd - l1 ) in hcc is associated with poor prognosis.43,44 in a phase i / ii study , pd-1 blockade with nivolumab showed complete responses in two out of 39 patients ( 5% ) and prs in seven ( 18% ) patients . current studies in adult cancer are ongoing , combining immune checkpoint inhibitors with targeted therapy or chemotherapeutic agents such as anthracyclines or gemcitabine since these can also modulate t - cell proliferation and promote immunogenic cell death.41,46,47 this makes sense as monotherapy with targeted agents demonstrated an improved response rate but a limited time to tumor progression , whereas checkpoint blockade monotherapy seems to have a lower response rate but an extended pfs.48 unfortunately , information about the feasibility or effects of those new agents is not available for childhood hcc . therefore , phase i / ii trials are urgently needed in childhood hcc the indication for liver transplantation in adults is restricted to the milan criteria , ie , the evidence of a single tumor < 5 cm in size or no more than three foci with each not exceeding 3 cm and no vascular invasion or extrahepatic involvement.49 the practice guidelines of the american association for transplantation and the north american society for pediatric gastroeneterology , hepatology , and nutrition recommend that the indication for liver transplantation in childhood hcc must be discussed individually for each patient . in principle , liver transplantation should be considered in children with no extrahepatic tumor or gross vascular invasion on radiological imaging , irrespective of the size of the lesions or the number of the lesions.50 successful transplantations have been done in children with more liberal criteria , even in patients with large , multifocal hcc , microscopic blood vessel involvement , or limited extrahepatic tumor.5153 a meta - analysis showed lower relapse rate and longer efs and os in patients treated with sirolimus compared with calcineurin inhibitor following liver transplantation for hcc.54 to conclude , the milan criteria are not applicable for children with hcc . back in 2,000 , malogolowkin et al55 reported that all eleven children ( 18 months14 years old ) with unresectable , chemotherapy - resistant liver tumors ( three with hcc ) responded five ( one with hcc ) went on to surgical resection and three survived . thus , tace could be offered to patients with chemotherapy - resistant liver tumor for palliative care or even with the goal of achieving surgical resectability and cure . research has to be done to characterize the molecular and genomic mechanisms of pediatric hcc to support the development of novel therapeutic approaches and the implementation of personalized medicine . at the moment , it would be worth initiating clinical studies to evaluate bevacizumab combined with standard chemotherapy ( plado or gemox with sorafenib ) , c - met inhibitors like cabozantinib in tumors with high met expression , and immune checkpoint blockade agents like nivolumab .

the circadian clock is an autoregulatory network that regulates behavioral and metabolic programing in the context of a 24 h light - dark ( ld ) cycle . body temperature is one of the representing benchmarks of circadian patterning , which peaks in animals while awake and troughs while asleep . brown adipose tissue ( bat ) is a major site for rodent thermogenesis , due to its involvement in controlling circadian thermogenic rhythms and influencing adaptability to environmental temperature challenges . a previous study has revealed rhythmic expression patterns of over 5,000 genes in murine bat , including genes associated with the circadian clock , adipose function , and metabolism . moreover , the inhibitor of dna binding 2 ( id2 ) gene encodes a helix - loop - helix ( hlh ) transcriptional regulator , which is rhythmically expressed in many mammalian tissues and involved in the input pathway , core clock function , and output pathways of the circadian clock [ 69 ] . our previous studies have shown that id2/ mice exhibit lower levels of locomotor activity , extended nighttime activity patterns of feeding and locomotor activity , and sex- and age - dependent enhanced glucose tolerance and insulin sensitivity . moreover , an energy - rich diet is able to rescue the disturbances to metabolic homeostasis and survival in the id2/ mice sex - specifically . importantly , id2/ mice show a sex - dependent elevated glucose uptake in interscapular bat ( ibat ) . id2 also plays a role in white adipose tissue ( wat ) adipogenesis [ 1012 ] . however , the role of id2 on temperature homeostasis regulation and its influence on bat physiology remain unknown . therefore , we investigated the function of id2 in the regulation of temperature rhythms under normal and thermoneutral conditions in a sex - specific manner and also profiled the expression of genes involved in insulin signaling and adipogenesis in bat of id2/ mice , sex - specifically . the generation of id2/ mice and genotype determination were performed as described previously [ 7 , 10 , 11 ] . mice were maintained on a regular chow diet and sterile water containing antibiotic ad libitum [ 7 , 10 , 11 ] . all mice were housed in laboratory cages at normal temperature ( 21c 1c ) and humidity of 5065% under a 12 : 12 light : dark ( ld ) cycle with lights on at zeitgeber time ( zt ) 0 and lights off at zt12 . animal experiments were approved by the university of notre dame animal care and use committee ( protocol number 14 - 02 - 1559 ) and performed in accordance with nih guidelines for the care and use of laboratory animals . temperature measurements were carried out on 2-month1.5-year- ( 5.5-month median ) old male and female id2/ mice and wt littermates , housed individually in a climate - controlled room set to either normal ( 21c 1c ) or thermoneutral ( 30c 1c ) temperature . body temperature sampling was conducted at 3 h intervals over the 24 h ld cycle . for thermoneutral conditions measurement , all wt and id2/ mice used in the studies were allowed to acclimate to thermoneutral temperature for 1 week before temperature measurement . core body temperature was measured using subcutaneously surgically implanted telemetric transmitters positioned proximal to the ibat ( iptt 300 transponders , bio medic data systems , seaford , de ) following isoflurane anesthetization . after a week of recuperation , ibat tissue was harvested at zt16 ( id2 mrna circadian rhythm in ibat has a broad peak phase between zt8 and zt16 ) . id2/ and wt male ( wt = 8 , id2/ = 6 ) and female ( wt = 6 , id2/ = 4 ) mice from 39 months ( 6.1-month median ) were sacrificed and ibat tissue was frozen in liquid nitrogen and stored at 80c until analyzed . we also measured ibat weight of these and additional mice ( 310-month - old id2/ mice and wt littermates ; 6.3-month median ; male , wt = 15 , id2/ = 7 ; female , wt = 10 , id2/ = 9 ) as described previously [ 10 , 11 ] . rna integrity was assessed using a bioanalyzer 2100 ( agilent technologies , santa clara , ca ) . rna was subjected to a dnase i treatment , and cdna was synthesized by rt first strand kit ( sabiosciences ) . relative mrna expression of 168 genes involved in insulin signaling and adipogenesis pathways was determined by using the mouse pcr arrays ( pamm-030zc-24 and pamm-049zc-24 , sabiosciences ) . quantitative real - time pcr was performed using an applied biosystems 7500 system with rt2 sybr green rox qpcr master mix reagent ( qiagen ) . pcr array data were calculated by the comparative cycle threshold method and analyzed by web - based free pcr array data analysis software provided by sabiosciences . normalization of expression was to housekeeping genes provided on each array ( actb , b2 m , gapdh , gusb , and hsp90ab1 ) . clock controlled genes ( ccgs ) were identified from the circa database of mouse 1.ost brown adipose ( affymetrix ) ( http://bioinf.itmat.upenn.edu/circa/ ) where we defined ccgs as a jtk_cycle algorithm determined q < 0.1 value and a period length of 2028 h as described previously [ 1315 ] . the linear regression of ibat temperature - body weight relationship was generated and analyzed using prism 5.0 graphpad software . pcr array data were analyzed using the web - based free pcr array data analysis software provided by sabiosciences ( student 's t - test ) . the discovery of a diurnal rhythm of glucose uptake in mice ibat and a sex - dependent elevated glucose uptake in ibat of id2/ mice prompted us to investigate whether id2 contributes to thermoregulation [ 5 , 10 ] . at normal ambient temperature conditions ( 21c ) , ablation of id2 reduced core body temperature across the 24 h day , in both male and female mice ( figure 1(a ) ) ( males , wild types ( wts ) = 14 , id2/ = 14 , anova , time ( t ) , p < 0.001 , genotype ( g ) p < 0.001 , interaction ( i ) , n.s . ; females , wts = 18 , id2/ = 17 , anova , t , p < 0.001 , g , p < 0.05 , i , n.s . ) . considering the possibility of any confounding genetic background contribution and partial stimulation of bat activity occurring under normal temperature conditions , id2/ mice core body temperature was also measured under thermoneutral conditions ( 30c ) [ 3 , 16 ] . consistently , at thermoneutrality , id2/ mice displayed a reduced core body temperature ( figure 1(a ) ) ( males , wts = 19 , id2/ = 20 , anova , t , p < 0.001 , g , p < 0.01 , i , n.s . ; females , wts = 18 , id2/ = 17 , anova , t , p < 0.001 , g , p < 0.01 , i , n.s . ) . under both conditions and in both sexes , no interaction between time and genotype was discovered , suggesting a generalized effect of the null mutation on core body temperature rather than a time - of - day specific contribution of the gene deletion . regression analysis of time - of - day representative core body temperatures ( day or night ) revealed no significant relationships between temperature and body mass for either id2/ or wt mice . however , id2/ mice of both sexes showed consistently lower y - intercept lines compared to wt mice when examined during either the daytime ( zt5.5 ) or nighttime ( zt17.5 or zt20.5 ) , thus confirming the consistently lower temperature of the id2 null mice ( figure 1(b ) ; supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/6785948 ) . lastly , id2/ mice exhibited no statistically significant difference in ibat weight and ibat to body weight ratio compared to wt controls ( figures 2(a ) and 2(b ) ) ( two - factor anovas , ibat weight , g , n.s . , sex ( s ) , p < 0.01 , i , n.s . ; ibat / body weight ratio , g , n.s . , s , n.s . the mean and sem body mass of wt and id2/ mice for both ibat weight and body temperature experiments are shown in supplementary table 2 : note that both male and female id2/ mice had on average a lower body mass compared to wt counterparts ( two - factor anova , g , p < 0.001 , s , p < 0.001 , i , n.s . ) . our previous results showed sex - dependent enhanced insulin sensitivity and glucose uptake in ibat of id2/ mice . in the current study we observed a decreased core body temperature in id2/ mice as described above . to fully evaluate the impact of ablation of id2 on bat gene - regulation , we performed a gene expression analysis using rt profiler pcr arrays of bat derived from id2/ mice and their wt littermates collected at the same time of the 24 h day ( specifically zt16 ) . deferentially regulated genes involved in insulin signaling and adipogenesis are shown in tables 1 and 2 , respectively . thirty of 168 genes examined were identified as differentially expressed when analyzed as a cohort or as individual sex - specific groups . using the circa database as a resource , six genes were identified as clock controlled genes ( ccgs ) , of which four oscillate in proximal phase with the rhythm of peroxisome proliferator activated receptor alpha ( ppar ) , peaking during the middle of the day ( ~circadian time ( ct ) 6 ; ct12 = onset of night in prior ld cycle ) . of importance for insulin signaling , glucose-6-phosphatase , catalytic ( g6pc ) , was upregulated in id2/ females and the related g6pc family member g6pc2 downregulated in id2/ males ( p = 0.079 , approaching significance ) compared to wts . insulin receptor substrate 1 ( irs1 ) was upregulated in both male and female id2/ mice . insulin - like growth factor 2 ( igf2 ) was downregulated in female id2/ mice , while fbp1 , a rate - limiting enzyme in gluconeogenesis , and shc1 , a component in the igf-1-regulated pathway , were upregulated . for adipogenesis , bone morphogenetic protein 4 ( bmp4 ) was elevated 1.7-fold ( n.s . ) in male and 1.6-fold in female id2/ mice . nuclear receptor coactivator 2 ( ncoa2 ) , pr domain containing 16 ( prdm16 ) , ppar , and twist homolog 1 ( twist1 ) were downregulated , in grouped analysis of male and female id2/ mice . fatty acid synthase ( fasn ) , lipase , hormone sensitive ( lipe ) , and peroxisome proliferative activated receptor , gamma , coactivator 1 beta ( ppargc1/pgc-1 ) were all downregulated in male id2/ mice . female id2/ mice displayed a downregulation of proliferative activated receptor , gamma , coactivator 1 alpha ( ppargc1/pgc-1 ) . a small 1.2-fold downregulation of peroxisome proliferator activated receptor gamma ( ppar ) was detected in id2/ males , where the p value was approaching significance ( p = 0.061 ) . note that the thermogenic protein , uncoupling protein 1 ( ucp1 ) , was present on both the insulin signaling and adipogenesis arrays , but its levels of expression were not significantly altered in the id2/ mice . in the present study , we discovered a reduced core body temperature in id2/ mice , and this effect was not found to be dependent upon the time - of - day . moreover , from the ibat of id2/ mice , genes involved in insulin signaling and adipogenesis were differentially regulated in a sex - dependent manner . these results reveal a role of id2 in the regulation of thermogenesis and bat metabolic functions . our previous study revealed that id2/ mice exhibit less activity as demonstrated by daily counts of general activity and the wheel running activity , which could partially explain the reduced core body temperature , since less physical activity would generate less heat . moreover , id2/ mice show a reduced body mass and less gonadal adipose deposits [ 6 , 10 , 11 ] . as the subcutaneous and intradermal fat functions as thermal insulation for mice to preserve heat loss , id2/ mice with low fat content might tend to lose heat more readily than wt mice . furthermore , the reduced body temperature associated with lower fat content might contribute to the high death rate observed previously ( mice housed under normal temperature ) , which was rescued by a high fat diet that resulted in increased total body fat . specifically in male id2/ mice ibat we observed increased glucose uptake and reduced tg accumulation , suggesting alterations in its metabolic programing . interestingly , our results suggest that the role of id2 in thermoregulation is opposite to the function of another member of this hlh family , id1 , whose deficiency results in higher thermogenesis and an elevated bat expression of thermogenic proteins . notably , id1 has a distinct and opposite function in wat adipogenesis compared to id2 , despite both id1 and id2 null mice exhibiting reduced adiposity [ 1012 , 17 ] . lastly , we examined the relationship between body mass and body temperature in id2/ mice by regression analysis and revealed a limited relationship between the two variables . no significant relationship was observed between body mass and body temperature at any time of the day or in the two sexes . however , as can be seen with the y - intercept of the regression lines , both id2/ male and female mice expressed a consistently lower temperature compared to wt controls , irrespective of body mass , and this feature was observed during both the day and night phases of the ld cycle . these results suggest a role for id2 in the regulation of core body temperature . in this study we also measured ibat mass and ibat / body mass ratio . while there was a tendency for higher ibat / body mass in both id2/ male and female mice , this was not determined to be a significant difference . note that the average body mass of id2/ mice used for both the ibat weight and body temperature experiments was found to be significantly lower , consistent with our previous studies [ 7 , 10 ] . important is the fact that a lower body mass , found for some of the id2/ mice and for males in particular , does not correlate with a lower body temperature , and body mass in this situation is therefore an independent factor when predicting core body temperature . it is important to note that while the objective of examining body temperature using the implanted thermometers was to record core body temperature , the position of the implants may not give an exact measure of true core body temperature . however , in a comparable study of mouse body temperatures , temperature measurements were similar whether derived from similarly subcutaneously implanted thermometers in the interscapular region of wt and rev - erb mutant mice or as determined using dataloggers that were implanted within the abdomen . id2 is rhythmically expressed in bat [ 4 , 15 ] amongst other tissues [ 6 , 7 ] . id2 protein has also been observed to be rhythmic in its abundance over the 24 hr diurnal / circadian cycle within the liver and heart ( ward , fernando , hou , and duffield , unpublished data ) . a role for id2 has been established as a mediator of circadian clock output and control of expression patterns of clock controlled genes ( ccgs ) within the liver . it is for this reason that we examined whether any of the genes identified as differentially expressed in ibat were in fact known ccgs . using the circa database [ 14 , 15 ] , 5 of the 17 differentially genes associated with adipogenesis were found to be ccgs ( e.g. , ppar and pgc-1 ) , and so a possible role for id2 is in mediating circadian regulatory effects on these genes within bat . the observation that few of the differentially regulated genes involved in insulin signaling are ccgs ( 1 out of 13 genes ) suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output . in order to explain how id2 deficiency has an impact on bat insulin signaling and adipogenesis the nuclear receptor ppars are fundamentally important for energy homeostasis and id2 plays a role in interfacing with the molecular pathways upstream or downstream of these transcriptional factors . expression of two members of the ppar subfamily of ligand - activated nuclear receptors , ppar and ppar , was downregulated in our study . ppar is highly expressed in bat and considered a marker of bat ; it also plays an important role in the overall regulation of lipid metabolism ; and its target genes are involved in mitochondrial and peroxisomal -oxidation of fatty acids ( fas ) [ 2022 ] . moreover , ppar regulates the expression of uncoupling protein 1 ( ucp1 ) , which confers on bat its thermogenic capacity . pgc-1 ( downregulated in our study ) is a transcriptional coactivator involved in the control of energy metabolism and critical for bat thermogenesis and enhancing overall mitochondrial oxidative activity . ppar can induce pgc-1 gene expression and contributes to the thermogenic activation of brown fat . prdm16 exhibits a brown fat selective expression pattern and regulates the thermogenic gene program in brown and beige adipocytes . the observation of reduced prdm16 expression in id2/ mice is consistent with the role of prdm16 as a transcriptional regulator of pgc-1 . ppar is essential for adipocyte differentiation , and ppar alone generates a fat phenotype that is common to both wat and bat . the ccaat enhancer binding protein beta ( c / ebp ) and pgc-1 are critical for controlling ppar expression in bat and for determining bat - specific programs [ 29 , 30 ] . the ppar thermogenic effect in bat it has been observed that overexpression of id2 associates with ppar expression , id2 acts upstream of ppar , and c / ebp induces id2 expression during the adipogenesis process [ 12 , 31 ] . cofactors such as ncoa2 ( downregulated in our study ) can interact directly with ppar to initiate its own transactivation . moreover , lipe ( downregulated in our study ) could modulate adipose metabolism by reducing the availability of ligands for ppar , since gene knockout of lipe in mice attenuates activation of ppar . lipe is also able to hydrolyze stored tgs in adipose tissue and to mobilize free fa from adipose tissue . furthermore , ppar is a direct target of the transcription factor sterol response element binding protein 1 ( srebp1 ) , whose transcriptional activity is modulated by id2 and which regulates downstream lipid metabolism genes such as lipe and fasn [ 35 , 36 ] . additionally , irs1 ( upregulated in our study ) plays essential roles in the differentiation of brown adipocytes and expression of ppar [ 37 , 38 ] . previous studies have revealed irs1-regulated id2 gene expression , although in the current study it is unclear whether this is a direct effect or a feedback response . as for the mechanism by which irs1 is elevated in id2/ ibat , bmp4 ( upregulated in our study ) is able to induce the white to brown transition of adipose cells , which could indirectly regulate ppar activation [ 40 , 41 ] . the elevated bmp4 expression in the context of reduced ppars is surprising since bmp4 upregulation is associated with increased bat adipogenesis and the wat browning effect . interestingly , the id2 gene promoter has bmp - response elements and has been shown to be a target of bmp signaling . ppar plays an integral role in transcriptional network regulation of fat - burning genes and brown fat metabolism . twist-1 ( downregulation in our study ) encodes a basic hlh transcription factor , and overexpression of twist-1 is associated with id2 expression . note that the gene encoding the thermogenic protein , ucp1 , was present on the pcr arrays , but no difference in its expression was observed between genotypes of either sex . interestingly , in the id1 null mouse , ucp1 gene expression is elevated in ibat , and this is associated with an increased core body temperature phenotype . thus , it is surprising that in the id2/ mouse that exhibits a reduced body temperature phenotype we do not observe a reduction in ucp1 gene expression . of course , this does not exclude , however unlikely , the possibility of an altered ucp1 protein abundance through a posttranscriptional / posttranslational process . hypoxia , while not a focus of the current study , is known to reduce body temperature in mammals and contribute to the thermogenic activity of bat . it is noteworthy that in a recent study of human glioblastoma cells / tissue , an important role was established for id2 in modulating the cellular effects of hypoxia and its activation of the hif2 pathway . the id2 gene is also a target for hif1 and hif2 [ 47 , 48 ] , making it part of a positive feedback loop mechanism , at least in models of brain tumor . it is plausible that the hypoxic effects on bat function might also include a contribution from id2 , and this would be an important pathway to examine in future experiments in this context . it is a somewhat contradictory finding that elements of the thermogenic pathway are reduced ( e.g. , pgc1- ) or unaltered ( ucp1 ) in id2/ mice but that id2/ male ibat exhibits increased glucose uptake ( pet - fdg ) coupled with reduced ibat triglyceride levels and a systemwide enhanced insulin sensitivity [ 10 , 11 ] and that core body temperature is consistently reduced in both id2/ male and female mice . id2/ mice also exhibit an altered 24 hr locomotor activity and feeding profile and an overall reduction in nocturnal locomotor activity , the latter of which suggests a reduction in energy expenditure / increased energy conservation . clearly the bat adipogenic program is altered in both male and female id2/ mice , as is wat adipogenesis [ 1012 ] , and it is likely that a change in bat function contributes to the reduced temperature phenotype . due to the nature of whole body knockout of id2 , it is possible that the temperature phenotype and ibat gene changes observed are secondary to whole body metabolic changes . clearly additional experiments are required to elucidate the relative contributions of these and other potential components in generating the altered core body temperature phenotype . our previous study showed enhanced glucose uptake in ibat of id2/ mice , and the present study reveals a reduced core body temperature in id2/ mice . this discrepancy could partially be explained by the differential regulation of irs1 , lipe , ppars , and pgc-1s . it has been reported that degradation of irs1 leads to impaired glucose uptake in adipose tissue . therefore , upregulation of irs1 might explain the increased glucose uptake we observed before , whereas downregulation of lipe , ppars , and pgc-1s might contribute to reduced fa oxidation , impaired adipogenesis , and a lower body temperature . inactivation of ppars is associated with insulin resistance [ 50 , 51 ] , yet paradoxically id2/ mice show enhanced insulin sensitivity with downregulated ppars [ 10 , 11 ] . it has been suggested that mice that lack one allele of the ppar gene are more sensitive to insulin , which could partially explain the enhanced insulin sensitivity we observe in id2 null mice [ 10 , 51 ] . furthermore , the differential regulation of genes specifically in female mutant mice , such as fbp1 , a rate - limiting enzyme in gluconeogenesis , shc1 , a component in the igf-1-regulated pathway , and igf2 , suggests a sex - specific physiological program for id2 in bat . inhibitor of dna binding 2 is rhythmically expressed in bat [ 4 , 15 ] , and the observation that few of the differentially regulated genes involved in insulin signaling are ccgs suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output . overall , id2 seems to be an important coordinator of energy homeostasis including insulin signaling , adipogenic programing , and thermoregulation . in conclusion , our finding that id2 contributes to the regulation of body temperature and energy homeostasis presents the possibility that id2 could be a potential therapeutic target for metabolic disease . further , these data emphasize the influence of id2 on bat molecular signaling and physiology in a sex - specific manner .

inhibitor of dna binding 2 ( id2 ) is a helix - loop - helix transcriptional repressor rhythmically expressed in many adult tissues . our previous studies have demonstrated that id2 null mice have sex - specific elevated glucose uptake in brown adipose tissue ( bat ) . here we further explored the role of id2 in the regulation of core body temperature over the circadian cycle and the impact of id2 deficiency on genes involved in insulin signaling and adipogenesis in bat . we discovered a reduced core body temperature in id2/ mice . moreover , in id2/ bat , 30 genes including irs1 , ppars , and pgc-1s were identified as differentially expressed in a sex - specific pattern . these data provide valuable insights into the impact of id2 deficiency on energy homeostasis of mice in a sex - specific manner .

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

the circadian clock is an autoregulatory network that regulates behavioral and metabolic programing in the context of a 24 h light - dark ( ld ) cycle . body temperature is one of the representing benchmarks of circadian patterning , which peaks in animals while awake and troughs while asleep . brown adipose tissue ( bat ) is a major site for rodent thermogenesis , due to its involvement in controlling circadian thermogenic rhythms and influencing adaptability to environmental temperature challenges . a previous study has revealed rhythmic expression patterns of over 5,000 genes in murine bat , including genes associated with the circadian clock , adipose function , and metabolism . moreover , the inhibitor of dna binding 2 ( id2 ) gene encodes a helix - loop - helix ( hlh ) transcriptional regulator , which is rhythmically expressed in many mammalian tissues and involved in the input pathway , core clock function , and output pathways of the circadian clock [ 69 ] . our previous studies have shown that id2/ mice exhibit lower levels of locomotor activity , extended nighttime activity patterns of feeding and locomotor activity , and sex- and age - dependent enhanced glucose tolerance and insulin sensitivity . moreover , an energy - rich diet is able to rescue the disturbances to metabolic homeostasis and survival in the id2/ mice sex - specifically . importantly , id2/ mice show a sex - dependent elevated glucose uptake in interscapular bat ( ibat ) . id2 also plays a role in white adipose tissue ( wat ) adipogenesis [ 1012 ] . however , the role of id2 on temperature homeostasis regulation and its influence on bat physiology remain unknown . therefore , we investigated the function of id2 in the regulation of temperature rhythms under normal and thermoneutral conditions in a sex - specific manner and also profiled the expression of genes involved in insulin signaling and adipogenesis in bat of id2/ mice , sex - specifically . the generation of id2/ mice and genotype determination were performed as described previously [ 7 , 10 , 11 ] . temperature measurements were carried out on 2-month1.5-year- ( 5.5-month median ) old male and female id2/ mice and wt littermates , housed individually in a climate - controlled room set to either normal ( 21c 1c ) or thermoneutral ( 30c 1c ) temperature . body temperature sampling was conducted at 3 h intervals over the 24 h ld cycle . for thermoneutral conditions measurement , all wt and id2/ mice used in the studies were allowed to acclimate to thermoneutral temperature for 1 week before temperature measurement . core body temperature was measured using subcutaneously surgically implanted telemetric transmitters positioned proximal to the ibat ( iptt 300 transponders , bio medic data systems , seaford , de ) following isoflurane anesthetization . we also measured ibat weight of these and additional mice ( 310-month - old id2/ mice and wt littermates ; 6.3-month median ; male , wt = 15 , id2/ = 7 ; female , wt = 10 , id2/ = 9 ) as described previously [ 10 , 11 ] . relative mrna expression of 168 genes involved in insulin signaling and adipogenesis pathways was determined by using the mouse pcr arrays ( pamm-030zc-24 and pamm-049zc-24 , sabiosciences ) . clock controlled genes ( ccgs ) were identified from the circa database of mouse 1.ost brown adipose ( affymetrix ) ( http://bioinf.itmat.upenn.edu/circa/ ) where we defined ccgs as a jtk_cycle algorithm determined q < 0.1 value and a period length of 2028 h as described previously [ 1315 ] . the discovery of a diurnal rhythm of glucose uptake in mice ibat and a sex - dependent elevated glucose uptake in ibat of id2/ mice prompted us to investigate whether id2 contributes to thermoregulation [ 5 , 10 ] . at normal ambient temperature conditions ( 21c ) , ablation of id2 reduced core body temperature across the 24 h day , in both male and female mice ( figure 1(a ) ) ( males , wild types ( wts ) = 14 , id2/ = 14 , anova , time ( t ) , p < 0.001 , genotype ( g ) p < 0.001 , interaction ( i ) , n.s . considering the possibility of any confounding genetic background contribution and partial stimulation of bat activity occurring under normal temperature conditions , id2/ mice core body temperature was also measured under thermoneutral conditions ( 30c ) [ 3 , 16 ] . consistently , at thermoneutrality , id2/ mice displayed a reduced core body temperature ( figure 1(a ) ) ( males , wts = 19 , id2/ = 20 , anova , t , p < 0.001 , g , p < 0.01 , i , n.s . under both conditions and in both sexes , no interaction between time and genotype was discovered , suggesting a generalized effect of the null mutation on core body temperature rather than a time - of - day specific contribution of the gene deletion . however , id2/ mice of both sexes showed consistently lower y - intercept lines compared to wt mice when examined during either the daytime ( zt5.5 ) or nighttime ( zt17.5 or zt20.5 ) , thus confirming the consistently lower temperature of the id2 null mice ( figure 1(b ) ; supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/6785948 ) . lastly , id2/ mice exhibited no statistically significant difference in ibat weight and ibat to body weight ratio compared to wt controls ( figures 2(a ) and 2(b ) ) ( two - factor anovas , ibat weight , g , n.s . the mean and sem body mass of wt and id2/ mice for both ibat weight and body temperature experiments are shown in supplementary table 2 : note that both male and female id2/ mice had on average a lower body mass compared to wt counterparts ( two - factor anova , g , p < 0.001 , s , p < 0.001 , i , n.s . ) our previous results showed sex - dependent enhanced insulin sensitivity and glucose uptake in ibat of id2/ mice . in the current study we observed a decreased core body temperature in id2/ mice as described above . to fully evaluate the impact of ablation of id2 on bat gene - regulation , we performed a gene expression analysis using rt profiler pcr arrays of bat derived from id2/ mice and their wt littermates collected at the same time of the 24 h day ( specifically zt16 ) . deferentially regulated genes involved in insulin signaling and adipogenesis are shown in tables 1 and 2 , respectively . thirty of 168 genes examined were identified as differentially expressed when analyzed as a cohort or as individual sex - specific groups . using the circa database as a resource , six genes were identified as clock controlled genes ( ccgs ) , of which four oscillate in proximal phase with the rhythm of peroxisome proliferator activated receptor alpha ( ppar ) , peaking during the middle of the day ( ~circadian time ( ct ) 6 ; ct12 = onset of night in prior ld cycle ) . of importance for insulin signaling , glucose-6-phosphatase , catalytic ( g6pc ) , was upregulated in id2/ females and the related g6pc family member g6pc2 downregulated in id2/ males ( p = 0.079 , approaching significance ) compared to wts . insulin - like growth factor 2 ( igf2 ) was downregulated in female id2/ mice , while fbp1 , a rate - limiting enzyme in gluconeogenesis , and shc1 , a component in the igf-1-regulated pathway , were upregulated . in male and 1.6-fold in female id2/ mice . nuclear receptor coactivator 2 ( ncoa2 ) , pr domain containing 16 ( prdm16 ) , ppar , and twist homolog 1 ( twist1 ) were downregulated , in grouped analysis of male and female id2/ mice . fatty acid synthase ( fasn ) , lipase , hormone sensitive ( lipe ) , and peroxisome proliferative activated receptor , gamma , coactivator 1 beta ( ppargc1/pgc-1 ) were all downregulated in male id2/ mice . female id2/ mice displayed a downregulation of proliferative activated receptor , gamma , coactivator 1 alpha ( ppargc1/pgc-1 ) . a small 1.2-fold downregulation of peroxisome proliferator activated receptor gamma ( ppar ) was detected in id2/ males , where the p value was approaching significance ( p = 0.061 ) . note that the thermogenic protein , uncoupling protein 1 ( ucp1 ) , was present on both the insulin signaling and adipogenesis arrays , but its levels of expression were not significantly altered in the id2/ mice . in the present study , we discovered a reduced core body temperature in id2/ mice , and this effect was not found to be dependent upon the time - of - day . moreover , from the ibat of id2/ mice , genes involved in insulin signaling and adipogenesis were differentially regulated in a sex - dependent manner . these results reveal a role of id2 in the regulation of thermogenesis and bat metabolic functions . our previous study revealed that id2/ mice exhibit less activity as demonstrated by daily counts of general activity and the wheel running activity , which could partially explain the reduced core body temperature , since less physical activity would generate less heat . moreover , id2/ mice show a reduced body mass and less gonadal adipose deposits [ 6 , 10 , 11 ] . furthermore , the reduced body temperature associated with lower fat content might contribute to the high death rate observed previously ( mice housed under normal temperature ) , which was rescued by a high fat diet that resulted in increased total body fat . specifically in male id2/ mice ibat we observed increased glucose uptake and reduced tg accumulation , suggesting alterations in its metabolic programing . interestingly , our results suggest that the role of id2 in thermoregulation is opposite to the function of another member of this hlh family , id1 , whose deficiency results in higher thermogenesis and an elevated bat expression of thermogenic proteins . notably , id1 has a distinct and opposite function in wat adipogenesis compared to id2 , despite both id1 and id2 null mice exhibiting reduced adiposity [ 1012 , 17 ] . lastly , we examined the relationship between body mass and body temperature in id2/ mice by regression analysis and revealed a limited relationship between the two variables . no significant relationship was observed between body mass and body temperature at any time of the day or in the two sexes . however , as can be seen with the y - intercept of the regression lines , both id2/ male and female mice expressed a consistently lower temperature compared to wt controls , irrespective of body mass , and this feature was observed during both the day and night phases of the ld cycle . these results suggest a role for id2 in the regulation of core body temperature . note that the average body mass of id2/ mice used for both the ibat weight and body temperature experiments was found to be significantly lower , consistent with our previous studies [ 7 , 10 ] . important is the fact that a lower body mass , found for some of the id2/ mice and for males in particular , does not correlate with a lower body temperature , and body mass in this situation is therefore an independent factor when predicting core body temperature . it is important to note that while the objective of examining body temperature using the implanted thermometers was to record core body temperature , the position of the implants may not give an exact measure of true core body temperature . however , in a comparable study of mouse body temperatures , temperature measurements were similar whether derived from similarly subcutaneously implanted thermometers in the interscapular region of wt and rev - erb mutant mice or as determined using dataloggers that were implanted within the abdomen . id2 is rhythmically expressed in bat [ 4 , 15 ] amongst other tissues [ 6 , 7 ] . id2 protein has also been observed to be rhythmic in its abundance over the 24 hr diurnal / circadian cycle within the liver and heart ( ward , fernando , hou , and duffield , unpublished data ) . it is for this reason that we examined whether any of the genes identified as differentially expressed in ibat were in fact known ccgs . the observation that few of the differentially regulated genes involved in insulin signaling are ccgs ( 1 out of 13 genes ) suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output . in order to explain how id2 deficiency has an impact on bat insulin signaling and adipogenesis the nuclear receptor ppars are fundamentally important for energy homeostasis and id2 plays a role in interfacing with the molecular pathways upstream or downstream of these transcriptional factors . ppar is highly expressed in bat and considered a marker of bat ; it also plays an important role in the overall regulation of lipid metabolism ; and its target genes are involved in mitochondrial and peroxisomal -oxidation of fatty acids ( fas ) [ 2022 ] . moreover , ppar regulates the expression of uncoupling protein 1 ( ucp1 ) , which confers on bat its thermogenic capacity . pgc-1 ( downregulated in our study ) is a transcriptional coactivator involved in the control of energy metabolism and critical for bat thermogenesis and enhancing overall mitochondrial oxidative activity . the observation of reduced prdm16 expression in id2/ mice is consistent with the role of prdm16 as a transcriptional regulator of pgc-1 . the ccaat enhancer binding protein beta ( c / ebp ) and pgc-1 are critical for controlling ppar expression in bat and for determining bat - specific programs [ 29 , 30 ] . the ppar thermogenic effect in bat it has been observed that overexpression of id2 associates with ppar expression , id2 acts upstream of ppar , and c / ebp induces id2 expression during the adipogenesis process [ 12 , 31 ] . moreover , lipe ( downregulated in our study ) could modulate adipose metabolism by reducing the availability of ligands for ppar , since gene knockout of lipe in mice attenuates activation of ppar . lipe is also able to hydrolyze stored tgs in adipose tissue and to mobilize free fa from adipose tissue . furthermore , ppar is a direct target of the transcription factor sterol response element binding protein 1 ( srebp1 ) , whose transcriptional activity is modulated by id2 and which regulates downstream lipid metabolism genes such as lipe and fasn [ 35 , 36 ] . previous studies have revealed irs1-regulated id2 gene expression , although in the current study it is unclear whether this is a direct effect or a feedback response . as for the mechanism by which irs1 is elevated in id2/ ibat , bmp4 ( upregulated in our study ) is able to induce the white to brown transition of adipose cells , which could indirectly regulate ppar activation [ 40 , 41 ] . the elevated bmp4 expression in the context of reduced ppars is surprising since bmp4 upregulation is associated with increased bat adipogenesis and the wat browning effect . interestingly , in the id1 null mouse , ucp1 gene expression is elevated in ibat , and this is associated with an increased core body temperature phenotype . thus , it is surprising that in the id2/ mouse that exhibits a reduced body temperature phenotype we do not observe a reduction in ucp1 gene expression . hypoxia , while not a focus of the current study , is known to reduce body temperature in mammals and contribute to the thermogenic activity of bat . it is noteworthy that in a recent study of human glioblastoma cells / tissue , an important role was established for id2 in modulating the cellular effects of hypoxia and its activation of the hif2 pathway . it is plausible that the hypoxic effects on bat function might also include a contribution from id2 , and this would be an important pathway to examine in future experiments in this context . , pgc1- ) or unaltered ( ucp1 ) in id2/ mice but that id2/ male ibat exhibits increased glucose uptake ( pet - fdg ) coupled with reduced ibat triglyceride levels and a systemwide enhanced insulin sensitivity [ 10 , 11 ] and that core body temperature is consistently reduced in both id2/ male and female mice . id2/ mice also exhibit an altered 24 hr locomotor activity and feeding profile and an overall reduction in nocturnal locomotor activity , the latter of which suggests a reduction in energy expenditure / increased energy conservation . clearly the bat adipogenic program is altered in both male and female id2/ mice , as is wat adipogenesis [ 1012 ] , and it is likely that a change in bat function contributes to the reduced temperature phenotype . due to the nature of whole body knockout of id2 , it is possible that the temperature phenotype and ibat gene changes observed are secondary to whole body metabolic changes . clearly additional experiments are required to elucidate the relative contributions of these and other potential components in generating the altered core body temperature phenotype . our previous study showed enhanced glucose uptake in ibat of id2/ mice , and the present study reveals a reduced core body temperature in id2/ mice . this discrepancy could partially be explained by the differential regulation of irs1 , lipe , ppars , and pgc-1s . it has been reported that degradation of irs1 leads to impaired glucose uptake in adipose tissue . therefore , upregulation of irs1 might explain the increased glucose uptake we observed before , whereas downregulation of lipe , ppars , and pgc-1s might contribute to reduced fa oxidation , impaired adipogenesis , and a lower body temperature . it has been suggested that mice that lack one allele of the ppar gene are more sensitive to insulin , which could partially explain the enhanced insulin sensitivity we observe in id2 null mice [ 10 , 51 ] . furthermore , the differential regulation of genes specifically in female mutant mice , such as fbp1 , a rate - limiting enzyme in gluconeogenesis , shc1 , a component in the igf-1-regulated pathway , and igf2 , suggests a sex - specific physiological program for id2 in bat . inhibitor of dna binding 2 is rhythmically expressed in bat [ 4 , 15 ] , and the observation that few of the differentially regulated genes involved in insulin signaling are ccgs suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output . overall , id2 seems to be an important coordinator of energy homeostasis including insulin signaling , adipogenic programing , and thermoregulation . in conclusion , our finding that id2 contributes to the regulation of body temperature and energy homeostasis presents the possibility that id2 could be a potential therapeutic target for metabolic disease . further , these data emphasize the influence of id2 on bat molecular signaling and physiology in a sex - specific manner .

barrett s esophagus ( be ) is a metaplastic premalignant disorder in which the normal stratified squamous epithelium of the lower esophagus is replaced by a columnar lined epithelium with intestinal differentiation . be generally occurs in the context of chronic gastroesophageal reflux disease ( gerd)1 that can induce metaplastic change of the distal esophagus whereby the normal squamous epithelium is substituted by a columnar epithelium.2 be represents the primary risk factor for the development of esophageal adenocarcinoma ( oac ) and is associated with an increased risk of cancer by about 0.4%1% a year;3,4 the prevalence increases with age , comprising 1% of the population over 60 years of age . the disease is more common in men than in women ( 2:1),5 it is rare in childhood , and it is estimated that the median age for the onset of the disease is approximately 40 years , although the median age at the time of diagnosis is approximately 60 years.6 the risk of developing cancer is 30125 times higher in be patients as compared to the general population.7 it has been clearly established that gerd is the main risk factor for the development of be;8 caucasian males over the age of 50 with longstanding gerd are at higher risk of developing be and oac.5,9 the second most common condition associated with barrett s esophagus and oac is central obesity.10,11 nonetheless , since only a minority of subjects exposed to these environmental factors are found to have columnar metaplasia in the distal esophagus , be is considered to be a complex disease in which the environment interacts with an individual s genetic predisposition.2 over the last three decades , evidence has accumulated suggesting the presence of an inherited genetic component impacting on an individual s predisposition to developing be.12 the age of occurrence of familial cases was found to be about 10 years younger with sporadic be , and inheritance patterns of these families was consistent with an autosomal dominant disease with incomplete penetrance . to date , van lieshout et al13 studied the frequencies of polymorphic variants in glutathione s - transferase p1 ( gstp1 ) among 247 blood donors , 98 be patients , and 34 subjects with oac . they found an association between the gstp1 polymorphic variant , gstp1b , and an increased risk of both be and oac . in this study , subjects with be and oac were recruited through a single center rather than using a population - based approach , increasing the risk of selection bias . abbas et al14 carried out a case - control study of esophageal cancer in northwest france . they studied the frequency of the same gstp1 variant but found no association between gstp1 and risk of oac in 27 patients . a number of factors , including inappropriate control group , lack of population - based dna collections , and small study size , probably accounted for the discrepancies between the published studies . in further studies , x - ray repair cross - complementing protein 1 ( xrcc1 ) and the increased susceptibility for developing esophageal squamous cell carcinoma have been evaluated . however , the authors found that the xrcc1 399 arg / arg was found more frequently in patients with esophageal cancer who were alcohol drinkers ; therefore , the results were not statistically significant . 15 in addition , the authors observed the genetic polymorphisms in xrcc1 associated with an increased risk of developing squamous cell carcinoma in a chinese population . several other studies have reported an association between dna repair genes and the pathogenesis of be and oac with a higher frequency of xeroderma pigmentosum complementation group c ( xpc ) , poly at insertion / deletion in oac , a lower frequency of xeroderma pigmentosum complementary group d ( xpd ) , and xrcc1 homozygous variants in be , as compared with normal controls.1517 although the data noted in the literature are not always in accordance amongst several populations , the findings prompted us to carry out similar genetic studies in an italian cohort of 74 patients with be . this preliminary study aimed to assess the association between genetic polymorphisms in dna repair genes and genes with detoxifying enzymes on the development of we studied six polymorphisms in four genes ( xpc , xpd , xrcc1 , gstp ) in 74 cases of patients with be and 67 controls . the group of patients consisted of 74 individuals with a confirmed diagnosis of be as per an identical protocol coming from six gastrointestinal units in italy . inclusion criteria were to be older than 18 years old and to have acquired a diagnosis of be through endoscopy and histology . all of the cases belong to a wider epidemiological study aimed at identifying individual and environmental risk factors for be.18 a total of 67 healthy donor controls were recruited both from the gastrointestinal units described previously and from niguarda hospital ( milan , italy ) . the selection criteria for control subjects included having no individual or family history of cancer . controls were matched to patients for sex , age , and the same geographic origin . the mean age was 63.08 years ( sd = 13.74 ) for cases ( 53 male ) , and 58.57 years ( sd = 11.16 ) for controls ( 43 males ) . dna was extracted from whole blood using the qiamp blood mini kit ( qiagen srl , milan , italy ) . six different single nucleotide polymorphisms ( snps ) were investigated : xrcc1 arg194trp ; xrcc1 arg399gln ; xpc poly at insertion / deletion indicated as pat ; xpd arg156arg ( allele c or a ) ; gstp1 ile105val ; and ala114val . the xpc pat polymorphism was analyzed by polymerase chain reaction ( pcr ) and then resolved on a 2% agarose gel stained with gel red 10000x ( biotium , hayward , ca ) . homozygous pat / genotypes were represented by a 266 bp dna band , whereas homozygous pat + /+ genotypes were represented by a 344 bp fragment ; heterozygous + / were represented by a 266 bp plus a 344 bp dna fragment . the polymorphisms in xpd and in xrcc1 were analyzed by pcr combined with restriction fragment length polymorphisms . the xpd pcr product was digested with thermus filiformis , whereas restriction enzymes proteus vulgaris 2 and methylation - specific pcr 1 were used to detect exon 6 and exon 10 polymorphisms in the xrcc1 gene , respectively ( new england biolabs gmbh , frankfurt , germany ) . the gstp1 polymorphisms in exon 5 and in exon 6 were assayed according to the methods and enzymes previously described.19 brief pcr amplifications were followed by enzymatic digestion with bsmai and acil , respectively ( new england biolabs gmbh ) ( figure 1 ) . each record corresponded to a known clinical condition ( case ) or to a sample population ( control ) . these data comprised the variables that corresponded to the six snps , each of which could have three genotype classes : wild - type , heterozygous , and homozygous status . those genetic markers for which only one genotype was present both in cases and controls were removed from the database . the association of each tested variable ( genetic polymorphisms , sex , and smoker status ) with be was tested using descriptive statistics including linear correlation and by using artificial neural networks ( anns ) . the models we used aimed to correctly classify the subjects according to one of two classes : barrett s esophagus patients ( cases ) or healthy subjects ( controls ) . anns are able to build a model with a strong genetic basis by collecting all the information included within the snp without any a priori definitions . the mathematical approach of anns consists of measuring the general dependence of random variables related to a group of subject without making any assumptions about the nature of their underlying relationships.20,21 advanced intelligent systems , which are based on novel coupling of artificial neural networks and evolutionary algorithms , have also been applied in the present study . supervised anns are networks which learn by examples and calculate an error function during the training phase and adjust the connection strengths in order to minimize the error function.22 the learning constraint of the supervised anns makes their own output coincide with the predefined target . the general form of these anns is : where y is the dependent variable ; f is the mathematical function ; x is the independent variables ; and w the set of parameters that best approximates the function . data analysis was performed using a re - sampling system named twist , developed by the semeion research center ( rome , italy ) . the twist system consists of an ensemble of two previously described systems : training and testing ( t and t ) and input selection ( is).23 the t and t system is a robust data re - sampling technique that is able to arrange the source sample into sub - samples that all possess a similar probability density function . in this way , the data is split into two or more sub - samples in order to train , test , and validate the ann models more effectively . the is system is an evolutionary wrapper system able to reduce the amount of data while conserving the largest amount of information available in the dataset . the combined action of these two systems allows us to solve two frequent problems in managing anns ( ie , the optimal splitting of the data set in training and testing subsets containing a balanced distribution of outliers , and the optimal selection of variables ) with the maximal amount of information relevant to the problem under investigation . both systems are based on a genetic algorithm , the genetic doping algorithm developed at semeion research center.24 the twist system has been previously applied in different medical contexts;25 additional data are given . after this processing , the features that were most significant for the classification of patients into either the be or control category were selected , and at the same time the training set and the testing set were created with a function of probability distribution similar to the one that provided the best results in the classification . a series of supervised multilayer perceptrons , with four hidden units , were then used for the classification task . the final anns , which were trained and tested on the new data set generated by the twist system , are virgin , and operate independently and blindly from each other and from the twist system . back - propagation anns25 were then applied to the results obtained from the twist approach using a validation protocol . this is a procedure that is used to verify the model s ability to generalize the results reached in the testing phase . among the different protocols reported in literature , the selected model is the protocol with the greatest generalizability on data unknown to the model itself . the procedural steps in developing the validation protocol are : ( 1 ) subdividing the dataset randomly into two sub - samples the first called the training set , and the second called the testing set ; ( 2 ) choosing a fixed ann ( and/or organism ) which is trained on the training set . in this phase , the anns learn to associate the input variables with those that are identified as targets ; ( 3 ) saving the weight matrix produced by the anns at the end of the training phase and freezing it with all of the parameters used for the training ; ( 4 ) showing the testing set to the anns so that in each case the anns can express an evaluation based on the training just performed . this procedure takes place for each input vector , but every result ( output vector ) is not communicated to the anns . in this way , the anns are evaluated only in reference to the generalizability that it acquired during the training phase ; and ( 5 ) constructing a new ann with identical architecture to the previous ann and repeating the procedure from point 1 . this general training plan has been employed twice , obtaining two independent classification experiments : the first by training anns on subsamble a and testing them on subsample b , and the second by training anns on subsample b and testing them on subsample a. the group of patients consisted of 74 individuals with a confirmed diagnosis of be as per an identical protocol coming from six gastrointestinal units in italy . inclusion criteria were to be older than 18 years old and to have acquired a diagnosis of be through endoscopy and histology . all of the cases belong to a wider epidemiological study aimed at identifying individual and environmental risk factors for be.18 a total of 67 healthy donor controls were recruited both from the gastrointestinal units described previously and from niguarda hospital ( milan , italy ) . the selection criteria for control subjects included having no individual or family history of cancer . controls were matched to patients for sex , age , and the same geographic origin . the mean age was 63.08 years ( sd = 13.74 ) for cases ( 53 male ) , and 58.57 years ( sd = 11.16 ) for controls ( 43 males ) . dna was extracted from whole blood using the qiamp blood mini kit ( qiagen srl , milan , italy ) . six different single nucleotide polymorphisms ( snps ) were investigated : xrcc1 arg194trp ; xrcc1 arg399gln ; xpc poly at insertion / deletion indicated as pat ; xpd arg156arg ( allele c or a ) ; gstp1 ile105val ; and ala114val . the xpc pat polymorphism was analyzed by polymerase chain reaction ( pcr ) and then resolved on a 2% agarose gel stained with gel red 10000x ( biotium , hayward , ca ) . homozygous pat / genotypes were represented by a 266 bp dna band , whereas homozygous pat + /+ genotypes were represented by a 344 bp fragment ; heterozygous + / were represented by a 266 bp plus a 344 bp dna fragment . the polymorphisms in xpd and in xrcc1 were analyzed by pcr combined with restriction fragment length polymorphisms . the xpd pcr product was digested with thermus filiformis , whereas restriction enzymes proteus vulgaris 2 and methylation - specific pcr 1 were used to detect exon 6 and exon 10 polymorphisms in the xrcc1 gene , respectively ( new england biolabs gmbh , frankfurt , germany ) . the gstp1 polymorphisms in exon 5 and in exon 6 were assayed according to the methods and enzymes previously described.19 brief pcr amplifications were followed by enzymatic digestion with bsmai and acil , respectively ( new england biolabs gmbh ) ( figure 1 ) . each record corresponded to a known clinical condition ( case ) or to a sample population ( control ) . these data comprised the variables that corresponded to the six snps , each of which could have three genotype classes : wild - type , heterozygous , and homozygous status . those genetic markers for which only one genotype was present both in cases and controls were removed from the database . there were 20 remaining variables in total in cases and controls . the association of each tested variable ( genetic polymorphisms , sex , and smoker status ) with be was tested using descriptive statistics including linear correlation and by using artificial neural networks ( anns ) . the models we used aimed to correctly classify the subjects according to one of two classes : barrett s esophagus patients ( cases ) or healthy subjects ( controls ) . anns are able to build a model with a strong genetic basis by collecting all the information included within the snp without any a priori definitions . the mathematical approach of anns consists of measuring the general dependence of random variables related to a group of subject without making any assumptions about the nature of their underlying relationships.20,21 advanced intelligent systems , which are based on novel coupling of artificial neural networks and evolutionary algorithms , have also been applied in the present study . supervised anns are networks which learn by examples and calculate an error function during the training phase and adjust the connection strengths in order to minimize the error function.22 the learning constraint of the supervised anns makes their own output coincide with the predefined target . the general form of these anns is : where y is the dependent variable ; f is the mathematical function ; x is the independent variables ; and w the set of parameters that best approximates the function . data analysis was performed using a re - sampling system named twist , developed by the semeion research center ( rome , italy ) . the twist system consists of an ensemble of two previously described systems : training and testing ( t and t ) and input selection ( is).23 the t and t system is a robust data re - sampling technique that is able to arrange the source sample into sub - samples that all possess a similar probability density function . in this way , the data is split into two or more sub - samples in order to train , test , and validate the ann models more effectively . the is system is an evolutionary wrapper system able to reduce the amount of data while conserving the largest amount of information available in the dataset . the combined action of these two systems allows us to solve two frequent problems in managing anns ( ie , the optimal splitting of the data set in training and testing subsets containing a balanced distribution of outliers , and the optimal selection of variables ) with the maximal amount of information relevant to the problem under investigation . both systems are based on a genetic algorithm , the genetic doping algorithm developed at semeion research center.24 the twist system has been previously applied in different medical contexts;25 additional data are given . after this processing , the features that were most significant for the classification of patients into either the be or control category were selected , and at the same time the training set and the testing set were created with a function of probability distribution similar to the one that provided the best results in the classification . a series of supervised multilayer perceptrons , with four hidden units , were then used for the classification task . the final anns , which were trained and tested on the new data set generated by the twist system , are virgin , and operate independently and blindly from each other and from the twist system . back - propagation anns25 were then applied to the results obtained from the twist approach using a validation protocol . this is a procedure that is used to verify the model s ability to generalize the results reached in the testing phase . among the different protocols reported in literature , the selected model is the protocol with the greatest generalizability on data unknown to the model itself . the procedural steps in developing the validation protocol are : ( 1 ) subdividing the dataset randomly into two sub - samples the first called the training set , and the second called the testing set ; ( 2 ) choosing a fixed ann ( and/or organism ) which is trained on the training set . in this phase , the anns learn to associate the input variables with those that are identified as targets ; ( 3 ) saving the weight matrix produced by the anns at the end of the training phase and freezing it with all of the parameters used for the training ; ( 4 ) showing the testing set to the anns so that in each case the anns can express an evaluation based on the training just performed . this procedure takes place for each input vector , but every result ( output vector ) is not communicated to the anns . in this way , the anns are evaluated only in reference to the generalizability that it acquired during the training phase ; and ( 5 ) constructing a new ann with identical architecture to the previous ann and repeating the procedure from point 1 . this general training plan has been employed twice , obtaining two independent classification experiments : the first by training anns on subsamble a and testing them on subsample b , and the second by training anns on subsample b and testing them on subsample a. this study included a total of 74 patients with be and 67 population controls , representing 52.5% and 47.5% of the overall sample , respectively . we failed to find xpc pat + / and xrcc1 trp194trp in both cases and controls , whereas gstp1 val114val was found in among the control group only . the distributions and confidence intervals ( 95% ) of sex , smoking history , and polymorphisms among cases and controls are reported in table 1 ; there were no statistically significant differences among the two groups . the r - value from the linear correlation allowed us to discriminate between possible protective factors and possible risk factors as reported in table 2 ; however , the results are without statistical relevance . the results that were obtained provided a strong rationale by which to employ anns . in using advanced intelligent systems like twist,21 it was possible to reach the best predictive accuracy to discriminate between cases and controls . indeed , the application of twist system allowed for the selection of a subgroup of nine variables , as reported in table 3 . this new data set has been analyzed with back - propagation anns employing a rigorous validation protocol . table 4 summarizes the results obtained with back - propagation anns applied 10 times on the final data set ; a mean global accuracy of 60% was reached , and was as high as 65.88% . figure 2 shows the area under the curve ( auc ) of the receiver - operating characteristic ( roc ) , auc of the two anns classifications , and the average roc auc . barrett s esophagus is a relatively common , benign , and asymptomatic disorder , the clinical importance of which relates to its role as a precursor lesion to esophageal adenocarcinoma ; the condition appears to be a complex disease due not only to multiple genes / genetic variants , but also caused by environmental factors . heavy , remote smoking , for example , has been associated with an increased risk of be , suggesting a long latency period between exposure and development of the disease , even after smoking cessation.26 among the risk factors associated with the disease , case reports and pedigree studies suggest a heritable component , albeit with complex and variable expressions . a prominent feature of most cancers including barrett s adenocarcinoma is genetic instability , which is associated with the development and progression of the disease . indeed , genetic instability has been shown to increase in patients with be , involving less than 2% of the genome in the early stages to over 30% in later stages.27 to safeguard the integrity of the genome , humans have developed a complex set of dna repair systems . defects in dna repair have been demonstrated to be a critical mechanism in human carcinogenesis.28 in addition , a reduced dna repair capacity caused by genetic polymorphism is associated with an increased cancer risk.29,30 in this sense , numerous dna polymorphisms have been identified in dna repair genes , and many of them have been shown to contribute to genetic instability and error accumulation due to reduced protein activity.31 these proteins are implicated in four major dna repair pathways , including ner , ber , double - strand break repair , and mismatch repair.32 recently , the role of oxidative dna damage , dna repair , glutathione s - transferase mu 1 , superoxide dismutase 2 , and 8-oxoguanine dna glycosylase polymorphisms for individual susceptibility to be have been investigated among 40 patients with be . even though the authors failed to find an association , the results of that study pointed to a role of oxidative dna damage in be.33 to date , a large number of case - control studies to explore the association between dna repair gene polymorphisms and the increasing risk of cancer have been performed . several reports have shown an association between polymorphisms in the xpc and xpd genes and the increased risk of developing different types of cancer , with some xpd allelic variants related to an increased risk of lung cancer,34 squamous cell carcinoma of the head and neck,35 and breast cancer.36 several studies have also found associations between genetic polymorphisms in some ber genes , such as xrcc1 , and an increased risk of cancer . ber genes play a key role in removing dna damage from oxidation , deamination , and ring fragmentation,37 and exposure to tobacco smoking induces oxidative damage by generating reactive oxygen species;38 polymorphisms in ber genes have been shown to be associated with lung cancer.31 furthermore , given that exposure of esophageal epithelium to luminal toxic agents likely plays a crucial role , several studies have analyzed the association between polymorphisms in the detoxifying enzyme glutathione s - transferase and the risk of developing be or oac . gsts comprise four main classes : a , m , p , and t , which are present in many species and tissues . among them , the gstp1 enzyme is the most important form found in the esophagus . decreased gstp1 enzyme activity has been detected in be , suggesting that these alterations may contribute to an increased cancer risk in association with this disease.39 significantly lower gst enzyme activity was found more often in patients with be and patients with oac , indicating that these genetic changes may contribute to the development of both be and oac.13 all the above data prompted us to investigate the xpc , xpd , xrcc1 , and gstp genetic polymorphisms in relation to barrett s esophagus in a cohort of italian subjects . we report here results from an exploratory study involving 74 be cases . as far as we know all the samples were collected in gastrointestinal centers from the northern , central , and southern parts of italy , and the clinical diagnoses have been unequivocally established.17 of the epidemiological data collected , only smoking status was analyzed together with the genetic data derived from analysis of the snps . indeed we believe that a non - conventional method , such as using advance intelligent systems , could successfully identify a genetic background ( if present ) that predisposes an individual to developing be . results from this study failed to find an association among the tested snps and be phenotype . we can hypothesize that the 74 cases tested here did not provide a large enough sample to find a statistically significant association given that the analysis may have been influenced by the number of tested variables and by the number of analyzed subjects . on the contrary , advanced intelligent systems such as twist system and back propagation are really able to handle the disease complexity , not treating the data with reductionist approaches unable to detect multiple genes of smaller effect in predisposing to the disease . with such an approach , we were able to identify nine variables within the genes involved in the ner and ber dna repair pathways and in a gene coding for detoxifying enzymes gstp1 . interestingly , the xpc pat variable lies within chromosome 3p , a fragile region recently found to be involved in the early stages of be.40 however , back - propagation analysis on the variables selected by the twist system was not able to exceed a mean sensitivity of 78.71% with a mean specificity of 53.05% . we can postulate that the genetic variables analyzed here do not represent the factors that make an individual susceptible to developing be ; however , our results suggest a strong positive correlation between genetic background and be does not exist . recently , residual embryonic cells have been proposed as a precursor of barretts - like metaplasia , suggesting that this precancerous lesion originates not from genetic alterations , but from competitive interactions between cell lineages driven by opportunity.41

backgroundbarrett s esophagus ( be ) , a metaplastic premalignant disorder , represents the primary risk factor for the development of esophageal adenocarcinoma . chronic gastroesophageal reflux disease and central obesity have been associated with be and esophageal adenocarcinoma , but relatively little is known about the specific genes that confer susceptibility to be carcinogenesis.methodsa total of 74 patients with be and 67 controls coming from six gastrointestinal italian units were evaluated for six polymorphisms in four genes : xpc , xpd nucleotide excision repair ( ner ) genes , xrcc1 ( ber gene ) , and glutathione s - transferase p1 . smoking status was analyzed together with the genetic data . statistical analysis was performed through artificial neural networks.resultsdistributions of sex , smoking history , and polymorphisms among be cases and controls did not show statistically significant differences . the r - value from linear correlation allowed us to identify possible protective factors as well as possible risk factors . the application of advanced intelligent systems allowed for the selection of a subgroup of nine variables . artificial neural networks applied on the final data set reached mean global accuracy of 60% , reaching as high as 65.88%.conclusionwe report here results from an exploratory study . results from this study failed to find an association among the tested single nucleotide polymorphisms and be phenotype through classical statistical methods . on the contrary , advanced intelligent systems are really able to handle the disease complexity , not treating the data with reductionist approaches unable to detect multiple genes of smaller effect in predisposing to the disease.impactto detect multiple genes of smaller effects in predisposing individuals to barrett s esophagus .

Impact Background Materials and methods Patients Genotype analysis Database Statistical analysis Results Discussion

barrett s esophagus ( be ) is a metaplastic premalignant disorder in which the normal stratified squamous epithelium of the lower esophagus is replaced by a columnar lined epithelium with intestinal differentiation . be generally occurs in the context of chronic gastroesophageal reflux disease ( gerd)1 that can induce metaplastic change of the distal esophagus whereby the normal squamous epithelium is substituted by a columnar epithelium.2 be represents the primary risk factor for the development of esophageal adenocarcinoma ( oac ) and is associated with an increased risk of cancer by about 0.4%1% a year;3,4 the prevalence increases with age , comprising 1% of the population over 60 years of age . the disease is more common in men than in women ( 2:1),5 it is rare in childhood , and it is estimated that the median age for the onset of the disease is approximately 40 years , although the median age at the time of diagnosis is approximately 60 years.6 the risk of developing cancer is 30125 times higher in be patients as compared to the general population.7 it has been clearly established that gerd is the main risk factor for the development of be;8 caucasian males over the age of 50 with longstanding gerd are at higher risk of developing be and oac.5,9 the second most common condition associated with barrett s esophagus and oac is central obesity.10,11 nonetheless , since only a minority of subjects exposed to these environmental factors are found to have columnar metaplasia in the distal esophagus , be is considered to be a complex disease in which the environment interacts with an individual s genetic predisposition.2 over the last three decades , evidence has accumulated suggesting the presence of an inherited genetic component impacting on an individual s predisposition to developing be.12 the age of occurrence of familial cases was found to be about 10 years younger with sporadic be , and inheritance patterns of these families was consistent with an autosomal dominant disease with incomplete penetrance . to date , van lieshout et al13 studied the frequencies of polymorphic variants in glutathione s - transferase p1 ( gstp1 ) among 247 blood donors , 98 be patients , and 34 subjects with oac . they found an association between the gstp1 polymorphic variant , gstp1b , and an increased risk of both be and oac . in this study , subjects with be and oac were recruited through a single center rather than using a population - based approach , increasing the risk of selection bias . a number of factors , including inappropriate control group , lack of population - based dna collections , and small study size , probably accounted for the discrepancies between the published studies . however , the authors found that the xrcc1 399 arg / arg was found more frequently in patients with esophageal cancer who were alcohol drinkers ; therefore , the results were not statistically significant . 15 in addition , the authors observed the genetic polymorphisms in xrcc1 associated with an increased risk of developing squamous cell carcinoma in a chinese population . several other studies have reported an association between dna repair genes and the pathogenesis of be and oac with a higher frequency of xeroderma pigmentosum complementation group c ( xpc ) , poly at insertion / deletion in oac , a lower frequency of xeroderma pigmentosum complementary group d ( xpd ) , and xrcc1 homozygous variants in be , as compared with normal controls.1517 although the data noted in the literature are not always in accordance amongst several populations , the findings prompted us to carry out similar genetic studies in an italian cohort of 74 patients with be . this preliminary study aimed to assess the association between genetic polymorphisms in dna repair genes and genes with detoxifying enzymes on the development of we studied six polymorphisms in four genes ( xpc , xpd , xrcc1 , gstp ) in 74 cases of patients with be and 67 controls . the group of patients consisted of 74 individuals with a confirmed diagnosis of be as per an identical protocol coming from six gastrointestinal units in italy . all of the cases belong to a wider epidemiological study aimed at identifying individual and environmental risk factors for be.18 a total of 67 healthy donor controls were recruited both from the gastrointestinal units described previously and from niguarda hospital ( milan , italy ) . the mean age was 63.08 years ( sd = 13.74 ) for cases ( 53 male ) , and 58.57 years ( sd = 11.16 ) for controls ( 43 males ) . six different single nucleotide polymorphisms ( snps ) were investigated : xrcc1 arg194trp ; xrcc1 arg399gln ; xpc poly at insertion / deletion indicated as pat ; xpd arg156arg ( allele c or a ) ; gstp1 ile105val ; and ala114val . the xpd pcr product was digested with thermus filiformis , whereas restriction enzymes proteus vulgaris 2 and methylation - specific pcr 1 were used to detect exon 6 and exon 10 polymorphisms in the xrcc1 gene , respectively ( new england biolabs gmbh , frankfurt , germany ) . the gstp1 polymorphisms in exon 5 and in exon 6 were assayed according to the methods and enzymes previously described.19 brief pcr amplifications were followed by enzymatic digestion with bsmai and acil , respectively ( new england biolabs gmbh ) ( figure 1 ) . these data comprised the variables that corresponded to the six snps , each of which could have three genotype classes : wild - type , heterozygous , and homozygous status . those genetic markers for which only one genotype was present both in cases and controls were removed from the database . the association of each tested variable ( genetic polymorphisms , sex , and smoker status ) with be was tested using descriptive statistics including linear correlation and by using artificial neural networks ( anns ) . the mathematical approach of anns consists of measuring the general dependence of random variables related to a group of subject without making any assumptions about the nature of their underlying relationships.20,21 advanced intelligent systems , which are based on novel coupling of artificial neural networks and evolutionary algorithms , have also been applied in the present study . in this way , the data is split into two or more sub - samples in order to train , test , and validate the ann models more effectively . the combined action of these two systems allows us to solve two frequent problems in managing anns ( ie , the optimal splitting of the data set in training and testing subsets containing a balanced distribution of outliers , and the optimal selection of variables ) with the maximal amount of information relevant to the problem under investigation . both systems are based on a genetic algorithm , the genetic doping algorithm developed at semeion research center.24 the twist system has been previously applied in different medical contexts;25 additional data are given . after this processing , the features that were most significant for the classification of patients into either the be or control category were selected , and at the same time the training set and the testing set were created with a function of probability distribution similar to the one that provided the best results in the classification . the final anns , which were trained and tested on the new data set generated by the twist system , are virgin , and operate independently and blindly from each other and from the twist system . among the different protocols reported in literature , the selected model is the protocol with the greatest generalizability on data unknown to the model itself . the procedural steps in developing the validation protocol are : ( 1 ) subdividing the dataset randomly into two sub - samples the first called the training set , and the second called the testing set ; ( 2 ) choosing a fixed ann ( and/or organism ) which is trained on the training set . in this phase , the anns learn to associate the input variables with those that are identified as targets ; ( 3 ) saving the weight matrix produced by the anns at the end of the training phase and freezing it with all of the parameters used for the training ; ( 4 ) showing the testing set to the anns so that in each case the anns can express an evaluation based on the training just performed . this procedure takes place for each input vector , but every result ( output vector ) is not communicated to the anns . this general training plan has been employed twice , obtaining two independent classification experiments : the first by training anns on subsamble a and testing them on subsample b , and the second by training anns on subsample b and testing them on subsample a. the group of patients consisted of 74 individuals with a confirmed diagnosis of be as per an identical protocol coming from six gastrointestinal units in italy . all of the cases belong to a wider epidemiological study aimed at identifying individual and environmental risk factors for be.18 a total of 67 healthy donor controls were recruited both from the gastrointestinal units described previously and from niguarda hospital ( milan , italy ) . controls were matched to patients for sex , age , and the same geographic origin . the mean age was 63.08 years ( sd = 13.74 ) for cases ( 53 male ) , and 58.57 years ( sd = 11.16 ) for controls ( 43 males ) . six different single nucleotide polymorphisms ( snps ) were investigated : xrcc1 arg194trp ; xrcc1 arg399gln ; xpc poly at insertion / deletion indicated as pat ; xpd arg156arg ( allele c or a ) ; gstp1 ile105val ; and ala114val . the xpd pcr product was digested with thermus filiformis , whereas restriction enzymes proteus vulgaris 2 and methylation - specific pcr 1 were used to detect exon 6 and exon 10 polymorphisms in the xrcc1 gene , respectively ( new england biolabs gmbh , frankfurt , germany ) . the gstp1 polymorphisms in exon 5 and in exon 6 were assayed according to the methods and enzymes previously described.19 brief pcr amplifications were followed by enzymatic digestion with bsmai and acil , respectively ( new england biolabs gmbh ) ( figure 1 ) . these data comprised the variables that corresponded to the six snps , each of which could have three genotype classes : wild - type , heterozygous , and homozygous status . those genetic markers for which only one genotype was present both in cases and controls were removed from the database . there were 20 remaining variables in total in cases and controls . the association of each tested variable ( genetic polymorphisms , sex , and smoker status ) with be was tested using descriptive statistics including linear correlation and by using artificial neural networks ( anns ) . the mathematical approach of anns consists of measuring the general dependence of random variables related to a group of subject without making any assumptions about the nature of their underlying relationships.20,21 advanced intelligent systems , which are based on novel coupling of artificial neural networks and evolutionary algorithms , have also been applied in the present study . the combined action of these two systems allows us to solve two frequent problems in managing anns ( ie , the optimal splitting of the data set in training and testing subsets containing a balanced distribution of outliers , and the optimal selection of variables ) with the maximal amount of information relevant to the problem under investigation . after this processing , the features that were most significant for the classification of patients into either the be or control category were selected , and at the same time the training set and the testing set were created with a function of probability distribution similar to the one that provided the best results in the classification . the final anns , which were trained and tested on the new data set generated by the twist system , are virgin , and operate independently and blindly from each other and from the twist system . among the different protocols reported in literature , the selected model is the protocol with the greatest generalizability on data unknown to the model itself . in this phase , the anns learn to associate the input variables with those that are identified as targets ; ( 3 ) saving the weight matrix produced by the anns at the end of the training phase and freezing it with all of the parameters used for the training ; ( 4 ) showing the testing set to the anns so that in each case the anns can express an evaluation based on the training just performed . this procedure takes place for each input vector , but every result ( output vector ) is not communicated to the anns . this general training plan has been employed twice , obtaining two independent classification experiments : the first by training anns on subsamble a and testing them on subsample b , and the second by training anns on subsample b and testing them on subsample a. this study included a total of 74 patients with be and 67 population controls , representing 52.5% and 47.5% of the overall sample , respectively . we failed to find xpc pat + / and xrcc1 trp194trp in both cases and controls , whereas gstp1 val114val was found in among the control group only . the distributions and confidence intervals ( 95% ) of sex , smoking history , and polymorphisms among cases and controls are reported in table 1 ; there were no statistically significant differences among the two groups . the r - value from the linear correlation allowed us to discriminate between possible protective factors and possible risk factors as reported in table 2 ; however , the results are without statistical relevance . in using advanced intelligent systems like twist,21 it was possible to reach the best predictive accuracy to discriminate between cases and controls . indeed , the application of twist system allowed for the selection of a subgroup of nine variables , as reported in table 3 . table 4 summarizes the results obtained with back - propagation anns applied 10 times on the final data set ; a mean global accuracy of 60% was reached , and was as high as 65.88% . figure 2 shows the area under the curve ( auc ) of the receiver - operating characteristic ( roc ) , auc of the two anns classifications , and the average roc auc . barrett s esophagus is a relatively common , benign , and asymptomatic disorder , the clinical importance of which relates to its role as a precursor lesion to esophageal adenocarcinoma ; the condition appears to be a complex disease due not only to multiple genes / genetic variants , but also caused by environmental factors . heavy , remote smoking , for example , has been associated with an increased risk of be , suggesting a long latency period between exposure and development of the disease , even after smoking cessation.26 among the risk factors associated with the disease , case reports and pedigree studies suggest a heritable component , albeit with complex and variable expressions . a prominent feature of most cancers including barrett s adenocarcinoma is genetic instability , which is associated with the development and progression of the disease . indeed , genetic instability has been shown to increase in patients with be , involving less than 2% of the genome in the early stages to over 30% in later stages.27 to safeguard the integrity of the genome , humans have developed a complex set of dna repair systems . defects in dna repair have been demonstrated to be a critical mechanism in human carcinogenesis.28 in addition , a reduced dna repair capacity caused by genetic polymorphism is associated with an increased cancer risk.29,30 in this sense , numerous dna polymorphisms have been identified in dna repair genes , and many of them have been shown to contribute to genetic instability and error accumulation due to reduced protein activity.31 these proteins are implicated in four major dna repair pathways , including ner , ber , double - strand break repair , and mismatch repair.32 recently , the role of oxidative dna damage , dna repair , glutathione s - transferase mu 1 , superoxide dismutase 2 , and 8-oxoguanine dna glycosylase polymorphisms for individual susceptibility to be have been investigated among 40 patients with be . even though the authors failed to find an association , the results of that study pointed to a role of oxidative dna damage in be.33 to date , a large number of case - control studies to explore the association between dna repair gene polymorphisms and the increasing risk of cancer have been performed . several reports have shown an association between polymorphisms in the xpc and xpd genes and the increased risk of developing different types of cancer , with some xpd allelic variants related to an increased risk of lung cancer,34 squamous cell carcinoma of the head and neck,35 and breast cancer.36 several studies have also found associations between genetic polymorphisms in some ber genes , such as xrcc1 , and an increased risk of cancer . ber genes play a key role in removing dna damage from oxidation , deamination , and ring fragmentation,37 and exposure to tobacco smoking induces oxidative damage by generating reactive oxygen species;38 polymorphisms in ber genes have been shown to be associated with lung cancer.31 furthermore , given that exposure of esophageal epithelium to luminal toxic agents likely plays a crucial role , several studies have analyzed the association between polymorphisms in the detoxifying enzyme glutathione s - transferase and the risk of developing be or oac . decreased gstp1 enzyme activity has been detected in be , suggesting that these alterations may contribute to an increased cancer risk in association with this disease.39 significantly lower gst enzyme activity was found more often in patients with be and patients with oac , indicating that these genetic changes may contribute to the development of both be and oac.13 all the above data prompted us to investigate the xpc , xpd , xrcc1 , and gstp genetic polymorphisms in relation to barrett s esophagus in a cohort of italian subjects . we report here results from an exploratory study involving 74 be cases . as far as we know all the samples were collected in gastrointestinal centers from the northern , central , and southern parts of italy , and the clinical diagnoses have been unequivocally established.17 of the epidemiological data collected , only smoking status was analyzed together with the genetic data derived from analysis of the snps . results from this study failed to find an association among the tested snps and be phenotype . we can hypothesize that the 74 cases tested here did not provide a large enough sample to find a statistically significant association given that the analysis may have been influenced by the number of tested variables and by the number of analyzed subjects . on the contrary , advanced intelligent systems such as twist system and back propagation are really able to handle the disease complexity , not treating the data with reductionist approaches unable to detect multiple genes of smaller effect in predisposing to the disease . with such an approach , we were able to identify nine variables within the genes involved in the ner and ber dna repair pathways and in a gene coding for detoxifying enzymes gstp1 . interestingly , the xpc pat variable lies within chromosome 3p , a fragile region recently found to be involved in the early stages of be.40 however , back - propagation analysis on the variables selected by the twist system was not able to exceed a mean sensitivity of 78.71% with a mean specificity of 53.05% . we can postulate that the genetic variables analyzed here do not represent the factors that make an individual susceptible to developing be ; however , our results suggest a strong positive correlation between genetic background and be does not exist . recently , residual embryonic cells have been proposed as a precursor of barretts - like metaplasia , suggesting that this precancerous lesion originates not from genetic alterations , but from competitive interactions between cell lineages driven by opportunity.41

the manipulation of electrical surface charge in a rapid and reversible manner is of great importance for a wide variety of biosensing applications , since electrostatic interactions influence the affinity and residence time of biological molecules in the sensing zone . charge manipulation in nanoscale sensors , such as nanopores , nanochannels and nanotubes , is particularly useful because of the scale of these devices . solid - state nanopores are a prototypical class of nanoscale sensors , employing electrophoretic forces to thread and translocate charged single nucleic acids or proteins through a nanoscale aperture in an ultrathin membrane . nanopore surface charge can affect its direct , coulombic interactions with charged biomolecules , and can indirectly influence sensing by generating electro - osmotic flow . this net flow can modulate molecular capture rate and molecular residence time in the sensing vicinity via hydrodynamic drag . this effect has previously been characterized using optical tweezers and by varying the nanopore s zeta potential by changing the solution s ph . the ability to modulate the surface charge of a nanopore and , consequently , the associated electro - osmotic flow could allow control of biomolecule translocation speed . this could in turn lead to the development of advanced single molecule , low - cost characterization techniques for a broad spectrum of clinical samples , including genomic dna , rnas and proteins . this challenge must be met without compromising the ability of the sensor to probe extremely dilute analytes in a reasonable time frame . recent efforts towards this goal have primarily involved the fabrication of metallic electrodes at the nanopore surface to induce an electrical field . yet to date , there has been no experimental report of in situ , opto - electrical manipulation of high - density surface charge in nanopores or other nanoscale sensors . we show here that an off - the - shelf , low - power visible laser beam can be used to directly manipulate the surface charge of a solid - state nanopore . first , it allows the translocation speed of dna to be tuned without chemically modifying the nanopore surfaces or altering the buffer properties . second , it can be used to reliably unblock clogged nanopores , keeping them functional over prolonged periods of time . third , it permits the detection of extremely low molecular weight proteins , which otherwise are nearly invisible to the resistive pulse sensing technique . a silicon nitride membrane containing a single nanopore was scanned in a custom - made confocal microscope with a tightly - focused green laser ( 532 nm ) , as illustrated in figure 1a . the continuously recorded ionic current ( i ) flowing through the pore at a fixed voltage reveals an increase when the laser spot overlaps with the nanopore location . figure 1b illustrates this effect with an intensity surface plot of the ionic current i flowing through this pore as a function of laser spot position . a line scan through the image reveals a clear symmetric peak in the ionic current , more than double the baseline current level ( fwhm ~500 nm , right panel ) . this effect is evident in all tested pores ( 4 - 20 nm diameter ) even for laser powers of just a few mw . although a tightly focused ir laser at high power ( ~1 w ) can produce a small increase in current by locally heating the solution , laser - induced heating can be ruled out here for two reasons : the electromagnetic absorption of water at 532 nm is extremely small , and the laser powers used in our experiments are only a few mw . a temperature increase of ~35k would be required to produce the observed increase in i , yet heating is estimated to account for a mere 0.005k per mw of focused light based on measurements performed at 800 nm . we thus conclude that direct interaction of light with silicon nitride must cause this increase in the ion current via a photo - conductive effect . permanent nanopore blocking by small molecules , tiny air bubbles , or other nanoscale particulates is a common limitation for nanopore sensors , severely reducing their functional lifetime . in some cases , a series of electrical pulses clears the pore , but often the system must be disassembled and re - cleaned . we find that in many of these cases a short exposure to a ~5 mw focused laser beam will immediately clear the nanopore . figure 1c ( left ) shows a raster scan ( bottom left to top right ) of a fully blocked nanopore ( i 0 at v = + 300 or 300 mv ) . as soon as the laser beam reaches the nanopore location , the pore is cleared . this is shown chronologically ( figure 1c , right ) as a time trace of i during the laser scan . we have found that light - induced nanopore unblocking is a highly robust and efficient tool to extend the functional lifetime of solid - state nanopores from a few hours to several days , as detailed in the supporting information ( si ) . over 1,000 translocation events of 10 kbp dna were acquired in a nanopore ( 5.4 nm ) in the dark ( laser power p=0 ) . at t=150 s the laser radiation was switched on ( p=2 mw , pre - aligned with nanopore ) as the software continued to record translocation events . three features are immediately apparent : ( i ) the open pore current ( io ) and the current during translocation ( ib ) both increased when the laser was turned on , ( ii ) the average event amplitude i = io ib remained at the same level of 0.780.10 na under both conditions , and ( iii ) the mean event dwell - time td increased by roughly a factor of 10 under illumination , as compared to dark conditions . these features are illustrated in figure 2b , which shows io and i as a function of time throughout the experiment ( top panel ) . a 150-event running window average over all translocations ( bottom panel ) indeed shows a tenfold increase in the translocation time from ~1 ms to ~10 ms under laser illumination . we define the retardation factor ( rf ; here rf=10 ) as the increase in mean translocation time under laser light relative to darkness . upon switching off the laser illumination at t=300 s , io returned to base level ( 3.6 na ) and the mean translocation dwell time was restored to ~1 ms , suggesting that this effect is completely reversible . this experiment was repeated for nanopores of various sizes , and in all cases we observed a marked retardation of translocation time under laser illumination ( see si for analysis ) . previous studies have sensed proteins such as bovine -lactoglobulin or avidin in solid - state nanopores , and antibodies such as anti - biotin fab fragments or anti - biotin in lipid coated nanopores . however , the detection of small proteins ( mw < 10 kda ) in their native state remains a major challenge , since these proteins translocate through the pore too quickly to produce a resolvable signal given current bandwidth limitations . we selected ubiquitin ( mw ~ 8.5 kda , 4 nm diameter ) as a representative small protein , relevant to a broad range of biological processes , to demonstrate that the photo - conductive effect can slow small protein translocations and enable their detection and characterization . under dark conditions , purified wild - type ubiquitin ( see si ) translocating through a ~5 nm pore yielded sporadic , brief downward spikes ( red trace , figure 2c ) . the dwell time of these spikes was estimated to be < 12 s , but neither this nor the current amplitude of ubiquitin translocations could be accurately determined due to bandwidth constraints even at 100 khz , suggesting that the true translocation dwell time of these proteins is shorter than estimated . laser illumination of the same nanopore at p=4 mw resulted in a marked increase in both the frequency and average dwell time of detected ubiquitin events ( blue trace , figure 2c ) . the average translocation time increased by at least two orders of magnitude , allowing full characterization of both amplitude and dwell times for ubiquitin translocations . the increase in event rate under illumination may be attributed to events that were previously undetectable under dark conditions due to bandwidth limitations . a typical dwell time distribution and fractional blockade current ( ib ) for illuminated ubiquitin translocations are shown in figure 2c ( n > 500 ) . two prominent timescales are observed for ubiquitin translocation ( 3405 and 89070 s ) , as well as two peaks in the blockade currents ( 0.88 and 0.78 ) , approximated by a sum of two gaussians . we hypothesize that the two peaks observed in these distributions may be associated with different orientations of the asymmetric mushroom - like ubiquitin ( inset ) during translocation . this hypothesis is consistent with previous reports for other protein / dna complexes . to elucidate the physical origin of light - induced retardation of translocation , we first characterized the pore current i at varying laser intensity p for a range of nanopore sizes ( 4 - 20 nm diameter , d ) . figure 3a displays ionic current enhancement ( i(p)/i(0 ) ) , for four representative pores ( d = 5 , 10 , 15 , 20 nm ) . in all cases linear fits for p<5 mw characterize the response of each pore to light as the initial slope i/p . a plot of i/p as a function of diameter for 14 pores ( 4 - 20 nm ) exhibits a clear linear dependence on d ( figure 3b ) . this suggests that when the sinx thickness l and nanopore drilling conditions are maintained , the pores response to light per their surface area ( dl ) is a constant . from figure 3b the linear dependence of i/p on nanopore diameter suggests that photo - conductance current enhancement is a surface phenomenon rather than a volumetric one ( which would exhibit d dependence ) . solid - state nanopores possess weak surface charge , which can dominate pore conductivity at salt concentrations lower than ~100 mm . however , it has been theorized that high charge densities on nanopore walls can affect electrical conductivity even under high salt conditions . surface charges induce a diffuse double layer containing an excess of oppositely charged ions , of thickness comparable to the debye screening length . ionic current is enhanced by the electrical double layer in two ways : ( i ) by creating a local imbalance of counter ions within , which ( ii ) drags water molecules with its motion , creating an electro - osmotic flow ( eof ) which in turn pulls along additional current ( see figure 4a ) . the total ionic current for a nanopore with a charged surface is : ( 1)i = ibulk+idl+ieof where ibulk is the current due to ions outside the electrical double layer , idl is the current due to ions forming the double layer , and ieof is the current due to net water flow induced by net movement of ions within the double layer . ieof is proportional to the product of the flow velocity field and the net charge density (r ) , which is only nonzero within . for small compared to the pore diameter , ( i.e. 0.3 nm at 1 m kcl at 21c ) , only ibulk scales with cross sectional area ( ibulk d ) whereas idl and ieof scale with circumference ( idl , ieof d ) . thus , surface charges can , in principle , explain increased i as well as its linear scaling with d. moreover , if the analyte remains mostly within the bulk pore volume where there is no charge imbalance , distance away from the pore walls , we expect i to be independent of surface charge and thus independent of p. this is consistent with our observations ( figure 2 ) . previous theoretical work has predicted that surface charge on a nanopore will affect ( i ) translocation times and ( ii ) polymer capture rate . a negatively charged nanopore produces an eof towards the cathode ( cis chamber , figure 4a ) , increasing drag on a negatively charged translocating polymer , resulting in longer translocation times and reduced polymer capture rates . our results ( figure 2 ) indicate that the rf for dna translocation grows with laser intensity . moreover , dna capture rate was reduced by 30% , from 11 s to 8 s , for 2 mw of laser light ( see si ) . these observations lead us to hypothesize that visible light can induce negative surface charges on silicon nitride , which in turn slows dna translocation through the drag created by eof moving in the opposite direction of dna . to validate this hypothesis we approximate that for low laser powers the surface charge density ( ) grows linearly with laser intensity , such that = p where is the photo - reactivity of the pore ( c m w ) . the total ionic current i can be obtained as a function of surface charge density using the following three individual contributions ( see si for full derivation ) : ( i ) ibulk is the electrophoretic movement of each ion species through the nanopore ; ( ii ) idl may be determined from the net charge distribution for a given wall potential , derived analytically from the poisson - boltzmann equation and from the boltzmann distribution , assuming a cylindrical nanopore and using the debye - hckel approximation ; ( iii ) ieof is a product of and the eof velocity profile described by the navier - stokes equation . the grahame equation may be substituted to give as a function of wall potential . the contributions to the ion current are : ( 2.1)ibulkenkcl(k+cl)d24lv ( 2.2)idl2dklsinh(ep2)v ( 2.3)ieof2d2l2e2[sinh1(ep2)]2v for elementary charge e , permittivity of aqueous solution , parameter = 1/kbt ( boltzmann constant kb , absolute temperature t ) , pore length l , applied voltage clamp v , and solution viscosity . the number density of potassium or chloride ions is nkcl , with electrophoretic mobilities k and cl ( see si for numerical values ) . nanopore diameter d can be determined from tem images , enabling a quantitative estimation of the nanopore photo - reactivity via eq.2 ( see si figure s7 ) . we additionally measured the dependence of i on kcl concentration ( 0.01 m to 1 m ) at different p and found it consistent with the model ( see si figure s8 ) . additional dna translocations were performed using a range of dna lengths , nanopores , and laser powers to determine the role of surface charge in slowing translocation speed . the results are summarized in figure 4b ( left ) , where rf is plotted against p for five nanopores ( 4.3 , 5.4 , 5.6 , 6.1 , and 7.4 nm diameters ) and four dna lengths ( 0.4 , 3.5 , 5 , and 10 kbp ) . td was measured for > 1,000 events per laser intensity , and the td histograms were fitted with exponential functions as previously described . these measurements suggest that rf per mw of light widely varies with pore size and dna length . 2 as described above to obtain for each of the pores , we then transform the dna rf into surface charge density using the relationship = p. remarkably , all data points collapse onto a single curve ( figure 4b , right ) . thus , light - induced slowing is dependent only on the induced surface charge density . controlling the rf value can be achieved either by selecting a highly optically reactive pore ( high ) under low laser intensity , or using a less optically reactive pore under higher laser intensity . drilling nanopores in sinx with an electron beam reduces the n / si ratio around the pore due to preferential ablation of n atoms . moreover , the principal defects in silicon - rich cvd amorphous sinx materials are si dangling bonds , which can trap electrons or holes to become charged . when the n / si ratio approaches 0.8 , the band gap is within visible light energies . when the n / si ratio of the material surrounding the pore is low enough , we propose that visible light can excite electrons from the ground state across the band gap , trapping them in si dangling bonds . eventually , trapped electrons would recombine with holes , but a high density of arriving photons could maintain a steady state of negatively charged si dangling bonds , creating a net surface charge density . we tem - drilled four additional nanopores , all with similar diameters , in a freshly deposited sinx membrane . two were exposed to the e - beam for 60 s , and two were exposed for 500 s , producing different local n / si stoichiometry ( see si ) . as expected , the pores drilled with a low e - beam dose had relatively low photo - reactivity ( =27 c m w ) , whereas the pores exposed to a high e - beam dose had much higher photo - reactivity ( =70 c m w ) , establishing a direct correlation between e - beam dosage and optical reactivity . over 1,000 translocation events of 10 kbp dna were acquired in a nanopore ( 5.4 nm ) in the dark ( laser power p=0 ) . at t=150 s the laser radiation was switched on ( p=2 mw , pre - aligned with nanopore ) as the software continued to record translocation events . three features are immediately apparent : ( i ) the open pore current ( io ) and the current during translocation ( ib ) both increased when the laser was turned on , ( ii ) the average event amplitude i = io ib remained at the same level of 0.780.10 na under both conditions , and ( iii ) the mean event dwell - time td increased by roughly a factor of 10 under illumination , as compared to dark conditions . these features are illustrated in figure 2b , which shows io and i as a function of time throughout the experiment ( top panel ) . a 150-event running window average over all translocations ( bottom panel ) indeed shows a tenfold increase in the translocation time from ~1 ms to ~10 ms under laser illumination . we define the retardation factor ( rf ; here rf=10 ) as the increase in mean translocation time under laser light relative to darkness . upon switching off the laser illumination at t=300 s , io returned to base level ( 3.6 na ) and the mean translocation dwell time was restored to ~1 ms , suggesting that this effect is completely reversible . this experiment was repeated for nanopores of various sizes , and in all cases we observed a marked retardation of translocation time under laser illumination ( see si for analysis ) . previous studies have sensed proteins such as bovine -lactoglobulin or avidin in solid - state nanopores , and antibodies such as anti - biotin fab fragments or anti - biotin in lipid coated nanopores . however , the detection of small proteins ( mw < 10 kda ) in their native state remains a major challenge , since these proteins translocate through the pore too quickly to produce a resolvable signal given current bandwidth limitations . we selected ubiquitin ( mw ~ 8.5 kda , 4 nm diameter ) as a representative small protein , relevant to a broad range of biological processes , to demonstrate that the photo - conductive effect can slow small protein translocations and enable their detection and characterization . under dark conditions , purified wild - type ubiquitin ( see si ) translocating through a ~5 nm pore yielded sporadic , brief downward spikes ( red trace , figure 2c ) . the dwell time of these spikes was estimated to be < 12 s , but neither this nor the current amplitude of ubiquitin translocations could be accurately determined due to bandwidth constraints even at 100 khz , suggesting that the true translocation dwell time of these proteins is shorter than estimated . laser illumination of the same nanopore at p=4 mw resulted in a marked increase in both the frequency and average dwell time of detected ubiquitin events ( blue trace , figure 2c ) . the average translocation time increased by at least two orders of magnitude , allowing full characterization of both amplitude and dwell times for ubiquitin translocations . the increase in event rate under illumination may be attributed to events that were previously undetectable under dark conditions due to bandwidth limitations . as with dna translocations , this effect was completely reversible . a typical dwell time distribution and fractional blockade current ( ib ) for illuminated ubiquitin translocations are shown in figure 2c ( n > 500 ) . two prominent timescales are observed for ubiquitin translocation ( 3405 and 89070 s ) , as well as two peaks in the blockade currents ( 0.88 and 0.78 ) , approximated by a sum of two gaussians . we hypothesize that the two peaks observed in these distributions may be associated with different orientations of the asymmetric mushroom - like ubiquitin ( inset ) during translocation . to elucidate the physical origin of light - induced retardation of translocation , we first characterized the pore current i at varying laser intensity p for a range of nanopore sizes ( 4 - 20 nm diameter , d ) . figure 3a displays ionic current enhancement ( i(p)/i(0 ) ) , for four representative pores ( d = 5 , 10 , 15 , 20 nm ) . in all cases linear fits for p<5 mw characterize the response of each pore to light as the initial slope i/p . a plot of i/p as a function of diameter for 14 pores ( 4 - 20 nm ) exhibits a clear linear dependence on d ( figure 3b ) . this suggests that when the sinx thickness l and nanopore drilling conditions are maintained , the pores response to light per their surface area ( dl ) is a constant . from figure 3b the linear dependence of i/p on nanopore diameter suggests that photo - conductance current enhancement is a surface phenomenon rather than a volumetric one ( which would exhibit d dependence ) . solid - state nanopores possess weak surface charge , which can dominate pore conductivity at salt concentrations lower than ~100 mm . however , it has been theorized that high charge densities on nanopore walls can affect electrical conductivity even under high salt conditions . surface charges induce a diffuse double layer containing an excess of oppositely charged ions , of thickness comparable to the debye screening length . ionic current is enhanced by the electrical double layer in two ways : ( i ) by creating a local imbalance of counter ions within , which ( ii ) drags water molecules with its motion , creating an electro - osmotic flow ( eof ) which in turn pulls along additional current ( see figure 4a ) . the total ionic current for a nanopore with a charged surface is : ( 1)i = ibulk+idl+ieof where ibulk is the current due to ions outside the electrical double layer , idl is the current due to ions forming the double layer , and ieof is the current due to net water flow induced by net movement of ions within the double layer . ieof is proportional to the product of the flow velocity field and the net charge density (r ) , which is only nonzero within . for small compared to the pore diameter , ( i.e. 0.3 nm at 1 m kcl at 21c ) , only ibulk scales with cross sectional area ( ibulk d ) whereas idl and ieof scale with circumference ( idl , ieof d ) . thus , surface charges can , in principle , explain increased i as well as its linear scaling with d. moreover , if the analyte remains mostly within the bulk pore volume where there is no charge imbalance , distance away from the pore walls , we expect i to be independent of surface charge and thus independent of p. previous theoretical work has predicted that surface charge on a nanopore will affect ( i ) translocation times and ( ii ) polymer capture rate . a negatively charged nanopore produces an eof towards the cathode ( cis chamber , figure 4a ) , increasing drag on a negatively charged translocating polymer , resulting in longer translocation times and reduced polymer capture rates . our results ( figure 2 ) indicate that the rf for dna translocation grows with laser intensity . moreover , dna capture rate was reduced by 30% , from 11 s to 8 s , for 2 mw of laser light ( see si ) . these observations lead us to hypothesize that visible light can induce negative surface charges on silicon nitride , which in turn slows dna translocation through the drag created by eof moving in the opposite direction of dna . to validate this hypothesis we approximate that for low laser powers the surface charge density ( ) grows linearly with laser intensity , such that = p where is the photo - reactivity of the pore ( c m w ) . the total ionic current i can be obtained as a function of surface charge density using the following three individual contributions ( see si for full derivation ) : ( i ) ibulk is the electrophoretic movement of each ion species through the nanopore ; ( ii ) idl may be determined from the net charge distribution for a given wall potential , derived analytically from the poisson - boltzmann equation and from the boltzmann distribution , assuming a cylindrical nanopore and using the debye - hckel approximation ; ( iii ) ieof is a product of and the eof velocity profile described by the navier - stokes equation . the grahame equation may be substituted to give as a function of wall potential . the contributions to the ion current are : ( 2.1)ibulkenkcl(k+cl)d24lv ( 2.2)idl2dklsinh(ep2)v ( 2.3)ieof2d2l2e2[sinh1(ep2)]2v for elementary charge e , permittivity of aqueous solution , parameter = 1/kbt ( boltzmann constant kb , absolute temperature t ) , pore length l , applied voltage clamp v , and solution viscosity . the number density of potassium or chloride ions is nkcl , with electrophoretic mobilities k and cl ( see si for numerical values ) . nanopore diameter d can be determined from tem images , enabling a quantitative estimation of the nanopore photo - reactivity via eq.2 ( see si figure s7 ) . we additionally measured the dependence of i on kcl concentration ( 0.01 m to 1 m ) at different p and found it consistent with the model ( see si figure s8 ) . additional dna translocations were performed using a range of dna lengths , nanopores , and laser powers to determine the role of surface charge in slowing translocation speed . the results are summarized in figure 4b ( left ) , where rf is plotted against p for five nanopores ( 4.3 , 5.4 , 5.6 , 6.1 , and 7.4 nm diameters ) and four dna lengths ( 0.4 , 3.5 , 5 , and 10 kbp ) . td was measured for > 1,000 events per laser intensity , and the td histograms were fitted with exponential functions as previously described . these measurements suggest that rf per mw of light widely varies with pore size and dna length . 2 as described above to obtain for each of the pores , we then transform the dna rf into surface charge density using the relationship = p. remarkably , all data points collapse onto a single curve ( figure 4b , right ) . thus , light - induced slowing is dependent only on the induced surface charge density . controlling the rf value can be achieved either by selecting a highly optically reactive pore ( high ) under low laser intensity , or using a less optically reactive pore under higher laser intensity . drilling nanopores in sinx with an electron beam reduces the n / si ratio around the pore due to preferential ablation of n atoms . moreover , the principal defects in silicon - rich cvd amorphous sinx materials are si dangling bonds , which can trap electrons or holes to become charged . when the n / si ratio approaches 0.8 , the band gap is within visible light energies . when the n / si ratio of the material surrounding the pore is low enough , we propose that visible light can excite electrons from the ground state across the band gap , trapping them in si dangling bonds . eventually , trapped electrons would recombine with holes , but a high density of arriving photons could maintain a steady state of negatively charged si dangling bonds , creating a net surface charge density . we tem - drilled four additional nanopores , all with similar diameters , in a freshly deposited sinx membrane . two were exposed to the e - beam for 60 s , and two were exposed for 500 s , producing different local n / si stoichiometry ( see si ) . as expected , the pores drilled with a low e - beam dose had relatively low photo - reactivity ( =27 c m w ) , whereas the pores exposed to a high e - beam dose had much higher photo - reactivity ( =70 c m w ) , establishing a direct correlation between e - beam dosage and optical reactivity . we have shown that just a few mw of visible laser light focused on a nanopore can induce surface charge densities up to ~1 c m , much larger than the weak native charge of sinx . increased produces net charge near the pore walls , which , under external voltage , creates an electro - osmotic flow moving in opposition to an anionic translocating molecule . this flow slows translocating dna and proteins , allowing detection and characterization of small analytes , such as ubiquitin . previous approaches to slowing analyte translocation have introduced permanent modifications to the nanopore walls , inducing stronger interactions of analytes with the pore surface during translocation . by contrast , the optoelectronic effect allows completely reversible , in - situ control of and translocation speed without permanently altering the surface properties of the nanopore or buffer . moreover , ultra - fast tuning of surface charge by light completely decouples the capture process from subsequent translocation . solid - state lasers , as well as other light sources , can be readily switched on / off in sub-s timescales , and hence induce current jumps in the nanopores within milliseconds ( see si ) . the dynamic range of the photoconductive effect is determined by local n / si stoichiometry , which in turn depends on e - beam exposure during nanopore drilling . we predict that this method will facilitate a broad range of emerging nanopore sensing applications , including genotyping using peptide nucleic acid ( pna ) probes and quantification of epigenetic markers . ultra - fast and reversible modulation of surface charge is fundamentally important for the development of novel nanosensors . by exploiting the intrinsically high sensitivity of the nanopore ionic current to small modulations of surface charge , we were able to explore and characterize a new optoelectronic effect , model the increase in electric conductance under visible laser illumination , and propose an underlying mechanism . we have shown here three useful applications of the technique : ( i ) clearing blocked nanopores and thus significantly extending their useful lifetime ; ( ii ) slowing and tuning the translocation speed of dna ; and ( iii ) detecting and characterizing translocation events of very small proteins , such as ubiquitin . together , these milestones broaden the range of molecules that can be probed by solid - state nanopores , enhance nanopore sensitivity , and extend the lifetime and throughput of individual pores . nanopores were drilled on low - stress ( silicon - rich ) , amorphous lpcv - deposited sinx membranes 30 nm thick . chips were then cleaned using piranha solution and kept in water until mounted on the teflon holder , rendering two independent chambers connected by the nanopore and filled with an electrolytic solution ( 1 m kcl , 20 mm tris , ph 8.0 , 21 c ) . a pair of ag / agcl electrodes was used to apply an electrostatic potential difference across the chambers separated by the insulating sinx membrane . the resulting ionic current through the nanopore was recorded using a patch - clamp amplifier ( axopatch 200b , molecular devices cooperation ) . the whole apparatus was shielded from external electromagnetic noise by a faraday cage . the electrical readout from the amplifier was filtered with a 10 khz low - pass bessel filter and digitized using a 16 bit a / d card ( national instruments ) controlled by a custom - written labview ( national instruments ) program ( see si for a detailed diagram of the apparatus ) . the nano - chip was secured to a closed loop nano - positioner ( physik instrumente ) with sub - nm accuracy , also controlled by the labview program . the nano - positioner was mounted in a custom - built confocal setup , in order to illuminate the nanopore with a focused laser beam . a 532 nm line from a laser diode ( new focus ) was cleaned by a glan - thompson polarizer ( thorlabs ) , with the final power adjusted on demand by a half - wave plate mounted on a motorized rotating holder . this resulted in a final dynamic range of 2 w to 17 mw with 500 intermediate values . the laser beam was expanded to completely fill the back aperture of a 60 , 1.2 na water immersion objective ( olympus uplanapo ) . the emission side contained a long - pass filter ( chroma hq560lp ) to remove any elastically scattered light , followed by a motorized mirror that acted as a shutter for the apds and allowed direct visualization of the membrane using a ccd camera ( thorlabs dc111 ) .

nanopores can be used to detect and analyse biomolecules . however , controlling and tuning the translocation speed of molecules through a pore is difficult , limiting the wider application of these sensors . here we show that low - power visible light can be used to control surface charge in solid - state nanopores and can influence the translocation dynamics of dna and proteins . we find that laser light precisely focused at a nanopore can induce reversible negative surface charge densities as high as 1 c / m2 , and that the effect is tuneable on sub - millisecond timescales by adjusting the photon density . by modulating surface charge , we can control the amount of electro - osmotic flow through the nanopore , which affects the speed of translocating biomolecules . in particular , a few mw of green light can reduce the translocation speed of double - stranded dna by more than an order of magnitude and the translocation speed of small globular proteins such as ubiquitin by more than two orders of magnitude . the laser light can also be used to unclog blocked pores . finally , we discuss a mechanism to account for the observed optoelectronic phenomenon .

Introduction The photo-conductive phenomenon in solid-state nanopores Light-induced retardation of DNA and proteins Light-induced modulation of electro-osmotic flow Nanopore photo-reactivity depends on Conclusions Methods Supplementary Material

the manipulation of electrical surface charge in a rapid and reversible manner is of great importance for a wide variety of biosensing applications , since electrostatic interactions influence the affinity and residence time of biological molecules in the sensing zone . charge manipulation in nanoscale sensors , such as nanopores , nanochannels and nanotubes , is particularly useful because of the scale of these devices . solid - state nanopores are a prototypical class of nanoscale sensors , employing electrophoretic forces to thread and translocate charged single nucleic acids or proteins through a nanoscale aperture in an ultrathin membrane . nanopore surface charge can affect its direct , coulombic interactions with charged biomolecules , and can indirectly influence sensing by generating electro - osmotic flow . this effect has previously been characterized using optical tweezers and by varying the nanopore s zeta potential by changing the solution s ph . the ability to modulate the surface charge of a nanopore and , consequently , the associated electro - osmotic flow could allow control of biomolecule translocation speed . this could in turn lead to the development of advanced single molecule , low - cost characterization techniques for a broad spectrum of clinical samples , including genomic dna , rnas and proteins . yet to date , there has been no experimental report of in situ , opto - electrical manipulation of high - density surface charge in nanopores or other nanoscale sensors . we show here that an off - the - shelf , low - power visible laser beam can be used to directly manipulate the surface charge of a solid - state nanopore . first , it allows the translocation speed of dna to be tuned without chemically modifying the nanopore surfaces or altering the buffer properties . second , it can be used to reliably unblock clogged nanopores , keeping them functional over prolonged periods of time . the continuously recorded ionic current ( i ) flowing through the pore at a fixed voltage reveals an increase when the laser spot overlaps with the nanopore location . a line scan through the image reveals a clear symmetric peak in the ionic current , more than double the baseline current level ( fwhm ~500 nm , right panel ) . this effect is evident in all tested pores ( 4 - 20 nm diameter ) even for laser powers of just a few mw . although a tightly focused ir laser at high power ( ~1 w ) can produce a small increase in current by locally heating the solution , laser - induced heating can be ruled out here for two reasons : the electromagnetic absorption of water at 532 nm is extremely small , and the laser powers used in our experiments are only a few mw . a temperature increase of ~35k would be required to produce the observed increase in i , yet heating is estimated to account for a mere 0.005k per mw of focused light based on measurements performed at 800 nm . in some cases , a series of electrical pulses clears the pore , but often the system must be disassembled and re - cleaned . we find that in many of these cases a short exposure to a ~5 mw focused laser beam will immediately clear the nanopore . as soon as the laser beam reaches the nanopore location , the pore is cleared . we have found that light - induced nanopore unblocking is a highly robust and efficient tool to extend the functional lifetime of solid - state nanopores from a few hours to several days , as detailed in the supporting information ( si ) . over 1,000 translocation events of 10 kbp dna were acquired in a nanopore ( 5.4 nm ) in the dark ( laser power p=0 ) . three features are immediately apparent : ( i ) the open pore current ( io ) and the current during translocation ( ib ) both increased when the laser was turned on , ( ii ) the average event amplitude i = io ib remained at the same level of 0.780.10 na under both conditions , and ( iii ) the mean event dwell - time td increased by roughly a factor of 10 under illumination , as compared to dark conditions . these features are illustrated in figure 2b , which shows io and i as a function of time throughout the experiment ( top panel ) . upon switching off the laser illumination at t=300 s , io returned to base level ( 3.6 na ) and the mean translocation dwell time was restored to ~1 ms , suggesting that this effect is completely reversible . previous studies have sensed proteins such as bovine -lactoglobulin or avidin in solid - state nanopores , and antibodies such as anti - biotin fab fragments or anti - biotin in lipid coated nanopores . however , the detection of small proteins ( mw < 10 kda ) in their native state remains a major challenge , since these proteins translocate through the pore too quickly to produce a resolvable signal given current bandwidth limitations . the dwell time of these spikes was estimated to be < 12 s , but neither this nor the current amplitude of ubiquitin translocations could be accurately determined due to bandwidth constraints even at 100 khz , suggesting that the true translocation dwell time of these proteins is shorter than estimated . the average translocation time increased by at least two orders of magnitude , allowing full characterization of both amplitude and dwell times for ubiquitin translocations . solid - state nanopores possess weak surface charge , which can dominate pore conductivity at salt concentrations lower than ~100 mm . however , it has been theorized that high charge densities on nanopore walls can affect electrical conductivity even under high salt conditions . ionic current is enhanced by the electrical double layer in two ways : ( i ) by creating a local imbalance of counter ions within , which ( ii ) drags water molecules with its motion , creating an electro - osmotic flow ( eof ) which in turn pulls along additional current ( see figure 4a ) . the total ionic current for a nanopore with a charged surface is : ( 1)i = ibulk+idl+ieof where ibulk is the current due to ions outside the electrical double layer , idl is the current due to ions forming the double layer , and ieof is the current due to net water flow induced by net movement of ions within the double layer . ieof is proportional to the product of the flow velocity field and the net charge density (r ) , which is only nonzero within . thus , surface charges can , in principle , explain increased i as well as its linear scaling with d. moreover , if the analyte remains mostly within the bulk pore volume where there is no charge imbalance , distance away from the pore walls , we expect i to be independent of surface charge and thus independent of p. this is consistent with our observations ( figure 2 ) . previous theoretical work has predicted that surface charge on a nanopore will affect ( i ) translocation times and ( ii ) polymer capture rate . moreover , dna capture rate was reduced by 30% , from 11 s to 8 s , for 2 mw of laser light ( see si ) . these observations lead us to hypothesize that visible light can induce negative surface charges on silicon nitride , which in turn slows dna translocation through the drag created by eof moving in the opposite direction of dna . the total ionic current i can be obtained as a function of surface charge density using the following three individual contributions ( see si for full derivation ) : ( i ) ibulk is the electrophoretic movement of each ion species through the nanopore ; ( ii ) idl may be determined from the net charge distribution for a given wall potential , derived analytically from the poisson - boltzmann equation and from the boltzmann distribution , assuming a cylindrical nanopore and using the debye - hckel approximation ; ( iii ) ieof is a product of and the eof velocity profile described by the navier - stokes equation . nanopore diameter d can be determined from tem images , enabling a quantitative estimation of the nanopore photo - reactivity via eq.2 ( see si figure s7 ) . additional dna translocations were performed using a range of dna lengths , nanopores , and laser powers to determine the role of surface charge in slowing translocation speed . the results are summarized in figure 4b ( left ) , where rf is plotted against p for five nanopores ( 4.3 , 5.4 , 5.6 , 6.1 , and 7.4 nm diameters ) and four dna lengths ( 0.4 , 3.5 , 5 , and 10 kbp ) . td was measured for > 1,000 events per laser intensity , and the td histograms were fitted with exponential functions as previously described . 2 as described above to obtain for each of the pores , we then transform the dna rf into surface charge density using the relationship = p. remarkably , all data points collapse onto a single curve ( figure 4b , right ) . controlling the rf value can be achieved either by selecting a highly optically reactive pore ( high ) under low laser intensity , or using a less optically reactive pore under higher laser intensity . moreover , the principal defects in silicon - rich cvd amorphous sinx materials are si dangling bonds , which can trap electrons or holes to become charged . when the n / si ratio of the material surrounding the pore is low enough , we propose that visible light can excite electrons from the ground state across the band gap , trapping them in si dangling bonds . eventually , trapped electrons would recombine with holes , but a high density of arriving photons could maintain a steady state of negatively charged si dangling bonds , creating a net surface charge density . two were exposed to the e - beam for 60 s , and two were exposed for 500 s , producing different local n / si stoichiometry ( see si ) . over 1,000 translocation events of 10 kbp dna were acquired in a nanopore ( 5.4 nm ) in the dark ( laser power p=0 ) . three features are immediately apparent : ( i ) the open pore current ( io ) and the current during translocation ( ib ) both increased when the laser was turned on , ( ii ) the average event amplitude i = io ib remained at the same level of 0.780.10 na under both conditions , and ( iii ) the mean event dwell - time td increased by roughly a factor of 10 under illumination , as compared to dark conditions . upon switching off the laser illumination at t=300 s , io returned to base level ( 3.6 na ) and the mean translocation dwell time was restored to ~1 ms , suggesting that this effect is completely reversible . previous studies have sensed proteins such as bovine -lactoglobulin or avidin in solid - state nanopores , and antibodies such as anti - biotin fab fragments or anti - biotin in lipid coated nanopores . however , the detection of small proteins ( mw < 10 kda ) in their native state remains a major challenge , since these proteins translocate through the pore too quickly to produce a resolvable signal given current bandwidth limitations . the dwell time of these spikes was estimated to be < 12 s , but neither this nor the current amplitude of ubiquitin translocations could be accurately determined due to bandwidth constraints even at 100 khz , suggesting that the true translocation dwell time of these proteins is shorter than estimated . the average translocation time increased by at least two orders of magnitude , allowing full characterization of both amplitude and dwell times for ubiquitin translocations . to elucidate the physical origin of light - induced retardation of translocation , we first characterized the pore current i at varying laser intensity p for a range of nanopore sizes ( 4 - 20 nm diameter , d ) . solid - state nanopores possess weak surface charge , which can dominate pore conductivity at salt concentrations lower than ~100 mm . however , it has been theorized that high charge densities on nanopore walls can affect electrical conductivity even under high salt conditions . ionic current is enhanced by the electrical double layer in two ways : ( i ) by creating a local imbalance of counter ions within , which ( ii ) drags water molecules with its motion , creating an electro - osmotic flow ( eof ) which in turn pulls along additional current ( see figure 4a ) . the total ionic current for a nanopore with a charged surface is : ( 1)i = ibulk+idl+ieof where ibulk is the current due to ions outside the electrical double layer , idl is the current due to ions forming the double layer , and ieof is the current due to net water flow induced by net movement of ions within the double layer . ieof is proportional to the product of the flow velocity field and the net charge density (r ) , which is only nonzero within . thus , surface charges can , in principle , explain increased i as well as its linear scaling with d. moreover , if the analyte remains mostly within the bulk pore volume where there is no charge imbalance , distance away from the pore walls , we expect i to be independent of surface charge and thus independent of p. previous theoretical work has predicted that surface charge on a nanopore will affect ( i ) translocation times and ( ii ) polymer capture rate . moreover , dna capture rate was reduced by 30% , from 11 s to 8 s , for 2 mw of laser light ( see si ) . these observations lead us to hypothesize that visible light can induce negative surface charges on silicon nitride , which in turn slows dna translocation through the drag created by eof moving in the opposite direction of dna . the total ionic current i can be obtained as a function of surface charge density using the following three individual contributions ( see si for full derivation ) : ( i ) ibulk is the electrophoretic movement of each ion species through the nanopore ; ( ii ) idl may be determined from the net charge distribution for a given wall potential , derived analytically from the poisson - boltzmann equation and from the boltzmann distribution , assuming a cylindrical nanopore and using the debye - hckel approximation ; ( iii ) ieof is a product of and the eof velocity profile described by the navier - stokes equation . the contributions to the ion current are : ( 2.1)ibulkenkcl(k+cl)d24lv ( 2.2)idl2dklsinh(ep2)v ( 2.3)ieof2d2l2e2[sinh1(ep2)]2v for elementary charge e , permittivity of aqueous solution , parameter = 1/kbt ( boltzmann constant kb , absolute temperature t ) , pore length l , applied voltage clamp v , and solution viscosity . nanopore diameter d can be determined from tem images , enabling a quantitative estimation of the nanopore photo - reactivity via eq.2 ( see si figure s7 ) . additional dna translocations were performed using a range of dna lengths , nanopores , and laser powers to determine the role of surface charge in slowing translocation speed . the results are summarized in figure 4b ( left ) , where rf is plotted against p for five nanopores ( 4.3 , 5.4 , 5.6 , 6.1 , and 7.4 nm diameters ) and four dna lengths ( 0.4 , 3.5 , 5 , and 10 kbp ) . td was measured for > 1,000 events per laser intensity , and the td histograms were fitted with exponential functions as previously described . these measurements suggest that rf per mw of light widely varies with pore size and dna length . 2 as described above to obtain for each of the pores , we then transform the dna rf into surface charge density using the relationship = p. remarkably , all data points collapse onto a single curve ( figure 4b , right ) . thus , light - induced slowing is dependent only on the induced surface charge density . moreover , the principal defects in silicon - rich cvd amorphous sinx materials are si dangling bonds , which can trap electrons or holes to become charged . when the n / si ratio approaches 0.8 , the band gap is within visible light energies . when the n / si ratio of the material surrounding the pore is low enough , we propose that visible light can excite electrons from the ground state across the band gap , trapping them in si dangling bonds . two were exposed to the e - beam for 60 s , and two were exposed for 500 s , producing different local n / si stoichiometry ( see si ) . we have shown that just a few mw of visible laser light focused on a nanopore can induce surface charge densities up to ~1 c m , much larger than the weak native charge of sinx . increased produces net charge near the pore walls , which , under external voltage , creates an electro - osmotic flow moving in opposition to an anionic translocating molecule . this flow slows translocating dna and proteins , allowing detection and characterization of small analytes , such as ubiquitin . by contrast , the optoelectronic effect allows completely reversible , in - situ control of and translocation speed without permanently altering the surface properties of the nanopore or buffer . moreover , ultra - fast tuning of surface charge by light completely decouples the capture process from subsequent translocation . solid - state lasers , as well as other light sources , can be readily switched on / off in sub-s timescales , and hence induce current jumps in the nanopores within milliseconds ( see si ) . the dynamic range of the photoconductive effect is determined by local n / si stoichiometry , which in turn depends on e - beam exposure during nanopore drilling . ultra - fast and reversible modulation of surface charge is fundamentally important for the development of novel nanosensors . by exploiting the intrinsically high sensitivity of the nanopore ionic current to small modulations of surface charge , we were able to explore and characterize a new optoelectronic effect , model the increase in electric conductance under visible laser illumination , and propose an underlying mechanism . we have shown here three useful applications of the technique : ( i ) clearing blocked nanopores and thus significantly extending their useful lifetime ; ( ii ) slowing and tuning the translocation speed of dna ; and ( iii ) detecting and characterizing translocation events of very small proteins , such as ubiquitin . together , these milestones broaden the range of molecules that can be probed by solid - state nanopores , enhance nanopore sensitivity , and extend the lifetime and throughput of individual pores . the resulting ionic current through the nanopore was recorded using a patch - clamp amplifier ( axopatch 200b , molecular devices cooperation ) . the electrical readout from the amplifier was filtered with a 10 khz low - pass bessel filter and digitized using a 16 bit a / d card ( national instruments ) controlled by a custom - written labview ( national instruments ) program ( see si for a detailed diagram of the apparatus ) . the laser beam was expanded to completely fill the back aperture of a 60 , 1.2 na water immersion objective ( olympus uplanapo ) . the emission side contained a long - pass filter ( chroma hq560lp ) to remove any elastically scattered light , followed by a motorized mirror that acted as a shutter for the apds and allowed direct visualization of the membrane using a ccd camera ( thorlabs dc111 ) .

in the post - war era , there was a pronounced trend towards state intervention in the establishment of universal public healthcare systems in market - based advanced industrial countries . this movement was particularly marked in western europe , but was also evident in canada , australia , and new zealand . however , organised medicine often opposed this development , worrying that its closer proximity to the state through changes in payment systems might , over time , reduce physician income and diminish professional autonomy . between 1960 and 1968 , there were a total of eight doctors strikes in response to the state s increased role in healthcare.1 two of the most sustained and threatening strikes occurred in canada and belgium . in 1962 , canadian doctors organised a twenty - three - day strike , while two years later , belgian doctors launched an eighteen - day strike . in both countries , these strikes re - shaped the subsequent design of universal healthcare , preserving the centrality of a fee - for - service contractual model of physician engagement with patients and the state as opposed to a salaried - employment model . a comparative analysis of the canadian and belgian strikes elucidates some of the common elements of these more extreme cases of physician resistance to collectively funded and administered healthcare.2 these included a commitment to what we call medical liberalism , an individualistic philosophy that was opposed to at least some of the more collectivist assumptions underpinning governing parties that were expanding the welfare state . drawing from the canadian and belgian cases , patient confidentiality , free choice of doctor by the patient , non - interference by state administrators in a doctor s individual clinical judgment , and fees set by individual doctors ( as opposed to the state ) in order to preserve the privacy and privileged nature of the doctor patient relationship . to protect their interests , physicians established new organisations , or refashioned existing organisations , into trade unions capable of opposing governments through collective action including the credible threat of strike . disciplined in their public messaging , these new organisations were also prepared to punish non - compliant physicians in order to ensure solidarity . in summary , they came to perform an overtly political function that differed substantially from the existing self - regulatory bodies focused on the licensing , insuring , and disciplining of members for not meeting professional standards of care and conduct . for physicians , the moral dilemma of whether a strike was consistent with their oaths to provide needed medical care to all patients was ever - present , and explains in part why physician strikes have been the exception rather than the rule in all countries.3 finally , the fact that such collective action also went against the grain of the individualistic underpinning of medical liberalism , also explains the challenge faced by doctors in initiating and sustaining a strike . comparing movements or phenomena across countries , particularly when drawing lessons from history , can be hazardous unless relevant institutional differences as well as similarities are taken into account.4 one important similarity is the fragmented nature of political bodies including organised medicine due to long - standing ethno - linguistic divisions within the respective societies : english french in canada and french dutch in belgium.5 another commonality is the extent to which the medical profession has protected fee - for - service remuneration from reform efforts to adopt salary and capitation forms of remuneration in order to improve the quality of primary healthcare . as a result , the fee - for - service method of payment became embedded in the public healthcare systems of canada and belgium.6 there were also significant differences between the two countries in the 1960s . canada was ( and remains ) a relatively decentralised federation , such that the original flashpoint for the confrontation between doctors and the state occurred in one province , although this conflict had a determinative influence on future national developments . conversely , as a unitary state in the 1960s belgium only became a constitutional federation in 1993 the conflict between organised medicine and the state was at the national level . another important difference is the more statist nature of canada s tax - based , governmental health system relative to belgium s more private , though more corporatist , social insurance system.7 the 1950s marked a critical ideological , sociological , and material transition for organised medicine in both canada and belgium . the medical establishment in both countries resisted key aspects of universal public healthcare . in the first phase immediately following the second world war , this elite discovered that it had less influence in shaping public healthcare policy than it felt it deserved : organised medicine in belgium was even barred from the negotiations that led to compulsory health insurance in 1945 . as physicians improved their economic position in the post - war prosperity of the 1950s , medical organisations in both countries became more ideologically opposed to the welfare state s expansion in general , and to increasing government intervention in the funding and administration of medical care in particular . by the late 1950s , doctors actively resisted the extension and deepening of universal healthcare policies . in canada , a twenty - three - day strike in 1962 forced major compromises from the government , granting doctors the option to work outside the public medical care insurance scheme , and entrenching an arms - length and contractual fee - for - service model of primary and specialised care . in belgium , an eighteen - day strike in 1964 forced a major retreat by the belgian government and the privileging of the fee - for - service model of remuneration . in june 1944 , the first social democratic party to be elected in canada formed the government in the province of saskatchewan and remained in power for the next twenty years.8 with a population of less than a million people , the majority of whom lived in medically under - serviced rural areas , the province of saskatchewan may have seemed an unlikely candidate to initiate north america s first major experiment in universal health insurance . however , it was precisely the rural nature of saskatchewan and the challenge in attracting physicians and providing hospital care that led to the implementation of salaried doctor schemes and publicly owned hospitals at the local government level earlier in the twentieth century.9 moreover , t.c . ( tommy ) douglas , as the provincial head of the social democratic party ( known as the co - operative commonwealth federation , or ccf ) and the premier of the new government , was personally committed to the incremental introduction of comprehensive public healthcare . for this reason , he assumed the additional role of health minister for the first six years of his administration . during this first period , douglas introduced universal hospital coverage for all provincial residents.10 in 1958 , saskatchewan received shared cost financing for its hospital plan from the government of canada , thereby freeing the resources douglas needed to offer universal physician care coverage . both his cabinet and the civil service were mobilised to produce an entirely tax - funded scheme with the government acting as the single payer for all medically necessary physician services.11 however , the government faced significant opposition from doctors to this plan . while physicians had largely co - operated with the provincial government s introduction of universal hospital care insurance in the late 1940s , the profession , and the mindset of its leaders , changed dramatically during the course of the 1950s . in the 1940s , physicians supported the principle of state payment of hospital bills in part because of their own experience with destitute patients and unpaid bills during the great depression , when almost one - third of all provincial residents were on relief due to the collapse of the wheat - based economy . as their incomes rose in the 1950s , many doctors could not see the same benefit flowing from universal medical care insurance.12 by the end of the decade , some 250 out of the 750 practising doctors in saskatchewan were also recent immigrants from the united kingdom , self - styled refugees from the national health service ( nhs ) with a deep antipathy to public healthcare.13 saskatchewan doctors also had collective interests to protect . through their provincial - based organisation they were responsible for two physician - based medical care insurance carriers with 308,000 provincial residents as clients , roughly one - third of the entire provincial population.14 for historical reasons , physicians in saskatchewan were organised differently from most provinces.15 to save money during the great depression , the regulatory administration ( the college ) was fused with the political organisation ( the association ) under a single college of physicians and surgeons of saskatchewan.16 this arrangement forced all members to pay dues to cover both functions , and physicians could not register any potential disagreement with the political goals and strategies of the college by opting out of the political organisation.17 in october 1959 , after months of private discussions within its policy - making subcommittee , the college finally released its judgment on douglas s plan for government - administered , single - payer medical care insurance . at its annual meeting on 29 october 1959 , the college passed a resolution unanimously supported to oppose the introduction of a compulsory government - controlled province - wide medical care plan , and declare our support of , and the extension of health and sickness benefits through indemnity , and service plans.18 stating that doctors would not look kindly on any plan that is introduced without the say of the voters , the college demanded a province - wide referendum so that those opposed to the government plan would have ample opportunity to voice their case , arguing that many doctors would not work under a scheme that was brought in without a referendum.19 on 16 december 1959 , douglas announced his government s plan to introduce a single - payer , compulsory , and universal plan , but only after consultations had been conducted by a committee that included four physicians , three of whom were drawn from the college . he left it to this appointed committee to design a programme that met his requirements , yet somehow be acceptable to a majority of doctors despite the growing resistance from organised medicine in saskatchewan and the rest of canada against single - payer , compulsory , and universal insurance plans.20 operating under the assumption that the college was merely negotiating for better terms under the proposed government plan with what he perceived as exaggerated rhetoric , the douglas government continued on its course , confidant that a majority of doctors would come to accept , if grudgingly , the inevitability of universal medical care insurance.21 douglas publicly stated that his government had no intention of pushing some pre - conceived plan down the doctors throats . i am convinced we will get it.22 he had hoped to secure this co - operation through a representative committee , appointed by the government with college participation , mandated to provide the government with a consensus report . instead , the college used its position on the committee to delay government action , and ultimately prepared a minority report that insisted on government subsidisation of private insurance ( mainly the physician - sponsored insurance carriers ) rather than the introduction of a state - sponsored single - payer plan.23 similar miscalculations were made by the belgian government in an effort to transform its fragmented social health insurance system into a british - style national health system in the early 1960s . the reforms targeted the five health insurance funds ( mutualits ) that had been transformed into compulsory funds by the belgian government in the immediate aftermath of the german occupation during the second world war . these funds were organised in political - ideological groupings , including liberal , socialist , and christian funds . although physicians had argued for decades in favour of neutral funds without alliances to political parties , their views were largely ignored . as a social elite , belgian doctors resented being disregarded , but their own organisational and political weaknesses left them unable to influence the shape of the ultimate plans and prevent an erosion of their unfettered right to set their own fees . instead , the umbrella public institution for the five funds known as lassurance maladie - invalidit , or ami took control of setting fees for both specialists and general practitioners . although there was considerable discussion about reform of belgian social health insurance due to rising costs in the years that followed , it was not until 1959 that reforming the deficit - ridden ami was seriously contemplated . in that year , edmond leburton , the new chair of the socialist health insurance fund , began his campaign for a single , national health service similar to the nhs in britain . in his view , there were two reasons for the growing deficits . the first was the high administrative costs associated with the division of health insurance into five separate and competitive funds . the second was the inflationary impact of fee - for - service practice . in his inaugural speech at the socialist party congress in september 1959 , leburton criticised doctors as profiteers : i can not help thinking they are galeazzi - lisi , the doctor of pius xii , who filmed the last hours of the pope to assure himself of a good income . how many doctors place their own ego above all considerations?24 in pursuit of his dream of a belgian nhs , leburton persuaded lon servais , the minister of social healthcare , to establish a parliamentary working group to re - examine the fundamental principles of public healthcare . five members of parliament from the three major political parties ( socialist , christian and liberal ) negotiated together . as was the case in 1945 , the doctors were not consulted.25 by 1960 , belgium was suffering a recession . the government responded by tightening its belt , which triggered massive protests throughout the country . the catalyst for this upheaval was the loi unique ( single law ) introduced by the christian - liberal coalition government to counter the economic crisis . from opposition benches , the socialist party contested the economic measures incorporated in the loi unique . at the close of 1960 and beginning of 1961 , riots and strikes erupted in belgium . despite this concerted resistance , the loi unique was ratified on 13 january 1961.26 some of the new budgetary constraints directly affected doctors . to counter the abuses of the ami , the loi unique placed the five health funds under stricter administrative controls that fixed the fees for all physician consultations . further , article 52 of the loi unique stipulated that doctors who did not comply with the maximum rates would be subject to fines and , in extreme cases , jail sentences.27 enraged by these measures , belgian physicians founded the contact commission to present their complaints in a single voice to the government , but the commission s objections were ignored . on 26 march 1961 , national elections yielded a new socialist christian coalition government , and leburton was appointed minister of social healthcare.28 this regime change precipitated an even more hostile relationship between the belgian government and organised medicine . it would also signal to physicians the necessity of establishing a more effective political organisation if they were to have any chance of preventing the introduction of socialised medicine . in this , they found an ally in the liberal party , now in opposition to the coalition , but the doctors ultimately concluded that they needed to be prepared to go it alone in the coming struggle with the government . though not identical , the more immediate factors precipitating the mobilisation of physicians in canada and belgium were similar . in the canadian case , it was the determination of the social democratic government of saskatchewan to implement universal , single - payer medical care insurance . in the belgian case , it was the determination of the socialist christian coalition government and its socialist minister of health to reform public healthcare without input from the medical profession . although physicians in saskatchewan , assisted by the canadian medical association , already had a disciplined political organisation to battle the government , physicians in belgium had to create the political structure for waging a disciplined and effective campaign against the government . in both cases , the governments presumed that the doctors would not have the cohesiveness and discipline to conduct a sustained extra - parliamentary struggle against their respective health system reforms . to accomplish this , organised medicine in both countries employed remarkably similar strategies and tactics which included establishing : a commanding and controlling political organisation ; pyramidal communications with members ; public information campaigns ; centralised fund raising ; and effective penalties on hesitant or dissident physicians . by the end of the 1950s , doctors in the rest of canada realised that saskatchewan was about to become the battleground that would determine the practice of medicine within a redesigned payment system administered by the state . as a consequence , the canadian medical association ( cma ) , the national umbrella organisation for the provincial medical associations , provided considerable financial and in - kind support to saskatchewan doctors in their struggle against the provincial government.29 the college spent more on the 1960 campaign than either the social democrats ( known as the co - operative commonwealth federation or ccf ) or the opposition liberal party . in addition , the ontario medical association the largest and most powerful of the provincial chapters of the cma sent its public relations guru to saskatchewan to assist the college in its media blitz . indeed , the struggle in saskatchewan was perceived to be the frontline of a larger north american war , and the american medical association ( ama ) also provided financial support to saskatchewan physicians . more important was what the college in saskatchewan and the cma learned from the ama s experiences in its long struggle against health system reform in the united states . in particular , the college would adopt the ama s key man system , creating cells of up to ten doctors under the control and direction of a well - known and trustworthy doctor ( hence the key man ) who would be solely responsible for receiving and sending communications up and down in the organisation.30 premier douglas of saskatchewan recognised the high stakes when he restated his determination to implement universal , compulsory , single - payer medical care insurance by 1961 : if we can do this and i feel sure we can then i would like to hazard the prophecy that before 1970 almost every other province in canada will have followed the lead of saskatchewan as we shall have national health insurance programs for [ canada as a whole].31 although douglas had intended his proposed scheme of medical care insurance to be the leading issue in the provincial election of june 1960 , he did not anticipate the extent to which the college was willing to turn the election into a referendum on this single issue.32 the college levied a mandatory fee of $ 100 on all its members to fund its extensive public relations campaign for the election . it delivered publicity kits to all doctors to ensure that the media and the public received consistent messages from its members.33 one example of the college s approach was a draft speech it sent to its members on 5 may 1960 . while individual doctors had the flexibility to change wording into their own phraseology , the college warned members not to stray from the hard line message : saskatchewan s 930 doctors are unalterably opposed to the introduction of a government controlled compulsory prepaid medical care plan , or in plain language , state medicine . our main reasons for opposition are based on two factors : compulsion , and government control . we are convinced that these factors would be responsible for lowering of the standards of medical care for our patients . our position may be defined as unalterable.34 saskatchewan s 930 doctors are unalterably opposed to the introduction of a government controlled compulsory prepaid medical care plan , or in plain language , state medicine . our main reasons for opposition are based on two factors : compulsion , and government control . we are convinced that these factors would be responsible for lowering of the standards of medical care for our patients . our position may be defined as unalterable.34 the college also purchased advertisements in local community newspapers similar to this notice in the humboldt journal : we would all like to continue practising in humbolt as your private family physician ; however , if state medicine is forced upon us by any government , you can see that we would have no choice other than to move to a province which would allow us our freedom.35 there was general approval for a tax - funded medical care insurance plan , despite a large and vocal dissenting minority a national poll suggested that sixty per cent of canadians supported tax - funded medical care insurance in 1960.36 this popular support helped carry douglas and his social democratic party to victory in the election . believing he had received a mandate from the people of the province to move ahead with his plan , douglas demanded a report from the deadlocked advisory planning committee . sympathetic to the government plan and in support of salary or capitation replacing fee - for - service remuneration attempted a last compromise with the college minority , and accepted fee - for - service remuneration for doctors.37 though the college representatives on the committee acknowledged the concession , the effort ultimately produced a majority minority report that was delivered in september 1961.38 beyond universal and compulsory coverage , the majority also recommended that the lion s share of funding come from general tax revenues , with only a small share raised through flat tax premium payments.39 the douglas government responded swiftly to the report . in october 1961 , the saskatchewan medical care insurance act , 1961 , was introduced into the provincial legislature without consultation with the college . although the government had correctly determined that further negotiation with the college would only lead to further delay and deadlock , the doctors were able to point to the lack of consultation as evidence of the government s unwillingness to compromise , thereby creating a further rift between the profession and the government . in the provincial legislature , tommy douglas led the debate on the bill , but just before the law was passed , he resigned as premier to become the first national leader of the newly formed social democratic party the new democratic party ( ndp ) . the national ndp , coupled with its provincial branches and organised labour unions throughout canada , now had a major political stake in successful health system reform in saskatchewan . in the same way that the college could depend on the active support of the cma , the insurance industry , and business lobbies throughout the country , the saskatchewan government could rely on the national ndp and ndp oppositions to provincial governments as well as affiliated trade unions throughout canada.40 woodrow lloyd replaced douglas as party leader and premier of saskatchewan . he and his newly appointed minister of health tried to reopen discussions with the college s leaders , but they mistook lloyd s willingness to negotiate as an indication of weakness that only hardened their position . however , by april 1962 , the lloyd government appeared ready to give way on two key issues . in a letter to the college , lloyd suggested that the physician - based private insurance plans might be allowed to continue under some strict understandings concerning the package of benefits , the amount assessed for premiums , and the remuneration of doctors . second , despite his grave concerns about the potential impact on the principle of universality , lloyd would allow doctors to opt out of the plan and directly charge patients . this was , in his own words , a desperate measure to prevent the threat of withdrawal of services on july 1st.41 although the government had seemingly conceded the principle of single - payer administration , the college rejected the compromise as not fundamentally altering the legislation , and offered its own final offer. the college insisted that the only satisfactory plan would involve government subsidies to private insurance carriers , in which doctors would be permitted to bill all patients ( except indigents).42 in order to force the government to withdraw its law , the college threatened a province - wide doctors strike . to demonstrate membership support , the college asked all available doctors in the province to attend a two - day emergency meeting in regina in early may 1962 . almost 600 doctors two - thirds of the province s 900 physicians closed their offices in order to attend . realising the danger posed by organised medicine s escalating militancy , premier lloyd asked if he could also address the meeting : the college leaders agreed , expecting that the doctors attending would impress upon lloyd the profession s readiness to strike if their demands were not met . the raucous , anti - government tenor of the meeting was buoyed considerably by the news that a cabinet minister a former minister of health no less had suddenly resigned from the government . when lloyd took the stage , he was hissed and jeered . immediately following lloyd s hour - long appeal to the unreceptive audience , the college s president called for a standing vote on a motion stating physicians would refuse to practise under the government s new scheme . all but five doctors stood and applauded enthusiastically in support.43 it was now evident that a majority of doctors were prepared to go on strike . a similar mass meeting in brussels in 1964 would also publicise the extent to which the majority of belgian doctors supported strike action . unlike the situation in canada , however , before this could occur , a more radical and pan - belgian political organisation for doctors needed to be forged.44 it is significant that most belgian doctors were deeply opposed to the health system reforms proposed by the new christian although the fdrations mdicale belge ( belgian medical federation , or fmb ) was a century old , it was ineffective as a political organisation because of linguistic strife and professional conflicts . for example , a separate flemish organisation ( algemeen syndicaat der geneesheren van belgi , or as ) had been established in 1954 , followed by a separate organisation for specialists that same year , and one for generalists in 1959.45 facing edmond leburton as minister of health , belgian physicians began to construct a more unified and organised opposition . unable to influence the government directly , the doctors formed a new national organisation , the association gnrale des mdecins belges ( general association of belgian physicians , or agmb ) , that clustered the old fmb with the younger , breakaway organisations.46 in july 1961 , the agmb published a white paper criticising the loi unique as well as the parliamentary working group s recommendations . the doctors also issued their own ami reform proposal , and demanded to be involved in all future governmental deliberations on the ami . the physician leaders also warned the parliamentary working group of the consequences that might flow from health system reforms that ignored the position of the doctors as articulated in the white paper.47 the parliamentary working group initially dismissed the agmb s requests , but as tensions mounted , leburton reluctantly entered negotiations with five agmb representatives . however , the protocol of 20 october 1961 was signed by the five agmb negotiators without the knowledge , much less the consent , of the majority of their members . most belgian doctors learned of the pact through the media.48 stunned , the doctors attacked the credibility and legitimacy of the negotiators after the public announcement . the christian health fund also opposed the protocol because it had been excluded from the negotiations.49 these objections convinced the rest of the cabinet to abandon leburton by not approving the protocol . the consequences were even more severe for the future of the agmb and its negotiators . the generational divide among belgian doctors helped fuel a revolt within the ranks of organised medicine . a younger group of doctors rejected the old guard s leadership and its efforts to create a more effective political organisation at the national level . in 1960 most of these doctors had been obliged to interrupt their studies because of the second world war , when many of them were anti - german resisters in the belgian underground or the maquis as it was known.50 these experiences would come to be formative in the decision to strike and in the deployment of effective strike tactics.51 in the summer of 1961 , thirty - six of these younger doctors held two secret meetings in luxembourg.52 these meetings would mark the beginning of a fundamental reconstitution of organised medicine in belgium.53 while the older doctors were more amenable to working out an accommodation with the government , their junior colleagues were unwilling to compromise , preferring to launch a general strike in order to force the government to concede . to circumvent future compromise , the self - styled action committee of younger physicians prepared a letter instructing all doctors to not sign anything from the agmb . in the view of the young activists , the time for negotiation had ended , and the time for battle had arrived.54 on 30 may 1962 the first ( lige - luxembourg ) of five syndical chambers of medicine was officially founded.55 these chambers were more overtly political and militant than any previous organisation , borrowing heavily from methods of the belgian resistance in the second world war . despite their underground nature , the five chambers would successfully recruit a majority of doctors such that , on the eve of the 1964 strike , they represented ninety per cent of belgian doctors . the chambers had two essential features.56 first , the members were divided into cells of ten doctors . each cell had one representative who was in direct contact with the direction committee and acted as an intermediary . this structure was derived from the cell structure of the belgian underground in the second world war , inspired in turn by the communist - led partisan movement.57 second , all critical communications were funnelled through a pyramidal telephone structure in which the chambers leaders would communicate with the appropriate cell representative who would then relay messages to the cell members . although personal meetings and written communications were generally avoided , any circular letters were prepared in both french and dutch to ensure solidarity across the language divide . in addition , generalists and specialists were treated equally within the chamber . unlike organised medicine in canada , previous physician organisations in belgium did not have an expert central office to manage finances and raise external funds , or manage internal communications and external publicity . the chambers addressed this weakness by establishing a technical bureau directed by a secretary - general and staffed by lawyers , economists , tax specialists , and public relations advisors . this remarkable shift went unnoticed by the government , and cloaked the older organisations particularly the agmb that continued to operate as if nothing had changed . in fact , the government upheld the status of the agmb as the legitimate bargaining agent for doctors , even though real authority and legitimacy had shifted to the syndical chambers . in september 1962 , when leburton reopened negotiations through a new parliamentary working group , he invited the same five agmb representatives with whom he had drafted the stillborn protocol of 1961 . moreover , when belgian doctors formally dissolved the agmb two months later , the government interpreted this action as an indication of fragmentation and disorganisation among physicians that could be exploited by ignoring the profession when formulating a new bill.58 the most important objective of the government was cost containment . to achieve this , the government insisted on regulating doctors fees through a state institution , the institut national dassurance maladie - invalidit ( national institute for sicknesss and disability , or inami ) . the regulations , due to come into force on 1 january 1964 , stipulated that any doctors audited by the inami would be required to provide full information concerning their patient billings.59 when the draft bill was introduced , the chambers discovered that the five agmb representatives had once again approved the reform.60 in its place , the chambers created a new national emergency organisation , the national committee for common action ( comit national daction commune , or cnac ) , representing all existing medical associations.61 henceforth , the cnac would be the doctors official representative in negotiations , but still keeping the chambers largely hidden from the government . by august 1963 , while the chambers had no interest in further negotiations , they did everything possible to delay passage and implementation of the law . meanwhile , the government continued to negotiate with the old physician organisations , a convenient distraction while the chambers prepared their members for a nationwide strike.62 on 19 october 1963 , the first mass meeting of belgian doctors protested the loi leburton . more than 4,500 doctors ( out of a total of 12,665 doctors in belgium at that time ) attended from every region of the country , all stating they could never agree to the loi leburton.63 governments in canada and belgium were unprepared for the cohesiveness and effectiveness of the doctors strikes . doctors were organised in cells in order to facilitate rapid and confidential communications , while isolating potential dissidents and minimising their influence . they also used modern communications techniques in order to stay on message when dealing with government , media , and the general public . finally , organised medicine in both countries used social ostracism as a weapon against dissident doctors who refused to toe organised medicine s line or , even worse , engaged in active strikebreaking . following their mass meeting in may 1962 , saskatchewan doctors , aided and abetted by organised medicine in the rest of canada , prepared for a general strike . the college publicly announced that , if the government did not accept its final demand for a voluntary system of private medical care insurance supplemented by public subsidy , the majority of doctors would go out on strike on 1 july 1962.64 the college s leadership selected the doctors who would exit and the doctors who would remain to provide emergency services . the college also determined which of a minority of hospitals would remain staffed with doctors , thus precipitating the temporary closure of the rest.65 between april and july , a number of keep our doctors ( kod ) committees were established in order to support the doctors . originally started by married women concerned about the possible loss of doctors in their communities and the impact this would have on their families , these kod committees mushroomed in number and visibility in the weeks leading up to the strike . these committees may have managed to recruit almost ten per cent ( 74,000 people ) of the population by the time of the strike.66 the kod committees were quickly infiltrated by anti - government forces including business organisations , the college , as well as opposition parties . kod rallies and meetings were held regularly in the weeks just before the strike , elevating the growing hysteria about the impending strike deadline . although trade unions and church groups that supported the government s position began to set up their own pro - reform civil society organisations , premier lloyd discouraged them from engaging too visibly or directly for fear that this would simply elevate the already dangerously high level of public hysteria.67 they complied , but their absence from the field only exaggerated the anti - government voice , making the college even more confident that the government would back down on the eve of the strike , a major miscalculation.68 realising that a strike was highly likely , the government began to recruit foreign doctors to take the places of the saskatchewan doctors . calculating that it could only rely on the support of between fifty and one hundred saskatchewan doctors , and that the majority of canadian doctors outside the province would be hesitant to provide relief because of the influence of the cma , the saskatchewan government began to recruit doctors abroad , particularly in the united kingdom . through its influential contacts in the british medical association ( bma ) , the cma did everything it could to discourage british doctors from emigrating to saskatchewan . on 17 may 1962 , for example , the general secretary of the cma wrote to the under secretary of the bma : our colleagues in saskatchewan are as near unanimous as it is possible to be that they can not practise under the act and retain professional freedom . i think it would be unkind and misleading for british applicants to be subjected to these conditions of practice and i am afraid that they would be regarded as strike breakers by saskatchewan doctors.69 our colleagues in saskatchewan are as near unanimous as it is possible to be that they can not practise under the act and retain professional freedom . i think it would be unkind and misleading for british applicants to be subjected to these conditions of practice and i am afraid that they would be regarded as strike breakers by saskatchewan doctors.69 the government also made emergency plans to airlift doctors from other countries , including the united states , in the event of a prolonged strike . even before the strike , doctors began arriving from the united kingdom , generating even more animosity between the government and the majority of doctors in the province . these new doctors also deepened the cleavage within the profession between those who supported the government and the majority against , a bitter division that was accentuated with the establishment of community clinics in those cities and towns in which pro - reform citizens opened offices for doctors who were willing to practise during the strike . to help set up the clinics , the government provided seed money to community groups to rent new offices and recruit doctors supportive of health system reform.70 despite an attempt at mediation by the saskatchewan hospital association in the last days of june , the strike began as planned on 1 july 1962 . up to this point , the events leading up to the strike had been followed closely by the provincial media and , to a lesser extent , by the media in the rest of canada . on the first day of the strike , however , media from the rest of the world , particularly american and british newspapers , magazines , and medical journals had their own journalists cover the confrontation.71 in the words of the london observer , saskatchewan has become a battlefield for the whole north american continent in the struggle of the big medical organisations against socialised medicine.72 there was a clear divide in the media with the majority of the provincial media community newspapers and local radio and television stations supporting the doctors , and only a minority supporting the government . in the rest of canada , however , the majority of newspapers and the national television stations leaned in favour of the government position , arguing that the doctors had no right to strike against a democratically elected government and its decision to reform its health system . they were joined in this view by a number of international publications , including the lancet which , while it had been sympathetic enough to the saskatchewan doctors to bar the government from placing recruitment advertisements for british physicians to practise in saskatchewan , nonetheless stated its objections to the college acting as a state within a state.73 as the media tide turned against the college , a small group of clerical leaders in the province met with premier lloyd and his inner cabinet ministers in an effort to come up with a compromise they could sell to the college in order to bring the strike to an end . distinct possibilities , but in return the college had to be prepared to get its members to return to work immediately.74 however , the doctors were not prepared to consider ending the strike until after the failure of a major rally sponsored by the kod on 11 july in front of the legislative assembly in the capital city of regina . although the kod groups and the college had expected between 25,000 and 50,000 to come to regina , only 5,000 people actually appeared on the day of the protest , deflating the spirit of the doctors and adding resolve to the position of the government . at a press conference towards the end of the protest , premier lloyd thanked the protestors for avoiding violence even while expressing their disagreement with the government . however , he castigated the kod for impugning the motives and competencies of doctors from the united kingdom who had left busy practices at home on short notice , often at personal sacrifice and accused the college of exercising its policing powers under the medical professions act as an offensive weapon against practitioners who were not supporting the strike and announced the establishment of a royal commission to investigate the college s actions.75 after the protest of 11 july , some physicians returned to work , and the college realised that its ability to continue the strike was waning . for its part , the government invited lord stephen taylor , a physician , member of the british house of lords , and a former labour party minister , to saskatchewan to advise on a solution . although not invited by the college , lord taylor quickly gained the trust of the doctors and acted as an intermediary between the college and the government . listening patiently to the criticisms of the doctors and rewording the concessions previously made by the government while refusing to let either side deal with the other directly to avoid what he felt would be inevitable misunderstandings lord taylor helped facilitate a deal that became known as the saskatoon agreement . signed by both parties on 23 july 1962 , the compromise protected the right to fee - for - service remuneration and allowed the physician insurance carriers to process cheques but not underwrite insurance on behalf of doctors refusing to engage with the government directly.76 with these two concessions , the government obtained what it wanted tax - based , universal medical care insurance . although the doctors could argue that they had won a victory in terms of the continued legal existence of their private insurance carriers , the new payment mechanism was , in reality , a single - payer public system . twenty - one months after the strike in canada , the doctors strike in belgium would take a very similar course , though the outcome would be interpreted very differently . as in the canadian case , the strike focused on the new law . though the belgian government continued to seek the approval of physicians for the loi leburton , even negotiating with organisations such as the cnac , these efforts would only backfire . instead , physicians used these desperate efforts at conciliation to buy time to prepare for a major strike . despite the fact that most of organised medicine welcomed the delay , some doctors were not willing to wait . to protest the law , 1,270 doctors from lige resigned from the order of doctors , preventing them from practising medicine.77 they hoped that their action would be followed in the rest of the country but they had miscalculated . it was only a matter of months , perhaps weeks , before the government realised that a doctors strike was being planned . once this occurred , the government was more than capable of scaring off the less militant doctors by setting out the sanctions that would be meted out to physicians who joined the strike.78 at the same time , the chambers still needed to recruit more members in regions of the country where they were weaker , particularly in flanders . in january , the cnac was abolished and the five chambers went above ground as the political representative of the doctors . by the end of march 1964 , following a second mass meeting of eight thousand doctors , the chambers revealed that they had recruited ninety per cent of belgian doctors.79 the show of strength convinced the government to delay passing the bill . in a final attempt to avoid a general strike , the government held a marathon meeting with the doctors on 31 march in order to obtain a compromise , but to no avail.80 the doctors had no intention of negotiating a settlement and had already committed to packing their suitcases in the early hours of 1 april 1964 to leave en masse for towns just beyond the belgian border , such as clervaux and mondorf in luxembourg , valkenburg in holland , and valenciennes in france.81 at four in the morning , dr thon , on behalf of the five chambers , released a press statement announcing that belgian doctors in the country had commenced total and unlimited strike.82 having managed to keep their strategy and preparations secret , the chambers quickly discovered that their general strike shocked the government , the press , and the public.83 on the ground , physicians were divided into three categories : 1 ) the majority who went on vacation ; 2 ) the commandos who had to remain in place in order to carry out any orders from the leadership including forcing recalcitrant doctors to go on vacation , and treating those that refused or disappeared as deserters;84 and 3 ) the guards who had to remain on active duty at the hospitals in order to decide whether patients needed emergency care during the strike . the bureaux of the chambers were responsible for supervising the strike as well as co - ordinating publicity and the printing of pamphlets . as in the canadian case , the belgian doctors strike was subject to saturation reporting by the media . each day , nir , belgium s public television station , broadcast daily news and images of the strike . on radio stations , proponents and opponents of the strike confronted and debated each other . reporting constantly on the strike , the majority of newspapers including the extensive catholic press took an editorial position against the strike , although a minority in particular the liberal press supported the doctors.85 the strike would have a direct impact on the lives of most belgians . no longer able to consult their own physicians , they had to rely on doctors providing emergency care in hospitals . , such guards generally erred on the side of caution in terms of admitting patients . the end result was that the hospitals were overcrowded within two days of the strike . in response , the government opened the military hospitals , over which it had control , to the general population . for those belgians who did not need or seek medical help ambulances raced through the cities with blaring sirens while the media focused on individual incidents highlighting the danger to health and life posed by the strike.86 with little or no evidence to back up their respective claims , both sides used such incidents to prove the extent to which either the strike or the government was endangering belgians through their respective actions.87 despite public pressure on both sides , the reopening of negotiations seemed impossible in the first week of the strike . both sides issued ultimatums , the government insisting that it would only negotiate once the doctors returned to work , and doctors insisting on major revisions to the law before they would call an end to the strike . then , on 9 april , the rectors of the four belgians universities intervened , offering to mediate in an effort to trigger negotiations . the parties agreed to sit down and talk on the weekend of 1112 april but this first effort failed due to one careless remark to the media by prime minister tho lefvre : jespre que lon ne devra pas appeler ce temps , le temps des assassin [ i hope we will not have to call this period , the time of assassins].88 when this comment was passed on to all doctors by telephone , they threatened to abolish guard duties at the hospital in retaliation.89 in response , the government panicked , and drew up new military guidelines facilitating the conscription of doctors in order to bring the strike to a rapid end . the new orders demanded that all doctors who were part of the belgian military reserve had to report in uniform to their respective commanding officers in order to be assigned for hospital duty . , the five chambers advised the doctors being called up not to obey orders that were meant to sabotage the strike.90 the government s desperate ploy marked a turning point in the strike . although its position had initially been accepted by a majority of the public and media , the national government faced increasing criticism as the strike continued . at the same time , however , the pressure of the strike began to produce cracks within the leadership and among the membership of organised medicine . for some , the purpose was to achieve their objective in ensuring that any health system reform did not change existing freedoms in terms of billing . for others , these weaknesses pushed both sides back to the bargaining table with the four university rectors the following weekend , 1718 april , at the end of which the government retreated on its position and the doctors agreed to end the strike.91 since the weekend settlement met most of the doctors demands , it was interpreted as a victory for the chambers and a major defeat for the government.92 however , as in the case of canada where the opposite was perceived to have occurred , the long - term consequences were more nuanced and less favourable to the perceived victors . both versions of medical liberalism defended the right of doctors to determine the nature of their relationship with patients , especially their untrammelled right to set their own fees , and attacked more collectivist philosophies that viewed healthcare as a public good and right of citizenship rather than a market service provided by physicians to clients or consumers . rooted in a nineteenth - century conception of the freedom to contract and a limited state , the tenets of medical liberalism clashed with the more collectivist philosophies propounded by social democratic , democratic ( non - revolutionary ) socialist , and similar centre the 1960s marked the apex of the expansion of the welfare state in the advanced industrial countries of the west , an expansion led by centre while this trend was resisted by organised medicine in numerous countries , the most formidable resistance came in the form of the lengthy and bitter doctors strikes in canada and belgium . these respective strikes would mark both polities and shape the evolution of public healthcare in the future by ensuring that doctors remained private , independent contractors with the state . in both countries , fee - for - service remuneration remains the dominant system of payment for physicians , and a semi - permanent obstacle to government - led reforms of primary healthcare . these longer - term consequences seem to clash with more contemporary evaluations of the outcomes of the strike in both countries . in canada , the government was generally perceived to have won the strike . however , in reality , organised medicine was able to improve the economic position of its members even while it preserved the contractual system of remuneration and private practice , protected the role of physicians at the centre of the healthcare system , and prevented major changes to primary healthcare.93 in a manner similar to the british medical association s ability to exploit divisions within successive british governments , the canadian medical association and its provincial chapters were effective in consolidating their gains and preventing subsequent governments from initiating further reform , in part because of the fear of another strike by physicians.94 in belgium , the doctors were perceived to have won the strike . in an important sense , this was an accurate perception in that belgian physicians , like their canadian counterparts , preserved the contractual system of fee - for - service remuneration and private practice and protected the role of physicians as the central players in the belgian health system . in particular , organised medicine in belgium had prevented a british - style national health system from being erected . however , unlike the canadian case , the doctors were unable to prevent the belgian government from placing controls on physician remuneration in order to contain cost , and in this respect , could not supplant the continued supremacy of the social insurance funds .

organised medicine in a number of advanced industrial countries resisted the post - war trend toward more state involvement in the funding and organisation of medical care . while there were eight doctors strikes during the peak of reform efforts in the 1960s , two of the most prolonged and bitter struggles took place in canada and belgium . this comparative analysis of the two strikes highlights the philosophy , motives , and strategies of organised medicine in resisting state - led reform efforts . although historical and institutional contexts in the two countries differed , organised medicine in canada and belgium thought and responded in very similar ways to the perceived threat of medical insurance reform . while the perception of who won and who lost the respective doctors strikes differed , the ultimate impact on the trajectory of public healthcare on the medical profession was remarkably similar . in both countries , the strike would have a long - standing impact on future reform efforts , particularly efforts to reform physician remuneration in order to facilitate more effective primary healthcare .

Introduction From Ineffective Resistance to Effective Opposition, 194559 Mobilising Forces and Preparing for Strike The Conduct of the Doctors Strikes Conclusion and Consequences

in the post - war era , there was a pronounced trend towards state intervention in the establishment of universal public healthcare systems in market - based advanced industrial countries . between 1960 and 1968 , there were a total of eight doctors strikes in response to the state s increased role in healthcare.1 two of the most sustained and threatening strikes occurred in canada and belgium . drawing from the canadian and belgian cases , patient confidentiality , free choice of doctor by the patient , non - interference by state administrators in a doctor s individual clinical judgment , and fees set by individual doctors ( as opposed to the state ) in order to preserve the privacy and privileged nature of the doctor patient relationship . for physicians , the moral dilemma of whether a strike was consistent with their oaths to provide needed medical care to all patients was ever - present , and explains in part why physician strikes have been the exception rather than the rule in all countries.3 finally , the fact that such collective action also went against the grain of the individualistic underpinning of medical liberalism , also explains the challenge faced by doctors in initiating and sustaining a strike . comparing movements or phenomena across countries , particularly when drawing lessons from history , can be hazardous unless relevant institutional differences as well as similarities are taken into account.4 one important similarity is the fragmented nature of political bodies including organised medicine due to long - standing ethno - linguistic divisions within the respective societies : english french in canada and french dutch in belgium.5 another commonality is the extent to which the medical profession has protected fee - for - service remuneration from reform efforts to adopt salary and capitation forms of remuneration in order to improve the quality of primary healthcare . as a result , the fee - for - service method of payment became embedded in the public healthcare systems of canada and belgium.6 there were also significant differences between the two countries in the 1960s . another important difference is the more statist nature of canada s tax - based , governmental health system relative to belgium s more private , though more corporatist , social insurance system.7 the 1950s marked a critical ideological , sociological , and material transition for organised medicine in both canada and belgium . the medical establishment in both countries resisted key aspects of universal public healthcare . in the first phase immediately following the second world war , this elite discovered that it had less influence in shaping public healthcare policy than it felt it deserved : organised medicine in belgium was even barred from the negotiations that led to compulsory health insurance in 1945 . as physicians improved their economic position in the post - war prosperity of the 1950s , medical organisations in both countries became more ideologically opposed to the welfare state s expansion in general , and to increasing government intervention in the funding and administration of medical care in particular . while physicians had largely co - operated with the provincial government s introduction of universal hospital care insurance in the late 1940s , the profession , and the mindset of its leaders , changed dramatically during the course of the 1950s . in the 1940s , physicians supported the principle of state payment of hospital bills in part because of their own experience with destitute patients and unpaid bills during the great depression , when almost one - third of all provincial residents were on relief due to the collapse of the wheat - based economy . as their incomes rose in the 1950s , many doctors could not see the same benefit flowing from universal medical care insurance.12 by the end of the decade , some 250 out of the 750 practising doctors in saskatchewan were also recent immigrants from the united kingdom , self - styled refugees from the national health service ( nhs ) with a deep antipathy to public healthcare.13 saskatchewan doctors also had collective interests to protect . through their provincial - based organisation they were responsible for two physician - based medical care insurance carriers with 308,000 provincial residents as clients , roughly one - third of the entire provincial population.14 for historical reasons , physicians in saskatchewan were organised differently from most provinces.15 to save money during the great depression , the regulatory administration ( the college ) was fused with the political organisation ( the association ) under a single college of physicians and surgeons of saskatchewan.16 this arrangement forced all members to pay dues to cover both functions , and physicians could not register any potential disagreement with the political goals and strategies of the college by opting out of the political organisation.17 in october 1959 , after months of private discussions within its policy - making subcommittee , the college finally released its judgment on douglas s plan for government - administered , single - payer medical care insurance . at its annual meeting on 29 october 1959 , the college passed a resolution unanimously supported to oppose the introduction of a compulsory government - controlled province - wide medical care plan , and declare our support of , and the extension of health and sickness benefits through indemnity , and service plans.18 stating that doctors would not look kindly on any plan that is introduced without the say of the voters , the college demanded a province - wide referendum so that those opposed to the government plan would have ample opportunity to voice their case , arguing that many doctors would not work under a scheme that was brought in without a referendum.19 on 16 december 1959 , douglas announced his government s plan to introduce a single - payer , compulsory , and universal plan , but only after consultations had been conducted by a committee that included four physicians , three of whom were drawn from the college . he left it to this appointed committee to design a programme that met his requirements , yet somehow be acceptable to a majority of doctors despite the growing resistance from organised medicine in saskatchewan and the rest of canada against single - payer , compulsory , and universal insurance plans.20 operating under the assumption that the college was merely negotiating for better terms under the proposed government plan with what he perceived as exaggerated rhetoric , the douglas government continued on its course , confidant that a majority of doctors would come to accept , if grudgingly , the inevitability of universal medical care insurance.21 douglas publicly stated that his government had no intention of pushing some pre - conceived plan down the doctors throats . instead , the college used its position on the committee to delay government action , and ultimately prepared a minority report that insisted on government subsidisation of private insurance ( mainly the physician - sponsored insurance carriers ) rather than the introduction of a state - sponsored single - payer plan.23 similar miscalculations were made by the belgian government in an effort to transform its fragmented social health insurance system into a british - style national health system in the early 1960s . though not identical , the more immediate factors precipitating the mobilisation of physicians in canada and belgium were similar . in the belgian case , it was the determination of the socialist christian coalition government and its socialist minister of health to reform public healthcare without input from the medical profession . to accomplish this , organised medicine in both countries employed remarkably similar strategies and tactics which included establishing : a commanding and controlling political organisation ; pyramidal communications with members ; public information campaigns ; centralised fund raising ; and effective penalties on hesitant or dissident physicians . in particular , the college would adopt the ama s key man system , creating cells of up to ten doctors under the control and direction of a well - known and trustworthy doctor ( hence the key man ) who would be solely responsible for receiving and sending communications up and down in the organisation.30 premier douglas of saskatchewan recognised the high stakes when he restated his determination to implement universal , compulsory , single - payer medical care insurance by 1961 : if we can do this and i feel sure we can then i would like to hazard the prophecy that before 1970 almost every other province in canada will have followed the lead of saskatchewan as we shall have national health insurance programs for [ canada as a whole].31 although douglas had intended his proposed scheme of medical care insurance to be the leading issue in the provincial election of june 1960 , he did not anticipate the extent to which the college was willing to turn the election into a referendum on this single issue.32 the college levied a mandatory fee of $ 100 on all its members to fund its extensive public relations campaign for the election . sympathetic to the government plan and in support of salary or capitation replacing fee - for - service remuneration attempted a last compromise with the college minority , and accepted fee - for - service remuneration for doctors.37 though the college representatives on the committee acknowledged the concession , the effort ultimately produced a majority minority report that was delivered in september 1961.38 beyond universal and compulsory coverage , the majority also recommended that the lion s share of funding come from general tax revenues , with only a small share raised through flat tax premium payments.39 the douglas government responded swiftly to the report . in the same way that the college could depend on the active support of the cma , the insurance industry , and business lobbies throughout the country , the saskatchewan government could rely on the national ndp and ndp oppositions to provincial governments as well as affiliated trade unions throughout canada.40 woodrow lloyd replaced douglas as party leader and premier of saskatchewan . in a letter to the college , lloyd suggested that the physician - based private insurance plans might be allowed to continue under some strict understandings concerning the package of benefits , the amount assessed for premiums , and the remuneration of doctors . in 1960 most of these doctors had been obliged to interrupt their studies because of the second world war , when many of them were anti - german resisters in the belgian underground or the maquis as it was known.50 these experiences would come to be formative in the decision to strike and in the deployment of effective strike tactics.51 in the summer of 1961 , thirty - six of these younger doctors held two secret meetings in luxembourg.52 these meetings would mark the beginning of a fundamental reconstitution of organised medicine in belgium.53 while the older doctors were more amenable to working out an accommodation with the government , their junior colleagues were unwilling to compromise , preferring to launch a general strike in order to force the government to concede . in the view of the young activists , the time for negotiation had ended , and the time for battle had arrived.54 on 30 may 1962 the first ( lige - luxembourg ) of five syndical chambers of medicine was officially founded.55 these chambers were more overtly political and militant than any previous organisation , borrowing heavily from methods of the belgian resistance in the second world war . more than 4,500 doctors ( out of a total of 12,665 doctors in belgium at that time ) attended from every region of the country , all stating they could never agree to the loi leburton.63 governments in canada and belgium were unprepared for the cohesiveness and effectiveness of the doctors strikes . finally , organised medicine in both countries used social ostracism as a weapon against dissident doctors who refused to toe organised medicine s line or , even worse , engaged in active strikebreaking . the college also determined which of a minority of hospitals would remain staffed with doctors , thus precipitating the temporary closure of the rest.65 between april and july , a number of keep our doctors ( kod ) committees were established in order to support the doctors . originally started by married women concerned about the possible loss of doctors in their communities and the impact this would have on their families , these kod committees mushroomed in number and visibility in the weeks leading up to the strike . although trade unions and church groups that supported the government s position began to set up their own pro - reform civil society organisations , premier lloyd discouraged them from engaging too visibly or directly for fear that this would simply elevate the already dangerously high level of public hysteria.67 they complied , but their absence from the field only exaggerated the anti - government voice , making the college even more confident that the government would back down on the eve of the strike , a major miscalculation.68 realising that a strike was highly likely , the government began to recruit foreign doctors to take the places of the saskatchewan doctors . calculating that it could only rely on the support of between fifty and one hundred saskatchewan doctors , and that the majority of canadian doctors outside the province would be hesitant to provide relief because of the influence of the cma , the saskatchewan government began to recruit doctors abroad , particularly in the united kingdom . up to this point , the events leading up to the strike had been followed closely by the provincial media and , to a lesser extent , by the media in the rest of canada . on the first day of the strike , however , media from the rest of the world , particularly american and british newspapers , magazines , and medical journals had their own journalists cover the confrontation.71 in the words of the london observer , saskatchewan has become a battlefield for the whole north american continent in the struggle of the big medical organisations against socialised medicine.72 there was a clear divide in the media with the majority of the provincial media community newspapers and local radio and television stations supporting the doctors , and only a minority supporting the government . they were joined in this view by a number of international publications , including the lancet which , while it had been sympathetic enough to the saskatchewan doctors to bar the government from placing recruitment advertisements for british physicians to practise in saskatchewan , nonetheless stated its objections to the college acting as a state within a state.73 as the media tide turned against the college , a small group of clerical leaders in the province met with premier lloyd and his inner cabinet ministers in an effort to come up with a compromise they could sell to the college in order to bring the strike to an end . distinct possibilities , but in return the college had to be prepared to get its members to return to work immediately.74 however , the doctors were not prepared to consider ending the strike until after the failure of a major rally sponsored by the kod on 11 july in front of the legislative assembly in the capital city of regina . twenty - one months after the strike in canada , the doctors strike in belgium would take a very similar course , though the outcome would be interpreted very differently . as in the canadian case , the strike focused on the new law . once this occurred , the government was more than capable of scaring off the less militant doctors by setting out the sanctions that would be meted out to physicians who joined the strike.78 at the same time , the chambers still needed to recruit more members in regions of the country where they were weaker , particularly in flanders . in a final attempt to avoid a general strike , the government held a marathon meeting with the doctors on 31 march in order to obtain a compromise , but to no avail.80 the doctors had no intention of negotiating a settlement and had already committed to packing their suitcases in the early hours of 1 april 1964 to leave en masse for towns just beyond the belgian border , such as clervaux and mondorf in luxembourg , valkenburg in holland , and valenciennes in france.81 at four in the morning , dr thon , on behalf of the five chambers , released a press statement announcing that belgian doctors in the country had commenced total and unlimited strike.82 having managed to keep their strategy and preparations secret , the chambers quickly discovered that their general strike shocked the government , the press , and the public.83 on the ground , physicians were divided into three categories : 1 ) the majority who went on vacation ; 2 ) the commandos who had to remain in place in order to carry out any orders from the leadership including forcing recalcitrant doctors to go on vacation , and treating those that refused or disappeared as deserters;84 and 3 ) the guards who had to remain on active duty at the hospitals in order to decide whether patients needed emergency care during the strike . reporting constantly on the strike , the majority of newspapers including the extensive catholic press took an editorial position against the strike , although a minority in particular the liberal press supported the doctors.85 the strike would have a direct impact on the lives of most belgians . for those belgians who did not need or seek medical help ambulances raced through the cities with blaring sirens while the media focused on individual incidents highlighting the danger to health and life posed by the strike.86 with little or no evidence to back up their respective claims , both sides used such incidents to prove the extent to which either the strike or the government was endangering belgians through their respective actions.87 despite public pressure on both sides , the reopening of negotiations seemed impossible in the first week of the strike . both sides issued ultimatums , the government insisting that it would only negotiate once the doctors returned to work , and doctors insisting on major revisions to the law before they would call an end to the strike . the parties agreed to sit down and talk on the weekend of 1112 april but this first effort failed due to one careless remark to the media by prime minister tho lefvre : jespre que lon ne devra pas appeler ce temps , le temps des assassin [ i hope we will not have to call this period , the time of assassins].88 when this comment was passed on to all doctors by telephone , they threatened to abolish guard duties at the hospital in retaliation.89 in response , the government panicked , and drew up new military guidelines facilitating the conscription of doctors in order to bring the strike to a rapid end . at the same time , however , the pressure of the strike began to produce cracks within the leadership and among the membership of organised medicine . for others , these weaknesses pushed both sides back to the bargaining table with the four university rectors the following weekend , 1718 april , at the end of which the government retreated on its position and the doctors agreed to end the strike.91 since the weekend settlement met most of the doctors demands , it was interpreted as a victory for the chambers and a major defeat for the government.92 however , as in the case of canada where the opposite was perceived to have occurred , the long - term consequences were more nuanced and less favourable to the perceived victors . rooted in a nineteenth - century conception of the freedom to contract and a limited state , the tenets of medical liberalism clashed with the more collectivist philosophies propounded by social democratic , democratic ( non - revolutionary ) socialist , and similar centre the 1960s marked the apex of the expansion of the welfare state in the advanced industrial countries of the west , an expansion led by centre while this trend was resisted by organised medicine in numerous countries , the most formidable resistance came in the form of the lengthy and bitter doctors strikes in canada and belgium . in both countries , fee - for - service remuneration remains the dominant system of payment for physicians , and a semi - permanent obstacle to government - led reforms of primary healthcare . these longer - term consequences seem to clash with more contemporary evaluations of the outcomes of the strike in both countries . however , in reality , organised medicine was able to improve the economic position of its members even while it preserved the contractual system of remuneration and private practice , protected the role of physicians at the centre of the healthcare system , and prevented major changes to primary healthcare.93 in a manner similar to the british medical association s ability to exploit divisions within successive british governments , the canadian medical association and its provincial chapters were effective in consolidating their gains and preventing subsequent governments from initiating further reform , in part because of the fear of another strike by physicians.94 in belgium , the doctors were perceived to have won the strike . however , unlike the canadian case , the doctors were unable to prevent the belgian government from placing controls on physician remuneration in order to contain cost , and in this respect , could not supplant the continued supremacy of the social insurance funds .

systemic lupus erythematosus ( sle ) is a chronic inflammatory autoimmune disease most common in women of reproductive age , characterized by a relapsing / remitting course and the involvement of multiple organs , including skin , kidneys , and central nervous system . pathophysiologically is characterized by the dysfunction of t , b , and dendritic cells ( dc ) , skewed cytokine production , breakdown of immunological tolerance , and by the production of antinuclear autoantibodies [ 25 ] . in sle bone marrow mononuclear cells , nakou et al . identified central gene regulators implicated in disease pathogenesis which include activation of multiple kinase pathways ( mapk / extracellular regulated map kinase ( erk ) , signal transducer and activator of transcription ( stat ) , akt , and pi3-kinase ( pi3 k ) ) . akt1 serine / threonine kinase is a key downstream target of pi3 k signaling pathway , and plays a role in the differentiation of peripheral b cells and in t cell homeostasis [ 710 ] . akt / mammalian target of rapamycin ( mtor ) axis has been successfully targeted with rapamycin for treatment of sle patients . mapk / erk kinases play a significant role in immune - mediated inflammatory responses and are involved in the maintenance of t - cell tolerance that fails in sle patients [ 4 , 12 ] . conversely , other studies documented a reduced mapk activity in human sle t cells . in mice , a defect in lupus t cell erk pathway signaling that can cause epigenetic abnormalities in t cells by inhibiting dna methylation has been reported [ 12 , 14 ] . in addition , in sle t cells , alterations in kinases pathways such as the spleen tyrosine kinase ( syk ) signaling patterns have been documented by krishnan et al . . the imbalance in cytokine activities is associated with human autoimmune and autoinflammatory diseases , and it has been reported to play a central role in regulating sle development [ 1620 ] . there is a key collaboration and regulation between key cytokines in activation / induction of transcription factors during the process of t - helper-(th)-cell differentiation towards th1 , th2 , th17 , and induced regulatory t ( itreg ) cells lineages [ 21 , 22 ] . changes in th - specific transcription factors gene expression ratios have been used as a marker of th - cytokine balance [ 20 , 2326 ] . moreover , increased inflammation mediators s100a8/s100a9-(calprotectin ) serum and/or plasma levels have been reported in sle patients [ 2729 ] . damage - associated molecular pattern molecules ( damp ) s100a8 and s100a9 members of the calcium - binding s100-family make a heterotetrameric form , s100a8/s100a9-(calprotectin ) . the aim of this study is to assess the gene expression levels of intracellular kinases akt1 and mapk1 in peripheral blood mononuclear cells ( pbmc ) from sle patients with inactive or mild disease and to analyze whether there was any correlation with th - transcription factors gene expression , with gene expression and plasma levels of a comprehensive panel of cytokines ( th0- , th1/th17- , and th2/treg - type ) , with plasma inflammation mediators s100a8/a9-calprotectin and clinical parameters . the study protocol was approved by the scuh committee and csic review board and ethics committees . written informed consent was obtained from all participating patients and controls , according to the helsinki declaration . clinical data and treatments of patients thirteen sle outpatients ( eleven women and two men ) with inactive or mild disease ( sle disease activity index ( sledai ) score 4 ) ( 0 ( 03 ) ( median ( 25% and 75% percentile ranges ) , see table 1 ) that fulfilled at least four of the revised sle criteria of the american college of rheumatology ( acr ) for the diagnosis of sle [ 30 , 31 ] were evaluated . age- and gender - matched healthy controls ( c ) included thirteen volunteers subjects , without known autoimmune disease ( eight women and five men ; 34 ( 3051.5 ) years old ; differences versus sle patients were not statistically significant , mann - whitney , p = 0.1435 ) . likewise , no significant differences in gender distribution were observed between patients and controls ( fisher test : p = 0.3783 , for sle patients versus c ) . medications taken by the patients at the time the blood was drawn were recorded ( table 1 ) . the majority of sle patients ( n = 12 ) were treated with hydroxychloroquine ( 200 mg / day ) ; nine sle patients were also treated with prednisone ( 5 mg / day ) , and six of them were additionally treated with one of methotrexate , azathioprine , or mycophenolate mofetil ( table 1 ) . blood was collected ( bd vacutainer system , k2-edta tubes , bd diagnostics , franklin lakes , nj , usa ) and plasma and pbmc were separated using density gradient centrifugation . total rna was isolated from pbmc from thirteen sle patients , and four controls , using rneasy plus mini kit ( qiagen , hilden , germany ) . rna quality was assessed by an experion automated electrophoresis station ( bio - rad , hercules , ca , usa ) . rt reaction , primer sequences , and gene - specific primers used in this studio are indicated ( table 2 and supplementary materials ) . glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was used as an endogenous control . to quantify cytokine mrna expression , a rt profiler pcr array system ( sabiosciences , qiagen ) the pcr array layout contained gene - specific primers for ten cytokines : interleukin ( il)-1b , il-2 , il-5 , il-6 , il-10 , il-12a , tumor necrosis factor ( tnf)- , interferon ( ifn)- , transforming growth factor beta ( tgfb)-2 , and tumor necrosis factor - related apoptosis - inducing ligand or trail ( tnfsf)-10 ; two internal loading gene - specific primers controls were included for standardization between samples ( gapdh and -actin , table 2 ) . proportion of transcript present in the samples was calculated using the relative quantification 2 scheme . results represented the relative amount of amplicon in patient 's sample ( fold change ) to the mean level of the transcripts in the control samples . bio - plex precision pro human cytokine 10-plex kit assays were used to simultaneously test 10 cytokines in plasma : il-1 , il-2 , il-4 , il-5 , il-6 , il-10 , il-12(p70 ) , il-13 , ifn- , and tnf- , in according with manufacturer 's protocol ( bio - rad ) . plasma concentration , a noncovalently associated calprotection , in the presence of calcium , was measured in duplicate using a sandwich elisa kit ( no . data are given as median and 25% and 75% percentile ranges , or otherwise indicated . relative mrna expression levels of the genes in patients ' pbmc versus controls were evaluated using the nonparametric wilcoxon signed rank test and comparing the medians against a hypothetical median ( value equal 1 ) . determinations of significant differences between groups were compared using the non - parametric mann - whitney u test . differences were considered statistically significant for p values < 0.05 ( garphpad prism v.5.01 , graphpad , ca , usa ) . akt1 ( 7.82 ( 2.6111.45 ) ) , mapk1 ( 19.47 ( 8.7133.45 ) ) , and zap70 ( 1.98 ( 1.143.66 ) ) relative gene expressions levels were significantly augmented in pbmc from sle patients ( n = 13 ) as compared with controls ( wilcoxon signed rank test : p = 0.0064 , p = 0.0012 , and p = 0.0024 , resp . ) cd38 ( 1.22 ( 0.771.95 ) ) and cd3zeta ( 1.15 ( 0.541.83 ) ) immunoreceptors relative gene expression levels were also evaluated ; not significantly differences were observed versus controls ( wilcoxon signed rank test : p = 0.0805 , p = 0.7869 , resp . ) ( figure 1(a ) ) . in addition , the th17-associated cc chemokine receptor ( ccr)-6 relative gene expression was significantly decreased in sle 's pbmc versus controls ( 0.50 ( 0.330.95 ) , wilcoxon signed rank test , p = 0.0398 ) ( figure 1(a ) ) . in pbmc from patients and controls , relative gene expression of t - box transcription factor ( t - bet or tbx21 ) and signal transducer and activator of transcription ( stat)-4 , both for th1 cells ; gata3 , a member of the gata family of zinc finger proteins for th2 ; retinoic acid - related orphan receptor ( ror)-c , for th17 cells and forkhead / winged helix transcription factor ( foxp)-3 for treg cells [ 33 , 34 ] were assessed and th - transcription factors gene expression ratios calculated . foxp3 relative gene expression was significantly increased in sle patients versus controls ( 18.3 ( 1.3944.14 ) , n = 13 , wilcoxon test , p = 0.0012 ) . however , there were no statistically significant differences between medians of the t - bet ( 5.94 ( 0.4463.98 ) ) , stat4 ( 1.09 ( 0.602.07 ) ) , rorc ( 4.49 ( 0.6030.56 ) ) and gata3 ( 2.80 ( 0.798.19 ) ) relative gene expressions levels , respectively , versus controls ( wilcoxon signed rank test , p = 0.0681 ; p = 0.3054 , p = 0.0681 , p = 0.0574 , for t - bet , stat4 , rorc , and gata3 , resp . ) . in sle patients , gene expression ratios of both foxp3/rorc ( 1.90 ( 1.414.85 ) ) and foxp3/gata3 ( 2.17 ( 1.677.22 ) ) were significantly increased versus controls ( wilcoxon signed rank test , p = 0.0042 and p = 0.0017 , resp . ) ( figure 1(b ) ) . while , ratios of t - bet / rorc ( 1.48 ( 1.091.95 ) ) , t - bet / gata3 ( 1.24 ( 0.646.77 ) ) , and rorc / gata3 ( 1.25 ( 0.793.23 ) ) were evaluated and no significantly differences were observed between sle versus controls ( wilcoxon signed rank test , p = 0.0503 , p = 0.1099 , and p = 0.0942 , resp . results indicated that il-10 relative gene expression was significantly increased versus controls ( table 3 ) . in addition , ten cytokines were simultaneously assessed in each plasma sample using a bio - plex ( table 4 ) . results indicated that plasma levels of proinflammatory cytokines il-1 , il-6 ( both th17 ) , il-12(p70 ) , ifn- ( both th1 ) together with il-5 ( th2 ) were significantly augmented in sle patients versus controls ( table 4 ) . in sle ( n = 13 ) patients , il-1 and il-6 plasma levels were positively correlated to il-1b and il-6 gene expression levels , respectively ( table 5 ) . il-6 gene expression also positively correlated with th1 ( il-12(p70 ) , ifn- , and tnf- ) , th0 ( il-2 ) , th17 ( il-1 ) , and il-10 plasma cytokines , respectively . there was a positive correlation between il-10 gene expression and plasma tnf- ( table 5 ) . while there was a negative correlation between il-10 and tnf- gene expressions ( r = 0.6905 , p = 0.009 ) . there were significant positive correlations in the il-2 , il-6 , il-10 , il-12a , and tgfb2 gene expressions , which were correlated to one another ; except for il-6 and tgfb2 ( table 5 ) . in addition , ifn- and il-12a gene expressions were positively correlated ( table 5 ) . in sle patients , there were significant correlations between mapk1 , akt1 , t - bet , rorc , and gata3 relative gene expressions , respectively ( table 6 ) . additionally , akt1 gene expression was correlated to both rorc ( table 6 ) and cd38 expressions ( table 8) , respectively . moreover , there were significant correlations in the t - bet , stat4 , gata3 , rorc , and foxp3 gene expressions , which were correlated to one another ( table 6 ) . both akt1 and mapk1 gene expressions were correlated with gene expression levels of treg-(tgfb2 ) and th2-(il-5 ) cytokine - related genes , respectively ( table 7 ) . there was also a correlation between akt1 and tnfsf10 ( trail ) gene expressions ( table 7 ) . additionally , mapk1 gene expression was correlated to il-10 and il-12a gene expressions ( table 7 ) . furthermore , significant correlations between il10 relative gene expression and foxp3 , rorc , gata3 , t - bet , ( table 7 ) , and cd38 gene expressions ( table 8) , respectively , were observed . correlations between foxp3 gene expression and th1 ( il-12a , ifn- ) , th0 ( il-2 ) , treg-(tgfb2 ) , th2-(il-5 ) , and il-6 gene expressions , respectively , were observed ( table 7 ) . plasma levels of inflammation mediator calprotectin were augmented in sle patients ( 175.6 ( 138.9229.2 ) ng / ml , n = 13 ) , as compared with controls ( 143.3 ( 133.4151.9 ) ng / ml , n = 11 ) although differences were not statistical significant ( mann - whitney , p = 0.0637 ) . our data are consistent with previous reports [ 2729 , 35 ] . in sle patients , in addition , significant correlations were obtained between disease - marker c - reactive protein ( crp ) and plasma cytokines type th1-(ifn- , tnf- ) , th0-(il-2 ) , th2-(il-13 ) , th17-(il-1 , il-6 ) , and il-10 , respectively ( table 8) . patients ' age was correlated with gene expression levels of il-2 , il-10 , il-12a , tgfb2 , and gata3 relative gene expressions , respectively ( table 8) . moreover , in sle patients , c4-complement levels were correlated to ccr6 ( lymphocyte trafficking ) and stat4 gene expressions levels , respectively , which were correlated to one another ( table 8) . in mice , ccr6 was recently identified by wei et al . among the stat4-occupied genes . lastly , lymphocytes numbers negatively correlated with tnfsf10 ( trail ) gene expression ( table 8) . in this study , we observed that akt1 and mapk1 gene expressions were upregulated in sle patients with mild or inactive disease and correlate with gene expressions levels of th17-(rorc ) , treg-(tgfb2 ) , and th2-(il-5)-related genes , respectively . pi3 k / akt and mapk pathways are regulated by extensive crosstalk at different levels [ 9 , 37 ] . akt effect on the generation and function of treg and th17 cells is not well understood . reported that rorc was necessary , but not sufficient , for il-17 production by human memory t cells , and they proposed that rorc synergized with common chain - cytokines mediated pi3 k and akt signaling to transactivate il-17 expression . recently , kurebayashi et al . demonstrated in mice that the suppression of pi3 k - akt - mammalian target of rapamycin complex ( mtorc)-1 axis impaired th17 differentiation in vitro and in vivo in s6 kinases 1 and 2-dependent fashion . in mice , sauer et al . documented that t cell receptor ( tcr ) signaling via pi3kp110 , p110 , akt , and mtor controls pi3 k - akt axis has also been reported augments clonal expansion of th1 and th2 cells [ 41 , 42 ] . in mature t cells , constitutively active akt could induce growth and survival of cd4 ; and a direct role of akt in cd8 t cells has been reported , regulating whether their differentiation leads them to either memory or effector fate . in sle patients , our results show in sle patients that akt1 expression positively correlates with tnfsf10 ( trail ) expression , and trail expression negatively correlates with lymphocytes numbers . mapk / erk and pi3 k / akt signaling pathways are activated by trail . up - regulation of trail mrna expression in pbmc from active sle patients was reported . trail - induced apoptosis by binding to death receptors but also can enhance t cell proliferation after tcr engagement via transducing a costimulation signal . our results show that mapk1 gene expression also correlates with gene expression levels of th1-(il-12a , t - bet ) , th2-(gata3 ) , and il-10 genes . mapk / erk signaling pathways integrate cytoplasmic signals to produce changes in transcription associated with differentiation , proliferation , and survival in multiple stages of t cell development [ 4749 ] . the results show a clear increase of il-10 relative gene expression in sle pbmc , although il-10 plasma levels are not significantly augmented . meta - analysis of microarray data indicated that one of the biological pathways consistently enhanced in sle patients was the il-10 signaling . il-10 has been identified as one of the risk loci for sle in a recent genome - wide association study . despite that no correlation of il-10 gene expression with sledai was observed , il-10 expression positively correlates with plasma tnf- ( table 8) , and with expression levels of th0-(il-2 ) , th1-(il-12a , t - bet ) , th2-(gata3 ) , treg-(tgfb2 , foxp3 ) , pro - inflammatory il-6 , and th17-(rorc)-cytokine related genes , respectively . il-10 is a key cytokine regulating immune responses , and the absence results in spontaneous inflammatory disorders as well as exacerbated inflammation in t cell responses during host defense or autoimmune inflammation ( see reviews [ 52 , 53 ] ) . il-10 is expressed by cells of the adaptive immune system ( th1 , th2 , treg cells , and regulatory b cells ( bregs ) and by cells of the innate immune system ( dcs , macrophages , mast cells , nk , eosinophils , and neutrophils ) [ 54 , 55 ] . il-10 provides a highly regulated feedback loop to avoid the extremes of excessive inflammation or chronic infections and also allow a protective response to pathogens . in mice , saraiva et al . reported that development of il-10-producing th1 cells required high tcr ligation , sustained erk1 and erk2 mapks phosphorylation , and il-12-induced stat4 activation , indicating that erk1 and erk2 activation was a common pathway required for the production of il-10 by th1 , th2 , and th17 cell subsets . . indicated that like ifn- , il-10 expression was induced by il-2 and il-12 stimulation . it has been reported by avagyan et al . that tgf-2 specifically enhanced signaling by the hematopoietic growth factor fms - related tyrosine kinase ( flt)-3 in hematopoietic stem cells . in activated human t cells , astier et al . furthermore , a relevant role of il-10 and tnf- genotypes in sle has been reported , and il-10 cytokine inhibits transcription elongation of the human tnf gene in primary macrophages . our results also show that il-10 gene expression in sle patients correlates with cd38 ( activation , maturation , and trafficking marker ) gene expression ; cd38 expression correlates with akt1 expression . in human t and b cells , upon receptor stimulation cd38 leads to pkb / akt and erk activation [ 63 , 64 ] . moreover , in a kinase - dead mutant of pi3kp110 mice , patton et al . reported that pi3kp110 kinase regulates expression of cd38 on treg . documented that cd19cd24cd38-b cells exhibit regulatory capacity in healthy individuals while the same b cells from sle patients produced less il-10 and lacked the suppressive capacity . our data show a clear increase in foxp3-(treg ) gene expression levels in sle patients and positively correlate with il-10 gene expression . recent reports provided new data about the mechanisms linking il-10 and treg cells by demonstrating that il-10 directly signals in th17 and treg cells to maintain control of th17 cell - mediated inflammation [ 67 , 68 ] . in addition , il-10-producing b cells , also known as regulatory b cells ( bregs ) , play a key role in controlling autoimmunity . reported that chimeric mice specifically lacking il-10-producing b cells developed an exacerbated arthritis compared with chimeric wild - type b cell mice . moreover , a significant decrease in the absolute numbers of foxp3 regulatory t cells ( tregs ) , in their expression level of foxp3 and a marked increase in inflammatory th1 and th17 cells were detected in chimeric mice lacking il-10-producing b cells compared with wt b - cell mice . how treg cell function and foxp3 expression are regulated is an important question under intensive investigation . increased gene expression of foxp3 compared to healthy controls has been reported . in sle patients , several reports indicated that increased proportions of cd4cd25foxp3 t cells were correlated with the clinical disease activity and the daily cortisone dose [ 72 , 73 ] . in our study , we can not exclude that treatments received by sle patients could have an effect on the results . although , a recent report from sbiera et al . documented that short - term glucocorticoid therapy in vivo either in immunologically uncompromised humans or in mice did not induce an increased in the frequency of circulating treg cells . balance between foxp3 and rorc function has been postulated that determines cd4 t cell fate and the type of immune response that will be generated . several studies reported in active sle patients an imbalance between treg and th17 cells [ 7779 ] . wang et al . demonstrated an essential role for gata3 , in controlling treg cell homeostasis by stabilizing expression of foxp3 . our results show an increased balance of both foxp3 ( treg)/rorc ( th17 ) and foxp3 ( treg)/gata3 ( th2 ) gene expression ratios in pbmc from sle patients with inactive or middle disease , suggesting a trend towards treg polarization . in addition , our results show no significant increases compared with controls in the t - bet ( th1)/rorc ( th17 ) , t - bet ( th1)/gata3 ( th2 ) , and rorc ( th17)/gata3 ( th2 ) gene expression ratios in pbmc from sle patients . our data also show a low relative gene expression levels of th17-associated ccr6 ( is a trait of tissue - homing effector t cells lymphocyte trafficking ) in pbmc from the sle patients with inactive or middle disease . ccr6 expression on cd4 t cells has been considered as a marker of disease activity in sle patients . in addition , sallusto et al . reported that following tcr stimulation , human memory / effector t cells down - regulate receptors for constitutive chemokines ( including ccr6 ) . they suggested that following tcr stimulation effector / memory t cells transiently acquire responsiveness to constitutive chemokines . as a result , t cells that are activated in tissues may either recirculate to draining lymph nodes or migrate to nearby sites of organized ectopic lymphoid tissues . several studies have shown a trend towards increased th17 cells and th17/th1 ratio in sle patients with active disease [ 82 , 84 ] . furthermore , reports on function and numbers and treg cells in sle have been contradictory ( reviews [ 75 , 85 ] ) , suggesting that not all foxp3 t cells in sle have protective suppressive activity . plasticity of cd4foxp3 t cells has been documented [ 34 , 86 ] , and it has been reported that conversion of human foxp3 tregs into il-17-producing cells can be enhanced in the context of an inflammatory cytokine milieu ( il-1 and il-6 ) [ 75 , 76 , 86 , 87 ] . nevertheless they are also able to adapt in response to stimuli by homing to sites of inflammation and exerting suppressive functions . it has been reported that treg are able to express other transcription factors normally associated with other th cell subtypes in order to better control immunopathology ( see review ) . in our study , plasma levels of proinflammatory cytokines il-1 , il-12(p70 ) , ifn- , and il-6 are significantly augmented in sle patients with inactive or middle disease compared with controls . in sle it has been suggested that aberrant control of immune cell responses to inflammatory cytokines may disrupt the delicate balance between immunity and self - reactivity [ 2 , 3 , 88 ] . il-6 has been studied in human sle , and il-6 was found to reflect disease activity [ 19 , 8993 ] . il-6 contributes to host defence against acute environmental stress , and continuous deregulated il-6 production plays a significant pathological role in systemic autoimmune diseases . il-6 has been known to have a key role in the maturation of b cells , as well to be one of the members of cytokines that drives the acute inflammatory response together with tnf - alpha and il-1 . additionally , plasma il-5 ( th2 ) was significantly augmented in sle patients versus controls . il-5 is produced by natural helper cells and is involved in antibody production [ 95 , 96 ] . il-5 is a critical growth factor for b1 cells present in the peritoneal cavity and plays an important role in innate - type immune responses by producing natural antibodies [ 9799 ] . wen et al . reported that deregulated , continuous high expression of il-5 in sle - prone mice may directly or indirectly mediate a skewed signaling of proliferation / differentiation of self - antigen - activated b1 cells , leading to suppression of autoimmune disease , but instead of aberrant expansion of b1 cells , giving rise to b - cell chronic lymphocytic leukaemia ( b - cll ) . moreover , despite no correlation with sledai , our results indicate that th17-(il-1 , il-6 ) , th0-(il-2 ) , th1-(ifn- , tnf- ) , th2-(il-13 ) , and il-10 plasma cytokines are positively correlated with the inflammation mediator crp . during inflammation these results are consistent with those of other studies [ 102 , 103 ] . moreover , our results show a moderate increase of zap70 tyrosine kinase gene expression in sle 's pbmc and zap70 correlate with gene expressions levels of tnf- ( table 8) . syk and zap-70 kinases also associated differently with key molecules involved in cytoskeletal and calcium signaling in sle t cells . in human mature t cells , meinl et al . demonstrated a key role of zap70 in cd2-mediated phosphorylation of cd3zeta chain of tcr , in ca influx , proliferation , and production of ifn- and tnf-. furthermore our data show that increased inflammation mediator s100a8/s100a9-calprotectin plasma levels in sle patients positively correlate with neutrophils numbers . s100a8/s100a9-calprotectin is expressed by neutrophils , monocytes , activated macrophages and released in neutrophil extracellular traps . these results also accord with our earlier observations , which showed in sle pbmcs that increased s100a9 levels , correlated positively with the abnormal presence of low - density granulocytes detected by flow cytometry in the mononuclear cells . despite the fact that the study has been performed in a small number of patients , the results and the positive correlations found suggest biological functions and associations that need to be further confirmed in a larger cohort of patients . sle patients with inactive or mild disease presented a systemic immunoinflammatory activity with augmented akt1 and mapk1 gene expressions , plasma proinflammatory cytokines , and calprotectin , together with increased gene expression of treg - related genes , suggesting an ongoing regulatory feedback opposing the inflammatory activity .

kinases have been implicated in the immunopathological mechanisms of systemic lupus erythematosus ( sle ) . v - akt murine - thymoma viral - oncogene - homolog 1 ( akt1 ) and mitogen - activated - protein - kinase 1 ( mapk1 ) gene expressions in peripheral mononuclear cells from thirteen sle patients with inactive or mild disease were evaluated using quantitative real - time reverse - transcription polymerase - chain - reaction and analyzed whether there was any correlation with t - helper ( th ) transcription factors ( tf ) gene expression , cytokines , and s100a8/s100a9-(calprotectin ) . age- and gender - matched thirteen healthy controls were examined . akt1 and mapk1 expressions were upregulated in sle patients and correlated with th17-(retinoic acid - related orphan receptor ( ror)-c ) , t - regulatory-(treg)-(transforming growth factor beta ( tgfb)-2 ) , and th2-(interleukin ( il)-5)-related genes . mapk1 expression correlated with th1-(il-12a , t - box tf-(t - bet ) ) , th2-(gata binding protein-(gata)-3 ) , and il-10 expressions . il-10 expression was increased and correlated with plasma tumor necrosis factor ( tnf)- and th0-(il-2 ) , th1-(il-12a , t - bet ) , gata3 , treg-(forkhead / winged - helix transcription factor- ( foxp)-3 ) , and il-6 expressions . foxp3 expression , foxp3/rorc , and foxp3/gata3 expression ratios were increased . plasma il-1 , il-12(p70 ) , interferon-(ifn)- , and il-6 cytokines were augmented . plasma il-1 , il-6 , il-2 , ifn- , tnf- , il-10 , and il-13 correlated with c - reactive protein , respectively . increased calprotectin correlated with neutrophils . conclusion , sle patients presented a systemic immunoinflammatory activity , augmented akt1 and mapk1 expressions , proinflammatory cytokines , and calprotectin , together with increased expression of treg - related genes , suggesting a regulatory feedback opposing the inflammatory activity .

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

systemic lupus erythematosus ( sle ) is a chronic inflammatory autoimmune disease most common in women of reproductive age , characterized by a relapsing / remitting course and the involvement of multiple organs , including skin , kidneys , and central nervous system . pathophysiologically is characterized by the dysfunction of t , b , and dendritic cells ( dc ) , skewed cytokine production , breakdown of immunological tolerance , and by the production of antinuclear autoantibodies [ 25 ] . identified central gene regulators implicated in disease pathogenesis which include activation of multiple kinase pathways ( mapk / extracellular regulated map kinase ( erk ) , signal transducer and activator of transcription ( stat ) , akt , and pi3-kinase ( pi3 k ) ) . akt1 serine / threonine kinase is a key downstream target of pi3 k signaling pathway , and plays a role in the differentiation of peripheral b cells and in t cell homeostasis [ 710 ] . mapk / erk kinases play a significant role in immune - mediated inflammatory responses and are involved in the maintenance of t - cell tolerance that fails in sle patients [ 4 , 12 ] . there is a key collaboration and regulation between key cytokines in activation / induction of transcription factors during the process of t - helper-(th)-cell differentiation towards th1 , th2 , th17 , and induced regulatory t ( itreg ) cells lineages [ 21 , 22 ] . changes in th - specific transcription factors gene expression ratios have been used as a marker of th - cytokine balance [ 20 , 2326 ] . moreover , increased inflammation mediators s100a8/s100a9-(calprotectin ) serum and/or plasma levels have been reported in sle patients [ 2729 ] . the aim of this study is to assess the gene expression levels of intracellular kinases akt1 and mapk1 in peripheral blood mononuclear cells ( pbmc ) from sle patients with inactive or mild disease and to analyze whether there was any correlation with th - transcription factors gene expression , with gene expression and plasma levels of a comprehensive panel of cytokines ( th0- , th1/th17- , and th2/treg - type ) , with plasma inflammation mediators s100a8/a9-calprotectin and clinical parameters . clinical data and treatments of patients thirteen sle outpatients ( eleven women and two men ) with inactive or mild disease ( sle disease activity index ( sledai ) score 4 ) ( 0 ( 03 ) ( median ( 25% and 75% percentile ranges ) , see table 1 ) that fulfilled at least four of the revised sle criteria of the american college of rheumatology ( acr ) for the diagnosis of sle [ 30 , 31 ] were evaluated . age- and gender - matched healthy controls ( c ) included thirteen volunteers subjects , without known autoimmune disease ( eight women and five men ; 34 ( 3051.5 ) years old ; differences versus sle patients were not statistically significant , mann - whitney , p = 0.1435 ) . the majority of sle patients ( n = 12 ) were treated with hydroxychloroquine ( 200 mg / day ) ; nine sle patients were also treated with prednisone ( 5 mg / day ) , and six of them were additionally treated with one of methotrexate , azathioprine , or mycophenolate mofetil ( table 1 ) . total rna was isolated from pbmc from thirteen sle patients , and four controls , using rneasy plus mini kit ( qiagen , hilden , germany ) . to quantify cytokine mrna expression , a rt profiler pcr array system ( sabiosciences , qiagen ) the pcr array layout contained gene - specific primers for ten cytokines : interleukin ( il)-1b , il-2 , il-5 , il-6 , il-10 , il-12a , tumor necrosis factor ( tnf)- , interferon ( ifn)- , transforming growth factor beta ( tgfb)-2 , and tumor necrosis factor - related apoptosis - inducing ligand or trail ( tnfsf)-10 ; two internal loading gene - specific primers controls were included for standardization between samples ( gapdh and -actin , table 2 ) . bio - plex precision pro human cytokine 10-plex kit assays were used to simultaneously test 10 cytokines in plasma : il-1 , il-2 , il-4 , il-5 , il-6 , il-10 , il-12(p70 ) , il-13 , ifn- , and tnf- , in according with manufacturer 's protocol ( bio - rad ) . relative mrna expression levels of the genes in patients ' pbmc versus controls were evaluated using the nonparametric wilcoxon signed rank test and comparing the medians against a hypothetical median ( value equal 1 ) . akt1 ( 7.82 ( 2.6111.45 ) ) , mapk1 ( 19.47 ( 8.7133.45 ) ) , and zap70 ( 1.98 ( 1.143.66 ) ) relative gene expressions levels were significantly augmented in pbmc from sle patients ( n = 13 ) as compared with controls ( wilcoxon signed rank test : p = 0.0064 , p = 0.0012 , and p = 0.0024 , resp . ) cd38 ( 1.22 ( 0.771.95 ) ) and cd3zeta ( 1.15 ( 0.541.83 ) ) immunoreceptors relative gene expression levels were also evaluated ; not significantly differences were observed versus controls ( wilcoxon signed rank test : p = 0.0805 , p = 0.7869 , resp . ) in addition , the th17-associated cc chemokine receptor ( ccr)-6 relative gene expression was significantly decreased in sle 's pbmc versus controls ( 0.50 ( 0.330.95 ) , wilcoxon signed rank test , p = 0.0398 ) ( figure 1(a ) ) . in pbmc from patients and controls , relative gene expression of t - box transcription factor ( t - bet or tbx21 ) and signal transducer and activator of transcription ( stat)-4 , both for th1 cells ; gata3 , a member of the gata family of zinc finger proteins for th2 ; retinoic acid - related orphan receptor ( ror)-c , for th17 cells and forkhead / winged helix transcription factor ( foxp)-3 for treg cells [ 33 , 34 ] were assessed and th - transcription factors gene expression ratios calculated . foxp3 relative gene expression was significantly increased in sle patients versus controls ( 18.3 ( 1.3944.14 ) , n = 13 , wilcoxon test , p = 0.0012 ) . however , there were no statistically significant differences between medians of the t - bet ( 5.94 ( 0.4463.98 ) ) , stat4 ( 1.09 ( 0.602.07 ) ) , rorc ( 4.49 ( 0.6030.56 ) ) and gata3 ( 2.80 ( 0.798.19 ) ) relative gene expressions levels , respectively , versus controls ( wilcoxon signed rank test , p = 0.0681 ; p = 0.3054 , p = 0.0681 , p = 0.0574 , for t - bet , stat4 , rorc , and gata3 , resp . ) in sle patients , gene expression ratios of both foxp3/rorc ( 1.90 ( 1.414.85 ) ) and foxp3/gata3 ( 2.17 ( 1.677.22 ) ) were significantly increased versus controls ( wilcoxon signed rank test , p = 0.0042 and p = 0.0017 , resp . ) while , ratios of t - bet / rorc ( 1.48 ( 1.091.95 ) ) , t - bet / gata3 ( 1.24 ( 0.646.77 ) ) , and rorc / gata3 ( 1.25 ( 0.793.23 ) ) were evaluated and no significantly differences were observed between sle versus controls ( wilcoxon signed rank test , p = 0.0503 , p = 0.1099 , and p = 0.0942 , resp . results indicated that plasma levels of proinflammatory cytokines il-1 , il-6 ( both th17 ) , il-12(p70 ) , ifn- ( both th1 ) together with il-5 ( th2 ) were significantly augmented in sle patients versus controls ( table 4 ) . in sle ( n = 13 ) patients , il-1 and il-6 plasma levels were positively correlated to il-1b and il-6 gene expression levels , respectively ( table 5 ) . il-6 gene expression also positively correlated with th1 ( il-12(p70 ) , ifn- , and tnf- ) , th0 ( il-2 ) , th17 ( il-1 ) , and il-10 plasma cytokines , respectively . while there was a negative correlation between il-10 and tnf- gene expressions ( r = 0.6905 , p = 0.009 ) . there were significant positive correlations in the il-2 , il-6 , il-10 , il-12a , and tgfb2 gene expressions , which were correlated to one another ; except for il-6 and tgfb2 ( table 5 ) . in addition , ifn- and il-12a gene expressions were positively correlated ( table 5 ) . in sle patients , there were significant correlations between mapk1 , akt1 , t - bet , rorc , and gata3 relative gene expressions , respectively ( table 6 ) . additionally , akt1 gene expression was correlated to both rorc ( table 6 ) and cd38 expressions ( table 8) , respectively . moreover , there were significant correlations in the t - bet , stat4 , gata3 , rorc , and foxp3 gene expressions , which were correlated to one another ( table 6 ) . both akt1 and mapk1 gene expressions were correlated with gene expression levels of treg-(tgfb2 ) and th2-(il-5 ) cytokine - related genes , respectively ( table 7 ) . there was also a correlation between akt1 and tnfsf10 ( trail ) gene expressions ( table 7 ) . additionally , mapk1 gene expression was correlated to il-10 and il-12a gene expressions ( table 7 ) . furthermore , significant correlations between il10 relative gene expression and foxp3 , rorc , gata3 , t - bet , ( table 7 ) , and cd38 gene expressions ( table 8) , respectively , were observed . correlations between foxp3 gene expression and th1 ( il-12a , ifn- ) , th0 ( il-2 ) , treg-(tgfb2 ) , th2-(il-5 ) , and il-6 gene expressions , respectively , were observed ( table 7 ) . plasma levels of inflammation mediator calprotectin were augmented in sle patients ( 175.6 ( 138.9229.2 ) ng / ml , n = 13 ) , as compared with controls ( 143.3 ( 133.4151.9 ) ng / ml , n = 11 ) although differences were not statistical significant ( mann - whitney , p = 0.0637 ) . in sle patients , in addition , significant correlations were obtained between disease - marker c - reactive protein ( crp ) and plasma cytokines type th1-(ifn- , tnf- ) , th0-(il-2 ) , th2-(il-13 ) , th17-(il-1 , il-6 ) , and il-10 , respectively ( table 8) . patients ' age was correlated with gene expression levels of il-2 , il-10 , il-12a , tgfb2 , and gata3 relative gene expressions , respectively ( table 8) . moreover , in sle patients , c4-complement levels were correlated to ccr6 ( lymphocyte trafficking ) and stat4 gene expressions levels , respectively , which were correlated to one another ( table 8) . lastly , lymphocytes numbers negatively correlated with tnfsf10 ( trail ) gene expression ( table 8) . in this study , we observed that akt1 and mapk1 gene expressions were upregulated in sle patients with mild or inactive disease and correlate with gene expressions levels of th17-(rorc ) , treg-(tgfb2 ) , and th2-(il-5)-related genes , respectively . reported that rorc was necessary , but not sufficient , for il-17 production by human memory t cells , and they proposed that rorc synergized with common chain - cytokines mediated pi3 k and akt signaling to transactivate il-17 expression . documented that t cell receptor ( tcr ) signaling via pi3kp110 , p110 , akt , and mtor controls pi3 k - akt axis has also been reported augments clonal expansion of th1 and th2 cells [ 41 , 42 ] . in sle patients , our results show in sle patients that akt1 expression positively correlates with tnfsf10 ( trail ) expression , and trail expression negatively correlates with lymphocytes numbers . our results show that mapk1 gene expression also correlates with gene expression levels of th1-(il-12a , t - bet ) , th2-(gata3 ) , and il-10 genes . the results show a clear increase of il-10 relative gene expression in sle pbmc , although il-10 plasma levels are not significantly augmented . meta - analysis of microarray data indicated that one of the biological pathways consistently enhanced in sle patients was the il-10 signaling . despite that no correlation of il-10 gene expression with sledai was observed , il-10 expression positively correlates with plasma tnf- ( table 8) , and with expression levels of th0-(il-2 ) , th1-(il-12a , t - bet ) , th2-(gata3 ) , treg-(tgfb2 , foxp3 ) , pro - inflammatory il-6 , and th17-(rorc)-cytokine related genes , respectively . il-10 is expressed by cells of the adaptive immune system ( th1 , th2 , treg cells , and regulatory b cells ( bregs ) and by cells of the innate immune system ( dcs , macrophages , mast cells , nk , eosinophils , and neutrophils ) [ 54 , 55 ] . indicated that like ifn- , il-10 expression was induced by il-2 and il-12 stimulation . that tgf-2 specifically enhanced signaling by the hematopoietic growth factor fms - related tyrosine kinase ( flt)-3 in hematopoietic stem cells . furthermore , a relevant role of il-10 and tnf- genotypes in sle has been reported , and il-10 cytokine inhibits transcription elongation of the human tnf gene in primary macrophages . our results also show that il-10 gene expression in sle patients correlates with cd38 ( activation , maturation , and trafficking marker ) gene expression ; cd38 expression correlates with akt1 expression . our data show a clear increase in foxp3-(treg ) gene expression levels in sle patients and positively correlate with il-10 gene expression . increased gene expression of foxp3 compared to healthy controls has been reported . in sle patients , several reports indicated that increased proportions of cd4cd25foxp3 t cells were correlated with the clinical disease activity and the daily cortisone dose [ 72 , 73 ] . demonstrated an essential role for gata3 , in controlling treg cell homeostasis by stabilizing expression of foxp3 . our results show an increased balance of both foxp3 ( treg)/rorc ( th17 ) and foxp3 ( treg)/gata3 ( th2 ) gene expression ratios in pbmc from sle patients with inactive or middle disease , suggesting a trend towards treg polarization . in addition , our results show no significant increases compared with controls in the t - bet ( th1)/rorc ( th17 ) , t - bet ( th1)/gata3 ( th2 ) , and rorc ( th17)/gata3 ( th2 ) gene expression ratios in pbmc from sle patients . our data also show a low relative gene expression levels of th17-associated ccr6 ( is a trait of tissue - homing effector t cells lymphocyte trafficking ) in pbmc from the sle patients with inactive or middle disease . several studies have shown a trend towards increased th17 cells and th17/th1 ratio in sle patients with active disease [ 82 , 84 ] . furthermore , reports on function and numbers and treg cells in sle have been contradictory ( reviews [ 75 , 85 ] ) , suggesting that not all foxp3 t cells in sle have protective suppressive activity . plasticity of cd4foxp3 t cells has been documented [ 34 , 86 ] , and it has been reported that conversion of human foxp3 tregs into il-17-producing cells can be enhanced in the context of an inflammatory cytokine milieu ( il-1 and il-6 ) [ 75 , 76 , 86 , 87 ] . in our study , plasma levels of proinflammatory cytokines il-1 , il-12(p70 ) , ifn- , and il-6 are significantly augmented in sle patients with inactive or middle disease compared with controls . il-5 is a critical growth factor for b1 cells present in the peritoneal cavity and plays an important role in innate - type immune responses by producing natural antibodies [ 9799 ] . reported that deregulated , continuous high expression of il-5 in sle - prone mice may directly or indirectly mediate a skewed signaling of proliferation / differentiation of self - antigen - activated b1 cells , leading to suppression of autoimmune disease , but instead of aberrant expansion of b1 cells , giving rise to b - cell chronic lymphocytic leukaemia ( b - cll ) . moreover , despite no correlation with sledai , our results indicate that th17-(il-1 , il-6 ) , th0-(il-2 ) , th1-(ifn- , tnf- ) , th2-(il-13 ) , and il-10 plasma cytokines are positively correlated with the inflammation mediator crp . moreover , our results show a moderate increase of zap70 tyrosine kinase gene expression in sle 's pbmc and zap70 correlate with gene expressions levels of tnf- ( table 8) . furthermore our data show that increased inflammation mediator s100a8/s100a9-calprotectin plasma levels in sle patients positively correlate with neutrophils numbers . these results also accord with our earlier observations , which showed in sle pbmcs that increased s100a9 levels , correlated positively with the abnormal presence of low - density granulocytes detected by flow cytometry in the mononuclear cells . sle patients with inactive or mild disease presented a systemic immunoinflammatory activity with augmented akt1 and mapk1 gene expressions , plasma proinflammatory cytokines , and calprotectin , together with increased gene expression of treg - related genes , suggesting an ongoing regulatory feedback opposing the inflammatory activity .

the evaluation of matrix elements between many - electron wave functions expanded in different orbital basis sets or over different molecular geometries is a task where the full complexity of these wave functions becomes apparent . not only the expansion of the wave functions into individual configurations and the construction of the molecular orbitals have to be taken into account , but also the explicit determinantal form , as required by the pauli principle , comes into play . furthermore , possible variations of the molecular geometry and the atomic basis functions have to be considered explicitly . the focus of this work is the simplest of these matrix elements , the wave function overlap . a new algorithm for the computation of wave function overlaps is presented , which is distinguished by enhanced computational performance reached through extensive reuse of recurring intermediate quantities . at the same time , a flexible formalism is used allowing for the computation of wave function overlaps for varying wave function expansions , molecular orbitals ( mos ) , basis sets , and molecular geometries . wave function overlaps are widely used in the field of nonadiabatic dynamics , where they allow the evaluation of state - to - state transition probabilities without the need of computing nonadiabatic coupling vectors . aside from the fact that this strategy provides a technical advantage for methods where coupling vectors are not available , it has been shown that , in the case of highly peaked nonadiabatic couplings , wave function overlaps can provide superior numerical stability , in particular when a locally diabatic propagation of the wave functions is carried out . in the simplest case , the overlap is approximated as a scalar product of the configuration interaction ( ci ) vectors to provide a qualitative description of changes in wave function character , possibly after a diabatization of the orbitals . beyond this , exact overlap terms have been derived under a number of assumptions ; implementations are available for semiempirical methods , plane wave expansions , single - reference methods with atom - centered basis sets , and multireference methods . these developments were used as a basis for excited state dynamics simulations with a wide range of electronic structure methods , including time - dependent density functional theory , complete active space self - consistent field ( casscf ) and multireference ci ( mrci ) , cas perturbation theory , and correlated single - reference methods . despite this wide interest , the practical use of wave function overlaps is hampered by high computational demands , especially when multiconfigurational wave functions are used , which are necessary for the correct description of many nonadiabatic processes . moreover , the numerical stability of the results with respect to truncation of the wave functions and the consequences of displaced orbitals in the case of varying molecular geometries have received almost no attention so far despite the fact that these can have a crucial impact on the computed results . the purpose of this work is to present a new general algorithm for the efficient computation of wave function overlaps and to address some related numerical questions . we first discuss the general theory of wave function overlaps in the framework of slater determinant expansions . using this foundation , specific algorithmic improvements are explained , which allow for significant enhancement of the efficiency of the code . we then discuss properties of the overlap matrix and outline the application of overlaps for nonadiabatic interactions . as a next step , a path integral over the coupling vector in coordinate space is computed , and the results are compared to standard nonadiabatic theory . in addition , the results are verified against two previous implementations . in order to give practical advice for future applications , the numerical stability of the results with respect to wave function truncation and atom displacements is discussed , and we show how orthogonalization of the overlap matrix can significantly improve the results . finally , the performance and parallel scaling are evaluated . in the following , we use the notation | to denote antisymmetric many - electron wave functions constructed as linear combinations of slater determinants . two sets of electronic wave functions { |i } and { |j } are constructed . the only requirement for the relation between these wave functions is that they contain the same number of and electrons , but they are otherwise allowed to vary in the wave function expansion , the mos , the basis set , and the molecular geometry . i and j are arbitrary indices for the individual sets . typically , i , j = 1 would refer to the ground state and higher indices to the excited states , but there are no formal restrictions with respect to their meaning . in this section , we will derive a general expression for computing terms of the form1and analogously the whole overlap matrix s between the two sets of states . as a first step , the expansion into slater determinants23is invoked . here , { |k } and { |l } denote two distinct sets of slater determinants used in the expansions , dik and djl are the ci coefficients forming the ci vectors , and nci and nci are the number of elements in these ci vectors . it should be noted that the formalism described here is based on slater determinants rather than on spin - adapted configuration state functions ( csf ) , which are often used in ci calculations . however , it is always possible to perform the conversion to the slater determinant basis . we write the slater determinants , which are constructed from four potentially different sets of spin orbitals { p } , { q } , { r } , and { s } , in the form56where the notation k(i ) is used to denote the index of the orbital that is at position i in slater determinant |k , n is the number of electrons , n the number of spin electrons , and the bar marks the spin orbitals . in the above equations , it is assumed that the orbitals are positioned in front of the orbitals in the slater determinant . obtaining this arrangement is always possible but care has to be taken to preserve the correct sign when the columns of the determinants are rearranged . the overlap of the two slater determinants is given by the determinant of the matrix containing all mutual orbital overlaps ( see appendix a and refs ( 9 and 21)).7using the arrangement chosen in eqs 5 and 6 , the matrix becomes block diagonal due to the fact that overlaps between orbitals of different spin vanish . the two blocks can , in turn , be evaluated individually8it is important to realize at this point that the two factors and are not unique to the determinant pair |k and |l , but that they reappear for other determinants with the same or spin occupation pattern . as will be described below , precomputing and storing these factors is one of the main points responsible for efficiency . to evaluate eq 8 , it is assumed that the mos are given in terms of atomic orbitals ( aos)910where the mo coefficients cp and the aos are both allowed to vary between the bra and the ket . consequently , the mo overlaps are given as11linear combinations of the mixed ao overlap integrals |. the overlaps p|q of the orbitals are computed analogously in the case of an unrestricted mo basis . two special situations can occur here : the cases of identical aos and identical mos in the two wave function expansions . if the aos on both sides are the same , i.e. , = , then it is not necessary to compute mixed ao overlap integrals , but the | terms are simply the overlap integrals | already computed in the standard quantum chemistry job . furthermore , as discussed in ref ( 22 ) , it is not necessary to use these integrals at all ( e.g. , if they are not available for technical reasons ) . whenever the mo - coefficient matrix c is square and nonsingular , the ao overlap matrix can be constructed as12 in the second case , when the mos on both sides are the same , i.e. , p = p , the whole formalism is greatly simplified , yielding13and the overlap computation ( eq 4 ) reduces to a simple scalar product of the ci vectors . assuming eq 13 to be approximately valid allows for computing the overlap as a simple scalar product between ci vectors ( expanded either in a slater determinant or csf basis ) , yielding a strategy that has indeed been applied successfully for dynamics simulations ( see , e.g. , refs ( 4 and 5 ) ) . however , inspection of the above equations shows that the k|l terms do not only depend on the resolution of quasi - degenerate orbitals , present for example during charge and energy transfer processes , but are even affected by the phases of the individual orbitals . for this reason , special care has to be taken when such a strategy is applied and a prior diabatization of the orbitals may be necessary . finally , it is worth noting that an alternative strategy for computing the slater determinant overlaps lies in a transformation of the orbitals yielding biorthogonal orbitals and consequently biorthogonal slater determinants , avoiding the necessity for computing the determinants of eq 8 . this can be achieved either by a singular value decomposition of the mixed mo overlap matrix shown in eq 11 to obtain the corresponding orbitals or by a somewhat more involved formalism involving nonunitary orbital transformations as implemented in the molcas 8.0 suite . however , an orbital transformation could significantly enlarge the ci expansion and appears practical only for specific wave function classes . we are not aware of any implementation of such a formalism that allows for the generality aimed at here . another way of computing matrix elements between nonorthogonal slater determinants proceeds by a generalized wick theorem . the algorithm presented above requires the computation of two determinants for every pair of bra and ket slater determinants , see eq 8 . without further considerations , this would lead to a formal scaling on the order of where npair = nci nci , and n is the number of electrons . this steep scaling shows that an efficient implementation is of utmost importance if the code should be generally applicable . the most important realization is that the factors and are not unique to a determinant pair |k and |l. assuming , for example , that four determinants are given asit follows that |1 and |2 share the same spin part . this leads to the situation that and are identicaland it similarly holds that as well as and . in summary , out of the 2 npair = 8 spin determinants , there are only nfac = 4 unique factors , cutting the required computational effort in half in this toy model . this reduction occurs independently in the and spin spaces and thus also applies for unrestricted mos . for larger wave function expansions , in particular , when a large number of simultaneous and excitations are present , the reduction can be dramatic . using the casscf(12,12 ) case as an extreme example , the reduction from 2 npair = 7.8 10 spin determinants to nfac = 1.3 10 unique factors exceeds 5 orders of magnitude . in general , it can be worked out for large casscf wave functions that , showing that casscf wave functions profit optimally from this reduction . the determinants are first individually sorted according to their and parts , and the repetitive blocks are identified . in a next step , . the final overlap computation amounts to a contraction step of the form14where the outer sum over k is implemented as an explicit loop , and the inner sum over l is realized as a matrix - vector product using the blas ( basic linear algebra subprograms ) library . if the number of unique factors is given as nfac , then the scaling of the determinant computations is reduced to . by contrast , the contraction step ( eq 14 ) considers the full number npair = nci nci of ci coefficients and scales as times the number of pairs of states . in practical calculations , either one of these steps can be the time - critical one depending on the ratio npair / nfac , the number of electrons n , and the number of states . the downside of precomputing and storing the factors is the high memory demand . however , in general we find that whenever the computation is feasible with respect to cpu time , memory restrictions do not play a significant role . furthermore , an additional algorithm was implemented , which rearranges the ci vectors in a way that the factors can be computed on - the - fly , whereas only the factors have to be precomputed . this semidirect algorithm allows for cutting the memory demands in half while showing similar performance with respect to storing all factors in memory . a notable loss of performance is only observed when the available memory is reduced even further . even when the and factors are precomputed , the computation of determinants is still a time critical step . to make this computation as fast as possible , we combine two prominent methods of calculating determinants . as one option , determinants can be computed by laplace s recursive formula15where ij denotes the matrix a with the ith row and the jth column deleted . a full recursive implementation of this equation would lead to an undesired factorial scaling . therefore , lu factorization is used as the main tool because it allows for computing determinants with scaling . taking a close look at eq 15 , however , one realizes that it allows the reuse of intermediates . especially in the context of ci expansions , it is observed that many determinants differ only with respect to the last orbital . in such a case , it is beneficial to perform a laplace expansion along the last column and precompute the cofactors ( 1)|in| to be used for all related determinants . in the present implementation , an automatic algorithm is used to decide case - by - case which determinants are computed directly and which ones via their cofactors , as given in eq 15 . the cofactors are in turn computed by lu decomposition , and no further recursion is carried out . this procedure is particularly efficient for wave functions constructed as single excitations out of one or a few references , and it is therefore complementary to the application of the factors , which are applicable for higher excitation levels . an alternative to this approach would be to use the matrix determinant lemma as discussed , e.g. , in ref ( 25 ) . however , the advantage of the present approach is that it does not require any matrix inversions or other numerically unstable steps . in addition to the previous algorithmic changes that do not affect the computed result , we also want to discuss two approximation schemes that do affect the results . first , a threshold t for a truncation of the ci vector is introduced . the elements dik are sorted by their magnitude , and then all the elements beyond a given index kt are discarded . the index kt in turn is determined as the smallest number giving16the overlaps are computed with respect to the truncated wave function17where , following eq 16 , the squared norm of this wave function is greater or equal to t18this approximation allows for significantly reducing the number of terms to be computed while recovering the major part of the wave function . truncation of the wave functions will generally lead to an underestimation of the overlaps . to overcome this problem if it can be assumed that the angles between the original and truncated wave functions are approximately equal19then the overlap between the normalized functions |i and |j can be approximated by the renormalization20the assumption of eq 19 should be valid if the same general wave function model is applied for |i and |j. in cases where very accurate values are required , the convergence of the results should be studied by varying the threshold values . below , an orthogonalization procedure is discussed , which has a similar effect on truncated wave functions but also allows for the correction of terms stemming from orbital displacements . the second approximation is based on discarding a number ncore of frozen core orbitals . for these orbitals , which are required to be occupied in all the determinants , it is assumed that21and consequently that they are also orthogonal to all noncore orbitals . under this assumption , these orbitals can simply be eliminated from the calculation leading to smaller determinants in eq 8 . furthermore , as discussed below , discarding core orbitals can improve the numerical stability of the computation if atoms are moved , because displacements of atoms with tight core orbitals can introduce numerical artifacts . four types of input quantities are needed : the mixed ao overlaps , the mo coefficients , the occupation strings of the determinants , and the ci coefficients . the mixed ao overlaps have to be computed explicitly in cases where the molecular geometry or the basis set are varied . technically , this step is most readily performed by using a standard integral code and computing the overlap integrals for a formal double molecule . the mo coefficients are usually directly available in ascii format . with respect to the ci vectors , three preparation steps are necessary : conversion from configuration state functions to slater determinants , extraction of the determinant strings , and rearrangement of the orbitals to comply with eqs 5 and 6 . after the input is read , the ao overlaps are combined with the mo coefficients according to eq 11 to compute the mixed mo overlaps . these are in turn used in connection with the determinant strings to compute the unique factors ( eq 8) . in the final step ( eq 14 ) , these factors are contracted with the ci coefficients to give the overlap . the code is written in a modular fashion , which allows for an easy interface to various quantum chemical programs and file formats when reading the input quantities shown on the left in scheme 1 . currently , interfaces to the columbus 7.0 and molcas 8.0 program packages are available for accessing the ci vectors , mo coefficients , and binary integral files . we want to inspect the properties of the overlap matrix s under the assumption that the two sets { |i } and { |j } are individually orthonormal . then , a wave function |i of the first set can be expanded using the resolution of the identity22here , |i is used to denote the component of |i that belongs to the orthogonal complement of the space spanned by the { |j}. this suggests to think of a decomposition of the wave function |i in terms of the individual projection components and the missing part belonging to the orthogonal complement . projection of the above equation onto i|23shows that the combined weight of these components is normalized to unity and consequently that the sum of the squared overlap values along a column ( or equivalently a row ) of the overlap matrix is less than or equal to one . if the two sets { |i } and { |j } span the same space , then the orthogonal component |i vanishes , and s becomes the transformation matrix between the two sets . furthermore , a projection of eq 22 onto a function of the first set k| immediately shows that s is an orthogonal matrix24 in practical applications , the calculated overlap matrix could deviate from orthogonality due to different reasons . for example , when modifying the molecular geometry during a dynamics simulation , such a deviation could be an indication of interactions with external states . in this case , the i value contains important nontrivial information . on the other hand , nonorthogonality of the matrix could simply be present for numerical reasons , e.g. , from the wave function truncation or from displaced basis functions . then , the stability of the results can benefit from an orthogonalization of the raw overlap matrix . for this purpose , a symmetric ( lwdin ) orthogonalization first , a singular value decomposition of the overlap matrix is performed25to determine the transformation matrices u and v along with the singular values 1, ... ,n . following numerical tests in ref ( 3 ) , we suggest applying this procedure for dynamics simulations , preferably in connection with the local diabatization formalism . when a comparison of wave functions constructed with different models is performed , the i term is an integral component of the discussion as it allows for quantifying discrepancies in the description . in such a case , the orthogonalization procedure is not expedient , but a renormalization ( eq 20 ) can be carried out to correct for the wave function truncation . the application of wave function overlaps for nonadiabatic dynamics has been discussed in detail elsewhere . therefore , we shall only present some relations immediately relevant for the following discussion . the symbol |i ( r) is used to denote the parametrical dependency of the electron wave function on the nuclear geometry r , whereas the electronic coordinates are considered only implicitly . in this nomenclature , the nonadiabatic coupling vector between states i and j is defined as27coupling vectors can be computed by response theory using a similar formalism to the computation of gradients . aside from the optimization of conical intersections , they are indeed widely used in nonadiabatic dynamics simulations . however , there are a number of issues that can come into play for different application areas . first , coupling vectors are only available for a limited number of methods and program packages . second , the computation of the required one- and two - electron derivative integrals can be the overall time - limiting step if large molecules or extended basis sets are used in connection with restricted wave function expansions . third , the convergence of the coupled - perturbed mcscf equation system is not trivial as discussed , e.g. , in ref ( 7 ) . finally , numerical problems can arise in the case of highly peaked coupling vectors . for these reasons , it is beneficial to have an efficient and general alternative available , which is provided by wave function overlaps . to understand the connection between overlaps and coupling vectors , eq 27 is written in the form28where the symbol is used to denote the gradient vector with respect to the r coordinates . the directional derivative with respect to a displacement vector rd is evaluated as29replacing the limit with a fixed small value of t and setting r= trd yields the discrete approximation30although this expression reduces to the exact directional derivative in the limit of |r| 0 , the linear approximation is not necessarily stable when r is increased . in such cases , the use of a somewhat more extended formalism using a locally diabatic wave function propagation is recommended for dynamics simulations . in this section , we investigate the accuracy and performance of the new implementation of wave function overlaps . then , two crucial numerical questions that have not received much attention so far despite the wide application for wave function overlaps in nonadiabatic dynamics simulations will be addressed . these are concerned with the consequences of wave function truncation as applied to keep the computational effort at an acceptable level and with numerical artifacts stemming from displaced atoms and orbitals . finally , the performance and the parallel scaling of the individual computation steps are examined . before proceeding to more specific numerical and performance issues , we want to verify the general numerical accuracy of the present code . for this purpose , we will first show that the results are consistent with general nonadiabatic theory by computing a path integral in molecular coordinate space and then proceed to a comparison of the results with respect to two different implementations . following previous work by some of us , the example molecule used throughout much of this work is selenoacroleine , shown in figure 1 . we will first discuss the torsion around its c = c bond , using results computed at the mr - ci level with single excitations ( mr - cis ) . in figure 2a , the energies of the lowest two triplet states are plotted with respect to this torsion . both states have their minimum energy at the planar geometry ( = 0 ) . at this point , t1 is of n * character , whereas t2 is a * state . as the torsion angle is increased , the t1 ( n * ) energy increases strongly , whereas the t2 ( * ) state shows a flatter profile . at around 55 , the states exhibit an avoided crossing , and at larger torsion angles , they exchange their state character . the path integral over the coupling vector was computed by numerical integration using eq 30.31where r are intermediate geometries . the value of the line integral over the 0 to 90 rotation amounts to approximately /2 . by symmetry , it is clear that the full rotation over 360 amounts to four times this value . accordingly , the integral around a closed path gives32a multiple of , in line with general considerations . we have therefore shown that the wave function overlaps computed here give not only a qualitatively but also a quantitatively correct picture of a passage through an avoided crossing , justifying the application of these quantities for nonadiabatic dynamical simulations . molecular structure of the selenoacroleine molecule and indication of the torsion angle . torsion of selenoacroleine along the cc double bond : ( a ) energies of the t1 and t2 states computed at the mr - cis / ano - rcc - vdzp level and ( b ) line integral over the nonadiabatic coupling vector converging against /2 . as a next step , the numerical results will be compared to two different previous implementations , a general overlap code that has been extensively used for surface hopping dynamics within the newton - x(14,17,40 ) and sharc(4143 ) packages and an implementation based on the state interaction formalism that is part of the molcas 8.0 program package . furthermore , results deriving from a simple scalar product of the ci vectors will be presented . to allow for a quantitative numerical comparison of results obtained with different program packages , meticulous control of all wave function parameters is necessary , and therefore , only a few selected examples are discussed in the following . we again choose selenoacroleine and compute the overlap between wave functions constructed at two different geometries around the avoided crossing with = 50 and 55 representing the geometries that could be present in two subsequent time steps in a dynamics simulation . first , casscf computations are carried out considering 6 electrons in 5 active orbitals and state - averaging over only the two triplet states , denoted casscf(6,5 ) . as this approach is implemented in molcas 8.0 as well as in columbus 7.0 , we can compare all of the above methods for overlap computation , and the results are presented in table 1 . there are nci = nci = 90 slater determinants contained in the casscf(6,5 ) wave function , which in turn means that npair = nci nci = 8100 pair - determinant computations are necessary , each of which requires the computation of the and spin - factors and . although all of these are computed explicitly in the code of ref ( 12 ) , only the nfac = 200 unique ones of them are evaluated with our new methodology . for this small case , almost perfect numerical agreement is observed with respect to the program of ref ( 12 ) , and the differences are below 1 10 . a comparison with the state interaction computation shows a semiquantitative agreement , giving a difference of 0.0010.002 with respect to the previous results . this discrepancy probably derives from the fact that , as to our understanding , the overlap terms of displaced aos | are neglected in the state interaction implementation . the ci vector dot product is also qualitatively correct here , showing however a somewhat larger discrepancy on the order of 0.005 . the above calculation including only triplet states was performed for technical reasons , as it allows a comparison to the implementation in molcas 8.0 , which only supports state - averaging within a spin multiplicity . we will now proceed to state - averaging over two singlets and two triplets simultaneously , i.e. , the casscf(6,5 ) level , which is the level used in the remaining part of this work . as seen in table 1 , the overlap elements are modified significantly by the different orbitals present due to the altered state - averaging , e.g. , the t1|t1 element is lowered from 0.924 to 0.687 . this shows the critical impact that state - averaging can have on the resulting wave functions . moving from casscf to mr - cis increases the computational time significantly . we start with a smaller active space of 4 electrons in 3 orbitals . for these mr - cis(4,3 ) computations , perfect agreement between the new code and the implementation of ref ( 12 ) is observed , and at the same time the computational time is reduced by a factor of 1000 , i.e. , from half a day to half a minute . a factor of approximately 40 of this speedup derives from the reduction in the number of spin factors from 1.7 10 to 4.6 10 , and a similar effect derives from the additional algorithmic improvements , as discussed above . the largest calculation here is mr - cis(6,5 ) , requiring the computation of 5 10 pair determinants . by contrast , it was not possible for us to compute the exact overlap with the code of ref ( 12 ) , and a screening formalism was used to reduce the number of spin factors to 1.2 10 , yielding a computation that could be finished in 2 days . the discrepancy in this case is on the order of 0.001 , and the error of a simple ci vector overlap is 1 order of magnitude larger . although a quantitative agreement with ref ( 12 ) is obtained , the results could be accelerated by 3 orders of magnitude , thereby significantly expanding the scope of problems that can be treated . npair and nfac denote the number of slater determinant pairs in the expansion and the number of and spin factors actually computed , respectively . as a second example , selected results on the model iridium complex fac - tris(3-iminoprop-1-en-1-ido)iridum [ ir(c3h4n)3 ] , as shown in figure 3 , will be presented , a system that some of us have studied in detail recently . here , we want to verify our results against the state - interaction implementation in molcas . to construct a job that can be properly compared between the two implementations , the geometry and active space are left unaltered between the bra and ket states . the only parameter varied is the number of singlet states in the state - averaging procedure using values of 1 , 4 , and 10 . the strong impact that state - averaging exerts on the resulting wave functions is apparent . switching from one to four states , the overlap between the 1a ground states only amounts to 0.892 , and there is also some non - negligible overlap of 0.044 between this state and the excited 2a state . by contrast , the ground state wave functions are almost equivalent between four and ten states , showing that the higher excited states require similar orbitals as the lower ones . from a methodological point of view , a quantitative agreement with deviations below 1 10 between our implementation and ref ( 13 ) is observed . in table 2 , these are qualitatively consistent with the actual wave function overlaps but exhibit significantly larger deviations . overlap terms of the 1a and 2a states of ir(c3h4n)3 between casscf(12,9 ) wavefunctions considering different numbers of singlet states nav , nav in the state - averaging procedure . in table 2 , these signs derive from the overall phases of the wave functions as computed by molcas . although the phases possess no physical meaning in isolated calculations , it is crucial to control them in dynamics simulations to obtain smoothly varying matrix elements along a trajectory . here , wave function overlaps offer a clear way to monitor wave function phases , independent of any orbital rotations or orbital phase changes . indeed , consistent sign information is obtained between the current implementation and the one of ref ( 13 ) . by contrast , a simple scalar product between ci vectors yields the opposite signs for the off - diagonal elements in this example . despite the significant algorithmic improvements reported above , it is necessary to allow for a truncation of the wave function to keep the computational cost acceptable for large wave function expansions . for this purpose , the threshold t ( eq 16 ) is used , pertaining to the minimal squared norm of the truncated wave function . selenoacroleine is considered , and overlaps are computed between the two geometries at = 50 and 55 torsion . computations are performed at the mr - cis(6,5 ) and mr - cisd(6,5 ) levels of theory , and the threshold t is varied systematically . the value for the overlap between the t1 state at 50 and the t2 state at 55 torsion is depicted in figure 4a . the mr - cisd values ( red ) differ significantly from the mr - cis values ( black ) : whereas the former indicate strong nonadiabatic interactions with overlap elements above 0.8 , the latter values are below 0.2 . this type of discrepancy , which derives from slightly altered potential surfaces due to dynamic electron correlation , is well - known in the literature ( see , e.g. , ref ( 45 ) ) and will not be discussed in more detail here . the current focus is an analysis of the numerical stability of the results within a chosen computational protocol . for this purpose , three values are considered : ( i ) the raw overlap between the truncated wave functions , ( ii ) the renormalized overlap according to eq 20 , and ( iii ) the orthogonalized overlap value . at the mr - cis level of theory , a stronger deviation exists for the smallest value of 0.90 . however , even in this case , qualitative agreement is found , and all of the wave function phases are reproduced correctly . furthermore , it is observed that the raw overlaps are always somewhat smaller than the renormalized ones , which are in turn smaller than the orthogonalized ones . the first effect derives from the wave function truncation , and the second derives from the geometric displacement , as will be analyzed in the next section . the convergence of the raw mr - cisd results is significantly worse when compared to mr - cis . figure 4b presents the number of slater determinant pairs ( npair , filled symbols ) and the number of actually computed determinant factors as shown in eq 8 ( nfac , empty symbols ) . there is a steep increase in the number of terms to be computed as the threshold goes against 1 . in the case of mr - cisd , npair goes up to 1.7 10 , whereas in the case of mr - cis , this value reaches 2.4 10 . however , not all of these terms are unique , and the number of factors computed ( nfac ) is significantly lower than npair , differing by approximately 2 orders of magnitude in most cases . figure 4c shows the computation time expended and the memory needed . in the mr - cis case , all of the computations are finished in at most a few minutes , and the memory requirements never exceed one gigabyte ( gb ) . by contrast , for mr - cisd , a steep scaling of time and memory with the threshold becomes apparent . the largest computation shown here requires over a million core - seconds , which amounts to approximately 10 h on 32 cores . storing all 7.4 10 factors occurring in this case would require 567 gb . to somewhat reduce this workload , a semidirect algorithm is implemented , which reduces the memory requirements by approximately one - half . the mr - cis wave function calculation in columbus 7.0 takes approximately 1 min . the computation of the nonadiabatic coupling vectors adds another 2 min , and the overlaps are computed in only 12 s ( t = 0.99 ) . in the case of mr - cisd , two and a half hours are required for the wave function calculation and , again , 2 min are added for the coupling vectors . in this case , the coupling vectors are cheaper than overlaps , which require at least 16 min of computation time ( t = 0.95 ) . still , in both cases , the overlaps can be computed in less time than the wave function calculations . in general , the question of whether nonadiabatic coupling vectors or overlaps are cheaper will depend on the wave function model , the basis set , and the number of electrons , and it is beneficial to have both methods available . the above results show that qualitative information about wave function character and phase can be obtained in a few seconds when using a threshold value of t = 0.9 . almost converged results are obtained at t = 0.95 or 0.97 while still allowing for favorable computation times when compared to the effort of the actual mr - ci computation . enhanced numerical stability is obtained if the results are additionally renormalized or orthogonalized . for quantitative applications , we suggest using 0.95 as a minimal threshold value . however , in many cases , larger thresholds are affordable , and for smaller wave function expansions , such as in casscf calculations , truncation becomes unnecessary . performance of the wave function overlap code for the t1|t2 element at the mr - cis(6,5 ) and mr - cisd(6,5 ) levels of theory in the case of selenoacroleine between geometries with 50 and 55 torsion using varying screening thresholds t : ( a ) the overlap considering raw , renormalized , and orthogonalized results , ( b ) the total number of determinant pairs ( npair ) and the number of unique and factors ( nfac ) , and ( c ) the computation time and memory requirements . whenever atoms are displaced , as is the case most prominently in dynamics simulations , the overlaps are not only affected by the actual nonadiabatic interactions of interest but also by more trivial consequences of the displacement , e.g. , the shift of the orbitals in space . as a consequence , the step size in nonadiabatic dynamics simulations will not only affect the general numerical stability of the wave function propagation , but when overlaps are applied , the specific effect of displaced orbitals should be taken into account , as well . it is worth noting that this problem is not a consequence of the use of atom - centered basis functions , but that it also exists in the complete basis set limit . computation of the casscf(6,5 ) t1|t2 overlap element for a selenoacroleine molecule displaced in the x - direction ( = 50 ) with respect to a stationary one ( = 55 ) . raw overlaps are given as dotted lines , and results after orthonormalization are given as solid lines ( all overlapping ) . the number of discarded core orbitals ( ncore ) is indicated by the color . to construct a controlled test for this issue , we again consider the selenoacroleine molecule and its two geometries at = 50 and 55. while one geometry ( = 55 ) is kept stationary in space , the other ( = 50 ) is translated in the x - direction ( cf . figure 1 ) up to a displacement of 0.1 . from a physical point of view , the nonadiabatic interactions should not be modified by this translation , and any modulations of the overlap elements are therefore unwanted artifacts . the t1|t2 overlap element is computed at the casscf(6,5 ) level of theory using different settings . on the one hand , three numbers of discarded core orbitals are used : 0 , 5 ( se-1s , se-2s , se-2p ) , and 12 ( also se-3s , se-3p , 3 c-1s ) . on the other hand , the results are presented in figure 5 . at zero displacement , all values agree , giving a value of 0.71069 for the raw overlaps and once the molecule is displaced , a very steep decline of the raw overlaps ( shown as dashed lines ) is observed . this effect is particularly pronounced with zero discarded core orbitals , whereas the results are somewhat more stable when this number is increased to 5 or 12 . to understand this effect , it is instructive to regard the size of the 1s core orbitals , which can be estimated as a(z ) = a0/z , where a0 is the bohr radius and z is the nuclear charge . the smallest orbital in the system is the se-1s orbital with a(34 ) = 0.016 . indeed , this distance corresponds approximately to the displacement where the dashed black curve reaches half its maximum . once the se-1s orbital and the other core orbitals are discarded , the decay of the overlap is less steep but still significant . considering , for example , a displacement of 0.1 , which amounts to only a small fraction of an interatomic bond distance , the values for 0 , 5 , and 12 discarded core orbitals are 9 10 , 0.009 , and 0.057 , respectively . this decay is problematic for dynamics simulations because even the smallest translation of the molecule , which might occur because of numerical inaccuracies , can affect the computed overlap values . a similar effect , albeit more difficult to quantify in contrast to the raw overlap values , excellent numerical stability is observed after orthogonalization . even in the case of 0.1 displacement , the orthogonalized overlap matrix elements are all 0.71882 irrespective of the number of discarded core orbitals . the good performance of the orthogonalization process can be understood by the fact that all elements of the overlap matrix are scaled down uniformly by the translation and that the orthogonalization then simply amounts to renormalizing these elements . two important conclusions can be drawn from figure 5 : first , core orbitals can have an unwanted effect on the overlap values despite not being involved in the nonadiabatic process . discarding them improves the numerical stability while at the same time saving computational effort . second , orthogonalization of the overlap matrix is a powerful tool to dispose of unwanted effects deriving from the molecular displacement . whereas the focus of this investigation was concerned with the effects of discrete displacements , it would be of interest to evaluate whether a similar procedure can be applied to eliminate the lack of rotational and translational invariance of nonadiabatic coupling vectors . however , this question is out of the scope of this work . in a next step , the general performance of the new implementation is examined . for this purpose , five distinct wave function expansions are chosen for the selenoacroleine molecule . aside from the mr - cis(6,5 ) and mr - cisd(6,5 ) methods discussed before , casscf(10,10 ) and casscf(12,12 ) computations are also performed to represent the case of larger active spaces , all using the ano - rcc - vdzp basis set . furthermore , the mr - cis(4,3)-1x expansion ( see computational details ) is chosen as a case with only two references in connection with the larger ano - rcc - vtzp basis set . various values of the threshold t were used to produce a set of 20 data points . in figure 6 , the computation times are plotted against the number of pair determinants npair = nci nci. all these data points are roughly on a straight line , highlighting the uniform performance characteristics of the code with respect to this diverse set of wave functions . in a simple direct algorithm , formal linear scaling of the timings with respect to npair is expected ( see section 2.2 ) . by contrast , the effective scaling behavior seen here , obtained as the slope in the logarithmic plot , is close to . it is observed that the above algorithm is particularly efficient for large casscf expansions owing to the fact that these allow for the strongest reduction in the number of spin factors . in the case of casscf(12,12 ) , there are npair = 3.9 10 terms to be computed that can be represented by only nfac = 1.3 10 spin factors . indeed , in this case , the computation time is determined by the final contraction ( eq 14 ) , whereas the primary determinant computation ( eq 8) requires less than 1% of time . the mr - cis expansions profit from the one - step laplace recursion of eq 15 , which again allows for efficient computation of the determinants . by contrast , the mr - cisd results are somewhat above the remaining data points , showing that further speedup would be possible through a more extended use of the laplace recursion ( eq 15 ) or a similar formalism . however , also in these cases , 1020% of the computation time is used for the contraction step ( eq 14 ) , setting a clear limit for the effect of any possible improvement in the determinant computation . performance of the wave function overlap code for varying wave function expansions : computation time plotted against the number of determinant pairs ( npair ) . the parallel performance in shared memory is tested in the case of an extended mr - cisd(6,5 ) calculation on selenoacroleine covering npair = 6.7 10 determinant pairs ( i.e. , the t = 0.995 case in figure 4 ) . this figure presents the good scalability of the code , showing in fact superlinear scaling . in figure 7b , the total computation time is dissected into the different relevant steps . the core hours consumed are plotted against the number of cores , a representation where perfect parallel scaling amounts to a horizontal line . the two major time - consuming tasks are the determinant computations ( eq 8) and the final contraction of the factors with the ci vector ( eq 14 ) . as seen in figure 7b , almost ideal scaling is obtained in the case of the determinant computations as this is a cpu - time - limited step with little memory overhead . by contrast , the contraction step shows somewhat erratic behavior , even providing a superlinear speedup for an intermediate number of cores . this behavior is probably a consequence of the fact that this step is limited by memory bandwidth and the precise usage of various cache levels . figure 7b also presents timings of the sorting step with a parallelization up to four cores and the sequential i / o step . the relative contributions of these steps increase linearly with the number of cores . however , even at 32 cores , these amount to only 1% of the total computation time , and further parallelization is not necessary . parallel performance of the overlap code for a selenoacroleine mr - cisd(6,5 ) computation requiring the evaluation of 6.7 10 slater determinant overlaps : ( a ) speedup from 1 to 32 cores and ( b ) computation times for the different steps as discussed in the text . a new algorithm for the computation of wave function overlaps between many - electron wave functions is presented . by virtue of an optimized algorithm , which makes extended use of recurring intermediates , a highly efficient code was generated , which allows for computations with mr - cisd wave functions , with large active space , and with extended basis sets . because of the general formalism employed , there are no restrictions with respect to the wave functions except that they have to be given in a slater determinant expansion based on restricted or unrestricted molecular orbitals . consequently , it is possible to vary the wave function model , the orbitals , the one - electron basis set , and the molecular geometry . the code is directly applicable to models producing explicit wave functions , i.e. , the configuration interaction and multiconfigurational scf methods . extensions to time - dependent density functional theory , coupled cluster , and other response theory methods are straightforward using approximations that have been described previously . the code was verified against general nonadiabatic theory by computing a circular path integral in coordinate space and against two existing implementations by using appropriate example computations , showing excellent agreement . furthermore , the effects of using truncated wave functions were studied , and it was found that values of t = 0.95 or 0.97 for the squared norm of the truncated wave function could already provide satisfactory results . in addition , attention was devoted to understanding unwanted effects deriving from discrete displacements of atoms and orbitals , which naturally occur in dynamics simulations . for both cases , wave function truncation and orbital displacement , it was found that an orthogonalization of the overlap matrix can improve the results dramatically . the wave function overlap code has been interfaced to the sharc program package with the focus of performing nonadiabatic dynamics simulations . because of the general formalism employed , the code is certainly not limited to this application , and other tasks can be envisaged where the new code will be beneficial , for example , the comparison of wave functions constructed at different levels of theory and probing the initial electronic states after -decay or after the photoionization of core - electrons . furthermore , the computation of dyson norms , as required for the simulation of photoelectron spectra , can be achieved by a slight modification of the code . most calculations on selenoacroleine were performed using an active space containing 6 electrons in 5 active orbitals ( 2 , nse , 2 * ) , i.e. , casscf(6,5 ) and mr - ci(6,5 ) , in connection with the ano - rcc - vdzp basis set including state - averaging over the lowest two singlet and triplet states . the active space was enhanced for casscf(10,10 ) and casscf(12,12 ) computations , whereas a smaller active space of 3 orbitals ( , nse , * ) was used for mr - cis(4,3 ) and mr - cis(4,3)-1x computations . in the latter case , the maximum number of reference excitations into the * orbital was restricted to 1 , resulting in only two reference configurations , and the computation was performed using the larger ano - rcc - vtzp basis set . scalar relativistic effects were taken into account in these all - electron calculations by using the second - order douglas kroll unless otherwise specified , 12 core orbitals corresponding to the s and p orbitals in the first , second , and third shells on se and the 1s orbitals on c were frozen in the mr - ci computations and discarded in the wave function overlap computations . for the triplet / triplet overlaps , generally , the ms = 1 wave functions were considered , as these contain fewer determinants than the ms = 0 ones , allowing for speedup of the computations while not affecting the results . casscf computations on ir(c3h4n)3 were performed by including 12 electrons in 9 orbitals ( 3 , 3 d , 3 * ) . the iridium atom was described by the lanl2dz effective core potential ( ecp ) , in its small - core version , and the corresponding basis set for the active ( 5s , 5p , 5d , 6s , 6p ) orbital shells , whereas for the remaining atoms , the 6 - 31 g * basis set was employed . the mr - ci computations were carried out with the columbus 7.0 program package using its parallel mr - ci implementation , whereas molcas 8.0(27,59 ) was applied for the integrals and most of the casscf calculations . in the cases of comparing different overlap programs , generally no frozen core orbitals were considered to allow for a clear comparison . the benchmark calculations for varying wave function models ( figure 6 ) were performed on one core of an intel xeon e5 - 2650-v3 cpu at 2.3 ghz , whereas the parallel performance tests ( figure 7 ) were carried out on an hp dl580g7 server with 512 gb of main memory and 4 intel e7 - 4850 ( westmere ) cpus at 2.0 ghz with 10 cores each .

a new algorithm for the computation of the overlap between many - electron wave functions is described . this algorithm allows for the extensive use of recurring intermediates and thus provides high computational efficiency . because of the general formalism employed , overlaps can be computed for varying wave function types , molecular orbitals , basis sets , and molecular geometries . this paves the way for efficiently computing nonadiabatic interaction terms for dynamics simulations . in addition , other application areas can be envisaged , such as the comparison of wave functions constructed at different levels of theory . aside from explaining the algorithm and evaluating the performance , a detailed analysis of the numerical stability of wave function overlaps is carried out , and strategies for overcoming potential severe pitfalls due to displaced atoms and truncated wave functions are presented .

Introduction Theory Accuracy and Performance Conclusions Computational Details

the evaluation of matrix elements between many - electron wave functions expanded in different orbital basis sets or over different molecular geometries is a task where the full complexity of these wave functions becomes apparent . not only the expansion of the wave functions into individual configurations and the construction of the molecular orbitals have to be taken into account , but also the explicit determinantal form , as required by the pauli principle , comes into play . a new algorithm for the computation of wave function overlaps is presented , which is distinguished by enhanced computational performance reached through extensive reuse of recurring intermediate quantities . at the same time , a flexible formalism is used allowing for the computation of wave function overlaps for varying wave function expansions , molecular orbitals ( mos ) , basis sets , and molecular geometries . wave function overlaps are widely used in the field of nonadiabatic dynamics , where they allow the evaluation of state - to - state transition probabilities without the need of computing nonadiabatic coupling vectors . aside from the fact that this strategy provides a technical advantage for methods where coupling vectors are not available , it has been shown that , in the case of highly peaked nonadiabatic couplings , wave function overlaps can provide superior numerical stability , in particular when a locally diabatic propagation of the wave functions is carried out . in the simplest case , the overlap is approximated as a scalar product of the configuration interaction ( ci ) vectors to provide a qualitative description of changes in wave function character , possibly after a diabatization of the orbitals . beyond this , exact overlap terms have been derived under a number of assumptions ; implementations are available for semiempirical methods , plane wave expansions , single - reference methods with atom - centered basis sets , and multireference methods . despite this wide interest , the practical use of wave function overlaps is hampered by high computational demands , especially when multiconfigurational wave functions are used , which are necessary for the correct description of many nonadiabatic processes . moreover , the numerical stability of the results with respect to truncation of the wave functions and the consequences of displaced orbitals in the case of varying molecular geometries have received almost no attention so far despite the fact that these can have a crucial impact on the computed results . the purpose of this work is to present a new general algorithm for the efficient computation of wave function overlaps and to address some related numerical questions . we first discuss the general theory of wave function overlaps in the framework of slater determinant expansions . in order to give practical advice for future applications , the numerical stability of the results with respect to wave function truncation and atom displacements is discussed , and we show how orthogonalization of the overlap matrix can significantly improve the results . in the following , we use the notation | to denote antisymmetric many - electron wave functions constructed as linear combinations of slater determinants . the only requirement for the relation between these wave functions is that they contain the same number of and electrons , but they are otherwise allowed to vary in the wave function expansion , the mos , the basis set , and the molecular geometry . the overlap of the two slater determinants is given by the determinant of the matrix containing all mutual orbital overlaps ( see appendix a and refs ( 9 and 21)).7using the arrangement chosen in eqs 5 and 6 , the matrix becomes block diagonal due to the fact that overlaps between orbitals of different spin vanish . assuming eq 13 to be approximately valid allows for computing the overlap as a simple scalar product between ci vectors ( expanded either in a slater determinant or csf basis ) , yielding a strategy that has indeed been applied successfully for dynamics simulations ( see , e.g. this can be achieved either by a singular value decomposition of the mixed mo overlap matrix shown in eq 11 to obtain the corresponding orbitals or by a somewhat more involved formalism involving nonunitary orbital transformations as implemented in the molcas 8.0 suite . the algorithm presented above requires the computation of two determinants for every pair of bra and ket slater determinants , see eq 8 . the cofactors are in turn computed by lu decomposition , and no further recursion is carried out . this procedure is particularly efficient for wave functions constructed as single excitations out of one or a few references , and it is therefore complementary to the application of the factors , which are applicable for higher excitation levels . the index kt in turn is determined as the smallest number giving16the overlaps are computed with respect to the truncated wave function17where , following eq 16 , the squared norm of this wave function is greater or equal to t18this approximation allows for significantly reducing the number of terms to be computed while recovering the major part of the wave function . to overcome this problem if it can be assumed that the angles between the original and truncated wave functions are approximately equal19then the overlap between the normalized functions |i and |j can be approximated by the renormalization20the assumption of eq 19 should be valid if the same general wave function model is applied for |i and |j. below , an orthogonalization procedure is discussed , which has a similar effect on truncated wave functions but also allows for the correction of terms stemming from orbital displacements . furthermore , as discussed below , discarding core orbitals can improve the numerical stability of the computation if atoms are moved , because displacements of atoms with tight core orbitals can introduce numerical artifacts . then , a wave function |i of the first set can be expanded using the resolution of the identity22here , |i is used to denote the component of |i that belongs to the orthogonal complement of the space spanned by the { |j}. furthermore , a projection of eq 22 onto a function of the first set k| immediately shows that s is an orthogonal matrix24 in practical applications , the calculated overlap matrix could deviate from orthogonality due to different reasons . then , the stability of the results can benefit from an orthogonalization of the raw overlap matrix . for this purpose , a symmetric ( lwdin ) orthogonalization first , a singular value decomposition of the overlap matrix is performed25to determine the transformation matrices u and v along with the singular values 1, ... ,n . when a comparison of wave functions constructed with different models is performed , the i term is an integral component of the discussion as it allows for quantifying discrepancies in the description . in such a case , the orthogonalization procedure is not expedient , but a renormalization ( eq 20 ) can be carried out to correct for the wave function truncation . the application of wave function overlaps for nonadiabatic dynamics has been discussed in detail elsewhere . the symbol |i ( r) is used to denote the parametrical dependency of the electron wave function on the nuclear geometry r , whereas the electronic coordinates are considered only implicitly . in this nomenclature , the nonadiabatic coupling vector between states i and j is defined as27coupling vectors can be computed by response theory using a similar formalism to the computation of gradients . second , the computation of the required one- and two - electron derivative integrals can be the overall time - limiting step if large molecules or extended basis sets are used in connection with restricted wave function expansions . in such cases , the use of a somewhat more extended formalism using a locally diabatic wave function propagation is recommended for dynamics simulations . in this section , we investigate the accuracy and performance of the new implementation of wave function overlaps . then , two crucial numerical questions that have not received much attention so far despite the wide application for wave function overlaps in nonadiabatic dynamics simulations will be addressed . these are concerned with the consequences of wave function truncation as applied to keep the computational effort at an acceptable level and with numerical artifacts stemming from displaced atoms and orbitals . as a next step , the numerical results will be compared to two different previous implementations , a general overlap code that has been extensively used for surface hopping dynamics within the newton - x(14,17,40 ) and sharc(4143 ) packages and an implementation based on the state interaction formalism that is part of the molcas 8.0 program package . to allow for a quantitative numerical comparison of results obtained with different program packages , meticulous control of all wave function parameters is necessary , and therefore , only a few selected examples are discussed in the following . we again choose selenoacroleine and compute the overlap between wave functions constructed at two different geometries around the avoided crossing with = 50 and 55 representing the geometries that could be present in two subsequent time steps in a dynamics simulation . as this approach is implemented in molcas 8.0 as well as in columbus 7.0 , we can compare all of the above methods for overlap computation , and the results are presented in table 1 . there are nci = nci = 90 slater determinants contained in the casscf(6,5 ) wave function , which in turn means that npair = nci nci = 8100 pair - determinant computations are necessary , each of which requires the computation of the and spin - factors and . as seen in table 1 , the overlap elements are modified significantly by the different orbitals present due to the altered state - averaging , e.g. switching from one to four states , the overlap between the 1a ground states only amounts to 0.892 , and there is also some non - negligible overlap of 0.044 between this state and the excited 2a state . by contrast , the ground state wave functions are almost equivalent between four and ten states , showing that the higher excited states require similar orbitals as the lower ones . for this purpose , the threshold t ( eq 16 ) is used , pertaining to the minimal squared norm of the truncated wave function . computations are performed at the mr - cis(6,5 ) and mr - cisd(6,5 ) levels of theory , and the threshold t is varied systematically . the value for the overlap between the t1 state at 50 and the t2 state at 55 torsion is depicted in figure 4a . the current focus is an analysis of the numerical stability of the results within a chosen computational protocol . for this purpose , three values are considered : ( i ) the raw overlap between the truncated wave functions , ( ii ) the renormalized overlap according to eq 20 , and ( iii ) the orthogonalized overlap value . at the mr - cis level of theory , a stronger deviation exists for the smallest value of 0.90 . however , even in this case , qualitative agreement is found , and all of the wave function phases are reproduced correctly . there is a steep increase in the number of terms to be computed as the threshold goes against 1 . the computation of the nonadiabatic coupling vectors adds another 2 min , and the overlaps are computed in only 12 s ( t = 0.99 ) . in the case of mr - cisd , two and a half hours are required for the wave function calculation and , again , 2 min are added for the coupling vectors . still , in both cases , the overlaps can be computed in less time than the wave function calculations . however , in many cases , larger thresholds are affordable , and for smaller wave function expansions , such as in casscf calculations , truncation becomes unnecessary . performance of the wave function overlap code for the t1|t2 element at the mr - cis(6,5 ) and mr - cisd(6,5 ) levels of theory in the case of selenoacroleine between geometries with 50 and 55 torsion using varying screening thresholds t : ( a ) the overlap considering raw , renormalized , and orthogonalized results , ( b ) the total number of determinant pairs ( npair ) and the number of unique and factors ( nfac ) , and ( c ) the computation time and memory requirements . as a consequence , the step size in nonadiabatic dynamics simulations will not only affect the general numerical stability of the wave function propagation , but when overlaps are applied , the specific effect of displaced orbitals should be taken into account , as well . from a physical point of view , the nonadiabatic interactions should not be modified by this translation , and any modulations of the overlap elements are therefore unwanted artifacts . at zero displacement , all values agree , giving a value of 0.71069 for the raw overlaps and once the molecule is displaced , a very steep decline of the raw overlaps ( shown as dashed lines ) is observed . to understand this effect , it is instructive to regard the size of the 1s core orbitals , which can be estimated as a(z ) = a0/z , where a0 is the bohr radius and z is the nuclear charge . this decay is problematic for dynamics simulations because even the smallest translation of the molecule , which might occur because of numerical inaccuracies , can affect the computed overlap values . the good performance of the orthogonalization process can be understood by the fact that all elements of the overlap matrix are scaled down uniformly by the translation and that the orthogonalization then simply amounts to renormalizing these elements . discarding them improves the numerical stability while at the same time saving computational effort . all these data points are roughly on a straight line , highlighting the uniform performance characteristics of the code with respect to this diverse set of wave functions . the mr - cis expansions profit from the one - step laplace recursion of eq 15 , which again allows for efficient computation of the determinants . however , also in these cases , 1020% of the computation time is used for the contraction step ( eq 14 ) , setting a clear limit for the effect of any possible improvement in the determinant computation . performance of the wave function overlap code for varying wave function expansions : computation time plotted against the number of determinant pairs ( npair ) . parallel performance of the overlap code for a selenoacroleine mr - cisd(6,5 ) computation requiring the evaluation of 6.7 10 slater determinant overlaps : ( a ) speedup from 1 to 32 cores and ( b ) computation times for the different steps as discussed in the text . a new algorithm for the computation of wave function overlaps between many - electron wave functions is presented . by virtue of an optimized algorithm , which makes extended use of recurring intermediates , a highly efficient code was generated , which allows for computations with mr - cisd wave functions , with large active space , and with extended basis sets . because of the general formalism employed , there are no restrictions with respect to the wave functions except that they have to be given in a slater determinant expansion based on restricted or unrestricted molecular orbitals . consequently , it is possible to vary the wave function model , the orbitals , the one - electron basis set , and the molecular geometry . furthermore , the effects of using truncated wave functions were studied , and it was found that values of t = 0.95 or 0.97 for the squared norm of the truncated wave function could already provide satisfactory results . in addition , attention was devoted to understanding unwanted effects deriving from discrete displacements of atoms and orbitals , which naturally occur in dynamics simulations . for both cases , wave function truncation and orbital displacement , it was found that an orthogonalization of the overlap matrix can improve the results dramatically . because of the general formalism employed , the code is certainly not limited to this application , and other tasks can be envisaged where the new code will be beneficial , for example , the comparison of wave functions constructed at different levels of theory and probing the initial electronic states after -decay or after the photoionization of core - electrons . furthermore , the computation of dyson norms , as required for the simulation of photoelectron spectra , can be achieved by a slight modification of the code . scalar relativistic effects were taken into account in these all - electron calculations by using the second - order douglas kroll unless otherwise specified , 12 core orbitals corresponding to the s and p orbitals in the first , second , and third shells on se and the 1s orbitals on c were frozen in the mr - ci computations and discarded in the wave function overlap computations . for the triplet / triplet overlaps , generally , the ms = 1 wave functions were considered , as these contain fewer determinants than the ms = 0 ones , allowing for speedup of the computations while not affecting the results . the mr - ci computations were carried out with the columbus 7.0 program package using its parallel mr - ci implementation , whereas molcas 8.0(27,59 ) was applied for the integrals and most of the casscf calculations . the benchmark calculations for varying wave function models ( figure 6 ) were performed on one core of an intel xeon e5 - 2650-v3 cpu at 2.3 ghz , whereas the parallel performance tests ( figure 7 ) were carried out on an hp dl580g7 server with 512 gb of main memory and 4 intel e7 - 4850 ( westmere ) cpus at 2.0 ghz with 10 cores each .

in the past , the cost and effort of developing a new drug has largely confined successes to large pharmaceutical companies or otherwise well - funded research institutions . although development and use of computer - aided drug design ( cadd ) techniques has provided numerous benefits to the overall process , the expertise required to create powerful commercial software packages has resulted in high licensing costs , thus limiting access to academic groups . fortunately , this trend has started to shift with the emergence of freely available software , such as autodock and several other packages , largely developed by the academic computational chemistry community . however , for the most part , these software packages require familiarity with cadd methodologies and are better suited for computer savvy users that are at least comfortable if not familiar with the computational component of drug discovery . this has hampered the proliferation of cadd tools into less computationally minded drug discovery laboratories . the need for intuitive and easy to use cadd solutions has largely been met by the commercial software companies such as accelrys , schrdinger , and others that have incorporated full - featured graphical user interfaces ( gui ) into their programs . however , as alluded to above , the cost of these packages is typically prohibitive to academic groups and/or institutions . further , it has proven increasingly difficult to strike a balance between software that is user - friendly yet incorporates a wide range of advanced functionality and customizability . for example stand - alone software that requires local installation on every computer may find less use in today s world where researchers expect both the application and the data to be accessible from any machine on any platform from any location . another hurdle , faced by the nonexpert , to incorporating computational modeling into drug discovery efforts is the difficulty of obtaining reliable small molecule parameters . most widely used and well - tested force fields have been developed with proteins and nucleic acids rather than small molecules in mind . until recently this has meant that drug - like molecule parameters have been less reliable , with assignment often arbitrary . lately , however , there has been a significant amount of effort devoted to improving the reliability of small molecule parameters and developing efficient protocols to generate them for a much greater and more diverse chemical space . the charmm interface and graphics ( charmming ) is a web interface to the popular macromolecular modeling package charmm . the goal of the charmming project is to provide a platform - independent web - based front - end that allows its users to set up and perform a wide variety of molecular modeling tasks . charmming s users range from small academic laboratories that benefit from the portal s functionality to educators that include molecular modeling in their curricula and use the portal to facilitate their teaching . moreover , the open source nature of the project allows outside developers to utilize the framework and build on the existing infrastructure , further expanding the range of features it includes . the framework can be easily installed on a private network or adopted into a new web - based interface ; this approach was utilized when developing a virtual target screening ( vts ) server . herein , we describe a similar effort using the charmming infrastructure ( i.e. , built on a python - based django framework with a mysql database ) ; the implementation of a new drug design module that incorporates a fragment - based docking protocol includes a diverse set of drug - like compounds and facilitates creation of charmm friendly protein small molecule systems for further modeling studies . we also assess the performance of the newly implemented docking protocol coupled to charmm s new generalized force field ( cgenff ) by reproducing a series of co - crystallized protein ligand complexes and comparing the results against a leading commercially available docking package . target proteins begin their preparation via charmming s structure submission section . here , tasks such as the addition of hydrogens , identification of any nonprotein moieties , and assignment of final parameters are carried out ( using the latest charmm36 protein force field ) . co - crystallized small molecules ( i.e. , ligands ) are automatically parametrized using the cgenff . specifically , ligand atom - typing and parametrization is performed by sequentially attempting several automated parametrization tools . the default order is ( 1 ) paramchem , ( 2 ) match , ( 3 ) antechamber , and ( 4 ) genrtf . as an alternative to the default order , a user can specify the exact build procedure to use for parametrization . ligand set details page allows the user to manage custom ligand sets . the user can define and describe a custom ligand set as well as add ligands to it from any of the other sets including the preloaded public library . charmming docking module provides a preloaded library of drug - like compounds for virtual screening experiments . the library consists of approximately 8000 molecules from the maybridge hitfinder set ( www.maybridge.com ) . all of the provided molecules have been atom typed according to cgenff convention to comply with charmm requirements and confirmed to decompose into at least three sufficiently sized fragments to meet the fragment - based docking criteria . charmming also allows users to upload ligands by providing a coordinate file in mol2 format . upon uploading , the ligand and corresponding parameter , topology , and structure files are then saved on a disk as well as cataloged in the database . unlike the preloaded compound library , any user - uploaded ligands are restricted to their account only and are not visible to other users . the user is also given the ability to create custom sets of molecules based on any preloaded or user - uploaded compounds . this can be done via the ligand sets section ( figure 1 ) of the docking module . any custom or preloaded set can be docked in its entirety or by selecting individual molecules on the docking submission page ( figure 2 ) . to provide maximum flexibility with respect to job setup , the first approach identifies the binding pocket using the position of a co - crystallized ligand that may be present . in this case , when launching a docking job , a user is presented with a list of all co - crystallized small molecules along with their 2d structural representations . once the desired small molecule is chosen , the binding site is defined via proximity to the aforementioned small molecule . in cases where no co - crystallized ligand is present , or if a user simply wishes to investigate alternative binding sites , we have implemented an interactive and graphical binding site definition tool ( figure 3 ) . to use this tool , two residues should be selected that roughly correspond to the edges of the desired binding region . the midpoint between these residues is then determined and defined as the approximate center of the binding site . on the basis of a user - defined radius , a list of all residues within this distance is compiled and both visually highlighted and presented as a list . the user can then add or remove residues to / from this list by either modifying the text of the residue list , changing the specified search radius , or modifying it via graphical selection ( i.e. , clicking ) . ultimately , all user - defined binding sites are saved and presented as options with any existing co - crystallized ligands at the docking job setup page . page presents the user with the ability to select the target coordinates for docking , define the binding pocket ( vide infra ) , and select ligands to dock from the list of available small molecules . native ligands and ligands available for docking can be visualized in 3d using the embedded visualization application . docking algorithms used in this protocol are based on the popular grid - based paradigm used by most current docking programs . in this approach , the solvent accessible surface area of the target and the ligand as well as the target s binding site the lattice then either stores information about the atoms enclosed by a cubic unit of the grid or contains the potential contributions projected onto the grid s vertices . precomputed grids allow for efficient calculation of both van der waals and electrostatic contributions to the scoring function , facilitating rapid evaluation of ligand placements within the binding site . binding site selection page provides the user multiple ways to select a custom binding site . this can be done either by manually typing in the residue numbers , graphically selecting residues , or defining the centroid and specifying the radius in . the docking procedure consists of several steps where different programs perform distinct tasks . to streamline the communication between the programs and ensure compatibility of input and output data , a series of scripts were written in python , perl , and linux shell scripting languages . the openbabel file conversion utility was used to interconvert between different representations of the protein and compound structures . the program match was used to generate cgenff compatible topologies and parameters . the fragment - based docking protocol implemented in charmming is outlined in figure 4 and described as follows : ( 1 ) each compound to be docked is first broken down into fragments . a fingerprint describing chemical richness is generated for each fragment and its parent compound . the three most chemically rich , but not necessarily different , fragments are identified to serve as anchors for docking . these steps are carried out by the program daim ( decomposition and identification of molecules ) . the user then identifies the binding site to be used in the docking job . all nonprotein nonsolvent compounds present in the submitted target structure are displayed on the submit docking job the user selected compound , the proximal residues are identified and the binding site defined . ( 3 ) the previously identified anchor fragments ( step 1 ) are then docked into the binding site using the program seed ( solvation energy for exhaustive docking ) . the placement of fragments within the binding site is determined by matching either the direction of polar vectors between ligand and receptor atoms to form a hydrogen bond or the apolar vectors on the solvent accessible surface area of the receptor . the seed score , used in fragment placement , accounts for the solvent effects by including terms for both receptor and fragment desolvation as well as a solvent - screened receptor - fragment electrostatic interaction term . ( 4 ) the docked fragments are reconnected into the original ligand while undergoing refinement using the ffld ( fragment - based flexible ligand docking ) program . ffld uses a genetic algorithm that generates and evaluates populations of conformations and positions them within the binding site , as guided by fragment anchor locations . the fitness of a placed conformation is evaluated using a scoring function that is aimed at approximating the steric effects as well as hydrogen bonding contributions of the protein ligand van der waals interaction terms as well as polar contributions based on the number of hydrogen bonds and unfavorable donor donor and acceptor acceptor interactions . ( 5 ) poses generated by ffld that are within a user - defined energy cutoff ( 10 kcal / mol by default ) are then clustered using a leader clustering algorithm implemented in the program flea ( ffld leader clustering ) . ( 6 ) following the clustering , the protein ligand complex is converted to native charmm format and saved . using these files , in addition to the charmm protein and generalized force fields ( i.e. , charmm36 and cgenff ) , protein structure ( psf ) and coordinate ( crd ) the minimized protein ligand complexes are then scored using seed and ffld in their evaluation only mode , producing their own estimation of electrostatic , van der waals , and total energy contributions for each pose . the final ranking of the docked poses is performed using a consensus approach . for this , energies ( i.e. , interaction energy from charmm and total energies from seed and ffld ) are used to create three lists in which individual poses are sorted and ranked . the final rank of each pose is then set to the median of the three ranks as assigned in the individual lists . the consensus approach to scoring or ranking compounds when performing molecular docking or virtual screening studies has been shown to be more accurate than single scoring methods . schematic of the fragment - based docking protocol implemented into the charmming web user interface . depicted are the three main stages of the docking : decomposition by daim , fragment docking by seed , and ligand placement by ffld . when a docking job is launched , the pbs ( portable batch system)-based queuing system torque accepts the job as a wrapper shell script that controls the entire docking procedure . using the interface , a job can be monitored in real time as it progresses and generates final poses for each docked compound . basic job statistics such as submission time and job status can be monitored along with the output file reflecting the job progression ( figure 5 ) . in addition , important files associated with job progress and results ( e.g. , final docked ligand poses , job output , etc . ) protein , ligands , compounds in the library , and final docked poses can all be visualized directly in charmming . the 3d structure of each of the above elements can be rendered with the jsmol or glmol visualization tools . structures can be visualized using a variety of representations to highlight important structural features or interactions of the molecules and their complexes . page provides general job information as well as the list of docked poses and their respective scores . the docked poses can be visualized in 3d within the binding pocket of the protein using the embedded visualization application . an archive of the job directory can also be downloaded from this page for execution on local resources . a walk - through outlining the entire process of performing a redock on a sample system is included in the tutorial covering basic charmm and charmming functionality at www.charmmtutorial.org . additionally , a docking lesson that guides a user through the redocking procedure has been added to the lessons section of the charmming web site . currently , all docking jobs executed via the web interface are carried out sequentially . however , after initial setup of the docking job , all necessary files are available for download and execution on local computational resources . to improve performance of this procedure , we have developed a protocol that can be carried out in parallel as outlined in figure 6 . this is achieved by spawning a new execution branch for each of the most time - consuming steps in the protocol via a user - modifiable job queuing command . for example , each fragment of each molecule is docked ( step 3 , vide supra ) as a separate submitted job . once all of a molecule s anchor fragments are docked , the placement of a ligand within the binding site by ffld is also spawned as a series of separate jobs . furthermore , to increase sampling by ffld and improve performance , the protocol performs multiple docking iterations per ligand , again each as a separate job . thus , instead of one docking job that attempts to sequentially sample a large conformational space per ligand , multiple shorter iterations with different random seeds are run in parallel , taking less real time and still sufficiently sampling ligand conformational space . the number of iterations per ligand as well as the amount of energy evaluations per iteration are all user modifiable parameters . parallelization of the docking protocol is achieved by spawning new job execution threads at both the fragment docking ( i.e. , one per fragment ) and ligand placement ( i.e. , one per iteration per ligand ) steps . clustering and scoring threads are also spawned for each docked ligand . in order to execute a job on local resources , the following programs need to be downloaded and installed : vmd , daim , seed , ffld , flea , match , and charmm . except for charmm , daim , seed , ffld , and flea can be obtained from the university of zurich s computational structural biology lab ( www.biochem-caflisch.uzh.ch/download ) . further , a more general description of the installation process is included as part of the charmm tutorial and can be found at the following web address : www.charmmtutorial.org/index.php/installation_of_charmming . once the job directory is downloaded and the software is installed on local resources , the provided settings file should be used to specify the location of program executables . in addition , job details ( e.g. , protein file name , number of docking iterations , clustering energy cutoff , etc . ) can be modified via the settings file . this file is also where pbs / torque commands can be modified for local resources . because there is no limit to the number of possible parallel processes spawned , the protocol checks for available resources and will wait for current processes to complete if the queue is full . the protocol will automatically take advantage of all available resources to speed up job completion while at the same time adhering to the local queuing system policies . to assess the performance of the docking protocol , a diversity set was constructed from the publicly available ccdc / astex test set containing high - resolution x - ray complexes and an augmented version of that set , which has been used to compare the performance of a number of docking programs . our final set contained 24 protein ligand complexes with x - ray resolutions ranging from 1.502.30 . in particular , we selected complexes where the ligand could be decomposed into three fragments ( i.e. , at least three rotatable bonds ) using the default settings of daim , as the ultimate goal was to evaluate the implementation of the decomposition - based approach . redock validation involved removing the co - crystallized ligand from the complex , redocking it via the fragment - based protocol , and comparing the docked pose to that of the original crystal structure . each complex was processed using charmming s submit structure section that downloads the structure based on the pdb code , adds hydrogen atoms , and prepares the structure for modeling using charmm . further , each system containing the protein , solvent , and ligand molecules was briefly minimized for 100 steps using the steepest descent method followed by 1000 steps of abnr using charmming s calculations module . using the ligand upload section of charmming s docking module , the previously downloaded ligand was processed . the docking calculation for each minimized system was set up by selecting a native ligand to define a binding pocket and user - uploaded ligand for docking , all from the the progress of each job was monitored using the job monitoring section of the docking module . to assess the performance of the dockings , root - mean - square deviation ( rmsd ) between the heavy atoms of the docked poses and the crystal structures redockings were performed using schrdinger s glide standard precision ( sp ) docking protocol . glide s sp protocol attempts to dock multiple conformations of a ligand into a receptor grid , subsequently calculating the effective ligand conformational sampling of the ligand is achieved via varying torsion angles around rotatable bonds . prior to docking , each target the preparation included removal of solvent molecules , addition of hydrogens , and brief minimization . as glide is also a grid - based docking protocol , the grids , similar to charmming s procedure , were built using the co - crystal ligand to define the binding region . the native ligand was removed and redocked using default parameters of the sp docking protocol . the poses with the best docking scores were used to calculate their respective rmsd from the crystal structure using vmd . table 1 reports the rmsd of poses generated by charmming s fragment - based docking protocol and glide sp docking ( w.r.t . results reported from charmming s fragment - based docking protocol correspond to the pose closest to the crystal structure . this set yields a 71% success rate using rmsd < 2.0 as the metric ; this criteria is commonly employed for evaluating the performance of docking algorithms . this clearly shows that the protocol can successfully recover the crystal pose in the majority of the cases . we are currently optimizing a consensus scoring function based on this diversity set ; results of that effort will be reported in a subsequent publication . nevertheless , virtual screening is known to suffer from a high false - positive rate , which does not diminish its value in drug discovery as the unfit compounds are screened out during the experimental stages of the discovery campaigns . regardless , we are encouraged by the success of fragment - based docking , which shows approximately the same performance as widely used docking programs , i.e. , within the range of 4090% . best rmsd glide sp rmsd is of the top scoring pose of glide s standard precision docking . the fragment - based approach that was implemented into charmming yields a substantial amount of information about the characteristics of each docked pose . at each step , from decomposition to minimization of docked poses , users have the ability to closely analyze results . the binding modes of each individual fragment can be inspected , and a number of modifiable parameters , such as decomposition criteria , can be used to optimize the protocol . moreover , information gained from docking a fragment library into a particular target can be used to mine large libraries for compounds containing those fragments that form the most favorable interactions with the target . there are potentially a number of improvements that can be made to improve the performance and usability of charmming s docking protocol . the most obvious limitation is the current requirement of three fragments to be used as anchors . as shown by the number of ligands eliminated from the original benchmarking set , this limits the applicability of this protocol in its current form to medium- to large - sized molecules with a sufficient number of rotatable bonds . although partially this problem can be alleviated by decreasing the fragment richness threshold at the decomposition step , this will only increase the eligibility rate of molecules by a small margin . alternatively , when docking these small and/or rigid molecules is desired , the decomposition step could be omitted , at which point the molecules would undergo docking only by seed . this however will require prior conformation sampling step as seed currently does not sample the internal conformation of docked fragments . the conformational sampling of the fragments is an obvious improvement to the docking protocol even in its current state . this addition will help ensure that larger fragments sample their orientations within the binding site while varying their internal geometry , thus ensuring greater enrichment of anchor positions for the final ligand placement . we have implemented a fragment - based docking protocol into the charmming web interface . the protocol allows users to perform docking and virtual screening calculations online as well as generates self - contained scripts to execute these in parallel on local hpc resources . the performance of the docking protocol was evaluated by carrying out a series of redockings and comparing the results against a top commercial docking package . the fragment - based docking protocol yielded results comparable to both the commercial package used herein and a wide variety of additional docking software . specifically , the rate of recovering the correct x - ray pose with charmming s protocol was 71% , well within the 4090% range that numerous benchmarking studies have reported . while the scoring function can still be improved , the tool lays substantial groundwork for allowing academic laboratories to set up and perform molecular docking and virtual screening studies . it is important to note that the protocol is able to create charmm - formatted protein ligand systems giving users the ability to access the wide range of functionality that exists in charmm . for example , docked poses can easily be refined with md simulations , and predocked proteins can be coupled with simulations or normal - mode analysis to proceed via an ensemble docking approach .

web - based user interfaces to scientific applications are important tools that allow researchers to utilize a broad range of software packages with just an internet connection and a browser.1 one such interface , charmming ( charmm interface and graphics ) , facilitates access to the powerful and widely used molecular software package charmm . charmming incorporates tasks such as molecular structure analysis , dynamics , multiscale modeling , and other techniques commonly used by computational life scientists . we have extended charmming s capabilities to include a fragment - based docking protocol that allows users to perform molecular docking and virtual screening calculations either directly via the charmming web server or on computing resources using the self - contained job scripts generated via the web interface . the docking protocol was evaluated by performing a series of re - dockings with direct comparison to top commercial docking software . results of this evaluation showed that charmming s docking implementation is comparable to many widely used software packages and validates the use of the new charmm generalized force field for docking and virtual screening .

Introduction Implementation Details Results and Discussion Conclusions

although development and use of computer - aided drug design ( cadd ) techniques has provided numerous benefits to the overall process , the expertise required to create powerful commercial software packages has resulted in high licensing costs , thus limiting access to academic groups . fortunately , this trend has started to shift with the emergence of freely available software , such as autodock and several other packages , largely developed by the academic computational chemistry community . however , for the most part , these software packages require familiarity with cadd methodologies and are better suited for computer savvy users that are at least comfortable if not familiar with the computational component of drug discovery . the need for intuitive and easy to use cadd solutions has largely been met by the commercial software companies such as accelrys , schrdinger , and others that have incorporated full - featured graphical user interfaces ( gui ) into their programs . further , it has proven increasingly difficult to strike a balance between software that is user - friendly yet incorporates a wide range of advanced functionality and customizability . most widely used and well - tested force fields have been developed with proteins and nucleic acids rather than small molecules in mind . the charmm interface and graphics ( charmming ) is a web interface to the popular macromolecular modeling package charmm . the goal of the charmming project is to provide a platform - independent web - based front - end that allows its users to set up and perform a wide variety of molecular modeling tasks . charmming s users range from small academic laboratories that benefit from the portal s functionality to educators that include molecular modeling in their curricula and use the portal to facilitate their teaching . moreover , the open source nature of the project allows outside developers to utilize the framework and build on the existing infrastructure , further expanding the range of features it includes . the framework can be easily installed on a private network or adopted into a new web - based interface ; this approach was utilized when developing a virtual target screening ( vts ) server . herein , we describe a similar effort using the charmming infrastructure ( i.e. , built on a python - based django framework with a mysql database ) ; the implementation of a new drug design module that incorporates a fragment - based docking protocol includes a diverse set of drug - like compounds and facilitates creation of charmm friendly protein small molecule systems for further modeling studies . we also assess the performance of the newly implemented docking protocol coupled to charmm s new generalized force field ( cgenff ) by reproducing a series of co - crystallized protein ligand complexes and comparing the results against a leading commercially available docking package . target proteins begin their preparation via charmming s structure submission section . here , tasks such as the addition of hydrogens , identification of any nonprotein moieties , and assignment of final parameters are carried out ( using the latest charmm36 protein force field ) . , ligands ) are automatically parametrized using the cgenff . specifically , ligand atom - typing and parametrization is performed by sequentially attempting several automated parametrization tools . the default order is ( 1 ) paramchem , ( 2 ) match , ( 3 ) antechamber , and ( 4 ) genrtf . as an alternative to the default order , a user can specify the exact build procedure to use for parametrization . the user can define and describe a custom ligand set as well as add ligands to it from any of the other sets including the preloaded public library . charmming docking module provides a preloaded library of drug - like compounds for virtual screening experiments . the library consists of approximately 8000 molecules from the maybridge hitfinder set ( www.maybridge.com ) . all of the provided molecules have been atom typed according to cgenff convention to comply with charmm requirements and confirmed to decompose into at least three sufficiently sized fragments to meet the fragment - based docking criteria . charmming also allows users to upload ligands by providing a coordinate file in mol2 format . upon uploading , the ligand and corresponding parameter , topology , and structure files are then saved on a disk as well as cataloged in the database . this can be done via the ligand sets section ( figure 1 ) of the docking module . any custom or preloaded set can be docked in its entirety or by selecting individual molecules on the docking submission page ( figure 2 ) . to provide maximum flexibility with respect to job setup , the first approach identifies the binding pocket using the position of a co - crystallized ligand that may be present . once the desired small molecule is chosen , the binding site is defined via proximity to the aforementioned small molecule . in cases where no co - crystallized ligand is present , or if a user simply wishes to investigate alternative binding sites , we have implemented an interactive and graphical binding site definition tool ( figure 3 ) . to use this tool , two residues should be selected that roughly correspond to the edges of the desired binding region . the midpoint between these residues is then determined and defined as the approximate center of the binding site . the user can then add or remove residues to / from this list by either modifying the text of the residue list , changing the specified search radius , or modifying it via graphical selection ( i.e. ultimately , all user - defined binding sites are saved and presented as options with any existing co - crystallized ligands at the docking job setup page . page presents the user with the ability to select the target coordinates for docking , define the binding pocket ( vide infra ) , and select ligands to dock from the list of available small molecules . native ligands and ligands available for docking can be visualized in 3d using the embedded visualization application . docking algorithms used in this protocol are based on the popular grid - based paradigm used by most current docking programs . in this approach , the solvent accessible surface area of the target and the ligand as well as the target s binding site the lattice then either stores information about the atoms enclosed by a cubic unit of the grid or contains the potential contributions projected onto the grid s vertices . precomputed grids allow for efficient calculation of both van der waals and electrostatic contributions to the scoring function , facilitating rapid evaluation of ligand placements within the binding site . the docking procedure consists of several steps where different programs perform distinct tasks . to streamline the communication between the programs and ensure compatibility of input and output data , a series of scripts were written in python , perl , and linux shell scripting languages . the openbabel file conversion utility was used to interconvert between different representations of the protein and compound structures . the program match was used to generate cgenff compatible topologies and parameters . the fragment - based docking protocol implemented in charmming is outlined in figure 4 and described as follows : ( 1 ) each compound to be docked is first broken down into fragments . the three most chemically rich , but not necessarily different , fragments are identified to serve as anchors for docking . the user then identifies the binding site to be used in the docking job . ( 3 ) the previously identified anchor fragments ( step 1 ) are then docked into the binding site using the program seed ( solvation energy for exhaustive docking ) . the placement of fragments within the binding site is determined by matching either the direction of polar vectors between ligand and receptor atoms to form a hydrogen bond or the apolar vectors on the solvent accessible surface area of the receptor . ( 4 ) the docked fragments are reconnected into the original ligand while undergoing refinement using the ffld ( fragment - based flexible ligand docking ) program . the fitness of a placed conformation is evaluated using a scoring function that is aimed at approximating the steric effects as well as hydrogen bonding contributions of the protein ligand van der waals interaction terms as well as polar contributions based on the number of hydrogen bonds and unfavorable donor donor and acceptor acceptor interactions . using these files , in addition to the charmm protein and generalized force fields ( i.e. , charmm36 and cgenff ) , protein structure ( psf ) and coordinate ( crd ) the minimized protein ligand complexes are then scored using seed and ffld in their evaluation only mode , producing their own estimation of electrostatic , van der waals , and total energy contributions for each pose . the final ranking of the docked poses is performed using a consensus approach . the final rank of each pose is then set to the median of the three ranks as assigned in the individual lists . the consensus approach to scoring or ranking compounds when performing molecular docking or virtual screening studies has been shown to be more accurate than single scoring methods . schematic of the fragment - based docking protocol implemented into the charmming web user interface . depicted are the three main stages of the docking : decomposition by daim , fragment docking by seed , and ligand placement by ffld . using the interface , a job can be monitored in real time as it progresses and generates final poses for each docked compound . basic job statistics such as submission time and job status can be monitored along with the output file reflecting the job progression ( figure 5 ) . protein , ligands , compounds in the library , and final docked poses can all be visualized directly in charmming . the 3d structure of each of the above elements can be rendered with the jsmol or glmol visualization tools . structures can be visualized using a variety of representations to highlight important structural features or interactions of the molecules and their complexes . the docked poses can be visualized in 3d within the binding pocket of the protein using the embedded visualization application . an archive of the job directory can also be downloaded from this page for execution on local resources . a walk - through outlining the entire process of performing a redock on a sample system is included in the tutorial covering basic charmm and charmming functionality at www.charmmtutorial.org . additionally , a docking lesson that guides a user through the redocking procedure has been added to the lessons section of the charmming web site . currently , all docking jobs executed via the web interface are carried out sequentially . however , after initial setup of the docking job , all necessary files are available for download and execution on local computational resources . to improve performance of this procedure , we have developed a protocol that can be carried out in parallel as outlined in figure 6 . this is achieved by spawning a new execution branch for each of the most time - consuming steps in the protocol via a user - modifiable job queuing command . for example , each fragment of each molecule is docked ( step 3 , vide supra ) as a separate submitted job . once all of a molecule s anchor fragments are docked , the placement of a ligand within the binding site by ffld is also spawned as a series of separate jobs . parallelization of the docking protocol is achieved by spawning new job execution threads at both the fragment docking ( i.e. clustering and scoring threads are also spawned for each docked ligand . in order to execute a job on local resources , the following programs need to be downloaded and installed : vmd , daim , seed , ffld , flea , match , and charmm . except for charmm , daim , seed , ffld , and flea can be obtained from the university of zurich s computational structural biology lab ( www.biochem-caflisch.uzh.ch/download ) . further , a more general description of the installation process is included as part of the charmm tutorial and can be found at the following web address : www.charmmtutorial.org/index.php/installation_of_charmming . can be modified via the settings file . because there is no limit to the number of possible parallel processes spawned , the protocol checks for available resources and will wait for current processes to complete if the queue is full . the protocol will automatically take advantage of all available resources to speed up job completion while at the same time adhering to the local queuing system policies . to assess the performance of the docking protocol , a diversity set was constructed from the publicly available ccdc / astex test set containing high - resolution x - ray complexes and an augmented version of that set , which has been used to compare the performance of a number of docking programs . , at least three rotatable bonds ) using the default settings of daim , as the ultimate goal was to evaluate the implementation of the decomposition - based approach . redock validation involved removing the co - crystallized ligand from the complex , redocking it via the fragment - based protocol , and comparing the docked pose to that of the original crystal structure . each complex was processed using charmming s submit structure section that downloads the structure based on the pdb code , adds hydrogen atoms , and prepares the structure for modeling using charmm . further , each system containing the protein , solvent , and ligand molecules was briefly minimized for 100 steps using the steepest descent method followed by 1000 steps of abnr using charmming s calculations module . using the ligand upload section of charmming s docking module , the previously downloaded ligand was processed . the docking calculation for each minimized system was set up by selecting a native ligand to define a binding pocket and user - uploaded ligand for docking , all from the the progress of each job was monitored using the job monitoring section of the docking module . to assess the performance of the dockings , root - mean - square deviation ( rmsd ) between the heavy atoms of the docked poses and the crystal structures redockings were performed using schrdinger s glide standard precision ( sp ) docking protocol . glide s sp protocol attempts to dock multiple conformations of a ligand into a receptor grid , subsequently calculating the effective ligand conformational sampling of the ligand is achieved via varying torsion angles around rotatable bonds . prior to docking , each target the preparation included removal of solvent molecules , addition of hydrogens , and brief minimization . as glide is also a grid - based docking protocol , the grids , similar to charmming s procedure , were built using the co - crystal ligand to define the binding region . the native ligand was removed and redocked using default parameters of the sp docking protocol . table 1 reports the rmsd of poses generated by charmming s fragment - based docking protocol and glide sp docking ( w.r.t . results reported from charmming s fragment - based docking protocol correspond to the pose closest to the crystal structure . this clearly shows that the protocol can successfully recover the crystal pose in the majority of the cases . we are currently optimizing a consensus scoring function based on this diversity set ; results of that effort will be reported in a subsequent publication . nevertheless , virtual screening is known to suffer from a high false - positive rate , which does not diminish its value in drug discovery as the unfit compounds are screened out during the experimental stages of the discovery campaigns . regardless , we are encouraged by the success of fragment - based docking , which shows approximately the same performance as widely used docking programs , i.e. , within the range of 4090% . best rmsd glide sp rmsd is of the top scoring pose of glide s standard precision docking . the fragment - based approach that was implemented into charmming yields a substantial amount of information about the characteristics of each docked pose . the binding modes of each individual fragment can be inspected , and a number of modifiable parameters , such as decomposition criteria , can be used to optimize the protocol . moreover , information gained from docking a fragment library into a particular target can be used to mine large libraries for compounds containing those fragments that form the most favorable interactions with the target . there are potentially a number of improvements that can be made to improve the performance and usability of charmming s docking protocol . as shown by the number of ligands eliminated from the original benchmarking set , this limits the applicability of this protocol in its current form to medium- to large - sized molecules with a sufficient number of rotatable bonds . the conformational sampling of the fragments is an obvious improvement to the docking protocol even in its current state . we have implemented a fragment - based docking protocol into the charmming web interface . the protocol allows users to perform docking and virtual screening calculations online as well as generates self - contained scripts to execute these in parallel on local hpc resources . the performance of the docking protocol was evaluated by carrying out a series of redockings and comparing the results against a top commercial docking package . the fragment - based docking protocol yielded results comparable to both the commercial package used herein and a wide variety of additional docking software . specifically , the rate of recovering the correct x - ray pose with charmming s protocol was 71% , well within the 4090% range that numerous benchmarking studies have reported . while the scoring function can still be improved , the tool lays substantial groundwork for allowing academic laboratories to set up and perform molecular docking and virtual screening studies . it is important to note that the protocol is able to create charmm - formatted protein ligand systems giving users the ability to access the wide range of functionality that exists in charmm . for example , docked poses can easily be refined with md simulations , and predocked proteins can be coupled with simulations or normal - mode analysis to proceed via an ensemble docking approach .

heterologous synthesis of selected ntts and mutant proteins in e. coli the heterologous synthesis of pamntt1 and of two atp / adp transporters from the rickettsia - related bacteria caedibacter caryophilus ( ccntt ) and holospora obtusa ( hontt ) was performed on the basis of existing pet16b constructs ( 13 , 22 ) , which were transformed into blr cells ( merck ) . heterologous expression of the full - length cdna encoding the plastidial atp / adp transporter from arabidopis thaliana ( atntt1 ) led to very low amounts of recombinant transporters in blr cells ( data not shown ) . however , the truncation of the n - terminal extension ( the putative leader peptide of 87 amino acid residues ) and application of rosetta2 ( de3 ) plyss expression cells resulted in an increased synthesis of atntt1 . the truncated atntt1 sequence was amplified from the expression construct ( 34 ) by pcr using pfu polymerase and the following oligonucleotides : atntt1shortndei_sense , 5-ggccgcggctcatatggacggagctg-3 , and the standard primer t7-term . the pcr construct contained one ndei restriction site inserted by the sense primer and the xhoi restriction site of the plasmid . the amplificate was cloned into an appropriately ( ndei / xhoi ) restricted pet16b vector . for generation of k446 mutant proteins , the pamntt1 encoding sequence ( inserted in pet16b ) was modified by site - directed mutagenesis using the quikchange site - directed mutagenesis kit ( stratagene ) . the triplet encoding lysine 446 ( aaa ) was substituted by the triplets cgc , cag , and gaa , respectively , by pcr . to eliminate possible pcr - caused mistakes in the amplified expression vector , we excised the mutated sequences by using bamhi ( 13 ) and inserted them into the correspondingly prepared original pet16b vector . the correctness of all constructs was proven by complete sequencing ( nanobiocenter , tu kaiserslautern , germany ) . briefly , transformed bacterial cells were grown at 37 c in tb medium under selective conditions . at an a600 of 0.5 , heterologous expression four hours post - induction the cells were harvested by centrifugation ( 5,000 g , 10 min , 20 c ) , resuspended in buffer medium ( 1 mm edta , 15% glycerol , 10 mm tris , ph 7.0 ) , and immediately frozen in liquid n2 . purification and reconstitution of heterologously expressed ntts enrichment of e. coli membranes and purification and reconstitution of heterologously expressed ntts were conducted as described by trentmann et al . the recombinant , histidine - tagged proteins were purified by immobilized metal affinity chromatography with nickel - sepharose 6 fast flow ( ge healthcare ) according to the manufacturer 's instructions . buffers used for washing and elution contained 0.5% n - dodecyl--maltoside . to rule out pi incorporation during reconstitution , we omitted pi not only in the elution buffer but also in the last washing step . additionally , the remaining liquid at the immobilized metal affinity chromatography material and in the column tip was removed thoroughly prior to elution . 50 l of the eluate were reconstituted into liposomes loaded with the given interior substrates ( 10 mm atp or adp or 5 mm pi ) . protein concentrations of the purified ntts were quantified in a bio - photometer ( eppendorf , hamburg , germany ) according to the method described by bradford ( 35 ) . purity of the recombinant ntts was analyzed by sds - page ( 3% stacking and 12% separating gel ) as described by laemmli ( 36 ) . import of radioactively labeled substrates into proteoliposomes uptake studies were performed as previously described ( 32 ) . for transport measurements , proteoliposomes were incubated at 30 c in presence of the given concentrations of -p - labeled nucleotides or radioactive pi [ p ] ( perkinelmer life sciences ) . optionally , import was conducted in the presence of the given concentrations of nonlabeled nucleotides or pi . removal of substrates by anion exchange chromatography ( dowex 1 8 cl , 200400 mesh ; sigma ) was used to terminate import . liposomes were eluted , and internal radioactivity was quantified in a scintillation counter ( canberra - packard , rsselsheim , germany ) . pi influences nucleotide exchange the observation that pamntt1 mediated adenine nucleotide exchange is electroneutral ( 32 ) was surprising . this carrier obviously transports a counterion that compensates for the charge difference of the hetero - exchange . the catalytic activity of bacterial and plastidial atp / adp transporters in contrast to bacterial proton - driven ntts is not affected by the proton gradient across the membrane ( 1215 , 1923 ) . , we analyzed the influence of diverse salt compositions on pamntt1-mediated nucleotide transport in the liposomal system . we identified that the comparably low rates of adp import into atp loaded vesicles ( adpim / atpex ) were increased by 500 m external pi ( 3.7 times ) and also by its structural analogue , arsenate ( 2.6 times ) ( supplemental table s1 ) . higher concentrations ( 2 mm ) of latter compounds further increased the stimulatory effects . the addition of other components had lower stimulatory or even inhibitory influences on the corresponding transport ( supplemental table s1 ) . figure 1.influence of the presence of pi in the liposomal lumen on pamntt1 mediated adenine nucleotide hetero - exchange . atpim / adpex transport and adpim / atpex transport was analyzed in absence ( light gray bars , set to 100% ) and in presence of internal pi ( dark gray bars , calculated according to the transport in absence of pi ) . for this , pamntt1 was reconstituted into liposomes loaded with 10 mm adp or atp or into vesicles containing 5 mm pi plus 10 mm adp or 5 mm pi plus 10 mm atp . import of 50 m -p - labeled atp or adp was allowed for 5 min and stopped by anion exchange chromatography . the data represent net values calculated by subtraction of uptake into vesicles lacking counter - exchange nucleotides and are the means of three independent experiments . the presence of pi and adenine nucleotides at the liposomal interior and exterior is graphically displayed . influence of the presence of pi in the liposomal lumen on pamntt1 mediated adenine nucleotide hetero - exchange . atpim / adpex transport and adpim / atpex transport was analyzed in absence ( light gray bars , set to 100% ) and in presence of internal pi ( dark gray bars , calculated according to the transport in absence of pi ) . for this , pamntt1 was reconstituted into liposomes loaded with 10 mm adp or atp or into vesicles containing 5 mm pi plus 10 mm adp or 5 mm pi plus 10 mm atp . import of 50 m -p - labeled atp or adp was allowed for 5 min and stopped by anion exchange chromatography . the data represent net values calculated by subtraction of uptake into vesicles lacking counter - exchange nucleotides and are the means of three independent experiments . the presence of pi and adenine nucleotides at the liposomal interior and exterior is graphically displayed . in a previous study we demonstrated that only those reconstituted pamntt1 proteins , which were inserted in the native orientation ( right side out ) , displayed catalytic activity ( 32 ) . to mimic the presence of pi in the bacterium , we tested whether pi application at the liposomal interior also influences adenine nucleotide hetero - exchange . when compared with the control ( 0 mm luminal pi ) , the addition of internal pi stimulated atp import in exchange with adp ( atpim / adpex ) 4-fold , whereas adpim / atpex transport was not highly affected by internal pi application ( fig . 1 ) . accordingly , not only external but also internal pi supports atp / adp exchange . however , stimulation of the hetero - exchange exclusively occurred when adp and pi were present at the same side of the liposome ( adp plus pi ) ( supplemental table s1 and fig . 1 ) . it had been suggested that pi plays a regulatory role in atp / adp exchange across the rickettsial membrane ( 37 ) . this based on the observation that high pi concentrations marginally enhanced atp but highly stimulated adp import into isolated bacteria cells . furthermore , only in the presence of pi , adp was able to compete with atp for import , leading the authors to the assumption that pi might increase ( at least ) the affinity of the rickettsial atp / adp transporter for adp ( 37 ) . to investigate the role of pi in ntt - mediated adenylate exchange in more detail and separated from the complex cellular metabolism , we analyzed the effect of rising external pi concentrations on adenine nucleotide import into pamntt1-proteoliposomes loaded with interior pi plus the given counter - exchange nucleotides . figure 2.influence of rising exterior pi concentrations on adenine nucleotide hetero - exchange mediated by pamntt1 . pamntt1 was reconstituted into liposomes loaded with 5 mm pi plus 10 mm of the given nucleotides . the effects of rising external pi concentrations on atp import into adp plus pi - loaded proteoliposomes ( light gray bars ) and on adp import into atp plus pi - loaded vesicles ( dark gray bars ) were analyzed . import of 50 m -p - labeled atp or adp was stopped after 5 min . the applied external pi concentrations ( in mm ) are displayed at the x axis . km and vmax values ( in parentheses ) were calculated for adenine nucleotide hetero - exchanges in the absence ( 0 mm ) and in the presence of 5 mm external pi , respectively , and are given above the bars of the corresponding exchanges . the data represent net values calculated by subtraction of uptake into vesicles lacking counter - exchange nucleotides and are the means of three independent experiments . standard errors of the kinetic parameters were below 12% and of adpim / atpex transport in absence of external pi ( 0 mm ) were at 20% . in the graphic depicting the presence of pi and nucleotides at the liposomal interior and exterior , influence of rising exterior pi concentrations on adenine nucleotide hetero - exchange mediated by pamntt1 . pamntt1 was reconstituted into liposomes loaded with 5 mm pi plus 10 mm of the given nucleotides . the effects of rising external pi concentrations on atp import into adp plus pi - loaded proteoliposomes ( light gray bars ) and on adp import into atp plus pi - loaded vesicles ( dark gray bars ) were analyzed . import of 50 m -p - labeled atp or adp was stopped after 5 min . the applied external pi concentrations ( in mm ) are displayed at the x axis . km and vmax values ( in parentheses ) were calculated for adenine nucleotide hetero - exchanges in the absence ( 0 mm ) and in the presence of 5 mm external pi , respectively , and are given above the bars of the corresponding exchanges . the data represent net values calculated by subtraction of uptake into vesicles lacking counter - exchange nucleotides and are the means of three independent experiments . standard errors of the kinetic parameters were below 12% and of adpim / atpex transport in absence of external pi ( 0 mm ) were at 20% . in the graphic depicting the presence of pi and nucleotides at the liposomal interior and exterior , changes in external pi availability had low impact on atp homo - exchange parameters and only slightly affected adp homo - exchange ( 4-fold increased vmax at 5 mm external pi ) ( data not shown ) . in contrast to the homo - exchanges , the hetero - exchanges were substantially influenced by pi addition ( fig . as observed in the effector analysis ( supplemental table s1 ) adpim / atpex hetero - exchange was significantly stimulated by exterior pi . the highest rise in stimulation was obtained between 50 m and 2 mm pi ( fig . 2 , dark gray bars ) . increasing the external pi concentration above 2 mm caused a further but comparably slight stimulation of the adp import rates and suggests the beginning of a pi saturation phase . determination of the kinetic parameters revealed that the stimulatory influence of external pi on adpim / atpex exchange can be traced back to an enhanced affinity ( from 0 to 5 mm : 14-fold lower km ) and an increased vmax ( from 0 to 5 mm : 3-fold higher vmax ) of pamntt1 for adp . concentrations of more than 2 mm pi in the transport medium led to a substantial reduction of the atpim / adpex transport rates . the vmax of this exchange was high in the presence as well as in the complete absence of external pi , and thus the lowered import rates were mainly caused by a decreased affinity of pamntt1 for atp import ( from 0 to 5 mm : 27-fold lower km ) ( fig . 2 ) . it has to be mentioned that the transport rates of optimal atpim / adpex exchange ( 0 mm external and 5 mm internal pi , 1000 nmol mg protein 5 min ) were much higher than that of pi stimulated adp import into atp plus pi - loaded vesicles ( 5 mm external and 5 mm internal pi , 350 nmol mg protein 5 min ) ( fig . it might be assumed that pi in the vesicle lumen hindered optimal atp export . however , depletion of luminal pi did not result in a further relevant stimulation of adpim / atpex exchange ( data not shown ; see also adpim / atpex in fig . the opposed effects of external pi on adpim / atpex versus atpim / adpex exchange could be explained by ( i ) a cotransport of adp and pi and by ( ii ) a possible competition of pi with atp during hetero - exchange . pi is substrate of pamntt1 but only in conjunction with adp to analyze whether pamntt1 is able to transport pi , we performed import studies using radioactively labeled pi ( 50 m p ) . pi import was conducted with proteoliposomes loaded with interior pi , pi plus adp , or pi plus atp . in absence of exterior nucleotides no pi uptake into the vesicles was measurable when only pi , or pi plus atp were internally present ( fig . 3a , white circles and black diamonds ) . to our surprise pi was imported into liposomes loaded with pi plus adp ( fig . 3a , gray squares ) . in a similar experiment we tested the effect of externally added adp ( fig . application of external adp did not markedly affect the rates of pi import into pi plus adp loaded liposomes ( fig . 3 , compare a and b , gray squares ) but stimulated pi import into liposomes containing solely pi , or pi plus atp ( fig . 3 , compare a and b , white circles and black diamonds ) . external atp led to no or to comparably low pi import ( compare the import rates of fig . the latter effect further argues for a possible competition of pi and atp for import . our results show that pi indeed is a substrate of pamntt1 but only when adp is present at least at one side of the liposomal membrane . the observation that pi transport occurs also in the absence of adenine nucleotide counter - exchange ( fig . 3 ) suggests that pamntt1 catalyzes , in addition to a possible pi plus adp cotransport in exchange with atp , an adp - dependent pi homo - exchange . to provide further evidence for a pi homo - exchange , we analyzed pi import into adp - loaded liposomes lacking luminal pi . the measured pi import rates were 10-fold lower than the corresponding rates obtained with liposomes containing adp plus pi ( data not shown ) . this is a further argument supporting the postulated pi homo - exchange . to analyze whether pi homo - exchange is accompanied by a simultaneous unidirectional translocation of adp , we additionally investigated [ -p]adp import into proteoliposomes solely loaded with pi . because in the absence of counter - exchange nucleotides pi but not adp import occurred ( data not shown ) , we concluded that adp - dependent pi homo - exchange is not associated with a unidirectional adp transport . pamntt1 catalyzes pi homo - exchange and cotransport with adp in a further study we analyzed adp and pi transport in more detail to address two important questions . first , is pamntt1 capable of transporting pi as cosubstrate of adp during hetero - exchange , and second , what is the stoichiometrical ratio of the pi plus adp cotransport . figure 3.analysis of pi import mediated by reconstituted pamntt1 . time - dependent import of 50 m radioactive pi into pamntt1-proteoliposomes loaded with 5 mm pi or 5 mm pi plus the given adenine nucleotides ( 10 mm ) . pi import was allowed for the given time spans and stopped by anion exchange chromatography . pi uptake studies were performed in the absence of external nucleotides ( a ) , in the presence of 50 m nonlabeled external adp ( b ) , or in the presence of 50 m nonlabeled external atp ( c ) to investigate a possible influence of external adenine nucleotides on pi transport . pi uptake into proteoliposomes loaded with 5 mm pi ( open circle ) , 5 mm pi plus 10 mm adp ( gray square ) , or 5 mm pi plus 10 mm atp ( black diamond ) . time - dependent import of 50 m radioactive pi into pamntt1-proteoliposomes loaded with 5 mm pi or 5 mm pi plus the given adenine nucleotides ( 10 mm ) . pi import was allowed for the given time spans and stopped by anion exchange chromatography . pi uptake studies were performed in the absence of external nucleotides ( a ) , in the presence of 50 m nonlabeled external adp ( b ) , or in the presence of 50 m nonlabeled external atp ( c ) to investigate a possible influence of external adenine nucleotides on pi transport . pi uptake into proteoliposomes loaded with 5 mm pi ( open circle ) , 5 mm pi plus 10 mm adp ( gray square ) , or 5 mm pi plus 10 mm atp ( black diamond ) . atp import in strict stoichiometrical exchange with one adp plus one pi ( h2po4 , with one negative charge ) would be electroneutral . in this context it is important to mention that under the applied conditions an important part of pi carries one negative charge . unfortunately , determination of the adp plus pi export parameters of the reconstituted pamntt1 was impossible . we were not able to measure the slight decrease of the high internal label . however , we demonstrated above that exterior pi stimulates adpim / atpex transport ( supplemental table s1 and fig . 2 , black bars ) and that similarly interior pi stimulates atpim / adpex transport ( fig . , it seems justified to assume that pi acts as cosubstrate of adp during hetero - exchange independent of the transport direction ( adpim+piim / atpex or atpim / adpex+piex ) . accordingly , we investigated the coimport of pi and adp . for this , import measurements were performed with radioactively labeled adp in the presence of nonlabeled pi as well as with labeled pi in the presence of nonlabeled adp . one set of experiments was done with liposomes containing atp plus pi , and one was done with vesicles loaded with solely atp ( to disconnect the adp - dependent pi homo - exchange from coimport with adp ) . for determination of the adp to pi coimport stoichiometry , we calculated pi import in relation to adp import ( adp import was set as 1 ) . the presence of pi in the liposomal lumen led to a high pi to adp import ratio [ 4.03 ( 0.45 ) pi to 1 adp ] ( supplemental fig . s1 ) . therefore , pi homo - exchange substantially exceeds coimport with adp . however , depletion of luminal pi led to a significantly reduced pi homo - exchange and allowed the calculation of a nearly one - to - one stoichiometry for pi and adp coimport ( 1.21 ( 0.16 ) pi to 1 adp ) in exchange with interior atp ( supplemental fig . it has to be mentioned that pi contaminations in the liposomal lumen or a marginal unidirectional pi transport might have caused a slight overestimation of the exact pi - to - adp ratio . determination of the kinetic properties and identification of the phosphate binding center of pamntt1as demonstrated above , already low external atp concentrations inhibited pi import ( fig . 3 , compare b and c ) , whereas high exterior pi concentrations were required to substantially reduce atpim / adpex transport ( fig . these characteristics suggest that the two anions , atp and pi , compete for uptake and that atp is the preferred import substrate . on the other hand , pi exhibits positive effects on adp transport ( import and export ) rather than competitive interference ( figs . 1 and 2 ) . accordingly , pi and adp simultaneously fit into the binding center , whereas pi probably interacts with the ntt domain , which is otherwise occupied by the -phosphate of atp . in a previous study we showed that mutations of a lysine residue at position 527 in the plastidial atp / adp transporter from a. thaliana ( atntt1 ) reduced atp transport to a higher extent than adp transport ( 34 ) . remarkably , this lysine residue is conserved in all plastidial and bacterial atp / adp transporters ( supplemental fig . s2 ) . to test whether this amino acid residue is not only important for atp but also for pi transport , we generated different pamntt1 mutant proteins and analyzed their biochemical properties in the liposomal system . this was mandatory because e. coli possesses endogenous pi transporters that obscure ntt mediated pi translocation . we substituted the positively charged lysine by a positively charged arginine ( k446r ) , by the neutral glutamine ( k446q ) , or by the negatively charged glutamate ( k446e ) to investigate a correlation between the charge of the amino acid residue 446 and the kinetic properties ( km and vmax values ) of pamntt1 . the biochemical parameters of unmodified pamntt1 for atp and adp transport were in the same range as previously described ( 32 ) . furthermore , the reconstituted wild type protein exhibited moderate affinities ( km in the range of 140 m ) and high vmax values ( 916 mol mg protein h ) for adp - dependent pi transport ( table 1 ) . application of internal atp slightly and of external atp highly reduced the affinity of pamntt1 for pi uptake . table 1comparison of the kinetic constants of reconstituted pamntt1 and k446 mutant proteins kinetic parameters of nucleotide or pi exchanges ( exterior substrates / interior substrates ) mediated by reconstituted pamntt1 and the three mutant proteins ( pamntt1-k446r , pamntt1-k446q , and pamntt1-k446e ) were determined by application of rising concentrations of the respective labeled import substrates ( 51,500 m ) . the used proteoliposomes contained 5 mm internal pi and the given nucleotides ( 10 mm ) . kinetic parameters of pi import were analyzed in the absence or presence of 50 m nonlabeled external nucleotides ( added external nucleotides in squared brackets ) . km values are given in m , and vmax values ( in parentheses ) are in mol mg of protein h , respectively . transport was allowed for time spans in the linear phase of the corresponding import at 50 m . standard errors of the low affinity and low velocity imports were below 20% and for the remaining imports below 14%.exchanges pamntt1 pamntt1 mutant proteins k446rk446qk446e atp / atp + pi 95 ( 27.6 ) 123 ( 0.5 ) 187 ( 6.5 ) 620 ( 2.3 ) atp / adp + pi 8.6 ( 31.3 ) 66 ( 1.0 ) 274 ( 7.2 ) 552 ( 2.4 ) adp / atp + pi 1401 ( 5.5 ) 179 ( 0.3 ) 68 ( 4.5 ) 29 ( 1.6 ) adp / adp + pi 120 ( 7.1 ) 96 ( 0.7 ) 44 ( 4.0 ) 159 ( 15.4 ) pi / adp + pi 125 ( 13.6 ) 271 ( 2.1 ) pi[+adp]/pi 154 ( 10.0 ) 152 ( 0.5 ) pi[+adp]/adp + pi 139 ( 15.9 ) 220 ( 1.9 ) 544 ( 0.4 ) pi[+adp]/atp + pi 295 ( 9.0 ) 275 ( 0.7 ) 550 ( 0.4 ) pi[+atp]/adp + pi 506 ( 13.3 ) 598 ( 2.0 ) comparison of the kinetic constants of reconstituted pamntt1 and k446 mutant proteins kinetic parameters of nucleotide or pi exchanges ( exterior substrates / interior substrates ) mediated by reconstituted pamntt1 and the three mutant proteins ( pamntt1-k446r , pamntt1-k446q , and pamntt1-k446e ) were determined by application of rising concentrations of the respective labeled import substrates ( 51,500 m ) . the used proteoliposomes contained 5 mm internal pi and the given nucleotides ( 10 mm ) . kinetic parameters of pi import were analyzed in the absence or presence of 50 m nonlabeled external nucleotides ( added external nucleotides in squared brackets ) . ( in parentheses ) are in mol mg of protein h , respectively . transport was allowed for time spans in the linear phase of the corresponding import at 50 m . standard errors of the low affinity and low velocity imports were below 20% and for the remaining imports below 14% . the conserved exchange of lysine 446 ( k446r ) led to a remarkable reduction of the vmax , in particular of the atp homo - exchange ( table 1 ) . furthermore , the affinity for atp import in exchange with interior adp plus pi was reduced , and that for adp import into atp plus pi - loaded vesicles was enhanced . nevertheless , the basic properties of the mutant protein pamntt1-k446r , such as the affinities for ( adp - dependent ) pi import or atp and adp import during homo - exchange , as well as the preference of the atpim / adpex transport over adpim / atpex exchange , still resemble that of the unmodified pamntt1 . in comparison with the wild type protein , the mutant protein with the neutral substitution ( pamntt1-k446q ) exhibited reduced affinities ( and vmax values ) for atp import , whereas the affinities for adp import , in particular in exchange with interior atp , were significantly enhanced ( table 1 ) . these changed biochemical characteristics led to a general adjustment of the import rates , the atp homo - exchange equals atpim / adpex transport , and also the high difference between adp homo - exchange and adpim / atpex transport of the wild type protein was reduced in pamntt1-k446q ( table 1 and supplemental fig . s3 ) . because pi transport of pamntt1-k446q was nearly completely diminished ( supplemental fig . solely in the presence of external adp and internal pi plus adenine nucleotides , very low affinities and vmax values for pi import were calculable ( table 1 ) . interestingly , the replacement of the positively charged lysine by the negatively charged glutamate completely blocked adp - dependent pi uptake ( supplemental fig . s3 and table 1 ) . in comparison with the unmodified pamntt1 and the mutant pamntt1-k446q , the mutation in pamntt1-k446e led to a further reduction of the km and vmax values of the atp import ( table 1 ) . furthermore , the affinity for adp import into atp - loaded proteoliposomes was enhanced , whereas the vmax of this exchange was reduced . the moderate affinity for adp import into adp plus pi - loaded vesicles , however , was accompanied by the highest vmax . generally , the observed differences in the biochemical characteristics between the wild type protein pamntt1 and the mutant pamntt1-k446q are more pronounced in the mutant pamntt1-k446e . our analyses of the mutant proteins revealed that a positively charged amino acid residue at position 446 is required for proper pi import , for the preference of atpim / adpex transport , and for the discrimination of adpim / atpex exchange . pi is substrate of several nonmitochondrial atp / adp transporters the fact that pamntt1 accepts pi as third substrate might resolve inconsistencies in the phosphate metabolism of p. amoebophila . to analyze whether also phylogenetically more distantly related atp / adp transporters from other organisms possess the capacity to transport pi , we investigated two carriers from the rickettsial species c. caryophilus ( ccntt ) and h. obtusa ( hontt ) and one plastidial transporter from the higher plant a. thaliana ( atntt1 ) . these representative rickettsial and plastidial ntts were heterologously expressed in e. coli , purified ( supplemental fig . s4 ) , and reconstituted into liposomes . all recombinant carriers mediated atp and adp transport and therefore were functional in the liposomal system ( supplemental fig . generally , hontt exhibited highest net uptake rates that suggest a high activity of this reconstituted protein ( fig . 4 and supplemental fig . import studies with radioactive pi showed that atntt1 and also the two selected rickettsial ntts were able to import pi . in fig . 4 the time - dependent pi uptake in presence of exterior adp is presented . pi import into vesicles loaded with adp plus pi exceeded pi import into proteoliposomes loaded with atp plus pi or with solely pi . the rates of atntt1- and ccntt - mediated pi homo - exchange were lower than the rates of pi ( plus adp ) import into vesicles loaded with atp plus pi , whereas hontt exhibited higher rates for pi homo - exchange than for pi import in presence of exterior adp and interior atp plus pi . apart from slight differences in the substrate preference pattern , our results clearly demonstrate that atp / adp exchanging ntts from distantly related organisms are capable for an adp - dependent pi transport , which resembles that of pamntt1 ( figs . 3 and 4 ) . figure 4.pi import mediated by phylogenetically distantly related ntt - type atp / adp transporters . heterologously expressed and purified atp / adp transporters from a. thaliana ( atntt1 ) , c. caryophilus ( ccntt ) , and h. obtusa ( hontt ) were reconstituted into liposomes loaded with 5 mm pi or with 5 mm pi plus the given nucleotides ( 10 mm ) . time - dependent import of 50 m radioactive pi in presence of 50 m nonlabeled adp mediated by atntt1 ( a ) , ccntt ( b ) , and hontt ( c ) . import into proteoliposomes loaded with pi ( open circle ) , pi plus adp ( gray square ) , or pi plus atp ( black diamond ) . the given values are net values ( calculated by subtraction of pi import in absence of external and internal nucleotides ) . the data are the means of at least three independent experiments , and the standard errors are displayed . pi import mediated by phylogenetically distantly related ntt - type atp / adp transporters . heterologously expressed and purified atp / adp transporters from a. thaliana ( atntt1 ) , c. caryophilus ( ccntt ) , and h. obtusa ( hontt ) were reconstituted into liposomes loaded with 5 mm pi or with 5 mm pi plus the given nucleotides ( 10 mm ) . time - dependent import of 50 m radioactive pi in presence of 50 m nonlabeled adp mediated by atntt1 ( a ) , ccntt ( b ) , and hontt ( c ) . import into proteoliposomes loaded with pi ( open circle ) , pi plus adp ( gray square ) , or pi plus atp ( black diamond ) . the given values are net values ( calculated by subtraction of pi import in absence of external and internal nucleotides ) . the data are the means of at least three independent experiments , and the standard errors are displayed . metabolically impaired energy parasites depend on atp import from the host cell , and also plant plastids rely on energy supply from the surrounding cytosol when photosynthetic activity is reduced or missing . atp / adp exchange would lead to substantial pi accumulation in the organelle or cell if no interacting export mechanism for phosphate exists . in this study we identified that pi transport is a previously not identified intrinsic feature of nonmitochondrial atp / adp transporters ( figs . 3 and 4 ) . pi is transported simultaneously with adp , and this cotransport is facilitated in a one pi to one adp stoichiometrical exchange with atp ( supplemental fig . the cotransport of one h2po4 ( with one negative charge ) would be in complete agreement with the recently documented electroneutrality of pamntt1-mediated atp / adp exchange ( 32 ) . during hetero - exchange , pi could act as a counterion compensating for the generation of a charge difference across the membrane . a stimulatory influence of high pi concentrations ( in the millimolar range ) on adp import into isolated rickettsia prowazekii cells had been observed more than three decades ago ( 38 ) . although a regulatory effect of pi on the transport characteristics was discussed , the exact role of pi in atp / adp exchange was not known ( 37 ) . interestingly , the simplest explanation , a possible cotransport of pi and adp , was never suggested for ntts . detailed analyses of pamntt1 ( figs . 2 and 3 and supplemental fig . 4 , b and c ) suggest that the regulatory principle of pi is tightly associated to its function as a cosubstrate of adp . the addition of pi indeed increased the vmax and the affinity of pamntt1 for adp transport into atp loaded proteoliposomes . it is imaginable that a simultaneous entry of adp and pi might mimic the presence of a triphosphorylated adenine nucleotide ( atp ) , which is the preferred import substrate . the capacity of the phylogenetically different ntts to transport adp plus pi in exchange with atp ( i ) is in line with the electroneutrality of nucleotide exchange ( 32 ) , ( ii ) prevents harmful pi accumulation in intracellular living bacteria and plant plastids , and ( iii ) implies that the phosphate exporter that was proposed for a long time in these bacteria and in plastids is represented by the very recent analyses demonstrated that energy parasitism is not only restricted to chlamydiales and rickettsiales but also occurs in further important mammalian pathogens ( 23 , 24 ) . ntt - type carriers reside in the plasma membrane and in the mitochondrial relict ( the so called mitosome ) of the intracellular protist encephalitozoon cuniculi ( 24 ) . by catalyzing a highly specific atp / adp exchange , these ntts provide energy to the cell or to the mitosome that lacks atp synthesis . it is tempting to speculate that also these eukaryotic ntts catalyze a pi coexport with adp . to our surprise , ntt - type atp / adp transporters mediate a nucleotide exchange - independent but adp - induced homo - exchange of pi in addition to the pi cotransport with adp ( figs . 3 and 4 , white circles ) . the pi homo - exchange was induced by external as well as by internal adp ( fig . we propose a scenario that could explain the induction of pi homo - exchange independent of the side of adp application . adp and pi enter the binding center , for example at the proteoliposomal lumen , and are transported to the opposed side ( fig . , adp stays at the binding center , whereas the nonlabeled pi is displaced by radioactive pi . a subsequent reversely directed translocation of the two bound substrates and replacement of radioactive pi by nonlabeled pi at the interior causes the observed pi homo - exchange ( fig . 5 ) . this hypothesized mechanism presupposes that only one single binding center exists for import and export that opens from one side of the membrane to the other where nucleotides and/or pi are displaced by other substrates . by the help of mutant proteins we identified a correlation between the charge of the amino acid residue at position 446 and the transport properties of pamntt1 . a positive amino acid residue at position 446 allowed the insertion of atp or pi ( plus adp ) , a neutral amino acid residue or even more a negative amino acid residue stimulates adp but suppresses pi or atp entry ( table 1 ) . substitution of lys by arginine ( k446r ) entailed substantially lowered import rates for all substrates . we assume that structural differences between lysine and arginine rather than an unfavorable charge might have caused the decreased transport velocity ( table 1 ) . the biochemical properties of the mutant proteins suggest that lysine 446 is an essential component of the atp and pi binding center or translocation pathway . we conclude that the positively charged lysine 446 interacts with the negative charge introduced either by the -phosphate of atp or alternatively by pi . figure 5.model explaining the adp - dependent pi homo - exchange mediated by pamntt1 . adp ( ado - p - p , p - p - ado ) supports entry of interior pi ( oval 1 ) , adp plus pi are translocated ( white arrow ) to the exterior . pi is displaced by radioactive pi ( pi ) , whereas adp remains at the binding center ( oval 2 ) . the substrate complex is translocated ( white arrow ) to the interior and radioactive pi is displaced by nonlabeled pi ( oval 3 ) . adp ( ado - p - p , p - p - ado ) supports entry of interior pi ( oval 1 ) , adp plus pi are translocated ( white arrow ) to the exterior . pi is displaced by radioactive pi ( pi ) , whereas adp remains at the binding center ( oval 2 ) . the substrate complex is translocated ( white arrow ) to the interior and radioactive pi is displaced by nonlabeled pi ( oval 3 ) . the fact that atp import was highly reduced , whereas atp export still occurred ( adpim / atpex transport ) in the mutant protein pamntt1-k446e might argue against a single binding center for atp import and export . however , the vmax of adpim / atpex exchange was remarkably low when compared with that of adp homo - exchange ( table 1 ) . this characteristic could be explained by an impaired atp export capacity leading to a return transport of previously imported adp . this would be in agreement with the characteristics of the mutated plastidial atp / adp transporter , which exhibited a highly reduced capacity for both atp import and atp export ( 34 ) . it becomes clear that additional studies are required to get more insights into structure / function relationships of ntts , and it will be interesting to focus on this topic in the future . furthermore , we wish to analyze transport parameters under conditions mimicking the energy state of intracellular living bacteria in their host cells . however , for this it is a precondition to determine pi , atp , and adp concentrations in the bacterium and the infected cell .

chlamydiales and rickettsiales as metabolically impaired , intracellular pathogenic bacteria essentially rely on energy parasitism by the help of nucleotide transporters ( ntts ) . also in plant plastids ntt - type carriers catalyze atp / adp exchange to fuel metabolic processes . the uptake of atp4- , followed by energy consumption and the release of adp3- , would lead to a metabolically disadvantageous accumulation of negative charges in form of inorganic phosphate ( pi ) in the bacterium or organelle if no interacting pi export system exists . we identified that pi is a third substrate of several ntt - type atp / adp transporters . during adenine nucleotide hetero - exchange , pi is cotransported with adp in a one - to - one stoichiometry . additionally , pi can be transported in exchange with solely pi . this pi homo - exchange depends on the presence of adp and provides a first indication for only one binding center involved in import and export . furthermore , analyses of mutant proteins revealed that pi interacts with the same amino acid residue as the -phosphate of atp . import of atp in exchange with adp plus pi is obviously an efficient way to couple energy provision with the export of the two metabolic products ( adp plus pi ) and to maintain cellular phosphate homeostasis in intracellular living energy parasites and plant plastids . the additional pi transport capacity of ntt - type atp / adp transporters makes the existence of an interacting pi exporter dispensable and might explain why a corresponding protein so far has not been identified .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

heterologous synthesis of selected ntts and mutant proteins in e. coli the heterologous synthesis of pamntt1 and of two atp / adp transporters from the rickettsia - related bacteria caedibacter caryophilus ( ccntt ) and holospora obtusa ( hontt ) was performed on the basis of existing pet16b constructs ( 13 , 22 ) , which were transformed into blr cells ( merck ) . optionally , import was conducted in the presence of the given concentrations of nonlabeled nucleotides or pi . figure 1.influence of the presence of pi in the liposomal lumen on pamntt1 mediated adenine nucleotide hetero - exchange . influence of the presence of pi in the liposomal lumen on pamntt1 mediated adenine nucleotide hetero - exchange . to mimic the presence of pi in the bacterium , we tested whether pi application at the liposomal interior also influences adenine nucleotide hetero - exchange . when compared with the control ( 0 mm luminal pi ) , the addition of internal pi stimulated atp import in exchange with adp ( atpim / adpex ) 4-fold , whereas adpim / atpex transport was not highly affected by internal pi application ( fig . however , stimulation of the hetero - exchange exclusively occurred when adp and pi were present at the same side of the liposome ( adp plus pi ) ( supplemental table s1 and fig . furthermore , only in the presence of pi , adp was able to compete with atp for import , leading the authors to the assumption that pi might increase ( at least ) the affinity of the rickettsial atp / adp transporter for adp ( 37 ) . figure 2.influence of rising exterior pi concentrations on adenine nucleotide hetero - exchange mediated by pamntt1 . the effects of rising external pi concentrations on atp import into adp plus pi - loaded proteoliposomes ( light gray bars ) and on adp import into atp plus pi - loaded vesicles ( dark gray bars ) were analyzed . km and vmax values ( in parentheses ) were calculated for adenine nucleotide hetero - exchanges in the absence ( 0 mm ) and in the presence of 5 mm external pi , respectively , and are given above the bars of the corresponding exchanges . in the graphic depicting the presence of pi and nucleotides at the liposomal interior and exterior , influence of rising exterior pi concentrations on adenine nucleotide hetero - exchange mediated by pamntt1 . the effects of rising external pi concentrations on atp import into adp plus pi - loaded proteoliposomes ( light gray bars ) and on adp import into atp plus pi - loaded vesicles ( dark gray bars ) were analyzed . km and vmax values ( in parentheses ) were calculated for adenine nucleotide hetero - exchanges in the absence ( 0 mm ) and in the presence of 5 mm external pi , respectively , and are given above the bars of the corresponding exchanges . in the graphic depicting the presence of pi and nucleotides at the liposomal interior and exterior , changes in external pi availability had low impact on atp homo - exchange parameters and only slightly affected adp homo - exchange ( 4-fold increased vmax at 5 mm external pi ) ( data not shown ) . our results show that pi indeed is a substrate of pamntt1 but only when adp is present at least at one side of the liposomal membrane . the observation that pi transport occurs also in the absence of adenine nucleotide counter - exchange ( fig . 3 ) suggests that pamntt1 catalyzes , in addition to a possible pi plus adp cotransport in exchange with atp , an adp - dependent pi homo - exchange . to analyze whether pi homo - exchange is accompanied by a simultaneous unidirectional translocation of adp , we additionally investigated [ -p]adp import into proteoliposomes solely loaded with pi . because in the absence of counter - exchange nucleotides pi but not adp import occurred ( data not shown ) , we concluded that adp - dependent pi homo - exchange is not associated with a unidirectional adp transport . pamntt1 catalyzes pi homo - exchange and cotransport with adp in a further study we analyzed adp and pi transport in more detail to address two important questions . first , is pamntt1 capable of transporting pi as cosubstrate of adp during hetero - exchange , and second , what is the stoichiometrical ratio of the pi plus adp cotransport . unfortunately , determination of the adp plus pi export parameters of the reconstituted pamntt1 was impossible . , it seems justified to assume that pi acts as cosubstrate of adp during hetero - exchange independent of the transport direction ( adpim+piim / atpex or atpim / adpex+piex ) . one set of experiments was done with liposomes containing atp plus pi , and one was done with vesicles loaded with solely atp ( to disconnect the adp - dependent pi homo - exchange from coimport with adp ) . therefore , pi homo - exchange substantially exceeds coimport with adp . however , depletion of luminal pi led to a significantly reduced pi homo - exchange and allowed the calculation of a nearly one - to - one stoichiometry for pi and adp coimport ( 1.21 ( 0.16 ) pi to 1 adp ) in exchange with interior atp ( supplemental fig . it has to be mentioned that pi contaminations in the liposomal lumen or a marginal unidirectional pi transport might have caused a slight overestimation of the exact pi - to - adp ratio . accordingly , pi and adp simultaneously fit into the binding center , whereas pi probably interacts with the ntt domain , which is otherwise occupied by the -phosphate of atp . in a previous study we showed that mutations of a lysine residue at position 527 in the plastidial atp / adp transporter from a. thaliana ( atntt1 ) reduced atp transport to a higher extent than adp transport ( 34 ) . remarkably , this lysine residue is conserved in all plastidial and bacterial atp / adp transporters ( supplemental fig . to test whether this amino acid residue is not only important for atp but also for pi transport , we generated different pamntt1 mutant proteins and analyzed their biochemical properties in the liposomal system . we substituted the positively charged lysine by a positively charged arginine ( k446r ) , by the neutral glutamine ( k446q ) , or by the negatively charged glutamate ( k446e ) to investigate a correlation between the charge of the amino acid residue 446 and the kinetic properties ( km and vmax values ) of pamntt1 . furthermore , the reconstituted wild type protein exhibited moderate affinities ( km in the range of 140 m ) and high vmax values ( 916 mol mg protein h ) for adp - dependent pi transport ( table 1 ) . table 1comparison of the kinetic constants of reconstituted pamntt1 and k446 mutant proteins kinetic parameters of nucleotide or pi exchanges ( exterior substrates / interior substrates ) mediated by reconstituted pamntt1 and the three mutant proteins ( pamntt1-k446r , pamntt1-k446q , and pamntt1-k446e ) were determined by application of rising concentrations of the respective labeled import substrates ( 51,500 m ) . standard errors of the low affinity and low velocity imports were below 20% and for the remaining imports below 14%.exchanges pamntt1 pamntt1 mutant proteins k446rk446qk446e atp / atp + pi 95 ( 27.6 ) 123 ( 0.5 ) 187 ( 6.5 ) 620 ( 2.3 ) atp / adp + pi 8.6 ( 31.3 ) 66 ( 1.0 ) 274 ( 7.2 ) 552 ( 2.4 ) adp / atp + pi 1401 ( 5.5 ) 179 ( 0.3 ) 68 ( 4.5 ) 29 ( 1.6 ) adp / adp + pi 120 ( 7.1 ) 96 ( 0.7 ) 44 ( 4.0 ) 159 ( 15.4 ) pi / adp + pi 125 ( 13.6 ) 271 ( 2.1 ) pi[+adp]/pi 154 ( 10.0 ) 152 ( 0.5 ) pi[+adp]/adp + pi 139 ( 15.9 ) 220 ( 1.9 ) 544 ( 0.4 ) pi[+adp]/atp + pi 295 ( 9.0 ) 275 ( 0.7 ) 550 ( 0.4 ) pi[+atp]/adp + pi 506 ( 13.3 ) 598 ( 2.0 ) comparison of the kinetic constants of reconstituted pamntt1 and k446 mutant proteins kinetic parameters of nucleotide or pi exchanges ( exterior substrates / interior substrates ) mediated by reconstituted pamntt1 and the three mutant proteins ( pamntt1-k446r , pamntt1-k446q , and pamntt1-k446e ) were determined by application of rising concentrations of the respective labeled import substrates ( 51,500 m ) . furthermore , the affinity for atp import in exchange with interior adp plus pi was reduced , and that for adp import into atp plus pi - loaded vesicles was enhanced . nevertheless , the basic properties of the mutant protein pamntt1-k446r , such as the affinities for ( adp - dependent ) pi import or atp and adp import during homo - exchange , as well as the preference of the atpim / adpex transport over adpim / atpex exchange , still resemble that of the unmodified pamntt1 . in comparison with the wild type protein , the mutant protein with the neutral substitution ( pamntt1-k446q ) exhibited reduced affinities ( and vmax values ) for atp import , whereas the affinities for adp import , in particular in exchange with interior atp , were significantly enhanced ( table 1 ) . solely in the presence of external adp and internal pi plus adenine nucleotides , very low affinities and vmax values for pi import were calculable ( table 1 ) . in comparison with the unmodified pamntt1 and the mutant pamntt1-k446q , the mutation in pamntt1-k446e led to a further reduction of the km and vmax values of the atp import ( table 1 ) . our analyses of the mutant proteins revealed that a positively charged amino acid residue at position 446 is required for proper pi import , for the preference of atpim / adpex transport , and for the discrimination of adpim / atpex exchange . pi is substrate of several nonmitochondrial atp / adp transporters the fact that pamntt1 accepts pi as third substrate might resolve inconsistencies in the phosphate metabolism of p. amoebophila . to analyze whether also phylogenetically more distantly related atp / adp transporters from other organisms possess the capacity to transport pi , we investigated two carriers from the rickettsial species c. caryophilus ( ccntt ) and h. obtusa ( hontt ) and one plastidial transporter from the higher plant a. thaliana ( atntt1 ) . pi import into vesicles loaded with adp plus pi exceeded pi import into proteoliposomes loaded with atp plus pi or with solely pi . the rates of atntt1- and ccntt - mediated pi homo - exchange were lower than the rates of pi ( plus adp ) import into vesicles loaded with atp plus pi , whereas hontt exhibited higher rates for pi homo - exchange than for pi import in presence of exterior adp and interior atp plus pi . figure 4.pi import mediated by phylogenetically distantly related ntt - type atp / adp transporters . pi import mediated by phylogenetically distantly related ntt - type atp / adp transporters . metabolically impaired energy parasites depend on atp import from the host cell , and also plant plastids rely on energy supply from the surrounding cytosol when photosynthetic activity is reduced or missing . atp / adp exchange would lead to substantial pi accumulation in the organelle or cell if no interacting export mechanism for phosphate exists . in this study we identified that pi transport is a previously not identified intrinsic feature of nonmitochondrial atp / adp transporters ( figs . pi is transported simultaneously with adp , and this cotransport is facilitated in a one pi to one adp stoichiometrical exchange with atp ( supplemental fig . during hetero - exchange , pi could act as a counterion compensating for the generation of a charge difference across the membrane . it is imaginable that a simultaneous entry of adp and pi might mimic the presence of a triphosphorylated adenine nucleotide ( atp ) , which is the preferred import substrate . the capacity of the phylogenetically different ntts to transport adp plus pi in exchange with atp ( i ) is in line with the electroneutrality of nucleotide exchange ( 32 ) , ( ii ) prevents harmful pi accumulation in intracellular living bacteria and plant plastids , and ( iii ) implies that the phosphate exporter that was proposed for a long time in these bacteria and in plastids is represented by the very recent analyses demonstrated that energy parasitism is not only restricted to chlamydiales and rickettsiales but also occurs in further important mammalian pathogens ( 23 , 24 ) . ntt - type carriers reside in the plasma membrane and in the mitochondrial relict ( the so called mitosome ) of the intracellular protist encephalitozoon cuniculi ( 24 ) . to our surprise , ntt - type atp / adp transporters mediate a nucleotide exchange - independent but adp - induced homo - exchange of pi in addition to the pi cotransport with adp ( figs . we propose a scenario that could explain the induction of pi homo - exchange independent of the side of adp application . a subsequent reversely directed translocation of the two bound substrates and replacement of radioactive pi by nonlabeled pi at the interior causes the observed pi homo - exchange ( fig . this hypothesized mechanism presupposes that only one single binding center exists for import and export that opens from one side of the membrane to the other where nucleotides and/or pi are displaced by other substrates . by the help of mutant proteins we identified a correlation between the charge of the amino acid residue at position 446 and the transport properties of pamntt1 . we conclude that the positively charged lysine 446 interacts with the negative charge introduced either by the -phosphate of atp or alternatively by pi . the fact that atp import was highly reduced , whereas atp export still occurred ( adpim / atpex transport ) in the mutant protein pamntt1-k446e might argue against a single binding center for atp import and export . this would be in agreement with the characteristics of the mutated plastidial atp / adp transporter , which exhibited a highly reduced capacity for both atp import and atp export ( 34 ) . however , for this it is a precondition to determine pi , atp , and adp concentrations in the bacterium and the infected cell .

the jpad trial was a multicenter , prospective , randomized , open - label , blinded , end point trial conducted at 163 institutions throughout japan . this trial was performed according to the declaration of helsinki and approved by the institutional review board at each participating hospital . written informed consent was obtained from each participant before the trial . the detailed design of the jpad trial has been previously described ( 10 ) . in brief , we enrolled 2,539 patients , aged between 30 and 85 years , with type 2 diabetes and no history of cardiovascular disease . the participants were randomly assigned to the aspirin group or the no - aspirin group . the 1,262 patients in the aspirin group were assigned to take 81 mg or 100 mg of aspirin once daily . the 1,277 patients in the no - aspirin group were allowed to use antiplatelet therapy , including aspirin , if needed . all patients were allowed to use any concurrent treatments . in this subanalysis , we classified all participants into three subgroups according to the diabetes management at baseline : insulin , oha , or diet alone . a total of 193 patients were lost to follow - up , and data for those patients were censored at the day of last follow - up . the consolidated standards of reporting trials ( consort ) diagram for the jpad trial was presented previously ( 10 ) . the primary end point was the occurrence during the follow - up period of any atherosclerotic event , which was a composite of the following : death from coronary , cerebrovascular , and aortic causes ; nonfatal acute myocardial infarction ; unstable angina ; newly developed exertional angina ; nonfatal ischemic and hemorrhagic stroke ; transient ischemic attack ; and nonfatal aortic and peripheral vascular disease . all potential end points and hemorrhagic events ( gastrointestinal bleeding and hemorrhagic stroke ) were adjudicated by an independent committee that was blinded to the group assignments . hemoglobin a1c values were converted from the japanese diabetes society ( jds ) values used in the main analysis of the jpad trial ( 10 ) into national glycohemoglobin standardization program ( ngsp ) equivalent values . ngsp equivalent values were calculated using the following formula : ngsp equivalent value ( % ) = jds value ( % ) + 0.4 ( 15 ) . categorical variables were expressed as number and percentage and were compared with the test . continuous variables were expressed as mean values sds unless otherwise indicated . based on their distribution , continuous variables were compared using the student t test or wilcoxon rank sum test for two - group comparisons and anova or kruskal - wallis test for three - group comparisons . cumulative incidence was estimated by the kaplan - meier method , and differences were assessed with the log - rank test . efficacy comparisons were performed on the basis of time to the first event according to the intention - to - treat principle , with all patients included in the group to which they were randomized and with patients lost to follow - up censored at the day of the last visit . cumulative incidences of end points were estimated by the kaplan - meier method , and differences between groups were assessed with the log - rank test . we used the cox proportional hazard model to estimate the hazard ratio ( hr ) of aspirin use along with the 95% ci . we also developed multivariable cox proportional hazard models to assess the effects of aspirin on atherosclerotic events adjusting for age ( 65 years ) , hypertension , dyslipidemia , and history of smoking to evaluate the robustness . statistical analyses were conducted by an independent statistician ( t.m . ) with the use of jmp 8.0 ( sas institute , cary , nc ) software and sas 9.2 ( sas institute ) . the jpad trial was a multicenter , prospective , randomized , open - label , blinded , end point trial conducted at 163 institutions throughout japan . this trial was performed according to the declaration of helsinki and approved by the institutional review board at each participating hospital . written informed consent was obtained from each participant before the trial . the detailed design of the jpad trial has been previously described ( 10 ) . in brief , we enrolled 2,539 patients , aged between 30 and 85 years , with type 2 diabetes and no history of cardiovascular disease . the participants were randomly assigned to the aspirin group or the no - aspirin group . the 1,262 patients in the aspirin group were assigned to take 81 mg or 100 mg of aspirin once daily . the 1,277 patients in the no - aspirin group were allowed to use antiplatelet therapy , including aspirin , if needed . all patients were allowed to use any concurrent treatments . in this subanalysis , we classified all participants into three subgroups according to the diabetes management at baseline : insulin , oha , or diet alone . a total of 193 patients were lost to follow - up , and data for those patients were censored at the day of last follow - up . the consolidated standards of reporting trials ( consort ) diagram for the jpad trial was presented previously ( 10 ) . the primary end point was the occurrence during the follow - up period of any atherosclerotic event , which was a composite of the following : death from coronary , cerebrovascular , and aortic causes ; nonfatal acute myocardial infarction ; unstable angina ; newly developed exertional angina ; nonfatal ischemic and hemorrhagic stroke ; transient ischemic attack ; and nonfatal aortic and peripheral vascular disease . all potential end points and hemorrhagic events ( gastrointestinal bleeding and hemorrhagic stroke ) were adjudicated by an independent committee that was blinded to the group assignments . hemoglobin a1c ( hba1c ) values were converted from the japanese diabetes society ( jds ) values used in the main analysis of the jpad trial ( 10 ) into national glycohemoglobin standardization program ( ngsp ) equivalent values . ngsp equivalent values were calculated using the following formula : ngsp equivalent value ( % ) = jds value ( % ) + 0.4 ( 15 ) . categorical variables were expressed as number and percentage and were compared with the test . continuous variables were expressed as mean values sds unless otherwise indicated . based on their distribution , continuous variables were compared using the student t test or wilcoxon rank sum test for two - group comparisons and anova or kruskal - wallis test for three - group comparisons . cumulative incidence was estimated by the kaplan - meier method , and differences were assessed with the log - rank test . efficacy comparisons were performed on the basis of time to the first event according to the intention - to - treat principle , with all patients included in the group to which they were randomized and with patients lost to follow - up censored at the day of the last visit . cumulative incidences of end points were estimated by the kaplan - meier method , and differences between groups were assessed with the log - rank test . we used the cox proportional hazard model to estimate the hazard ratio ( hr ) of aspirin use along with the 95% ci . we also developed multivariable cox proportional hazard models to assess the effects of aspirin on atherosclerotic events adjusting for age ( 65 years ) , hypertension , dyslipidemia , and history of smoking to evaluate the robustness . statistical analyses were conducted by an independent statistician ( t.m . ) with the use of jmp 8.0 ( sas institute , cary , nc ) software and sas 9.2 ( sas institute ) . at baseline , 326 patients were treated with insulin , 1,750 with ohas , and 463 with diet alone ( table 1 ) . the mean age was slightly lower in the insulin group ( 62 years ) than the other groups ( 65 years ) . mean bmi was lowest in the insulin group ( 23 kg / m ) and highest in the diet - alone group ( 25 kg / m ) . median duration of diabetes was longest in the insulin group : 13.0 years in the insulin group , 7.2 years in the oha group , and 3.5 years in the diet - alone group . the prevalence of diabetic microangiopathies was highest in the insulin group ( retinopathy , 43% ; nephropathy , 20% ; neuropathy , 32% ) and lowest in the diet - alone group ( retinopathy , 4% ; nephropathy , 8% ; neuropathy , 4% ) . baseline characteristics by diabetes management data are mean ( sd ) or n ( % ) ; duration of diabetes data are median ( interquartile range ) . hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or therapy with antihypertensive agents . the frequency of other cardiovascular risk factors was also investigated at baseline ( table 1 ) . the prevalence of hypertension and dyslipidemia was significantly higher in the diet - alone group ( hypertension , 72% ; dyslipidemia , 56% ) than in the other two groups . the use of statins was low in all groups : 18 , 27 , and 24% in the insulin , oha , and diet - alone groups , respectively . smoking history and family history of cardiovascular diseases were similar among groups . the incidence of atherosclerotic events was 26.6 , 14.6 , and 10.4 cases per 1,000 person - years in the insulin , oha , and diet - alone groups , respectively ( supplementary table 1 ) . both coronary artery and cerebrovascular events occurred most frequently in the insulin group ( coronary artery events , 12.1 ; cerebrovascular events , 12.1 cases per 1,000 person - years ) . there was a low incidence of peripheral vascular events among the three groups ( insulin , 2.4 ; oha , 1.7 ; diet alone , 1.6 cases per 1,000 person - years ) . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group than those in the other groups ( log - rank test , p = 0.0013 ; fig . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group ( log - rank test , p = 0.0013 ) . participants were randomly assigned to the aspirin group or the no - aspirin group ( supplementary table 2 ) . in the insulin group , low - dose aspirin did not affect the incidence of atherosclerotic events ( hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; fig . similar results were observed in the oha group ( hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ; fig . , low - dose aspirin significantly reduced atherosclerotic events despite the fact that this group had the lowest atherosclerotic event rate ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ; fig . adjusting for age , hypertension , dyslipidemia , and history of smoking , low - dose aspirin significantly reduced atherosclerotic events in the diet - alone group ( hr 0.20 [ 0.060.68 ] ; log - rank test , p = 0.0099 ) but not in the insulin or oha groups ( insulin : hr 1.0 [ 0.502.00 ] , log - rank test , p = 1.0 ; oha : hr 0.77 [ 0.521.14 ] , log - rank test , p = 0.20 ) . efficacy of low - dose aspirin on primary prevention of atherosclerotic events in each diabetes management . a and b : in the insulin ( a ) and oha ( b ) groups , low - dose aspirin did not affect the incidence of atherosclerotic events ( insulin : hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; oha : hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ) . c : in the diet - alone group , low - dose aspirin significantly reduced atherosclerotic events despite the lowest event rates ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ) . the number of gastrointestinal bleeding and hemorrhagic strokes was very low overall and seemed similar between the aspirin and no - aspirin groups in each diabetes management . the number of gastrointestinal bleeding was one in the insulin group ( this patient took aspirin ) and seven in the oha group ( four in the aspirin group and three in the no - aspirin group ) . in the diet - alone group , no patients suffered from gastrointestinal bleeding . the number of hemorrhagic stroke was two in the insulin group ( one in the aspirin group and one in the no - aspirin group ) , nine in the oha group ( five in the aspirin group and four in the no - aspirin group ) , and two in the diet - alone group ( all in the no - aspirin group ) . at baseline , 326 patients were treated with insulin , 1,750 with ohas , and 463 with diet alone ( table 1 ) . the mean age was slightly lower in the insulin group ( 62 years ) than the other groups ( 65 years ) . mean bmi was lowest in the insulin group ( 23 kg / m ) and highest in the diet - alone group ( 25 kg / m ) . median duration of diabetes was longest in the insulin group : 13.0 years in the insulin group , 7.2 years in the oha group , and 3.5 years in the diet - alone group . the prevalence of diabetic microangiopathies was highest in the insulin group ( retinopathy , 43% ; nephropathy , 20% ; neuropathy , 32% ) and lowest in the diet - alone group ( retinopathy , 4% ; nephropathy , 8% ; neuropathy , 4% ) . baseline characteristics by diabetes management data are mean ( sd ) or n ( % ) ; duration of diabetes data are median ( interquartile range ) . hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or therapy with antihypertensive agents . the frequency of other cardiovascular risk factors was also investigated at baseline ( table 1 ) . the prevalence of hypertension and dyslipidemia was significantly higher in the diet - alone group ( hypertension , 72% ; dyslipidemia , 56% ) than in the other two groups . the use of statins was low in all groups : 18 , 27 , and 24% in the insulin , oha , and diet - alone groups , respectively . smoking history and family history of cardiovascular diseases were similar among groups . the incidence of atherosclerotic events was 26.6 , 14.6 , and 10.4 cases per 1,000 person - years in the insulin , oha , and diet - alone groups , respectively ( supplementary table 1 ) . both coronary artery and cerebrovascular events occurred most frequently in the insulin group ( coronary artery events , 12.1 ; cerebrovascular events , 12.1 cases per 1,000 person - years ) . there was a low incidence of peripheral vascular events among the three groups ( insulin , 2.4 ; oha , 1.7 ; diet alone , 1.6 cases per 1,000 person - years ) . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group than those in the other groups ( log - rank test , p = 0.0013 ; fig . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group ( log - rank test , p = 0.0013 ) . participants were randomly assigned to the aspirin group or the no - aspirin group ( supplementary table 2 ) . in the insulin group , low - dose aspirin did not affect the incidence of atherosclerotic events ( hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; fig . similar results were observed in the oha group ( hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ; fig . , low - dose aspirin significantly reduced atherosclerotic events despite the fact that this group had the lowest atherosclerotic event rate ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ; fig . adjusting for age , hypertension , dyslipidemia , and history of smoking , low - dose aspirin significantly reduced atherosclerotic events in the diet - alone group ( hr 0.20 [ 0.060.68 ] ; log - rank test , p = 0.0099 ) but not in the insulin or oha groups ( insulin : hr 1.0 [ 0.502.00 ] , log - rank test , p = 1.0 ; oha : hr 0.77 [ 0.521.14 ] , log - rank test , p = 0.20 ) . efficacy of low - dose aspirin on primary prevention of atherosclerotic events in each diabetes management . a and b : in the insulin ( a ) and oha ( b ) groups , low - dose aspirin did not affect the incidence of atherosclerotic events ( insulin : hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; oha : hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ) . c : in the diet - alone group , low - dose aspirin significantly reduced atherosclerotic events despite the lowest event rates ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ) . the number of gastrointestinal bleeding and hemorrhagic strokes was very low overall and seemed similar between the aspirin and no - aspirin groups in each diabetes management . the number of gastrointestinal bleeding was one in the insulin group ( this patient took aspirin ) and seven in the oha group ( four in the aspirin group and three in the no - aspirin group ) . in the diet - alone group , no patients suffered from gastrointestinal bleeding . the number of hemorrhagic stroke was two in the insulin group ( one in the aspirin group and one in the no - aspirin group ) , nine in the oha group ( five in the aspirin group and four in the no - aspirin group ) , and two in the diet - alone group ( all in the no - aspirin group ) . the major findings of this subanalysis were as follows : japanese patients with type 2 diabetes receiving insulin therapy had a high incidence of atherosclerotic events and low - dose aspirin reduced atherosclerotic events not in patients receiving insulin therapy but in patients treated with diet alone . the patients baseline characteristics categorized by diabetes management obviously demonstrated the difference in diabetes status ( table 1 ) . the insulin group was characterized by the youngest mean age ( 62 years ) and the longest median duration of diabetes ( 13.0 years ) , which meant that patients in the insulin group developed diabetes at the youngest age among the three groups . in contrast , patients in the diet - alone group developed diabetes in recent years and at older age . the slightly but significantly low bmi in the insulin group ( mean bmi , 23 kg / m ) might be a reflection of insufficient insulin secretion . the glycemic control was worst in the insulin group ( mean levels of hba1c and fpg , 8.1% and 8.77 mmol / l ) and best in the diet - alone group ( mean levels of hba1c and fpg , 6.7% and 7.22 the prevalence of diabetic microangiopathies was highest in the insulin group ( 20% ) and lowest in the diet alone group ( < 10% ) . there are no definitive criteria defining clinical stages of diabetes ; however , it is usually classified according to the extent of vascular complications or the level of glycemic control . thus we could regard the insulin group as demonstrating an advanced clinical stage of diabetes and the diet - alone group as demonstrating a less advanced or earlier clinical stage of diabetes than the insulin and oha groups . our results might indicate that low - dose aspirin was most beneficial in early clinical stages of diabetes , despite the lowest event rates . recently , we reported that a subanalysis of the jpad trial showed a difference in low - dose aspirin effect on primary prevention of atherosclerotic events in renal function ( 16 ) . in the study , reduced renal function in diabetic patients was significantly associated with high incidence of atherosclerotic events . low - dose aspirin did not prevent atherosclerotic events in patients with an estimated glomerular filtration rate ( egfr ) < 60 ml / min/1.73 m ( hr 1.3 [ 95% ci 0.762.4 ] ) . on the other hand , in the patients with an egfr of 60 to 89 ml / min/1.73 m , low - dose aspirin significantly reduced atherosclerotic events , despite low event rates ( hr 0.57 [ 0.360.88 ] ) ( 16 ) . as in the current study , low - dose aspirin was not beneficial in patients with advanced stages of renal dysfunction and with high event rates but rather in those with early stages of renal dysfunction and with low event rates . these results suggest that progression of diabetes and diabetes complications ( renal dysfunction ) might attenuate the effect of low - dose aspirin . the current recommendation of the ada , aha , and accf regarding the preventative use of aspirin in high - risk diabetic patients is based on the number of cardiovascular events prevented by low - dose aspirin ( relative risk reduction of 10% ) being greater than the number of hemorrhagic events ( 15 cases per 1,000 person - years ) ( 12 ) . therefore , low - dose aspirin use depends on individual cardiovascular risk . because diabetic patients with higher cardiovascular risk should have greater absolute benefit from low - dose aspirin , our findings were unexpected and inconsistent with the recommendation . to confirm whether low - dose aspirin is really effective in high - risk or low - risk diabetic patients , one possible mechanism of our findings is that events in patients with advanced clinical stages of diabetes are unpreventable because of overly advanced atherosclerotic lesions . aspirin resistance is often described as a phenomenon whereby patients develop cardiovascular disease despite aspirin intake . the details surrounding aspirin resistance remain obscure , but it is thought to be affected by underlying interindividual variability ( 17 ) . several studies have reported that aspirin s antiplatelet function is lower in diabetic patients than in nondiabetic patients ( 19 ) and that the frequency of aspirin resistance is positively correlated with the levels of hba1c and fpg ( 20 ) . higher aspirin doses have been shown to improve aspirin resistance in patients with diabetes ( 19 ) . in our study , poor glycemic control in the insulin group aspirin dosage may improve antiplatelet function , but there are insufficient data regarding the effect of high - dose aspirin on the prevention of cardiovascular events at this time ( 12 ) . combination therapy with aspirin and other antiplatelet agents , such as clopidogrel , suppresses platelet aggregation more strongly ( 19 ) , but the benefit of dual antiplatelet therapy on primary prevention is also inconclusive ( 21 ) . the differing prevalence of other cardiovascular risk factors among patients in the various diabetic treatment groups might alter the low - dose aspirin effect on primary prevention of atherosclerotic events . in the diet - alone group , a high prevalence of hypertension ( 72% ) and dyslipidemia ( 56% ) , high age ( mean age , 65 years ) , mild overweight ( mean bmi , 25 kg / m ) , and mild glucose intolerance ( mean levels of hba1c and fpg , 6.7% and 7.22 mmol / l ) might indicate that these patients could be considered to have multiple cardiovascular risk factors rather than overt diabetes . in fact , previous clinical trials of primary prevention in patients with multiple cardiovascular risk factors showed that low - dose aspirin reduced cardiovascular events ( 57 ) , whereas no trial provided a definitive conclusion regarding patients with diabetes ( 911 ) . the primary prevention project ( ppp ) trial reported a difference in the effect of low - dose aspirin on primary prevention between patients with and without overt diabetes ( 22 ) . the ppp trial enrolled 4,495 patients with at least one major cardiovascular risk factor , including 1,031 patients with overt diabetes ( fpg 7.8 this trial showed that low - dose aspirin lowered the incidence of cardiovascular events by 33% in total ( 7 ) ; however , in the diabetic group , low - dose aspirin did not reduce cardiovascular events despite 50% higher event rates ( 22 ) . these reports might confirm that low - dose aspirin was beneficial in the diet - alone group , as a subset of patients in the early clinical stages of diabetes and with comorbid other cardiovascular risk factors . recent meta - analyses , including data from the jpad trial , reported that low - dose aspirin is not effective in the primary prevention of cardiovascular events in patients with diabetes ( 9,23 ) . patients treated with insulin are at high risk for cardiovascular events ; however , our findings indicated that low - dose aspirin reduced atherosclerotic events not in the insulin group but in the diet - alone group . these results suggest that low - dose aspirin therapy is most beneficial in early clinical stages of diabetes . when clinicians use low - dose aspirin in diabetic patients to prevent cardiovascular events , they should take into account not only conventional cardiovascular risk factors but also the clinical stage of diabetes . further studies are needed to assess the effect of low - dose aspirin at each clinical stage of diabetes . our project is the first report to estimate the effect of low - dose aspirin on the primary prevention of atherosclerotic events in groups undergoing various diabetes management strategies . our results suggest that the clinical impact of low - dose aspirin depends on the clinical stage of diabetes . however , this subanalysis has several limitations in addition to those reported in the main jpad report ( 10 ) . such changes could be especially frequent in the diet - alone and oha groups as they begin to require more intensive treatment , and as a result the subgrouping might not be valid . second , the small number of patients in each treatment group , especially in the insulin group , limited the certainty with which we could formulate conclusions regarding aspirin s effect . larger studies are needed for definitive evaluation of the effect of aspirin in patients treated with insulin . third , several ohas are reported to be beneficial for preventing cardiovascular events ( e.g. , metformin and pioglitazone ) , but we analyzed the oha group without taking specific drug properties into account . since most patients ( 81% ) in the oha group took sulfonylurea at baseline , we could not assess the single - agent effects of the cardioprotective ohas . finally , the number of hemorrhagic events was very low in both the aspirin and no - aspirin groups ; however , the sample sizes were too small to estimate aspirin s side effects in the jpad trial . therefore , we could not make firm conclusions about the safety of low - dose aspirin based on our results . our project is the first report to estimate the effect of low - dose aspirin on the primary prevention of atherosclerotic events in groups undergoing various diabetes management strategies . our results suggest that the clinical impact of low - dose aspirin depends on the clinical stage of diabetes . however , this subanalysis has several limitations in addition to those reported in the main jpad report ( 10 ) . such changes could be especially frequent in the diet - alone and oha groups as they begin to require more intensive treatment , and as a result the subgrouping might not be valid . second , the small number of patients in each treatment group , especially in the insulin group , limited the certainty with which we could formulate conclusions regarding aspirin s effect . larger studies are needed for definitive evaluation of the effect of aspirin in patients treated with insulin . third , several ohas are reported to be beneficial for preventing cardiovascular events ( e.g. , metformin and pioglitazone ) , but we analyzed the oha group without taking specific drug properties into account . since most patients ( 81% ) in the oha group took sulfonylurea at baseline , we could not assess the single - agent effects of the cardioprotective ohas . finally , the number of hemorrhagic events was very low in both the aspirin and no - aspirin groups ; however , the sample sizes were too small to estimate aspirin s side effects in the jpad trial . therefore , we could not make firm conclusions about the safety of low - dose aspirin based on our results .

objectiverecent reports showed that low - dose aspirin was ineffective in the primary prevention of cardiovascular events in diabetic patients overall . we hypothesized that low - dose aspirin would be beneficial in patients receiving insulin therapy , as a high - risk group.research design and methodsthis study is a subanalysis of the japanese primary prevention of atherosclerosis with aspirin for diabetes ( jpad ) trial a randomized , controlled , open - label trial . we randomly assigned 2,539 patients with type 2 diabetes and no previous cardiovascular disease to the low - dose aspirin group ( 81 or 100 mg daily ) or to the no - aspirin group . the median follow - up period was 4.4 years . we investigated the effect of low - dose aspirin on preventing atherosclerotic events in groups receiving different diabetes management.resultsat baseline , 326 patients were treated with insulin , 1,750 with oral hypoglycemic agents ( ohas ) , and 463 with diet alone . the insulin group had the longest history of diabetes , the worst glycemic control , and the highest prevalence of diabetic microangiopathies . the diet - alone group had the opposite characteristics . the incidence of atherosclerotic events was 26.6 , 14.6 , and 10.4 cases per 1,000 person - years in the insulin , oha , and diet - alone groups , respectively . in the insulin and oha groups , low - dose aspirin did not affect atherosclerotic events ( insulin : hazard ratio [ hr ] 1.19 [ 95% ci 0.602.40 ] ; oha : hr 0.84 [ 0.571.24 ] ) . in the diet - alone group , low - dose aspirin significantly reduced atherosclerotic events , despite the lowest event rates ( hr 0.21 [ 0.050.64]).conclusionslow - dose aspirin reduced atherosclerotic events predominantly in the diet - alone group and not in the insulin or oha groups .

RESEARCH DESIGN AND METHODS Study design Definition of atherosclerotic events Presentation of hemoglobin A Statistical analyses RESULTS Baseline clinical characteristics Atherosclerotic events in each diabetes management Efficacy of low-dose aspirin in each diabetes management Hemorrhagic events CONCLUSIONS Study strengths and limitations Supplementary Material

in brief , we enrolled 2,539 patients , aged between 30 and 85 years , with type 2 diabetes and no history of cardiovascular disease . the participants were randomly assigned to the aspirin group or the no - aspirin group . the 1,277 patients in the no - aspirin group were allowed to use antiplatelet therapy , including aspirin , if needed . in this subanalysis , we classified all participants into three subgroups according to the diabetes management at baseline : insulin , oha , or diet alone . the primary end point was the occurrence during the follow - up period of any atherosclerotic event , which was a composite of the following : death from coronary , cerebrovascular , and aortic causes ; nonfatal acute myocardial infarction ; unstable angina ; newly developed exertional angina ; nonfatal ischemic and hemorrhagic stroke ; transient ischemic attack ; and nonfatal aortic and peripheral vascular disease . efficacy comparisons were performed on the basis of time to the first event according to the intention - to - treat principle , with all patients included in the group to which they were randomized and with patients lost to follow - up censored at the day of the last visit . we also developed multivariable cox proportional hazard models to assess the effects of aspirin on atherosclerotic events adjusting for age ( 65 years ) , hypertension , dyslipidemia , and history of smoking to evaluate the robustness . in brief , we enrolled 2,539 patients , aged between 30 and 85 years , with type 2 diabetes and no history of cardiovascular disease . the participants were randomly assigned to the aspirin group or the no - aspirin group . the 1,277 patients in the no - aspirin group were allowed to use antiplatelet therapy , including aspirin , if needed . in this subanalysis , we classified all participants into three subgroups according to the diabetes management at baseline : insulin , oha , or diet alone . the primary end point was the occurrence during the follow - up period of any atherosclerotic event , which was a composite of the following : death from coronary , cerebrovascular , and aortic causes ; nonfatal acute myocardial infarction ; unstable angina ; newly developed exertional angina ; nonfatal ischemic and hemorrhagic stroke ; transient ischemic attack ; and nonfatal aortic and peripheral vascular disease . we also developed multivariable cox proportional hazard models to assess the effects of aspirin on atherosclerotic events adjusting for age ( 65 years ) , hypertension , dyslipidemia , and history of smoking to evaluate the robustness . at baseline , 326 patients were treated with insulin , 1,750 with ohas , and 463 with diet alone ( table 1 ) . mean bmi was lowest in the insulin group ( 23 kg / m ) and highest in the diet - alone group ( 25 kg / m ) . median duration of diabetes was longest in the insulin group : 13.0 years in the insulin group , 7.2 years in the oha group , and 3.5 years in the diet - alone group . the prevalence of diabetic microangiopathies was highest in the insulin group ( retinopathy , 43% ; nephropathy , 20% ; neuropathy , 32% ) and lowest in the diet - alone group ( retinopathy , 4% ; nephropathy , 8% ; neuropathy , 4% ) . the prevalence of hypertension and dyslipidemia was significantly higher in the diet - alone group ( hypertension , 72% ; dyslipidemia , 56% ) than in the other two groups . the use of statins was low in all groups : 18 , 27 , and 24% in the insulin , oha , and diet - alone groups , respectively . the incidence of atherosclerotic events was 26.6 , 14.6 , and 10.4 cases per 1,000 person - years in the insulin , oha , and diet - alone groups , respectively ( supplementary table 1 ) . both coronary artery and cerebrovascular events occurred most frequently in the insulin group ( coronary artery events , 12.1 ; cerebrovascular events , 12.1 cases per 1,000 person - years ) . there was a low incidence of peripheral vascular events among the three groups ( insulin , 2.4 ; oha , 1.7 ; diet alone , 1.6 cases per 1,000 person - years ) . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group than those in the other groups ( log - rank test , p = 0.0013 ; fig . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group ( log - rank test , p = 0.0013 ) . participants were randomly assigned to the aspirin group or the no - aspirin group ( supplementary table 2 ) . in the insulin group , low - dose aspirin did not affect the incidence of atherosclerotic events ( hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; fig . similar results were observed in the oha group ( hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ; fig . , low - dose aspirin significantly reduced atherosclerotic events despite the fact that this group had the lowest atherosclerotic event rate ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ; fig . adjusting for age , hypertension , dyslipidemia , and history of smoking , low - dose aspirin significantly reduced atherosclerotic events in the diet - alone group ( hr 0.20 [ 0.060.68 ] ; log - rank test , p = 0.0099 ) but not in the insulin or oha groups ( insulin : hr 1.0 [ 0.502.00 ] , log - rank test , p = 1.0 ; oha : hr 0.77 [ 0.521.14 ] , log - rank test , p = 0.20 ) . efficacy of low - dose aspirin on primary prevention of atherosclerotic events in each diabetes management . a and b : in the insulin ( a ) and oha ( b ) groups , low - dose aspirin did not affect the incidence of atherosclerotic events ( insulin : hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; oha : hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ) . c : in the diet - alone group , low - dose aspirin significantly reduced atherosclerotic events despite the lowest event rates ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ) . the number of gastrointestinal bleeding was one in the insulin group ( this patient took aspirin ) and seven in the oha group ( four in the aspirin group and three in the no - aspirin group ) . in the diet - alone group , no patients suffered from gastrointestinal bleeding . the number of hemorrhagic stroke was two in the insulin group ( one in the aspirin group and one in the no - aspirin group ) , nine in the oha group ( five in the aspirin group and four in the no - aspirin group ) , and two in the diet - alone group ( all in the no - aspirin group ) . at baseline , 326 patients were treated with insulin , 1,750 with ohas , and 463 with diet alone ( table 1 ) . mean bmi was lowest in the insulin group ( 23 kg / m ) and highest in the diet - alone group ( 25 kg / m ) . median duration of diabetes was longest in the insulin group : 13.0 years in the insulin group , 7.2 years in the oha group , and 3.5 years in the diet - alone group . the prevalence of diabetic microangiopathies was highest in the insulin group ( retinopathy , 43% ; nephropathy , 20% ; neuropathy , 32% ) and lowest in the diet - alone group ( retinopathy , 4% ; nephropathy , 8% ; neuropathy , 4% ) . the prevalence of hypertension and dyslipidemia was significantly higher in the diet - alone group ( hypertension , 72% ; dyslipidemia , 56% ) than in the other two groups . the use of statins was low in all groups : 18 , 27 , and 24% in the insulin , oha , and diet - alone groups , respectively . the incidence of atherosclerotic events was 26.6 , 14.6 , and 10.4 cases per 1,000 person - years in the insulin , oha , and diet - alone groups , respectively ( supplementary table 1 ) . both coronary artery and cerebrovascular events occurred most frequently in the insulin group ( coronary artery events , 12.1 ; cerebrovascular events , 12.1 cases per 1,000 person - years ) . there was a low incidence of peripheral vascular events among the three groups ( insulin , 2.4 ; oha , 1.7 ; diet alone , 1.6 cases per 1,000 person - years ) . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group than those in the other groups ( log - rank test , p = 0.0013 ; fig . survival analysis showed that the rate of atherosclerotic events was significantly higher in the insulin group ( log - rank test , p = 0.0013 ) . participants were randomly assigned to the aspirin group or the no - aspirin group ( supplementary table 2 ) . in the insulin group , low - dose aspirin did not affect the incidence of atherosclerotic events ( hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; fig . similar results were observed in the oha group ( hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ; fig . , low - dose aspirin significantly reduced atherosclerotic events despite the fact that this group had the lowest atherosclerotic event rate ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ; fig . adjusting for age , hypertension , dyslipidemia , and history of smoking , low - dose aspirin significantly reduced atherosclerotic events in the diet - alone group ( hr 0.20 [ 0.060.68 ] ; log - rank test , p = 0.0099 ) but not in the insulin or oha groups ( insulin : hr 1.0 [ 0.502.00 ] , log - rank test , p = 1.0 ; oha : hr 0.77 [ 0.521.14 ] , log - rank test , p = 0.20 ) . efficacy of low - dose aspirin on primary prevention of atherosclerotic events in each diabetes management . a and b : in the insulin ( a ) and oha ( b ) groups , low - dose aspirin did not affect the incidence of atherosclerotic events ( insulin : hr 1.19 [ 95% ci 0.602.40 ] ; log - rank test , p = 0.61 ; oha : hr 0.84 [ 0.571.24 ] ; log - rank test , p = 0.38 ) . c : in the diet - alone group , low - dose aspirin significantly reduced atherosclerotic events despite the lowest event rates ( hr 0.21 [ 0.050.64 ] ; log - rank test , p = 0.0069 ) . the number of gastrointestinal bleeding was one in the insulin group ( this patient took aspirin ) and seven in the oha group ( four in the aspirin group and three in the no - aspirin group ) . in the diet - alone group , no patients suffered from gastrointestinal bleeding . the number of hemorrhagic stroke was two in the insulin group ( one in the aspirin group and one in the no - aspirin group ) , nine in the oha group ( five in the aspirin group and four in the no - aspirin group ) , and two in the diet - alone group ( all in the no - aspirin group ) . the major findings of this subanalysis were as follows : japanese patients with type 2 diabetes receiving insulin therapy had a high incidence of atherosclerotic events and low - dose aspirin reduced atherosclerotic events not in patients receiving insulin therapy but in patients treated with diet alone . the insulin group was characterized by the youngest mean age ( 62 years ) and the longest median duration of diabetes ( 13.0 years ) , which meant that patients in the insulin group developed diabetes at the youngest age among the three groups . in contrast , patients in the diet - alone group developed diabetes in recent years and at older age . the glycemic control was worst in the insulin group ( mean levels of hba1c and fpg , 8.1% and 8.77 mmol / l ) and best in the diet - alone group ( mean levels of hba1c and fpg , 6.7% and 7.22 the prevalence of diabetic microangiopathies was highest in the insulin group ( 20% ) and lowest in the diet alone group ( < 10% ) . thus we could regard the insulin group as demonstrating an advanced clinical stage of diabetes and the diet - alone group as demonstrating a less advanced or earlier clinical stage of diabetes than the insulin and oha groups . our results might indicate that low - dose aspirin was most beneficial in early clinical stages of diabetes , despite the lowest event rates . recently , we reported that a subanalysis of the jpad trial showed a difference in low - dose aspirin effect on primary prevention of atherosclerotic events in renal function ( 16 ) . in the study , reduced renal function in diabetic patients was significantly associated with high incidence of atherosclerotic events . low - dose aspirin did not prevent atherosclerotic events in patients with an estimated glomerular filtration rate ( egfr ) < 60 ml / min/1.73 m ( hr 1.3 [ 95% ci 0.762.4 ] ) . on the other hand , in the patients with an egfr of 60 to 89 ml / min/1.73 m , low - dose aspirin significantly reduced atherosclerotic events , despite low event rates ( hr 0.57 [ 0.360.88 ] ) ( 16 ) . as in the current study , low - dose aspirin was not beneficial in patients with advanced stages of renal dysfunction and with high event rates but rather in those with early stages of renal dysfunction and with low event rates . these results suggest that progression of diabetes and diabetes complications ( renal dysfunction ) might attenuate the effect of low - dose aspirin . the current recommendation of the ada , aha , and accf regarding the preventative use of aspirin in high - risk diabetic patients is based on the number of cardiovascular events prevented by low - dose aspirin ( relative risk reduction of 10% ) being greater than the number of hemorrhagic events ( 15 cases per 1,000 person - years ) ( 12 ) . because diabetic patients with higher cardiovascular risk should have greater absolute benefit from low - dose aspirin , our findings were unexpected and inconsistent with the recommendation . to confirm whether low - dose aspirin is really effective in high - risk or low - risk diabetic patients , one possible mechanism of our findings is that events in patients with advanced clinical stages of diabetes are unpreventable because of overly advanced atherosclerotic lesions . in our study , poor glycemic control in the insulin group aspirin dosage may improve antiplatelet function , but there are insufficient data regarding the effect of high - dose aspirin on the prevention of cardiovascular events at this time ( 12 ) . the differing prevalence of other cardiovascular risk factors among patients in the various diabetic treatment groups might alter the low - dose aspirin effect on primary prevention of atherosclerotic events . in the diet - alone group , a high prevalence of hypertension ( 72% ) and dyslipidemia ( 56% ) , high age ( mean age , 65 years ) , mild overweight ( mean bmi , 25 kg / m ) , and mild glucose intolerance ( mean levels of hba1c and fpg , 6.7% and 7.22 mmol / l ) might indicate that these patients could be considered to have multiple cardiovascular risk factors rather than overt diabetes . in fact , previous clinical trials of primary prevention in patients with multiple cardiovascular risk factors showed that low - dose aspirin reduced cardiovascular events ( 57 ) , whereas no trial provided a definitive conclusion regarding patients with diabetes ( 911 ) . the primary prevention project ( ppp ) trial reported a difference in the effect of low - dose aspirin on primary prevention between patients with and without overt diabetes ( 22 ) . the ppp trial enrolled 4,495 patients with at least one major cardiovascular risk factor , including 1,031 patients with overt diabetes ( fpg 7.8 this trial showed that low - dose aspirin lowered the incidence of cardiovascular events by 33% in total ( 7 ) ; however , in the diabetic group , low - dose aspirin did not reduce cardiovascular events despite 50% higher event rates ( 22 ) . these reports might confirm that low - dose aspirin was beneficial in the diet - alone group , as a subset of patients in the early clinical stages of diabetes and with comorbid other cardiovascular risk factors . recent meta - analyses , including data from the jpad trial , reported that low - dose aspirin is not effective in the primary prevention of cardiovascular events in patients with diabetes ( 9,23 ) . patients treated with insulin are at high risk for cardiovascular events ; however , our findings indicated that low - dose aspirin reduced atherosclerotic events not in the insulin group but in the diet - alone group . these results suggest that low - dose aspirin therapy is most beneficial in early clinical stages of diabetes . when clinicians use low - dose aspirin in diabetic patients to prevent cardiovascular events , they should take into account not only conventional cardiovascular risk factors but also the clinical stage of diabetes . further studies are needed to assess the effect of low - dose aspirin at each clinical stage of diabetes . our project is the first report to estimate the effect of low - dose aspirin on the primary prevention of atherosclerotic events in groups undergoing various diabetes management strategies . such changes could be especially frequent in the diet - alone and oha groups as they begin to require more intensive treatment , and as a result the subgrouping might not be valid . second , the small number of patients in each treatment group , especially in the insulin group , limited the certainty with which we could formulate conclusions regarding aspirin s effect . larger studies are needed for definitive evaluation of the effect of aspirin in patients treated with insulin . finally , the number of hemorrhagic events was very low in both the aspirin and no - aspirin groups ; however , the sample sizes were too small to estimate aspirin s side effects in the jpad trial . our project is the first report to estimate the effect of low - dose aspirin on the primary prevention of atherosclerotic events in groups undergoing various diabetes management strategies . our results suggest that the clinical impact of low - dose aspirin depends on the clinical stage of diabetes . such changes could be especially frequent in the diet - alone and oha groups as they begin to require more intensive treatment , and as a result the subgrouping might not be valid . larger studies are needed for definitive evaluation of the effect of aspirin in patients treated with insulin . finally , the number of hemorrhagic events was very low in both the aspirin and no - aspirin groups ; however , the sample sizes were too small to estimate aspirin s side effects in the jpad trial .

recent data suggest that approximately 1 110 000 men and women in the united states have a history of cancer of the colon and rectum . colectomy , often used to treat colorectal cancer , can be performed by various techniques . an open approach is the most frequent , but laparoscopic techniques are also used and well accepted . the rate of laparoscopic techniques is increasing particularly in urban centers , in which laparoscopic colectomies are performed at a higher rate than in other settings . within the large premier hospital database , from 2009 through the second quarter of 2011 , approximately one - third of segmental colectomies ( cecectomy , right hemicolectomy , left hemicolectomy , and sigmoidectomy ) were identified as having a laparoscopic procedural code . laparoscopic techniques have minimized the perioperative morbidity associated with many types of surgery , including colectomy . several prospective randomized trials have shown that laparoscopic colectomy has equivalent oncologic outcomes to the traditional open surgical approach . additional advantages with regard to pain , blood loss , return of bowel function , length of hospitalization , and overall recovery time have been shown . in addition , resource use is lower for laparoscopic colectomy , including reduced length of stay , fewer readmissions , and less use of skilled nursing facilities . robotic - assisted surgery is an emerging approach in the field of laparoscopic colorectal surgery . currently , there is only one commercially available robotic device cleared by the us food and drug administration for laparoscopic procedures ( da vinci surgical system ; intuitive surgical , sunnyvale , california ) . several authors have published their experiences and case series related to robotic - assisted laparoscopic colectomy . although no specific large randomized controlled trials have evaluated robotic - assisted versus traditional laparoscopic colectomies , clinical outcomes suggest that robotic - assisted laparoscopic surgery is equivalent to conventional laparoscopy when considering important endpoints such as conversion to open surgery , hospital stay , and recovery time . in this era of comparative effectiveness and health care reform in the united states , and with concerns about optimal resource utilization at the forefront , the use of robotic - assisted laparoscopic surgery deserves further evaluation . given this background , this study examined clinical and economic outcomes ( cost and utilization ) in patients undergoing laparoscopic colectomy performed with and without robotic assistance . this database contains complete patient billing , hospital cost , and coding histories from more than 600 health care facilities throughout the united states . the data from which this study was derived were extracted from more than 25 million inpatient discharges and 175 million hospital outpatient visits from acute care facilities , ambulatory surgery centers , and clinics across the nation . a protocol describing the analysis objectives , criteria for patient selection , data elements of interest , and statistical methods was submitted to the new england institutional review board , and exemption was obtained . eligible patients were aged 18 years and had undergone a laparoscopic colectomy during the period from 2009 to the second quarter of 2011 . patients were categorized according to the following 4 types of laparoscopic segmental colectomies : laparoscopic cecectomy ( 17.32 ) , laparoscopic right hemicolectomy ( 17.33 ) , laparoscopic left hemicolectomy ( 17.35 ) , and laparoscopic sigmoidectomy ( 17.36 ) . these procedures were chosen because the laparoscopic approach has been shown to have equivalent oncologic outcomes with documented perioperative morbidity benefits compared with open surgery . other procedures , such as low anterior resection , where traditional laparoscopy has not yet been established to be equivalent to open surgery , were not included . laparoscopic colectomy procedures using robotic technology were identified if one of two conditions was met : ( 1 ) a robotic international classification of diseases , ninth edition ( icd-9 ) procedure code accompanied the primary procedure code of interest or ( 2 ) text fields were found when mining the hospital charge master file for each patient indicating use of the robot . for all eligible patients , elements describing hospital cost , surgery time , length of stay , use of the robot , colectomy type , and indication for colectomy were obtained from the data . cost analysis ( calculation ) reflected the cost to the hospital for the colectomy procedures but did not include capital costs . this analysis was limited to the total cost per patient episode and did not break out costs at the level of disposables , operating room time , or other patient care costs . the preoperative all patient refined diagnosis related groups severity level was used as an index of comorbidity . the 3 m all patient refined diagnosis related groups classification system is a widely adopted proprietary risk - adjustment classification tool that uses information from routine claims data to produce valid and reliable severity measurement and risk - adjustment scores . it is used to account for differences related to an individual 's severity of illness or risk of death in large datasets . comorbid conditions that might influence procedure selection or outcomes of interest , such as the presence of cardiovascular or pulmonary disease , cancer , or diabetes mellitus , were obtained by use of icd-9 diagnosis codes . comorbid conditions were grouped into 11 categories based on the medical expenditure panel survey data lists : arthritis , malignant neoplasms , mental disorders , metabolic diseases , diseases of the digestive system , diseases of the genitourinary system , diseases of the circulatory system , diseases of the musculoskeletal system and connective tissue , diseases of the nervous system , diseases of the respiratory system , and diseases of the cutaneous system . appendix a provides a detailed listing of all icd-9 codes for each condition within each category . information on sociodemographic characteristics and health insurance status was also included , as were descriptors of the care setting , namely census region , urban or rural setting , teaching hospital status , and facility bed count . adverse events identified by icd-9 codes that occurred intraoperatively and 30 days postoperatively , which included pulmonary , cardiac , vascular , neurologic , and other , were flagged and included in the analysis . the other category encompassed shock and perforations or fistulae of organs or vessels not included in the aforementioned organ systems . minor and major bleeding was categorized by icd-9 diagnosis as well as procedure codes related to hemorrhage and transfusions . a detailed list of each event and the corresponding icd-9 code is found in appendix b. each specific adverse event identified by icd-9 code was organized as either major or minor categories based on clinical experience . these were then evaluated and characterized based on whether they were related to the surgical technique ( bleeding , abscess , wound infection , and so on ) . information on adverse events among matched data by analysis groups appears in appendix b. stoma procedures were identified and treated separately from the complications ( appendix c ) . because of limitations of the dataset , it could not be determined whether these were planned stomas or due to a complication . the study objective was to use the premier hospital database to compare clinical and economic outcomes in patients undergoing laparoscopic colectomy with and without the use of robotic assistance . outcomes of interest included adverse events ( minor , major , and surgical ) , whether a stoma was performed , hospital costs , length of stay , and surgery time . a quasi - randomization method for limiting bias called propensity scoring was used to create groups of analyzable patients who were well matched . covariates on which to match were selected based on their availability in the premier hospital database , as well as their general acceptance as factors associated with the outcomes of interest . the goal of this propensity matching analysis was to find pairs of patients receiving and not receiving a robotic laparoscopic colectomy who share like propensities for candidacy for the procedure based on the matching variables . nearest - neighbor matching on the estimated propensity scores to choose matches for the patients who had a robotic procedure . propensity scores were calculated for receipt of robotic procedures for each of the patients included in the analysis based on a nonparsimonious multivariable logistic regression model . patients were matched on the following 13 characteristics : age , gender , race , insurance type , primary icd-9 procedure code , region of facility , urban versus nonurban classification of facility , teaching status of facility , number of beds at facility , and presence or absence of 4 comorbid conditions that were shown to be statistically significant before matching the robotic and nonrobotic patients were randomly ordered , and the nonrobotic patient with the propensity score closest to that of the first robotic patient was chosen . assessment of residual bias was conducted by evaluating the differences in the distribution of patient characteristics before and after matching . to assess the extent to which the propensity matching reduced confounders , the distributions of several variables before and after matching were compared including age , gender , race , insurance type , health status , region , location , facility type , primary icd-9 procedure code , comorbid conditions , and cancer versus noncancer based on the top 10 most frequently occurring icd-9 diagnosis codes among the patients in the cohorts . group comparisons were made by use of t tests and tests after confirmation of approximately symmetric distribution of the variables and comparable variability before and after the match . we used t tests to test for differences between the matched cohorts in the 3 continuous variables of interest : hospital cost , surgery time , and length of stay . logistic regression models were used to test for significant differences between the two groups and to generate odds ratios on the following categories of adverse events and complications : major , minor , and surgical and whether the patient also received a stoma . residuals and akaike information criterion were checked for goodness of fit of the logistic regression models . analyses were performed with sas , version 9.2 ( sas institute , cary , north carolina ) . this database contains complete patient billing , hospital cost , and coding histories from more than 600 health care facilities throughout the united states . the data from which this study was derived were extracted from more than 25 million inpatient discharges and 175 million hospital outpatient visits from acute care facilities , ambulatory surgery centers , and clinics across the nation . a protocol describing the analysis objectives , criteria for patient selection , data elements of interest , and statistical methods was submitted to the new england institutional review board , and exemption was obtained . eligible patients were aged 18 years and had undergone a laparoscopic colectomy during the period from 2009 to the second quarter of 2011 . patients were categorized according to the following 4 types of laparoscopic segmental colectomies : laparoscopic cecectomy ( 17.32 ) , laparoscopic right hemicolectomy ( 17.33 ) , laparoscopic left hemicolectomy ( 17.35 ) , and laparoscopic sigmoidectomy ( 17.36 ) . these procedures were chosen because the laparoscopic approach has been shown to have equivalent oncologic outcomes with documented perioperative morbidity benefits compared with open surgery . other procedures , such as low anterior resection , where traditional laparoscopy has not yet been established to be equivalent to open surgery , were not included . laparoscopic colectomy procedures using robotic technology were identified if one of two conditions was met : ( 1 ) a robotic international classification of diseases , ninth edition ( icd-9 ) procedure code accompanied the primary procedure code of interest or ( 2 ) text fields were found when mining the hospital charge master file for each patient indicating use of the robot . for all eligible patients , elements describing hospital cost , surgery time , length of stay , use of the robot , colectomy type , and indication for colectomy were obtained from the data . cost analysis ( calculation ) reflected the cost to the hospital for the colectomy procedures but did not include capital costs . this analysis was limited to the total cost per patient episode and did not break out costs at the level of disposables , operating room time , or other patient care costs . the preoperative all patient refined diagnosis related groups severity level was used as an index of comorbidity . the 3 m all patient refined diagnosis related groups classification system is a widely adopted proprietary risk - adjustment classification tool that uses information from routine claims data to produce valid and reliable severity measurement and risk - adjustment scores . it is used to account for differences related to an individual 's severity of illness or risk of death in large datasets . comorbid conditions that might influence procedure selection or outcomes of interest , such as the presence of cardiovascular or pulmonary disease , cancer , or diabetes mellitus , were obtained by use of icd-9 diagnosis codes . comorbid conditions were grouped into 11 categories based on the medical expenditure panel survey data lists : arthritis , malignant neoplasms , mental disorders , metabolic diseases , diseases of the digestive system , diseases of the genitourinary system , diseases of the circulatory system , diseases of the musculoskeletal system and connective tissue , diseases of the nervous system , diseases of the respiratory system , and diseases of the cutaneous system . appendix a provides a detailed listing of all icd-9 codes for each condition within each category . information on sociodemographic characteristics and health insurance status was also included , as were descriptors of the care setting , namely census region , urban or rural setting , teaching hospital status , and facility bed count . adverse events identified by icd-9 codes that occurred intraoperatively and 30 days postoperatively , which included pulmonary , cardiac , vascular , neurologic , and other , were flagged and included in the analysis . the other category encompassed shock and perforations or fistulae of organs or vessels not included in the aforementioned organ systems . minor and major bleeding was categorized by icd-9 diagnosis as well as procedure codes related to hemorrhage and transfusions . a detailed list of each event and the corresponding icd-9 code is found in appendix b. each specific adverse event identified by icd-9 code was organized as either major or minor categories based on clinical experience . these were then evaluated and characterized based on whether they were related to the surgical technique ( bleeding , abscess , wound infection , and so on ) . information on adverse events among matched data by analysis groups appears in appendix b. stoma procedures were identified and treated separately from the complications ( appendix c ) . because of limitations of the dataset , it could not be determined whether these were planned stomas or due to a complication . the study objective was to use the premier hospital database to compare clinical and economic outcomes in patients undergoing laparoscopic colectomy with and without the use of robotic assistance . outcomes of interest included adverse events ( minor , major , and surgical ) , whether a stoma was performed , hospital costs , length of stay , and surgery time . a quasi - randomization method for limiting bias called propensity scoring was used to create groups of analyzable patients who were well matched . covariates on which to match were selected based on their availability in the premier hospital database , as well as their general acceptance as factors associated with the outcomes of interest . the goal of this propensity matching analysis was to find pairs of patients receiving and not receiving a robotic laparoscopic colectomy who share like propensities for candidacy for the procedure based on the matching variables . nearest - neighbor matching on the estimated propensity scores to choose matches for the patients who had a robotic procedure . propensity scores were calculated for receipt of robotic procedures for each of the patients included in the analysis based on a nonparsimonious multivariable logistic regression model . patients were matched on the following 13 characteristics : age , gender , race , insurance type , primary icd-9 procedure code , region of facility , urban versus nonurban classification of facility , teaching status of facility , number of beds at facility , and presence or absence of 4 comorbid conditions that were shown to be statistically significant before matching the robotic and nonrobotic patients were randomly ordered , and the nonrobotic patient with the propensity score closest to that of the first robotic patient was chosen . assessment of residual bias was conducted by evaluating the differences in the distribution of patient characteristics before and after matching . to assess the extent to which the propensity matching reduced confounders , the distributions of several variables before and after matching were compared including age , gender , race , insurance type , health status , region , location , facility type , primary icd-9 procedure code , comorbid conditions , and cancer versus noncancer based on the top 10 most frequently occurring icd-9 diagnosis codes among the patients in the cohorts . group comparisons were made by use of t tests and tests after confirmation of approximately symmetric distribution of the variables and comparable variability before and after the match . we used t tests to test for differences between the matched cohorts in the 3 continuous variables of interest : hospital cost , surgery time , and length of stay . logistic regression models were used to test for significant differences between the two groups and to generate odds ratios on the following categories of adverse events and complications : major , minor , and surgical and whether the patient also received a stoma . residuals and akaike information criterion were checked for goodness of fit of the logistic regression models . analyses were performed with sas , version 9.2 ( sas institute , cary , north carolina ) . ninety - eight percent of all laparoscopic colectomies included in this analysis were performed without the use of robotic assistance ( n = 25 210 ) . robotic assistance was used in 548 procedures , or approximately 2% of the total colectomies . the procedural breakdown was as follows : laparoscopic cecectomy , 12 ; right hemicolectomy , 203 ; left hemicolectomy , 42 ; and sigmoidectomy , 291 ( table 2 ) . apr - drg = all patient refined diagnosis related groups neop , ub = neoplasm , uncertain behavior . before matching , distributions were similar for age , gender , insurance , and most primary diagnosis codes for patients in both groups ( table 2 ) . furthermore , few differences in comorbidities or illness severity index were noted between the robotic and nonrobotic groups . the characteristics of the 364 hospitals with colectomy procedures were similar with regard to census region and location ( urban vs rural ) . there were notable differences , however , in teaching versus nonteaching and bed count , with most robotic procedures being performed in teaching hospitals with > 200 beds , as compared with nonrobotic procedures , with the majority coming from nonteaching hospitals with greater variation in bed size ( table 3 ) . patient characteristics , comorbid conditions , and hospital characteristics after matching are represented in table 4 . after matching , patients were balanced with respect to demographics , comorbid conditions , and hospital characteristics , with the exception of hospital location ( urban vs nonurban ) , which was statistically significantly different between the two groups ( p = .017 ) . hospital demographics based on patient counts apr - drg = all patient refined diagnosis related groups ; copd = chronic obstructive pulmonary disease ; gerd = gastroesophageal reflux disease ; mi = myocardial infarction ; na = not applicable . after matching , clinical endpoints and adverse events occurring in the postoperative period 30 days after discharge were tabulated and grouped into 4 categories : major , minor , surgical , and stoma related . complications ( major , minor , and surgical ) and stoma procedures were not significantly different between the robotic and nonrobotic surgery cohorts , regardless of whether they were examined within a perioperative 30-day period or only within the original perioperative hospital stay ( table 6 ) . hospital costs , surgery time , and length of stay after matching adverse events after matching ci = confidence interval . major : acute respiratory failure , spontaneous tension pneumothorax , atelectasis / pulmonary collapse , empyema , bronchopleural fistula , air leak and other pneumothorax , chylothorax , pneumonia , other pulmonary infections and inflammation , acute myocardial infarction , acute heart failure / pulmonary edema , acute pulmonary embolism / infarction , acute deep venous thrombosis of extremities , acute cerebrovascular accident ( stroke ) , transient cerebral ischemia / transient ischemic attack , intracranial hemorrhage ( includes hemorrhagic stroke ) , dehiscence , perforations of organ or vessels , in - hospital death , sepsis , other postoperative complications , accidental puncture or laceration during procedure , other postoperative infection , peritoneal abscess , other retroperitoneal abscess , abscess of intestine , fistula of intestine , excluding rectum and anus , ureteral fistula , intestinoureteral fistula , intestinovesical fistula , digestive genital tract fistula , female , persistent postoperative fistula , other specified intestinal obstruction , unspecified intestinal obstruction , intestinal or peritoneal adhesions with obstruction ( postoperative ) , peritonitis ( acute ) , generalized , other suppurative peritonitis , other retroperitoneal infections , unspecified peritonitis , iatrogenic pulmonary embolism and infarction . minor : hematoma / seroma complicating procedure , cellulitis , other postoperative infection , including other ( non - cellulitis ) wound infection , other digestive system complications , paralytic ileus , perioperative autologous transfusion of whole blood or blood components , transfusion of previously collected autologous blood , other transfusion of whole blood , transfusion of packed cells , hemorrhage complicating procedure , hematoma complicating procedure . surgical : chylothorax , dehiscence , hematoma / seroma complicating procedure , cellulitis , other postoperative infection , including other ( non - cellulitis ) wound infection , perforations of organ or vessels , in - hospital death , sepsis , other postoperative complications , other digestive system complications , paralytic ileus , accidental puncture or laceration during procedure , other postoperative infection , peritoneal abscess , other retroperitoneal abscess , abscess of intestine , fistula of intestine , excluding rectum and anus , ureteral fistula , intestinoureteral fistula , intestinovesical fistula , digestive genital tract fistula , female , persistent postoperative fistula , other specified intestinal obstruction , unspecified intestinal obstruction , intestinal or peritoneal adhesions with obstruction ( postoperative ) , peritonitis ( acute ) , generalized , other suppurative peritonitis , other retroperitoneal infections , unspecified peritonitis , perioperative autologous transfusion of whole blood or blood components , transfusion of previously collected autologous blood , other transfusion of whole blood , transfusion of packed cells , hemorrhage complicating procedure , hematoma complicating procedure . enterostomy : colostomy and enterostomy complication unspecified , infection of colostomy or enterostomy , mechanical complication of colostomy or enterostomy , other complication of colostomy or enterostomy , exteriorization of large intestine , colostomy , not otherwise specified , temporary colostomy , permanent colostomy , exteriorization of small intestine , ileostomy , not otherwise specified , temporary ileostomy , continent ileostomy , other permanent colostomy , other enterostomy . cohorts were also tested for differences in average hospital costs , surgery time , and length of stay ( table 5 ) . the average length of stay of the two cohorts was not statistically different ( 5.74 days for robotic vs 6.09 days for nonrobotic , p = .344 ) . the inpatient surgery time was significantly longer for robotic - assisted procedures ( 4.37 hours ; 95% confidence interval [ ci ] , 4.244.51 hours ) than for nonrobotic procedures ( 3.34 hours ; 95% ci , 3.233.46 hours ) ( p < .001 ) . hospital costs were substantially higher for robotic - assisted laparoscopic colectomy than for procedures without robotic assistance ( $ 17 445 vs $ 15 448 , p = .001 ) . this study showed that in a real - world setting , one - third of all segmental colectomies are performed by a minimally invasive approach , the vast majority without robotic assistance ( 98% ) . when well - matched cohorts are compared , the results of laparoscopic colectomy with and without robotic assistance are similar with respect to clinical outcomes ( length of stay ) and when considering perioperative complications . the findings related to higher hospital costs associated with robotic surgery are consistent with similar studies in the literature evaluating other laparoscopic surgical procedures . although there is a difference in hospital charges versus costs , charges are directly correlated to costs , and the trend is still the same , with robotic surgery consistently costing more . for example , rodgers et al compared the cost of robotic - assisted tubal reanastomosis with mini - laparotomy and also found that the cost of the robotic procedure was higher , with a median cost difference of $ 1446 ( 95% ci , $ 1112$1812 ; p < .001 ) . although not all of these studies examined colectomies specifically , these results do provide directional understanding of cost comparisons for other robotic - assisted minimally invasive procedures . two other clinical studies have directly compared robotic - assisted and laparoscopic left- and right - sided colectomies ( table 7 ) . rawlings et al found an increase in mean operative time , similar mean length of stay , and similar mean total hospital cost for right - sided colectomies . the reported comparison for sigmoid colectomies showed a similar mean operative time , mean length of stay , and mean total hospital cost . in a retrospective review , deutsch et al showed similar means for operative time and length of stay . there was a difference in operative time and a similar length of stay for left - sided colectomies . right and left colectomies : operative parameters data are shown as mean sd . in this premier dataset , before matching for right- and left - sided procedures , the right - sided procedures showed a significant difference in operative time and a similar length of stay . the left - sided procedures also showed a difference in operative time and a similar length of stay . for both robotic and traditional cases , there was a considerable reporting difference between the reported operative time and length of stay of the retrospective cases series by deutsch et al and rawlings et al compared with those reported in the premier dataset . this may reflect the differences between a single site , surgeon and hospital learning curves , and heterogeneity in patient populations . further analysis around the clinical and economic outcome differences between aggregated payor reporting database outcomes and historic single - center series may provide future insight into the complexities of clinical outcomes research , especially when assessing new and evolving technologies . in highly complex or technically challenging cases , robotic technology may offer the potential for advancing minimally invasive surgery . however , this research indicates that the traditional laparoscopic approach achieves similar clinical outcomes for segmental colon resections at a significantly decreased cost to the hospital . although subsequent generations of robotic technology may represent the future , economically , it is difficult to justify the uptake in robotic surgery for procedures such as routine colectomies . important strengths of this analysis included the prospectively developed protocol that directed the analysis , the quasi - randomization propensity scoring methodology that was used , the broad geographic and demographic representation of us hospitals included in the sample , and the fact that these data are relatively recent and represent the real - world setting . because the data were mined from a hospital administrative database used for billing purposes , certain data points were unable to be captured or could not be clearly identified . examples include body mass index , patient behaviors such as smoker versus nonsmoker , and complications resulting in an unplanned enterostomy or specific complications related to anastomotic leaks . enterostomies could not be identified as being planned or related to some complication and thus were evaluated separately from complications . because there is no specific icd-9 code for anastomotic complication , this analysis had to rely on existing diagnosis codes , which often result from anastomotic complications but are not exclusive or specific . furthermore , data regarding the precision of robotic versus nonrobotic procedures , including surgical margins and adequacy of lymph node dissection , could not be evaluated . the analysis was limited to a 30-day perioperative period , which limits analysis related to long - term survival or potential long - term complications . other limitations of this analysis include lack of comparison between rates of conversion to an open approach and differentiation between hand - assisted and total laparoscopic approaches . however , these limitations are inherent to the data source and could be rationalized to impact both cohorts similarly . as a result , the risk of bias in one cohort is lessened . finally , surgeon and institutional learning curve relative to using robotic technology could not be evaluated . this study represents the most up - to - date and expansive analysis of cost and effectiveness outcomes associated with robotic - assisted laparoscopic segmental colectomy in a real - world setting . these findings show few clinical differences in perioperative adverse events . coupled with the increased per - case cost of the robot and increased operative times , the results suggest that further consideration is warranted before using this technology for segmental laparoscopic colectomies when standard laparoscopic means yielding comparable results are available . future studies evaluating cost relative to robotic - assisted case volume and prospective randomized controlled studies focusing on comparative effectiveness between traditional and robotic - assisted laparoscopic segmental colectomy procedures are needed .

background and objectives : laparoscopic colectomies , with and without robotic assistance , are performed to treat both benign and malignant colonic disease . this study compared clinical and economic outcomes for laparoscopic colectomy procedures with and without robotic assistance.methods:patients aged 18 years having primary inpatient laparoscopic colectomy procedures ( cecectomy , right hemicolectomy , left hemicolectomy , and sigmoidectomy ) identified by international classification of diseases , ninth edition procedure codes performed between 2009 and the second quarter of 2011 from the premier hospital database were studied . patients were matched to a control cohort using propensity scores for disease , comorbidities , and hospital characteristics and were matched 1:1 for specific colectomy procedure . the outcomes of interest were hospital cost of laparoscopic robotic - assisted colectomy compared with traditional laparoscopic colectomy , surgery time , adverse events , and length of stay.results:of 25 758 laparoscopic colectomies identified , 98% were performed without robotic assistance and 2% were performed with robotic assistance . after matching , 1066 patients remained , 533 in each group . lengths of stay were not significantly different between the matched cohorts , nor were rates of major , minor , and/or surgical complications . inpatient procedures with robotic assistance were significantly more costly than those without robotic assistance ( $ 17 445 vs $ 15 448 , p = .001 ) . operative times were significantly longer for robotic - assisted procedures ( 4.37 hours vs 3.34 hours , p < .001).conclusion : segmental colectomies can be performed safely by either laparoscopic or robotic - assisted methods . increased per - case hospital costs for robotic - assisted procedures and prolonged operative times suggest that further investigation is warranted when considering robotic technology for routine laparoscopic colectomies .

INTRODUCTION MATERIALS AND METHODS Data Source Statistical Analyses RESULTS DISCUSSION CONCLUSION

colectomy , often used to treat colorectal cancer , can be performed by various techniques . the rate of laparoscopic techniques is increasing particularly in urban centers , in which laparoscopic colectomies are performed at a higher rate than in other settings . within the large premier hospital database , from 2009 through the second quarter of 2011 , approximately one - third of segmental colectomies ( cecectomy , right hemicolectomy , left hemicolectomy , and sigmoidectomy ) were identified as having a laparoscopic procedural code . additional advantages with regard to pain , blood loss , return of bowel function , length of hospitalization , and overall recovery time have been shown . in addition , resource use is lower for laparoscopic colectomy , including reduced length of stay , fewer readmissions , and less use of skilled nursing facilities . robotic - assisted surgery is an emerging approach in the field of laparoscopic colorectal surgery . currently , there is only one commercially available robotic device cleared by the us food and drug administration for laparoscopic procedures ( da vinci surgical system ; intuitive surgical , sunnyvale , california ) . several authors have published their experiences and case series related to robotic - assisted laparoscopic colectomy . although no specific large randomized controlled trials have evaluated robotic - assisted versus traditional laparoscopic colectomies , clinical outcomes suggest that robotic - assisted laparoscopic surgery is equivalent to conventional laparoscopy when considering important endpoints such as conversion to open surgery , hospital stay , and recovery time . in this era of comparative effectiveness and health care reform in the united states , and with concerns about optimal resource utilization at the forefront , the use of robotic - assisted laparoscopic surgery deserves further evaluation . given this background , this study examined clinical and economic outcomes ( cost and utilization ) in patients undergoing laparoscopic colectomy performed with and without robotic assistance . this database contains complete patient billing , hospital cost , and coding histories from more than 600 health care facilities throughout the united states . the data from which this study was derived were extracted from more than 25 million inpatient discharges and 175 million hospital outpatient visits from acute care facilities , ambulatory surgery centers , and clinics across the nation . a protocol describing the analysis objectives , criteria for patient selection , data elements of interest , and statistical methods was submitted to the new england institutional review board , and exemption was obtained . eligible patients were aged 18 years and had undergone a laparoscopic colectomy during the period from 2009 to the second quarter of 2011 . patients were categorized according to the following 4 types of laparoscopic segmental colectomies : laparoscopic cecectomy ( 17.32 ) , laparoscopic right hemicolectomy ( 17.33 ) , laparoscopic left hemicolectomy ( 17.35 ) , and laparoscopic sigmoidectomy ( 17.36 ) . laparoscopic colectomy procedures using robotic technology were identified if one of two conditions was met : ( 1 ) a robotic international classification of diseases , ninth edition ( icd-9 ) procedure code accompanied the primary procedure code of interest or ( 2 ) text fields were found when mining the hospital charge master file for each patient indicating use of the robot . for all eligible patients , elements describing hospital cost , surgery time , length of stay , use of the robot , colectomy type , and indication for colectomy were obtained from the data . comorbid conditions that might influence procedure selection or outcomes of interest , such as the presence of cardiovascular or pulmonary disease , cancer , or diabetes mellitus , were obtained by use of icd-9 diagnosis codes . comorbid conditions were grouped into 11 categories based on the medical expenditure panel survey data lists : arthritis , malignant neoplasms , mental disorders , metabolic diseases , diseases of the digestive system , diseases of the genitourinary system , diseases of the circulatory system , diseases of the musculoskeletal system and connective tissue , diseases of the nervous system , diseases of the respiratory system , and diseases of the cutaneous system . information on sociodemographic characteristics and health insurance status was also included , as were descriptors of the care setting , namely census region , urban or rural setting , teaching hospital status , and facility bed count . adverse events identified by icd-9 codes that occurred intraoperatively and 30 days postoperatively , which included pulmonary , cardiac , vascular , neurologic , and other , were flagged and included in the analysis . minor and major bleeding was categorized by icd-9 diagnosis as well as procedure codes related to hemorrhage and transfusions . a detailed list of each event and the corresponding icd-9 code is found in appendix b. each specific adverse event identified by icd-9 code was organized as either major or minor categories based on clinical experience . these were then evaluated and characterized based on whether they were related to the surgical technique ( bleeding , abscess , wound infection , and so on ) . information on adverse events among matched data by analysis groups appears in appendix b. stoma procedures were identified and treated separately from the complications ( appendix c ) . because of limitations of the dataset , it could not be determined whether these were planned stomas or due to a complication . the study objective was to use the premier hospital database to compare clinical and economic outcomes in patients undergoing laparoscopic colectomy with and without the use of robotic assistance . outcomes of interest included adverse events ( minor , major , and surgical ) , whether a stoma was performed , hospital costs , length of stay , and surgery time . covariates on which to match were selected based on their availability in the premier hospital database , as well as their general acceptance as factors associated with the outcomes of interest . propensity scores were calculated for receipt of robotic procedures for each of the patients included in the analysis based on a nonparsimonious multivariable logistic regression model . patients were matched on the following 13 characteristics : age , gender , race , insurance type , primary icd-9 procedure code , region of facility , urban versus nonurban classification of facility , teaching status of facility , number of beds at facility , and presence or absence of 4 comorbid conditions that were shown to be statistically significant before matching the robotic and nonrobotic patients were randomly ordered , and the nonrobotic patient with the propensity score closest to that of the first robotic patient was chosen . to assess the extent to which the propensity matching reduced confounders , the distributions of several variables before and after matching were compared including age , gender , race , insurance type , health status , region , location , facility type , primary icd-9 procedure code , comorbid conditions , and cancer versus noncancer based on the top 10 most frequently occurring icd-9 diagnosis codes among the patients in the cohorts . we used t tests to test for differences between the matched cohorts in the 3 continuous variables of interest : hospital cost , surgery time , and length of stay . logistic regression models were used to test for significant differences between the two groups and to generate odds ratios on the following categories of adverse events and complications : major , minor , and surgical and whether the patient also received a stoma . analyses were performed with sas , version 9.2 ( sas institute , cary , north carolina ) . this database contains complete patient billing , hospital cost , and coding histories from more than 600 health care facilities throughout the united states . the data from which this study was derived were extracted from more than 25 million inpatient discharges and 175 million hospital outpatient visits from acute care facilities , ambulatory surgery centers , and clinics across the nation . a protocol describing the analysis objectives , criteria for patient selection , data elements of interest , and statistical methods was submitted to the new england institutional review board , and exemption was obtained . eligible patients were aged 18 years and had undergone a laparoscopic colectomy during the period from 2009 to the second quarter of 2011 . patients were categorized according to the following 4 types of laparoscopic segmental colectomies : laparoscopic cecectomy ( 17.32 ) , laparoscopic right hemicolectomy ( 17.33 ) , laparoscopic left hemicolectomy ( 17.35 ) , and laparoscopic sigmoidectomy ( 17.36 ) . these procedures were chosen because the laparoscopic approach has been shown to have equivalent oncologic outcomes with documented perioperative morbidity benefits compared with open surgery . other procedures , such as low anterior resection , where traditional laparoscopy has not yet been established to be equivalent to open surgery , were not included . laparoscopic colectomy procedures using robotic technology were identified if one of two conditions was met : ( 1 ) a robotic international classification of diseases , ninth edition ( icd-9 ) procedure code accompanied the primary procedure code of interest or ( 2 ) text fields were found when mining the hospital charge master file for each patient indicating use of the robot . for all eligible patients , elements describing hospital cost , surgery time , length of stay , use of the robot , colectomy type , and indication for colectomy were obtained from the data . comorbid conditions that might influence procedure selection or outcomes of interest , such as the presence of cardiovascular or pulmonary disease , cancer , or diabetes mellitus , were obtained by use of icd-9 diagnosis codes . comorbid conditions were grouped into 11 categories based on the medical expenditure panel survey data lists : arthritis , malignant neoplasms , mental disorders , metabolic diseases , diseases of the digestive system , diseases of the genitourinary system , diseases of the circulatory system , diseases of the musculoskeletal system and connective tissue , diseases of the nervous system , diseases of the respiratory system , and diseases of the cutaneous system . information on sociodemographic characteristics and health insurance status was also included , as were descriptors of the care setting , namely census region , urban or rural setting , teaching hospital status , and facility bed count . adverse events identified by icd-9 codes that occurred intraoperatively and 30 days postoperatively , which included pulmonary , cardiac , vascular , neurologic , and other , were flagged and included in the analysis . a detailed list of each event and the corresponding icd-9 code is found in appendix b. each specific adverse event identified by icd-9 code was organized as either major or minor categories based on clinical experience . these were then evaluated and characterized based on whether they were related to the surgical technique ( bleeding , abscess , wound infection , and so on ) . information on adverse events among matched data by analysis groups appears in appendix b. stoma procedures were identified and treated separately from the complications ( appendix c ) . because of limitations of the dataset , it could not be determined whether these were planned stomas or due to a complication . the study objective was to use the premier hospital database to compare clinical and economic outcomes in patients undergoing laparoscopic colectomy with and without the use of robotic assistance . outcomes of interest included adverse events ( minor , major , and surgical ) , whether a stoma was performed , hospital costs , length of stay , and surgery time . covariates on which to match were selected based on their availability in the premier hospital database , as well as their general acceptance as factors associated with the outcomes of interest . nearest - neighbor matching on the estimated propensity scores to choose matches for the patients who had a robotic procedure . patients were matched on the following 13 characteristics : age , gender , race , insurance type , primary icd-9 procedure code , region of facility , urban versus nonurban classification of facility , teaching status of facility , number of beds at facility , and presence or absence of 4 comorbid conditions that were shown to be statistically significant before matching the robotic and nonrobotic patients were randomly ordered , and the nonrobotic patient with the propensity score closest to that of the first robotic patient was chosen . to assess the extent to which the propensity matching reduced confounders , the distributions of several variables before and after matching were compared including age , gender , race , insurance type , health status , region , location , facility type , primary icd-9 procedure code , comorbid conditions , and cancer versus noncancer based on the top 10 most frequently occurring icd-9 diagnosis codes among the patients in the cohorts . we used t tests to test for differences between the matched cohorts in the 3 continuous variables of interest : hospital cost , surgery time , and length of stay . logistic regression models were used to test for significant differences between the two groups and to generate odds ratios on the following categories of adverse events and complications : major , minor , and surgical and whether the patient also received a stoma . analyses were performed with sas , version 9.2 ( sas institute , cary , north carolina ) . ninety - eight percent of all laparoscopic colectomies included in this analysis were performed without the use of robotic assistance ( n = 25 210 ) . the procedural breakdown was as follows : laparoscopic cecectomy , 12 ; right hemicolectomy , 203 ; left hemicolectomy , 42 ; and sigmoidectomy , 291 ( table 2 ) . before matching , distributions were similar for age , gender , insurance , and most primary diagnosis codes for patients in both groups ( table 2 ) . furthermore , few differences in comorbidities or illness severity index were noted between the robotic and nonrobotic groups . the characteristics of the 364 hospitals with colectomy procedures were similar with regard to census region and location ( urban vs rural ) . there were notable differences , however , in teaching versus nonteaching and bed count , with most robotic procedures being performed in teaching hospitals with > 200 beds , as compared with nonrobotic procedures , with the majority coming from nonteaching hospitals with greater variation in bed size ( table 3 ) . patient characteristics , comorbid conditions , and hospital characteristics after matching are represented in table 4 . after matching , patients were balanced with respect to demographics , comorbid conditions , and hospital characteristics , with the exception of hospital location ( urban vs nonurban ) , which was statistically significantly different between the two groups ( p = .017 ) . after matching , clinical endpoints and adverse events occurring in the postoperative period 30 days after discharge were tabulated and grouped into 4 categories : major , minor , surgical , and stoma related . complications ( major , minor , and surgical ) and stoma procedures were not significantly different between the robotic and nonrobotic surgery cohorts , regardless of whether they were examined within a perioperative 30-day period or only within the original perioperative hospital stay ( table 6 ) . hospital costs , surgery time , and length of stay after matching adverse events after matching ci = confidence interval . cohorts were also tested for differences in average hospital costs , surgery time , and length of stay ( table 5 ) . the average length of stay of the two cohorts was not statistically different ( 5.74 days for robotic vs 6.09 days for nonrobotic , p = .344 ) . the inpatient surgery time was significantly longer for robotic - assisted procedures ( 4.37 hours ; 95% confidence interval [ ci ] , 4.244.51 hours ) than for nonrobotic procedures ( 3.34 hours ; 95% ci , 3.233.46 hours ) ( p < .001 ) . hospital costs were substantially higher for robotic - assisted laparoscopic colectomy than for procedures without robotic assistance ( $ 17 445 vs $ 15 448 , p = .001 ) . this study showed that in a real - world setting , one - third of all segmental colectomies are performed by a minimally invasive approach , the vast majority without robotic assistance ( 98% ) . when well - matched cohorts are compared , the results of laparoscopic colectomy with and without robotic assistance are similar with respect to clinical outcomes ( length of stay ) and when considering perioperative complications . the findings related to higher hospital costs associated with robotic surgery are consistent with similar studies in the literature evaluating other laparoscopic surgical procedures . although there is a difference in hospital charges versus costs , charges are directly correlated to costs , and the trend is still the same , with robotic surgery consistently costing more . for example , rodgers et al compared the cost of robotic - assisted tubal reanastomosis with mini - laparotomy and also found that the cost of the robotic procedure was higher , with a median cost difference of $ 1446 ( 95% ci , $ 1112$1812 ; p < .001 ) . although not all of these studies examined colectomies specifically , these results do provide directional understanding of cost comparisons for other robotic - assisted minimally invasive procedures . two other clinical studies have directly compared robotic - assisted and laparoscopic left- and right - sided colectomies ( table 7 ) . rawlings et al found an increase in mean operative time , similar mean length of stay , and similar mean total hospital cost for right - sided colectomies . the reported comparison for sigmoid colectomies showed a similar mean operative time , mean length of stay , and mean total hospital cost . in a retrospective review , deutsch et al showed similar means for operative time and length of stay . there was a difference in operative time and a similar length of stay for left - sided colectomies . in this premier dataset , before matching for right- and left - sided procedures , the right - sided procedures showed a significant difference in operative time and a similar length of stay . the left - sided procedures also showed a difference in operative time and a similar length of stay . for both robotic and traditional cases , there was a considerable reporting difference between the reported operative time and length of stay of the retrospective cases series by deutsch et al and rawlings et al compared with those reported in the premier dataset . this may reflect the differences between a single site , surgeon and hospital learning curves , and heterogeneity in patient populations . further analysis around the clinical and economic outcome differences between aggregated payor reporting database outcomes and historic single - center series may provide future insight into the complexities of clinical outcomes research , especially when assessing new and evolving technologies . however , this research indicates that the traditional laparoscopic approach achieves similar clinical outcomes for segmental colon resections at a significantly decreased cost to the hospital . although subsequent generations of robotic technology may represent the future , economically , it is difficult to justify the uptake in robotic surgery for procedures such as routine colectomies . important strengths of this analysis included the prospectively developed protocol that directed the analysis , the quasi - randomization propensity scoring methodology that was used , the broad geographic and demographic representation of us hospitals included in the sample , and the fact that these data are relatively recent and represent the real - world setting . the analysis was limited to a 30-day perioperative period , which limits analysis related to long - term survival or potential long - term complications . other limitations of this analysis include lack of comparison between rates of conversion to an open approach and differentiation between hand - assisted and total laparoscopic approaches . finally , surgeon and institutional learning curve relative to using robotic technology could not be evaluated . this study represents the most up - to - date and expansive analysis of cost and effectiveness outcomes associated with robotic - assisted laparoscopic segmental colectomy in a real - world setting . coupled with the increased per - case cost of the robot and increased operative times , the results suggest that further consideration is warranted before using this technology for segmental laparoscopic colectomies when standard laparoscopic means yielding comparable results are available . future studies evaluating cost relative to robotic - assisted case volume and prospective randomized controlled studies focusing on comparative effectiveness between traditional and robotic - assisted laparoscopic segmental colectomy procedures are needed .

targeted therapy relies on cell - surface receptors to direct anticancer agents to tumors . because of the selectivity to cancer cells , this type of treatment has shown improvements in patient quality of life by reducing unwanted side effects of nontargeted drugs , such as those utilized in chemotherapy . however , cancer cell populations within the same tumor are often diverse and exhibit various cell - surface receptors ; hence , different types of treatment are usually necessary to eradicate all of the cancer cells . recurrence rates of cancers after targeted treatment can be as high as 50% because of the regeneration of cancer cells that can not be eliminated . one solution to overcome this problem is to target a cell - surface receptor that is ubiquitous . nucleolin has recently been investigated as a surface receptor that can also transport targeting molecules from the plasma membrane to the nucleus of cancer cells . the overexpression of nucleolin on the cell surface and in the cytoplasm of exponentially growing cells suggests that this protein , whose one of the primary roles is to shuttle molecules between the nucleus and the cytoplasm , can function as a receptor for cell - type independent targeted cancer treatment . monoclonal antibodies ( mabs ) are the key anticancer agents employed in targeted therapy ; however , off - target effects and immunogenic responses can result in severe side effects . one emerging alternative to mabs is aptamers , synthetic oligonucleotides that can fold and bind to specific moieties on targeted molecules . the dna aptamer as1411 ( 26-mer , 7.8 kda ) has shown high binding affinity to nucleolin ( kd is in pm to low nm range ) via its g - quadruplex structure . one important function of nucleolin is binding to the au - rich components in the 3-untranslated region of the antiapoptotic gene bcl-2 mrna as protection against mrna degradation . free as1411 can bind to nucleolin in the same area where nucleolin stabilizes bcl-2 mrna ; hence , destabilization and subsequent down - regulation of bcl-2 by as1411 can result in apoptosis . as1411 has been tested in clinical trials for leukemia and renal cell cancer ; however , there are some concerns based of fast clearance and premature degradation ( half - life 5 h ) . to improve the stability of as1411 , we recently reported a strategy to attach as1411 ( apt denotes the homodimer form ) to the surface of gold nanostars ( auns ) . we found that high loading densities of apt on the auns increased the overall stability of aptamers in physiological environments , similar to other reports of nucleic acids on spherical gold particles . we evaluated aptamer - loaded auns ( apt - auns ) in hela ( cervical ) cancer cells as a model system and were able to observe effects from the nanoconstruct interacting with the cell nucleus . apt - auns was trafficked by nucleolin from the cell surface to the perinuclear regions and induced folding of the nuclear envelope . we demonstrated that these changes in nuclear phenotype could be directly correlated with disruption of cellular function , including increased double - stranded dna breaks in the nucleus as well as escalation of caspase 3/7 activity and cell death . the potential of apt - auns as a general anticancer agent , however , has not been tested . here , we report that apt - auns can function as a nanoconstruct resulting in in vitro efficacies superior to that of free aptamer biomolecules in a wide range of cancer cells . this study is the first to show that g - quadruplex aptamer homodimer - loaded nanoparticles can be effective in four major cancer subcategories . we found that nucleolin was abundant in plasma membrane and cytoplasm extracts of a 12-cancer cell panel . expression of surface nucleolin was also higher in cancer cells compared to normal cells . incubation of the cancer - cell panel with apt - auns resulted in cellular uptake that was quantified by inductively coupled plasma - mass spectrometry ( icp - ms ) . the reformulation of as1411 by grafting to auns enhanced the anticancer effects in all cancer cell lines with a 17% higher average cell death compared to free as1411 exceeding 10 times the concentration . we also discovered that downregulation of bcl-2 mrna expression was a contributing factor to apoptosis . furthermore , the in vitro efficacy of apt - auns was improved by detaching aptamers from the auns nanocarrier inside cancer cells . using ultrafast laser light to trigger the release of apt , we showed that the average percentage of cell death increased to 65% , which is 55% higher compared to that of free as1411 at over 10 times the concentration . because overexpression of nucleolin in the plasma membrane and cytoplasm is crucial for cellular uptake and trafficking of as1411 , we first determined the non - nuclear nucleolin ( cytoplasmic and plasma membrane ) levels in a 12-cancer line panel using immunoblotting ( supporting information , materials and methods ) . the cancer panel consisted of four types of subcategories : carcinoma , sarcoma , melanoma , and glioblastoma . we also included a control panel with three lines : hs-27 ( skin fibroblast ) , wi-38 ( lung fibroblast ) , and mcf-10a ( epithelial mammary cell ) . we observed that the levels of full - length nucleolin ( 106 kda ) and its proteolysis product ( 98 kda ) in fibroblast - like cancer cells ( ht-1080 , fibrosarcoma and sk - mel-2 , melanoma ) were up to four times higher compared to normal fibroblast cells ( hs-27 and wi-38 ) ( figure 1 ) . regarding epithelial cells , the expression of nucleolin in cancer cells was 10 times higher on average than that of mcf-10a cells ( figure 1 ) . the relative amounts of nucleolin were normalized to the expression of the housekeeping protein , -actin . although minimal expression of nucleolin was found in mcf-10a , high nucleolin levels in the non - nuclear extracts of hs-27 and wi-38 fibroblasts were seen ( figure 1b ) . this observation suggests that a normal cell panel containing both fibroblast and epithelial cells is necessary to assess effects of apt - auns on different types of tissues . ( a ) full - length ( 106 kda ) and the proteolysis product ( 98 kda ) of nucleolin appear in the non - nuclear lysates of all cancer cells . ( b ) mcf-10a cells show minimal expression of nucleolin , while fibroblast cells show nucleolin expression comparable to cancer cells . apt - auns is a biocompatible nanoconstruct that has demonstrated excellent in vitro efficacy in hela cells . the auns carrier was synthesized by reducing a gold precursor ( haucl4 ) in hepes buffer , where hepes acts as both a reducing agent as well as a shape - directing agent . the absence of surfactant in the auns synthesis is a major advantage over typical syntheses of gold particles , which require citrate ions or cetyl trimethylammonium bromide ( ctab ) for stabilization ; however , these molecules are toxic to healthy cells . in addition , auns contains multiple branches ( typically 29 ) that range in length from 10 to 65 nm ( figure 2a b ) . the average hydrodynamic diameter measured by dls was 39.2 6 nm ( table s1 ) . because of this unique shape , extinction spectra of the nanoparticle suspension revealed that the localized surface plasmon resonance of auns was centered around 800 nm ( figure s1 ) . this resonance can be tuned within the near - infrared ( 700860 nm ) region by controlling the size and shape of the gold nanostars by varying the concentration of hepes buffer relative to haucl4 . important for potential in vivo work , the simplicity of synthesis , which only involves two reagents , can be easily scaled to 5 l ( figure 2c ) . ( b ) higher magnification tem image of a representative auns shape , whose average hydrodynamic diameter is ca . ( c ) scaled - up production of apt - auns indicating that up to 5 l of apt - auns can be synthesized in an hour . to synthesize the nanoconstructs , thiolated as1411 was attached to the auns surface via gold sulfur bonds in a 2-day salt - aging process ( supporting information , materials and methods ) . the average hydrodynamic diameter of apt - auns increased from 39.2 to 44.6 nm , and the surface charge decreased from 33.1 to 29.4 mv . although we might have expected the surface charge to increase , the high concentrations of sodium ions surrounding the nanoconstructs tend to screen the negatively charged dna . using a fluorescence calibration assay with cy5-labeled as1411 ( supporting information , materials and methods ) , we determined that there were ca . we found that apt - auns was stable in aqueous solutions ( e.g. , di water , pbs , hepes buffer ) at room temperature over two months ( figure s1 ) . previous work revealed a 7 h incubation time with 0.3 nm apt - auns was sufficient to deliver in vitro ic50 dosages of the nanoconstruct to hela cells . therefore , we used the same conditions to screen the 12-cancer cell line panel . to visualize uptake by confocal fluorescence microscopy , we labeled the 5-end of apt with cy5 dye prior to attaching the aptamer to the auns ( cy5-apt - auns ) . figure 3a shows localization of the cy5-apt - auns ( red fluorescence ) in the cytoplasm and near the dapi - stained nucleus ( blue fluorescence ) in all cancer cells . also , surprisingly , we observed cy5-apt - auns signals in the cytoplasm of normal cells , especially the fibroblasts ( figure 3b ) . this uptake of apt - auns in fibroblasts is consistent with a recent report that found high uptake of free as1411 was possible in hs-27 via a different endocytosis mechanism from that in cancer cells . uptake of apt - auns in 12-cancer cell line panel and 3-normal cell line panel . cy5-labeled apt - auns ( red ) in the cytoplasm and near dapi - stained nuclei ( blue ) in ( a ) cancer cells and ( b ) normal cells suggest that apt - auns can be internalized . cancer cell panel : hct-116 ( colon ) , ht-1080 ( connective tissue ) , a-549 ( lung ) , hela ( cervix ) , mcf-7 ( breast ) , u-87 ( brain ) , du-145 ( prostate ) , mda - mb-231 ( breast ) , sk - mel-2 ( skin ) , skov-3 ( ovary ) , panc-1 ( pancreas ) , a-498 ( kidney ) . normal cell panel : mcf-10a ( epithelial mammary ) , wi-38 ( lung fibroblast ) , hs-27 ( skin fibroblast ) . to quantify cellular internalization of apt - auns in the panel , we measured the au content using icp - ms ( supporting information , materials and methods ) . the highest level of au content ( 24 ppt / cell ) was found in panc-1 ( pancreatic cancer ) cells , which was 12 times higher than that in normal mcf-10a ( 2 ppt / cell ) ( figure 4 ) . the au content in fibroblasts ( 10 ppt / cell ) was also much higher than that in mcf-10a cells ( figure 4 ) . these quantitative results were in agreement with confocal microscopy images ( figure 3 ) , where significantly higher cy5 signals were observed in cancer cells and fibroblasts compared to mcf-10a cells . we expected that increased nucleolin expression would correlate with increased au content across the cancer cell panel ; however , no clear correlation between these two factors was observed . we hypothesize that cellular uptake can also depend on other factors , such as the doubling time of the cells , since cells tend to take up different amounts of particles at different stages of the cell cycle . for example , hct-116 cells have a much shorter doubling time ( 15 h ) compared to ht-1080 cells ( 27 h ) . thus , there is more time for ht-1080 cells to accumulate nanoconstructs , and uptake of apt - auns was not directly proportional to nucleolin expression . apt - auns uptake and expression of nucleolin in plasma membrane and cytoplasmic in cancer and normal cells . higher au content and expression of non - nuclear nucleolin was found in cancer and fibroblast cell lines compared to mammary epithelial cells . to determine whether there were differences in how apt - auns were internalized by fibroblast cells and cancer cells , we used immunoblotting to compare the levels of nucleolin in the plasma membrane of normal and cancer cells . although the non - nuclear nucleolin levels in the fibroblasts were comparable to that in cancer cells , plasma membrane extracts of hs-27 were much lower than those in hela and ht-1080 cells ( figure s2 ) . hence , the lack of nucleolin in plasma membranes of normal cells suggests that uptake is independent of surface - nucleolin and occurs through other endocytosis pathways , similar to previous reports . as described previously , as1411 can bind to nucleolin and result in the downregulation of bcl-2 in mcf-7 and mda - mb-231 breast cancer cells . using quantitative real - time polymerase chain reaction ( rt - pcr ) , we examined whether apt - auns could also reduce bcl-2 expression in other types of cancer cells by binding to nucleolin ( supporting information , materials and methods ) . compared to untreated cancer cells , the levels of bcl-2 mrna were reduced by at least two times in ht-1080 cells and up to four times in panc-1 cells after incubation with apt - auns ( figure 5 ) . these decreases in gene expression are similar to that reported in breast cancer cells ( mcf-7 and mda - mb-231 ) after continuous treatment with 5 m of free as1411 for 72 h. importantly , the levels of bcl-2 mrna in all normal cells remained unchanged despite high apt - auns uptake by the fibroblasts . although apt - auns can enter both normal and cancer cells , we expect the uptake to occur by different mechanisms based on previous reports of free as1411 . we hypothesize that these differences can help explain why a reduction in bcl-2 mrna expression is observed in cancer cells but not in normal cells after treatment with apt - auns ( figure 5 ) . in effect , the nucleolin confined in and near the nucleus is not interacting with the nanoconstruct , and the levels of bcl-2 will remain unchanged . downregulation of antiapoptotic bcl-2 gene in cancer cells after treatment with apt - auns . a 400% reduction of bcl-2 mrna expression was observed in panc-1 cancer cells after treatment with 0.3 nm apt - auns ( 33 nm as1411 ) compared to untreated cancer cells . gene expression was reduced further ( 200% to 1500% ) in cancer cells after a single treatment with apt - auns + h. only seven out of 12 cancer cells ( hct-116 , ht-1080 , hela , mda - mb-231 , panc-1 , sk - mel-2 , and u-87 ) treated with 450 nm free as1411 decreased by minimal amounts . ( * ) and ( * * ) indicate p < 0.05 and p < 0.1 , respectively . because bcl-2 mrna functions as an antiapoptotic gene that prevents cancer cells from entering programmed cell death , downregulation of bcl-2 expression can trigger apoptosis . we measured the caspase 3/7 activity of the 12-cancer cell panel ( supporting information , materials and methods ) and found that the caspase activities increased by ca . note : we define the time after this 7 h incubation and after removal of the nanoconstructs as time t = 0 . at t = 72 h , when control populations were close to 100% confluent , the activity of the proteases increased up to four times in hct-116 ( colon cancer ) cells relative to untreated cells ( figure 6 , blue bar ) . cell viability was measured using a cell - titer blue assay to determine the percentage of live cells in the population ( supporting information , materials and methods ) . seventy - two hours after apt - auns incubation , the average cell viability decreased by 25% ( figure 7 , blue bar ) . previously , we discovered that conjugating as1411 onto auns carriers resulted in reduced dosages for efficacy in hela cells ; low nm concentrations of apt - auns produced anticancer effects similar to m of free as1411 . we were interested in whether the in vitro efficacy of 0.3 nm apt - auns ( 33 nm of apt , ca . 110 strands per auns ) in the 12-cancer cell lines would also be superior to free as1411 at over 10 times the concentration . first , we observed an average reduction of 2 times of bcl-2 mrna expression in cancer cells treated with apt - auns compared to those treated with free as1411 ( 1.5 times ) ( figure 5 and table s1 ) . these results suggest that apt - auns can increase the destabilization of bcl-2 mrna and require less than a tenth of the aptamer dose . in addition , the caspase 3/7 activity levels in cancer cells incubated with apt - auns increased by 1.4 times compared to those with free as1411 ( figure 6 ) . cell viability assays also indicated an average of 17% higher cancer cell death 72 h after incubation with apt - auns than with free as1411 ( figure 7 and table s1 ) . we attribute the superior biological effects of apt - auns over free aptamer to two main factors : ( 1 ) reformulation of as1411 as apt - auns . the nanoconstruct is more stable in physiological environments and the tight packing of apt on the auns surface makes the aptamer less susceptible to degradation by serum or dnase compared to free apt . hence , the effects of apt - auns could persist up to 72 h. ( 2 ) high local concentration of as1411 uptake . since each nanoconstruct can transport over 100 strands of as1411 into cancer cells , high local concentrations of apt can be internalized by cancer cells . elevation of caspase 3/7 activity in cancer cells after treatment with apt - auns and apt - auns + h. higher increases in caspase activities ( up to 6-fold ) are observed in all cancer cells treated with apt - auns + h. up to a 4-fold increase of caspase 3/7 activities after treatment with apt - auns indicates that large populations of cancer cells entered apoptosis . negligible increase in caspase 3/7 activities in cancer cells from treatment with 450 nm as1411 ( less than 1.5-fold ) . notably , there are no significant adverse effects observed in normal cells after exposure to laser irradiation . light - triggered release of apt from auns increases the percentage cell death . cancer cells treated with 450 nm of free as1411 showed reduced effects on cell death ( less than 10% ) compared to those treated with 0.3 nm apt - auns ( 33 nm as1411 ) and three times lower than that from light - triggered treatment . ( * ) and ( * * ) indicate p < 0.05 and p < 0.1 , respectively . light - triggered release of apt from auns near the cancer cell nucleus was found to increase in vitro therapeutic efficacy . our hypothesis is that this tandem treatment , where cancer cells are first incubated with apt - auns and then exposed to fs pulses for 2 s ( apt - auns + hv ) could also enhance in vitro efficacy compared to apt - auns only in the cancer cell panel . first , we evaluated whether apt - auns + hv interfered with nucleolin - bcl-2 mrna interactions . we found an average of 3.6 times reduction of bcl-2 mrna expression , and this reduction was as high as 15 times in panc-1 cells ( p < 0.05 ) ( figure 5 , hatched bar ) . the expression of bcl-2 mrna in cancer cells treated with apt - auns + h was 1.6 times lower than that with only apt - auns ( figure 5 and table s1 ) . these significant reductions in bcl-2 mrna expression suggest that intracellular release of high local concentration of apt can further degrade the mrna . the intracellular release of apt also showed superior anticancer effects , where the average amount of cell death induced by apt - auns + h increased by 55% compared to free as1411 and 40% compared to apt - auns only . 70% cell death in u-87 , mcf-7 , panc-1 and a-498 ( renal cancer ) cells ( p < 0.05 ) ( figure 7 , hatched bars ) , which is comparable to the levels of cell death achieved in mcf-7 cells three days after a single dose of 10 m free as1411 . furthermore , the caspase activities of all cancer cells treated with apt - auns + h increased by an average of 3.4 times compared to 1.6 times with apt - auns alone ( p < 0.1 ) ( figure 6 and table s1 ) . this drastic increase of efficacy can be attributed to a highly localized concentration of apt detached from nanocarriers by light - triggered release . therefore , this tandem strategy can result in potent effects that require only minimal amounts of as1411 ( 33 nm ) and that surpass results from m concentrations of free as1411 . apt - auns that exhibits in vitro anticancer effects in a 12-cancer cell line panel with four major subcategories . we showed that the protein nucleolin is a viable surface target for apt - auns by measuring expression in the plasma membrane and cytoplasm of the cancer cells . by binding to surface nucleolin , importantly , the loading of as1411 onto auns nanocarriers significantly improved the in vitro efficacy as a result of increased aptamer ( drug ) stability and presentation of high local concentrations of as1411 . similar to free as1411 , apt - auns resulted in downregulation of antiapoptotic bcl-2 gene expression , elevation of apoptosis signals , and increased cell death in the panel . light - triggered release further enhanced the in vitro efficacy by making available high local concentrations of apt near the nucleus . since apt - auns shows anticancer effects on the 12-cancer cell line panel mediated by the ubiquitous protein nucleolin , we anticipate that this nanoconstruct can act as a platform for a new class of cell - type independent agents that could address some current challenges in targeted therapy . all cultures were grown in a humidified incubator maintained at 37 c with 95% air/5% co2 . gold nanostars ( auns ) were synthesized by reducing au ( iii ) chlorate in hepes buffer to create biocompatible , surfactant - free gold nanoparticles for in vitro studies . the resonance wavelength of the auns and apt - auns was measured using uv particle size was determined using transmission electron microscopy ( tem ) and dynamic light scattering ( dls ) . cy5-labeled aptamer 5-(c6-s - s - c6)- cy5-ttg gtg gtg gtg gtt gtg gtg gtg gtg g-3 ( cy5-apt ) cy5-apt - auns were treated with potassium cyanide ( kcn ) overnight to dissolve the au core of the nanoconstruct and release cy5 aptamer into the solution . the cy5 fluorescence intensity of the kcn solution was measured using a nanodrop spectrophotometer , and the concentration of as1411 was determined based on the intensity of the cy5 signal . this fluorescent assay indicated that approximately 110 strands of as1411 were conjugated on a single auns . cancer and normal cells were harvested from cell culture flasks . to determine expression of cytosolic nucleolin in cancer and normal cells , we lysed the cells on ice for 15 min in 5% np40 lysis buffer ( invitrogen ) , followed by centrifugation at 10 000 g for 20 min . the supernatant contained the cytosolic extract , and the pellet contained the nuclear components . for plasma membrane extraction , cancer and normal cells were lysed in homogenized buffer ( abcam ) and homogenized 50 times on ice using a dounce homogenizer . protein concentrations after the extractions were determined by the bradford assay ( pierce ) . aliquots of the cytosolic extracts containing 20 g of protein were electrophoresed on a 10% tris - hcl gel ( bio rad ) and transblotted using polyvinylidene fluoride ( pvdf ) membranes ( millipore ) . antihuman nucleolin mab ( clone ms-3 ) ( santa cruz biotechnology , inc . ) and antimouse actin polyclonal antibody ( sigma - aldrich ) were used to label nucleolin and the housekeeping protein -actin . the bands were developed using enhanced chemifluorescence substrate ( ge healthcare ) and visualized by typhoon phosphorimager ( ge healthcare ) . the amount of each protein in the blots was determined by counting the total number of pixels in each band ( integrated density value ) . values of nucleolin that were within the linear range of the assay were normalized to -actin for the cytosolic extracts . cells were incubated with 0.3 nm cy5-labeled apt - auns for 7 h and then washed three times with pbs . a drop of prolong gold antifade reagent containing dapi ( invitrogen ) was used to mount each coverslip on a glass slide for confocal imaging . confocal imaging was performed on an inverted zeiss axio observer z1 confocal microscope with a 40 objective and zen acquisition software . the cells were incubated with the nanoconstructs for 7 h at 37 c in 5% co2 environment . after 7 h , excess apt - auns were removed from the wells , and the cells were washed twice with ice cold pbs ( invitrogen ) . the cells were then harvested and suspended in 100 l of phosphate - buffered saline ( pbs ) . the cells were counted before being digested for 4 h at 75 c in acid mixture containing 30% hcl ( sigma - aldrich ) and 70% hno3 ( sigma - aldrich ) . after complete digestion of the auns , the solution was diluted with millipore water . real - time polymerase chain reaction ( qpcr ) was used to quantify mrna expression in cancer and normal cells . the assay was conducted using power sybr green cells - to - ct kit ( invitrogen ) . identified primers were purchased from idt dna and tested for the amplification of a single uniform amplicon through analysis of sybr melting curves for two cell lines ( hela and a-549 ) . each sample was run in triplicate with the iq5 qpcr system ( bio - rad ) . each plate probed the expression of bcl-2 and actb genes in the target cell lines . real - time pcr data were analyzed using the comparative ct method , also known as the 2 method , in which the expression of bcl-2 mrna was normalized to the expression of the housekeeping gene actb . apo - one homogeneous caspase-3/7 assay kit ( promega ) and cell - titer blue cell viability assay ( promega ) were used to measure caspase 3/7 activity and cell viability , respectively , in cancer and normal cells after treatment with apt - auns , apt - auns + h , and 450 nm free as1411 .

we report the design of a nanoconstruct that can function as a cell - type independent agent by targeting the ubiquitous protein nucleolin . gold nanostars ( auns ) loaded with high densities of nucleolin - specific dna aptamer as1411 ( apt - auns ) produced anticancer effects in a panel of 12 cancer lines containing four representative subcategories . we found that the nanoconstructs could be internalized by cancer cells and trafficked to perinuclear regions . apt - auns resulted in downregulation of antiapoptotic bcl-2 mrna expression by ca . 200% compared to cells without the nanoconstructs . the caspase 3/7 activity ( apoptosis ) and cell death in cancer cells treated with apt - auns increased by 1.5 times and by ca . 17% , respectively , compared to cells treated with free as1411 at over 10 times the concentration . moreover , light - triggered release of aptamer from the auns further enhanced the in vitro efficacy of the nanoconstructs in the cancer line panel with a 2-fold increase in caspase activity and a 40% decrease in cell viability compared to treatment with apt - auns only . in contrast , treatments of the nanoconstructs with or without light - triggered release on a panel of normal cell lines had no adverse effects .

Introduction Results and Discussion Conclusions Experimental Section

however , cancer cell populations within the same tumor are often diverse and exhibit various cell - surface receptors ; hence , different types of treatment are usually necessary to eradicate all of the cancer cells . recurrence rates of cancers after targeted treatment can be as high as 50% because of the regeneration of cancer cells that can not be eliminated . nucleolin has recently been investigated as a surface receptor that can also transport targeting molecules from the plasma membrane to the nucleus of cancer cells . the overexpression of nucleolin on the cell surface and in the cytoplasm of exponentially growing cells suggests that this protein , whose one of the primary roles is to shuttle molecules between the nucleus and the cytoplasm , can function as a receptor for cell - type independent targeted cancer treatment . the dna aptamer as1411 ( 26-mer , 7.8 kda ) has shown high binding affinity to nucleolin ( kd is in pm to low nm range ) via its g - quadruplex structure . one important function of nucleolin is binding to the au - rich components in the 3-untranslated region of the antiapoptotic gene bcl-2 mrna as protection against mrna degradation . free as1411 can bind to nucleolin in the same area where nucleolin stabilizes bcl-2 mrna ; hence , destabilization and subsequent down - regulation of bcl-2 by as1411 can result in apoptosis . to improve the stability of as1411 , we recently reported a strategy to attach as1411 ( apt denotes the homodimer form ) to the surface of gold nanostars ( auns ) . we found that high loading densities of apt on the auns increased the overall stability of aptamers in physiological environments , similar to other reports of nucleic acids on spherical gold particles . we evaluated aptamer - loaded auns ( apt - auns ) in hela ( cervical ) cancer cells as a model system and were able to observe effects from the nanoconstruct interacting with the cell nucleus . apt - auns was trafficked by nucleolin from the cell surface to the perinuclear regions and induced folding of the nuclear envelope . we demonstrated that these changes in nuclear phenotype could be directly correlated with disruption of cellular function , including increased double - stranded dna breaks in the nucleus as well as escalation of caspase 3/7 activity and cell death . here , we report that apt - auns can function as a nanoconstruct resulting in in vitro efficacies superior to that of free aptamer biomolecules in a wide range of cancer cells . we found that nucleolin was abundant in plasma membrane and cytoplasm extracts of a 12-cancer cell panel . expression of surface nucleolin was also higher in cancer cells compared to normal cells . incubation of the cancer - cell panel with apt - auns resulted in cellular uptake that was quantified by inductively coupled plasma - mass spectrometry ( icp - ms ) . the reformulation of as1411 by grafting to auns enhanced the anticancer effects in all cancer cell lines with a 17% higher average cell death compared to free as1411 exceeding 10 times the concentration . we also discovered that downregulation of bcl-2 mrna expression was a contributing factor to apoptosis . furthermore , the in vitro efficacy of apt - auns was improved by detaching aptamers from the auns nanocarrier inside cancer cells . using ultrafast laser light to trigger the release of apt , we showed that the average percentage of cell death increased to 65% , which is 55% higher compared to that of free as1411 at over 10 times the concentration . because overexpression of nucleolin in the plasma membrane and cytoplasm is crucial for cellular uptake and trafficking of as1411 , we first determined the non - nuclear nucleolin ( cytoplasmic and plasma membrane ) levels in a 12-cancer line panel using immunoblotting ( supporting information , materials and methods ) . we observed that the levels of full - length nucleolin ( 106 kda ) and its proteolysis product ( 98 kda ) in fibroblast - like cancer cells ( ht-1080 , fibrosarcoma and sk - mel-2 , melanoma ) were up to four times higher compared to normal fibroblast cells ( hs-27 and wi-38 ) ( figure 1 ) . regarding epithelial cells , the expression of nucleolin in cancer cells was 10 times higher on average than that of mcf-10a cells ( figure 1 ) . ( a ) full - length ( 106 kda ) and the proteolysis product ( 98 kda ) of nucleolin appear in the non - nuclear lysates of all cancer cells . apt - auns is a biocompatible nanoconstruct that has demonstrated excellent in vitro efficacy in hela cells . this resonance can be tuned within the near - infrared ( 700860 nm ) region by controlling the size and shape of the gold nanostars by varying the concentration of hepes buffer relative to haucl4 . to synthesize the nanoconstructs , thiolated as1411 was attached to the auns surface via gold sulfur bonds in a 2-day salt - aging process ( supporting information , materials and methods ) . the average hydrodynamic diameter of apt - auns increased from 39.2 to 44.6 nm , and the surface charge decreased from 33.1 to 29.4 mv . we found that apt - auns was stable in aqueous solutions ( e.g. previous work revealed a 7 h incubation time with 0.3 nm apt - auns was sufficient to deliver in vitro ic50 dosages of the nanoconstruct to hela cells . to visualize uptake by confocal fluorescence microscopy , we labeled the 5-end of apt with cy5 dye prior to attaching the aptamer to the auns ( cy5-apt - auns ) . figure 3a shows localization of the cy5-apt - auns ( red fluorescence ) in the cytoplasm and near the dapi - stained nucleus ( blue fluorescence ) in all cancer cells . also , surprisingly , we observed cy5-apt - auns signals in the cytoplasm of normal cells , especially the fibroblasts ( figure 3b ) . this uptake of apt - auns in fibroblasts is consistent with a recent report that found high uptake of free as1411 was possible in hs-27 via a different endocytosis mechanism from that in cancer cells . uptake of apt - auns in 12-cancer cell line panel and 3-normal cell line panel . cy5-labeled apt - auns ( red ) in the cytoplasm and near dapi - stained nuclei ( blue ) in ( a ) cancer cells and ( b ) normal cells suggest that apt - auns can be internalized . to quantify cellular internalization of apt - auns in the panel , we measured the au content using icp - ms ( supporting information , materials and methods ) . these quantitative results were in agreement with confocal microscopy images ( figure 3 ) , where significantly higher cy5 signals were observed in cancer cells and fibroblasts compared to mcf-10a cells . apt - auns uptake and expression of nucleolin in plasma membrane and cytoplasmic in cancer and normal cells . higher au content and expression of non - nuclear nucleolin was found in cancer and fibroblast cell lines compared to mammary epithelial cells . to determine whether there were differences in how apt - auns were internalized by fibroblast cells and cancer cells , we used immunoblotting to compare the levels of nucleolin in the plasma membrane of normal and cancer cells . using quantitative real - time polymerase chain reaction ( rt - pcr ) , we examined whether apt - auns could also reduce bcl-2 expression in other types of cancer cells by binding to nucleolin ( supporting information , materials and methods ) . compared to untreated cancer cells , the levels of bcl-2 mrna were reduced by at least two times in ht-1080 cells and up to four times in panc-1 cells after incubation with apt - auns ( figure 5 ) . these decreases in gene expression are similar to that reported in breast cancer cells ( mcf-7 and mda - mb-231 ) after continuous treatment with 5 m of free as1411 for 72 h. importantly , the levels of bcl-2 mrna in all normal cells remained unchanged despite high apt - auns uptake by the fibroblasts . although apt - auns can enter both normal and cancer cells , we expect the uptake to occur by different mechanisms based on previous reports of free as1411 . we hypothesize that these differences can help explain why a reduction in bcl-2 mrna expression is observed in cancer cells but not in normal cells after treatment with apt - auns ( figure 5 ) . downregulation of antiapoptotic bcl-2 gene in cancer cells after treatment with apt - auns . a 400% reduction of bcl-2 mrna expression was observed in panc-1 cancer cells after treatment with 0.3 nm apt - auns ( 33 nm as1411 ) compared to untreated cancer cells . gene expression was reduced further ( 200% to 1500% ) in cancer cells after a single treatment with apt - auns + h. only seven out of 12 cancer cells ( hct-116 , ht-1080 , hela , mda - mb-231 , panc-1 , sk - mel-2 , and u-87 ) treated with 450 nm free as1411 decreased by minimal amounts . because bcl-2 mrna functions as an antiapoptotic gene that prevents cancer cells from entering programmed cell death , downregulation of bcl-2 expression can trigger apoptosis . we measured the caspase 3/7 activity of the 12-cancer cell panel ( supporting information , materials and methods ) and found that the caspase activities increased by ca . cell viability was measured using a cell - titer blue assay to determine the percentage of live cells in the population ( supporting information , materials and methods ) . seventy - two hours after apt - auns incubation , the average cell viability decreased by 25% ( figure 7 , blue bar ) . previously , we discovered that conjugating as1411 onto auns carriers resulted in reduced dosages for efficacy in hela cells ; low nm concentrations of apt - auns produced anticancer effects similar to m of free as1411 . we were interested in whether the in vitro efficacy of 0.3 nm apt - auns ( 33 nm of apt , ca . 110 strands per auns ) in the 12-cancer cell lines would also be superior to free as1411 at over 10 times the concentration . first , we observed an average reduction of 2 times of bcl-2 mrna expression in cancer cells treated with apt - auns compared to those treated with free as1411 ( 1.5 times ) ( figure 5 and table s1 ) . these results suggest that apt - auns can increase the destabilization of bcl-2 mrna and require less than a tenth of the aptamer dose . in addition , the caspase 3/7 activity levels in cancer cells incubated with apt - auns increased by 1.4 times compared to those with free as1411 ( figure 6 ) . cell viability assays also indicated an average of 17% higher cancer cell death 72 h after incubation with apt - auns than with free as1411 ( figure 7 and table s1 ) . since each nanoconstruct can transport over 100 strands of as1411 into cancer cells , high local concentrations of apt can be internalized by cancer cells . elevation of caspase 3/7 activity in cancer cells after treatment with apt - auns and apt - auns + h. higher increases in caspase activities ( up to 6-fold ) are observed in all cancer cells treated with apt - auns + h. up to a 4-fold increase of caspase 3/7 activities after treatment with apt - auns indicates that large populations of cancer cells entered apoptosis . negligible increase in caspase 3/7 activities in cancer cells from treatment with 450 nm as1411 ( less than 1.5-fold ) . light - triggered release of apt from auns increases the percentage cell death . cancer cells treated with 450 nm of free as1411 showed reduced effects on cell death ( less than 10% ) compared to those treated with 0.3 nm apt - auns ( 33 nm as1411 ) and three times lower than that from light - triggered treatment . light - triggered release of apt from auns near the cancer cell nucleus was found to increase in vitro therapeutic efficacy . our hypothesis is that this tandem treatment , where cancer cells are first incubated with apt - auns and then exposed to fs pulses for 2 s ( apt - auns + hv ) could also enhance in vitro efficacy compared to apt - auns only in the cancer cell panel . first , we evaluated whether apt - auns + hv interfered with nucleolin - bcl-2 mrna interactions . we found an average of 3.6 times reduction of bcl-2 mrna expression , and this reduction was as high as 15 times in panc-1 cells ( p < 0.05 ) ( figure 5 , hatched bar ) . the expression of bcl-2 mrna in cancer cells treated with apt - auns + h was 1.6 times lower than that with only apt - auns ( figure 5 and table s1 ) . these significant reductions in bcl-2 mrna expression suggest that intracellular release of high local concentration of apt can further degrade the mrna . the intracellular release of apt also showed superior anticancer effects , where the average amount of cell death induced by apt - auns + h increased by 55% compared to free as1411 and 40% compared to apt - auns only . 70% cell death in u-87 , mcf-7 , panc-1 and a-498 ( renal cancer ) cells ( p < 0.05 ) ( figure 7 , hatched bars ) , which is comparable to the levels of cell death achieved in mcf-7 cells three days after a single dose of 10 m free as1411 . furthermore , the caspase activities of all cancer cells treated with apt - auns + h increased by an average of 3.4 times compared to 1.6 times with apt - auns alone ( p < 0.1 ) ( figure 6 and table s1 ) . this drastic increase of efficacy can be attributed to a highly localized concentration of apt detached from nanocarriers by light - triggered release . therefore , this tandem strategy can result in potent effects that require only minimal amounts of as1411 ( 33 nm ) and that surpass results from m concentrations of free as1411 . apt - auns that exhibits in vitro anticancer effects in a 12-cancer cell line panel with four major subcategories . we showed that the protein nucleolin is a viable surface target for apt - auns by measuring expression in the plasma membrane and cytoplasm of the cancer cells . by binding to surface nucleolin , importantly , the loading of as1411 onto auns nanocarriers significantly improved the in vitro efficacy as a result of increased aptamer ( drug ) stability and presentation of high local concentrations of as1411 . similar to free as1411 , apt - auns resulted in downregulation of antiapoptotic bcl-2 gene expression , elevation of apoptosis signals , and increased cell death in the panel . light - triggered release further enhanced the in vitro efficacy by making available high local concentrations of apt near the nucleus . since apt - auns shows anticancer effects on the 12-cancer cell line panel mediated by the ubiquitous protein nucleolin , we anticipate that this nanoconstruct can act as a platform for a new class of cell - type independent agents that could address some current challenges in targeted therapy . gold nanostars ( auns ) were synthesized by reducing au ( iii ) chlorate in hepes buffer to create biocompatible , surfactant - free gold nanoparticles for in vitro studies . the resonance wavelength of the auns and apt - auns was measured using uv particle size was determined using transmission electron microscopy ( tem ) and dynamic light scattering ( dls ) . cy5-labeled aptamer 5-(c6-s - s - c6)- cy5-ttg gtg gtg gtg gtt gtg gtg gtg gtg g-3 ( cy5-apt ) cy5-apt - auns were treated with potassium cyanide ( kcn ) overnight to dissolve the au core of the nanoconstruct and release cy5 aptamer into the solution . aliquots of the cytosolic extracts containing 20 g of protein were electrophoresed on a 10% tris - hcl gel ( bio rad ) and transblotted using polyvinylidene fluoride ( pvdf ) membranes ( millipore ) . after 7 h , excess apt - auns were removed from the wells , and the cells were washed twice with ice cold pbs ( invitrogen ) . apo - one homogeneous caspase-3/7 assay kit ( promega ) and cell - titer blue cell viability assay ( promega ) were used to measure caspase 3/7 activity and cell viability , respectively , in cancer and normal cells after treatment with apt - auns , apt - auns + h , and 450 nm free as1411 .

with increasing emphasis on bio - based fuels and chemicals , the cellulase market is expected to increase dramatically . to create a sustainable bioeconomy , cellulases need to be produced cost - effectively and possess excellent biocatalytic properties . solid - state fermentation ( ssf ) offers a low - cost alternative for producing cellulases using natural polymers derived from agroindustrial residues [ 3 , 4 ] . ssf is defined as a discrete solid phase in which microorganisms grow on the surface of moist particles as well as inside and between them . gas phase in ssf is strongly affected by the size , shape , and tortuosity of a network of gas - filled pores . the air- or gas - filled pores are referred to as bed porosity , which is defined as the volume of gas contained in the system at any given time ( void fraction ) . availability of spaces between particles ensures availability of oxygen that improves enzyme production in aerobic fungal cultures [ 79 ] . showed that by blending rice bran with wheat bran resulted in substantial improvement in the morphology of rice bran which improved protease production during solid - state culturing of a. oryzae . the increase in bed porosity of the substrate could be the reason behind improved production ; however , no attempts were made to measure bed porosity to show its relationship to enzyme production . several authors in the past have suggested the merits of open porous solid beds but no explicit investigation has been conducted yet that relates bed porosity with enzyme production in ssf . in industrial scale ssf processes , bed porosity is essential but not sufficient for complete process control . other parameters , such as microbial cell physiology , composition of the solid substrate , and substrate reactivity also could influence the productivity of the process [ 11 , 12 ] . substrate reactivity , especially in case of cellulosic substrates , is influenced by physicochemical characteristics of the substrate at different levels . at microfibril level it is crystallinity of cellulose , and at fiber level it is specific surface area ( characterizing pore size or degree of swelling ) [ 1315 ] . the increase in cellulase reactivity due to increase in specific surface area is attributed to the creation of surface openings or internal slits , voids , or spaces , by the removal of cell wall components , that enhances the direct physical contact between the enzymes and the substrate . during growth on complex substrates , propagation of fungal mycelium it has been proposed that the hydrolysis occurs efficiently when the pores within the substrate are large enough to accommodate both large and small enzyme components to maintain the synergistic action of the enzyme system [ 14 , 17 , 18 ] . on the other hand , the crystalline nature of the carbon source used to induce cellulolytic expression in many species of fungi significantly influences the hydrolytic potential of the enzyme preparation . evans et al . showed that crystalline - cotton - induced cellulolytic complex derived from submerged t. reesei cultures exhibited higher potential in hydrolyzing crystalline cellulose than solka - floc - induced cellulases . the extent of catabolite repression depends on the rate of glucose formation , which in turn depends on the secretion of enzymes that degrade cellulose . [ 22 , 23 ] , and , more recently , ciolacu et al . and hall et al . have shown that the rate of cellulose degradation is dependent on crystallinity of the cellulosic substrate . in other words , crystallinity of cellulosic sample could alter not only the quality of enzymes ( the proportion of various activities with cellulolytic enzyme complex ) but also the quantity of enzymes produced . thus , studies delineating the effects of crystallinity on enzyme production in ssf are of significant interest . the growth of fungi in natural substrates is usually slow and this limitation must be overcome by suitable mechanical and chemical pretreatment of the raw substrate . however , pretreatments are known to induce structural changes in cellulosic substrates , which could alter the physicochemical properties of the substrate . the effect of pretreatment methods on physicochemical characteristics of substrate and its repercussions on cellulolytic enzyme productivity in fungal solid - state fermentation has not been investigated so far , which is evident from the recent reviews on ssf [ 3 , 27 ] . an in - depth understanding of the role of physicochemical characteristics of substrate on cellulase production in ssf would provide a framework for comprehensive analysis of critical design issues that should facilitate cellulase production with enhanced biocatalysis . the present study aimed to determine the role of pretreatment techniques in altering the physicochemical characteristics bed porosity , volumetric specific surface , and crystallinity of solid - state substrate . in addition , the effect of change in physicochemical attributes on enzyme production in fungal solid - state fermentation was studied with respect to type of fungal species and different cellulolytic enzyme activities . the pretreatments were carefully chosen to limit the effect on the chemical compositional changes of solid substrate , which would otherwise diminish the role of physicochemical attributes . since crystallinity is critical to this study , a new method of measuring crystallinity of complex cellulosic substrate untreated ground soybean hulls ( purchased from archer daniels midland , salina , ks , usa ) , herein referred to as native soybean hulls , had a geometric mean diameter , dgw , of 0.61 0.002 mm . native soybean hulls were subjected to four different treatments before being used for production of the cellulolytic enzyme system : ( 1 ) steam pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in distilled water was pressure cooked at 121c for 60 min ; ( 2 ) hydrochloric acid pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in 1 n hcl was kept on a gyratory shaker ( 150 rpm ) for 24 h at ambient temperature ; ( 3 ) sulfuric acid pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in 1 n h2so4 was kept on a gyratory shaker ( 150 rpm ) for 24 h at ambient temperature ( 4 ) sodium hydroxide pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in 1 n naoh was kept on a gyratory shaker ( 150 rpm ) for 24 h at ambient temperature . after acid and alkali pretreatments , treated soybean hulls were collected by filtration and extensively washed with distilled water . all treated substrates were dried overnight at 45c in a forced - draft oven ( fisher scientific , usa ) . dried substrates were used for compositional analysis , analysis of physicochemical characteristics , and production of enzymes . two fungal cultures t. reesei ( atcc 26921 ) and a. oryzae ( atcc 12892 ) were used for ssf of native and pretreated soybean hulls . cultures were used as both mono and mixed ( 1 : 1 ) . native and pretreated dried soybean hulls ( 5 g ) were adjusted to 70% ( wet basis ) moisture content ( mc ) by using mandels media of ph 5 and were sterilized in a vertical sterilizer ( 121c/15 psi gauge ) for 30 minutes . cultures were added as spore suspensions ( 10 spores / ml - suspension ) at the loading of 0.1 ml per gram dry substrate . flasks containing two cultures in the ratio of 1 : 1 were labeled as mixed . the conditions of temperature , ph , moisture ( 70% ) , and incubation days of the ssf process used in this study were optimized previously . following incubation , porosity ( ) of the samples was computed from the values of true density and bulk density by using the relationship described in as follows : ( 1 ) =(1bt)100 . true density ( t ) was determined using a standard liquid pycnometer by determining the volume of the sample at various moisture contents . volume ( v , cm ) was calculated from the following relationship : ( 2)v=(mpsmp)(mptsmt)tol , where mt is mass of the pycnometer filled with toluene , mps is the mass of pycnometer and sample , mp is mass of the pycnometer , mpts is mass of the pycnometer filled with toluene and sample , and tol is the density of toluene . knowing v , the true density ( g / cc ) then can be calculated from the following expression : ( 3)t=(mpsmp)v . bulk density ( b ) is estimated by weighing the samples ( 70% mc ) after pouring in a vessel of known volume ( 10 ml ) . volumetric specific surface is defined as external surface area per unit volume of the samples . samples were sieved using usa standard testing sieves stacked in order of decreasing aperture size above the collection pan placed in ro - tap sieve sifter ( laval lab inc . , canada ) . weight of oversize generated during sieving was used to compute geometric mean diameter ( dgw ) and geometric standard deviation ( sgw ) according to the following equations : ( 4)dgw = log 1 ( (wilog di)wi)sgw = log 1 ( [wi(log dilog dgw)2]wi ) , where di is the diameter of the ith sieve in the stack and wi is the weight fraction on the ith sieve . using dgw and sgw , surface area per gram was calculated as follows : ( 5a)s ( cm2/g)=svexp ( 0.5 ln 2 sgwln dgw ) . volumetric specific surface ( sa , cm ) can then be obtained from ( 5a ) by multiplying it with specific weight ( ) ( g / cm ) , that is , ( 5b)sa ( cm1)=svexp ( 0.5ln 2 sgwln dgw ) , where s is the shape coefficient for calculating surface area of particles ( fixed at 6 ) and v is the shape coefficient for calculating volume of particles ( fixed at 1 ) . wide - angle x - ray diffraction ( xrg 3100 x - ray generator , phillips electronics instrument inc . , tx , usa ) was used to estimate the crystallinity of native and pretreated soybean hulls . the x - rays from a cu tube operating at 35 kv and 20 ma were collected by an energy dispersive detector that is able to resolve cuk line . counts were collected at a step size of 0.02 at a series of angles between 5 and 40. speed of count collection was 0.6/min . the raw diffractograms were subjected to a fitting procedure using a nonlinear least squares numerical procedure . the deconvolution method separates amorphous and crystalline contributions to the diffraction spectrum under curve - fitting process by selecting a shape function . in this method it is very important to understand the major sources that contribute to the shape function of the observed x - ray profile h(2 ) , which is a convolution ( ) of the intrinsic specimen profile f(2 ) with the spectral distribution ( w ) and the instrumental function ( g ) superimposed over the background b , as given below : ( 6)h(2)=[(wg)f](2)+b . the voigt function , which is a convolution of gaussian and lorentzian peak functions , would include both gaussian intrinsic broadening of the specimen along with the lorentzian instrumental profile that considers the background from amorphous scattering . the voigt function , therefore , appropriately takes into account the peak broadening due to diffusive scattering [ 35 , 36 ] . using the voigt function intensity of the reflection is represented by following equation : ( 7)f(2)=ao(exp ( (2)2)/(al2+((xac)/ag2)2))d(2)(exp ( ((2)2))/(al2+(2)))d(2 ) , where ao is the amplitude of the peak , ac is the center of the peak , al is the width of the lorentzian component , and ag is the width of the gaussian component of the peak . the major reflective planes in cellulosic material from plant sources correspond to the following miller indices ( hkl ) : 101 , 101 , 002 , 021 , and 040 , with 002 as the prominent reflection representing crystalline cellulose ( sometimes resolved into 021 plane as well ) . x - ray peaks were fitted using voigt function as profile shape function using peakfit ( seasolve software inc . the program was rerun locking these planes ; consequently , five voigt functions were fitted . the fitted peaks were used to evaluate degree of crystallinity ( xcr ) of the sample per ( 8) described by wada et al . , ( 8)xcr ( % ) = i002+i021i101+i101+i002+i021+i040100 , where i followed by a subscript represents the integrated intensity of the particular bragg plane . crystallinity , therefore , represents the fraction of -cellulose represented by planes 002 and 021 present in a particular sample . the lignocellulosic composition of soybean hulls was determined with an ankom 200 fiber analyzer ( ankom technology , usa ) . neutral detergent fiber ( ndf ) , acid detergent fiber ( adf ) , and acid detergent lignin ( adl ) were analyzed per procedure specified by the manufacturer ( http://www.ankom.com ) . protein content ( n 6.25 ) was determined by the kjeldahl method after digestion and distillation with an autoanalyser ( leco fp-2000 , leco corporation , mi , usa ) . all moisture measurements were carried out using denver infrared moisture analyzer ( model ir35 ; fisher scientific , usa ) . crude cellulases were extracted from various production steps described in section 2.4 by adding 30 ml of citrate buffer ( 50 mm , ph 5 ) to each flask and shaking the contents at 150 rpm for 30 minutes . contents were filtered using coarse filter paper ( fisher scientific , p-8 coarse grade ) , and the filtrate obtained was centrifuged at 10,000 g for 15 minutes at 4c ( sorvall rc-6 , thermo scientific , usa ) . g - ds ) , endocellulase ( iu / g - ds ) , -glucosidase ( iu / enzyme activities were reported as units per gram of dry substrate ( g - ds ) . statistical analysis was carried out using the glm procedure in sas software version 9.1 ( sas institute , cary , nc , usa ) . effects of various pretreatments on compositional changes in soybean hulls are shown in table 1 . data is represented only to outline holocellulose ( cellulose + hemicellulose ) , lignin , protein , and ash content of soybean hulls , and not necessarily embody composition fully . soybean hulls are known to contain appreciable amount of pectin ( ~15% ) and lipids ( < 4% ) as well [ 38 , 39 ] . both acid and alkali pretreatments enriched the cellulosic fraction and extracted a small part of the hemicellulosic fraction . steam - pretreated soybean hulls , on the other hand , had a composition similar to that of native soybean hulls . an interesting finding was that holocellulosic content was fairly constant ( no significant difference , p < .05 ) across the spectrum of treatments used in this study ( table 1 ) . total cellulosic content may be useful to consider because both cellulose and hemicellulose are implicated in induction of cellulolytic enzyme complex . henceforth , subjecting soybean hulls to mild pretreatments preserved the holocellulosic composition of native soybean hulls . there was a substantial increase in the bed porosity ( table 2 ) , estimated at 70% mc , for pretreated soybean hulls compared with native soybean hulls . the increase in bed porosity is likely due to modification of the internal structure of soybean hulls that led to redistribution and partial solubilization of hemicellulose and swelling of the substrate . volumetric specific surface ( cm ) , on the other hand , was similar for pretreated and native soybean hulls . notably , volumetric specific surface measurements were the outcome of particle size analysis that accounted only for external surface area ; however , fibers have lumen characterized by hollow space . internal porosity that has capability of accommodating large enzyme molecules and leads to thorough digestibility . unfortunately , finding a simple technique to determine lumen internal surfaces is difficult , and volumetric specific surface incorporating external particle diameter is unable to capture the internal specific area , which characterizes microfibrillar spaces . this was evident in the current study when the volumetric specific surface of pretreated and untreated soybean hulls were not significantly different ( p < .05 ) . apparently , it seems essential to identify or modify current techniques that can easily implement rapid and routine analysis of internal surface area , and therefore warrants future investigations . wide - angle x - ray diffraction has been used extensively to measure the crystallinity of cellulosic substrates . crystallinity in the polymeric sample may be measured in several ways from an x - ray diffractogram ; the most common is the peak intensity method . the method requires amorphous material to diffract with the same intensity at 18 ( ~101 plane ) and 22 ( 002 plane ) , and does not account for peak shifting or overlap . further , this method assumes highest peak ( 002 ) as the only determinant of the cellulose crystallinity , which is certainly not the case as five planes have been identified responsible for the characteristic reflection . finally , lignocellulosic substrates contain appreciable amounts of hemicellulose and lignin that lead to diffusive x - ray scattering ( reflection ) , a hallmark of paracrystalline substances [ 44 , 45 ] . given these drawbacks of the peak intensity method , a sophisticated technique using deconvolution was successfully applied in our studies to x - ray spectra of both native and pretreated soybean hulls for crystallinity measurements . this method is relatively new in the arena of lignocellulosic biofuels research , although it is routinely used in polymer science research . the fitted x - ray diffractograms using voigt function are shown in figures 1(a)1(e ) for both native and pretreated soybean hulls . fit was assessed using r. almost all diffractograms using this scheme had r > 0.95 . also , featured in the table 2 r ) and root mean square error ( rmse ) of the fit . the higher value of adjusted r and lower rmse further confirmed the goodness of fit . notice the five peaks corresponding to identified lattice planes and gradual evolution of peaks in pretreated soybean hulls compared to native soybean hulls indicating increase in degree of crystallinity due to pretreatments . degree of crystallinity was calculated from ( 8) , and the values are listed in table 2 . the steam , acid , and alkali pretreatments all resulted in a significant increase in degree of crystallinity compared to native soybean hulls . the enhancement in crystallinity is due to enrichment in the -cellulose fraction in the pretreated samples due to reduction in the interlocking amorphous cellulosic chains and plausible correction in lattice defects of cellulose during pretreatments [ 46 , 47 ] . the -cellulose fraction is the crystalline cellulose of plant polymers and is responsible for the characteristic x - ray diffraction . additionally , due to the mild nature of pretreatments , enrichment in -cellulose fraction was possible by selective reduction of the amorphous phase . the outcome could have been different if harsh chemical pretreatments ( using high temperature and pressure ) were employed . production of a cellulolytic enzyme system was assessed through measurement of four leading activities : filter paper units ( fpu / g - ds ( dry substrate ) ) , -glucosidase ( iu / g - ds ) , endocellulase ( iu / g - ds ) , and xylanase ( iu / g - ds ) . inspection of figure 2 reveals that enzyme production in both mono and mixed cultures of t. reesei and a. oryzae was significantly reduced in alkali - pretreated soybean hulls compared to native , steam- , and acid - pretreated substrates . . stated that an important aspect of alkali pretreatment is that biomass itself consumes some of the alkali . as a result , changes brought about by alkali pretreatment can cause solubilization , distribution , and condensation of lignin and hemicellulose and modification of cellulosic structure . aiello et al . showed that alkali - pretreated sugarcane bagasse in liquid fermentation of t. reesei ( qm 9414 ) significantly decreased cellulase yield over untreated bagasse . cellulolytic enzyme production in hcl- and h2so4-pretreated soybean hulls was significantly ( p < .05 ) lower as well for both cultures compared to production in both native and steam - pretreated substrates . acid pretreatment of lignocellulosics is known to generate inhibitory compounds as result of sugar and lignin degradation during the treatments [ 50 , 51 ] . though the acid pretreatment may result in increased digestibility of lignocellulosic substrate , the inhibitory compounds have deleterious effects on enzyme and microbial activity . steam pretreatment resulted in significant ( p < .05 ) and substantial enhancement in production of all cellulolytic activities in t. reesei culture compared to production in untreated soybean hulls . the production of xylanase , though , was not significantly ( p < .05 ) different . steam - pretreated soybean hulls had about 4 fpu / g - ds compared with 0.75 fpu / g - ds in native and endocellulase of 45 iu / g - ds compared with 7.29 iu / g - ds in native . -glucosidase activity also improved significantly ( p < .05 ) in steam - pretreated compared with native soybean hulls . the preponderance of these results is apparent from the fact that both native and steam pretreated soybean hulls had compositional similarity ( table 1 ) but significantly different enzyme production ( figure 2 ) . this is a key indication that in ssf , in which fungal mycelium is in direct contact with the substrate particles , the physicochemical nature of the substrate is important in addition to its composition . in a. oryzae no significant differences ( p < .05 ) occurred in enzyme production between steam - pretreated and native soybean hulls except in endoglucanase levels . in steam - pretreated soybean hulls , a. oryzae produced a significantly higher amount of endoglucanase ( 47 iu / g - ds ) compared to that in native substrate ( 31 iu / g - ds ) . mixed culture had similar results as in a. oryzae , where production in steam - pretreated soybean hulls was not significantly different ( p < .05 ) compared to native soybean hulls ( figure 2 ) . the foregoing indicated steam pretreatment had disparities in enzyme production , which were both enzyme and culture specific . to relate the trends in enzyme production with physicochemical characteristics of the substrate in the two fungal cultures , t. reesei and a. oryzae , additional statistical analysis was performed . the interaction of crystallinity and porosity was modeled using the general linear model of sas with the following expression : ( 9)yijk=+abij+ijk , where is one of the enzyme activities as the dependent variable , is the grand mean ( n = 4 ) , abij is the interaction effect of crystallinity and porosity , and ijk is random error with mean 0 and experimental error variance as its variance . both composition ( holocellulose ) and volumetric specific surface were excluded as they were nearly constant across pretreatments ( tables 1 and 2 ) . in addition , only native and steam pretreated substrates were considered in our analysis because enzyme production in acid- and alkali - pretreated substrates was lower due to their inhibitory effects on microbial propagation . crystallinity and porosity were considered together because both of them were simultaneously altered when substrates were subjected to pretreatments . broadly speaking , the model represented by ( 9 ) is more reflective of one - way variance analysis than factorial variance analysis . examination of data ( table 3 ) shows that for t. reesei , with an increase in crystallinity and porosity due to steam pretreatment , all cellulolytic enzyme activities increased significantly except xylanase . in a. oryzae fermentation , significant improvement was noticed only in endoglucanase production whereas , in mixed culture fermentation , significant decrease occurred in filter paper units at p < .01 and endoglucanase at p < .05 as a result of increased crystallinity and porosity . it is plausibly implicated in the propagation of fungal cultures and , therefore , affects enzyme production . [ 8 , 9 ] explained this phenomenon by using various model substrates that differed in the amount of open spaces for production of -amylase in solid - state cultures of a. oryzae and explicitly showed that model substrates with more porous structure had better enzyme production compared to less porous substrates . therefore , decrease in filter paper and endoglucanases activities in mixed culture compared to t. reesei could be attributed to another factor that is , increase in crystallinity . it is apparent from the literature that t. reesei cellulases are particularly active towards crystalline cellulose [ 20 , 52 , 53 ] ; however , enzymes from aspergillus spp . mixed culture fermentation wherein a. oryzae was dominant , filter paper and endocellulase activities were reduced due to the inability of a. oryzae to digest crystalline substrate . this is further confirmed by observing the data of a. oryzae fermentation , where no improvement in cellulolytic activities in steam - pretreated soybean hulls over native substrate was observed except in endoglucanase activity . evidently , results highlighted that effect of crystallinity was specific for type of culture as it brought enhancement in cellulolytic activities of t. reesei , and this enhancement was not particularly observed in a. oryzae . the analysis also showed that within the spectrum of cellulolytic activities studied not all activities got altered on exposure to crystalline substrate . the results are interesting in view of the fact that pretreatments due to their ability to induce changes in physicochemical attributes resulted in altered enzyme production in fungal ssf of soybean hulls . for the first time , current work demonstrated that mild pretreatment methods could significantly alter the physicochemical attributes of the substrate ( soybean hulls ) without significant changes in holocellulosic composition . the altered physicochemical attributes due to pretreatment had significant effects on the production of cellulolytic enzyme activities , and these effects were both culture and enzyme specific . a sophisticated deconvolution method was used to determine x - ray crystallinity from raw diffractograms of both treated and untreated substrates . this method takes into account diffusive scattering due to paracrystalline nature of celluloses found in plant material , and therefore provides consistent and reliable measurements . steam pretreatment significantly increased both porosity and crystallinity of soybean hulls , and production of all the three cellulase activities in t. reesei culture ( i.e. , filter paper , -glucosidase , and endocellulase ) compared to untreated substrate . xylanase production , however , remained unaltered . while using a. oryzae culture , significant improvement was observed only in endocellulase whereas in the mixed culture fermentation , filter paper , and endocellulase activities decreased in steam - pretreated soybean hulls . further study of porosity and crystallinity and their effects on enzyme production is necessary if we are to understand fully the effects of physiochemical attributes . our studies highlighted the effects of pretreatment methods , changes in the physiochemical characteristics of substrates , and choice of fungal culture in ssf on enzyme production . experimental methods to enhance enzyme production are imperative for the success of the biofuels industry , which uses enzymatic and microbial fermentation platform .

we investigated the effect of pretreatment on the physicochemical characteristics crystallinity , bed porosity , and volumetric specific surface of soybean hulls and production of cellulolytic enzymes in solid - state fermentation of trichoderma reesei and aspergillus oryzae cultures . mild acid and alkali and steam pretreatments significantly increased crystallinity and bed porosity without significant change inholocellulosic composition of substrate . crystalline and porous steam - pretreated soybean hulls inoculated with t. reesei culture had 4 filter paper units ( fpu)/g - ds , 0.6 iu / g - ds -glucosidase , and 45 iu / g - ds endocellulase , whereas untreated hulls had 0.75 fpu / g - ds , 0.06 iu / g - ds -glucosidase , and 7.29 iu / g - ds endocellulase enzyme activities . in a. oryzae steam - pretreated soybean hulls had 47.10 iu / g - ds endocellulase compared to 30.82 iu / g - ds in untreated soybean hulls . generalized linear statistical model fitted to enzyme activity data showed that effects of physicochemical characteristics on enzymes production were both culture and enzyme specific . the paper shows a correlation between substrate physicochemical properties and enzyme production .

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

solid - state fermentation ( ssf ) offers a low - cost alternative for producing cellulases using natural polymers derived from agroindustrial residues [ 3 , 4 ] . ssf is defined as a discrete solid phase in which microorganisms grow on the surface of moist particles as well as inside and between them . gas phase in ssf is strongly affected by the size , shape , and tortuosity of a network of gas - filled pores . the air- or gas - filled pores are referred to as bed porosity , which is defined as the volume of gas contained in the system at any given time ( void fraction ) . showed that by blending rice bran with wheat bran resulted in substantial improvement in the morphology of rice bran which improved protease production during solid - state culturing of a. oryzae . the increase in bed porosity of the substrate could be the reason behind improved production ; however , no attempts were made to measure bed porosity to show its relationship to enzyme production . several authors in the past have suggested the merits of open porous solid beds but no explicit investigation has been conducted yet that relates bed porosity with enzyme production in ssf . in industrial scale ssf processes , bed porosity is essential but not sufficient for complete process control . other parameters , such as microbial cell physiology , composition of the solid substrate , and substrate reactivity also could influence the productivity of the process [ 11 , 12 ] . at microfibril level it is crystallinity of cellulose , and at fiber level it is specific surface area ( characterizing pore size or degree of swelling ) [ 1315 ] . the increase in cellulase reactivity due to increase in specific surface area is attributed to the creation of surface openings or internal slits , voids , or spaces , by the removal of cell wall components , that enhances the direct physical contact between the enzymes and the substrate . showed that crystalline - cotton - induced cellulolytic complex derived from submerged t. reesei cultures exhibited higher potential in hydrolyzing crystalline cellulose than solka - floc - induced cellulases . [ 22 , 23 ] , and , more recently , ciolacu et al . thus , studies delineating the effects of crystallinity on enzyme production in ssf are of significant interest . however , pretreatments are known to induce structural changes in cellulosic substrates , which could alter the physicochemical properties of the substrate . the effect of pretreatment methods on physicochemical characteristics of substrate and its repercussions on cellulolytic enzyme productivity in fungal solid - state fermentation has not been investigated so far , which is evident from the recent reviews on ssf [ 3 , 27 ] . an in - depth understanding of the role of physicochemical characteristics of substrate on cellulase production in ssf would provide a framework for comprehensive analysis of critical design issues that should facilitate cellulase production with enhanced biocatalysis . the present study aimed to determine the role of pretreatment techniques in altering the physicochemical characteristics bed porosity , volumetric specific surface , and crystallinity of solid - state substrate . in addition , the effect of change in physicochemical attributes on enzyme production in fungal solid - state fermentation was studied with respect to type of fungal species and different cellulolytic enzyme activities . the pretreatments were carefully chosen to limit the effect on the chemical compositional changes of solid substrate , which would otherwise diminish the role of physicochemical attributes . since crystallinity is critical to this study , a new method of measuring crystallinity of complex cellulosic substrate untreated ground soybean hulls ( purchased from archer daniels midland , salina , ks , usa ) , herein referred to as native soybean hulls , had a geometric mean diameter , dgw , of 0.61 0.002 mm . native soybean hulls were subjected to four different treatments before being used for production of the cellulolytic enzyme system : ( 1 ) steam pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in distilled water was pressure cooked at 121c for 60 min ; ( 2 ) hydrochloric acid pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in 1 n hcl was kept on a gyratory shaker ( 150 rpm ) for 24 h at ambient temperature ; ( 3 ) sulfuric acid pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in 1 n h2so4 was kept on a gyratory shaker ( 150 rpm ) for 24 h at ambient temperature ( 4 ) sodium hydroxide pretreatment , in which a 5% ( w / v ) slurry of soybean hulls in 1 n naoh was kept on a gyratory shaker ( 150 rpm ) for 24 h at ambient temperature . after acid and alkali pretreatments , treated soybean hulls were collected by filtration and extensively washed with distilled water . all treated substrates were dried overnight at 45c in a forced - draft oven ( fisher scientific , usa ) . dried substrates were used for compositional analysis , analysis of physicochemical characteristics , and production of enzymes . two fungal cultures t. reesei ( atcc 26921 ) and a. oryzae ( atcc 12892 ) were used for ssf of native and pretreated soybean hulls . native and pretreated dried soybean hulls ( 5 g ) were adjusted to 70% ( wet basis ) moisture content ( mc ) by using mandels media of ph 5 and were sterilized in a vertical sterilizer ( 121c/15 psi gauge ) for 30 minutes . the conditions of temperature , ph , moisture ( 70% ) , and incubation days of the ssf process used in this study were optimized previously . volume ( v , cm ) was calculated from the following relationship : ( 2)v=(mpsmp)(mptsmt)tol , where mt is mass of the pycnometer filled with toluene , mps is the mass of pycnometer and sample , mp is mass of the pycnometer , mpts is mass of the pycnometer filled with toluene and sample , and tol is the density of toluene . volumetric specific surface is defined as external surface area per unit volume of the samples . weight of oversize generated during sieving was used to compute geometric mean diameter ( dgw ) and geometric standard deviation ( sgw ) according to the following equations : ( 4)dgw = log 1 ( (wilog di)wi)sgw = log 1 ( [wi(log dilog dgw)2]wi ) , where di is the diameter of the ith sieve in the stack and wi is the weight fraction on the ith sieve . volumetric specific surface ( sa , cm ) can then be obtained from ( 5a ) by multiplying it with specific weight ( ) ( g / cm ) , that is , ( 5b)sa ( cm1)=svexp ( 0.5ln 2 sgwln dgw ) , where s is the shape coefficient for calculating surface area of particles ( fixed at 6 ) and v is the shape coefficient for calculating volume of particles ( fixed at 1 ) . , tx , usa ) was used to estimate the crystallinity of native and pretreated soybean hulls . using the voigt function intensity of the reflection is represented by following equation : ( 7)f(2)=ao(exp ( (2)2)/(al2+((xac)/ag2)2))d(2)(exp ( ((2)2))/(al2+(2)))d(2 ) , where ao is the amplitude of the peak , ac is the center of the peak , al is the width of the lorentzian component , and ag is the width of the gaussian component of the peak . crystallinity , therefore , represents the fraction of -cellulose represented by planes 002 and 021 present in a particular sample . the lignocellulosic composition of soybean hulls was determined with an ankom 200 fiber analyzer ( ankom technology , usa ) . contents were filtered using coarse filter paper ( fisher scientific , p-8 coarse grade ) , and the filtrate obtained was centrifuged at 10,000 g for 15 minutes at 4c ( sorvall rc-6 , thermo scientific , usa ) . g - ds ) , endocellulase ( iu / g - ds ) , -glucosidase ( iu / enzyme activities were reported as units per gram of dry substrate ( g - ds ) . effects of various pretreatments on compositional changes in soybean hulls are shown in table 1 . data is represented only to outline holocellulose ( cellulose + hemicellulose ) , lignin , protein , and ash content of soybean hulls , and not necessarily embody composition fully . both acid and alkali pretreatments enriched the cellulosic fraction and extracted a small part of the hemicellulosic fraction . steam - pretreated soybean hulls , on the other hand , had a composition similar to that of native soybean hulls . total cellulosic content may be useful to consider because both cellulose and hemicellulose are implicated in induction of cellulolytic enzyme complex . henceforth , subjecting soybean hulls to mild pretreatments preserved the holocellulosic composition of native soybean hulls . there was a substantial increase in the bed porosity ( table 2 ) , estimated at 70% mc , for pretreated soybean hulls compared with native soybean hulls . the increase in bed porosity is likely due to modification of the internal structure of soybean hulls that led to redistribution and partial solubilization of hemicellulose and swelling of the substrate . volumetric specific surface ( cm ) , on the other hand , was similar for pretreated and native soybean hulls . notably , volumetric specific surface measurements were the outcome of particle size analysis that accounted only for external surface area ; however , fibers have lumen characterized by hollow space . unfortunately , finding a simple technique to determine lumen internal surfaces is difficult , and volumetric specific surface incorporating external particle diameter is unable to capture the internal specific area , which characterizes microfibrillar spaces . this was evident in the current study when the volumetric specific surface of pretreated and untreated soybean hulls were not significantly different ( p < .05 ) . apparently , it seems essential to identify or modify current techniques that can easily implement rapid and routine analysis of internal surface area , and therefore warrants future investigations . the method requires amorphous material to diffract with the same intensity at 18 ( ~101 plane ) and 22 ( 002 plane ) , and does not account for peak shifting or overlap . further , this method assumes highest peak ( 002 ) as the only determinant of the cellulose crystallinity , which is certainly not the case as five planes have been identified responsible for the characteristic reflection . given these drawbacks of the peak intensity method , a sophisticated technique using deconvolution was successfully applied in our studies to x - ray spectra of both native and pretreated soybean hulls for crystallinity measurements . the fitted x - ray diffractograms using voigt function are shown in figures 1(a)1(e ) for both native and pretreated soybean hulls . notice the five peaks corresponding to identified lattice planes and gradual evolution of peaks in pretreated soybean hulls compared to native soybean hulls indicating increase in degree of crystallinity due to pretreatments . the steam , acid , and alkali pretreatments all resulted in a significant increase in degree of crystallinity compared to native soybean hulls . production of a cellulolytic enzyme system was assessed through measurement of four leading activities : filter paper units ( fpu / g - ds ( dry substrate ) ) , -glucosidase ( iu / g - ds ) , endocellulase ( iu / g - ds ) , and xylanase ( iu / g - ds ) . inspection of figure 2 reveals that enzyme production in both mono and mixed cultures of t. reesei and a. oryzae was significantly reduced in alkali - pretreated soybean hulls compared to native , steam- , and acid - pretreated substrates . as a result , changes brought about by alkali pretreatment can cause solubilization , distribution , and condensation of lignin and hemicellulose and modification of cellulosic structure . showed that alkali - pretreated sugarcane bagasse in liquid fermentation of t. reesei ( qm 9414 ) significantly decreased cellulase yield over untreated bagasse . cellulolytic enzyme production in hcl- and h2so4-pretreated soybean hulls was significantly ( p < .05 ) lower as well for both cultures compared to production in both native and steam - pretreated substrates . steam pretreatment resulted in significant ( p < .05 ) and substantial enhancement in production of all cellulolytic activities in t. reesei culture compared to production in untreated soybean hulls . steam - pretreated soybean hulls had about 4 fpu / g - ds compared with 0.75 fpu / g - ds in native and endocellulase of 45 iu / g - ds compared with 7.29 iu / g - ds in native . -glucosidase activity also improved significantly ( p < .05 ) in steam - pretreated compared with native soybean hulls . the preponderance of these results is apparent from the fact that both native and steam pretreated soybean hulls had compositional similarity ( table 1 ) but significantly different enzyme production ( figure 2 ) . in a. oryzae no significant differences ( p < .05 ) occurred in enzyme production between steam - pretreated and native soybean hulls except in endoglucanase levels . in steam - pretreated soybean hulls , a. oryzae produced a significantly higher amount of endoglucanase ( 47 iu / g - ds ) compared to that in native substrate ( 31 iu / g - ds ) . mixed culture had similar results as in a. oryzae , where production in steam - pretreated soybean hulls was not significantly different ( p < .05 ) compared to native soybean hulls ( figure 2 ) . the foregoing indicated steam pretreatment had disparities in enzyme production , which were both enzyme and culture specific . to relate the trends in enzyme production with physicochemical characteristics of the substrate in the two fungal cultures , t. reesei and a. oryzae , additional statistical analysis was performed . the interaction of crystallinity and porosity was modeled using the general linear model of sas with the following expression : ( 9)yijk=+abij+ijk , where is one of the enzyme activities as the dependent variable , is the grand mean ( n = 4 ) , abij is the interaction effect of crystallinity and porosity , and ijk is random error with mean 0 and experimental error variance as its variance . both composition ( holocellulose ) and volumetric specific surface were excluded as they were nearly constant across pretreatments ( tables 1 and 2 ) . in addition , only native and steam pretreated substrates were considered in our analysis because enzyme production in acid- and alkali - pretreated substrates was lower due to their inhibitory effects on microbial propagation . examination of data ( table 3 ) shows that for t. reesei , with an increase in crystallinity and porosity due to steam pretreatment , all cellulolytic enzyme activities increased significantly except xylanase . in a. oryzae fermentation , significant improvement was noticed only in endoglucanase production whereas , in mixed culture fermentation , significant decrease occurred in filter paper units at p < .01 and endoglucanase at p < .05 as a result of increased crystallinity and porosity . it is plausibly implicated in the propagation of fungal cultures and , therefore , affects enzyme production . [ 8 , 9 ] explained this phenomenon by using various model substrates that differed in the amount of open spaces for production of -amylase in solid - state cultures of a. oryzae and explicitly showed that model substrates with more porous structure had better enzyme production compared to less porous substrates . therefore , decrease in filter paper and endoglucanases activities in mixed culture compared to t. reesei could be attributed to another factor that is , increase in crystallinity . it is apparent from the literature that t. reesei cellulases are particularly active towards crystalline cellulose [ 20 , 52 , 53 ] ; however , enzymes from aspergillus spp . mixed culture fermentation wherein a. oryzae was dominant , filter paper and endocellulase activities were reduced due to the inability of a. oryzae to digest crystalline substrate . this is further confirmed by observing the data of a. oryzae fermentation , where no improvement in cellulolytic activities in steam - pretreated soybean hulls over native substrate was observed except in endoglucanase activity . evidently , results highlighted that effect of crystallinity was specific for type of culture as it brought enhancement in cellulolytic activities of t. reesei , and this enhancement was not particularly observed in a. oryzae . the analysis also showed that within the spectrum of cellulolytic activities studied not all activities got altered on exposure to crystalline substrate . the results are interesting in view of the fact that pretreatments due to their ability to induce changes in physicochemical attributes resulted in altered enzyme production in fungal ssf of soybean hulls . for the first time , current work demonstrated that mild pretreatment methods could significantly alter the physicochemical attributes of the substrate ( soybean hulls ) without significant changes in holocellulosic composition . the altered physicochemical attributes due to pretreatment had significant effects on the production of cellulolytic enzyme activities , and these effects were both culture and enzyme specific . this method takes into account diffusive scattering due to paracrystalline nature of celluloses found in plant material , and therefore provides consistent and reliable measurements . steam pretreatment significantly increased both porosity and crystallinity of soybean hulls , and production of all the three cellulase activities in t. reesei culture ( i.e. , filter paper , -glucosidase , and endocellulase ) compared to untreated substrate . while using a. oryzae culture , significant improvement was observed only in endocellulase whereas in the mixed culture fermentation , filter paper , and endocellulase activities decreased in steam - pretreated soybean hulls . further study of porosity and crystallinity and their effects on enzyme production is necessary if we are to understand fully the effects of physiochemical attributes . our studies highlighted the effects of pretreatment methods , changes in the physiochemical characteristics of substrates , and choice of fungal culture in ssf on enzyme production .

protozoan parasites , such as leishmania , trypanosoma , and plasmodium species are the cause of a large array of diseases hampering the lives of people all over the world . control of such diseases depends on a rather small number of prophylactic or therapeutic antiparasite drugs , many of which are highly toxic and/or inefficient [ 25 ] . in addition , an increasing number of parasites develop resistance towards several of the frontline drugs [ 69 ] . this has created an urgent need for novel compounds to prevent and cure diseases caused by protozoan parasites . species - specific inhibition of parasitic enzymes has been suggested as one promising approach in the development of new therapeutics . one family of enzymes that have attracted considerable interest as potential targets for antiparasitic therapeutics are the dna topoisomerases ( topos ) [ 1 , 11 ] of which the human counterparts are well - known targets in anticancer treatment . dna topos are ubiquitous enzymes needed to overcome the topological stress arising in dna during replication , transcription , recombination , and repair . this is achieved by the enzymes introducing transient breaks in the dna in a reaction that restores the energy of the broken phosphodiester bond in a covalent phosphotyrosyl cleavage intermediate . based on their mechanism of action topos are divided into two main classes [ 13 , 14 ] . the type i topos are with few exceptions monomers and relax dna by breaking only one strand of the double helix , while type ii topos are mainly homodimers or heterotetramers and break both strands of the dna simultaneously . type i topos are further classified into two structurally unrelated families denoted the type ia and type ib topos , defined by the polarity of their strand cleavage . the type ia topos are prevalent in prokaryotic species and create a 5-phoshotyrosyl linkage and a free 3-oh dna end during cleavage . type ib topos are mainly found in eukaryotic species and generate a 3-phosphotyrosyl linkage and a free 5-oh dna end during cleavage . the class of type ii topos are subdivided into the type iia and type iib families , of which all members are structurally related and characterized by the formation of a 5-phoshotyrosyl linkage and a free 3-oh dna end during cleavage . typically the eukaryotic members of this group are homo - dimers while the prokaryotic enzymes are heterotetramers . the type iib group encompasses topovi of extreme thermophilic archaebacteria . besides their important biological functions , dna topos from the various groups are well - known targets of both antibacterial and anticancer therapeutic agents . hence , bacterial type iia topos , such as dna gyrase and topoiv , are targets of clinically important antibiotics active against a wide spectrum of bacterial pathogens [ 16 , 17 ] . human type ib and iia topos are targets of several anticancer compounds , exemplified by camptothecin and etoposide , respectively , of which synthetic derivatives are routinely used in systemic treatment of different cancer types [ 12 , 18 ] . of relevance for the treatment of protozoan - caused infectious diseases , structural and/or subtle mechanistic differences between protozoan and host topos can be exploited for the rational design of novel therapeutic compounds . indeed , the unusual heterodimeric topoib of kinetoplastid parasites , such as leishmania donovani gives hope for the development of drugs targeting parasite topoib without interfering with the function of the monomeric topoib in the human host [ 11 , 19 , 20 ] . as another example , the apicomplexan parasite plasmodium falciparum contains apicoplast dna , which requires bacterial - type dna gyrases ( type iia topo ) for replication , thus providing a unique drug target absent in the host [ 21 , 22 ] . in addition , the high expression rate of topoib and topoiia in rapidly growing parasites , compared to the expression levels of these enzymes in the host , may be exploited for the development of topo - targeting protocols that specifically kill the parasites . synthetic peptides have been prophesied to be the ideal inhibitors of enzyme activity either alone or in combination with small - molecule drugs [ 10 , 23 ] . however , high synthesis costs and great challenges regarding delivery , intracellular targeting and clearance half - life of peptides have until recently hampered the interest of most pharmaceutical companies in developing peptide - based drugs . new efficient synthesis strategies and low monomer prices have led to a renewed interest in therapeutic peptides . indeed , compared to small - molecule drugs , which are currently dominating the pharmaceutical market , peptide - based therapeutics offer several advantages , such as high specificity , lower accumulation in tissues , lower toxicity , and biological diversity [ 2427 ] . the potential for synthetic peptides as efficient species - specific inhibitors even of discrete steps of topo catalysis is highlighted in several studies by nagaraja and co - workers describing the identification and characterization of species - specific antibodies with inhibitory activities against particular steps of mycobacteria dna gyrase or topoi catalysis [ 2830 ] . peptides with similar inhibitory activities and potential in future antituberculosis treatment are likely to be derived from such antibodies [ 3134 ] . relevant for the potential development of peptide - based drugs targeting eukaryotic topos , almost a decade ago , segall and co - workers identified a series of hexapeptides inhibiting various catalytic steps of the tyrosine recombinases ( bacteriophage -int and cre ) [ 35 , 36 ] . tyrosine recombinases share so important structural and mechanistic features with the type ib topos that they can be considered a subbranch of the type ib topo family [ 37 , 38 ] . it was therefore not surprising , that several of the hexapeptides selected on basis of -int inhibition also inhibited dna relaxation by the type ib topo of vaccinia virus ( vvtopoi ) , although less potently . a rescreening of the peptide combinatorial library ( used for selection of the above - mentioned peptides ) specifically against vvtopoi resulted in the identification of three new peptides , wycrck , kccrck , and wrwycrck with high activity against this enzyme . of these , wrwycrck was the most potent inhibitor of the type ib topos tested . this peptide inhibited vvtopoi with an ic50 value of 0.10.25 m and -int with an ic50 value of 0.015 m , while the structurally unrelated type ia topo , e. coli topoi was inhibited only to a limited extent ( ic50 value of 5.5 m ) . using these peptides as a starting point , it may in longer terms be possible to develop peptide - based topoi targeting inhibitors with therapeutic activity against protozoan pathogens . as an initial investigation of this possibility , we address , in the present study , the effect of the peptides wycrck , kccrck , and wrwycrck on the activity of the recently cloned and purified recombinant topoi ( pftopoi ) from the malaria - causing parasite plasmodium falciparum . we find that wrwycrck inhibits dna relaxation and cleavage by pftopoi whereas neither wycrck nor kccrck have any effect on pftopoi activity . molecular docking of the three peptides in the noncovalent pftopoi - dna complex shows wrwycrck to be located in the minor groove of the dna in proximity of the enzyme active site , while wycrck and kccrck are positioned far from the enzyme active site . the plasmid , ppft100 ( the cloning of pftopoi is to be published elsewhere ) , containing the pftopoi gene ( plasmodb accession number pfe0520c ) ( codon optimized for expression in s. cerevisiae ( geneart , germany ) ) , was transformed into the yeast s. cerevisiae top1 strain rs190 ( a kind gift from r. sternglanz , state university of new york , stony brook , ny , usa ) according to standard procedures , and pftopoi enzyme was expressed and purified as previously described for human topoisomerase i ( htopoi ) . u is the amount of enzyme needed to fully relax 200 fmol of negatively supercoiled pbr322 plasmid dna at 37c in 30 min in 10 mm tris ( ph 7.5 ) , 1 mm edta , 150 mm nacl , 5 mm mgcl2 and 5 mm cacl2 . wycrck , kccrck , and wrwycrck were purchased from genscript usa inc . , usa . the lyophilized peptides were dissolved in h2o . dna relaxation reactions included 1 u pftopoi in the absence or presence of peptide ( wycrck , kccrck , or wrwycrck ) at the following concentrations : 1.3 m , 2.5 m , 5 m , 7.5 m , 12.5 m , 25 m , or 50 m and 200 fmol negatively supercoiled pbr322 plasmid in 20 l of 10 mm tris ( ph 7.5 ) , 1 mm edta , 150 mm nacl , 5 mm mgcl2 and 5 mm cacl2 . the plasmid was preincubated with the peptide for 5 min at 37c prior to addition of enzyme . reactions were incubated at 37c for 30 min before being stopped by addition of 0.2% ( w / v ) sds and proteolytically digested with 0.5 g / ml proteinase k for another 30 min at 37c . samples were subjected to gel electrophoresis on 1% agarose gels in tbe buffer , and dna bands were stained with ethidium bromide and visualized by illumination with uv light . oligonucleotides for assembly of dna suicide cleavage substrates and dna ligation substrates were purchased from dna technology , denmark and purified by denaturing polyacrylamide gel electrophoresis . the sequences of the substrates are as follows : ol19 : 5-gcc tgc agg tcg act cta gag gat cta aaa gac tta ga-3 , ol27 : 5-aaa aat ttt tct aag tct ttt aga tcc tct aga gtc gac ctg cag gc-3 , and ol36 : 5-aga aaa att ttt-3. the oligonucleotide representing the scissile strand ( ol19 ) was 5-radiolabeled by t4 polynucleotide kinase ( new england biolabs , usa ) using [ -p ] atp as the phosphoryl donor . to prevent ligation of the 5-oh from the bottom strand ( ol27 ) , these ends were 5-phosphorylated with unlabeled atp . the oligonucleotides were annealed pairwise with a 2-fold molar excess of the bottom strand over scissile strand as previously described . the cleavage reactions were carried out in 20 l reaction volumes by incubating 20 nm of the duplex ol19/ol27 with 500 fmol of pftopoi or htopoi enzyme at 37c , in 20 mm tris ( ph 7.5 ) , 10 mm mgcl2 , and 10 mm cacl2 . the dna substrate was preincubated with peptide wrwycrck at concentrations varying from 0 to 75 m for 5 min at 37c prior to addition of enzyme . after 30 minutes of incubation , the reactions were stopped with 0.1% ( w / v ) sds . for the ligation reactions , 20 nm ol19/ol27 was incubated with 500 fmol of pftopoi for 30 min at 37c in 10 mm tris ( ph 7.5 ) , and 5 mm mgcl2 , 5 mm cacl2 . after preincubation of the cleavage samples with the peptide at concentrations varying from 0 to 12.5 m for 5 min at 37c , ligation was initiated by the addition of a 200-fold molar excess of oligonucleotide ol36 over the duplex ol19/ol27 . samples were incubated at 37c for 60 min , and reactions were stopped with 0.1% ( w / v ) sds . cleavage and ligation samples were precipitated with ethanol , resuspended in 10 l of 1 mg / ml trypsin , and incubated at 37c for 30 min . reaction products were analyzed by gel electrophoresis on 12% denaturing polyacrylamide gels , and radioactive bands were visualized by phosphorimaging . densitometric quantification of gel bands was performed using quantity one v4.6.3 software ( bio - rad , usa ) . the relative cleavage was calculated by the following equation : relative cleavage = ( ic bi)/(ic bi + is bi ) , where ic denotes the intensity of the band(s ) representing the cleavage product(s ) , is denotes the intensity of the band representing the substrate , and bi denotes the background intensity . restriction digests were performed in 20 l reaction volumes by incubating 3 g puc19 plasmid with ecori or pvuii ( both from new england biolabs ) in the reaction buffers provided by the manufacturer in the absence or presence of peptide wrwycrck ( 12.5 m , 25 m , or 50 m ) . the plasmid was preincubated with the peptide for 5 min at 37c prior to addition of enzyme . for both restriction endonucleases , the lowest amount of enzyme , able to fully digest the plasmid within the timeframe of the experiment , was used . reactions were incubated at 37c for 30 min before being stopped by addition of 0.2% ( w / v ) sds and proteolytically digested with 0.5 g / ml proteinase k for another 30 min at 37c . samples were subjected to gel electrophoresis on 1% agarose gels in tbe buffer , and dna bands were stained with ethidium bromide and visualized by illumination with uv light . the three - dimensional structure for residues pro140-phe839 of pftopoi was obtained through homology modeling using the swissmodel server and the crystal structure of htopoi ( 1k4 t and 1a36 pdbs ) as a template [ 45 , 46 ] . the alignment was performed with the tcoffee server , using the sequences having the swissprot code q26024 and p11387 for the pftopoi and htopoi protein , respectively . the 22-base - pair dna present in the noncovalent htopo1-dna complex crystal structure 1k4s was fitted into the putative pftopoi active site in the 3d protein model to obtain the pftopoi - dna noncovalent complex that was used for the docking experiment . the bases are numbered from 1 to 22 starting from the 5 end of the scissile strand and from 23 to 44 starting from the 5 end of the intact strand . the structure of the octapeptide wrwycrck and of the two hexapeptides wycrck and kccrck was designed with the sybyl v. 6.0 program ( tripos , http://www.tripos.com/ ) creating a disulphide bond between the two cys3 and cys5 cysteines in all the peptides ( this was done since the experimental data confirmed that disulfide bridging was necessary for the inhibitory effect of the peptide ) . the structure of the peptides was minimized in vacuum using the powell algorithm implemented in the sybyl program and then simulated in a rectangular box filled with water molecules using the gromacs 4.0 package for 2 ns in order to regularize the structure . 250 docking runs were performed using the autodock 4.2 program using the lamarckian genetic algorithm . the structures of the ligands ( wrwycrck , wycrck , or kccrck ) and the receptor ( pftopoi - dna complex ) were first prepared using the autodocktools v. 1.5.2 suite , building a cubic box able to contain the cap and cat domains of the protein and the dna bases . the contacts between the ligand and the receptor were identified using a cutoff of 3.5 applying a modified version of the g_mindist tool present in the gromacs 3.3.3 package for molecular dynamics analysis . the inhibitory potency of the peptides wycrck , kccrck , and wrwycrck on pftopoi activity was investigated in a standard plasmid relaxation assay . the assay was performed with the minimum amount of pftopoi that sufficed to fully relax the plasmid dna ( i.e. , convert fast - mobility supercoiled plasmid to slow - mobility relaxed plasmid forms ) in the absence of added peptide within the timeframe ( 30 min ) used in the experiment ( data not shown ) . as evident from figure 1 , the peptide wrwycrck inhibited pftopoi relaxation activity in a dose - dependent manner , with an ic50 of 2.55 m . the peptides wycrck , kccrck had no effect on the relaxation activity of pftopoi , even at concentrations up to 50 m . moreover , consistent with previous reports of inhibition of vvtopoi by wrwycrck , the peptide only retained its inhibitory effect in the absence of dtt ( data not shown ) , suggesting that the active form of the peptide involves disulfide bridging . dna relaxation by type ib topos involves two discrete transesterification reactions that is , a cleavage reaction , in which the active site tyrosine attacks the phosphodiester bond of the dna backbone to generate a 3-phosphotyrosyl cleavage intermediate and a free 5-oh end , and a ligation reaction in which the 5-oh acts as a nucleophile on the phosphotyrosyl bond to restore intact dna . it was previously demonstrated that the inhibitory effect of the peptides wycrck , kccrck , and wrwycrck on dna relaxation by vvtopoi and -int could be ascribed to a specific inhibition of the cleavage and not the ligation step of catalysis [ 39 , 40 ] . to address which steps of pftopoi catalysis are affected by peptide wrwycrck , that inhibited relaxation by this enzyme we used a synthetic partially single - stranded suicide dna substrate containing a preferred type ib topoi cleavage sequence . this substrate , that was originally developed to investigate cleavage by htopoi , acts as a mechanism - based inactivator of nuclear type ib topos by allowing dna cleavage , while the subsequent religation reaction is prevented due to diffusion of the generated 5-oh end ( see figure 2(a ) ) . prevention of religation , however , is only conditional and this step of catalysis can be initiated by the addition of a surplus of a 5-oh - containing ligator strand with a sequence matching the protruding noncleaved strand of the generated cleavage complexes ( figure 2(a ) ) . first , the ability of pftopoi with cleave the suicide dna substrate was tested in comparison to cleavage by htopoi . the two enzymes were incubated with substrate radiolabeled at the 5-end of the cleaved strand ( to allow visualization of the cleavage products ) , the products were ethanol precipitated , trypsinated , and separated on a denaturing polyacrylamide gel prior to visualization by phosphorimaging . as evident from figure 2(b ) , pftopoi cleaved the substrate and gave rise to cleavage products ( marked cl1 and cl2 ) with approximately the same gel electrophoretic mobilities as those of cleavage products generated by htopoi ( compare lanes 1 and 2 ) . these products were retained in the slot of the gel if trypsin digestion was omitted ( data not shown ) , confirming their identity as covalent pftopoi - dna or htopoi - dna complexes . as previously reported in , even after trypsin digestion , the cleavage products of both pftopoi and htopoi were retarded in the gel due to the covalent attachment of short protease - resistant peptides to the radiolabeled strand of the dna substrate . for htopoi , the major cleavage product cl1 was previously demonstrated to result from cleavage at the preferred site ( indicated by an arrow in figure 2(a ) ) , while the minor cl2 product arises from cleavage two nucleotides upstream to the cleavage site . the gel electrophoretic mobility of cleavage products generated by pftopoi suggests that this enzyme cleaves the utilized substrate at the same positions as does htopoi . to test the effect of peptide wrwycrck on pftopoi mediated cleavage , increasing concentrations of the peptide were incubated with the above - described suicide dna substrate prior to addition of pftopoi . the reactions were performed essentially as described above and the percentage of substrate converted to cleavage product shown as a function of peptide concentration ( figure 2(c ) ) . as previously reported for vvtopoi and -int , peptide wrwycrck inhibited dna cleavage by pftopoi in a dose - dependent manner , although the observed cleavage inhibition was less potent than that observed for dna relaxation . using the suicide substrate system , the effect of peptide wrwycrck on pftopoi - mediated religation was investigated . in this experiment , preformed cleavage complexes were incubated with increasing concentrations of wrwycrck prior to addition of the ligator strand shown in figure 2(a ) . consistent with previous results obtained for vvtopoi and -int the peptide did not affect ligation by pftopoi ( figure 2(d ) ) . the three peptides tested for activity against pftopoi in the present study were previously demonstrated to inhibit vvtopoi and -int activity with ic50 's of 0.0152.3 m , while more unrelated enzyme activities such as e. coli type ia topo and restriction endonucleases were hardly affected by any of the peptides . although far from being species - specific , the peptide inhibitors appear rather sensitive to even subtle structural differences between the different target enzymes . this is evident from the different inhibition pattern of pftopoi observed here ( only wrwycrck inhibits pftopoi ) relative to that of the above mentioned topoib type enzymes ( inhibited by wycrck , kccrck , and wrwycrck ) . to further address the specificity of the pftopoi active inhibitor wrwycrck , we tested the effect of this peptide on the two restriction endonucleases ecori and pvuii . increasing concentrations of the peptide were incubated with the test plasmid ( puc19 ) before addition of either of the restriction enzymes . as evident from figure 3 , and consistent with previously published results , the peptide had no or only very modest effect on the cleavage activity of these enzymes , confirming the specific action of wrwycrck . all three peptides , wycrck , kccrck , and wrwycrck have previously been shown to possess an unspecific dna binding capacity , which was confirmed in the present study ( data not shown ) . however , the lack of inhibition of endonuclease activity and the inhibition of pftopoi activity only by wrwycrck and not by wycrck and kccrck even at very high concentrations argues against peptide inhibition being the result of a simple competition for noncovalent dna binding . indeed , for vvtopoi and -int , all peptides were demonstrated not to affect noncovalent dna interaction and it was suggested that inhibition was a result of the peptides preventing the transition from noncovalent to covalent binding , that is , dna cleavage , by interfering with the enzyme - dna interphase . docking experiments have been carried out to identify the preferential binding site of the wrwycrck octapeptide on the noncovalent pftopoi - dna complex . 250 docking runs were done and the best complex , having a free energy value of 14.0 kcal / mol , was selected and analyzed . this complex shows that the peptide is located in the minor groove cavity in front of the active site ( see figure 4(a ) ) , establishing many contacts with both the protein and the dna bases , as reported in table 1 . concerning the dna contacts , interesting interactions occur between the peptide and gua12-ade15 and thy32-thy34 on the scissile and intact strand , respectively . the optimal positioning of the octapeptide in the minor groove is due either to a good geometrical fitting between the two molecules , or to the high number of electrostatic interactions between the positively charged residues of the peptide and the negatively charged dna phosphates . as far as the protein is concerned , interesting interactions occur between trp3 and cys5 of the peptide and arg310 of pftopoi and between tyr4 and asp513 of pftopoi ( see table 1 ) . residues arg310 and asp513 of the plasmodium protein correspond to residues arg364 and asp533 of the human enzyme , which are known from the 3d structure of the ternary drug - dna - enzyme complex to directly interact with the camptothecin drug [ 55 , 56 ] . the peptide then , positioned in the minor groove of the dna just in front of the protein active site , exerts an inhibition of the cleavage process thus providing an explanation for the experimental results reported in figure 2(c ) . an identical docking experiment has been performed also for the two hexapeptides wycrck and kccrck , not having any inhibitory effect on pftopoi relaxation . the best docked complexes , having a free energy value of 11.36 and 11.04 kcal / mol , are reported in figures 4(b ) and 4(c ) for the wycrck and kccrck peptide , respectively . both peptides are found in a region different from the one found for the octapeptide . the two hexapeptides are located in proximity of the major groove in a region far from the enzyme active site and , in contrast to what was observed for the octapeptide , they are not able to interact with arg310 and asp513 , providing a structural explanation for their lack of inhibition . during recent years , bioactive peptides have been suggested as an alternative or complement to traditional small - molecule drugs in the combat against protozoan parasites [ 10 , 24 , 55 , 57 , 58 ] . one of the suggested advantages of peptide drugs in antiparasite treatment relies on the ease by which such drugs can be selected or modified to target desired biological pathways using nature 's own selection mechanisms or large throughput in vitro screening and/or directed evolution setups . another advantage relies on the relatively large interphase between peptide drugs and their target , possibly facilitating an increased specificity of peptide drugs compared to small - molecule drugs [ 25 , 59 ] . until recently , high synthesis costs have hampered the possibilities of developing peptide - based drugs against various relevant targets . however , with new synthesis strategies and lowered monomer costs the interest in developing peptide drugs has markedly increased [ 2427 ] . one of the very promising strategies was first presented by nagaraja 's research group , who had taken advantage of antibodies raised by the natural immune response of mice injected with the desired target , in the reported cases , mycobacteria dna gyrase or topoi [ 2830 ] . as a result remarkably , these antibodies appear extremely specific and show no activity against the e. coli counterparts of the mycobacteria topoisomerases . hence , these antibodies hold great promise for the further development of mycobacteria - specific peptide drugs based on the amino acid sequence of the active parts of the antibodies . indeed , several studies highlight the feasibility in deriving active peptides with specificity retained from the antibodies from which they originate [ 3134 ] . another reported strategy was based on selecting peptides with activity against the topoi related -int from a large library . as a result of this study , some of these , wycrck , kccrck , and wrwycrck , also inhibited the relaxation activity of vvtopoi [ 39 , 40 ] . in the present study , we demonstrate that of these peptides , wrwycrck but not wycrck , or kccrck inhibits dna relaxation mediated by pftopoi . as previously reported for the peptide inhibition of -int and vvtopoi , it is specifically the cleavage reaction of pftopoi that is inhibited by wrwycrck , while ligation is largely unaffected by the peptide , possibly due to the peptide being unable to bind to the covalent pftopoi - dna cleavage complexes . the inhibition on cleavage appears to be dependent on cysteine bridging since the addition of dtt counteracts the peptide effect . for -int and vvtopoi it was demonstrated that although the peptide does bind dna unspecifically , the inhibitory effect of active peptides on dna cleavage could not be ascribed to a simple competition preventing noncovalent dna interaction of the topos . although this was not addressed experimentally for pftopoi the inhibition of this enzyme by only one of the peptides , wrwycrck , argues for a specific inhibition rather than merely an unspecific competition for dna binding . this notion is further supported by molecular docking experiments in which the molecular mechanism for the inhibition exerted by the octapeptide was analyzed . this analysis allowed us to predict the preferential interaction interface between the noncovalent pftopoi - dna complex and the peptide itself . this is in agreement with the peptide being able to prevent the transition from noncovalent to covalent binding . hence , the complex with the lowest free energy , that is , the best complex , is represented by the peptide inserted in the dna minor groove , near the active site ( figure 4 ) , where it impedes the catalytic tyrosine to produce the nick on the scissile strand , as demonstrated by the cleavage assay ( figure 2 ) . indeed , the peptide interacts with two residues in proximity of the active site , arg310 and asp513 , which are the plasmodial counterpart for the human residues arg364 and asp533 that in the 3d structure of the human enzyme are in direct contact with the camptothecin drug . the peptide is stabilized by numerous contacts to either the protein or the dna , confirming that it represents an efficient inhibitor of the enzyme . docking of the two noninhibiting peptides , wycrck and kccrck , into the noncovalent pftopoi - dna complex revealed that these peptides were located far from the active site of pftopoi , which may explain why they do not inhibit pftopoi . although , until now , no species - specific peptide inhibitors of parasitic topos have been reported , we believe that the presented studies demonstrate the feasibility of inhibiting topos relevant in antiparasite treatment and that molecular docking may pave the road for the rational development of species - specific inhibitors .

control of diseases inflicted by protozoan parasites such as leishmania , trypanosoma , and plasmodium , which pose a serious threat to human health worldwide , depends on a rather small number of antiparasite drugs , of which many are toxic and/or inefficient . moreover , the increasing occurrence of drug - resistant parasites emphasizes the need for new and effective antiprotozoan drugs . in the current study , we describe a synthetic peptide , wrwycrck , with inhibitory effect on the essential enzyme topoisomerase i from the malaria - causing parasite plasmodium falciparum . the peptide inhibits specifically the transition from noncovalent to covalent dna binding of p. falciparum topoisomerase i , while it does not affect the ligation step of catalysis . a mechanistic explanation for this inhibition is provided by molecular docking analyses . taken together the presented results suggest that synthetic peptides may represent a new class of potential antiprotozoan drugs .

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

protozoan parasites , such as leishmania , trypanosoma , and plasmodium species are the cause of a large array of diseases hampering the lives of people all over the world . control of such diseases depends on a rather small number of prophylactic or therapeutic antiparasite drugs , many of which are highly toxic and/or inefficient [ 25 ] . this has created an urgent need for novel compounds to prevent and cure diseases caused by protozoan parasites . species - specific inhibition of parasitic enzymes has been suggested as one promising approach in the development of new therapeutics . dna topos are ubiquitous enzymes needed to overcome the topological stress arising in dna during replication , transcription , recombination , and repair . the type i topos are with few exceptions monomers and relax dna by breaking only one strand of the double helix , while type ii topos are mainly homodimers or heterotetramers and break both strands of the dna simultaneously . the class of type ii topos are subdivided into the type iia and type iib families , of which all members are structurally related and characterized by the formation of a 5-phoshotyrosyl linkage and a free 3-oh dna end during cleavage . hence , bacterial type iia topos , such as dna gyrase and topoiv , are targets of clinically important antibiotics active against a wide spectrum of bacterial pathogens [ 16 , 17 ] . human type ib and iia topos are targets of several anticancer compounds , exemplified by camptothecin and etoposide , respectively , of which synthetic derivatives are routinely used in systemic treatment of different cancer types [ 12 , 18 ] . indeed , the unusual heterodimeric topoib of kinetoplastid parasites , such as leishmania donovani gives hope for the development of drugs targeting parasite topoib without interfering with the function of the monomeric topoib in the human host [ 11 , 19 , 20 ] . as another example , the apicomplexan parasite plasmodium falciparum contains apicoplast dna , which requires bacterial - type dna gyrases ( type iia topo ) for replication , thus providing a unique drug target absent in the host [ 21 , 22 ] . in addition , the high expression rate of topoib and topoiia in rapidly growing parasites , compared to the expression levels of these enzymes in the host , may be exploited for the development of topo - targeting protocols that specifically kill the parasites . indeed , compared to small - molecule drugs , which are currently dominating the pharmaceutical market , peptide - based therapeutics offer several advantages , such as high specificity , lower accumulation in tissues , lower toxicity , and biological diversity [ 2427 ] . the potential for synthetic peptides as efficient species - specific inhibitors even of discrete steps of topo catalysis is highlighted in several studies by nagaraja and co - workers describing the identification and characterization of species - specific antibodies with inhibitory activities against particular steps of mycobacteria dna gyrase or topoi catalysis [ 2830 ] . a rescreening of the peptide combinatorial library ( used for selection of the above - mentioned peptides ) specifically against vvtopoi resulted in the identification of three new peptides , wycrck , kccrck , and wrwycrck with high activity against this enzyme . of these , wrwycrck was the most potent inhibitor of the type ib topos tested . as an initial investigation of this possibility , we address , in the present study , the effect of the peptides wycrck , kccrck , and wrwycrck on the activity of the recently cloned and purified recombinant topoi ( pftopoi ) from the malaria - causing parasite plasmodium falciparum . molecular docking of the three peptides in the noncovalent pftopoi - dna complex shows wrwycrck to be located in the minor groove of the dna in proximity of the enzyme active site , while wycrck and kccrck are positioned far from the enzyme active site . the plasmid , ppft100 ( the cloning of pftopoi is to be published elsewhere ) , containing the pftopoi gene ( plasmodb accession number pfe0520c ) ( codon optimized for expression in s. cerevisiae ( geneart , germany ) ) , was transformed into the yeast s. cerevisiae top1 strain rs190 ( a kind gift from r. sternglanz , state university of new york , stony brook , ny , usa ) according to standard procedures , and pftopoi enzyme was expressed and purified as previously described for human topoisomerase i ( htopoi ) . the plasmid was preincubated with the peptide for 5 min at 37c prior to addition of enzyme . samples were subjected to gel electrophoresis on 1% agarose gels in tbe buffer , and dna bands were stained with ethidium bromide and visualized by illumination with uv light . the sequences of the substrates are as follows : ol19 : 5-gcc tgc agg tcg act cta gag gat cta aaa gac tta ga-3 , ol27 : 5-aaa aat ttt tct aag tct ttt aga tcc tct aga gtc gac ctg cag gc-3 , and ol36 : 5-aga aaa att ttt-3. for the ligation reactions , 20 nm ol19/ol27 was incubated with 500 fmol of pftopoi for 30 min at 37c in 10 mm tris ( ph 7.5 ) , and 5 mm mgcl2 , 5 mm cacl2 . after preincubation of the cleavage samples with the peptide at concentrations varying from 0 to 12.5 m for 5 min at 37c , ligation was initiated by the addition of a 200-fold molar excess of oligonucleotide ol36 over the duplex ol19/ol27 . the relative cleavage was calculated by the following equation : relative cleavage = ( ic bi)/(ic bi + is bi ) , where ic denotes the intensity of the band(s ) representing the cleavage product(s ) , is denotes the intensity of the band representing the substrate , and bi denotes the background intensity . restriction digests were performed in 20 l reaction volumes by incubating 3 g puc19 plasmid with ecori or pvuii ( both from new england biolabs ) in the reaction buffers provided by the manufacturer in the absence or presence of peptide wrwycrck ( 12.5 m , 25 m , or 50 m ) . for both restriction endonucleases , the lowest amount of enzyme , able to fully digest the plasmid within the timeframe of the experiment , was used . samples were subjected to gel electrophoresis on 1% agarose gels in tbe buffer , and dna bands were stained with ethidium bromide and visualized by illumination with uv light . the bases are numbered from 1 to 22 starting from the 5 end of the scissile strand and from 23 to 44 starting from the 5 end of the intact strand . the structure of the octapeptide wrwycrck and of the two hexapeptides wycrck and kccrck was designed with the sybyl v. 6.0 program ( tripos , http://www.tripos.com/ ) creating a disulphide bond between the two cys3 and cys5 cysteines in all the peptides ( this was done since the experimental data confirmed that disulfide bridging was necessary for the inhibitory effect of the peptide ) . the structure of the peptides was minimized in vacuum using the powell algorithm implemented in the sybyl program and then simulated in a rectangular box filled with water molecules using the gromacs 4.0 package for 2 ns in order to regularize the structure . , convert fast - mobility supercoiled plasmid to slow - mobility relaxed plasmid forms ) in the absence of added peptide within the timeframe ( 30 min ) used in the experiment ( data not shown ) . as evident from figure 1 , the peptide wrwycrck inhibited pftopoi relaxation activity in a dose - dependent manner , with an ic50 of 2.55 m . the peptides wycrck , kccrck had no effect on the relaxation activity of pftopoi , even at concentrations up to 50 m . moreover , consistent with previous reports of inhibition of vvtopoi by wrwycrck , the peptide only retained its inhibitory effect in the absence of dtt ( data not shown ) , suggesting that the active form of the peptide involves disulfide bridging . dna relaxation by type ib topos involves two discrete transesterification reactions that is , a cleavage reaction , in which the active site tyrosine attacks the phosphodiester bond of the dna backbone to generate a 3-phosphotyrosyl cleavage intermediate and a free 5-oh end , and a ligation reaction in which the 5-oh acts as a nucleophile on the phosphotyrosyl bond to restore intact dna . it was previously demonstrated that the inhibitory effect of the peptides wycrck , kccrck , and wrwycrck on dna relaxation by vvtopoi and -int could be ascribed to a specific inhibition of the cleavage and not the ligation step of catalysis [ 39 , 40 ] . to address which steps of pftopoi catalysis are affected by peptide wrwycrck , that inhibited relaxation by this enzyme we used a synthetic partially single - stranded suicide dna substrate containing a preferred type ib topoi cleavage sequence . prevention of religation , however , is only conditional and this step of catalysis can be initiated by the addition of a surplus of a 5-oh - containing ligator strand with a sequence matching the protruding noncleaved strand of the generated cleavage complexes ( figure 2(a ) ) . first , the ability of pftopoi with cleave the suicide dna substrate was tested in comparison to cleavage by htopoi . the two enzymes were incubated with substrate radiolabeled at the 5-end of the cleaved strand ( to allow visualization of the cleavage products ) , the products were ethanol precipitated , trypsinated , and separated on a denaturing polyacrylamide gel prior to visualization by phosphorimaging . as previously reported in , even after trypsin digestion , the cleavage products of both pftopoi and htopoi were retarded in the gel due to the covalent attachment of short protease - resistant peptides to the radiolabeled strand of the dna substrate . for htopoi , the major cleavage product cl1 was previously demonstrated to result from cleavage at the preferred site ( indicated by an arrow in figure 2(a ) ) , while the minor cl2 product arises from cleavage two nucleotides upstream to the cleavage site . to test the effect of peptide wrwycrck on pftopoi mediated cleavage , increasing concentrations of the peptide were incubated with the above - described suicide dna substrate prior to addition of pftopoi . using the suicide substrate system , the effect of peptide wrwycrck on pftopoi - mediated religation was investigated . consistent with previous results obtained for vvtopoi and -int the peptide did not affect ligation by pftopoi ( figure 2(d ) ) . the three peptides tested for activity against pftopoi in the present study were previously demonstrated to inhibit vvtopoi and -int activity with ic50 's of 0.0152.3 m , while more unrelated enzyme activities such as e. coli type ia topo and restriction endonucleases were hardly affected by any of the peptides . although far from being species - specific , the peptide inhibitors appear rather sensitive to even subtle structural differences between the different target enzymes . this is evident from the different inhibition pattern of pftopoi observed here ( only wrwycrck inhibits pftopoi ) relative to that of the above mentioned topoib type enzymes ( inhibited by wycrck , kccrck , and wrwycrck ) . to further address the specificity of the pftopoi active inhibitor wrwycrck , we tested the effect of this peptide on the two restriction endonucleases ecori and pvuii . as evident from figure 3 , and consistent with previously published results , the peptide had no or only very modest effect on the cleavage activity of these enzymes , confirming the specific action of wrwycrck . all three peptides , wycrck , kccrck , and wrwycrck have previously been shown to possess an unspecific dna binding capacity , which was confirmed in the present study ( data not shown ) . however , the lack of inhibition of endonuclease activity and the inhibition of pftopoi activity only by wrwycrck and not by wycrck and kccrck even at very high concentrations argues against peptide inhibition being the result of a simple competition for noncovalent dna binding . indeed , for vvtopoi and -int , all peptides were demonstrated not to affect noncovalent dna interaction and it was suggested that inhibition was a result of the peptides preventing the transition from noncovalent to covalent binding , that is , dna cleavage , by interfering with the enzyme - dna interphase . this complex shows that the peptide is located in the minor groove cavity in front of the active site ( see figure 4(a ) ) , establishing many contacts with both the protein and the dna bases , as reported in table 1 . concerning the dna contacts , interesting interactions occur between the peptide and gua12-ade15 and thy32-thy34 on the scissile and intact strand , respectively . the optimal positioning of the octapeptide in the minor groove is due either to a good geometrical fitting between the two molecules , or to the high number of electrostatic interactions between the positively charged residues of the peptide and the negatively charged dna phosphates . as far as the protein is concerned , interesting interactions occur between trp3 and cys5 of the peptide and arg310 of pftopoi and between tyr4 and asp513 of pftopoi ( see table 1 ) . residues arg310 and asp513 of the plasmodium protein correspond to residues arg364 and asp533 of the human enzyme , which are known from the 3d structure of the ternary drug - dna - enzyme complex to directly interact with the camptothecin drug [ 55 , 56 ] . the peptide then , positioned in the minor groove of the dna just in front of the protein active site , exerts an inhibition of the cleavage process thus providing an explanation for the experimental results reported in figure 2(c ) . an identical docking experiment has been performed also for the two hexapeptides wycrck and kccrck , not having any inhibitory effect on pftopoi relaxation . the two hexapeptides are located in proximity of the major groove in a region far from the enzyme active site and , in contrast to what was observed for the octapeptide , they are not able to interact with arg310 and asp513 , providing a structural explanation for their lack of inhibition . during recent years , bioactive peptides have been suggested as an alternative or complement to traditional small - molecule drugs in the combat against protozoan parasites [ 10 , 24 , 55 , 57 , 58 ] . one of the suggested advantages of peptide drugs in antiparasite treatment relies on the ease by which such drugs can be selected or modified to target desired biological pathways using nature 's own selection mechanisms or large throughput in vitro screening and/or directed evolution setups . another advantage relies on the relatively large interphase between peptide drugs and their target , possibly facilitating an increased specificity of peptide drugs compared to small - molecule drugs [ 25 , 59 ] . however , with new synthesis strategies and lowered monomer costs the interest in developing peptide drugs has markedly increased [ 2427 ] . hence , these antibodies hold great promise for the further development of mycobacteria - specific peptide drugs based on the amino acid sequence of the active parts of the antibodies . as a result of this study , some of these , wycrck , kccrck , and wrwycrck , also inhibited the relaxation activity of vvtopoi [ 39 , 40 ] . in the present study , we demonstrate that of these peptides , wrwycrck but not wycrck , or kccrck inhibits dna relaxation mediated by pftopoi . as previously reported for the peptide inhibition of -int and vvtopoi , it is specifically the cleavage reaction of pftopoi that is inhibited by wrwycrck , while ligation is largely unaffected by the peptide , possibly due to the peptide being unable to bind to the covalent pftopoi - dna cleavage complexes . for -int and vvtopoi it was demonstrated that although the peptide does bind dna unspecifically , the inhibitory effect of active peptides on dna cleavage could not be ascribed to a simple competition preventing noncovalent dna interaction of the topos . although this was not addressed experimentally for pftopoi the inhibition of this enzyme by only one of the peptides , wrwycrck , argues for a specific inhibition rather than merely an unspecific competition for dna binding . this notion is further supported by molecular docking experiments in which the molecular mechanism for the inhibition exerted by the octapeptide was analyzed . this is in agreement with the peptide being able to prevent the transition from noncovalent to covalent binding . hence , the complex with the lowest free energy , that is , the best complex , is represented by the peptide inserted in the dna minor groove , near the active site ( figure 4 ) , where it impedes the catalytic tyrosine to produce the nick on the scissile strand , as demonstrated by the cleavage assay ( figure 2 ) . indeed , the peptide interacts with two residues in proximity of the active site , arg310 and asp513 , which are the plasmodial counterpart for the human residues arg364 and asp533 that in the 3d structure of the human enzyme are in direct contact with the camptothecin drug . the peptide is stabilized by numerous contacts to either the protein or the dna , confirming that it represents an efficient inhibitor of the enzyme . docking of the two noninhibiting peptides , wycrck and kccrck , into the noncovalent pftopoi - dna complex revealed that these peptides were located far from the active site of pftopoi , which may explain why they do not inhibit pftopoi . although , until now , no species - specific peptide inhibitors of parasitic topos have been reported , we believe that the presented studies demonstrate the feasibility of inhibiting topos relevant in antiparasite treatment and that molecular docking may pave the road for the rational development of species - specific inhibitors .

the nuclear factor of activated t cells 5 ( nfat5 , also known as tonebp or orebp ) is a rel family transcriptional activator . nfat5 is among the very few mammalian transcription factors activated by hypertonic conditions . it regulates the transcription of membrane transporters and synthetic enzymes , and its activation causes the adaptive accumulation of organic osmolytes in the cell . in addition , nfat5 also can regulate other important cellular processes including the migration of carcinoma cells and atherosclerotic lesion formation . nfat5 belongs to the nfat family of transcription factors and its dna binding domain shares a high sequence identity with the nfat14 proteins . however , unlike other nfat14 proteins which can bind dna as monomers , nfat5 binds to dna as a dimer . experimental data shows that it likely forms a dimer in solution even in the absence of dna and dimerization is necessary for dna binding and transcriptional activity . the dna affinity of nfat5 is much lower than that of nfat14 ; however , it has a slower dissociation rate than another rel dimeric transcription factor , nf - kb . the nfat5 gene encodes multiple isoforms of which nfat5a and c have been most extensively studied . nfata and c have identical sequences , but nfatc has an additional 76 amino acids at the n - terminus . in this work , we use the amino acid numbering of the nfat5 isoform a but include notation of the corresponding amino acids in isoform c. the crystal structure of an nfat5 region containing amino acids 170470 ( 246546 isoform c ) in complex with dna was resolved about 10 years ago . this region of nfat5 has two domains : an n - terminal rel homology domain ( rhd ) which makes most of the contacts with dna and a c - terminal ipt domain which mediates interactions between the two monomers and has limited contacts with dna . the crystal structure of the nfat5 complex with dna remains the only available structure of this protein . according to this structure , one monomer of nfat5 makes specific contacts with the conserved dna consensus nucleotides ( tggaaa ) of the nfat5 cognate dna element , ore ( osmotic response element ) . the other monomer binds to other , nonconserved nucleotides of ores which differ among nfat5 target genes . the detailed comparative analysis of nfat5 and nfb structures showed that dimerization interfaces formed by the rhr - c domains of nfat5 are very similar to those observed in nfb . dna complex has a second dimer interface which is formed by the ef loop of the rhr - n domain . nuclear translocation of nfat5 in response to change in tonicity has been studied and several regions have been identified . a nuclear export sequence ( nes ) is unique to isoform c , whereas a nuclear localization sequence ( nls ) and an auxiliary export domain ( aed ) are common to all nfat5 isoforms . moreover , several studies point to a possible connection between nuclear translocation and dna binding . namely , different mutations introduced in the dna - binding loop and dimerization interface of the c - terminal domain were found to be important for dna binding and some of them greatly decreased the nfat5 nuclear cytoplasm ratio . however , the mechanism of nfat5 nuclear translocation and the effect of other protein or dna binding on retaining nfat5 in the nucleus are largely unknown . mutations represent a convenient way to decouple dna - binding specific events from other effects , and here we model the effect of different mutations in the dna - binding loop on protein dynamics and binding . we find that the specifically bound chain makes more contacts with the dna molecule and its binding affinity is higher compared to the nonspecifically bound chain . these contacts , coming from the dna - binding loop and a few other regions , confer binding specificity . moreover , the nonspecific chain is characterized by higher flexibility than the specifically bound chain . we show that the nfat5 complex without dna is much more flexible than the complex with dna and the binding energy to dna significantly exceeds the energy of stabilization of the dimer without dna . we investigate the effects of two sets of mutations from the dna - binding loop experimentally analyzed previously , and observe that different nfat5 mutants have different mechanisms of specific and nonspecific binding to dna . the r217a / e223a / r226a ( r293a / e299a / r302a in isoform c ) mutant is characterized by significantly compromised binding to dna and higher complex flexibility , whereas the t222d ( t298d in isoform c ) mutation , on the contrary , makes the overall complex more rigid and does not affect dna binding much . our analysis also suggests that it is unlikely that a fully formed nfat5 dimer can form prior to nuclear binding to dna without significant structural rearrangements . we used the only available x - ray structure of the nfat5 homodimer / dna complex ( pdb code : 1imh ) . by removing the dna molecule coordinates , we also produced a model system of the dimer in the absence of dna ( native_withdna , native_nodna ) . altogether we created six systems for further molecular dynamics ( md ) simulation analysis . we introduced a t222d mutation ( t298 in isoform c ) in both chains ( chain c and chain d ) with and without dna ( t222d_withdna , t222d_nodna ) . separately , we created a triple mutant in each chain by substituting residues r217 , e223 , and r226 ( r293 , e299 , and r302 in isoform c ) into alanine ( 3m_withdna and 3m_nodna ) . the mutants were made using the mutator plugin of the vmd molecular dynamics software , and the hydrogen atoms were added with the vmd program . all models were immersed into rectangular boxes of water molecules extending up to 10 from the protein in each direction . to ensure an ionic concentration of 150 mm ( concentration reported in the original paper of the nfat5 structure ) and zero net charge , na and cl ions were added by vmd . six systems mentioned in the previous section were optimized and equilibrated using energy minimization and the md simulation protocol . first , a 4000-step energy minimization was carried out using the steepest descent method , with harmonic restraints ( force constant = 10 kcal / mol / ) applied on the backbone atoms of all residues , followed by an 8000-step energy minimization on the whole system . the systems were then heated to 300 k over 300 ps with harmonic constraints applied on protein backbone atoms . consequently , the systems were subject to a 25 ns unconstrained md simulation performed in the npt ensemble . the langevin piston nos hoover method was used to control temperature and pressure with temperature t = 300 k and pressure p = 1 atm . periodic boundary conditions and a 12 cutoff distance for nonbonded interactions were applied to the systems . the particle mesh ewald ( pme ) method was used to calculate the long - range electrostatic interactions . lengths of hydrogen - containing bonds were constrained by the shake algorithm , and the coordinates of the systems were saved every 2 ps during md simulations . the energy minimization and md simulation were carried out with namd program version 2.9 using the charmm27 force field and the tip3p water model . principal component analysis ( pca ) was applied to extract the dominant modes corresponding to the collective motions of atomic groups . to eliminate translational and rotational motions and isolate only the internal motions of the system , the pca analysis was applied to the cartesian covariance matrix which was diagonalized to obtain a set of eigenvectors and corresponding eigenvalues . hydrogen bonds and salt bridges were identified using the charmm program . to define a salt bridge , binding energies were calculated using the mmpbsa method that combines the molecular mechanical energies with the poisson boltzmann continuum representation of the solvent calculated using the charmm force field . we extracted 60 snapshots of the last 6 ns of all md trajectories for six systems ( after stripping all water molecules and ions ) to calculate the protein and protein the binding energy g was expressed as g = gvdw + gelec + gsa . here gvdw corresponds to the van der waals interaction energy in the gas phase and gelec and gsa are polar and nonpolar solvation energies , respectively . all energy terms were calculated as the difference between the complex and each monomer ( protein or dna ) in a solvent . boltzmann ( pb ) method , while gsa was approximated according to the formula gsa = 0.00542*sa + 0.92 , where sa stands for the area of the molecular surface . in addition to an unweighted binding energy expression , we also used a weight of 0.5 on the van der waals interaction energy term , as it was found to be more suitable to describe the experimentally measured ph dependency of the effect of mutations on the dissociation constants . for the pb calculation , dielectric constants of = 1 , 2 , and 4 were used for the protein interior and the dielectric constant for the exterior aqueous environment was set to = 80 . = 4 produced the smallest energy fluctuations among 6 ns snapshots and was applied in further analysis . all pb calculations were performed with the pbeq module of the charmm program . the atomic born radii were previously calibrated and optimized to reproduce the electrostatic free energy of the 20 amino acids in md simulations with explicit water molecules . we also used a simplified binding energy calculation implemented in the foldx method which calculates the effect of mutations using an empirical force field . foldx optimizes the side chain configurations but does not estimate the effects produced by backbone conformational movements . the native complex of nfat5 with dna represents a dimer with two identical chains of 281 residues long each . chain c makes specific contacts with the dna molecule through the conserved nucleotides of the ore , its dna binding site . chain d binds dna less specifically mostly through contacts with the phosphate backbone of the non - consensus nucleotides of ores . we performed md simulations for six systems for 25 ns ( native_withdna , native_nodna , 3m_withdna , 3m_nodna , t222d_withdna , and t222d_nodna ) . the time dependence of root - mean - square deviation ( rmsd ) of the main chain atoms from the minimized structure is shown in figure 1 . as one can see from comparing parts a and b of figure 1 , complexes without dna deviate from the initial minimized nfat5 structure considerably more than complexes with the dna . all systems with dna seem to reach equilibrium after 25 ns of simulation . to ensure convergence , we ran additional md simulations for the native with dna and t222d mutation systems . although the systems without dna have relatively large fluctuations , the rmsd does not increase any further after about 15 ns for these systems . we used the time - averaged coordinates within the last 6 ns as a representative of the final equilibrated structures ( the native average nfat5 structure for the first md trajectory will be referred to as native hereafter ) , and calculated the binding energy between protein and dna , between chains c and d within the last 6 ns . backbone rmsd ( with respect to the minimized structure ) for six systems as a function of simulation time for ( a ) systems with dna and ( b ) systems without dna . to identify the most flexible regions , next we analyzed the root - mean - square fluctuations ( rmsfs ) per residue . figures 2a and 3b show the rmsf values calculated for c - alpha atoms based on the superposition of the whole protein complexes . these rmsf values correspond to the whole complex movements including the relative movements between chains . figures 2b , 3b , and s4 ( supporting information ) depict rmsf values based on the superpositions of chains c and d separately , which allows us to compare the flexibilities of the two chains . as one can see from these figures , chain d ( rmsf of 1.35 0.05 , mean value and 95% confidence intervals are listed , standard deviation is 0.47 ) in the native complex with dna is more flexible than chain c ( rmsf of 1.25 0.06 , standard deviation is 0.51 , wilcoxon test p - value < 0.01 ) . this observation is supported by the larger number of hydrogen bonds and salt bridges formed between chain c and dna ( figure 3a and table s1 ( supporting information ) ) and its higher dna binding affinity ( tables 1 and s3 ( supporting information ) ) . the dna binding ab loop in the n - terminal domain ( residues 217227 ) is particularly rigid in both chains . ( b ) frame alignments are based separately on chains c and d. ( a ) the number of contacts ( hydrogen bonds and salt bridges ) between the protein complex and dna , chain c and dna , and chain d and dna for the three systems with dna . ( b ) mean values and 95% confidence intervals ( error bars ) of rmsf . for complex , the alignment is based on the whole protein complex , and for chain c and chain d , the alignments are performed separately for each chain . the binding energy of the native complex was scaled to 10 kcal / mol , the experimental value obtained previously . standard deviations are listed in parentheses for energies calculated within 60 frames of the last 6 ns . different weights ( = 1 and = 5 ) are used for the van der waals term of the binding energy expression . the nfat5 dimer without dna is characterized by much higher flexibility than the complex with dna ( figure s1 ( supporting information ) , wilcoxon test p - value 0.01 ) . this difference is especially pronounced for the rhd domain of chain c and can be explained by chain c making specific contacts with the dna molecule . the large difference between complexes with and without dna is also evident from the pca analysis ( figures 4 and s5 ( supporting information ) ) and is consistent with the binding energy calculations , according to which the binding affinity of two monomers in the dimer is considerably lower than the binding energy between protein and dna ( table 1 ) . when we compare actual native dimer structures with and without dna after md simulations ( structures are averaged over the last 6 ns ) , we found that the dimer structure without dna has changed compared to the native structure with dna ( only 149 residues on one chain could be structurally superimposed within 0.5 on the 562-residue dimer ) . as can be judged from the structural superposition , almost half of all contacts ( hydrogen bonds and salt bridges ) with the dna molecule which are present in the dna complex are lost in a dimer without dna . therefore , even if a dimer can be formed prior to dna binding , significant conformational adjustments may take place when it binds to dna . next we analyzed the effects of two series of mutations on nfat5 dynamics which revealed two different outcomes produced by mutations from the same dna - binding loop . next we investigated the effect of three mutations r217a , e223a , and r226a which were experimentally shown to reduce the nuclear according to the ibis database , r217 and r226 residues make contacts with the dna coding strand and these contacts are invariant in many nfat transcription factors , whereas an invariant e223 residue binds a noncoding dna strand . a comparison of native and triple mutant complexes with dna shows that , while three residues in each chain lose their specific and nonspecific contacts with dna ( all three mutations occur in the dna - binding loop ) , the structure undergoes local conformational changes to maximize the contacts with dna and to make new contacts which are not observed in the native complex ( table s1 ( supporting information ) ) . overall , the mutant complex loses nine contacts ( hydrogen bonds or salt bridges ) with the dna molecule . namely , it loses 12 contacts in chain c and gains three contacts in chain d , some of which are located far away in sequence and structure from the specific dna - binding loop ( figures 3a and 6 and table s1 ( supporting information ) ) . interestingly , the loss of specific contacts with dna by chain c and the gain of contacts by chain d make the mutant complex more symmetric with respect to dna orientation ( figure 6 and table s1 ( supporting information ) ) . this results in a considerable drop in binding energy from 10 to 6.79 kcal / mol for the whole complex with dna for native compared to the triple mutant ( table 1 ) . although three mutations in the dna - binding loop are introduced in both chains , they make chain c somewhat more flexible compared to the native complex ( 1.49 0.06 and standard deviation of 0.52 compared to1.24 0.06 , standard deviation of 0.51 , p - value < 0.01 ) and have almost no effect on chain d ( figures 2b and 3b ) . the dna - binding energy drops from 3.50 to 2.82 kcal / mol and from 2.83 to 2.29 kcal / mol for chains c and d , respectively . moreover , loop 322332 which contains several lysines and makes partial contacts with dna in the native complex ( figure 6 and table s1 ( supporting information ) ) becomes more flexible and undergoes conformational movements in chain c ( rmsf 2.27 compared to 1.70 in native protein ) ( figure 2b ) . native ( blue ) and triple mutant ( green ) structures are averaged over the last 6 ns and structurally superimposed . side chains of residues making contacts with dna are shown in blue and green . next we examined the effect of the phosphomimetic mutation t222d on dna binding in the presence of dna . previously , scansite software was used to predict that phosphorylated t222 site is a potential binding site for 1433 proteins . we checked if t222 can be phosphorylated using additional two programs , and it was indeed predicted to be phosphorylated by kinasephos and gps though not with the disphos program . according to the ibis database , t222 makes contacts in both chains with the noncoding dna strand and these contacts are invariant among nfat family members . the effect of t222d located in the same dna - binding loop is drastically different from the impact of mutations described in the previous section . as can be seen from figures 1 and s3 ( supporting information ) , the rmsd deviations from the initial structure are constrained for the t222d mutant complex with dna for two different md trajectories ; in fact , they are even lower than for the native structure almost everywhere along the simulation time . the t222d mutation makes the overall complex more rigid than native ( rmsf is 1.53 0.05 and standard deviation is 0.55 for mutant compared to 1.75 0.05 and standard deviation of 0.58 for the native complex ; p - value < 0.01 , figures 2a and 3b ) . if we compare individual chains , the loss of flexibility is less pronounced , which points to the reduction in relative movement between the two chains in mutant . while the ab loop does not show a noticeable conformational change , other regions undergo movements which lead to the formation of additional contacts with the dna molecule ( figure 3a and table s1 ( supporting information ) ) . for example , arg276 on chain c makes a contact with the dna phosphate backbone upon t222d mutation ( table s1 ( supporting information ) ) . while the loop 322332 on chain c is more flexible in the 3m_withdna system , on the contrary , it becomes more rigid in the t222d_withdna mutant ( figure 2b ) . moreover , a new salt bridge is formed between the substituted t d222 of chain d and lys329 of chain c , which restrains the motion between the two monomers ; the distance between the side - chain nitrogen of lys329 and the gamma - oxygen of thr222 was 11.9 in the native structure compared to 2.7 between the side - chain nitrogen of lys329 and the side - chain oxygen of asp222 in the mutant ( figure 7 and table s1 ( supporting information ) ) . all these contacts were conserved between structures corresponding to two md trajectories ( table s1 ( supporting information ) ) . although the overall binding affinity to dna almost does not change upon this mutation using the mmpbsa method , the binding between chain c and dna seems to be tighter than that of the native structure ( tables 1 and s3 ( supporting information ) ) . the mutated residue t222d makes a salt bridge with k329 in the mutant while displaced 11.9 away in the native structure . the eigenvalue is calculated as the mean - square fluctuation in the direction of the principal mode , and the largest eigenvalue corresponds to the most dominant collective mode . as can be seen from figure 4 , the first two or three eigenvectors contribute the most to the system motion and eigenvalues decrease in the following order : 3m_nodna > t222d_nodna > native_nodna > native_withdna 3m_withdna > t222d_withdna . similar to previous observations , dna binding makes the protein complex more rigid , especially for the t222d mutation . moreover , mutant complexes without dna are characterized by higher flexibility than the native complexes without dna . from comparing the eigenvalues of chains c and d ( see figure s2 ( supporting information ) ) , it is evident that chain c is more rigid than chain d in the native_withdna and especially in the t222d_withdna systems . for the triple mutant , the situation is contrary and chain c is characterized by more extensive motions than chain d. projection of trajectories on the first two principal components is shown in figure 5 . as apparent from this figure , complexes without dna , especially 3m_nodna and t222d_nodna , sample much larger conformational space than the nfat5 complexes with dna . furthermore , projections for native and mutant complexes with dna are quite different from each other and the t222d mutant samples much smaller regions of the conformational space . the specific recognition of dna sequences by proteins is governed by the formation of hydrogen bonds with specific bases ( mostly occurring in the major groove ) , and by varying the dna shape and its electrostatic potential ( this type of recognition may happen in major as well as minor dna grooves ) . the nfat5 dimer represents a convenient system to study the difference between specific ( maintained by chain c ) and less specific or nonspecific dna binding ( maintained by chain d ) . structural analysis of the nfat5 native complex and md simulations show that the dimer does not significantly change its overall orientation with respect to dna in specific compared to nonspecific chains , which is consistent with previous experimental results on other dna - binding proteins . the specifically bound chain makes more contacts with dna , and its binding affinity is found to be higher compared to the nonspecifically bound chain ( the energy difference between specific and nonspecific binding is about 23 kt ) . some of these contacts , coming from the dna - binding loop and a few other regions , are different between the two chains and therefore may confer binding specificity . moreover , our results show that the nonspecific chain has a much higher flexibility in the native complex than the specifically bound chain . this can be explained by the extensive sampling of different protein conformations on the dna surface by the nonspecific chain which might be required before the specific chain binds dna . indeed , according to the energy landscape theory of protein dna binding , nonspecific protein dna complexes might have more rugged energy landscapes , whereas specific complexes can be characterized by the funnel - like energy landscape guiding the search to the native state . on the other hand , protein flexibility may facilitate binding and provide kinetic advantages via the fly casting mechanism . in the latter scenario , the unfolded flexible chain binds weakly at larger distances and undergoes a disorder - to - order transition as the protein recognizes the dna through specific binding . we studied different nfat5 mutants and found that they had distinctly different effects on specific and nonspecific binding to dna . the r217a / e223a / r226a mutant was characterized by significantly compromised binding to dna ( by 58 kt ) . this result is consistent with a previous experimental observation of several authors of this paper that these mutations reduce high nacl - induced nuclear translocation . although all of these mutations were introduced within the same dna - binding loop on both chains , a different effect on specific and nonspecific chains was obvious . namely , the triple mutations made the specific chain more flexible with almost no effect on the nonspecifically bound chain . since the nfat5 dimer forms a complete circle around the dna molecule , the loss of specific contacts with dna by chain c makes the orientation of the mutant dimer more symmetric inside the inner protein ring . on the contrary , unlike the three previously mentioned mutations , which enhanced the mobility of the specific chain and decreased the binding to dna , the t222d mutation made the specific chain / dna complex less flexible and more tightly bound . the overall effect of this mutation on dna binding was much less pronounced than the effect of the triple mutations ( even if scaled down by the number of mutations ) . this result points to the possibility that reduced binding to dna can not fully explain the reduced nuclear t222 is predicted to be phosphorylated by several methods although not yet confirmed by experiments . one might hypothesize that the t222d mutation can prevent the reversible potential phosphorylation which in turn might disrupt the balance between nucleic and cytoplasmic forms of nfat5 . recently , an energy landscape approach was used to model the effect of multiple phosphorylation in the nfat1 protein regulatory region . the authors showed that phosphorylation increased the helical propensity and rigidified the structure of the phospho - peptides similar to the effect of the t222d mutation studied here . it was suggested that the cytoplasmic form of nfat1 needed a well - defined structure to perform its function , while the more flexible regulatory region was characteristic for the nuclear form . further experimental characterization is needed of residue 222 s effect on nfat5 dynamics and binding . overall , the decreased binding of the nfat5 dimer to dna for triple mutant might explain its reduced nuclear localization reported earlier . however , it was proposed recently that nfat5 dimerization might be required for the nuclear transport , whereas dna binding might not . we observed a higher flexibility of mutant dimers compared to native dimers without dna . in addition , our binding energy calculations point to the marginal stability of dimers in the absence of dna , with the predicted dissociation constant in the mm range . our results allow us to conclude that dna binding considerably stabilizes the dimer complex and certain mutations may destabilize the binding to dna . all of this appears to contribute to the mechanism of nuclear cytoplasm transport .

the nuclear factor of activated t cells 5 ( nfat5 or tonebp ) is a rel family transcriptional activator and is activated by hypertonic conditions . several studies point to a possible connection between nuclear translocation and dna binding ; however , the mechanism of nfat5 nuclear translocation and the effect of dna binding on retaining nfat5 in the nucleus are largely unknown . recent experiments showed that different mutations introduced in the dna - binding loop and dimerization interface were important for dna binding and some of them decreased the nuclear cytoplasm ratio of nfat5 . to understand the mechanisms of these mutations , we model their effect on protein dynamics and dna binding . we show that the nfat5 complex without dna is much more flexible than the complex with dna . moreover , dna binding considerably stabilizes the overall dimeric complex and the nfat5 dimer is only marginally stable in the absence of dna . two sets of nfat5 mutations from the same dna - binding loop are found to have different mechanisms of specific and nonspecific binding to dna . the r217a / e223a / r226a ( r293a / e299a / r302a using isoform c numbering ) mutant is characterized by significantly compromised binding to dna and higher complex flexibility . on the contrary , the t222d ( t298d in isoform c ) mutation , a potential phosphomimetic mutation , makes the overall complex more rigid and does not significantly affect the dna binding . therefore , the reduced nuclear cytoplasm ratio of nfat5 can be attributed to reduced binding to dna for the triple mutant , while the t222d mutant suggests an additional mechanism at work .

Introduction Methods Results Discussion

the nuclear factor of activated t cells 5 ( nfat5 , also known as tonebp or orebp ) is a rel family transcriptional activator . nfat5 is among the very few mammalian transcription factors activated by hypertonic conditions . experimental data shows that it likely forms a dimer in solution even in the absence of dna and dimerization is necessary for dna binding and transcriptional activity . the dna affinity of nfat5 is much lower than that of nfat14 ; however , it has a slower dissociation rate than another rel dimeric transcription factor , nf - kb . in this work , we use the amino acid numbering of the nfat5 isoform a but include notation of the corresponding amino acids in isoform c. the crystal structure of an nfat5 region containing amino acids 170470 ( 246546 isoform c ) in complex with dna was resolved about 10 years ago . this region of nfat5 has two domains : an n - terminal rel homology domain ( rhd ) which makes most of the contacts with dna and a c - terminal ipt domain which mediates interactions between the two monomers and has limited contacts with dna . the crystal structure of the nfat5 complex with dna remains the only available structure of this protein . nuclear translocation of nfat5 in response to change in tonicity has been studied and several regions have been identified . moreover , several studies point to a possible connection between nuclear translocation and dna binding . namely , different mutations introduced in the dna - binding loop and dimerization interface of the c - terminal domain were found to be important for dna binding and some of them greatly decreased the nfat5 nuclear cytoplasm ratio . however , the mechanism of nfat5 nuclear translocation and the effect of other protein or dna binding on retaining nfat5 in the nucleus are largely unknown . mutations represent a convenient way to decouple dna - binding specific events from other effects , and here we model the effect of different mutations in the dna - binding loop on protein dynamics and binding . these contacts , coming from the dna - binding loop and a few other regions , confer binding specificity . moreover , the nonspecific chain is characterized by higher flexibility than the specifically bound chain . we show that the nfat5 complex without dna is much more flexible than the complex with dna and the binding energy to dna significantly exceeds the energy of stabilization of the dimer without dna . we investigate the effects of two sets of mutations from the dna - binding loop experimentally analyzed previously , and observe that different nfat5 mutants have different mechanisms of specific and nonspecific binding to dna . the r217a / e223a / r226a ( r293a / e299a / r302a in isoform c ) mutant is characterized by significantly compromised binding to dna and higher complex flexibility , whereas the t222d ( t298d in isoform c ) mutation , on the contrary , makes the overall complex more rigid and does not affect dna binding much . our analysis also suggests that it is unlikely that a fully formed nfat5 dimer can form prior to nuclear binding to dna without significant structural rearrangements . by removing the dna molecule coordinates , we also produced a model system of the dimer in the absence of dna ( native_withdna , native_nodna ) . we introduced a t222d mutation ( t298 in isoform c ) in both chains ( chain c and chain d ) with and without dna ( t222d_withdna , t222d_nodna ) . separately , we created a triple mutant in each chain by substituting residues r217 , e223 , and r226 ( r293 , e299 , and r302 in isoform c ) into alanine ( 3m_withdna and 3m_nodna ) . consequently , the systems were subject to a 25 ns unconstrained md simulation performed in the npt ensemble . in addition to an unweighted binding energy expression , we also used a weight of 0.5 on the van der waals interaction energy term , as it was found to be more suitable to describe the experimentally measured ph dependency of the effect of mutations on the dissociation constants . we also used a simplified binding energy calculation implemented in the foldx method which calculates the effect of mutations using an empirical force field . as one can see from comparing parts a and b of figure 1 , complexes without dna deviate from the initial minimized nfat5 structure considerably more than complexes with the dna . to ensure convergence , we ran additional md simulations for the native with dna and t222d mutation systems . although the systems without dna have relatively large fluctuations , the rmsd does not increase any further after about 15 ns for these systems . backbone rmsd ( with respect to the minimized structure ) for six systems as a function of simulation time for ( a ) systems with dna and ( b ) systems without dna . as one can see from these figures , chain d ( rmsf of 1.35 0.05 , mean value and 95% confidence intervals are listed , standard deviation is 0.47 ) in the native complex with dna is more flexible than chain c ( rmsf of 1.25 0.06 , standard deviation is 0.51 , wilcoxon test p - value < 0.01 ) . the dna binding ab loop in the n - terminal domain ( residues 217227 ) is particularly rigid in both chains . ( b ) frame alignments are based separately on chains c and d. ( a ) the number of contacts ( hydrogen bonds and salt bridges ) between the protein complex and dna , chain c and dna , and chain d and dna for the three systems with dna . the nfat5 dimer without dna is characterized by much higher flexibility than the complex with dna ( figure s1 ( supporting information ) , wilcoxon test p - value 0.01 ) . this difference is especially pronounced for the rhd domain of chain c and can be explained by chain c making specific contacts with the dna molecule . the large difference between complexes with and without dna is also evident from the pca analysis ( figures 4 and s5 ( supporting information ) ) and is consistent with the binding energy calculations , according to which the binding affinity of two monomers in the dimer is considerably lower than the binding energy between protein and dna ( table 1 ) . when we compare actual native dimer structures with and without dna after md simulations ( structures are averaged over the last 6 ns ) , we found that the dimer structure without dna has changed compared to the native structure with dna ( only 149 residues on one chain could be structurally superimposed within 0.5 on the 562-residue dimer ) . as can be judged from the structural superposition , almost half of all contacts ( hydrogen bonds and salt bridges ) with the dna molecule which are present in the dna complex are lost in a dimer without dna . therefore , even if a dimer can be formed prior to dna binding , significant conformational adjustments may take place when it binds to dna . next we analyzed the effects of two series of mutations on nfat5 dynamics which revealed two different outcomes produced by mutations from the same dna - binding loop . next we investigated the effect of three mutations r217a , e223a , and r226a which were experimentally shown to reduce the nuclear according to the ibis database , r217 and r226 residues make contacts with the dna coding strand and these contacts are invariant in many nfat transcription factors , whereas an invariant e223 residue binds a noncoding dna strand . a comparison of native and triple mutant complexes with dna shows that , while three residues in each chain lose their specific and nonspecific contacts with dna ( all three mutations occur in the dna - binding loop ) , the structure undergoes local conformational changes to maximize the contacts with dna and to make new contacts which are not observed in the native complex ( table s1 ( supporting information ) ) . namely , it loses 12 contacts in chain c and gains three contacts in chain d , some of which are located far away in sequence and structure from the specific dna - binding loop ( figures 3a and 6 and table s1 ( supporting information ) ) . interestingly , the loss of specific contacts with dna by chain c and the gain of contacts by chain d make the mutant complex more symmetric with respect to dna orientation ( figure 6 and table s1 ( supporting information ) ) . this results in a considerable drop in binding energy from 10 to 6.79 kcal / mol for the whole complex with dna for native compared to the triple mutant ( table 1 ) . although three mutations in the dna - binding loop are introduced in both chains , they make chain c somewhat more flexible compared to the native complex ( 1.49 0.06 and standard deviation of 0.52 compared to1.24 0.06 , standard deviation of 0.51 , p - value < 0.01 ) and have almost no effect on chain d ( figures 2b and 3b ) . the dna - binding energy drops from 3.50 to 2.82 kcal / mol and from 2.83 to 2.29 kcal / mol for chains c and d , respectively . moreover , loop 322332 which contains several lysines and makes partial contacts with dna in the native complex ( figure 6 and table s1 ( supporting information ) ) becomes more flexible and undergoes conformational movements in chain c ( rmsf 2.27 compared to 1.70 in native protein ) ( figure 2b ) . next we examined the effect of the phosphomimetic mutation t222d on dna binding in the presence of dna . the effect of t222d located in the same dna - binding loop is drastically different from the impact of mutations described in the previous section . as can be seen from figures 1 and s3 ( supporting information ) , the rmsd deviations from the initial structure are constrained for the t222d mutant complex with dna for two different md trajectories ; in fact , they are even lower than for the native structure almost everywhere along the simulation time . the t222d mutation makes the overall complex more rigid than native ( rmsf is 1.53 0.05 and standard deviation is 0.55 for mutant compared to 1.75 0.05 and standard deviation of 0.58 for the native complex ; p - value < 0.01 , figures 2a and 3b ) . while the ab loop does not show a noticeable conformational change , other regions undergo movements which lead to the formation of additional contacts with the dna molecule ( figure 3a and table s1 ( supporting information ) ) . while the loop 322332 on chain c is more flexible in the 3m_withdna system , on the contrary , it becomes more rigid in the t222d_withdna mutant ( figure 2b ) . moreover , a new salt bridge is formed between the substituted t d222 of chain d and lys329 of chain c , which restrains the motion between the two monomers ; the distance between the side - chain nitrogen of lys329 and the gamma - oxygen of thr222 was 11.9 in the native structure compared to 2.7 between the side - chain nitrogen of lys329 and the side - chain oxygen of asp222 in the mutant ( figure 7 and table s1 ( supporting information ) ) . although the overall binding affinity to dna almost does not change upon this mutation using the mmpbsa method , the binding between chain c and dna seems to be tighter than that of the native structure ( tables 1 and s3 ( supporting information ) ) . as can be seen from figure 4 , the first two or three eigenvectors contribute the most to the system motion and eigenvalues decrease in the following order : 3m_nodna > t222d_nodna > native_nodna > native_withdna 3m_withdna > t222d_withdna . similar to previous observations , dna binding makes the protein complex more rigid , especially for the t222d mutation . moreover , mutant complexes without dna are characterized by higher flexibility than the native complexes without dna . for the triple mutant , the situation is contrary and chain c is characterized by more extensive motions than chain d. projection of trajectories on the first two principal components is shown in figure 5 . as apparent from this figure , complexes without dna , especially 3m_nodna and t222d_nodna , sample much larger conformational space than the nfat5 complexes with dna . furthermore , projections for native and mutant complexes with dna are quite different from each other and the t222d mutant samples much smaller regions of the conformational space . the specific recognition of dna sequences by proteins is governed by the formation of hydrogen bonds with specific bases ( mostly occurring in the major groove ) , and by varying the dna shape and its electrostatic potential ( this type of recognition may happen in major as well as minor dna grooves ) . the nfat5 dimer represents a convenient system to study the difference between specific ( maintained by chain c ) and less specific or nonspecific dna binding ( maintained by chain d ) . structural analysis of the nfat5 native complex and md simulations show that the dimer does not significantly change its overall orientation with respect to dna in specific compared to nonspecific chains , which is consistent with previous experimental results on other dna - binding proteins . the specifically bound chain makes more contacts with dna , and its binding affinity is found to be higher compared to the nonspecifically bound chain ( the energy difference between specific and nonspecific binding is about 23 kt ) . some of these contacts , coming from the dna - binding loop and a few other regions , are different between the two chains and therefore may confer binding specificity . moreover , our results show that the nonspecific chain has a much higher flexibility in the native complex than the specifically bound chain . this can be explained by the extensive sampling of different protein conformations on the dna surface by the nonspecific chain which might be required before the specific chain binds dna . indeed , according to the energy landscape theory of protein dna binding , nonspecific protein dna complexes might have more rugged energy landscapes , whereas specific complexes can be characterized by the funnel - like energy landscape guiding the search to the native state . in the latter scenario , the unfolded flexible chain binds weakly at larger distances and undergoes a disorder - to - order transition as the protein recognizes the dna through specific binding . we studied different nfat5 mutants and found that they had distinctly different effects on specific and nonspecific binding to dna . the r217a / e223a / r226a mutant was characterized by significantly compromised binding to dna ( by 58 kt ) . although all of these mutations were introduced within the same dna - binding loop on both chains , a different effect on specific and nonspecific chains was obvious . namely , the triple mutations made the specific chain more flexible with almost no effect on the nonspecifically bound chain . since the nfat5 dimer forms a complete circle around the dna molecule , the loss of specific contacts with dna by chain c makes the orientation of the mutant dimer more symmetric inside the inner protein ring . on the contrary , unlike the three previously mentioned mutations , which enhanced the mobility of the specific chain and decreased the binding to dna , the t222d mutation made the specific chain / dna complex less flexible and more tightly bound . the overall effect of this mutation on dna binding was much less pronounced than the effect of the triple mutations ( even if scaled down by the number of mutations ) . this result points to the possibility that reduced binding to dna can not fully explain the reduced nuclear t222 is predicted to be phosphorylated by several methods although not yet confirmed by experiments . recently , an energy landscape approach was used to model the effect of multiple phosphorylation in the nfat1 protein regulatory region . the authors showed that phosphorylation increased the helical propensity and rigidified the structure of the phospho - peptides similar to the effect of the t222d mutation studied here . it was suggested that the cytoplasmic form of nfat1 needed a well - defined structure to perform its function , while the more flexible regulatory region was characteristic for the nuclear form . overall , the decreased binding of the nfat5 dimer to dna for triple mutant might explain its reduced nuclear localization reported earlier . however , it was proposed recently that nfat5 dimerization might be required for the nuclear transport , whereas dna binding might not . in addition , our binding energy calculations point to the marginal stability of dimers in the absence of dna , with the predicted dissociation constant in the mm range . our results allow us to conclude that dna binding considerably stabilizes the dimer complex and certain mutations may destabilize the binding to dna . all of this appears to contribute to the mechanism of nuclear cytoplasm transport .